Molecular and Supramolecular Chemistry of Natural Products and Their Model Compounds

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DieWahrheit der Ahnlichkeit istdieAnalogie.DieKrafi,solche Ahnl~chkeitenwahrzunehmenist der komb~natorischeGeist.DieSphure des kombinatorischen Geistes ist ~urchaus unbestim~t. aber es mu@eine ~ e t h ode geben, nach welcher dabei ve~ahrenwird. Diese~ e t h o d wird e Experiment~erensein.Wernachdieser ~ e t h o d e v e ~ u h r tder , darfsichdie k~hnstenVersuche erlauben. Er wird gewiJ3 auf Realitut stoJ3en. (The truth of similarity is found in analogy. The power to recognize such similarities is the combinatorial spirit. Its domain is undeter~ined,but its method clearly consists of experimentation. He who follows this eth hod is allowed the boldest enterprises. At the end he will certainly pounce upon reality.) Friedrich Sc~legel,1800

In 1800, in the age of early romanticism in Germany, it was known thatthe materials of biological tissues were difficult to isolate in pure form, that they decomposed readily and that it was impossible to synthesize them from simple help to unprecursors. A “vital force” of nonphysical character was invented aas derstand the complexity of life. Nevertheless, it was clear to the romantic spirit, which is a combinatorial spirit, that experimentation, not abstract speculation, was the only way to proceed withany artistic or scientific enterprise. With respect to natural compounds, experimentation concentrated on isolation, purification, and elemental analysisof carbon compounds. ning of the twentieth centuryall of the major natural products had been isolate~7 and the stereochemistryof amino acids and carbohydrates had become known in detail. The vitalforce had lost its reputation in chemistry textbooks. ~ i t h i the n last 100 years isolation andderi~atization,as well as synthesis of carbon compounds, have been perfected. Today, all the structuresof impo~ant natural products are known in great static and dynamic detail and many are commercially available.As discussed in this book, thousandsof reactions are known

...

iv

related to synthesis, decomposition, and functionof natural products in biological systems as well as in vitro. The term c o ~ ~ i ~ ~has, t o in~ the i ~ meantime, Z become a purely statistical oneinchemistry. It hasnothing to do with a combi~ationof different spirits in chemistry in orderto reach a universal accord. On the contrary,one could notbe more specialized and isolated than one is today at the frontiersof research. Combinations are, however7 possible andfruitful on the borderline between well-known terrains. Established knowledge and experimental expertise in natural product chemistry can be combined very well with established photophysics, the physical chemistry of intermolecular and surface forces, and all kinds of p h ~ a c o l o g i c a and l biological activities of membran~ systems. This book discusses the most important reaction-type in living systems: the reversible formation of noncovalent molecular complexes and membranes. Formation of cells, catalysis in enzyme~oenzyme-substrate complexes, charge separation in organized redox chains, the interaction of hormones with glycoproteins and proteins-all of these processes depend in first approximations on selective weak interactions betweennaturalproducts. The understandingand mastery of these processes are sometimes thought to be the most impo~antaims of contemporaryorganicchemistry. The reversible s y n ~ e s i sof noncovalent compounds is called synkinesis here. It employs the molecules of nature and their simplified model compounds sasy ~ t ~ (= o s~ ys ~ ~ i ~for o the ~ sconstruction ) of defined and functional molecular assemblies. In comparison with other textbooks on natural compounds, this book gives strongemphasistowater,membranes,andsolidsurfacesasreactionmedia. ~ y n ~ i n e sisi smuch more efficient and useful there than it is in homogeneous organic solutions. Furthermore, electrons, protons, oxygen, and water are taken as the most important reagents in synkinetic assemblies, the not various carbanions and carbonyl compoundsof synthetic chemistry. Why should one mimic partial aspects of the organization and chemistry of living orga~sms?Three major motivationscan be given. 1. Synkinesis opens a pathway to produce functional molecular assemblies from easily accessible compounds. Organic synthesis, which is drifting toward more and more exotic antibiotics, would find an extension that would make it possible to use the conventional, better known natural compounds as building blocksor synkinons. Natural products provide a large variety of properties, which makes them useful as molecular modulesfor the construction of functional assembliesby combination. Typical contrasts, which can be exploited and which are often found within a single molecule, consist, for example, of hydrophilic-hy~ophobic, soluble-insoluble, rigid-~e~ible, plus-minus charged, hydrogen bond donor-acceptor, D- and L-configured, and

fiat-curved or smooth-rugged surfaces. Intelligent experiments with like and unlike components and careful analysis of successful combinations will show what fits and stays together. Theory will help us to understand what has been found.The stepwise application of combinatorial assemblies beyond today’s liquid crystal and gasoline additive technology willjustify the efforts. 2. It hasbecomepossiblewithin the lastdecadeor so toanalyze supramolecularassemblies by using new techniquessuchassolid state NMR, scanning and transmission microscopy with nanometer resolution and UVNIS and IR- spectroscopy of molecular monolayers. 3. It is to be expected that within the next century renewable resources consisting of the primary and secondary products of photosynthesis, will become the basis of chemical technology. It is therefore time to learn about the organic chemistryof natural products in water as well as on the surface of semiconductor chips. Vectorial reaction chains running in membrane systems in aqueous media may allow construction of nanometer-sized devices, which are ableto produce hydrogen and oxygenfrom water and sunlight and be continuously repaired by synkinesis. Chiral membrane-type carrier systems may permit organselective drug transport. On anindustrial scale, molecular assemblies of cellulose degradation products may become the major basis of plastics. This book is intended for advanced studentsof chemistry and biochemistry who are interested in the fundamental structural properties and reactivity patterns of cornrnon natural products. Biological and material aspects guided the choice of subjects. The book may also be of interest to research chemists, because much work done within the last decade is described. For more than three decades I (J.-H. F.) had the pleasure of teaching and learning the noncovalent chemistry of natural products. I have to thank my students and co-workersfor never-ending trust and enthusiasmon slippery ground. Seemingly endless syntheses of analogs, diastereomers, and enantiomersof amphiphiles and bola-amphiphiles finally allowed synkineses of well-defined molecular assemblies. Their molecular structures were often accessible only through the development of scrupulous staining procedures and tedious cryo-electron microscopy in connection with solid state IMMIX spectroscopy. Elucidating crystal structures of the waxy materials helpedus to gain a footing. Mrs. Regina Stuck typed the manuscript several times and was incredibly patient. Mrs. Andrea Schulz organized the references, whose tracks I had often lost. I am verygrateful to both of them, to the whole of the institute of organic chemistry of the Free University of Berlin and its funding agency FNK,

i

as well asto the Deutsche ~orschungsg~~einschaft, which supported our work generously. The book was writtenby one of us (J.-H. F.); the other (C.E.) producedthe drawings. The author of the text is most indebted to his wife, Jutta, without whose constantlove and patience this book would not have been written.

iii

Preface S

1.1 ~ntrod~ction 1.2 Resources, Structures, and Confo~ationsof Natural Products l .3Synthesis 1.4 Reactivity 1.5 Synkinesis and Analysis of Molecular Assemblies

1 3 20 24 35

2.1 Introduction 2.2 Resources, Structure, and Physical and Physiological Properties 2.3 Isolation and Syntheses 2.4 Reactivity 2.5 Fluid Synkinetic Membranes 2.6 Surface onol layers 2.7 Edge Amphiphiles asPores in and Harpoonsas Disruptors of Lipid Membranes

61 61 81 86 94 113

3.1 ~ntroduction 3.2 Structure and Physical and Physiological Properties 3.3 Synthesis 3.4 Reactivity 3.5 Synkinesis 3.6 Surface Monolayers

129 129 140 147 156 163

121

viii

4.1 Introduction 4.2 Resources, St~cture,and Physical and Physiological Properties 4.3IsolationandSynthesis 4.4 Reactivity 4.5 Synkinesis Using Monosacch~desas Guests in Water or Amphi~hileswith Carbohydrate Headgroups 4.6 Monolayers 5 l Introduction 5.2StructureandSpectra 5.3ResourcesandSynthesis 5.4 unction andReactivity 5.5 Syn~neticReactions

167 169 201 214 224 237

243 244 256 259 259

265 6.2 The Electronic Structures of Porphyrins and Metalloporphy~ns 6.3 Synthesis 6.4 Reactivity 6.5 Photochemistry 6.6SynkineticReactions 6.7Reactive Metallopo~hy~ns olecules, Molecular Monolayers, and Multilayers on Water and Solid Surfaces 6.9EnergyConversion

7.1 Introduction 7.2RedoxCatalysts-Occurrence,Structure,and Physiological Properties 7.3 Activation of Small Molecules

Introduction 8.1 8.2IsolationandStructure 8.3 Reactivity of Inte~ucleotideLinkages in DNA and RNA and Analytical Methods 8.4 Synthesis

266 283 293 305 310 321 327 331

37 337 338 372

3 395 396 411 417

nte

i

8.5 Reactivity 8.6 Synkinesis

428 437

9.1 In~oduction 9.2Occurrenceand Structure 9.3 Synthesis 9.4 Reactivity 9.5 Synkinesis with Amino Acid Derivatives 9.6SynkinesiswithProteins

463 464 480 49 1 501 504

~efe~en~es

Index

527 585

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The classic sequence of experiments with natural compounds in the twentieth century has been as follows: isolation frombiological sources, purification, elucidation of molecular structure, and finally total synthesis. These tasks of organic chemistry were fulfilled by the end of the century asfar as the major components of higherorganismsareconcerned.Onlycompoundsthatarecentraltothe growth of their tissues arethe subject of this book. Many individual compounds of this kind of naturalproduct,namelylipids,steroids,carbohydrates, carotenoids, porphyrins, vitamins, nucleic acids, and proteins, are today commercially available, and theirstructural and dynamic analysishas reached an accuracy and diversity that leaves little to be desired (Karrer, 1954; Fieser and Feiser, 1960; Tedder et al., 1972; Nuhn, 1981; Fuhrhop, 1982; Beyer and Walter 1988; Fuhrhop and Penzlin, 1994; Mann et al., 1994). The most impo~antby-product of the analytic and synthetic work accomplished so far is knowledge about the stereochemistry and reactivity of natural compounds. There is an enormous potentialfor the chemists of the twenty-first century lying in the mastery and applicationof this knowledge in order to produce organized and finally functional materials. Typical contemporary examples include surface monolayerson metals and colloids made of fatty acid and steroid derivatives, the regio- and stereoselective assembly reactions between steroids and carbohydrates, coupled redox chains of metalloporphyrins and vitamins, noncovalent fibers made of amino acids, nucleotides, and saccharides, and the functionalization of proteins by incorporation of reactive molecules.The field of supramolecular or noncovalent natural compound chemistryhas been scientifically f ~ i t f ufor l several decades andis presently exploited for the development of useful molecular devices and machines as wellforasmedical applications.

The eight chapters of this book deal witheight classes of natural products common to biological organisms and simple model compounds. Each chapter willfirstdescriberelevantstructuralanddynamic details, whichshould be known to the architects and engineers using molecules as architectural building blocks or modulesfor machinery. Sections on important synthetic strategies and typical reactions will follow. The latter are often describedin the contextof biological activities of the molecules. Poisons and antibiotics, which constitutethe most important target molecules of contemporary synthetic chemistry, will be mentioned only occasionally.We concentrate here on functional compounds and building blocks, noton molecules, which block productive interactions. The last part of each individual chapter then assumes that one central goal of contemporary natural compound chemistryis the production of noncovalent molecular assemblies and membranes. Natural products and their model compounds are considered here as synkinons in synkinesis, which is the reversible synthesis of complex molecular assemblies. Examples for contemporary target assemblies of synkinesis are chiral cholesteric phases for color display and carbohydrate recognition, molecular machinery for light-induced charge separation based on lipid membranes and porphyrins, and compact nucleic acids with receptor-specific lipid coatings for the transportof genetic material (Fendler, 1982; Fuhrhop, 1982; Fuhrhop and Koning, 1994). Possible applications range from cell surgery by membrane-coated enzymes, antibiotics,or nucleic acids to combinatorial synthesesof millions of drug molecules on the surface of silicon chips, from ultrathin battery layers to stereoselective ultrafiltration membranes. Biological, pharmaceutical, and medical applications will, however, only be mentioned occasionally, when simple physical or supramolecular effects are involved. More complex mechanisms involvingenzyme control, di~erentiation,and neural nets are not within the scope of this book (Metzler, 1977; Jungermann and Mohler, 1980; Albertset al., 1986; Forth et al., 1987;Voet and Voet, 1990). The pathway of the book as a wholeleads from less reactive,chiral molecules,whichareusefulasmembranecomponents(lipids,steroids,carbohydrates), to molecules that react reversibly with light and electrons are helpful and in energy conversion (carotenes, porphyrins, redoxactive vitamins), and finally to helical and reactive biopolymers (nucleic acids, proteins), which are used as frameworks for molecular machinery. Natural compounds that do not form important supr~olecularassemblies or have not been used extensively as model compounds (e.g., alkaloids, antibiotics, metabolites) are not treated in separate chapters, but appear occasionally. All chaptersare subdivided into at leastfour sections: Isolation and structure Synthesis Reactivity Synkinesis

A monolayer section and some special sections are then occasionally added (e.g., catalysis inthe case of porphyrins). This first, introducto~chapter is intendedasareview of somebasic knowledge about thesefour aspects of natural compound chemistry.

rc The major sources of natural products are cattle (lipids, protopo~hyrin,bile, pigments,nucleicacids,collagen,enzymes),eggs(lecithin,cholesterol), microorga~sms(vitamins, proteins, nucleic acids), and plants (fats, phytosterols, carotenes, carbohydrates). Synthetic natural products usuallydo not come from total synthesis, but are industrially obtained by partial syntheses starting with related natural products (e.g., steroids from cholesterol, ascorbic acid from glucose). So far all these compounds stem from renewable resources, not from mineral oil. Total synthesis basedon mineral oil has been commercially successful in only a few cases. One example is given the carotenes, where the Wittig reaction made things easy. Another example is the steroid norgestrel (used in birth control pills), in which a nonbiological angularethyl group providedthe unique effectiveness of an oral drug. Model compounds,on the other hand, are mostly made by total synthesis. They are often preferred by chemists and physicists because they are easyto obtain on a gramscale from commercial precursors. Even easily accessible natural products are replacedby model compounds. Protoporphyrin and chlorophyll,for example, canbe isolated on a large scale fromcattle or spinach. Their substitution pattern is, however, irregular, and chemical reactions lead to complicated mixtures. Furthermore, they contain chemically labile substituents, such as conjugated vinyl substituents, an isocyclic ring with a p-ketoester group, and a water-labile magnesium central ion. Substitution and redox reactions are much simplerto perform with ~-octaethylporphyrin;for large synkinetic assemblies ~ e ~ ~ - t e t r a p h e n y l p o ~ hderivatives yrin are more suitable.

0.96 (OH) Covalent bonds in biological carbon compounds vary in length from to 1.54 (CC). In other words: the variability is not more than *20%. The rnedian length is about 1.25 (Table 1.2.1). Longer bond lengths are only found in nonbiological bonds (e.g. 2.14 in CI) or in metal complexes and oxides (e.g., 2.1 A in Fe(I1)O). The bond anglesof carbon-carbon bondsrise with the multiple bond character. The angle is 109" for single bonds, and somewhat larger for double bonds (1120").It disappears inthe triple bond (180°), which does not occur in common

Typical Covalent Bond Lengths Ain SingleTriple H-C H-N H-0 C-C C-"

c-0

C-Cl C-Br C-I

1.09 C=C 1.00 0.96 1.54 1.47 1.43 1.76 1.94 2.14

Double

1.35 C=N 1.30 C = 0 1.22

CEC 1.20 CEN 1.16

The all-anti or zigzag conforrnation ofan oligornethylene chain.

naturalcompounds. In zigzagconfigured chains of n carbon-carbon single bonds, the C-C distance in the chain directionis 1.25 A and the end-to-end distance is 1.25n A.The chain configurationis, however, flexible,and in a statistical coil containing all kinds of conformers(seenextsection)theaverage distance d,, between chain endsis equal to 1 . 2 5 G Covalent bond energies are close to 100 kcallmol in single bonds, 150 kcallmol in double bonds, and 200 kcallmol in triple bonds in natural compounds (Table 1.2.2). Hetero atom-hetero atom single bonds (e.g., in hydrazine, peroxides, disulfides, and elementary halogen molecules) have bond energies of less than 50 kcallmol. They tend to homolyze spontaneously, giving two radical moieties with an unpaired electron each. Themal energy is expressed in kcal/mol or is multiplied by a factorof 4.2 to yield U/mol. Electrical energyis given in electron volts (eV), which is directly connected to the electrochemical potential in volt (V) for one-electron transfers. The volt scaleof electrochemistry is connected to the hydrogen electrode, which differs from the electrical eV scale by a constant of 4.5 V. This diaerence is, however, of no

ivit Covalent Bonds in Larger Molecules (in kcaVmo1)

importance if one considers only differences of electrochemical potentials, not the absolute values.If n electrons are exchanged in an electrochemical reaction, the potential difference must be multipliedn by in order to obtain the energy diEerence in electron volts. Photochemical energy is best expressed in frequencies (cm'; cles per second). Useful approximate conversion factors are (for one mole): 1 eV = nV for a transferof n electrons = 23 kcal = 96 H = 8000 cm1 Furthermore, [cm1]= lo7I [nm] Consider the following two examples: 1. One mole of light quanta corresponding to a wavelength of 500 nm (= 20,000 cm1) containsthe same energy as 1 mol of a one-electron redox pair with a potential difference of 250 mV and may cleave one mol of a molecular complex with a bond energy of 58 kcaVmo1. 2. In the reaction of molecular hydrogen and oxygen, four electrons are exchanged and the difference in oxidation potentials is about 1 V, producing 92 kcaVmol or one molof 3 13 nm light quanta.

Carbon-carbon single bonds cause flexibility of molecular shapes, since rapid rotations around the single bond occur at room temperature and change the relative positions of substituents to each other.h infinite number of conformers is formed. Dynamicrecognitionprocessesbetweenlipids,steroids,proteins,andcarbohydrates strongly depend on conformational changes in their carbon skeletons and the accompanying reorientationsof substituents. ~ e m b r a n epermeability for ions, enzyme catalysis in surface clefts, and signal activity of steroids and neurotransmitters

are quite often a consequence of enforced strained conformationsin molecular ass defined semblies (Frelog, 1971; Quinkert et al., 1995).All attempts ats y ~ n e s iof molecular assemblies (section 1.5) must therefore develop analytical tools for and take careof confo~ationalpreferences occurringin the anticipatede n v ~ o ~ e n t . onf formations are formally described for four connected atoms in the order AXYE A and B are those substi~entsat centers X and Y that c m the highest priority in theC~-Ingold-Frelog(=CIF) notation.It is then the torsion angle8 separating A and B that describes the conformer.If 8 is not known exactly, the Newman projectionis used for denominations in the followingway: the circleis divided into six sections correspondingto the close e n v ~ o ~ ~ofnperipZanar ts and clinal confo~ations. ~eripZanar @eri = around, about) means that A, X, U, and B are all in a planeor only slightly away fromit and cZinaZ that B is rotated far away from the A,X,Y-plane (clinal = inclined). Flus and minus signs as well as syn and anti designations are usedto define left- and right-hand sensesof the B displacement againstA, the latter always being fixed at the12 o'clock position. Syn and anti indicate thatB is either in the same upperhalf as Aor in the lower half of the circle. As a result, one obtains one pairof periplanar conformations, namely syn~eripzanar(=sp; close to ecliptic) andantiperiplanar (=ap; close to anti). If A andB are chemicallythe same groups, one often uses trans instead of B are about60"apart: two ecliptic anti. There arefour conformers in which A and ( f tac) and two staggered confo~ations(ftsc). The Greek word anticZina2 also indicates a saddle and ~nclinaZmeans in a groove. Both pictures together imply inclining slopes and relate nicely to the energy states of these co~ormers(Fig. 1.2.2). In the case of open-chain biological compounds, one only needs to consider ASC and kap confo~ations.The conformations approaching the e c Z i ~ s e ~ cases, namely ASPand -c, are to be taken as high-energy transition states and occur only as shortlived intermediates in rotational isomerizations. One cannot decide on a preference between the favorable ~ S or C ap conformations on an a priori basis. These two cases must be distinguished by experiments, usually by measuring coupling constants in ~~R spectra (see Fig. 1.2.14). X-ray analysisis of limited use,since crystal forces often enforce linear conformations of polyfunctional chains, which are not found in solution or in nonc~stallinemolecular assemblies. Intermolecular forces are invariably more important incrystals than in curved supr~olecularstructures. For butane and most compounds of the form YCH2CH2X,antiperiplanar conformers are the most stable ones.The long alkyl chainsof fatty acids areusually also aZZ-anti or aZ1-trans conformers in molecular assemblies (Fig. 1.2.3). If the substituents are, however, electronegative atoms, in particular oxygen, the syncZi~aZ(= gauche) form usually dominates. Important gauche effects (= the tendency to adopt confo~ationswith the mum number of g a ~ c h einteractions between adjacent electron pairs and/or polar bonds) are found in peroxides,

tterns in Natural ~ o m p o u n ~ s

tive

negative

0"

180" Derblanar

s (SYn) a (anti) C (ciina~) P (~erip~anar) periplanar 1.2.2 The description of conformers of an A-X-Y-B system. First row: definition of torsion angle Cp. Second row: definition of left- and right-handed as well as of syn(cis) and anti (trans).Third row: definition of defined conformers, always relatingto the relative position of A and B.

Energydiagramfortheconformers of butane,Themostprominent are given below. stretched and cyclic (Fischer) conformers

carboxyl acid derivatives, openchain carbohydrate derivatives, and several related compounds (see Fig. 4.2.6).

cli If n-hexane is bent to an aZZ-ecZipse~(=Fischer) conformation, one obtains a planar cycle with overlapping endings. Formation of cyclohexane by formation of a carbon-carbon bond between these endings would lead to a widening of the bonding angles from 109" to 120" (Baeyerstrain) as well as to strong steric interactions between the substituents, which would be all in the same plane and therefore in ecEiptic positions (Pitzer strain; Fig. 1.2.4). In order to release the Baeyer and Pitzerstrains, cyclohexane folds in away that all hydrogen and carbon atoms slip into a gauche conformation. The six n-butane units, which now make up the cyclohexane ring, are inthe +sp conformation. At each carbon atom one finds oneaxial and oneequatoriaZ hydrogen atom. If the hydrogen atoms in cyclohexane are replaced by large substituents, the conformer with mostof them in equatoriaZ positions is preferred. The chair conformer is about 5 kcal/mol more stable than corresponding twisted confomers. The activation energy needed to get these high-energy conformersis about 12 kcal/mol (Fig. 1.2.5). Non-chair conformers therefore never exceed a molar fraction of about 0.1% at room temperature, but they may be enforced in molecular complexes (see Figs. 9.6.6 and 9.6.7). The situation changes drastically if a double bond occurs. In cyclohexene

109"

Conversion of open-chain,all-anti hexane to the helical all-cisconformer and to the (non-existent) planar and chair closed-ring Conformers.

The major confo~ationsof the cyclohexane ring. From chair, high-energy twist boat,boat, low-energy twist boat, chair.

left to right:

The major half-chair conformers of cyclohexene.

there are two e ~ ~ a t o ~(e) i aand l axial (a)bonds at C4 and C5. The allylic carbon substituents are called ~ s e ~ ~ o e ~ u a(e’) t o and ~ i ~~ Zs e u ~ o (~ a’)i.The ~ Z activation energy of conversion drops from 12 kcallmol in cyclohexane to 6 kcallmol in cyclohexene (Figure 1.2.6). Two major half-chair conformations occur. The first corresponds to a chair with respect to C4 and C5, the other to a pseudo-boat, if one looks at C3 and C6. Confo~ationalenergy differences of substituents in cyclohexene units are in general considerably lower than in cyclohexane derivatives. The same holds true for cyclohexanones,where a methyl group at C3 has only a AG value of 1.3 kcallmol instead of 1.8 kcallmol. A planar cyclopentane ring is without angle or Baeyer strain becauseC-C the

.7 The major conformers of cyclohexanone. Twist boat and boat forms become stable conformers.

bond angles are 109" in a planar pentangle. It suffers, however, much Pitzer strain because allsubsti~entshave ecEipsed positions. The ring relieves this strain by positioning oneof the five carbon atoms 0.5 A above or below the plane to form either an enveZope-shapedor a~ a ~ - cconformer. ~ i r In both forms, however, itis not always the same atom that moves out of the plane, but the distortion migrates by a vibrating of thecarbonatomsthroughthering. 'Ibis cyclicprocessiscalled "pseudorotation" and knows no energy barriers at room temperature. The carbon atoms in cyclopentane move freely up and down. One major of this effect pseudorotation phenomenonis the difficulty of cyclopentane derivatives to pack in crystals and consequently their extreme solubility. Two examplest eare ~ ~ y d r o (m) ~an andproline. "Wmixeswithwater in allproportions,whereasopenchaindiethylether, with only two more hydrogen atoms, separates from water. Proline is by far the most soluble amino acid, although it has one methylene group more ala- than nine and noOH group like serine (see Table 9.2.3). The splitting of DNA double helices depends on the flexibility of the five-membered furane ring. The most frequent cyclopentane conformations show carbon atoms that carry two substituents equally disposed above and below the ring plane (Fig. 1.2.7). These are called isocZina2 (i). Substituted cyclopentanes usually do not show significant conformational preferences.cis- 1,3-Dimethylcyclopentane,for example, is only 0.5 kcaVmol more stable than thetrans isomer, compared with an approximate 2k c a h o l difference betweenthe 1,3-dimethyl-cyclohe~anes.

a ur chair (right).

Themajorconformers

of thecyclopentane ring: envelope (left) andhalf-

Molecules and molecular assemblies undergo energy transitions at defined resonance energies upon irradiation with appropriate electromagnetic waves. The theoretical and experimental detailsof the various spectroscopies are discussed in many modern textbooks and reviews. We shall limit the discussion here to a few remarks on those aspects of the final spectra that are importantto the analysis of conformational changesof natural products and their intermolecular interactions in assemblies. Spectra are arrays of absorption bands. Such bands are at first characterized by three numerical values, namely position in an energy scale, relativeintensity,andlinewidth(Fig. 1.2.9). Thesenumericalcharacteristicsareoften supplemented by a description of the form or splitting of the band. Energy and intensity of absorption bands are specific properties of molecules and of func-

Characteristics of resonance absorption bands in spectra. A s an example the band correspondingto an electronic excitation of a n;-system withlight is given. E = Extinction coefficient; h, = wavelength of absorption peak; h,,,= linewidth at half maximal absorption. In NMR spectra one would speakof chemical shifts, integrals, and linewidths instead.

ivit

tional groups and vary only upon strong electronic interactions of the chromophores with other molecules in the environment. Broadening of absorption lines, on the other hand,is a quite general consequenceof aggregation. 1.2.5.1

UVNis Spectra

Excitation of conjugated x-electron systemsby light is the most significant single interaction of living systems with their environment. Photosynthesis and vision are the most obvious examples (Hoppe et al., 1982). UVlvis or electronic spectra are obtained by measuring the relative absorption of monochromatic light in dependence of wavelength. There are two major types of organic chromophores: polyene and aromatic n-systems.AZZ-trun~configured polyenes typically produce two or three broad bands within a spectral range of about 100-200 nm with extinction coefficients around 104-105 and halfwidths of about 100 nm. Symmetrydisturbances by cis-double bondslead to extra short-waveleng~ bands. Aromatic chromophores,in particular porphyrins, benzene, and pyridine derivatives, produce spectra with an intense short-wavelength band and several well-separated bands at longer wavelengths (Fig. 1.2.10).The extinction coefficient E of the short-wavelength band is in the order of 2 X 105/mol, whilethe longer wavelength bands have intensities that are 10 times weaker. The half width of the absorption bandsis much narrower than with polyenes andis usually around15-30 nm. The sharpening of the bandsof aromatic compounds with respect to those of polyenes is caused by rigidity of the cyclic compounds: the planar conformation of the ground and excited states is the same in aromatic chromophores, whereas a population of different bent and twisted conformers may occur in the polyenes. The intensity difference between high-and low-energy bandsof the aromatic compounds occurs because the highest occupied molecular orbitals (HOMO) and lowest unoccupied molecularorbitals ( L ~ are~ ) both degenerate.This leads to an allowed transitionat high energy and some forbidden transitions at lower energy. The degeneracy of the outer orbitals is also responsible for the validityof the Huckel rule, which states that cyclic and planar conjugated n-systemsare particularly stableif they contain 4n+2 n-electrons. 4n n-electrons are on the degenerate orbitals; the +2 comes from the lowest energy level, which is not degenerate. 4n+2 n;-electrons thus produce fully occupied binding orbitals. Cyclic systems with 4n n-electrons have a single electron on each of the degenerate HOMO levels. These are unstable biradicals or triplet states and do not occw in the ground statesof natural products. The wavelengths of n,n* transitions are not only determined by the type and extensionof conjugation pathway butalso by the presence of terminalelectron donors and electron sinks. Long-wavelength or bathochromic shifts occur when the chromophore contains an electron donor at one end and an acceptor at the other end. Typical electron donors are amino groups; typical acceptors are carbonyl groups. In the absence of special effects, about 10 conjugated double

\ \ \

l

E

1 c

-

l l

\ \

Polyene-type spectrum of sorbaldehyde showing only one broad band for the n-system and-aromatic-type spectrum of benzene. An intense absorption band at low wavelength (high energy) is followed by several less intense (“forbidden”) absorptions at longer wavelengths. Broadening of absorption bands usually either indicates more flexible or distorted or aggregated chromophore. Such a broadening of the bands leads to smaller extinction coefficients, whereas the area of the absorption bands remains about constant for a given chromophore.This area is related to the so-called transition moment, which can be approximated by the product of extinction coefficient and linewidth.

.’l ’l The color of indigo is causedby a double-crossed conjugation systemof two NH electron donors and two CO electron acceptors. The benzene rings contribute very little. bonds are needed to produce a dye absorbing above 400 nm. There also exist many dyes with much shorter conjugated Iz-systems. Special interactions between chromophores, e.g., cross-conjugation of two dienes or trienes, or electron repulsion then lower the transition energy.The most p r o ~ n e nnatural t product of this kind is indigo, with two crossconjugated amino-ketone chromophores (Fig. 1.2.1 1) (Sawicki,1970; Hesse et al., 1984). The absorption by a dye in solutionat any wavelength is directly proportional to the concentration of the dye (Beer’s law). The proportional factor is called the extinction coefficient,or E. Deviations from Beer’s law point to aggregation phenomena. 1.2.5.2

Circular ~ i c h r oSpectra i~~

ITV/vis light passing a solutionof a chiralChromophore becomes ellipticallypolarized. Circular dichroism (CD) spectra correspond to the difference of the absorption coefficients E,&, of left- and right-handed polarized light. The unit of CD spectra is molar ellipticity and is given in deg cm2/dmol. Single molecules with chiral centers close to the chromophore have molar ellipticities of a few hundred to thousands;helicalchromophores or helicalassemblies of chromophores reach several millions (Hesse et al., 1984).

l

I

0 CD spectraof B- and C-DNA (see Chapter8).

1.2.5.3 FluorescenceSpectra

If a rigid chromophoreis excited to its singlet state,it may emit light when the excited electron returnsto the ground state. Some energyis lost in this process, and fluorescence emission occursat longer wavelengths than the longest wavelength absorptionof the given chromophore. Typical examples are found in metalloporphy~ns.The longest wavelength absorption occurs at around 550 nm and fluorescence emission around 620 and 670 mm. Absolute intensities of fluorescence bands in the sense of a molar emission constant cannot be given, since self-quenching occurs at higher concentrations and eer’s law is not followed. elative band intensities or “fluorescence yields” at onstant, very low concentration (
ctivi

increase in luminescence when molecular oxygen is removed from the solution. This phenomenon usually means that the radiation comes from an excited triplet state, andit is then called phosphorescence. Whereas strong absorption bands are detectable down to concentrations of about M, fluorescence spectra can often be detected at concentrations as low as lom9 M. Picograms of a dye can thusbe routinely characterized. Fluorescence spectra are most useful for the detection of inte~olecularinteractions between a fluorescing and a quencher molecule. Another important application is in the analysis of molecular interactions within large assemblies of fluorescing dyes (see Sec. 1.5). 1.2.5.4

~ ~ f r a Spectra re~

The thermal vibrationsof atoms in molecules leadto absorption bands inthe infrared (IR) region (Bellamy, 1964; Colthup et al., 1964; Hesse et al., 1984). I bands are most intense if a dipole is induced by the vibration (OH, NH, C C=O, C=N). The mass of the interactingatoms M1 and M2and the bond strengths definedby a force constant f deterrnine the wave number n or energy of d absorption band: v = ~ ( f ~ ~ ) where 1 ' 2 , the reduced mass is and K is a constant conversion factor. The frequency ation is around 2900 cm1, for C=O close to 1700 c bonds lead to a broadening and low-frequency shift of OH and (3400 cm1-+ 3200 cm'). The relative intensities of I bands are indicated by three letters: namely S ,and W (weak). Quantitative extinction coefficients are not C=O, etc. bands show essentially identical values in very Exchange of protons for deuterons leads to shift a to higher frequencies by about 300 cm'. Other isotope effects are much less pronounced the mass ratios are smaller. odern computerized IR spectrometers can measure and average out several thousands spectra within a few rninutes and are therefore very sensitive. olecular monolayerson a square centimeterof solid substrates can be routinely measured (Ulman, 1991;see also Sec. 1S ) . The amount of ater rial needed to spectrum is in the orderof nanograms,

a1 compounds are IH and 13C with nuclear spins 14N with spin +l and 0 (Noggle 197 1;Jackman unter,1978;Kaplan amson1989;Sander emical shift with respec

(a) Partial 400 MHz 1H-NMR spectrum of the methine protons of testosterone. A hopeless case for assignment. (b)Decoupling and~nuclearOverhauser enhancement effects can be usedto disect the multiline, one-dimensional spectruminto a variety of t~o-dimensionalspectra. Assignment of signals to individual hydrogen , atoms can thus be achieved. (From Sanders and ~ u n t e r 1993.)

atural ~ o r n ~ o u n ~ s

carbon signals of the reference compound tetramethylsilane, Si(CH3)4. These shifts are proportional to the applied magnetic fieldof the spectrometer. The resonance frequency of modern spectrometers is usually around 100-600 megahertz (MHz). The exact chemical shift in parts per million (ppm)of the applied frequency with respect tot e t r ~ e t h ~ l s i l a nthen e depends on the chemical environment of the atom. Protons on saturated carbon atoms, for example, appear between 1 and 2.5 ppm when there are no heteroatoms on the same carbon atom. Increasing the elec~onegativityof a substituent causesa shift of the proton signal to the left (downfield or paramagnetic shift) by up to about 10 ppm. Ring current effectsof aromatic compounds are considered in Sec. 1.5.7.4. In the case of simplecompoundswithwell-differentiatedhydrogenandcarbonatoms,one may take the chemical shifts from the literature and then assign signals to specific atoms by simple comparison (Jackman and Cotton 1975). If, however, a

"14 elations ship between the coupling constant of protons on adjacent carbon atoms to the dihedral angleCp.

compound produces many signals in a narrow region (Fig. 1.2.13a), the spectrum must be dissected. This is achieved by a large variety of two-dimensional R techniquesusingdecouplingandnuclearOverhauser e~ancement (=NOE) effects and rsHunter, 1993). Afterthesignalshavebeenassigned,onecanapplythe curve (Fig. 1.2.14) to deduce molecular conformations from the hyperfine coupling constants arising from interactions with neighboring protons.The separation of peaks in a multiplet signal is zero if the interacting protons at adjacent carbon atoms open a dihedral angle of 8 = 90". Itis about 10-15 Hz at 8 = 180". In open-chain compounds the rotation about single bonds may lead to a population of several different conformations andthe separations of peaks corresponds to an average of several coupling constants. In such case a computer simulation of the expe~mentalspectrum using established programs like ALTONA provides a direct means to calculate the fractions of the different conformers (see, e.g., measurements routinely need a few mg of substance, but 100g! may also be sufficient.

rganic synthesis achieves the f o ~ a t i o nof carbon-carbon bonds in order to prouce larger carbon compounds from smaller ones (Fuhrhop and Penzlin, 1994). The formation of thelinearcarbon-carbonskeletons of lipids and carotenes usually depends on the reaction of the electropositive carbonyl carbon atoms of aldehydes or ketones with carbanions. Most popular is the ~ i t t i greaction between a phosphorus ylene as obtained from alkylbromides and an aidee to form alkenes. Catalytic hydrogenation then converts alkenes to alkanes. e reaction has been adjusted to all kinds of functional groups, which can be connected to both educts, namely aldehydes and bromides (Scheme 1.3.1). The major structural motif of steroids is the decalin unit, whichis readily accessible by the Diels-Alder reaction.A famous early exampleis its maste~ul .€3.~ o o d w ~Ad methyl . quinone was reacted with butadiene to cis-decalone, a possible precursor for bile acids and after base treatment the t h e ~ o d y n ~ i c a lmore l y stable trans diastereomer, whichmay be applied in h o ~ o n esyntheses. Another classical approach to substituted decalins is the binsonannelation(Scheme 1.3.2), consisting of asequence of aldoland chael additions. onomeric saccharides, nucleic bases, and amino acids are in general ~ommerciallyavailable. The major synthetic problem with these natural compounds lies in the development of high-yield condensation reactions, which allow automated syntheses of poly(aceta1s) or polysaccharides, poly(phos-

Br

phodiesters) or polynucleotides, and polyamides or proteins. Qpical condensation examples are given in Scheme 1.3.3. They do not include synthetic reactions (CC bond formation), but correspond to reversible group inversions; a carboxylic acid is changed to an ester, an amide, etc. The synthons of p o ~ h y r i nsyntheses are the pyrroles, which in turn must be made from 194-difunctional synthons. These carbon skeletons are available by an aldol-type condensation of the enol of a 1,3-diketone with an ~-nitrosylated acetoacetate ( norr pyrrole synthesis, cheme 1.3.4). The final reductive ring closure by Schiff base formation is again a reversible condensation reaction. After dehydration, however, a stable n-el formed, which gives the res~ltingpyrrole "aromatic,' stability. this enami~ecan now only occur in very strong acid. In water ty it isperfectly stable. rin synthesis then relies on the reaction of the electronrich pyrrole units (six It-electrons for five atoms) with ~ - p y r r o l carbon i~ substituents a goodleavinggroup,e.g.,benzyl-typehydroxylor amino s u b s ~ ~ e n tfor s , example, split off water in the presence of p produce reactive carbenium ions. No strong Lewis acids are needed as catalysts-acetic acid is sufficient. The ~-carbenium group i pyrrole molec~lesleading to m~thy1ene"bridgedpyrrole polymerization grade of four is reached, intram~lecularcondensation becomes much more likely than further oligomerization (ne and a strain-free macrocyclic tetrapyrrole unit named formed in high yield. This is then oxidized by molecular o *

0

H

+

0ch3

2 H3C-OH

H

0ch3

0

R-0

R-0

i-‘R-OH

‘pNo

no‘o’n

+

R

R

’R-NH;!

O

0 NOH

0

0

H3N-R

0

OH

+

0

“R

The quantitatively mostimpo~anttypes of biological reactions are condensation reactionsandhydrolysis ( F u ~ h o p ,1982; Fuhrhop andOning, 1994). The reagent is water. As a chemical it is quite inert, but canshift protons and dissolve salts. Its exchange rates from the first coordination sphere of a metal ion vary e2+>and finally lo9 S-' !As a nucleop~lein organic chemistry, howactions mustbe catalyzed; eventhe h y ~ a t i o ~ of the extremely electrop~liccarbon atomsof aldehydes needsthe help of extra on the formyl oxygens before they add water. itrogen of amines, on the other hand, is quite a good nucleophile pro-

tlvi

vided it is not protonated. Amidation is never executedvia the ammonium salt as suggested by Scheme 1.3.4. The acidic hydroxyl group is rather replaced by a X should be good leaving groupX (e.g., acetate, sulfide, phosphate, or chloride). able to carry a negative charge atpH 7; hydroxyl and amide ions cannotdo this (Scheme 1.4.1). Nonenzymatic amide hydrolysis is, except at extremes of p and temperature, a very slow process (Smith and Hansen, 1998). The half-life for a typical peptide bond hydrolysis in water at pH 7 and 25°C is in the range of 143-8 17 years! The enzymatic arnide formation and hydrolysis is well understood, but convincing model systemsdo not exist. Thereis no recipe so far that describes how to lead the educts efficiently along the assumed reaction pathway and to hydrolyze an arnide at neutral pH. eterocycles are almost invariably formed by inter- and intramolecul~ Schiff baseor lactam formation, We cite here the classical b o r r pyrrole synthesis (see Scheme 1.3.4) and Baeyer's barbituric acid synthesis, where the amide nitrogen atoms of urea are nucleophilic enough to add to malonic acid esters (Scheme 1.42).

+

R'

R*"-

NH2

-4-

H"R2

x=

-61

~ 0 P O 3 H ~ O ~ O 3 H - 0 ~ O 3triphos~hate H2

HX

In~oductionof a good leaving groupis, however, notthe only way to steer a reaction.In enzymatic and intramolecular reactions, neighbor group eEects are as important. The pathway chosen by the anGno nitrogen to the C=O double bond in addition reactions has, for example, been reconstructed from crystal structures. The intr~oleculardistance between the nucleophilic nitrogen atom and the electrophilic carbonyl carbon atom varies in three alkaloids selected from the Cambridge data file (Fig. 1.4.1) from 2.56 A (no binding) to 1.16 A (covalent bond). The relative position of both groups, however, does not change. The final tetrahedral angleis kept at all distances from3 to 1.5 A.The weak IV -+ CO interaction in protopin has the same geometrical ordering effect as the covalent bond in retusamin. iological as well as organic syntheses apply various activation mechanisms in order to force the two educts along a single reaction pathway. arbo on-carbon f o ~ a t i o nbetween enols and acceptor carbonyl carbon atoms can, however, be accelerated, if at a neutralpH one simply applies an enamine as donor. E n ~ n eare s

0

6

0.116 nm

Clivorin 7 7 15

Schematic molecular structures of three alkaloids in which nitrogen apof the intermediateof the amiproaches from the back to a carbonyl group. The geometry dation reactionis kept in all three cases, although theNC distances are different.

readily formed by condensation of an aldehydeor ketone with a secondary amine. In Scheme 1.4.3 two propanal molecules are condensed, but the reaction between A. and enamine two different aldehydes can also be achieved between aldehyde ita am in-catalyzed biosyntheses often follow such pathways.In organic synthesis the enol can also be stabilized and activated by silylation (not shown). onosacch~descan be synthesizedstereoselectively by sequences of Wittig reactions and Sharpless epoxidations (see Scheme 4.3.1). This is, however, usually not necessary, because a large selection of ~ o n o s a c c h ~ d is es comercially available. Carbohydrate reactions then only involve intro~uction of protectinggroupsandpolymerization by condensation,e.g.,formation of oligosaccharides (see Sec. 4.3.4) or polymeric phosphodiesters (nucleotides;see Secs. 8.4.1 and 8.4.2). Inall cases the condensation depends on the nucleophilicity of amino or hydroxyl functions and the electropositive character of carboxyl carbon or phosphateor phosphite phosphor atoms.

.ste

~ ~ ~ o l e c uesterification lar (lactonef o ~ a t i o nis) fastest if the OH-CO angle is 98". Smaller and larger angles lead to much smaller kinetic constants. Since 1,4bridged cyclohexane boat conformers are some of the most rigid carbon skeletons, such reactions between neighboring substituents have extensively been studied with rigid terpene derivatives. The velocity constants KE1given in Scheme1.4.4 are relative to the i n t e ~ o l e c ~reaction ar between ethanol and acetic acid as a standard. of magnitude have been observed. Accelerations by more than four orders

l

on confor~~tion)

ivi

oducts are, with the exch single collision between a donor and acceptor leads to a reaction ((diffusion controlled (reaction) and velocity constants are inthe order of k = 1O1Omol s-l. Proton exchange between water molecules is an example of such a reaction. It has no m e c h a ~ s ~ and obeysthe thermodynamic laws of reversible processes. Removal of an electron from the HOMO of an organic moleculeis an equally cation radical and correspondsa one-electron to oxidation. fast reaction. It leadsato Addition of oneelectrontothe 0 produces an anionradical.Inordertobe fully reversible, the energy difference between the LUMO or OM0 and the nonbonding energy level should not exceed 1 V, since very strong oxidants or reductants havea high tendency to undergo irreversible secondary reactions. Low redox potentials in organic molecules are often found,if at least 10 lone-pair yz- and Eelectrons are present in the conjugation pathway. Such redox-active compounds

Reduction and oxidation of organic molecules occurs on thefrontier orbitals, namely the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO)

usually have absorption bands in the near-W or visible region(2280 nm). A notable exception is ascorbic acid (see Sec. 7.2.3). Oxidants or reductants may be other dyes or inorganic reagents such as metal ions (Sn2+, Fe3+,Cu+),iodide, sulfides, or oxygen (Fig. 1.4.2). xcited states of dyes, as obtained by absorption of light quanta, react as idants and reductants. The excited electron in the LUMO acts as strong reductant, the electron hole in the HOMO as oxidant. The oxidation and reductentials are again given by the energy digerence between HOMO or and the nonbonding energy level (Fig. 1.4.3). A few important redox potentials of ground state molecules and ions are reproduced in Table 1.4.1. The acid strengthof proton acid-base pairs (Bronsted acids) is given by the pk,value (Table 1.4.2). At the pH that corresponds to the pk,, half of the molecules are in the acid form, theother half in the form of the co~espondingbase. Each pH unit thenshifts the ratio by a factor of 10. Phenol, for example, is 50% phenolate and50% phenol atpH 10, corresponding to a 1:1 ratio at its pk,. At pH 7 the ratioof pheno1:phenolate is then 1000:1. The free energy of acidbase reactions can be directly calculated from the pk, values. The pk, of water (14 at a concentration of 10' M) is also drastically changed by coordination to metal ions, e.g., to 10 in the coordination sphereof Zn2+.

1

---.( n ~ n ~ o nlevel) ~ i n~--------

(lo~-ener~y Redox reactions of the ~ ~ o t o c h e ~ c aexcited l l y singlet state.

Redox Potentialsof Some Important Reactions

E = E,

+ 2 . 3R-*1T0 g ~ 'red

Reaction

Eo [vlat

pH

7

O2 + 4H@ + 4e'

2 H20

0.815

Fe3@ f e@

Fe2@

0.771a

Chlorophyll @ + e@

Chl

0,32a

02

0.295

+ 2H@ + 2e@ Hydroquinone

Dehydroascorbic acid f 2 H'

f

2 e@

~lutathione-(S-S) + 2 H@ + 2 e@ Pyruvate + 2 H'

+ 2 e@

f

-0.10 -0.1 9

Ethanole

-0.20

Dihydroribo~avin

-0.2ga

+H2 + 2 e(3

NADH + H

-0,32a

2e@

H2

-0.414

Riboflavin

' H 2

F2 Glutathione-SH

0.05~

Lactate

Acetaldehyd~+ 2 H'

NAD'

Ascorbic acid

f

+ 2 e@

2 H@ + 2 e@

CO;! + 2 H @ + '2,

Hemine + e@

0

0 WC00 Heme

-0.42

-0.48a

aFastand reversible reactions; the other reactions slow are and often irreversible.

Intramolecular proton transferis called tautome~zation.A few pka values of important tautomers are reproduced in Scheme 1.4.5. In c~boaromaticsystems the enol and enamine tautomers invariably prevail, butin nitrogen heterocycles impo~antdifferentiations take place: oxygen tendsform to lactams, while nitrogen prefers enamines. Furthermore, there is a general trend to replace intramolecular hydrogen bonds by intermolecular ones if several electron withdrawinggroupsarepresentin the originalaromaticsystems.Intramolecular hydrogen bonds,on the other hand, stabilize enols.

pka Values of Important Acid-Base Pairs

AG = -2.3R * I log K = 5.9* ApK [W * mol-1] Acid

P&

Base

20

cr;,

R-CO-N-RI

16

H3C"OH

H3C-O

16

H20

OH

@

@

15.7

@

ROOC-CH-COOR

13.5

H3C-CO-CH2~COOR

@

HCO3

H~CG-OH HCN

H2c03

@

@

Pyridine

Pyridine-H

H3C--COO

HCOOH

6.37

HCO3

3.75

HCOO@

0.65

Pyrimidine

@

5.21 4.75

tivi

no tau~omeri~tion

r l

3

3 YO

A final equilibriumof synkinetic interest concerns the reversible hydration of carbonyl and iminium groups. In both cases electron-wi~~awing groups strongly favor the hydration of C=O and C=N bonds. Scheme 1.4.6 shows the approximate hydration upon dissolution of the carbonyl compound in water. Acid catalysisis usually requiredto establish equilibria. tiVi

The oxygen moleculeis, next to water, the most important reagent in nature.Its electronic formula is 4kO*,where the dots indicate single electrons in antibinding orbitals. They reduce the binding energy of the triple bondto the binding energy of a double bond and give the oxygen molecule in its ground state the character of a biradical or triplet state. The oxidation potential of oxygen is around 1-V,which is similar to the oxidation potentialof bromine. Nevertheless, oxygen does not react spontaneously with the strongly reductive media of biological organisms, because a radical is not allowedto react with spin-paired molecules (Hund's rule).The body fluidsof humans, for example, have an oxidation potential of 30 mV, which is very close to the hydrogen electrode. Bromine with paired electrons only (BrBr)is therefore an extremely potent poison. Intake of a few breaths can lead to destruction of the lung tissue and death. Oxygen does not have this effect because its biradical or triplet nature prevents direct reactions. Catalysis by other radicals such as transition metal ions or quinone radicals is

needed to allow reactions between molecular oxygen food. and Such radicals are located in membrane-separated mitochondria, where the oxidation of food molecules can take place without harming the tissues of the body. The existence of a kinetically inert oxidant is essential to life, and the fact that this oxidantis a gas and is made continuouslyby photosynthesis makeslife very easy. The final oxidation products of organic compounds are invariably acetic andcarbonicacids,whichreleaseprotons.Biologicalfluidsmust,however, maintain a constantpH of 7.4. A few milligrams of burned food would lower the pH of the body fluids of 70 a kg man to 6, and if the acids were released into the blood the resultantchangedproteinconformationsandsubsequentswelling would be fatal. This does not happen becausethe acetic acid is i ~ e d i a t e l yesterifiedandthemajoroxidation product-carbonicacid-dehydrates spontaneously in an aqueous environment to form the gas carbon dioxide. This is breathed outinto the atmosphere, andno protons are released in the body. Another magic molecule is hydrogen cyanide, HC=N, It is formed when ammonia and methane or similar carbon and nitrogen compounds react with each other inelectric discharges in the gas sphere. This may have happened, under prebiotic conditions, when the earth’s crust was formed, and it happens in corresponding model systems The carbon atom of HCN carries a partial positive charge, which changesto negative upon deprotonation and formation of cyanide anions, I C ~ N-I.If both carbons react with each other, a carbon-carbon bondis formed to give N=CCH=NH, which is converted to glycine, the most simple amino acid, by hydro~enationof the Schiff base and hydrolysis of the nitrile. This sequence of reactions can be considered as the mother reaction of biosynthesis.The rest is repetition and modification. CN is a strong poison because it adds to the positively charged iron of heme and blocks the transport of oxygen in the blood. A similar effect is seen with carbon monoxide,-lC=OI+,in which again the carbon atom carries a negative charge and the heteroatom, here oxygen, is more positive. This abnormal charge distribution and the stable triple bond preventsat first the addition of water to giveformic acid and then leadsto strong interaction with empty d-orbitals of metal complexes. Carbon monoxide is thus another acid anhydride that is stable in water, andit is a poison that blocks the oxygenation of hemoglobin.

Non-covalent bonds in molecular complexes and membranes are formed only under defined conditions (e.g., in water at pH 7 andat room temperature). Under different conditions (e.g., in ethanol or at pH 4 in water or at 70°C in water) complete decomposition may occur. Such decomposition is, however, usually fully reversible.Since reversible reactions have small activation energies, synki-

netic reactions arefast. A first major difference between non-covalent and covalent compounds is therefore the relative speed and reversibilityof the noncovalent assembly reaction. A synkinetic polymer fiber of defined stereochernist~ and a molecular weight of several rnillionmay be formedor destroyed in a cooperative process within seconds, a covalent polymerization would be formed very slowly or need an optimized catalysts. onc covalent assemblies of covalent or noncovalent polymers, e.g., the gel of the vitreous humor (Figure 1.5.1), may remain unchanged for several decades under favorable conditions but may also decompose upon simple heatto relatively low temperatures(e.g. 60°C) or application of modest mechanical pressure. The classic namefor the formation of molecular assemblies in water from amphiphiles is “self-~~ga~zation.’~ This name is only descriptive of the formation of m i n i ~ a surface l molecul~ asse~blies of ~ p ~ e ~orc planar al shape. In such aggregates the combination of nondirected, repulsive h ~ ~ a t i forces o n between the head groups of amphiphiles withstatistical entropic effects and nondirectedvan der Waals interactionsbetween hydropho~icskeletonsleadsto particleswith the smallestpossible surface. The “self’ in the organization process then just means entropy controlled. If, however, linear-binding forces, asymmet~caldistribution of head groups in bolaamphiphiles, and chirality determine a well-defined molecular orderingasymme~cal in assemblies, we speak of synkinesis in analogy to covalent synthesis. The noun implies an essentially vectorial assembly process. A proton donor center, for example, reacts regio- and st~reo-selectivelywith a proton acceptor group (see Fig. 4.5.8). Large and small headgroups combine selectively only with themselves, but not with the head-

Molecular modelof the vitreous humorof the human eye.

group of different size, to give a curved,asymmetricalmembrane(seeFig. 2.5.14). Tetraanionic porphyrins form stable heterodimers with fitting tetracationic porphyrins in water, and polymerization is prevented by ethyl substituents at the right place (see Fig. 6.6.2). The reaction pwtners of such molecular complexes must beas carefully planned and optimized as the synthonsof reactions in covalent synthesis. There is no such thing as self-organization, but careful retrosynkinesis and synkinesisis needed. The name comes from putting molecules together (syn = together) under conditions where total reversibility of allits reaci~ by changing one parameter (e.g., tempertions is possible ( ~ i n e=~movement) counterions). The termsynkinesis also impliesthat s ~ c t u r a l l y mpounds of molecular or bimolecularthicknessareformedfrom monomeric molecules-the synkinons. Three-dimensional crystals are considered as bulk materials, not as synkinetic molecular assemblies. formations in synkinetic assemblies are uniform and usually different from those found in crystals, since packing forces arepartiallyovercome by ~ y ~ a t i o n forces as well as by thermal motions (undulations)of the molecular layers.

pica1 target assemblies of synkinesis are noncovalent dimers, charge-transfer complexes, hetero-dimers and -trimers, cyclic or helical oligomers, inherently asymmetrical or helical membranes in the form of les, spheres, tubules, and rods, as well as macroscopicsurfacemonolayers.anddomainsinvesicle membranesandgaps of molecular size insurfaclayersareothersynkinetic targets. They may function as receptors or reactive, enzymeclefts (Fendler, 1982; Fuhrhop 1982; Israelachvili, 1992; Fuhrhop an

olecular lnter~ct~ons hile covalent compounds, which are synthesized by regioselective connection of functional groupsin any combinationof educts, noncovalent compoundsusually depend on the fitting of molecules. Five binding effects forces or or a combination of them connect such molecules. The ~ y ~ r o ~ hE,'ect. o ~ i c The medium for synkinetic reactions is usually bulk water, the surfaceof water, or the surfaces of appropriate solids in the presence of water. Water is an unusual and poorly understood liquid. Its first extraordinary property is a density anomaly. If solid water or ice is heated above its melting point, the density first increases, reaches a m ~ ~ density u m at about 4"C, and then decreases. Ice thus swims on liquid water, and living organisms in lakes are not killed ~mensions two using by a sinking ice cover in winter. Simple model calculations in as tools reproduce enz logoas the water model and small computers

this phenomen perfectly. The structure and t h e r m o d y n ~ c sof the ice phase are

dominated by hydrogen bonding. Optimal hydrogen bonding leads to perfect hexagons in two dimensions. Ice melts when the thermal isenergy sufficient to break the hydrogen bonds, broadening the dis~butionof H-bond angles and lengths. Van der Waals ~teractionsnow become more important and favor systems of higher density. Liquid water becomes denser than ice. Hydrogen-bonded clusters, however, remain present in water, resulting in an enormous dielectric c o n s ~oft E = 80. This is the second magic and important property of water. Ion pairs are solvated not only by single molecules, but also by water clusters. The distance between of opposite ions charges thus becomes very large and strong Coulomb forces can be overcome. Water is by far the best solvent for ions at room temperature. Nerve and muscle excitation by water-dissolved electrolytesis thus made possible. Third, water has a high heat capacity, because the H bonds in the clusters are disrupted in a stepwise manner by thermal energies. The high water content of the human body thus greatly simplifies maintenanceof a constant body temperature.A fourth important experimental finding relates to the addition of nonpolar solutes to water. This is strongly unfavorable because water clusters are disrupted, it is strongly opposedby entropy at room temperature, andit is accompanied by a large positive heat capacity. The comb~ationof these three properties constitutes the “hydrophobic effect” (Fig. 1.52).The large positive heat capacity of inse~ionof nonpolar solutes results from an extra ordering of water molecules around the solute. Two-dimensional modeling indeed showed such an effect. The local tetrahedrality of water molecules increases around h y ~ ~ ~ b o n - tsolutes y p e and causes the transfer enthalpies and entropies to

/

The hydrophobic effect. Apolar organic compounds are hydrated by water (left), but there is no interaction stronger than the water-water interaction in water clusters. Entropy effects therefore favor large assemblies of apolar compounds in water. (For the hydrationof ~eadgroupsand surface repulsion, see Sec. 2.5.)

be negative. At a higher temperature, close3'7°C to (body temperature), the transfer entropy changes sign and the cluster of shell water molecules around the hydrophobic solute breaks up (Silverstein et al., 1998). Organic solvents can also be used for synkinesesand are mentioned occasionally. The useof organic solvents and solvophobic effects often allow the direct synkinesis of hydrogen-bonded dimers and reverse micellar systems, but thereis relatively little interest in gels containing massive amounts of organic solvents. The sameor closely related systems can usually also be obtained in aqueous membrane systems, and applicationof organocolloids to solid surfaces produces nothing but problems. Even electron microscopy becomes dif'ficult because the carbon gridsareattacked by solvents.Onenotableexception is c~o-microscopyin toluene. Solvent useis in general limited to the application of monomolecular solutions in self-assembly processes, which is the essentially irreversible chemisorption of ~ p h i p h i l e from s monomolecular solutions by reactive surfaces. ~ i t t i ~ofg ~ o Z e c ~ Z Shapes. a~ Planarsurfaces,e.g.,boardlikecellulose fibers, protein sheets, or disklike porphyrins, as well as concave-convex pairs, e.g., cyclodextrin and polymer fibers, tend to form molecular assemblies (Figs.

Fitting of (a) rods into tubules to form inclusion compounds and right- and left-handed helicesto form sheets.

(b)

Formation of alternating stacks and heterodimersby charge interactions of planar, charged dye molecules. The ethyl groups prevent the formation of large stacks.

1.5.3a and 4.5.5). The most striking example is the formation of dipolar urea tubules around linear hydrocarbons (see Fig. 2.5.1’1). F u ~ e r m o r emixtures , of right-andleft-handedhelicalassemblies of enantiomersoftenprecipitateas sheets (Figs. 1.5.3b and 4.5.9). ~ittingof Charges. Well-definedheterodimersandstacksareformed with the placement of at least two chargeson the synkinons. Stack formation can be stopped at the dimer stage by alkyl substituents (Figs.1.5.4 and 6.6.2) ~ y ~ r o g Bonding. en Hydroxyl, amino, and carboxyl protons form hydrogen bonds with basic heteroatoms, e.g., nitrogen and carbonyl oxygen atoms. The strongest hydrogen bond chain is formed between secondary amides (Fig. 1.55). A typical property of hydrogen bonds is a sharp melting point caused by cooperative formation and degradation processes. Directed hydrogen bondsoften overcome repulsive nondirectional hydration forces and lead to a regio- and stereoselectiveconnectivityinsupramolecularassemblies.Multivalentmetal ions can alsobe used insteadof protons, butthe insolubility of salts and chelates often leadsto illdefined precipitates (Jeffrey and Saenger, 1991). Charge-~ransfer Interact~on. Hydroquinone-quinone pairs are connected by hydrogen bridges and thus form ground state heterodimers. Electron

tivi

S

0.36 nrn

O-H-N Waals O-H-N distance der van distance

tems.

in ~ydrogenbondings

Binding energy, distance, and geometryof typical hydrogen-bonded sys-

Electron Electron donor acceptor D A

Kation radical

D8

0""""" H

Anion radical A?

H0

O@

2

o..."."..H0

Fe I'

Fe 4

2

0. Fe 'I

0.

?

Energy diagramof a light-induced charge-transfer system and two examples: the quinhydrone dimer is formed by hydrogen bonding and dispropo~ionatesinto radicals upon deprotonation; a pyrazine-bridged heme dimer shows a 800-nm charge transfer band indicating an electron transfer from one porphyrin to the other. (From Fuhhop et al., 1980.)

transfer from the hydroquinone to the quinone then occurs in the excited state, and long-wavelength absorption bands are observed. Electron pairing of two planar radicalsin a face-to-face dimer has similar effects, and metal complexes can also be dimerized via ligands. (Fig. 1.5.6)

urface Forces The interaction between two macroscopic bodies decays much more slowly with distance than it does for two molecules. Four long-range forces (Israelachvili, 1992)dominate the interactions betweenmacroscopicparticlesatdistances above about3 nm: 1. Van der ~ a ~forces l s are always attractive and large and act not only when the bodies are in contact. If they were the only force between particles, stable colloids would not exist, Large dissolved particles would coagulate immediately. 2. ~lectrostatic forces can be repulsive or attractive. processes suchas adsorption of ions to electroneutral surfaces, e.g.,of Ca2+to z~itterioniclecithin bilayers, release of protons from surface carboxyl groups to form carboxylate ions, or addition of protons to surface amino groups, introduce repulsive charges to the surface of particles. This charge is balanced by an equal number of counterions to form a diffuse electric double layer. Two similarly charged surfaces will repel eachother by a double layer repulsion force. Neutralization of charges by tightly bound counterions, e.g., perchloratefor ammonium head groups, will lead to precipitation. 3. ~ y ~ r a t or i os~~ l v e forces ~t can be attractive, repulsive, or oscillatory. These additional forces depend on the medium encounte~gthe particle as well as the chemical and physical propertiesof the surfaces. Amorphous or crystalline, smooth or rough, rigid or fluid-l~e-all of these differences havea large effect on short-range interactions (<3 m)in liquid media. Crystalline polar surfaces will lead to ordered, continuous ordering of polarliquidsatlargedistances,butatsmallerdistances oscillatory solvation forces dominate. Only on smooth surfaces will the liquid molecules appear in ordered layers; on rough or highly curved surfaces oscillationsas well as ordering will be eliminated. In fluid surfaces, e.g., in lipid mono- or bilayers of vesicles, oscillations are evened out and any longer-range structural force collapses. M a t remains is a much shorter- ranged monotonic hydration repulsion, which can be overcome by hydrogen bonding between the groups on the surface. Linear ordering will then enforce the formation of fibers. 4. Stericforces occur if chain molecules adsorbed to the particle surface protrude into the solution like the m s of an octopus. A repulsive en-

tropic force results, caused by the overlap of the polymer molecules, which is called “steric repulsion.” In a good solvent for the polymer the sidechains appear as a brush on the surface and prevent coagulation. Small amounts of water-soluble polymers (e.g., proteins) may thus solubilize hydrophobic colloids in water by adsorbing or grafting them on the surface of the particles. At low temperature or very low coverage of the colloidal particles by the polymer, one observes the adverse effectof “bridging” by the sidechains.The large free surfaces of the particles are then connected with each other by polymer strings rather than separatedby solvated polymer brushes

eterodimers and heterotrimers are formed in water if planar cations and anions are mixed and if polymerizationis impeded by ethyl groups or larger alkyl substituents (Fig. 1.5.4). Other possibilities are the isolation of hydrophobic C-G or A” pairs in small micelles (Fig. 2.5.4), formation of racemates (Figs. 4.2.7, 8.6.1, and 9.5.3), and formation of pseudo-racemates (Fig. 4.5.10) between enantiomeric ~ ~ h i p ~ l e s with chiral head groups and the s m e or similar hydrophobic skeletons.

Micelles (see Sec. 2.5.2) made of charged ~ p h i ~ h i l have e s typical lifetimes of milliseconds (Fendler, 1982), but micelles with a lifetimeof several seconds can be made by selfor~a~zation of single long-chain mphiphiles with elec~oneu~al head groups. Defined micellar fibers of bimolecular thickness (E4 m)and many micrometer lengths are best made of ~ p h i p h i l e sin which the hydrophobic tail and hydro~hilicheadgroup is connected by a secondary amide link. Such fibers are often stable for months in aqueous suspensions and can be isolated in dry form, when the head group is chiral, e.g. an amino acid or a carbohydrate (see Sec. 2.5.3) (Fig. 1.5.7). Isolable spherical micelles have, to the best of our knowledge, only been achieved once using large metal complex head groups (Draeger, 1999).

Schematic modelsof a) spherical and b)t u b u l ~micelles.

tivi

Schematic modelsof (a) spherical and (b) tubular vesicles.

pherical vesicles (seeSec. 2.5.4) are madeby the same kindof amphiphiles that form micelles. Highly soluble amphiphiles (e.g., sodium salts of fatty acids or soaps) form micelles; badly soluble amphiphiles (e.g., free fatty acids) give vesicles or crystallize. ~ m p ~ p h i lmonomers ic with two or three long alkyl chains are often totally waterinsoluble as monomers but dissolve well as vesicular assemblies. Vesicles usually collapse upon drying (Fig. 1.5.8a), but one isolable monolayer vesicle made of rigid carotenoid bolaamphiphiles has also been reported (Fig. 5.5). ydrogen bond chains convert spherical vesicles to tubules. Such tubules can again be isolated in the dry form and can be stored. They are p ~ i c u l a r l ystable if monolayer membranes are used (Fig. 1.5.8b).

surface layer. The error in these measurements was too large to determine the area coveredby a single molecule. The first ~uantitativemeasurement leadingto a molecular areaof about 20 L&* per fatty acid molecule and a monolayer thickness of 23 L& came from a young German amateur named Agnes

used small, absolutely grease-free troughs with a limiting mobile barrier instead of lakes and applied highly dilute organic solutionsof amphiphiles insteadof oil droplets. The solvent evaporated from the water surface and quantification became simple.In 1917 Irving Langmuir published the first systematic study using the Pockels trough. A typical surface pressure-surface area isotherm as measured with the Pockels trough first shows no surface pressure atSmall, all. separated domains of molecular monolayers occur, which move independently of each other. Upon movingthe barrier, a plateau of intermediate surface pressure, typically 5-20 mN/m, is often observed. Plateaus are typical for dou~leheaded bolaamphiphiles (see Sec. 2.2) and indicate the removal of one of the head groups from the water surface. In case of single-headed am~hiphilesplateaus usually indicate the beginning of the formation of coherent monolayers with tilt angles of the molecules much smaller than 90". Finally, whenthe monolayer is (more or less) closed to a continuous single layer, the surface pressure rises to 60-80 mN/m. The tangent to this final isotherm slope cuts the xaxis at the molecular area value (Fig. 1.5.9).The concentration and volumeof the spreading so-

collapsed

0

10 0

0

10

20

30

4.0

50 Area I molecule compre~sio~ of barrier

Typical surface pressure isotherm with a plateau. The arrow shows the molecular area. mN= millinewton

lution as well the area of the confined film must,of course, be known in order to calculate molecular areas. In1935KatherineBlodgetttransferredmono-andmultilayers of fatty if the surface pressure acids on solid substrates. Thisis in general successful only of the solid monolayer reaches values above 60 Nlm. In the 1970s Hans Kuhn optimized this technique by introducing multilayers in X, U,and Z-orientations on solid surfaces (Figure .S. 1 10) (Fendler,1982; Ulman, 1991;Tredgold, 1994). Similar mono- and multilayers on solid surfacesmay also be obtained by self-assemblyfromorganicsolvents(self-assembledmonolayers, or SAMs). are monolayersof organic molecules which are bound by chemical bonds head group of the ~ p h i p h i l eto the atoms on the surface of the solid or fluid(in the case of mercury)subphase. A carboxylicacid,anazide, or a trichlorosilane dissolved in a nonpolar solvent will, for example, irreversibly bind to hydroxyl or amino groups of a hydrophilic surface, e.g., glass, mica, metal oxide surfaces, polyallylamine layers. Hydrosul~desbind to gold. Upon washing with a good solvent for the amphiphile, only the self-assembled monolayer remains (Fig. 1 .S.11). T~io-1SAMson gold exposed to air for periods of several days oxidize to sulfinates or sulfonates. They then desorb upon rinsing a fresh thiol with solvents, or they rapidly exchange with thiols when placed in solution. Ozoneis probably the primary oxidant in ambient laboratory air. Cyclic v o l t ~ e t e r yof Ru(N J3+, however, shows that withina few hours of air oxidation the blockingcharacte~sticstoward electron transfer are retained even after exposureto solvents (Schoenfisch, 1998). oly(dimethy1 siloxane) (PD S) stamps with prot~dingfeatures (e. pm lines or 10 pm squares on a side) may be inked with hexadecyl amine pressed on smooth substrates covered with carboxylic acid a n h y ~ d esurfaces. These in turn are obtainableby S S of fatty acids and subsequent dehydration. decylarnide p a t t e ~ sare thus formed on carboxylate surfaces. ~nreacted still contain a ~ y d r i d e for s further self-assembly processes to fillthe voids (“micrometer or nanometer ~rinting’,thiols or

most direct andi n f o ~ a t i v method e of analyzing thes t ~ c t u r of e supramoler assembliesis microscopy. icrosco~yis used to studyainboundaries of Langmuirms and,inconnectionwithterangletechniques(see Fi ~ i c r u s c o ~achieves y inprinmonolayers onwater. FZuore ciple the same, since dissolved dyes usually give strongly fluo which disappearat higher surface pressuresor rnigrate to the ed is squeezed out of bilayer crystals. Vesicles with a diameter in the rnicro~eter range canalso be seen underthe light microscope. ~ e m b r fusion ~ e and undula-

ivit

tion as well as changes of shape upon osmotic stress have been studied by light microscopy and video cameras. ~rans~ission electron ~icroscopy requires thatthe thickness of the molecular assembliesis in the rangeof 10-300 A.Thicker assemblies appear as black objects;for thinner objects resolutionis usually not good enough. the methodof choice for the visuali~ationof all artificial membranest~cturesas they occur in micelles (Fig.2.5.3),vesicles (Fig. 1.5.12), micellar and vesicular fibers (Figs, 2,5.8,4.5.8,4.5.9, and 6.5.4), and disks (Fig.6.5.7) in bulk aqueous solution. Uranyl salts or phosphotungstate is often added to the solution before the drying process takes place on the electron microscope grid. These heavy metal saltsform a thin glassy film around lipid membranes, which then appe white stripes in black coatings in TEM (negative staining) (Fig. 1.5.12). may also rapidly freeze aqueous probes to obtain them in vitreous, noncrystalline ice. The probe holder has thento be cooled with liquid nitrogen in order to avoid water evaporation in high vacuum. Drying artifacts are avoided with this technique, called cryo-microscopy. The method canbe combined with tive staining, if the stain is added to the aqueous solution,or work without Careful defocusingof the TEMlens is then used to enhancethe contrast. electron microscopy(S ,usually called atomic .These can be applied under~ondestructive conditions in air and under water. The height and surface shapeof a sample on a smooth surface is scanned when it passes a stationary tip spring. The diameterof the tip is in the orderof 10 nm or larger;A larly usefulfor the ch~acteri~ation of assemblies on smooth gold, mica, silicon, or polymer surfaces. Orthogonal displacements in the orderof S 1 A can be measured accurately usinga variety of laser optical techniques.This allows measurement of forces in the ran e between lo1' and 108N, co~espondingto a weight lecular adhesion forces can thus be obtained. The latbetween 1 ng and 1 pg. ~~~~~

Schematic drawingof a Pockels trough usedto form compact molecular surface. The depositiont&es place on the downward stroke if hydrophobic interactionis responsible for deposition (hydrop~obi~ surface) and on the upward stroke if hydrophi~csurface- onol layer interactions are morei m p o ~ n tIf. the deposition area is equalto the lossof the monolayer on the airiwater interface (deposition ratio equal to I), it is assumed that perfect deposition has taken place. If the ratiois near unity in both upward and downward strokes, the material is deposited in the Y mode, which is the most stable multilayer structure (b). If the deposition ratiois near unity in the down stroke and zero on the up stroke, the deposition is in theX mode (a). The surfaceis hydrophobic and arniles are bound in the A orientation. The converse situation leads to Z-mode ~ultilayers rientation). X-and Z-types often rearrange to Y-type multilayers. a water

ter 1

The self-assembly process of surface monolayersfrom monomolecul~ to sulfide. solutions is based on irreversiblechemical binding, here from gold

Typical electron micrographof a bilayer vesicle membrane negatively stained with uranyl acetate (black regions). The white membrane corresponds to the hydrophobic lipid bilayer. Its width is about 40 A.

era1 resolution depends onthe tip diameter (as small as a single atom, but usually not smaller than10 nm) and the widthof the valleys inthe probe. In general it is quite easy to elucidate the structure of molecular elevations accurately, whereas nanometer indentations are hardly detected at all and cannot be characterized in their full depth (Fig. 1S.13). A technique called scanning near field opticalmi-

ill

i

im 1.5.13 The tip in scanning atomic force scanning microscopy has a typical diameter of 10 nm. Height measurements are possible witha resolution of 1 W.The lateral resolution is much better with elevated objects than with engraved objects.

croscopy ( S N O ~works ) with laser light and fluorescence insteadof force measurements. It may in the long run allowus to measure fluorescence and perform photochemical reactions within molecular assembly with a nanometer resoluSNO tion. This is, however, outof today's experimental reach, where the nique is hardly more useful than a good fluorescence light microscope. irect measurements of the forces between planar surfacesat separations molecular dimensions are possible by the useof surface force a p p ~ a ~ s )(Israelachvili, 1992). Most frequently two molecular monoor bilayers on mica are opposed. The rnica is glued on cylindrical silica lenses (radius,r, is 20 nm). The surface separation, D,in nanometers is measured by m u l t i p l e ~ b ein~ terferomet~.The distance zerois set at a positionof contact. The force F is then d e t e ~ n e dfromdeflection of a double-cantileverspring(springcon 100 N/m) on which one surfaceis mounted. When the gradi positive and exceeds the stiffness, K, of the spring (dF/dD curs and the surfaces jump into contact. The pull-off force is then a measure of the adhesionforce between two surfaces.The a ~ e s i o energy, n related to F and D by the Derjaguin equation: E = F/2nR. Fo measuring sensitivityfor F of about lo8 N, the sensitivity inmeasurin~adhesion energy is about lo6 J m2. An example for ~easurementsof molecular interactions for nucleic basesis given in Sec. 8.7. fi:

~~stallography does not help directly in synkinetic chemistry, since the molecular confo~ationsin t~ee"dimensionalcrystals are invariably di~erentand in general much more simplethan in s u p r ~ o l e c uassemblies. l~ aclsing forces u s u ~ l yflatin molecules, which are all-important in highly hydrated and curved rystals should, however, be used as referencesfor the inte~retation which do not provide directly any i n f o ~ a t i o n ful are crystal structures of cocrystals of two difpossible to identify and characterize the major 9; Luger, 1980; Glusteret al., 199 mers anda s s e ~ b ~in e swater. ow" spectrQscopictec~niquesare p ~ i c u l a r l yuseful for the identification of molecularinteractionsinsolutionandlorin 'd state:UV/vis,fluorescence, infrared, and nuclear magnetic resonance briefly here, and some examples will be gi

f o ~ a t i o nof molecular complexes of defined stoichiomet~in solution is usually accompanied by isosbestic points in ti~ationspectra and by changes of

ivi 0.5

0.4 0.3 A

0.2 0.1 0.0

100% A I

-4000

1

1:1

l00%B I

100YoA I

-4000

2:1

,

:,

I

I

I

I

1OO? B

Titration of a porphyrinA + l3 hetero dimer formation and the corresponding Job plot (see Fig.6.5.2). A& corresponds to the loss of absorption at 516 or557 m.

linewidth, and extinction coefficient with respect to the addition of the comlots of absorptions against molar ratios (Job plot) show the maximal absorptionloss or gain for both monomer spectraat the stoichiomet~~ ratio of the given molecular complex (e.g.,1:l or 12)(Fig. 1.5.14). olecular assemblies of dyes often show hypsochromic or bathochromic shifts of the main absorption bandsif the n; systems are touching each other 4 A)or if the distance is less than 6-7 A.This shift can be explained by the exciton model,whichassumesresonanceinteractionbetweenthedipoles of excited states. The most simplecase is the interaction between two such dipolesof excited state of linear dyes, e.g., carotenoids. The interaction of two stacked chromophores then leads to a short wavelength shift (stack or aggregate) of the monomer abso~tionbands; the interactionof two chromophores in lateral position (Scheibe or Jelly or J ag~regate)leads to a long-wavelength shift (Fig, 1.5.15). The exciton effect in aromatic chromophores, e.g., porph~rins,is more complex since excitation leads to two dipoles of the same energy in perpendicular orientations. In stacks one still finds only shortwavelength shifts, but

h,,,

(m

0

a

0

0

Exciton shifts in the UV/vis-abso~tionbands of linear polyene chromophores and their dependence on the directionof the excitedstate dipoles as well as on the relative Orientation of the molecules. J aggregates here their name from Jelley, the discoverer, H points to the formof stacks.

in lateral assemblies both long- and short-wavelength shifts occur. The magnitude of the shifts again depends on the distance and angles of chromophores (Fig. 1.S.16).

If a chromo~horeA fluoresces, this can usually be quenchedby an appropriate c ~ o ~ o p h o B, r e if A and B formstableheterodimers.This is calledstatic quenching andis quantitatively describedby the SternVolmer relationship: l=K[B]

" @OF

@F

where (h?is the fluorescence yield in the absence of the quencher molecule,@E in presence of the quencher, K, the quenching constant, and[B], the concentration of the quencher. Formally the same equation is also valid in the case where

stacked

es,= es,= 90" a = 0"

lateral

es,= 0" es,= 90"

es,= es,= 90"

a = 0"

a # 0"

E%

EP

Ea

monodi-mer hypsochromic

monodi-mer

broaden in^

or splitting

monodi-mer monodi-mar hypsochromic and bathochromic E

I

h

H (stack) and J (lateral) shifts of aromatic chromophores, in particular p o ~ h y ~with ~ s two , orthogonal dipoles of the excitedstates.

does not form a stable complex with the fluorescing dye A, but reacts with the excited state A* only in a collision process (dynamic quenching). K is then equal to the equilibrium constant of the reaction A + B "3AB. Static and d y n ~ i c quenc~ingcan be distinguished by the measurement of fluorescing lifetimes at different concentrations. It remains constant only in case the of static ~uenching. uorescence quenching does usually not occur in homodimersh or e t einr o ~ ~ e r s , where bothc ~ o ~ o p h o rhave e s very similar redox potentials. The most i m p o ~fluorescing ~t natural compounds are po~hyrins,flavins, and reducednicotina~ide.The most i ~ p o ~ aquenchers nt aretriplet oxygen, redox active dyes, radicals, and transition metal ions. In thecase of polymeric dye assemblies, one observes several complex, illunderstood fluorescence phenomena. Very often it is observed that monomeric and dimeric aromatic dyes fluoresce strongly, whereasthe fl~orescencein large

molecular assemblies may or may not be quenched. Strong exciton interactions and fluorescence quenching sometimes go together, sometimes not. In the case of artificial complexes madeof cyanine dyes, one finds that the monomer hardly fluoresces, whereas fibers consisting of thousands of non-covalently connected molecules fluoresce strongly. In the case of a porphyrin monolayerin bulk water showing an enormous exciton splitting, both the mo~omersand the monolayers fluoresce strongly (seeSec. 1.5.7.1; Fig. 6.6.7). In both molecular assemblies the ~uorescenceexcitation spectra-which means measu~ngrelative fluorescence yields by excitation at all wavelengths of the spectrum-are similar to the absorption spectraof the assemblies, notof the monomers, which are often in equilibrium with the fibers. Furthermore, the addition of one quencher molecule to fluorescing fibers or monolayers usually extinguishesthe fluorescence of many dye moleculesby energy transduction over a few nanometers.

ourier transform infrared spectra of molecular monolayers can routinely be measured, They are particularly useful to characterize the conformation of oligomethylene chains andthe orientation of C=Q bonds with respect to solid subphases. If the bonds are oriented parallel to the surface of the subphase,no absorption bands are detectable;if they are orthogonal to the surface, strong bands are found. StaS. Anothereffect of tisticalorientationsgivethesamespectraasisotropic S aggregation concerns hydrogen bonds involving OH groups. They lead to a broadening and low-energy shiftof the QH stretching bands (3400+ 3200 cm'). This effect is, however, useful in analysis. Amide hydrogen bonds in proteins, on the other hand, lead to characteristic variations of the amide carbonyl absorption band pattern, which helps to differentiate helices from sheets. I band near 1680 cm1 involves primarily the C=Qstretching. Its shape and wavelength depend heavily on the conformation of proteins and supramolecular assemblies. A. bandat1680cm1indicatesahelix. ~ - S ~ c t u r eabsorb s at wa~enumbersaround 1660 cm1. Another characteristic amide I1 band appears near 1550 cm1 and involves NH bending and CN stretching vibrations. This band is characteristic for monosubstituted amides. Furthermore, there is an amide A band near 3450 cmi for the NH stretching mode, which shifts to 3300 cm' and broadensuponhydrogenbonding.Anotherindicativeinfraredbandshift for supramolecular assembliesis the replacement of the CQQH absorption at 1730 cm' to 1560 cm' upon deprotonation (for examples, see Secs. 2.5.3 and 2.6).

The detailed s t ~ c t u r e sof dimers and heterodimers containi~gporphyrins or other aromatic systems insolution have been solved ingreat detail by the quan-

titative evaluation of ring current effects. This is shown for benzene in 1.5.1'7 for protons at a distance Y above the center of the ring and a lateral distance X away from the center of the ring. The ring current effects have been most extensively applied inthe evaluation of porphyrin and chlorophyll assernblies (see Fig. 6.6.8).

" " " " "

I I I

l

istance x [cm] ~ 0 ~ 1~cm1= 0. s Typical ring-currentshifts of protons lying above andin the plane of a benzene ring. (Taken fromR. J. Bible, 1965)

In ~M~ spectra of solids, the chemical shift anisotropy is not averaged to its isotropicvalueasinsolution. 13C and 'H nucleiinpolycrystallinematerials therefore produce a range of chemical shifts and broad, uncharacteristic N M R signals. The dominant broadening effectis caused by dipole-dipole interactions within the solid. The angular dependenceof the shift anisotropy goes with a factor of 3cos28-1, where 8 is the angle between dipoles. At 8 = 54.7" the shift anisotropy is reduced to zero (magic angle). If one rotatesthe sample against the of the crystallite axes will be magnetic field at this angle, the average orientation equal to the magic angle and the signal will become sharp (magic angle spinning, MAS).F u ~ h e ~ o rthe e , line broadening of 13Csignals by dipolar coup~ing to protons can be removed by a heteronuclear decoupling method, and at the same time the intensity of the I3Csignals can be enhanced by a factor of about three by cross-relaxation from protons to carbon atoms. This experiment also increases the rateat which spectra can be measured and is called cross-pol~ization (CP). The cross-pol~izatio~dipol~ decoupling sequence combined with magic angle spinning (CPMAS 13C-NM[R)then produces sharp I3C peaks of solid carbon compounds containing 13C in its natural abundance of 1.5%. Only single lines are, however, observedby this technique, becauseall couplings have been removed in order to obtain sharp signals. The lack of coupling constants prevents a conformational analysis. This can be overcomeby an analysis of chemical shifts, whichalso depend on confo~ation.A detailed case history, which provides an example of NM conformational analysis in solution and the solid state, for glyconamides is given in Sec. 4.2 .

V o l t a ~ e t r ydeals with the variation of potential of a polarized electrode in an electrolysis cell on the current that flows through it. pping mercury,a rotating platinumdisk, ind ury allows only measurements below 200 mV versus a saturated calomel electrode(SCE; 240 mV higher thanthe hydrogen electrode).The other elec~odesusually work in the range of about &l V. Spectroelectroche~stry, are measured during the electrochemical oxidation or rewhere ~ V / v i spectra s uction of the redox system, is then used to identify the products formed at a cyclic volt am met^, which usually works with an unsti~edelectrode, varied with a typical sweep rate of 0. -100 Vs. A trian sweep is applied and the currents are in the range from femto- to m i l l i ~ p ~ r e s (10~5-103 A). An oscilloscope or a fast X-Y recorder can sweepa cyclic voltam-

Typical cyclic voltammogram of a reversible one-electron oxidation (lower trace) and reduction (upper trace). A Ep is 60 mV.

mogram within a few seconds (Fig. 1.5.18). On the forward scan one may observe an oxidation peak, on the reverse scan a reduction peak. Their position is not identical.If a separation of 59/11 mV independent of the sweeprate is found, and if the steps inforward and back reactions areof equal height, then one has a reversible, purely diffusion-con~olledelectron transfer reaction. Typical electron transfer rate constants for the reversible oxidationof organic dyes (e.g., metallopo~h~rins) are in the orderof k" = 0.1 cmls, Irreversible adsorption or very slow diffusion processes lead toa partial or total loss of CV peaks (Fig. 1.5.18) (Lipkowski andRoss, 1994). For Referencesfor Chapter 1, see pages 527-529.

This Page Intentionally Left Blank

Satisfied humans depend on fats in their food in order to experience a smooth “mouthfeel” and the rich aroma of fat-dissolved food. This feeling is, however, paid for with ‘?U&”calories inthe food. Chemists can help to getof rid digestible fat without disturbing the pleasure derived from both mouthfeel and aroma. Most successful has been the application of lipids on solid surfaces. (Figure 2.1.1.)~olymeri~ation of linseed oil allows oil painting. Thin lecithin or fat layers on the skin prevent its dehydration. Molecular monolayers of long-chain amines protect the surfaces of hair, teeth, and motor cylinders. Organic chemists rely on arnphiphilic lipids to build up membranes in water-the only organic reaction medium of nature. Biological molecular machinery is based on lipid membrane potentials. Artificial models so far do not work like cell membranes in vectorial transport and charge separation chains, but theylook good underthe electron microscope.

Oils and fats are of vegetable (55%) or animal (40% land, 5% marine) origin. Naturally occurringfats contain about 9’7%triglycerides withat least two different fatty acids. Milk and coconutfats contain a high proportion of C,“,, fatty acids and have thereforea relatively low melting point, Tallow and lard contain predominantly C,, and C,, fatty acids and have high melting points, The lower

/

Typical surfacemodi~cationof a solid by a~phiphiles(left) or bolaamphiphiles (right). Typical wetting anglesof a water droplet on apolar (center) and polar (left) surfaces are shown. Hy~ophobichydrocarbon surfaces on the tongue produce a good mouthfeel and favor the development of aroma. ~ y d r o p ~ lend i c groups may be used for the selective adsorption or desorption of aqueous solutes on the surface of solids. Gaps of m o l e c u l ~size may act as enzymelike reaction centers (right; see Figs. 9.6.9 and 9.6.10).

melting point of vegetable oils and m a r g ~ n eis, on the other hand, caused by high proportions of C,, unsaturated fatty acids localized preferentially in the 2-position of the glyceride (Kaudy et al., 1987). The most c o ~ o saturated n fatty acidsconsist of an unbranched chainof an uneven number of methylene groups and one terminal carboxyl group. Unsaturated fatty acids with one Z-configured(=cis) double bond, in particular oleic acid, are also common. Polyunsaturation usually means two, three, or four Z double bonds separated each time by a single and particularly reactive allylic CH, group. ~olyunsaturatedfatty acids constitute one out of three groups of essential food or growth stuffs that are needed for the growthof human cells. They cannot be produced by the human body, and must be therefore be supplied esternally. The main chain of natural saturated fatty acidsis occasionally ~ethylated or hy~oxylated,thus producing aninternal chiral center.The melting points and solubility depend on both chain lengths and number of Z-configured double bonds. Two methylene groups enhance the melting point by about 68°C and lower the water solubilityby a factor of about two.The cis double bond in oleic acid lowers the melting point by 50"C, because the CH groups allow for more rotational freedom of neighboring CH, groups than theCH, groups in saturated fatty acids (Table2.2.1).

.l

Structure, Nomenclature, andSome Physical Properties of Natural Fatty

Acids Solubility in water bp. ("C) (mg1100 at g ("at X: mm) 20°C)

Trivial name Systematic name ("C) mp. Saturated Butyric acid

Butanoic acid(4:O)

Isovaleric acid

3-~ethyl-butanoic acid (3-CH3,4:O) Pentanoic acid(5:O) -34.5 3700 Hexanoic acid (6:O) -3.4 868 Heptanoic acid(7:O) -7.5 244 Octanoic acid(8:O) 68 16.7 Nonanoic acid (9:O) 26 12.5 Decanoic acid(1O:O) 31.6 15 Undecanoic acid 9.3 29.3 (11:O) Dodecanoic acid 5.5 44.2 (l2:O)

-7.9.

Completely 163.5 miscible

Valeric acid Caproic acid Enanthic acid Caprylic acid Pelargonic acid Capric acid

Lauric acid

Myristic acid

Palmitic acid

Margaric acid Stearic acid

Tridecanoic acid (13:O) Tetradecanoic acid (14:O) Pentadecanoic acid (15:O) Hexadecanoic acid (16:O)

3.3 41.5 2.0 54.4

52.3 0.72 62.9

Heptadecanoic acid (17:O) Octadecanoic acid 0.29 (18:O)

Tuberculostearic 10-Methyl-octadecaacid noic acid(10-CH,;) (18:O) Nonadecanoicacid (19:O) Arachinic acid Icosanoic acid (20:O) Henicosanoicacid(21 :0)

61.3 69.6

68.7 75.4 74.3

187 205.8 223.0 249.7 255.6 270 284 299.2 225loo 1 301 236loo 1401 318.0 25O1O0 149' 202.5'O 158l 353.8 2681° 167l 22010 175l 370 2135 184l

204l

1.2

0.42

Saturated Docosanoic Behenic acid acid

80.0

(22:O) Tricosanoic 79.acid (23:O) Lignoceric Tetracosanoic acid 84.2 acid (24:O) Pentacosanoic 83.5acid (25:O) 87.7 Hexacosanoic acidCerotic (26:O) Heptacosanoic 87.6acid (27:O) 90.9Octacosanoid Montanic acid (28:O) Nonacosanoic 90.4acid (29:O) 93.6 Triacontanoic acid Melissic (30:O)

l

Unsaturated Undecylenic 10-Undecenoic acid 275 24.5 acid (1O:l 10) Palmitoleic 9-Z-Hexacedenoic acid 0.5 acid (16:1 9c) 9-Z-Octadecenoic acid Oleic acid ( l 8: l 9c) Elaidic 9-E-Octadecenoic acid acid (18: 1 9t) Nervonic 15-Z-Tetracosenoic acid 42.5 acid (24:l 1%)

360 and 16.3 13.4 43.7 28

23415 8loo

-43

Polyunsaturated (essential)

Z- 9-25, acidLinoleic

Linolenic acid 9-Z,

Arachidonic acid 5-Z,

2021.4 Octadecadienoic acid (18:29c, 12c) 12-2, 15-Z-1 l .o Octadecatrienoic acid (18:3 9c, 12c, 1%) 8-2, 11-Z, 14-49.5 2-Icosatetraenoic acid (20:4 5c, 8c, llc, 14c)

Wote smalleven-odd effects, which disappear at high carbon numbersFig. (see2.7.4).

The molecular and crystal structures of fatty acids usually show double lay(ap) conformation, and the terminal ers of alkyl chains in an aZZ-a~tiperi~Za~a~ carboxyl groups are connected by bifurcated hydrogen bonds to neighbor molecules (Fig. 2.2.la). The carbonyl oxygen atoms are basic act as strong proton acceptors, the hydroxyl groups as strongly acidic donors. a1 salts of fatty acids (soaps)canusuallynotbecrystallized,sincethecarbogroupsnowrepel ion-anion bonds are not directed; the metal ions behave like hard rystal planes become too slippery. There are, however, a few notable exceptions. Potassium palmitate has even been analyzed by single crystal diffraction. The opposing carboxylate groups are arranged inhot-hole a mode, and the potassium ions lie in the same plane as the carboxylate groups. The alkyl chains in the free acid (Fig, 2.2.lb) are in a perpendicular o~entationto the carboxylate plane, but the chains in the potassium carboxylate are tilted. Tilting er thinner, H atoms from one molecule enter the depression beMS in an adjacent molecule (Schwartz, 1992). Possible tilt angles are 34.5,31.5,27,19 and 0". Suspended stearate crystals are also used to produce a pearlescent effect in shampoos and liquid soaps. This effect often masks the fact that thef o ~ u l a t o is r unable to make the product clear. Fatty acid salts of bivalent metal ions are normally too insoluble to be crystallized, but soft cadmium ions provoke the formation of crystalline monolayers on water. Bivalent organic piperaziniumdications mayconnecttwocrystalsheetsandthusproducestable

C

Typical crystal structures of (a) a fatty acid with terminal hydrogen bond connections (stearic acid), (b) a soap with a monovalent, inorganic counterion (potassium palmitate) and, (c) a doublechain organic counterion (piperazinium palmitate). The bilayer becomes inter digitate^' like folded hands.

t~ee-dimensionalcrystals. Four MH200C hydrogen bonds provide strong intera similar manneras found in the free acid crystals. These salts plane connections in are well soluble in the heat, because the hydrogen bonds melt and crystallize upon cooling (Fig. 2.2.IC).In this case, it is also found that the allsyl chainsinterdigitate, so that the bilayer thickness shrinks essentially to the thickness of a monolayer (Fig. 2.2.1~).Formation of a sy~-cZi~aZ conformation of the oligomethylene chain

Typical packing of fatty acid type molecules in crystals and in surface monolayers seen from the top (see Sec. 2.6).

close to the carboxyl group is a typical mecha~smfor the bending of the chain. et al., 1986). (Gunstone, 1986a, b; Larsson, 1986; McConnell The crystal packing of the oligomethylene chains may be triclinic, orthorhombic (mean volume per CH2 groupin each case: 24 A3; surface area: 19 A,), or hexagonal (26 t i 3 and 20 A2 per CH,; Fig. 2.2.2). In the molten state the volume perCH, group risesto 30 A3,the molecular surface area 23 to A2 (Abe et al., 1966; ?relog, 1971). Mixtures of fatty acid salts are used as soaps. Sodium palmitate-stearate mixtures are solid at room temperature, and the corresponding ~otassiumsalt mixtures are fluid, although only potassium palmitate has been crystallized at room temperature, Metal carboxylates hydrolyze in water and release hydroxyl ions on theskin’s surface. Soaps with fewer than 12 carbon atoms therefore bite. This happens with nonpurified soaps as obtained fromfats containing Cge1,fracas tions.Longeralkylchainsproducesoftsoaps,sincetheyarenotsoluble monomers in water and the surface liquids of the skin (sebum, sweat). Sulfonates, on the other hand,do not show such differences because they are always present as fully dissociated saltsat physiological pH values and produce no hydroxyl ions. Allergic reactions to commercial soaps are mostly not caused by the fatty acids butby additives, suchas perfumes. Polyvalent metal salts(Ca,Ba, Zn) are called metal soaps. They are water insoluble and are applied as lubricants. Fatty acids themselves do not have any cleaning activity in water (see Fig. 2.5.7), since theydo not dissolve. Unsa~ratedfatty acids belong to the group of ~nctionalfatty acids with pronounced reactivity (see Sec. 2.4). The main importance of the Z-configured double bondsof natural fatty acids in membrane structures, however, consists of the lowering of melting points (Table 2.2.1).The corresponding destabili~ation of crystals is caused by the rise in flexibility of the methylene groups neighboring the methine groupsof double bonds,as mentioned before, and, more imporconformations next to the co con figured tant, by the enforcement of ~yncZi~aZ double bonds. This can be seen most clearly in crystal structures. Oleic acid retains a molecular structure withall CH, groups ina~ti~eri~Za~ar confo~ations, which then means that the central Z double bond leads to a U-conformation of the whole molecule (Fig. 2.2.3a). In biological membranes, where saturated and unsaturated alkyl chains are mixed in a statistical manner, such a U-conformer would leave a huge gap, which is of course not tolerated.The usual way out is instead of then given bya rotation of neighboring CH, groups to form syn-cZi~~Z anti-~eri~zanar conformers.Onefindssuch a compensationincrystals of linoleic acids, wherethe large U-curve is straightened to a flat S-curve connecting two more or less linear alkyl chains (Fig. 2.2.3b). Z double bonds thus disturb crystallinity of membrane structures, make them more fluid, and thereby improve their dissolution power, but they do not open large cavities.An admix-

Typicalpacking of unsa~ratedfatty acidsin (a) oleic acidand (b) linoleic acid crystals. (From Emst et al., 1979).

ture of short-chain amphiphileshas in principle the same effect, it but also drastically raises the solubility of the amphiphile in water. The membrane becomes more fluid andis at the sametime endangered by micelles formedby dissociated amphiphiles. Oleamide (~~~-9-octadecenamide; Scheme 2.2. l)is a brain constituent that accumulates under conditionsof sleep deprivation and disappears upon sleep recovery. ~ a n o ~ o lquantities ar induce physiological sleep when oleamide is injected intravascul~ly.An integral membrane oleamide hydrolase then catalyzes its degradation (Cravattet al., 1996; Patterson et al., 1996).

Tetraalkylammoniumand ~-alkylpyridiniumamphiphilespossessapproximately half the solubilities of their anionic counterparts. Protonated ammonium salts can be precipitated by bases, quaternary a ~ o n i u msalts by iodide or perchlorate ions. The latter anions are large and hardly fitinto solubilizing water clusters around cationic head groups of amphiphiles. They are therefore called “chaotropic.” The crystal structure of d o d e c y l t r i ~ e t h y l - a ~ o nium bromide (not shown) has the same side-by-side arrangement of ions and counterions as potassium palmitate and the same interdigitation pattern seen in piperazinium bis-dodecanoate (see Fig. 2.2.1). Double-chain tetraalkylammonium salts, on the other hand, show the usual head-to-tail arrangement (not shown). Invert soaps are active as bacteriocides. They are insensitive to hard waterand are standard detergents in shampoosandwashingpowder. Their magic lies in their ability to change the surface properties of anionic colloids and polymers (see Sec. 2.7). a,w~ifunctionalamphiphiles are called bolaamphiphiles. The name originates from the shape of a South American sling called a bola, which consists of two wooden balls connected by a string. a,wDicarboxylic acids and a,wdiamines provide no particular new structural elements. A Z Z - a ~ t i ~ e ~ i ~ Z a ~ a ~ conformations dominate. The most interesting molecular interactions are, however, found inthe zwitterionic ~-ammoniumcaproate(Fig. 2.2.4). ThreeNhydrogen bonds are formed between NH,+ and COO-groupsof n e i g h b o ~ nmol~ ecules in the crystal I. O(2) participates in two hydrogen bonds, 0(1) in only one. The molecular packing shows two sets of molecular chains inclined toward each other at 55”.If in such crystals polycondensation is enforced by heating, nylon4 is formed with antiparallel polyamide chains. In the course of this reaction, the chains change their relative orientations in order to become parallel to each other. A rotation of 55”occurs, but the relative positions of the NH,+ and COO-groups are retained: N(2) reacts with COO(l), N(3)with COO(4), etc. In nature one also finds chiral fatty acid or fatty alcohol derivatives with a hydroxyl or aminosubsti~entat the alkyl chain.Cornmon examples are given in Table 2.2.2 of thefatty acids in cheap comercial castor oil(a fat mixture) consist of ricinoleic acid(l2-~-hydro~y-9-~-octa~ecene-carboxylic acid). The hydrogenation product (R)-l2-hy~oxy-ste~c acid is the cheapest chiral fatty acid (about $lOO/g). Sphingosin (2-S-amino-4-E-octadecene-173-R-&ol) is a c h i d a-amino($5,OOO/g). Chirality is expendiol, whichis found in large amounts in nerve tissue sive, if it is localized witbin the hydrophobic chain. The most common lipids in the human body are phospholipids and glycolipids, which are based on glycerol diesters (glycerides) or sphingosine amides (s~hingosides;Table 2.2.3). Naturalamphiphiles in membraneformationcontaincarboxylicacid

7

0

0

(a) Crystal structure of m-aminocaproic acid. Eight neighboring molecules in twocrystal sheets are given. Each nitrogen atom is connected by three hydrogen bridges to neighboring oxygen atoms.(b) Hydrogen bond patternin the polyamide made of m-aminocaproic acid. (From Bodor et al., 1967.)

a1 Fatty Acids and Alcohols

OH

0

R-l 2-~ydroxy-9-Z-octadecenoic acid

OH

R-l 2-Hydroxy-stearic acid

OH

Cerebronic acid

and/or phosphate ester bonds; occasionally amides are found. These bonds are more or less susceptible to hydrolysis at low pH or high temperature. Archaebacteria live at high temperature (thermophilic bacteria) and low pH (acidophilic bacteria), whereat least the ester bonds would have no chance to survive. Evolution has led here to ether-linked lipids, which appear amphip~les in as well as in bola~phiphiles(Langworthy, 1977; Langworthy et al., 1982; Ourisson et al., 1984; Schnabel, 1984). Table 2.2.4 gives some examples of natural double- hai in ether lipids isolated from archaebacteria. It is by no means necessary to use natural lipids in order to form membranes. Modern bioorganic chemistry rather tends to develop new molecules, which allow production of membrane materials with properties unknown in nature (e.g., ultrathin asymmetrical membranes with different headgroups on the in- and outsides, polymeric membranes, and membranes that can be isolated and stored without water; see Sec. 2.5). Table 2.2.5 reproduces a9few useful artificial amphiphiles derived from simple fatty acids and fatty alcohols. Many biologicalcells contain fat and wax droplets madeof lipids without

*,R3= Saturated or u n s a t u ~ t ~ lkyl cham of a fatty acid

lycolipid :R’ = Ca~ohydrate

hydrated headgroups, Fats are glycerol triesters with three fatty acids; waxes are fatty acid monoesters of large, hydrophobic alcohols. We shall discuss the wax cholesteroyl octanoate in Sections 3.1 and 3.2. Natural glycero or sphingosine lipids contain chiral centers, whose role in membrane structures is not known. The description of these stereocenters in the conventional nomenclature of glycerolipids and sphingosines varies in the literature and is somewhat confusing. The correct name for L-a-glyceropbosphate, for example, is ~-glycerol-l-phosphateand is equivalent to L-glycerol3iphosphate. This confusion arises because Fischer rules do not differentiate between different substituents of chiral alcohols. The difficulty is, of course,

Archaebacterial Arnphiphiles and ~olaarnphiphilesContaining Ether Linkages

H t i

H

OH

avoided if the universal RIS system is adopted in the usual way. Fischer’s conventions are modified by additional preference rules. One starts numbering glyc~rophosphatidesin a Fischer-type projection with the phosphatidyl carbon as C1 (Fig. 2.2.5). The lipids then obey the same Fischer rules as introduced for amino acids and carbohydrates. This is the best nomenclature when conformational changes at the asymmetrical center are considered. Both, the modified DIL and the pure RIS nomenclatures lead, however, very often to changes of configurational prefixes when ester bonds are formed or hydrolyzed, although none of the four bonds at the chiral center is changed. Phospho~lation of S-1,2-dipalmitoylglycerol,for example, yields an phosphatidic acid. This disturbing effect is overcome by the stereospecific numbering of the sy1 nomenclature. It accepts that the two primary alcohols are hardly ever identical in the usually disymmetrical structures of lipids. The numbers 1 and 3 can no longer be used interchangeably for the same carbinol carbon atom, but remain fixed. In the case of glycerolipids it is laid down that if the secondary hydroxyl group is to the left of C2, the carbon atom above is called Cl. An sn prefix is used to indicate this convention. No formal inversion can now take place as long as the four bonds of C2 remain intact. The chirality of C2 is thus not be expressed directly, but the optical antipodes are described as sn-3 or sn-l. The conformation of the carbon atom carrying the phosphate diester group

Synthetic Membrane-Fo~ing~olaamphip~les R' = R2: -SO3Na R2

R

R' = R2: --S-C,H-COONa H2C-COONa R': -S03Na

R2: --S-C,H-COONa H2C-COONa

0

H00

COOH

x = S;s=o;s,(,O,

is best described by Klyne-Prelog designationsof the torsion angles at thethree constituent chains. The torsion angle may change from +sc to up, whereby the head-group changes froman orientation parallelto the crystal layer (A-form) to pe~endicular(B-form). This situation is, however, energetically unfavorable, because the P-N dipole tends to keep all plus and minus charges within one plane. The separation of all negative and all positive charges into two separate layers requires energy.The most favorable conformationof the glycerol oxygen atoms bearingthe fatty acid chains is ~ S Csince , the sp rotamer does not allow a proper parallel alignment of the hydrocarbon chains. This conformer is invariably found in crystalline glycerolipids (Fig. 2.2.6). Therefore one finds a rigid part of the molecule bound to the acyl chain combined with a flexible head group. The alkyl chains areall-up and slightly tilted (78", not shown).The extra

0

. *

. Conventional designationsof the stereocentersof glycerolipi~s(top) and sphingosides (bottom).

Crystal structure of di~y~stoyl-phosphatidyl-choli~e (DMPC). (a) Molecular structure,A- and B-forms; (b) bilayer crystal sheets, A form..

Components of Sebum andIts Natural Moisturing Factor ~

" ~

Sebum: 28% fatty acids ((216, CIS) (Ser,Clu,Pro), 32%fats, 14% waxes (fatty acid esters of fatty alcohols), 13% hydrocarbons(5% squalene), 13% steroids (mostly cholesterol),pH L: 5.5 Natural Moisturizing Factor: 50% aminoacids, 12% lactate, 7% urea, 20% inorganic salts (Na+,K+, Cl, etc.)

substituent in the sphingosine-derived headgroup, on the other hand, enforces strong gauche bendings of the hydrocarbon chains in order to bring them into packing contact. Such bendingsusually occur next to the ester groupsand nowhere else in the alkyl chain (not shown). Sphingosines andrelated ceramides (Table 2.2.3)are therefore differentiated from the glycerolipids by strong binding interactions between the headgroups (more crystallinity) and more disturbed alkyl chains (less crystallinity). Although the commercially most important triesters of glycerol, the fats, hardly play a role in supramolecular chemistry, they must be mentioned here. Fats are stored in organisms as sources for energy production by com-

0

a

lestra

A

.

.

\ \

\

\ \

\ \ \ \ \

\

Crystal structureof a model fat (see text).

bustion (700 kcall100 g) and as precursors for the synthesis of membranous amphiphiles. Furthermore, they play a role as lubricants in joints and as a main component of the thin isolating layer on the skin surface (sebum), which hinders evaporation of water from the body. The sebum (Table 2.2.6) is also the only human fluid that contains free fatty acids. This acid mantle has a pH value of around 4.2 and prevents the growth of microorganisms (“inflammations”). Furthermore, the sebum contains a variety of low molecular weight molecules and ions, which help to stabilize the thin layer of the acid mantle emulsion by lowering its vapor pressure. These compounds comprise the socalled natural moisturizing factor (NMF), which is occasionally copied in cosmetics. Life insurance statistics show a significant inverse relationship between lifetime expectancy and highfat consumption, whichis mostly motivatedby the pleasant mouthfeel andthe release of fat-dissolved aroma onthe tongue. Dietary experiments with large numbersof rats fed with5-10% of their food as fat produced animals that lived for an average of 700 days; a 20% fat intake lowered this valueto 450 days. Oneway out of this dilemmais the useof indigestible fat derivatives. The best studied contemporary exampleis the fatty acid sucroseester, or “olestra,” containing eight indigestible stearic acid side chains (Scheme 2.2.2). Its taste and mouthfeel are similar to those of fat, but its calorie content is close to zero. The molecular and crystal structures of fats are e~emplifiedhere by a small, crystallizable model compound with two C, chains at carbon atoms l and 2 of glycerol anda C, chain onC3 (Fig. 2.2.7). Chainsl and 3 run parallel inaZEtrans confo~ationsexcept for the usual gauche bent next to the ester bond on C,. Chain 2 runs in the other direction, is also aZZ-trans, and is parallel to the two chains of neighboring molecules.The a ~ a n ~ e m e of n t the molecules as a whole within a crystal plane is uniformly antiparallel. Such uniform fats do not occur to any extent in nature. Natural fats are statistical mixtures of glycerol triesters with three different fatty acids usually containing 12-20 carbonatomsand c~s-con~gured doublebonds. A. rulethat seems oftento be followedis that the few percentof unsaturated acids in animal fats are locatedon the central carbon atomof glycerol, whereas in plant fats their position is rather onC l or C3. Crystals of single-chain ammonium salts(inverse soaps) with azobenzene units in the centermay contain interdigitatedor noninterdigitated chains depending on even- or odd-numbered alkyl chains, The relative positions of the chromophores and their inte~olecularinteractions are different in both cases, and one obtains abso~tionbands either at 300 or 390 nm in the crystals with otherwise identical chro~ophores(Fig. 2.2.8). Similar orientation effects influence the poly~erizationof molecular monolayersof fatty amine derivatives containing diacetylene units.

tacked

Crystal structure of synthetic even and odd amphi~hilescontaining an azobenzene unit in the center. Thecrystal on the left corresponds to a stack of dyes and absorbs at 300 nm; in the second case a lateral arrangement of dyes is more prominent and the crystal absorbs at 390 nm. (From Okuyamaet al., 3988.)

Oil seeds are dried at first to a water content of about 10% to avoid enzymatic hydrolysis. Then they are "expelled" (pressed)to a fat content of 20%. The expeller cake is extracted with h ocarbons, usually hexane, and is later from the oil with water steam xane solubility in water is only 0.1% ing of the raw oils occurs with aluminum silicates containing montmorillonite at approximately 60°C. ~eodorizationis the final step in the purification of the fatty raw materials from plants and occurs essentially by steam distillation. Milk is defatted by cen~fugation,animal tissuesby heating and sedimentation. ~ a ~ o ~ i f i c a tisi ono n longer accomplishedby sodium hydroxidetreat~ent, but by active earth-catalyzed hydrolysisclose to neutralpH values.

(11) Wittig long chain fatty acids

QY

CH0 11

1

or dicarboxylic acids

'*

The chemistry of fatty acidsis well developed, and structural modifications are c o m o n in industry. Functional group inversionsto produce fatty acids from fatty alcohols(RuO,-KIO, in acetone-wateror permanganate~rownether in benzene), fatty nitriles (acid or alkali hydrolyses), or unsaturated fatty acids (chlorination and dehydrohalogenation to form acetylenic acids) are of minor practical importance, since saturated fatty acids can either be purchased or are accessible by ozonolysis of undecylenicacid or aldehydefollowedbyWittig-typecarbonchain extension. Hydrogenation of double bonds as obtained in Wittigtype syntheses yield selectively deuterated carbon atoms within the alkyl chainNfor U R studies, but it is necessary to use homogeneous catalysts. Heterogeneous catalysis leads to a s c r ~ b l i n gof deuterium atoms. Long-chain amines are made by the reductionof nitriles, which are accessible from the oximes of the above aldehydes and dehydration. Terminal trideuteromethyl groups may be introduced into (iodonitriles with deuterated dimethyl lithium cuprate (Scheme 2.3.1). Acetylenic and, more interesting as polymerizable monomers, diacetylenic fatty acids and their derivatives have been made by oxidative radical coupling. Using copper(I) salts as catalysts almost any head-group, including carbohydrates, amines, and arnino acids, survive the conditions of such coupling reactions. Combination of two acetylenes with different headgroups gives asymmetrical compounds if one of the acetylenes is brominated first. A stunning reaction sequence leads to a-hydroxy-o-alkynes. It starts with an alkyne, to which formaldehyde is added. The resulting a-alkynol is isomerized with a strong base to form the a-alkyne-o-ol in 60-80% yield even when 16 carbon atoms have to be rearranged in a series of protonationdeprotonation reactions (Scheme 2.3.2). carboxylic acids canbe extended by six carbon atomsby an enamprocedure of cyclohexanone. The carbonyl groups in the center may be reduced by a Clemmensen or Wol~-Kishnerreduction or used for the introduction of side chains.An easily accessible macrocycle with two maleic ester units has been converted to various a,o-bolaamphiphiles with identicalor different headgro~ps.Tetrasulfide nzacrocycles are easily accessible from a,@-diols and thiodiethanol.The sulfide groupsmay then be oxidized to sulfoxides or sulfones and work as perfect headgroups in monolayered ~embranes.These arethe onlymacrocyclictetraethersavailableasmodels for archaebacteriallipids (Scheme 2.3.3;see Table 2.2.4) (Fuhrhop, 1988). Unsaturated fatty acidesters as obtainedby Wittig reactions as well as saturated ones have been dimerized to gemini-diesters by deprotonation and subsequent oxidation to radicals (Scheme 2.3.4). Total synthesesof glycerolipids using protective groupsfor both the glycerol andphosphategroupsusuallystartwith 1,2-isopropylidene-~ his is readily obtained by oxidative cleavage of the bis-acetonide o

MeOOC

H -

cat. CuCI, DBU Pyridine,O2

MeOOC COOMe

HOOC N H

OH

tol. The free alcohol onC3 is first esterifiedeither with an activated fatty acid or phosphoric ester derivative. The aceto~de-protectinggroup is then removed, and the alcohol groups are tosylated. Since primary tosylates react much faster than secondary ones, the large leaving group also serves as a kinetic protecting group. The three glycerol oxygen atoms can thus be connected to three different acids or alkyl groups. Asymmetrical phosphodiesters with two different alcohols were prepared from the diphosphate via monoalkyl dihydrogenphosphate in two steps (Scheme2.3.5). Uniform model compounds of glycerophospholipids with a large variety of headgroups are available on the gram scale by transphosphatidylation of diwith almost any chosen alcohol oleoyl- or dimyristoyl-~-phosphatidylcho~ne

coox

H 0

-

0

-

0 0

H

H

L

coo COO^^

COOMe

ha

0 0 O+X2 0

0 ol,o.J-xl bH

84%

-0

85%

28%

52%

H

I

-0

-0

I

H2N-Ser-Gly-Val-OMe

H2N-Ser-OMe

82%

OH

0

32%

31%

OH

69%

and commercial phospholipaseD as a catalyst. Scheme 2.3.6 gives a small, typical collection.

The biological oxidationof fatty acids starts with an ~ , ~ ~ e h y d r o g e n a tofi ofatty n acid-CoA thioesters followed by a hydration and oxidation of the resulting p-alcohol. The resulting p-ketoacylCoA looses acetylCoA (retro-Claisen condensation) and the cycles start again (Scheme 2.4.1). Fatty acids are thus split up in acetic acid units, which is one of the most time-consu~ngprocesses in the body's me~bo~sm. Several model reactionsfor the dehydrogenation of acid anionsvia the enolate have been worked out (Scheme 2.4.2). Regioselective hydroxylation of the nonactivated p-carbon atom, which is standard in biochemistry, has not been achieved in nonenzymatic model reactions. Heteroatoms (e.g., Br) can only be introduced at the (position with external reagents (Scheme 2.4.2).

S

an

R

SCoA

H

R

SCoA

W

H

-

retro Claisen R

R SCoA

H

0

0

0 Na0

0 Na0 Li

0

R

H

0 0

0 Na0

0

0

0 OH

OH

The situation changes drastically if long-chain oxidants are applied. One example of a successful regioselective oxidation of a nonactivated carbon atom comes from the use of long-chain tertiary amines as catalysts. They are first oxidized to an aminoxide with hydrogen peroxide in the presence of iron@) salts, These radical-type oxidants form to molecular complexes with long-chain alcohols

in polar solvents (e.g., in trifluoroacetic acid) and transfer their oxygen atom regioselectively. I-Octanol is, for example, oxidized to 19’7-octanediol in ’70%yield (Scheme 2.4.3). Another technique is to bind carboxylic acid to ~ u m i n and u ~ then to chlorinate them with a bulky radical oxidant, Only terminal alkyl groups are then attacked. Similar regioselectivities have been achieved with intramolecular oxidation of long-chain esters. enzophenone forms biradicals upon whicheliminatehydrogetomsfromsaturatedhydrocarbons. esters prefer intramolecular attack, and a cetylester is oxidized at 614 in 66% yield (Scheme2.4.4). The general schemeof this remote oxidation proce beenappliedevenmoresuccessfully to rigidsteroidskeletons (see 3.4.3). Saturated fatty acids belong to the most stable organic compounds under sunlight inthe oxygen atmosphereof earth. Unsaturated acids,on the other hand, are very vulnerable to chemical oxidation under atmospheric conditions. react to form peroxides at the allylic methylene groups, especially in the presence of paramagnetic metal ionsandor microorganisms. The reaction is autocatalytic, becausethe primary reaction products, the hy~operoxides,decompose to form radicalsagain. The chain reaction stops when two radicals combine to form ~ ~ ) to ketones (rancid butter). Analogous alsolid oligomers( v ~ r ~ori dehydrate lylic peroxidations can be achieved in high yields using selenium dioxide and bulky peroxides. Subsequent activation with chloroforrniate and palladiumcatalyzed substitution with all kinds of alcohols (e.g., p ally protected carbohydrates)yieldcomplexdouble-chainbolaamphiphiles. c r o b i o l o ~ i coxidation ~

OH

0 OH

OH

0 0

of 3-hexenoic acid not only gives the expected allylic hydroxylation, but also leads to the introductionof an extra acetate unit (Scheme2.4.5). Degradation by oxygen is particularly fast if two double bonds frame a methylene group as in linoleic acid. Oil paints containing linseed oil and diet margarine contain such methylene groupsin large concentrations. up to 60%linoleic and linolenic acids. In oil paints the allylic oxidation is the basis of polyme~zation(“drying”) of the oil and is accelerated by p~amagnetic metal ions of the suspended inorganic pigments.In diet margarine, on the other hand, autoxidation has to be prevented. The most important aspectis growth of m i ~ r o o r ~ a ~ s which m s , attackthe active sites by peroxidation. This is prevented on of ultrafine waterfat emulsions with water droplets smaller roorganisms need larger drops for growth and duplication, Antioxidants (vitamin E, carotenes) are added to margarines contai~ngessential fatty acids in order to trap oxygen. Citric acid in the water micro-droplets binds heavy metal ions, which would also catalyze autoxidation. Such margarine contains only compounds that are common the in diet anyway, andit remains stable for monthswithout ref~geration.The aroma of butter is thensimulated by adding copies of the major butter flavor components. Food chemists have thus

R

R2

+

R2

rancy butter

COOMe

(I) 0.5 Eq. Se02 2 Eq. t-Bu00H * . C02Me

OC02Me

1

6

HOOC

1

COOH

1

HOOC

OOH

10.3 gll

accomplished oneof the greatest triumphsof bioorganic chemistry inthe twentieth century, namely a healthy and stable margarine of perfect mouthfeel (cold and creamy) and acceptable flavor. The introduction of two neighboring asymetrical carbon atoms in the center of fatty acids is best achieved by the dihy~oxylationof natural unsaturated acids using osmium tetroxide-type catalysts in presence camphonic acid for enantiomeric purity (Scheme2.4.6). Fluid cis-configured unsaturated fatty acids can easily be isomerized to the solid ~~~~s-diastereomers (e.g., oleic acid to elaidic acid)by radicals such as nitrogen oxides. The ~~~~s-diastereomers are much more stable towards oxygen, because they co-crystallize with saturated neighbor chains and solid fats do not dissolve as much oxygen.'They are, however, worthless as an essential foodstuff (Scheme 2.47).

OH

(l) HPLC II KOHl MeOW COOMe

COOMe (+)(R-Q,S-IO)

(-)(S-Q,R-10)

inally, the amphiphilic character of fatty acids can be removed by elec,which leads to decarbo~ylationand dimerizationin high yield (Scheme 2.4.8). The chemist^ of complex lipids is dominated by re~ioselectivehydr reactions of (1) the glyceryl fatty acid esters and (2) phosp~atediesters. types of reactions are routinely performed with the corresponding esterases. A ge variety of lipid active tr sferase enzymes is also c o ~ e r c i a l l yavailable. ospholipases A,, A,, C, and ,for example, split anyof the four ester bonds of hospholipid regioselectively.The product withouta fatty acid sidec glycerol is called a lysophos~holipid*~ecithin-chol rol-acyltransferase OUP at C3 of chotransfers the fatty acid at C2, often linoleic acid, to the 0

electrolysis 2 90%

lesterol. Other transferases catalyze similar transeste~ficationsof amino acids, nucleosides, etc. with fatty acids (not shown here; see Scheme 2.3.6) Macrocyclic model lipids for the formation of vesicles often contain four ester bonds-two on each head group. It has been found that theseester bonds are exceptionally stable against acid-catalyzed hydrolysis in water.The reasons probably lie in the partial hydrophobicity of the ester group’s environment, the macrocycle’s order, andthe reversibility of ester hydrolysis. Partially hydrolyzed tetraesters are presumably i ~ e d i a t e l yreformed in vesicular membra~estructures beforethe second, third, andfourth ester group reacts (Scheme2.4.9). A large variety of standard procedures exists for the exchange of heteroatoms in simple headgroups. Hoffmann and Curtius elimination allowinthe troduction of terminal amines insteadof carboxyl groups (not shown). Terminal sulfide groups needed in selfassembly reactions on gold are usually introduced by substituting a terminalhalideatom by thiocarbonicacid,which is then cleaved by Nd3H4 in the presenceof amines (Scheme2.4.10).

HOOC

Br

@OOC

SW

Fluid syn~neticmembranes are formedby self-organization of water-insoluble amphiphiles (see Sec. 1.5) Theyare characterized by extensive hydration of the a~phiphile’sheadgroups and a loosely organized hydrophobic interior, which serves as good solvent for water-insoluble substances. If one considers an amphiphile as a solvent molecule, its solubilization power in fluid membranes is comparable to chlorofom, one of the best organic solvents.

Cell membranes (elemental membranes, ~iomembranes)are madeof a molecular bilayer Figure 2.5.1) of water-insoluble lipids (solubility 10loM!), cholesterol (3~50-m01%)and membrane proteins (20-50% of the membrane space). Cell membranes are stable only in bulk water. Organic solvents as well as lyophilization or other drying processes and contact with solid walls lead d eto s ~ c t i o nIn . archaebacteria, one also finds molecular monolayers made of tetraether bolaam~ ~ p h i l (Table es 2.2.4). The t?&kness of biological cell membranes ranges usually from4 to 6 m. ~iomembranesare not only the only organicsolvents in nature. They also allow the const~ctionof vectorial reaction systems. Itis relatively easy,for example, to localize a photoactiveelectron donor (e.g., chlorophyll)on one side of a membrane and an acceptor (e.g., a quinone) on the other side. Visible light may then ecxcite the chlorophyll moleculeto produce an energy-richelectron, which may travelinnanoseconds to the quinone. The back-reactionbetween the formed cation and anion radicals through the membrane may, under circum-

ran

D

amphiphil~s (= ~ ~ m b r a nproteins) e

Model of a biological membrane (see text).

stances that have been optimized in evolution, beso slow that the oxidized and reduced dyes can undergo further chemical reactions. Oxidized chlorophyll molecules produce oxygen from hydroxide ions of water; semiquinones reduce protons to hydrogen atthe end of long reaction chains. This process is fu~damental to biological photosynthesis andits realization by synkinetic systems the major dream of many chemists, A large variety of totally artificial membranes accessible by s y ~ n e s i s have structures unknown to biological systems. One may, for example, prepare membranes as thin as 2 nm containing photoactive groupsin various positions, and they may show a totally unsymmet~cal dis~bution of two different headgroups on both surfaces of a curved membrane (see Sec. 2.5.3). Natural membranes, on the other hand, are incredibly functional. They perform the complex energyconversionandreproductionprocesses of life withunsurpassedefficiency and reliability. In the following we describe simple chemical models that be cananalyzed on an atomic level and that have been developedto perform single, useful tasks in dailylife and inthe lab.

The word 66"miceZZe9' indicates nothing but the ultimate smallness of a spherical molecular bilayer (Latin: mica, grain; + eZZa, diminutive suffix). Micelles are loose a~gregatesof amphiphiles in water that form above a certain temperature point) and concentration (critical micellar concentration, cmc). temperature, clear micellar solutions become turbid and h crystals are formed and often precipitate, In the case of electroneu~almicelles the adverse effect may be observed: at temperatures above the Bafft point precipitation may occur because the headgroups are dehydrated; at lower temperatures h y ~ a t i o noccurs and the solution clarifies.elowthe cmc micelles degrade to forma mixture of small aggregate com~oundsprecipitate below or above the cmc. ~ b o v the e crnc micellescontaining a detergent-specific numbern of moleformed. For typical fatty acid salts (soaps), the cmc is of the order of d the aggregation number n is between 50 and 70 molecules per miy for amphiphiles containing twoamm m groupsconnectedby a short spacer the cmc drops to values as low as 10The diameter of a micelle is typically twice the molecular length. A cross se through a typical micelle igure 2.5.2. The center of the micelle (20% of the volume) is relatively free of water; the rest is rugged and wet, The alkane chains of the wet region are predominantlyin an aZZ-a~ticonformation, whereas the inner segments are ~uid-likewith a predominance of gauche rotamers.

Sterochernical modelof a short-lived micelle.There are no binding interactions between the ~ o n o ~ e rAl sl.headg~oupsare separated by hydration spheres(repulsive hydration forces).

icelles made of soaps break upand reform within milliseconds.The individual lifetime of a charged micelle is very short. Nevertheless, eicosane sulfate micelleshavebeentrapped by rapidcryo-fixationatliquidnitrogen then be seen under the transmission re important than any other: they solubilize organic compoundsin water. A single sodium dodecyl sulfate( celle, for example, dissolves up to 40 benzene molecules or a single p o ~ h y r i n molecule or onehy~ophobizedAT pair (Fig.2.5.4). Very often micelles madeof long-chain sulfonates are chosen as solubilizers instead of the natural carboxylates, because carboxylates tend to precipitate with bivalent metal and ammonium counterions. Sulfonate micelles are much more hydrated and remain, for

Electron micrographof a cryo-fixated eicosane sulfate micelle.

(a) One porphyrin molecule or (b) two ~ p h i p h i l i cnucleic bases may be dissolved within one rnicelle.No more! The electron spin resonance (esr) spectra at the bottom of (a) were talcen from the modeled micellarSDS solution (left) and the aqueous solution withoutSDS (right). Themultil~esignal corresponds to a copper(II) monomer; the singleline spectrum isfor an aggregate. The diameter of the micelleis about 5 nm.,

example, soluble in the presence of the calcium ions of hard water. Sodium lauryl sulfate produces a creamy foam in shampoos, which is stabilized by fatty acids (RCQQH) and diethanolamides[RCQN(CH2C~20H)~]. SDS micelles have also been used as basis a for light-induced charge sepaporphy~nate) ration processes.A hydrophobic, photooxidizable dye (e.g., a zinc was, for example, dissolved in an anionic SDS micelle with copper(I1) counterions. Upon excitation with visiblelight an electron was transferred first and very fast to the copper(I1)coating, which then was reoxidized by anionic ferricyanide in the bulk water phase.The reduced ferrocyanide ion formed did not react with the oxidized porphyrin, becausethe anionic micelles and reductant repelled each other and the ferrocyanide was highly diluted by ferricyanide (Fig. 2.5.5). The energy of sunlight has thus initiated a simple vectorial reaction in a primitive membranous system. Nature avoids micelles made of soaps because they first integrate into biomembranes and then degrade them. Steroidalbile acids are used insteadfor the dissolution of water-insoluble foodstuffs in water (see Sec. 3.5). Micelles can also be made in organic solvents. The usual a ~ p ~ p h iofl e choice is a branched dioctyl sulfosuccinate called AQT di-(2-ethylhexyl)-sodiumsulfosuccinate; AerosolT; (Fig. 2.5.6). AQT micelles in organic solvents are inverted micelles in which the hydrocarbon chains point into the bulk medium, while the sulfonate headgroups stabilize the water droplets. The inverted micelles dissolve enzymes in isooctane, benzene, and similar solvents contain in^ about 10% of water. C h y m o ~ p s i ndissolved in tiny water droplets remains, for example, very efficient in the hydrolysis of hydrophobic peptides (e.g.,N-glutql-Lphenylalani~e-p-nitro~lide) or in ~ ~ s e s t e r i ~ c a t reactions ion of hydrophobic esters with amino acids. The most important economic application of soaps and analogous deter-

re Scheme of a vectorialelectrontransport lar system (seetext).

or chargeseparationin

a micel-

Model of inverted micellesfor the dissolutionof enzymes and hydrolysis of a hydrophobic peptide.

gents is, of course, the washing process. Soaps are very efficient in dissolving grease (Fig. 2.5.7). The alkyl chains dissolve in fattystains and the headgroups introduce chargesto its surface. The fatty layer is thereby t r a n s f o ~ e dto watersoluble fat droplets. What appliesto conformationally soft grease works neither with stacked multilayersof dyes (e.g., sulfonated aniline blue of ink or heme of blood stains) nor with proteins containing rigid crystallines e c ~ n d ~ structures y (e.g., the helices of hemoglobin). Such organic compounds are soluble in water, but once they are adsorbed to solid surfaces, they can be removed neither by purewaternorbymicelles.Theyhave to be destroyed by strongoxidants (bleaching) or by enzymatic degradation (hydrolysis). ~rintingand writingcan be seen as a contrast to washing. They dependon inks. Color printing inks are made primarily of linseed oil or soybean oil or a

Model of various types of co~poundsadsorbed to solid surfaces and their interaction with flexiblea~phiphilesof soap character.

heavy petroleumdistillate as the solvent (“vehicle9’) combined with aromatic and heteroaromatic salts or “pigments” (insoluble dyes). ~ r i t i n ginks, on the other hand, contain solutions of dyes because pigment particles tend to clog the pen tip. Linseed inks dry by air oxidation leading to cross-linked polymers in the same way as oil paint (see Scheme2.4.5). Inks with an alcohol- or hydrocarbontype solvent dryby evaporation, sometimes assistedby heating the paper. inks do not rub off easily. Newspapers, however, are generally printed a at thousands of meters per minute, andthe ink is only adsorbedby the inner cellulose fibers of the sheet of paper, not dried. The vehicle does not evaporate pletely and the ink therefore partially rubs off. A journal of about 50 p printed with 1 m1 of ink, most of it consisting of fluid fats (Ritter, 1998).

on covalent fibers do notoccur in nature.Theyare of synkineticure onlyusescovalentpolymers. The mostsimplesynkineticroute to covalent assemblies applies fatty acidsof intermediate solubility and variationsof and counterions. Myristic acid with l 4 carbon atoms is most suitable, An “acid soap” consistingof 50% sodium myristate and 50% myrist~cacid crystal-

lizes, for example, in thef o m of thin platelets from water at pH 9, which is four orders of magnitude abovethe pKaof 5 of myristic acid!The high concentration of negative chargeson the surface of the crystals leads to this enormous change of the pK with respect to solutions. At higher pH values hairylike crystals of light microscopic dimensions or “curds” are fomed, which are isolable in dry form and can be spun to form soap tissues (Fig. 2.5.8a). Neither the acid soap platelets nor the fibrous soap materials dissolve anything. They behave like crystals, notlike micelles. Eihedrine as a counterion has difTerent a effect. It induces the formation of fluid fibers at a concentration of approximately lo3M. These fibers havenow the thickness of a few molecular bilayers only (Fig. 2.5.8b) and dissolve achiraldyes (e.g., po~hyrins)in achiral packing mode. Strong circular dichroism is observed for the fiber-dissolved dyes, whichis presumably caused by stacking interactions of the aromatic counterions.The f l u i ~ t yof the fibers is presumably related to the gliding of these hydrated counterions on each other (Trager et al., 1997). Sodium ions cannot form such aggregates, and the corresponding myristate fibersare hard and crystalline. Similar assemblies have been extensively characterized for the ion pair cetyl~methyla~onium-salicylate. In both cases the micellar fibers produce slightly viscous solutions and the effect of viscoelasticity is observed: if one rotates such a solution and suddenly stops the rotation, small particles in the solution (e.g., air bubbles) bounce back. While the bulk wateris still rotating in the nonviscous solutions, the inertiaof the high molecular weight threads builds up an elastic wall for the suspended particles and pushes them back. Lithium and sodiumricinolatesproducehelicalmicellarfibers of opposingchirality in toluene (Tachibaona, 1970, 1978).

Vesicles (Latin: vesicula, bladder) are sealed, extremely thin(8. Upon acidififor example, micelles with an aggregation number cation to pH 4-5 only half ofthe soap molecules are converted to fatty acid molecules, which are then connected by hydrogen bonds to the carboxylate anions. Large clusters are thusfomed, which then rearrangeto give vesicles. At pH values below 3 all molecules become protonated and electroneu~al,planar bilayers precipitate. This observation can be generalized:wellwater-solubleamphiphiles form sho~livedmicelles with the smallest possible radius of one molecular length; less soluble amphiphiles buildup planar molecular bilayers or spherical

(a) Fibrous crystalsof sodium myristate (light micrograph) and (b) fluid fibers madeof ephedrinium myristate (cryo-electron micrograph).

0th types of assemblies are metastable intermedi evertheless, they may survive for years in water. live forever because rnicelles are in e~uilibriumwith monomers and dissolve crystallites. Vesicle membranes, on the other hand, undulate and not stick together. If the monomers of micelles become insoluble nsoluble salts) they precipitate. If vesicle memb~anesbecome by dissolution of massive amountsof steroids or c~otenes)they

order thein

co~elationbetween the shapes of an amphiphile (e.g.,ratio of skeleton volumes)a d the shape of its polymeric asysical model calculations (Israelachvili, 1992) have m o l e c ~terms. l ~ (Israelachvili, 1992, see chapt~r1) Vesiclesenclosewatervolumesbetween and m3, and theycanbe 0 or concen~ated(up to large (diametersfiom a p p r o ~ a t e l y30 to 3 ~ 0 m) ids) that upto a few percentof the bulk water volume becomes en~appe~ iochemists also use the name liposome (fat particle) A g ~ e g a ~ onumber§ n are usually of 104"1Os~ o l e ~ u l e -lived, have critical low

Schematic drawing of molecularbilayer(left) vesicle ~ e m b r ~ e s .

and monolayer (right)

concen~ations( 4 0 5 M), and very few monomers are correspondingly found in equilibrium. Although the overall structure is well ordered and the radius of the vesicles is well defined (Fig. 2.S.9), the aggregation state of the vesicle membrane is again essentially of fluid character. All kinds of hydrophobic molecules, even the largest and most rigid dyes, dissolve within vesicle membranes. Aqueous suspensions of egg yolk, which contain massive amounts of lecithin, contain vesicles and are good cleansing solutions. Insoluble bilayer lipid membranes made of the waterinsoluble lecithin will, however, remain on the cleansed surfaces. The bilayer lipid membrane (I3 ) is typically 4-6 nm thick. ds of a hy~ophobicco phiphiles with a water-soluble headgroup on . Membranes as thin as 1. can form thinner monolayer lipid membranes nm have been obtained (Fig. 2.5.9). Vesicles can be used to entrap water-soluble compounds, which cannot pass the membrane within the inner water volume. Inorganic salts or organic polyelectrolytes are typical examples. Protic acids pass fluid membranes within a minute or so, and membranes containing SO% of cholesterol stabilize a p dient of two units for about 30 minutes. Hy~ophobiccompounds are dis within the membrane. Rigid or polar, poorly water-soluble compounds are mostly localized on the outer or inner surfaces of vesicles (Fig. 2.5.10). Charged monolayer membrane vesicles are the most stable. They stay in solution for years and do not grow with measurable speed. Fusion of such vesicles is impeded by the d i ~ c u l t yof the inner c drophobic membrane (Fig. 25.11). The outer 0th types of vesicles, major methods 1. ~ltrasonicationof dispersions of ~ater-insolubleamphiphiles. vesicles with diameters ranging from 20 to 40 nm are obtained. id precipitation of water-soluble amp~philesby a change of addition of approp~atecounterions or by injection of organi tions of the amphiphiles into water. The same uni~ormsmall vesicles diameters from 4 to 10 nm as above can thus be made. ssurized injection of an emulsion of ~ p h i p h i l i clipids into water ugh microporous filters or slow hydrolysis of fatty acid anhydrides in aqueous media. Giant vesicles are obtained by these methods. They have diameters of several pm and can be observed under the light microscope.

Vesicles have molecular masses in the order of 106-108 daltons. Van der als attraction between such gigantic solutes is very large. Nevertheless, vesicles do not precipitate but stay in solution, because the ultrathin membranes undulate.

Typical dissolution pattern in the different regions of vesicle membranes: the entrapped water volume9 may contain an ionic dye, which can be separated from dyes in the bulk water phase by gel chromatography. Theh e a ~ g r o u 3 ~ sand 7 may consist of redox systems, e.g., quinones in different oxidation states. The aqueous and me~braneoussurface regions2,8 and 4,6 may enrich polar or charged compounds. Large and fiat hydrophobic molecules (e.g., porphyrins) prefer the same regions. central The region 5 is thought take up some hydrophobic steroids and carotenoids.

This thermal movement dissipates them after collisions and leadsoftotheone most

~ p o ~ aparadoxes nt of natural compound chemistry: one single lecithin molecule is t o t ~ water y insoluble M), but an assembly of morethan lo6l e c i t mole~ cules is quite well soluble(>lom3 M), provided the assembly remains ultrathin! The IBLM vesicles are usually prepared from a~phiphileswith two long alkyl chains. This leads to 32 or so CH, goups per molecule, whichis enough to produce the desired insolubility. Typical examples are natural phos~holipids(see Table 2.2.3) and dimethyldioctadecyla~o~um bromide (DODAB). The inner

Inmonolayer lipid membranes the inner and outer headgroups are covalently connectedto each other. In all investigated casesso far, the molar ratioof inner to outer head groupsis 1:1. This indicates that no bendingof the hydrophobic core occurs. F ~ t h e ~ o rfusion e , of charged vesicle membranes does not occur easily.

and theouter halves of the vesicle’s bilayer are not identical.In a vesicle with an outer radius of 40 nm and a bilayer thickness 5ofnm, the outer halfof the membrane contains almost twice as many molecules as the innerhalf, and the outer surface is convex, the inner surface concave, with respect the to water volumes. Dissolved molecules therefore have a tendency to concentrate either in the inner half close to the ends of the alkyl chains orclose to the outer surface. These differences in space filling are, however, not pronounced enoughto isolate solutes in a defined region (Fig.2.5.12). MLM vesicles can be made of bola~phiphileswith two identical headgroups or two different headgroups. Water-soluble b i p y ~ d i n i ~ tetrabromide m

Stereochemical models of (a) a small section of a monolayer (MLM) and (b) bilayer lipid me~brane(BLM).The inner partof the BLM has fewer molecules than the outer part. The empty spaces are preferred but not exclusive regions for solutes. In MLMs they are located onlyat the outer surface, in BLMs also in the center.

bolaamphiphiles form vesicles spontaneously if two equivalents of perchlorate ipyridinium perchlorateis water insoluble, andthe one-sided precipitation directly inducesthe curvature needed to f o m vesicles (Fig.2.5.13). A macrocyclic and unsymmetrical bolaamphiphile with a large succinic acid headgroupon one end and a smaller sulfonate headgroup on water soluble at pH >8 and foms vesicles upon acidification to case, however, the precipitated large succinic acid headgroups are located on the outer surface,all small sulfonate headgroupson the inner surface.This has been

Model of a symmetrical monolayer lipid membrane with an asymmetrical distribution of counterions. The empty spaces for solutes are exclusively on the outer surface.

proven by an application of the so-called metachromatic effect: methylene blue cations tendto assemble on the surface of anionic polyelectrolytes and then form E--stacks in water.A s a result, one observes a blue shift of their main absorption band. Very small percentages of anions in a membrane surface can be detected by this effect (Fig. 2.5.14). On the outer, electroneutral succinic acid surfaceno shift is observed, but the addition of a few percentof SDS i ~ e d i a t e l yproduces it. One can thusbe sure, that >99% of the sulfonate groupsof the bola~phiphile are located at the inside of the vesicle. R-shift reagents give similar results

Asymmetricalmonolayeredvesiclemembraneshavebeenobtained from the two bolaamphip~lesshown. (a) All large headgroups are on the outer surface, all small headgroupsat the insideof the vesicle. This phenomenonis by no means universal. It has to be tested for each individual asymmetrical bola~phiphile.In most cases there is only a small difference in the localization of different headgroups. In (b)the h eta chromatic effect of polyanions on methylene blue aggregation on the sulfonated membrane outsideis indicated (seetext above).

neutral cationic

+ DN

Organic polyelectrolytes (e.g., DNA) and inorganic colloids with negative surface charges are efficiently coated by cationic-neutral bola~phiphiles.The neutralized colloid with the soft membrane surface does not precipitate and dissolves organic compounds on the surface.

but are much less sensitive. Such a totally unsymmetrical distribution of functionalgroupsandchargeshasneverbeenobservedinbiological BLMs or MLMs. Even the chiral bola~phiphilesfrom archaebacteria do not form defined vesicle structures. In nature only integrated or associated membrane proteins providethe membrane asymmetry neededfor vectorial reaction sequences. o l a a m p ~ p ~ l ewith s onecationic t e t r a a ~ y l ~ o n i u and m oneelectroneutral carbohydrate headgroup at the ends have been used to entrap DNA and inorganic colloids (e.g., manganite MnOOH) as well as to dissolve porphyrins (Fig. 2.5.15). Such systems may in principle be for used light-induced charge separation, but this useful functionality has not been demonstrated for any of the artificial vesicular systemsso far. Back-reactions between the separated charges are in general too fast to allow coupling of oxidized porphyrinsand reduced quinones with useful chemical reactions. Rigid protein structures in which well-defined conformational changes can follow the charge separation process are obviously much more efficient here than the soft membrane systems tested so far.

Not only do complex lipids form vesicles, but lecithin-type bilayer crystallites also swell in water to form helical multilayers (Fig. 2.5.16a). The resulting fibers change their shapes continuously and are also of fluid character.

H

Typical exarnples of fibrous assemblies made of water-insoluble mphiphiles. (a) The fluid type with a time-dependent shape, which cannot isolated be from be by lyophili~ationand water. @,c) The solid type of constant shape, which can isolated stored for years in thedry state.

If artificial phospholipids with diacetylenic units are sonicated in water, long-lived fluid tubules are formed. "he stiff diacetylene partof the chain obviously provides enough ordering force to rearrange vesicular spheres into short tubules (not shown). Solid rods and tubules, the on other hand, are formed when strong hydrogen-bon~ngamide or phosphate groups are introduced into the headgroups oralkane chains, One exampleis the solid vesicle tubule made of an amino acid lipid shown in Figure 2.5.16-b,c. These fibers are indefinitely stable and can be isolated in solid form by lyophili~ationof aqueous solutions. Fluid micelles and vesicles, on the other hand, degradeto form powders upon the removal of water. More examples of such fibers are given the carbohydrate in (Sec. 45.3, p o ~ h y r i n(Sec. 6.6), nucleic acid (Sec. 8.6), and amino acid (Sec. 9.5) chapters.

Urea inclusion compounds (e.g., with fatty acids) are only characterized

in the crystalline state, but they are formed inaqueous solutions. They provide an indus-

trial storage form for pure polyunsaturated fatty acids. Within urea c h a ~ e lthey s are not oxidized by oxygen.

Atomic force scanning microscopy (AFM) imagesof a cadmium arachidate monolayers. (a) Molecular dimensions of a fatty acid monolayer as obtained from cadmium arachidate monolayercrystal on a water surface. (b) Picture of a hole in a cadmium arachidate monolayer on graphite.

the alkyl chains, andit has been found that tilt angles varyfrom 0 to 33" corresponding to molecular areas from 19.8 to 23.7 A2, An upright, ~ZZ-transconfigured alkylchain has a suxface area of about 20 A2.Methylesters usuallyform the same kindof monolayers as carboxylic acids. The orientation and rigidityof the monolayers is best studied by grazing-angle (382") infrared spectroscopy using p-polarized light. Methylene groups in an ~ Z Z - t ~orientation ~ns will give no signal if the chainis in an orthogonal position to the reflecting subphase.The intensity of the peaks at 2918-2920 and 285 1 cm1 willbe a direct function of the tilt angle. In fluid monolayers shift a little to higher wavenumbers, e.g, 2924 and 2855 cm1(Porter et al., 1987). Charged carboxylate end groups often render the monolayers unstable, since micelles can be formed and disappear in the subphase. Cadmium arachidate, on the other hand, forms perfect surface monolayers, because the cadmium ion is highly polarizable ("soft'*), the binding constant to two carboxylates is high, and large hydration spheres and micelles can therefore not form. Such a crystalline monolayer has beenimaged by atomic force microscopy with atomic resolution (Fig. 2.6.1a). Holes as small as 10 nm have been seen (Fig. 2.6.lb). Double chain lipids are much less water soluble than the single-chain fatty acids and form beautiful domains on water at low surface pressure. These monolayers can be studied under the light microscope if polarized light is used and if the water surface is observed under the Brewster angle of 53". No light is then reflected from the water surface, but monolayers appear as bright spots (Fig. 2.6.2). Chiral S-dipalmitoyl phosphatidyl-~holinedomains containing 29% of cholesterol appear as chiral monolayer crystals on water. The shapes of such domains are determined either by strong line tension, which favors compact, circular shapes, or by long-range electrostatic repulsion, favoring thin stripes. Chiral amphiphiles then often introduce spirals of uniform direction within these domains, whereas enantiomers produce mirror images. Amide hydrogen bonding and a cis double bond in sphingolipids provide tighter packing than the hydrocarbon chains of glycerolipids. Myelin structures in nerve membranes are typical examples of such tightly packed sphingosine-containing membranes. This can also be demonstrated with molecular surface-pressure isotherms of monolayers. ~-Octadecanoyl-sphingosine, for example, gives more crystalline monolayers than its hydrogenated derivative (Fig. 2.6.3). The fluidity of oleic acid has thus a surprising condensing effect in the final membrane. Cationic detergents (invert soaps) form strong solid complexes with anionic silicates (e.g., montmorillonite), Depending on the concentration and sols

C k a l surface monolayers as observed by (a) Brewster angle light microscopy at low surface pressure(DMPE; 0.52 m2) and (b) fluorescence microscopy ata higher surface pressure at which the dye has been squeezed out into the aqueous phase (DPPC;2% cholesterol).

S u ~ a ~ e - p r e s s uisotherms ~e of (A) ~ ~ * s p ~ i n g o s(i n e gosine.

vent, the cationic soaps may be bound in either an orthogonal or a parallel fashion with respect to the silicate planes (Fig. 2.6.4). nvert soaps do not appear in nature but are impo~ants y n ~ n o n sin preparation of artificial ~ e m b r a n es ~ c t u r e sThe . most c o ~ o application n such mon~layersis a cosmetic one, For centu~espeople smeared shiny, but the hair then stuck together. Invert s roteins and provide them with the elegant luster of a and the ful~nessof non reasy and non-polar hair. carbon monolayer on surface looks i~esistiblyshiny, fluffy, and bi~atio~ with s polymers~such as silicones, proteins, and poly(viny1 to build even more body in leave-in conditioning pro~ucts. yisobutene with a t e ~ ~ naa ~l n group o is s ~ ~ a c of e sc y l i ~ ~ ein r s ~ o t o of ~ scars. ~ n u t amounts e to gasoline, it is sprayed into the it forms a molecular film on the deposits. The polymer film sticks for a short time and then moves slowly to the b u r ~ n groom, where it is

Alkyl chains with cationic headgroups intercalate mont~o~llonite in silicate layers.No such swelling effects have been observed with fatty acids (see2.3). Sec.

stroyed. A s a result of this film formation and rapid cleansing, the motor lasts longerandgasolineconsumption is reduced.bythelubrificationeffect. The monolayer is constantly renewed fromthe gasoline ( The best way to produce well-organized multilayers on surfaces (e.g., silicate spheres or surface oxidized silicone wafers) is to replace the carboxylate end group of fatty acids by phosphonates, which are readily available from alkyl bromides and trialkyl phosphites. Phosphonates carry one negative charge and form very stable salts with zirconyl(1V) salts. The negative surface charge is thereby changed to a positive one and may again be combined in quantitative yield with another phosphonate bolaamphiphile. Up to 10 monolayers have thus been assembled on silicone surfaces in perfect order (Fig 2.6.5). The magic of the zirconyl phosphonateslies in the perfect geometrical fitting between the zircony1 cation and the phosphonate anion. This s i ~ a t i o nis similar to cadmium carboxylates, which give the most perfect fatty acid monolayers (see Fig. 2.6.1). Lipid monolayers on solid phases become totally impermeable to small,

-Si-

“

PO?

i

Pop

i

i

Cl

At least 10 layers (total thickness: 20 nm) of phosphonate bolamphiphiles have been assembled in perfect order silicon. on

membrane-soluble compoundsif two amide hydrogen bond chains are present at both ends of the hydrophobic core. The membrane thereby becomes a twodimensional crystal with no free space for solutes. Methylamine, for example, cannot substitute an azide group at the inner side of the membrane, although it reacts quantitatively with the same groupat the outer surface within minutes.A membrane of 1.5 nm thickness becomes “amine-tight” (Bohme, 1995). Furthermore, diamide bola~phiphilesshow strong odd-even effects. An oligomethylene chain with evenor odd numbers of methylene groupsis used to connect an a-mercapto with an m-acetamide group. The bolaamphiphileis then bound to gold with the terrninal SH group.The even-numbered bolaamphiphile forms a solid, tight monolayerby self-assembly. The odd homolog gives a fluid membrane under the same conditions. Thisis caused by a tilt of the molecules against the gold plane. This tilt allows for two parallel amide hydrogen-bond chains only in the even case. The monolayer is crystalline and hard. In the odd case one of the amide NH andCO groups pointsto a neighboringC to a neighboring COor NH group. The surface of this monolayeris fluid and soft as shownby M M .Infrared spectraof CH, groups inamidecontainin~monolayers are slightly shiftedto higher wa~enumbers.In the odd monolayer this effect is 12 cm’; in the even homologs only 5 cm’. The crystalline even monolayers

Odd-even effects in (a)crystalline bilayers in fatty acid crystalsand (b) b o l a ~ p h i p ~ l monolayers ic on gold. The “odd” homolog form less stable assemblies in both cases, and both effects are caused by a tilting of the layers (see text).

view

side

top vi

Model of a flat self-assernbled monolayer with three amide hydrogenbond chains.

show no NH or amide I absorptions, becausethe CO and IVH bonds are parallel to the gold subphase, whereas the odd monolayer has these bonds in statistical orientations and therefore produce both bands. In bilayers onealso observes odd-even effects because of tilt. fitting of terminal methyl groups in the even case that causes,for example, better solubility and higher melting points of even-numbered fatty acid crystals as compared to odd-numbered homologs. Crystalline SAMs with three amide hydrogen bond chains have been produced on gold (Fig. 2.6.7). Infrared spectra show a narrow amideI1 band at 1563 cm1, s u p p o ~ the ~ g assumptionof uniform hydrogen bonding (Clegg et al., 1998).

So far only amphiphiles and bolaamphiphiles with terminal headgroups have been taken into account. Linear and macrocyclic carbon compoundscontaini~g

several oxygen or nitrogen functionsmay also appear as edge amphiphiles. The co~espondingnatural compounds and their models do not belong to the fatty acid derivatives-they are antibiotics. We summwize their properties atthe end of the lipid chapter because they function as ion pores and transport agents in lipid membranes and because this book does not contain an antibiotics of chapter its own. There are many methods for the ~easurementof ion transport through membranes. We shall describe here two physical methods that directly measure ion flow in black lipid membranes,large vesicular, and cell membranes as well as a method that works with a large population of vesicles of any size and entrapped fluorescence dyes. We begin with the classical black lipid membrane method and surnmwize

Schematic black lipid membranesetupforconductivityandspectroscopic m e a s ~ e m e ~ t s .

some results obtained with A it.hairbrush soaked witha decane solutionof some water-insoluble lipidis led over a 1-2 mm hole in a teflon disk. A lipid droplet then covers the hole.The teflon disk is then placed as a separating wall between two water volumes. The droplet is repelled by both water surfaces and flattens spontaneouslyto form a bilayer lipid membrane in the center of the hole. This appears black from the side because BLM the is much thinner thana quarter wavelength of visiblelightandfullyextinguishinginterferencesoccur for light reflected from both BLlWwater interfaces. The membrane is held by the thick lipid ring andmay be stablefor a few hours provided there are no mechanical vibrations (Fig. 2.7.1). Radioactivity measurementsof radioactive compounds in the right and left c o m p ~ m e n t showed s that water passes the membrane witha velocity of about 10-100 m d s . The permeability of this BLM for water is exactly the sameas for S. The pera hydrocarbon layerof the same 5 nm thickness, namely 20 mol/cm2 meability for polar molecules such as glycerol, glucose, or urea is slower by a factor of 50-100. Small ions penetrate the black BLM extremely slowly: permeabilitydrops by a factor of approximately 10l1(Na+: 0.3 X mol/cm2 S) as compared to water. Organic molecules forming complexes with sodium ions in water, such as monensin, speed up ion transport by factors of about lo8, molecules that form tunnels by factors of 1O1O and more. Scheme 2.7.1 depicts two typical structures of ion transport agents, namely monensin and filipin. ~ramicidine, on the other hand, is thought to form a pore in membranes. The only clear-cut distinction between an ion pore and an ion transporter is that a pore can be closed and reopened by stopper molecules, whereas ion transport will always occur as long as complexation in membranes and decomplexation in water can occur, The protein channels in biological systems are usually cation-selective (e.g., sodium passes much faster than potassium) as well as cation-controlled. Two acetylcholine molecules, for example, may be adsorbed to two neighboring membrane proteins and push them a few ~ngstromsapart for a few milliseconds. As a result, 104-105 ionsmove across themembrane. The large ampli~cationfactor resulting from the movement of 105ions per pore-opening event allows the measurement of ion flowby the so-called “patch-clamp” method, which is applicable to biological cells and vesicles large enough to be handled under the light microscope (Fig. 2.7.2). A micropipette with an inner diameter of less than 1 mm is used to suck in and holda small patch of a cell or vesicle membrane containing a few pores. The ion flow may also be triggered by application of a voltage of about 100 mV/5 nm (equal to 105V/cm !);currents in the order of 10-l2A flow through a single pore again co~espondingto several thousand ions.

HQQC ti

H

OH

There are now countless publications that demonstrate ion flow through vesicle membranes after addition of all kinds of compounds. This is in perfect a g ~ e e ~ ewith n t expectation, because all kinds of crystalline domains causeleakiness of fluid me~branes. any reports claim, however, that le ation of water-filled pores within the membrane. This is clearly not warranted as long as these pores have not been closed and reopened by appropriate stopper molecules. Edges of crystalline domains are noto~ouslybad barriers, and occasionalbreakt~oughof electrolytes may always occur. The third methodfor the detectionof ion flow through vesicle membranes works with the large populationof a vesicular solution andis based on fluorescence measurements of calcein or similar water-soluble, ion-binding dyes. A macrocyclic ~ , ~ - d i s u l f o ngives e extremelylong-lived,electroneutralvesicle

A disulfone bola~phiphileforms vesicles, which are perforated by a tetraamino edge amphiphile containing two carboxylate end groups. The amine presumably occurs in the confo~ationgiven. It assembles in vesicle membranes to form pores that let iron(II) ions pass the membrane. Large organic ionsclose the pore. EDTA in the bulk water phase cannot pass through the pore and sucks iron ions out of the vesicle. (From Fuhrhop etal., 1988.)

membranes. It has been efficiently perforated by a large variety of bolaamphiphilic oligoethylene (PEG) derivatives, but these pores have never been efficientlysealed. Of all artificialmembranepores,onlythosewitha cationic o l i g o a ~ o n i u mcore andanioniccarboxylateendgroupscouldbesealed (Scheme 2.7.13). Charge interactions between the ~ m o n i u mdomain in the membrane and anionic stoppers closes the pore. Upon neutralization of the stopper's charge by metal ionsor protons the pore is opened again. In such a system the vesicles are first formed by ultrasonication of a water-insoluble~ , ~ d i s u l f o n e b o l a ~ p h i p ~ in l epresence of calcein and the oligoarnine. then removedby gel chromatography of the vesicles. Upon additionof equimolar amounts of iron(I1) ions the fluorescence of the vesicle entrapped dye is totally quenched, because iron ions pass the membrane through the pores. Addition of equimola~~ o u n t of s EDTA recovers the fluorescence, because stronger bindingconstant to iron than calcein and sucks the iron ou cle. An excess of iron then again leads to quenching. Only when large anions h as taurine are added are the vesicle pores closed, and addition of neither TA nor iron salts has any effect. Charge interaction has,so far, beenthe only chemical means of successfully opening andclosing membrane pores in synkiemblies (Fig. 2.7.3). onensine ~ , ~ - d i c ~ b o x y is l aonly t e 2 nm long and has been used in connection with a macrocyclic C,, disulfone-bola~p~phile. Six molecules of the monensine-bolaamphiphile per vesicle made of about lo4lipid bola~phiphile molecules are sufficient to produce a pore for lithium ions. If a dicationic bolaam hi hile is added during sonication, no salts are released, Upon lowerin e carboxylic acid groups are neutralized and the stopper disappears i ater. The pore is opened. This bola~phiphilicstopper cannot, howe hole again (Fig.2.7.4). -gated channels are also known. 'This physical st~eringpreends on dipole reorienta~onof membr~e-dissolvedmolecules and presumably always involves reversible domain formation (Fyles et al., 1998). It is of interest not onlyto perforate vesicle membranes but also to destroy r they have served their purpose as transport vehicles,in p ~ i c u l a for r 50% cholesterol. DNA. Natural vesicles, so-called endosomes, contain about The disruption of such cholesterol-cont~ninglipid bilayers by Triton sodium deox~cholate,examples of artificial and natural detergents, re leaky membran~at low concent~ationand in acatastrophic rupture process above the cmc of the amphip~iles.Vesicles made of fluid phospholipid bilayers devoid of cholesterol showed only leakiness under the same conditions. Amphiphiles with a carboxylate end group and a very bulky hy~ophobicend (e.g., groups) disrupt membranesat pH 5 and haveno effect above r an example,see Figure 6.5.3.

Steroids: Rigidity and Flexibility in Carbocycles

3.1 INTRODUCTION Steroidal hormones steer protein synthesis in biological cells. Muscles grow, inflammations fade away, and fertilization is controlled by the action of a few micrograms 11f steroid hormones. In synkinesis steroids are useful in solution as spacers between two reactive sites bound ti) steroid wbstituents and as matrices on solid surfaces. The magic of the steroidal skeleton lies in its extreme variability of stiffness and Aexibility accompanied by consistent intramolecular distances (Fig. 3, I . I ). Steroids also form hydrophobic and amphiphilic domains with pronounced stereocheniic d coiitrol and selective solubility in membrane structures.

3.2 STRUCTURE AND PHYSICAL AND PHYSIOLOGICAL PROPERTIES The hasic skeleton (“gonane” o r “sterme” nucleus) of steroids withoul substituents consists of 17 carbon atoms arranged in three six-membered rings. A-C (perhydro phenanthrene). and a terminal five-membered ring, D. An I 8-carbon atom C-I8is always present in natural steroids in the form of an uxiul methyl gruup a( rings C and D. C,, steroids contain a phenolic A ring and are called rstrunc’s. And,-osrurics c m y an additional uxiul methyl group at one of the carbon atoms common to rings A and B. The typical shape of steroids is thus a plane of two parallel ull-truns alkane chains with cross-links and tw11 methyl groups perpendicular to the chnins. The plane carrying the methyl group poles is called the !.%plane. Both estrane and androstane steroids are without carbon substituents at C17. the top of the five-memhered ring. Pregnanes. cholanes. and chulesltanes. 129

The matrix character of steroids: several steroids with a hydroxy or carbonyl groupat C3 combined withaxial or equatorial functional groups at C7, C11, or C17 are commercially available. The cyclopentene ringD is always flexible; ring A becomes flexible when double bonds are present. Nevertheless, the overall shape steroids of hardly changes with molecular movements. The distance betweenC3 and C17, for example, is always close to 11 A.

the other three important classes of steroids, have side chains at C17 with two, five, and eight carbon atoms, respectively (Scheme 3.2.1) (Fieser and Fieser, 1972, 1989). Unique names canbe assigned to steroids defining composition,constitution, and configuration. The most important carbon skeletons including numberings have been given above; a and p refer to the two faces of the steroid nucleus: p is the top face as viewed inthe orientation shown in Scheme 3.2.2 and a is the unde~eathface. As a typical exampleof IUPAC nomenclature, the comercial catabolicum flumethasoneis depicted in Scheme 3.2.2. C,,-C2,steroids,e.g.,androstanederivatives,mayactashormones. Steroids with longer alkyl chains on C17 areinactive as messengersof biological information. Relatively large amountsof C,,-C,, steroids are ubiquitous in cell membranes. Typical foodstuffs and human tissues contai~ngmassive amounts of cholesterol (animals) or phytosterols (plants) are given in Table 3.2.1. The nucleus of steroids is almost planar, when all rings are connected by two trans-configured carbon bonds (old name:aZZu-series) or contain an sp2-hybridized atom at one of the junctions. The nucleus shape changes to an L when rings A and Bare cis-connected (old name:numal series), and it gives the overall impression of a bowl if both rings A-B and C-D are cis-connected (Barton and Morrison, 1961). Examplesof all three kinds of con~gurationsare given in Schemes 3.2.1-3.2.3. The unique kindof A-B and C-D cis-steroids found in digitalis plants is active on cardiac and extracardiac receptors, which regulate the contractility of the heart and the rhythm of the heartbeat. They are comercia1

holestane C27

Amount of Cholesterol in 100 g of Biological Tissuesor Fluids Total amount cholesterol Free Cholesterol ( W ) (mg> Foodstuff Beef Beef 136liver Pork Bacon Chicken Butter Cheese Milk E s 284 Olive oil So oil 188 Hair, suprarenal gland Spinal cord Brain Nerves Aorta, skeleton muscle, liver,

ester (W)

262 61 28

93 140-170

120-150

1159 396 802

fat tissue, blood plasma

254 65 20 0 356 0

5000 4000 3000 1500 200-400

bestsellers. Such steroids are often connected to carbohydrates via the 3p-0 group and are therefore called heart glycosides. The most impo~antdigitalis steroid structures arealso given in Scheme 3.2.3. Zoosterols, mainly cholesterol, occur in all fatty tissues, in particular in egg yolk (Table 3.2.1). They are more easily resorbedby the body than the phytosterols of plant oils, because the latter contain a more branched carbon side chain. In order to transport free cholesterol in body fluids,it is dissolved in bile acid micelles (see Fig. 3.5.1 and 3.5.2) orin high molecular weightfat droplets c o n t a i ~ proteins ~g (chylomycrons).The latter biological assemblies have an approximate diameter of 1 pm and a half-life time of 20 minutes and may lead to cloudy blood. They are dangerous clogging agents for arteries. Most cholesterol in blood is, however, chemically bound to low-density lipoproteins (LDL)in the form of esters at the 3-OH group. Normal concentrations of such esters are 1.4-3.3 g&, but values up to 10 g& may be reached. High concentrations of cholesterol in blood may lead to cholesterol deposits in arteries leading,for example, to heart attacks and gallstones (Moudgil, 1987).

COOH

H0

~ndrosterol

bHacid

Cholic

H0

Cholesterol 16p-OW :~itoxi~enin

,g-Sitosterol

Steroidal hormones belonging to the estrane, androstane, and pregnane classes act as steering agents for protein synthesis (sex hormones, cell growth, anabolica, and catabolica) and for the regulation of salt concentrations in body fluids (Scheme 3.2.4). Steroid hormones are active in pg quantities and in general act on protein receptor molecules covering the DNA of the cell nucleus. Hydrocortisone (cortisol) is efficientasacatabolicumandconstitutesavital hormone. Total deficiency afterillness or removal of the suprarenal glandleads to death within days (Addison disease). Excessive cortisol, on the other hand,

Steroidal Hormones

Synthetic

0

2

Testosterone male sex hormone and anabolicum

Methandrostenolon, Dianabol 17a-Methyl-17~si-3-onandrosta-A' 4-dien anabolicum (oral)

gluco- and mineral corticoid

H

Estradiol female sex hormone (estr~en) 0

Nandrolon decanoate Strabolen~(~njected)

Estrone female sex hormone (est~gen)

~thinyiestradiol (estrogen, oral)

3

H

CH

Progesterone female sex hormone (ges~gen)

Norgestrel (gestagen, oral)

causes less protein biosynthesis connected with an inhibition of growth and excessive glucose (gluconeogenesis). It is therefore called a glucocortocoid. The most significant male sex hormone is testosterone. The stimulation of the growth of prostata in rats and of the combs of cocks have been taken as indicationsof “androgenic” hormone activities. Anabolic effects can be evaluated by an analysis of the ratio between nitrogen contents of food taken in and excrement (nitrogen retention). Strong nitrogen retention means development of muscles. Several anabolica with relatively little androgenic activity have been synthesized; the structure of the classical anabolicum methandrostenolone is given in Scheme 3.2.4. Two major female sex hormones called estrogens and gestagens regulate the menstruation cycle. Estrogens stimulate the production of eggs, gestagens the formation of the endometrium, a zotty tissueinto which the fertilized egg is embedded. The natural estrogenis estradiol, an orally active analog used in birth control pills is the nondigestible 17-acetylene adduct. The natural gestagen is progesterone; its much more active synthetic analog in the pill is norgestrel. Acetylene or fluor substitutionis a standard pharmacological method used to obtain pharmaceuticals, which survive bacterial attacks in the intestines and can therefore be given orally. Bacteria “don’t h o w ” triple bonds or fluorine and therefore do not digest them. Both hormones are given continuously The perhydrophenanthrene units constitute the rigid part of the steroid nucleus, whichis responsible for the fact that steroids crystallize from waxy or fluid lipid mixtures as extracted from biological materials with chloroform or hydrocarbons. This tendency to solidify is unusual for a lipid and gave the steroids their descriptiveandsterochemicallyappropriatename(Greek: stereos, solid). The five-membered D ringis, on the other hand, much more flexible and the C17 side chain is as mobile as the hydrocarbon chain of fatty acids. The D-ring end is therefore more solubleand membrane active than the rings A-C. A direct demonstration of these important flexibility di~erentiationscomes from crystal structures of liquid crystalline steroid derivatives, such as cholesteryl nonaoate (Fig. 3.2.1). The thermal ellipsoidsof the six-membered rings are smaller than those of the five-membered D ring and the neighboring carbon atoms in ring C. The openchain substituentsat C3 neighboring the three cyclohexane units in the crystal are rigidified by their neighbors, whereas the C17 substituent shows much freedom and produces fluid-like channels within the three-dimensional crystal. The cyclopentane ring withits functional groupsis presumably the active center in most hormone-receptor interactions (Altonaet al., 1968; Duaxet al., 1976). Receptors for steroids and their models may work in membranes or in organic solvents. isIt to be expected that protein receptor sites should be as flexible as the cylopentane unit of the steroid hormones. A long-chain substituent at C17 prevents recognition processes not only at C17, but also at the nearby carbon atoms 11,14, and 18, which explains the hormonal inactivity of cholesterol and phytosterols. Sudace properties of eroids can be characterized in computer modeling

Crystal structure of the liquid crystal cholesteryl-3p-nonaoate showing differentiated thermal motions. Fluid parts with large thermal mo~ementare separated on C17 rigidifies in the~eighborhoodof the rigid from rigid parts. Note: The alkyl chain cycloh~xaneunits.

by electrostatic and hy~ophobicitypotentials. Density maps with10 points per A2 on the van der als surface have been calculated. Experimental affinities of 3 1 steroids to a corticosteroid-binding globulin were then successfully correlated with the three-dimensional models obtained.~ x p e ~ m e n tdata a l and model calculations separatedthe same steroidsinto the same three activity classes (Figure 3.2.2) ( ~ a g e n eet r al., 1995). The critical function of steroids is thought to induce structural changesof fluid regions in the receptor proteins on the surface of cell nuclei, thereby uncov~A-intera~tive residues. This steroid action depends criticallyon stereochemical details, which have been studied, for example, with various 4-ene-3-one derivatives. This is a typical arrangement in seral hormonal steroids inclu~ng testosteron~,progesterone, and cortisol. The ellipsoids of sp3- carbon atoms close to double bonds are larger than those for carbon atoms with aliphatic neighbors of six-m~mberedrings then become as flexi(see also Fig. 3.2.4). Carbon atoms ble as thoseof the cyclopentane units.The 4-ene-3-one group, as the most significantexample,exhibitsahighdegree of flexibilityrangingfroma la,2P half-cha~to an la sofa, in which carbon atoms 2 and 4 are in the same plane (Fig. 3.2.3). In several crystal structures one also finds an inverted 1P,2a h a l f - c h ~ conformation, which is favored by the following group effects: a nonstandard 9a,10P configuration in whichthe 19-methyl group is below the plane, a bulky 2p substituent, an additional C9-ClO double bond, a loss of the 19 methyl group, and an ester group onC 17. These features rarely occur in natural steroids but are commonly found in semi-synthetic steroids havin high ~ n i t for y Some her-

ity

ility i

(a) Classic model of a steroid-proteininteraction, which only shows relative positions of functional groups. (b) Calculated point representation of the electrostatic potential on the van der Waals surface of the steroid. A similar surface potential distribution can be calculated for the protein receptor site, and both models can then be fitted.

P

n

x

Two hundred and thirty-seven crystal structures of 4-en-3-one steroids are known, 191 of which have conformations ranging between the la,2P half-chair and the l a sofa. The remaining 46 steroids have an inverted half-chair conformation.

mone receptors. The energy difference between the normal and inverted halfchairs lies inthe order of 1-2 kcal/rnol. The confo~ationof an unsaturated ring A. thus clearly dependson substituents or double bonds asfar away as C17. This suggests that the crystallization process preferentially accommodates molecular c o n f o ~ e r sat the global minimum. Steroid-receptor interactions may follow a similar rule, butthe global minimum now also implies the protein co~ormation. Although ody steroids ~ i t ~ ano extended ~ t C17 chain are active as hormones, it was found that association between the receptor andD the ring does often not occuror is not stereospecific. The key to receptor binding then lies A ring, in the e.g., the presenceof a 4-ene-3-one ring, which can be twisted into the inverted conformation as discussed above or a planar phenolic ring in some sex hormones. There are, however, exceptions this to rule. h androgens, for example, the l’7p-hydroxy-substituted D ring is most critical to ~ g h - ~ n ibinding. ty The acetyl side

vi

Two Newman projectionsof the major conformersof the C20X17 bond in progesterone. The flat conformer with the carbonyl oxygen almost in the cyclopentane plane is slightly less energetic and is also the biologically active conformer.

chain of progesterone has been studied by circular dichroism spectroscopy in solution and by a Comparison of 175 pregn-20-one crystal structures. The torsion y angle is usually -30", whichis 1.1 kcdmol lower in energy than they = -90" conformer, The y = -30" conformer is also the biologically active one. The 90" conformer is, however, enforced by 16-methyl group subs~tuents(Fig. 3.2.4) (DUGet al., 1994). The conformations of saturated long side chains such as observed in cholesterol need not be considered here, because no receptors are knownfor them. "he extra ethyl group of phytosterols, however, makes difference a even ininteractions with vesicle membranes (see Sec. 3.5). 1~,25-Dihydroxyvita~n D,, the hormonally active form of cholecalciferol (vitamin D,), is responsible for intestinal calcium absorption and bone calcium mobilization. In crystals it occurs either in a steroid-like s-cis ( S = single bond) conformation or in an unfolded s-trans conformation. Rotation aboutthe C6-C7 single bondis facile. In solution one finds onlythe more extended form. Calculations,however,showthat the s-cis conformationinwhichring A is folded over ring Clis the global energy~ n i m u mThe . la-OH group is then in close proximity to the C l l position, a functional area known be to critical for biological activity of glucocorticoids. Molecular mechanics calculations indicate t a ~the n folded form becomes the that in synthetic l1~ - ~ u o r o - l ~ - h y d r o x y v iD, preferred conformation with an 01-F11 distanceof 2.8 A in line with an OH-F hydrogen bond (Fig. 3.2.5) (Kirk and Hartshorn, 1968; Zhu et al., 1994).

Two conformers of a seco-steroid, namely 1 1p-fluoro- 1a-hydroxyvitamin D,. The s-trans conformer shows lesssterical repulsion, but theS-& conformer may be stabilized by 1-1 1 hydrogen bonds.

Table 3.3.1 lists a few commercial steroids, in particular those that can be used as inexpensive starting materials. Most industrial syntheses of steroids are based on these naturally occurring compounds. Only norgestrel is made by total synthesis andis therefore expensive. The major problem in partial syntheses starting from abundant natural products is the selective degradationof the 17 side chain. Cholesterolis present in animal tissues (14%) and in wool fat (15%) and may be isolated by extraction with chlorinated solvents. Purification is often not simple, because lanosterol and other triterpenes are present in the extracts. Diosgeninis a most useful precursor since its side chain is particularly easyto degrade. Oxidative degradation of an intermediateenol ether produces a ketone and ester an group and, after dehydration, 16-dehy~opregnenoloneacetate in 60% (!)overall yield. Today diosgenine is, however, more expensive than pregnenolone since the plants are no longer competitive with soybeans as a natural source. Within the last few decades evenbile acids from animals for slaughter became a competitive starting materialfor cortisone synthesis. ~tigmasterolsfrom soybeans are available limitless in quantities, but those with degradable olefinic side chains are difficult to separate from the saturated ones. Once isolated, the olefinic side chain can be ozonolyzed to form an aldehyde, since the double bond in ring B is deactivated by isomerization, which brings it in conjugation with a keto group at C3. The aldehyde is then transformed into an enamine and ozonolyzed to give progesterone, again in 60% overall yield. e cheapest of all sterols, however, is cholesterol, followed by p-sitosth steroids have saturated side chains. Their chemical degradation is complicated and expensive and has no commercial value today. Biotechnology solvedthisproblem. Fe~entationwith ~ y c o b a c t e r phki, i ~ ~ ~ u c a r ~ or ~a, Art~rubacte~ in the presence of various inhibitors (e.g., nickel sulfate, cobalt chloride, 8-hy~oxyquinoline)gives androstadiene-dione. From that point one can get anywherein steroids by simple chemical modification. Only the degrada-

Some Readily Available Steroids (numbers give approximate prices per

log) cholesterol, $1.5; deoxycholic, cholic, and dehydrocholic acids, $4 stigmasterol, $0.30 (mixtures of phytosterols are cheap); sarsasa~ogenin,

S:

tion of bile acids still follows the old much-trodden chemical path, since no efficient ~ c r o o r g a ~ sisminterested in them. The 7a-hydroxy group is removed first, then the 12-hydroxy groupis shifted to C11 by dehydration and microbial hydroxylation, and finallythe 3-keto-4,s-dehydrostructure is introduced by conventional procedures removing the cis-configured AB-unit (not shown). Scheme 3.3.1 shows the industrial degradation procedures for phytosterois and cholesterol to produce hormones. In the long run,total syntheses of steroids may perhaps become more competitive withthe enzymatic degradation procedures applied today. The major potential of total synthesis comes from the greater variability in target structures, which may lead to products with higher binding constants and fewereffects, side Norgestrel is the best exampleof such an industrial product. Its total synthesisis given at the end of this section. Three major reaction sequences or “strategies” dominate steroid syntheses: (1) Diels-Alder reactions, (2) all kinds of aldol and Michael addition reactions, and (3) “bio~metic”syntheses corresponding to polyene cyclizations. We shall give a few examplesof each of these approaches, starting withthe polyene cyclizations(AkhremandTitov, 1970; Anand et al.,1970;Blickenstaff et al., 1974). In biosynthesis the enzyme lanosterol synthase catalyzes the conversion of )-2,3-oxidosqualene through a sequenceof four cyclizations and four 1,2-hyogen and methyl group ~ g r a t i o n s(Scheme 3.3.2). The primary tetracyclization has been evaluated by 14C and tritium-labeling studies and is res sum ably triggered by the opening of the epoxide by a proton. Subsequent attack of the C2-carbonium ion by C7 gives a new carbonium ion at C6, which is then attacked in a trans fashion by the n;-electron of C11. In order to bring C11 close enough toC6, the squalene epoxide mustbe folded in away that the future ring appears in a boat conformation. All four steroid rings could thus be formed as “prot~sterol’~ in a concerted process and the positive charge removedby a final deprotonation of a tertiary hydrocarbon (Coreyet al., 199s). The original model of a concerted cyclizationof squalene epoxide on lanosterol synthase is presumably wrong, because tricyclic inte~ediateshave been detected and isolated (last formula inScheme3.3.2a).Neverthelesssuccessfultotalsyntheses of androstene-3,17-dione andof progesterone were basedon the biological electrocyclization scheme (Johnson, 1976). A cylopentene-2-01 with an unsaturated side chain was dehydrated to form an allylic carbonium ion and tricyclized to give tetracyclic steroid precursors in78 and 72% yield. The linear vinyl cation were intercepted either with trifluoroacetate or with the strongly nucleophilic carbonyl oxygen of ethylene carbonate, The latter decomposed upon work-upto give the enolate and the 17-acetyl group;the enol trifluoracetate, on the other hand, was isolated as such and was ozonolyzed only in ring A (seealso Scheme 3.3.4 for continuation). Androstanes and estranes (see Scheme 3.3.4) were thus accessible

ACOWA

A

0'

fOl Of fOl

v

V

a) ~ i o s y ~ t ~ e s i s boat type~ n ~ o ~ ~ t i o n I

H

I

protosterol

lanosterol

tricycle (see text)

H

)c~emicalmodel

0

see scheme 3.3.4

by slight variations ofthe last step in total synthesis. A double Heck reaction USing palladium-catalyzed cyclizationof styrene halides has been used to synthesize estrone and estradiol derivatives (not shown) (Tietze et al., 1998) The classical total synthesis of cholesterol by R. .Woodward published in 1951152 begins with the formation of the future cyclopentane ring D as aCYclohexane unit by a Diels-Alder cyclization. The cis-fused ring was formed at first, butthe desired trans structure was obtainable through enol-me~iatedequilibration. Uields were low since the thermal Diels-Alder cyclization of methylated enes occurs only slowly andis reversible. It was reported in 1939by Dane that 2-methyl-cyclopentene-~-onedid not react at all with a diene, whereas the nonmethylated cyclopentenone gave no problems. Later it was shown that Lewis acids catalyze such reactions very efficiently and give high yields. The nature of the Lewis acidhas even been used to determine the positionof the double bond in the final styrene derivative: the more acidic titanium tetrachloride leadsto proton migration inthe primary cyclohexene product andto a tetrasubstituted double bond, which is difficult to realize directly (Quinkert et al., 1991). Modern variations of Diels-Alder reactions often apply regio- and stereoselective ring opening reactionsof two 0-quinodimethanes and immediate reaction with neighboring enes in chiral a environment (Scheme 3.3.3). A regio- and stereoselective Robinson annelation(a sequence of an intermolecular Michael and an intramolecular aldol addition reaction) regulated by the angular methyl group wasalso applied in Woodward’s cholesterol synthesis. ~ e f o r ~ y l a t i oisnanalogous to the spontaneous decarboxylation of acetoacetic acid with ethylformiate activatedC8. Then the C8 enolate was added to ana$unsaturated ketone in a Michael-type addition, followed by an aldol-type condensation to the ringketone. The angularmethyl group directed the large substituent to the trans position, the proton co~espondinglyto the cis. The cyclohexene D ring was later converted by periodate to the open-ch~ndialdehyde, and another aldol condensed it tothedesiredcyclopentene. The anionwas formed preferentiallyon the upper, methylene group because the other one leads to an unfavorable steric crowding.A similar reaction sequence was appliedin a progesterone synthesis to go in the other direction: a five-membered, ring was converted into the cyclohexenone ring A.For this purpose the methylated ene was ozonolyzedto the diketone and aldol condensation at relatively modest base strength gavethe desired condensation reaction in 51% yield (Scheme 3.3.4). As an example of a steroid total synthesis, the industrial prockdure used to malse norgestrel, the gestagen used in many contraceptives,is sketched bere.Its basis is the Torgov reaction, a variationof the Robinson ,armelation, which runs essentially neutral media. The carbanion of 1,3-dioxo compounds addsto allylic alcohols, e.g., 2-ethylcyclopentanol- 1,3-dione, under weakly basic conditions. 0th the dione and the styrene double bond are stable under these condi~ions. egio- and stereoselective microbial reductionof one of the keto groups, acid-

0

Me0

0

Me

#..

un

no

Me0

I"

.x

/Triton B

H saccharo-

myces

n,co

(l) ~ U N H ~ P h ~ H ~ ~ ~ C (11) butanone [AI(OPr')&A

(l) &[Pd-CaCOd (11) 1 M ~ O ~ M a O H

(l) LIC %CH *EDA

(11) dil.&SOdO (111) c. HCI

16% overall yield

(+)-norgestrel

catalyzed cyclization, and dehydration followed by a few standard reactions then. produce the most successfulof all artificialhormones (ethylat C13, no methylat ClO) (Scheme 3.3.5).

Many stereoselective substitution reactions (S$) were first studied and optimized with steroidal substrates, because class this of compounds provides a large variety of pure enantiomers with just one reactive chiral center. Furthermore, the

e~uatoriaZand axiaZ substituents of the decalin skeleton are exceptionally easy to ~fferentiate.The best known examples arethe inversions of configurations in .Zreactions on C3 substituents, the more rapid removal of e~uatoriaZhydrogen atoms as compared to axiaZ ones, andthe relatively fast acetylation of equatorial hydroxy groups. The preference of the reagents is always the same: they attack e~uator~aZ substituents. This leads, of course, to different results with respect to a and p substituents incis- and trans-configuredsteroids (Scheme 3.4.1) 1950; Fried and Edwards, 1972; Kirk and ~ ~ s h1968). o ~ , ore sophisticated examples are provided by substitution reactions, which are in~uencedby a remote double bond. The 3p-hydroxy group of cholesterol, for example, can be substituted by chloride with PCl, or, after tosylation, by methoxide. In bothcases almost quantitative yields of p-substituted compo~nds are observed. All 3p-hydroxy steroids with a 5,6-double bond give these reactions. The homoallylic carbonium ionat C3 and its cyclization after neutralizationatthe y carbonatomhavethusbeenestablishedaswellasthe t h e ~ o ~ y n preference a~c of e~uatoriaZ substitutionincyclohexaneunits egioselective oxidation of saturated carbon centers in steroids is difficult when. theyare not activated by neighbo~ngcarbonyl groups. atoms show identical inertness to weak oxidants and vulnerabili The method of intramolecular remote oxidation overcomes this problem by a iginal solution was found with a eta-biphenylcarboxygroup at C3.A terminal iodine substituent was boundto hich would be fixed in a benzene solution in a position parallel to the steroid by van der interactions. The iodide substituent c o ~ l dbe chlorinated to forrn a high y I-C1 bond. UV irradiation then produced chlorine radicals, which attacked the nearest lying carbon center, namely the desired C17-target with a yield of SO% (Scheme 3.4.2). n, a fewre~angements,and ozonolysis produced useful hormone precursors er intramolecular oxidation depends on the presence nitrous or hypochlo~cacids. hich are esterified with 1cal cleavageagainyieldsradicals,whichpreferably ~trosate hyl groups (Scheme 3.4.4). in.duce tumors in animals, a fact that p r o ~ a ~ depends ly on the chol-type A rings. Arene oxides and/orse~quinonesmay then be formed, which react as cross-linkers inthe same way as p ~ l y c y chydrocarl~~ ns, such as benzop~rene,which is oxidized in the body to a diol-epoxide. ctroreduction of the correspondingort~o-~uin.one to the s e ~ q u i n o n radical, e ct that would be formed upon oxygen oxidationof the catechol, of adenine, does indeed produce a covalent adduct in. 14% yield

+

Tos

Me0

11%

I

I

OH

OH

cycles

1 1 ~-nitrosoxy-pregnadiene

~en~o[aJp~rene-7,8-diol-9,1O-epoxide a known carcinogen

Cathodic reduction

0

ti

..

.. ..

Catechol steroid

I

I

\

The flexible characterof steroidal hormones andthe corresponding receptor sites on proteins hasso far prevented x-ray analysisof crystals. The mechanism of steroid desorptions from their transport systems by cell surfaces remains obscure. In orderto shed some lighton the molecular recognition betweena hora way that is commone and their protein receptors, the steroid was modified in patible with its interaction with the receptor and, at the same time, improve X-ray di~raction. ~rganometallics, for example, can be complexed to the 17aposition carrying an acetylene substituent (Scheme 3.4.6). Measurements of relative binding affinities to lamb uterine cytosol showed thata rhenium complex in combination with an 11~-chloromethylsubstituent binds tightly to the receptor protein. Such a totally artificial steroid substituentmay not only allow better xray analysis. It may also be useful in therapy as well as diagnosis using radioacOH

OH U3

OH

e. Both emit y-photons of high energy and can be detected with a yray camera (Top et al., 1995 ). The regio- and stereoselective reduction of a s t e r o i ~ a l 1 , ~ 7 - d ~ ~ ewit^ tone l i t ~ i ~ ~ a l has u ~been i ~ achieved ~ ~ with ~ y the ~ raid ofi polymeric ~ e matrices. Polyacrylatewasesterifiedwith17-ste a ss-linkedwithdivinylbenzene, sterol removed by Li treatment and extraction with was then attached available hydroxy groups in the empty molecular cavities and unreacted LiAlH4 was washed away with T An excess of the diketone was added and its reduced products analyzed. Seventy percent of the ketone was convertedto a monoalcohol and the product mixture contained 15% 3p-OH, 80% 17a-OH, and 5% 17 a-OHas compared to 99% as the hydrogenation product in solution (Fig. 3.4.1). The original -binding site is obviously more accessible than the a-site, where the proton of the imprint molecule was situated. Severalother i using steroids and cross-linked polymers have been rep 1993; ~ i t c o et~al.,~ 1995). e Their success is always c ference between the sterically hindered, methylated p-surfaces and the freely accessible a surfaces of steroids. An aryl-to-ketone intramolecular singlet-singlet energy transfer has been used to achieve the selective remote photoreduction in a steroidal 11, 17"diketone. The dimethylphenylsiloxy group was boundto the hydroxyl group on C3 and used as an antenna chromophore. The 11-ketone quenches thearyl fluorescence yield and acts as a singlet-triplet switch.The l 1-keto tripletis then transferred to the 17-keto group. The excited triplet is reduced by added triethyl amine to the alcohol in 87% yield, whereasthe 11-ketone does not react, because it is to quickly deactivatedby the chromophore at C3. The interchromophore distance betweenthe 3 - q 1 group andthe 11-ketone is 6.4 A,to the 17-ketone 10.2 A,and only the far-away keto group is thus photoreduced (Scheme 3.4.7). The flexibility of both the cyclohexanone (see Fig. 1.2.7) and cyclopentanone rings (see Fig. 1.2.8 for cyclopentane) does disturb the effect, because the distances are hardly effected by thermal vibrations within the covalently fixed steroid skeleton. The stereochemistry of vitamin D, was introduced in Fi ring-opened steroidis formed in nature from the cyclohexadiene terol. Since six electrons-four n and two S electrons-are involved, the photoThis . is atfirst e x e ~ p l i ~ eind c h e ~ i c a ring l openingshould be c o n r ~ t a t o ~ Scheme 3.4.8 with the conversion of 2,6-tra~s-4-cis-octatrieneto trans-2,'l"imethylcyclohexadiene; trans remains trans. In ergosterol a subsequent thermal r e a ~ a n g e ~ e nnt ,~ e l ay 1,cl-hydrogen shift,is favored by a helical ~ a n g e m e n t ly. 3.4.8). The ring-opened of rings A and C and occurs ~ ~ t a r a ~ a c i a z(Scheme product then forms the linears-trans conformer (Sec. 2.2), and the reaction becomes essentially irreversible.

tc

Model of the bindingof the steroidal diketone B to the cavityin a crosslinked polymer formedby the imprint moleculeA.

0

h * v/266nm

DPS

*

DPS intra singlet-singlet energy transfer

intersyst@m

DPS

l

DPS intra tripl@t-triplet energy transfer

DPS

The bulkiness and stereochemistry of large steroid substituents allows stereoselective hydrolyses of unnatural enantio~ers.Cholesterol together with cholesterol esterase have been used to resolve binaphtols and other diols into their enantio~ers : the diasteromeric dicholesteryl-diesters are hydrolyzed selectively by the crude enzyme in two steps and the final diol is a pure enantiomer.

h*v

conrotato

h*v

conrotato

n

n

iochemical reactions take place in the hydrop~obiccenters of membranes or e n ~ y m eclefts. Aqueous compartments of biological cells are usually taboo media for theformation of small molecular assemblies or covalent compounds. here is, however, one notable exception, namelythe f o ~ a t i o nof inclusion

complexes of bile acids (Davis, 1993). Deoxycholic and cholic acids form watersoluble assemblies in the presence of water-insoluble compounds, e.g., fats. These assemblies are held together (1) by the hydrophobic effect acting on the fat droplet and the nonpolar steroid surface and (2) by hydrogen bridges between the steroidal hydroxy groups in axial a positions (choleic acids). They bind benzene derivatives, open-chain hydrocarbons, and fats on the outer surface, which has been characterized by NMR spectroscopic ring current effects. Futhermore, they carry small water droplets inside the steroid cavity, which is made of about 8-12 molecules. This inverted rnicelle cannot be integrated within the BL biological cell membranes, nor does it dissolve them. The edge-amphiphile character of cholic acid also determines the crystal structures of its derivatives. A beautiful channel structure has, for example, been found in crystals of a cholic acid diglucoside (Fig. 3.5.1) Model building of deoxycholeic acid assemblies and x-ray studies of inclusion compound fibers (choleic acids) show that about eight deoxycholic acid molecules may entrap one palmitic acid molecule (Fig. 3.5.2). The extraordinary property of the deoxycholate micelles to absorb fatty

Typical structure of a curved cholic acid assembly, namely a porous crystal structure of cholic acid diglycoside with a benzylic side chain on C3. (From Cheng et al., 1991.)

Schematic structure of a choleic acid, an inclusion compound of a fatty acid in deoxycholic acid assemblies in water (From Conte et al., 1984.)

molecules on the hydrophobic outer surface renders the water-insoluble molecules directly accessible to water-dissolved reagents and enzymes. Fu~ermore, the micelles consistof small rigid helices, whichdo not interact with biological bilayer membranes. &-Configured steroids are thus extremely suitable for the c o n s ~ c t i o nof small water-filled caxriers with large curvature, Nevertheless, one also finds planar assemblies, as is the case with other amphiphiles. Two co-crystal structures of cholic acid with 2-fluorobenzyl alcohol or with water show the aromatic amphiphiles aligned with the steroidal edge amphiphile, whereas water is located exclusively at the hydrophilic edge.The benzyl alcohol guest can be replaced by water in the solid state without destroying the crystal. No a m o ~ h o u s inte~ediatewas detectable. The intramolecular hydrogen pattern between the cholic acid monomers together with the strong van der Waals interactions between the rigid hydrophobic steroid edges obviously stabilize both the crystalline host and channels for guest molecules. A classical method to separate 3p-hydroxy steroids from 3 a epimers is the precipitation of (water-soluble) digitonin (Scheme 3.5, l ) from 90% ethanol with an ethanolic solution of the steroid. Only the p epimer is found in the complex digitonide, which is precipitated by the addition of ether and can be separated by filtration. Boiling pyridine is used to release the pure 3p-hy~oxysteroid. The molecular complexesof the 3 a epimers are presumably also formed but are not precipitated by ether. Although this stereoselective precipitation reaction was discovered in 1907, the structureof the digitonides is still not known, and it remains unclear whythe substituent onC3 is so important for digitonide solubility, whereas the C17 side chains ranging from OH (testosterone) to C,H,,(cholesterol) make no difference (Fieser and Fieser, 1959). In Scheme 3.5.1 a hydrogen bond stabilized a van der Waals complex, which may be stabilized by an ice-like water ~ a n g e m e nin t the encircled volume (compare withFig. 3.6.2). These locally immobilized water volumes would be destroyed by 3 a steroids. Natural vesicles, in particular endosomesfor the transport of proteins and nucleotides throughcell membranes, contain about50 mol% cholesterol in a bilayer where the lipids closely resemble the mixtures found in egg lecithins. Typ-

ity

hydrophii~c

01

possible

ical phos~holipid-cholesterolmixtures in plasma membranes, endosomes, and eggs are 30% phosphatidylcholine, 15% phosphatidylethanol~ine,10% phosphatidylserine, 5% phosphatidylinositol, 15% sphingomyelin, and2040% cholesterol. The stiff cholesterol moiety presumably occurstheinform of crystalline domains, and the borderlines between rigid steroid and fluid phospholipid regions are vulnerableto disruption. Upon additionof long-chain benzene derivatives with two tert-butyl groups on the hy~ophobicbenzene end (“harpoons”), the cholesterol vesicles break open and release entrapped electrolytes (see Fig. 6.6.3). On the other hand, cholesterol also helps to trap electrolytes. Ionic dyes are usually released within a few minutes from fluid egg lecithin membranes, whereas the same membranes with a high cholesterol content entrap the same dyes for several days (Nagawa and Regen,1991; Chung and Regen,1993; Naka et al., 1993). Cholesterol has, in general,the effect of making fluid membrane structures manageable and selective. Edge ~ p h i p h i l e sform channels in vesicle membranes containing cholesterol. It has been shown that efficient pore formation for

potassium ions is only accomplished in the presence of ent-cholesterol, but not in presence of the natural enantiomer. Thisis a perfect example of stereoselective domain induction, The natural product squalamine and a model compound, both containing oligoammonium as well as sulfonate substituents, show antibacterial activity. They constitute ionophores, or possibly ion pore formers, and have been found to combine preferably with negatively charged phospholipid membranes of vesicles as compared to electroneutral ones (Scheme 3.5.2) (Deng et al., 1996; Funasaki et al., 1990; Cholesteric estersof fatty acids, e.g.,3~-cholesterylnonaoate(for a crystal structure see Fig.3-21), form thermotropic and lyotropic mesophases, an aggregation state also called liquid crystalline. ~ e ~ o t r o pmesophases ic are formed upon melting of t~ee-dimensionalcrystals, whereas lyotropic mesosystems are fixtures of solid material with a small percentage of solvent, usuallywater. Thermotropic cholesteric phases consist of large helical aggregatesof molecules with pitches in the rangeof hundreds of nanometers corresponding to the wavelength of visible light. They reversibly melt in a narrow temperature range to form isotropic liquids with a total enthalpy of only 2-3% of t~ee-dimensional-crystal melting and have been used repeatedly as chiral solvents and for stereocontrolled thermal reactions, in particular Diels-Alder reactions (Kansui et al., 1996). Cholesteric liquid crystals, e.g., those of cholesteroyl~onaoate(see Sec. 3.2), produce a Bragg-type scattering, which depends on temperature and angles of incidence and observation. Either total reflection or total transmission of circular polarized light is observed, which effect provides the basis of the darkbright liquid crystal display in the Schadt-Helf~chcell (Fig. 3.5.3) as well as color reflection.

Chiral liquid crystals without a chromophore may reflect colors because of light-scattering effects in helices of 400-700 m pitches. Models of the cholesteric phase and theSchadt-Helf~chcell for liquid crystal displays are given. Two perpendicular polarization filterslet light pass only ifits direction of polarization has been rotated by the liquid crystals. If the liquid crystals are destroyed by an electric field, nolight is transmitted, because the crossed polarizers quench it.

The unique character of liquid crystals allows the sensitive change of molecular orientation withboth temperature and chemical environment. Steroidal crown compoundsentrapped in a mixture of cholesteryl nonaoate and cholesterol thus forrn a liquid crystalline phase whose helical pitch can be modified by different complexed metal salts (Fig. 3.5.4). Potassium R-mandelate, for example, induces a green assembly color, potassium S-mandelate a blue one. For the detection of carbohydrates by steroidal borates, see Figure 45.1 (Shinkai et al., 1991). One percent (w/w) hydrophobic cholesteryl4-(2-ant~lo~y) butyrate in hydrocarbons and long-chain alcohol solvents produces solid gels. Weak CD spectra indicate helix formation, A steroid with a piperidine D-ring dissolved in cyclohexane shows a fiber network under the polarizing light microscope (Fig. 3.5.5).

0

0.0

l

0.l

rol n o n ~ n o ~ t e / hloride mixture Chiral substituents and bound metal ions modify the pitches and reflection colorsof cholesteric phases.

Electron micrograph of m aqueous gel madeof the given amino steroid.

Cholesterol forms stable monolayers on water. The surface pressureis 45 d / m , the molecular surface about40 A2. Cholesterol thus standspe~endicularto the water surface (Fig. 3.6.1). The crystalline Langmu~-~lodgett film on gold is, however, not uniform. Cyclic voltammetry of ferricyanide and~ t h e n i u mions in the bulk water volume shows about 30% of the current of the naked electrode.A more or less quantitative blocking of the currents is obtained when 3p-thiocholesterol is used. The steroid is now covalently bound to the gold subphase and cannot be washed away with dichloromethane. Mixed monolayers of cholesterol or lithocholic acid with lecithin showed that cholesterol has a much stronger condensing effect than the bile acid. The thiol group of spironolactone steroid is located in the center of the steroid nucleus andis axial. Covalent bindingto gold thusleads to a parallelorientation of the steroid. Only about 50% of the gold surfaceis covered by self-assembly. Fe~cyanideions in bulk water phase still migrate freely to the gold surface. Since the sulfur bondfrom the steroid to gold is protected by the bulky steroid, self-assembly of octadecanethiol on the remaining gold surface occurs without any loss of the steroid. The metal ion current to the gold surfaceis now reduced by about SO%, since the fiat steroids have no significant isolating effect.

IO0

(a) Area-pressure isothermof cholesterol on water.(b)A 3P-thiocholesterol LB monolayer on gold isolates the electrode surface from ferricyanide ions in bulk water as shownby cyclic v o l t ~ m eusing t ~ the naked or steroid-covered electrodes.

0

SH

At firsta steroid is covalently boundto a gold subphase, then the remaining gaps are filled with octadecyl-thiol. A closed membrane is thus formed with6 x 14 gaps causedby the steroidal bottom. The gold electrodeis then submersedin an aqueous ferricyanide solution, and a strong cyclovolt~metricsignal is observed. Upon addition of molecules that fit into the immobile waterstructure within the hy~ophobicgap (=hydrophobic water), the ion flow is blocked. ~ ~ ~ ~ s - C y c l o h e ~ a with n e d i two o l equatorial hydroxy groups is optimal; glucose and rhamnosealso work. cis-Cyclohexanediol with an axial OH groupis much less efficient. The entrapped molecules do not equlibrate with the bulk water. Only upon acidification with HCl to pH 5 3 are the entrapped molecules released. (From Fuhrhopet al., 199’7.)

1

Uponaddition of l,2-tr~~s-cyclohexanediol, the membranegapwiththe steroidal bottomis almost completely blockedto ferricyanide transport. 1,2-cisCyclohexanediol has very little isolating effect, andD-glucose is about half aseffective. Although all three compounds are highly water soluble, washing with water does not restore the gold-surface accessibility to ions. The pore remains closed. Since it is generally assumed that hydrophobic as well as hydrop~ilic walls always fixate about three water molecules by surface forces (Vossen and Forstmann, 1994), the steroidal gap with a width of 6 A should be filled totally with an immobile water volume. It is therefore thought that the cyclic polyols with the e ~ ~ ~ t o rhydroxyl i ~ Z groups freeze into the ice-likewaterstructure (Franks, 198’7)and are thereby(l)removed from equilibration with the bulk water phase and (2) prevent h y d r ~ ~ oofn external ferricyanide ions (Fig. 3.6.2). Open-chain compounds and cyclohexanediol have no stabilizing effect on the immobile water structure and therefore do not impede ferricyanide transport. Acidi~cationto pH 3 with HCl also destroys the immobile water structure; the cyclohexanediol is released andthe ion current flows. For References for Chapter 3,see pages 535-537.

The name carbohydrateis suggestive: it stands for the stoichiometryC11(H20)n, it indicates biosynthesis from water and a carbon source, namely carbon dioxide, and it suggests thatthere is nothing more closely relatedto water in nature than carbohydrates. The suggestionsareborneout byreality. The equatorialOH groups of glucose, for example, fit perfectlyinto the tetrahedral water clusters of fluid or crystalline water, glycosylation helps to carries water-insoluble compounds into the blood stream, and the carbohydrates of glycoproteins on the surface of cells function as the letter boxes for messages from solutes in biological water volumes. For organic chemists two major motivations for working with carbohydrates prevail: l. Adjust polymer technology to cellulose. The most important property

of cellulose is quantity. About loi1 tons (an orgy of mass) are produced on earth every year by photosynthesis. This compares to about 3 X lo9 tons of mineral oil products, more than 85% of which is burned. The remaining 15%is mostly convertedto polymers. There is no reason, exceptfor price, that cellulose and fat chemistry cannot be applied to produce both fluid energy sources and polymers. 2. Use the chiral pool in synthesis and synkinesis. The common monosaccharides provide three or four asymmetrical carbon atoms per unit (an orgy in precious chiral center at very low cost). Carbohydrates together with amino acids therefore constitute the principal components of the “chiral pool,” the most rewarding and renewable source of fine chemicals and stereoselective membrane surfaces. 7

ode1 of cellulose microfibrils, which are produced as a biomass of about 10" tons per year (From Bever, 1986.) (b) Illustration of the carbohydrate chiral pool asit is usedin modern synthesis. (c) Model of a carbohydratetree on cell surfaces as part of a glycoprotein. It is recognized here by lectin-type proteins and polypeptides. (Prom Sharon).

istr

Monosacch~des(sugars)arepolyhydroxyalcoholswithaprimaryalcohol group (alditols or glycitols) or an aldehyde group on C l (aldoses or glycoses) (Binkley, 1988; Collins, 1987; Ferrier and Collins, 1972; Hall et al., 1980; IUC, 1969, 1971; Kennedy and White, 1983; Kennedy, 1988; Lehmann 1976, 1996; Pigman and Horton, 1972; Stoddart, 197 1). A few monosacch~descarry a keto group on C2 (ketoses) insteadof the formyl group atl.CMost natural carbohydrates areof the aldose group type with the molecular formula (C are therefore also called carbohydrates. Upon pyrolysis they decomposeto carbon and water; the reductive and high-pressure conditionsof soil also favor formation of saturated hydrocarbons (mineral oil).Clycitols are reduction products of aldoses, and ketosesare rearrangement productsof aldoses. Fischer projections choose the cyclic and planar,all-eclipsed conformation of the carbon skeletonsof sugars as a basis, then pull the cycle straight to form a line and orient it so that the more highly oxidized endC l is located at the top. This projection procedure remainedalive for almost a century because inherent symmetry properties in carbohydrates become apparent here in the most simple way. Always remember: Fischer projection formulas are the short notations of all-eclipsed cyclic confo~ations,although theylook linear (Fig. 4.2.l). The terminal chiral center, C5 in hexoses, is usually R- or, in the Fischer notation, configured. The conformations of acyclic carbohydrate derivatives in crystals never correspond to a cyclic conformation in which mostof the substituents are in unfavorable eclipsedpositions.It is mostnoteworthy that the opticallyactive alditols provide examples of a single conformation in crystals. Confo~ational polymorphism is not observed, although there may be more than one crystal structure (e.g., in thecase the straight all-a~ticonformation of D-mannitOl (Andr6 et al., 1993, 1995; Jeffrey and Takagi, 1978; Jeffrey Wood, and 1982; Jeffrey, 1990; Panagiotopoulus et al., 1974). D-Clucitol crystallizes with a bent chain and a g a ~ C2-C3-C4-C5 c ~ ~ torsion angle (Berman et al., 1968; Park et al., 1971). The conformational differences between both polyols are caused by the repulsive 1,3-syn interactionof the hydroxyl groups in glucitol, whichis not present in the mannitol (Fig. 42.2). Open-chain carbohydrate derivatives are ideal objectsfor conformational analysis bylH -NMR solution spectroscopy. The OH-proton signals are well separated from CH-methine proton signals, and decoupling experiments allow easy signal assignments. Polar carbohydrate derivatives and the carbohydrates themselves are soluble in dimethyl sulfoxide(DMSO), which destroys all inter-

-

D Giy~flna~ehyde

W OH

I

H-C-OH

I I H,C”oH

i-

G

H-C-OH

Threose

All

gladly

altruists

-

Fischer 2’s’ *&l ~ ~ o ~ a t [all i o anti, n a// trans) (a//-ecliptic)

-

-

make tanks

gum gallon

in

chair

Common naturalalditols. First row: R-and D- glycerolaldehydes, second row: R-and D-ribose; third row: D-tetroses; fourth row: D-pentoses; fifth row: D-hexoses together witha mnemonic rule for Americans. This sentence will help to memorize the sequence of D-hexose names, whichare built up in the“carbohy~ate ~itting” pattern: (C5) eight right, this establishes the D series; (C4) four right-four left; (C3)two right-two left and (C2) one right-one left.; sixth row: various f o ~ u l a €or s D-ribose and D-glucose.For furanose and pyranose hexniacetals, see Figure 4.2.8.

COOH OH

H0 H0

H0

OH OH CH2OH

CH2OH

-

D Gluconic acid COOH

COOH

OH H0

H0

OH H0

OH

H0

OH

-Glucuronic

OH

COOH

COQH

acid

-

D Glucaric acid

COOH

-

D Galactaric acid

Top: Fischer projections of a few common glycitols and glyconic acids. Note the symmetricalstructure of D-mannitol. Bottom: Molecularstructures of (a) linear D-mannitol, (b) bent D-glUCitOl, (c) D-glUCariC acid, and (d) crystal structure of water-insoluble D-galactaric acid. In thelatter structure the inter~olecularhydrogen band pattern is indicated.

and intramolecular hydrogen bonds.OH signals thus become very sharp inthis solvent, and OH-CH coupling constants can be measured. Most helpful in interpretation is the fact that the effectsof repulsive, dipolarinteractions lead to relativelylargechemicalshiftdifferencesbetweendifferentconformers. The conformations of five ~-octyl-D-glyconamideshave, for example, been unequivocally assigned and NOE measurements combined with computer simulations of the individual coupling constants even allowed to differentiate the populations of different conformersat each single C-C bond.Two of the original spectra andthe results of the conformational analysis are reproduced in Figure 4.2.3. It turns out that three out of five glycon head groups prefer the planarall-trans (P) conformation, whereas only glucose and talose show one gauche-bent in DMSO solutions (Svensonet al., 1994~). In single crystals ~-octyl-D-gluconamideis aZZ-trans configured with a head-to-tail orientation of the crystal layers and a homodromic hydrogen bond cycle between neighboring molecules (Fig. 4.2.4a). In ~-0ctyl-D-gulonamidethe usual head-to-head and tail-to-tail arrangement of amphiphilic crystal sheets, hydrogen bonds between neighboring sheets, anda similar aZZ-tran~ conformation are found (Fig. 4.2.4b,e) (Svenson et al., 1994a). In gluconamide the 1,s-repulsion of the two hydroxy groupsat C2 and C4 is overcome by crystal forces; the poly01 chain is straight. In the diateromeric gulonamide the same 1,5-repulsion between hydoxyl groups leads to a strong distortion of the poly01 chain. The well-ordered, rigid gluconamide chain in crystals enforces an identical orientation of all molecules. The molecular ordering of the micelles in water is not taken. The bentgulonamidecannotpackastightlyandacceptsthebilayer formed in spherical rnicelles also in the crystal planes. In Figure 4.2.5 the 13C-CPMASsolid-state NMR spectra of both crystals are reproduced together with the one of D,L-gluconamide microcrystals and Dgluconamide fibers. The assignments of the singlet signals have been achieved by heteronuclear shift correlation of 'H and 13Csignals in DMSO solution (Fig. 4.2.3) and the I3C signals in the solid state (Fig. 4.2.5). It i ~ e d i a t e l ybecomes evident that the racemic D&-gluconamide crystals are in the same tail-to-tail arrangement asthe gulonamide crystals.The chemical shift of the terminal C group, which highly depends on the polarity of its environment, is the same in tail-to-tail gulonamide crystals and the racemic gluconamide crystallites. It is quite different in head-to-tail D-gluconamide crystals. Comparisons of solutionandsolid-state N R spectraaswellas NMR spectra from solid supramolecular assemblies and single crystals allowed the exact determination of four conformations of ~-octyl-gluconamidein three solid materials,namelymonolayer single crystals, bilayer r e microcrystals,and D-enantiomerbilayerquadruplehelicesaswellas in solution (Fig, 4.2.6, inserts) (Svenson et al., 1994a). The most important effects that stabilize certain conformations of polysub-

) N

OH

-

H

-~luconamide(all-trans-conformer)

4tt3

I

412

I

) The major

318

314

conformer^ in D "2

70%

I

6 [ppA]

c-3

OH H

C-6

OH H

OH

100% c-5

90% OH

c-2 C-6 L

2-H

(a) IH-NMR spectra of ~-octylgluconamidein DMSO and an irradiation experiment at 3CH, which removes the doublet at 30H; assignment of proton signals is possible by a systematic series of such irradiation experiments. (b) The most important conformer is gauche-oriented at the C4-C5bond (90%).(c) Cross-correlation betweenlH and I3Cchemical shifts given on the y- and x-axes allows the assignment of 13C signals and is later needed for theinterpretationof solid-state spectra (Fig.4.2.5).

Crystal structure of (a) N-octyl-~-glucona~de (monolayer) and (b)No c t y l - ~ - ~ ~ l o n(bilayer). a ~ d e Comparewith zig-zag confo~ationin Figure 4.2. I. (From Svenson et al., 1994a,b,)

stituted carbonchains over others conformationsaxe the attractive gauche (Juaristi, 1979) andthe repulsive 1,3-syn-diaxial effects (Stoddart, 1971). The gauche effect has two meanings: (1) in open-chain polyols it is usually observed that 1,4-o~ygen-oxy~en attraction makes the gauche confo~ationmuch morefavorablethan the anti conformation,and(ii)thesame l,4-gauche interaction of heavy atoms causes an upfieldshift of the connecting carbon atoms in comparison to the co~espondinganti conformation.Botheffectsarereproducedin model calculations but are difficult to rationalize by simple ~guments,The result is often an edge-amp~philecharacter of polyols: OH groups tend toform a hydrophilic edge, CH protons a hydrophobic one. A similar efYect is observed in synthetic polyethylene glycols (

= octyl

c-l

172.2

Solid-state ~ ~ ~ ~ S spectra - 1 of 3 (a)N-octyl-D-glucon~de, C - ~ ~ (b)gulona~de, (c)-D,L-~lucon~de crystals, and (d) -D-gluconamide micellar fibers (see Fig. 4.5.6). The correspond in^ molecular confo~ationsare given. Note: Soft race~ate crystallites (c) and micellar fibers (d) have the same beadgroup confo~ation.

wheremeanderconformersdominate:ethylenegroups form a hydrophobic bridge on one edge of the molecule, the ether oxygens hydrophilic tips on the other. PEGS therefore tend to aggregate side-by-side in water and do not mix with polysacch~desand proteins in water. Such aqueous solutions may therefore form two phases and are usedfor the separation of polymers with different overall shapes (Albertsson, 1986). Hydration not only influencesthe confomation of organic molecules, but the action of dissolved organic molecules also determines the packing of the water clusters!The 1,3-syn diaxial repulsion, on the other hand, is a direct result of an overlap of van der Waals radii of large substituents in these positions in the ~ Z Z - t r confomation ~~s (Fig. 4.2.6). What happens if both end groupsof a polyol chain are highlypolar? This does not occur often in natural hexose derivatives, but tartrates are important examples in thetetrose series. Ever since Pasteur separated tartrate enantiomers by c~stalli~ation, this compound has remained the heart of organic stereochemistry. Herewe just point to the recent eventof stereoselective and catalytic epoxidation procedures steered by tartrates and discussed in Section 4.3 in context with car~ohydratesyntheses. Inthe crystal structures of D-tartaric acid one finds relatively weak hydrogen bond chains between hydroxyl and carboxyl groups

Two effects determine thecon~gurationsof open-chain carbohydrate derivatives: (a) gauche confi~ationsare favored in 1,2-diolsover anti confo~ations,and (b) 1,3-syn diaxial interactions between hydroxyl substituents are repulsive and often lead to distortions of the aZZ-anti chain.

(Fig. 4.2.7a). D- and tartaric acids are both easily soluble in water (133 g L ) , because there is no special stabilization of the crystals. D,L-Tartaric acid (racemic acid), on the other hand, contains a strong hydrogen bond cycle between the hydroxyl groups of opposite enantiomers (Fig. 42.717). As a result, the racemate is This pheless soluble than the enantiomers by a factor of 12 (10.6 g/100 EL)! nomenon is quite c o ~ o n~: i ~ o r - i m a molecules ge often tend to stick together

Crystal structures of (a) D-tartariC acid with hydrogen bonds including COOH groups and (b) racernic acid showing OH-OH hydrogen bond cycles.

more tightly than pure enantiomers. There are, however, exceptions, and Pasteur found one very early in the history of organic chemistry. Below 30°C ammonium-sodium racemate separates spontaneouslyinto two different crystals with m~or-imageshapes, whereas above 30°C the racemate crystallizes as single crystals. Ammonium-carboxylateversus OH hydrogen bond shifts are responsible for these spectacular findings. Lipophilic tartaric esters also react with other chiral hydrogen-bonding molecules (Prefoget al., 1989).

lic The aldehyde and keto groups cyclize spontaneously withthe hydroxy groups in monosaccharides to form half-acetalsor half-ketals with a “glycosidic” hydroxy group. Glycosidicis a trivial narne meaning “sugarlike”. Six-membered rings are called pyranoses; five-membered rings are furanoses. In Figure 4.2.8, typical s ~ u ~ ~ofr cyclic e s monosaccharides are given in the stereochemically correct cyclohexane chair notation. Most common in nature are glucose units with all substituents ine ~ ~ a t u ~positions, ial galactose units with one OH group at C4mi d , and mannose with one axial OH group at C2.For comparison, the monosaccharides are also reproduced in the Haworth projection. This neither indicates the most important D-L mirror-image relationship correctly, nor does it say anything about the conformation of the ring and sterical interac~onsbetween substituents. Since these classical projections neglect the most important stereoche~caldetails without simplifyingsymme~y obse~ations in the manner of the Fischer projections, one should avoid using them. Different pyranose conformations are designated as follows: C for chair, B for boat, S for twist-boat, and H for half chair. The numbering starts with the oxygen atom taken as zero and continues with the aldehyde carbon atomAas 1. reference plane containingfour ring atoms is chosen so that the lowest-number carbon atom inthe ring is displaced from it. Ring atoms thatlie above the plane are written as superscripts and precede the letter. Atoms below the plane follow behind and below the letter. The 4 C , ( ~conformer ) is enantiomeric to the ‘C,(L) conformer. Descriptors should therefore be used in referenceto the L- or D-series (Fig. 4.2.9).There are two chair conformations possible, namely 4C1 and,C1 ‘C4. means a chair in which C4 is above and C l is below the plane containing the oxygen atom. IC, is the other chair conformer. If two identical sugars are compared, a change from one chair conformer to the other converts all equatorial substi~entsinto axial ones and vice versa. The activation energyof a rearrangement reaction with equal numbersof axial and equatorial groupsis in the order of 10 kcal/mol, unfavorable conformers with twoor more axial groups, such as the all-axial glucose molecule, are not formedallatif a more equatorial choice is av~lable.This situation only changes if one succeeds in co~necting stituents by covalent links (Stoddart, 1971;~uinkert,1995).

CH.tOH HOH2C

H

1 OH

H

OH

’ p-L-Glucopyranose

P-D-Glucopyranose p-L-Glucopyranose CH,OH

OH

H

.H0

H0

p-D-Galactopyranose

OH

\

ow

H6

OH

p-L-Galactopyranose

H OH

p-D-Galactopyranose p-L-Galactopyranose CH20H H

H OH OH

p-D-Mannopyranose

p-~Mannopyranose p-L-Mannopy~no~ HOH&

H0

OH

H

H0

H0

HOH&

p-L-Glumfuranose

pD-Glumfuranose p-L-Glumfuranose

CHpOH

HOHzC H0

D-Glucose a s reference

i

(p: allequatorial I) 2 D-AIIow

3

only difference atC-3 Ure (a>Structure ofsomeD-aldohexosesandL-aldohexoses in thepyranose comform andof D- and L-glucosein the furanose form and the Haworth’s formula afor parison. Don’t use thelatter! (b) ~ssignmentof a name to a given pyranose formulastarting from a comparison first with the glucopyranose or glucofuranose and then with the simple Fischer projections.In the example in the second row only the30H group is different from D-glucosein the pyranoses and furanoses drawn. Going to the Fischer projections inFigure 4.2.1, it is i ~ e d i a t e l yobvious that thea-$-glycosidesgivenare a-,p-alloses.

in a ~ r o arr~n~ement; n ~ n u ~ b must ~ ~bendoc ~

1

Different chair conformersof D- and L-glucose.

The crystal structures of cyclic hexoses showa cyclohexane chair with the As an examplefor a glucose deprimary alcohol group in an equatorial position. rivative, thecrystal structure of l-decylsubstituents are in equatorial positions, but alcohol side chain on the glycosidiccarbonatom is axial ( a ) ,Thecyunithasaboutdoublethe width of the alkyl chain and the latter therefore interdigitate in the crystal( he extra or dinar^ stability of a-con~guredhemiacetals (epimers) with the xyl group in the sterically hinderedaxial position has led to the assumption of an “anomeric effect,” name1 electrostatic repulsion between the oxygenatom of thecyclohexane chair theequatorialoxygen (~uaristiand aqueoussolutionthiseffect i el co~pensatedby hydraas also found that in a anomers than inthe

Crystal structure of l-decy1"-D-glucopyranoside.

p anomer, which substantiates the repulsiveeffect. Furthermore, the bond angle OrinLZ-C 1-Omeis widened to 112" in the a anomer as compared to 108" in the a th findings canalso be assigned to a stronger sp'kharacter of the ring in the p anomer (Fig. 4.2.1 1). Model calculations showed that in vacuo glucose follows thegauche effect of the 0-C-0-C linkage and prefers the a con~gurationby 0.5-1 kcaVmol. Upon solvation, however,the equilibrium is n al., g 1998). Furthermore the driven to the p form by -0.3 kcaVmol ( ~ i ~ e r l i et a anomer binds water molecules more tightly (Molteni and Parrinello, 1998), thus disrupting water clusters. The reverse anomeric effect occursif the first atom boundto Cl is either positivelycharged ( p y ~ d i ~ ~orm carries ) apartialpositivecharge(car.M~tarotationcan be conveniently followed by measurements of ectra. The hemiacetal methine proton appears at lower field than the protons of the carbohydrate (e.g.,at d = 4.97 ppm for an a-pyranose), Furthermore,large coupling constants(J= 6-9 Hz) indicate an ~ i a l - ~ x i a Z tion of the protons at C l and C2, whereas small coupling constants (J = )point to equato~al-equatorialor ~ i a z - e ~ ~ a t orelationships r~al between these protons (Fig. 4.2.12). The most clear-cut assignments of l chemical shifts in aldopyranosides come from the determinationof 13C-1H spin-coupling constantsin a large number of methyl aldopyranosides with different single sites of I3C e ~ c ~ e nIt t . ring protons havethe same chemical shift in water for 03 k 0.041; H2 4.002 rt 0.058; H3: 4.180rt. 0.064; S of axial protons are much moretyp 1 4.555 A 0.192; H2: 3.6430.218; :3.789 f 0.176). The major con~butions

1.6

0.4

3 3

l

0

3 Anomers of glucosides. Bond lengths are givenpm. in

to chenxical shifts in methyl glycosides of aldopyranoses are the trans-l ,2- and the 1,3-diaxial effects. Other stereo~hemicali n f o ~ a t i o nthathasbeendrawn the C-Cfrom l3C-IH coupling constants include the anomeric con~gu~ation, coupling pathway involving the carbon atom neighboring the13Csite, the differentiation of diastereomers, andthetorsion gle betweentheterminalcarbon atom and a P-proton. A model study on 13C-l pin coupling in furanoses yielded detailed predictions about the conformational dependence of the co~esponding

Reverse anomeric effect as shown b y l H - ~ Rspectra of N-glycoside cations. The concentration of the (-anomer is small and becomes even smaller upon Nprotonation. (Prom Perrinet al., 1993.)

coupling constant values(lJCH), but experimental verification could only be obtained with the constrained model systems of ribonucleosides. The assignments of 'H signals then allows the quantitative d~temination of all foms of monosacch~desin aqueoussolutionasreproducedinFig. 4.2.13. The most impo~anthexoses, namely glucose, mannose, and galactose, all occur as pyranoses only.The axial 3-OH group of allose, however, interacts repulsively with the a-OH group on C-1 and thereby overcomes the anomeric effect. The combination of both repulsive effects then also leads to 12% furanose. The latter effect is even more pronounced in altrose and ribose (Stoddart,1971) (Fig. 4.2.13). In the almost planar furanoses, small coupling constants mean cis, large coupling constants trans con~guration.In pyranoses only 1,2-~iaZ-axi~Z inter-

H

OH

H

H

7%

bn

OH I

OH
33%

OH

OH

7%

OH

OH

HOH2C OH

OH

0%

H

13% H0

OH

~ q u i l i b ~ umixtures m of cyclic hexosesand ribose in water.

OH

actions of methine protons, corresponding to equatorial large couplings.Pyranose-furanoserearrangementscan niently followed by lH-NMR spectroscopy. The relative stability of a pyranose chair conformation depends onthe position of the hydroxyl substituents. In stable conformations most hydroxyl groups should occupy equato~alpositions. The aldoses and ketosesexist in aqueous solution as a mixture of four isomers, a- and p-pyranoses, anda- and p-furanoses.There is no evidence for any significant propo~ionof the acyclic configuration in solution, with the possible exception of idose. In general, only one of these four isomers crystallizes at a given condition (e.g., a-glucopyranose from water at high temperature or p-~luco~yranose at low temperature). It is either the predominant species that crystallizes or the least soluble one in the solvent used. Crystallization then gives rise to optical mutarotation, which is caused by establishment of the equilibrium mixture in a solution from which one component is removed. In the literature crystal structures of pyranoses predominate with only a few samples of crystalline furanoses. The furanose isomers ofD-ribose and 2-deoxy-~-ribose,for example, have never been isolated and crystallized since configurational interconversion of cyclic compounds in solution tends to inhibit crystallization. For this reason, the l-0-methyl acetals, which cannot isomerize, crystallize more readily than half-acetals. Unsaturated pyranoid cyclohexene rings occuras half-chairs (symbol: The 4H, and ,H, forms of triacetyl-D-glucal are shown in Figure 4.2.14. Their equilib~umis strongly influenced by the “allylic effect,” which means that the ,H, conformation with syn-axial 1,3-repulsion is of similar conformational energy as ,Hs, in which all substituents are equatorial.The binding energy between the lone electron pairs of the oxygen atoms and nthe electrons of the carbon-carbon double bond presumably providethe estimated 0.8 kcal/mol neededto pull the acetoxy groupsinto the same plane~ H o ~ m a n1989; n , Johnson, 1968). Pyranose rings are thermodynamically favored over furanose rings. The formation of the unfavorable furanose ring becomes, however, dominant in pentoses whenthe terminal primary alcohol group is esterified with bulky acids (AltonaandSundaralingam, 1972; Lehmann,1976,1996). The largesubstituent then remains partof an open-chain structure and is not squeezed in at the periphery of a cyclohexane unit. Prominent examples are the phosphorous esters found in nucleotides (see Chapter9) and the 5-tritylether (Figure 4.2.15). Five-membered furanoid rings occurin envelope (E) or twist (T) conformations, which are ina rapid equilibriu~at room temperature. Both conformers help to avoid energy-rich eclipsed orientation of neighboring substituents, which would occur in the planarconfomations. The preferredconfomation in methyl furanosides is [E in which C1 is above the oxygen-C(2,3,4) plane and the hydroxymethylgroup is inaxialposition. This conformer is enforced bythe

The allylic effect. Both conformers have about the same energy, because the n-electronsof the enolic double bond attract the n-electrons of the allylic oxygen atom.

Large substituentson C5 of ribose shift the equilibrium from pyr~oses to furanoses.

anomeric effect as well as oxygen-oxygen repulsion between the substituents in the alternative2T,conformer (Fig. 4.2.16) (Stoddart, 1971). Another effect of the ring flattening of furanose in respect to pyranose is importantinrespecttoDNAstructure: the torsionanglebetween l,4-substituents in cyclohexane chairs is always close to the ideal 60°, whereas in cyclopentane it is muchlargerandusuallyapproaches 80”. This makeslinear strands of chiral furanose units impossible; helicityof the polymer backboneis enforced (see Sec. 8.2). Some reducedand oxidized monosaccharides areof outstanding commercial value. D-Glucitol (diabetic sugar), in which the aldehyde functionof glucose is reduced to an alcohol, is the sweet component of rowan berries. It occurs as linear poly01 or as bicyclic anhydride. D-Mannitol and galactitol, a meso compound, as well asD-glutamine are also readily available. Cyclitols (e.g.,the cyclohexane hexaolsor inositols), are carbocyclic derivativesof monosacch~des. They bear hydroxy groups at each carbon atom. Most important are those that occur in muscles (“growth factors”) as well as in yeast and fruits. Furthe~ore, they occur together with their phosphate esters in all animal tissues. Deoxy carbohydrates are usually aldoses. The most common deoxyhexoses are L-configured(!)[e.g., 6-deoxy-~-mannose(rhamnose) and 6-deoxy-g~actose (fucose)]. The all-important deoxypentoseof DNA is 2-deoxy-~-ribose. The terminal aldehyde groupof aldoses may also be oxidized in natureto give a carboxylic acid, which then cyclizes to form lactones. D-Gluconic-, Dgalactonic, and L( !)-mannonic acids are the most prominent examples. Important 1,6-dicarboxylic acids are glucuronic and galacturonic acids. The dehydrolactone called ascorbic acid is discussed in Section 7.2.3. Finally, an arnino acid carbohydrate with an unusual nine carbon atoms should be mentioned, namely n e u r a ~ n i c acid (5-a~no-~,~-dideoxy-~-glycero-D-ga~acto-~-nonu~osonic acid), the parent compound of the sialic acids (Fig. 4.2.17).

OW

Major confo~ationsof furanosides. E = Envelope, T = Twist. Position of numbers relates to position of plane shaded in gray.

CH OH

CHz.NH2

OH

H0 H

O

~~~

~

H

O

OH ~

H

H0 OH OH

OH

CH2OH CH20H

CH2OH

CH20H

D-Glucitol D-Giucamine D-Galactitol D-Mannitol H0

Cyclitols

OH

H0 OH

OH

OH

OH

cis

&H

epi

allo

OH H0

OH OH

OH

neo

muco

I

\

OH

OH

my0 H0

H H

H OH

H0

L-chiro scyllo

D-chiro

"-\2%.

OH H0

OH bH

~-L-~hamnose2-Deoxy-D-ribose f3-L-Fucose COOH

COOH

H0

CH

H0 H0

H0

OH

D-Galactonic acid

OH

H0 CH20H

D-Gluconic acid

L-Mannonic acid

COOH OH OH COOH

D-Glucaric acid

Neuraminic acid

H

H0 OH

OH CHzOH

0

H H0

COOHo

D-Galactaric acid

~

OH OH

L-Ascorbic acid

Special naturalmonosaccharidederivative andcyclitol analogs.

Dimers of glucose are formed by removal of the half-acetal hydroxyl group of one molecule and condensation with oneof the six hydroxyl groups of another one. Six regioisomerseach in form of two diasteromers can thusbe formed between twoD-glucose molecules by a single condensation reaction! The most important glucose dimers are(1->4)-1i&ed cellobiose and maltose. In cellobiose the glycosidic (carbohydrate acetal) linkage is bis-equatorial or linear; in maltose it is a ~ i a Z - ~ ~ u a t oor r i ~orthogonal. Z If both glycosidic hydroxyl groups are pconnected, one has p,/%trehalose.These three diglucosides are sweet, but the (1->6)-1i&ed regioisomer gentiobioseis bitter. Another common disaccharideis sucrose, whereD-glucose is a-connected to fructose, a common furanose. The carbohydrate of mother milk lactoseis similar to maltose but has an axial hydroxy groupin the first sugar (D-galactose). Disaccharide crystals are mostly obtained from aqueous ethanol solutions by slow evaporation over months. In P-cellobiose,the cellulose monomer unit, both rings are the in strainless trans form and all available OH groups are used in hydrogen bonds, namely eight per molecule (Brown, 1966).One of these is an intramolecular linkingof the 3’-OH proton with the ring oxygen 05, 1’OH-20H and 2OH-5’ helical system. The other five form a linear hydrogen bond chain. In maltose monohydrate crystals, the monomer unit of starch. The monomer shows an intramolecular hydrogen bond between 02 and 03’ (Quickley et al., 1970).Two infinite hydrogen bondchains are formed in the crystal. Neutron diffraction shows that all hydroxy groups are involved in donor both and acceptor hydrogen bonds. The a-glycosyl group has a gauc~e-trans,the P-rz-glucose residue a gauchegauche (1-5c)orientation. Bothrings are folded towards eachother in a roof-like manner(NealandGoring,1970).Thermodynamicmeasurements in solution showed that maltoseis also folded in aqueous solution. The inner sideof the roof is obviously more hydrophobic than the outer side. The period in the c direction is 10.6 A, which coincides with the fiber repeat in pamylose of 10.4-10.6 A. The packing of maltose molecules almost duplicates the ~ a n g e m e nof t glucose residues in two contiguous strands of pamylose (Blackwell et al., 1969). maltose molecule thus represents two glucose residues in one turn of the starch helix, and the equivalent maltose inthe adjacent cell in the c direction may represent the residues in the next turn. The helical motifof starch is, however, much better expressed in the trehalose dihydrate monomer in than maltose (Taga et al., 1972). Both glycopyranosides are connected by a glycosidic a,a-bond and by hydrogen bonds 02-06’, which need two water molecules as relays. Again a roof with a hydrophobicinside is formed. Intermolecular hydrogen bondsnow form infinite spiral chains about a twofold screw axis; the water molecules build up another helical ~ a n g e m e n tGentiobiose . is the most flexible and most soluble

ter

CH OH H

#O *H HOHzC OH

Molecular and some crystal structuresof disaccharides.

H

CHzOH

CH OH H

OH

OH

OH

The exo-anomeric effect. The gray shading indicates the most favored conformers.

d i s a c c h ~ d ebecause the glycosidic bond to the primary alcohol group on C6 is the most mobile one.Its molecular structure resembles that of cellobiose, butintramolecul~hydrogen bonds are not found (Rohrer et al., 1981).The final diglucoside is sucrose, in whicha pyranose is a-linked with an furanose unit, again in a connection of two half acetal-ketal type hydroxy functions (Brown and

et al., 1973). In this disaccharide there are two intramolecu02-01’ and 05-06’. The crystal is held together by three hyne is parallel to the bc plane, while the other two are perpendicular to it. The 4-OH group of p-lactose (Hirotsu and Shimada, 1974) is axial, and both monosaccha~derings twist into the same plane. The hydrogen bond present in the twisted cellobiose analog disappears ( The conformations of the glycosidic link by two an ence to the hydrogen atoms at both sides of the glycosidi 1,4-li&ed carbon disaccharides exist in the gauche 0-C 1-Cex0-Cor 0-C 1-Oex0-C ~ a n g e m e n t srather than the anti ~ a n ~ e m e n(Figure t. 4.2.19) ( ~ a n get al., 1992). The preference for axial anomers to exist as conformers with no syn-axial lone pair interactions and of equatorial anomers with a single syn-axial lone pair interactions has been called the exo-anome~ceffect. The effect is of the order of 4 kcaVmol in glycosides. The observation of only one type of conformer, namely the type where both units are twisted against each other by 60°, is especially puzzling in the case of the C”g1ycosides.It has so far only be solved by ab initio quantum mechanics in the sense that slight electrostatic and bond angle changes common than the monomeric and dimeric carbohydrates are oligoh the monosaccharide half-acetal hydroxy groups (glycosidic hydroxy group) are replaced by other carbohydrates, glucose is by far the most important monomer, but thousands of polysaccharides made of all kinds of monosaccharides can be produced on a large scale by fermentation or farming from ~croorganismsor plants.

Cyclic oligomers of amylose are called cyclodextrins (Wenz, 1994). on the number of glucose units y2 = 6-9, the prefixes a-6 are used. The inner diameter of the cycles are 4.9 A for a-cyclodextrin, 6.5 A for p-cyclodext~n,and 7.9 A for y-cyclodextrin. The height of all cyclodextrin tori is 7.8 A. The torus carries twice as many hydroxy groups on the outer surface as on the inner surface (Fig. 4.2.20). All glucopyranoses are connected by hydrogen bands and a rotation around the C1-0-C4’ bond is impossible for sterical reasons. In crystals a-cyclodextrin binds 6 or 8 water molecules, whereas the larger cyclodextrins all bind about 12 water molecules.

Trivial names of polysaccharides often reflect an origin or a property: cellulose stems from cells and starch from stercan, meaning to stiffen. The term glycan is derived from glycose, meaning monosacch~de;polyglycan or simply glycan is

Structure of P-cyclodextrin.

a synonym for polysaccharide. D-GlUCaII is a homoglycan madeof D-glucose;aD-galacturonan is a polymer made of D-galacturonic acid units. Branched polymers can be named in the same way,e.g., (1"+2)--~, ( 1 4 6 ) " ~ branched mannan. Although the indiuidual structures of polysacch~desare extremely complex, there are three fundamental motifs in the chain c o n f o ~ a t i o nof homopolymers. A useful concept to describe them is to regard any c o n f o ~ a t i o n as a helix made of rz monomers per turn with a projected length h of each monomer unit on the helix axis. Three types of c o n f o ~ e r sare then possible (Fig. 4.2.21)

6 cycle n=5h-0

rib~on

helix

n=2

n=4-10

coiI

l fincipal structures of polyglycans (Source: Kennedy, 1988 see text for details).

1. Extended or crumbled ribbons with n from 2 to k4 and n close to the absolute length of the monomer (negative n-values indicate a lefthanded helix). 2.“Crumbled”indicatesirregularclusterformationwithintheribbons and is typical for (1+2)-glycans, where linear ~ a n g e m e n t sare disturbed by repulsive sterical interactions (not shown). A helix with n = 2 to*l0 and h approaches zero. The above two conformers are found in all common polysaccharides.

3. A flexible coil typical for (1+6)-glycans. The primary alcohol group allows extreme mobility, and these gentiobiose-type polymers promise to be most useful in molecular architecture provided there are somestrong hy~ogen-bondingcenters in strategicpositions,e.g., NH;, COO-,CQQH, etc. The principal food reserve starch occurs in cereal, grain, potatoes, and other plantsin the form of granules. These granules maybe loosened in their matrix by swelling with cold water and can then be isolated without destruction. Starch occurs mainly as a (1->4)-linked chain of a-r>-glucopyranosyl residue (amylose), which tends to form right-handed double helices up to 20% (w/w) (Fig. 4.2.22). Jcions, which are charge transfer complexes between iodide and iodine, can be entrapped within these helices and form extended chains. The charge transfer transition now produces a blue color. In nature amylose helices may entrap various lipids: about six glucose molecules form one turn. These complexes are somewhat similar to the urea inclusion complexes described in

V

x Secondary structuresof starch molecules.

Figure 2.5.17. The same chain with (l->6) links is called amylopectin. It does not form helices, but flexible coils. The extra bond that separatesthe rings means more flexibilityfor the polymer, as exemplified by the dissaccharide gentiobiose (see Fig. 4.2.17). Cellulose, the most abundant organic compound found in nature, forms the principal constituentof cell walls in higher plants. It accounts for 91% of cotton fibers and40-50 of wood. It is a linear chain made of up to lo4(1->4)-linked pD-glucose molecules. The characteristics of cellulose result fromthe tendency of the individual chains to form linear, extended ribbons, which assembleto form highly ordered, hy~ogen-bondedmicrofibrils. If the ribbons are laid down in sheets in a parallel manner such all that reducing terminalsoccw at the same side of the bundle, it is called cellulose I. This is the most common natural material (Fig. 4.2.23). emicelluloses or polyoses rank as the second most abundant class of organic compounds onearth and occur incell walls of plants. They are differentiatedfromcellulose by alower polymeri~ation grade of50-250, extensive branching, and the presenceof a large variety of hexoses and pentoses in addition to glucose. Typical examples for these highly branched molecules are polymeric L-arabino-D-galact~s,D-mannans, D-galacto-D-mannans, D-xylans, etc. A common acidic xylan from hardwood consistsof a main chain (1->4)-1i~edpD-xylopyranose, where ~-methyl--D-glucuronic acid is attached to about one the same main chain but twice as out of 10 xylose C2 atoms. Softwood xylan has many acid side chains. F u ~ h e ~ o rthe e , hardwood xylan carries acetyl groups, whereas the softwood analogs are substituted with arabinose and other monosaccharides at C2 (not shown). The major hemicellulose of hardwoods, however, consists of (l->4)-linked p-~-glucopyranoseand ~-D-m~nopyranose residues

4

:

Secondary structure of cellulose. (Source: Kennedy,1988)

(glucom . Galactose is frequentlya substi~entin the sidechain ( 4.2.24). elluloses do not f o m stable ~crofibrilsandaremorereadily drolyzed than cellulose. One of the few hemicelluloses that have found industri~ use is arabino-galactan from larch trees. Itis highly branched, very water soluble, and surface active. Its aqueous solutions have a very low viscosity and are useful as an emulsifier, e.g., for mineral oil recovery. Pectins make up13%of the dry weightof apples and 30% of the rinds of citrus fruits. They are subdividedinto pectinic acids, containing partially me lated D-galactopyranosyl-uronicacid chains, and nonesterified pectic acids. majority of pectins contain various proportions (10--25%) of neutral monosaccharides, of which only L-rhamnoseis found in the main chain, as, for example,

H0

-pD-Galp-( 1-+3)-P-D-Galp-( 1 -+3)-P-D-Galp-( 1-+3)-p-Malp-( l“+3)-fMl”alp-( 1 +3)6 6 6 6 1‘ t f f 1 i l i .1 D-D-Galpln a-D-Galp p-D-GlcpA P-D-Galp a-L-Araf 6 or (a-L-Araf) f 1 i i P-D-Galp CL-L-A~P P-D-Galp

4 4

r6

H0

\

.24 Typical structures of hemicelluloses or polyoses: hardwood D-Xylan and g l u c o ~ a n and n ~ softwood galactan.

in the pectin of white willows. Other neutral sugar residues are attached as side chains (not shown). Pectinic acids are easily extracted with water and possess considerable gelling power.In contrast, the pectic acids usually contain calcium as counterion andare only solubilizedby EDTA solutions. The plant cell wall of wood is built up by the three components discussed above:rigid,water-insolublecellulosemicrofibrils,hemicellulosemolecules, which are presumably fixated by hydrogen bonds on the fibril’s surface, and pectin calcium salts, which form an aqueous connecting gel where side-arm carbohydrates act as hydrogen-bonded linkers (Fig. 4.2.25). The plantgums are essentiallyheteropolysaccharidescontaininghexuronic acid salts and electroneutral monosaccharide residues in highly branched structures. Gum arabic,for example, contains interiorchains of (1->3)-linked pD-galaCtOpyranOSyl residues to which chains comprised of various furanosyland py~anosyl-uronicacid residues are linked. These gums are extruded as viscous aqueous solutions when the tree’s bark is cut and produces hard nodules upon dryingto seal the site of injury. Unilayers areof similar structure and form gels in seeds, which retain their own weight of water over a wide rangeof concentrations. They probablyact as reservoirsof water and protect seeds and roots from irreversible desiccation (Bever, 1986). Algae, plants without roots, contain (1->3)- or (1->4) linked p-D-galactopyranosyl polymers (e.g., agar and carageenans) carrying negative charges (uronic acids, sulfates). Alginic acid, onthe other hand, is a linear polymer and

2 5 Molecularmodel of wood. (From Bever, 1986)

Structure and Source of Common Homo- andHeteropolysacch~des

Common name

Linkage D-FruCtanS (2+l)--~-linear (2+6)--~-linear

Dandelions, dahlias, Jerusalem artichokes Various grasses

Inulin

Red seaweeds Plant pectic substances Penicillin mold

C~ageenan

Plant pectic substances

Pectic acid

Agrobacteria Brown seaweeds, plants plants, algae, fungi, and yeasts Plants Fungi Animals, plants, and ~croorganisms Plant cell walls Bacteria Lichens

Laminaran, callose, curdian, pachyman Amylose Pullulan Glycogen, amylopectin Cellulose Dextran Pustulan

Levans

D-

,( l +4)--~-linear (1+4)--~-linear

(1+2)--D-linear (1+3)--~-linear (1-34)--~-linear (1-34)--~, (1+)--D-linear (1+4)"-~, (1+(i)--~-branched (1-+4)--~-linear (1-+6)--~~ (1+3)--~-branched 1-36 -"+-linear

Galactocarolose

o ~ ~ - ~ - ~ ~ ~ ~ a ~ s

linear (1+6)--~-branched (1+4)--~-linear D-

Crab and lobster shells, fungi

Chitin

Yeast Seaweeds, plants

S

Green seaweed ( l +3)--~-linear Plant cell walls ( l +4)--~-linear Red seaweeds DL-Galactose, linear D-Galactose, 2-amino-2Cornea deoxy-D-glucose, linear 2-&nino-2-deoxy-~Cornea, cartilage galactose? D-glucuronic acid, linear 2-Amino-2-deoxy-~-galactose, Skin L-iduronic acid, linear D-Glucose, D-mannose, Coniferous linear woods, seeds, ~ - ~ ~ n o - ~ - d e o x y - ~ - g ~ u c o s eAnimal , and D-glucuronic acid, linear m a ~ a l i a tissues n L=Guluronic acid, Bacteria, brown D-mannuronic acid, linear seaweeds Source: Kennedy, 1988.

~odymenan Agarose Keratan sulfate Chondroitin, chondroitin sulfates Dematan sulfate

Hylauronic acid Alginic acid

istr

consists ofD-manno- and L-gulo-pyranosyluronic acids. It may contain crystalline regions of pure domains interrupted by amorphous regions containing a mixture of both residues.The algae polysaccharides are nontoxic to microorganisms and cannotbe degraded by most ofthem. Bacterial cell surface polysaccharides frequently carry pyruvic or lactic acids linkedto a sugar residue. They act as antigenic determinants. Their conformation has been characterized by NMR spectroscopy (Severn and 1993). DextransfromLeuconostocbacteriahaveramifiedstructuresmade of (1-+6)-1inked, sometimes (1-+3)-linked a-D-glucopyranosyl chains. Water-soluble dextrans form filmsby evaporation of dilute solutions that are composedof microscopic filaments. Electron microscopy and molecular assemblies with proteins suggest that many of these dextran assemblies remain associated indilute solution.Computersimulation of dextranhelicessuggestedtwo to seven residues perturn, which is also in agreement with the extended, ribbon-like conformation given in Figure 4.220. Dextrans are used, for example, as artificial blood plasma (6% solution) and in glues. Cross-linked dextrans are carriersfor pharmaca, molecular sieves,c~omatographymaterials, and plastersfor wetting injuries. Pullulan (10 g: $300) is a water-soluble a-( 1-+6)-~-maltotriosepolymer with some a-(1-+3) links from fungi. It forms foils that are quite resistant to oxygen diffusion and are therefore used as packing material for food. Table 4.2.1 names a few common polysaccharides from natural sources.

Only a few monosaccharides occur in nature in measurable quantities. Approximately 0.1% D-glucose is important in human blood, and this concentration is controlled by the peptide hormones insulin and glucagon. Lower values lead to hypoglycemic shock. The usual wayof obtaining D-hexoses is hydrolysis of more abundant biopolymers. In the case of glucose, mannose, and galactose even the L-enantiomers are accessibleon a commercial scale.The most common amino hexoses areglucosamine, m ~ ~ o s a m i n eand , galactosamine,inwhich the 2-hydroxy group is replaced by an amino group. D-Pentoses [(CH,O),] are all available at reasonable prices; the cheapest enantiomer is L-arabinose. Tetroses and glycerinaldehyde arepro~bitivelyexpensive, butthe latter unstable compoundis readilyavailable from mannitol by periodatedegradation of thediacetonide. Fructose occurs in somefruits (e.g., apples). The sugars D-glucose, D-galactose, D-mannose, D-fmctose, and D-XylOSe are attractive starting materials, which can be isolated in quantity from natural

sources. A few examples of isolation procedures will be given in this chapter. Then syntheses by isomerization will be explored, followed by carbon-chain elongation and shortening sequences. Functional group interconversions (FGI) and the use of carbohydratesas chiral synthons(chirons)(Hanessian,1979, 1983) will be introduced in the final sections about monosaccharides.

D-Glucose (10 g: $ 0.20) is made by acid-catalyzed hydrolysis of starch from potatoes or corn. The process usually runs in autoclaves at 140-145°C and an HCl concentration of 0.03-0.09 M. After neutralization, the mixture is centrifuged in order to remove proteins and fatty acids and filtered.The clear dark brown solution is then clarified withcharcoal and evaporatedto a concentration of 75-78 % ( ~glucose. ~ The) solution is mixed with some crystalline glucose and cooled from46 to 120°C within 2 days. The crystalline sludgeis further dried to a water contentof 8%. This is less than the 9.1% that would correspondto the desired monohydrate. This procedure leads to fine crystals within several days(!) and prevents the formationof lumps. The crystallization canbe achieved within 10 hours if sodium chloride is added and the glucose X NaC1 double crystal is isolated first. Another possibility is enzymatic hydrolysisof starch by glucoamylase. Enzymatic degradation has the major advantage that small changes in the fabrication scheme and using different enzymes allows one to produce not only glucose, but also a large varietyof branched and nonbranchedoligosacch~des (Fig. 4.3.1). Fructose (10 g: $4) is a relatively harmless sugarfor diabetics. In order to obtain it from sucrose, the latter is first "inverted" by HCl. Sucrose is thereby split into two fructose molecules. Concentration and crystallization processes are then similar to those described for D-glucose. There is also a polyfructanoside called inulin that occurs in chicory roots, butisolation its is difficult. L-Sorbose (10 g: $1.60), the starting material for vitan& C, is fabricated by microbiological oxidationof D-sorbitol. D-sorbitol(log: $1SO) or D-glUCitOl, on the other hand, is easily obtained by chemical reduction of D-glucose (e.g., with sodium amalgam). (L-Sorboseis an L-configured carbohydrate becausethe last OH group counted from the keto group is L-configured!) D-Mannitol is much less soluble in ethanollwater than sorbitol (glucitol) because it is symetrical and therefore crystallizes more easily. Separation by crystallization is simple. Electroreductionof D-glucose under alkaline conditions gives a mixtureof 80% glucitol and20% mannitol (10 g: $0.80). D-Gluconic acid is usually obtained asthe calcium (10 g: $1) or potassium salt by oxidative f e ~ e n t a t i o nin the presence of CaCO, or NaOH. Metal gluconates are usefulin pharmacology for the injection of metal ions, e.g., iron.

0

'

exo-l,4-pD-Glucosidase

.l Schematicrepresentation of theenzymatic de~adationof starchfrom (o), Reducing glucosyl residues endplants, fungi and bacteria, and cellulose from plants. no~educingglucosyl residues corresponding to full acing with a half-acetal unit; etals; ( ) , crystalline regions of cellulose; amorphous regions of cellulose. (From Kennedy, 1988.)

e),

e**),

D-Glucosamine (l0 g: $2) is obtained from the chitin of lobster shells by HCl hydrolysis under similar conditions as described for glucose. D-Lactose (~-(~-D-g~actos~do)-D-g~ucose; 10 g: $1) is made from defattened milk powder from which the lactone is extracted withethanol, the solvent is removed, and the lactone recrystallized from water. One thousand liters of milk yields about30 kg of pure lactose. D-Lactitol (4-O-(~-galactosyl)-~-glucitol) is an interesting and expensive (l0 g: $300) glycoside madeof a cyclic galactopyranose and an open-chain glucitol. It is obtained by reduction of lactose with sodium borohydride or, technically, by catalytic hydrogenation. Itis useful as sweetenerfor diabetics and does not induce dental caries. Cyclodextrins are preparedon a commercialscale by the enzymatic degradation of starch. The co~espondingcylodextrin transferases are nonspecific with respect to ring size. Pure a-6 cyclodextrins are precipitated selectively, andthe transferases then establish a new equilibrium of all four cyclodextrins. a-Cyclodextrin can be obtained in40% yield in one step by precipitation with l-decanol, p-cyclodextrin in 60% yield by precipitation with toluene, and ~ - c y c l o d e x ~inn 50% yield with cyclohexadec-8-en-one. extrans (10 g :$5-20) are producedby Leuconostoc bacteria growing on a sucrose substrate. Polydextrose is an artificial product formedby citric acid-catalyzed poly~erizationofD-glUCOSe in the presence of sorbit. The cross-linked polymer is only partially digestible and 60% from food is excreted by humans. It is not sweet but improvesthe texture and flavorof low-caloric anddiabetic foods. Several carbohy~atesare inexpensive and can therefore be usedas starting mate~alsin syntheses of chiral products. Theyconstitute major components of the “chiral pool.” In the following, we list some carbohydrates thatcost less than one dollar per gram.

A general synthesis for all diastereomeric L-hexoses, as an example for monosaccharides that oftendo not occur inthe chiral pool, has been worked out. The epoxidation of allylic alcohols with tertiary butyl hydroperoxide in presenceof titanyl tartaric ester catalysts converts the carbon-carbon double bond stereoselectively to a diol and is thus ideally suitedfor the preparationof carbohydrates. The procedure is ~articularlyuseful as a repetitive two-carbon homologization in total syntheses of higher monosaccharides and other polyhydroxy compounds. It starts with a Wittig reaction of a benzylated a-hydroxy aldehyde with (triphenylphosphoran-y1idene)acetaldehydeto produce the olefinic double bond needed for epoxidation. Reduction with sodiu~-borohydride

ss

gives the allylic alcohol, which is used as a substrate for a Sharpless epoxidation. The resulting epoxide is regio- and stereoselectively opened by the adjacent hydroxyl group in the presence of strong base and the resulting epoxide again openedwithbenzenethiolate.Thediol is thenprotectedwithacetone,the (pheny1~o)methylgroup oxidizedto the sulfoxide and converted to the aldehyde by an acid-catalyzed Fumrnerer rearrangement with or without inversion of the stereocenter neighboring the aldehyde group (KOet al., 1983) (Scheme 4.3.1). Wittig reaction with a benzylated a-hydroxy aldehyde then starts another cycle. In all cases exarnined, the E-diastereomers of the allylic alcohols reacted satisfactorily, but 2-isomers were slow and nonselective. The e ~ ~ ~ ~ ~ - i s o m e r s from the E-diastereomerscan, however, be epimerized in 95% yieldto the ther-

~utOOH~(OPri)~

(+)or (-)diethyl tartrate; -20°C

x

threo

0 0

1

1

x p p

)3Ph

R-CH-CH-CW2.SPh eryUlro

x

L

0

0

i

I

‘0 Ac

R-CH-CH-CH0

0

\

R = Ph2CHO-CH2CH- or ONc/

H2

threo Bui2AlH~oluen~; -78°C

erythro = cis

threo = trans (> 95%)

no ~~limination

modyna~callymore stable threo-isomersby treatment with potassium carbonate. The competitive P-elimination is suppressed in the acetonides because the enolate and P-alkoxy groups are forced into orthogonal positions and cannot reach the t r a n s - ~ e r i ~ z ~~naanr g e m e n t which , is necessary for base-catalyzed eli~nations(Scheme 4.3.2). In contrast to stepwise and highly stereoselective syntheses of hexoses and higher monosaccharides, carbohydrates(CH,O), have also been made by basecatalyzed oligomerizationof formaldehyde in presence of calcium hydroxide.A total yield of 55% sugars was obtained comprising about 50% hexoses. Glyceroaldehyde phosphate produces hexoses in the presence of sodium hydroxide alone and gives pentoses if formaldehyde is added (not shown).

Carbohydrates are usually synthesized from natural carbohydrates, which involves massive applicationsof protective groups and activationof the anomeric center. A large variety of books and review articles is available, each of which advocatesadifferentapproach.Individualschemesmustbeexperimentally checked for new target molecules; the applicability of general rules is by no means guaranteed. Base treatment of aldoses gives 1,2-enediols, which then tautome~zeto

7

H0 H

i

OH CbOH

D -Glucose

,

CHpOH

CbOH ,

CbOH

CH20H

CHpOH

D - ann nose

the 2-ketose. Ketoses,on the other hand, have a low tendency to isomerize to aldoses. Ketoses are therefore obtained in 30-90% yield from the corresponding aldoses. Lower yields occur; when simple bases are used such as calcium hydroxide, 90% can be achieved with bases, which form complexes with the ketose (e.g., sodium aluminate). In this so-called de Bruyn-van Ekenstein rearrangement, the epimerof the original carbohydrate is, of course, also obtained.D-ClUcose thus produces not only fructose, but also some mannose (Scheme 4.3.3) (Lehrnann 1976,1996; Binkley, 1988). Similar stereoisomerizationsat C2 are successful with 2-acetamido sugars and aldonic acids. In the case of fructose or other ketoses, the chiral centerat C3 can be isomerized effectively (Scheme 4.3.4). In orderto suppress competing reactions,one often hasto protect the other hydroxylgroups.6-0-Benzyl-2,3, 4,5-di-O-isopropylidene-aldehydo-~-talose, for example, is converted by base into a mixture containing95% of the protected L-galacto diastereomer.This isomer is formed because the 2,3-acetal ring with trans substituents is the~odynamicallymore stable than the original ring with cis substituents (Scheme 4.3.5).

H

H0 OH

0

H0

OH OH OH

CHzOH

-

D Fructose

D -Psicose

In glycosylation reactions the component that contributes the arromeric carbon of the resulting glycoside is described as the glycosyl donor I). (Scheme 4.3.6) Glycosyl donors are glycosyl halides, trichloroacetimidates, or glycals, The corresponding glycosyl acceptor A. furnishes the osygen of the glycoside and usually consists of a free hydroxyl group in an otherwise fully protected carbohydrate. Scheme 4.3.6 portrays globally the classical strategy of ~lycosylation, fully osygenated pyranose donors and acceptors to produce the prodisaccharide and second the glycals, which have been activated by hile E+ (e.g., I+).Yields with both methods usually do not exceed 60%, and solid-state synthesis of oligosaccharides is therefore problematic (Wulff and Rohle, 1974; Paulsen, 1982; Schmidt, 1986; Kafine et al., 198 Toshima and Tatsuta, 1993; Seeberger and Danishefsky, 1998 computeri~e %hang, 1999.

P

HQ

P

x + Po

op'

PO g i y ~ s ydonor i D

g l y ~ s yacceptor i A

DA disa~haride

lycai a p p r o a ~ P P

donor glycal

+

D

H

P

P

P

acceptor glycal

CIA d i s a ~ a r i d e

A

-

P = protective group,see schemes 4.3.7 4.3.12 X = activating group, e.g. F, Cl,0 C ( ~ ~ ) C C i ~ E = electro~hil, g. e. 0,I'

QP

An important factor in glycoside synthesis with respect to the glycosyl acceptor is sterical hindrance.l’wo rules areof general significance: 1. Axial hydroxyl groups usually react more slowly than equatorial hydroxyl groups. 2. In the most important glucopyranosides, where hydroxyl groups in the ring and the 6CH,OH group are all equatorial, the order of reactivity is 6CH,OH >> 30H > 20H > 40H. The special situation at C6 can, however, be exploited in regioselective synthesis. Partial protection of monosaccharides, which leaves only one OH group free,is usually a necessity, and examples are described in Section 44.2. Apart from these rules, each glycosidation must be considered as an individual synthetic problem, and the literature should be consulted extensively. Success depends upon the ability to form glycosidic bonds stereoselectively and to apply efficient protecting-group strategies. The common Koenigs-Knorr synthesis of glycosides consists of the reaction of an appropriate glycosyl halide with the alcohol in the presence of an acid acceptor such as silver oxide or silver tsiflate. Glycosyl halides are prepared by reaction the corresponding hydrogen halide with aper-0-acyl- dose or a per-o-benzyl-aldose, in which only the l-0-substituent is replaced by halogen. Under acidic conditions the a anomer always p r e d o ~ a t because ~s of the powerful anomeric eBect. The relative reactivities increase in the order F<J. Chlorides and bromides have been used most extensively, but fluorides oBer the advantage of stabilit in silicagel clxomotography. A neighboring trans-acetoxyl group at C2 participates in the halidee l ~ n a t i o nand leads to a cyclic1,2-acyloxoni~cation. Acetoxl groups are therefore considered as p ~ i c i p a ~ groups, g whereas benzylether groups do not form cyclic intermediates and are called nonparticipa~ng.Participating groups on C2 invariably lead to high yields of 1,2-trans glycosides (Scheme 4.3.7).

A

A

Me

A

iom~ssand ~ter~ochemistry

1,2-cis-Glycosidescanbemade from a-D-glucopyranosidesand a - ~ galactopyranosides provideda nonp~icipatinggroup is present at C2 (i.e., benzyloxy or acetonide). The P-D-glycopyranosylhalideaffordsthe a-glycoside when reacted with an alcohol, silver perchlorate, and sym-collidine in ether. The a anomer of the glycopyranosyl bromideis first convertedby tetraethyl ammonium bromide to the p anomer in dimethyl formmide in presence of a bulky base, and thenthe alcohol and catalysts are added (not shown). Another comon route to 1,2-cis-glycosides applies solid catalysts, to which the a-halide is adsorbed first (Scheme 4.3.8). If the usual Koenigs-Knorr syntheses using chlorides and bromides are not stereoselective enough, it is advisable to use stereochemically pure fluorides or t~chloroaceti~idates as educts (Schmidt, 1986; Toshima and Tatsuta, 1993). The use of glycosyl fluorides requires a ~uorophilicSnC1,-AgClC), activator and leads preferably to retention of configuration. Trichloro-acetimidates are madeof the otherwise protected half-acetal of a carbohydrate and tric~loroacetonitrile. Glycosidation in the presence of silylated trifluoromethanesulfonate leads again preferably to glycosides with retention of configuration. Treatment of the a-glycosides with base may, however, convert them to the P diastereomer, provided there is a participating substituent on C2 (Scheme 4.3.9) Z-~eoxy--D-glycopyranosides are accessible from peracetylated glycals

OR

r

ROH, Ag2C03or Ag-imidazole

Cl AKSH C

B

B

OR

SPh

SPh

H SnQ,

-AgCI04

T

ed

HOH C

B OMe

OBn

“am

B

‘ I L ,

( m 4 : 1)

Base

b

“ a

(m 1 :1)

B

NH

OH BnO

A H A

AcOH C

AcOH C

A

Bu&nH

A

B

QPent

62%, alp = I l l

.

OPent

OPent

SEt

t SEt

(see Scheme 4.4.10) (Seeberger et al., 1997). Iodination leads to P-iodonium ions, which react withalcohols to a-glycosides almostexclusively(Scheme 4.3.10). Reduction with tin butyl hydride then gives the a-D-glycosides. 2-Deoxy--D-glycopyranosides are formed on the surface of silver silicate or silver zeolite. Another glycosylation method uses 4-pentenyl glycosides (see Scheme 4.4.3) with different protecting groups as donors and acceptors. It was that found 4-pentenyl glycosides with acyloxy groups at C2 were much less reactive than glycosyl donors having a benzylether group at the same position. The deactivated 2-acyloxy donor was called a “disarmed” sugar, the activated benzyletber an “armed” sugar. Carbohydrates with the same leaving group, in particular 0pentenyl,S-pentenyl, or S-phenyl,werecombinedin a r m e d - d i s ~ e dpairs. Upon oxidationof the armed pentenyl-or thio-glycoside with iodonium dicollidine perchlorate(IDCP) the pentenyl or thiol groups are activatedby forming acetal cations (see Scheme 4.4.3). They become glycosyl donors. They are mixed with the unreactive, disarmed partnercont~ninga free alcohol group, which is then the only nucleophilein solution. Yieldsof diglycosides typicallyrange between 60 and 80%, but stereoselectivity of glycosidation is often low (Scheme 4.3.11) (Toshima and Tatsuta,1993; computerized: Zhang, 1999).

onosaccharides have been extensively used aschiral synthons in synthesesof chiral polyhydroxy compounds and their derivatives. The reader is referred to co~espondingbooks and review articles, because the target molecules of these compounds are mostly out of the scope of this book (Hanessian 1979, 1983; Fuhrhop, 1994) Theacid-catalyzed ~ranose-pyranoseequilibria havealreadybeendepicted in Scheme 4.2.14. ~lycosylationwas sketched in the previous synthetic section. In the following, we discuss the hydrolysis of glycosides, protection and deprotection reactions of glucose and galactose hydroxyl groups,glycal formation, andthe introduction of amino and sulfide groups.

Acid-catalyzed hydrolysis of glycosides occurs between the glycosidic oxygen atom andC 1. lsO”abeled water produces an180-labeledhalf-acetal, and a resonanc~-stabilize~ carboxonium ion occurs as an intermediate. Since secondary c ~ b e n i u mions are more stable than primary ones, the expe~men~al finding that ketosides are much more sensitive to hydrolysis than aldosides corresponds to expectation (Scheme 4.4.1; McPhail et al., 1992).

assand ~ t e r e o c ~ e HOH2C

Aldosides

HOH2C

HOH C

,

H

H

+ HOCH,

Ketosides H

CHzOH

Fast_l -

~

,H

H20H HOCH3

0

H

+ HOCH,

With strong reservation onemay suggest the following order of OH reactivities in glycosides: 6-OH>>>open-chain OH (e.g., 5-OHin furanosides) 2OH > 3-OH = 4-OH and e~~atorial OH >axial OH. These rules are, however, by no means reliable. Dilute acids lead to the hydrolysis of the glycosidic bond; forcing conditions are required with alkali (e.g., 1’70°C for the cleavage of methyl furanosides). Aryl glycosides are more susceptible to alkali. Few reactions show an anomeric selectivity. Pyranoid glycosides with e~~atorial aglycone groups give, for example, aldonic esters with ozone in acetic anhydride, whereas the axial anomers areinert to ozone. The reaction of monosaccharides withethylmerca~tanein the presenceof HCl produces mainly open-chain dithioacetals, which can be decomposed quantitatively with HgC1, in humid acetone. This reaction provides a simple way to go from cyclic to open-chain monosaccharides and vice versa (Scheme 4.2.2; Biddey, 1988; Nambiar et al., 1989). The reaction of glycosides with open-chain amines leads to Schiff bases and N-glycosides at first, but a varietyre~angements of (Amadori, see Scheme 4.4.9 for an example) and condensation reactions may occur later and usually lead to mixtures of exotic molecules. These reactions shall not be described here. Pyridine- and imidazole-type heterocycles, however, f o m stable N-glycosides. They are discussed in Chapter 8. N-Pentenyl acetals are often used in glycoside synthesis since they can be readily hydrolyzed under neutral conditions by halonium ions, e.g., ,CN/H,O mixtures ( ootoo et al., 1988). This hydrolysis of an acetal without the use of an acid is driven by a cascade of ionic intemediates 4.4.3). starting withthe bromonium adductto the terminal double bond (Scheme

ups are usually masked as half-acetals in natural monosacch~des. chemical properties of individual monosaccharides are dominated blocking and deblocking procedures of the OH groups of most standard monosacch~deshave been worked out, and it is often possible to block all but one hydroxy group. This free group may then be oxidized to a keto group and submitted to a Wittig reaction, substituted by an amino group or changed in other ways (Fu~rhop,1994; nkley, 1988; Lehmann, 1976, 1996).

In the following, it is shown how each individual OH group of D-glucose and D-galactose can be exposed as a single or, in unfavorable cases, as a pair in this way. Most of this reactions are selective, but far from specific. Depending on the experience and patienc of the student of carbohydrate chemistry, they may work close to quantitative, satisfactory, or not at all. The classical blocking reaction for carbohydrate hydroxyl groups is acetal and ketal formation. Five rules are followed: (1) acetone and other ketones form 1,3-dioxolanrings, (2) benzaldehyde and other aldehydes give 1,3-dioxane rings (benzylidene derivatives), (3) ketones react only with neighboring cis-hydroxy groups, (4) bridged bicycles are not formed at all, and (5) the maximal number of OH groups are converted to acetals and lcetals (Scheme 4.4.4). To memorize

Amtylation

3

Oxidation

5

Su-choIestan-3~-0l via cholestano

epicholestanol 30%

OH

Redox: cis

HO

OH

ter

AcOH~C, CH CHOlHCl AcOH OR

A

I OR OH

A

HPd

H

A

R

these rules one just has to remember the reaction between glucose and acetone. It gives thefurane diacetonide shown inthe Scheme, not the pyranose derivatives. group of half-acetals is easy to exchangeby acid- catalyze^ substituti ous types of glycosidation procedures without blocking of the other S, provided the alcohol used is monovalent. group of glucose and galactose has been selectively activated in form of an iodide. 2-Iodides are obtained in good yields from co~esponding the

glycal (see next section)by oxidation with ~ - i o d o s u c c i n i ~ dCatalytic e. hydrogenation of the 2-iodides then gives the 2-deoxy monosaccharides, Grignard reactions yield carbon-carbon bonds and substitution with azides and subsequent hyd~ogenolysisleads to2-deoxy-2-aminosaccharides.Another,more direct method to produce 2-a~no-monosacch~des from acetylated glycals uses the electrophilic additionof nitrosylchloride. A dimeric nitroso-glycopyranosylchloride is f o ~ e dwhich loses HC1 anddecomposestomonomeric2-hydroxya1of the oxime with acetaldehyde gives the 2-keto compound group of glucose is particularly easy to isolate, Simple acidization gives the 1,2,5,6-diacetonide in form of the glucofuranose and the 3-0 group remains free (see Scheme 4.4.4). ~ e n ~ y l i d e n e derivatives contain g an acetal of the primary alcohol at C6 are more rapidly h?drolyzed in the presence of Lewis acids than acetals of secondary alcohols. In the case of glucose and galactose both ~ r a ~ ~ - c o n ~ 20Hgur~d free together in an otherwise protected group is in general more accessible than can be selectively glycosylated. The base-catalyzed migrationof acetyl groups from sterical more hindered to less hindered positions can be used to prepare a triacetylated glucose with a group (Scheme 4.4.7).The axial 40H group of galactose reacts only slowly with benzylchloride in presence of pyridine and can therefore easilybe kept free and reactive selectively (Scheme 4.4.7). The groups of hexosescanonlybeattackedselectivelyinfurano-

N

Et

H

OR

OR

OAc

H0

OR OAc

ti0

H0

OR

+ BzCl

BZO

OR

OH

sides (e.g., in the partially deblocked acetonide of glucofuranoside). The primary alcohol may then be blocked afterwards with tritylchloride, butthe 30H group also remains unprotected (Scheme 4.4.8). The open-chain OH group is kinetically favored overthe furanose substituents in subsequent reactions. Selective reactions at the primaryalcohol, the group,areagaineasy to perform. Several procedures are known. A tetrame~ylsilyle~er of glucose, for example, whichis accessible in almost quantitative yield from glucose or its glycosides and trimethylsilylchloride, hydrolyzes selectively at 06 with weak bases (Scheme 4.4.9). Treatment of alactose with acetone yields the 1,2,3,4acetonidewitha free roupdirectly(Scheme4.4.9). ective derivatization cyclodextrins occurs first at the priost easily accessible, and at 2 0 or bridging substituents then enforce uniform selective mono-,di-, or t~substitutedproducts (Figure 4.4.1).

T

T

HO CH

H0

OH

OH

CO

Regioselective derivatizationof cyclodextrin.

Peracetylated or benzylated glycopyranosyl halides react with zincin acetic acid in high yields toform 1,2-deoxy carbohydratesor glycals (Scheme 4.4.10).The enolic double bondof glycals is highly polarized and undergoes all kinds of proton-catalyzed addition reactionsin which the proton always ends up on C2. Oxidationwithnitrosylchloride co~es~ondingly brings the electropositive NO+ group to C2, the chloride to Cl. The nitrogen radical then dimerizes (see Scheme

islr

ass

A

MC1

Cl C

A

t al., 1997; Lehmann, 1976, 1996). Glycals are versatile syny- and 2-a~ino-glycos and Anderson, 1980). AiBN) produces preferentially the 2deoxy-(-~lycoside. The nonen~ymaticglycosylation of certain proteins may start a cascade of chemical reactions, which finally lead to irreversible cross-linking of neighbo~ng proteins.Advanced glycosidation endproducts (AGE) aremostly yellowishbrown and they fluoresce. One AGE structure, namely 2-furanyl-4(5)-2-furanyl-

H H

H " 0 H H

I

Schiff base

H

example of a c r o ~ - l i n k ~protein, d

occur which does not in p r ~ ~ of e :n ~ Arnadoriproduct

H2N-NH~NH NH2

1H-imida~ole,has been identified. Itmay come from a condensationof glucose re~angementto form relawith an amino groupof protein side chains, Arnadori tively longer-lived a-aminoketones from the water-sensitive Schiff base, crosslinking with a second protein, and addition of another glucose molecule. In model experiments, it was found that collagen forms spontaneously several cross-links if glucose is added to its solution. If, however, the strong base and nucleophile aminoguanidine was added, cross-linking was almost completely inhibited, presumably because it adds irreversibly to the Amadori product (Scheme 44.11) (Cerarni et al., 1987).

The most general approachesto carbo ate fixationsare by boronic acid derivatives,calixarenes,andoligoarnines.nicacidhasbeenattached to cholesterolderivativeswhichformhelicalliquidcrystals.Binding of various monosacch~idesto these liquid crystals induces changes of the helical pitches. The color of reflected light changes. A boric urethane cholesteric phase appears, for example, red upon addition of D-glucose, because the ~ve-mernberedboric ester ring points downwardinto the cholesteric helix, whereasthe corresponding L-glucose borate points up and induces a pitch that corresponds to a blue colorof the reflectedlight (Fig. 4.5.1 James 1993). Boric esters havealsobeenbound to porphyrinsbearingalcoholsub-

. . . . . . . . . . . . . . . . . . . . . . .

The cholesteric borate amide foms liquid crystals, which react stereoselectively with stereoisomers of monosaccharides, e.g., the enantiomers of glucose. Double ~borationmaybeinvolved in therecognitionprocess(comparewithFigs.3.2.1, 3.5.3, and 3.5.4) (from James, Harada, Shimkin, 1993).

stituents. These borates are water soluble at basic pH and precipitate at p Upon addition of ~onosacc~arides9 howev the resulting po~~yrin-sugar complexes dissolveat neutral or slightly acid Carbohydrateshavebeen solubilize c solvents(e.g.9 CCl,) with a macrocyclic oligoamine ( s t o i c h i o ~ e t ~:1) as well as with a noncyclic oligoamine bearing hydroxyalkyl chains. th hosts provide adjustable core and are therefore not selective.No detectable water was transportedinto the organic phase (Figure45.12). One of the long-standing and presumably unsolvable problems of sugar c h e ~ ~ tisr ythe structural originof sweetness. Early infrared work on sweet and

I

R

0 hopen-chain and a cyclic amino ligand for monosacch~desin water.

nonsweet sugars suggested(1) that vicinal hydroxyl groups in the staggered glycol orientatio~formed the saporous (sweet) unit and (2) that sweetnessis hampered by intramolecular hydrogen bonds. Later, when large a variety of nonsugar sweet compounds had been evaluated, it became clear that a hydrogen bond donor and an acceptor in a distance of about 4 A was common toall of them.The observation that some D-an6no acids are sweet whereas the L-enantiomers are not indicates a third binding site on the protein receptor, which probably involves a “dispersion” interaction since aromatic amino acids are much sweeter than aliphatic ones. The “tripartite” model for sugars and dipeptide sweetness and some sweet compounds is reproduced in Figure 4.5.3. Probably there is more than one single receptor protein for sweetness recognition, andthe overall picture is complicated. Cyclodextrins havea h y d r o ~ ~ louter i c surface anda hydrophobic interior. If the guest moleculeis entrapped inside the cyclodextrin torus, one speaks of an inclusion compound.The guest may be covered partlyor totally by the carbohy-

AH

AH

B

I

I

OH L-Alanine

Cl Cl AH

Saccharin

Chloroform AH

B

Cyclarnic acid

AH

-300 pm

B

The tripartite model of protein receptors for various compounds with a sweet taste.

drate mantle.A long thin guest formsaxial inclusion compounds,a round guest enforces sand~ich-like st~ctures, a flat, long-edge amphiphile may act as a cover (Fig.4.5.4) (Wenz, 1994).Per-6-a~no-cyclodextrinand per-6-~ioglycolic P-cyclodextrin strongly interact at pH 7 and form a capsule-like dimer with an average molecular distance of 0.6 nm (not shown) (Hamelin et al., 1998). The primary 6-amino groupof an aminoglycoside antibiotic (paromomycin) waslabeled with ~uorescingdyes containing carboxyl groups and then used in RNAbinding studies(not shown) (Tok and Rando, 1998). Cyclodextrins havealso been assembled on polymer chainslike pearls on a string. Polyethyleneoxide is entrapped by a-cyclodextrin, but not by p-or ycyclodextrin. Water-soluble (stringsof pearls( have been made of polymers with a l t ~ m a t i nhydrophobic ~ oligornethylene and ~ y ~ o p h i l i c a l k y l - a ~sego~um ments or on polyethyleneglycols (PEGS) with amino endings. The assembly of such chains takes days, and up to70% of the polymeric ol~~omethylene chains are then covered bya-cyclodextrin molecules madeof six glucose(or three maltose) units. The driving force for the string formation is an enthalpy gain of

Topology of cyclodextrin adducts.(a) A fitting cylinder is en~appedin an axial position, (b) a sphere rnay be s ~ d w i c ~ ebetween d the wide ends, and (c) a large

disk-like molecule rnay act as cover.

F Q2N

Q2

Q2

Formation and fixation of a string of cyclodextrin “’pearls” on a PEG fathom.

about 20 kJ/mol. The process can be made irreversible by reacting the free ends of the fathom with bulky stopper molecules (Figure 4.5.5) (Wenz 1994). Cyclodextrin inclusion compoundshave found application in p h ~ a c o l ogy, the food industry, and cosmetics. Typical examples are the solubilization of insulin and steroids,the removal of cholesterol from egg yolk, and the conservation of basis notes in perfumes by slow release rates of rose oil and similar ingredients, Chemical stabilization of oxygen-sensitive compounds, e.g.,the sulfides of onions andgarlic flavor, andof vitamins is also achieved by cyclodextrins.

Above a given melting temperature, usually around 50°C glycosides of carbohydrates with long alkyl chain alcohols form “thermotropic liquid crystals”: upon heating, first the hy~ocarbonchains disengage from the crystal lattice (“‘melting”), whereas the carbohydrates separate at a higher temperature (“clearing”). etween melting and clearing point there is a liquid crystallinestate with disordered hydrocarbon chains and hydrogen-bonded carbohydrates. At room temperature, however, carbohydrate amphiphiles often produce well-defined crystals with small thermal ellipsoids in the x-ray structures. In octyl-l-O-~-gluco~yranoside, the carbohydrate moiety forms extensive hydrogen bond nets and the alkyl chains interdigitate(see Fig. 4.2.10) Long-lived supramolecular assemblies

of cyclic carbohydrate acetals or esters with fatty alcoholsor acids are virtually unknown in water, Only a few organogels (e.g., of cellobiose octa(decanoate) in hexadecane)havebeencharacterized.Flexibleopen-chaincarbohydratesare much more suitable educts for the synkinesis of molecular assemblies in water. 1,3,2,4-~i-O-benzylidene-~-sorbitol (DBS)is a polyol derivativethat forms gels in liquids ranging from hexadecane to ethylene glycol-water mixtures. Electron microscopy shows filamentsof about 10 nm width and practically infinite length entangled in net structures (Fig. 4.5.6). The co~espondingmono-benzylidene derivative also forms fibers but crystallizes rapidly, whereas the apolar tribenzylidene derivative never formsgels but crystallizes immediately.The complex b~tterflyshape obviously hinders crystallization, its chirality promotescurvature (racemates do not give fibers!), andits polar unit leads to a linear backboneby formation of hydrogen bonds (organogels; Terech, 1997). Benzylidene-sorbitol has also been introduced in polymer matrices and its gel structure characterized in detail (Thierryet al., 1990; Yamasaki and Tsutsumi, 1994). Open-chain N-octyl gluconamide crystal structures show an interesting hydrogen bond pattern including a homodromic cycle aswellasstrongamide chains (see Secs, 4.2.2.1 and 4.2.2.2). These binding interactions cause insolubility in water. Cyclic glucose amphiphiles, on the other hand, give interdigitated bilayers in crystals (Fig. 4.2.10), whichare soluble as micelles in waterat room temperature. Glucopyranosides are standard detergents for the solubilizat~onand crystallization of membrane proteins and protein assemblies at room temperature, such as photosystems I and 11, whereas secondary amide hydrogen bond chains render gluconamides practically waterinsoluble (ca. l00 mgL) at room temperature. Only upon heating above70°C do the amide hydrogen bonds melt and the N-octyl- and ~-dodecyl-gluconamidesbecome very water soluble (ca. S00 gL). Upon cooling, the amphiphiles do not precipitate ast~ee-dimensionalcrystals, which they had been before dissolution, but form gels. These gels again melt around70°C (Fig. 4.5.7). Trans~ssionelectron microscopy of such a gel shows micellar fibers, which are several micrometers long and about 8 nm wide. Image analysis reveals regular quadruple helices of molecular bilayer rods or ribbons (Fig. 4.5.8). In cryo-electron microscopy the single helices appear somewhat broader (Fuhrhop et al., 1988; Koning et al., 1993). These fibers are held together by binding interactions between the headgroups and can, in contrast to spherical micelles and vesicles, be isolatedfrom the aqueous phase by lyophilization. The resulting solids still have the intact t e t r ~ e ~ c structure, al as shown by redispersion in cold water after months and electron microscopy. The fibers have been analyzed by 13C-Cl?MAS solid-state spectroscopy (see Fig. 4.2.5), and the conformation of the glucon headgroup was foundto be similar to the one found in racemic bilayer crystallites. A

Electron ~ c r o g r a p hof diben~yliden~-D-sorbitol fibers in glycol-water mixtures. This gelis commercially used in antiperspirant gels.

50

60

70 80 Temp. [“C]

90

Differential scanning calorimetry (DSC) of an aqueous suspension of Noctyl-D-glucon~degels.

(a) Transmission electron micrograph and (b)computer image of an NO ~ t y l - D - ~ l u c o n quadruple ~de helical fiber.

ss a

istry

gauche bent next to the amide group occurs, which presumably allows hydration in the aqueous phase. The dried fiber, however, does not contain any water. ~ - 0 c t y l - L - g l u c o n ~ fibers d e gave a right-handed helix in contrast to the left-handed helix observed for the D-enantiomer. In racemic mixtures of both compounds, no gel or fiber formation whatsoever was observable. This fact together with the observation that the crystals of the pure enantiomer showed a head-to-tail a~angementof the crystal sheets (see Fig. 4.2.4) lead to the formulation of a “chiral bilayer effect.” Many ~ p h i p h i l e swith chiral centers form longlived helical bilayer fibers with enormously large surface areas (each single molecule is in direct contact with water!) because the re ~ a n g e me n to t monolayer crystals is unfavorable in water. Head-to-tail crys are formed because only here are all chiral centers ordered in the same way within a single layer, thus allowing tight crystal packing. h racemates, on the other hand, mirror-image packring leads to a perfect ordering chiral centers in planar sheets, and there is no reason to form assemblies with high surface energies (Figure 4.5.9). Chirality of the headgroups thus induces curvature in fibers and prevents crystallization. Since all living organisms consists mainly of aqueous gels to which

I I

r I

I I I I I I I I 1 I

one en~ntiomer

P

r

I

I

fast

The chiral bilayer effect. Chirality in headgroups of ~ p h i p ~ l enes hances the lifetime of fibers if there are strong intermolecul~binding inetractions between the headgroups in the crystal and the fiber.

D- and L-Cluconamides with different alkyl chains first separate into right- and left-handed helices (above) and later unite tofom pseudo-race~iccrystallites (below).

Electron micrographof (a)~-0ctyl-D-mannon~de scrolls, (b) N-octylD-gdactonamide twisted ribbons, and (c) g ~ a c t o n - g l u c o n ~ d1:e1 mixture tubules.

Schematic representation of Gram staining. Crystal violet monomers diffuse only into porous gram-positive membranes and are trapped here after the formation of polymeric charge transfer complexes.

crystallization processes would be deadly, the chirality and the chiral bilayer effect can be considered as an important condition of life. Crystallization of ultrathin membrane structures in nature is also hindered by the presence of molecules with different chain lengths within these molecular assemblies. In the case of gluconamides, pseudo-racemates with alkyl chains of different lengths ( e g , C , , and C,), first separate to produce left- and righthanded helices, which only later pair and rearrange to form racemic platelets within about 30 minutes (Fig. 4.5.10). Diasteromeric glyconamides form different bilayer structures in water. NOctyl-D-galactonamide and N-octyl-D-mannonamideshave all 1,3-OH groups in ga~c~e-positions in the stretched all-anti conformer and form planar bilayers in water, not curved rods. In the case of mannonamide, they roll up to form scrolls; in the almost insoluble galactonarnide one finds twisted ribbons. A 1:1 mixture of glucon and galactonamide, however, produces long hollow tubules (Fig. 4.5.11); neither the helices nor twisted ribbons of the pure compounds are observed. A co-fiber has been formed in which the glucon headgroup is probably forced into the same alZ-a~ticonformation as the galacton diastereomer (Fuhrhop, 1990, 1991, 1993). Cell walls of bacteria are divided into two classes according to the results of Gram staining. Gram-positive bacteria possess cell walls containing rigid peptidoglycans, negatively charged phosphodiester teichoic acids, and other polysaccharides, which are mostly interconnected by cross-links. As a result, the cell wall is porous and crystal violet is adsorbed to heat-denatured cells. Upon addition of iodine, a polymeric charge transfer complex is formed and fixated within the peptidoglycan pores (Fig. 4.3.12). Washing with ethanol or acetone does not remove it. Gram negative bacteria, on the other hand, are covered by a thin cell envelope, which acts as a permeability barrier to crystal violet as well as many antibiotics. The polymerized dye is not entrapped and can therefore be washed away with ethanol.

igid calixarene Langmuir- lodgett monolayers with long alkyl chains fixated at the indium-tin oxide ( I ~ O electrodelwater ) interface bind various monosaccharides from bulk water with high and differentiated binding constants. Changes of electrode potentials are detected at carbohydrate concentrations in water as low as molar (Fig. 4.6.1). In bulk organic solvents the same binding process is dominated by hydrogen bonding (Kobayashi et al., 1992); on electrode surfaces in aqueous environments the special polarity of the electrodehater interface is presumably responsible for the favorable adsorption of the carbohydrates ( K u n i t ~ e1997). ,

~ p ~ p h i lcalixarenes ic form monolayers on tin oxide electrodes, which then differentiate between water-dissolved monosaccharides (fromleft to right: ribose, galactose, glucose).

The hydrophobic calixarene was substituted with e t h y l a ~ n on e the phenol units and condensed with lactose lactone to give an polyp-like structure. This compound is tightly adsorbed by quartz surfaces. Water cannot remove it, but aqueous aminesolutions lead to desorption. The expe~mentaloccupation areais 3.5 nm2/molecule, and saturation binding is achieved within 10 S. The cluster c o ~ p o u n dobviously forms a closely packed monolayer onquartz. The quartzads or be^ polypbinds 8-a~linona~hthalene1-sulfonate )from waterwith a binding constantof 2 x lo5l", which was shown by nm (Figure 4.6.2) (Fujimoto et al., 1997, 1998). It has also been shown that ble monosacch~desare tightly bound to hydrophobic gaps of steroid monolayers (see Figure3.6.2). (1-+3)--~-Glucans, which are substitut~on every third unit byfk"1-+6)D-glucose (sclerogluc~),produce triple helices with a pitch of1.8nm and six

n

The calixarene polyp witheight lactose arms binds tightly to quartz surfaces and binds the water-solublea~nonaphthalene-sulfonatefluorescence dye.

backbone glucose residues per helical turn in each strand. The triple helix is very stiff, and the glucons are useful as aqueous viscosity control agents. The triple helix decomposes inDMSO to form a single-strand coil. ~enaturationin wateris incomplete and leads to circular polysaccharide structures with diarneters of many nano~eters,which arere~niscentof DNA toroids (see Fig.8.6.21). The proposed m e c h ~ ~ sofmformation, namely formation of linear clustersand subsequent SUIface d i ~ n u ~ by o nrolling up, is also similar, the glucose substituent playing the role of the main-chain connecting link( cIntire and Brant, 1998) (Figure 4.6.3).

H0 CH20

H0

Repeating unit of scleroglucan and a model of a triple helix in water and of a toroid formed after denaturation in DMSO and rendt~ationin water.

Molecular grid made of cellulose hairy rods

Cellulose fibers that are partially hydrophobized by alkyl chainsas well as cellulose itself align on water surfacesto form two-dimensi~nalcrystals, which are quite regular, inthe direction of the polymer chain. In case of alkylated cellulose (“hairy rods”) multilayer grids of molecular size can be synkinesized if the direction of the LB plate is changed by 90” in each application (Fig.4.6.4). Similar hydrophobized polysacch~deshave also been usedas hydrogel n a n o ~ ~ i cles to bind albumin protein in water (~ish~aw et aal., 1996) For Referencesfor Chapter 4,see pages 537-542.

This Page Intentionally Left Blank

Carotenes are simple polyenes with conjugated double bonds as their only functional groups. Their chemistry is quickly summedup: they absorb light,cis-trans isomerize, polymerize, and take up oxidants in allylic positions. Nevertheless, there are more open questions in carotene chemistry than anywhere else in bioorganic chemistry. Some examples of such questions are as follows: 1. Carotenesarewaterinsoluble.Neverthelessp-carotene, C,, curs in large amounts in carrots, which consist almost exclusivelyof water (80%) and cellulose(20%). p-Carotene is, therefore, not partof a functional membrane or enzyme system in these roots and occurs in the form of dead microcrystals. Also, carrots grow the in earth in total darkness. Why should there be a crystalline dye and how can it be formed? Can crystals play a biological rolethat would justify the pain it takes to synthesize them? 2. Bixin, a bolaamphiphilewithonemethylesterandone carbo~ylate end group, appears in the waxy cover of seed granules of the ~ o u t h American and African treeB i m orellanu. This lively colored coating of the seed proteins may protect them against attack by ~ ~ r o o r g a n isms. Why is an unsymmetricalbolaamphi~hileneeded, if the protective role can presumably also be played by p-carotene? Why is a cis-double bond introducedat one end? 3. p-Carotene is digested by humans and animals and degraded oxidatively to colorless retinal (h,,, 320 nm). This white compound is then coupled with a white protein to allow color vision in the range of 400-700 nm. How did nature know that a colorless dye would eventually become useful in connection with a protein and sunlight?

4. Why is the color of goldfish, of grilled “golden nuggets” of chicken, of orange and tomatojuice received so positively when the colors of life are red for respiration and green for photosynthesis? Chicken or egg yolks that are not yellow-orange cannot be sold; noodles and margarines mustbe carotinized in order to stimulate appetite.If eggyolks or noodles get aslight touch of red (too muchcarotene), they immediately become dead stock. Orange-yellow is commercially the most desirable. Is it thesunlikeappearance that makesorangesuchan optimistic color? The answer to this psychological question cannot be determined scientifically. Nevertheless, weshall look into the chemistry of the carotenoid dyes with some special anticipation.

If carotenes are cut in half by oxygen, colorless retinal is formed and the story of carotenes becomes even more interesting. Imagine a rabbit digs a carrot out of the dark9wet soil and eats it. In the rabbit’s mitochondria the p-carotene is bleached by molecular oxygen. Colorless retinal is formed and bound to a colorless protein called rhodopsin. It thereby becomes red, green, or yellow depending on the p ~ i c u l a localization r of the protein in the rabbit’s retina.. The rabbit can now “see” the light coming from the sun, which means that the sunlight’s energy is somehow converted to change the calcium ion currents running through the membranes of the brain of the rabbit. (Fig. 5.1.1). The magic of the molecules of nature is nowhere more obvious than in the case of simple retinal.

Carotenoids are conjugated polyenes (carotenes) or oxygenated derivatives (xanthophylls, carotene acids) containing 40 carbon atoms or eight isoprene units (Table 5.2.1). The polyene skeletons are often s y ~ e ~ i cabout a l the central 15,15’-bond (tail-to-tail ~angement).Prefixes in carotenoid names have folthe lowing signification: characterize the position of the double bonds in terminal cyclohexene units. 2. y is for carotenes without twotenninal cyclohexene units. 3. 6 6 A p ~designates 99 the carbonatomatwhichacarotenechainwith fewer than40 carbon atoms ends. The S spectrum of carotenesusuallyshows three bandsbetween 450 and 500 n an a~ditionalcis band a r o u n ~330 nm, when there is acis-conureddoublebondpresent(Isler,1971 ).The l spect~m just shows illd e ~ n ~ultiplets e~ for methine, methylene,and methyl protons (Fig. 5.2.1)

_ . ( . I _

r

V

A short story about carotenoids, which are only supporting actors in the to grow from the sun (b), but chemical play called life(a).The carrot receives the energy its major massis produced in the dark soil. Nevertheless, the carrot is excessively colored by p-carotene. Since the carrot consists predo~nantlyof water and cellulose, the polyene hydrocarbon crystallizes out (c). As such and in suchan environment the chemically very vulnerable p-carotene molecules become stable. The rabbit digests the p-carotene to give colorless 11-cis-retinal. Thisis integrated intoa membrane protein called rhodopsin. The color comes back again and appears now in different shades covering the whole visible range. Upon sunlight i ~ a d i a ~ i oitnisomerizes and thereby closes ion-con~uctingmembrane gaps. The brain nerves of the rabbit enable the rabbit to see the i n c o ~ n glight (d).

Structural Formulasof Some Carotenoids Name 1

@-Carotene

2

a-Carotene

4

Retinole

9

12

13

f3,~-Carotene-3,3’-&01 Lutein

3,3’”~~y~oxy-f3,f3’ carotene-

4,4’-&one A s t ~ ~ ~ i n

9’-cis-6,6’-Diapo-4,4’carotenedioic acid-6’-monome~yl ester

Bixin

l

1

OH

W

1

1

h

I

H7,7', 8, 8'

I

(a) UV/vis spectra of some p-carotene ~iastereomersand (b)'H-NMR spectrum of uZZ-tru~sp-carotene.

The crystal structures of thecarotenes are dominated by the packing a ~ a n ~ e m eof n tthe methyl groups. Optimal density is reached whenthe polyene chains are oriented parallel by turning the methyl groups of neighboring molecules away from each other. T e ~ i n acyclohexene l units render this type of packing d i ~ c u l t The . molecules are therefore highly-soluble in apolar solvents or

membranes, In apo-carotenes, on the other hand, the linear polyene chains can be packedperfectlyandcrystallizationbecomesmore favorable (Cilardi et a1.,1971,1972). cis-Retinal is the only polyene c~omophoreused in vision and in lightinduced proton transportof bacteria. The isolated dye has sixconjugated double bonds and absorbsat 360 mm. The cis-band at 250 nmis very weak (Fig. 5.2.3). Concentrated organic solutions of retinal are only slightly yellow, which is not very promising for “color vision’’ in the wavelength region between 400 and 700 nmusingrodsand cones ~ounteringonly small amounts of the chromophore. Innature,however, retinal is boundasaSchiffbase to proteins (rhodopsins). all-trans-Retinal butyl imine, as a simple model, absorbs at 364 nm, and protonation with HCl in dry solvents gives a ~ a x i r n uat ~462 nm, to the conjugation system already carries Adding a positively charged end group the pigment’sabsorption into the visibleregion.It is nowasorangeas pcarotene with only half the conjugation path length.If one then takes into consideration that 11-cis-retinalis twisted because of the strong sterical interaction between the C 13 methyl group and the C10 methine proton (Fig. 5.2.4), then one can approach a negative charge, e.g., a carboxylate group from the rhodopsin side chains, in a very close distance (3 A) to the twisted polyene system. This ad-

Molecular structure of canthaxantin and partial crystal s t ~ c t u ~ofe pcarotene.

UVhis spectra of some diastereornericretinals.

Hypothetical conformations and charge interactions with rhodopsin side chains of (a) retinal and(b)11,~2-dihy~oretin~.

ditional charge then shiftsthe 462 peak to 500 nm (Peters et al., 1977; Honig et al., Sheveset a1.;1979, Rando,1990; Singhet a1.,1990). A shift of this magnitude from 270 to 3 15 nm was also found for 11,12-dihydroretinal b u t y l i ~ n ehydrochloride when it was transferred from a methanol solution to rhodopsin.Of all dihydro derivatives, only this regioisomer showed such a large shift upon binding to the protein, since only this isomer could form a conformer of a shape that resembled the one of 1l-cis retinal and only this isomer’s chromophore was attached close to the protein’s negative charge. The energy of the ground state with a positively charged end group and a negative charge close to the polyene’s most disturbed site, the cis double bond, thus approaches the excited state. Excitation becomes as favored as in pigments with an electron-donating and an electron-accepting substituent at both endsof the chromophore. Isomerization of 1l-cis-retinal to theaZZ~transdiastereomer is sterically favored because of the sterical repulsion between the 13-methyl and 10-H substituents in a planar polyene. In solutionsof retinal or retinyl ester in which all stereoisomers are e~uilibratedby the reversible addition of iodine or trifluo% of the 1l-cis diastereomer. The energy differroacetic acid one finds only 0.1 ence between aZ2-trans- and 11-cis- retinalis 4.1 kcalhol. The crystal structure of 1I-cis-retinal shows a highly twisted polyene at C 12-C 13. The methyl group

Molecular structuresof (a)cis- and (b) trans-retinal.

r

at 613 is turned away from the polyene substituents (Fig. 5.2.5) (Gilardi et al., 1971). A retinoyl glucuromicle also occurs in nature (Zile, 1980). Upon cis-trans isomerization, the polyene moves its end by about 0.7 m and carries the rhodopsin protein with it. Although the aZZ-trans c~omophoreof rhodopsin (496 m)contains less sterical energy than the cis isomer, the product of the isomerization (540 nm) is richer in energy by 36 kcal mo1-1(67% of the 57 kcal of a mole of 500 nm light quanta). This is presumably caused by a charge separation between the Schiff base cation and the protein carboxylate anion (Fig. 5.2.6). The light energy is thus not lost but is used to produce an energy-rich protein containing an energy-poor chromophore. In order to explain the extremely high speed of this light-induced isomerization, it was hypothesized that the cis-trans isomerization of the 11-cisdouble bond may be accompaniedby a concerted twist of the neighbo~ng single bonds. cis-trans Isomerization can occur within a few picoseconds, and the quantum yield is close to 1. Single photons can thus be detected by the naked human eye. A few minutes after cis-trans isomerization the 11-&-retinal is recovered by an isomerase and recombined with a rhodopsin protein. The isomerization all-trans =-+11-cis is a dark reaction and occurs with activated retinol esters (Fig. 5.2.7). A nucleophilic group of an enzyme is reversibly added in a reaction to GI 1 and the 5 kcal needed for the unfavorable isomerization comes from the cleavage of the activated ester. The resulting diene with the nucleophile added to the terminal allylic position is then hydrated and the nucleophile eliminated again. The trans-cis isomerization has thus been achieved in a controllable nucleophilic addition-elimination cycle, which is typical for biological reactions. The stereochemistry of the system is not disturbed. The other chemically plausible isomeri~ation,namely via homolysis of the double bond and biradical formation, is not used in biological systems because it may lead to uncont~olledpolymerization and side reactions with the protein (Rando, 1990). Although carotene radical cations are too short-lived to determine their reversible reduction potentials directly, the (relative ease( of electron transfer for seven carotenoids has been determined by pulse radiolysis. In the series astaxanthin > p-a~~-8-8’-carotenal > canthaxantin > lutein > zeaxanthin > pcarotene > lycopene, lycopene is the strongest reductant. It can, for example, reduce lutein cation radicals to lutein, whereas p-carotene cannot (Edge et al., 1998). The electronic spectra of carotene radicals in triton X micelles produce a strong band at 850 nm ( ~ a t s u o1989; , Hill, 1995) (Fig. 5.2.8). Po~hyrin-carotene dyads, in which the carotene is supposed to donate electrons in charge sep~ation processes, give similar absorption bands and are discussed in Chapter 6 (Debreczeny, 1997). ~arotenoidpigments, in particular p-carotene, photoprotect systems in

0c3

0

N-Protein

Pc3

Hypotheticalconcerted cis-trans isomerizationsand confomational changes of retinal in rhodopsin. The final trans-retinal would also have high confomational strain, because the methyl group on C9 interacts with the proton onC12.

which visible light is absorbed by a dye, which sensitizes the conversion of triplet to singlet oxygen. p-Carotene may interact directly with singlet oxygen to yield an excited state, which then is deactivated to the carotenoid ground state and energy. At first sight one would expect the formation of a carotene triplet state, which would keep the multiplicity in the reaction constant: IO2 + karotene -+3carotene + 302. Another possibility is the deactivation of triplet states of the sensitizing dye molecule by other dye molecues in the ground state. This would againlead to carotene triplets. Carotenoids, in particular p-carotene, are well known as free radical and triplet quenchers. S h o ~ - l i v e(k~ = 2 X 105 S-I)radical cations have been characterized by near-infrared spectroscopy. They absorb around 900 nm and can be selectively generated in nanoseconds by peroxy1 radicals (Conn et a1.,1993). The mechanism of energy dissipation after the reaction with singlet oxygen, however, remains unknown, because it has not been possible so far to detect the carotenoid radicals in reactions with triplet oxygen. Computational studies indicated that the energy-transfer process, corresponding to catalytic physical quenching of singlet oxygen, is the most favored route. Several low-energy barrier reactions may, however,also lead tothe supposed diradical systems, which may attack then not only the singlet oxygen formed andother carotenes, but also deactivated triplet oxygen (Garavelli et al., 1998). Incrystals or membrane domains polymerization may occur via carotene triplets (~omatsuet a1.,1997) (see Figure 5.5.3). p-Carotene minimizes the appe~anceof ~ i f i c i induced ~y skin cancer in mice andis a chemo-~rotectingor c h e m o - p o s ~ o agent ~ g of promise in the areas of aging, ~ u n deficiency, e senile cataracts, and several types of cancer. is It the triad of natural a n t i o ~ d ~ tnamely s, p-carotene, vitamin E, and vitamin C, that seems to fight mosteficiently singlet oxygen and radicals, both of which may damage DNA and proteins (Roels et al.,1969; ~ a ~ e w s - R o t1991). h , There are several other clinical applications of carotenoids (e.g., prevention of lung cancer), all of which presumably rely on the scavenging of radicals in the apolar env~onmentof cell membranes. Only carotenes are active here, not retinol. p-Carotene, added in the form of beadlets to the diet, i ~ b i t e the d developmentof breast cancer in rats by ~ - m e t h y l - ~ - ~ ~ o sp-Carotene o ~ e a . and retinol are finally important precursors of the eye pigment retinal. Since polyenes cannot be produced by humans, im-they are portant vitamins (Lucy,l969; Roels al.,l969; et B a a and Olson,l987). An interesting way to produce a p-carotene diradical would involve a 90" twist of one of the double bonds, e.g., the central 15,15'-bond. The energy to generate such a 90" diradical should be connected directly to the intrinsic enthalpy of the radicals formed on both sides of the original double bond, e.g., the energy of two nonatetraenyl radicals. The calculated enthalpy for the direct activation of the central bond was 25 kcal/mol and therefore much too high produce a thermal equilibrium mixture containing the 90" diradical. Rates of thermal cis-trans rea~angementsamong all-trans-, 15-cis-, and13-cis--

carotene have been measured at temperatures from 39 to 69°C. Only the 15cis- and 13-cis-”carotenes were kinetically competitive with the all-trans compound at these temperature (Fig. 5.2.9).Human tissues contain, correspondingly, 90% all-trans-”carotene and 10% of a 1:6 mixture of the 15-cis and 13-cis diastereomers (Doering et al.,1995).

all-trans-6-Caroten

15% 13-cis-P-Carotene

l

H

H

15-cis-P-Carotene 8.5%

Cis-transisome~~ation of polyenes may occur not only light, by but also by thermal twisting and resultingdiradical formation. The processis, however, very slow at room temperature.

Caroteneshave little or nofluorescencethemselves,butp-carotene quenchesthefluorescence of bacte~ochlorophyll, magnesiumoctaethylporphyrin, and similar dyes efficiently. The eEective energy transferfrom carotenes to chlorophylls observed in plants depends on the contact between both molecules and orbital overlap (Dexter ~ e c ~ a n ~ Ass a~ consequence, ). all Ends of photooxygenations [e.g., the formation of fo~ylbiliverdinatefrom magnesium porphyrins (see Fig. 6.5.1)] are prevented by catalytic quantities of p-carotene (Fuhrhop9 1971).The most significant effectof p-carotene in plants is not energy transport,but the protection of chlorophyllagainstphotooxygenation. pCarotenes quench singlet oxygen and then become oxidized themselves. If one therefore extracts plants in the evening one finds large amounts of epoxides, which carry the oxygen at the terminal cyclohexene units. During the night, these carotenes are re-reduced to p-carotene. Protection against photooxidation also works effectivelyfor porphyria patients.For every thousand molecules of pcarotene involved in singlet oxygen quenching only one is oxidized, e.g.,to canthaxanthin or epoxides. Retinoic acid, on the other hand, is not reduced in the body to retinal or retinol. It remains stable, for example, on the skin and is used for treatment of atthews-Roth, 1991). n inherently important property of conjugated 7c systems is electric conductivity. Metals have typical conductivities of 102-106Siemens cm-l (S rocal Ohms), semiconductors 10-6-10 S cm-l, and insulators cm-*. Organic dyes and polymers lie typically in the order of 10-14-10-10S cm-l and are therefore insulators. AEE-trans configured polyacetylene, (CH)x however, is a semiconductor comparable to silicon (lo4 Siemens), and intelligently connected carotenes may also reach such values. This has, however, not yet been demonstrated (Vogtle, 1989; Wegmann et al., 1989) (see Sec. 5.5).

~ - ~ a r o t e n are e s producedby extraction of dried plant mate~alwith petrolether and/or acetoneor simple ~hro~atographic procedures. Charcoal adsorbsall oxidized carotenes, which may then be extracted with hexane-isopropanol rnixtures. The chlorophylls are finally desorbed with hexaneltoluene rnixtures. Total synthesis is applied for carotenoids on a lo4 t scale per year. One usually starts with the cheapest carbonyl components (formaldehyde, acetone) and carbanions (acetylide, acetoacetate, cyanide, Wittig ylides) available. A typical industrial synthesis of retinol acetate (vitamin A) is outlined in Scheme 5.3 1. L

l) NBS H) CzH50H

(l) NBS (11) C2HeOH

0

0

lor

p-Carotene should be used in an oxidized form in food in order to be readily digestible. “Golden chicken” with nonoxidized p-carotene may lead to a “golden liver” for the consumer, which means hard, crystalline tissues vulnerable to polymerization and sudden death. Canthaxanthines, on the other hand, which have cyclohexenone end rings, are readily metabolized and are 1.4 times as active as vitamin A precursors when compared to p-carotene. The carotenoid ketone group is obviously readily removed in the reductive medium of living orga~sms. Oxidation of p-carotene to canthaxant~neis carried out in two steps with N-bromosuccini~de(Scheme 5.4.1). Oxidation with peracids gives epoxides, which can be re-reduced with lithium aluminum hydride (Scheme 5.4.2). Another typical carotene reaction is rapid oxidative or reductive bleaching, which may also occur in the solid state. Cross-linked polymers of unknown structure are formed (see Fig. 5.5.3). With age, fluorescent pigments accumulate in the retinal pigment epithelium. The major chromophore of this particular pigment contains a pyridinium ring with two polyene side chains. It can be synthesized from two retinal molecules and ethanolamine via the enamine of retinal and condensation with a second retinal molecule (Scheme 5.4.3) (Eldred and Lasky,1993; Sakai et a1.,1996). Besides the allylic oxidation, no high-yield reaction of the carotene polyene systems is known. The large variety of totally regioselective and often reversible biochemical reactions of the carotenoids and retinal have, so far, not been matched by chemists.

Zeaxanthin (C,J has been incorporated in DMPC and egg lecithin vesicles. This a,m-bipolar carotenoid reinforces the DMPC vesicle with respect to mechanical stability and water permeability but has no effect on fluid egg lecithin membranes (Lazrak et al., 1987). Electron-poor derivatives with electron-withdrawing carboxyl or pyridinium end groups should reversibly take up electrons in a type of reversible Michael reaction and then act as organic wires. There are, however, no reports on stable anion radicals of such chromophores in the literature. Claims of electron transport through vesicle membranes are very probably erroneous. It has been shown by reduction of an entrapped indigo dye that bixin derivatives in DPPC vesicle membranes favor the transport of borohydride and dithionite ions through the membrane rather

R

R

OH

than taking up and releasing electrons (Fig. 5.5.1). Carotenoids in membranes are prone to form crystalline domains that often polymerize in light. In any case these domains destroy the fluid membrane character in their environment leading to leakiness, whereas cholesterol usually diminishes membrane permeability. Retinene (3?4-didehydroreti~ol)forms a molecular complex with tryptophan in HCl-saturated methanol.The absorption band at 390 nm of retinene is then replaced by a 540 nm band. The carboxyl group of tryptophan (pk, 2.5) must be protonated and the formationof the complex takes about an hour. Canth~anthin-proteincomplexes found in the shell of lobsters and eggshell pi ments are often blue, green, or red (Figure 5.5.2) (Cheesman et a1.,1967). The terminal keto group of this carotene is equally well suited for polarization by protein charges as retinal (see Fig. 5.2.4) andis suitable for model work.The ketone group shouldbe more stable than the aldehyde of retinal, and spectroscopic changes would mostly occur in the visible range. A ~e~u-tetranilidopo~hyrin with four bixin side chains leads us to the ma-

0

.l Crystalline domains of a bis-bixin phenylenedi~neallowtherapid transport of dithionite and borohydride ions together with sodium counterions through vesicle membranes. No electron transport through hypothetical “electron wires” occurs. ‘This is shown by the reduction products (leukoindigos) of the entrapped water-soluble indigo. They show the typical differentiated absorption spectra of leukoindigos as obtained by chemical reduction inho~ogeneoussolution. If electron transport was responsiblefor the reduction inside the vesicle, only one type of reduction product would be formed, not two as observed.

UV/Vis spectra of a protein complex of canthaxanthin -( retinene-~ptophanmolecular complex in HCl-methanol(-----).

) and a

jor dye class of nature, the tetrapyrrole pigments. This porphyrin dissolves in water upon ultrasonication and yields perfect monolayered vesicle membranes with a thickness of 4.7 nm. The electron micrograph in Figure 5.5.3 shows the extreme stifhess of these polyene membranes, sincethere is no flattening at the points where they touch. Fluid vesicle membranes tend to merge like soap bubbles, but these vesicles remain perfectly round. The vesicles areso rigid that they survive even drying and deposition on a solid surface. AI?" shows rigid spheres for many minutes beforethe monomolecular balls finally collapse. Upon irradiation with visible light the carotene monomers of the vesicle surface polymerize to form a single molecule. The monomer vesicles are porous, osmotically inactive, and therefore stable in 1 M sodium chloride. The polymeric vesicle membranes behave similarly but are in addition also fully stable in ethanol or other organic solvents. They have also been isolated 'in the dry state (Komatsu et a1.,1997). Such polymerization reactionsof carotenes in light and under various oxidative and reductive conditions are typical for all polyenes if they are aggregated. The polymerizationcanonly be hindered by largesubstituents(e.g., tert-butyl groups), andthis would mean prohibitively tedious polyene syntheses. Wittig reactionsdo not like sterical hindrance. Carotenes are therefore not likely to be applicable as electron donors, lightswitches for membrane pores or elec-

100 nrn

The te~abixinatopo~hy~n produces stiff, isolable vesicles. Upon irradiation with visiblelight the bixin chromophores bleach out quantitatively and totally insoluble polymers of vesicular shapeare formed. The polymer structure given is hypothetical.

tron wires (Lazralc et al., 1987, Effenberger et al., 1988; ~ e g m a n net a1.,1989; ingh et al., 1996). Combined with their tendencyform to crystalline domains in fluid membranes (Andreoli, 1968; Milon, 1986),all negative properties of reactive dyes seem tobe combined polyenes. Onlyfor the preparationof white polymeric coatingsof solid surfaces do they seemto be ideal. Thisis of no interest to anisms, butthe carboxyl groupsof bixin may be useful infunc~onalizing organic and inorganic surfaces with insoluble an monolayer coating. or References for Chapter 5, see pages 542-544.

Life means energy consumption and has for over 4 billion years on earth. The only energyhas come from the sun, and porphyrins constituted its major chemical link to the earth. Chemists began to learn slowly from natural photosynthesis and triedto split water into h y ~ o g e nand oxygen usingthe sun's radiation as an energy source. Porphyrin chemistrymay be a good starting point.The dominating porphyrin derivative in photosynthesisis chlorophyll a, which provided the optimal chromophorefor sunlight absorption as well as its conversion to chemical energy. Its spectrum fitted the emission spectrum of the sun on the earth's surface (chloro~hyllsa,b) or in water (bacte~ochlorophylls,various bile pigments in algae); the photochemically excited states reversibly release electrons, yielding stable radicals. The chemical energy of these radicals is then bundled and migratesinto the most abundant substance onearth, namely water, whichis split into h y ~ o g e nand oxygen. This mixture of gases is the most useful and harmless chemical means of storing energy because the combustion product is again water. Plants expel oxygen into the gas atmosphere on earth. They avoid autoxidation or self-destruction and retain only hydrogen in the form of reductive carbohydrates. But the process of photosynthesis thereby becomes wasteful. Half of the stored light energy disappears into the atmosphere.An iron porphyrin, heme, was therefore developed. The central iron ion able to transport and activate the oxygen of the atmosphere in living organisms and the respiration process was developed. "he carbohydrates and molecular oxygen produced by plants from water, carbon dioxide, and light are now used by animals in respiration to produce thermal energy (Fig.6.1.1). Plants and oxygen are recycled. Much of the plant material was deposited over billions of years under the

H 0 reaction redox transfer energy

Chi

E reaction center light harvesting

C02

f

H20

.+~ PS,h*v

resplratlon

Primary reaction(Catalysts:NADH, Mn4 clusters) CH20 + 02

Total reaction

.l .l Schematic reaction cycleof plant photosynthesis(PS I, I1 = photosystems I and 11) and its overall stoichiometry.

earth in the form of solid coal andor gaseous and fluid hydrocarbons.The mineral oils and their solid and gaseous companions did not harm anybody for billion of years, but caused explosions in population and transport in the twentieth century. Life became free and demanding, consuming more energy than ever before. A more efficient useof solar energy will soon become ~andato~, In synkinetic systems porphyrins should play the same role as in nature: they can be applied to convert sunlightinto chemical or electrical energy andto use activated molecular oxygen as the cleanest of all oxidants (Fischer and Orth 1934, 1937; Fischer and Sterns, 1940; Lemberg et al., 1949; Vernon and Seely, 1966; Smith,1975; Longo, 1979; Dolphin, 1978a-d, 1979a-c; Falk, 1989;Scheer and Katz, 1995). Nowadays they are used as sensitizers in phototherapy, and their various metal complexes show the most versatile and interesting chemistry of all dyes known to chemistry.

Chlorophylls, heme, and bile pigments are made of four pyrrole units connected by methine bridges.We first introducethe electron-rich pyrrole unit and then dis-

cuss the consequences of a methinebridgebetweentwopyrroleringsin pyrromethene. Pyrrole is a five-membered aromatic heterocycle containingsix n electrons. Five impo~antresonance hybrid structuresd i s ~ b u t the e n-electron pairof the nitrogen atom over the four carbon atoms, and the dipole momentof pyrrole (1.84 daltons; similarto water) in the gas e is directed in. a way to make the ni~ogen atom the positive endof the dipole. le carbon atoms are thereforeextraor~narilyrichinelectrons(Gossauer, 198 l H - spectrum ~ ~ is of theexpected type (Figure6.2,1); the chemicalshifts are sinilar to the onesof benzene. If one connects twoa-pyrrolic positions via a methylene group, one obtains a pyrromethane, which easily autoxidizes in air toform a dipyrromethene, Thisis the f u n d ~ e n t aunit l of a porphyrin: a six x-electron pyrrole ring is connected viaa methine bridge toa five x-electron pyrrolenine unit. The central double isbond Zc o n ~ ~ u r eind the macrocyclic porphyrin but maybe either E or Z in open-chain oligopyrromethene-~pe chromop~ores. ~hotoisomerizationsof both diasteromers are discussed at the end of this section for the biologically i m p o ~ a cases ~ t of bil~ubinand phytoc~ome,The spectrum of themodelcompound hexmNH tautomers are involved in an extremely fast ethylpyrromethene shows that the tautomeric equilibrium. Only one signal at6.7 ppm is found; the methine proton

a

P I

A

A

l H

H

P

a

"

X

TM

" . . I

7.0

I

6.0

l

5.0

I

I

.O 3.0 IPPml

The 'H-NMR spectrum of pyrrole.

I

2.0

I

1.0

I

0.0

appears at 10.2 ppm. In the 15N spectrum one does not find a pyrrole-nitrogen signal at -230 ppm and a pyrrolenine nitrogen signal at -130 ppm, but only one avere signal at-162 ppm. This result is reproduced in the 13C spectrum (C=N:165 p p ; C=NH: 135 ppm; found: 15 1 ppm). The 5% and 6n systems are thus in rapid exchange even in the noncyclic dipyrromethenes. The stable confo~ationin solutions is usually the 2-syn-periplanar geometry given in Figure 6.2.2 (F&, 1989). Further condensation reactions of a-substituted pyrroles lead to macrocyclic

2.3

6.7 1

1 10.2

1

ethyl groups A 6:6:6 2.1 1.9

pprn

Methyl groups 13

15

5.1

-162.0

I

14.4 9.6 9.3 ppm

PPm

Schematic NMR spectra of h e x ~ e ~ y l d i p y ~ o m e ~1H, e n e13C, . and 15N spectra do not differentiate between pyrrole and pyrrolenine units. Proton exchange is too rapid.

porphyrinogens with four methylene bridges in high yield (see Sec. 6.3). Autoxidation of thesecolorlesscompoundsthengivesred po~hyrinsinwhichtwo pyrromethene units are again connected by two additional methine bridges. The po~hyrinmacrocycle contains two electron-rich pyrrole units with an each and two electron-de~cientpyrrolenine units without an tons are again in a rapid e~uilibriumon theNR/I[Rtime-scale. Only attempera~es below -53°C have two differentNH ~utomersbeen detected (Braunet al., 1994). The overall D, symetry in free base porphyrinsand the D,, s y ~ e t r in y metal complexes produce an aromatic character in the macrocycle. All bonds have about 50% double bond and 50% single bond nature.This does not, however, mean that all bonds have the same length. Within the inner 16-member A, and C-Cbondstypicallymeasure1.41 slightly shorter, namely 1.37A. The pyrrolic C-C bonds are, however, different: the Ca-Cp bond is 1.46 A ,the Ca-CP bondonly1.38 A long (Fig. 6.2.3) Lauher and Ibers,1973; Scheidt and Lee, 1987).

1.37

1.35

The bond lengths in porphyrins are more or less alternating, feigning polyene character, if the pyrrolic carbon-carbon bonds are included. The inner ring cont ~ ~ the n pyrrole g andpy~oleninenitrogen is, however, fully aromatic. The bond lengths are all close to 1.37 A.It is this macrocycle in connection with the electron-donating-or with~awingpower of the central metal ions that determines both the redox and photochemistry of met~loporphyrins.The bond lengths in thecenter correspond to NH bonds.

the UVlvis and lH-~MR spectra are similar to the benzene spectra. The S spectrumshowstheusual intense sho~-wavelengthbandandacouple of less intense bandsat longer wavelengths.The intense band occursat about 400 nm, its extinction coeficient is in the order of 1.5-4 X lo5, and the linewidth at half heightis 15 m (~onomers)or 30 nm (dimers). Itis called Soret band and originates from the allowed n;,n;transition between the degenerated highest occupied and lowest unoccupied molecular orbitals ( H ~ M~ ~ , ~The four ~ visible bands of the free base porphyrins and the two visible bandsof metalloporphy~natesare called Q bands and occur in the region between500 and 630 nm. They correspond to forbidden transitions involving changes of s y m m e t ~ from the ground to the excited state. 18 n; electrons and is isoelecThe inner 16-membered macrocycle contains tronic with the 16-annulene dianion. The bond lengths in this inner ring are all very close to the one in benzene, as shown in Figure 6.2.3.The UVlvis spectra of both compounds are as similar as one could have be predicted by the analogy n, ig. 6.2.4) ( ~ o u t e ~ a1978). The fact that the inner conjugation pathway dominateselectronic the spectra of symmet~calp o ~ h y ~ nand s metalloporphyrins does notmeanthat ppyrrolicsubstituents or centralmetalionshave no influenceontheUVlvis

0

The electron conjugation pathways of the inner macrocycle macrocycle of magnesium-octa~~ylpo~hyrin (ethyl groupsnotshown)andthe16-annulene 16--annuhe dianiondianion with with sodium counterions. The electronic spectra are as similar as one could predict from the the €€tickelmodel of aromaticity.

)

spectra. On the contrary, many typical substituent and metal effects have been identified and characterizedin great detail The visible spectra of porphyrins are therefore outstandingly meaningful and easy to analyze by analogy. The two vinyl groups of protoporphyrin, for example, cause a 6-nm-long wavelength shift of Soret and visible bands; a p-formyl group leads to a 6-nm b a t h o c ~ o shift ~ c of all bands andto an additional broad charge transfer band at 640 nm. Large substituentsat the methine bridgesor large metal ions inthe center tend to cause ruffling of the porphyrin macrocycle leading to long wavelength shifts as wellas broadening of the Q-bands. Central metal ions with low-energy por d-orbitals also strongly influence the LJVhis spectra. In regular metalloporphyrins with no electronic interaction between the porphyrin and metal %-orbitals, one observes only two the visible region (0,O and 0,l transitions) instead of four in the fre phyrin because the s y ~ e t r is y raised from D,, to D,. Metal ions without delectrons (Mg2+,AP+, Sn4+)and those with closed shells (Zn2+)are examples of such regular metalloporphyrins. Hypso metalloporphyrins, on the other hand, contain d6-d9metal ions and showa blue shift of the Q-bands.This is caused by bac~-bondingfrom the filled metal orbitals to empty porphy Co3+).The spectra of p-type (Pb2+,Sn2+)andd-type(Fe3+, phyrins, on the other hand, are disturbed by strong porphyrin donation. This leads to new bands in the visible andUV spectra as well as to unpredictable shifts of the Soret band ( ~ o u t e ~ a 1978). n , Fhorescence is usually only observed withfree base and regular metalloporphyrinates (see Sec. 6.5). ydrogenation of a 3,4-pyrrolicdoublebondproduceschlorins,the mother chromophores of chlorophyll, and the whole aromatic porphyrin spectrum changes to a polyene-type chlorin spectrum. It consists mainly of two bands of comparable intensity at 4.00 and 650 nm and several smaller bands in between. (see Fig. 6.2.9) (Cox et al., 1974; Smith, 1975; outerm man, 1978). Infrared spectra of ~ o r p h y ~ do n s not provide much structurali n f o ~ a t i o n except for the usual group frequencies of side chains. Pyrrole NH bands appear from 3310 to 3326 cm-l, protons of bridge and vinyl group hydrogens around 3 100 cm-l. Vinyl groups in protoporphyrin derivatives arealso characterized by aninfraredband at 1620 cm-I andresonanceamanbands (e~citationinthe Soret band region) at 1630 and 1621 cm-l. The intensity of the 1630 band dees monotonically relative to the 1620 band as the temperature is lowered, bands have been assignedto torsional conformers, in whichthe vinyl double bond lies in theporphy~nplane. In c o n f o ~ e Ir the terminal hydrogenis cis to the neighboring p-pyrrolic methyl group, whereas confor~er in TI it is next to the methine proton (Fig. 6.2.5). Another characteristic infrared absorption is a strongbandinthe m 1940 to 1970 cm4 for carbonmonoxidebound to ns exhibit high po~arizabilityby interactions with heme derivatives. central metal ions or electron-rich substituents. This then leads to shifts of in-

2.26A

0

90

140 180

z (deg)

The p-vinyl groupsof protopo~hyrinderivatives are conjugated with the porphyrin E-system and tendto lie parallel to the ring.Two conformations dominate and show characteristic Raman resonance bands. (From Kalsbeck et al., 1995.)

frared absorption bands in the fingerprint region upon complexation. Such shifts are, however, only useful in comparisons between many metallopo~hyrinates with the same ligands (Alben, 19’78). Mass spectra are dominated by theextremestability of theporphyrin macrocycle’s molecular ion and by benzylic P-cleavage of substituents. The pyrrole units can, however, be fragmented by electron impact when the methine bridges are oxidized to carbonyl groups (xanthoporphinogens) or reduced to methylenes (porphyrinogens;see Scheme 6.4.2). A wealth of structural i n f o ~ a t i o nhas been obtained by ‘H-NMR spectroscopy of porphyrins, This circumstance arises fromthe large ring currenteffect in strong magnetic fields, which functions as built-in chemical shift reagent and spreads proton signals over the large range of 15 ppm. The NI3 protons occur typically at “4ppm, the methine protonsat 10-1 1 ppm, and substituent protons within thisrange (Fig. 6.2.6) (Janson and Katz,19’72; Smith, 1975). Diamagnetic central metal ions barely influence the porphyrin ring current. ~aramagneticionsusuallybroadenallsignals,butin the case oflow-spin IH-NM iron(II1) porphyrins, one often observes well-resolved over more than 50 ppm leading to enhanced resolution (Fig. phyrin interactions that influencethe ~ ~ - N Mspectra R can be split into two major components: leakage of unpaired spins into the porphyrin’s nuclei (“contact shifts”)anddipole-dipolecouplings(“pseudocontact shifts”). ~seudocontact shifts occurring through space correspond to the actionof shift reagents and provide valuable information aboutthe porphyrin e n v i r o ~ e n as t well asthe num-

I l

10.0

l

8.0

I

6.0

I

I

4.0 2.0 6 [PPml

I

0.0

NH

-210

410

R spectrum of p r o t o p o ~ h y ~IX n dimethylester in CDC13

'H-NMRspectrum of ferricyanide p r o t o p o ~ h y ~ nCompare . methyl group signals with those of the freep o ~ h y ligand ~ n in Figure6.2.6, ber and conformatio~sof substitue~tsand a x i d ligands. Contact shifts, on the other hand, provide useful informationon spin densities and spin transfer mechanisms within the porphyrinn: system. The most dramatic effectis observed for methyl group substituents, which reflect high electron density at the adjacent carbons ( ~ u ~ c1970; h , Smith, 1975).

The spin states of iron and manganese porphyrins are determined by the oxidation states as well as by the ligand fields. Unpaired electrons on the central metal ions, [e.g., in the d5ions Fe(II1) and Mn(II), d7ion cobalt(II), and d9 ion copper(II)] produce metal ion ESR signals and often destroy the ligand NMR signals by broadening effects. The spin state is determined by the ligand field, which reflects and is dominated to a great extent by axial ligands. Low-spin complexes usually have two identical ligands, whereas high-spin metalloporphyrins have one strongly bound ligand (e.g., cyanide) and one weak ligand (e.g., water). For nitrogen substituents, the ligand field usually increases with the pKa of the nitrogen base. ~iamagnetictransition metals, in particular d6-Fe(I1) in hemes, produce shifted l H - N ~ Rspectra, where the chemical shifts of the porphyrin signals also strongly depend on the axial ligands (Fig. 6.2.8) (Wuthrich, 1970). Two examples of well-resolved electron spin resonance (ESR) spectra of frozen high-spin Fe(II1)-porphyrin derivatives with one axial chloride counterion or two imidazole ligands plus one distant chloride ion are reproduced in Figure 6.2.8. The measured g-values of the high-spin chloride with go, = 6 and gpar= 2 are considered a fingerprint of a strong tetragonal ligand field with axial asymmetry. Three lines in the di-imidazolide of iron(II1) p o ~ h y ~ n a t e s around g = 2, on the other hand, point to a more symmetrical, rhombic field (Subramanian, 1975). The UV/vis spectrumof the chlorophylls is a combination of the spectrum of an aromatic system with a relatively intense (E= 1 x lo5)Soret band and a polyene spectrum represented byanalmostequallyintenselong-wavelength band (y= 660 nm, E = 8 X lo4) (Fig. 6.2.9). The spectrum is understandable be-

X

X-Band ESR spectra of (a) chlorodeutero-he~n ~ime~ylester and (b) the corresponding bis-imidazole complex in dimethyl f o ~ a ~ atd77e K.

1

0 500 600 700 h [nm] green yellow orangered Absorption spectrum and structure of chlorophylls a and b and emission spectrum of the sun) " ( on the surfaceof the earth.

cause the inner conjugation pathway of 18~c-electronsis still present, but the original symetry of the porphyrin macrocycleis disrupted by the reduction of one of the p y r r o l e ~ units ~ e to a pyrroline ring andby the introductionof an isocyclic ring E containing an electron-with~awing P-ketoester grouping. changes introduce a strong dipole moment into the macrocyclicn. system move the degeneracyof the outer orbitals. The major reason for which nature selected the complicated chlorophyll chromophore derived from protoporphy~n IX rather than the porphyrin itself is adjustement of the absorption spectra of photosynthetic organisms tothe emission spectrumof the sun on theearth's surface (Figure6.2.9) (Vernonand Seely,1966;Smith,1979; Scheer andKatz, 1995). Long-wavelength chromophores based on aromatic systems often contain strong a s y ~ e t r i e within s the aromatic cycle, whereasfor polyenes an appropriate polar environment is sufficient to induce enormous long-wavelength shifts (see Chapter 5) The W N M R spectrum of magnesium-andphytol-freechlorophyll a (pheophorbide a) shows two methine protons at 9.8 and 9.6 ppm and I? at -1.7 ppm. The ring currentis diminished in comparison to porphyrins but still very strong. Only the methine proton at C20 neighboring the pyrroline Dring is strongly shifted upfieldto 8.9 ppm.

Since the chlorophyllsalso contain three chiralcenters, intense CD spectra are produced by these chromophores (Fig. 6.2.10). The major bands appear in the Soret band region, but Q-bands are also found. InCD spectra of chiral porbands may or may not appear. densities in %-cation radicals of the chl~rophyllsare unevenlydistributed. A combination of ESmethodologiescalled ENDOR (electronnucleusdoubleresonance) spe ygives high~resolutionspectra and provides a wealth of information on charge and spin densities in chlorophyll c~omophores.A, typical ENDOR spectrum and a resulting electron density map is reproduced in Figure 6.2.11 Such spectra provide apower€ul starting pointfor investigations of chlorophyll-protein interactions (Kurreck, 1988). Electronic and steric effects of porphyrin substituents always become app ~ e n in t both electronic and I ~ - spectra. ~ R any thin^ disturbing the aromaticity (electron-with~awingeffects, steric repulsion and consequent ruffling of the macrocycle, asymmetry) leadsto a long-wavelen~thshift andbroade~ng of the absorption bands as well toasan upfieldshift of methine proton~R signals. Since essentially all porphyrin publications contain detailed descriptions of new f synthetic electronand NMR spectra, it is usuallyeasy to ass' po~hyrinsand reaction products by visible and combined with mass spectra and elemental analysis alone.

0 Circular dichroism spectrumof two diastereomers of a pheophorbide a dervative methoxyl~tedat the isocyclicring. (From Wolf et a1.,1967.)

Qpical ENDOR spectrum of the bacterioc~lorophylla cation radical, Large spin densityis found at all nitrogen atoms, on the reduced pyrrole ringsI1 and IV, and on methyl groups of the pyrrole units.

The analytical situation becomes much more complicated with open-chain as bile pigments in nature (Falk, 1989). Their tetrapyrrole pigments, which occur conformation is by no means limited to planaror ~ ~ e d - p l a n abut r , all kinds of c ~ s - t ~ a ~ s ~ o n f i gdiastereomers ured are known and produce extremely differentiated and often complicated UV/vis spectroscopic shifts and extreme changes in solubility, causedby enormous changes in neighbor group interactions. The latter are best analyzedby NOE (nuclear Overhauser effects) (Sanders, 1996). In biliverdins four pyrrole units are connected by three methine bridges. These pigments occur in massive amounts in urine, feces, and algae, they colorize bird eggs, and they act in microgram quantities as hormones in higher plants, where theyregulate photomo~hogenesis.Two diasteromers, namely the red phytochrome Pr absorbing at 660 nm andthe far-red pigmentP, with its major abso~tionpeak at 730 nm, are in photoequilibrium. The inactive 7 3 0 - n ~ pigment is activated during the sun set by 7 3 0 - light, ~ which only occurs when the days become longer after a winter period. An active 660-nm pigment appears and then acts as a plant growth hormone. Protein synthesis is switched on and seeds starts putting out shoots. This effect is reversed if a 660-nm light pulse is applied right after the 730-nm light. The chemistry behind hormone activation and inactivation is a c i s - t ~ a isomerization ~s of the bilatriene. The allc o n f i ~ ~ r ecyclic d conformer of a model bilatriene absorbing at 636 nm shows indeed a fully ~ight-reversibleconfo~ationalchange to an former absorbing at shorter wave ,12). All bile pigm are, of course, of the polyene type diger, 1969; Falk, son et al., 1994.).

ter

0

H

H

n

0

l

Structure of phytochrome and a model bilatriene showing a photoreversible c i s - t r isomerization ~~~~ uponirradiation with monochromaticlight. The spectrum given belongs to natural phytochrome; wavelengths and structures for the model comPredshould also be Z,Z,Z-configured, whereas P,re(, should pound are given at the bottom. be E,Z,Z.

A related light-induced isome~~ation involving a biladiene with a central group named bilirubinis of i m ~ o ~ a nin c enewborn babies. Starting with the seventh monthof pregnancy, fetal hemoglobin is rapidly degraded in the fetus’s body and replaced by adult hemoglobin. If bilirubin, the degradation product, is not properly solubilizedby enzymatic glycosylation, then water-insoluble biliru-

bin may buildup in the fatty skin tissue and the brain of the baby. Jaundice of the brain may produce catastrophic consequences if the pigment is not removed quickly. It has been found accidentally that not only glycosylation renders the bilirubin more water soluble, but that modest irradiation with visible light also helps. Detailed investigations of the initially water insoluble bilirubin and its most water-soluble irradiation product have shown that insolubility is caused by intramolecular hydrogen bonds between the propionic acid side chains and pyrrole nitrogen atoms. These bonds, which counteract hydration, are removed if the cis double bondis photoisornerized totrans (Figure 6.2.13)( ~ a c ~ o n a and gh Palma, 1982a,b). Lightis thus very helpful to remove bilirubin from the baby’s body, not by bleaching but by solubilization. Now that we have dealt with the chromophores of natural tetrapyrrole pigments anda few model compounds, we consider the most important substituents that determine synkinetic reactions (see Sec. 6.4). (For vinyl groups see Fig. 6.2.5.) The most easily accessible model porphyrins are ~eso-tetraphenyl the porphyrins (TPP). The phenyl rings cannot orient themselves theinporphyrin plane and form a conjugated YE system with the the porphyrin. The steric repulsion between the ortho-phenyl protons and the @”yrrolic protons is too large. Angles between 60 and 80” are common (Scheidt and Lee, 1987). Meso-phenyl substituents do not allow a close approach of the porphyrin chromophores in solution or in crystals. As a result, one observes the narrowestSoret band possible, corresponding to a porphyrin monomer in solution.The half-width is around 15 nm, the extinction coefficientclose to 4 X lo5.Crystal structures are d o ~ n a t e d by interactions between the phenyl rings of neighboring porphyrins. The electron-rich para-methine group of one phenyl substituent pointsinto the electronpoor center of a neighboring one. Both phenyl rings are then orthogonal to each other similar to the situation found in benzene crystals (Fig. 6.2.14). porphyrin distances are in the order of 8 A with respectto these plane crystals therefore act as sponges for small molecules. CO-crystallization fills up the large spaces between the linear TPP chains (Byrnet al., 1993). substituted porphyrins, which are found in all natural compounds, show a 3O-n~-wideSoret band and extinction coefficients between 1.3 and 2 X lo5. This is typical for face-to-face dimers. Porphine and octamethylporphyrin a gate further to form insoluble polymeric aggregates. These porphyrinsdis badly in c h l o r o f o ~slightly ; pink solutions can be obtained with boiling pyriacid. ~-~ctaethylporphyrin, however, is quite solublein ,because aggre~ationbeyond the dimer is prevente~by the away from the first partner molecule (Fig.6.2.1 f natural porphyrins suchas propionic acids, but not methyl or vinyl groups, have the same effect. The most common aggregation stack of

15" 2'-H 18'-H

22-H' 182-H'

The Z,Z-isomer of bilirubin contains intramolecular hydrogen bonds, which are converted by upon photoisomerization to the E,E-isomer. 'Hspectra allow the ch~acterizationof both diastereomers. Only thelatter isomer is water soluble andp~ysiologicallyharmless. I n t ~ ~ o l e c u lhydrogen ar bonding renders the2,Zdiastereomer water insoluble. It then migrates into lipid m e ~ b r ~ eins ,particular brain tissue.

(a) Crystal structure of ~ e ~ ~ - t e t r a p h eporphyrin. nyl The para- hydrogen atom of the phenyl group on the right of the left porphyrin is in van der Waals distance to the perpendicular phenyl ringat the top of the right porphyrin. The perspective does not suggestthis. (b)Go-crystal with 1,3-xylene.

substituted porphyrins in organic solventsor membranes is the shifted face-toface dimer found incrystal structures (Fig. 62.1%). Here one usually finds that an electron-rich pyrrole ringof one porphyrin sits atop the electron-poor center of the other porphyrin. Water-soluble substituents, in particular the most common acetic and propionic acid side chains, lead to amphiphilic bilayers (Fig. 62.1%) (Fuhrhop, 1976; unter and Sanders, 1990). Lateral interactions between porphyrins are induced by hydrogen bonds or hydrophobic effects,as discussed in Section 6.4.

1..

-

0.7 nrn-

..

Crystal structure of the usual p-substituted porphyrin dimers with (a) shifted, unrotated porphyrin planes, (b)a p-alkyl chain,and (c) acid side-chain effects.

If one cuts the macrocycle of p o ~ h ~ by ~ oxidation, n s helical structures becomedominant. The most spectacularcasehasbeenfoundwithzinc octaethylfo~~l-biliverdinate. At neutral the central zinc ion is h~dratedand ed with a disturbed square ar ligand field and one ~ i water ~ Z n acidification, however, the water molecule is r~movedand the zinc ion enforces a tetrahedral ligand field by binding to two di~erentfomylbiliverdinate molecules, r e ~ a n g i nto~ f o m a double-helix. Upon neutr~ization, hydration takes place again and the planar, ~ o n o m e ~ monohyd~ate c is refomed in q~antitativeyield. 0th the helical ~ o n o m e and r dimer st~ctures have been solved by singlecrystal analysis (Fig.62.16).

A

I

Schematicx-ray structures of (a) zinc octaethylfor~ylbiliverdi~ate monohydrate and (b)its dehydrated, bis-helical dimer. (FromStrucheier et al., 1976.)

Natural po~hyrinogens,i.e., cyclic tetrapyrrolesconnected by methylene ne called ~orphobilinogen bridges, are made from an ~ - a ~ n o - m e t h y l e pyrrole by removal of four a ~ o n i molecules. a Four isomericp o ~ h y ~ n o g e I-IV n s are obtained, which are differentiated by the ~ a n g e m e n t sof p-acetic acid and ppropionic acid side chains on each pyrrole unit (Mauzerall, 1960). ~ o ~ h y r i n o gens thenau~oxidizein air to form the corresponding porphyrins (not shown; see Schemes 6.3.5-6.3.7 for analogous syntheses). The most simple method of obtaining a p o ~ h y r i non the gram scale is to reflux a dilute benzaldehyde-pyrrole mixture in propionic acid (141"C) for 30 minutes, cool, andfilter. A 20% yield of crystalline por~hyrinis easy to achieve (Scheme 6.3.1) (Lindsey et al., 1987, 1994; rath ha pan et al., 1993). Even pentamers have been obtained by such a reaction in one step from a porphyrin benzaldehyde building block and pyrrole. One just has to carry out the primary formation of the porphyrinogen at a relatively low concentration ( )and under nonoxidative conditions, which allow the rearrangem~ntof undesired polymers. Up to 50% of the colorless po~hinogensare obtained by acid-catalyzed condensation of pyrrole and aromatic aldehydes under nitrogen'

The reaction proceeds because it is more probable that the ends of a tetramer react with each other rather than that the addition of a fifth monomer occurs (Fuhrhop, 1974). Dimer and trimer precursors, on the other hand, cannot be folded so that their ends can be covalently connected. In the second step, the porphyrinogen is then oxidized to the desired porphyrin with chloranil {Scheme 6.3.1). Condensation with aldehyde mixtures of two components A and B gives six different porphyrins: two parent porphyrins and four hybrid porphyrins (cis- and trans- A, 2, AB,, and A,B). Direct access to trans-A,B, porphyrins is best achieved with an aldehyde reacting on a-freedipy~omethanes. Capped porphyrins can be obtained from dialdehydes and pyrroles. Even a huge bis-steroidal dialdehyde yields the corresponding porphyrin in 7% yield (Scheme 6.3.2) Linear porphyrin dimers are co~espondinglyobtained from p o ~ h y r i n ~ i -

DDQ

4

CH0

CH0

aldehydes and pyrromethenes. Covalent stacks of tetraphenyl porphyrins have been synthesized by similar procedures starting withu~~~u-terephthalaldehydes zig-zagoligomers(notshown) from the ~ e ~ ~ - s u b s t i t u isomers t e d (Scheme 6.3.3) (Nagataet al., 1990;Asahi et al., 1993). All these porphyrin one-step syntheses workquite well with benzene-derived aldehydes, but yields drop dramatically with aliphatic aldehydes. Undecylenealdehyde, for exarnple, gives a yield of only 2% of the porphyrin withfour C,, chains. In the case of u~~~u-disubstituted aldehydes, one obtains extremely dense 11; stacks with Soret band absorptions at 370 nm, whereas ~e~~-disubstituted analogs give a lateral packing characterized by 450-nm Soret bands (Fig. 6.3.1). Such covalent porphyrin oligomers are useful references in structure elucidation of corresponding supramolecular assemblies, where one cannot hope for a

onc

+

R

3

completely uniform ~ a n g e m e n of t the monomers and where analysis is often difficult. The most common P-substituted model porphyrin is octaethylpo~hy~n, which is available by a orr pyrrole synthesis, followed by hy~ogenationof a @-acetylgroup, leadtetraacetate oxidation of the ethyl group, acid-~atalyzed decarboxylation,andsubsequentcyclizationanddehydrogenation(Scheme 6.3.4). ~ctaethylpo~hyrin can thus be made routinely on a 10-g scale within 3 weeks (~nhoffenet al., 1966a). A similar synthesis to give octaacetic acid porphyrin is much less productive (Chiusoli et al., 1989). Highly substituted porphyrins with eight p-pyrrolic ethyl groups and four phenyl groups in the mem positions have been obtained similarly. The latter, highly “overcrowded” porphyrins(see chlorophyll synthesis) are totally stable (Evanset al., 1977). ~nsymmetricallysubstituted dipyrromethanesare obtained from ~ - u n s u b ~ stituted pyrroles and ~-(bromomethyl)py~oles in hot acetic acid within a few

.l Absorption spectra of the covalent porphyrin stacked and lateral pentamers shown in Scheme 6.3.3. Stacks show only short-wavelength shifts; lateral assemblies often give, although not in the present case, both short- and long-wavelength Soret bands.

minutes. These reaction conditions are mild enoughto avoid acid-catalyzed re~ a n g e m e n t s of the dipyrromethanes (“jumbling,” “redistribution”). All tetrapyrroles containing methylene bridges (e.g.,, bilirubins or porphy~nogens) are accessible under these reaction conditions. Heteroatoms on the a-methyl groups of pyrroles can also be replaced under mildly nucleophilic conditions to yield better leaving groups. Highly resonance-stabilized a-pyrrolyl~ethe~ium (azafulvenium) intermediates occur (Scheme 6.3.5) (Gossauer, 1984). a-Free and a-diformyl pyrromethanescanbecoupledunderreductive conditions (HJ/AcOH)to give, for example, uroporphyrin111, coproporphyrin I, and ~-tetrapyridiniump-tetraethyl porphyrin II from porphodimethenes in high yield (Scheme 6.3.6a). The problem of ‘~umbling”is best avoided by utilizing pyridine-like po~hodimethenesas first tetrapyrrolicinte~ediatesy which do not addprotons to carbon(6.3.6b).Yieldsare,however,oftenlowbecausethe dipyrrolic inte~ediatescombineonlyslowly(Smith, 1979; Endisch et al., 1996) ostfrequently,however, one obtainsnatural p o ~ h y ~ nfrom s hemin, which is comercially available or can be produced on a kg scale from fresh blood from a slaughterhouse. Useful protoporphyrin derivatives are discussed in Section 2.4.2. Only one sequence of a complex porphyrin synthesis is described here, namely the path leading from a substituted porphyrin to a chlorine containing

HOOC,

,OH

+ H

the substituted pyrroline ring D of chlorophyll a. When R. (1960, 1990) originated chlorophyll synthesis, he doubted Hans Fischefs structure, which was derived from degradation studies only. Woodward analysis led at carbon atoms 13,15, and to the postulateof m “overcrowded” porphyrin plane 17.A prohibitive “stereoche~calstrain’’ shoulddisturb the planarity of the inner p o ~ h y r i nconjugation pathway tothe extent that aromaticityis destroyed. Then it was realized that the sp3 carbons16 and 17 could ease the strain. Chlorophyll

COOEt

COOEt

PhHZCOOC

>

>

>

I

COOH

COOH

COOH

COOH

l

X ROOC

H

ROOC

X = NH2, OH, OAc, OTos, ha1 ROOC d

synthesis was consequent].ybasied on the assumption of a tendency to “sterteochemical strain release.”The hydrogenation pattern in ring should establish itself if large substituents on C 15 andC 17 repel each other. The most adventurous endeavor developed as follows: an overcrowded p o ~ h ~ r with i n three carbon substituents at C 17, C15, and C 13 was prepared. ings 111 and IV twisted out of the plane. Hydrogenation (hoping for addition

ha

H00

H00

> f COOH

COOH

u r o p o ~ h y 111 ~ n(60%)

COOH COOH

0

COOH COOH

COOH

coproporphyrin I (50%)

of hydrogen atoms at ring C) was carried out but gave a blue “phlorin” instead of the anticipated green chlorin (not shown). The methine bridge carbons of porphyrins turned out to be much more reactive than p-pyrrolic double bonds and were hydrogenated first. Spontaneous dehydrogenation of a propionic acid side chain gave acrylate and a phlorin intermediate. Totally regioselective electrocyclization with ring D (ring C was deactivated by an electronwithdrawing ester substituent), photoo~ygenation of the cyclopentene ring with the porphyrin as a “self’-sensitizer, and removal of the labile a-keto-car-

(l) Claisen (11) Knorr pyrrole synthesis (Ill) Benzylic alcoholelNa

Nicotinic acid ethyl ester

(I) H$Pd

boxylate led quickly to a stereochemically perfect pyrroline ring aldehyde group on C15. The latter then allowed the synthesis of the isocyclic ring E (not shown) (Scheme 6.3.7). The anticipated direct hydro~enation based on the concept of “stereochemical strain release” was thus replaced by a reversible C-C bond formation. Newly discovered porphyrin reactions (phlorin formation, photoo~ygenation,electrocyclization) were not used to produce publications but were immediately integrated into the synthetic plan. And, most important, success, high yields, and elegance characterized Woodward’s unique achievements in natural product synthesis (includin~cholesterol, vitamin D, and vitamin BIZ).Rarely in the history of ch intelligent speculation been rewarded by so much real success. ward known that “overcrowded” porphyrin planes have very unspectacular properties, as was realized later, he may not have been so courageous (see Schemes 6.3.4,6.4.4, and 6.7.1). Syntheses of “expanded, contracted and isometric porphyrins” have been comprehensively overviewed in a recent book by Sessler and Weghorn (1997).

(l) mild acidic

(11) strong acidic (Ill) 12; AQO

regioselective coupling

/

AcHN

Med

Me0

Phlorin AcHN

(l) AcOH;A

(It) HClfMeOH (Ill) Me SO NaOH

KOHIMeOH

b ~ e rac lsopurpurin 5

ethyle ester

The reactivity of the porphyrins and metalloporphyrins will be described mainly using octaethylporphy~nand protoporphyrin derivatives. Reactionsof TPP and chlorophyll a derivatives are mentioned only on the occasion of ~-formylation and the reduction of nitro groups, which yield important educts for syn~neticreactions. The reversible reactions describedfor ~ - s u b s t i ~ t reactions ed also work with ~e~~-tetraphenylporphyrins.

Three types of fully reversible reactions are known in the chemistry phyrins (Scheme 6.4.1):

of por-

1. Protonation and deprotonationof the central nitrogen atoms 2. Metalation of the central nitrogen atoms 3.One-electronoxidationandreduction Addition of acids to aqueous, micellar,or organic solutions of po~hyrins leads to diprotonated dications, which show only two absorption bands in the visible region (S50 and 592) for ~-octalkylporphyrinsinstead of four for the free bases (approximately 500, 530,570, and 620 nm). The pka as measured in micelles lies between 4 and S (Falk, 1964).In the case of TPP derivatives, however, the porphyrin solution turns green upon protonation anda strong phlorintype band around 700 nm appears. X-ray structures of the TPP dication reveal the reason for this abnormal change:the porphyrin ring becomes puckered by repulsion of the central positive charges and one of the phenyl groups rotatesinto a position almost parallelto the pyrrole units (Fig. 6.4.1). It is conjugated with the disturbed porphyrin conjugation pathway and its spectroscopic nature changes from aromatic to polyene-like (Fleischer and Stone, 1967; Stone and Fleis 1968). The planarity and aromaticityof TPP derivatives is easily destroyed. is insharpcontrastto the natural ~-octalkylporphyrinsandtheirsynthetic analogs. Deprotonation of porphyrins is possible with strong bases (e.g., sodium hydride) in pyridine or other polar, nonprotic solvents (not shown) (Falk, 1964). The corresponding sodium salts are formed, which produce similar two-banded visible spectra as the diprotonated dications. Little is known about the dianion chemistry. They cannot, however, be oxidized to dehy~oporphyrinswith only 16 n-electrons in the inner conjugation pathway. Such a porphyrin wouldbe “anti aroma ti^'^ and appear asa triplet state.It has not been detectedso far. The most rapid metal complexation reactions of porphyrins occur with Pb2+)are two-valent metal ions. Metal ions of low electronegativity (Mg2+, Ca2+, introduced in refluxing pyridine, metal ions of intermediate electronegativity and

+2H+ -2H'

protonation

12

L

I'

one-el~ctron oxidation

ionic radius are readily incorporated in c h l o r o f o r ~ m e t h ~ mixtures ol (Zn2+, Cu2+,Ni2+),large, kineticallyinert, or redox active metal &cations are best introduced in acetic acid, where redox active metals are then immediately oxidized by air (Pd2+,Pt2+,A13+,Fe2+to Fe3+,Sn2+to Sn4+).Most of these metalation reactions are fully reversible with hydrochloric acid (except for Ni, Pd, Pt), but higher~valentmetal ions must first be reduced with borohydride or dithionite [e.g., Sn(1V) to Sn(II)] (Smith, 1979). Axial ligands, such as H20, Cl-, and Er, can undergo exchangeat the usual fast rates, but CO,RS-, and CN- form rather inert M-Lbonds. A uniqueproperty of met~oporphyrinatesis thegeneralstabilityand longevity of %-cation radicalsas obtained in organic or aqueous solutions by oxidation with iodine, bromine,~-bromosucci~mide (NEB), iron (HI)salts, or cathodic oxidation. Titrationof magnesiumporphyrinates inc ~ o r o f o ~ m e t h a nwith o l NBS or iodine, for example, gives a clean andqu~titative conversion to the %-cation rad-

60

661 I

50

I \ I I I f

I I I I I I

40

z

c?

W

f

30

l l l

2c

C I

I I I

515

f

608

I I I J

1

\ \

\ \

\ t \ l I \

l I

I \

l 1,

I \ 1 \ \ \ \

L

.4.1 Molecular structure and UVrlvis spectrum of thediprotonated mesotetraphenyl-po~hyrindication.

ical, and addition of iodide as an reductant reproduces the original ~ ~ e s i uporrn phyrinate, again in quantitative yield (Figure 6.4.2) (Fuhrhop ~and a u ~ e r a 1968, ll, 1969; Fuhrhop, Kadish etal., 1973; Fuhrhop, 1974; Felton, 1978). Highly electropositive ions deactivate the porphyrin towards oxidants. The Sn(1V)-porphyrinates present an extreme here. They are totally stable against molecular bromine but can easily be photoreduced to form porphyrin anion radicals and, in proton-contai~ngsolvents, phlorins (Fuhrhop and Lumbantobin~, 1970). Both porphyrin7c-anion radicals and the protonated neutral phlorin radicals usually produce absorption bands around 850 nrn (see Sec. 6.5). The lifetimes of a-anion radicals of metalloporphy~nsas obtained by pulse radiolysis are also dependent on the central metal ions. Sb(V) and Sn(IV) produce stable

Spectroscopic titration of ~agnesi~m-octaethyl-po~~yrinate with NB§ in chlorofo~~methanol to yield the corresponding It-radical. Here the cation radicals reversibly add methoxide anionsto give electroneutral phlorinradicals.

anion radicals; zinc porphyrinate anion radicals decay within ~lliseconds( choux and Neta, 1986). A plot of the midpoint one-electron oxidation and eduction potentials against eachother yield linear correlation, indicating that metalloporphyrins that asy to oxidize are difficult to reduce and vice versa (Figure 6.4.3) (Fu~hop, sh, et al., 1973). Figure 6.4.3also shows two exceptions to the rule depicted in Figure 6.4.4, namely manganese and molybd~numporph t and extremely weak Soret band :437 and 355 nm. These metal1 states are mucheasier to oxidize than they should be in respectthe to ligand’s a system, which is obviously caused by electron donation from the metal ions to S , the metal-ligand interaction is purely of the a-cation radical. In the regu electrostaticcharacter:boththandthe LUMO areraised by twovalent metal ions of low electroneg sulting in electron-rich porphyrins, and both “frontier” orbitals are lowered by electron-withdrawing central ions. The difference between HOMO and LUMO remains constant as is indicated unchan~ingwavelengths of the Soret and visible absorption bands inall of the metalloporphyrins (Fig. 6.4.4) with the exceptions of

Sb

Ca

l

l I

Plot of oxidation and reduction potentials of various metallopo~hynnates. Metalloporphy~nsthat easily form cation radicals with weak oxidants cannot be chemically reduced (e.g., the Zn- and Mg-porphyrinates). Metallopo~hynnsthat easily form anion radicals upon reduction [e.g., Sn(IV)], on the other hand, cannot be chemically oxidized, Only electrochemical reactions are possible at potentials far above 1 V or far below -1 V

tioned above. They belong to the hyperporphyrins (see Sec. 6.1.l) with strong interactions betweenthe n; orbitals of the porphyrin andthe d-metal orbitals. If one substitutes the porphyrin ligand by chlorophyll or bacteriochlorophyll type ligands containing P-dihydropyrrole units, the nitrogen atoms become more basic and the ligand more electronegative. The oxidation potentialof metallochlorins is therefore about 100 mV lower than the potential of the corresponding metallopo~hyrins,and of bacteriochlorinsagain100 mV lower (Fuhrhop, 1970; Noy et al., 1998). A typical magnesium chlo~n-chlorin cation radical oxidation potentialis around 300-400 mV only.The situation in homogeneous solution is thus well understood. In natural photosynthetic systems,hovvever, a "special pair" of chlorophyll a has an oxidation potentialof about l.l V. acteriochlorophyll potentialsof up to 900 mv have also been observed, which is at least 600 mV higher than anything that can be achieved in homogeneoussolution. The explanation given in theliterature includes massive hydrogen bonding of a m e m b r ~ protein e to the chromophore substituents, buthow that could have such a massive effect remains poorly understood (Czarnecb et al.).

z

I.

MgOEP

"

4.

2

I.

~n(~V)OEP(

h(Soret)= 411 n m h(Soret)= 410 n m ~ O ~ O - energies L ~ Oin zinc and tin(1V) porphyrins, the redox potentials, and the Soretband wavelengths.

~etallo-chlorinsand bacteriochlorins havelower oxidation potentials than the corresponding metalloporphyrins.The replacement of a pyrrole by a hydrogenated pyrroline ring lowersthe potentials by about 100 mW. Furthermore, the oxidation potentials are generally somewhat lower in polar solvents or in micelle and vesicle membranes than in apolar solutions because the charged oxidation and reduction products become more solvated in polar media. It is thus possible to change the redox propertiesof metalloporphyrins by (a) change of central metal ions, (b) hydrogenation of pyrrole units, and (c) change of the e n v i r o ~ e n t ' spolarity. Furthermore, chlorins, bacteriochlorins, biliverdinates, and also synthetic pentapyrrole pigments such as texaphyrins can in principle be used as light-harvesting materialsfor red and near-idrared light. uch compoundsmay be useful in laser surgery of malignant cells because laser surgery should work withthe relatively inexpensive andnondes~uctiveneodym YAG laser emitting in the near-infrared. (Sessler and Weghom, 1997).

P-Substituted porphyrins react at the methine bridges first irrespective of reagents. Strong reductantshy~ogenateall four methine bridges to produce porphyrinogens without touching the pyrrole rings at all (Talk, 1964). Strong oxidants oxidizeall methine bridges to form keto groups and ~e~~-tetraoxopo~hyrinogens (xanthopo~hyrinogens)(Inhoffen, 1966) are formed (Scheme 6.4.1). Half-hy~ogenatedor oxygenated porphodimet~eneswith aroof-like conformation are easily accessible intermediates. The oxodiporphodimethenes do

not behave as quinones. These compounds have been efficiently dimerized to form the possibly most hindered tetrasubstituted alkene known (Scheme 6.4.2) coupling of 5,15-dibro(Fuhrhop et al., 1981). ~alladiu~copper-catalyzed mides with malononitrile anion gives the corresponding 5,15-dialkylideneporphyrin of bright green color. Its zinc complex tightly bound two axial pyridine rings, which was attributed to a greater positive charge inthe center of the electron-poor porphodimethene as compared to porphyrins (not shown) ( al., 1998). ~urthermore,porphyrins react like extremely electron-rich aromatic compounds, comparableto phenol, aniline, or pyrrole. Central metal ions, in particular zinc(I1)andcopper(II), further activatetheminelectrophilicsubstitution reactions. Typical examplesof the di- and te~achlorination,mono- and dinitration, and most important Vilsmeier mono meso-formylation are given (Scheme 6.4.3) (Bonnett and Stephenson, 1965). Vilsmeier formylation of tetrapheny1po~phyrins gives mono P-formyl products, which have been used to attach side chains to an imidazole end. Five coordinated metal complexes thus become accessible (not shown). The monoformyl porphyrins have been efficiently dimerized by titaniumcatalyzed reduction (McMurry condensation)to give disubstituted alkenes.The ted 6.4.4). S,S?-Butadiyneresulting products canbe cis- or t ~ ~ ~ s - o r i e n(Scheme bridged octaethylporphyrin dimers and heterodimers with different central metal ions have been produced by oxidative coupling of meso-acetylene porphyrins (not shown). Both covalent dimers show almost identical redox and spectroscopical behavior to the corresponding monomers ( h o l d and Heath, 1993; Senge et al., 1994). In another covalent porphyrin dimer two monocarboxylatetetraphenylporphyrin units are connected by a pentanediol diester linkage. The central zinc ions of this dimer clamp chiral diamines, and weak CD effects are induced (not shown) (Huanget al., 1998). Formyl groups can be efficiently elongated by Wittig reagents andmines. P-Formyl porphyrins as obtained from protoporphyrin derivatives (see below) are generally more reactive than meso-formyl analogs (Scheme 6.4.5) (Witte and Fuhrhop, 1975). The vinyl groupsof protoporphyrin canbe efficiently removedby heating inphenols (“deuteroporphy~n”).Hydrogenation to ethyl groups(“mesoporphyrin”) by catalytic hydrogenation takes place without attacking the methine using hydrogen perbridges. Oxidation toa secondary diol (‘~hemato~orphyrin~’) oxide or ozonization toform the diformyl compoundis possible in acidic media only. Diprotonation of the porphyrin raises its oxidation potential to a level reached neither by ozone nor by peroxide (Scheme 6.4.6) (Smith, 1975, 1979; Fuhrhop and Lehmann, 1984). A po~hyrin-speci~c reaction sequence of the propionic acid side chains consists of P-oxidation with thallium trifluoroacetate, followed by acid-catalyzed

and monomeric and dimeric byproducts

ming HNOdAcOH; 0"

cyclization to form an isocyclic ring. Standard reactions imply transfo~ationof the carboxyl groups to amines by Ho~mannor Gurtius degradation and condensation reactions. Steroid, carbohydrate, and other chiral esters and amides areof considerable interest in s y ~ n e t i chemistry c (Scheme 6.4.7) (Fischer andStern, 1940; Kenner et al., 1974). In chlorophyll chernistry,it is often appropriateto remove the ma~nesium ion, which is vulnerable to water, andthe phytol, which gives a waxy character

0

C1BH33-P(C6H5)3

+

+ n-BuLi C15H31

I

to the chlorophyll and prevents~rystalli~ation. The magnesium- and phytol-free ligand is called pheophorbide. The ester group on the isocyclic ring forms a pketoester functionality with the carbonyl group on the same ring. ~hlorophyll forms there an extremely reactive enolate anion under basicconditions, which is rapidly oxidized to a mixture of ill-de~ned,brown c o ~ ~ o u n in d sair (" is is avoided by sapo~i~cation and r e ~ o - ~ ~ a icondensatio~ sen tableand c ~ s ~ l l i nco~pounds e called chlorin e6 andpyS obtained,which are oftenthe chlorop e has three carboxyl groupson the so very water soluble; the othe has a reactive isocyclic ring with a reactive keto group on it.If one leaves the hytol chain on the molecule, onehas pheophyti~a, which is useful in ~ e ~ b r a work n e (Scheme6.4.8) (Fischer, 1940; Tre~bs,1971).

(l) K O H ~ e O H / P y ~ d i n e (11) partial reesterfication (Ill) S O C I ~ (W)~ ~ ~ ~ C ~ ~ M e / N a H (V)C F S O O H (VI) reesterfication

I

COOMe

~hodoporphyrin XV dimethyl ester

(l) 2 TI(CF&00)3 (11) h*v (111) SOz/HCI

6OOMe

(I)c. H2SOJ65X oleum

(11) r ~ s t e ~ ~ t i o n

\

(l) NaOH (11) NEt3/CIOOC2H5 (Ill) NEt $1

~

~

(111) MeOH/A ( W ) HCI (V) KOH

~

~lu~samine

~

~

~

O

~

H

~

R = Me : Pheaph~in-a R = CHO : Pheoph~in-b

R = Me : Chlorin-e~tri~ethyiester R = CHO : Rhodin-~~ trimethylester

The prominent electronic transition of porphyrins and me ta llo p o ~ h y ~ is n sthe n+n* ~ a i i s i t i oassociated ~ with the p o ~ h y r i nring. In general free base porphyrins, chlorins, and some bile pigments show strong fluorescence at room temperature and both fluorescence and phosphorescence in rigid glasses. In Table n sgeneral interest 6.5.1 the lurninescence properties of a few m etallo p o ~ h y ~of are s u m m ~ z e dFree . base p o ~ h y rin susually show fluorescence peaks around kmaX = 650 nm co~espondingto singlet energies of 45 kcaVmol and phos~horescence near 750 nm or 40 kcal/mol triplet energies. The energies of excited metall o p o ~ h y ~ are n s somewhat higher than for the free bases and therefore occur at s two axial lower wavelengt~s(Hopf and itt ten, 1975). Silicon p o ~ h y rin with alkyl ligands show a nonplanar ring and produce diradicals with a lifetime of several days upon excitation (Zheng et al., 1998).

Typical Effectsof Central Metal Ions to the Luminescence ~ o p e ~ iof es Porphyrins at Room Temperature

inesc~nt:Ni(II).Sn(II),Ru(II)L~yCu(II),Co(~) ~o~escence only: free b a s e , ~ g ( ~ ) , Z n ( I I ) , S n ( I ~ ) , ~ ~ ( ~ I ) d(~)¶Pt(II),Ru(~)~~ L = Axial ligand.

Table 6.5.1 exemplifies the general findings that only closed shell metals allow fluorescence at room temperature, with the exception of the ~ t h e ~ u m ( I 1 ) carbonyl complex. Very few redox active central ionslet phosphorescence happen, and only Pd, Pt,and Ru(I1)CO give both. Ru(I1) with ligands or Ru(II1) are nonlu~nescent* These latter complexes show very a small singlet-triplet separation, which makes them attractive as sensitizers and as possible energy-conversion systems, High intersystem crossing yields together with long triplet lifetimes are sometimes accompanied by efficient energy transfer to electron acceptors having x40 kcal/mol triplet energies, R u t h e ~ u ~ ~ o ~ h yand ~ nthe a t high e potential ~ t h e n i u mtrisbipy~dinatep h o t o c h e ~ stherefore t~ allow the catalysis of a large variety of ~hotoreactions.A small selectionis given in the next section. One of the most prominent energy transfer processes involving po~hyrin triplets involvesthe conversion of triplet oxygento singlet oxygen: 3p*

+30,

”+OP

+ lo,*

The po~hyrin-s~nsitized oxygenationhasbeenusedin ~oodw~d’s c~orophyllsynthesis to convert an exocyclic double bond to an aldehyde and a ketone group (see Sec. 6.3) and in the photooxygenationof magnesium and zinc p o ~ h y ~ n a t etos forrn fo~ylbiliver~inates. A dioxetane including a methine bridge and an a-pyrrole carbon atom is formed in the latter case and the cyclic peroxide opens spontaneously. Isosbestic points of spectra taken in the time course of photooxygenation indicate that only one product, namely zinc forrnylbil~verdinate,is formed in ~uantitativeyield (Figure 6.5.1). The zinc complex of substituted 5 - h y ~ o x y - o c t a e t h y l p o ~ ~reacts y ~ n in a ~ o m ~ l e t e ~ y d i ~manner. e r e n t It loses carbon monoxide uponi~adiationand cyclizes to forrn anoxoniapo~hyrin.The TJVlvis spectrum of the zinc oxoniapor(E2 30,000) at 400 and 660 pbyrin is of the polyene type with two strong bands nm (Fuhrho~et al.,1975),but the Rmethineproton signals appezrabove 6 = 9 ppm,indicatingstrongringcUponreversible (!)hy~olysis,azinc inate is f o ~ e from d the o x o n i a p o ~ h y ~ n .l) (Fuhrhop and ,1977). The x-ray structure shows an essen ligand and an axial water molecule (seeFig. 6.5.1). *

*

h * v10

ur Irradiation of benzenesolutions of magnesiumoctaethylporphyrinateunder air leads to the short-lived dioxetane and thento the fo~ylbiliverdinatein quantitative yield as shown by the spectroscopic changes within 30 minutes. The zinc ion in the center carries an axial pyridine or waterligand. Upon acidification withHG1, this ligand is removedanda bis-helical dimer is formedinquantitativeyield.Back-titrationwith sodium hydroxide reforms the monomer. (From Wasser and Fuhrhop, 1973; S~uc~eier et al., 1976.) ~ ~ o r o p h yoxidation ll in nature also leads to formylbiliverelin derivatives (Wautler and Matile, 1999).

Amphiphilic porphyrins, e.g., hematoporphyrins (photofrin) or chlorin e6 (aspartyl amide) derivatives, are used in photodynamic therapy of tumors using red laser light. They tend to accumulate in tumor cells when injected intravenously, can be regioselectively irradiated via thin optical fibers, and then produce toxic singlet oxygen upon irradiation. Most useful are porphyrins with

.th absorptions, because wavelengths > 600 m better penetrate duct of photosynthetic water splitting, y ~amines n sto by the ~ ~ o t o h y ~ o ~ e n aoft tin(1V) i o n ~ ~ ~ hwith lorins and their reversible ~ehydrogellationwith colloidal ms haveusedviologenradicalsa ors between porcatalytic hydrogen ~roduction. (1V)po~hyrinate 10,000 molecules molecule can set free about versibly bleached ( cheme 6.5.2) ( F u ~ h and o~ The ultrafastelectrontransferfromchlorophyll or ~acte~ochlorophyll '

through lipid membranes to quinones (30 ps) and the very slow (300 ps) recombination of charges observed in biological photosyntheses have no c o u n t e in ~~ synthetic systems so far. There have, however, been many successful attempts to realize fast vectorial, light-induced charge separations. A few covalent porphyrin-quinone model systems are outlined in Chapter 7. Instead of quinones one may use other p o ~ h y ~ as n selectron acceptors. Prominent covalent p o ~ h y r i ndimers are connected by ~ e ~ ~ - a c e t y l eunits. ne Strong lateral exciton effects are observed in electronic spectra (Fig. 6.5.2) (Officer et al., 1996; Ruhlmann and Giraudeau, 1996). High yields (>95%) of energy transfer, an impo~antrequisite for light

R

R

CuCVnVlEDAf CHzCl$o~

-.--

n Monomer

Polymer

R=

Electronic spectra o f ~ o ~ h dimers y ~ nconnected by acetylenic links.

I

harvesting, was achieved incovalent porphyrin mays with diarylethyne linkers (Scheme 6.5.3). These arrays show a 20 A separation of po~hyrins,no strong exciton coupling, and high photostability. The electronic interaction is weak, and the oxidation potentials of zinc porphyrins in dimers and trimers areesseneklectron hopping in all monocation radicals is rapid, Other light-h~estingarrays include eight boron-dipy~nsubstituents (Li et al., 1998) and four catechol-type dendrimers (Jiang and Aida,1998) around one central, tetraphenyl-type porphyrin. Fast energy transfer from the periphery to the p o ~ h y r i ncore was invariably observed.It should, however, be d i ~ c u lto t collect stored energy from the center of the dense dendrimer sphere. A linear carotene-po~hy~n-fullerene triadgives a chargeseparatedcarotene(+)-porphyrin-fullerene(-.) state, which decays in 170 ns to a carotene triplet state at room temperature. At 5 an applied magnetic field enhancesthe lifetime of the charge separated state (Kuciauskaset al., 1998).

odel ~~~~-tetraphenylporphyrins are ~onomericin solution and produce a nmow Soret band with a half-width of 15 nm. ~ - O c t a a l ~ y l p o ~ h ye.g., ~ n soc, taethylporphyrin or protopo~hyrinderivatives, dimerize in solution down to a concentration of about M and show a Soret band witha half-width of 30 m,

The typical porphyrin dimer consists of two parallel,face-to-facemacrocycles in which an electron-rich pyrrole ringof one porphyrin sits atop the electron-poor cavity of its neighbor (see Fig. 6.2.5). Only in porphyrin cation radical dimers (binding energy: 17.5 kcallmol) do the porphyrin chromophores cover each other completely. An extremely strong charge transfer band at 900 nm is observed for these dimers, and theface-to-face dimers are diamagnetic (Fig. 6.6.1) (Fuhrhop et al., 19’72; Song et al., 1989). In aqueous solution or in water (methanol 1:l), tetracationic porphyrins form molecular assemblies with tetraanionic porphyrins, e.g., ~es~-tetraphenylsulfonato-with ~es~-tetr~ethylpyridinium porphyrin.Suchassembliesare, however, ill-defined with respect to structure and aggregation number because both surfaces of the chromophores behave identically. They tendto polymerize and precipitate, Only when stereochemically fitting porphyrins are combined by charge interactions and polyme~~ation is prevented by alkyl substituents does one obtain well-defined heterodimers. This has been realized with an isomer mixture of four ~ - p y r i d i n i u m - ~ t hporphyrins yl (solubilityE 10-1 tetraphenylsulfonato porphyrin (same solubility) (Scheme 6.6.1). In this case, the porphyrins are forcedinto close proximity by four charge-charge interactions and into a face-to-face position by the ~ a n g e m e nof t cllarges. The inability of

A

olecular s t ~ c t u r of e zinc porphy~natecation radical dimers in solution and the ~ ~ / v ispectra s ~ ofI the ~ zinc o c t a e ~ y l p o ~ h y ~ ncation a t e radical(- --)and its diamagnetic dimer(”----).(From Fuhrhop et al., 1972; Songet al., 1989.)

M = 2H or Cu(ll) or Zn(l1) or Sn(lV)CI2

the four isomeric tetracation-tetra~ionpairs to crystallize makes even the elecand thereby ~ 1 0 clean ~ s spectroneutaldimerverywater soluble (2 x on of the electroneutral trophotometry of the d i ~ e ~ ~ a tprocess. ion N dimers occurs in water below concentrations Fu~hermore,the binding ence~uenching occurs when a slight constant is hi h lo7 M) and total flu 4" is added to a 10 solution of the free baseisomermix(Endisch et al., 1996).

0

A *

.

-3

0

..

.

* i.

I)

I)

*

. . .. 0

*

.

0.0 500

600

500

600

rel. fiuomscence intensity

(a) Spectrophotomet~ctitration curve of the tetracationic porphy~ns e tetraanionic porphyrinate TPPS”- (5)in water/meth~ol1:1. Pure water can also be used as a solvent, but the absorption bands then are slightly broader, the isosbetic less sharp. (b) The co~espondingJob plot. (c) Fluorometric titration of the isomer mixture with CuWPS4 in water.The assumed molecular structure of the dirner is also indicated. (From Endisch et al., 1996.)

This quenching process has been applied in measurements of vesicle entrapment volumes and permeability. One of the two te~aionicporphyrin components is sonicatedtogetherwiththe vesicle-fodng component,e.g.,egg lecithi~cholesterol1:1, and the fluorescence of the solution is measured (usually after a dilution of 1:100). The copper-containing counterion is then added in a -foldexcess.Only the fluorescence of the porphyrininbulkwater is quenched; the vesicle’s interior is not reachedby the chargedpo~hyrins.The ratio between the remaining fluorescenceis then the same as the ratio between the internal vesicle and bulk volumes. Addition of all kinds of membrane-disrupting agents (e.g., harpoons) (seeSec. 2.7) leads to a leakiness of the vesicles and can be directly detected by the disappearance of the remaining fluorescence (Fig. 6.6.3). Efficient harpoons are usually ~ p h i p h i l e swith a bulky hydrophobic headgroup, which dissolves in the membrane and disrupts its s t ~ c t u r eand long f moderate water solubility, e.g., polyethyleneglycol, orphyrins substituted with a guanine side chain interact in aprotic solvents or liquid crystals with cytosine-substituted quinonesor another porphyrin to form a 1:1 lateral heterodimeric system (Scheme 6.6.2). Such pairs photoinduced charge separations with lifetimesof a few rnicroseconds rotopo~hyrinIX and its amphiphi~icderivatives have not been crystalater or any other solvent so far, although hydrophobic derivatives, e.g., the ~imethylester,rapidly crystallize from chlorofor~methanol. p o ~ h y r i nIX rather gives fibers in aqueous solution and shows a split Soret band at 450 and 370 nm. It has, however, not been possible to isolate these fibers and to produce electron micrographs of defined molecular assemblies. This situation improves drastically if the anioni ropionate side chains are replaced by cationic propyl ammonium groups. th the ammonium salts and the half-neutralized amine-ammonium forms produce well-develo ed fibers. n the case of the diammonium salts (p 4), a short-wavelength 3’70 nm) and the electron micrograph ( ellar fiber, diameter: a 7 rphyrin stack. Typical lengths are in e pm range, which corre9, the fibers r e ~ a n g to e 3’70 nm, and electron miS now show shorter vesicular tubules instead of micellar rods. between amine and a ~ o n i u m groups obviously has chan to a lateral arran~eme nm signal upon flashing, inpresumably resulting from lght-in~ucedcharge separation. of chiral fibers, and strong

0~ t r o n ~~i uy o r e s c ~porphyrin nt

harpoon

nt copper p o r p ~ y ~ n (a) Model of the vesicle permeability test using fluorescence quenching in a p o ~ h y heterodimer ~n made of two charged porphyrins, which (b) cannot pass the intact vesicle membrane. (c) Typical pathway of the corresponding hetero~i~erization through a vesicle membrane perturbed by a membrane disrupter. (Endisch et al., 1999.)

of metal ions. There it was lithium-sodium in the reversed bilayer, here it is copper-zinc in the porphyrin center of a normal bilayer (Fig. 4.6.5). meso-Tetra-pphenolpor~hyrinhas been tetraglycosylated and shows long-wavel~ngt~ shifts of the Soret band upon titration with heavy metal ions ( ohata et al., 1994; et al., 1995). Two ~ i a substitue~ts 2 in tin(1V) porphyrinates prevent stacking. case of basic cyanate ions, however, the nitrogen atoms of the axiaE ligands can be laterally connected with small carboxylic acids dissolved in bulk water. Using

1.0

0.0

300

~ 0 0 500

~ 0 0

7

Acid titration of the a m i n o p o ~ h y ~ n c h athe n ~ elateral s vesicular assemly with a 450 m Soret band into a micellar stack with a 380 nm Soret band. ( ~ u ~ h o p , indig, et al., 1993, 1994.)

i7H

O 'H

CDspectra of zinc-and copper-po~hyrinateglucona~idesin water. (Fuhrhop, Demoulin, etal.,3992).

and L-lactic acids,one obtains mirror imageCD spectra (Fig.6.6.6). The Soret bandat 470 nmindicates the expected lateral assembly,whichfluoresces strongly. Electron micrographs show thick tubules made of rolled-up monolayers (notshown). If amixture of ~ e ~ ~ - t e ~ a p y r i d i nand i u m~e~~-tetraphenylsulfonato porphyrins is adsorbed to the surfaces of poly-D- or ~Oly-L-~lutamates, one obseves strongCD effects at pH 3.6 due to the chiralityof the glutamate helix. If the helix is destroyed by raising the pH to 12, the porphyrin CD effect remains, indicating that the shape of the mold has been retained. The porphyrin assembly shows a memory effect (Bellacchio et al., 1998). Isomer 11of ~-tetraethyl--tetrapyridinyl porphyrin is the least soluble of all four isomers (1 X 10". M). It is a bola~phiphilewith h y ~ o p ~ lnorth i c and south edges and hy~ophobiceast and west edges (see Scheme 66.1). In dilute aqueous hydrochloric acid it dissolves as a tetracation (no protonation at the pyrrolenine nitrogens!) at pH 1 and assembles to form monolayered leaflets at to bind, redoxpH 2.5 (Fig. 4.67). These leaflets provide the unique opportunity active anions, e.g., Fe(CN)," or ascorbate, on the cationic surfaces and porphyrin anions to the porphyrin faces. Electron donors and traps can then be affixed to the fluorescing porphyrin assemblyin water or on electrode surfaces Similar leaflets have been made at neutral pH with N-methylated pyridinium substituents and various counterions. Benzene with six porphyrin substituents, each bearing one long alkyl chain, assemble on carbon or glass substrates to

D-

Model and CD -spectra of t i n ( I ~ ) - p o ~ h ~ ~ n - d i c y ainn aaqueous te solution. L-Lactic acid functions as a connecting link betweenaxial the ligands. (Rosengarten, 1998).

mesoscopic rings with a diameter of about 1 mm (not shown) ( ~ i e ~ a et n sal., 1998). ~hlorophyllforms a highly organized cyclic assembly in biological protein matrices. In vitro, one finds a~alogousassemblies in organic solvents, but electronmicrographs do notshowtheimagespredictedfromfluorescence, NMR, and scattering data. Terrace-like assemblies with water bridges between the central magnesiumions and the carbonyl group of the isocyclic ring consti-

3

Monolayer porphyrin leaflets in water made of 1. The leaflets are rolled up here becauseof salt effects. (a) Electronic (- --) and fluorescence excitation ( ” 1 spectra (b) electron micrograph, and (c) model. (From Endisch et al., 1996.)

tute the most interesting assembly and are also shown in the “magic dimer” (Figure 6.6.8). Similar dimers have been found in active centers of bacterial and plant photosynthesis. ~ o ~ h y and ~ nmetalloporphy~ns s have also been integrated and functionalized for investigations in vesicle membranes. ~ o l y e t h y l e n e ~ m i n ~ - lmani~ed ganese porphyrins, for example, have been used for transmembrane electron transfer reactions. The water-soluble, cross-linked polymer was combined via hy~ocarbon spacers of limited lengths with the manganese(~1~)mesote~aphenyl-typeporphyrins. The porphyrin was dissolved on both sides of a large egg lecithin vesicle membrane. Since the cross-linked polymer could not enter the membrane, the minimal distance between inside and outside manganese porphyrins was regulatedby the length of the spacer assumed in an extended conformation and varied between zero and 28 nly in the case where 4 A could a reduced outside indigothe twop o ~ h y ~ were n s separated by zero or

Models of two chlorophyll assemblies in apolar environment as deduced from I ~ - spectroscopic ~ M ~ shifts: (a) the terrace-like assembly, (b)the magic dimer. (From Scheerand Katz, 1995; Vernon and Seely, 1966.)

tetrasulfonic acid dye reduce ferricyanideions, which were entrapped in the inner vesicle volume.At a larger distance between the porphyrin centers,no acceleration of electron transportwasobserved.Electrontunnelingbetweenthe manganese porphyrins thus occurred only over a distance of 4 A at A Eoof t-350 mV between ferricyanide and the manganese porphyrins and wasthere already 10 times less efficient thanat a distanceof zero A (Dannhauser et al., 1986).

icket fence iron(I1) porphyrins with a hydrophobic crownof tert-butyl groups above the porphyrin centers cannot add imidazole on the protected side.The central iron(I1) ion ther~foreonly forms ~ve-coor~nated a complex with thisligand. Oxygen, on the other hand, is soluble in the hydrophobic center and a stable, crystallineoxyheme has beenobtained. Its x-raystructureshowsanend-on bound oxygen molecule in the heme center at an angleof about 45".This finding confirmesPauling's40-year-old structural prediction of oxygenbindingand backbinding to form Fe=Q a double bond (Figure 6.7. l), which camefrom magnetic measurements indicating a diamagnetic oxyhemoglobin (Collman, 1977; GerothanassisandMomenteau, 1987; Gerothanassis et al.,1989).Analogous tetra-arylurea atropisomers bindchloride anions in DMSO (Jagessar et al., 1998) and stiff ~ , ~ - d i c ~ b o x y l afixate t e s twoextra iron ions above the porphyrin plane (Zheng et al., 1998) Cytochrome P-450 is a familyof about 300 heme enzymes, which is used to activate oxygen for theburning processes of respiration or,in chemical terms, the oxidation of nonactivated hydrocarbon.The carbon monoxide adduct of P-450 shows aSoret band at450 nm, whichgave the enzyme its name. These enzymes catalyze monooxy~enationreactions by transfer of a single oxygen atom from a ferry1 oxygen (Fe=O) adduct of the heme unit. At first a substrate RH binds to the ferric form, which is reduced to ferrous gen then addsto form a ferrous dioxygen species. Model compounds show it to be end-on bound (Schappacher et an spectra show the Q-Q stretching frequency at 1140 cm-l.A second oneron reduction leads to a dou t ground state ferrous dioxygen species, the structure of which is not known. th oxygen complexes produce a split Soret band due to mixing of sulfur d-orbitals and porphyrin a-orbitals. The reduced dioxygen complex then adds two protons to form the ferryl(V) c o ~ p o u n dand water. The axial ligand of the P-450 heme is a cysteine sulfur, which catalyzes the heterolytic cleavage of oxygen molecules inapolar environments, and water is split OB.The c~ntraliron ion becomes thereby oxidizedto the Fe(V)=Q state under the reductive conditions of living organisms. Alternativelythe porphyrin

ter

W

X-ray structureof picket fence heme with heme-coordinated oxygen and ~-nlethylimidazole,Nitrogen atoms are black, the oxygen molecules are white.

ligand may also be oxidized to a n-cation radical and the central iron atom may be Fe(V)=O. The diatomic unit can then be written as PEe(II1)0"f-~.P+Fe(IV) 0- f-~.PFe(V) 02-or PFe(V)=O, Such a compoundhas been characterized in the case of an oxidation-inert perhalogenated iron(II1) p o r p ~ y ~ n a t(Scheme e 6.7.1). The iron-bound oxygen atom is of comparable reactivity to the carbon atoms of carbenes and is therefore called oxenoid oxygen, synonymous to atomic oxygen.The most typical and spectacular reaction of such oxygen atoms is the hydroxylation of nonactivated alkanes to alcohols (Fig. 6.7.2).This reaction has not yet been realized in model systems. Iodosobenzene, OIG,H,, as a distantly related model of oxygen shows some activation by porphyrins and then gives some reactions similar to atomicoxygen (Ogoshi andMizutani, 1996)

F"

F 0

0

OH

+

~Fe~'i(~or)]

-

heterolysis

[F~lv=O(~or)]o'

+

OH

Practically all metal ionsof the periodic system have been introduced into the electron-rich center of the porphyrin macrocycle, and a large variety of strange reactions has been discovered.We give examplesof ruthenium, rhodium, and manganese complexes. Ruthenium(I1) octa~thylporphyrindimerizes upon in vacuo sublimation and a Ru=Ru double bond is formed. The red, diamagnetic monomer becomes green, showing a broad absorption stretching from 600 to 800 nm and paramagnetic (S=l). Upon oxidation (-0.11 V vs. AglAgCl) a radical is formed, which produces an upfield shift in the lH-NMR spectrum, but only little line broadening. The unpaired electron is presumably located between the metal ions. Removal of another electron [Oh2 V vs. Ag/Ag(I)] leads to a Ru-Ru triple bond. "he chemical shift of the methylene proton pointing towards the Rushifts from 26 to 9 and then to4.5 ppm (Fig. 6.7.3). Additionof oxygen, water, or pyridine cleave the Ru-Ru bond (Antipas et al., 1978; Collman et al., 1981, 1986, 1988, 1993; Balch et al., 1988). A ~thenium(TPP)-molybdenum(~~P) heterometallic and heteroporphyrin dimer has also been crystallized, and the metal-metal distance is as short as 0.22 nm corresponding to a bond order of 3. The a s y ~ e ~ cunit a l of the crystal shows twodistinct conformers: one has totally eclipsed porphyrin macrocycles,in the other they are staggeredat 45" (not shown) (Collmanet al., 1998).

H

cys alkane.

Reaction scheme of the cytochrome P-45O-catalyzed hydroxylationof an

o d i u m ( ~ ) p o ~ h y ~ n aform t e s similar dimers, which split upon heating add methane as well as hydrogen by splitting single bonds (Scheme goshi andM i z u t a ~1996). , ganese(II1) porphyrinates show electronic spectra and redox behavig. 6.4.3), which indicate strong elect~onic inte~actions between the metal d- and ligand n-orbitals. Manganese porphyrins catalyze the stereoselective epoxidation of olefins in presence of hydrogen peroxide, oxygen, and r oxidants. Mn(V) = 0 complexes act as catalytic oxidants. analogs produce only peroxides, not molecular oxygen. A. few typical oxidation reactions realized with manganese p o r ~ h ~ are ~ n summas rized in Scheme 6.7.3 (Woo and Goll, 1989; A.rasasin~hamet al., 1993; orokin et al., 1993).

x 3

n

t

4

m

l H - ~ spectra ~ R or ruthenium porphyrin dimers. Top: (~u(II)OE~), in in~CD,Cl,; bottom: (Ru(III)OE~),in CD,CI,. benzene-d,; middle: Ru(1I)OE~R u ( ~ ) O E

+H2

-- base

' H

rate lirnitin

"oxene" insertion

oxidation

radical chain products

+

rearranged epoxide products

rearran~ed products

Porphyrin monomers and organized monolayers on conducting or semiconducting surfaces provide the unique possibility to convert the energy of the excited state directly into photocurrents. Furthermore,the large porphyrin surface allows

the construction of large gaps in lipid membranes on surfaces, which may be applied as analogsof enzyme clefts (compare with Fig.3.6.2).Third, porphyrin multilayers have been used ascatalytic electrode surfaces. Typical systemswill be introduced here in the following order: (1) monomers, (2) pure monolayers, (3)monomers with admixed lipids, (4) reactive multilayers. ~ubpha$eswill be arranged inthe following order: water, gold, graphite, silicon, silicate.

he isothiocyanate of the monoanilino porphyrin (Scheme 6.8.1) binds covaently in the formof single molecules to gold( 11l).The porphyrin moleculeslie flaton the surface.revealedpreferentiallocalizations on thissurfaceleadto an appearence of “string of pearls.” The isothiocyanate bond to gold is heat and oxidationr e s i s ~ n(Tao ~ et al., 1995). Tetracatio~c~e~o-tetramet~ylpyridinium porphyrin and the anionic lipid ~-~imy~stoylphosphatidic acid mixed in a ratio of 1:4 form a monolayer on glass. It shows the spectrum of porphyrin monomers, again lying flat on the surface, whereas on water surfaces they appear as stacked dimers. Only those molecules that had been directly attached to the anionic lipid layer on water were transferred to the glass plate (Fig. 6.8.1).The tendency of porphyrin monomers

N

li

Porphyrins tend to form dimers on water surfaces. Lipid monolayers may be superposed. Upon transfer to solid surfaces, of half the porphyrins are frequently lost.

to lie flat on solid surfacesis, however, overcomeby an admixture of long alkanes, e.g.,CH3(CH*)~~CH3, to LB films. The alkane monolayers enforce an almost perpendicular orientationof porphyrin monomers (Azumi, 1995a,b).

~orphyrinswith small substituents,e.g., four meso-phenyl or eightP-ethyl groups, do not form stable films on water in pure form. Such porphyrins have, however, been dissolved either in surface monolayers of fatty acids or similar lipids: at low surface pressure they are often perfectly integrated. When the domains of the lipid monolayers merge to f o m a solid film, the porphyrins are squeezed outof the monolayer and form microcrystallites at the edges of the old domains. Calculated molecular areas of hydrophobic porphyrins lying flat on the water surface vary from 60 for porphin bearing only hydrogen atoms in the periphery to 250 A*in the case of the tetracarotenoid ester of Figure 5.5.3. The hydrophobic porphyrin edge in a perpendicul~orientation has a surface of about 50 A2. If one calculates the surface area of the flat-lying porphyrin under

consideration and measures the experimental surface area, then the angle 0 of the porphyrin macrocycle against the water surface is: arc cos 0 = model surface area in A2 n h u s 50 A2 divided by the experimental surface area A2 minus 50 A2.~mphiphilicporphyrins often start flat on the surface at low surface pressures and then reach 0 values of 70-85" at highs pressures. Symmetrical porphyrins with four charges remain flat on the surface but may form stacked dimers or trimers at higher pressures. Long-chain substituents, in particular those that carry a water-soluble end group, are often adequate to stabilize pure orthogonal porphyrinfilms.Extrapolatedmolecularareasarethenusually around 50 A2, and collapse occurs at pressures around 40 InWm. Films maintained at surface pressures above 15 mN/m tend to develop visible folds and wrinkles over time (ca. 30 min), indicating a slow collapse of the monolayers. This collapse may be inhibited by addition of fitting, more flexible su~actants, e.g., 4-(l-octyloxy)ben~aldehydein a molar 1:l ratio (Schick et a1.,1989). The exciton effects observed inabsorption spectra of porphyrin monolayers are often different from those in stacked or lateral fibrous assemblies. One usually findsexclusively long-wavelength shifts of the Soret bands or no effect at all. Porphyrins either lie parallel to each other on the subphase (no shift) or are oriented in a slipped stack-of-cards configuration tilted by 10-20" angles against the subphase surfaces, whichproduce the observed 20-100nm red shifts (Fig 6.8.2). ~e~~-Te~aphenylporphyrins, e.g., pyridiniumand carboxylate salts,lie flat on the water surface, and heterodimers are formed ina lateral o~entationin 1:1

Typical configurationof a~phiphilicporphyrin stacks on a water surface. (FromSchick et d.,1989.)

mixtures. No exciton interactions are detectable if one builds up monolayers of alternating porphyrins, although mixed LB layers of copper complexes of rnesopyridi~umandcarboxylateporphyrinatesshowCu-Cuinteractionsin ESR spectra, similar monolayers made of free base and copper porphyrin mixtures fluorescestrongly.Theinterchromophoredistance is toolarge for fluorescence quenching, but the d-electrons of the copper ions still interact. The critical distance for fluorescence-quenching inorthogonal porphyrin stacks lies obviously in the range between and 6 8 A, but no theory is available to rationalize this. Cobalt rneso-~-anilinopo~hyrins with one or two long-chain W-thiol carboxamide side chains are bound to gold surfaces as filmswithin a tilted or parallel orientation (see Figs. 6.8.1 and 6.8.2). Theyreplace butanthiolate films on gold within about 18 hours. Mixed films are obtained in shorter periods. The tilted, single-chain porphyrin shows exciton splitting of the Soret band, whereas for the double-bonded disulfide only a small red shift was observed (Tao et al., 1995). Myoglobin and acytochrome P450 bound to gold electrodes are electroactive. A second monolayer shows third a of the electroactivity of the first; a third monolayer is redox-inactive. Inert separating polymers such as poly(styrenesu1fonate) or poly(dimethy1 diallyl) a m o n i u m chloride do not substantially inhibit electron transfer if the heme protein is separated by less than 4 nm (Lvov et al., 1998).

~ is o ~a~mema s~ ~ The photosynthetic reaction center of ~ ~ o ~ o ~ s e ~ ~vo r n brane-spanning protein.A bacteriochlorophyll (BChl)special pair is located in a hydrophobic region inside the membrane, another BChl in a more hydrop~lic environment, and a magnesium-freebacte~opheophytin(BPh) comes next,The electron-accepting menaquinone (Q) is on the outer side of the membrane at a distance of about 30 A from the BChl pair.A cytochrome is connected to the pair outside the membrane. The electron transfer from the BChl pair to the quinone takes less than a nanosecond, the back-reaction about 300 p . Energy has been lost in the forward reaction, and the stepwiseway back via electron conducting dyes is not available (Deisenhoferet al., 1985).This light-induced vectorialelectron transport is the basis of photosynthesis. The electron hole on the BChl and the electron on the semiquinone are able to undergo further redox reactions; the light energy of the sunis converted to chemical energy (Fig. 6.9.1). This biological membrane systemis ideal for porphyrin-based energyconversion systems. For energy storage by p~otoinducedvectorial charge separation, the electron mustbe removed faster from the excited porphyrin than about 10 PS in order to avoid deactivation. Thereafter,the electron should be kept at a

~

s

Schematic drawingof the photosynthetic reaction center within the membrane of ~ ~ o d o - ~ s e u d viridis ~ ~ o nand ~ approximate s times of electron-transfer reactions.

high energy to allow for useful dark reactions. The distance of 30 A from the to the thicknessof a typichlorophyll donor to the quinone acceptor corresponds cal hy~ophobicbilayer minusthe radii of enclosed dyesat the membrane's surface. It shouldtherefore be possibletorealizecharge-separatingsystems in bilayers (see Fig. 1.5.9), where metalloporphyrins occupy the inner surface, quinone the outer surface, and somehy~ophobicporphyrins are localized in the membrane center. Such organized assemblies have been realized, but they are not successful. Invariably the back-reaction is found to be approximately as fast as charge separation; energy conversion has been impossible so far (Siggel et al., 198'7). A more promisingway would use a porphyrin producing hydrogen directly in solution using a Shilov-type system (see Scheme 6.5.2). The oxidative part, which should give oxygen from water as in plant photosynthesis, would be then

separated byan appropriate membrane system. An antenna leading the light quanta to the reactive porphyrin center would also be required for efficiency (Fig. 6.9.2). Ll3 chlorophyllmonolayers(25 d i m ; 1.4. nm2/molecule) on Pt electrodes showed low photoactivity, possibly caused by a quenching of excited states by the metal electrodeor by total reversibility of electron exchange. Addition of electron acceptors, e.g., quinones, had no effect. The optically transparent tin oxide semiconductor electrode proves to be a much better subphase for the generation of photocurrents. Chlorophyll-coated SnO, combined with a platinum electrode gave approximately 100 nA/cm2. Similar results were obtained with photovoltaic systems of the form mercury drople~uffersolutio~chlorophylla monolayer/electronacceptormonolayer/aluminum(Fig.6.9.3). The quantum yield of such monolayer arrangements never exceededIOm3in any of these systems and is thus far away from competitive inorganic semiconductor cells (Norris and Meisel, 1989). Better photovoltaic cells are based on thicker solid porphyrin filmsaluon ninum surfaces. The porphyrin acts as organic semiconductor may and be of the N-type (~e~o-tetraphenyl porphyrin) or the n;-type (zinc ~e~o-tetraphenyl porphyrin). This means that organic n;-njunctions canbe made solely from porphyrin parts. As a cell one often uses the sandwich type [metal (l)/porphyri~metal(2)~ or ( M 1 ~ ~imersed 2 ) in an aqueous solutionof a redox pair, e.g., KI/I2. is usually aluminum, metal2 may be indium-tin oxide (ITO),Pt, or Au. "he current passing fromA1 to IT0 (or Au or Pt) through thecell is taken to be positive. The action spectraof short-circuit photocurrentsfor the I T 0 ~ ~ y P / cell A l suggest that only the light absorbed near I the T0 contact is effective in producing charge carriers and that a high resistance contact (Schottky barrier) is formed at the

Hy~otheticalmodel of a synkinetic system producing hydrogen and oxygen from water andlight. (From Balzani and Scandola, 1996.)

1111111111111111

TTTTTTTTTTTTTTTT 1111111111111111

vacuum level

0

7-2

conduction band

Chl

Model of an Al-quinone(A)-chlorophyll a@)-Hg photovolt~ccell. Hg electrodes give perfect contact; oxide-coatedA1 does not quench excitedstates.

e. The photocu~entincreases in the presence of hydrogen as current obtained in vacuo and decreases under air, "his means S up electrons from A1 and carries them to It acts as an n-type and, produces more r (Yamashita et al., 1989).ZnTPP, on the o photocurrent in anIT0 (or Au or ~ ) / ~ n T P P / A cell l if air is present. The oxidizable zinc complexis therefore an;-type semiconductor.The photocu~entflows, of course, alwaysin the same directionfrom A1 t ~ o u g hTPyP or ZnTPP toIT0. If both po~hyrinsareusedtopreparean IT0/ZnTP Al s ~ d w i c hcell, the organic K-njunction leads to shortstrong vacuo asin as the cu~entswere the in presence of pure ecomponents. S , phthalocyanines, dimethylviologen, and other dyes can be ded other electrode materialsysicalvapordeposition,spinlytic micelle dis~ption(E Only the lastprocedure is of chemical interest, and it is also the most efficient. Aqueous micellar solutions of the dye, e.g. ,zinc t e t r a ~ ~ e n y l p o ~ h y ~ nina t11 e-ferrocenyl-undec~lt~deca-eth, yleneglycol ether (EFPE6) are obtained by sonication, stirring, and c e ~ ~ f u g a tion. Theclear solution is electrolyzed at0.50 V vs. SCE to give porphyrin films

of 0.1-0.6 pm thickness on ITO; scanning electron micrographs show rod-like crystals of about 500 nm in length. The film forrnation very likely involves oxidation of ferrocene moieties to Fe2+and subsequent destruction of the micelles

(Fig. 6.9.4).A sandwich electrodeis assembled with aluminumor platinum, and the photocurrents obtained reach several hundred nanoamperes. ZnTPP on layers IT0 obtained by EMD methods are about 0.14.3 pm thick and quite uniform under the electron microscope. They show narrow-lined UVlvis spectra. It is likely that in caseof the TPyP not onlyis the ease of reduction of the porphyrin macrocycle important, but the pyridyl rings (at least are three necessary) give extra anion stabilization in the course of n-type conductance. Dramatic increases in the quantum yields of photovoltaic cells of ITO/zinc (~-decoxyethy1)porphyri~TO wereobservedwhen a moltenporphyrin wax was solidified in the presenceof an applied electric field. Space charges or ions are thus frozenin, polar molecules are aligned, and charge separation and transport promoted. Short-circuit currents were increased by a factor of >l0 and quantum efficiencies 4, (electrons per incident photon) of up to 8.4% were obtained with400 nm light. Without applicationof a bias voltageof 2000 V/cm, 4, was only0.7%. Inactivated cells could be repaired several timesby heating-cooling cycles, which suggests that isolated impurities or dust rather than breakdown of the porphyrin was responsible for the short-circuit currents. Impurity ions may cause the build-up of an electric field, thus increasing the probability of light-induced charge separation and charge movement. In accordance with this ~ypothesis,the direction of the short-circuit current Iscwas always determined by the polarity of the bias voltage, whereas in ITO/porphyri~TOcells prepared

on metal modes using micelles with

without an applied voltage, it is always the i~adiatedelectrode that takes up electrons. 1% decayed within 24 hours to the value of nonpoled layers (Liu arnd ard, 1998). The only commercially successful photovoltaic cell based on multilayer technology today is the so-called Gratzel cell. It uses ~ t h e ~ ucomplex m monolayers on nanocrystallineTiO, films. The porous film with a very large surface is somewhat analogous to the grana structure of chloroplasts; electron injection m the ruthe~umchromophore into the TiO, se~conductortakes only 7 PS agfeldt and Gratze1,1995). For Referencesfor Chapter 6, see pages 544-558.

Redox-active quinones and dialkylviologensform long-lived radicals upon oneelectron reduction. These perfect storage molecules for single electrons help in light-induced charge sep~ationreactions (photosynthesis). They also catalyze the oxidation of diamagnetic organic compounds by p~amagneticmolecular oxygen (respiration). Contemporary attemptsof organic chemists to mimic biochemistry (e.g., inthe photochemical hydrogen evolutionfrom water orthe oxidation of hydrocarbons) often imply quinones and their nitrogen analogs, the viologens. ~ r o u p - ~ a n s f e vitamins ~ng may in thefar future be of interest for synthesis in aqueous mediac ~ n t a i nartificial i~~ membrane systems. Vitamins and essential amino acids make up two thirds of food and feed additive products (Table 7.1.1). The other third includes mainly antioxidants, enzymes, preservatives, and growth promotors (McCoy, 1998). Vitamin C helps to sustain the low oxidation potential of body fluids in an oxidative environment, Vitamins E and K keep the body’s radical concentration low and thus prevent peroxidation and cross-linking aging processes. Vitamin A provides the basisof vision and is also an efficient antioxidant. VitaminD helps to build bones from water-soluble calcium salts and phosphate in hydroph~bic an environment. ions must be keptawayfromthebody fluids, because p~ecipitatin calcium would clog up arteries and calcium ion conducting pores. vitamins help in the metabolism of small organic molecul ups,carbondioxide,and fo~aldehyde(Lang,1974; Tsler and rubacher, 1982; lsler et al., 1988; Murakamiet al., 1996).

Vitamins-Structure, Function, and Daily Requirements EssentialVitamins A D E K

structure Polyene 9,10-Seco steroid Polyprenyl hydroquinone Naphthoqui~one

Ene-diol Thiazolium Isoalloxazine ~ridinium aldehyde Cobald corrine B,, Pteridine Folic acid P~tothenic Hydroxycarboxylic acid acid Niacinamide Py~diniumamide Cyclic urea Biotin

Function

Daily requirement 0%)

Vision, skin development Ca,(PO,), resorption Antioxidants Protein synthesisin blood; coagulation Redox catalyst, scorbut Decarboxylase, transferase Oxidoreductases, respiration Amino acid metabolism

1.5 0.03 30 0.15 100 1.5 2 2.5

ethyla at ion, re~angements C metabolism, blood formation Acyl transfer (CoA)

0.01 20 l0

Oxidoreductases, respiration CO, transfer

20 0.3

The steroid- and carotene-derived vitamins D and A,or retinol, have been discussed in previous chapters. Here we discuss first the participants in photosynthesis and respiration redox chains as they occur in mitochon~riaand chloro~lasts.These are the membra~e-solublequinones (vitamins E and and the water-soluble riboflavin and nicotine amide derivatives. These compounds participate in hydrogen and oxygen productio~and oxygen activation, In model systems the ~uinonepart is often played by viologens, which are also presente~here. Furthermore, we briefly discuss the reactivity of the tyrosine radical and of manganese complexes in connection with o~ygen-evolving systems. Ascorbic acid is the final member of this group. It acts as a general reducing agent, which helps to maintain the low oxidation potential of the body fluids, which is very close to that of the hydrogen electrode (approxi30 mV) in an oxidative environment (approximately 900 mV for oxyinally the activation of small molecules in hydrophilic media will be discussed.

Plants obtain theirlife energy from the photochemical cleavage of water into hydrogen and oxygen (photosynthesis), animals from the combustion of the diamagneticcompounds of foodwithparamagneticoxygen(respiration).Both processes are primarily sustained and catalyzed by metallopo~hyrins(see Chapter 6). Quinones, nicotinamides, flavins, and lipoic acid play, however, important roles as electron transfer and protecting agents, which are tightly connected to the reactions of chlorophyll and heme. The main absorption band of benzoquinones appears around 260 nm in nonpolar solvents and at 280 nm in water. Extinction coefficients are 1.3-1.5 x 104 l. Upon reduction to hydroquinones, a four times smaller band at 290 nm is found. The most important property of quinones and related moleculesis the relative stability of their one-electron reduction products, the semiquinone radicals. "he parent compound 194-benzoquinoneis reduced by FeCl,, ascorbic acid, and many other reductants to the semiquinone anion radical which becomes protonated in aqueous media (pka= 5.1). Comparisons of the benzaldehyde reduction potential with some of the model quinones given below show that carbonyl anion radicals are much stronger reductants than semiquinone radicals and orthothat and parabenzoquinonesthemselvesareevenrelativelystrongoxidantscomparableto iron(III) ions in water (Table 7.2.1). Thisis presumably causedby the repulsive interactions between two electropositive keto oxygen atrns, which are separated only by a carbon-carbon double bond. When this positive chargebecan distributed into neighboring n: systems, the oxidation potential drops significantly (Lenaz, 1985). The semiquinone has a high tendency to disproportionate and to form a hydrogen-bondedquinone-hydroquinonedimer(quinhydrone),althoughthe monomer is almost completely deprotonatedat pH 7. Crystalline benzoquinone is yellow, the hydroquinone colorless.The quinhydrone co-crystalof both is red, which is caused by a charge transfer bandat 450 nm. The heterodimer is stabilized by hydrogen bonds between the phenolic protons and the basic ketone oxygen atoms (Figure 7.2.1), but it is not stable in dilute solution M),The van der Waals interactions between the small benzene rings are not strong enough. More stable charge transfer complexes are found with macrocyclic p o ~ h y ~ n s and tricyclic flavins. The most common quinones in nature are the ubiquinonesof plants. They are tetrasubstituted benzoquinones with to up10 isoprene units in the alkene side chain (Scheme 7.2.1). Their names are either derived from the number of isoprene units (UQ- 1, UQ-2, UQ-3, etc.) or the number of carbon atoms within these side chains (U-5, U-IO, U-15, etc.). The transient formation of ubisemiquinone radicals can only be observed (445 nm) in nonpolar solventsby stopped flow techniques. In water and membrane systems the hydrophobic radicals are extremely short-lived (Kitagawa et al., 1990; Lipshutz et al., 1996).

Redox Potentialsof Some ortho- and para-Quinones

-0.91

0.79

0.72

0.48

0.46

0.40

0.15 0

Tocopherol carries six methyl groups:three on the hy~oquinonering and three on the side chain.The three chiral centers are all of R configuration (2The most significant chiral center at C2 is as stable to oxidative the remote centersat C4’ and C8’, but it disappears, of course, upon o~idationto the quinone (see Scheme 7.2.1). The UV spectrum shows a maxinm (E = 30,000) in petrol ether and at 292m nation in food occursby extraction with ether, S to Fe(I1). The iron(I1) concentrationis then determined colorimetrically with 2,2’-dipyridyl (Amax= 520 nm) or 4,7-diphenyl-I, 10-phenanthrolin. Solutions of free tocopherol fluoresce in theUV(!), whereas neither tocopherol acetatenorthequinoneshowsanyfluorescence(Lang, 19’74; Islerand phenomenon is not understood. inhibits the auto~idationof organic molecules and acts as a specific quencher of peroxyl radicals ( 0) (Nagaoka and Ishihara, 1996):

quinhydrone

~miquinone se~iquinone

tadical

Equilibria and crystal structureof q u i ~ y ~ o n e .

.-+ ROOH I-TOC,The x-ray structureof tocopherol showsthe flexspectrum of tocopherol acmethylated side chain. Inthe lH-N~R etate in CDCl,, the three aromatic methyl groups appearat 1.98, 2.02, and 2.33 PPm* Tocopherol can be isolated from plant oils, but pu~ficationis tedious. Several total syntheses, including a few indust~alones, are similar to carotene syntheses involving the repeated applicatio f the Wittig reaction. The side chain of ubiquinonesand vitamins E andhasbeen introduced by using vinylketones and benzyl chlorides as educts in the presence of a ~ i (c a~t a)~ ~ s t . Pd(0) doesnotwork! Electron-rich and electron-poor aromatics react with equal facility. The most interesting reactions of the natural are (a) their reversible one-electron redox reactions, quinones, and (c) the formation of polyquino~es.Oxid eC1, leads to a cleavage of the enol ether and quinone f o ~ a t i o n(Scheme 7.2.2). There is also a natural ortho-quinone, namely a py~oloquinoli~e quinone or methoxatin (Scheme 7.2.3).PQQ occurs in bacterial dehydroge-

Ubiquinones (coenzyme Q ) n = 6 - 10

cheme 7.

nases and mammalian enzymes in the form of quinoproteins. This quinone forms charge transfer complexes with aromatic amino acids (Ishidaet al., 1989). Hydrophobic quinones can be chemically transformed into water-soluble compounds or amphiphilesby Michael additions and subsequent oxidation. The redox-activeamphiphilescanthen be i~tegratedinto lipid vesicles(Scheme 7.2.4) (Fieser and Turner, 1947; Fuhrhop, 1990). ~oly(quinones)are an interesting class of redox polymers with high charge

Vitamins-Versati~e Assistants COOH

I

cherne 7.2.3

0

+

H0

HS-COOH

00 H0

densities. They are typically prepared by chemical or electrochemical oxidation of hydroquinones, but large amountsof byproducts occur. Only polymerization of water-soluble ~ydro~inones givesuniformproducts: ~-glucoro~dase catalyzesthemonoglucosylation of hydroquinonetogiveglucose--+-hydroquinone butin in). This water-solublecompoundthengiveswater-soluble oligomers upon radical formation witha degree of polyme~zationof 6-12? in a yield of 170%. ~eglucosylationwithHClyieldstheoligohydroxyquinonein

OH OH

OH

"H

NO,

HCI/HzO

- -Glucose Hd

quantitative yield, and this canbe oxidized to the oligoquinone in 0. l M H,SO, or acetonitrile (Scheme 7.2.5)(Wang et al., 1995). Synkinetic assemblies of quinones in bulk water include monolayer vesicles, where a negatively charged largequinone carboxylate is localized outside and a fatty acid carboxylate group inside. Upon addition of sodium borohydride exactly 50%of the quinone is reduced to the hy~oquinone,because the borohydride anion cannot pass the membrane to react inside quinoneheadgroups (Scheme 7.2.6) (Fuhrhop,1990). uinones play an important role in both covalent (synthetic) and noncovalent (syn~netic)assemblies, which are supposed to allow electron transport through membranes and light-induced charge separation processes. With respect to their ability to carry electrons through lipid membranes, quinones with a side chain of more than two isoprene units are far more efficient than homologs with shorter chains. In the reduction of vesicle-entrapped f e ~ c y a n i d e ions by external dithionite, for example, the long side chain may act as the rod of a pendulum (Fig, 7.2.2) leading to fast electron transfer. Tocopherol and other branched-chain hydrocarbons (e.g., phytol,) also disturb egg lecithin

0

OH

0

Donor

lipid ~ e ~ b r a n eAcceptor

ur Hydrophobicquinonesarelocalized in thecenter of lipid me~branesand mayefficiently transfer electrons by pendulu~swinging. (Prom Futami et al., 1979; Pileni and Gratzel, 1980.) membranes and allows fast transport of hydrated metal salts (e.g., Pr3+ salts), which act as ~ ~ R - s h ireagents. ft Open-chain terpenes obviously move freely in hydrocarbonmembranesand introduce ruptures into the fluid ~ e ~ b r a n ~ structure. Long-term charge separation has so far not been achieved in vesicular quinone systems (Leidner and Liu, 1989). In one example,~thraquinone an bo-

hapter 7

l a ~ p h i p h i l ewas entrapped in the center of various vesicle bilayers, and electron-donating porphyrins were adsorbed to the surface of the vesicle. Upon flash photolysis, charge separation occurred, but the recombination of charges was even faster (Fig. 7.2.3). The fluid environment in lipidvesicle membranes is obviously no more suitablefor the stabilizationof reductant-oxidant pairs thanthe solvation sphere in homogeneous solutions.

DODAB

Static entrapment of a bolaamphiphilic a ~ ~ r a q u i n o in n e a vesicle membrane. Light-induced elctron transfer and the dark back-reaction both occur with high speed in this system (within nanoseconds). Surface charge of the vesicle has onlya small effect. (From Siggelet al., 1987.)

Severalhundreds of covalentquinone-quinone and porphyrin-quinone compounds have been synthesized and tested for light-induced charge transfer by fluorescence quenching, fluorescence lifetime, and flash photolysis experiments. Themostsimpleartificialsystemconsistsofcofacialbis-quinonecyclophanes in which the two quinones have different redox potentials. Extreme differences in relative orientations have been realized with orthothe and g e ~ i isomers ~ ~ Z in which a tetracyanoquinomethane oxidant and dimethoxy hydroquinone reductants are connected by two propylene bridges (Fig. 7.2.4).A strong broad charge transfer band(600-1000 m)was observed for the geminal isomer (E= 3500) and a weaker one for the ortho isomer (E= 120), indicating the importance of the relative orientation of electron donors and acceptors (Staab and h a u s , 1979). One example is a cofacial quinone-capped porphyrin (Scheme 7.2.7). It

UVNis spectra of two donor-acceptor cyclophanes.

shows 60% fluorescence quenching withM = 2H. Flash photolysisin polar solPS), vents yields a long-lived transient band at 415 nm (growth: 15 ns; decay: 1.4 which is assigned to the charge-separated ZnP+Q-(activation energy: 6 .kJ/mol) (Lindsey et al., 1988; Sessler et al., 1988; Sakata et al., 1989). The most efficient charge separation in terms of lifetime occurs in porphyrin-hydroquinone pairs connected by two secondary amide groups and an oligomethylene linker. They occur in two sets of conformers. In a “complexed” set the porphyrin and the quinone are close and interact electronically. Optical line broadening and diminished fluorescence lifetimes are typical for this family of conformers. The other conformers are “extended.”No line broadening occurs. Oxidation of the porphyrin-linked hy~oquinoneto the quinone does not significantly affectthe absorption or fluorescencespectra, but causes strong quenching of fluorescence and diminutionof lifetimes. On the basisof the assumption that the shortening of the fluorescence lifetimes are due entirely to electron transfer, apparent electron-transfer ratesat room temperature have been calculated. They range from 108-7 x lo9s-l. Quenching in both sets of conformers is thermally activated and strongly forbiddenin frozen matrices. Upon irradiationthe diamide given in Figure 7.2.5 developed EPR signals in frozen methylene chloride after irradiation for minutes with visible light.The lifetime of this signal was several minutes. This is in contrast to the reversible signals observedfrom corresponding diester-lin~edcompound. The obtained EPR spectrum correspondsto a 1:1 complex of P+and Q- radicals (Fig. 7.2.5). Quinone union radicals give EPR signals at g = 2.0047, porphyrin cation radicals at g = 2.0025 (Fig. 2.75,d). The observed signals after photoinduced charge separation were either s y ~ e ~ i c a l sharp signals at g= 2.0037, causedby the desired intramolecular charge separation (Fig. 7.2.5a,b), or asymmetrical signals at g = 2.0032 correspondingto intermolecular charge transfer (Fig. 7.2.5e). Onlyl-2% of the PQ molecules can be converted to P+Q- pair, andthe yield decreases when four, not three, methylene groups separate the porphyrin from the quinon amides. Experiments with “reCO amides” showed that the electron transfer rate in the covalently connected redox pair (9.9 X lo9 s-l) is 25 times faster as in the “normal CONH amides.” No direct molecular interpretation of these findings has been given, but they are highly suggestive of the important role of vectorial amide polarity:the sequence electron donor-NHCO-acceptor is better than the donor~ONH-acceptor sequencefor electron transfer, and two amide groups may play the roleof ode1 systems for charge separation should always consider amide effects. An amusing nucleic acid derivativeis also known: a PE~-solubilizedzinc porphyrin with an guanine side chain combines with a cytosine derivative of benzo~uinone(Scheme 7.2.8). Electron transfer occurs in dichlorometh tions (4 X 108S”), but lifetime of the charge-separated pairis short man et al., 1992; Sessler et al., 1993).

X=OorNH I

.5 Light-induced electron transfersinamide-andester-linkedporphyrinquinone pairs. The resultinglong-lived ESR spectrum shows porphyrin as well as semiquinone anion radical signals. (a)n = 2, x = VH; (b) n = 3, x = NH observed signals for in~amolecularcharge transfer; (c, d) calculated spectra for the sum of porphyrin and quinone radicals; (e) asymmetrical signal for intramolecular charge separation. (From McIntosh et al., 1983.)

OR

More complex “triad” porphyrin compounds containing a carotenoid or chloro~hyll-li~e substituent in addition to the quinone have also been synthesized. One example is given in Scheme 7.2.9. Li~ht-inducedcharge separations are fast in such molecules (101o-lO1l S-’), but the recombination rates are almost equally fast (109-1010SI). Neither carotenes nor chlorophylls help in the stabilization of charge-separated states. Laser flash photolysis of chloro-

Vita~ins-Versatile Assistants

1

phyll a-benzoquinone mixtures in fluid egg lecithin vesicle membranes produced chlorophyll triplets, which were then quenched by the quinone. Radicals are formed, which decay with a half-life of microseconds (Janzen and Bolton, 1979; Gust et al., 1987; Johnson et al., 1993; MacPherson et al., 1995). Similar triads C-P-QCOO-of an electroneutral carotene C,a porphyrin P, and a carboxylated naphthoquinone QCOO- dissolve in an asymmetrical manner in a vesicle membrane: QCOO-is on the outside of the membrane, P-C spansit. Water-insoluble 2,5-diphenyl-benzoquinone(Qmemb)is dissolved in the membrane as an electron as well asa proton shuttle. Upon irradiation intramolecular electron transfer occurrs from the carotene to the quinone to give C+-P-Q-COOwith a quantum yield of 0.15. Since the membrane-dissolved diphenylquinone Qmembr is a stronger oxidant than the Q-COO- of the triad, it is reduced by the triad and now acts as electronand proton shuttle. The oxidized Q-COO-is then rereduced only onthe outer surface by external dithionite. Its proton is simultaneously released also on the outer surface and a pH gradient of about 0.5 pH units is detected by entrapped fluorescence indicators. The system thus changesa given redox gradientinto a pH gradient by light. The system is complicated and difficult to sustain because lipid membranes are notoriously permeable to protons., but careful control experiments have been reported (Steinberg-Yfrach et al., 1997). Quinones are firmly established in photosynthesis models. But how about vitamins E and K? How do quinones work in animals? First of all they transport electrons in a similar way as in photosynthesis (Metzler, 1977; Voet, 1990). Second, tocopherol is known to act asan antioxidant or radical quencher. The radical chain starting with the decomposition of unsaturated lipid peroxides, for example, is inhibited by tocopherol, which produces longlived semiquinone radicals (Scheme 7.2.10). Vitamin E prevents, for example, sterility in rats fed rancid lipids. Vitamin E in connection with carotenes is also used as a stabilizer for diet margarines containing large amounts of essential fatty acids. Another possible activity of tocopherol is participation in "oxidative phospho~lation":a hydroquinone is mono-esterified withphosphoric acid to form a quinol phosphate and then oxidized to the quinone. The cationic quinone-phosphate adduct then reacts with anionic phosphate to form pyrophosphate (Scheme 7.2.10), (Wang, 1969; Breslow, 1980; Isler and Bmbacher, 1982). The most efficient organic conductor material consists of co-crystals of tetracyano-~-quinodimethane,an electron-poor quinone analog, and tetrathiafulvalen, an extremely potent electron donor. The crystals are green and have a conductivity of CT = 1.5 x lo4 Siemens cm-' at 66 K as compared to metallic copper with a CT = 6 x lo5 Siemens cm-' at 298 K. In order to obtain such high conductivity, organic charge transfer complexes must not appear as face-toface dimers in crystals. In such cases, the acceptor takes up an electron and

0

+

+

+

H20

0

OH

cannot give it back to the next donor molecule in the crystal. Alternating donor-acceptor layers are therefore insulators. The acceptor and donor molecules must rather appear in neighboring, separated stacks. If the acceptor stacks are partially reduced and the donor stacks partially oxidized (Figure 7.2.6),the negatively charged acceptor stacks as well as the cationic donor stacks build up conducting valence bands in a similar way to metals (Wudl, 1984).

N

N

amm

Nd

D+A Isolator

A@

D+A

Conductor

Conducting double layer of tetracyano-~-quinodimethane (A) and t e ~ a t h i a f u l ~ ~(D). e n eThe separation of donor and acceptor layers is presumably caused by the geometrical nonfitting of the molecules, the co-c~stallizationby strong lateral charge-transfer interactions.

In plant photosynthesis molecular oxygen is produced from water. In this process tyrosine molecules carry electron holes from oxidized chlorophyll molecules to manganese-protein complexes. Short-lived tyrosine radicals keep the photoexcited chlorophyll molecules apart from reactive oxygen intermediates, such as peroxides and oxygen atoms (Calvin, 1978; Tommos et al., 1995, 1998) In a simple model system a modest oxygen evolution. has been achieved with a dimeric manganese(II1) Schiff base complex. A bis-(salicylaldimine) ligand plays the role of the photoexcitable dye as well as of the tyrosine radical. Oxygen is liberated, and added n-benzoquinone is reduced to hydroquinone when the aqueous mixture is irradiated with visible light. The linking chains that connected the Schiff base ligands were ethylene, propylene, and butylene diamines, and the active manganese complex containing three methylene groups exhibited a band at 590 nm. This band was absent in inactive complexes connected by shorter or longer methylene chains and was traced back to a water-bridged manganese dimer (Fig. 7.2.7). This model is extremely inefficient: the 590 dimer band is not easy to reproduce and the mech-

2+

S

$20 0

10

260

300

460

500

600

0

100

200

300

400

500

.7 A s~icylaldimine-manganese(~1) complex dimerizes and is protonated. The visible spectrum shows a small charge-transfer band close to belonging to the 600 nm dimer. ~ a d i a t i o nwith visible light produces some molecular oxygen. Its formation may al., occur in the way indicated by the given stoichiometric equation. (From Ashmawy et 1985, andAwad and Anderson, 1989.)

anism of oxygen evolution remains obscure. Nevertheless, this artificial system is, to the best of our knowledge, the only organic manganese system that produces oxygen upon irradiation with visible light.

L-Ascorbic acid is a vitamin necessary for humans, who lack a gulono-oxidase, which is needed for its biosynthesis from glucose. The daily r e q u ~ e ~ efor n t vitamin C is extraordinarilyhigh: 45 mg/dayisaminimum,but 1,~0-~0,000 mglday can be ingested without causing harm, provided there is no dispositionof the individual to produce gallstones, in which case vitamin C intake should be limited to 2 g/day. (Linus Pauling took 19,000 mglday!) Most mammals (e.g., dogs, cats, rats) produce L-ascorbic acid D-glucose from themselves in daily quantities of about 30-300 mg per kilogramof body weight. The resorption of vitamin C by the human body diminishes with raising doses: 100 mg are resorbedto 7'0%

(70 mg) by a stereoselective transport system in the small intestine, but12 g are only resorbedto 16% (2 g). In human organs ascorbic acidOCCWTSin the reduced 80%. Only in erythrocytesand in form to more than 97%, in blood to more than the kidney, where atmospheric oxygenis close, does more than50% of dehydro C can easily be co-crystallized with a ascorbic acid appear (Lang, 1974). Vitamin variety of water-soluble compounds, e.g., arginine (Sudhakar and Vijayan, 1980). Vitamin C is another enol-based redox system like the hy~oquinonesand tyrosine, butit has no aromatic character. The enediol component is stabilized by a conjugated lactone group. The oxidation(Ela= "58 mV) occurs in two one-electron steps, but the ascorbate radical is not as stable as the semiquinone radical (Bielski, andRichter,1977).Theanionradicaldisproportionatesviaaninitial dimerizationinasimilarwayassemiquinoneradicals(Bielski et al.,1981; Sawyer, et al., 1982). The lifetimeof the radical is in the order of microseconds. The acidityof ascorbic acid (pk,= 4.2) stems from the OH group in thep position to the lactone carbonyl group. It corresponds to the OHofgroup a vinylogous carboxylic acid (Scheme 7.2.11). ItsUV maximum occursat 260 nm (E = 1 X 10"). Dehydroascorbicacid is anunstable1,2-diketone,whichreadilyreacts with superoxide anionsto give a dioxetaneradical anion and a dianion. This then decomposes to oxalic and threonic acid anions together with triplet oxygen molecules. Several standard reactionsare also summarized in Scheme 7.2.12.

Ob

OH

0 OH

Scheme 7.2.11

0

0

H

OH

H

OH

OH

OH

COOH

CH20H

L-ldonic acid

H0 H0

AcetobacterlO2

H0

H

H0 CH20H L-Sorbose

D-Sorbitol

CH OH H0

0 OH OH

H0 CH20H

2-Oxo-L-gulonic acid methylester

Industrial synthesis of ascorbic acid follows Reichstein’s classic pathway (Scheme 7.2.13): D-glucose is first reduced to sorbitol (glucitol) and then oxidized to L-sorbose by Acetobacter. Acid-catalyzed acetonization leads to a furanoside with the free primary alcohol group of C6. Subsequent treatment with acidified methanol, calcium carbonate, and weak acids yields ascorbic acid.

Nicotinamide, together with viologen (sec. 7.2.5) and flavins (sec. 7.2.6), constitute the counterpart of electron-donating tetrapyrrole pigments. Nicotinamideis

an extremely electron-deficient pyridinyl redox systems, which picks up electrons and nucleophiles from the environment.This tendency is strengthened by substituents on the pyridinyl nitrogen atom, which introduce a positive charge, and by electron-with~awingamide groups. Nicotinamide-adenine dinucleotide (NAD+) andits 2"-phosphate (NADP) are coenzymes in a varietyof redox enzymes (Scheme 7.2.14).NAD+ occurs in two preferred conformations: a macrocyclic conformerAwithanoverlap of theheterocyclesand a stretched confo~ationB. Conformer A dominates in solution, as shown by'H-NMR spectra; conformerB is found incrystal structures. The NAD+ chromophore absorbsat 260-270 nm and does not fluoresce, NADH, on the other hand, has an additional absorption band at 340 nm (Fig. 7.2.8) and a fluorescence band at 350 nm, NADH fluorescence has been used to follow NAD+-catalyzed reactions down to a detection limit of mol L-l. The midpoint potentia1 of the NAD+/NADH pair lies -113 mV lower than the 2H+/H, pair. NADH is the strongest reductantof nature (Table 7.2.2). Nicotinamide cations(NAD') add all kinds of nucleophiles inpara or ortho positions. NADH, its reduced counterpart, reacts with oxidants as well as with a few electrophiles (Scheme 7.2.15).

0

0 @ O " O -

II

I

0

I

@O-"O

I

0

0

OH OR

eheme 7.2.14

0

0

35

Vit~m~ns-Vers~tile Assistants

15

12 9 6

3 0

h inml UV spectra of NAD+ and NADH. Redox Potentialsof Biological Redox Pairs Eo[mV]Half-reaction 02+4H++4e-=2H20 Fe3++ e- = Fe2+ Fe(CN),3- (fe~icyanide)+ e- = Fe(CN),40,+2H++2e-=H,02 7c-Quinone + 2 H++ 2 e-= hydroquinone Cytochrome c (Fe3+)+ e- = cytochrome c (Fe2+) Ubiquinone + 2 H++ 2 e- = ubiquinone H, Dehydroascorbic acid+ 2 H++ 2 e-= ascorbic acid S+2H++2e-=H2S Lipoic acid+ 2 H++ 2 e-= dihydrolipoic acid NAD+ + H++ 2 e-= NADH ~ b o ~ a v+i 2n H++ 2 e- = d i h y d r o ~ b o ~ a ~ i n 2Hi+2e-H, Hemin 2heme

pH 7 +8 15 +77 1 +360 +295 +285 +254 + 10 + 58 -274 + 29 -32 208 -4 14 -480

Na"

h,, l

x=

355 nm

R

R-

h,, p 330 nrn

One-electron oxidants often react faster than two-electron oxidants with NADH.Theporphyrexidradical is particularlyefficient.Suchobservations m&e it likely that several apparent nicotina~detwo-electron reactions may also mn via radicals (Scheme7.2.16).

Nicotinamide is located at the reducing, hydrogen-evolving endof photosynthesis andthe respiratory redox chain. Although NADH may be used as a strong reductant in synkinetic assemblies, it is most often replaced here by more versatile and stable dimethylviologen (4,~"dimethyl dipyridiniumbromide) or other alkylated viologens. One major advantage of the d i p y ~ ~ n i uinmcontrast to the monopy~diniumredox systemis the stability of the one-electron reduction prod-

0

HO NH

+ NAD@

2 HN

porphyrexid radical

ucts. Another is its absorption band in the visible range. The radicals appear as violet cation radicals (Fig. 7.2.9), which live for hours in the absence of oxygen (Sumers, 1980). Methyl viologeri, under the name of “paraquat,” is a major herbicide. It is water soluble, is readily destroyed by air and sunlight, and is used early in the spring to kill weeds before crop plants appear. Toxicity is caused by the fomation of viologen radicals in the reducing medium of living plants and by the formation of superoxide anions from molecular oxygen, although 0,- is fairly harmless to aerobic organisms (Summers, 1980). With positively charged metalloporphy~ns, electrostatic repulsion of MV2+allows only for short-lived encounter complexes. Electron transfer is slow,

UVNis spectra of (a) dimethyl viologen and (b) its one-electron reduction product.

but quantum yields for M V + production is high, because the reduced acceptor diffuses quicklyaway. Anionic porphyrinsare much less photoactive. In model systems for the light-induced reduction of water, monocationic viologen radicals produce hydrogenin the presence of colloidal platinum. Any system that photoreduces viologento the radical with visible light can therefore serve as a modelof the reductive part of photosynthesis. The first modelof this kind used the photochemically excited state of a ruthenium(I1) bipyridinate as a reductant. The viologen radical was then used to produce hydrogen, andoxithe dized ruthenium was rereduced chemicallyby EDTA or ascorbic acid (Scheme 7.2.17). The protons of water were thus reduced to molecular hydrogenby a relatively weak reductant and sunlight. Quantum efficiencies are, however, low (approx. %). Mixed inorganic systems are far more efficient. In the presence of the strong sacrificial oxidant RuO,, ~ t h e n i u mbipyridinate complexes can also photooxidize water to 0, (Harriman, 1981). Covalent triads of ruthenium bipyridinates, an electron donor (phenothiazine), and paraquat as acceptor produce a charge-separated system with a half-decay time of 108 ns (~ecklenburg, 1993). Scheme 7.2.17 contains another very interesting bipyridyl system, namely the ruthenium(I1) complex of 2,2’-bipyridine. In this complex the d-orbitals of the ruthenium ion and themorbitals of the bipyridine are similar in energy and mix. As a result, there is almost no redox quenching of the excited singlet and triplet states and the compound fluoresces strongly, whichis very unusualfor a transition metal complex. It cannot only be used to produce hydrogen with the aid of viologen, but the oxidation product ~ t h e ~ u m ( I I 1 ) b i p y ~is ~ nsuch atea

hv

strong oxidant thatit may also oxidize hydroxyl ions to molecular oxygen without sacrificial oxidants (Scheme 7.2.18). It is therefore hoped that ~ t h e ~ u r n bip~ridinatemay split waterto hydrogen and oxygen under favorable conditions (Balzani, 1997). Viologen also forms radicals without light at high pH. The reaction between hydroxide anions and viologen is complex, but one major pathway is given below. It leads to a 1:1 mixture of the radical and the pyridone (Scheme 7.2.19). With methoxide insteadof hydroxide a clean reactionto formaldehyde and the radical (yield2- 90%) was observed.The Eovalue is lower in water (-430 mV) than in solutions containing 80% ethanol (-250 m~)(Summers7 1980). The corresponding 2,2’-bipyridiniurn salt occurs predominantly in the anti conformation, but the cis conformation can be enforced by a connection of the nitrogen. atoms via anethylene bridge (diquat). Only the latter compound gives stable radicals upon reduction (Scheme 7.2.20) (Lmm:378 nm;E: = 307000)(Summers, 1980). When dimethyl viologen dicationis entrapped in DPPC vesicles and irradiated in the presence of external K4[Fe(CN),], the viologen is reduced to the radical monocation and iron is oxidized. Light-induced membrane potential is thus built up (Calvin, 1978). Electrons can be stored and released in viologenbenzoquinone polymers onelectrode surfaces. At first the quinoneis reduced to the hydroquinone at low pH. Two electrons are entrapped per hydroquinone monomer. Upon raisingthe pH to 6-9, the viologen becomes reducedby the hydroquinone anions and delivers the electronsthe to electrode (Smith et al., 1989)

-

H3C-

+ OH@

2

/ /

W

n

OH "CH3

Ftiboflavin (lactoflavin) and flavin nucleotides are yellow, water-soluble redox systems based on isoalloxazine chromophores (Scheme 7.2.21) (Hemerich, 1977). In riboflavin (7,8-dimethyl- 10-(R)-ribityl isoalloxazine), the poly01 ribitol is connected via an amino bond to the chromophore. Riboflavinis not an N-glycoside. Accordingly, flavin mononucleotide(FMN, riboflavin S’-phosphate) and flavin adenine dinucleotide (FAD; riboflavin 5’-adenosine diphosphate) are hydrolyzable only inthe nucleotide part (Scheme 7.2.22). H

0

-

P

“ + “ -Adenosine (A)

0

0

Flavins are mostly synthesized from aniline derivatives by routine condensation reactions (Scheme 7.2.23)(Isler et al., 1988). Similar to quinones andNAD+the flavins are one- and two-electron redox systems (Fig. 7.2.loa). Upon half-reduction a quinhydrone-typedimer appears, producing a charge transfer bandat 820 nm (Fig. 7.2.1Ob). The noncovalent interactions of flavins arise from electrostatic attractionbetweenelectron-rich donor atoms,in particular the basic oxygen atomsof amide groups, and electrondeficient aromatic systems, the inner conjugation system of oxidized flavins (Breinlinger et al., 1998). This is rerninescent of the interactions between porphyrin macrocycles (seeFig, 6.2.15). A peculiarity of the flavins is, however, the changing basicityof the nitrogen atoms.The imine nitrogens of the flavins are not basic (pk, = 0), whereas the radicals (pk,= 6.5) bind protons and metalions in water. Curiously, the basicdihydroflavins (pk, = 6.5) again do not react with two-valent metal ions (Scheme 7.2.24). The lactam oxygen in the radical is presumably more electronegative and therefore responsiblefor the complexation of the radicals (Lauterwein et al., 1975). A flavin-bearing crownether ring has been used as a redox active model compound with peripheral metal cations (Shinkai et al., 1984). The extraordinary stabilityof the radical metal complexes as compared to

OH H0

OH

OH OH

1. H,; -Aniline M@

Ribose

7

0 E

2.5 2.0

1.5 1.o 0.5

- -

0

~~

UV-'Vis/NIR spectra of flavins upon reduction. (a)Glucose oxidase pH 5.8 (...)and ( ) ; pH 9.2 (-.-).(From Massey, 1966.) (b) Riboflavin titration. (From Hemmerich, 1977.)

stable only in aprotic solvents

Mstable in water

Red

no stable metal complexes known

7.

7.

the instabilityof the complexes of the other oxidation states leads to symproportions steeredby the metal ions (Scheme 7.2.25). Flavins decompose rapidly in light. In particular, the N-Cbondof the ribitylamines is cleaved or the carbohydrate is photoreduced to form a methyl group (Scheme 7.2.26). NADH directly transfers a hydride ion to flavinsand analogs (e.g., deazaflavin), indicating hydrogen transfer within a molecular complex (Scheme 7.2.27). Hydrophobic NADH derivatives with anammonium side chain have been bound to a PEG substituent of a flavin and are then oxidized AD+ than without complex formation. Chiral deazaflavino-

n

I iboflavin 0

0

NADH

NAD@

DO

I R

+

FH3 0

phanes oxidize chiral NADH model compounds in an asymmetrical manner (ee 60%), (Levine and Kaiser, 1978, 1980). Reduced flavins are also good catalysts for the reductive cleavage of strained carbon bonds via radical formation. The cyclobutane unit of pyrimidine dimers in UV-damaged DNA, for example, has been photochemically cleaved by reduced flavin substituents. The quantum yield was up to 14, = 0.06 in water and the cis-syn isomer was most vulnerable (Epple et al., 1997) (not shown); the trans-syn diastereomer was 10 times more stable. Another specialtyof the flavinsis the reversibleperoxida~onwith molecular oxygen. The angular peroxides are decomposed in high yields with fo~aldehyde to give forrnic acid, oxidized flavin products, and light (chemoluminescence): This system is not a very efficient one and is replaced in artificial luminescent devices by various activated carboxylic acids with an a-methine proton or a double bond.The reactive chromophores react with normal triplet oxygen in the ground state; the corresponding reaction of alkenes depends on light-activated

37

PH

HCHO

v

+ light

OOH

Scheme 7.2.28

0

2 *

R

R

0

0

c" 2 0 R E

NO

I

NH NW2

0

0

Ox

N.

I

NO NH2

0

NO NW2

0

Luminoi

singlet oxygen. In both cases, a dioxetane is formed, which decomposes to form a ketone in its excited state and which then emits light (Scheme '7.229). A simple system consistsof the lumina~3-~ino~hthalate pair. These peroxides have been N+-0 and -S+-0oxides used to oxidize amines and sulfides to the corresponding ( ~ u r ~ a s heti nal., 1989).

ita am ins-Wersatile ~ssistants

71

The artificial systems are noncatalytic. For each light quantum one large dye molecule is lost. In the related natural systemsof fireflies, however,the luminescing lactam-thiolact~is decomposed to a nitrile and thioacetic acid.The nitrile is then condensed withcysteine in order to recover the oxidizable methine proton next to the carboxyl group. In thisway only the CHNH2 group of a cysteine is irreversibly oxidized in order to produce light, and the other reactions can all run in a cycle (Scheme 7.2.30). Flavinophanes and 5-deaza~avinophanesin which N, and 0,' are linked by (CH2)nchains (Scheme 7.2.31) have been resolved into the enantiomers by liquid c~omatography(Shirikai et al., 1989a,b). Short-chain enantiomers did not

h v

Luciferin cheme 7.2.30

0

X = N or CH; n = 6,7,8,10 or I 2

racemize below 40"C, but the attachment of ropes longer than(CH,),, racemized invariably when they were reduced the to 1,5-dihydro forms and then reoxidized (Scheme 7.2.31). The coupling of (irreversible) oxidation reactions with (reversible) condensation reactionsin the biological luminescencecycle exemplifies a major advantage of livingsystems over synkineticlightreactionchains.Only in biological systemsis the renewal of decomposed dyes realized. All useful model reactions developedso far are limited to totally reversible reactionsof the dyes, whereas naturealso optimizes the interplay between irreversible degradation and biosynthetic reactions.

Thissectiononredox-active catalyst systems is terminatedwithadisulfide called lipoic acid. It does not participate directly in the all-important photosynthesis or respiration chains, but constitutes a nice and very simple reversible system that produces sulfide radicals. Lipoic acid contains a disulfide group in a five-memberedisring. less staIt ble than the corresponding open-chain disulfide. Ring strainis introduced by the large hetero atoms. A weak absorptionm ~ i m u m at 333 nm (E= 120) is observed. In the presence of sensitizers (e.g., porphyrins) visible light produces diradicals, which form sulfenic acids with water. It has been postulated that the disulfenic acid formed may split off hydrogen peroxide, which would be equivalent to the catalytic light oxidation of water. But this most exciting reaction has not been demons~atedyet (Scheme 7.2.32) (Barltrop et al., 1954; Doi and Musker 1981). 1,5-Dithiacyclooctane is a closely related model system with interesting properties. Moleculariodine oxidizes it to the 1-sulfoxide atpH 5.5. Upon acidification to pH 1.5, the starting dithioether is formed back in quantitative yield. This reaction cycle occurs with the probable intermediacyof a dicationic thionium analogof lipoic acid (Scheme 7.2.33) (Doi and Musker, 1981).

~olutionsto the problemof continuous catalyst repair in artificial reaction chains are not apparent, but they lie buried in the chemistry of the coenzymes, which activate small carbon molecules. In the following, we briefly s u ~ a r i z ethe c h e m i s ~of some coenzymes. Theyare also mostly vitamins. This time we do not deal withelectrons and protons, but with small, carbon-containing molecules such as carbon dioxide, formic acid, acetic acid, and a-amino acids. Some of these coenzymes interact very strongly with protein receptors, which also makes them useful in synkineses of reactive molecular complexes with peptides.

caan

2

H202

S-S

2

2

R

+

R S-S

R

+

R HS

Ski

HS

SH

+

R

0

Biotin is a hetero-bicycle with urea a and a thioether unit. It has three chiral centers. In synthesis one often starts with tartaric acid derivatives in order to produce the chiral sites at the ring junctions, ~es~-~ibromotartaric ester gives the desired cis configuration of a symmetrical bicycle, which becomes chiral upon introduction of the acid side chain. In one procedure the CH, groups of the thioether were activated by oxidation of the sulfur to the sulfoxide. Alkylation and removalof protecting groups gives the racemate. Separation of enantiomers occurs readilyvia the avidine molecular complex. CO, must be activated in order to add to biotin. Nature usesATP and presumably produces carboxyphosphate, the mixed anhydride of phosphoric and carbonicacids.Inmodelreactions chloromethylfo~iateand2-imidazolone were used. Saponificationof the urethane gavethe anion of N-carboxy-2-imidazolone, which decarboxylated again. Attemptsto use the evolving carbon dioxide in the carboxylation of carbanions failed, even if it wasbound to the imidazolone. More successful was the intramolecular attackof a carbanion bound to a neighboring thioenoether,The positive charge at the neig~boringnitrogen atom obviously facilitates the re~angement. The most important synkinetic reaction of biotin is its strong interaction with avidine, a glycoprotein from egg white (MW 66,000; K, = M(!)), and streptavidin, a nonglycosylated protein from ~ ~ ~ e ~ a v~ i ~~ i ~~(MW i iy c60,000; e s K, = IO-18).The avidin-biotin complex can be neither digested nor resorbed. Raw egg white therefore leadsto a biotin deficiencyof the body (egg white injury).Avidine has four binding sites and has been spin-labeled with a nitroxide. One biotin molecule gives the usual triplet of a nitrogenradical, but avidine saturated with four spin-labeled biotin molecules displays strong coupling effects (Fig. 7.3.1). Two lines show a splittingof 14 gauss, from which a spin-spin distance of 16 A has been calculated. Two biotin pairs of this kind are thus ob-

H COOH

Scheme 7.3.2 0

KOH, 20°C; -CbOH

H

H

H

H

.

. .

ESR spectra of (a) a monomeric biotin nitroxide and (b) avidine labeled with four such molecules.

served, and theydo not interact with each other (Weberet al., 1992; Katz et al., 1996). Since both avidin and biotin are readily available, they arefor used affinity c~romatography.Any protein, amino acid, nucleotide,or nucleic acid thatis covalently labeled with biotin can, for example, be adsorbed specifically on an avi~in-sepharosecolumn and subsequently be removed witha concentrated bi-

Synkinetic membrane assembly on a water surface: the biotinlipid forms a monolayer, which adsorbs streptavidin from the aqueous subphase.A biotinylated Fab fragment (see Sec.9.6.9) then bindsto the remaining binding sitesof streptavidin.(From Herron et al., 1992.)

otin solution. In synkinesis, biotinylated amphiphiles have been used to form crystalline arrangements withthree different, highly connected layers: at first the biotin surface monolayeris saturated with streptavidin inthe aqueous subphase, and then a biotinylatedi ~ u n o ~ l o b u lisi nbound to the distant binding sitesof streptavidin (Fig. 7.3.2)

Folic acidis a pteridine derivative thatis found in green leaves. Para-aminobenzoic acid(PABA) and glutamic acid are the other constituents. Folic acid derivatives are availableby standard condensation reactions (not shown) and react with formaldehyde at the aromatic and nonaromatic nitrogen atoms form to N5,N10 methylene derivatives (Benlsovicet al., 1973). In enzymes,folic acid catalyzes the N-formylation of amines and the 5-hy~o~ymethylation of uracil. None of these reactions has been performed in enzyme-free systems.

COOH

2-Am~no~-me~~+ = " -~ x o - 3 , ~ i h y d r o pteridine acid acid

benzoic

( S ~ ~ l u ~+.r n i ~

Pteroinicacid

Folic acid

rl

B.

c ~ e m e7.3.5

hypot~etic~i ~annichbase

Structure of the cyclic folate tetramer.

The self-organization of alkaline folate aqueous solutions leads to cyclic tetramers, which further assemble to form rods (Fig. 7.3.3), similar to guanine is formed in presenceof an assemblies (see Fig. 8.6.5).A chiral cholesteric phase excess of sodium ions (Spada, 1991). Folates are alsoof interest to the medical community.The antibiotic activity of sulfonamides is caused by their action as PABA acid replacements (see Scheme7.3.5).Folicacidcannot be formedintheirpresence,andbacterial growth is inhibited,

Coenzyme A (for acetylation) contains four structural units:ADP, pantoic acid(a vitamin), p-alanine, and P-mercapto ethylamine (cysteamine) (Scheme 7.3.7). Coenzyme A analogs are best synthesized by a combined chemical and enzymatic method. Pantothenic acid was first converted to the thiophenol ester and then the terminalOH group was phosphorylatedvia the dimethyl phosphatetriester. Thiol exchangewithethanedithiolgave the ~-thiol--phosphate. This compound was first enzymatically combined with ATP and then p h o s p ~ o ~ l a t e d at 3-OH’ (Scheme 7.3.8). A wide range of CoA analogsis thus accessible (Martin and Drueckhammer, 1992). CoA is enzymatically acetylated in nature at the terminal S drolysis of this thioester gives a free energy of 34 kJ mol-l, which is comparable to the hydrolysis of ATP to ADP (see Sec. 8.5). Nevertheless, thioesters are as stable in water as the corresponding alcohol esters. The very special reactivity

n

.N

W

0

I 00

s-

Ade~in~iphosphate~-phospho~c acid

~ a ~ t oacid ic

OH

~ a n t o ~ e nacid ic

SPh

0 SPh

00

I

~~~~o

(l) dil. aq. LiOH II HS H SH

NH

(I) Dephospho~oAp ~ p h o s p h ~ a sATP e, (11) D ~ ~ h o s kinase, p ~ A+ATP, -ADP

n 0

of thioesters only becomes apparent with nucleophilic amines and imines as wellas carbanions. Imidazole, for example, accelerates the hydrolysis of thioester by a factor of lo6,whereas in alcohol esters the factor is less than lo2 (Scheme7.3.9)(HersfieldandSchrnir,1973). Sterically hinderedGrignard reagents do not react with alcohol esters, but lead to Claisen condensation with thioesters. The peculiarityof thioesters is caused by the labilityof C=S double bonds, which means thatthe carbonyl groupof the thioesters behaveslike the carbonyl group of ketones: the carbon is very electrophilic andthe neighboring CH, group is activated.

+

MgBr

+

MgBr

no re~ction

in

Thiamine hydrochloride is the usual comercial form of vitamin B,. The reactive part is the thiazolium cation; its reactive center is C2. This carbon atom is extremely electropositive.Its C13 signal appears in the carbonyl region at 179.3 ppm. The double bond is, co~espondingly,easy to hydrogenate; the methine

H2ffd OH

H

Thionia-aza form

OH

6 = 179,3 ppm X, x= 235,267 nm

OH

-H20 +H20

OH

H

R = Pyrimidin base

+H@

-H@

fh

OH

proton is relatively acidic(pk, = 18).In strongly basic solutions, deprotonation is usually followed by a reversible ring opening or condensation with the amino group of the neighboring pyrimidine unit. Sulfite cleaves the immonium-carbon bond (Isleret al., 1988). The synthesis of the 1,3-thiazole nucleus starts with a-chloroketone an and thiofo~amide.The nucleophilic sulfur (seeSec. 7.3.3) substitutes the chloride, and the nucleophilic nitrogen atom condensates with the carbonyl group. The pyrimidine unit is obtained via a malonic ester aldehyde condensation with acetamidine. Thiamine anions add to aldehydes and ketones (e.g., acetaldehyde, carbohydrates, and pyruvic acid). Pyruvic acid adducts decarboxylate with a half-life of 24 hours in water and 3.2 minutes in ethanol, since ethanol cannot stabilize the intermediate zwitterion as well as water. After acidification, acetaldehyde is split off. In the adduct between acetaldehyde and thiamine, the electrophilic carbon atom of the aldehyde undergoes an ‘ ‘ ~ ~ ~ o to 2 a~ resonance-stabilized ~ g ” enolate carbon atom.The thiazole-bound acetaldehyde then functions as carbanion in Michael additions under mildly basic conditions. Retro-aldo reactions are observed, when 1,3-thiazolium ions react with the carbonyl groupsof carbohy-

0

H

CH0

OH

I

OH

OH

c-

M

""I

* " " " "

H

H

drates, e.g., the keto groupof fructose (Breslow, 1958; Crosby et al., 1970; Stetter and Kuhlenau,1974; Jordan, 1976) Innature,lysine-bound lipoic acid is used to oxidize thia~ne-bound acetyldehyde to acetic acid. The reduced dithiol is then reoxidized to the disulfide by a flavin.The lysine oligomethylene chainis thought to act asa pendulum string, when lipoic swings back and forth betwen thiamine and flavin coenzymes

of the dehydrogenase. This is somewhat similar to the mode of action of the isoprene side chainsof the bioactive quinones (see Fig. '7.2.5).

Pyridoxal phosphate(PLP) is the reactive centerof several enzymes.Its reactive group is the formyl group, whichis positionedparu to a pyridine nitrogen. Schiff baseswithaminoacidsarestabilized by the 3-hydroxygroup,andtwo quinonoid structuresmay occur. Pyridoxal absorbsat 320 nm (E= 4000), and the

Pyridoxol

R = H: Pyridoxal (PL) R = P03W2: Pyridoxalphosphate(PLP)

Py~d~xol

coo0 N

h,,

7.

= 490 nm

3

short-lived quinonoid intermediates can be detected by an absorption peakclose to 490 nm. The Schiffbaseformation of aminoacidswithpyridoxallabilizes all bonds of the amino acid. Thisis exemplified by Scheme 7.3.15 for serine to acarbon atom decomposition, which can be further catalyzed by copper(II) ions (Marcello et al., 1975;Tatsumoto and Martell, 1978). ~yridoxal-cont~ning enzymesacceptonlya single enantiomer of an amino acid. The bond oriented perpendicular to the7c system of the Schiff base becomes most labile as shown in Figure 7.3.4 for deprotonation. Enzymesmay not only control the stereochemistry of chemical reactionby orienting the amino acid substituents, butalso select one outof the five reactions sketched in Scheme 7.3.15. Oligoamines, such as 1,4-dia~inobutane(putresane), spermidine, and spermine, are major nucleophiles in biological cells, whose concentrations

HCHO

HO

H0

cherne 7.3.15

+

COOH

7 R

R

H

H

H

cooc3

re 7.3.4 Coenzyme-bound amino acid often react stereoselectively, e.g., only amino acids are deprotonated.

L-

may reach l mM. The isolation of a reduced Schiff base of PLP with spermidine from sodium borohydride-treated urine suggest its formation in body fluids, and substituted geminal diamines are presumed intermediates inmany enzymatic reactions. In model reactions between PLP and diamines, it has been demonstrated that cyclic diamines are readily formed from diaminopropane at pH 8, but not as readily from diaminoethane systems (Robitaille et al., 1989).

The macrocycle found in vitamin B,, (cobalamin) (Scheme '7.3.16) is called a corrin. In biological microorganisms it is derived from uroporphyrin. Seven of the eight carboxyl acid side chains are preserved but appear as primary amides, the peripheral double bonds are hydrogenated or methylated, and the 20-methine bridge is removed. The stable aromaticn system of the porphyrin is replaced by a chemically labile hexaene system, and a central cobalt(I1) ion is extremely tightly boundin the center. Its fifth coordinationsite is occupied by an imidazole nitrogen; the sixth coordination site may contain any anion, including carbanions such as CH; (Ioatlcy et al., 1995). It is the reactive center of vitamin B,2. Vita-

hapter 7

3

min B,, forms the only organometallic compounds of nature (Schrauzer, 1968; Pratt, 1972). Alkylcob(II1)al~nchemistry and reactivity is dominated by the lability of the C-CO bond with a bonding energy of about 31 kcaVmol. Photolysis yields ageminateradicalpairconsisting of c o b ( I 1 ) a l ~ nand5'-deoxyadenosine (HCH,-Ado). This process is reversible. Within 2 nsafter a photolyzing pulse, 75% of the geminate radical pair recombines.A large fraction of the remaining 25% of the radical pair recombines on a slowertime scale, giving an overall recombination fraction of >90% (Chagovetz and Grissom, 1963).The CO-C bond has a lengthof 2.1 A in the Co(1) oxidation state.The bond is cleaved by nucleophiles, hydrogen, oxygen, or light, and all these reactions have been demonstrated with a simple glyoxime-cobalt complex (cobaloxime) (Scheme 7.3.17). Ethyl radicals dehydrogenate to form ethylene and hydrogen; methyl malonate bound to Co(1) rearranges to give succinic acid.The latter reaction is also found in biological organisms (Schrauzer, 1968). The most s ~ a i g h t f o ~ asynthesis rd of the corrin nucleus applied an enamine as d2-synthonfor one pyrrolidine ring bearing the prospective methine bridge as an exocyclic methylene group. ~eprotonationof the enamine nitrogen produced the necessary carbanion.A s an al-acceptor, an alkylated imidicester was chosen. This was availableby alkylation of a pyrrolidone lactam oxygen with the

0

H

R = H,CN,not Alkyl ~ c h @ m7.3.1 e 8

triethyloxonium tetrafluoroborate (Meenvein reagent)(Scheme 7.3.18). It was not possible to use the d2-synthon with a naked ethylidene group to produce a methylated bridge, but a cyano group as methine substituent worked fine (Eschemoser, 1970). Essentially the same reaction was then used to combine the east and west halves of corrin chromophores regioselectively. Donor (enamine anion) and acceptor (imidicester) were both located on the western half.The eastern half carried an imidic ester as acceptor. Upon deprotonation of the western half, it was deactivated toward self-dimerization, because two anions would not react with each other. It rather reacted withthe eastern half's imidic ester to form an openchain tetrapyrroline. Complexation with nickel (template effect) and treatment with base (removalof an allylic proton) then led to the desired nickel corrinate in 80% yield (Scheme 7.3.19) (Eschenmoser, 1974; Pfaltz et al. 1997). Another type of cyclization reaction of tetrapyrrolidine pigments, which

N

KOBU',A

then HCI

I

CN

Scheme 7.3.19

COOMe

COOMe

7.

connected rings A and D directly and left a methyl group at Cl, was photoinduced electrocyclizationof a fluorescing cadmium complex. Irradiation with vishydrogen shift fromthe methylene group ible light first induced ana~tara~aciaZ neighboring the e n a ~ n group e of ring Dl to Cl of ring A.The resulting conjugated zwitterion cyclized spontaneouslyat room temperature. The first reaction is an exampleof a sigmatropic18 x-electron rearrangement inducedby light; the second reaction is a 16 x-electron thermal rearrangement (Scheme7.3.20). The overall yield was 90%; stereoselectivity was 295%. For References for Chapter 7, see pages 558-563.

This Page Intentionally Left Blank

DNA is the central object of research in recent biochemistry or molecular biology. New and apparently limitless areas of research and fields of applications concerning cell manipulations are continuously discovered.The frontiers of the biosciences have been pushed forward with an incredible speed for more than4.0 years now. How can organic chemists contribute? How can synthetic DNA derivatives and analogs become of value in the treatment of diseases and genetic malfunction? How can DNA be used in synkinesis?There are several possibilities, five of which are mentioned here: 1. Nucleosidesconnected by amideinstead of phosphodiesterbonds may yield stable antagonists to DNA as well as work as direct links between DNA and proteins. 2. The recognition properties of nucleic bases, nucleosides, and nucleotides can be used to connect functional parts of synkinetic dye assemblies in aqueous and organic environments and on solid surfaces. (Fig. 8.1.1). 3. Upscale solid-state synthesesof small DNA oligomersto gram quantities may be possible. 4. Sensitive spectroscopic methods can de developed to analyze nanogram quantities or smaller. 5. Compactionprocedurescanbedevelopedto stabilize longDNA strains and to transport them through cell membranes.

n

Addition of linking DNA duplex

n14 .l Non complementary DNA oligomers capped with thiol groups are bound an oligonucleotide duplex with complementary “sticky to 13-nm gold spheres. When ends” is gold-grafted, then the particles self-assemble into aggregates. Heating above the dissociation temperature annealsthe macroscopic materials. (From Mirkinet al., 1996,)

The human body consists of about 6 x 1013( ‘ ‘ ~ O OM”) - ’ ~ cells, each containing about 6 X g of nucleic acids or 5.5 x lo9base pairs. This would mean a total of about 350 g of nucleic acids per 7’0 kg of human being or 0.5% of the body weight. Nucleic acid strands of human chromosomes have typical lengths of

about 3 cm and a thickness of 1 nm. This length-to-diameter ratio is the same as in a l-mm wire 30hlong. In cells the DNA is compacted by a factor of at least lo4,but the enormous length-to-diameter ratio of more thanlo7makes the structure very vulnerable to rupture (Calladine and Drew, 1992). Gentle lysis with enzymes and uncharged detergents must be used in order to isolate high molecular weight chromosomal DNA (up to lo6 base pairs) from cells. RNA is removed by enzymatic degradation; proteins associated with DNA are denatured with phenol and extracted. Microgram quantitiesof DNA can be electroelated from electrophoresis agarose plates (see next section)byoradsorption toa suspension of glass powder. Purified DNA can then be precipitated from aqueoussolutionswith ethanol or2-propanol.SmallerplasmidDNA(about 103-104 base pairs) is much more stable to shearing forces than chromosomal DNA and usually recovers after adsorption processes on glass powdersor equilibrium centrifugation in cesium chloride~thidiumbromide gradients. Largescale HPLConhydroxyapatiteoranionexchangecolumns is sometimes possible; reversed-phase chromatography and gel filtration are also used. (102-103base pairs)is isolated by similar methods, but no phenol is used, dissolves small amountsof RNA. Proteins are here removed by proteases or precipitation at the waterlchlorofom boundaryphase.Oligo(dT)celluloseefficiently removes mRlVA from nucleic acid mixturesby affinity chromatography, because mRNA possesses a poly(A) tail (Elverset al., 1991; Shabarova and Bogdanov, 1994). Nucleotides are polymeric phosphoric acid diesters, or phosphodiesters, of nucleosides. 3’ and 5’-Hydroxyl groups of D-ribose (RNA) or D-2-deoxyribose (DNA) provide the alcohol part of the esters as well asthe most important chiral centers of the polymer strand. The phosphate group is also chiral, because it bears three different substituents on the phosphoryl (P=O) group (Lowe, 1983). Nucleosides are N-glycosides of D-ribose or 2-deoxy-~-ribosewith one out of fivenucleicbases,namelythepyrimidinederivativesuracil(Ura),thymine (Thy), or cytosine (Cyt) and the purine derivatives adenine (Ade) and guanine (Gua). The lactam and aromatic structures of the nucleic bases given below have been determinedspec~oscopically.NMR and IR spectra as well ascrystal structures clearly show that the given tautomers predominate in aqueous and organic solutions, as well as in fibers and three-dimensional crystals. The tautomers have also been characterizedby ab initio calculations. Three of the bases occurring in DNA are lactams (U, C, G) and behave like ureas. One is an aromatic molecule with aniline-like properties. It is thus generally found that oxygen pulls electhe trons out of aromatic p y r i ~ d i n eand purine derivatives, whereas nitrogen substituents leave the double bonds within the heterocycle (Fig. 8.2.1). N generally act as proton donors in hydrogen bonds; oxygen and nitrogen atoms thathavedoublebondsto a carbonatomareprotonacceptors(Ts’o,1974; Saenger, 1984).

osine

ciI

Structures of the four DNA bases and theRNA base uracil. Only adenine is aromatic; theother bases are polar lactams orbis-lactam.

t-WAs with a molecular weight of about 26,000 daltons are crystallized from water by addition of salt or alcohols. Their crystals contain 30-80% solvent 50 (Pb2+) and can be considered as ordered gels. Heavy atoms with more than electrons must be introduced to allow x-ray data collection.The reliability index R (R-factor) is 0.3 or larger; the best t - M A crystals diffract only to 2.5 W resolution, Individual atoms are not resolved, only the position of base and ribose can be determined. The helix can often be recognized by a cross-like diffkaction pattern, but even the diffraction between single and double or double and triple bodies has not been successful in a number of cases. The crystallization aseither A, B, or D DNA depends on the humidity: the larger the relative h u ~ d i t y ,the larger the pitch and the number of base pairs per pitch. The shape of the grooves also depends on water content (Saenger,1984). The structure of the phosphate group differs from thatof the carboxylate group. The planar carboxyl group shows a strong polarization of the n-bond, which canbe strengthened by electron-with~awinggroups of substituents, e.g., CF,. The n;-bonding of the phosphoryl (P=O) group, on the other hand, is distributed unevenly amongall oxygen atomsof the phosphate group.Furthe~ore,the oxygen atoms are more nucleophilic than the carboxylate oxygens, and the phosphorus atomis more electrophilic thanthe carbon in carboxylate groups. The latter property is most pronounced in lower oxidation states of phosphor, e.g., P(II1) in phosphite esters. Neighbor group effects, on the other hand, are much less pronounced in phosphates than in carboxylates. The proton of a neighboring

- olar, Flexi~ie,andFragileMatrix CH, group, for example, is hardly activated. The most important difference, however, is the unique propertyof the phosphorus atomto use vacant 3d orbitals in additional bonds.This leads to relatively long-lived transition states in nucleophilicsubstitutionreactionsandallowstheir r e s t r u c ~ ~ n(pseudorotation). g Such processesultimatelyaffect the stereoselectivity of SN2-type reactions where the nucleophile and the leaving group were originally in opposite axial positions (Fig. 8.2.2). Several SN1-typesubstitutions at phosphorus atoms are also known: first oneof the substituentsX is eliminated with intermediate formation of the corresponding metaphosphate, then the nucleophile Y- is attached to it (Shabarova and Bogdanov, 1994). Variations of DNA and RNA polymers are caused by steric interactions between the base and carbohydrate units in combination with hydration. They lead to rotations around the glycosidic bond. In a bird's-eye view down the

x

x

I

J

l

I

I

l

I

l

Nucleophilic substitutionson the phosphate group compared to carboxylate and rearrangementsof the phosphate intermediate.

glycosidic bond towards the ribose ring with Cl’ at the center of the circle, the m o w represents the Nl-C2 bond in pyrimidine nucleosides and the N9-C4 bond in purine nucleosides. If the arrow points toward the center the conformation is syn, otherwise it is anti (Fig. 8.2.3). For pyrimidines, the anti conformation is strongly preferred because two oxygen atoms repel each other in the syn conformation. For purines anti is generally preferred, but not strongly. If the imidazole ring carries a bromine atom or similar substituents on C8, the carbon atom between the nitrogen atoms, then rejection by the ribose oxygen atom favors the syn conformation. The anti conformation is usually found in nucleosides and nucleotides, but substitution at C8 of purines often leads to syn, which is an inhibitor of DNA-directed RNA polymerase. Restriction of rotation about the glycosidic bond may be responsible for the antimetabolic activity of subsL,“uted nucleic bases in nucleosides (Fig. 8.2.3) (Saenger, 1984; Hagerman, 19f ’; Calladine and Drew, 1992). The loc structure of 2’-deoxynucleosides is a determining factor in DNA structure. X-ray studies show that four carbon atomsof the deoxyfuranose units are usually in a plane and that the rings occur either in C3’-endo or C2’-endo conformations, depending upon whether C3’ or C2’ is displaced outof the plane toward C5’ (Fig. 8.2.4a).The three major DNA conformations A, B, and2show, for example, different endo-puckerings of the deoxyribose units:C2’-endo for BDNA, C3’-endo for A-DNA, and C3’-endo for purines and C2’-endo for pyrimidines in 2-DNA (Dickerson, 1992). Conformational transitions between these three families are always accompanied by changes in deoxyribose puckering (pseudorotation) and different hydration spheres.At 92% humidity native DNA adopts the €3 form in the crystalline state and has 10 base pairs per pitch. Although it does not persist in solution, it is generally believed to represent the DNA occurringin living organisms.At 75% relative humidity the B-DNA crystal rarranges to the A-DNAform, which is less hydrated and has 11 nucleotides in one turn.The phosphate-phosphate distance shrinksfrom 7.0 W in the B-form to 5.9 W.The A’-form, which prevailsin alcohol or highsalt (Gabb and Harvey, 1993), gives left-handed ill-helices upon concentration-induced crystallization. The repulsion between the phosphate groupsis reduced by high salt concentration as well asby spermine or divalent cations It contains12 nucleotide pairs per pitch. The line connecting phosphorus atoms in ill-helices does not have the smooth appearance of doublehelices, but it follows a zig-zag course. Thisis so because neighboring sugar units point in opposite directions. Homo-DNA (see Scheme 8.4.2) with a cyclohexane-type glucose derivative instead of the cyclopentane-analogous deoxyriboseS connecting link of the nucleotides does not undergo such conformational changes. It falls into a conformationaltrap leading to a homo-DNA with a helix structure similar to A-DNA (Fig. 8.2.4~). Ribose and deoxyribose rings display different co~ormations:in ribose there is sterid repulsion between substituents at Cl’,C2’, C3’, and C4’, in deoxyribose

H0

R

H0

Preferred confor~ationsof the bases in nucleosides as indicated by the lengths of the e q u i l i b ~ uarrows. ~

Chapter

3”endo

6 [degrees] ure Nucleotide confo~ationsat(a) C2’ and(b) C5’. (c)Thevariation of the in and RNA. The absolute value of total energy of the deoxyribose and ribose rings DNA the energy is arbitrary. The dashed curveshows an estimate of the corresponding homoDNA (From Levitt and Warshel, 19’78;Eschenmoser, 1991.)

lexible, and Fragil

only between C3’ and C4’. The more sterically demanding the nucleobase is, the more C2’ becomes endu, making the base more “equatorial.” More electronegative substituents at C2 of pyrimidine pull C3’ into an endu conformation. This sugar puckering mayalso tune the syn-anti orientations of the bases. Finally there are several different conformers possible at the 5’-hydroxymethyl group, where the polymericbackbone of thephosphodiestersismostflexible.Nucleosidesand nucleotides commonly adopt the gauche-gauche conformation given (Fig. 8.2.4b). Figure 8.2.4b also demonstrates the usualgauche effect observed in glycosides: the C2’-Cl’-N7-C2 in pyrimidine N-glycosidic bonds are gauche oriented with respect to the heteroatoms. Nucleoside analogs that lack not only the 20H’ but also the 3’-hydroxy group have been studied as potential drugs for the treatment of AIDS. Their S’triphosphates inhibit HIV- 1 reverse transcriptase (Mitsuya,1991). All molecules have the dihydrofuran ring in a nearly planar conformation with a slight tendency toward O4’-endu. The glycosidic bond is often observed in the unusul high-anti conformation = 90”). The increased flexibilityof the molecules may make them more amenable for phosphorylation by making the “active site” conformation. more accessible. This is an unusual furanose ring conformation with a C3’-exu/C4’-endu twist. Endu ande m refer to displacements of the atoms above and below the plane.This conformation forcesC5’ into an extreme “high”-axial position = -90”), which should facilitate phosphorylation. Even greater flexibility was observed with cyclopentene analogs withC2’-C3’ a double bond(Van Roey et al., 1993). C3’-exu/C4’-endu furanose ring puckering andhigh-anti glycosydic link geometryis common. Hu~dity-controlledsingle crystal transitions between disodiumATP dihydrate and trihydrate are, for example, fully reversible because the ribose moiety is so flexible even in the solid state. The conformation of the ribose unit changes upon hydration: it is C3’-endu and the C4’-C5’ torsion is G+ in the dihydrate, whereas it is C4’-endu and the C4’-C5’ torsion is G- in the trihydrate. The torsional angles around theP-0 ester bonds also change comon characteristics to both crystals on the stacking arrangement of the adenine bases and the helicity of the pyrophosphate linkage assembly along the c-axis (Sugawara et al., 1991). RNA and DNAare polyanions at pH 7. The pkaof the phosphate OH group base always paired is close to 1. DNA is a double helix in which a large purine is with a small pyrimidine base. Only AT and CC pairs occur. AT and GC pairing are favoredby optimal hydrogen bonds, which connect the base pairsthe in hydrophobic center (Fig. 8.2.5a). Monomeric AT and GC basesdo not pairin water but they do pair in organic solvents.The double helices are destroyed reversibly (they “melt”) if the hydrogen bonds are thermally disruptedat temperatures between 70 and 80°C. In UV spectra one then observes a loss of intensity of the bands around 270nm and a small short-wavelength shift. Each DNA has a characteristic ratio (G+C) (A+T), called the coefficient ofspecifity,whichvaries

(x

(x

a) DNA double helix

0 1

Antiddon

major groove

minor groove

P

I

CH3 C=O

I

NH2

I

N

I

N

C L

?H2

minor groove

ase pairing in DNA and RNA. (a) B-DNA;(b)tRNA; (c) m pairs) (d) chemical environmentin major and minor groovesof DNA as produced by the ~ e ~ e r o a tand o ~ s u b ~ t i ~ e noft sthe GCand AT pairs.

from 0.3 to 2.8. The substituentsof the GC and AT pairs also determine the basiscity and binding properties of the DNAperiphery-the major and minor grooves. The minor groove is somewhat more polar, with 40% of lactam oxygens compared28% in the major groove. For tRNA, the same AT and GC pairs occur, although large parts of the polymer may occur as single-strand loops (“cloverleaf’) (Fig. 8.2.5b). mRNA, on the other hand,is a short-lived single-strand DNA. Only some 4% of the total RNA is mRNA (Fig. 8 . 2 . 5 ~ whose )~ sizes show the expectedlarge variations in length, depending on the proteins for which they code. The stereochemistry of the code presumably depends on the ribose units. may One then say that D-ribose codes for L-amino acids. So far we have only put the building blocksof double helices in order: a charged phosphodiester backbone and hydrophilic ribose esters form the hydrophilic outer surface, N-glycoside formationat the opposite ribose corner, and hydrogen bonds leadto a hydrophobic centerof a cylinder. Butwhy a helix? The first important reasonis that the phosph~diesterunit is longer thanthe width of a stacked base pair. A gap of 2.7’ A would appear in an orthogonal ladder structure. These gaps disappear, however, in a skewed ladder in which the carbohydratephosphate chains are tilted 60” from the axis (Fig. 8.2.6). This effect produces tilt, but no helix. A helix is, however, formed when the gap is not closed by means of a base pair shift but by 30”twisting. Now 2 X 11phosphate diesters can wrap around12 base pairs within one helical turn of 360” (Fig. 8.2.7).This twisting is partly reversed when hydrophobic molecules, e.g., ethidium bromide, are forced betweenthe individual base pair (Fig. 8.2.8). The spontaneous entrapment of hydrophobic dyes (“intercalation”) (see Sec. 8.6.2) indicates already an instability of the inner hydrophobic assembly in DNA helices. This instability is mainly caused by the fact that along the base pair stack of DNA purine bases and pyrimidine bases may lie on top of each other and that cross-chain stacking of purines may occur. Optimization of van der Waals interactions may then lead to a slide of the base pairs. Such a purine base slide usually enforces a rolling motion (Fig. 8.2.9). Extreme forms of DNA helices are known as linear (no roll or slide) or hi h1 twisted A helices (roll angle 12”, slide 1 A) (Fig. 8.2.9) have rotation and slide values between these extremes. ANTT steps will be close to form many GG/CC steps prefer the high slide ether, the roll-stick-twist model is indispensable in order unwinding9 coiling processes, and their interaction with proteins. All kinds of intermediate twists are possible. ~nwindingof helices is easiest in regions containing many AT pairs that are connected only by two hydrogen bonds as compared to three hydrogen bonds in the CG pair (Fig 8.2.10).

5' Pyrimidine

3.3A

"

Purine base

3'

5'

- b o o

,P+

Pvrimidil

IT"

P

Stereochemical skewed ladder model of DNA with nogaps between base pairs. (Adapted from Calladineand Drew, 1992.)

Now, if we want to roll double helical DNA around protein loops, then ANTT sequences are known to prefer low roll, R = O", while steps such as CClGC prefer high roll, with R = 5-10". If a given sequence of DNA has the right degree of flexibility, it canbendaround a protein and maythenform enough hydrogen bonds to allow "docking" of DNA to cationic proteins, e.g., positively charged nucleasesor histones (Fig. 8.2.11) Apart from the covalent phosphodiester bonds,four molecular interactions stabilize the DNA double helix:

"

1.Stronginteraction of Mg2+counterionswithtwoneighboringphosphate ions. Both Mg2+and the oxygensof phosphate are hard ions and therefore f o m a very insoluble salt.

Polar, Fiexibie, and Fragile Matrix

07

,3 A

Figure 8.2.7 Sugar phosphate cylinder around twisted base pairs, the most simplehelical DNA model. (Adapted from Calladine and Drew, 1992.) I

Intercalation of ethidium bromide (see Sec. 8.5.3) converts the double helix to a ladder. (Adaptedfrom Calladine and Drew, 1992.)

Uniform rolling orsliding motions upon helix formation leadto linear B helices, twisted A helices, and several related structures. (Adapted from Callidine and Drew, 1992.)

Unwinding of DNA double helices occurs most easily in weakly and very weakly bound base pair regions. (Adapted from Calladine and Drew, 1992.)

A DNA molecule wrapping a cationic protein. (Adapted from Calladine and Drew, 1992.)

Stacking of the nucleicbases.All four bases are essentially flat; methylgroups do not interfere withstacking(comparewithoctamethyl porphyrin, Fig. 6.2.15). The hydrophobic effect onthe base pairs. Between the hydrated phosphate-deoxyribose diesters lie thehydrophobicedges of the base pairs. Hydrogen bonding betweenthe polar edgesof the nucleic bases, Hydration of the phosphodiester backbone, nonmatching base pair thickness and phosphodiester bondlengths, and, aboveall, flexibility of the furanose ring tend to disintegrate the double helix. Any diminution of binding efTects jeopardizes it and rendersthe phosphodiester links more vulnerable to hydrolysis. Destabilization occurs, for example, by (a) removal of Mg2+ions, (b) disturbance of stacking interactions andthehydrophobiceffect by addition of heteroatomsor charges to the hydrophobic edge of the nucleic bases (e.g., hydrazine, methylation; see Sec. 8.4), and (c) meltingof the hydrogen bondsby heating or addition of urea. Interdigitationof large dyes, polycationic counterions, or covalent crosslinking of base pairs,on the other hand, stabilize the double helix. Some sequences in DNA havea marked propensity to adopt altered conformations withinthe double helix.Homopurine-homopyri~dinesequences, 5’-+”ACAGA.--3’ 3’--CTCTCT--5’ although clearly double-stranded, are hypersensitiveto S1 nuclease, an enzyme that ordinarily only cleaves DNA single strands. The hypersensiti~itymay be

......

...

Hairpin and s i d e - m formation in DNA by palindromic sequences. (Adapted from Calladine and Drew,1992.)

owned by excess hydration and a conversion of the usualI3 to an A helix. Alternating pu~ne-pyrirni~ne sequences, on the other hand, hadto adopt the Z-conformation, e.g.: 5’--GCGCGC--3’ 3’--CGCGCG--5’ Most significantfor the stability and structureof double helices are palindrornic sequences, in whichthe sequence of bases is the same when it is led in the s”to-3’ directionof each strand. Such sequences may extrude from the linear duplex to form intrastrand segments called cruciforms (Fig. 8.2.12). Such regionsareagainhypersensitive to avariety of enzymesinboth the singlestranded loop andthe lower stem region (see Fig. 8.3.3). The most remarkablefeature of the DNA double helixis that it suggests a

- alar, F l e x i ~ ~and e , Fra simple mechanism for copying genetic i n f o ~ a t i o n(see Sec. 8.3.) The vulnerability of the DNA double helix allows separation of strands; the the cooperativity of the binding effectsleads to perfect duplication.

The analysis of DNA sequences relies on poly(acry1~de)gel electrophoresis (PAGE) (Westermeier, 1990). When placed in an electrical field, DNA polyanions move towards the anode. In the viscous medium of polymer gels, the is re-motion tarded by drag. The smaller the DNA, the faster it moves. DNA fragments are therefore separatedon the basisof length. The effects of helix differentiation can be removed by using melting conditions. This may involve either temperatures above 70°C or urea concentrations above8 M in “sequencing” gels,As markers for double helices, one uses fluorescing intercalating dyes for large DNA pieces or radioactive phosphate containing 32P at5‘the end. 32Pis introduced withan enzyme called kinase. DNA kinase transfers (e.g., radioactive) phosphate from AV to a free end of the nucleotide, usually 5‘. ATP with a 32P-labelled y-phosphate group has a half-life of just over a week, emitting 1MeV P-particles, It can be detected by autoradiography on electrophoresis plates andis used in the analysisof DNA degradation. The phosphate can also be chemically removed again with alkaline phosphatase, which only splits terminal monoesters. The chemical reactivityof the DNA and RNA polyphosphordiester bonds is very different. First of all, DNA is not hydrolyzed in alkaline media. It is as stable as monomeric dialkylphosphatesat pH 13 (see Sec. 8.5.3). When treated, however, with acid, DNA is not only hydrolyzedat the phosphodiester positions, but the N-glycosides are also destroyed. Purines react here faster than pyrimidines. Chemical hydrolysisof DNA is thus useless as an analytical tool because either the monomers are destroyed or no reaction at all occurs. RNA, on the other hand, is readily hydrolyzed by alkali. Simultaneous cleavage of internucleotide bonds overthe entire length of the polymer chain occurs (Scheme 8.3.1) (Fung andYeung, 1995). Simple hydrolysis of natural DNA does not lead anywhere in analysis.If the acid hydrolysis is, however, preceded by base-modi~cationreactions, one obtains Nobel prize-winning differentiations.This is documented in the simplified protocol developedby Maxam and Gilbert (Scheme 8.3.2). Dimethyl sulfate methylates guanineat N7 five timesfaster than N3 at adenine. Noneof the other bases reacts with dimethylsulfate if reaction times are kept short and the temperature low. Reaction conditions are selected in away that only 1-2 % of the nucleobasesreactina ratio of guanine:adenine of 5: l. Thesebasesare now

positively charged, and their N-glycosidic bonds are cleaved most rapidlypHat 7 and 90°C. C reacts faster than G @>A). With 0.5 M HCl, however, the more basic adenosine nucleotide reacts almost exclusively (A>C). The purine bases can thus be differentiated by the intensity of electrophoresis spots in two independent experiments. The pyrimidine bases are differentiatedby their reactions with hydrazine.In distilled water, both bases react equally well (C+T), but upon NaCl the cleavage of the thymine glycoside is selectively suppressed becauseit is less hydrophilic than cytosine(C). The phosphodiesters that have lost their nucleobases are then selectively hydrolyzed by alkali or piperidine (Scheme 8.32). Electrophoresis of the fragments with 32Pat the 5'-end are a ~ e ~ a r separated ds into four independent lanesby electrophoresis. Forall four bases, there are two redundant spots appearing on autoradiographs together with two nissing spots. Thesefour independent resultsfor each oligomer obtainedby four independent chemical degradation procedures starting from the 5'-end eliminate most sources of errors. A weak A>G band in the reaction mixture together with a strongG>A band and noC and CcT band proves,for example, C in positions 2 and 6 in the example given in Figure 8.3.1, in which the first 17 nucleotides couldbe assigned directly. An even more sensitive method introduced by anger is called the chain-

Fragment3

0"O

I I

I

0"

0

0 O=&O"

? I

0-1-0"

d

d

I

Fregmenl,

iragmenk l

Phosphate buffer WC (A G )

-

Pipendine/HzO

I o=A-o

I oo

.3.

termination reaction and applies the unchanged probe DNA as a matrixfor the synthesis of a mixtureof partial sequences (Sanger,1988).A primer, theKlenovv fragment of DNA polymerase I and mixtures of deoxyribonucleosides triphosphatescontaining32P-dATPasamarkerandasmallamount of the four dideoxyribonucleoside triphosphate analogs are used as starting materials. The polymerase reaction then starts but is intempted every time the dideoxyribose derivative is built into the polymer. Since each of the four dideoxy analogs is used in a separate vial, one obtains four independent hints asto the presence of a given base at a given position: three negative ones (no oligomer of the given

A>GG>A

C C+T

I -

-G-G -G

3.1 Electrophoresis of the four mixtures of DNA fragments as obtained by the reactions of the hydrolysis scheme (Scheme8.3.2). The arrow runs from the starting point to the front. The smallest fragments run on the front.

polymerization number) and one positive (the oligomer ending with the added dideoxy nucleotide). The chain-termination methods yield, of course, the sequence of the complementarystrain(Scheme 8.3.3), whereas the chemical degradation gives the original sequence (Fig.8.3.2) Smith et al., 1980). The last example for the enormous utility ofPAGE procedures in the analysis of pg quantities of DNA is given for the synthesis of quadrilateral or rectangle DNA molecules and of three intermediates using palindromes (Fig. 8.4.7).

In nucleo~idesthe positions of 3iP(spin = 1/2) resonances in N M R spectra depend on the charges on the phosphates and therefore change with pH. The intracellular pH in vivo can thereforebe measured. Furthermore,l 8 0 atoms bound to phospho~syield upfield shiftsof the 31Psignals, andthe enzymatic reaction between "0-labeled ADP and 180-labeled Q ~ ~ ~ ~ - p h o s pinhcells a t e can be followed by 31P-NMR.A useful property of the I7O nucleus (spin = 5/2) is the drastic shortening of 31Prelaxation times leadingto line broadening to the point where the 31Psignal may virtually disappear.In this context,it is also of impo~ancethat

olar, Flexible, and Fra

5'-A-T-C-G-G-C-T-A-C-3' 5'-A-T-C-G-G-C-T"A-3' 5'-A-T-C-G-G-C-T-3' 5'-A-T-C-G-G-C-3' 5'-A-T-C-G-G-3' 5'-A-T-C-G-3' 5"A-T-C-3' 5'-A-T-3' 5'-A-3'

The four electrophoresis lanesof synthetic DNAs containing oneof four dideoxy terminators and a mixture of all four synthesis mixtures. The arrow runs from the starting point to the frontof the electrophoresis.

dR

s[ppm]

I

201 203

I

202

I

I

200

I 199

15N-NMRspectra of d[CGT("Nl)ACG] acquired at 3.3"C in (a) 100% D,O and (b) 20% D,O after 18 hours. The spectroscopicsplitting of a singlet into a group of five resonances indicates two different kinds of exchangeable protons in the AT base pair. Deuterium substitution at thymine N3 gives a downfield shift of 0.34 ppm, the same substitution at adenine N6 an upfield shift of 0.17 ppm. These effects indicate that the thyminehydrogenatom interacts withadenineviadipolarhydrogenbondingonly, whereas for the adenine amino hydrogens through-space interactions predominate. "N shifts are useful indicatorsof DNA interactions with molecules addedto the DNA periphery as well as for mismatching withinDNA the double helix.

phosphates with one substituent and three different oxygen atoms (160,1 7 0 , and W) are chiral. Such phosphates canbe made from the thiophosphonates of chiral diols derived from mandelic acid.The stereochemistry of enzymic reactions on nucleoside triphosphates can thus be eludicated by 31P-NMR (Lowe, 1983; Byrd et al., 1986). The helices formedby double-stranded nucleic acids are not rigid entities, but display flexibility chracterized by numerous thermal motions of phosphate units. These motions have been measured by 31P-NMR and are veryfast. Relaxation times occur in the nanosecond time range (Bolton and James, 1980). The local motions are, however, not strongly coupled to the confo~ationof the nucleic acid, and intercalated dyes are reoriented on a slower time scale.

Hydrogen exchange in the hydrophobic center of DNA occurs in a slow, so-called opening reaction leadingto a state with disrupted pairs. Hydrogen-exchange experiments between water and base imino protons, for example, point to a millisecond timescale, which has been confirmedin model calculations using a Brownian dynamics simulation (Briki et al., 1991). The slow opening of the base pairis not relatedto inherently slow motions in the DNA center, but reflects the statistical nature of the building-up process of energy, which is necessary to break a base pair and to allow hydrogen-exchange reactions. Self-repairof small disturbances, whichis so favorable in DNA, evenretards proton exchange. Nitrogen NMRof specifically15N-labelednucleotides(Goswamiand Jones, 1991) provide unique information about protonation and hydrogen bonding. The chemical shift of an sp2 nitrogen is strongly affected by protonation. AdenineN1protonation, for example, causes anupfieldshiftof 70 ppm (~uchanan,1989). lH-15N co~elationspectra ~ ~ a t i c a lreduce ly the time required to obtain W chemical shifts relative to direct 15N measurements. In d[CG(I5Nl-A)GAnCCCGL] in D,O at pD 5.0 and pD 7.8the 15Nresonance of A is, for example, shifted upfield by 66 ppm upon protonation. A pka of 6.6., which is more than two units higher than that for the monomeric A, indicated an AC “mispair” instead ofAT, in which the proton is strongly fixated (Muller, 1979;Wang et al., 1991a).Since the nitrogen chemicalshift of the adenine N1 in base pairsis also influencedby the hydrogen isotope present in the H-bond, one may obtain complex spectrafor a singleNH or ND proton and detailed information on abnormal strutures within DNA oligomers can be deduced (Fig. 8.3.3) (Wang et al., 1991b).

We start with a short summary of some essential biochemistry in order to provide a background for the synthetic and synkinetic chemistry described in this and the following chapter, More detailed and sometimes beautifully illustrated descriptions are found in biochemistry textbooks (Voets, 1992; see Chapter 1 references) and review articles (Wu et al., 1979, 1983, 1987; Berger et al., 1987; Marx, 1988; Sambrooket al., 1989).

The manipulation of DNA has been revolutionized by a combination of enzymatic synthesis and solid-state chemical synthesis (“glue technology”). Two enzyme types are involved in DNA synthesis. DNA ligases catalyze the formation of a new phosphodiester bond when given ATP as phosphate donor and nicked double-stranded oligonucleotides as

substrates. Mostligases require that the5’ end of DNA bephospho~latedin advance. Ligases are appliedto covalently connect several small oligonucleotides whichhave before self-assembled in solutionviastickyends,andonlyone strand needs to be phospho~latedat the 3’ end. Several synthetic DNA fragments can thus be ligated in one step to give a polynucleotide without upper limit to size. Blunt endligation is also possible. It merely requires more ligase than the connection of sticky end terminals. DNA and RNA polymerases catalyze the template-directed polymerization of deoxyribonucleoside or ribonucleoside triphosphates. DNA polymerase allows one to copy any DNA molecule starting from the 5‘ end. RNA polymerase makes possible the preparation of any RNA molecule complement^ to any DNA molecule (“transcription”),The DNA segmentto be transcribed must follow a segment of DNA (“promoter”) that serves as a recognition site for the RNA polymerase.The following sequenceis obeyed: 1. DNA single strands always grow in the 5’+3’ direction, because they are synthesized exclusively from 5’-triphosphates (“leading strand”) by polymerase-catalyzed condensation. The second single strand of the double helix (“lagging strand”) is replicated in the 3”-35’ direction. Ligase, on the other hand, connects sticky ends of DNA singlestrand fragments (Fig. 8.4.1). 2.DNAreplicationinvolvesunwinding of thedoublehelix (see Fig. 8.4.1) and needs a short RNA primer as a starter before the nucleotide triphosphates can be added (Fig. 8.4.2). 3. DNA is then used to produce RNA with i n t e r m e d i ~mRNA (“transcription”). There are 20 different RNAs for 20 amino acids, and the terminal 3’-OH group is always esterified with the carboxyl groupof the amino acid (Fig. 8.4.3). 4. The final reaction, namely protein synthesisor “translation” of mRNA information into amino acid sequences, then occurs via a specific “anticodon” of specific RNASs (Fig. 8.4.4). A triplet of nucleotides codes for a single amino acid, e.g.,UUC or U W for Phe. The DNA-controlled protein synthesis thus starts with the synthesis of a short-lived, complementary messengerRNA (mRNA) strand. d W A then associates to ribosomes inthe cytosol and triggers protein synthesis. Since mRNA is quickly synthesized and as quickly decomposed in vivo, it allows the cells to react quickly on changes in the food supply. Escherichia coli cells, for example, produce enzymes for the hydrolysis and membrane transport of lactose only when lactose is present in the environment.The short-lived mRNAsingle helix provides unique opportunitiesto organic chemists to block specific protein syntheses in living organisms, which are critical for certain diseases to flourish. In principle, it should be possibleto block mRNA action in aqueous cytosol media completely with small amounts of RNAs complementaryto mRNA for the given protein. Since the rnRNA template is known as the “sense” strand, the comple-

DNA-A

Polar, Flexible,and Fragile Matrix lagging end

5’

5’ leiding and

I

3’ Triphosphate polymerase

I

DNA polymerase

3’

6

l

DNA ligase

double strandDNA

.l (a)Schematic modelof DNA replication. The DNA double helix is partially split into two single strands, the Watson-Crick pair is formed to the separated single strand and the new DNAs are proofiead. This enormously complicated multitask is fulfilled bya single enzyme named DNA polymerase I.(b)DNA fragments with “sticky ends” r~co~bine spontaneously and are then condensed to polymeric phosphodiesters by ligase. rnentary strand is called “antisense” ( U h l r n ~ nand Peyrnann 1990; ~ e i n t r a u b , 1990). A typical antisense mRNA would be 17-25 nucleotides long and would destroy the target mRNA by binding to it and invoking ribonucleaseH activity. The synthetic RNAs are, however, also quickly destroyed by other ribonucle-

-0 I -o-y=o

c:

-0-7=0 -0-

DNA matrix

Chain growth from the 5’ to the 3’ end at a DNA matrix. Antisense DNA

Sense DNA

Sense RNA

Sequence- and strain-specifictranscriptio~of DNA into an mRNA sequences. Single-strand sense mRNA is formed. Antisense mRNA could form a double strand with sucha molecule.

ases, and for medical application more stable derivatives are needed. One polymer backbonerecognized by RNAse H, whichis not destroyed quickly by other RNAses, consists of phosphorthioates, where one of the phosphor osygens is replaced by sulfur (Cohn, 1982; Eckstein, 1983) (see Scheme 8.6.1). Since the antisense activity depends largely on the base sequence and not so much on the polymer backbone connecting them, it is also possible to use amide-connected

tRNA

‘0 I o=P-0 l 0

0

R

Enzymatic aminoacylationof a specific tRNA is the key step in“transthe lation” of nucleic base triplet sequences of DNA into amino acid sequences of proteins.

nucleic acids (De Mesmaeckeret al., 1994) as relatively long-lived but quickly digestible carrier systems for the phosphorthioate antisense drugs.

esi Automated solid-phase DNA syntheses start with a deoxynucleoside linked to the solid support through the 3’ or 5’ hydroxy group (Fig. 8.4.5). Cross-linked poly(~,~-dimethyl acrylamide) gels or glass beads can be used. In case of the glass beads,the silanol residueis connected with the3’ end of a protected nucleoside via a propylamine spacer by a stable amide and a base-labile ester bond. The protecting 4,4’-dimethoxy tritylium group is then removed with (Ama = 498 nm) servesas a trichloroacetic acid, and the released tritylium cation monitor for the success of this first step and later for monitoring the success of each cycle. Acid treatment must be brief inorder to prevent depurination. Instep 2, a protected deoxynucleoside 3’-phosphor~diteand tetrazole are added simultaneously. Tetrazole (pk, = 4.8) protonates onlythe amidite nitrogen and cona good leaving group.The P(II1) cation is now a very good verts the amidite to electrophile and reacts with the 5’-OH group toform the desired 5’ +3‘ internucleotidiclinkage. The phosphite also carries a methyl or 2-cyanoethyl ester group in order to prevent self-condensation (Sim et al., 1993). Yields are between 95 and 99%. Nonreacted nucleosides on the polymer surface are now capped (step 3) by acetylation in order to preventlater reactions with phosphoramidites and productionof two short nucleotides. The phosphite triester linkage is unstable to acids and bases and is therefore immediately oxidized withiodine to the phosphate triester (step 4). One cycle is now closed, anda new cycle starts with the release of tritylium cations from the growing nucleotide branch on the polymer surface. In the final steps, all protecting groups are removed from the

Chapter

4

I

1. step CFaCOOH

OMe

ure 8.4.5 Scheme of the automated DNA synthesis on a polymer support (glass bead; see text).

phosphate linkages, in particular themethyl ester by benzenethio~~iethyl amine in dioxane and the cyanoethyl groups by ammonia, which also cleaves the anchoring 3 ’ - s u c c i n ~ cester. Up to 30 rng of a 20-base DNA can thus routinely be obt~ned,although at very high material cost (Narang,1987; Cmthers et al., 1987; Englisch and Gauss, 1984.) A large variety of modified DNA molecules has been synthesized (Olgivie et al., 1987). Amides andt~ophosphateshave already been mentioned. Another obvious variation is, of course, to convert the polyanionic DNA into a polycationic analog.This was achievedby the phosphoramidite chemistry described in Figure 8.4.5. Phosphodi~ste~ links were generated as usual and then oxidatively coupled with appropriate diamines to obtain cationic 2-aminoethyl links using tetra~hloro~ethane (Scheme 8.4.la) (Letsinger et al., 1988).The most spectacular example of polycationic DNA arethe polyguanidiniu~or poly-S-methy1~-

olar, Flexible, and Fragile Matrix

P Scheme 8.4.1

iourea derivatives, which contain as many positive charges as monomeric units at all pH values. Synthesis involves at first a condensation reaction between 3’a isothiocyanate and a 5’-amino nucleoside affording the 3’ ”+ 5’ thiourea-li~ed dimer. The positive charge was introduced by S-methylation of the oligomer. Urea thymidyl thymidyl oligomers with poly(dA) gave a triple helix with a poly(dA) core and two thioureaT strands (Scheme8.4.lb). Hydrolytic cleavage of DNA or RNA without a preceding methylationor oxidation generates fragments that are chemically competent for ligation toother oligonucleotides by enzymaticreactions.Covalentlylinkednucleoside-2,2’bipyridine Cu(I1) complexes were,for example, introducedinto oligonucleotides by the phosphoramidite method. They are able to hydrolyze RNA at 37°C and neutral pH (not shown) (Modarket al., 1991). The second important variation concerns the chiral center, consisting of the deoxyribose units of DNA. It wasreplaced by a more rigid 2,3-dideoxy-~ - ~ ~ ~ ~ ~ ~ - h e x o punit y r aton produce ose a “homo-DNA” with an extra CH, group in the carbohydrate unit. The automated phosphoramidite method was again used with almost no variations. Base pairing was also observed in double-helical homo-DNA, but it was found that homodimers became more stable than heterodimers (Scheme 8.4.2) (Eschenmoser, 1991; Bohringer et al., 1992). The major difference between single-strand DNA and homo-DNA is the much more rigid folding of the latter, which favors formation of stable double helices.

/I

-

Watson Crick

H

anti anti antiparallel

(1)

KzCOdOMF

(11) NBS Hydrolysis

depmtection d(GTTATCCGCTCAC) further 5 cyclespurification

I

The final variation concerns the drastic variationof nucleic bases. A sitespecifically modified oligodeoxynucleotide was synthesized as a simple mutagen model. Anthracene was chosen to study carcinogenic effectsof a 13-base singlestrand complement^ DNA to a bacteriophage DNA. A 9-anthracenyl moiety was attached to N2 of guanosine, the eighth nucleotide in the 13-mer. Since N2 of guanosine cannot be alkylated directly, a tricyclic precursor was chosen and the auxiliary ring removedby bromination and hydrolysis. Poor coupling yields were observed with this bulky nucleotide and long reaction times were necessary to obtain the octanucleotide in 70% yield. All coupling steps afterwards proceeded in the usualway (Scheme 8.4.3) (Casale and McLaughlin, 1990).

The polymerase chain reaction (PCR)cycle is used to multiply any DNA molecule by factors of 106 and more. It begins by heat denaturing picomoles of a double-stranded DNA of any origins (e.g., archeological dinosaur pieces) to give two single-stranded templates.The 3' end is analyzed, and a complementary20mer is made by chemical synthesis. This primer is attached to the single strand, and DNA polymerasein the presence of nucleotide triphosphates then produces a complementary DNA molecule. The double helix is again denatured, new primer and polymeraseis added, and the cycle begins again. Aftern cycles one has produced 2" copies. About25 cycles are usually possible;10-12 moles have become X 225 = 1O-l2x 3.4 x lo7= 34 p o l e s (Figure 8.4.6) (Mullis, 1990). Double strand DNA

+ primer

1+dATP,dGTP, dCTP, dTTP DNA-polymerase,

The PCR cycle consistsof three steps: heat denaturingof the DNAto be multiplied, primer annealing, and DNA polymerase I-catalyzed copying.

In living cells, DNA is found almost exclusivelyin the form of linear duplex molecules; theymay be supercoiled, stressed,or even knotted (Whiteet al., 1987), butthe helical axis is unbranched. DNA branchedjunction structures occur as interme~atesin the process of reco~bination,but they arei ~ e r e n t l yunstable because of thetwofoldsequencesymmetry,which occurs innatural DNAs. ~ligonucleotideswith stable branchings can, however, be made if the sequence symmetry is minimized. Solid-state synthesis (Chen and Seeman, 1991; ZhangandSeeman,1992,1994)thenallowstheconstruction of complex supr~oleculeswith the hm-thick DNA double strands. The procedure contains the following steps:

1. Attachment of the first unit to a solid support. 2. Restriction of one or twohairpinstoexposestickyends.Cohesive ends are liberatedby the controlled removalof DNA hairpins utilizing restriction endonuclease digestion. 3. Additive or cyclizing ligation to form the next edge. 4. Exonuclease destruction of failure products. 5. Removalfromthesupport. 6. Annealing the product to covalency. Steps 2, 3, and4 are repeated in order to build up the target molecule. Figure 8.4.7 illustrates the strategy of these ligation steps for the synthesis of a qua~lateralmolecule. Purificationof minute amountsof product is impossible; all unwanted side products, namely thoseof unsuccessful restriction, must therefore be removed. A “restriction rescue procedure’’ works as follows (Zhang and Seeman, 1992): the successfully ligated productis covalently closed andfailure products are destroyed with an exonuclease. This t r e a ~ e nist analogous to “capping” in solid-phase oligonucleotide synthesis.DNA cubes (Chen and Seeman, 1991) and octahedrons (Zang and Seeman, 1994) have also been constructed as geometrical bodieson a nanometer scale. Each double-helical m designated to become an edge is protected first as a hairpinloop, which is then removed by a r ~ s ~ i c t i oendonuclease, n thereby esposing a ligatable sticky end. The only analytical data for intermediatesand end productsin pg quantitiesare electrophoretic autoradiograms and comparisons with DNA molecules of known length as markers (Fig. 8.4.7).

DNA libraries are obtained by a combination of the techniques of chemical solid-state synthesis and the combinatorial methods described later for peptides (see Sec. 9.3.2.2). DNA should, however, first be transformed to RNA libraries in order to yield useful recognition systems. Starting from a large pool of random-sequence DNA molecules, RNA sequences are obtained (see Fig. 8.4.3),

Polar, Flexible, and Fra

1

2

3

4

5

6

7

8

9

J -4%;

267

184

-124 -104 -89 -80 -64 -57 -51

CG GC TA AT CGCATGT A T A ACA T A T G TGCGTqCACGACGAGG T CG TA CG CG AT

CG

CG T

CG T

GC

A.7 (Top) Electrophoresis lanes of single synthetic DNA molecules, which are characterized here onlyby a comparison with DNAsof known polymerization grade at the rightof the plate. On the left, two examples illustrate cutting along a given, shaded base triplet (restrictionR) and one examplefor sticking ends ligation L. (Bottom) Model of R,L-synthesis of the DNA rectangle. If the synthetic DNAs, e.g., the rectangle, are made by combination of sufficiently large fragments or by fragmentation of larger DNAs with specific restriction enzymes, electrophoresis may be sufficient to measure molecular mass and thereby prove structure. Differences of 40-60 base pairs between educt and product are very easy to detect.

which are specifically recognized by amino acids bound to a chromatographic phase. The amino acid may differ by only one stereocenter to produce a phase that is active in affinity chromatography. D-Tryptophan bound to agarose (D-trpAga) is, for example, clearly differentiated from L-trp-Aga by an RNA molecule, which has been isolated by affinity chromatography on an D-trp-Aga colum. A random-sequenceRNAlibrarycontaining 5X (!)differentsequenceswas obtained from a 169-meric DNA consisting of 120 random deoxynucleotides

ha

flanked by two defined primer binding sites for PCR amplification. Of the 700 pg of synthetic DNA, only 5% were amplified by PCR. This mixture was then transcribed to yield a pool of 32P-labeled RNA molecules. Repeated affinity chromatography on D-trp-Aga, reverse transcription and PCR amplification of the cDNA finally yielded an RNA pool awith defined sequence. It was tightly bound to D-trp-Aga but not to L-trp-Aga. This procedure has been named “systematic evolution of RNA ligands by exponential enrichment” (SELEX) and has been applied to several proteins, amino acids, and nucleotides. From a pool of different RNA sequences randomized in 74 positions, individual RNA molecules have been selected that bind specifically to Lcitrulline in aqueous solution. Citrulline is an analogue of arginine, in which the terminal imido function is replaced by carbonyl. The selection led to the enrichment of one common structural motif that consists of two consense sequences with a length of 10 and 6 bases. A triple mutant of this motif bound arginine, but had entirely lost its specifity for citrulline. L-citrulline is presumably fixated by hydrogen bonds, L-arginine in addition by strong electrostatic interactions. In both cases good enantioselectivity has been observed. Seven fold decreases in the binding constants were typical (Famulok and Szostak, 1992a,b; Famulok, 1994).

Strong bases hydrolyze nucleic bases slowly and ammonia is released. Acids, however, often stabilize the urea-type heterocycles.The basic lactam oxygen is protonated and converts the bases to aromatic systems (compare with Scheme 8.5.4). The C=C double bonds can usually be removedby catalytic hydrogenation or Michael additionof hydride or other nucleophiles (Scheme 8.5.1). Oxidationwithperacids,ontheotherhand,usuallygivesN-oxidesratherthan epoxides (Suhadolnik, 1979; Shabarova and Bogdanov, 1994). Hydroxylamine reacts exclusively with cytosine at neutral or acid pH to give a Michael adduct and amino group substitution (Scheme 8.5.2). The reaction rate reaches it maximum at pH5-6.

NHzOH NHOH

I

I

R = Ribose residue

NH,

The nucleic bases are all weak acids with pkaY values from10 to 12. N protons of imidazole N9 or of the lactam nitrogens dissociate by 50% at p 10-12. Mercury or silver salts react with these nitrogen atoms to form salts (Scheme 8.5.3). Nucleosides are formed if the mercury or silver salts are combined with halosidesof deoxyribose. Nucleosides are formed from benzyl ethers of ribose or deoxyribose by replacement of the acetal benzyl groups by bromide and coupling with the mercury salts of nucleic bases. The benzyl ether groups are removed by treatment with ammonia (~ischer-Helferichmethod; not shown). In the case of pyrimidine nucleosides, one usually converts the lactams first to aromatic ditrimethylsilane ethers, which then react in high yields with protected halogenoses (Scheme 8.5.4.) Nucleosidesynthesis is a rareprocedurenowadays for nucleotide chemists, because a large choice of high-purity nucleosides is commercially

ha

6z

OBz 062

che

available. Only when uncommon carbohydrates and nucleobase derivatives are desired does one need to come backto monomer synthesis. The amino groups of cytosine and adenine are converted to lactam oxygens by diazotization with nitrite and subsequent hydrolysis. ~uanosinereacts faster than adenosine and cytidine (gua >ade >cyt). The base sequence in DNA may thus be changed by the “mutagen” nitrite. The nitrogen atoms of nucleobase are also easily alkylated by various reagents (e.g., diazomethane, epoxides, aziridines), which again may either change or destroy genetic material.

olar, Flexible, and Fragile Matrix

R = Ribose

Guanine derivatives are alkylated most readily; inDNAN7of guanine the residues are most susceptible. Nonenzymatic alkylating reagents often create DNA lesions with carcinogenic consequences. E. coli employs the so-called Ada protein to repair alkylated DNA products by irreversible methyl group transfer to specific cysteine residues. Cys-321 repairs base adducts; Cys-69 repairs methylphosphotriesters.Cysteines are usually bound to a zinc ion here in order to maintain the §-nucleophilicityat physiological pH. Modelexperiments with inorganic zincbenzenethiolates and PO(OCH,), suggest an anionic moiety in Ada. Presumably only anionic sulfur(s-) is reactive, not §H (Scheme 8.5.5; Wilker and Lippard, 1995). A few typical examples of electrophilic and oxidative reactions are summarized in Schejne 8.5.6:b r o ~ n a t i o nusually gives substitution rather than addition products, whereas in nitration reactions one also observes electrophilic substitutionand the ribosemust be protected.Hydroxymethylationwith formaldehyde occurs preferrablyat C5 of uridine. The reactivity of nucleophiles with nucleosidesis often surprising and always unpredictable. Hydrazine,for example, reactsat pH 7 only with cytosineto give mono- and disubstituted hydrazines; hydroxylaminegives the same substitution reaction plus an addition to the C=C double bondof cytosine at C6 (see Scheme 8.5.2). With P,§, only the lactam oxygen, not the ureic oxygen, is replaced.Thesulfuratomcanthen be replaced by amines to formimidates (Scheme 8.5.7). The classical irradiation (UV, x-ray, etc.) damage to DNA consists of the formation of pyrimidine photodimers. A photolyase enzyme may cleave these

5 oLN

HzNNHz

I

N

OAN

HN-NH N

T

I

'' i

+OAN

i

R

P

N

NAO

I

R

R = Ribose

covalent dimers in order to repair the DNA double helix (Burdi and Begley, 1991).

The N-glycosidic bonds of nucleosides are usually highly stable in neutral and alkaline media and prone to acid hydrolysis. Purine derivative hydrolysis is then much faster than that of pyrimidine derivatives (Table 8.5.1). The glycosidic carbon-oxygen bondin pseudouridine (AC-glycoside!) can hardly be broken by acids. It undergoes isomerization tothe a- pyranoside because the ether bond is reversibly opened by acids, whichform short-lived carbenium ions. Such a-pyranosides are not observed with the N-glycosides in nucleosides (Scheme 8.5.8).

Rate Constantsfor Acid Hydrolysisof Purine and Py~midineDerivatives" Compound Adenosine Guanosine Cytidine Uridine Deoxyadenosine Deoxyguanosine Deoxycytidine Deoxyuridine

k,,s-l 3.6 10-7 9.36 10-7

1 10-9 1 10-9 4.3 10-4 *

8.3

*

lo4

1.1 10-7 1 10-7

H

H !H+ NH

“he acid-base behaviorof nucleotides is dominated by the acidity of the nucleic bases and the phosphate groups (Table8.5.2). Some remarkable changes in pka values are observed when going from hydrophobic to hydrophilic derivatives (e.g., it is more difficultto protonate theNH, of adenosine than thatof adenine), the acidity of the anomeric pentose proton (typicalplca, values: 12-13), and the pk, value of the phosphate close to ’7.At high pH values, the 20H group of ribose can also be deprotonated (Shabarova and Bogdanov, 1994). The rate of hydrolysis of the most simple monoalkyl phosphates passes

pk, Values for Nucleotides and Their Components PhosphatePentose

Adenine Adenosine Deoxyadenosine 3.8 Adenosine-5’”phosphate 3.7 Adenosine-2’-and3’-phosphate3.7 Thymine Deoxythymidine Deoxythy~idine-5’-phosphate 10.0 12.2 4.6(NH2); Cytosine Cytidine Deoxycytidine Cytidine-5’-phosphate 4.5 H) 3(NH); 3.0(NH2); Guanine 2.1; Guanosine 9.33 Deoxyguanosine 2.4; Guanosine-5’-phosphate 2.9; 9.6

Base

NH2);

12.4 13.1

0.9; 6.05 0.9; 6.1

12.9 1.6: 6.5

0.8; 6.3

12.3 0.7; 6.3

434

Chapter

through a maximum somewhere between pH 3 and 5. At alkaline pH values,the hydrolysis rate drops sharply, The maximum rate corresponds to the highest monoanion concentration in solution; the dianion is not active because of electrostatic repulsionof incoming hydroxyl ions.The methyl phosphate monoanion hydrolysis is much faster than that of the dimethylphosphateanion. Dialkylphosphates are among the least reactive esters, and their hydrolysis rates drop linearly from pH 0 to pH 4, Above pH 5 these esters are stable in water, which is indicative of extremely slow hydrolysisof dialkyl phosphate monoanions. Exceptionsinclude phosphates of 1,2-diols7with hydroxyl groups adjacent to the phosphate ester. RNA can therefore be degraded by alkaline, whereas DNA cannot (see Fig. 8.3.1). The stability of the diesters comes fromthe combination of negative charge, which repulses the hydroxide anion, the andweak nucleophilicity of water, which cannot attack the phosphorus atom if it carries a negatively charged group. Trialkyl phosphates undergo hydrolysis in both alkaline and acid media.At alkaline pH values, a nucleophilic attackof the hydroxide ion takes place at anaxid position of a trigonal bipyramid. Optically active phosphates undergo inversion (see Fig. 8.2.2). In neutral and acid solutions, the C-0 rather thanthe P-0bond is broken as shownby H,I*O labeling. Unlikesimple diakyl phosphates,five-membered cyclic dialkylphosphates are extremely reactive. Ethylene phosphate is hydrolyzed 107-108 times faster than dimethyl phosphate. Onlythe P-0bond is broken. This special reactivity is simply causedby ring strainof five-membered phosphate rings. Six- and seven-membered phosphatediesters are as stable asthe open-chain analogs. Alkyl phosphates containing electronegative cyano groups at the p-position of one of the alkyl groups readily undergo hydrolysis in alkalinebymedia p-elimination (Scheme 8.5.9). Another important neighboring effect concerns ribonucleotides. bon nucleoside 3’- or 2’-phosphates are, for example, ~uan~tatively hydrolyzed by lanthanum hydroxide at pH 6, whereas 2-deoxyribonucleotides are not dephospho~latedunder these conditions, The role of the hydroxyl group is not known in this case. Heavy metal catalysis of phosphate ester hydrolysis is probably caused by complexationof the metal ions, which renders the phosphorus atom more electrophilic.

Flexible, Polar,

and Matrix Fragile

35

Adenosyltriphosphate (ATP) is a tetraanion atpH 7 and hydrolyzesto the in water. Its free enthalpy is -34.5 diphosphate ADP and inorganic phosphate (Pi) H/mol, and it is able to phosphorylate alcohols and carboxylic acids. Again,is P, first split off as metaphosphate in an S,1 reaction. Bivalent metal ions (e.g., Cu2+)often catalyze the hydrolysis of ATP, but Mg2+ makes a notable exception. It only binds to the outer two phosphate groups (Son et al., 1975; Granot and Fiat, 1977). Stereoselective phosphorylationsof chiral phosphorothioatesis also possible using adenylate kinase and Mg ATP (Jang et al., 1991) Figure 8.5.1). Examples of “bioorganic” reactionsof metaphosphate as liberatedby ATP are the phosphorylationof alcohols in the presenceof zinc salts, the imidationof esters, and the reversiblephosphorylation of hydroquinone,whichactsasa phospho~latingagentin the presence of oxidants(“oxidativephosphorylation”), (Cooper et al., 1968; Sigman et al., 1972). The chemical synthesis of mono-, di-,tri-, and te~aphosphatesusually starts with unprotected nucleosides to produce posphoarnidates at first. The monophosphate may be treated either with morpholine and DCC to afford the phosphoromorpholidate or with carbonylbis(imid~o1e)to yield the phosphor imidazolidate. A third method uses diphenylphosphorochloridate to give a pyrophosphate triester (Scheme 8.5.10). All three intermediates react with o~ophosphate,diphosphate, or A n to yield di-,tri-, or te~aphosphates,respectively. Amides of nucleotides are, like carboxamides, morestable than the corresponding esters. Their hydrolysis rate depends on pH the of the medium and the nitrogen substituents. Aliphatic amine derivatives are unstable belowpH 4, hydrolyze slowly at pH 4-6, and are virtually stableat or above pH 7. The conjugated acid is thus readily hydrolyzed; the neutral forms and the anion are not. Aromatic amines react slower in acid media than the alkyl derivatives. In both cases, one frequently observes SN1mechanisms correspondingto metaphosphate formation (Scheme 8.5.1 1). There is no selectivity towardthe nucleophile.

ADP3@ + HP042@+ Mg2@

+ H@

+ 34.5 kJlmol

.5.1 ~ a g n e s i uions ~ bind regioselectively to the outer diphosphate group of

ATP.

0

I

+H2POd@,

Poiyphosphates

Models of a metal ion-catalyzed phospho~lation,the imidiation of an ester, and formationof reactive metaphos~hate moiety by oxidation.

R=HorOH

@O-H

II

R O ~ P ~ N H

I

OH

fast

0

II RRO-P-NHR I OH slow

0

II

RO-P-NHR

stable R = Alkyl; R

I

60 = Alkyl,

Aryl

The destructive mutagenic and carcinogenic effects of ionizing radiation on living matter is due to DNA changes in the cell nucleus. Pulse radiolysis or laser photolysis has been analyzed by ESR spectroscopy. It becomes evident that the negative charge produced by ionization is trapped by cytosine, a d the positive charge by guanine, a purine base (§teenken ge tends thus to migrate in DNA polymers until it is loIntrapair proton and electron transfers then lead to a CG radical pair. This may lead to a break of the double strand. of C leads to an enhanced probability of oxidative damage at G an vice versa. This is true in fast reactions in the cold. If, however, the DNA solution is warmed to room temperature, the thymine radical that is also formed becomes irreversibly protonated at C6, which does not occur in cytosine radicals, and the burial site of the electron, identical to the site of irreversible damage, will be T. Formaldehyde introduces statistic cross-links in synthetic DNA d with some preference for the 5’-d(AT) pair. Site-specific cross-linking o without structural distortion of the double helix has been achieved with dithiobis(ethane) units coupled to an adenine at position nine at position 5 in a complement^ decamer ( spectra indicatedan essentially undis r melting point, which was stable in millimolar mercaptoethanol solutions ntz et al., 1993). Thionucleotides canbe used as efficient cross-linkers, because they can be coupled efficiently with N-mercaptophthalimides carrying reactive end groups. The thiolated nucleobase is best introduced as cyanoethyl ether (Cole~an,1991), because thiocarbonyl groups do not survive solid-phase synthesis conditions. U in acetonitrile is then used to free the thiolactam. Attachment of pendantgroupasdisulfiurswiththe N-mercaptophthali~des in ~ua~titative yield(Coleman,1992lfidebridging of thioethylatedadenosine has also been used aassite-specific cross-linking reaction (Ferentz and Verdine 1991).

A plethora of molecul~ comple~es of nucleic bases, nucleosides, nucleotides, and polynucleotides witha large variety of mono~ersattached by charge inter,or hydrophobic intercalation have been reporte ,metal complexes, and polymers have been prep scussed in the following order:(a) nucleic base an peptides and related model compounds, (c) metal ssible a~plicationswill be mentioned, but for the es connected to many of these assemblies, the reader is referred

In contrast to helical proteins(see Fig. 9.5.3) and glyconamides(see Fig. 4.5.9) L-enantiomeric and D-enantiomeric pieces of DNA [e.g., d(CGCGCG)] do not form racemic sheet structures upon mixing (chiral bilayer effect). One rather finds that the L-enantiomeric right-handed double helix and the ~-enantiomeric left-handed duplex remainstable and stick together end-to-end in an alternating manner. A pseudohelixis thus formed in the crystal, which contains a half-turn of right-handed and a half-turn of left-handed helix (Doiet al., 1993; Hashimoto et al., 1993) (Fig. 8.6.1). L-dA,, is stable to phosphodiesterase; the mesa-compound L-D-dA,, with alternating stereochemistry is slowly hydrolyzed. Diasteromeric 2’,5’-linked deoxynucleotides show the same self-association properties asthe natural 3’,5’-phosphodiesters. L-Configured RNA is also known (Ashley, 1992). L-DNAs do not act as antimessenger oligonucleotides. NMR, UV, CD, and mobility shift assays on PAGE clearly showed thatthe L-DNAsdo not recognize D-DNAs in solution (Garbesi, 1993). Both L-dA,, and the mesa compound LD-A,, are resistant to phosphodiesterases, although LD-A,, decomposed very slowly (Hashimotoet al., 1993). The DNA triple helix is as a structural motif first discovered in homop-

r------------

I

I I I I I I I

I

L,

I I I I I I I I I I I I I I I I I I I ” ” ” ” ” -

Structure of a double-helical co-crystal of a h e ~ ~ u c l e o t i dracemate. e Compare to the formationof racemic sheets in caseof racemic carbohydrate and peptide assemblies in Figures4.5.9 and 9.5.3.

urine-homopyrimidine DNA wherea third pyrimidine strand binds to the purine strand in the major groove. Recognition is achieved between protonated cytidines and guanines or between thymines and adenines leading to C+GC or T-AT triplets. Antiparallel TaAT triplets are also known. Later the triplex code was extended to include GeTA and TCG triplets. The x-ray structure of an intramolecular Y.RY triple helix, which containsa central GTA triplet flanked by two TsAT triplets and connectedby T-hairpins, showshow the three bases interact by hydrogenbonds(Fig.8.6.2)(comparewiththepicture of themajor NOE IH-NNIR spectra were usedto elugroove in Fig. 8.2.4). Two-dimensional dicate the solution structure of a central G-TA triplet and two flanking T-AT triplets in oligonucleotide, withinthe GaTA triplet the TA base pair retainsWatson-Crick hydrogen bonds.The third strand guanine adjusts its sugar pucker and basestackingto the existingB-DNA(Fig.8.2.13)(Felsenfeld et al, 195'7; Kiessling et al., 1992;Yoon et al., 1992; Koh and Dervan, 1992). Triple helix formationat a DNA contai~ngall four bases does not occur. It can, however, be enforced when nonnatural deoxyribonucleotides are introduced.

Molecular model of a DNA triple helix made of homo~ucleotides.The third base adds to the major groove (see also Fig. 8.4.2).

Furthe~ore,macrocyclic DNA with two opposing T-hairpins and or-image TC multiples bind both CC and A oligomers in the center to build up specific .8.6.3) (Bed and Dervan, 1992). A and RNA are usedas molecular targets in genetic medicine, x and antisense strategies. These are based on the that hopen l5-mers or largeroligonuclackthechosensegments of DNA(inthe appro triplex m impede willapproach) andnse ans script ion "f protein) of genes, selected be they of microbial, aberrant,or human origin. Use of an oligonucleotide to inhibit transcription (trip1 on the windingof the oligonucleotide around double-helica the control region of a chosen gene by triple helix formation with a synthetic oligonucleotide9the transcription factors cannot work. At neutral cleotides that contain C instead of C are most potent since the G more stable than the CCC triplet in triple helices.It was indeed shown that ina cell-free system triplex formation can prevent transcription of selected viral genesandreducanscription of variouscancergenes(Cohen,1994). Antisense IS usedincertainmicrobes as a tool for regulatinggene expression because theyinterfere with sense strand and block protein synthesis.

Triple helix formation of heteronucleotide helices can be enforced with complementary multiples acting on cyclic DNA.

In order to protect the antisense oligomers from enzymatic breakdown, one replaces an oxygen atom in normal phosphate groups with a sulfur atom (Scheme 8.6.1). These phosphorothioates(S-oligos) are takenupbydirectdiffusion across the cell membrane and by endocytosis, a process in which thecell membrane engulfs the nucleotide and forms a little transport vesicle. Antisense tAthennotonlyinterfereswiththereading of them-RNAsequence in translation, but also stimulates ribonuclease H, which destroys the deactivated m-RNA. The first S-oligosto reach the market will probably be applied to combat viral infections. S-oligos directed against the RNA productof a ing gene are being tested for AIDS treatment. Antisense drugs are also being tested for cancer of the blood and leukemia. 8.4.6) surprisingly doesnot react with counteriCationic DNA (see Scheme ons with identical base sequences, but it does bind stronglyto c o m ~ l e m e nant~ ionic sequences. Steric fitting of bases and hydrogen bonds within a duplex is thus moreimpo~antthan optimum ion pairing (Fig. 8.6.4) (Letsinger et al., 1988) " Zwitterionic, net charge-neutral DNA oligomers bearing pyrimi~ine5 aminohexyl substituents,on the other hand, bind to natural DNA as do the negativelyDNAstrandswitheachother.Suchdoublehelicesareinsensitive to changes of ionic strengthof the solution (Hashimotoet al., 1993a,b). Other pharmaceutically relevant modified oligonucleotides include bicyclic ribose derivatives(Christensen et al.,1998)andpeptidenucleicacidswithamidelinks instead of phophodiesters (Koppitz, 1998). Both ligate with DNA. ~uanosine,guanylic acid, but not guanine alone form gels in aqueous meof excess dia. No other nucleic acid or nucleoside shows this behavior. Addition

no binding

strong bi~din~

C o ~ p l e ~ e n t is ~ talso y retained in electroneutralDNAduplexesin which one strandis a polycationic, the other the usual polyanionic DNA.

0.1 M alkali saltis essential; withoutit guanosine produces well-defined crystals, but no gel. X-ray fiber diffraction studies of the gel indicate the formation of quadruple helices. (Fig. 8.6.5) (Saenger, 1984). Similar quadruple helices are also formedby corresponding DNA heelho house et al., 1998) Two hy~ophobystrategies have been utilizedto further promote G-quartet formation: 2’-0-methylationof the guanine residues and covalent attachment of cholesterol (Fig. 8.6.6)at a position distant from them. Duplexes and triplexes can alsobe stabilized by inters~andhydrophobic interactions. Such noncovalent tetraplexes even partially survive PAGB analysis, and they retain the ability of monomers to hybridize with acomplement^ RNA sequence, a crucial property for medical applications (Bonazziet al., 1991). y ~ o p ~ o b i z enucleosides d form complement^ pairs in micelles (NowChen, 1992; Nowicket al., 1993) and have been used as selective transportsystems(Tips) for complementarywater-solublenucleosidesthrough c ~ l o r o f oA~ . and U-Tips, for exarnple, haveno effect on guanosine transport, whereas G-Tips have an e~ancementeffect of about 100 and G-Tips of more than 1000. In SDS micelles one finds a 1:1 pair formation of ~ydrophobizedT a al., 1991). and A de~vatives(Fig. 8.6.7)( F u ~ t et An elegant and surprising artificial self-replicating system ~l amides on top of an ~ l l - ~ x1i,3,5-cyclohexyl-t~c~bo~ylate also bears methyl groups at the same carbons, not shown)on nucleic acid basis

Model of noncovalent guanosine quadruple helices in water. Compare to the quadruple helices of ~ - 0 c t y l g l ~ c o n ~in i dFigure e 4.5.8 and folic acid in Figure 7.3.3.

must also be mentioned (Fig. 8.6.8). For example, the coupling reaction of 5‘aminoadenosine with the penta~uoronaphthylester shows the following features: 1. Addition of the product, namely, a naphthylamide again connectedto Kemp’s acid, increasesthe coupling rate (“autocatalysis”). 2. The condensationproductformsdimerswitheductsandproducts (“self-com~lement~”). 3. The dimerdissociatespartiallyandworksagainastemplate(“selfreplicating”) Tjivikuaet al., 1990).

In artificial homo-DNAs the deoxyribose is replaced by 2,3-dideoxyglucose, the 4’,5’-phosphate ester substituents. The nucleic bases areall in e ~ ~ ~ t u r i d positions. One might have expected that the rigidityof the pyranose unit as compared to the furanose unit would cause a much morestable double helixand co~espondin~ly high melting points. This is not the case. Helix stabilities and melting points are similar. It is, however, found thatfour stable base pairs instead

P-NH(CH*)*NH-CO-O-choieste~l 3”CholllOmer 4x4 OMe Tetraplex 3”Chol Tetraplex

lOmer Tetraplex Single Strand2Omer

(~)n-Oligomersattached to cholesterol enforce a quadruple helix in water with four terminal cholesterol units. Tips0 ase

Tips0

A-Tips :Base =Adenine U-Tips :Base = Uracil C-Tips :Base = Cytosine G-Tips :Base = Guanine

OTips

Tips = Si(CH(CH3)& NHAc

The hydrophobic Tips derivatives transportmany water-soluble derivatives of c o ~ p l e m e n t ~ bases y into chloroform.

Self-replicationmodel cycle of the left molecule based on the formation of amide hydrogen bonds on a concave basis (Kemp’s acid).

of two are fomed, namely, ddG ddC > ddA ddA = ddG ddG > ddA ddT. In pariticular the purine bases have a high tendencyto f o m homodimers, and the formation of a double helix with a statistical ~ a n g e m e n of t all four nucleic bases is impossible. The structure of the most stable Watson-Crick pair d was given in Scheme 8.4.2 (Bohringer et al., 1992; Eschenmoser and 1992; Ottimg, 1993). The fomation of DNA double helices thus dependson the flexibility of the deoxyribose unit.The rigid pyranose unitfalls into a confomational trap, the movements depicted in Figure 8.2.9 become impossible, and life crystallizes out.

The interactions between proteins and nucleic acids are dominated(a) by trostatic interactions between cationic side chains of amino acids (Lys, and phosphate and(b) by water-mediated hydrogen bonds between the p-p ribbon’s amide groups and ribose04’ and 02’ of RNA and D interactions place the polypeptides into the minor groovesof

DNA. Since polylysine binds preferentially toAT-rich DNA, hydrogen bonding in addition to salt bridge formation is likely to occur. Complex formation between polylysine or polycytosine is also reversible and may lead to rod-like structures (Hayneset al., 1970). Small cationic peptides with an aromatic amino acid, e.g.,the tripeptide Lys-Tep.Lys, first addto double-stranded DNA and then force thearomatic side chain to intercalatebetweentwonucleicbasepairs. Bending of the DNAis then observed (Gabbayet al., 1973). The most interesting and evaluated interactions between oligopeptides and DNA concern antibiotics. Most of these natural compounds are different from the usual peptides. They contain amino acid units that are not found in proteins, in particular small heterocycles. Minor groove and partial intercalation models have been advanced to explain binding between antitumor peptide antibiotics and DNA. Unfused aromatic ring systems typically favor AT-rich regions and bind to the minor groove;fused aromatic rings, on the other hand,favor intercalation and tendto be GC specific. Bleomycin and distamycin are selected here as examples that demonstrate convincingly what a simple combinationof organic synthesis and synkinesis can achieve. Bleomycin is a complex glycopeptide with a spermine end (Kozarich et al.,1989).Onewould expect carbohy~ate-carbohydrate andphosphateo l i g o a ~ o ~ uinteractions m combined with some intercalationof the heterocycles. The results from structural analysis byNMR spectroscopyare quite different. At firsttheN-terminus or metal-bindingdomainbinds to doublestranded DNA (K = lo5 and produces lesions by oxidative dest~ctionof the pyrimidine nucleotides, initiated by C4’ H-atom abstraction from the deoxyribose moiety. ~ u ~ h e r m o rdownfield e, shifts are noted for some protons in the metal-binding domain of bleomycin. These include His 2,4 (t-0.16 and 0.09 ppm), p r o p i o n ~ i d ea,b (t-0.11 and 0.24 ppm), and Ala a (t-0.09 ppm). These shifts are consistent with minor groove binding since the shifted signals correspond to protonsthat lie attheperipherythisbindingsite.Chemical shift changes betweenthe proton signals of the free octanucleotide andits bleomycin A, complex were very small, but eight inte~olecularnuclear Overhauser effects )were observed.(Fig. 8.69). Most of the close neighborhoods were found to occur between hy~ophobicsites of the antibiotic, e.g., methylene and ethylene groups,withdeoxyriboseandbases ofDNA.Surprisinglyneither the cationic s p e r ~ n eends nor the carbohydrate units showed any interaction with the nucleotide (Mandervilleet al., 1995). eomycin B2 specifically cleaves double-stranded DNA at5’-6T-3’ and sites in the presenceof ferrous ions and oxygen.The bithiazole moiety is presumably responsible for the GC and GT selective intercalation or groove binding of bleomycin, the terminal arnino groups should act as metal binding domain and the carbohydrate, and guanidine groups presumably adsorb on the NA surface. The detailed mechanism of bleomycin-DNA binding and oxidation is unknown, but it has been shown thatthe L enantiomer L - d ( ~ G C G ~ G is ) not cleaved oxidatively, in contrast to the D enantiomer. The binding of both

ur Diagram showing the eight intermolecular nuclear Overhauser enhancement effects between zinc bleomycin A, and d(CGCTAGCG),.

enantiomers, D- and L-d(CGCGCG), to bleomycin B, as studied by 'H-N titration is, on theother hand, quite similar (Urataet al., 1993). Distamycin is an oligomide containing three ~-methylpyrrolecarboxamides. It is commercially available, but expensive (about$5 per mg). Several derivatives are based on a simple total synthesis of distamycin A starting with ~-methyl-2-carboxypyrrole. A p-nitro group is introduced with HNO, in acetic anhydride, the carboxy group esterified, and the nitro group reduced mine. to an The resulting 4-amino-2-carbomethoxypy~oleis then combined with the acid chloride of 2-carboxy-4-nitropy~ole,the nitro group is again reduced, and the amidation step repeated to give a pyrrole trimer in gram quantities. This trimer can then be used as the starting material for easy synthesis of a large varietyof distamycin derivatives ( ialer, 1978; Baker, 1989). Amide hydrogen bondsfix the distamycine in the minor groove of doublehelical DNA with a strong preference for A+T-rich regions as characterized by xray(Sriam,1992)and NMR (Faganand Wemmer, 1992). This sequence specifity is thought to result from hydrogen bonding between the amide groups of the antibiotic and the0 2 of thymine and N3 of adenine. As a whole, thedistmycine oligomer assumes a crescent shape and fills the minor groove of DNA (Fig. 8.6.10). 'HJ5N heteronuclear two-dimensional NM[R spectroscopy with a I5N-labeled nucleotide was applied to characterize the molecular complexes between

T= T=

A= A=

Netropsin

Binding schemesof distamycin A and netropsin to TA domains. 21 5 214

* ; : c

'85213 _ I

5:

212 21l

210

6 210110

"C

do io io

Temperat~re-dependent plot of the shift of N3-labeledadenosine in x d(CGCAATTCGCG), after addition of distamycin and netropsin. (A) Average N3 shifts in absence of drug; (0)A6N3 shift in netropsin complex shows no effect, except exclusion for of water; ( amycin complex-shows large sa the are complexes. These reeffect; (&A)A18N3 shiftsnetropsin sults led to hydrogen bonds sketched boldin Figure 8.6.10. (From Rheeet al., 1993.)

distamycin and the duplex ( d C ~ C ~ A * A T T C ~Adenosine C ~ ) ~ . (A*) was IsNlabeled at N3. The shorter oligoamide netropsin was also boundtothisnucleotide. lsN chemical shifts were then plotted against temperature (Fig. 8.6.11). The A17 and A5 signals show the same shifts as in the absenceof ligands. Upon addition of distamycin both nitrogen signals are shifted downfield, but they are now di~erentiated:A5 is hydrogen-bonded to NH1 of distamycin, A17 toMI122 The shorter netropsin molecule also wraps around the DNA helix, but since it produces a strong hydrogen bond between A17 andHNl8, it cannot react with is therefore not shifted downfield, but slightly the other lSNend (Fig. 8.6.10). A5

upfield. It seems tobe more hydrated thanthe ligand-free nucleotide.The spectroscopic data thus support a model in which there is no water involved in the DNA-ligand binding and where the helix is widened by ligand attachment. assumptions arealso in agreement with x-ray data of both complexes, The finalpyrroleunit of distamycinwasalsoexchanged by pyridine, which causeda different footprinting pattern (see Fig. 8.6.12). The pyridine carboxamide was bound to G+C-A+T mixed sequences in preference to pure stretches. This was rationalized with the assumption that NH, the group of sine binds with both the pyridine nitrogen and the amide groups in the same manner as metal ions do.Py~dine-carboxamidewould then direct towards G+C, the neighboring pyrrole carboxamide towards A+T (Wade et al., 1992). Imidazole ends have similar effects (Griffin et al., 1992; Traugeret al., 1996).A teminal pyridine unit in the netropsin ligand also moves it to a GC t e ~ n a of l an AT-rich region. TA and other metal complexes catalyzeoxidative cleavage reactions of DNA in the presence of oxygen and di~iothreitol,which generate hydroyl radicals (Fenton reaction) (Knapp et al., 1995). This reaction can be used for site-selective cleavageif the complexis connected to one end of a distamycin short spacer. For this preparation one out of four acetic acid side had to be selectively attached.The tetramethylester of EDTAinteracted with copper(I1) ions to give the complex[Cu-~eEDTA~I2+ in which two ester groups are boundto the metal ion in conjunction with two nitrogen donors. Hydrolysis of thetwocopper-bound ester groupsoccurredinthe pH range 4.1-5.4, whereas the secondester pair hydrolyzes only betweenpH 7.2 and 8.0. The metal ion thus activates the ester by weak interaction with the basic carbonyl

A= A=

A=

G=

Position of a pyridinyl distamycin derivativeon a DNA surface.

oxygen. If the copperis removed during hydrolysisby precipitation with hydrogen sulfide, the monoacid can be isolatedfrom the reaction mixture by crystallization (Hay and Nolan, 1975) (Fig. 8.6.13).The monoacid was then activated with ~ , ~ ' - c ~ b o n y l - d i i ~ d a zand o l condensed e with a distamycin analog DE and rnetalated to give the desired iron complex DE'Fe(I1). Since distamycin binds only to At-T-rich polynucleotide sequences with high affinity, the DNA chain is regioselectively disconnected in the neighborhood of those regions. A single cleavage productmay then be found (Fig. 8.6.13) (Schultz et al., 1982). On the other hand, it is also possible to protect specific DNA regions from cleavage reactions with molecules that bind to the surfaceof AT-rich regions. In this case, the noncomplexed DNA gives the usual regular PAGE pattern upon axam-Gilbert degradation, whereas the region involved in molecular complex formation shows no degradation products in this region. The white region in the sequencinglane is calledafootprint(Fig.8.6.14).Footprintingcan also be achieved by activation of nucleic bases toward pipe~dine-catalyzedhydrolysis. ethylatedguanosine (G-CH,), forexample, is cleavedbypiperidine-catalyzed hydrolysis.If such a baseis introduced into DNA, then the chain breaks at

f

G a) b) c)

G a) b) c)

8

A dista~ycin-Fe~11)EDTA derivative binds to the AT-rich regions of a nucleotide, and Fenton's reagent then cleaves theDNA next to this region upon addition of H202and dithiothreitol.

Maxam-Gilbert lanes untreated DNA (control) sample

I

l

A tightly bound DNAligand protect the region below from chemical degradation, e.g., following the Maxam-Gilbert protocol. Untreated DNAis not hydrolyzed at all.

all G-CH, sites, except the one protected by a strongly bound molecule, e.g, appro4 p-alanyl priate distamycin derivatives. Another distamycin derivative containing and 12 pyrrole carboxamide units binds one and a half windings ofin DNA the minor grooveor 16 base pairsof A-I-T. Ifthe EDTA was complexed with Fe(II), it catalyzedveryselectiveaffinitycleavingreactions, if, however,In(II1)wasused instead, it prevented DNA cleavage in this region. Oligonucleotides 15 bases in length equipped with a cleaving function EDTA-Fe at5’-end the caused sequencespecific double strand breaks at only one site in plasmid DNA, which was 4.0 kilobasepairsinsize. This is lo6 timesmoresequencespecificthanbiological restriction enzymes. This cleavage specificity was also realized in a bacteriophage genome at base pair positions 39138-39156 counted from the left end. Doublestrand cleavage at the 18-base-pair target afforded only one small DNA fragment (8.4 kilobases in size) and one large one. Autoradiogr~sshowed indeed only one clean small fragmentof 8.4 kilobases (Traugeret al., 1996). Even more interestingare distamycins with terminalEDTA complexes and complexing moietiesin the center. One example introduces chiral a tartaric acid moiety into the center, either as S,S- or R,R-enantiomer. Itis found that theS,Startaric acid-distamycin-EDTA-iron(I1) complex cleaves DNA at a concentration 10 timeslessthantheconcentrationneededwiththeR,R-tartaricacid analog. S,S-Tartaric acid derivatives show right-handed twists in crystal structures, whereas R,R-analogs show left-handed twists.If this tendency persists in solutions, one might conclude a “similar screw sense recognition” in this example (Griffin and Dervan, 1986). An 18-crown-6-ether boundto two terminal EDTA-iron(I1) complex moi-

eties and distamycin cleaves circular, double-helical DNA. At a concentration of - DTA distamycin just below where its ing activity started ( concentrations of Na+,K+, Mg2+,Ca2+ ,Ag”, Ni2+,Cd2+,or added and produced little DNA cleavage. However, additionof Sr2+or Ba2+ resulted in strong DNA cleavage. Sequencing gels reveal two well-resolved bindingsites,namely 5’-TATAGGTTAA-3’ and 5’-AATA-3’. The dependence of DNA cleavage efficiency upon variationof Ba2+ concentration indicated maximum activity at 10 mM:Ba2+.Above that concentration Ba2+began to replace Fe2+in the EDTA site and thereby inactivated the compound. This metallo regulation canbe interpreted in termsof a synthetic exampleof allosteric interaction. Complexation of Ba2+or Sr2+obviously induces a more favorablecrescent conformation of the distamycin ligand and thereby also favors chemical reactions of a far distant site of the molecule (Fig.8.6.15).

Nucleicacid-boundmolecules(ligands,drugs)showmarkedchangesinabsorbance and fluorescence as compared to those in free solution. E ~ ~ bromide u m r) is the most comonly used indicator for nucleic acid conformational

Only Sr- and Ba-crown ether complexes put the terminal iron in the right position for oxidative cleavage (see text).

changes.Itdisplays a bathoc~omic shiftand a loss of opticaldensity (hypoc~omicity)as well as a dramatic enhancementof its fluorescence whenit slips between twobase pairs (intercalates)of DNA. In aqueous solutiona nonradiative decay of the excited state of E+occurs, whichis coupled witha proton release from one of its amino groups to the solvent. However, when complexed to DNA this proton-transfer pathwayis virtually eliminated andthe lifetime of the excited state is lengthened from 1.8 ns in water to 23inns DNA. This then leads to the observed overall increase in molar fluorescence by about one order of magnitude (Fig. 8.6.16) (Thurston, 1990). The enhancedfluorescence of theethidium-DNAcomplexcan be quenched partially by the addition of a second ligand. Two mechanisms have been proposed to account for quenching, namely displacementof ethidium bromide by melting of the helix or electron transfer from external quencher molecules. The most efficient quenching occurs of course when both the quencher andethidiumbromidemoleculesarebound to DNA. This nondisplacement quenching is correlated with DNA-e~ancedelectron transfer, either from excited ethidium to an acceptor (methylviologen, copper(11) counterio~s)or from an donor to an excited ethidium acceptor. The DNA double helix works as a well-de~nedmatrix for an “organized” electron transfer.It enhances its yield by h (H20), which is often in theorder of 5 X lo5. ning nucleosides have also been incorporated into oligonucleoti~esusing standard solid-phase phosphoramidite chemistry. chemistry of the DNAis hardly changed. Upon hybridization of complement~y R#- and Os~-containingoligonucleotides distance-dependent “intraduplex” fluorescence quenchingof the excited Run is found (Hurleyand Tor, 1998).

lntercalator Minor groove binder Ethidium

H i

Major groove binder

Bis-intercalator \

H Ellipticine

Ethidium bromide andellipticine are typical intercalating dyes. Larger, cationic dye moleculesmay only bind into the major or minor grooves. An excessof the latter dyes would notsubstitute for ethidium bromide.

4

hapter

Single-stranded DNAs, e.g., dT20, on the surface of quartz fibers are applied as biosensors. complement^ DNA, e.g., dA20,is adsorbed at a detection limit of 86 ngfnd.,,yielding a200% fluorescence increase with hybridization (Piunno et al., 1995). Extremelyaccurate tests for DNA sequencesare based on the polymerase chain reaction and detection of the hybridized double strand by fluorescence changes. Modern tests use poly(thiophene), poly(pyrrole), or poly(acety1ene) wires to which a given nucleotide is covalently attached and bind the system to electrodes. Current intensity decreases drastically upon hybridization of the oligonucleotide single strand with the DNA to be detected. ~urface-attachedpeptide nucleic acids, which are commercially available, also give strong hybridization signalson carbon paste electrodes ( ~ a n et g al., 1996; Wilson, 1998). at er-soluble cationic porphyrinsinteractextensivelywithDNA. msTetra-~-methylpyridi~umporphyrin intercalates into calf thymus DNA under low ionic strength and converts to the exterior groove region as salt concentration is increased. In contrast,the corresponding nickel complex binds only as an intercalator at low concentrations, whereas the zinc complex, which contains an axiaE ligand, binds only externally. Intercalation leads to -0.9 a ppm shift in the 31P-NMRsignal of the phosphate backboneof DNA and induced circular dichroism peaks with molar ellipticities Q, = 1-2 x lo5. The Soret absorption band shows a 15 nm red shift and a 40% loss of intensity (hypochromicity).The formation of these intercalated complexes occurs only at CC pair sites, which show greater rigidity and stability towards bending relative to AT base pair regions. Cuanosine quadruplehelices(seeFig.8.6.5)andcorrespondingDNAs also stackwith mes~-tetramethylpyri~nium porphyrin eelho house et al.,1998). External complexes, on the other hand, are predo~nantlycoulombic in nature with minimalp o r p h y r i ~ a s eoverlap and presumably leadto a distortion of the helices, linearity. Induced circular dichroismalso occurs with the Soret band of porphyrins bound to the minor groove, but red shifts are small (8 nm), and hypochromicity (10%) and31Pshifts barely exist (Marzilli et al., 1986). The external binding occurs to AT sites. The binding of zinc tetramethylpyridinium porphyrinate to poly(dAT) is cooperative,suggestingawidening of the minor groove. End-on binding in the major grooveis also possible, but would not explain the observed cooperative (allosteric) transitions. Intercalation of copper and nickel porphy~nates,on the other hand, occurs at CC sites (see Scheme 8.6.1). Molar ellipticities are about x5 lo4, equilibrium constants around x5 M-l. Althoughaxiallysubstituted metalloporph~rins areoftennot bound, vanadyl meso-tetr~ethyl-pyridi~um porphyrinate binds stronglyto poly(dAT) surfaces and provides a usefulparama~neticprobe for AT regions (molar ellipticity: -5.5 X lo5deg cm2dm-1at 441 nm) (Pasternacket al., 1983). Porphyrins bound to the minor cleft have large effects on the DNA-enhanced fluorescence of intercalated ethidium ions (ET). Cationic porphyrinsare

capable of virtually quenching the ethidium fluorescenceat a distance of 25-30 A.An extremely efficient transition statedipole coupling within theE+-DNA-P+ complexhasbeenheldresponsible for thisextraordinarylong-rangeenergy transfer of the Forster type.Several porphyrins and model compounds absorbing above 700 nm adsorbed to DNA surfaces were screenedfor their photocleavage capabilities. The lutetium derivative was most efficient in cleaving plasmid DNA with light of wavelengths 2'700 m. Thefindingthat5,15-diphenyl10,20-dipyridi~um (trans-DPyP) porphyrins show a much greater tendency to aggregate with each other than the tetracationic analogsled to another type of binding to DNA, namely long-range porphyrin assemblies on the DNA surface. More intense CD spectra were found (molar ellipticities: 2-3 x lo5). They report the helical sense of DNA, namely U(+) or U(-) (Gibbs et al., 1988). DNA has also been appliedfor synkineses of molecular wires.One example applies aligned DNA fibers in which one acridine orange molecule intercalates per 10 base pairs. It shows photocurrents if a voltage is applied to the material placed between comb-form electrodes (Okahataet al., 1998). The dyeDNA complexes are probably not useful as parts of a charge separation system, but they clearly demonstrate extensive electronic c o ~ u n i c a t i o nbetween bound drug molecules-an influence that servesto raise the efficiency of transi~ondipole coupling at long distances. There is strong interest in the photodynamic therapyof cancer using porphyrins and other biode~adabledyes as sensitizers for DNA photolysis. Some evidence exists that porphyrins inhibit HIV-1, the v h s responsible for AIDS. Occasional medical applications and biological effects of DNA-porphyrin interactions result from sensitized p h o t o c h e ~ cDNA ~ cleavage reactions. The ellpiticine substituted porphyrin (Scheme 8.6.2) may, for example, be useful. The

ellipticine side chain intercalates, and the und porphyrin becomes toyisolated from thefluorescencequencystems of thenucleicbases lder et al., 1990). Artificial expanded porphyrins containing five rrole units photocleavage of DNA by irradiation above ’700 nm ganese(II1) porphyrinates with small axial ligands st minor groove, catalyze strand scission by potassium superoxide, and prevent strand scission by DNAase I (footprinting) in theAT regions. Large manganese ligands or bulkyperipheralsubstituentsblock close contactwiththeminor groove, and the manganese porphyrin-superoxide system also cleavesCC sites l.,1986). ~anganese(II1)-~es~-tetramethylpyridinium porphyriquite efficiently by hydroxylation of the 1’ and 5’ positions of its (Piti6 et al., 1993).The molecule becomes more efficient if one of the pyridinium substituents is replaced by para-benzoic acid, which is coupled to oligonucleotides acting as antisense-DNA.Such conjugates cleave a 35mer single-strand DNA regioselectively. Theymay work as efficient hibitors if thephosphategroups of the oligonucelotidesareneutralized by polylysine or replacedby phosphorthioate groups in order to enhance resistance toward nucleases. Nonenzymatic, photochemical cleavageof RNA has been achieved by the cooperative actionof carboxylate and ammonium ions bound to an intercalating anthraquinone moiety. The carboxylate presumably activates the phenolic 0 group of the quinone. Repulsion between the phosphate and reagent’s negative charges is compensated by the hy~ogen-bonded~ m o n i u mion (Endo et al., 1996). More elaborate enzyme mimetics have used acid-base pairs on the rim and in the center of cyclodextrin matrices with similar results. ~arcinogenicpolycyclicaromatichydrocarbons are metabolizedinthe body to diol epoxides, which are their ultimate carcinogenicforms. They exert their effectsby covalent attachmentof the exocyclic amino groupsof dA anddG to the benzylic carbon through both cis and trans ring opening of the epoxide. of these stereoisoSome stereoselectivityis observed in the tumorogenic activity mers (Lakshmanet al., 1991). Carcinogenic aflatoxins from fungi that frequently infest peanuts, corn, and other seed crops undergo enzymatic epoxidation in the body and become genotoxic (Baertschiet al., 1988; Iyeret al., 1994a,b). Another antibiotic that acts primarily on DNA is the dienediyne epoxide neocarcinostatin, which opens first to an annulene and then forms an aromatic biradical. It has been shown that this relatively long-lived annulene is the species that causes 95% of the observed DNA cleavage by reaction with the ribose moieties.

The first example is not a metal complex, but a metal colloid, to which DNA is covalently attached by a thiolate linker. Cold particles 13 nm in size connected

with 24-base single-strand DNA change color from red to purple when hybridized with complementaryDNA. The color changeis due to a red shift of the Au nanoparticlecolor by aggregationand is verysensitive to mismatches (Storhoff et al., 1998) (see Fig. 9.3.1). cis-Diamineplatinum(1I) (cisplatin)is one of the few chemical success stories in the treatmentof cancer. Its activity probably resides in a reaction with cellular DNA. The simple diaminedichloroplatinum, for example,prevents cell division in bacterial cell cultures. Two Cis amine groups are necessary and are, for example, also provided in ethylenediamine Pt complexes.Cisplatin, but not its trans isomer,allows the intrastrandchelation of twoneighboringpurine bases, where the AG- and CC-Pt chelates are strongly preferred. It is thought that Pt-induced DNA lesions are responsiblefor the antineoplastic activityof cist. Enzymatic digestionof a ~ra~s-platinum-DNA complex is much slower than with the correspondingcis adduct. Chromatographic analysisof enzyme digests point to a N7-N7 cross-link between adjacentGG monomers within one strand. A 31P"NM~ 3 ppm downfield shift of cross-linked guanosine signals also suggested a distortion (Admiraal et al., 1992), since similar shifts were observed when the stacking of nucleic bases was removed. Denatured single strands or hairpin formsof the Pt adduct (Fig. 8.6.1'7) have been proposed. The reason for the extraordinary stability of cisplatin-single strand complexes lies (a) in the a n t i s y ~ e t r i carrangement ~ of the amine ligands, which

3

G

Model and rearrangement of cisplatin DNA. Single strands and pin forms are induced closeto GG dimers.

allows the side-on attachment of platinum to DNA (Berners-Price et al., 1993; Takahara et al., 1996) and (b) the extreme kinetic stability of platinum complexes. Slow rates are also often responsible for the nonfo~ationof adducts. Platinum is h o w n to have a strongkinetic preference for binding to basic guanine over aromatic adenine. Closure to form the bifunctional adduct between DNA and cis-platin is more rapid for CC and AC than for GA (Davies et al., 1998). Quite often platinum-oligonucleotide complexes survive denaturing gel electrophoresis. The corresponding traris diastereomers of platinum diamines are also kinetically stable, but they have a tendency to replace hydrogen bonds in base pairs, e.g., AT (Fig. 8.6.18). This leads to a larger separation of the bases. Pt-NH lH-NMR signals of amine and amine ligandsin Pt(I1) complexes of guanine and adenine in aqueous solution also provided evidencefor the formation of hydrogen bonds with the 5'-phosphate group of nucleotides coordinated in the cis position (Qizanovic et al., 1993). The observed low-field shifts are largest and H-bondingis strongest when theNH protons are constrained in chelate rings (ethylene diamine), when purine rotation is slow (A slower than C), and when the phosphate group is fully deprotonated (OPO,2-). The temperature effects on the chemical shifts of Pt-NH proton signals are low (-5.6 x ppm k-l). Cis-platin diacetate binds to ethidium bromide and yieldst ha e ~ o c ~ o m i c 1:l complex. The transition at 490 nrn (orange) shifts to 640 nrn (blue) as the temperature is increased and a proton transfer occurs between the coordinated amino group of the dye and the acetate counterion (Fig. 8.6.19). Ternary ethidium-cisplatin-DNA complexes are also known (Ren et al., 1993). ~henanthrolinemetal complexes are chiral and also appear as L and D enantiomers (Bartonet al., 1982; Erikssonet al., 1992).The D isomer of the zinc complex becomes enriched as a result of dialysis of the ~nantiomermixture against DNA (Reedjik, 1987).The corresponding ~ t h e n i u mcomplex exhibits a similar kinetic stability as platinum complexes and is tightly bound to DNA. Again the right-handed DNA structure is matched better tothe asymmetry of the D isomer and somewhat precludes binding by the L isomer. Nevertheless,no intercalative binding of the penant~olinemoieties, which was at first impliedto explain results of fluorescence quenching by ferrocyanide, presumably takes place. In the complex with d(CGCGATCGCG), neither are the expected NOE cross-peaks seen nor does reduction of DNA sequential connectivity peaks occur. Furthermore, the observed binding kinetics are toofast for an intercalation

H

trans-Platin tends toseparate ~ydrog~n-bonded base pairs.

0.8 A 0.6

Spectroscopic shifts upon bindingof cisplatin to ethidium bromide.

5.0

3.0 1.0

g

0.0 -1.0 Q

-3.0

M 240

solutions ( ) .

260

280

300 h [nm]

Formula of L- or A-zinc phenanthroline andits CD spectrum in DNA Broken line is the co~es~onding spectrum of the D- or A-enantiomer.

process. Only minor groove binding occurs with the usual highest affinityfor the AT region (Fig.8.6.20). The oxidizing reaction mixture ~Fe11ERTA~2with H2Q2(Fenton reagent) does not interact directly with polyanionic DNA, rather it produces hydroxyl radicals, which abstract hydrogen ainstatistic manner from the deoxyribose ring leadingtosugar r e ~ a n ~ e m e nand t strandscission. A continuous ladder of oligon~cleotidesis thus detected in electrophoreses."he same cleavage patterns were obtained with y-radiolysis, whichare known to produce hydroxyl radicals inwater, Since neither the FeEDTA/H,Q, mixturenor the hydroxylradical reagent adsorbs to the surface of RNA or intercalate, they are ideally suitedfor

footprintingexperimentswithmolecularassemblies of DNA.Ruthenium(I1) complexes boundto DNA allow photophysical cleavage (Kumar et al., 1985). A binuclear cobalt(II1) complex with twocyclic tetrammine (cyclen) ligof ands efTectively catalyzes the hydrolysisof plasmid DNA. Rate enhancements lo1’at a concentrationof 1 M are obtained (Hettich, 1997).

M of the cationic surfactant cetyltrimethyl ~ m o n i u mbromide (CTAB) changes the characterof large DNA chains from random coil to a compact globule state in water. Ill-defined fat droplets are observed under the fluorescence light microscope (Radler et al., 1997). Oligocations, such as spermine, have a low binding cooperativity and fast exchange rates. Theylead to relatively homogeneous populations of small particles, but the salts with DNA do not survive physiological salt concentrations. Larger amino oligomers stabilize 30- to 100nm particles well (Blessinget al., 1998). The combination of spermine and uranyl acetate,on the other hand, yields well-defined toroidsof electron microscopic dimensions. Usually one finds only a few percent of such “compacted” DNA molecules within a large population of linear or circular DNA strands, but under carefully controlled conditions quantitative compaction to toroids of quite uniform size has been achieved (Fig. 8.6.21) (Bottcher, 1998). Chromium and cobalt ammonium complexes have similar effects, but compaction yields are usually low. The least poisonous system to compact DNA toroids consists of a hydrophobic tripeptide,narnely a (Val), derivative with a terminal naphthalene sulfonate headgroup (Vengerov et al., 1985).

Electron micrograph of DNA toroids formed by successive additionof oligoamines (e.g., spermidine) and heavy metal ions (e.g., uranyl),

air

A mixed-surface monolayerof adenine and guanidine amphiphiles binds to UMP, leading to short wavelength shifts of attached chromophores.

NA oligonucleotides of defined length and sequence ending with an §H group have been attached to 13-nm gold spheres or nanocrystals. On addition of complement^ single-stranded DNA templatesor duplexes with sticky ends (see Fig. 8.1. l), the nanoparticles stick together and form regular aggregates by base specific recognition processes. Upon heating the assemblies dissociate reversibly, and absorption changes at 260 nm (DNA) and ’700 nm (gold colloid) are observed (see Fig. 8.1.1). Upstanding DNA on gold colloids has been realizedby covering the gold surface with hexanol. The hydrated hydroxyl surface prevents the flat adsorption of the biopolymer (Levickyet al., 1998 process of DNA adsorptionon solid surfaces covered withcomplement^ or DNA-derived dendrimer sequences has also been followed quantitatively by quartz balancem~asurementsand on water surfaces at monolayers ( ~ et al.,~ ~ 1996;Alivisator et al., 1996;Wang and Jiang, 1998). Two-dimensionalrecognition of c o ~ p l e m e n nucleotides t~ hasbeen foundbetweenrnixed-surfacemonolayer guanidi~ium monolayers and mononucleotides in the aqueous subphase. P, as can be shown directly by V spectroscopic shifts of attached ure 8.7.1) (Sasaki et al., 1992; rihara et al., 1996). rfaceligation is alsoused for fu c“DNAcomputingongoldsurfaces.’, The “words97are 16-base nucleotides;T4 DNA ligase is used to join the

words. The “word append” or printing operation consists of the usual sulfide binding to gold. Words are then “marked” by hybridization of c o ~ p l e m e n t ~ molecules, whereupon they become double-stranded. Words that are not marked by adducts are then removedby exposure to exonuclease I; the mark canbe removed from the remaining DNA by washing with urea. Readout at the end is done by sequencing of the words that remain on the gold surface. It will countall the mark events and thus read the DNA word buriedin solution with the aid of a DNA library of 16“etter words onthe gold surface (Frutoset al., 1998). Nucleic acids connected by amide bonds have been attached to carbon electrodes and were used there as sequence specificDNA biosensors. They are more stable and DNA,which permits the useof shorter react more base specifically than normal probes or “words” (Wang et al., 1996). For References for Chapter 8,see pages 563-574.

Proteins arethe biopolymers thatconstitute the chassis and the motorof the molecular machines neededto organize as well as execute the processes of life. The name protein was introduced by Berzelius in18l 2 and is derived from the Greek protos, meaning “the first.” It should be seen in a logical connection with two other protos, namely protons and protoporphyrin, Intelligent differentiation, ordering, and i n f o ~ a t i o nprocessing come from the proteins; the energy of life processes comes from the sun andis transmitted by protopo~hyrinderivatives, which shuffle electrons, and by protons. In addition to the daily life polyamides (e.g., nylon and perlon), five major as~ gorganic synthesis and synkinesis: pects of protein chemistry make it~ t e r e s t to a-L-Amino acidsconstitute chiral and versatile synthonsfor chiral nitrogen compounds and synkinons for chiral membrane surfaces and pores when they are applied as headgroups of amphiphiles. In the “antigen active fragments” of antibodies (Fab), in synthetic model peptides, and in planar macrocyclic lactams, proteins provide specific recog~itionsites for all kinds of ions, small molecules, and polymers. ~ombinatorialsyntheses on the surfaces of solids allow the screening of thousa~dsof peptide-peptide, peptide-antibiotic,or peptide-nucleic acid recognition processes in a few days (Fig. 9.1.1). e catalytic activity of commercial enzymes can be widely applied m synthesis and synkinesis.

Combinatorial peptide libraries as obtained by Merrifield synthesis of peptides on membrane-covered goldparticles allow not only investigations of recognition processes between the surface receptors and water-soluble substrates.Systematic changes of the environment of the receptor also become feasible (see Secs. 9.3.2 and 9.6.10). Steps I,III, and V are coupling reactions; steps 11, IV, and VI are deprotection reactions, About 50 thiolate molecules, including dodecyl sulfide and three peptides, have been des . Temposited on a single gold cluster in statistical dist~butionsand ~ a n g e ~ e n t(From pleton et al., 1998.)

5. Artificial proteins with two very different recognition sites-one polar, the other apolar-may allow vectorial, light-induced charge transfer between identical chromophores. 6. The renewableproteins of animals(gelatin)andsoybeans maynot only be used as foodstuff, but may become a source for biocompatible materials. This chapter concentrates on the basic structures and chemistryof amino acids and on natural and synthetic proteins that have been applied as materials or in synkinesis. Biochemistry and molecular biology are touched upon occasionally. Metzler’s and Voet’s excellent biochemistry textbooks are recommended for these subjects.

Proteins are polyamidesmade of 20 different a-l-amino acids(Greenstein, 1961; Dickerson and Geis, 1969; Creighton, 1993). Digestible proteins occur in muscles (“‘meat”), egg white, milk products (except butter), beans, and wheat,to name only the most important sources of food proteins. During digestion the polyamides are hydrolyzedto yield amino acids, which are then usedto build up the proteinsof the body. Proteins that digestother proteins by catalytic hydroly-

sis (enzymes)and the animalorplantproteins,thataredigestedareboth polyamides containingthe same amino acids.In chemical hydrolysisby example, their hydrolysis rates arequite similar. In an organism, howev own proteins survive, while foreign proteins are split at very high rates. The of synthesis and degradation inthe same organism is c ~ ~ ~ ~ r t ~ iological catalystsfor synthetic reactions, oxidations, and reductions are always found tobe susceptible to self-dest~ctionby the reactions they catalyze. They work in specialized, separate en~ironmentslocalized in organelles such as m i t o c h o n ~ aand lysosomesin order to degrade food but not the material of the body. Eight of the 20 amino acids are “essential” food componentsfo They cannot be synthesized by the human body and therefore must be food. The minimum requirements and some important sources for these acids are given in Table 9.1. A kilogram of food proteins costs about$10, compared with about $1 for starch and$2 for a kilogramof fat. Proteins of high nutritious value are those that have an amino acid composition similarto that of human proteins.Inferior are those proteins that are diEerent from human proteins and, most important, that lack some essential amino acids. Animal proteins are superior to plant proteins. Digestible proteins usually consist of single helices and absorb water. Nondigestible proteins are those that consist of heavily cross-linked helices (e.g., skin, hair, horn) or of P-pleated sheets (e.g., silk). Whereas cattle hides typically contain65% water, 33% protein,2% fats, and 0.5% mineral salts, finished leather is more than 80% pure collagen. Collagenis a left-handed triple helix with a pitchof 8.6 A (see sec. 9.2.3). The most frequent repeat unit is glycyl-X-Y7 whereX and Y are often prolyl and hydroxyprolyl units. Close to 3X lo9heads of cattle, sheep, and goats are slaughtered worldwide per year and pro-

The Essential Amino Acids Daily requirement (mglday) Sources

(mg amino acid/g nitrogen)

Women Men WheatBeef Egg Milk Isoleucine Leucine Lysine Methionine ~he~ylalanine Threonine T~ptophan Valine

700407 1100630 800496 1100154 1100 500292 250 800440

450 620 500 550 1.120 311 305 157 650

90

428 565 396 196 368 3 10 106 460

332 515 540 154 256 275 75 345

262 442 126 78 322 174 69 262

Protein contents (dry weight): milk, 25%egg, 50% (100%in egg white);beef, 40-80%; wheat, 15%.

vide the raw material for this natural polymer material.Wool is mostly sheep’s and sometimes goat’s hair (angora and cashmere). It consists of several interwound right-handeda-helices (a-keratin, pitch 5.1A), which rearranges to form P-pleated sheets ~P-keratin)upon mechanical stretching in the wet state. Silk is pure P-keratin with alanine and glycine as the main monomer units.Wool contains all 20 standard amino acids,is heavily cross-linked by disulfide bridges as well as by a few amide bonds between acid and basicside chains. Upon stirring in boiling water, the wool fibers first swell and then clog up. Hydrogen bonds are loosened and rebuilt. After drying and pressing, felt is formed, which may be considered a “hydrogen-bond” materiallike cotton and paper. Amide hydrogen bonds are, however, much stronger and more directed than hydrogen bonds betweenhydroxygroups.Covalent S-S cross-links further strengthenproteinbased materials such as leather and wool. The world productionof wool is about 1.7 X 106 tons per year (Bever, 1986). Proteins inthe form of gelatin from skin and bonesof animals constitute a renewable sourceof raw materials.World production is currently small (125,000 t/yr) but could be raised by several orders of magnitude. The flexible backbone and variety of side chains in proteins present an optimal mixof contact and cohesive interactions with polar substrates. Proteins have therefore been used as carpenter’s glue and component of mortars throughout history. Today proteins are not considered as state-of-the-art adhesives because of their plasticizationin moist e n v i r o ~ e n t sThey . lose 90% of their tensile and shear strengths when wet. F u ~ h e ~ o rthey e , are sensitiveto extracellular proteasesof microorganisms in the moist state. Natural proteinaceous adhesives such as barnacle cement or mussel adhesive, however, set in presence of water and remain unaffected by proteolytic enzymes, because the proteins are heavily cross-linked. A possible near future of commercial applications may lie in intelligent cross-linking to yield biomedical adhesives. None of available synthetic polymer adhesives really adheres to soft or calcified gelatin with its DOPA units on the surface of bones on one side and muscles or tendons with lysine cross-links on the other side. Novel materials for large-scale applications (e.g., adhesivesfor metal surfaces) can alsobe obtained by the grafting of dihydroxybenzaldehydes onlysine residues of gelatin. The phenol residues are then activated withepichlor~y~in, and the resulting epi-gel is used as a starting materialfor adhesives, which react with hydroxyl and amino groups (Scheme 9.2.1) (Kaleem et al., 1987a,b).

All 20 amino acids have the common motif of a so called a-carbon atom bearing a proton, an amino, and a carboxyl group as nonvariable substituents. Furthermore, all amino acidsin the major proteins are L-configured (except glycine) and all of them occur as zwitterions in water. In the gas phase the neutral glycine

7 -NH*

Lysine

-OH

Serine or Threonine

0

0

.

Tyrosine

OH

OH

-

Mod gel

-

Epi gel

structure is lower in energy than the zwitterionic structure. Addition of two water molecules, however, is sufficient to shift the e~uilibriumtowards the zwitterion (Jensen andCordon, 1995). The fourth a-carbon substituent R is chemically extremely variable. Inertalkyl groups, highly redox-active phenol, thiol and d n o groups, acids, and bases varying inplca from 2 to 13 as well as photochemically active phenol andindole groups are found (Table 9.2.2). The most important properties in respect to protein structures (Richardson and Richardson, 1989) are, however, not so much related tochen6cal reactivity. Even more important is the variation in water solubility (Hutchens, 1976) and hydrophobicity (Sueki et al., 1984) of proteins. Table 9.2.3 gives the usual assigments of acidic, basic, and hydrophobic amino acids. Solubility in water turns out to be unpredictable from first principles. Amino acids with charged side groups R are, for example, not always moresoluble than those with electroneutral hydrocarbon substituents. On the contrary, by far the most soluble amino acid (1.5kg&!) is proline, withthee CH, groups in a pyrrolidine unit as only the substituent,Aspartic acid with an acetic acid side chain is less soluble by a factor of 250 (6gL). Cationic amino acids are, in general, much more soluble than their anionic counterparts; the effect of hydrophobic substituents (e.g., isobutyl, secbutyl, phenyl,or indole) is not very pronounced (Table 9.2.3). Hy~ophobicity,on the other hand, has a much more rational connection to both d n o acid and protein structures. Hydrophobicity numbers are calculated

Names, Structures,pKaValues andIsoelectric Points of the Common

Amino Acids

Amino Acid Codes,Solubilities,and Their Effectson the Stability of Secondary Protein Structures Solubility Hydro-"C 25 NO. water Codeation Name

Abbreviin (g/L)

1

Alanine

Ala

A

2 3 4 5 6 7

Arginine Asparagine Aspartic acid Cysteine Cystine Glutamine

A% Asn Asp CYS

R N D C

Cys-Cys Gln

Q

8

Glutamic acid

Glu

E

9 10 11

Glycine Histidine Isoleucine

G1Y His Ile

G H I

12

Leucine

Leu

L

13 14

Lysine Methionine

LYS Met

K M

15

Phenylalanine

Phe

F

16 17

Proline Gerine

Pro Ser

P S

18 19 20

Threonine Tryptophan Tyrosine

Thr TrP TYr

T W Y

21

Valine

Val

V

>1 = Former; -1 I ~ d i ~ e r e n
166.53 167 (DL) zz 150 (21°C) -4.5 zz(28°C) 35 = :4.5 (20°C) Freely soluble 2.5 0.112 (18°C) 26 = :26 (DL, 18°C) 8.64 20.54 (DL) 250 41.9 41.2 22.3 (DL) 24.26 9.91 (DL) Freely soluble 51 (20°C) 33.81 (DL) 29.6 14.11 (DL) 1620 362 (20°C) 50.23 (DL) Freely soluble 11.4 0.453 0.351 (DL) 88.5

Pp

pathy

Pa

1.8

0.97 1.45

-3.5 -3.5

0.79 0.73 0.98 0.77

-3.5

0.26 1.17

-3.5

1.23 1.53

-0.4 -3.2 4.5

0.53 1.24 1.00

0.81 0.71 1.60

3.8

1.34

1.22

-3.9 1.9

1.07 1.20

0.74 1.67

2.8

1.28 1.12

-1.6 -0.8

0.59 0.79

0.62 0.72

-0.7 -0.9 -1.3

0.82 1.14 0.61

1.20 1.19 1.29

4.2

1.65 1.14

0.90 0.65 0.80 1.30

from the statistical weighing of amino acids in globular proteins as obtained from resolvedcrystal structures. An amino acidis hydrophobic if it is found predo~inantlyin thecore of such micellar proteins;it is hydrophilic if it mostly occurs on the surface. It is generally found that nonpolar substituents produce strong hydrophobic indices(1.8.-4.5), whereas charged groups give negative indices (-1.3to -45). T~ptophan,tyrosine, and phenylalanine rings are at once hydrophobic and polar. Their n”1ectron systems may not only act as hydrogen bond acceptors, but also solvate cations. The amino acids, therefore, participate in a wide range of intra- and intermolecular interactions, and their hydrophobicity is considerably divergent in different proteic environments. In membrane proteins they tend to cluster near the water/membrane interface (Andersen et al., 1998). Another important amino acid propertyis a tendency to favor or disfavor a-helical or P-sheet structures.The corresponding “conformational parameters” Pa and Pbare again simply astatistical measure of the amino acid location inahelices or P-sheets comparedto the frequency averaged overall 20 amino acids. An accumulation of amino acids withPa > 1 in a protein sequence indicates high probability of helix fomation, whereas three outof five P-sheet fomers (Pb> 1) in a protein sequence stronglyfavor a partial P-sheet structure. Helix and sheet a or Pb c 0.75) tend to destroy both secondary protein structures. Achiral glycine is, as one would expect, a strong breaker of chiral helix structures. Proline cannotf o m strong i n ~ a m o l e c uhydrogen l~ bonds, andits flexible five-membered ringis extremely water soluble.The relatively acidic phenol and amide protonsof side chainsare, on the other hand, involved in extremely strong hydrogen bonds, which compete with the regular intramolecular hydrogen bonds of the main amide chains.Their helix-brea~ngactivity is thus also understandable (Scheraga, 1978).The helix-favoring effects of amino acids are more complex.Theseaminoacidshaveincommonthattheyare of intermediate water-solubility (except alanine) and are electroneutral (except glutamic acid) (see Table 9.2.3). Polylysine foms, co~espondingly,helices only at hi h H, polyglutamic acid only at low pH. Helices of alanine-rich peptides e or twolysine residues, on the other hand, are strongly stabili by packing of the lysine side chain againstthe helix barrel ( ~ i l l i a m et s al., 1998). The intermolecular assembly of crystals is ruled by packing forces. Here the molecules havelost most of the unique mobility andconfomational variability of biological or synkinetic tissues, Nevertheless, the preferences and orientations of binding interactions between amino acids can nowhere be studied and understood in moredetail than in crystal structures. The most simple chiral amino acid is L-alanine andits racemate is D,L-alanine. Their crystals already exhibit important structural details. Molecular interactions within L-alanine crystals are first dominated by hydrogen bonds between

ins

the ~ m o n i u mand carboxylate groups, second by the sterical requirements of the substituent, and thirdby chirality. There are three NH-OC bonds to three different molecules: two within one crystal plane anda third one to the neighboring plane. The arrangement of neighboring chains is antiparallel in order to avoid close contact between the methyl groups. In the racemate D,L-alanine, again two HN-OC hydrogen bonds are found between two independent L-alanine molecules within one layer. Neighboring layers are madeof the enantiome~cD-alanine and are connected with the L-alanine layer only by one hydrogen bond per molecule. The stronger bonding between the same enantiomers by two hydrogen bonds means that a small amount of crystalline material of one enantiomer can lead to a spontaneous resolutionof the racemate. Furthermore,the chains of the neighboring D- and L-enantiomer planes run parallel in the racemate and in alternating a~angementsto keep the methyl groups apart (Fig. 9.2.1). The structure of the racematemay be transformed to thatof the L-form by reflection of the Dconfigured molecules andby a 0.285 nm displacement alongthe c-axis. The ordering of the molecules within one sheet of an amino acid crystal may be straight or zig-zag (Fig. 9.2.2). In the straight arrangement, the molecules are all in the same orientation (translational symmetry); in the zig-zag ~ a n g e m e n t ,themoleculesareorderedon a screw-axis(helicalsymmetry).

Crystal structures of (a) L-alanine and (b) D,L-alanine. (From Levy and Gorey, 1941; Donohue, 1950; Sirnpson andMarsh, 1966.)

Ny3 I

\ \

2 I I

'plypicdl crystal structures of amino acids: (a) straight and (b)zig-zag ~ r a ~ g e m e n t(From s. Suresh and Vijayan, 1983; Soman et al., 1988.)

ost hydrophobic amino acid crystals produce two straight sequences in two es, whereas many hydrophilic amino acids have one plane with a straight and one plane witha zig-zag sequence.There is no obvious correlation between packing of the pure amino acids in crystals and favored secondary structures in proteins: helix bredcers may, for example, form helices in crystals of the pure amino acid. Arginine-asp~ic acid and arginine-glutamic acid pairs, as well as the correspondingdipeptides,havebeenco-crystallized in a systematicmanner. These crystal structures (Fig. 9.2.3) allow detailedinsight into side-chain interactions, odd-even chainlink numbers, and chirality. An odd-even effect differentiates the crystal structures of Arg-Asp and kg-Glu, containingone or two C groupsintheacidicaminoacidpart. In Asp-Clu the two terminal nitrogen atomsof the guanidyl group bind tothe carboxyl groupof the y-aspartate terminal; the a-amino acid terminalsof both acids also stick together ina linear array (Fig. 9.2.3a).In the shorter Arg-Asp pair, no chains arefound. Two differently oriented macrocyclesare formed by hydrogen bonding; the aspartate carboxyl groups bend towards the argine nitrogens. The cycles are then fitted together in a complicated ~ a n g e m e n (Fig. t 9.2.3b). Pronounced chirality effects are found in kg-Clu (both L-configured) and Arg-DGlu co-crystals, In the L-L case a perfect face-to-face matchingof the side-chain guanidyl nitrogens and carboxylate oxygen atoms occursand both amino acids appearinseparatelayers(notshown). In ~-Arg-~-Glu anirregularside-on arrangement of the oppositely chargedside chains is found and thea-amino acid ends appear as mirror images (not shown). A similar arrangement also occurs in -Arg-D,L-Glu co-crystal, but the twistof neighboring pairs no longer oc0th Arg-Glu pairs are turned around 180" (not shown).

Crystal structures of amino acid eo-crystals: (a)kg-Glu and (b) k g Asp. (From Panditet al., 1983;R a ~ ~ i s and ~ ~'Viswamitra, a n 1988.)

An interesting phenomenon concerns the co-crystallization of racemic hydrophobic amino acids, e.g., Val, Leu, or Ile.In all cases it was found that a mixture of pure L-Phe and the racemate (e.g., of L-Phe and D,L-Leu) dissolved well in alkaline water and co-crystallized upon acidification. The co-crystals always consistedof L-phe andthe D-enantiomerof the other amino acid, e.g., L-Phe-D-Leu (Shiraiwa, 1984). Pseudo-racemic head-to-head and tail-to-tail bilayers are formed. This is another striking example of the chiral bilayer effect (see Fig. 2.5.9 andSec. 9.5).

Proteins are regular polyamides with repetitive a-L-amino acid units (if one neglects the enormous variations of side chains). The main chain unit consists five functional groups,namely-C~-NH-~a-C~-NH-, where the torsion angle describes the conformation of the part left to Ca beginning withni~ogenand describes the conformation of the right cmboxyl carbon part. These two angles are sufficientfor the descriptionof protein conformers, because the amide group is planar and rotation occurs only around the Ca bonds. The barrier to rotationof secondary amide bonds in peptides is around 15-20 kcal mol"; the cis-isomer population is usually less than1% (Scherer, 1998). Three repulsive interactions strongly reduce the possible conformations of peptide chains: l. The 1,5-interaction between two nonhydrogen atoms on Ca and N or

CO (Newman strain) constitutes the most important repulsive interaction in proteins. One first draws the formal = C N double bond in the Newman projection with Ca up andN down. At 4, = 0 both carbonyl

carbons are inecliptic positions, leading to strong Newman strain. For the evaluationof the role of W,one draws theCa-CObond, again with Caup but this time with CO down. = 0 now means two nitrogen atoms inecliptic positions and again maximal Newman strain. 2. The 1,4 repulsive interaction between substituents inecliptic positions on Caand N or on Coland the oxygen atom of the CO group (Pitzer strain) is the second most important strain in proteins. 3. Theinteractionsbetweensubstituentsatthe(formal)C=Ndouble bond and the (allylic) substituents onCaare also repulsive (1,3-allylic strain). This strain is only zero at angles of = 120" and Q, = -120°, where the formal C=N double bond and the CaHbond are ecliptic. Figure 9.2.4 shows at first the ~ZZ-anti-conformer with = 120" and 4) = -120" andthe numbering used inthe conformational models. An example of a strongly strained conformation withQ, = 0" follows. The Newman projectionof the (formal) C=N bond and its substituents showsa strong 1,5-interaction between 01and C2 as well as Pitzer strain between C1 and C2 and allyl strain betweenC, and C,. It immediately becomes clear that in order to evaluate these repulsive interactionsby inspection, one needs both the three-dimensional drawing and the Newman projection. The = 0 case is similar. There are two fundamental conformations that are essentially strain-free, namely Q,= 120" and = 120" (Figs. 9.2.4 and 9.2.5; only linear conformation, sheet) and Q, = -60" (a-helix) (Fig. 9.2.6). They have minimal Pitzer and Newman strains, and the linear conformeris even free of allylic strain. Furthermore, interchain (intercatenar) NHCO-hydrogen bonding is optimal in the Q, = -120" and = -60" conformation. The allylic strain present in the latter conformer is overcome by this favorable dipole interaction in the right-handed helix (Fig. 9.2.6) (Ramachandran andS a s i s e ~ ~ a1968). n, Linear chains of proteins have conformation angles of Q, = -120" and = uch chains may orient in a parallel or an antiparallel manner, and the hyrogen bond angles are close to 160", with lengths of about 2-3 A (Fig. 9.2.5). uch intermolecul~hydrogen bond chains lead to P-pleated sheets, which are ble in water anddo not swell. They are favored when ~ p o ~ asecondary nt protein structure is the helix stabili~edby intra~olecularhydrogen bonds. In the right-handed a-helix made of a - ~ hydrogen bond is almost parallel to the axis of the helix. It group of one amino acid with the carbonyl oxy fourth amino acid. Each helical turn contains 3.6 amino acids or 13 atoms in in chain. The handedness of helice determine^ if one which turn is up and which is down ectron m i c r o ~ r a ~ h s ch information only after p l a t i n u ~

Q,

= 0'

dD = -60'

Y

= 0'

Y!

= 1200

Repulsive interactionsin proteins.

a?= -120", Y = 120" 6-Pleated-sheet protein structure.

left-handed

right-handed right-handed

&Helical protein structures.

~onnectivityin an a-helix. Eighteen amino acids form five windings of the screw; each amino acidis displaced by 100" from its neighbor. With the sixth in^ of the screw, a newcycle begins.

esc~ption of helices is end and displaces neighbo~ngamino acids 2 in eli it tin) (Fig. 9.2.7) (Juvvadi et al., obtains an align~ent ofthe amino acids along the circurnference of the total helix. In m e ~ b r a n erotei ins, such as melittin, one often obtains c ~ c u ~ e r e n where ~ e s oneside is hydrophobic or ~ e m b r a n eoriented and the or prone to domain and water-~lledpore f o ~ a t i o nin other side is hydro~hili~ ~embranes.

In contrast to a-helices and P-sheets,in which 4, and V are repetitive, nonrepetitive patterns in which consecutive amino acidseach have their own4, and V values are called “turns.” Figure 9.2.8 shows a P, turn forming a 10-membered ring. This turn is more tightly twisted than the a-helix containing 13membered rings, and the C=O group is therefore tilted further away from the helix axis The conformation of an artificial cyclic octapeptide containing an azobenzene unit changed from “extended” to “turn” when the trans azo linkage was changed tocis by near-UV light.l H - ~spectra M ~ showeda P-strand with bends at Pro, andthe amino acids adjacentto the azobenzene unit. This was changed to an antiparallel P-sheet with many intramolecular hydrogen bonds in thecis diastereomer(Ulysse et al.,1995).Anothertotallyartificialhairpincontains a ~ ~ ~ n ~ - o double l e ~ n ibond c in the loops (Haque et al., 1994;Cardner et al., 1995). The most common helical protein in humans is the interwound triple helix collagen, with an abnormal high number of proline and oxyproline units (Fig. 9.2.9) (Squireand Vibert, 1987).An isolated a-helix is generally unstable in water. It gains stability when it aggregates with other structures through apolar or ionic interactions.The packing of a-helices in collagentriple helices is achieved by tiltingthe a-helical axessuchthattheapolarresiduesthatappearin repetitively occurring positions in the sequence of one helix fit into the spaces between apolar residues (“holes”)of one or two other helices. Knobs and holes may also be side chains of different charge (e.g., arginine and glutamate). The hydrophobic residuesand the ion pairs are then buried within the double or triple helix (knobs-into-holes mode).The triple structure of collagen is mostly madeof a sequence glycine-X-Y, whereX and Y are frequently proline and hydroxyproline yielding flexibility of the main chain or almost any other amino acid (e.g., arginine and glutamic acid).The acid hydrolysis of 100 g of gelatin gives typically 27 g of glycine, 20 g of proline, 14 g of hydroxyproline, 20 g of glutamic and aspartic acid, and 15 g of arginine and lysine.The remaining 14g are evenly

A p-turncontaining a~ydrogen-b~dged loop.

Small section of a collagen triple helix. H ~ ~ o p h o band i c ion pairs appear in a hot-to-holefitting arrangement.

divided between alanine and several hydrophobic amino acids. Each collagen achain is terminated by short segments (“telopeptides”) without glycine. Furthermore, the carboxyl end contains a lysine molecule, which is cross-linked with the other a-helices and thereby provides mechanical strength and stability to the triple helix and larger assemblies of collagen molecules. About 30% of human and animal proteins are collagens. Figure 9.2.10 summarizesthe approximate geometrical shapesof proteins for hypothetical ideal proteins made of 300 amino acids. The longest structures are always helical. Sheet or globule formation as well as coiling leads to much shorter extensions. The NMR analysis of protein structuresis a far too complicatedissue to be covered here. One example of a simple modelp e ~ t i d e - ~complex N ~ is given in Figure 8.6.11 (Kessler, 1988; OscMnat et al,, 1988; Roderet al., 1988). odem mass spectrometric methods greatly helpto speed up degradation Vluious desorption studies byidenti~ingthe molecular mass of peptide fragments. tec~iques,which produce protonated protein ions in the gas phase, are applied. Prominent examples are fast-atom bombardment (FAB) and matrix- assisted laser desorptio~ionization(MALIIT). Most commonly these techniques provide only the mass of the molecular ion, but they can be coupled with a collision-induc~d ~ssociation(CD)procedure. The ions are activated by collisions with neutral target gases, and the peptides dissociate in the gas phase. The resulting mass spectra

0 l

5 l

15

10 l

20nm J

l

a-helix 45 nm long -1 .lnm diameter -80 turns

collagen triple helix -29 nm long

p-pleated sheet 797nm -0.5 nm thick

extended coil -0.5 nmwidth

\

globule 4.3 nm diameter

Typicalshapes (Adapted from Metzler, 1977.)

and sizes of proteinscontaining

300 amino acids.

then produce valuable information on internal reactions between amino acid side chains and the polyamide backbone. It has, for example, been shown that peptide s an exlinks containingan arginine unit are more stable than other peptide Alinks. planation, the internal formation of a hydrogen bond was postulated, which provides extra binding energy in the gas phase (Vachat et al., 1996). Protein-based, cross-linked polymers containing glutamic acid residues swell when the carboxylate (-COO-) moiety is present, and they contract with lower pH, when -COOH is formed instead. Lowering the pH may thus lead to mechanical work, e.g., the lifting of a weight, by hydrophobic folding (Urry and Peng, 1995). Such a molecular machine should respond sharply to small pH changes in order to allow efficient energy output when picking up protons. For this purpose, the system must be made hydrophobic overall. In this case the energy differences between a stretched (=-COO-) and contracted (-COO conformation becomes as large as possible and a “muscle model” can be realized. If one dips a collagen fiber into hot water (>8OoC), the hydrogen bonds melt and the fiber contracts to form a coil. The same eBect is observed at room temperature with a concentrated lithium bromide solution. Lithium ions are

Reversible contraction of collagen fibers in concentrated lithium bromide solutions ordistilled water.

a-helix OF

" + + " -

@-sheet

Typicalarrangement of sidechains of peptides.(From Voyerand Lamothe, 1995.)

particularly effective as hydrogen bond breakers, because they bind tightly to carbonyl oxygen. The contraction force corresponds to about 20 kg cm2of dry muscle cross-section. Upon washing with distilled water the original collagen fiber is restored (Katchalsky, 1951) Figure 9.2.11). The ~ n i m u m number of amino acids required to form a P-sheet is usually 6-7 and around 12-13 for an a-helix. For such small peptides it is possible to predict the specific secondary structure (a-helix, P-sheet, p-turn) as well as the orientation of side chains (Fig. 9.2.12), thereby allowing an efficient design. For shorter peptides the confo~ationalequilibrium is displaced towards the unordered coil, and for large peptides predictions are still impossible.

A large variety of L- and D-configured amino acids, protected derivatives, and oligopeptides is commercially available from the usual suppliers of chemicals

and a specialist manufacturer.The major motivations to synthesize amino acids in the lab come from the necessity of labeling for NMR work or the need for nonbiogenic amino acids They may be derived from natural onesor obtained by total synthesis. The classical Strecker-type synthesis involving the additionof cyanide to aldehydes, ammonolysisof the cyanohydrin, and hydrolysisof the nitrile, yields racemates. More reactive than the cyanide anion are the azolactones for aromatic aldehydes or acet~ido-malonateanions for Michael additions or alkylations (Scheme 9.3.1). Several methods toresolve racemic mixtures of a-amino acids have been worked out, including separation of diastereomeric salts by crystallization or amides by chromatogra~hy.Chiral HPLConphases carrying D- and L-proline-copper complexes has been scaled up to 20 g quantities of amino acid racemates. Resolution with imobilized amidases, which deacetylate only L-amino acid acetamides, and subsequent precipitation of the D-amino acid a c e t ~ i d ~ s work on a 500-kg scale. All kindsof labels (13C,15N, ID) can thus be introduced by chemical synthesis of racemates and subsequent large-scale resolution.

R-CH0

NHy'HCN

" W

H C'N

H

H

H@lHzO

" + R-C-CN

" + R-C-COOH

I

I

NH2

CH3 Hippuricacid azlactone

NH2

CH,

CH3

H /catalyst hidrolysis Ar (Phe, His, Trp)

~

~ ~ - C O O H

I

NH2

COOEt hydrolysisand

decarboxylation

COOEt COOEt H&CONH{a COOEt

COOEt

R"-CooH NH2

Syntheses of amino acids were mainly developed to obtain amino acids with nonnatural substituents. They usually start with chiralcyclic and derivatives of glycine anions and then apply alkylation reactions (Polt and Seebach, 1989). This approach is exemplified by an a-aryl-alanine synthesis from glycine using L-valine as a chirality steering agent. At first a cyclic bis-lactone is formed by phosgene activation of one component which is then converted with trimethyla~onium tetra~uoroborateto a bislactim ether (Scheme 9.3.2). Asymmetrical alkylation of the chiral glycine anion generated with n-butyllithium at -70°C derivatives occurs with high optical induction.The diastereomeric excess is often higherthan95%,becausethe cyclic anionadopts a folded confo~ationin which C6 can only be approached from the sidetrans to the dirnethoxy groups (Scholl~opfet al., 1982). The synthesis of chiral glycine derivatives was also achieved by direct condensationof pseudoephedrine with glycine methyl ester in the presence of BuLi and LiC1. Alkylation of R at -78°C gave severalD- and Lconfigured amino acids in large enantiomeric excess (Myers et al., 1995). Enantioselective hydro~enationhas also been applied(Burls et al., 1993).

k-CHzX

Q

HY-COOR"

Ar \L-&o "oR*

I

W

COOMe OCH, HaCO

0%

hydmlysis

NH2

4.

COOMe

Q

I

H0 DCC, DMAP

BFQEtzO

1. DMSO, (C0CI)z

2. DIPEA

"

COOMe

Another approach uses chiral amino acids as starting materials and t&es advantage of the reactivity of the side chain (see Sec. 9.4).The oxidation of the oxymethylene groupof serine to a formyl group,for example, opens theway to an application of Grignard and Wittig reactions.In order to achieve this oxidation without racemizationof the a-carbon, the acidity of the a-proton is at first reduced by converting the carboxylgroup to anonactivating ortho-ester (Scheme 9.3.3) (Blaskovich and Lajoie, 1993).

Protein synthesis implies only one reaction, namely amide formation by condensation of a carboxylic acid withan amine group of two different a-amino acids. The amino group of the carboxyl component and the carboxyl group of the amino component as well as reactive side chains mustbe protected. Condensation as well as deprotection conditions must be mild, since the a-carbon chiral center has a strong tendency toward r a c e ~ ~ a t i o Nowadays n. solid-phase machines or biotechnological techniques are applied and trace amounts of thousands of different peptides are simultaneously synthesized by "combinato~al synthesis" methodologies, 9.3.2.1

Solid-Phase Synthesis

The protecting groupof choice for thea-amino group is usually N--fluoren-9-ylmethoxycarbonyl (Fmoc) because it is stable to Lewis acid conditions and because Fmoc amino acid derivatives are often crystalline. Deprotection occurs either in acetic acid with hydrogen bromide or by catalytic hydrogenation. Carbobenzoxy groups react in the same manner, and allylcarboxy esters are easily removed by hydrogenation using homogeneous catalysis. The tert-butoxy group of carbamides, the BOC-group,is removed in chloroform solution with trifluoroacetic acid or by

simple pyrolysis (see Scheme 9.4.6). Lysineand arginine side chains are usually protected with bulky carbonic acid chlorides, which react only slowly with the aamino group of anino acids. The sarne holds truefor the protection of terminal caboxyl groups with bulky alcohols. The extremely reactive SH group in cysteine finally may be protected either by reversible dimerization or by ~phenylmeth~lation (Table 9.3.1), (Greenstein, 1961; Lubke, 1975; Gross, 1980). Acid activation occurs with DCC derivatives (Scheme 9.3.4).The N-Fmoc amino acid is at first attached via nucleophilic substitution tothe chloromethylated styrene-divinylbenzene copolymer beads. This polymer swells in organic solvents and is therefore accessible, butit does not dissolve. Porous glass beads canbeusedinstead.Treatmentwithacidremovesthcgroup;theresulting ammoniumsalt is neutralizedwithtriethylaminein The nextaminoacid Fmoc protectedat the amino group andby one of the protecting groupsof Table 9.3.2 is coupled to the free a o group using DCC as coupling reagent (Crowley,1976; ~eienhofer,l985 danszkyandBodanszky,1994). This concludes one deprotection-neutralization coupling cycle of solid-phasesynthesis.All threestepscan be driventototalyields of >99.5%whenlargeexcesses of mono~ersand coupling reagents are used.At the end of the reaction cycle, the polymer is filtrated and washed. A new cycle can begin without working-up problems. Final detachmentof the peptide bound benzyl ester occurs by acidolysis with TFA. Protecting Groups for the Amino Group of Amino Acids Deblocking Reagent group Protecting

Cbr

PhCH20-CO-CI HBrlAcOH

HzP4

or

4

NO-

protection

amino acidFmoc protected

neulralimtion NEtdDMF

final detachment complete daprotection

HBrilFA

n

After each cycle, the few amino groups that failed to couple are blocked with 3-nitrophthalic anhydride.The peptide growth is thereby t e ~ n a t e dand , a colored, negatively charged indicator is tagged to all incomplete peptide sequences in the mixtures. These are separated by chromatography on an anion-exchange resin. Merrifield syntheses produce homogeneous macrornolecules up to about 10,000 daltons (80-100 amino acids). The poor solubility of large protected fragmentsinorganicsolventsthensetsa limit. Unprotectedfragments may, however, be joined by chemical ligation. This procedure consistsof chemoselective reactions of mutually reactive functionalities on each segment. Useful examplesare(a)thioesterformationfromathioesterandabromideand(b)

ter Protecting Groupsfor Amino Acid Side Chains +wtional

Croup

Aminoacid

ent

Protected

derivative

Removal

~ - ~ n ~ l o x y(Cbz, ~ ~2) ~ n y l H2 X H, Cl,NO2

HBr I AcOH or I Pd I

)(NO3 I H2S04

N-Slitn, Cysteine -SH

A ~ ~ d o m e(Awn) ~ y l

+CH,

~ e ~ o ~ Sulfoxide n e

COOH

acid Aspartic Glutamic acid

e

Tyrosine

-OH

Ph&CI

Tripheny~~yl (trityl, Tit)

Serine Threonine

Benzyl and resp.

H2 I Pd

W: I Hfl I 12

H c A \ c HgW* HSCHzCOOH

Hz02 t-butyl esters or ethers, PhCHzBr;

HBr I 'ITA

I

A I BF3

0

Et20

(aminooxy)acetyl and ketone functionalities to fonn an oxime linkage. Other strategies of potential utility in the chemical ligation approach involve hydrazones,metalchalation,disulfides,andthioethers(Rose,1994;Canne et al., 1995, 1996; Dawson et al., 1997). Protein and nucleic acid syntheses are fully automatized procedures nowadays. Detailed instructions come with commercial apparatus. Problems that only an organic chemists can solve only occur if the biopolymers are needed in large quantitities. Gold clusters (10-20 nm) protected with alkanethiolate monolayers produce stable colloidal solutions. They have been used as support of Meri~eld-type multistep peptide syntheses (Fig. 9.1.1) (Templetonet al., 1998). Colorized and multifunctional colloids withsingle or coupled receptor moleculesare thus easilyaccessible.Enzyme-catalyzedfragmentcondensationusingsubtilisinand

Fmoc-protected peptide esters or glycopeptide esters is another efficient methodology in solid-phase synthesis (Witte et al., 1998)

9.3.2.2. ~om~inatorial Syntheses Combinatorialsyntheses of peptidesdeliberately create simultaneously a plethora of compounds (“a library”). They are usually fixed on small solid supports, namely Merrifield beads or p m 2 areas on gold electrodes, silicon wafers, and similar materials. Fluorescence or otherwise labeled substrates or fluorescing receptor protein solutions are then added. Reactive peptide sections become observable under the fluorescence microscope andare selected manually. Since all individual beads or surface areas can be identified by microscopy, one may quickly find out which peptide binds, for example, a protein, a metal ion, ora nucleic acid. Screening of thousands of peptides becomes a matter of days or weeks; analysis of a given dissolved analyte can be doneby submerging a chip containing thousandsof receptor domainson pm areas and scanningit under the fluorescence microscopeor by isolating tagged beads and analyzingextracts by mass spectrometry. Two examples of the organization and tagging of combinatorial libraries are given in Figures 9.3.1 and 9.3.2: photolithography and a divide, couple, and recombine (DCR) methodology. Photolit~ographyis used to activate a smooth glass or oxidizedsiliconwafersurfacecoveredwithphotolabileprotecting groups (e.g., W-sensitive polymers or o-nitrobenzyl groups). After irradiation of a masked surface leaving onlya l-pm strip free, amino acid 1 can be fixedby a d u k reaction on thisstrip, containing free hydroxy, carboxy, or amino groups. Moving the slit of the mask by 1 p n then allows one to fixate another amino acid strip with photolabile protecting groups. For example, 10 l-pm strips of anino acids witha photolabile end groupmay thus be bound to the solid subphase. TJV irradiation through a l-pm slit now lying perpendicular to strips 1-10 then removes the photolabile protecting group first from the firstmm of strips 1-10, and solid-phase synthetic procedures are used to make a dipeptide with amino acid 11, again containinga terminal photolabile protecting group.The process is repeated on each strip with a known amino acid. Ten strips with 10 different amino acids 1-10 and 10 different amino acids 11-20 then yield 100 digerent dipeptides in l p m 2 spots of defined and known position. Six reaction cycles would yield 100 hexapeptides. The polypeptides may then be mixed with enzyme solutions or fluorescing substrates, and the most active peptides are detected directly under the fluorescence microscope (Fig. 9.3.1) (Baum, 1994). A less physical and more chemical approach that applies polymer or glass beads is the DCR method. It is used most widely in the lab. The solid support beads are divided into as may portions as one intends to vary in the given position (e.g., three for three different amino acids). Each portion is then coupled with the given protected amino acid, after which the portions are again

amino

amino

\.

silicon wafer

Ten amino acids were insertedat positions 1 and 2 in a pentapeptide. After acetylation thearray of substrates was treated with the protease thermolysin.liberThe atedtenninalaminogroupswerelabeledwithfluoresceinisothiocyanateandthe fluorescing spots detected with a scanning fluorescence microscope. The pentapeptide with amino acid 1 in both sites was obviously most easily hydrolyzed, followed by the ones with amino acid1 in site 2 and amino acid7 or 8 in site 1.

recombined. The cycles are repeated, and all protecting groups are removed 9.3.2). The beads are covered at the end with decapeptides, if 10 cycles been applied. Depending on the number of different amino acids, one obtains thousands to billions of different peptides, but numbers around 103-105 are optimal. Each bead carries a single individual decaptide plus impurities from unsuccessful couplings. The fluorescence test with dye-labeled substrates is now carried out, and the active beads are collected (Lam et al., 1991; et al., 1994; Czarnik and Hobbs De Witt, 1996; Jung, 1996). The sequences of active peptides may be determined directly by mass spectrometry. Alternatively, the beadsmay be coded in each condensation step with a photolabile or oxidation-sensitive tag molecule, The tags cover only a small portion of the bead surface and are removed by i~adiationor oxidation and, after silylation, analyzed by gas chromatography (~hlmeyeret al., 1993; Jung, 1996). The active peptide is then synthesized by the usual methods on a larger scale and can be further evaluated.

A simple DCR with three amino acids and two cycles yields nine types of beads with nine different dipeptides.

iotechnolo~ic~l Syntheses ~iotechnologicalsyntheses of natural proteins or single-site mutants (one amino acid exchanged) occur via appropriate DNA polymers, since each of the 20 natural amino acidsis coded by a nucleotide triplet (e.g.,AGA codes for arginine). The most reliable and efficient syntheses of proteins rely on “recombinant bacteria” and “gene cloning.” At first the messenger RNA for the desired protein must be isolated from an appropriate biological system (e.g., human pituitary gland tissue for insulin messenger N A ) . Isolation as an electrophoretic spotby elution from the plate gives sufficient material. Reverse transcriptase, a viral enzyme,thenreadsandcoprnRNAsequencetoproduce a single-str (copyDNA;cDNA).Themplate is removed,DNApolymeraseand triphosphates are used to copy the new DNA template, and short sticky terminals, usually CCCC, are attached to both DNAs. As a second educt a nicked plasmid with sticky complement^ ends is prepared with the aidof a restriction endonuclease. These have been mentioned already DNA in syntheses. They recognize nucleotidesequences(e.g., A ~ G G A ~and ) cleavethemat a specificsite(e.g., between A and G). Short sticky ends, usually GGGG, are then to added both termi-

nals of the nicked DNA. Combination with the CCCC-ended double-strand DNA gives a complete plasmid, which carries the genetic i n f o ~ a t i o nof the desired protein (e.g., insulin). From one plasmid a clone of lo8copies can be obtained, and the yield of the desired natural product rises to comercially useful quantities. The technique is only hampered by the fact that yields of plasmid DNA intake by the cells are very low. The few bacteria containing the hybrid need to be sorted out, which corresponds to a purification procedure. This is done by integrationof extra genes, which code for antibiotic-de~adingenzymes (not shown). Only bacteria that contain, for example, penicillinase togetherwith the insulin gene ~agments will survivepenicil~ntreatment and be able to replicate (Fig. 9.3.3) (Voets, 1992). If a relatively short sequence of a protein gene (e.g., a 21-meric nucleotide for seven amino acids) is paired with the single-strand plasmid DNA containing the whole protein gene, then one mismatch will not disturb the pairing process at 10°C.

inverse trenscriptase

lalkali

~DNA polymerase

SI

nuclease

I

I

mixing, annealing

+

I

uptake by cells [CaCIz]i cellular DNA ligase repair enzymes

growth of colonies

e

ca. 108copies in a clone

Scheme of DNA synthesis for protein production. Thecells will excrete the foreign proteininto the fermentation broth.

’OoC

l

mutant

ur nucleic base.

Production of proteinsingle-sitemutantsbybase

mismatc~ingof a single

The binding energyof the correct 19 base pairs will overcome the one antibin~ng rnismatch and DNA polymerase will produce the wrong DNA copy. One “wrong” base means one wrong triplet or one wrong amino acid in the corresponding protein. Mutant clones canthus be produced besides the original clones. The mutants are identified and isolated by their ability to bind the mismatched 21 -mer not only at 10°Cbut also at 50°C. Only the mutants will multiply at 55°C (Fig. 9.3.4). The multiplicationof genetic material is,of course, not restricted to bacteria and plasmids. Well-growing cancer cells have, for example, been hybridized with human cells, and the hybrids produce antibody proteins typical for the human cells. ost biotechnologic~protein synthesis work is now in the of hand molecular biologists. Organic protein chemists should, however, learn to handle thec o ~ e r ~ i a l tools of molecular biologyas well. Their main duty should is scalingtrophoretic spots to quantities that can be crystallized andlor analy spectroscopy.Mass production of pure proteinsis another goal for chemists.

a-Amino acids (Greenstein, 1961;Lubke, 1975; Creighton, 1993) are protonated at the arnino group in water (pk, 9) and deprotonated at the carboxyl group (pk, = 1.7-2.7)(see Table 9.2.2). Insteadof protons, one may also bind bivalent metal

-

Crystalstructureofcopper(I1)(isoleucinate),hydrate. lengths are given.

A few bond

ions to the nitrogen and oxygen atoms. The fourfold coordinationof metal ions to very stable chelates. The binding (e.g., Cu2+)with two amino acids often leads constants of Cu(AA),, for example, are typicallyl O I 5 M for the first amino acid M solutions of thecoppercomplexes,onlyone and lo8 for thesecond.In third is dissociated (Fig. 9.4.1). The chiral center at the a-carbon atom is acid and base labile. The C,COOH bondis as sensitive as the C,H bond. Heating treatment with acetic anhydrideinboilingpyridine or oxidationwith ~-bromosucciminidealllead to racemization or decarboxylation (Scheme 9.4.1). Nitrosation of the a-amino group leads to a-hydroxy acids; the evolving nitrogen canbe used for quantitative amino acidd e t e ~ n a t i o n (van s Slyke test). Upon mild treatment with acetanhydrideone obtains chiral azlactones, in which the proton can be replaced by bromine with retention of configuration. Ethylchloroformate gives cyclic anhydrides (Leuch’s anhydrides), which polymerize to give quite uniform homo-oligopeptides (Scheme 9.4.2). The Strecker synthesisstartingfrom a-amino nitrilesandpotassium cyanate yields useful hydantoins as intermediates. They are hydrolyzedat neutral pH to hydantoic acids andto amino acids (not shown) (Scheme 9.4.3) For the qualitative and semiquantitative detection of amino acids on thin layer plates, one may use either the blue condensation product with ninhydrine or the more sensitive dye fluorescamhe, which becomes fluorescent only upon condensation with amine groups.The chromophore presumably becomes much more rigid by electron donation fromthe vinylogous lactam nitrogento its carboxyl oxygen atom (Scheme 9.4.4). a-Amino acids do not undergo intramolecular condensation reactions to form a-lactams; the angle or Baeyer strain is too large. They can only condense

R-CHz.NH2

R-CH

II

R-CH-c"

I

NH2

NH

I

0'

R-CBN

HN02 R-CH-COOH R-CH-COOH

I

NH2

R-CH0

l

f

N2

orescent

R-CH-COOH AH*

slrongly

nonfluores-cent

with another amino acid molecule to form a dipeptide. The dipeptide may then cyclize to yielda six-membered ring,a bis-lactam (Scheme 9.4.5) A classical protecting group for the amino function is the benzyloxcarbonyl (Cbz) group, whichis removed by hydrogenolysis (Pt/H,). Another group that can be removed at neutral pH is the allylster group. Allylsters rearrange with homogeneous rhodiumphosphine catalysts to enolsters, which then hydrolyze in water-ethanol at pH ’7 (Scheme 9.4.6; (Morrison, 1984; Rose, 1994; Bodanszky and ~ o d a n s z ~1994). y, ( Another popular protecting groupis ~e~~-butylo~ycarbonyl is eliminated as isobutene and carbon dioxide at acidicpH or elevated temperatures. In automatedsolid-statesynthesis,however,theFmoc(fluoren-9-ylmethoxycarbonyl) group has become the standard, because the cleav protecting group canbe followed spectrophotome~cally(Scheme 9.4 Activation of the carboxyl group in condensation reactions can occuran as azide or, most common, with dicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide. Neutral conditions are applied and no r e ~ z a ~ i occurs. on Application of these condensation reactionsis discussed in In proteins the reactivity of the a-amino acid groups disappear. Only the reac-

penyl ester

Allyl

tivity of the side chains and the amide chain remain. Coupling of s i d e - c ~ reactiv~n of proteins is most ity with~ain-chainsplitting may occur. Regioselective cleavage efficientnexttomethionine S , "he thioethergroups of me~onime is mucleophilic enough tosubsti~tethe bromide in bromocyanogenand to o ~ o salt. ~ This u in turn ~ reacts with the basic oxygen atom of nei groups in proteins to give a h o ~ o s e ~ lactone ne and the amimoacyl p C1 c y ~ o g e nbromide therefore splitsa protein at theC - t e ~ n aside l of each rnen e chain also reacts with trichl ne residue. "he m e ~ o ~ side e toxic sulfoximimes. Early inthis century NC1, was used a

for flour and the sulfoximines caused epileptic convulsions (spasms, attacks) in dogs (Scheme 9.4.8). The nutritious values of D- and L-methionine are surprisingly identical. Therefore, one synthesizes this commercially second important arnino acid (8 X lo4tlyr) in food only as a racemate. The SEI: group of cysteine is prone to oxidation to radicals and subsequent dime~zationto cystine (Fig. 9.4.2).All proteins on the surfaceof biological organisms (skin, hair, fingernails, hoofs) are cross-linked cystine derivatives (skeleton proteins) because they are in contact with oxygen. They do not react with mercury and c a ~ u m ions. All proteins inside of the reducing e n v ~ o ~ eof n tbiological cells contain only cysteineSH groups. They strongly bind to mercury and cadmium ions. Thiols are thereforealso called mercaptans, the mercury-precipitating principle. The S-S group are cleaved by reducing agents, in particular by thiols or ascorbic acid. The c o m o n reagentusedinthelab is water-soluble di~othreitol(Clelands reagent). The reversible cross-linkingof the thiol side chain has the most impo~ant role in making surface proteins (e.g.., skin, fingernails, and hair) hard, nonswellabl~, and quite inert to attack by heavy metal ions. It allows, for example, the of formation p e ~ a n e nwaves t in originally smooth hairby reduction of S-S groups with thiols, curling and fixation by reoxidation with hydrogen peroxide. New S-S bridges are thus formed ata different position. Peracids leadMtoe r oxidation to sulfonic acids (Scheme 9.4.9).mso-Cystine, the molecular racemate, hdf is as solublein water (57 m@) as the pure enantiomer (1mgL), 12 another example ofa chiral bilayer effect. The amino group of lysine is the second proteinside chain frequently applied by nature for the cross-linkingof proteins. In elastin, the elastic protein of

I

R

H ’

A

ca.

Molecular structureof cystine with.a long S-S bridge.

Hg-OH

Z- @ O O C a g - J

irreversible

fully

2 HS+

reversible

00“ r O s -Ns H r ”~o @ Cystine

ONH,

+ HoQ OH

Clelands reagent

connective tissue (e.g., aorta, lungs, skin, tendons), one finds a t~trasubstituted pyridinium unit called desmosin. It connects no fewer than four protein units. This is achieved by the enzymatic oxidationof two lysines to form a butyral side chain andby subsequent condensationof two amino groups with two aldehydes. A similar reaction occurs in the processof baking, where the arnine reacts with carbohy~ates,(e.g., fructose or glucose) to producea large varietyof condensation products (e,g., furosin and desmosin) (Scheme 9.4.10). Lysine is ~arti~ularly suitable for cross-linking reactions, because it has a long (CH,),-spacer group (Fig. 9.4.3). Lysine condensates are usually indigestible, whether theyofare biological or kitchen origin.

ca. n m

Molecular structure of lysine with a long alkyl chain.

Similar to cysteine and terminal amino groups, lysine can be marked in oteins withall reagents sensitive to nucleophilic attack, e.g., ~ u o r e s c a ~ and ne uordi~itrobenzene(see Scheme 9.4.4). ~henylalanineis oxidized nature in to give tyr The latter is then biopolymerized to yield the dyes of hair and S malignant monomeric oxidation product,a phenyl~etone,is formed when the side chainis attacked. ~ h e ~ c a ltyrosine ly canbe selectively nitrate^ in proteins usin itromethane. Its character then changes from an electron donor, e.g., in photosynthetic systems, toan electron acceptor (Scheme 9.4.11). In some cases, one finds amino acid side chains that arepa~icularlyreac-

0

\ no

00

no

.11

.....JQ.

tive inenzymes.Oneexample is theserinein serine proteases.Thishas a strongly nucleophilic oxygen reacting like an alcoholate and displaces fluorine in diisopropyl ~uorophosphate.Other serine residuesor the free amino acid do not react (Scheme9.4.12). Amino acids are practical chiral educts (chirons), and they also steer stereoselective reactions as catalysts. Phenylalanine-con~olledaldol condensations with 2-dialkyl-cyclopenta-l,3-dionegive just one enantiomer (Danishefsky and Cain, 1976) Intramolecul~cyclization. reactionsof serine yield chiral p-lactarns via an active hydroxyl amide and after protection of the amino group of serine. The addition of aldehydes to cysteine produces chiral thiazolidinec~boxylic acids in quantitative yield. Aspartic and glutamic acids have been converted to

0

0

Phenylalanine HCIO,

CrOSPY

~

OHCJt 79%

(I) PhSH (11)CHzNz (11) Raney Ni

NHCbz

83%

PhtN 0

the terminal aldehydes, which are versatile chirons in syntheses of heterocycles e 9.4.13) (Coppola and Schuster,1987). C o ~ e r c i a l(S)- and (R)-1-~ino-~-metho~ypyrrolidines (S ) can be synthesized from either prolines or g l u t a ~ cacids. sed for the stereoselectivea-alkylation of aldehydes. One first forms the P and then alkylates the inpresence of lithiuhydrazo~ewith S mamide atlow temperature. The optical purityis usually in the order of 90%, but e n ~ t i o m e ~excesses c of 95% may also be obtained with large alkylbro~des cbeme 9.4.14) (Coppola and Schuster,198’7).

hitesides, 1995), in nation, and chemic cult hydrolysis reactions. Cross-1in.ked e n ~ y m ecrystals acquire high stability while preserving their activity in aqu S electrolytes as well as in. organic solvents such as toluene or t-butanol. S has been shown for molysin, a protease used for peptide synt~esisusing amino acid

(I) HCOOCH3 CH31 (11) NeH, (Ill) 10% KOH, A

H

(I) HONO

(11) LiAIH4 H

H

CHzOCH,

I

donors, and for lipases, which form or degrade hydrophobic esters. One milligram of such a stabilized enzme crystal is able to produce 4.5 g of the secphenethylacetate from the phenethylalcohol and vinylacetate in toluene without any racemization (St. Clair et al., 1992; ~ersichettiet al., 1995; laf et al., 1996).

Amino acids are p ~ t i c u l ~ useful ly as chiral and reactive headgroups of synthetic amphiphiles and bolaamphiphiles and synkinetic membrane s ~ c t u r e des rived from them. The nucleophilic reactivityof cysteine has been exploited in tions with quinones. ne example is a water-soluble naphthoquinone, which has been entrappedin chlorophyll-contain in^ vesicles in order to study light-in electron transfer through a membrane from glutathione to the quinone 1983). Another example is an asymmetrical vesicle membrane made of a cysteine quinone carboxylate bolaamphiphile, where the quinone is localized on the outer surfaceof the vesicle (see Scheme7.2.6; ous dispersion of a t~ple-chainglutamicacid diester monoamide ers under the light~ c r o s c o p ewhich , the fibers melt andr e ~ a n g to e vesicl bons made of similar molecules with azobenzenein the hydrop upon UV irradiation is leads to thedisappearance o lan nine-de~ve~ m of s~rfaceadsorbethylorange. azobenzene units in the chain also form helical fibers in W of the me~~ylene groups in theou chro~ophorein the fiberis presu *

0

b)

3601

l , 0

,

*

6 7 8 9 10 11 'l2 13 chain length (n)

(a) ~ e l i c afibers l formed by double-ch~nglutamate amp~philesunder the light microscope ( ~ ~ a s heti al~., a1985) and (b) light absorption of twisted ribbons made of alanine-based, single-chain a ~ p ~ p ~with l e odds andeven-numberedalkyl chains. (From Yamada and Kawasaki, 1990.)

A bolaamphi~hilewith two central s e c o n d ~ yamide groups and t e ~ n a l lysine arnino groups assemble to u n i f o ~long , tubules.The inner diameteris 50 nm, the emb bran^ thickness 4.4 nm. These tubules were quantitatively isolated in solid form by lyophilization,Shorter bolaamphip~lesgive cell^ rods (Figure 9.5.2). wand ~-~oly(lysine) both occur ina helical confo~ationin aqueous solu10.A 1:1 rnixture of both helices in water prGcipitates in the form of r e g ~ l sills-like ~, P-sheets (Fig. 9.5.3). This is a poly~eptideexample of the chiral bilayer effect.It demonstrates thei m ~ o ~ a nof c echiral u~ifo~mity no for no~covalent,soft-membrane systems, butalso for covalent ~olyarnides. cal fibers in aqueous gelsdo not survive the additionof enantiome~cfibers with the single exceptionof DNA (see Fig. 8.6.1). The ~recipitationof enantiomeric of arnino acid has, however, not been repeptides containing more than one kind ported so far.

Electron micrographs of vesicular tubules and micellar rods made of single-chain lysine-amino bolaamphiphiles. (From Fuhrhopet al., 1993.)

Poly@-lysine)and poly(D-lysine) both form helicesat pH l0 in water, Upon 1987.) mixing, racemic &pleated sheets precipitate (chiral bilayer effect). (From FulXhop,

Model of a tubular stack of cyclic peptides. Note the presenceof L- and D-configured amino acids. (From Ghadiriet al., 1992.)

The peptide cyclo [(D-Ala- L-Clu- -Ala- L-Glu)J with an even number of alternating D- and L-amino acids adoptsa flat confo~ation. This macrocyclic synkinon forms extended stacks in water and vesicle membranes. A ~ontiguous p-sheet in theform of tubules may reach a length of a few hundred nanometers and an inner diameterof about '7-8 W in water (Fig.9.5.4). The tubules also condense to form bundles of about 100 parallel strands. In lipid bilayers these peptide nanotubesarealignedparallelto the hydrocarbonchainandactasion channels at theirrigid outer surfaces. Their regulation by molecular stoppers, applied potentials, etc. has not been achieved so far (Kim et al., 1998).

Assemblies of small molecules with proteins areubi~uitousin nature. give a few of the better known synkinetic examples containing natural products discussed inthe previous chapters.

any membrane-penetrating proteins contain hydrophobic segments, usually 18-24 amino acids long. A 21-mer coiled into an a-helix is just long enough to span a lipid bilayer. These apolar segments may also function as topogenic elements, which guidethe protein to integration sites of functional elements within the membrane. The role of the lipid heterogeneity of biological membranes (variation of chain lengths, and charge, fluidity, cholesterol) seems first of all appropriate to optimize its properties as a two-dimensional solvent for membrane proteins. It must be fluid enough to allow chemical reactions and conformationalchanges of the integrated proteins, and it must also serve as an ion-impermeable protein envelope. Negatively charged phospholipids, for example, may not only preventcell-cell contacts, but may also fix cationic protein sites. cis-Configured double bonds in the lipids leading to membrane fluidity may allow for large hydrophobic amino acid substituents of the protein. Amphiphilic cholesterol derivatives may increase membrane microviscosity and prevent rotation of a-helical strands. In synkinetic membranes maximumactivity of integrated proteins as receptors or enzymes is usually obtained with lipid mixtures involving charged and unsaturated lipids. Even the admixture of detergents may help (Shinitzky, 1993). Rapid ~ ~ A - s e q u e n c i methods ng have allowed to determine the sequences of several membrane proteins. The mean hydrophobicity of 40 A long amino acid sequences in helical proteins with respect to a membraneous e n v i r o ~ e n t can thus be calculated. ost hydrophobic segmentsjust correspond to the thickness of a bilayer membrane. The usual techniqueto determine the dynamic helix characterof the transmem~ranesegment is C spectroscopy. In bacteriorhodopsin, for example, the helical content of the protein changes drastically duringthe light-induced reaction cycle leading to proton transport (Grigorieff et al., 1996). A fatty acid binding protein has been crystallized, but x-ray analysis did not show the localization of the bound oligomethylene chains, nor wasthe protein structure changed significantly upon adsorptionand desorption of palmitic 1). uller-Fa~ow, l99 Two classical peptide pores for lipid membranes are gramicidine A and melittin. Gramicidine A is a peptide made of 15 alternating D- and L-amino acids. ~trangelyenough, this peptide produces “helices,” although the alternating direction of the side chains should destroy molecular screws. The gramicidine helix is indeed not a tightly wound a-helix, but a widely twisted P-ribbon, a rolled-up P-sheet.Correspondinglytheinner,water-filledroom is wide enough to allow the transport of large cations through membranes. All side chains of g r a ~ c i d i n eare hydrophobic, making it membrane-solub]e. Furthermore, the length of the twisted protein ribbon (“P-helix”) is only 26 A.TWO

ter Gramicidine A

Model of a gramicidine ion pore.

molecules musttherefore combine at the membrane-soluble end to f o m a metal ion-conducting pore. If one connects the inner amino ends covalently with a linker, the pore becomes continuously conducting. If gramicidine monomers are introduced in bothparts of a lipid bilayer,the pore is closed when thedimer dissociates (Fig. 9.6.1). Calciumions block the pore by complexation, with fluorinated t~ptophansreplacing the t~ptophanswithin gramicidine increase ion conductivity by electrostatic effects, although the pore is less hydrated (Andersen et al., 1998). The dissociation of the gramicidine porein lipid bilayers has been used to c o n s t ~ cat sliding ion gate and a biosensor. This molecular c o n s t ~ c ~ is o nbased on the gold supportof the membrane on fixating the inner gramicidine molecule and using the outer ~ a m i c i d i molecule ~e in a recognition process, which then inhibits ion currents (Comell, 199’7). The recognition process of a small hapten or a polymeric antigen moleculeis triggered by two s~eptavidinmolecules (see ec. 7.3.1), whichare bound to both a gramicidine molecule floating the in outer me~braneand to a bolaamphiphile fixed on the gold surface an streptavidin molecule is connected by noncovalent bonds ch have been developedby co~binatorialsynthesis (Lang, are condensed to the ends of binding fragments (Fab) of the antibodies for haptens or antigens as well as to the carboxyl end of the outer r a ~ c i d i n molecule. e Onenow has two binding sites for the antigen in theouter one fixatedto the subphase (bola~phiphile)and one mothis membrane comes in contact with anti~en an in the surase, the Fabs are connected and the movement of the outer a ~ c i ~ i molecule ne and potassiumflow stops, Since Fabs can be madefor al-

Sensor devicefor anti~ensand haptens.

c+++

+

most any analyte of biological and ~edicinalinterest, the stable bila~ermembrane s y s t e bolaamp~iphile-locked ~ to the goldelectrode sudace may ther be a quite general sensor device (Fig. 9.6.2). elittin, a toxin in bee venom, is a ~entacationicpeptide co~posedof 26 amino acids. Four of the basic residues occur in a cluster n r made of the enantiome~camino acids in a .l) forms helicesin water containing20% ~ e x a ~ u o r o a cosolvent. The spectra of L-melittinandretroenantiome ~ e ~ i t t iare n ) a p p r o ~ i ~ amirror t e ~ ~ images, but both are e~uallyeffective in their a n t i b a c t e ~activity. ~ The toxic actionof melittin can therefore not function viaa rotein in) receptor or en~yme-ty~e action, The only other possibility is f o ~ a t i o n of ion-conductingpores by self-gation (~uvvadi,1997). In eli it tin-treated planar ilayers theioncurrentstronglydepen

Electrostatic interactions

Random coil

++++

(+) ++c+

Applied

(4

ion-conducting pore

Antiparallel amphipathic helices

It has been proposed that melittin forms helices on the surface of lipid membranes, which become integrated pores upon application of a membrane potential. Solid-state NNIR measurements on 15N-, I3C-,or 31P-labeled membrane proteins have been used for thech~acte~zation of the protein-lipid ~ a n ~ e m e n t s .

applied voltage. This can be explained with a reordering of surface-adsorbed helices (Fig. 9.6.3) in the direction of the applied field leading to membrane-reversible dissolution and pore formation (Juvvadi, 1997).

The different conformations of the 4-ene-3-one A-rings of many hormonal steroids have been discussed in ection 3.2 (see Fig. 3.23). A 5-ene-3-one duces these steroid by a stereospecific transfer of a hydrogen to C6. The 4P- and GP-hydrogens are involved, and the reaction proceeds via an enolic intermediate. The isomerase is an elon~ateddimer in e steroid is entrapped in a barrel of eight P-strands (Fig. 9.6.4). any proteins in blood and various tissues bind steroids stereoselectively. A progesterone-bin~ingglobulin ( ,for example,produces a U'v di~erence 4-ene-3-one chrospecifi indicates which spectrum, mophore two and t~ptophanresidues. complex of indole and progesterone has been grown from a variety of solvents. tals to indole molecules are hydrogen-bondedto the ketone ,and oneindole is coplanar with the 4-en-3-one chromo teroids act as stimulus for protein synthesis (anabolica, cata~olica)in cells of di~er~ntiated organisms. Itis thought that theho~one-binding

Molecular structure of aproteinP-pleatedsheetbarrelentrappinga steroid. (From Duaxet al., 1994.)

Model of an end-on binding of progesterone by two indole moietiesof protein tryptophane side chains or in tryptophane-indoleeo-crystals. (From Duax et al., 1994.)

region of proteins that are bound to DNA prevent thestart of transcriptional activity. The addition of hormone would then relieve this inhibition (Gehring, 1987 a,b; Evans, 1988; Duax et al., 1994). There is no evidence for direct interaction between a steroid and DNAin biological cells.

The best characterized protein-carbohydrate interactions occur in plant lectins. Other carbohy~ate-bindingproteins include immunoglobulins and enzymes. Lectins recognize terminal monosaccharide units in covalent and noncovalent polymers. Extremelylarge substituents, such as proteins or vesicles, are accommodated. 4-Deoxy or 3-deoxy derivativesof the recognized sugar moieties have little binding activity to lectins. Itis this c h i d center, the edge opposite to the glycosidicsite, that pointsto the bulk water phase and is responsible for molecular recognition.C~stallographyshows that ina mannose-binding proteinthe 3- and 4-OH groups directly bindto a calcium ion andf o m an extensive hydrogen bond network (Lee and Lee, 1995). The binding affinity of lectins to saccharide monomers is in the order of millimolar. It rises to nanomolar when carbohydrate clusters are provided. The

most simple exampleis the smooth surfaceof vesicles with glucoseor mannose headgroups or with glutamate-based trivalent glycosides with spacers. the lectins alsoaggl~tinateerythrocytes, which gave them their early name “phytohemagglutinins.” Some lectins are glycoproteins, some are pure proteins. The best-known lectinis concanavalin A (ConA), which comes from soybeans. It is a tetramer withone binding site each for two ~ a r b o h y ~ amolecules te and manganese and calcium ions. More than one half of ConA is made of ppleated sheets, and it has been proposed that these sheets can also stereoselectively bind carbohydrate assemblies.This a r g u ~ e ncomes t from the finding that lipid vesicles with carbohydrate and with tetracetate carbohydrate headgroups agglutinate equally well and equally selectively with C o d . This finding can easily be a c c o ~ o d a t e dwith a planar bindingsite c o ~ e s p o n d i nto~a p-pleated sheet (Fig. 9.6.6) (Fuhrhop, 1987), not with a narrow cleft, where four protons and four acetyl groups shouldbe clearly distinguishable. ConAalso binds toglycolipid monolayers on gold surfaces withtwo binding sites man^ et al., 1998).

Dg1ucoo;e(Oh)

0.1

(a) Vesicles made of double-chain glycosides are precipitated stereoselectively byGonA; the turbidity of the solution rises within a few seconds. L-Glucose and D-galactose show no response, but the tetraacetatesof D-glucose and D-mannose are recognized like the parent alcohols. (b) Model of a planar binding site on Con A. (From ~ u h ~ h o1987 p , .)

X-ray studies of ConA-mannose complexes (Naismith, 1996; Loris, 1996) and calorimetric studies (Swamhathan, 1998) also indicate participation of several water molecules. The accepted mechanism of glycosidase activity involves protonation of the glycosidic oxygen with departureof ROW, a transient carbonium ion and a glycosylenzyme intermediate (Figs. 9.6.7, 9.6.8). This is similar to the proposed mechanism of lysozyme-catalyzed hydrolysis of a hexmeric unit of a 1 , 4 " glycosidic polysaccharide made of N-acetylglucosamine (NAG) and N-acetylmuramic acid(NANL). This unit is bound to the enzyme cleft by six cooperative noncovalent interactions (1-6 in Fig. 9.6.7), which are strong enough to press the glycosidicbond 4+5 into a contraction of the cleft. The pyranosering of (NLIM)~ is thereby deformedto a half-chair, a conformation that is close to that of a possible cationic oxonium intermediate of the glycolysis reaction. Neighboring glutamic acids and aspartate anions are thought to act as proton donors and acceptorsin the final hydrolysis step (Dickerson and Geis, 1969). The mechanism given aboveis supported by a series of classical enzyme experiments: the location of hydrolysis was determined by the use of W, I8O where all "80 wasthenfound at Cl of N (4); thebindingenthalpyof oligosacch~desin the series f r o m ( ~ A G )to~ (NAG), rose by 2-4 kcal/mol for each additional NAG unit, but when(NAG), was replaced by (NAG), it fell by 3.5 kcal/mol. This energy is sufficient to enforce the conversion of a chair to a half-chair confo~ation;if the sp2-hybl~di~ed lactone of NAG was bound to (NAG),instead of thehalf-acetal,the new tetramerwasbound 3600 times

antibinding areaS

active center binding binding area area

Asn 37

Trp 63 \

Asp l02

H

62

Trp

Asn 44

In lysozyme the glycosidic bond is cleaved by acid-base-catdyzed hydrolysis. They gray areais the active center (see Fig. 9.6.8).

hypotheticaloxonium intermediate

(a) The reactive oxonium intermediate presumably fits nicely cleft contraction of lysozyme, andso does (b) a stable lactone analog.

into the

tighter than (NAG),. The lactone has already the confo~ationof a half-chair ickerson and Geis, 1969). Plants and microorganisms produce a variety of monosacch~ide-likealkaloids, which are potent glycosidase inhibitors. Some show promisein treating diabetes or asantiviralandanticanceragents.Protonatednitrogenanalogs apparently mimic the gluco- or mannopyranosyl cations. An amidine analog of D-glucose combines the correct charge and conformation of glucosyl cations implied in Figure 9.6.8. Besides being a potent inhibitor of P-glucosidase and other mannose and galactose processing enzymes,the amidine, whichis stable to water, also binds to a cross sectionof other glycosidases. Recognition between enzyme and substratesis thus dominatedby favorable electrostatic interactions and much less by modest stereochemical scrimi in at ion. Potent examples for covalent i ~ i b i t o r sof glycosidases axe carbohydrate-derived epoxides, which are activated by protonation inthe first step of enzyme-assisted glycoside hydrolysis or 1 , l - d i ~ u o r a ~glycosides yl (not shown), which have a half-lifeof about a month in water and yet give 80% irreversible inactivationof yeast a-glucosidase. Synthesis of N- and 0-linked glycopeptides has been achievedby their s u ~ s e q ~ e ~ t treatment first with subtilisin and a dipeptide and then with P-194-galactotransferase and urindinyl galactase(Wong et al., 1993).

The best known polyene-protein complexes are, of course, rhodopsinand bacte~orhodopsin.The model for the long wavelength shifts of the retinal-lysine chiE bases have already been discussed in Chapter 5. A detailed electron micrograph image analysisis available for bacte~orhodopsin,which shows seven he-

lices crossing the membrane (Grigorieff, 1996). No direct data for retinal-protein weak interactions are available with atomic resolution. The same is true for other carotenopo~hyrins,where modelsfor long-wavelength shifts exist, but no crystal structure,

Po~hyrinsare entrapped in tightly fitting and relatively stiff molecular boxes in proteins. These boxes are not made of P-sheet-like structures, but of a-helices. In the caseof human deoxymyoglobin containing 146 amino acids, five light helices are foldedto f o m five walls around the central heme molecule[E, F, CD, and A(EF)], whereas the propionic acid south edge is in touch with the surrounding water. Oneclose imidazole unit fixes the porphyrinto the F helix; another remote imidazole comes from the E helix. All the helices of myoglobin are strongly hydrophobic on the inside, and the heme surfaces are tightly wrapped. Upon additionof oxygen to the sixth coordination place not much happens to the protein structure.The most obviouschange is a movementof the central iron ion from O S 5 fi out of the porphyrin plane towardsthe close imidazole axial ligand to only 0.26 A out of the same plane.The oxygen molecule forms a 121 * angle with the heme plane (Fig. 9.6.9). In hemoglobin, four similar units are connected aintetramer by hydrophobic interactions. Bindingof the first oxygen atom enhances the binding constants of the three remaining units. For such cooperative (“allosteric”) effects, which are typicalfor biological but outof reach for synkinetic systems, the reader is referred tob i o c h e m i s ~textbooks (Dickerson and Geis, 1969, 1983; Voets, 1990). The active site in myoglobin is a heme tightly bound to the proteins through about 80 hy~ophobicinteractions as well as by one close histidine and one more remote histidine interaction with the central iron ion. The ~ s y ~ e ~substituc a l tion with two imidazole ligands produces a high-spin Fe@) ion with relatively high

Schematic structures of myoglobin and oxymyglobin.

Helix

Loop Y

A

ca,&d stronglyhydrophilicarea "_;_- -j hydrophobic area repeated each turn c ,*$&p

\li",

L

,"C

Y

Helix \

" . + porphyrin-binding site

A synthetic heme pocket. Both the peptide and the porphyrin react as b o l a a ~ p h i p ~ l and e s as edgea~phiphiles.

electron density in the d(z2) orbital orthogonal to the porphyrin ligand. The hy~ophobicityof the pocket dlows dissolution of an oxygen molecule and prevents oxidation of the central iron(II) ion, because no negatively charged counterion can approach it.A myoglobin model features a synthetic four-helix bundle. The tetrahelical model protein was designed to coordinate a single heme molecule with two histid~eunits in the center of a four-helix bundle parallel to the helices. The carboxyl ends were connected aby Pro-Arg-kg loop tobind the hydrophilic propionic side chains of protoporphyrin E,and the N-terminal carried cystine units which t the apolar north edge of the porphyrin. provided a hydrophobic e n v i r o ~ e nfor The h ~ ~ o p h o bproduct ic around the porphyrin n system was realized with leucine units adjacent to the histidines in the helices (Fig. 9.6.10) (Choma et al., 1994). This synthetic protein fixates heme and zinc protopo~hyrinIX very well in water (K,,,, < 10, M).The heme's iron(I1) is a mixture of high spin and low spin, but no oxygen fixationis possible in water. Sucha relatively simple designed protein may, however, fixate various redox- and photoactive chromophores and may be combined with anionic redox systems at its open end. Fast electron transfer may be achieved. The redox potential of the Femmen pair drops by 90 mV if the peptide environment of the heme changes from h y ~ o toph y~~ o~~ h~o b iHydro~hobicc. ity increases the binding constants of the peptides to the heme iron, They scale with more negative reduction potentials(Hu~mannet al., 1998).

Heme can be extracted from horse heart metmyoglobinby acid-butanone mixtures. The apomyoglobin may then be reconstituted with iron(I1) protoporphyrins dissolved in pyridine/methanol(3:1) mixtures, which is added to aqueous myoglobin mixtures. Purificationis by gel chromato~raphy.The ratio of the absorbance at 408 nm (Soret band) to that at 280 nm (aromatic protein side chains) shouldbe close to 4.8, the valuefor native myoglobin.A p r o t o p o ~ h y ~ n derivative carrying two phenylboronic acid substituents on the propionic acid side chains has been integrated into apomyoglobin in the presence of various monosaccharides. Solubilization by complex formation then leads to a more or less rapid reconstitution (Hamacheet al., 199’7). Quadruple peptide helices have also been made by self-assembly in water. The designed amino acid 16-meric sequence ~ E L E E L L ~ ~ forms ~ L LanK ~ amphiphilic helix and should form a four-helix bundleor quadruple helixby assembly of the hydrophobic leucine segments. It was indeed found experimentally that the ellipticity in the CD spectrum increased with the fourth power of the peptideconcen~ation.The helices were only formed if there was a chance of te~amerization(Eisenberg et al., 1986). The design of linear protein sequences that are not merecopies of natural compounds has to address the major inherent problem of polypeptide chain folding, i.e., the competition between intra- and intermolecular interactions. To overcome such uncertainties, amphiphilic polypeptides were also attached as neighboring side chains to a carrier molecule or template assemblyprotein (TASP),namely A C C K A P ~ ~ A K ~ ~ Both H , .central (LysAlaLys = KAK) units have all NH, groups on the same side of the ring, and one can assemble

S

.l1 A q u a d ~ p l ehelix on a macrocyclic peptide containing few lysine (K) units.

Glutathione

four parallel helices on one main-chain TASP by solution peptide synthesis (Fig. 9.6.11) utter et al., 1992). The kinetics of protein folding triggeredby electron transfer in myoglobin and cytochromeb is much faster than in in cytochromec. Upon reductionof the central ironion, a-helices cluster around the hemeforming, for example, a fourhelix bundle, It appears that highly helical proteins have favorable energy landscapes for folding (Wolynes, 1996; Telford et al., 1998).

With respect to vitamin-protein molecular complexes, the structure of the enzyme glutathione reductase is most instructive. Catalysis occurs in two steps: first NADPH reduces an S-S bridge within the enzyme and secondthe released groups reducethe glutathione S-S bridge ( cheme 9.6.2). Glutathione (GSH) is the tripeptide g-L-glutamyl-cysteinylglycine, which occurs in muscles and acts as reductant and transport agentfor amino acids. It is the cheapeast commercial tripeptide. The basis of the catalysis of the splitting of the disulfide is presumably the n of a charge-transfer complexbetweenthetwo-electron donor (equivalent to a hydride anion) andthe acceptor flavin combined with proximity effects. 0th coenzymes, NADPH and FAD, are bound to the protein by adenosine phosphate-protein interactions, the substrate is loosely bound at the cleft between the units of a protein dimer (Fig. 9.6.12) (Schulz, 1983; Douglas, 1987). For interactions between avidin or streptavidin with biotin,, see Section 7.3.1, *

Tyr 197 Glu 472

His 467

Simple modelof an enzyme containing two interacting redox catalysts: NADPH and FAD+. (Adaptedfrom Voet and Voet, 1990.)

Several cationic proteins containing massive amounts of lysine and arginine bind to nucleic acids in chromosomes. They may be either in the center of a DNA superhelix (Hl) or attached to the DNA surface. Another possibility to introduce positive charges into proteins is to use zinc ions which are bound to histidine and cysteine side chains in proteins. The tetracoordinate zinc ions, an essential trace element, connect four amino acids and form a hydrophobic loop (zinc finger). These zinc fingers are then integrated into the major groove of RNA. A similar motif made an a-helical protein, which contains only leucine at one edge. These proteins form bis-helical dimers in aqueous solution. Zinc fingers, leucine-edge proteins, and similar protein motifs provide possibility for the formation of protein assemblies on DNA surfaces (Figure 9.6.13), which is a prerequisite for transcription, the enzymatic synthesis of DNA matrices.

Reading of information in peptides relies on interactions between different side chains. It is hampered by the lack of conformational order, so that only a few peptide-recognition processesof peptides in water have beeen found (Peczuh et

protein baclcbone

72 5

Molecular complexes of (a) peptides, (b) zinc-finger proteins, and (c) leucine peptides withDNA double helices.

reslow et al., 1998).A host peptide witha crown ether at the N-tern& nus and a large tetraal~ylammoniumgroup at the G-terminus showed no end-toend interactions whatsoever.The t e t r a ~ ~ y l a ~ o n igroup u m did not fitinto the crown, Other peptides, however, bind strongly to this with their onium and carboxylate end groups. Binding interactions between the amino acid in between support this process moreor less. Extraction experimentsof various tripeptides, for example, show exclusivelythe formation of 1:1 complexes and binding constants ranging from200 to 2000 in water (Hossain and Schneider, 1998). Antibody molecules or i ~ u n o g l o b u l i n sare made up of two identical heavy (H) and two identical light (L) chains, both held together by S-S bonds (Fig. 9.6.14.). They constitute large, folded polypeptides(MW = 2 x lo5daltons) that bind virtually any natural or synthetic protein, polysaccharide, or nucleic

Model of an antibody structure.

acid (antigen) with high affinity and exquisite selectivity. Antibodies protect organisms through their ability ~to s c ~ ~ nsharply a t e nonself molecules and cells from self. Although in general only biopolymers are recognized, antibodies can also be tangled with small organic molecules (haptens). For that purpose, the haptens are first covalently boundto proteins and then given alone (e.g., 2,4-nitrobenzene),Largepolymersarebound to P-sheetsurfacesthatcancover 0.6-0.8 nm2 ( 6 0 ~ 8 0 0A2), while haptens are typically boundclefts in of a fewA wide. Association constants range from lo4to l O I 4 h 4 - I (Lerner et al., 1991). Antibodies are synthesized in lymphocytes. The genes encoding each antibody are spliced together from a battery of gene segments, which enables an organism to mount a primary immune response of lo8 different antibody protein molecules. A complex screening system coupled with further cellular events such as mutation and maturation provides another 106-foldvariants. Up to l O I 4 variations of protein sequences and folding variants are thus produced biologically to find the optimal precipitating agent for a single type of foreign invader. Each single antibody molecule is, however, produced in negligible quantity, and the successful binding antibodies are destroyed quickly by a biological complement^ system. Nevertheless, it is possible to produce monoclonal cell systems (Kohler and Milstein, 1975) that yield single antibodies in quantities of several micrograms (typical price of a commercial monoclonal antibody: $300/10-6g, but see Fig. 9.6.15). The location of the antigen-combining sites can be demonstratedby electron microscopy in preparations of purified antibodies, which are mixed with a bolahapten cont~ningtwo dinitrophenylend groups. Dimers, trimers, tetramers, and pentamers are observed that have the shape of a rod, a triangle, a square, and a pentagon, respectively. This means that the hapten is bound end-onto the protein carrier. Haptensare small molecules thatare not immunogenic on their own, but will react with preformed antibodies induced by injections of the hapten linked to a protein carrier. Binding sites are composed of hype~ariableregions

of the heavy and the light chains. Their relative positions are fixated by neighboringP-pleated-sheetstructuresandcan be movedaround by oligoglycine links. Forat least five of the six hypervariable regionsof most i ~ u n o g l o b u l i n s there seemsto be only a small repertoireof main-chain conformations. Sequence variations modulate the chemical character of the surface,so that these structures present to the antigens or haptens. I ~ u n o g l o b u l i n sare formed by six peptide loopsattachedto a P-sheet.Theyalsorecognizecarbohydrates,but little is known about the specific interactions. The chemical interest of these trace amount proteins stems from the fact that they can be used as catalysts or enzyme analogs for almost any chemical reaction (Tramontano et al., 1986; Pollack et al., 1986; Lerner et al., 1991). The fundamental difference between antibodies and enzymes does not relate so much to the protein structures as to the structure and lifetime of the substrates. Antibodies selectively bind molecules in their ground state, whereas enzymes selectively produce and then bindmore strongly to short-lived transition states. Antibody-antigen complexes tend toward precipitation, whereas enz~me-transition state complexes react to enzyme-product complexes, which immediately dissociate. In both cases, however, the same noncovalent bonds are used. How can one now exploit the binding and specificityof antibody binding to make or break covalent bonds?The answer is by producing antibodies in the presence of ground state molecules that resemble transition states. Molecules known as efficient enzyme inhibitors are good candidates. The synkinetic antibodies may then be used as catalysts. Their binding site is sterically and electronically complementa~to the rate-determining transition state. Three examplesfor the enzymic activity of monoclonal, artificial antibodies are given in Scheme 9.6.3 and Figure 9.6.15. Transition state analogs are those molecules that are chemically similar to substrates bound to an enzyme.An antibody to synthetic peptide boundto a cobalt complex,for example, catalyzed the hydrolysis of amides with rate accelerations of approximately lo5 relative to background hydrolysis (Janda, 1988; Iverson, 1989). A iels-Alder reaction was accelerated by a factor of lo6by a hapten that resembled the transition state, although the educts looked very different (Tramontano, 1988). An N-methylated porphyrin triggered the formation of an antibody that would bend simple porphyrin bases ina way to makeit more accessible to incoming zinc ions. The antico~esponding body chelatase wasonlyabout 10 times less efficient than the enzyme (Fig. 9.6.14) (Cochran and Schultz, 1990). The regio- and stereoselective opening of a cyclopentyl-epoxide was achieved with the transition-state like chiral pipe~diniumanalog (not shown) (Sinha and Keinan, 1993). Since the educts so different, the rate e~ancementwas and transition states were structurally small (4X lo2),but enantioselectivity of the catalyzed reaction reached 98%. A model for the porphyrin metalation indicates its driving force: empty space for the metal ion is provided in the protein-porphyrin complex. Antibody 38C2 efficiently catalyzes a wide variety of ketone-ketone, ketone-aldehyde, aldehyde-ketone, and aldehyde-aldehyde intermolecular cross-aldol reactions.

Antigen

substrates

Products

COOH

COOH

Thee examples of antigens that correspondto an sit ion states in various reactions: (a) amide hydrolysis,(b) Diels-Alder cyclization,and (c) porphyrin metalation. In (d)amodel of thetransition state is also given. It implies distortion of the porphyrin macrocycle before metalation. Thisdistortion i s mimicked in the hapten byintroduction of a methyl group in the porphyrin center.

Inte~olecularreactions usually give single enantiomers with ee's up to 299% intramolecular reactions always leading to condensation products. tant is the fact that 100mg or even gram quantities can be produced and the that artificial antibodyis comercially available fromAdrich. The esoteric antibody catalysts thus become realistic tools in organic synthesis and in combinatorial synthesis ( H o ~ m et ~ al., n 1998) (Scheme 9.6.1) As usual, synkinetic chemists must not rely on natural biopolymers, which are only accessible in trace amounts. More easily accessible models must be constructed. For this purpose, one has to combine the knowledge about combinatorial peptide libraries and the structure of antibody-re~ognition sites. Although the overall structure of an antibody protein is complex, the variable peptides forming the binding sites for haptens at the tips of the Y-

donoi

Example of a gram-scale stereoselective synthesis using a commercial antibody. 90 mmTenta GelSNH2

Simple modelof an antibody-recognition site. (From Francis alet ., 1996.)

shaped protein consist simply of two juxtaposed polypeptides on the ends of fixated carrier proteins. Huge parts of the Fab fragments are not active in. binding biopolymers, but the specific fixation of haptens needs two optimized peptides. A simple model antibody applies two peptides (heavy and light chains) in close proximity to form an active site for metal complexing. Standard Merrifield and splittin~/poolin~ techniques of combinatorial synthesis yields thousands of face-to-face dipeptides with different end caps. Nickel(1I) and iron(II1) are bound to the library and then produced colorized adducts with dimethyl~lyoxime or thiocyanate. Nickel was exclusively bound by bis-histidine dipeptides with methyl or naphthyl caps, iron(II1) by a nonbiological dipeptide containing methionine in position 1 and iso-nicotic acid in position 2. No structural overlap between nickel and iron binders occurred (Francis et al., 1996) (Fig. 9.6.16). A receptor mimic and peptide guest based on the formation of a three-stranded, antiparallel p-sheet upon forming a 1:l complex has been optimized in a similar way be Gombinatorial chemistry of both receptor and the renz and Kelly, 1995) Effective receptors are characterizedby conformers having concave bindl dist~butionto their ing sites that are complement^ in size and e l e c ~ c acharge substrates. Electrical complementarity usually involves ion pairing andor hydrogen bonding.A key requirementfor high selectivityis a distinct conformerof low energy. Bindingsites that easily slip from one conformationinto another are

likely to bind many different substrates. Furthermore, in water the diving force for binding is usually the minimizationof hy~ophobicsurface areas. A stereoselective receptor of L-amino acids and peptides is, for exarnple, a macrolactarn made from (R,R)-cyclohexane-l,~-diamineand trimesic acid (Yoon and Still, 1993; Still, 1996).

Crystal morphologies of glycine can be controlledby using surfactant monolayers at the airfwater interface as templates. Catalyzed nucleationmay, for example, yield (010) pyramids of glycine in supersaturated aqueous phases below surface monolayersor at foam lamellae (Chen et al., 1998). The molecular mechanisms of the adsorption processof proteins on solid surfaces have an impact on fields as diverse as laudry, chromatography, immunoassays, and biocompatibility. In the great majorityof cases adsorption is irreversible, A detailedstudy is availableontheadsorption of the soluble, negatively charged tryptic fragmentof cytochrome b,. A highly cationic polyallylamine surface acted as a perfect sink. The adsorption to a highly negative stearate surface is much slower and fully reversible. Adsorption to a neutral phos~holipidbilayer finally was very slow and practically irreversible. Simple analysis based on net charge gives nom e ~ i n g f uresults, l as was shownby single-site mutants. The actual surface charge distribution, polar and apolar interfacial interactions, must be takeninto account (vanOss, 1988; Ramsden, 1995). SH-Bolaamphiphileson gold with polar and nonpolar end groups adsorb proteins at the waterfair as well as waterfcyclooctaneinterfaces. Small proteins only adsorb strongly to hydrophobic surfaces, whereas largerproteins adsorb to almost any surface, again with some preference for less wettable surfaces. A single exception was found: surfaces presenting hexa(ethy1ene glycol) groups to the approaching proteins did not adsorb any protein (Sigal et al., 1998). The deeply penetrating ordered water-PEG structures do not allow for hydrophobic rotei in-monolayer interactions, they behave too much like water. If, however, the PEG surface is coupled with receptor peptides, selective recognition processes may take place on an otherwise nonadhesive surface. The tripeptide Arg-Gly-Asp (RGD),for errample, is a ligandfor cell adhesion. Polyacrylamide gels as well as monolayers with PEG end groups were derivatized with this tripeptide and then became efficient inall attachment and spreading by binding to all surface receptors, whereas other proteins were rejected (Roberts et al., 1998) Figure 9.6.18) Amide bonds connecting electron donor-acceptor moieties also support electron transfer by o-tunneling superexchange (Tsai, 1998). Both forward and back-transfer of electrons proceed through bonds of oligoamide chains rather than through space (Slate, 1998).

Two-dimensional model of a self-assembled monolayer on gold presenting protein-rejecting PEG and cell-adhesive GRGD peptides to the solutes in bulk water.

S with COOH-te~natedalkanethiols (HS-(CH~)~-COOH) immobilize cytochrome c in a stable, electroactive state. With n 2 8 the standard electron-transfer rate depends exponentially on rz and electron-transfer occursby tunneling. The electron transfer rateis increased bya factor of >1000;the COOH-thiol monolayer is coassembled with OH-thiols. The mixed monolayer obviously allows direct contact with the heme edge, which does not occur in the pure COOH-thiol monolayer. F u ~ e ~ othe r esurfaceplca of8 of the pure monolayer shifts to more acidic values in the co-assembly. The monolayer may thus contain more negative charges to allow the higher binding of cytochrome c (El Kasmi et al., 1998).

So far only productive binding processes have been described. noncovalent destructionof ordered structures.The book finishes with an elementary way to destroy orderin proteins and todo this reversibly. Manyphmaceuticals are based on the selective and reversible destructionof protein structures by urea derivatives producingdipole moments in the order of 3.8 D as compared to 1.8 D for water.

Globular proteins unfold upon additon of excess amountsof urea. A cornmon explanationis that such reversible “denaturation” occurs by the breakup of water structure by the strong dipole of urea and its derivatives. Model calculationsindicate,however,thatureapreferentiallyadsorbsontochargedhydrophilic protein residues on the surface and gives rise to a swelling of the protein. Hydrophobic residuesin the center of the protein are now exposed and water onset into the interior takes place. Urea thus causes an “outside-in” re~ a n g e m e nand t lowers the hydrophobic effect within globular proteins (Wallquist and Covell, 1998). Monte Carlo calculations indicate that urea and guanidinium are unique among polar compounds: they are highly soluble in water, but structure water inthe same way as nonpolar compounds. They are able to bind to hydrophobic groupsof proteins and destroy water clusters on their surface. Distorted hydrogen bonds, which are characteristic for bulk water as well as for proteins, are depleted, leading to denaturation (Vanzi et al., 1998). Here could start a whole new storyof natural products: how natural products can be their job in living organisms, in particular in cancer cells, bacteria and viruses. This story could be written as a mirror image to the present book, starting with the disruptionof proteins and ending with the punchingof uncontrolled holesinto membranes. Nature also knows very well how to target natural products andhow to destroy functional molecular assemblies. For Referencesfor Chapter 9, see pages574-583.

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a,b-~ehy~ogenation of acid anions, 86 Acetaldehyde, 383 Acetic acid, 379 Acetylene adduct, 135 Acid soap, 102-103 Acid strength hydration, 34 p&, 32-33 tautomers, 32-34 A c i ~ o p ~ lbacteria, ic 71 Adenine, 398 Adenosyl~iphosp~ate (ATP), 435 Adhesives, 466 A-DNA, 400 AI?" = atomic force microscopy, 49-52, 114 fatty acids, 114 vesicles, 263-264 Agnes Pockels, 45 Alditols, l69 Aldol reaction, 141, 146146, 521 Alkaloids, S12 Alkanes, 20 A~ylphosphates,433-434 Allose, 184 Allosteric effects, 513 Allyl ester, 495 Allylic effect, 186 Allylic hydroxylation, 89-90

Allylic oxidation, 259 Allylic strain, 474 Alternating bond lengths, 269 Altrose, 184 Amadmi r e ~ ~ g e m e n215,224 t, Amid~-contai~ng monolayers, 119-1 21 Arnidine, S12 Amines, 117-118 Amine-tight m e m b r ~ e 119 , Amino acids, 466-473 abbreviation, 469 cationic, 467 essential, 465 hydropho~icity,465,467,470 metal complexes,491-492 pKa, 468 protecting groups, 484-486 reactions, 491-500 synthesis, 481-483 table, 468,469 water solubility,467,469 Aminoacylation, 42 1 Aminocaproic acid, 70 A~onium perchlorates, 43 Amylase, 203 Anabolic effect, 135 Androstanes, 129, 130-131 Androsterol, 133 1,6Androst-diene-3, 17-dione, 142

Anion radical, 355 16-Annulene dianion, 270 Anomeric effect, 180-183 Antarafacial 1,7 H-shift, 156 An~aquinone,345 Antiaromatic, 293 Antibodies, 5 17-523 Antigens, 5 17-523 Antisense, 419,441 AOT ,99-l00 Arabinose, 201 Arbutin, 343 Archaebacterial lipids, 82,94 Arginine, 428 Armed sugar, 214 Ascorbic acid,354-357,496 Atomic force microscopy (AFM),49-52, 114,263-264 ATP (Adenosyl~iphosphate),435 Autocatalysis, 443-445 Autoradiography, 4 1 1 Autoxidation, 340 Avidine, 374,376-377 Axial ligands, 273,294 Azides, 2 19 Azobenzene, 80,501-502 p-carotene, 243,254 P-cyclodextrin, 194 P-helix, 505 P-keratin, 466 P-plane in steroids, 129-130 P-pleated sheets, 466,470,475,520 p-sitosterol, 140 Bacterial cell surface, 201 Bacteriochlorophylls, 265 Bacteriorhodopsin, 512 Baeyer strain, 9-12,492 Barrier to rotation, 473 B-DNA, 439 Beer’s law, 16 Benzene, 279 Benzyl ether, 429 Benzylosy-carbonyl(Cbz), 494 Bicyclic ribose, 441 Biladiene, 278

Bilatriene, 277 Bilayers, 281 Bile pigments, 265,277,280-282 Bilirubin, 278,279,280 Biliverdin metal complex, 282 crystal structure, 282 Biosensors, 462,506-507 Biotin, 374-377 Bis-helical dimer, 307 Bixin, 243,264 Black lipid membrane (BLM), 122-123 Bleaching, 100 Bleomycin, 446-447 BLM (bilayerlipid membrane or black lipid membrane), 122-1 23 Blood, 35 Body fluids, 34 Bolaamphiphile, 82, 84, 104-110,502, 523 Bond angles, 3-4 Bond energies,4 Bond lengths, 3,270 Bones, 466 Brewster angle, 115-116 Bromination, 43 1 Bromine, 34 Cadmium aracfiidate, 114 Cadmium, 496 Calactan, 198 Calcein, 126-1 27 Calcium, 506 Calixarene, 237 Cancer, 254,440,456 Ca~th~anthines, 259 Capped porphyrins, 284 Carbohydrates, 167-241,509 allylic effect, 186 amphiphilic fibers, 229-237 conformers, 169-1 92 cyclic, 179-188 cyclic oligosaccharides, 194-201 disaccharides, 189-193 equilibrium mixtures, 184-188 furanose-pyranose, 184-1 88

[Carbohydrates] gauche-effect, 176 molecular complexes,224-229 monolayers polymers,261-264 open-chain, 169-178 oligosaccharides, 193-201 protection, 2 16-224 rearrangements, 206-208 repulsion, 176 resources, 169,201-209 stereochemis~,169-172 stoichiometry, 167 sweeteners, 226-227 Carbon dioxide, 35,374-375 Carbon monoxide,35 Carboxylate, 399 Carboxyphosphate, 374 Carcinogenic effects, 437 Carotenoids, 89,243-264 cis-trans isomerization, 244253 conformers, 249-252 nomenclature, 245-247 radicals, 253-254 spectra, 244-25 1,253 Cmot, 245 Catalysis, 500, 520 hydrolysis of amides, 5 11 Cationic DNA,441-442 Cbz (benzyloxy-carbonyl), 494 CD (circular dichroism), 15-16, 102, 314,455,505,507,515 Cell membranes,94 Cellobiose, 189, 191, 192 Cellulose, 167-168, 196-197, 199, 240-241,245 Celluloseacetate, 240 Cerebronic acid, 71 C-Glycosides, 432 Chain-termination reaction,412-413 Chaotropic, 69 Charge separation, 2,99,314,348 Charge transfer, 42,3 11 Chelatase, 520 Chemical hydrolysis ofW A and DNA, 411 Chemoluminescence, 369-37 1

Chinhydrone, 339-341 Chinon radical, 339 Chiral bilayer effect, 233-234,438, 502-503 Chiral pool, 16'7,204 Chlorin e6,303,307 Chlorophyll, 94,265,276,287-290, 301-302 Cholane, 131 Cholanes, 129,130-131 Cholastanes, 129, 130-1 3 1 Choleic acid, 157 Cholestane, 131 Cholesteric liquid crystals, 160-161 Cholesteric phases, 2 Cholesterol, 130, 133, 140, 142, 148, 159-160,163 Cholesteryl-3b-no~aoate,136, 160-162 Cholic acid, 133 Chylomycrons, 132 Circular dichroismspectra, 15-1 6, 102,314,454"455,505, 507,5 15 Cisplatin, 457-459 Citrulline, 428 Clone, 490 Cmc (critical micellar conce~tration),96 Cobalamin, 387-393 Cobalt, 520 CO-crystallization, 1 13 amino acids, 470473 porphyrins, 279-28 1 urea-fatty acid Coenzyme A, 379-381 Collagen, 465,477480 Combinatorial syntheses DNA, 426-428 peptides, 464,487489,522 Con A = Concanavalin A,5 l 0 Conductor material, 35 1-353 Confo~ational par~eters, 66,400,470 Conformations, 5-1 1,271 carbohydrates, 169-192 clinal, 6-8 cyclic compounds, 5 cyclohexane, 9-10

[Confo~ations] cyclohexene, 10 cylcohexanone, 11 cylcopentane, 11-12 energy, 8 fat, 78-79 fatty acid, 66-48 NAD, 358 nucleosides, 400-403 open chain compounds,5-9 perilanar, 6-8 proteins, 473-480 retinal, 248-249 steroids, 135-139 153-154 C o ~ o t a t o 156 ~, Contact shifts, 272 Coproporphyrin, 287 Gorrinates, 387-393 Corticosteroid, 136 Cortisol, 134, 136 Cortisone, 134 Coulomb forces, 38 Cross-linked, 479,500 Cross-linkers, 437,497 Crown ether,5 18 Crystal struc~res,279 alanine, 471 amino acids, 470-473 azobenzene amphiphile, 80 fkxa”tene, 248 biliverdinates,283 canthaxant~n,248 choleic acid diglycoside, 157 co-crystal urea fatty acid, 113 co-crystals, 472-473 Con A mannose, 5 11 cystine, 497 decyl-~-glucopyranose,180-1 8 1 dimy~stoylphosphatidylcholine,76 DNA, 397-398 DNA triple helix, 439 fat, 78 fatty acids, 65-70 general, 472

[Crystal structures] gluconamide, 174 gulonamide, 174 lysine, 498 lysozyme, 511-5 12 monolayers, 113- 115 octae~ylfo~yl-biliverdin, 282 palmitic acid proteins, 505 protein czbohydrate, 509 steroid, 509 RNA, 397-398 steroids, 129, 136-138 tartaric acid, 176-177 TPP dication, 293 tryptophane indole, 509 Curds, 102-103 Current intensity, 454 Curtius degradation, 301 Cyanogen bromide, 495 Cyclic voltammetry. 58-59 Cyclitols, 187-188 Cyclodextrins, 204,222,228 Cyclohexane 9-10 Cyclohexanediol, 165 Cyclohexanone 10 Cyclohexene l0 Cyclopentane 11-12 Cyclomaltodestrin,203 Cyclophanes, 347 Cysteine, 371,43l, 496 Cystine, 496,497 Cytochrome b5,523 Cytosine, 398 and L-amino acids,504 230 DBS (di-0-benzylidene-D-sorbitol), DCC (dicyclohexyl-carbodiimide), 484“485,494 DCR (divide, couple, recombine), 487-489 De Bruyn-van Ekensteinre~angement, 207 Deaza~avinophanes,368-369

D-

Deblocking of OH groups. 216-224 Dehydrogenases, 342 16-Dehydropregnenolone, 142 Deoxycholeic acid, 157 Deoxymyoglobin, 513 Deoxyribonucleic acid, 395-462 Deoxyribose, 400-402 Desmosin, 497-498 Desoxycorticosterone, 134 Deuteroporphyrin, 299 Dextran, 201 Dextrans, 204 Diabetic sugar, 187 5, 15-~ialkylideneporphyrin,299 Diamines, 69 Dianabol, 134 Dicarboxylic acids, 69 Dicyclohexyl-carbodii~de(DCC), 484485,494 Dielectric constant, 38 Diels Alderreaction, 20,22, 140, 144"146,520 Dienediyne, 456 diether, 126- 127 edge, 126-1 27 Di~erentials c a ~ i n gcalorimetry (DSc), 232 Digitonins, 158-159 Digitoxygenine, 133 Dihydroxyvit~nD3,139 Dimers, 517,519 antibody, 5 19 arginine-asp~icacid, 472-473 arginine-glutamic acid, 472473 bis-helical, 5 17-5 18 4,4'-Dimethoxy tritylium, 421 Dimethyl sulfate, 411 Dimethyldioctadec~la~onium bromide (DODAB), 106 Dimyristoylphosphatidylcholine,76 Di-0-ben~ylidene-D-sorbitol(DBS), 230 Diosgenine, 140, 142 1,3-Dioxane, 217 Dioxetane, 306, 370 1,3-Dioxolane, 217

5, 15-Diphenyl- 1 0 , 2 ~ i p y r i d i n i u ~ porphyrins, 455 Dipolar interactions, 172 Dipy~omethanes7 284 Dipy~omethene,267 Disaccharides, 189-193,209 Disarmed sugar, 214 Distamycin, 446452 Disulfenic acid, 372 Disulfide, 437,5 16 DNA (deoxyribonucleicacid), 395-462 A-form, 405,408 amino acid recognition, 426-428 amount in humans, 396 B-form, 405 B-helix, 408 bicyclic ribose, 44 1 branching, 426-427 cationic, 441-442 cationic proteininteraction,409 CD spectra, 16 cisplatin, 457-458 coating, 110 combinato~alsynthesis 426428 computing, 461-462 conformation, 400,402-410 cross-linking, 437 crownether, 452 cruciform, 410 degradation, 411-414 dyes, 452-456 electrophoresis, 41 1-414 energy transfer, 455 flexibility, 408-409 gold colloids, 461-462 hairpin,410 homo, 443,445 hydrogen exchange, 417 hydrolysis, 428429,433-436 ~ypersensiti~ity, 409 intercalation,406 isolation, 397 labeling, 411 L-enantiomer, 438 lagging strand, 4 18

[DNA (deoxyribonucleicacid)] leading strand, 4 18 length, 396-397 major groove,403-404 ~ a x ~ - ~ i l b e4 1r1-4 t , 13 melting point, 403 methylation, 4 11-413 methylthiourea, 422-4123 Mg counterion, 405 minor groove, 403-404 monolayers, 461-463 NMR, 414,416-417 opening, 4 17 orthogonal ladder,405-406 oxidation, 459-460 PCR, 428 phosphodiester reactivity, 411-414 polyamides, 445-452 pol~guanidinium, 422-423 polymerase, 4 18 porphyrins, 453-454 protonation, 4 17 q u a ~ p l helices, e 442-444 radiation damage, 437 reactivity, 428-437 rectangle, 426-427 replication, 4 19 roll, 404-405 Sanger, 412-415 skewed ladder,405-406 slide, 404-405 straight helix, 406 synthesis, 417428,437-461 thickness, 397 toroids, 239,460 translation, 42 1 triple helix, 438-441 twist, 405-409 DNA ligase, 46 1 DNA polymerase,4 13 DNA unwinding, 405 DOPA, 466,498 DSC (differential scanning calorimetxy), 232 D-Tryptophan,427

Edge amphiphiles, 121-128 EDTA, 126-1 27,449,45 1 Elaidic acid, 9 1-92 Elastin, 496-497 Electrical energy, 4-5 Electrocyclization, 290 Electron microscopy,49-50,3 14-3 15 c ~ b o h y ~ afibers, t e 230-235 micelles, 97 vesicles, 50 Electron transfer, 29-3 l, 59, 344-346, 348-35 1 Electron wires, 262 Electrophoresis, 411,489 Electrostatic forces, 43 Ellipticine,455-456 Enamines, 26,31 Endometrium, 135 Endo-puckerings, 400 ENDOR spectrum, 276 Endosomes, 127,158-159 1,2-Enediols, 206 Energy conersionfactors, 5 Energy transfer, 309,455 Enzymatic degradation, 100 Enzyme crystals, 500 Ephedrine 102-103 Epi-gel, 467 Epoxide, 520 EPR spectra hemin, 274 quinones, 348-349 spin-labelled protein, 374-377 EPR spectrum, 348 Ergocalciferol, 156 Ergosterol, 156 Erythrocytes, 5 10 Essential amino acids, 465 Essential food stuffs, 62 Estradiol, 134 Estranes, 129,131 Estrogens, 135 Estrone, 134 Ether linked lipids, 71 Ethidium bromide,452454,459 Ethinylestradiol, 134

Exciton coupling, 53, 309-310 Exo-anomeric effect, 192 Extinction coefficient, 13 Eye, 36 Fat isolation, 8 l Fats, 6 1,77 Fatty acids, 45,61,91-92 acetylenic, 82-83 allylic hy~oxylation,89-90 chiral, 69,71 chiral soaps, 102-103 conformation, 62,65-67 crystal structures,65-67 deuteration, 8 1-82 dimerisation, 92 elongation, 8 1-83 hydrogenation, 8 1-82 melting points, 62-64 microbial hydroxylation, 90 oxidation, 81-82 table, 63-64 Fenton reagent, 459 Ferricyanide protoporphyrin, 273 Fetal hemoglobin, 278 Fibers, 110-113 porphyrin, 314-316 Fibrous soap, 102-103 Filipin, 123-1 24 Fischer conformation, 170 Fischer projections, 169 Fischer rules, 73 Fischer-Hel~ch,429 Flash photolysis, 348, 366 Flavins, 365-372 Flumethasone, 131 Fluor substitution, 135 Fluoren-9-yl-me~oxycarbonyl (Fmoc), 494 Fluoresca~ne,498-499 Fluorescence, 127,256,305,314,348, 487-488 carotenes, 256 quenching, 1617,126-127, 312-314 NADH, 358

[Fluorescence] tocopherol, 340 yield, 16 Frnoc (~uoren-9-yl-rne~oxyc~bonyl), 494,484 Folic acid, 377-379 Footprinting, 449-45 1,456 Formaldehyde, 377,437 Formyl group, 271 Frontier orbitals, 296 Fructose, 201,202,208 Furanose ring, 409 Furanose-pyranose, 178-180 Furosin, 497-498 Galactose, 184,201,208 Galacturonic acid, 194 Gallstones, 132 Gel filtration, 397 Gelatin, 477 Geminidiesters, 82,85 Gene cloning, 489 Genes encoding,519 Gentiobiose, 189, 19 1 Gestagens, 135 Glucans, 238 Glucitol, 169-176, 187 Glucocorticoid, 134 Glucornannan, 198 Gluconamide, 11 1 Gluconeogenesis, 135 Gluconic acid, 202 Glucosamine, 204,208 Glucose, 180, 184,201,202,203 Glue, 417,466-467 Gluococortocoid, 135 Glutathione, 516-5 17 Glycals, 209,222-224 Glycerolipids, 72,73,75,82,85-86 Glycerophosphatides, 73 Glycitols, 169 Glycosides, 209-214 Glycosidase inhibitors, 512 Glycosyl acceptor, 209 Glycosyl donor, 209 Glycosyl halides, 209,210

Glycosylation, 279 Gold colloid, 461,486,456457 Golden chicken, 259 Golden liver, 259 Golden nuggets, 244 Goldfish, 244 Gonane, 129 Gram staining, 236-237 G r ~ c i ~ A, n 505-506 e Growth factors, 187 ~uanidinium,46 1 Guanine, 398 Guanosine, 441-442 Guanylic acid, 441442 Gums, 199-201 Hairpin, 410,439,477 airy rods, 240 Halides, 2 10 Haptens, 517-523 Harpoons, 127,314,159 Heart attacks, 132 Heartbeat, 130 Heavy metal catalysis, 434 Hematopo~hyrin,299,307 eme, 5 13-5 14 emicellulose, 196-198, 199 H~moglobin,513 Heterodim~rs,44 Heterotrimers, 44 High pressure liquidc ~ o m a t o ~ a p h y , 397 HIV, 403,456 Holelelectron hopping, 3 10 Holes, 477 H O ~ O 13,29,296 , Homo-DNA, 400,443,445 LC (high pressure liquid c~omatography),397

Hydantoic acid,492-493 Hydration, 34, 176 DNA, 398,400 forces, 43 ydrazine, 43 1

Hydrocarbons, 39 in urea, 112-113 Hydrocortisone, 134 Hydrogen bonding,4142,314 chains, 474 nucleic acids, 397 types, 4 1 4 2 Hydrogen cyanide, 35 Hydrogen exchange,4 17 Hydrogen peroxide, 372 Hydrogen, 265,308,362-363 Hydrolysis, 428429,433436,520 Hydrophobic effect, 37-39 Hydrophobic sudaces, 523 Hydrophobicity, 505 Hydrosulfides, 93-94 Hydroxy-9-Z-octadecenoicacid, 7 1 Hydroxyapatite, 397 Hydroxyl radicals, 459 H ~ d r o x y l ~ n e , 4 2 8 4 2 91, 4 3 Hypersensitivity of DNA, 409 Hypervariable regions,519 Immunoglobulins, 518-523 Indigo, 262 In~am~ations, 79 Infrared spectra, 17,56 amide monolayers, 12 1 DNA, 397 fatty acids, 115 ion pores, 121-128 monolayers, 1 15 porphyrins, 27 1-272 Inks, 100-101 Insulin, 489490 Intercalation,405 Invert soaps, 115, 117 Inverted micelles, 99 Ion conductivity of membranes, 123 Ion currents, 125 Ion pore, 506 Ionic radius, 294 Isoamylase, 203 Isocyclic ring, 301 Isoleucinate,492

Isoprene units, 344 Isosbestic point, 52-53, 306 J-assembly, 55 Jaundice, 279 Job plot, 53,313 Karplus curve, 20 Kemp’s acid, 442 Kinetically inert, 294 Kleenow fragment,4 13 Klyne Prelog designations,74 Knobs, 477 Oafft point, 96 Lactam, 494 Lactitol, 204 Lactose, 189, 191,204 Lactose arms, 239 L-amino acids, 404 Langmuir-Blodgett (LB) monolayers, 45-49 Lanosterol, 143 Lanthanum hydroxide, 434 Laser photolysis, 437 Ll3-monolayers, 113-121 amide bonds, 119-121 calixarene, 237-239 even-odd, 1 19-1 20 LDL (Low-densitylipoproteins), 132 Lecithine, 1 11 Lectins, 510 Leuch’s anhydrides, 492 Leukoindigos, 262 L-Glucose, 180 Lifetime expectancy, 79 Ligases, 4 18 Light-harvesting arrays, 3 10 Linoleic acid, 89-90 Linolenic acid, 89-90 Linseed oil,61 Lipids, 61-128 Lipoic acid, 372-373 Liquid crystals, 224-225, 136 Lithocholic acid, 163 Low-density lipoproteins (LDL), 132

Luminal, 370 LUMO, 13,29,296 Lutetium, 455 Lymphocytes, 5 19 Lysine, 384,497 Lysine cross-links,466 Lysophospholipid, 92 Lysozyme, 5 11-5 12 Macrocycles model lipids, 93 tetraesters, 82, 84 tetrasulfides, 82,84 tetraethers, 82, 84 Magnesium, 406,409,435 Magnesium-octae~ylporphy~, 270 Major groove, 404 Maltose, 189-190,203 Mandelic acid, 416 Manganese, 296 D-Mannitol, 85 Mannitol, 85, 169-170,202 M ~ o s a m i n e 208 , Mannose, 184,201 Margarines, 62,89,244,351 Mass spectra porphyrins, 272 x~thoporphinogens,272 Match, 154 M ~ a m - ~ i l b e r45 t, 1 McMurry condensation,299 Melittin, 476,507-508 Membrane disruption, 3 14 Membrane fluidity, 505 Membrane, 61-128 gap, 165-166 ion conductivi~,123 MLM coating, 109-1 10 monolayer (MLM), 107-110 rupture, 127 Mercury, 429,496 Merrifield syntheses, 485-487 M e s o p o ~ h y ~299 n, meso-Tetraphenyl porphyrins, 279 M e t a c ~ o m a ~effect, c 109 Metaphosphate, 435

Methandrostenolone, 134-135 Met~onine,496 Methoxatin, 342 Methylene blue, 109 Metmyoglobin, 514 Micellar fibers, 44, 101-103, 229-234, 3 14 Micelles, 44 confo~ations,96 critical concentration (cmc),96 definition, 96 deoxycholate, 157-158 dye solubilization,97-98 electron microscopy, 97 inverted, 99.100 Qafft point, 96 Michael addition, 141, 144-146,428, 501 Microscopy Brewster angle, 115-116 fatty acids, l 15-1 16 fluorescence, 115-1 16 fluorescence microscopy,115-1 16 Mineral corticoid, 134 Mineral oils, 266 Minor groove, 404 Mismatch, 154,490-491 Model compounds, 3 Molecular assemblies, see synkinesis Molecular wires, 455 Molisch test, 303 Molybdenum porphyrinates, 296 Monensine, 123-124,127-128 Monoclonal antibody, 5 19 Monoformyl porphyrin, 299 Monolayer coating manganite, 110 DNA, 110 Monolayers cadmium arachidate, 114 carotenoids, 264 DNA derivatives,461-462 saccharides, 206214,237-241 porphyrins, 23-24 pyrroles, 23 steroids, 21, 140-147

Monte Carlocalculations,525 Montmorillonite, 115 Mouthfeel, 79,91 m-RNA, 441 Muramic acid, 236 Muscle model, 479-480 Muscles, 466 Mutagenic, 437 Mutagens, 430-431 Mutarotation, 181 Myoglobin, 513-5 14 Myristic acid, 102 NADPH, 516-S 17 N-al~ylpyridiumium~phiphiles,69 Nandrolon decanoate, 134 Natural Moisturing Factor (NMF), 77, 79 Neighbor effects, 28 Neocarcinostatin, 456 Netropsin, 448-449 Newman projection, 474 Newman strain, 474 N-Glycosides, 432-434 hydrolysis, 432 Nicotinamide, 357-360 Nitrite, 430 Nitrogen oxides, 91 Nitrogen retention, 135 NMR-spectra, 17-20,56 anomeric effect, 181-183 p-carotene, 247 bilirubin, 280 bleomycin-DNA, 446-447 13C, 268 carbohydrates, 172-173, 183 chlorophyll, 276-278 coupling constant, 20 dimers, 56-57 distamycin-DNA, 446-448 DNA triple helix, 439 DNA-Pt complex, 457-458 dynamic, 269 ferricyanide protoporphyrin, 273 Karplus curve, 19-20 labeled amino acids, 481

[NNIR-spectra] 15N, 268 nuclear Overhauser effect(NOE), 17 nucleic acids, 397 nucleotide, 414,416417 oxygen, 254,265 phosphate, 411415,454 porphyrins, 272,276 protoporphyrin E, 273 py~omethane,268 py~omethene,268 ring current effect, 57 shift reagent, 109 solid state fibers, 230 solid state, 58, 172-173 solution, 17-20 tautomers, 269 testosterone, 18 thiamine, 382 tocopherol, 341 triple helix, 439 N-octyl-D-g~acton~ide, 235 N-octyl-~-gluconamide,172, 174, 175, 230 N-octyl-D-gulonamide, 172, 174 N-octyl-D-mannon~de,235 NOE (nuclear Overhausereffects), 170, 277,446 phytochrome, 277 Noodles, 245 Norgestrel, 134 Nuclear Overhauser effect (NOE), 446 Nucleic acids, 517, see DNA Nucleic base pair,98 Nucleic bases, 428 reactivity, 428433 hydrophobized, 442 Nucleotides, 433437, see DNA pKa-values, 433 Octaethylformyl-biliverdinate,282 Octyl gluconamide, 172, 174, 175,230 Octyl gulonarnide, 172,174 Odd eveneffect, 119-120 Oils, 61, 81 Oleamide, 68

Oleic acid, 91-92 Olestra, 77,79 Oligoamines, 386 Oligoglycine, 520 Oligosacch~desyntheses,209 Open-chain carbohydratederivatives, 169 Orbital degeneracy, 275 Organogels, 230 Osmium, 453 Oxodipo~hodimethenes,298-299 Oxoniaporphyrin, 306-308 Oxygen evolution, 353-354 Oxygen, 34-35,89,306,361,362,369 180xygen, 214,414,434 Oxygenation, 306 Ozonolysis, 148 n-cation radicals, 276, 294 P-ketoester, 275 electron transfer, 308 enolate anion, 303 isocyclic ring, 303 Uvlvis, 274 n,x* transitions, 13 PAGE, 411414,438,442,450 Palindromic sequences,4 10 Palmitic acid, 505 Paraquat, 361 Patch clamp, 125 PCR (polymerase chainreaction), 425 Pectins, 199 PEG, 127,174,228,314,368,523 Penant~oline, 458459 Penicillin, 490 Pentoses, 201 Perchlorate, 69 permeability, 3 14-3 15 Peroxides, 370 Phenylketone, 498 Phenol, 30,397,467 Phenylalanine, 470,498 Phenylboronic acid,5 15 Pheophorbide, 303 Pheophytin a, 303 Phlorin, 289,295

Phosphate, 397 chirality, 397 carboxylate comparison, 399 pKa, 403 Phosphite, 398-399,421-422 Phospholipases, 92 Phosphonates, 118-1 19 Phosphor~dite,42 , 1423-424 Phosphorescence, 305 Phosphorothioates, 441 Phospho~enta~ulfide, 43 1-432 Phospho~hioates,420 Photocurrents, 455 P h o t o d y n ~ therapy, c 455 Photoequilibrium, 277 Photofrin, 307 Photoisomerizations, 267 Photolithography, 487-488 Photolysis CO-Cbond, 390 Photooxygenation, 290,256 Photosterols, 130 Photosynthesis, 265-266,33 1-336 Phytochrome, 277 Phytol, 345 Piperazinium palmitate, 65 Pitzer strain, 474 pKa, amino acids,491 phenol, 30 porphyrins, 27 table, 32-33 water, 30 Plant gums, 199-201 Plasmid DNA, 397 Pockels trough,49 Poly(dA), 423 Poly(lysine), 502 Poly(~,N-dimethylacrylamide) gels, 42 1 Poly(viny1 pyrrolidone),117 Polyallyl~ine,523 Polydextrose, 204 Polyene cyclizations, 141, 143 Polyene spectrum, 274 Polyet~yleneglycol(PEG), 127, 174, 228,314,368,523

Polyguanidinium DNA, 422 Polyisobutene, 117 Polymerase chain. reaction(PCR), 425 Polyquinone, 341-344 Polyunsaturated fatty acids, 62 Pores, 121-128 stopper, 126, 128 Porphyrexid radical, 36 1 Porphyrins, 97,454 acetylenic, 309 5, 15-dialkylidene,299 5, 15-dioxo,299 amino, 3 15-3 16 anions, 311 anion radicals, 295 antimon, 295 p-octaacetic acid, 287 P-substituted, 286ff books, 266 capped, 284 carotenoids, 3 10 cation radicals, 296 cation radical dimer,3 11 charge transfer,3 1 1 charge separation, 309,310,314 charge separation in micelles,99 chemical shift, 12 covalent, 284 covalent dimers, 299-301 deprotonation, 293 dichloro, 299-300 diradicals, 305 electronic, 266 ESR, 274 exciton effects, 3 15-3 16 fibers, 3 14-3 16 fluorescence, 305-306 fomyl, 299-300 glycosylated, 3 15-3 16 guanine, 3 14 heterodimers, 3 11-3 14 high spin complexes, 274 homodimers, 3 11-3 12 in micelles, 97-98 integral, 12 iron, 274

[Porphyrins~ iron(III), 272 lateral assemblies, 3 15-3 16 lateral dimer, 287 ligand field effects, 274 line width, 12 low spin complexes, 274 manganese, 274,296 meso-tetrahydro, 298 meso-tetraoxo, 298 metallation, 293 ~olydenum,296 NNIR, 274 noncomlent dimers, 3 10-312 oligomers, 284ff overcrowded, 286ff oxidation, 293 oxonia, 306 oxy, 306 photodyna~ctherapy, 455456 photoreduction, 308 pol~zability,27 1 protonation, 293ff Q-bands, 271 radicals, 293 redox potenti~s,297-298 reduction, 293 reversible reactions, 293f reviews, 266 ruffling, 27 1 ruthenium, 306 silicon, 305 solubility, 279ff stacked dimer, 287 stacking assemblies,3 15 structure, 266 syntheses, 283ff tin(IV), 295,308, 315 zinc, 296 Po~hyrinogens, 283,298 otassiu~palmitate, 65 otassiu~R- delat ate, 162

Primer, 425

Progesterone, 134, 136, 142 Proline, 470 Propenyl ester, 495 Protecting groups, 216-224,484,486 Proteins, 463-525 carbohy~ates,509-5 I 2 carotenes, 5 12 catalysis, 500 channels, 123 combinatori~,464 denaturation,524-525 DNA interaction,409 in inverted micelles, 99-100 hobs, 477-478 lipids, 505-508 name, 463 nucleic acids, 517 pleated shut, 474-477 porphyrins, 5 13-516 proteins, 5 17-524 rejection by PEG, 523-524 repulsive interactions,473-474 secondary structures, 473-480 shape, 478479 side-chain ~angement,480 surface, 523 synthesis, 48349 1 triple helix, 465,478-479 turns, 477 urea, 524525 vitamins, 5 16 a-helix, 47-77 Proton shuttle, 351 Protoporphyrin IX, 271,273,287, 310, 314,515 Protosterol, 143 Pseudocontact shifts, 272 Pseudoephe~ne,482 Psicose, 208 Pullulan, 201 Pulse radiolysis, 295,437 Purines, 411 P y r ~ o s e - f ~ a n o s178-188 e, Pyridoxal, 385-387 Pyri~idines,4 1 1 ~ropheophorbide,303

I

Pyrrole, 269-27 1 electronic structure, 266-268 Pyrrolenine unit, 267ff Pyrroline ring, 275 Pyrromethane, 267 Pyruvic acid, 382 ~ u a ~ phelices, l e 230, 232,234,442 Quadruple peptidehelices, 5 13-5 IS Quinhydrone, 42 ~uinones42,94,337-353 spectra, 339-340,347 redox potential, 340 ~uinoproteins,342 R- 12-Hydroxy-stearicacid, 7 1 Rabbit, 245 Radicals, 34-35 P.500 Rancid butter, 88 Reactions, 2, 214 amidation, 26 amino acids, 491-500 bromination, 258-259 c~bohydrates,214-224 condensations, 24-28 dehydrogenation, 86-87 dimerization, 92 enamines, 26-27 fatty acids, 86-94 hydrolysis, 24-28 hydroxylation, 86-89 lactone formation, 28 oxidation, 260 pathway, 26 neighbor effects, 28 Recombinant bacteria, 489 Redox potentials, 30-3 1, 359 bacte~ochlorophyll,297 chlorins, 297 flavins, 366-368 porphyrins, 297 table, 31,359 Reliability index, 398 Remote oxidation, 88-89, 148 Remote photoreduction, 153

Renewable resources, 3 Repulsive 1,3-syn diaxial effect, 176 Repulsive interactions, 172,473-474 Respiration, 265 Restriction-rescue procedure, 426 Retinal, 243-244,5 12 Retinoic acid, 256 Retinol, 254 Retro D-melittin, 507 Retro-Claisen condensation, 303 Retroenantio melittin, 507 Reversed-phase chromatography, 397 mamnose, 197 Rhenium complex, 152 Riboflavin, 365 Ribonucleic acid, 397,418,419,440 antisense, 41 9-42 1 isolation, 397 L-enantiomer, 438 photoche~calcleavage, 456 Ribose, 184, 186,400402,405 Ring current, 272 RNA (ribonucleic acid), 397,418,419, 440,456 antisense, 4 19-421 L-enantiomer, 438 photochemical cleavage, 456 RNA polymerase,400,418 RNA sequences, 428 Robinson annelation, 21, 144-146 Roll, 405 Ruthenium, 362-363,453,458 SAM (self assembly of membranes), 45, 50, 113,524 SAMP, 500 S~sasapogenin,133 Scanning force microscopy(SFM),49 Scanning near fieldoptical microscopy (SNOM), 5 1-52 Schadt-Helfric~cell, 160-161 Scheibe complex,55-56 Scleroglucan, 240 Sebum, 67,77,79 Self organization, 36 Self-assembly (SAM),45,50, 113,524

Self-replicat~ngsystem, 442-445 Semiquinone, 148,339-341,355 Sense strand, 419 Sequencing gels, 411 Serine, 498-499 Shampoos, 99 Sharpless epoxidation, 205 Shift reagents, 109 Side-arm formation,4 10 Sigmasterol, 133 Silicon chips, 2 Silver, 429 Single-sitemutants, 491 Sitosterol, 133, 142 Skin, 466 Slide, 405 sn ~omenclature, 73,75 SN2-type reactions phosphate, 399 SNOM (scanning nearfieldoptical microscopy), 52 Soap curds, 102 Solid phase synthesis, 483-484 Solid state synthesis, 487-489 Solid-phase DNA syntheses,421 Solubility amino acids, 469 arabinogalactan, 197 c ~ b o h y ~ a t169,172,193-204 es fatty acids, 63-64 PEG, 176 proline, 11 polyquinones, 343 porphyrins, 279-28 starch, 195 Sorbose, 202 Soret band, 270,271,274,279. 296 Spec~ophotometrictitration, 3 13 S p e ~ d i n e386 , Spermine, 386,460 Sphingosine, 71-72, 115 Spin states, 274 Squalene epoxide. 143 Stacking, 403 Starch, 195-196,203 Starch-iodide-iodine complex, 195-196

Sterane, 129 Stereochemical strain, 288 Steric forces, 43 Steric repulsion, 279 Steroid-receptor, 138 Steroids, 129-166,508-509 4-ene-3-ones, 136 acetylene derivatives, 135 all0 series, 130 onium steroid, 162-163 binding model, 137 biosynthesis, 143 conformers, 135-139 degradation, 142 fluor derivatives, 135 function, 136 hormones table, 134 imprinting, 153-154 membrane gap, 165 monolayers, 163-165 nomenclature, 129-130 normal series, 130 photochemistry, 148-151,155-156 point charge, 137 price, 140 reactivity, 147-156 redox reaction, 148-151 resources, 132 rhenium complexes,152-1 53 seco, 139 separation, 158 SN2 reaction, 148-149 stereoselectivereduction, 153-154 synkinesis, 156-166 synthesis, 140-147 tumors, 148, 151 Sticky ends, 418,419 Sticky terminals, 489 Stigmasterols, 140, 142 Strabolen, 134 Strecker synthesis, 492-493 Streptavidin,506 Subtilisin,486 Sucrose, 189, 191 Sulfoxide, 372 Sulfoximines, 495-496

Sun light, 275 Surface monolayers, 113-1 2 l Surface potentialdist~butionof steroids, 137 Surface pressure isotherm, 46, 117 Surface proteins, 523 Sweat, 67 Sweeteners, 226-227 Syn, 7 ~ynclinal,6’7 ~ y n ~ n e s i2,41, s , 35-59, 259,437 amino acids, 501-504 barbital, 4 1 cell membranes, 94-95 charge fitting, 40 definition, 35-37 ~ ~ A - p o l y ~ i d445-452 es, eye, 36 gels, 36 hydrogen bonding,40-4 1 hydrophobic effect, 37-38 membr~es,38-40 micellar fibers, 101-102, 229-238 micelles, 35, 96-101 porphyrin dimers, 40 porphyrins 35 proteins lipids, 505-508 steroids, 508-509 carbohy~ates,509-5 12 cafotenes, 512-5 13 porphyrins, 513-5 16 vitamins, 516-5 17 nucleic acids, 517 proteins, 517-524 urea, 524-525 proteins, 504-525 quinone dimers,42 retinene complexes,261 -262 shape fitting, 39-40 steroids, 156-166 surface forces in, 43-44 urea, 39 vesicles, 94 ~ e m b r ~ e505 s,

Synthesis, 2,20-24 biotechnological protein, 48949 1 alkanes, 21 alkenes, 21 biotechnological, 48949 1 carotenoids, 256-258 co~binatorial, 426,487489,522 heterocycles, 25-26 lipids, 81-86 monosacch~des, 204208 nucleoside, 429-430 nucleotides, 417-428 oligosaccharides, 209-214 peptides 464-49 1 porphyrins, 21,23-24,283-292 solid state, 421424,483-489 steroids, 20,22 Tags, 488 Talose, 208 Tautomers, 31, 33-34,267,269,397 carbohydrates, 206-208 Telopeptides, 478 Tendons, 466 Testosterone, 134, 136 Te~aalkylammoniumamphiphiles, 69 Te~acyanoquinomethane,347 Tetraether bolaamphiphil~s,94 Te~apy~dinium (-tetraethylporphyrin, 287 Thallium tri~uoroacetate,299 Thermal energy,4-5 T ~ e ~ o p h i lbacteria, ic 7l The~otropicliquid crystals, 229 Thiamine, 382-385 Thiazolidinocarbo~ylicacids, 499-500 Thiocarbonic acid, 93-94 Thioesters, 38 1 T~ophosphonate~, 416 Thymine, 398 Tips derivatives,444 Tocopherol, 339-343 Torgov reaction, 144, 147 Toroids, 239,460 Torsion angle, 473 Transc~ption,418

Transition state, 520-523 Translation, 41 8,421 Trehalose, 189, 191 T~chloroacetimidates,209,2 11-213 Trichloroa~ne, 495496 Trifluoromethanesulfonate,21 1 Trimethylsil~eethers, 429 Tripartite model, 227 Triple helix, 438440,501 Triplet state, 310 t-RNA, 398,404-405 T~ptophan,470 Tubular stack, 504 Tumor phototherapy, 307 Turns, 477 Twisted proteinribbon, 505 Tyrosine, 353,498,470 ~ i q u i n o n e339-343 , Umpolung, 383 Uracil, 398 Uranyl acetate, 460 Urea inclusion compounds,112-1 13 Urea, 39,524-525 Uroporphy~n,287 Uvlvis spectra, 12-1 5 acetylenic porphyrins309-3 10 16-annulene, 27 benzene, 27 canthaxanthin, 263 carotene radical, 253 carotenes, 244,247 characteristics, 12 charge transfer complexes, 3 11 chlorophylls, 275 chromatic, 13-14 cross-conjugation, 15 disulfide, 372 excitation, 53-54 extinction coefficient, 12-13 flavin, 366-367 formyl biliverdinate, 307 H-stack, 55 indigo, 13 isosbestic points, 52-53,307 J-assembly, 55

[UV/vis spectra] Job plot, 53 leukoindigo, 262 line width, 12 magnesium octaethylporphyrin, 270 metalloporphyrins, 27 1 NAD+, 358-360 NADH, 358-360 nucleotides, 403 polyene, 13-14 porphyrin dimers, 287 porphyrin heterodimers, 13 3 porphyrin radicals, 296 porphyrin radical dimers, 11 3 porphyrins, 271,279 pyridoxal, 385-386 retinal, 249 Soret band, 274 TPP dication, 293,295 tritylium, 421 viologen, 361-363 wavelength, 12 UV-damaged DNA, 369 Van der Waalsforces, 43 Van der Waalsinteractions,405 Varnish, 88 Vegetable oils, 62 Vesicles, 45,50, 102, 103-110,314 aggregation number, 102 diameter, 104 en~apment, 104105,314 formation, 105 fusion, 107 mass, 105 model, 104, 106 monolayer, 107-1 10 paradox, 106 thickness, 102 unsymetric, 108-109 water in, 35-36 Vesicular fibers, 45,110-1 13,229-238 amino acids, 503 carbohydrates, 229-234 porphyrins, 314-3 16 Vinyl group, 299

Viologen, 337,360-364 Viscoelasticity, 102 Vitamins 337-381,516-517 A, 246249-252, B,, 382-385 B,, 365-372 BI2,387-393 Biotin, 374-377 C, 254,354-357 Dz,153 q , 139 E,89,254,339-34 Folic acid, 377-379 K,339-34 p y ~ d o ~ a385-387 l,

Washing process, 100-101 Water, 24,37-38,43, 165-166 Wires, 454 Wittig reaction, 20,204, 205-206,257 Wool, 466 X~thoporphyrinogens,298 Xylan, 198 Xylose, 201 Zinc fingers,5 17-5 18 Zinc formylbiliverdinate, 306 Zirconyl(1V) salts, 118-1 19 Zoosterols, 132 Zwitterion, 467