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Über dieses Buch

A continued interest in Peptide Chemistry prompted the revision of the first edition of this book. This provided an opportunity to update several details. I am grateful to colleagues who were kind enough to inform me of errors, typographical and other, they had discovered in the first edi­ tion. These have now been corrected, as were certain shortcomings in language and style pointed out by my daughter, Dr. Eva Bodanszky. In 1991 the excellent The Chemical Synthesis of Peptides by John Jones (Oxford University Press, 1991) appeared. It covers, in part, the same field, but is different enough from Peptide Chemistry, to justify publication of a revised edition of the latter. Princeton, July 1993 M. Bodanszky Preface to the First Edition Nature applied peptides for a great variety of specific functions. The specificity provided by the individual character of each amino acid is further ehanced by the combination of several amino acids into larger molecules. Peptides therefore can act as chemical messengers, neuro­ transmitters, as highly specific stimulators and inhibitors, regulating var­ ious life-processes. Entire classes of biologically active compounds, such as the opioid peptides or the gastrointestinal hormones emerged within short periods of time and it is unlikely that the rapid succession of discoveries of important new peptides would come to a sudden halt. In fact, our knowledge of the field is probably still in an early stage of development. Peptides also gained importance in our everyday life.

Inhaltsverzeichnis

Frontmatter

Introduction

I. Introduction

Abstract
The importance of proteins, substances responsible for primary functions in the living cell, need not be stressed any more. We may pay tribute to the nomenclator who from the Greek protos (first) or proteios (primary) coined the word protein. This foresight or intuition, usually attributed to Jac. Berzelius, remains vindicated in spite of the most impressive progress in nucleic acid chemistry and the emergence of DNA as the carrier of genetic information and RNA as template in protein biosynthesis. Nucleic acids provide the blueprint for the construction of complex machinery, the machines themselves are proteins. In fact, information encoded in DNA-s and in RNA-s is operative only in the presence of enzymes, that is proteins. It is clear therefore, that protein chemistry is one of the most important chapters of biochemistry, and can even stand by itself as the subject of a textbook. It is perhaps less obvious why peptide chemistry should be treated separately. The term “peptide” (from pepsis = digestion or peptones = digestion products of proteins) denotes relatively small compounds which are quite similar to proteins except that the latter are substances of higher molecular weight. The reasons for this distinction are not self evident. There is no distinct borderline between the two groups of materials; molecules built of 100 or more amino acid residues are usually regarded as proteins and those containing a lesser number of residues as peptides.
Miklos Bodanszky

Structure Determination

Frontmatter

II. Amino Acid Analysis

Abstract
The most revealing piece of information that can readily be obtained about a peptide is its amino acid composition. Yet, the results of amino acid analysis are really meaningful only if the sample consists of a single peptide. Analysis of mixtures is usually an unrewarding effort. Thus, purification should precede analysis, and this generalization is valid for most other methods of structure determination as well. Homogeneity as a prerequisite of analysis cannot be overemphasized. Purification is sometimes possible by crystallization but in most cases chromatography, electrophoresis or countercurrent distribution or a combination of these techniques is needed.
Miklos Bodanszky

III. Sequence Determination

Abstract
The order or sequence of individual amino acid residues along the peptide chain defines the covalent structure of the molecule. It is also called primary structure in order to make a clear distinction from the three dimensional geometry in peptides and proteins. The latter is generated by non-covalent forces such as hydrogen bonds between amide groups (secondary structure) and combination of polar and non-polar interactions and of disulfide bridges which result in chain folding (tertiary structure).
Miklos Bodanszky

IV. Secondary and Tertiary Structure

Abstract
The bond between the carbon atom of the carbonyl group and the amide nitrogen has partial double bond character that can be attributed to resonance:
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Accordingly, the carbonyl carbon and oxygen atoms, the amide nitrogen and hydrogen and the two adjacent α-carbon atoms He approximately in the same plane. Experimental evidence, mainly from x-ray crystallography, supports the near coplanarity of these six atoms in peptide backbones and also shows a relatively short distance between the carbonyl carbon and the nitrogen. The carbonyl oxygen and the amide hydrogen are on opposite sides of the (partial) double bond, at least in most peptide bonds. The cis arrangement is somewhat less stable and, since there is a considerable energy barrier between the cis and trans forms, the latter generally prevails.
Miklos Bodanszky

Peptide Synthesis

Frontmatter

V. Formation of the Peptide Bond

Abstract
In order to convert carboxylic acids into acylating agents their hydroxyl group must be replaced by an electron-withdrawing substituent (X) to enhance the polarization of the carbonyl group and thereby the electrophilicity of its carbon atom. Thus the nucleophilic attack by the amino group (of the amino acid to be acylated) is greatly facilitated:
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Miklos Bodanszky

VI. Protection of Functional Groups

Abstract
While it is necessary to mask the amino function of an amino acid or peptide during activation of the carboxyl group which participates in the subsequent coupling, it is equally important to select protecting groups that can readily be removed. Otherwise the integrity of the already formed peptide bonds would be endangered. If the peptide obtained in a coupling reaction has to be lengthened at its N-terminus, then an additional requirement limits the choice of the amine-blocking group: it must be removable under conditions which leave the masking of the carboxyl group and the protection of side chain functions intact. Thus, blocking of the α-amino function is generally transient while that of all other functions mostly semipermanent.
Miklos Bodanszky

VII. Undesired Reactions During Synthesis

Abstract
It might be nigh impossible to reduce peptide synthesis to a mere routine, because the intended transformations, such as blocking, activation, coupling and removal of protecting groups, are accompanied by numerous undesired reactions. Therefore, instead of a single product often a mixture of peptides is obtained and the target compound must be secured through purification, which may require several steps and sometimes tedious, time consuming operations. Consequently, the final yield on homogeneous material can be rather disappointing. Thus, it is absolutely necessary to recognize and to understand such undesired reactions, to anticipate and, when possible, to prevent them. Here we can discuss only those side reactions which are repeatedly encountered and reported. An important problem, racemization during synthesis, will be treated in a separate chapter.
Miklos Bodanszky

VIII. Racemization

Abstract
With the exception of glycine, in all amino acids that are constituents of proteins, the α-carbon atom is chiral. In threonine and isoleucine a chiral center is present in the side chain as well. In order to secure the target peptide in homogeneous form it is absolutely essential to start from enantiomerically pure amino acids and to insist on conservation of chiral homogeneity throughout the various operations of synthesis. Otherwise, instead of a single product, a mixture of stereoisomers will be obtained. Their number in a peptide with n chiral centers is 2n. Accordingly, if racemization is not prevented, even in the synthesis of a moderately large peptide a complex mixture will be produced and separation of the desired material from a multitude of similar compounds might turn out to be an at least arduous and sometimes overwhelming task. Therefore, the importance of racemization studies and of the measures that must be taken for the prevention of any loss in chiral purity can not be overemphasized. In fact, “strategies” of peptide synthesis, that is general planning of schemes for syntheses (Chapter IX) are dictated primarily by considerations concerning conservation of chiral homogeneity.
Miklos Bodanszky

IX. Design of Schemes for Peptide Synthesis

Abstract
In the strategical planning that must precede the synthesis of a larger peptide, racemization is one of the most important considerations. Therefore, it seems to be appropriate to discuss the various schemes of synthesis at this point. Due to the individuality of amino acid residues and to variations in the properties of blocked intermediates, it appears impractical to propose a general scheme (strategy) that would be applicable for any peptide. Peptide synthesis should be based on retrosynthetic analysis, starting with identification of the problems inherent in the sequence of the target compound.
Miklos Bodanszky

X. Solid Phase Peptide Synthesis

Abstract
By the conventional methods of organic synthesis, preparation of peptides containing more than just a few amino acids is an arduous task. Introduction of blocking groups, coupling reactions and deprotection steps entail a large number of operations, such as washing the reaction mixtures neutral after coupling, precipitation or crystallization of intermediates, collecting solid products by filtration or centrifugation followed by drying, etc. Thus, synthesis of peptide chains containing dozens of residues requires an almost heroic effort and proteins, even small ones, can be made by tour de force but certainly not routinely. The need for facilitation of the process was obvious for some time. The stepwise strategy, demonstrated in a novel synthesis of oxytocin (Bodanszky and du Vigneaud 1959) was, because of the repetitiveness of the operations, conducive to experimentation with techniques suitable for mechanization and automation of chain building. Attachment of the (N-blocked) C-terminal residue to an insoluble polymeric support (a “resin”), followed by deprotection and acylation of the exposed amino group with the penultimate residue and continuation of the procedure by similar cycles of deprotection and incorporation, absolve the practitioner from handling filters and separatory funnels, from washing and drying intermediates etc. Excess starting material and reagents, as well as byproducts of the reactions, are eliminated simply by washing the peptidyl polymer with appropriately selected solvents.
Miklos Bodanszky

XI. Methods of Facilitation

Abstract
Solid phase peptide synthesis is the only widely practiced technique of facilitation, but several other methods have also been proposed for the rapid construction of long peptide chains. Some of these alternative approaches have certain advantages over solid phase synthesis: reactions carried out in solution are not affected by the rate-limiting control of the gel. Also, where isolation of intermediates is possible, these can be analyzed and purified. A major disadvantage, however, common to the various techniques discussed below, is that they are less conducive to mechanization and automation than the Merrifield method.
Miklos Bodanszky

XII. Analysis and Characterization of Synthetic Peptides

Abstract
The molecular weight of most synthetic organic compounds is well below 1000 daltons. The products of peptide synthesis, however, frequently have molecular weights that exceed this number. Calculating with an average residue weight of 115, even a nonapeptide’s molecular weight is more than 1000 and a further increase is caused by the acids (e.g. acetic acid) associated with cations in the side chain of basic residues. In protected intermediates blocking groups contribute in a major way to the size of the molecule. While high molecular weight in itself could be the cause of some difficulties in analysis, the problem is compounded by the similar elemental composition of the amino acid constituents. Thus, incorporation of an additional residue might lead to only minor changes in the values of elemental analysis of an intermediate, or in its physical properties. Moreover, the by-products formed in various operations of synthesis are often quite similar in their composition to the desired peptide derivatives. These complexities obviously increase the need for thorough scrutiny of synthetic peptides, if possible by a battery of tests. In the following paragraphs we will attempt to point out some well established methods of analysis that are useful in this area.
Miklos Bodanszky

Backmatter

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