I Introduction

In the selection of examples of well established procedures a series of decisions had to be made in order to keep the material to be presented within practical limits. These decisions resulted in a seemingly distorted picture of peptide synthesis. The number of procedures for the introduction of protecting groups appears to be excessive or at least not commensurate with the number of methods applicable for their removal. Also, relatively few methods of coupling were rendered and still less coupling reagents. These choices, however, were made not without good reasons. There is indeed a definite need for a plethora of blocking groups. The simultaneous handling of numerous side chain functions in combination with various methods of coupling and with potential side reactions related to a particular sequence of amino acids is possible only if a whole gamut of masking groups is available. For the removal of protecting groups many proposals can be found in the literature, but a closer scrutiny reveals that most of these are variations on a few themes: reduction, e.g. hydrogenolysis, acidolysis and displacement by nucleophiles. A similar re­petitiveness can be discerned among coupling methods. Only exceptionally can one find methods for the formation of the peptide bond, which are not based on symmetrical or mixed anhydrides or on the aminolysis of reactive esters. Therefore, we confined ourselves mainly to the presentation of the few principal procedures. Coupling reagents deserve a special mention at this point. There seems to exist a certain fascination in connection with coupling reagents. The idea of adding some magic compound to the mixture of a carboxylic acid and an amine and thereby accomplishing the formation of an amide bond attracted many investigators. Yet, most coupling reagents simply cause the activation of a carboxyl group by converting it into an anhydride or an active ester. It is not obvious, however, why the carboxyl should be activated in the presence rather than in the absence of the amino component. Furthermore, a true coupling reagent should be completely inert toward amines. Unfortunately very few of the reactive materials proposed for this purpose have this necessary attribute. Carbodiimides stand out in this respect: the rate of their reaction with amines (to form guanidine derivatives) is usually negligible. This accounts for their undiminished popularity although more than a quarter of a century has passed since their introduction [1J in peptide synthesis. The use of carbodiimides, particularly in the presence of auxiliary nucleophiles [2J is, at this time, one of the most important approaches to

M. Bodanszky et al., The Practice of Peptide Synthesis© Springer-Verlag Berlin Heidelberg 1994

2 Introduction

peptide bond formation and had to be demonstrated in more than one example, but for the reasons just outlined, very few other coupling reagents are discussed.

So far no well-established process is available for the activation of the amino component and peptide bond formation is generally accomplished through reactive derivatives of carboxylic acids. This requires activation of the carboxyl group with more or less powerful reagents, such as alkyl chlorocarbonates, carbodiimides, etc. In more recent years the long standing desire [3J to avoid such aggressive chemicals in the preparation of peptides and to follow Nature in the use of enzyme-catalyzed reversible reactions led to the practical application of proteolytic enzymes for the formation of the peptide bond. Major progress has been made in this direction by the selection of suitable enzymes and pH ranges which are more favorable for the synthesis than for the hydrolysis of the amide bond. A most important contribution in this area is the addition of organic solvents [4J to the reaction mixture. To indicate the growing significance of this approach we include a few examples of enzyme­catalyzed syntheses and also some enzymatic methods of removal of protecting groups. Similarly, the semi synthesis of pep tides and proteins, the construction of large molecules from fragments of proteins, will be treated only briefly; a more detailed presentation would transcend the limits of this volume.

We had to give special consideration to techniques of facilitation such as the "handle" method [5J, to synthesis of pep tides attached to soluble polymers (or "liquid phase" peptide synthesis [6J), to the "in situ" technique [7J and particularly to the extremely popular method of solid phase peptide synthesis [8]. It seemed impractical to add examples of these promising and already very significant approaches in a number commensurate with the available literat­ure. Solid phase peptide synthesis itself requires a separate volume for proper presentation. The execution of solid phase synthesis has been rendered by Stewart and Young and an updated version of their book became available [9]. The extensive literature was assembled in review articles and books [10, 11J and also in a "user's guide" for the preparation of synthetic peptides by the solid-phase method [12]. We still deemed it necessary to include a few examples of syntheses in which techniques of facilitation were applied in the hope that the examples are sufficiently representative and will be found useful by the readers.

The word "technique" in connection with peptide synthesis calls to mind also some simple technical aspects of preparative work in organic synthesis. We mention here a seemingly trivial example, the separation of a solid intermediate from the solvent and from the by-products in solution. Only thoughtfully designed and carefully executed techniques of filtration provide an intermediate which can be used without purification in the next step of the chain building procedure. If the filter cake is not properly packed down, e.g. with the help of a sturdy glass rod with a flattened head, then it is not likely that

Introduction 3

the impurities are completely removed by displacement with a limited volume of the solvent Uudiciously) selected for washing. The authors apologize for this somewhat lengthy discussion of such minor points as a glass rod with a flattened head or a sinter-glass funnel. We are convinced, however, that the difference between success and failure in synthesis can hinge on such impon­derabilia. This is particularly true when in stepwise chain building poor solubility of an intermediate in the commonly used organic solvents (including dimethylformamide) prevents extensive purification by chromatography, elec­trophoresis or countercurrent distribution. Also, even when these efficient methods of purification are applicable, they are time consuming. If they are avoidable simply by washing of the intermediates with appropriate solvents, the tedium of synthesis is greatly reduced. For such reasons similar technical aspects are the subjects of comments throughout the book.

Other comments refer to health hazards. Operations with noxious materials such as liquid ammonia, liquid HF or HBr in acetic acid obviously demand a well ventilated hood. Enthusiasm about or dedication to our objectives can make us forgetful of the dangers surrounding our work. Therefore it may not be superfluous to add reminders which call the attention to some potential harm. This does not, however, absolve the researcher from the usual caution which belongs to the practice of organic chemistry, like the protection of the eyes, hands or protection of our colleagues in the laboratory.

In the ideal synthesis a single peptide is produced which requires no purification. Even if this objective is usually not achieved, every attempt has to be made to avoid the other extreme in which the target compound must be "fished out" from a mixture of numerous closely related peptides. Situations can (and not seldom did) arise where the peptidic material prepared by synthesis is an intractable mixture and the endeavor already progressed to a late stage has to be abandoned. The best way to prevent this is to select only methods which, at least in principle, give rise to a single product and to execute all operations in an unequivocal manner. Thus, the investigator must know the possible alternative pathways the reactants might follow. He must be well informed about possible side reactions. Reviews [13J on side reactions in peptide synthesis tried to provide some help in this respect, but the peptide chemist has to remember Murphy's law: whatever can go wrong, will. Such dangers, however, should not be considered deterrents: many already experi­enced side reactions notwithstanding, fairly complex peptides have been secured in high yield and in good quality by competent peptide chemists.

In addition to chemical factors which need to be considered in peptide synthesis, some of the physical properties of synthetic peptides can also cause difficulties. Peptides are often polyelectrolytes and if they have several cationic centers they can be adsorbed on glass, a polyanion. Losses of peptides were noted, particularly at high dilution, for instance in solutions used in pharmaco­logical assays. The hydrophobic regions in peptide chains can similarly lend

4 Introduction

themselves to adsorption, namely on polyethylene. A related and sometimes major problem is created by the self-association (aggregation) of peptides, the formation of insoluble particles and the consequent loss of valuable material. No simple remedies can be offered for these potential losses, but awareness should lead to solution of such problems; for instance, absorption on glass can be prevented by presaturation of the surface with polycations (e.g., serum albumin).

A few closing sentences must be dedicated to the analytical control of peptide synthesis. Because of the high molecular weight and complex structure of most biologically active pep tides, their analysis is more problematic than the analysis of many other products of organic synthesis. Thus, the execution of elemental analysis is hampered by the tendency of peptides to tenaciously retain water or solvents such as acetic acid. The ionic character of pep tides causes further complications in this respect: weak cationic centers, such as the nitroguanidino group or the imidazole nucleus, can remain associated with acids, e.g. trifluoroacetic acid used in preceding steps. In spite of such complications the practitioners of peptide synthesis should not abandon elemental analysis. Prolonged drying in good vacuum at elevated temperature might be necessary and the dried sample may require special handling, as if it were hygroscopic. The carefully prepared material, however, should give satisfactory values for the elements which constitute the peptide. Such a satisfactory analysis should be considered necessary but not sufficient evidence for the homogeneity of the synthetic product. Amino acid analysis of a carefully hydrolyzed sample will provide additional valuable information which can be further supplemented by u.v. spectra, if the peptide contains residues with chromophores in the side chain (tyrosine, tryptophan or nitro arginine). Infrared spectra are usually less informative, but in special cases, such as in peptides in which the tyrosine residue is esterified with sulfuric acid, the evidence provided by i.r. spectra is quite important. Infrared spectroscopy is more useful in the examination of starting materials, for instance active esters, anhydrides, insoluble polymeric supports. The value of nmr spectra cannot be overestimated, but again, they are less informative when used for large molecules. An important but not always available tool of analysis is the sequencing of intermediates or, even more importantly, of the final product. The synthetic intermediates can and, whenever possible, should be scrutinized on thin layer chromatograms and by high pressure liquid chromatography. The complexity of the products of peptide synthesis de­mands that not one analytical method should be used for their examination but as many as possible.

The authors attempted here to point out some of the more important factors which influence the outcome of an endeavor toward a synthetic peptide. Yet, like probably all human undertakings, peptide synthesis also requires some good luck, and this is what we wish our colleagues embarking on demanding ventures.

Introduction 5

1. Sheehan 1C, Hess GP (1955) 1 Am Chern Soc 77: 1067 2. Konig W, Geiger R (1970) Chern Ber 103: 1067 3. Waldschmidt-Leitz E, Kuhn K (1957) Chern Ber 84: 381 4. Homandberg M, Mattis lA, Laskowski M, 1r. (1978) Biochemistry 17: 5220 5. Camble R, Garner R, Young GT (1968) Nature 217: 247 6. Mutter M, Bayer E (1972) Nature 237: 512 7. Bodanszky M, Funk KW, Fink ML (1973) 1 Org Chern 38: 3565; Bodanszky M, Kondo M,

Yang-Lin C, Siegler GF (1974) ibid 39: 444 8. Merrifield RB (1963) 1 Amer Chern Soc 85: 2149 9. Stewart 1M, Young 1D (1984) Solid Phase Peptide Synthesis, Second Edition, Freeman, San

Francisco, CA 10. Birr C (1978) Aspects of the Merrifield Peptide Synthesis, Springer Verlag, Berlin,

Heidelberg New York 11. Atherton E, Sheppard RC (1989) Solid Phase Peptide Synthesis, A Practical Approach, IRL

Press at Oxford University Press, Oxford 12. Grant GA (1992) Synthetic Peptides. A User's Guide, Freeman, New York 13. (Former Ref 12) Bodanszky M, Martinez 1 (1983) In: Gross E, Meienhofer 1 (eds) The

Peptides, Academic Press, New York, Vol 5, pIlI