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In spite of the enormous progress in the synthesis of peptides and proteins using commercial peptide synthesizers and the immense technological possibilities of recombinant DNA technology, a C-N ligase is an indispensable tool for the racemization-free fragment condensation of peptides. Since activation of the C-terminal a-carboxyl group of a peptide segment could cause partial racemization, chemical condensations of peptide fragments are prone to racemization. For the synthesis of the huge number of peptides and proteins, however, nature has only developed the ribosomal peptidyltransferase, which exhibits its full catalytic function independent of the side-chain functions of the amino acids being coupled. However, its function requires coordination with numerous other ribosomal factors. Besides the limited possibilities of using multienzyme complexes of bacterial peptide synthesis systems, the only alternatives to peptidyltransferase are proteases, which, based on their in vivo function as hydrolases, cannot act as ideal ligases. However, by exploiting the intrinsic reversibility of hydrolytic reactions and by adjusting appropriate physicochemical reaction parameters, the protease acitivity can be used in the direction of ligation. Undoubtedly, the course of kinetically controlled, serine and cysteine protease-catalyzed reactions can be more efficiently influenced than the equilibrium-controlled protease-catalyzed synthesis. This article describes the influence of the enzyme specificity on the efficiency of kinetically controlled synthesis and points the way toward a broad exploitation of serine and cysteine proteases for the catalysis of C-N bond formation.

The stereospecific formation of peptide bonds under mild conditions and without side reactions is still a formidable task in peptide synthesis. One approach that springs to mind, namely the use of the naturally occurring catalyst involved in the biosynthesis of proteins, the ribosomal peptidyl transferase, cannot be realized in practice. The fact, however, that the natural cleavage of proteins is carried out by other enzymes, namely the proteases, together with the reversibility of these cleavage reactions in principle, has led to an interesting synthetic concept. Proteases normally catalyze the enzymatic degradation of proteins and peptides by hydrolytic cleavage of the peptide bond in an exergonic reaction. The use of physicochemical principles in order to influence the equilibrium, the concentration of products, and the kinetic parameters of the reaction results in the successful application of the catalytic properties of proteases to peptide synthesis. The purpose of this review is to describe and summarize the methods used in such approaches and to attempt a systematic categorization. The principles are applied to the synthesis of such practically relevant products as aspartame and human insulin.

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From the literature we collected all available quantitative data on the chymotrypsin-catalyzed hydrolysis of series of amino acid and peptide substrates. Utilizing this data base, we performed calculations on their quantitative structure/activity relationship (QSAR). The substrates were considered to be composed of fragments; log(k,,,/ K,) values for the substrates resulted from additive contributions of their fragments. Despite the fact that the kinetic constants in the data base were determined by different authors under various reaction conditions, the data are well described by the simple additivity model. Obviously, the intrinsic specificity of chymotrypsin dominates the influence of varying reaction conditions.Chymotrypsin is one of the most investigated enzymes [l]. Its three-dimensional structure has been elucidated [2]. There are hundreds of papers in the literature dealing with the kinetics of chymotrypsin-catalyzed hydrolysis reactions. In these publications different aspects of the enzyme specificity are investigated and the results are often difficult to compare. A systematic characterization of chymotrypsin specificity wouldCorrespondence to H.-D. Jakubke, Sektion Biowissenschaften der Universitlt Leipzig, Talstrasse 33, 0-7010 Leipzig, Federal Republic of Germany Abbreviations. Abu, 2-aminobutyric acid; Ac, acetyl; Ahe, 2-aminoheptanoic acid; An4Ac, 4-acetylanilide; An3C1, 3-chloroanilide; An4C1,4-chloroanilide; An4CN, 4-cyanoanilide; An4Me, 4-methylanilide; An3Me0, 3-methoxyanilide; An4Me0, 4-methoxyanilide; An4MeS02, 4-methylsulfonylanilide; An2N02, 2-nitroanilide; An3N02, 3-nitroanilide; Boc, tert-butyloxycarbonyl; Bz, benzoyl; OCH,CN, cyanomethyl ester; Cys(Bzl), S-benzylcysteine; Cys(Et), S-ethylcysteine; Dopa, 2,4-dihydroxyphenylalanine; FA, N -[3-(2-furyl)]acryloy1; For, formyl; Glt, glutaryl; H6Phe, hexahydrophenylalanine; Mal, maleyl; Mca, monochloracetyl; MeOSuc, methoxysuccinyl; Mes, methylsulfonyl; NH2, amide, Nic, nicotinyl; Nle, norleucine; Nva, norvaline; OBu', isobutyl ester; OBut, butyl ester; OCam, carboxamidomethyl ester; OEt, ethyl ester; OEtl RPh, (R)-( +)I-phenylethyl ester; OEtlSPh, (8-(-)1-phenylethyl ester; OMe, methyl ester; ONp, 4-nitrophenyl ester; ONb, 4-nitrobenzyl ester; OPh, phenyl ester; OPh4Ac, 4-acetylphenyl ester; OPh4C1, 4-chlorophenyl ester; OPh4F, 4-fluorophenylester; OPh4Me, 4-methylphenyl ester; OPh4Me0, 4-methoxyphenyl ester; OPh4NH2, 4-aminophenyl ester; OPh3N02, 3-nitrophenyl ester; OPr, propylester; OPri, isopropyl ester; 02RBu, (R)-( -)2-butyl ester; 02ROc, (R)-( -) 2-octyl ester; 02SBu, (9-( +)2-butyl ester; 02SOc, (q-( +)2-octyl ester; Om, ornithine; Phe4NH2, 4-aminophenylalanine; Phe4N02, 4-nitrophenylalanine; Phe20H, 2-hydroxyphenylalanine; Phe30H, 3-hydroxyphenylalanine; pNA, 4-nitroanilide; SBzl, thiobenzylester; SBzl4C1,4-~hlorobenzylthio ester; SEt, thioethyl ester; SPh4N02, 4-nitrophenylthioester ; SUC, succinyl; Tos, 4-toluenesulfonyl; Trp4For, 4-formyltryptophan; TrpNC, 2-(2-nitro-4-carboxyphenylsulfonyl)-tryptophan; Tyr3N02, 3-nitrotyrosine...