Protease‐catalyzed peptide synthesis using inverse substrates: The synthesis of Pro‐Xaa‐bonds by trypsin

Abstract

Benzyloxycarbonyl-L-proline p-guanidinophenyl ester is an "inverse substrate" for trypsin; i.e., the cationic center is included in the leaving group instead of being in the acyl moiety. This substrate can be used in trypsin-catalyzed acyl-transfer reactions leading to the synthesis of Pro-Xaa peptide bonds. The reaction proceeds about 20 times slower than reaction with similar alanine-containing substrates, but the ratio between synthesis and hydrolysis is more favorable. The investigation of a series of nucleophiles led to information about the specificity of the process. Nucleophiles differing only in the P(1)'-position show an increasing acyl transfer efficiency in the order Phe < Gly < Ley < Ser < Ala < lle. C terminal elongation of the nucleophiles is of minor influence on their efficiency. The formation of an H bond between the acyl-enzyme and the nucleophile seems to play an important role in the aminolysis of the acyl-enzyme.

Application of Inverse Substrates to Trypsin-Catalyzed Peptide Synthesis

“…Calculation of the partition constants was carried out for our case. The values were determined to be 8.6-5 mM for the typical examples in Table 3 (for cases which afford 70-80% yield), and these values were 10-times smaller than those of Schellenberger et al (4); i.e., ours are preferable for peptide synthesis. This is the reason why in our case a low concentration of acyl acceptor (one-third of that used by Schellenberger et al) is applied to the coupling.…”

“…Noting the characteristic feature of inverse substrate Schellenberger et al (4) have tested the applicability of p-guanidinophenyl ester as tool for trypsin-catalyzed peptide synthesis. The paper prompted us to report the results on the general use of our inverse substrate, p-amidinophenyl esters, to tryptic peptide synthesis in detail.…”

6.11 N-Acylation Reactions of Amines

“…273 The broad specificity profile of many proteases means that they may also be used to catalyze the N-acylation of natural and unnatural amino acids with activated ester donors to afford peptide isosteres. 274,275 Proteases with broad specificity profiles such as clostripain 276 have been employed for the synthesis of nonpeptidic carboxylic acid amides from the coupling of simple primary amines with activated acyl donors. 277 For example, chymotrypsin was used to couple unprotected L-Tyr and L-Arg together to prepare the potent analgesic Kyotorphin (L-Tyr-L-Arg) on a kilogram scale, 278 whereas industrial processes have also been developed that enable thermolysin to catalyze stereoselective coupling of the L-enantiomer of (rac)-phenylglycine methyl ester with N-Cbz-L-Asp to afford N-carbobenzoxy-L-Asp-L-Phe-OMe as a precursor to the sweetener aspartame.…”

How C&#945;-Fluoroalkyl Amino Acids and Peptides Interact with Enzymes:Studies Concerning the Influence on Proteolytic Stability, Enzymatic Resolution and Peptide Coupling

“…The advantage of this methodology is that the 4-guanidinophenylester (OGp) functionality mediates the acceptance of non-specific amino acid moieties in the specificitydetermining S 1 position of Arg-specific proteases such as trypsin, thrombin and clostripain, as well as the Phe-specific protease α-chymotrypsin ( Fig. 9) [85][86][87][88][89][90]. The substrate mimetic concept has been shown to be a powerful strategy for the incorporation of sterically demanding C α,α -dialkyl amino acids into the P 1 position of peptides [91].…”