Protease‐catalyzed peptide synthesis using inverse substrates: The influence of reaction conditions on the trypsin acyl transfer efficiency

Abstract

Benzyloxycarbonyl-L-alanine p-guanidinophenyl ester behaves as a trypsin "inverse substrate," i.e., a cationic center is included in the leaving group instead of being in the acyl moiety. Using this substrate as an acyl donor, trypsin catalyzes the synthesis of peptide bonds that cannot be split by this enzyme. An optimal acyl transfer efficiency was achieved between pH 8 and 9 at 30 degrees C.The addition of as much as 50% cosolvent was shown to be of minor influence on the acyl transfer efficiency, whereas the reaction velocity decreases by more than one order of magnitude. The efficiency of H-Leu-NH(2) and H-Val-NH(2) in deacylation is almost the same for "inverse" and normal type substrates.

Nonconventional amide bond formation catalysis: programming enzyme specificity with substrate mimetics

“…acceptors (28,29) and later extended by further examples (30,31). Our own studies demonstrated that, in addition to single amino acid derivatives, the 4-guanidinophenyl ester functionality also mediates coupling of nonspecific acyl moieties derived from peptides (Table 2) (27).…”

How Cα-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].…”

Proteases as Powerful Catalysts for Organic Synthesis

“…However, the range of amino acids serving as substrates is narrow, productivities are lower, mainly because of the low activity and stability of proteases in nonconventional media, and no general protocols for synthesis have been developed as opposite to the case of SPPS [60,188]. The broad specificity of proteases restricts their application in peptide synthesis, since the peptide product that accumulates during the reaction can be attacked hydrolytically by the proteases simultaneously with the synthesis reaction [199]. The broad specificity of proteases restricts their application in peptide synthesis, since the peptide product that accumulates during the reaction can be attacked hydrolytically by the proteases simultaneously with the synthesis reaction [199].…”