Dipeptide derivative synthesis catalyzed by Pseudomonas aeruginosa elastase

Abstract

Pseudomonas aeruginosa elastase was used to synthesize various N-protected dipeptide amides. The identity of the products was confirmed by FAB(+)-MS. After recrystallization, the yield of their synthesis was calculated, their purity was checked by RP-HPLC and their melting point was measured. With regard to the hydrolysis, it is well-established that the enzyme prefers hydrophobic amino acids in P'1 position and it has a wide specificity for the P1 position. This specificity was demonstrated to be quite unchanged when comparing the initial rates of peptide bond formation between different carboxyl donors (Z-aa) and nucleophiles (aa-NH2). The elastase, but not the thermolysin, was notably able to incorporate tyrosine and tryptophan in P'1 position. Furthermore, synthesis initial rates were at least 100 times faster with the elastase. To overcome the problematic condensation of some amino acids during chemical peptide synthesis, it has been previously suggested that enzymatic steps can combine with a chemical strategy. We demonstrated that the elastase readily synthesizes dipeptide derivatives containing various usual N-protecting groups. It was especially able to condense phenylalaninamide to Fmoc- and Boc-alanine. Increasing interest in peptides containing unnatural amino acids led us to try the elastase-catalyzed synthesis of Z-dipeptide amides including those amino acids in the P1 position. A synthesis was demonstrated with alphaAbu, Nle, Nva and Phg.

D- and Unnatural Amino Acid Substituted Antimicrobial Peptides With Improved Proteolytic Resistance and Their Proteolytic Degradation Characteristics

“…We speculate that the N-terminal Aib residue effectively protects this peptide from degradation by plasma proteases. As reported previously, Aib does not interact with the serine proteases elastase and thermolysin (Rival et al, 1999), and the introduction of Aib into a peptide backbone thus represents a useful strategy for enhancing its resistance toward proteases. Previous research has indicated that Aib tends to favor the formation of a helical conformation and this effect was reported to be more obvious when Aib is located in the inner region of the sequence (Conlon et al, 2007).…”

Effect of α,α-Dialkyl Amino Acids on the Protease Resistance of Peptides

“…The peptide synthesis was carried out by a stepwise solidphase method utilizing 9-‰uorenylmethoxycarbonyl chemistry. 9,10) Tryptic digestion assay. N-Tosyl-L-phenylalanyl chloromethyl ketone-treated trypsin (Worthington, Lakewood, NJ, U.S.A.) was used for the digestion assay.…”

“…In their enzymatic peptide synthesis studies, Rival et al have reported that Aib did not interact with these two proteases. 10) Apparently, these two proteases did not recognize the Aib residue in the peptide sequence. In the present study, we used tryp- sin, also one of the serine proteases, as a speciˆc endopeptidase for the Lys residues present in BKBA-20.…”

Strategic Single-Residue Substitution in the Antimicrobial Peptide Esc(1–21) Confers Activity against Staphylococcus aureus, Including Drug-Resistant and Biofilm Phenotype

“…Furthermore, stability data in serum revealed that ∼40% of the original amount of peptide 2 remained after 16 h from the addition of bovine serum (Figure ), highlighting a prolonged half-life compared to most gene-encoded AMPs including the same parent peptide 1 , the amount of which decreased to 21% after 5 h incubation with fresh human serum . This is likely due to the presence of the noncoded Aib that should protect the peptides from degradation by plasma proteases, by preventing their interaction with the serine proteases elastase and thermolysin . In support of this, Hirano and co-workers designed analogs of the Stripe peptide (21 residues long), carrying different α,α-disubstituted amino acids or side-chain stapling to stabilize their helical structures.…”