One‐Step C‐Terminal Deprotection and Activation of Peptides with Peptide Amidase from Stenotrophomonas maltophilia in Neat Organic Solvent

Abstract

Chemoenzymatic peptide synthesis is a rapidly developing technology for cost effective peptide production on a large scale. As an alternative to the traditional C!N strategy, which employs expensive N-protected building blocks in each step, we have investigated an N!C extension route that is based on activation of a peptide C-terminal amide protecting group to the corresponding methyl ester. We found that this conversion is efficiently catalysed by Stenotrophomonas maltophilia peptide amidase in neat organic media. The system excludes the possibility of internal peptide cleavage as the enzyme lacks intrinsic protease activity. The produced peptide methyl ester was used for peptide chain extension in a kinetically controlled reaction by a thermostable protease.

Versatile Peptide C-Terminal Functionalization via a Computationally Engineered Peptide Amidase

“…The catalytic mechanism involves formation of a covalent acyl (peptidyl)–enzyme intermediate, and we anticipated that it would allow alternative reactions if water can be replaced by other nucleophiles in the hydrolytic half-reaction or if the reaction is reversed, which might be facilitated by performing conversions under anhydrous conditions. The use of PAM for one-step peptide C-terminal amide–ester interconversion has been reported . However, the expansion of PAM-catalyzed reactions to broader peptide functionalization was restricted, because PAM has modest stability and low resistance to organic solvents while many modification reactions, e.g., functionalization of bioavailable peptide free acids, require a nonaqueous environment.…”

“…The use of PAM for one-step peptide C-terminal amide−ester interconversion has been reported. 15 However, the expansion of PAM-catalyzed reactions to broader peptide functionalization was restricted, because PAM has modest stability and low resistance to organic solvents while many modification reactions, e.g., functionalization of bioavailable peptide free acids, require a nonaqueous environment.…”

Traceless enzymatic protein synthesis without ligation sites constraint

“…Establishing compatibility between peptide-activating and peptide-coupling enzymes At the outset of our studies, we searched for a broadly applicable enzyme for C-terminal peptide esteri cation, to provide accessible reactive handles for Peptiligase. Accordingly, we explored the peptide amidase (PAM) from Stenotrophomonas maltophilia, which affords sequence-independent C-terminal peptide modi cation with absolute regioselectivity 24 . Using computational redesign, we signi cantly expanded the synthetic utility of PAM [25][26] .…”

Chemoenzymatic synthesis of polypeptides in neat 1,1,1,2-tetrafluoroethane solvent

“…Recently, the bioengineering of microbial proteases, amidases, and peptiligases has emerged in the sought for efficient peptide synthesis, macrocyclization and segment condensation in water. [9][10][11][12][13][14] Other approaches include the stability of these enzymes in neat organic solvents or co-solvent systems, thus improving substrates and products solubilities. 15 Additionally, it is worth mentioning recent advances on transpeptidation and macrocyclization of peptides by sortase and butelase ligase enzymes, although these require specic segment recognition.…”