Peptiligase, an Enzyme for Efficient Chemoenzymatic Peptide Synthesis and Cyclization in Water

Abstract

We describe a novel, organic cosolventstable and cation-independent engineered enzyme for peptide coupling reactions. The enzyme is a variant of a stable calcium-independent mutant of subtilisin BPN', with the catalytic Ser212 mutated to Cys and Pro216 converted to Ala. The enzyme, called peptiligase, catalyzes exceptionally efficient peptide coupling in water with a surprisingly high synthesis over hydrolysis (S/H) ratio. The S/H ratio of the peptide ligation reaction is correlated to the length of the peptide substrate and proved to be > 100 for the synthesis of a 13-mer peptide, which corresponds to > 99% conversion to the ligated peptide product and < 1% hydrolytic side-reaction. Furthermore, peptiligase does not require a particular recognition motif resulting in a broadly applicable and traceless peptide ligation technology. Peptiligase is very robust, easy to produce in Bacillus subtilis, and its purification is straightforward. It shows good activity and stability in the presence of organic cosolvents and chelating or denaturing agents, enabling the ligation of poorly soluble (hydrophobic) or folded peptides. This enzyme could be useful for the (industrial) synthesis of diverse (pharmaceutical) peptides. In addition, peptiligase is able to efficiently catalyze headto-tail peptide cyclization reactions.

Methodologies for Backbone Macrocyclic Peptide Synthesis Compatible With Screening Technologies

“…However, the yield of this reaction is not high, with it giving more or less 60% due to hydrolytic side reactions. Recently, a novel and robust subtilisinbased variant termed peptiligase was developed by introducing the same Ser-to-Cys and Pro-to-Ala mutations into a calciumindependent and stable variant of substilisin BPN' (Toplak et al, 2016). Peptiligase is easily accessible through recombinant expression from Bacillus subtilis.…”

“…polar amino acids are preferred at the P1, P4 position, and proline should be avoided at the P1 ′ , P2 ′ position) allows it to be used for footprint-free backbone cyclization, enabling a ligation reaction of unprotected peptides at ambient temperature with high yield in a short time (up to 90% in <1 h) in aqueous media with neutral to slightly basic pH. Additionally, organic co-solvent media (e.g., up to 50 vol% of DMF and DMSO) and denaturing agents (e.g., 2M urea or guanidium chloride) are tolerated in the ligation reaction, allowing poorly soluble peptides to be cyclized (Toplak et al, 2016). Omiligase-1 has a high catalytic efficiency (<0.0003 molar equivalents of enzyme required) and is commercially available.…”

A Thiolactone Strategy for Straightforward Synthesis of Disulfide‐Linked Side‐Chain‐to‐Tail Cyclic Peptides Featuring an N‐Terminal Modification Handle

“…Cyclisation of peptides is currently one of the major strategies to improve the pharmacokinetics of peptide drugs. Although various approaches for peptide cyclisation have been developed, including on‐resin amide‐bond formation, lactonisation, copper‐catalysed alkyne–azide cycloaddition (CuAAC), ring‐closing metathesis, native chemical ligation, or even enzymatic cyclisation, the formation of cyclic disulfides is an easy and straightforward way to reduce conformation flexibility of peptides . Though unstable in the intracellular reductive environment, the disulfide bond remains intact during screening assays and upon binding extracellular targets.…”

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.…”