Traceless synthesis of protein thioesters using enzyme-mediated hydrazinolysis and subsequent self-editing of the cysteinyl prolyl sequence

Abstract

Thioester-producing protocol featuring carboxypeptidase Y (CPaseY)-mediated hydrazinolysis and subsequent self-editing of tag affords protein thioesters in a traceless manner.

Greening the synthesis of peptide therapeutics: an industrial perspective

“…124,[142][143][144][145] Such C-terminal tags have been reduced to minimal sequences as Komiya et al recently developed an elegant method of hydrazinolysis of peptides bearing a cleavable C-terminal three amino acid motif (Cys-Pro-Leu) using biocatalysis and DKP formation with commercially available carboxypeptidase Y. 146 Similarly, Qiao et al used the cyanide donor 2-nitro-5-cyanatobenzoic acid (NTCB) to activate Cys residues on C-terminally tagged expressed proteins, with subsequent hydrazinolysis to generated peptide hydrazides, in a process denoted activated cysteine-based protein ligation (ACPL). 147 Such advances in C-terminal activation strategies are interesting from a manufacturing perspective as they appear to facilitate access to peptide thioesters from recombinant sources on large scale.…”

“…Potential sources for peptide thioester generation include (i) hydrazides, 165,166 (ii) N-acylureas, 139,140 (iii) bis(2-sulfanylethyl)amido (SEA) amides, 141,167 (iv) peptides fused with intein proteins, 145,168 and (v) peptides with C-terminal enzymatic recognition motifs or tags. 146,147,169 (B) Protein trans-splicing involves peptides fused with split intein sequences that self-assemble to form an active intein unit. The intein is then autocatalytically excised while ligating the peptides of interest.…”

Sequence-Independent Traceless Method for Preparation of Peptide/Protein Thioesters Using CPaseY-Mediated Hydrazinolysis

“…Substrate 9 with a varying number of CP sequences (n = 1, 2 or 3) was treated with CPaseY under previously optimized reaction conditions. 16) A hydrophobic Phe (F) or a hydrophilic Lys (K) was introduced at the X position. The time course of the conversion of 9 to 10 is summarized in Chart 3B.…”

“…does not leave extra sequences is thus required. [11][12][13][14][15][16] We recently developed a traceless preparation of peptide/protein thioesters by the use of carboxypeptidase Y (CPaseY) (EC 3.4.16.5), 16) which is a commercially available serine protease, easily isolated from baker's yeast (Chart 1A). 17,18) Treatment of a substrate 1 possessing a C-terminal Cys-Pro-Leu-OH (CPL) sequence with CPaseY in the presence of hydrazine and some additives gives the hydrazinolysis product 2 instead of a hydrolyzed product.…”

“…11 and 16 using CPaseY Typical procedure: As described in the previous paper,16) peptide 9 (0.050 μmol) in 50 μL aqueous solution with 0.1 μM CPaseY, 0.2 M H 2 NNH 2 •H 2 O and 60 mM cyclohexanone was incubated at 25 °C (pH 6.4). Before HPLC analysis, an aqueous solution of 6 M Gn•HCl, 200 mM MeONH 2 •HCl and 200 mM tris(2-carboxyethyl)-phosphine (TCEP)•HCl was added twice to the reaction mixture, and the resulting mixture was incubated at 37 °C for 10 min.…”