One-Pot In Vitro Ribosomal Synthesis of Macrocyclic Depsipeptides

Abstract

Here, we report a method for the one-pot ribosomal synthesis of macrocyclic depsipeptides. This method is based on a Ser-Pro-Cys-Gly (SPCG) motif discovered by in vitro selection of peptides for the function of self-acylation in the presence of a thioester acyl donor, which forms an O-acyl isopeptide bond via intramolecular S-to-O acyl transfer. Ribosomal synthesis of linear peptides containing the SPCG motif and a backbone "acyl donor" thioester at a downstream position results in spontaneous conversion to the corresponding cyclic depsipeptides (CDPs) in a nearly independent manner of ring size and sequence context. Mutational analysis of the SPCG motif revealed that the P and G residues are dispensable to some extent, but the arrangement of residues in SXCX is crucial for efficient acyl transfer, e.g., CPSG is much less efficient. Finally, one-pot ribosomal synthesis of macrocyclic depsipeptides with various ring sizes and sequences has been demonstrated. This synthetic method can facilitate the ribosomal construction of highly diverse CDP libraries for the discovery of de novo bioactive CDPs.

Inner residues of macrothiolactone in autoinducer peptides I/IV circumvent spontaneous S-to-O acyl transfer to the upstream serine residue

“…Dethioacylation. As previously reported, 11 5 µL of in vitro translated AIP-Ia analogs, 0.55 µL of 100 mM cysteine were added and incubated for 40 min at 25 ˚C. The reaction mixture was desalted and analyzed by MALDI-TOF MS.…”

“…In vitro expression of AIP-Ia and its analogs by the Trp-deficient FIT system A 5 mL in vitro translation was prepared by mixing a DNA template, 0.5 mM Trp-deficient 19 amino acids mix, 50 mM of pre-loaded HS Phe 4Cl -tRNA Pro1E2 CCA , and in-house PURE system 29 and incubated at 37 1C for 30 min. 11 The crude reaction mixture was desalted using solid-phase extraction (SPE) C-tip (Nikkyo Technos) and eluted with 80% acetonitrile, 0.5% acetic acid, half saturated with the matrix (R)-cyano-4-hydroxycinnamic acid (Bruker), and spotted on a MALDI Ground Steel 384 Target plate. MALDI-TOF-MS measurement was performed under linear or reflector positive mode using an UltrafleXtreme (Bruker) with external calibration (Peptide Calibration Standard II, Bruker).…”

“…10 We have recently discovered that when a SPCG (Ser-Pro-Cys-Gly) motif in peptides is S-acylated on the Cys's thiol side chain upon the incubation with a thioacyl donor molecule, and their acyl group spontaneously transfers to the Ser's hydroxyl side-chain via S-to-O acyl transfer in a site-specific manner. 11,12 This unprecedented chemistry has led us to in vitro ribosomal synthesis of side-chain-to-tail cyclized depsipeptide (CDP) in one-pot, where a nascent linear peptide with a combination of a SPCG motif and a backbone thioacyl donor at the downstream macrothiolactonizes to form the corresponding CTP. This CTP spontaneously undergoes macrolactonization via S-to-O acyl transfer, yielding the corresponding CDP (Fig.…”

“…Potassium adduct ( §), DTT adduct (2), and Cys adduct (3) were also observed. 2 and 3 are attributed to an in vitro translation system (see ref 11)…”

Site-specific nonenzymatic peptide S/O–glutamylation reveals the extent of substrate promiscuity in glutamate elimination domains

“…1c). A recent report from our laboratory 42 describes site-selective S-to-O acyl transfer to synthesize various Ser(O-acyl) peptides, including cyclodepsipeptides. Upon treating a peptide containing the reactive motif, which can be as short as Ser-Xaa-Cys, with a thioester at ambient temperature and pH, a Cys(S-acyl) intermediate forms, which further yields Ser(O-acyl)containing peptides upon a spontaneous S-to-O acyl transfer.…”