Amide-forming chemical ligation via O-acyl hydroxamic acids

Abstract

The facile rearrangement of "S-acyl isopeptides" to native peptide bonds via S,N-acyl shift is central to the success of native chemical ligation, the widely used approach for protein total synthesis. Proximity-driven amide bond formation via acyl transfer reactions in other contexts has proven generally less effective. Here, we show that under neutral aqueous conditions, "O-acyl isopeptides" derived from hydroxy-asparagine [aspartic acid-β-hydroxamic acid; Asp(β-HA)] rearrange to form native peptide bonds via an O,N-acyl shift. This process constitutes a rare example of an O,N-acyl shift that proceeds rapidly across a medium-size ring (t_1/2 ∼ 15 min), and takes place in water with minimal interference from hydrolysis. In contrast to serine/threonine or tyrosine, which form O-acyl isopeptides only by the use of highly activated acyl donors and appropriate protecting groups in organic solvent, Asp(β-HA) is sufficiently reactive to form O-acyl isopeptides by treatment with an unprotected peptide-^αthioester, at low mM concentration, in water. These findings were applied to an acyl transfer-based chemical ligation strategy, in which an unprotected N-terminal Asp(β-HA)-peptide and peptide-^αthioester react under aqueous conditions to give a ligation product ultimately linked by a native peptide bond.

Keywords: O-acyl hydroxamic acid; acyl shift; acyl transfer; chemical ligation; hydroxamic acid.

Fig. 1.

An O-acyl hydroxamic acid isopeptide derived from Asp(β-HA) rearranges to a native peptide bond in aqueous buffer. (A) Reaction scheme. (B) First-order rate constants for O-acyl isopeptide rearrangement as a function of pH, obtained at each of two isopeptide concentrations. Rate constants were derived from fits of LC-MS peak areas to an integrated rate expression, as described in SI Appendix, section 5.

Fig. 2.

Reaction of a peptide-^αthioester with an N-terminal Asp(β-HA)-peptide yields a peptide-^αO-acyl hydroxamic acid isopeptide under mild aqueous conditions. Reaction scheme and LC-MS data showing conversion of thioester 2 to O-acyl hydroxamic acid isopeptide 1, by treatment with N-terminal Asp(β-HA)-peptide 3, at pH 9.

Fig. 3.

Amide-forming chemical ligation of a peptide-^αthioester and N-terminal Asp(β-HA)-peptide, at pH 7. The MS inset for the 2-h timepoint corresponds to isopeptide 1; the MS inset for the 20-h timepoint corresponds to amide product 1′. An isolated yield of 61% was obtained for 1′ after isolation by preparative HPLC.

Fig. 4.

Conversion of an Asp(β-HA) residue to either Asn or Asp. (A) Reaction schemes. (B) LC-MS analysis of starting material and crude reaction mixtures, for a representative substrate.

Fig. 5.

Synthesis of a 58-mer peptide by Asp(β-HA)-mediated chemical ligation. Thioester VDNKFNKEQQ¹⁰NAFYEILHLP²⁰NA-^αCO-S-C₆H₄-CH₂-CO₂H 8 and hydroxamic acid D(β-HA)EEQRNAF³⁰IQSLKDDPSQ⁴⁰SANILLAEAKK⁵⁰LNDAQAPK⁵⁸-^αCONH₂ 9 were first combined at pH 8 for 30 min, to generate isopeptide 10. Then, the reaction mixture was acidified to pH 6; formation of amide product 10′ was confirmed after 4 h. The MS insets correspond to isopeptide 10 (30-min timepoint) and amide product 10′ (4-h timepoint), respectively. Product 10′ was obtained in 49% yield after isolation by preparative HPLC.