Amide-forming chemical ligation via O -acyl hydroxamic acids

Abstract

The facile rearrangement of "-acyl isopeptides" to native peptide bonds via ,-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, "-acyl isopeptides" derived from hydroxy-asparagine [aspartic acid-β-hydroxamic acid; Asp(β-HA)] rearrange to form native peptide bonds via an ,-acyl shift. This process constitutes a rare example of an ,-acyl shift that proceeds rapidly across a medium-size ring (t ∼ 15 min), and takes place in water with minimal interference from hydrolysis. In contrast to serine/threonine or tyrosine, which form -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-acyl isopeptides by treatment with an unprotected peptide-thioester, at low mM concentration, in water. These findings were applied to an acyl transfer-based chemical ligation strategy, in which an unprotected -terminal Asp(β-HA)-peptide and peptide-thioester react under aqueous conditions to give a ligation product ultimately linked by a native peptide bond.

Nγ‐Hydroxyasparagine: A Multifunctional Unnatural Amino Acid That is a Good P1 Substrate of Asparaginyl Peptide Ligases

“…For comparison studies, Asp‐RTD1 18 was obtained by oxidation of Asn(OH)‐RTD1 17 with NaIO 4 [14] (1 equiv) in 20 mM PBS (pH 7.2) at 0 °C, which quickly converted Asn(OH) to Asp in 5–10 min. Previously, Pentelute and co‐workers showed that NaNO 2 oxidation can also be used to convert Asn(OH) to Asp [9c] . Asn‐RTD1 19 was prepared in the same manner as Asn(OH)‐RTD1 17 by cyclization at the Asn residue using butelase‐1.…”

Solubilizing Trityl‐Type Tag To Synthesize Asx/Glx‐Containing Peptides

“…In this case, the formation of side products resulting from the internal cyclization of the peptides, such as aspartimide‐ or β‐Asp‐containing peptides, would be assumed to be more likely than that in the case of Glu(Dbz)‐containing peptides. In general, the synthesis of the desired Asp/Asn‐based peptides is expected to be more challenging than that of the synthesis of their Glu/Gln‐based counterparts, because aspartimide contains a five‐membered ring structure. Fortunately, under the same conditions as those used for the synthesis of Glu/Gln‐containing peptides, the Asp/Asn‐containing peptides were obtained without significant amounts of side products, such as aspartimide/β‐Asp(Asn)/D‐Asp(Asn), respectively (Figure and the Supporting Information).…”

The Application of Isoacyl Structural Motifs in Prodrug Design and Peptide Chemistry

“…In 2018, the group of Pentelute reported O ‐acyl‐Asn isopeptide mediated ligation. The ester bond was constructed on the side chain of hydroxyl‐Asn (aspartic acid–β‐hydroxamic acid) by capturing the peptide thioester with the hydroxamic acid . Subsequent O‐to‐N acyl transfer mediated by a seven‐membered cyclic intermediate produced a peptide bond.…”

“…The ester bond was constructed on the side chain of hydroxyl-Asn (aspartic acid-b-hydroxamica cid) by capturing the peptidet hioester with the hydroxamic acid. [54] Subsequent O-to-N acyl transfer mediated by as evenmembered cyclic intermediate produced ap eptideb ond. The hydroxyl-Asn residue of the ligatedp roduct could be reduced to asparagine by zinc/ascorbic acid or oxidized by sodium nitrite to aspartica cid,t hus realizing at raceless ligation (Scheme 3F).…”