Derivatized Amino Acids Relevant to Native Peptide Synthesis by Chemical Ligation and Acyl Transfer

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 (t1/2 ∼ 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.

Download Full-text

Peptide Bond Synthesis by a Mechanism Involving an Enzymatic Reaction and a Subsequent Chemical Reaction

Journal of Biological Chemistry ◽

10.1074/jbc.m115.700989 ◽

2015 ◽

Vol 291 (4) ◽

pp. 1735-1750 ◽

Cited By ~ 10

Author(s):

Tomoko Abe ◽

Yoshiteru Hashimoto ◽

Ye Zhuang ◽

Yin Ge ◽

Takuto Kumano ◽

...

Keyword(s):

Peptide Synthesis ◽

Chemical Reaction ◽

Enzymatic Reaction ◽

Amide Bond ◽

Acyl Transfer ◽

Native Chemical Ligation ◽

Specific Substrate ◽

Chemical Ligation ◽

Subsequent Chemical Reaction ◽

Subsequent Chemical

We recently reported that an amide bond is unexpectedly formed by an acyl-CoA synthetase (which catalyzes the formation of a carbon-sulfur bond) when a suitable acid and l-cysteine are used as substrates. DltA, which is homologous to the adenylation domain of nonribosomal peptide synthetase, belongs to the same superfamily of adenylate-forming enzymes, which includes many kinds of enzymes, including the acyl-CoA synthetases. Here, we demonstrate that DltA synthesizes not only N-(d-alanyl)-l-cysteine (a dipeptide) but also various oligopeptides. We propose that this enzyme catalyzes peptide synthesis by the following unprecedented mechanism: (i) the formation of S-acyl-l-cysteine as an intermediate via its “enzymatic activity” and (ii) subsequent “chemical” S → N acyl transfer in the intermediate, resulting in peptide formation. Step ii is identical to the corresponding reaction in native chemical ligation, a method of chemical peptide synthesis, whereas step i is not. To the best of our knowledge, our discovery of this peptide synthesis mechanism involving an enzymatic reaction and a subsequent chemical reaction is the first such one to be reported. This new process yields peptides without the use of a thioesterified fragment, which is required in native chemical ligation. Together with these findings, the same mechanism-dependent formation of N-acyl compounds by other members of the above-mentioned superfamily demonstrated that all members most likely form peptide/amide compounds by using this novel mechanism. Each member enzyme acts on a specific substrate; thus, not only the corresponding peptides but also new types of amide compounds can be formed.

Download Full-text

Going Round in Circles with N→S Acyl Transfer

Synlett ◽

10.1055/s-0036-1588789 ◽

2017 ◽

Vol 28 (13) ◽

pp. 1517-1529 ◽

Cited By ~ 4

Author(s):

Derek Macmillan

Keyword(s):

Transfer Reaction ◽

Peptide Sequence ◽

Acyl Transfer ◽

Native Chemical Ligation ◽

Full Potential ◽

Chemical Ligation ◽

Peptide Engineering ◽

Naturally Occurring ◽

Native Peptide ◽

Peptide Thioesters

It is not highly sophisticated, yet the N→S acyl transfer reaction of a native peptide sequence potentially fills an important technology gap. While several routes to synthetic peptide thioesters exist, only one is routinely applicable for biologically derived samples. Using the naturally occurring amino acid cysteine as the sole activator for N→S acyl transfer we have demonstrated transformation of synthetic and biologically derived precursors into thioesters for use in Native Chemical Ligation, providing a viable alternative for biological samples. Further refinement will be key to realising the full potential of this intriguing process, and increase the number of applications in peptide engineering and therapeutics.1 Introduction2 N→S acyl transfer in ‘normal’ peptide sequences3 Reduced reactivity of internal Xaa-Cys motifs as an advantage in head-to-tail peptide cyclisation4 Reduced reactivity of internal Xaa-Cys motifs as an advantage in modification and cyclisation of biologically produced precursors5 Hydrazinolysis of Xaa-Cys motifs and the acyl hydrazide as a stable thioester equivalent6 Rapid thioester formation via an N→Se acyl shift7 Outlook and conclusions

Download Full-text