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Across a concentration range of 10 to 200 mM of MPAA, NCLs proceeded at similar reaction rates. Although the intramolecular N-S acyl transfer of SEAlide peptides under neutral conditions was not observed in the absence of thiols, addition of a large excess amount of MPAA (100 equiv) to the solution of 1a in Gn{dot operator}HCl [6M, Na phosphate (0.2m), TCEP (30 mm), pH 7.4] induced the formation of the corresponding MPAA thioester in up to about 10% conversion ratio after 6 h.
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Replacement of MPAA (an arylthiol) with sulfanylethylsulfonic acid (an alkylthiol) induced side reactions in the presence of TCEP, including desulfurization at cysteine and SEAlide moieties. See: Q. Wan, S. J. Danishefsky, Angew. Chem. 2007, 119, 9408-9412; Angew. Chem. Int. Ed. 2007, 46, 9248-9252; and references cited herein.
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Note
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Although we have yet to elucidate the role of phosphate ions in the ligation, one potential explanation for the effect of phosphate is that the salt efficiently catalyzes proton transfer to accelerate the N-S acyl transfer, possibly involving a cyclic intermediate as shown below.
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45
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Note
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The N-S acyl transfer under acidic conditions (4M HCl in DMF) of a chiral amino acid-incorporated SEAlide peptide such as 1b is accompanied by partial racemization; however, the N-S acyl transfer followed by NCL under neutral conditions affords no racemization (Figure S7 in the Supporting Information).
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