Detection and prevention of urethane acylation during solid phase peptide synthesis by anhydride methods

Studies in solid‐phase peptide synthesis: A personal perspective

“…The presence of H-(Gly 4 )-OH could result from amino acid insertion as postulated by Brenner and/or a ''double insertion'' reaction observed with glycine derivatives under certain conditions of SPPS. 13 At any rate, the simple model system showed that amino acid insertions, though possible, are not significant side reactions under the usual conditions of SPPS. These early results were later substantiated when a sensitive mass spectrometric technique that showed no insertion peptides could be detected (<0.03%) in a 21-residue peptide prepared by SPPS.…”

“…The mechanism for this side reaction involves disproportionation of the mixed anhydride of BpocGly-OH to the symmetrical anhydride, and intramolecular rearrangement of the latter to form N-Bpoc-N a -(Bpoc-Gly)-Gly-OH, which is subsequently activated by anhydride interchange and reacts with Val-resin to yield N-Bpoc-N a -(BpocGly)-Gly-Val-resin. 13 Rearrangement is dependent on the temperature and time of mixed anhydride formation and is undetectable after activation at À158C (10 min) and coupling at À158C (2 h). No urethane acylation (<0.1 mol %) was observed during coupling of Bpoc-Gly-OH with Valresin when DCC was used under standard SPPS conditions.…”

“…Glycine was employed since insertion reactions should be most favored in the absence of bulky side chains. Large excesses (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) equiv) of Boc-Gly-OH and DCC were used to promote acylation of the peptide bond. An amino acid analyzer calibrated with H-(Gly n )-OH (where n ¼ 1 through 4) was used to detect the products obtained from the acidolytic cleavages (HBr-TFA) of peptide resin products 1 and 2.…”

“…The same finding was made in this study when it was shown that Bpoc-Gly-Val-resin was not acylated at the urethane nitrogen by symmetrical or mixed anhydrides of BpocGly, by Bpoc-Gly activated with dicyclohexylcarbodiimide, or by leucine-N-carboxyanhydride. 13 It was determined that urethane acylation occurred before the activated intermediate (Bpoc-Gly-OCO 2 Et) was coupled to the H-Val-resin in the original synthesis. The mechanism for this side reaction involves disproportionation of the mixed anhydride of BpocGly-OH to the symmetrical anhydride, and intramolecular rearrangement of the latter to form N-Bpoc-N a -(Bpoc-Gly)-Gly-OH, which is subsequently activated by anhydride interchange and reacts with Val-resin to yield N-Bpoc-N a -(BpocGly)-Gly-Val-resin.…”

“…The model peptide Leu-Ala-Gly-Val is used as a chromatographic system that separates all possible deletion and termination peptides from the parent peptide at a 0.1% detection level is available. 13 …”