Comparison of methods for the Fmoc solid‐phase synthesis and cleavage of a peptide containing both tryptophan and arginine

Abstract

A major side reaction which can occur during the synthesis of Trp‐containing peptides is modification of the Trp indole by reactive carbonium ion species released during acidolytic cleavage. [Asn²,Trp⁴]Dynorphin A‐(1–13), a sequence which is very susceptible to Trp modification, was chosen as a model peptide to compare the effectiveness of various methods proposed to minimize Trp modification during Fmoc solid‐phase synthesis. The peptide was synthesized with the side chain of Trp unprotected and cleaved by Reagent K [82.5% trifluoroacetic acid (TFA)/5% phenol/5% water/5% thioanisole/2.5 % ethanedithiol (EDT)] [King, D.S. et al. (1990) Int. J. Peptide Protein Res.36, 255–2661, Reagent R [90% TFA/5 % thioanisole/3% EDT/2% anisole] [Albericio, F. et al. (1990) J. Org. Chem.55, 3730–3743], TFA containing 20% EDT and 4% water [Riniker, B. & Hartmann, A. (1990) in Peptides: Chemistry, Structure, and Biology (Rivier, J.E. & Marshall, G.R., eds.), pp. 950–952, Escom, Leiden], and TFA containing trialkylsilane, MeOH, and ethylmethyl sulfide [Chan, W.C. & Bycroft, B.W. (1992) in Peptides: Chemistry, Structure, and Biology, Op. cit., pp. 613–614]. Cleavage with Reagent K, Reagent R and TFA containing 20% EDT and 4% water yielded similar results; in addition to the desired peptide, the crude product contained 22–30% of a side product which appeared to result from Trp modification by a Pmc group. Cleavage with the triakylsilane‐containing mixture gave the lowest recovery of the desired peptide and the highest levels of Pmc‐containing peptides. In contrast, synthesis of the peptide by Fmoc solid‐phase synthesis utilizing Fmoc‐Trp(Boc) and subsequent cleavage with TFA containing 20% EDT and 5% water yielded the desired peptide in essentially pure form with < 5% of the Pmc‐containing side product. Thus, in the Fmoc solid‐phase synthesis of [Asn²,Trp⁴]dynorphin A‐(1–13) protection of the indole nitrogen by Boc was the most effective method for suppressing the modification of Trp by Pmc. This demonstrates the potential for improving the yield and purity of peptides containing both Trp and Arg by utilizing Fmoc‐Trp(Boc) during the Fmoc solid‐phase synthesis of these peptides.