Formation of oligopeptides during the synthesis of 9‐fluorenylmethyloxycarbonyl amino acid derivatives

We wish to report a serious side reaction which occurs during the preparation of 9-fluorenylmethyloxycarbonyl (Fmoc) amino acid derivatives 1.Because of their base lability, Fmoc-amino acids are potentially amongst the most versatile intermediates for peptide synthesis, especially when used in conjunction with acid-labile sidechain protecting groups.lS2 1 However, when these compounds are prepared by standard pro~edures,~.3we have found that the majority of Fmoc-amino acids are contaminated by variable but often substantial amounts of Fmoc-oligopeptides.We have established this by HPLC, amino acid analysis, nmr, and the synthesis of a number of standards.The Fmoc-oligopeptides are likely formed via the intermediacy of relatively stable mixed anhydrides formed during Fmoc-chloride-mediated synthesis of the Fmoc-amino acid derivatives.'T3The oligopeptide contaminants were first noted during routine TLC analyses to assess the homogeneity of commercial samples of Fmoc-amino acids prior to their use.While the literature TLC systems' (e.g.ch1oroform:methanol:acetic acid, 9:l:0.1)did not necessarily separate the contaminants in all cases, we were able to demonstrate reproducible separations using the TLC system to1uene:acetic acid, 10:l.The impurities were invariably slower-moving in this TLC system, were strongly uv-absorbing, chlorine-tolidine4 positive, and also difficult or impossible to remove completely by recrystallization techniques.This suggested that the impurities were both Fmoc-blocked and amino-acid containing.One of the most severely contaminated amino acid derivatives, Fmoc-glycine, was chosen for careful, in-depth study.When Fmoc-glycine was synthesized by the standard literature procedure,'13 using Fmoc-chloride in dioxane/aqueous sodium carbonate, the product was found by TLC to be contaminated by a major (10-20%), slower-moving contaminant.Fractional crystallization using ethyl acetate/hexane provided a sample that was