Ene and Epoxide Impurities in Fluorenylmethoxycarbonyl (Fmoc) α-Substituted Amino Acids: Causes of Formation, Reactivity, and Means of Minimization

Carpino’s invention of fluorenylmethoxycarbonyl (Fmoc) as a base-labile amino protecting group (PG) ( J. Am. Chem. Soc . 1970, 92, 5748) has transformed peptide chemistry in a manner so profound that only Merrifield’s solid-phase peptide synthesis ( J. Am. Chem. Soc . 1963, 85, 2149) does not pale in its comparison. In fact, Fmoc is the most important PG in peptide chemistry with no viable alternatives in sight, enabling the assembly of a staggering variety of peptides including blockbuster therapeutics. Here, we report that α-Fmoc amino acids (Fmoc-AA-OHs) are susceptible to undergo formation of impurities differing from parent compounds by −2 and +14 Da, respectively. Based on tandem mass spectrometry (MSMS) analyses, we propose that these byproducts are ene and epoxide alterations of the Fmoc group, situated adjacent to the fluorene core of Fmoc. The generality of these Fmoc-ene/-epoxide byproducts was exemplified by detecting them in five Fmoc-AA-OHs by liquid chromatography high-resolution mass spectrometry (LC-HRMS), while based on the dependence of the rate of Fmoc-ene/-epoxide formation on temperature, air, and iron, we propose storage and handling protocols aimed at keeping the content of ene/epoxide Fmoc alterations to a minimum. Finally, we report that Fmoc-ene/-epoxide alterations are stable toward the coupling additive ethyl cyanohydroxyiminoacetate (Oxyma) and the Fmoc removal base piperidine, whereas exposure to the standard cleavage reagent trifluoroacetic acid (TFA) results in a breakdown of the Fmoc-ene/-epoxide moieties. Notably, exposure to TFA in the presence of triisopropylsilane (TIS)/H 2 O/dithiothreitol (DTT) as scavengers leaves the Fmoc-ene alteration unscathed, whereas Fmoc-epoxide is ring-opened to the corresponding alcohol, suggesting that whether Fmoc-ene/-epoxide peptide truncations will remain unaltered will depend on synthesis and cleavage protocols used. As peptide drugs are subject to stringent quality requirements, varying the content of Fmoc-ene/-epoxide impurities coupled with synthesis/cleavage protocols impacting Fmoc alterations in a varying manner may pose challenges during downstream processing, warranting that due care is paid to the formation and reactivity of Fmoc-ene/-epoxide alterations.