Enhanced Epimerization of Glycosylated Amino Acids During Solid-Phase Peptide Synthesis

Abstract

Glycopeptides are extremely useful for basic research and clinical applications, but access to structurally-defined glycopeptides is limited by the difficulties in synthesizing this class of compounds. In this study, we demonstrate that many common peptide coupling conditions used to prepare O-linked glycopeptides result in substantial amounts of epimerization at the alpha position. In fact, epimerization resulted in up to 80% of the non-natural epimer, indicating that it can be the major product in some reactions. Through a series of mechanistic studies, we demonstrate that the enhanced epimerization relative to non-glycosylated amino acids is due to a combination of factors, including a faster rate of epimerization, an energetic preference for the unnatural epimer over the natural epimer, and a slower overall rate of peptide coupling. In addition, we demonstrate that use of 2,4,6-trimethylpyridine (TMP) as the base in peptide couplings produces glycopeptides with high efficiency and low epimerization. The information and improved reaction conditions will facilitate the preparation of glycopeptides as therapeutic compounds and vaccine antigens.

Synthesis and cell-selective antitumor properties of amino acid conjugated tumor-associated carbohydrate antigen-coated gold nanoparticles

“…We employed coupling conditions that were determined previously by our laboratory to minimize racemization of the α-carbon of the amino acid. 85,86 Final stages for the preparation of the desired AuNP ligand included Fmoc deprotection with piperidine, acetylation of the resulting amino group with acetic anhydride in methanol and Zemplèn deprotection of all O- and S-protected acetates resulting in thiols 10a and 10b . For the preparation of the control ligand, compound 2 was processed directly to the thioacetate 11 and hydrolyzed to 12 ; this compound was used directly for AuNP synthesis.…”

COMU: scope and limitations of the latest innovation in peptide acyl transfer reagents

“…The combination of COMU ( 16 ) and Oxyma ( 15 ) is an alternative to maintain acceptable epimerization levels, which can also be used to decrease the risk of guanidylation. Moreover, COMU ( 16 ) and Oxyma ( 15 ) mixtures are reported to display higher acylation capacity than HDMA ( 11 )/HOAt ( 2 ) and HCTU ( 6 )/HOAt ( 2 ) .…”

Divergent Behavior of Glycosylated Threonine and Serine Derivatives in Solid Phase Peptide Synthesis

“…In addition, we recently demonstrated that many commonly used peptide coupling conditions produce high levels of epimerization for glyco-amino acids. 11 In fact, epimerization could produce as high as 80% of the unnatural epimer. At present, however, the factors that influence efficiency and epimerization are not well understood, and additional studies are needed to develop efficient and general peptide coupling conditions for glycopeptides.…”

“…11 In this study, we evaluated efficiency and epimerization when coupling glycosylated threonine derivatives. Non-glycosylated threonine derivatives are known to react more slowly than the corresponding serine derivatives in solid phase peptide coupling reactions.…”