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Research Article Open access CC BY 4.0

Stereochemical Stability of Phenylglycine in Peptide Synthesis: Stereoretentive Coupling and Deprotection Strategies

Zeping Wang, Shoko Ishikawa, Yuki Fukuda, Sayaka Yamada, Meika Inomoto, Desita Triana Aziz, Xueyu Yang, Zetry Puteri Tachrim, Takeyuki Suzuki, Yuta Murai, Makoto Hashimoto

Organics · pp. 27–27 · Published 3 Jul 2026

10.3390/org7030027

Abstract

Phenylglycine (Phg) is a nonproteinogenic α-amino acid found in various bioactive molecules. The C-terminal activation of N-acyl Phg is often accompanied by oxazolone-mediated racemization, arising from the direct attachment of the phenyl ring to the α-carbon. After peptide bond formation with another chiral amino acid, this stereochemical erosion is observed as Phg-site epimerization and diastereomer formation. N-acyl activated esters, particularly N-hydroxysuccinimide (OSu) esters, are widely used for peptide bond formation with proteinogenic α-amino acids. Our previous study on N-trifluoroacetyl phenylglycine (TFA-Phg-OH) revealed that Phg-site epimer formation could still occur when TFA-Phg-OSu was employed as an acyl donor for coupling with amino acid ester hydrochlorides (AA–OMe·HCl) in the presence of a soluble organic base. To address these issues, in this study, we report a base-limited one-pot coupling of TFA-Phg-OH with α-amino acid ester hydrochlorides (AA–OR·HCl; R = Me or tert-Bu) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCD·HCl), which effectively suppresses Phg epimerization. The resulting TFA-Phg–AA–OR dipeptides (AA = Ala, Val, Leu, Met, Phg) were all obtained at a >60% yield with a diastereomeric excess (de) ≥ 98.5%. Notably, reducing the amount of triethylamine further minimized epimer formation, while Ba(OH)2·8H2O and trifluoroacetic acid enabled stereoretentive deprotection of the N-TFA group and tert-butyl ester, respectively. This workflow provides practical access to both protected and deprotected Phg–AA motifs, thereby facilitating the preparation of unprotected Phg-containing peptide building blocks.

Chemistry Epimer Diastereomer Trifluoroacetic acid Peptide Triethylamine Stereochemistry Peptide bond

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