Separation of Peptide Diastereomers

Abstract

Diastereomeric peptides, isomers in which one or more of the chiral centers have been converted to the opposite configuration (R or S, D or L ), often possess different biological activities and/or conformational properties. Therefore, the separation and determination of diastereomeric peptides is important in life sciences and particularly significant to the pharmaceutical industry for quality control of peptide synthesis and stability as well as for regulatory requirements. Diastereomers differ in their physicochemical properties that can be utilized for their determination by numerous techniques. In contrast to many other methods such as optical rotation, circular dichroism, differential scanning calorimetry, or infrared (IR) spectroscopy, separation techniques such as chromatography or capillary electrophoresis (CE) allow the sensitive and simultaneous identification and quantification of peptide diastereomers. This chapter covers analytical‐scale separation of peptide diastereomers by chromatographic methods, including paper chromatography (PC), thin‐layer chromatography (TLC), gas chromatography (GC), high‐performance liquid chromatography (HPLC), and CE. HPLC is currently the predominantly used method for peptide diastereomer separations. However, owing to the high resolving power, particularly for polar compounds, CE has been applied to the separation of diastereomeric peptides in recent years. The chapter focuses on methods employing achiral stationary phases or buffers without chiral additives for the separations, although some examples of such separations, i.e. the use of chiral columns or chiral mobile phase additives, are mentioned. Moreover, only diasteromeric peptides containing natural amino acids will be discussed.

Catalytic Peptide Synthesis: Amidation of N-Hydroxyimino Esters

“…After optimization studies using several nonchiral hydrogenation catalysts, we found that the hydrogenation using Pd­(OH) 2 /C in 0.4 N HCl in CPME/AcOH (1:9) proceeded smoothly to afford 11n in 90% yield with 65:35 dr. When diastereoselectivity is not sufficiently high, separation of these diastereomers is usually carried out by recrystallization, including chiral resolution, chiral derivatization, or the use of HPLC technology, and we accomplished the separation of these diastereomers to obtain highly pure 11f , 11h , 11i , and 11l – 11n by practical techniques of recrystallization and/or normal phase silica-gel column chromatography without any chiral sources . All spectroscopic data pertaining to the produced di- and tripeptides 11a – 11q were accorded with those of the authentic samples prepared via a common synthetic method using 1-ethyl-3-(3-(dimethylamino)­propyl)­carbodiimide hydrochloride (WSC·HCl) and 1-hydroxybenzotriazole (HOBt)…”