Separation of diastereomers of phosphinic pseudopeptides by capillary zone electrophoresis and reverse phase high‐performance liquid chromatography

Abstract

Separation of diastereomers of phosphinic pseudopeptides by capillary zone electrophoresis and reverse phase high-performance liquid chromatography Capillary zone electrophoresis (CZE) and reverse phase high-performance liquid chromatography (RP-HPLC) were used for separation of diastereomers of phosphinic pseudopeptides in achiral separation media. A set of phosphinic pseudopeptides, i. e. peptides with one peptide bond substituted by phosphinic acid moiety -PO 2 --CH 2 -derived from the structure N-Ac-Val-AlaB(-CH 2 )Leu-His-NH 2 synthesized as a mixture of four diastereomers was used. Separations of diastereomers by CZE were carried out in Tris-phosphate background electrolytes in the pH range 1.1 -3.2 and at least partial separation of the four diastereomers of each pseudopeptide was achieved. A routinely used RP-HPLC method (C 18 -silica column and water/acetonitrile/trifluoroacetic acid mobile phase) was also capable of resolving the diastereomers. In addition, since individual diastereomers of majority of the pseudopeptides were isolated by RP-HPLC it was possible to check the purity of these RP-HPLC separated diastereomers and to compare the migration order of the diastereomers in CZE with their elution order in RP-HPLC. The results obtained by CZE and RP-HPLC demonstrate a complementarity of both methods in analysis and separation of phosphinic pseudopeptides including their diastereomers.

Recent developments in capillary electrophoresis and capillary electrochromatography of peptides

“…Successful separation of the diastereomers of phosphinic pseudopeptides, i.e., peptides with one peptide bond substituted by phosphinic acid moiety -PO 2 H-CH 2 -derived from the structure N-Ac-Val-Alac(PO 2 H-CH 2 )Leu-His-NH 2 has been achieved by CZE in achiral separation media, strongly acidic Tris-phosphate BGEs, pH 1.1-3.2 [91,292].…”

Stereoselective separations of chiral phosphinic acid pseudodipeptides by CEC using silica monoliths modified with an anion‐exchange‐type chiral selector

“…The phosphinate group in phosphinic pseudopeptides has a pK a -value of 1.5-2.0 [9]. Accordingly, the present hPhe-Phe phosphinic c-dipeptide should be retained on the tBuCQD selector primarily by an ion-exchange mechanism as at moderately acidic pH the quinuclidine moiety of the cinchona carbamate selector will be protonated and positively charged, and the analyte molecules will be retained primarily by ionic interactions with this fixed charge of the stationary phase.…”

“…Also HPLC separations have been described utilizing chiral cellulose or amylose phases [5] or anion-exchange-type cinchona alkaloid-derived chiral stationary phases (CSPs) [6]. Koval et al [7][8][9] used CE and RP-HPLC for the separation and physicochemical characterization of phosphinic pseudopeptide diastereomers.…”

Determination of pKa values of diastereomers of phosphinic pseudopeptides by CZE

“…The results were in agreement with the rule of thumb saying that separation of diastereomers is most likely to be achieved in BGEs of pH around the pK a values of the analyte. The summarized data of this paper and our previous work [19] reveal that, in general, diastereomers of phosphinic pseudopeptides were most successfully separated in strongly acidic BGEs. Slight differences of pK a values of the central phosphinate group between the diastereomers were more markedly reflected in resolution of the diastereomers than differences in pK a of the 3) and NVHA (Fig.…”