New biphasic solvent system based on cyclopentyl methyl ether for the purification of a non‐polar synthetic peptide by pH‐zone refining centrifugal partition chromatography

Abstract

A new type 1 ternary biphasic system composed of cyclopentyl methyl ether, dimethylformamide and water was developed, characterized and successfully used for the purification of a lipophilic, protected peptide by pH-zone refining centrifugal partition chromatography. The protected peptide is an 8-mer, key intermediate in bivalirudin (Angiomax®) synthesis and shows a very low solubility in the solvents usually used in liquid chromatography. All ionic groups, except the N-terminal end of the peptide, are protected by a benzyl group. The purification of this peptide was achieved with a purity of about 99.04% and a recovery of 94% using the new ternary biphasic system cyclopentyl methyl ether/dimethylformamide/water (49:40:11, v/v) in the descending pH-zone refining mode with triethylamine (28 mM) as the retainer and methanesulfonic acid (18 mM) as the eluter.

Cyclopentyl Methyl Ether (CPME): A Versatile Eco‐Friendly Solvent for Applications in Biotechnology and Biorefineries

“…Finally,A marouche et al [79] described the application of a ternary biphasic system composed of CPME/DMF/water (49:40:11v/v/v) for the purificationo falipophilic protected octapeptide, which is ak ey intermediate in the preparation of bivalirudin( Angiomax), ar eversible direct thrombin inhibitor.F or the purification of this octamer [H-Glu(OBzl)-Glu(OBzl)-Ile-Pro-Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu-OBz, in which all ionic groups except the N-terminal end of the peptidea re protected by a benzylg roup], the technique of displacementm ode is used; this technique, whichi su sed in centrifugal partition chromatography (CPC) [80] and counter-current chromatography (CCC), [81] implies the dissolution of an eluter-also named a displacer-in them obile phasea nd ar etainer or an ion-exchanger in the stationaryp hase. When an acid or ab ase is added to the stationary phase as ar etainer,t his technique is known as pH-zone refiningm ode.…”

Large‐scale separation of acetylcholinesterase inhibitors from Zanthoxylum nitidum by pH‐zone‐refining counter‐current chromatography target‐guided by ultrafiltration high‐performance liquid chromatography with ultraviolet and mass spectrometry screening

“…Additionally, pH‐zone‐refining CCC enables the detection of components by their pH, facilitating the separation of compounds with low or no ultraviolet absorption . Thus pH‐zone‐refining CCC has been successfully used to separate ionizable compounds with large scale, such as alkaloids , organic acids , peptide derivatives , and so on.…”

“…Thus pH-zone-refining CCC has been successfully used to separate ionizable compounds with large scale, such as alkaloids [26,27], organic acids [28], peptide derivatives [29], and so on.…”

Application of chromatography technology in the separation of active alkaloids from Hypecoum leptocarpum and their inhibitory effect on fatty acid synthase

“…The solvent system used in this study was selected in the typical way developed by Ito . Successful separation by pH‐zone‐refining CCC largely depends on the selection of suitable solvent system that should have an appropriate partition coefficient ( K ) values in both basic ( K base >>1) and acidic ( K acid <<1) conditions . According to the rules for the selection of the suitable solvent system described in the related references, the new quaternary biphasic solvent system composed of n ‐hexane/ethyl acetate/methanol/water at different volume ratios (5:5:5:5, 3:5:3:5, 3:4:2:5) was tested.…”