Human serum albumin as chiral selector in

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The displacement of (S)-oxazepam hemisuccinate (OXH) by diazepam. (DZP) and salicylic acid (SA) are representative for the behavior observed in the case of ...
Human serum albumin as chiral selector in enantioselective HPLC Daniele Tedesco*, Carlo Bertucci Department of Pharmacy and Biotechnology, Alma Mater Studiorum – University of Bologna, Italy

Site I Site II

PB (50 mM, pH 7.4)/1-PrOH 93:7 (v/v) 0.6 mL/min, 25 °C

+ SA + DZP

Competitive binding

Standard conditions PB (50 mM, pH 7.0)/1-PrOH 93:7 (v/v) 0.8 mL/min, 30 °C, λ = 240 nm

(S)-WFR

α (R)-WFR

HSA structure

HSA-based CSPs, obtained by anchoring the protein to a silica matrix, found successful applications for the enantioselective separation of acidic and neutral drugs, while less efficiency was obtained with basic drugs. HSA-based CSPs are usually employed with aqueous mobile phases; retention (k) and enantioselectivity (α) can be efficiently optimized by changing the nature, pH and concentration of the buffer in the mobile phase, the nature and the concentration of the organic modifier, and temperature, as for the enantioresolution of rac-warfarin (WFR) [5].

Site III

X-ray crystallographic structures of ligand/HSA complexes are widely used to characterize the binding sites of HSA and contributed to elucidate their ligands and their chiral discrimination mechanisms. The two main drug binding sites of HSA were first identified by Sudlow in sub-domains IIA (site I) and IIIA (site II) [2-3], while sub-domain IB (site III) was recently proposed as a third drug binding site on HSA [4].

The use of HSA ligands (displacers) as modifiers to the mobile phase can result in different chromatographic behaviors of the analyte [6]: ● independent binding: different binding sites for analyte and displacer; the affinity of the analyte is not affected (same retention). ● cooperative binding: different binding sites for analyte and displacer; the affinity of the analyte increases (higher retention times). ● anti-cooperative binding: different binding sites for analyte and displacer; the affinity of the analyte decreases (lower retention). ● competitive binding: same binding site for analyte and displacer; direct competition lowers the HSA affinity for the analyte (no retention at high concentrations of displacer). The displacement of (S)-oxazepam hemisuccinate (OXH) by diazepam (DZP) and salicylic acid (SA) are representative for the behavior observed in the case of competitive and anti-cooperative binding, respectively [7].

HSA ligands as displacers

Introduction

The development of chiral stationary phases (CSPs) with high selectivity for a wide range of chemical classes is one of the most important topics of research in the field of enantioselective HPLC (eHPLC). CSPs based on human serum albumin (HSA) have been extensively used as biochromatographic supports for the determination of the binding parameters of drugs to HSA; the chromatographic performance of HSAbased CSPs is directly related to the binding properties of the protein in solution, allowing a relatively easy prediction of the optimal chromatographic conditions necessary to achieve adequate selectivity for the enantioresolution of a chiral analyte. The present poster reviews the use of HSA as a chiral selector in eHPLC, with particular emphasis on the modulation of its chromatographic performance [1].

Mobile phase conditions

* e-mail address: [email protected]

Lithocholic acid (LCA), a site II binder, was used as a modifier to the mobile phase to improve the chromatographic performances of HSAbased CSPs in the enantioresolution of profens. LCA acts as direct competitor for both the enantiomers of the most popular profens, with consequent reduction of retention times; a greater displacement is observed for the less retained enantiomers, due to their lower binding affinities to the protein. As a net effect, the enantioselectivity increases for concentrations of lithocholic acid in the mobile phase up to 6 μM, as observed for rac-naproxen (NPX) [8].

PB (100 mM, pH 7.4)/1-PrOH 90:10 (v/v) + LCA 1.0 mL/min, 25 °C, λ = 260 nm

α

(S)-NPX

(R)-NPX PB (50 mM, pH 7.0)/1-PrOH 94:6 (v/v) + (S)-WFR 40 μM 0.8 mL/min, 30 °C, λ = 230 nm

Compound

Conditions

Temazepam Oxazepam Lorazepam N-(tert-butyl)norfludiazepam Temazepam hemisuccinate Oxazepam hemisuccinate Oxazepam pivalate Oxazepam acetate

A A A B B B B B

(S)-LZH + (S)-WFR

(S)-LZH + (R)-WFR (R)-LZH

k1

k2

α

−16 −15 −15 −23 −47 −52 −13 −17

−2 −10 −17 −26 −17 −36 −3 −17

+16 +6 −2 −5 +56 +35 +12 +1

A: PB (50 mM, pH 7.4)/1-PrOH 94:6 (v/v), 1 mL/min, 30 °C. B: PB (100 mM, pH 7.4)/1-PrOH 95:5 (v/v), 1 mL/min, 30 °C.

Acknowledgements: Financial support from MIUR and University of Bologna.

Cooperative binding

The covalent modification of HSA gives rise to a new chiral selector with different binding properties with respect to the native protein, and then with different chromatographic performances. An in situ selective modification of Lys199, located in site I, was obtained by reaction with aspirin. The acetylation affected the binding behavior of HSA for both site I and site II ligands; the acetylated HSA column showed a significant decrease in analysis time for drugs binding to site II, and, in most cases, an increase in enantioselectivity [10].

Conclusions

Acetylated HSA as CSP

rac-LZH

Allosteric interactions can also be exploited for improving the chromatographic performances of HSA-based CSPs. The most impressive example is the cooperative binding of (S)-lorazepam hemisuccinate (LZH) and (S)-warfarin (WFR); this allosteric interaction is very selective and is the only one occurring between the enantiomers of the two drugs. As a result, the retention of (S)-LZH increases markedly in the presence of (S)-WFR in the mobile phase, while the retention of (R)-LZH is not affected; the enantioselectivity increases from 1.40 to 2.47 when 10 μM (S)-WFR is added to the mobile phase [9].

A synergistic interaction between experimental techniques and computational investigations should lead to further progresses in the understanding of the molecular principles of chiral recognition phenomena by HSA, prompting new improvements of the chromatographic performance of HSA-based CSPs. The experimental conditions used with HSA-based CSPs are well suited to the hyphenation with highly sensitive and selective MS detection systems, thus allowing the application of the method even to complex matrices.

References: [1] Bertucci C, Tedesco D, Curr Med Chem 2016, submitted. [2] Sudlow G et al, Mol Pharmacol 1975, 11, 824. [3] Sudlow G et al, Mol Pharmacol 1976, 12, 1052. [4] Zsila F, Mol Pharm 2013, 10, 1668. [5] Bertucci C et al, Chirality 1999, 11, 675. [6] Honoré B, Pharmacol Toxicol 1990, 66, 1. [7] Ascoli GA et al, Biomed Chromatogr 1998, 12, 248. [8] Bertucci C et al, Chromatographia 2001, 53, 515. [9] Domenici E et al, J Pharm Sci 1991, 80, 164. [10] Bertucci C et al, Ann Chim 1997, 87, 45.