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Enantioselective separation and online affinity chromatographic characterization of R,R ‐ and S,S ‐fenoterol
Author(s) -
Beigi Farideh,
Bertucci Carlo,
Zhu Weizhong,
Chakir Khalid,
Wainer Irving W.,
Xiao RuiPing,
Abernethy Darrell R.
Publication year - 2006
Publication title -
chirality
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.43
H-Index - 77
eISSN - 1520-636X
pISSN - 0899-0042
DOI - 10.1002/chir.20317
Subject(s) - chemistry , fenoterol , enantiomer , chromatography , circular dichroism , membrane , racemic mixture , affinity chromatography , stereochemistry , organic chemistry , biochemistry , enzyme , medicine , asthma
Background: rac‐ Fenoterol is a β 2 ‐adrenoceptor agonist (β 2 ‐AR) used in the treatment of asthma. It has two chiral centers and is marketed as a racemic mixture of R , R ′‐ and S , S ′‐fenoterol (R‐F and S‐F). Here we report the separation of the R‐F and S‐F enantiomers and the evaluation of their binding to and activation of the β 2 ‐AR. Methods: R‐F and S‐F were separated from the enantiomeric mixture by chiral chromatography and absolute configuration determined by circular dichroism. β 2 ‐AR binding was evaluated using frontal affinity chromatography with a stationary phase containing immobilized membranes from HEK‐293 cells that express human β 2 ‐AR and standard membrane binding studies using the same membranes. The effect of R‐F and S‐F on cardiomyocyte contractility was also investigated using freshly isolated adult rat cardiomyocytes. Results: Chiral chromatography of rac‐ fenoterol yielded separated peaks with an enantioselectivity factor of 1.21. The less retained peak was assigned the absolute configuration of S‐F and the more retained peak R‐F. Frontal chromatography using membrane‐bound β 2 ‐AR as the stationary phase and rac‐ 3 H‐fenoterol as a marker ligand showed that addition of increasing concentrations of R‐F to the mobile phase produced concentration‐dependent decreases in rac‐ 3 H‐fenoterol retention, while similar addition of S‐F produced no change in rac‐ 3 H‐fenoterol retention. The calculated dissociation constant of R‐F was 472 nM and the number of available binding sites 176 pmol/column, which was consistent with the results from the membrane binding study 460 ± 55 nM (R‐F) and 109,000 ± 10,400 nM (S‐F). In the cardiomyocytes, R‐F increased maximum contractile response from (265 ± 11.6)% to (306 ± 11.8)% of resting cell length ( P < 0.05) and reduced EC 50 from −7.0 ± 0.270 to −7.1 ± 0.2 log[M] ( P < 0.05), while S‐F had no significant effect. Discussion: Previous studies have shown that rac‐ fenoterol acts as an apparent β 2 ‐AR/G s selective agonist and fully restores diminished β 2 ‐AR contractile response in cardiomyocytes from failing hearts of spontaneously hypertensive rats (SHR). Here we report the separation of the enantiomers of rac‐ fenoterol and that R‐F is the active component of rac‐ fenoterol. Further evaluation of R‐F will determine if it has enhanced selectivity and specificity for β 2 ‐AR/G s activation and if it can be used in the treatment of congestive heart failure. Chirality, 2006. Published 2006 Wiley‐Liss, Inc.