Enantioselective separation and online affinity chromatographic characterization of R,R- and S,S-fenoterol

Farideh Beigi, Carlo Bertucci, Weizhong Zhu, Khalid Chakir, Irving W. Wainer, Rui Ping Xiao, Darrell R. Abernethy

Research output: Contribution to journalArticle

Abstract

Background: rac-Fenoterol is a β2-adrenoceptor agonist (β2-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 β2-AR. Methods: R-F and S-F were separated from the enantiomeric mixture by chiral chromatography and absolute configuration determined by circular dichroism. β2-AR binding was evaluated using frontal affinity chromatography with a stationary phase containing immobilized membranes from HEK-293 cells that express human β2-AR and standard membrane binding studies using the same membranes. The effect of R-F and SF 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 β2-AR as the stationary phase and rac-3H-fenoterol as a marker ligand showed that addition of increasing concentrations of R-F to the mobile phase produced concentration-dependent decreases in rac-3H-fenoterol retention, while similar addition of S-F produced no change in rac-3H-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 EC50 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 β2-AR/Gs selective agonist and fully restores diminished β2-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 β2-AR/Gs activation and if it can be used in the treatment of congestive heart failure.

Original languageEnglish (US)
Pages (from-to)822-827
Number of pages6
JournalChirality
Volume18
Issue number10
DOIs
StatePublished - 2006

Keywords

  • Enantiomers
  • Fenoterol
  • β-adrenergic agonist

ASJC Scopus subject areas

  • Analytical Chemistry
  • Catalysis
  • Pharmacology
  • Drug Discovery
  • Spectroscopy
  • Organic Chemistry

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  • Cite this

    Beigi, F., Bertucci, C., Zhu, W., Chakir, K., Wainer, I. W., Xiao, R. P., & Abernethy, D. R. (2006). Enantioselective separation and online affinity chromatographic characterization of R,R- and S,S-fenoterol. Chirality, 18(10), 822-827. https://doi.org/10.1002/chir.20317