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
Externally publishedYes

Fingerprint

Fenoterol
Adrenergic Receptors
Membranes
Chromatography
Enantiomers
Cardiac Myocytes
Rats
Chemical activation
Affinity chromatography
Enantioselectivity
Dichroism
Binding sites
Ligands
HEK293 Cells
Inbred SHR Rats
Circular Dichroism
Affinity Chromatography
Asthma
Heart Failure
Binding Sites

Keywords

  • β-adrenergic agonist
  • Enantiomers
  • Fenoterol

ASJC Scopus subject areas

  • Analytical Chemistry
  • Drug Discovery
  • Organic Chemistry
  • Pharmacology

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

Enantioselective separation and online affinity chromatographic characterization of R,R- and S,S-fenoterol. / Beigi, Farideh; Bertucci, Carlo; Zhu, Weizhong; Chakir, Khalid; Wainer, Irving W.; Xiao, Rui Ping; Abernethy, Darrell R.

In: Chirality, Vol. 18, No. 10, 2006, p. 822-827.

Research output: Contribution to journalArticle

Beigi, F, Bertucci, C, Zhu, W, Chakir, K, Wainer, IW, Xiao, RP & Abernethy, DR 2006, 'Enantioselective separation and online affinity chromatographic characterization of R,R- and S,S-fenoterol', Chirality, vol. 18, no. 10, pp. 822-827. https://doi.org/10.1002/chir.20317
Beigi, Farideh ; Bertucci, Carlo ; Zhu, Weizhong ; Chakir, Khalid ; Wainer, Irving W. ; Xiao, Rui Ping ; Abernethy, Darrell R. / Enantioselective separation and online affinity chromatographic characterization of R,R- and S,S-fenoterol. In: Chirality. 2006 ; Vol. 18, No. 10. pp. 822-827.
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T1 - Enantioselective separation and online affinity chromatographic characterization of R,R- and S,S-fenoterol

AU - Beigi, Farideh

AU - Bertucci, Carlo

AU - Zhu, Weizhong

AU - Chakir, Khalid

AU - Wainer, Irving W.

AU - Xiao, Rui Ping

AU - Abernethy, Darrell R.

PY - 2006

Y1 - 2006

N2 - 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.

AB - 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.

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