CHIRALITY 421-23 (1992)
Stereoselective Effect of Phenprocownon Enantiomers on the Binding of Benzodiazepines to Human Serum Albumin ILONA FITOS AND MIKLOS SIMONYI Central Research Institute for ChemzStly, Hungarian Academy of Sciences, Bua'upest, Hungary
ABSTRACT The effect of phenprocoumon enantiomers on the stereoselective binding of 3-substituted 1,4-benzodiazepinesto human serum albumin WSA) was studied by chromatography on HSA-Sepharose column. (S)-Phenprocoumonexerts stereoselective allosteric interaction on the binding of benzodiazepines.The structural requirements of enhanced stereoseiectivities are similar to those found previously with (S)-warfarin.
KEY WORDS: protein binding, afKnity chromatography, resolution, allosteric interaction, increased enantioselectivity INTRODUCTION
MATERIALS AND METHODS
Phenprocoumon is a chiral drug and, like the structurally related warfarin (Scheme 1)the (- )-(S)enantiomer has higher anticoagulant activity.* It has a very strong binding to human serum albumin (HSA) with an association constant about twofold higher for the S enantiomer. Based on the induced cotton effects observed for the binding to HSA of phenprocoumonand warfarin enantiomers, different binding mechanisms were supposed2 for the two drugs; whereas the orientation of the 4-hydroxycoumarin nucleus is different for the bound warfarin enantiomers, it is the same for the phenprocoumon stereoisomers. In previous binding studies performed with different methods including affinity chromatography on HSA-Sepharose column, we reported s7 stereoselective allostericinteraction in the simultaneous binding of chiral 1,Cbenzodiazepines(Scheme 1) and warfarin. The most remarkable mutually increased binding was observed between (S)-warfarin and certain (S)-benzodiazepines, possessing ortho-chlorine in the 2'psition and lacking methyl substitution at the N(l) atom. This type of interaction was also revealed8 by HPLC technique on silicaimmobilized HSA. Since this phenomenon sensitively reflects structural features of the drug, it may be applied as a paradigm to test the above assumption2 based on CD results. Accordingly, if the binding mechanism of phenprocoumon enantiomers really differs from that of warfarin antipodes, the allosteric interaction is expected to reflect this difference.
ruc-Benzodiazepines were obtained as previously s7 described. Resolved enantiomers were investigated in cases of 3-Me-clonazepam and oxazepam hemisuccinate. Phenprocoumon enantiomers were kindly donated by Hoffman-LaRoche (Basel). Binding studies by chromatography were performed following the method of Lagercrantz et al. HSA in N 1YOconcentration was immobilized on CNBr-activated Sepharose 4B/Pharmacia Fine Chemicals (Uppsala, Sweden). The gel was filled into glass column (diameter 12 mm); the flow rate was about 1 ml/min. Samples of 2-5 pg were applied in a 10-20 p1 ethanol solution. Elution volumes were detected by UV or by radioactive liquid scintillation counting. The eluent was Ringer's buffer (pH 7.4) containing 0.01% sodium azid. For interaction studies, the eluent contained phenprocoumon enantiomers dissolved in buffer with 0.5% ethanol.
OH RI I
R1 i
n
'R
3
R7
Stereoselective Effect of Phenprocownon Enantiomers on the Binding of Benzodiazepines to Human Serum Albumin ILONA FITOS AND MIKLOS SIMONYI Central Research Institute for ChemzStly, Hungarian Academy of Sciences, Bua'upest, Hungary
ABSTRACT The effect of phenprocoumon enantiomers on the stereoselective binding of 3-substituted 1,4-benzodiazepinesto human serum albumin WSA) was studied by chromatography on HSA-Sepharose column. (S)-Phenprocoumonexerts stereoselective allosteric interaction on the binding of benzodiazepines.The structural requirements of enhanced stereoseiectivities are similar to those found previously with (S)-warfarin.
KEY WORDS: protein binding, afKnity chromatography, resolution, allosteric interaction, increased enantioselectivity INTRODUCTION
MATERIALS AND METHODS
Phenprocoumon is a chiral drug and, like the structurally related warfarin (Scheme 1)the (- )-(S)enantiomer has higher anticoagulant activity.* It has a very strong binding to human serum albumin (HSA) with an association constant about twofold higher for the S enantiomer. Based on the induced cotton effects observed for the binding to HSA of phenprocoumonand warfarin enantiomers, different binding mechanisms were supposed2 for the two drugs; whereas the orientation of the 4-hydroxycoumarin nucleus is different for the bound warfarin enantiomers, it is the same for the phenprocoumon stereoisomers. In previous binding studies performed with different methods including affinity chromatography on HSA-Sepharose column, we reported s7 stereoselective allostericinteraction in the simultaneous binding of chiral 1,Cbenzodiazepines(Scheme 1) and warfarin. The most remarkable mutually increased binding was observed between (S)-warfarin and certain (S)-benzodiazepines, possessing ortho-chlorine in the 2'psition and lacking methyl substitution at the N(l) atom. This type of interaction was also revealed8 by HPLC technique on silicaimmobilized HSA. Since this phenomenon sensitively reflects structural features of the drug, it may be applied as a paradigm to test the above assumption2 based on CD results. Accordingly, if the binding mechanism of phenprocoumon enantiomers really differs from that of warfarin antipodes, the allosteric interaction is expected to reflect this difference.
ruc-Benzodiazepines were obtained as previously s7 described. Resolved enantiomers were investigated in cases of 3-Me-clonazepam and oxazepam hemisuccinate. Phenprocoumon enantiomers were kindly donated by Hoffman-LaRoche (Basel). Binding studies by chromatography were performed following the method of Lagercrantz et al. HSA in N 1YOconcentration was immobilized on CNBr-activated Sepharose 4B/Pharmacia Fine Chemicals (Uppsala, Sweden). The gel was filled into glass column (diameter 12 mm); the flow rate was about 1 ml/min. Samples of 2-5 pg were applied in a 10-20 p1 ethanol solution. Elution volumes were detected by UV or by radioactive liquid scintillation counting. The eluent was Ringer's buffer (pH 7.4) containing 0.01% sodium azid. For interaction studies, the eluent contained phenprocoumon enantiomers dissolved in buffer with 0.5% ethanol.
OH RI I
R1 i
n
'R
3
R7
