General approach to chiroptical characterization of binding of prochiral and chiral 1,4-benzodiazepin-2-ones to human serum albumin

General interpretation of chiroptical characteristics of binding process of prochiral (Z) and chiral (Z¹) 1,4-benzodiazepin-2-ones to human serum albumin (HSA) is presented. Interpretation of binding of Z studied by circular dichroism (CD) measurements allows us to conclude that the CD within the bands of bound Z is mainly due to the chirality of the first sphere and that the greatest part of 3 is bound in the M-conformation. For Z¹ we conclude that (S)-1 binds on both binding sites of HSA in the M-conformation, while (R)-1 binds on two different binding sites in opposite conformations. Furthermore we conclude that binding via two nitrogens of the benzodiazepine, i.e. at two ends of the chromophoric system is rather more probable than via ring A.     

Binding of 3-alkyl-1,4-benzodiazepin-2-one stereoisomers to human serum albumin

The binding of both enantiomers of 3-alkyl-1,4-benzodiazepin-2-ones to human serum albumin (HSA) strengthens with increasing size of the 3-alkyl substituent. 3,3-Dimethylderivatives show diminished binding affinities although they bind in the favourable conformation assumed by the (S)-enantiomers. N(1)-Methyl substitution increases the enantioselectivity of binding by selectively enhancing the binding strength of (S)-antipodes.     

Tocainide analogues binding to human serum albumin: A HPLAC and circular dichroism study

A series of synthesised tocainide analogues were characterized for their human serum albumin (HSA) binding, using high-performance liquid affinity chromatography (HPLAC) and circular dichroism (CD). The synthesis and physico-chemical characterization of compounds 7a–7d is reported here. For the HPLAC investigation HSA was covalently immobilized to the silica matrix of the HPLC column, using an anchoring procedure, which allows the binding properties of the protein to be maintained. The HSA-based column was used for getting information on the high affinity binding sites of the tocainide analogues to HSA. According to the displacement chromatography approach, the retentions of the analytes were determined in the absence and in the presence of increasing concentrations of competitors known to bind to specific binding sites on the protein. The same system, drug/protein, was investigated in solution by CD.     

Binding of coumarin anticoagulants to human and bovine serum albumin. Circular dichroism studies

The interaction of acenocoumarin, coumachlor, phenprocoumon, and warfarin with human and bovine serum albumin was investigated by ultracentrifugation and circular dichroism measurements. Although all four drugs generate extrinsic Cotton effects when bound to human and bovine serum albumin, large differences in the signs and the intensities of the Cotton effects are observed. The differences in the induced Cotton effects suggest differential molecular binding mechanisms.     

Circular dichroism studies on the inhibiting effect of oleic acid on the binding of diazepam to human serum albumin

The effect of a free fatty acid (oleic acid) on the binding of a benzodiazepine derivative (diazepam) to human serum albumin (HSA)¹ has been studied using the technique of circular dichroism. Both qualitative and quantitative results suggest that oleic acid significantly affects the binding of diazepam, even at low molar ratios to albumin (below 1:1). It is suggested that the displacement of bound diazepam occurs primarily through an allosteric mechanism.     

Stereoselective binding of 3-acetoxy-, and 3-hydroxy-1,4-benzodiazepine-2-ones to human serum albumin. Selective allosteric interaction with warfarin enantiomers

Stereoselective binding of oxazepam, lorazepam, temazepam and methyl lorazepam as well as of their acetates to human serum albumin was investigated by different techniques. The 2'-chlorine and the N(1)-methyl substitution exert opposite effects on the antipodes. Enantiomers of oxazepam acetate (OAc) and lorazepam acetate (LAc) displace diazepam. Allosteric interactions with warfarin were manifested by either mutually increased or decreased binding depending on the structure of benzodiazepine and on the configuration of both benzodiazepine and warfarin. The most remarkable effect could be observed in the simultaneous binding of (S)-lorazepam acetate and (S)-warfarin.     

Stereochemical aspects of benzodiazepine binding to human serum albumin. I. Enantioselective high performance liquid affinity chromatographic examination of chiral and achiral binding interactions between 1,4-benzodiazepines and human serum albumin.

The displacement of a series of 1,4-benzodiazepine (BDZ) drugs from a chiral stationary phase, based upon human serum albumin, for high performance liquid chromatography was investigated. The different displacement patterns obtained using various mobile phase additives could not be interpreted in terms of binding of the solutes to a single site. The observations were better described by considering the attachment of the BDZs to several loci on the protein. Two main mechanisms of binding were discerned, a nonstereoselective mode, which affected all solutes and seemed to occur at a large number of locations on the protein, and a highly stereoselective mode, which involved only one enantiomer of chiral BDZs and presumably one conformation of certain achiral solutes. The stereoselective binding mode encompassed at least four different sites, each of which displayed slightly different structural requirements. It is suggested that the nomenclature currently used to describe drug binding to human serum albumin may be misleading. Rather than the use of site I or site II, it may be preferable to adopt the terms type I and type II binding, according to the displacement patterns of the compound concerned. This approach would retain the conceptual simplicity of the current notation, while avoiding misleading implications of the exact molecular locus of binding.     

Studies on the binding of benzodiazepines to human serum albumin by circular dichroism measurements

The binding of twenty-one different benzodiazepine derivatives to human serum albumin (HSA) has been studied by circular dichroism (CD) measurements in 0.1 M KCl and 0.005 M phosphate buffer at pH 7.4 and 25°. The binding has been related to the qualitative changes of the CD spectra of HSA between 250 and 350 nm and, in some representative benzodiazepine derivatives, to the electron distribution as calculated by the CNDO/2-method. The binding has also been quantitatively studied with a continuous CD titration technique. The data were numerically analyzed with computer programs based on one-site, two-sites and three-sites models. It is concluded that most derivatives will bind primarily to one site on HSA. It is moreover concluded that variations of the C₂-amino side chains will not influence the binding properties. Oxygens at C₂ or C₃ will increase the binding, while oxygens in both positions will decrease the binding. A derivative with a C₇-amino group will show only weak affinity for HSA, which might be explained by the positive character of the hydrogens in the amino group according to the CNDO/2-calculation. A C₇-nitro group will also impair the binding, as well as large substituents at N₁.     

Chiral 1,4-benzodiazepin-2-ones: Relationship between stereochemistry and pharmacological activity

Pure enantiomers of 3-substituted-1,4-benzodiazepin-2-ones, obtained by HPLC resolution on chiral stationary phases, show significant differences in their pharmacological activity. The occurrence of biotransformation during the pharmacological test is monitored using a new chromatographic method. The reliability of the pharmacological activity data is discussed.     

Azapropazone binding to human serum albumin

Summary Azapropazone, a new non-steroidal antiinflammatory drug, is strongly bound to human serum albumin. As revealed by Scatchard analysis, one high-affinity binding site with an association constant of about 1.2×10⁶ M^–1 and two low-affinity binding sites with association constants of about 0.05×10⁶ M^–1 were found. While the high-affinity binding site of azapropazone is clearly not identical with the diazepam or digitoxin binding sites of human serum albumin, contradictory evidence was found by optical measurements and displacement studies for the similarity of the azapropazone and the warfarin binding site of human serum albumin. At present, it is suggested that both drugs bind to different areas of the same binding site. Therefore, the pronounced effects of various disease states on the plasma protein binding of azapropazone can not be explained by a binding to an unusual binding site, but seem to be due to an extreme sensitivity of the azapropazone binding area to the putative endogenous binding inhibitors, present in the blood during those disease states.     

Circular dichroic investigations of the binding of salicylate and related compounds to human serum albumin

The binding of salicylate and some related compounds has been investigated by circular dichroism. The binding constants, where possible, were determined by direct titration, otherwise by their ability to displace salicylate. The binding constants for the first two sites for salicylate were found to be 1.05 × 10⁵ 1. mole^?1 and 5.10 × 10³ 1. mole^?1. The induced circular dichroism of salicylate was diminished by addition of acetate ions. Attempts to correlate binding constants with partition coefficients and Hammett σ values suggested hydrophobic and electrostatic forces to be involved in the binding of these benzoic acid derivatives. Aspirin, indomethacin and phenylbutazone appeared to share the primary site with salicylate.     

Circular dichroic investigations into binding of sulfonamides to serum albumin

The binding of twelve structurally related sulfonamides to serum albumins including human was investigated using a circular dichroic technique. Some differences of circular dichroic spectral characteristics were observed when sulfonamides were bound to the same albumin or when the drug was bound to several albumins. The differences in these circular dichroic characteristics may be due to various asymmetries. The Scatchard plots indicated that only the primary site was capable of inducing ellipticities of the drugs. The interaction with rabbit serum albumin showed significantly large binding constants and apparent anisotropy factors (g′, values), in comparison with other albumins. No significant correlation between the g′ values of the induced circular dichroic bands and partition coefficients or/and pKa values was observed. The induced ellipticities of the drug-albumin complexes decreased with pH. This pH dependence can be explained by the ionization of drug and albumin as well as the conformational change of the albumin.