Interactions between carbonic anhydrase and its inhibitors revealed by gel electrophoresis and circular dichroism

Structural properties, and especially the differential stability, of complexes between carbonic anhydrase (CA) and three sulfonamide inhibitors, acetazolamide, dorzolamide and methazolamide, were investigated by spectroscopic and electrophoretic techniques. These included denaturant gradient gel electrophoresis either across a urea or a steady-state transverse sodium dodecyl sulfate (SDS) gradient. Acetazolamide, the smallest and most hydrophilic of the sulfonamides, forms the most stable complex in the presence of urea, whereas dorzolamide, with a bulky and hydrophobic structure, is most stable against the effects of SDS. At pH 7.4, complexes with dorzolamide show minimal changes in mobility across the SDS gradient, as if unaffected by the detergent, both in the presence and in the absence of excess ligand in the gel. When bound to both acetazolamide and methazolamide, on the other hand, CA displays an increase in mobility above 0.05% SDS, lower in the presence than in the absence of excess ligand. The finding of a distinct pattern for the unliganded enzyme, however, suggests the complexes can still retain the ligand, although binding of the surfactant changes their charge density. Under saturating conditions and in the presence of SDS, the surface charge of all complexes is much lower than for unliganded, denatured CA. Circular dichroism (CD) spectra clearly indicate that the increase in secondary structure and the decrease in tertiary structure brought about in CA by the presence of low concentrations of SDS are largely prevented by complexing with the inhibitors. These observations point out peculiar properties of each CA inhibitor, of potential value in the definition of their biological activities and also in the potential development of novel antagonist molecules.     

Study of donepezil binding to serum albumin by capillary electrophoresis and circular dichroism

The interaction between human serum albumin (HSA) and the acetylcholinesterase inhibitor donepezil, has been studied by means of capillary electrophoresis frontal analysis (CE/FA) and circular dichroism. CE/FA enabled rapid and direct estimation of the quantity of free donepezil present at equilibrium with a physiological level of serum albumin (600 mol L^–1). Application of Scatchard analysis enabled estimation of the binding parameters of HSA towards donepezil, such as association constant and number of binding sites on one protein molecule. Furthermore, due to enantioseparation ability shown by HSA on donepezil in CE mode, displacement experiments were carried out using ketoprofen and warfarin as coadditives to the HSA based running buffer. The addition of these compounds reduced the enantioresolution of donepezil by HSA only when used at high concentration. These data were confirmed and corroborated by circular dichroism (CD) experiments. Using CD, bilirubin was also applied as a ligand specific to site III of HSA. The observed behaviour suggested that donepezil could be considered a ligand with independent binding to sites I and II; although site III is not the highest affinity site, indirect interaction (i.e. cooperative binding) can be assumed.     

Vibrational circular dichroism study for natural bioactive schizandrin and reassignment of its absolute configuration

Bioactive natural product (+)-schizandrin was assigned as (7S,8S) using NMR. Recently, we obtained (+)-schizandrin from TCM Schisandra sphenanthera Rehd. et Wils. Its planar structure was well established using NMR and HR-MS including the reported references. Its absolute configuration is assigned using vibrational circular dichroism (VCD). By careful VCD investigation of (7S,8S) and (7S,8R) using B3LYP/6-311+G(d) methods, absolute configuration of (+)-schizandrin is assigned as (7S,8R). Electronic circular dichroism (ECD) was used for the discussion too and it gave the same conclusion.     

Vibrational circular dichroism study of optically pure cryptophane-A

Vibrational circular dichroism (VCD) measurements and density functional theory (DFT) calculations were used to obtain the absolute configuration of optically pure cryptophane-A molecule. This large molecule (120 atoms) that possess a globular shape, but no chiral centers, exceeds the molecular size of published structures for which VCD has been used to determine the absolute configuration. VCD spectra recorded in CDCl(3) solution for the two resolved enantiomers are near mirror images, and very good agreement between the observed IR and VCD spectra and intensity calculations performed at the DFT (B3PW91/6-31G) level establish, besides the absolute configuration, the preferential anti conformation of the aliphatic linkers of the chloroform-cryptophane-A complex. Experiments performed in CD(2)Cl(2) and C(2)D(2)Cl(4) solutions show no significant modifications in the IR and VCD spectra, indicating that the conformation of the aliphatic linkers is similar for empty (C(2)D(2)Cl(4) solution) and encaged (CDCl(3) and CD(2)Cl(2) solutions) cryptophane-A molecules.     

Magnetic circular dichroism study of directly fused porphyrins.

The magnetic circular dichroism (MCD) spectra of doubly and triply linked fused bisporphyrins (2MD and 2MT, M = Ni, Zn, Cu, Pd, and H2) and triply linked higher oligomers (3ZnT and 4ZnT) have been measured, and their Q-bands assigned based on the results of INDO/s calculations. In contrast to the Faraday A term observed for the Q(0,0) band of Ni(II) tetraphenylporphyrin, a single positive Faraday B term was observed for the lowest energy transition of the fused systems. The calculations indicated that the molecular orbitals (MOs) of the directly fused porphyrins consist of linear combinations of the constituent monomeric MOs, and that the effect of lowering the symmetry is always larger on the lowest unoccupied molecular orbital (LUMO) than on the highest occupied molecular orbital (HOMO). On the basis of Michl's perimeter model, these features can be correlated with the observed positive MCD signs in the near infrared region. A weak absorption band at 600-700 nm for the fused dimers can be assigned to a short-axis polarized Q transition.     

秋水仙碱立体异构体电子结构及圆二色谱性质的理论研究

采用量子化学密度泛函理论(DFT)在B3LYP/6-311++G二水平上对秋水仙碱四个立体异构体分子几何构型进行了优化,在优化的基础上进行了振动圆二色谱(VCD),紫外-可见光谱(UV-Vis)和电子圆二色谱(ECD)研究.为模拟真实条件,以水为溶剂,计算其对分子电子结构和光谱性质的影响.研究结果表明:秋水仙碱四个立体...     

Induced circular dichroism of azulene

The π→π^* absorption bands of azulene show circular dichroism in the presence of (+)-nopinone.     

Photoacoustic detection of circular dichroism

An analysis is presented for an experimental technique involving the measurement of circular dichroism using polarization-modulated photoacoustic spectroscopy. The technique is referred to as photoacoustic circular dichroism (PACD). In the PACD experiment, a photoacoustic signal is induced by using polarization-modulated excitation light which is alternately left-and right-circularly polarized. Expression appropriate for analyzing the PACD experimental observables (signal strength and phase) in terms of sample circular dichroism are developed within the general framework of the Rosencwaig and Gersho model for the photoacoustic effect in solids and liquids. Calculations based on these expressions are reported and the applicability of PACD for measuring the circular dichronic properties of optically opaque samples is discussed.     

Capillary electrophoresis study on phase of mixed micelles and its role in transport phenomena of particles

In the present work comprehensive studies on electrophoretic effects induced by a phase of mixed micelles, that migrates surrounded with background electrolyte (BGE) and is denoted as the BGE/segment of mixed micelles/BGE system, were undertaken using capillary electrophoresis coupled with contactless conductivity or UV–vis detector. It was established that mixed micelles under electrophoresis are subject of evolution in terms of mobility, peak area and presence of sub-zones enforced by the composition of micellar phase, segment length and applied voltage. Established features allowed us to explain the electrophoretic behavior of nanoparticles in the system BGE/sample containing nanocrystals/segment of mixed micelles/BGE and it was postulated that a pseudomicellar state of nanoparticles can be useful term in analyzing the migration phenomena of nanoparticles within micellar environment. In contrast to the previous works, where transport of nanocrystals (NCs) within micellar segment or between two micellar segments was analyzed, the present work is focused on the transport of NCs from sample of NCs dispersed in BGE to phase of mixed micelles, i.e., to rear boundary between micellar zone and BGE. Based on these results, systematic studies on transport efficiency for nanoparticles in the system BGE/sample containing nanocrystals/segment of mixed micelles/BGE show that the system assures efficient transport of nanoparticles from BGE based sample to micellar phase and their efficient preconcentration at the micellar segment/BGE rear boundary.     

Capillary zone electrophoresis study of cyclodextrin--lipoic acid host-guest interaction

Lipoic acid is a naturally occurring compound which is being widely investigated for its therapeutic effects in the treatment or prevention of a variety of diseases associated with oxidative injury, particularly diabetes. The diversity of therapeutic applications of lipoic acid requires an appropriate formulation to control its bioavailability, site-targeting delivery and to overcome its inherent chemical instability. In this regard, cyclodextrins (CDs) are ideally suitable due to their well-documented ability to include in their cavity proper guest molecules and protect them from physical or chemical damages. Lipoic acid forms 1:1 inclusion complexes with betaCD as shown in a previous report of an extended investigation that also indicated the suitability of capillary zone electrophoresis (CZE) for the study of such host-guest interactions. In view of these possible applications, we extended the CZE analysis to determine the strength of binding, in a pH 9 phosphate buffer, of lipoic acid with other CD derivatives such as alphaCD, gammaCD and the alkylated derivatives of betaCD, namely (2-hydroxypropyl)-beta-CD (HPbetaCD), and heptakis(2,3,6-tri-O-methyl)-beta-CD (TMbetaCD). Once established that the easily available betaCD is the most suitable receptor for lipoic acid, we set up and here describe a simple and reliable procedure for the quantitative determination of lipoic acid in commercial dietary supplement tablets containing also other active substances and excipients.     

Cobaltocenium acetylsalicylate: synthesis and circular dichroism study of interactions with DNA

Russian Chemical Bulletin - Cobaltocenium acetylsalicylate was synthesized and characterized for the first time. The concentration dependences observed in its interaction with the double helix of...     

Synthesis and circular dichroism of tetraarylporphyrin-oligonucleotide conjugates

The preparation and circular dichroic (CD) studies of self-complimentary 8-mer DNA sequences with a porphyrin at the 3' end are presented. Electronic interaction between the two porphyrins (the interchromophoric distance is in the range of 28-40 A), attached to both ends of the double-stranded helix, gives rise to a long-range exciton-coupled CD in the visible region (400-450 nm). The porphyrin chromophores act as sensitive probes of geometrical changes in the DNA backbone and sensitively reflect the double-strand to single-strand transition. This study demonstrates the possibility of using exciton-coupled porphyrin CDs for conformational studies of DNA.     

DFT study of vibrational circular dichroism spectra of D-lactic acid-water complexes

This paper presents a discussion of the interaction energies, conformations, vibrational absorption (VA, harmonic and anharmonic) and vibrational circular dichroism (VCD) spectra for conformers of monomeric chiral d(-)-lactic acid and their complexes with water at the DFT(B3LYP)/aug-cc-pVDZ and DFT(B3LYP)/aug-cc-pVTZ levels. A detailed analysis has been performed principally for the two most stable complexes with water, differing by lactic acid conformation. The VCD spectra were found to be sensitive to conformational changes of both free and complexed molecules, and to be especially useful for discriminating between different chiral forms of intermolecular hydrogen bonding complexes. In particular, we show that the VCD modes of an achiral water molecule after complex formation acquire significant rotational strengths whose signs change in line with the geometry of the complex. Using the theoretical prediction, we demonstrate that the VCD technique can be used as a powerful tool for structural investigation of intermolecular interactions of chiral molecules and can yield information complementary to data obtained through other molecular spectroscopy methods.