Induced Circular Dichroism Spectra Research Articles

Self-organization of G-quadruplex structures in the hTERT core promoter stabilized by polyaminic side chain perylene derivatives

hTERT core promoter regulates telomerase transcription in human cells, thus its structural features are of large interest. We have found that the G-rich hTERT core promoter region, corresponding to the major DNase I hypersensitive site in chromatin organization, contains nine putative G-quadruplex forming sequences (PQS) and is unfavorable for nucleosome formation. Here we show that four PQS are effectively able to form stable parallel intramolecular G-quadruplexes, using PAGE and CD spectroscopy analysis. The PQS-region, as a whole, appears to be organized in three self-interacting G-quadruplexes, probably giving rise to a helicoidal superstructure, as shown by CD and polymerase stop assay. POL–HPDI drugs, that we previously found useful in selectively stabilizing telomeric G-quadruplex, are able to stabilize both the single intramolecular G-quadruplex and the PQS-region superstructure. The features of their induced CD spectra suggest that POL–HPDIs bind to single G-quadruplexes and to whole PQS-region superstructure, mainly by end-stacking interactions.

Design of benzo-15-crown-5 azoprobe/γ-cyclodextrin complexes for alkali metal ion recognition in water

Novel photosignal transduction systems of benzo-15-crown-5 (B15C5) azoprobe/γ-cyclodextrin (γ-CyD) complexes were designed. Four B15C5 azoprobes were synthesized by azo coupling of 4'-aminobenzo-15-crown-5 with phenol (for 15C5-Az-Ph) and N,N-dialkylanilines (for 15C5- Az-Cn). These azoprobes exhibited strong induced circular dichroism (ICD) by forming an inclusion complex with γ-CyD. Examination of the spectral responses and 1 H NMR analysis of the B15C5 azoprobe/γ-CyD complexes in the presence of alkali metal cations revealed that K + binding selectively induced dimer formation of the B15C5 azoprobes inside the γ-CyD cavity, which resulted in distinct changes in their UV-vis and ICD spectra in water. The effect of alkyl chain length of 15C5-Az-Cn (n1, 2, 4)/γ-CyD complexes was examined in 90% H2O/10% CH3CN (v/v). The highest K + ion response was noted for the 15C5-Az-C2/γ-CyD complex. The 15C5-Az-C4/γ-CyD complex was found to show abnormally high ICD responses for TMA + and Cs + .

Watching the Conformational Changes of Maleonitriledithiolate Chromophores Inside the Inclusion Complexes with Cyclodextrins: Probed by ICD Spectra and DFT Calculations

A series of inclusion complexes between cyclodextrins (alpha-, beta-, gamma-cyclodextrin and HP-beta-cyclodextrin, HP-beta-cyclodextrin = 2-hydroxypropyl-beta-cyclodextrin) and sodium maleonitriledithiolate (Na(2)mnt) were investigated by electronic spectra, induced circular dichroism (ICD) spectra, and quantum chemical studies. The inclusion complexes Na(2)mnt@alpha-cyclodextrin and Na(2)mnt@gamma-cyclodextrin did not show any ICD signals, whereas Na(2)mnt@HP-beta-cyclodextrin displayed two signs of splitting Cotton effects (CEs), with one positive CE couplet at 376 nm in the 365-410 nm region and the other negative at 277 nm in the 265-306 nm region. In addition, a dimeric host inclusion pattern of Na(2)mnt@HP-beta-cyclodextrin in solution was determined by the method of continuous variation. Density functional theory (DFT) was used to assist assignment of the ICD signals in inclusion complexes Na(2)mnt@beta-cyclodextrin and Na(2)mnt@HP-beta-cyclodextrin in combination with the well-known Harata's rule. The orientation of p --> pi* transition in Na(2)mnt chromophore was predicted by TD-DFT (time-dependent DFT) calculations to be along the C=C double bond instead of being perpendicular. Upon titrations with Zn(2+) solutions, reversals of the p --> pi* transition-relevant ICD peak and splitting CE were experimentally observed in the cases of Na(2)mnt@beta-cyclodextrin and Na(2)mnt@HP-beta-cyclodextrin, respectively, which strongly supported our hypotheses on their coconformations and the subsequent conformational changes of mnt chromophores occurring during the titration procedures. Therefore, on the basis of both the experimental data and TD-DFT calculations, the HP-beta-cyclodextrin dimer host in inclusion complex Na(2)mnt@HP-beta-cyclodextrin was disclosed, which in turn generated the exciton coupling between the two individually included guests and produced the splitting CEs as well as the reversals of CEs.

Selective binding interactions of deramciclane to the genetic variants of human α 1-acid glycoprotein

Background α 1-Acid glycoprotein (AGP) plays a decisive role in the serum protein binding of several drugs.Genetic variants of AGP have different ligand binding properties. The binding of deramciclane (DER), a chiral anxiolytic agent, has been studied on A and F1/S genetic variants of AGP. Methods The effects of DER and reference drugs on the binding of specific fluorescent and circular dichroism (CD) probes of AGP were determined. Dicumarol (DIC) binding was measured by CD and equilibrium dialysis. Results DER effectively displaced probes bound to variant A, while it was less effective at displacing probes bound to variant F1/S. DER increased the binding and inverted the induced CD spectrum of DIC in the solution of variant F1/S. This phenomenon could not be brought about by the enantiomer of DER. Conclusion DER has high-affinity binding ( K a ≥ 2×10 6 M -1) to variant A, while its binding to the variant F1/S is about thirty times weaker. During simultaneous binding of DER and DIC to variant F1/S a ternary complex having about four times higher affinity is formed, in which the opposite chiral conformation of DIC is favored. General significance The binding interactions found prove that AGP can simultaneously accommodate different ligand molecules. Even weakly bound ligands can provoke unexpected allosteric protein binding interactions.

The effect of pH on the initial rate kinetics of the dimeric biliverdin‐IXα reductase from the cyanobacterium Synechocystis PCC6803

Biliverdin-IXalpha reductase from Synechocystis PCC6803 (sBVR-A) is a stable dimer and this behaviour is observed under a range of conditions. This is in contrast to all other forms of BVR-A, which have been reported to behave as monomers, and places sBVR-A in the dihydrodiol dehydrogenase/N-terminally truncated glucose-fructose oxidoreductase structural family of dimers. The cyanobacterial enzyme obeys an ordered steady-state kinetic mechanism at pH 5, with NADPH being the first to bind and NADP(+) the last to dissociate. An analysis of the effect of pH on k(cat) with NADPH as cofactor reveals a pK of 5.4 that must be protonated for effective catalysis. Analysis of the effect of pH on k(cat)/K(m)(NADPH) identifies pK values of 5.1 and 6.1 in the free enzyme. Similar pK values are identified for biliverdin binding to the enzyme-NADPH complex. The lower pK values in the free enzyme (pK 5.1) and enzyme-NADPH complex (pK 4.9) are not evident when NADH is the cofactor, suggesting that this ionizable group may interact with the 2'-phosphate of NADPH. His84 is implicated as a crucial residue for sBVR-A activity because the H84A mutant has less than 1% of the activity of the wild-type and exhibits small but significant changes in the protein CD spectrum. Binding of biliverdin to sBVR-A is conveniently monitored by following the induced CD spectrum for biliverdin. Binding of biliverdin to wild-type sBVR-A induces a P-type spectrum. The H84A mutant shows evidence for weak binding of biliverdin and appears to bind a variant of the P-configuration. Intriguingly, the Y102A mutant, which is catalytically active, binds biliverdin in the M-configuration.

Electrochemical polymerization of helical poly(2-methoxyaniline) doped with β-cyclodextrin sulfate: pH driving the opposition of induced circular dichroism

Optically active poly(2-methoxyaniline) (PMOA) was synthesized by electrochemical polymerization of 2-methoxyaniline in the presence of β-cyclodextrin sulfate (CDS). The synthesized PMOA films were characterized by induced circular dichroism (ICD) and UV-vis spectra. PMOA films showed a mirror-imaged ICD spectrum while they were respectively prepared at pH 3 and lower pH values, which suggested that opposite one-perfected helical handedness was induced into PMOA. Nevertheless, post-treatments, such as dedoping, redoping and thermo-treating, seemed no influence on helical handedness of PMOA.

Selective plasma protein binding of antimalarial drugs to α 1-acid glycoprotein

Human plasma protein binding of six antimalarial agents of quinoline and acridine types was investigated by using spectroscopic techniques, affinity chromatography, ultrafiltration and HPLC methods. Induced circular dichroism (ICD) spectra showed binding of amodiaquine (AMQ), primaquine (PRQ), tafenoquine (TFQ), and quinacrine (QR) to α 1-acid glycoprotein (AAG), the serum level of which greatly increases in Plasmodium infections. Association constant ( K a) values of about 10 5–10 6 M −1 could be determined. Analysis of the ICD and UV spectra of the drug–AAG complexes suggested the inclusion of the ligands into the central hydrophobic cavity of the protein. Using the purified forms of the two main genetic variants of AAG, ICD data indicated the selective binding of AMQ and PRQ to the ‘F1/S’, while QR to the ‘A’ variant. Results of fluorescence experiments supported the AAG binding of these drugs and provided further insights into the binding details of TFQ and QR. Fluorescence and CD displacement experiments showed the high-affinity AAG binding of mefloquine ( K a ≈ 10 6 M −1). For this drug, inverse binding stereoselectivities were found with the ‘F1/S’ and ‘A’ genetic variants of AAG. HSA association constants estimated from affinity chromatography results lag behind (10 3–10 5 M −1) the similar values derived for AAG. In case of chloroquine, no significant binding interaction was found either with AAG or HSA. Pharmacological aspects of the results are discussed.

Species‐dependent stereoselective drug binding to albumin: A circular dichroism study

Drug binding to albumins from different mammalian species was investigated to disclose evidence of species-dependent stereoselectivity in drug-binding processes and affinities. This aspect is important for evaluating the reliability of extrapolating distribution data among species. The circular dichroism (CD) signal induced by drug binding to the albumins [human serum albumin (HSA), bovine serum albumin (BSA), rat serum albumin (RSA), and dog serum albumin (DSA)] were measured and analyzed. The binding of selected drugs and metabolites to HSA significantly differed from the binding to the other albumins in terms of affinity and conformation of the bound ligands. In particular, phenylbutazone, a marker of site one on HSA, showed a higher affinity for binding to BSA with respect to RSA, HSA, and DSA, respectively. In the case of diazepam, a marker of site two on HSA, the affinity decreased in order from HSA to DSA, RSA, and BSA. The induced CD spectra were similar in terms of energy and band signs, suggesting almost the same conformation for the bound drug to the different albumins. Stereoselectivity was high for the binding of ketoprofen to HSA and RSA. A different sign was observed for the CD spectra induced by the drug to the two albumins because of the prevalence of a different conformation of the bound drug. Interestingly, the same induced CD spectra were obtained using either the racemic form or the (S)-enantiomer. Finally, significant differences were observed in the affinity of bilirubin, being highest for BSA, then decreasing for RSA, HSA, and DSA. A more complex conformational equilibrium was observed for bound bilirubin.

An inclusion complex of β-cyclodextrin with mnt anion (mnt = maleonitriledithiolate) studied by induced circular dichroism

A new inclusion complex of β-cyclodextrin with sodium maleonitriledithiolate (Na2mnt) was investigated by electronic spectra, induced circular dichroism (ICD), and quantum mechanics (QM) methods. The orientation of the guest anion inside the host cavity was studied by ICD spectra and analyzed by structural optimization using PM3 quantum chemical method. Finally, the inclusion constant was determined by both a linear and a non-linear fitting methods, which were based on the variation of ICD signals of the guest upon inclusion complexation with the host. The inclusion constant of Na2mnt/β-cyclodextrin was estimated to be (2.45 ± 0.15) × 103 or (3.10 ± 0.11) × 103 M−1 in solution by these two fitting methods.

Synthesis and Helicity Induction of Poly(phenylacetylene)s Bearing Crown Ether Pendants. Reversible On‐Off Switching of the Induced Helical Chirality Tunable with Temperature

AbstractCis‐transoidal poly(phenylacetylene)s bearing 15‐crown‐5 (2a) and 18‐crown‐6 (2b) were prepared by stereoselective polymerization using Rh(nbd)BPh4. In the circular dichroism (CD) spectrum of 2a in the presence of the perchloric acid salt of L‐phenylglycine (L‐Pgly· HClO4), a characteristic induced CD (ICD) was observed in the absorption range corresponding to the polymer backbone. On the contrary, the 2b/L‐Pgly · HClO4 system did not show such an ICD spectrum. The ICD intensity of the 2a/L‐Pgly · HClO4 system drastically depended on temperature, i.e., the ICD disappeared at 30 °C or higher. Thus, the on‐off switching of the ICD of 2a was controllable by either the temperature modulation or the doping/undoping process of the chiral guest.

The drug binding site of human α 1-acid glycoprotein: Insight from induced circular dichroism and electronic absorption spectra

Human α 1-acid glycoprotein (AGP) is an important drug binding plasma protein which affects pharmacokinetical properties of various therapeutic agents. For the first time, interpretation of the induced circular dichroism (ICD) spectra of drug–AGP complexes is presented yielding valuable information on the protein binding environment. ICD spectra were obtained by novel ligands of which AGP induced optical activity have never been reported (primaquine, mefloquine, propranolol, terazosin, carbamazepine, rhodamine B) and by re-investigation of ICD spectra of protein-bound drugs published earlier (chlorpromazine, dipyridamole, prazosin). Spectroscopic features of the ICD and absorption bands of drugs combined with native AGP indicated chiral non-degenerate exciton coupling between the guest chromophore and the indole ring of an adjacent tryptophan (Trp) residue. Results of additional CD experiments performed by using recombinant AGP mutants showed no changes in the ligand binding ability of W122A in sharp contrast with the W25A which was unable to induce extrinsic CD signal with either ligand. Thus, these findings unequivocally prove that, likely via π–π stacking mechanism, Trp25 is essentially involved in the AGP binding of drugs studied here as well as of related compounds. Survey of the AGP binding data published in the literature support this conclusion. Our results provide a fast and efficient spectroscopic tool to determine the inclusion of ligand molecules into the β-barrel cavity of AGP where the conserved Trp25 is located and might be useful in ligand-binding studies of other lipocalin proteins.

Temperature-induced chirality reversal of induced circular dichroism of premicellar aggregates of acridine orange derivatives and dodecanoyl-l-threonine in aqueous solution

Circular dichroism (CD) and absorption spectra were measured for acridine orange derivatives: 3,6-bis(dimethylamino) acridine (AO), 3,6-bis(dimethylamino)-10-methylacridinium bromide (C1AO), and 3,6-bis(dimethylamino)-10-dodecylacridinium bromide (C12AO) in aqueous dodecanoyl-l-threonine (C12Thr) solutions at 30, 40, 50, and 60 °C and pH 8–9. CD spectra were not induced for AO and C1AO in C12Thr aqueous solutions. Induced circular dichroism (ICD) based on the exciton interaction was found for premicellar aggregates of C12AO with C12Thr, but the micellar aggregates failed to induce CD for solubilized C12AO at a higher concentration than C12Thr critical micelle concentration. The maximum ICD intensity was observed for 1:1.2 aggregates of C12AO and C12Thr. The ICD spectrum indicated positive chirality at 30 °C, but negative chirality at 50 °C. The chirality transition occurred at 40∼45 °C. The slow change in both the absorption and ICD spectra is ascribed partly to the rearrangement of dye alignment and partly to the growth of the aggregate; the system reaches final phase separation after a few days.

Binding of anti-prion agents to glycosaminoglycans: Evidence from electronic absorption and circular dichroism spectroscopy

The polyanionic glycosaminoglycans (GAGs) are intimately involved in the pathogenesis of protein conformational disorders such as amyloidosis and prion diseases. Several cationic agents are known to exhibit anti-prion activity but their mechanism of action is poorly understood. In this study, UV absorption and circular dichroism (CD) spectroscopic techniques were used to investigate the interaction between heparin and chondroitin-6-sulfate and anti-prion drugs including acridine, quinoline, and phenothiazine derivatives. UV band hypochromism of (±)-quinacrine, (±)-primaquine, tacrine, quinidine, chlorpromazine, and induced CD spectra of (±)-quinacrine upon addition of GAGs provided evidence for the GAG binding of these compounds. The association constants (∼10 6–10 7 M −1) estimated from the UV titration curves show high-affinity drug-heparin interactions. Ionic strength-dependence of the absorption spectra suggested that the interaction between GAGs and the cationic drugs is principally electrostatic in nature. Drug binding differences of heparin and chondroitin-6-sulfate were attributed to their different negative charge density. These results call the attention to the alteration of GAG-prion/GAG-amyloid interactions by which these compounds might exert their anti-prion/anti-amyloidogenic activities.

Site‐Selective Formation of Optically Active Inclusion Complexes of Alkoxo‐Subphthalocyanines with β‐Cyclodextrin at the Toluene/Water Interface

Several subphthalocyanine derivatives that contain an alkoxo substituent as an axial ligand (RO-Subpc, R = 9-anthracenemethyl, benzyl, phenyl, 3,5-dimethylbenzyl, 3,5-dimethylphenyl, 4-methylbenzyl, and 4-methylphenyl) were synthesized. The formation of inclusion complexes of RO-Subpc with beta-CD in DMSO and at the toluene/water interface was investigated by UV/Vis absorption spectroscopy, induced circular dichroism (ICD), and nuclear magnetic resonance (NMR) measurements. Interfacial tension measurements suggested that beta-CD adsorbed as a monolayer at the toluene/water interface and probably orientated towards the toluene phase with its primary face. The 1:1 composition of beta-CD.RO-Subpc inclusion complexes was confirmed in DMSO and at the toluene/water interface for BzO-Subpc, PhO-Subpc, MeBzO-Subpc, and MePhO-Subpc. A 2:1 inclusion complex of AnO-Subpc formed in DMSO. The observed ICD spectra of beta-CDRO-Subpc inclusion complexes are discussed with respect to molecular modeling and the simulation based on Tinoco-Kirkwood theory. Interestingly, the ICD spectra of beta-CD.BzO-Subpc and beta-CD.MeBzO-Subpc inclusion complexes exhibited a negative sign in DMSO and a positive sign at the toluene/water interface. This reversal of the ICD sign strongly suggests a difference in the structure of the inclusion complexes: beta-CD at the interface formed the inclusion complex with its primary face, whereas the secondary face of beta-CD bound favorably to RO-Subpc in DMSO.

Solvent- and Temperature-Tuned Orientation of Ferrocenyl Azide Inside β-Cyclodextrin

An induced circular dichroism (ICD) solution study on the orientation of ferrocenyl azide within the beta-cyclodextrin cavity is described. In DMSO, ferrocenyl azide prefers an axial inclusion, whereas in ethylene glycol and DMSO/H2O = 50/50 an equatorial alignment dominates. As shown by temperature-dependent ICD spectra, at lower temperatures ferrocenyl azide adopts preferentially an equatorial arrangement, whereas at higher temperatures an axial one is favored. Temperature and solvent effects on the co-conformation of ferrocene noncovalently bound to cyclodextrin have never been observed before.

Binding of the Pepper Alkaloid Piperine to Bovine β-Lactoglobulin: Circular Dichroism Spectroscopy and Molecular Modeling Study

The pepper alkaloid piperine is a nontoxic, natural dietary compound with a broad range of physiological activity. The present work is the first demonstration of its interaction with a mammalian protein. Circular dichroism (CD) spectroscopy was used to reveal and analyze the binding of piperine to a lipocalin protein. Induced CD spectra measured in pH 7.7 phosphate buffer at 37 degrees C demonstrated reversible, non-covalent association of piperine with bovine beta-lactoglobulin (BLG), the major whey protein in milk. The binding parameters (K(a) approximately 8 x 10(4) M(-1), n = 0.8) determined from the CD titration data showed no significant differences between the piperine binding properties of the two main genetic variants of BLG (A and B). The vanishing extrinsic CD signal obtained upon acidification of the piperine-BLG sample solution (Tanford transition) suggested that the ligand binds in the central hydrophobic cavity of the beta-barrel. The cavity binding concept was further supported by a CD displacement experiment using palmitic acid, the well-known hydrophobic ligand of BLG. Molecular docking calculations showed that piperine can be efficiently accommodated within the calyx of BLG. Additional molecular modeling calculations indicated that the beta-barrel of human tear lipocalin, human serum retinol binding protein, and human neutrophil gelatinase associated lipocalin might also accommodate a piperine molecule.

ΔΛ-Isomerism of Mixed 1,3-Diketonate Complexes of Ru(III)A Designed New Source of Chirality in Nematic Liquid Crystals

A high-power chiral dopant system involving 6-coordinate metal complexes was designed on the basis of a shape model. Accordingly, a series of mixed 1,3-diketonate complexes of Ru(III), [Ru(acac)2(L-n)], in which acac = acetylacetonate and L-n = dibenzoylmethanate substituted with n octyloxy groups (abbreviated as Ru-n hereafter), was prepared and optically resolved. Their performance as chiral dopants was evaluated in terms of helical twisting power (HTP) in a room-temperature nematic liquid crystal, N-(4-methoxybenzylidene)-4-n-butylaniline (MBBA), by measuring the helical pitch lengths and CD spectra for the induced chiral nematic phases. The Δ- and Λ-enantiomers induced macroscopic left-handed (M) and right-handed (P) helices, respectively, and the (absolute) values for HTP have proven to be remarkably large, e.g. βM = 1.8 × 102 μm-1 in the case of Λ-Ru-2. The induced CD spectra for the dilute MBBA* materials (the asterisk denotes in this paper that MBBA has been doped with a chiral substance) were fit...

Circular dichroism and absorption spectroscopic data reveal binding of the natural cis-carotenoid bixin to human α 1-acid glycoprotein

Using circular dichroism (CD) and electronic absorption spectroscopy techniques, interaction of the natural dietary cis-carotenoid bixin with an important human plasma protein in vitro was demonstrated for the first time. The induced CD spectra of bixin obtained under physiological conditions (pH 7.4, 37 °C) revealed its binding to the serum acute-phase reactant α 1-acid glycoprotein (AGP), a member of the lipocalin protein family. Spectral features of the extrinsic Cotton effects of bixin suggested the inclusion of a single, chirally distorted ligand molecule into the asymmetric protein environment. Compared with the absorption spectra obtained in ethanol and benzene, the strong red shift of the main absorption peak of AGP-bound bixin indicated that the proposed binding site was rich in aromatic residues, and also suggested that hydrophobic interactions were involved in the binding. Using the data obtained from the CD titration experiments, the association constant ( K a = 4.5 × 10 5 M −1) and stoichiometry of the binding (0.15) were calculated. The low value of the stoichiometry was attributed to the structural polymorphism of AGP. To the authors’ knowledge, the current study represents the first human lipocalin protein for which carotenoid binding affinity has been explored in vitro with these techniques.

A spectroscopic study of the inclusion of azulene by β- and γ-cyclodextrins

The inclusion of azulene (AZ) inside the cavities of β-cyclodextrin (β-CD) and γ-cyclodextrin (γ-CD) was studied using absorption, fluorescence and induced-circular dichroism spectroscopy. The inclusion of AZ into the cavity of β-CD has a stoichiometry of 1:1, whereas that of AZ/γ-CD complex is 1:2. The equilibrium constants for the formation of the two complexes were calculated to be 780 ± 150 M −1 for AZ:β-CD and (4.5 ± 0.86) × 10 5 M −2 for AZ:(γ-CD) 2. The latter is due to a stepwise equilibrium mechanism in which a 1:1 complex is formed with a binding constant of 775 M −1, followed by the formation of a 1:2 complex with a binding constant of 580 M −1. The difference between the two binding constant values is slight, indicating an almost equal contribution from each of the γ-CD molecules to the overall binding in AZ:(γ-CD) 2. From the induced-circular dichroism spectra, the inclusion of AZ was found to be axial in AZ:β-CD and nearly axial in AZ:(γ-CD) 2.

Induced Circular Dichroism and Structural Assignment of the Cyclodextrin Inclusion Complexes of Bicyclic Azoalkanes

The stoichiometries and binding constants of the host-guest complexes between the bicyclic azoalkanes 1-6 and alpha-, beta-, and gamma-cyclodextrins (CDs) and the induced circular dichroism (ICD) of the complexes were analyzed. Assisted by proximity relationships obtained from 2D ROESY NMR spectra, the signs and intensities of the ICD spectra are interpreted in terms of the solution structures (co-conformations) of the CD complexes. The ICD assignments are based on the orientation-intensity ICD rules of Harata and Kodaka, which relate the ICD signs and intensities to the relative orientation of the electric dipole transition moment of the n,pi azo chromophore to the CD axis. The influence of the size of the guest and the host is discussed and the effect of introducing an additional chromophore (either a phenyl or a second azo group) on the ICD spectra is demonstrated.