Gert van der Zwan

Excited state intramolecular proton transfer in some tautomeric azo dyes and schiff bases containing an intramolecular hydrogen bond

Photophysical properties of several basically important aromatic azodyes (1-phenylazo-2-naphthol and 2-phenylazo-1-naphthol) and Schiff bases (N-(2-hydroxy-1-naphthylmethylidene) aniline and N-(1-hydroxy-2-naphthylmethylidene) aniline) all containing an intramolecular hydrogen bond were studied by both steady-state and time-resolved fluorescence spectroscopy with temperatures down to 98 K. It was found that the fluorescence results from the quinone form (H-form) only. The enol form (A-form) undergoes rapid excited state intramolecular proton transfer (ESIPT) resulting in the excited H-form. The compounds have relatively low quantum yields at room temperature, which increase considerably at low temperatures. Lifetime data at the different temperatures indicate that a substitution by both acceptor or donor groups on the para position in the phenyl ring decreases the deactivation rate and hence results in increased lifetime.

Structural rationalization of novel drug metabolizing mutants of cytochrome P450 BM3

Three newly discovered drug metabolizing mutants of cytochrome P450 BM3 (van Vugt‐Lussenburg et al., Identification of critical residues in novel drug metabolizing mutants of Cytochrome P450 BM3 using random mutagenesis, J Med Chem 2007;50:455–461) have been studied at an atomistic level to provide structural explanations for a number of their characteristics. In this study, computational methods are combined with experimental techniques. Molecular dynamics simulations, resonance Raman and UV–VIS spectroscopy, as well as coupling efficiency and substrate‐binding experiments, have been performed. The computational findings, supported by the experimental results, enable structural rationalizations of the mutants. The substrates used in this study are known to be metabolized by human cytochrome P450 2D6. Interestingly, the major metabolites formed by the P450 BM3 mutants differ from those formed by human cytochrome P450 2D6. The computational findings, supported by resonance Raman data, suggest a conformational change of one of the heme propionate groups. The modeling results furthermore suggest that this conformational change allows for an interaction between the negatively charged carboxylate of the heme substituent and the positively charged nitrogen of the substrates. This allows for an orientation of the substrates favorable for formation of the major metabolite by P450 BM3. Proteins 2008.

Phosphorescence for Sensitive Enantioselective Detection in Chiral Capillary Electrophoresis

Enantioselective phosphorescence lifetime detection was combined with chiral cyclodextrin-based electrokinetic chromatography for the analysis of camphorquinone (CQ). A time-gated detection system based on a pulsed light-emitting diode for excitation at 465 nm was developed for the online lifetime determination. The background electrolyte for the chiral separation consisted of 20 mM alpha-cyclodextrin (alpha-CD), 10 mM carboxymethyl-beta-CD, and 25 mM borate buffer at pH 9.0. The separation of (+)-CQ and (-)-CQ is caused by a difference in association constants of these enantiomers with alpha-CD. Under the separation conditions, different phosphorescence lifetimes were obtained for (+)-CQ and (-)-CQ (tau = 384 +/- 8 and 143 +/- 5 micros, respectively), which could be used to distinguish the enantiomers. This selectivity in detection is based on a difference in protection of the enantiomers against phosphorescence quenching after their complexation with alpha-CD. Concentration detection limits were 2 x 10(-7) and 1 x 10(-6) M for (+)-CQ and (-)-CQ, respectively. After correction for the lifetime shortening by triplet-triplet annihilation at higher CQ concentrations, a linear dynamic range was obtained from the detection limit up to 2 mM. The system was used to determine the enantiomeric impurity levels of commercial samples of (+)-CQ and (-)-CQ; 0.2% and 0.1%, respectively.

Characterization of hybrid bilayer membranes on silver electrodes as biocompatible SERS substrates to study membrane-protein interactions

Hybrid bilayer lipid membranes (HBMs) were built on roughened silver electrodes exhibiting surface-enhanced Raman scattering (SERS) activity. The HBM consisted of a first layer of octadecanethiol (ODT) directly bound to the electrode surface, on which a second layer of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) was obtained by self-assembled phospholipid vesicle fusion. The electrochemical properties of the HBM were investigated in situ by cyclic voltammetry (CV), AC voltammetry and electrochemical impedance spectroscopy (EIS). The results indicate that our HBMs are well-formed, and their insulating properties are comparable to those observed for HBM supported by smooth metal substrates. The interaction between the bilayer and the human enzyme cytochrome P450 2D6 (CYP2D6) was investigated. Surface-enhanced resonance Raman scattering (SERRS) measurements in combination with AC and EIS, performed on the same electrode sample, proved that the CYP2D6 is immobilized on the HBM without evident alterations of its active site and without significant perturbations of the bilayer architecture. This study yields novel insights into the properties of HBMs built on roughened surfaces, providing in situ electrochemical characterization of a substrate which is suitable for studying peripheral membrane proteins with SERRS spectroscopy.

Temperature dependent absorption spectroscopy of some tautomeric azo dyes and Schiff bases

The spectral properties of several aromatic azo dyes of fundamental importance (4-phenylazo-1-naphthol, 1-phenylazo-2-naphthol and 2-phenylazo-1-naphthol) and Schiff bases (N-(2-hydroxy-1-naphthylmethylidene)aniline and N-(1-hydroxy-2-naphthylmethylidene)aniline) are investigated at temperatures down to 100 K. The observed spectral changes are interpreted in terms of the existing tautomeric equilibrium and hydrogen-bonding (H-bonding) with the solvent. In non-polar solvents the decrease in temperature leads, in all compounds except 4-phenylazo-1-naphthol, to a full transformation of the enol tautomeric form (A) to the quinone form (H). The temperature dependence of the equilibrium A ⇌ H exhibits a shift around 240 K. In ethanol, due to intermolecular H-bonding, in the case of the azo dyes full transformation was not observed, while the behavior of the Schiff bases is the same as in non-polar solvents. The thermodynamic parameters of the equilibrium A ⇌ H were estimated in both non-polar solvent and ethanol for all investigated compounds.

Substitution effects on the photophysical characteristics of the salicylic anion

Spectral and photophysical properties of substituted salicylic anions are investigated by steady-state and time-resolved fluorescence spectroscopy for a variety of electron donating substituents at the positions para to the hydroxyl and carboxylic groups. Next to the usual excited-state intramolecular proton transfer in aqueous solution, an excited-state intermolecular proton transfer is found to be responsible for the dual emission observed in the case of 5-aminosalicylic anions.

Active-site structure, binding and redox activity of the heme–thiolate enzyme CYP2D6 immobilized on coated Ag electrodes: a surface-enhanced resonance Raman scattering study

Surface-enhance resonance Raman scattering spectra of the heme–thiolate enzyme cytochrome P450 2D6 (CYP2D6) adsorbed on Ag electrodes coated with 11-mercaptoundecanoic acid (MUA) were obtained in various experimental conditions. An analysis of these spectra, and a comparison between them and the RR spectra of CYP2D6 in solution, indicated that the enzyme’s active site retained its nature of six-coordinated low-spin heme upon immobilization. Moreover, the spectral changes detected in the presence of dextromethorphan (a CYP2D6 substrate) and imidazole (an exogenous heme axial ligand) indicated that the immobilized enzyme also preserved its ability to reversibly bind a substrate and form a heme–imidazole complex. The reversibility of these processes could be easily verified by flowing alternately solutions of the various compounds and the buffer through a home-built spectroelectrochemical flow cell which contained a sample of immobilized protein, without the need to disassemble the cell between consecutive spectral data acquisitions. Despite immobilized CYP2D6 being effectively reduced by a sodium dithionite solution, electrochemical reduction via the Ag electrode was not able to completely reduce the enzyme, and led to its extensive inactivation. This behavior indicated that although the enzyme’s ability to exchange electrons is not altered by immobilization per se, MUA-coated electrodes are not suited to perform direct electrochemistry of CYP2D6.

Spectroscopy and kinetics of tyrosinase catalyzed trans-resveratrol oxidation

The spectroscopy and kinetics of the tyrosinase catalyzed trans-resveratrol oxidation were investigated by measuring both UV-vis absorption spectra over the 200-500 nm range and Raman spectra over the 600-1800 cm(-1) region. Room temperature UV-vis absorption spectra, as a function of time, showed the presence of two isosbestic points located at λ(1) = 270 nm and λ(2) = 345.5 nm delimiting two different regions: the reactant region around 300 nm, where the absorption decreased with time, and the product region over the low wavelength (λ < 260 nm) and high wavelength (λ > 390 nm) wavelength zone in which the absorption increased with time until, in both cases, constant values were achieved. A first-order kinetics was deduced with a rate coefficient of k(1) = (0.10 ± 0.001) min(-1), which turned out to be independent of substrate concentration over the 50-5 μM range; a feature that was rationalized by invoking the limiting case of the Michaelis-Menten scheme appropriate for substrate concentration much lower than the respective Michaelis constant. The observation of the distinct resonance enhanced Raman lines, specifically those peaking at 830 cm(-1), 753 cm(-1), and 642 cm(-1) together with their time evolution, permitted us to gain insight into some crucial features and steps of the catalytic reaction. Namely, that the formation of the so-called trans-resveratrol and tyrosinase (S)P complex with its O-O bridge plays a crucial role in the first steps of this enzymatic reaction and that the hydroxylation of the ortho C-H bond of the trans-resveratrol OH group occurs after O-O bond cleavage in the tyrosinase active site. The present study makes clear that a class of potential inhibitors of tyrosinase can be found in compounds able to bind the two Cu (II) ions of the enzyme bidentate form.

Absence of 633-nm laser irradiation-induced effects on glucose phosphorylation by hexokinase.

In a paper by Amat et al. (Modification of the intrinsic fluorescence and biochemical behavior of adenosine triphosphate ATP after irradiation with visible and near-infrared laser light, J. Photochem. Photobiol. B 81 (2005) 26-32) it was shown that the conversion of glucose to glucose-6-phosphate by hexokinase in vitro was accelerated when ATP, which supplies the reaction with energy, was priorly irradiated at non-resonant optical frequencies (NROF, i.e., 655 and 830 nm). Correspondingly, the authors postulated that NROF may lower the energy barrier for the dephosphorylation of ATPs terminal phosphate and thus accelerate the reaction rate through a more expeditious energy delivery. Next to the established photobiostimulatory influence of visible light on cells, which is mediated by cytochrome c oxidase through resonant effects of light, Amat et al. posited an interesting theory with which the same processes could be induced through non-resonant effects. To investigate the effects of NROF with respect to the hexokinase reaction in greater detail, the reaction rates were measured spectrofluorometrically after 633-nm laser irradiation of ATP, the ATP-Mg complex, hexokinase, and the entire reaction mixture at room temperature (22 degrees C) and at the optimal reaction temperature (30 degrees C). No differences in reaction rates between the NROF-irradiated and control groups were found at either temperature. The hypothesis that NROF enhances in vitro hexokinase activity by lowering the activation energy for the dephosphorylation of ATPs terminal phosphate by hexokinase was therefore disproven. Consequently, it is questionable, albeit not unequivocal, that NROF exerts an effect on other ATP-driven reactions in cell metabolic pathways through a direct impact on ATP.

Anomalous photophysics of H1 antihistamines in aqueous solution.

Electronic absorption, emission, and excitation spectra, and fluorescence lifetimes of two H1 antihistamines--tripelennamine and mepyramine--are investigated in detail to ascertain their usefulness as fluorescent probes for ligand binding to G-protein coupled receptors. The photophysical behavior of these compounds in aqueous solution is complex due to the presence of three protonable nitrogens, intramolecular hydrogen bonding, quenching due to the formation of a charge transfer state, and intramolecular fluorescence resonance energy transfer. At physiological pH values, anomalous photophysical behavior is observed: the compounds are found to be in a ground-state equilibrium mixture of two species, one with the alkylamine tail involved in an intramolecular hydrogen bond and a second without such a bond. This internal hydrogen-bonded tail has a profound effect on the ground and excited-state properties of both tripelennamine and mepyramine, which is further elucidated by comparing them to the reference compounds 2-aminopyridine and 2-(N,N-dimethylamino)pyridine.