Effects of methyl substitution on radiative and solvent quenching rate constants of [Ru(phen)2dppz]2+ in polyol solvents and bound to DNA

Methyl substituents on the distant benzene ring of the dppz ligand in the "light switch" complex [Ru(phen)(2)dppz](2+) have profound effects on the photophysics of the complexes in water as well as in the polyol solvents ethylene glycol, glycerol, and 1,2- and 1,3-propanediol. Whereas 11,12-dimethyl substitution decreases the rate of quenching by diminishing hydrogen bonding by solvent, the 10-methyl substituent in addition also decreases both the radiative and the nonradiative rate constant for decay to the ground state of the non-hydrogen-bonded excited state species. For both the 10-methyl and the 11,12-dimethyl derivatives, the effect of methyl substitution on the equilibrium of solvent hydrogen bonding to the excited state is due to changes in the entropy terms, rather than in the enthalpy, indicating that the effect is a steric perturbation of the solvent cage around the molecule. When intercalated into DNA, the effects of methyl substitution is smaller than those in polyol solvent or water, suggesting that the water molecules that quench the excited state by hydrogen bonding to the phenazine aza nitrogens mainly access them from the same groove as in which the Ru(II) ion resides. Since the Delta-enantiomer of [Ru(phen)(2)10-methyl-dppz](2+) has an absolute quantum yield of up to 0.23 when bound to DNA, a value 7000 times higher than in pure water solution, it is promising as a new luminescent DNA probe.     

Cobalt Ion Incorporation into Periodic Mesoporous Organosilica Materials Synthesized by Co-condensation of 1,2-Bistrimethoxysilylethane with 3-Glycidoxypropyltriethoxysilane

Bifunctional periodic mesoporous organosilica materials with and without cobalt ion incorporation were synthesized by co-condensation of 1,2-bistrimethoxysilylethane (BTME) with 3-glycidoxypropyltriethoxysilane (GPTS) in the presence of cetyltrimethylammonium bromide. Nitrogen gas adsorption on samples with varying ratios of BTME:GPTS revealed that increasing the amount of GPTS affects pore size, surface area and pore volume as well as shapes of the isotherms and hysteresis loops. The hysteresis loops of the Type IV isotherms obtained for GPTS-modified ethane silica materials (without cobalt ion) change from Type H3 to Type H4 with increasing GPTS content. There is a tendency for pore sizes to change from mesopore to micropore when the amount of GPTS is increased. Isotherms of cobalt ion incorporated GPTS-modified ethane silica materials change from Type IV to Type I with increasing GPTS content. The surface area, pore volume and pore diameter decrease with increasing loading of GPTS as well as after cobalt ion incorporation. Thermogravimetric analysis and differential thermal analysis show that the surfactant is removed by solvent extraction. Cobalt ion incorporation is confirmed by powder X-ray diffraction and Raman spectroscopy.     

Methods for research on immune complement activation on modified sensor surfaces

We have developed a methodological system consisting of a new surface sensitive quartz crystal microbalance with dissipation monitoring (QCM-D) sensor surfaces together with different surface modification methods for the investigation of surface associated complement activation in human sera. The QCM-D surface, 10 mm in diameter, was modified by spin-coating of poly(urethane urea) (PUUR) and polystyrene (PS). Some sensor surfaces were also sputtered with titanium (Ti) or modified by hydrophobic self-assembled monolayer (SAM) of an 18-carbon alkane thiol with a ---CH₃ end group. The amount of surface deposited complement protein was investigated by incubation of the modified sensor surfaces in human sera, followed by incubation with antibodies directed against complement factor 3c (C3c). The amounts of bound anti-C3c were then used as an arbitrary measure of surface induced complement activation. The order of complement activation of the different surfaces, as judged by three separate measurements per surface modification, was PUUR>PS=SAM>Ti. The Ti surface had a similar low degree of anti-C3c binding as the negative controls (heat inactivated sera). The novel QCM-D methodology was found to be very simple, accurate, sensitive and well suited as a screening method for complement activation and protein adsorption on different materials. We also compared the sensitivity of QCM-D method with surface plasmon resonance (SPR) for the quantification of protein adsorption and complement activation on gold sensor surfaces. The QCM-D method was equally sensitive as the SPR for the detection of protein adsorption from a solution independently if low flow rate (5 μl/min) was used. A slight increase in sensitivity was found at higher flow rate (30 μl/min). However, we found it difficult to use the SPR method on the Ti, PS and PUUR surfaces due to decreased light penetration of the modified SPR sensor chip.     

Micelle-enhanced dissociation of a Ru cation /DNA complex

Anionic surfactant monomers have a large catalytic effect on the dissociation rate constant of a Ru2(4+)-DNA complex, an effect further enhanced upon exceeding the critical micelle concentration. Electrostatic estimates are made of this effect, the effect of salt and temperature on the binding constant, and of the binding constant itself. The effects are compared with the experiment, and the calculated salt effect on the binding constant is compared with condensation theory. The results indicate that the catalytic effect is primarily nonelectrostatic (hydrophobic) in nature.     

Artificial neural network modification of simulation-based fitting: application to a protein-lipid system

Simulation-based fitting has been applied to data analysis and parameter determination of complex experimental systems in many areas of chemistry and biophysics. However, this method is limited because of the time costs of the calculations. In this paper it is proposed to approximate and substitute a simulation model by an artificial neural network during the fitting procedure. Such a substitution significantly speeds up the parameter determination. This approach is tested on a model of fluorescence resonance energy transfer (FRET) within a system of site-directed fluorescence labeled M13 major coat protein mutants incorporated into a lipid bilayer. It is demonstrated that in our case the application of a trained artificial neural network for the substitution of the simulation model results in a significant gain in computing time by a factor of 5 x 10(4). Moreover, an artificial neural network produces a smooth approximation of the noisy results of a stochastic simulation.     

Production of hybrid 16-membered macrolides by expressing combinations of polyketide synthase genes in engineered Streptomyces fradiae hosts

Combinations of the five polyketide synthase (PKS) genes for biosynthesis of tylosin in Streptomyces fradiae (tylG), spiramycin in Streptomyces ambofaciens (srmG), or chalcomycin in Streptomyces bikiniensis (chmG) were expressed in engineered hosts derived from a tylosin-producing strain of S. fradiae. Surprisingly efficient synthesis of compounds predicted from the expressed hybrid PKS was obtained. The post-PKS tailoring enzymes of tylosin biosynthesis acted efficiently on the hybrid intermediates with the exception of TylH-catalyzed hydroxylation of the methyl group at C14, which was efficient if C4 bore a methyl group, but inefficient if a methoxyl was present. Moreover, for some compounds, oxidation of the C6 ethyl side chain to an unprecedented carboxylic acid was observed. By also expressing chmH, a homolog of tylH from the chalcomycin gene cluster, efficient hydroxylation of the 14-methyl group was restored.     

Sensitive spectrophotometric determination of ascorbic acid in fruit juices and pharmaceutical formulations using 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (Br-PADAP)

A simple and very sensitive method has been developed for the determination of ascorbic acid based on the oxidation of ascorbic acid to dehydroascorbic acid by iron(III), followed by a complexation of iron(II) with 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol(Br-PADAP). The iron(II) complex is formed immediately, with absorption maxima at 560 and 748 nm and a molar absorptivity of 1.31 × 10⁵ l mole^–1cm^–1 and 5.69 × 10⁴ l mole^–1cm^–1, respectively. The ascorbic acid determination is possible with a linear range up to 2.4 μg ml^–1, a calibration sensitivity of 0.744 ml μg^–1 at 560 nm and 0.323 ml μg^–1 at 748 nm, and a detection limit of 15 ng ml^–1 and 44 ng ml^–1, respectively. The procedure was used for the ascorbic acid determination in several fruit juices and pharmaceutical formulations. The results demonstrated a good precision (R.S.D. < 1%) and are in agreement with those obtained with others methods. The Br-PADAP method proposed is six times more sensitive than the method using the iron(II)-1,10-phenanthroline system. Received: 7 May 1996 / Revised: 1 July 1996 / Accepted: 8 August 1996