Time-dependent pump-probe spectra of NeBr2

Time- and frequency-resolved pump-probe measurements on NeBr2 have been performed to better characterize its fragmentation dynamics on the B electronic state for vibrational levels in the energy region of the transition from direct vibrational predissociation to intramolecular vibrational relaxation dynamics. Above nu'=20 of the Br2 stretching mode, it was observed that the dependence of lifetime on the vibrational quantum number deviates from the energy-gap law by leveling off in the range of 10 psE transitions of the complex. These transitions are shifted 20 cm(-1) to lower energy from the free Br2 resonances, indicating an E state Ne-Br2 bond energy of 82 cm(-1). Measurements of NeBr2 vibrational predissociation via the delta nu=-2 channel were also performed for nu'=27, 28, and 29. The closing of the delta nu=-1 channel leads to an increase in the lifetimes of these vibrational levels. A new Nd:yttrium aluminum garnet pumped dual optical parametric oscillator/optical parametric amplifier system is described that allows us to conveniently record time-delayed pump-probe spectra with 2-cm(-1) spectral resolution and 15-ps time resolution.     

A fluorescent molecular imaging probe with selectivity for soluble tau aggregated protein

Soluble forms of aggregated tau misfolded protein, generally termed oligomers, are considered to be the most toxic species of the different assembly states that are the pathological components of neurodegenerative disorders. Therefore, a critical biomedical need exists for imaging probes that can identify and quantify them. We have designed and synthesized a novel fluorescent probe, pTP-TFE for which binding and selectivity profiles towards aggregated tau and Aβ proteins were assessed. Our results have shown pTP-TFE to be selective for early forms of soluble tau aggregates, with high affinity of dissociation constants (K_d) = 66 nM, and tenfold selectivity over mature tau fibrils. Furthermore, we found that pTP-TFE is selective for tau over Aβ aggregates and had good cell permeability. This selectivity of pTP-TFE towards early forms of aggregated tau protein ex vivo was also supported with studies on human brain tissue containing tau and Aβ pathology. To the best of our knowledge, this is the first fluorescent molecule to be reported to have this form of selectivity profile, which suggests that pTP-TFE is a unique probe candidate for imaging-based detection of early stages of Alzheimer''s disease and other tauopathies.

pTP-TFE imaging probe can distinguish soluble tau aggregated proteins from other aggregated proteins enabling earlier detection of neurodegenerative diseases.     

Chromatographic analysis of hydrocarbons in marine sediments and seawater.

The low concentration of hydrocarbons anticipated in pollution baseline studies necessitates the development of analytical techniques sensitive at the sub-microgram per kilogram concentration level. The method of analysis developed in this laboratory involves dynamic headspace sampling for volatile hydrocarbon components of the sample, followed by coupled-column liquid chromatography for the non-volatile components. These techniques require minimal sample handling, reducing the risk of sample component loss and/or sample contamination. Volatile sample components are separated from the matrix in a closed system and concentrated on a TENAX-GC packed pre-column, free from large amounts of solvent and ready for GC/GC-MS analysis. Non-volatile compounds, such as the benzpyrenes, may be extracted from large volumes of water and concentrated on a Bondapak C18 packed pre-column for coupled-column liquid chromatographic separation and analysis. Results of the application of these techniques to the analysis of samples from sites of known low level hydrocarbon contamination are presented and discussed.     

Novel methyl helianthrones as photosensitizers: synthesis and biological evaluation

A combination of light, oxygen and a photosensitizer is used to induce death of cancer cells by photodynamic therapy. In this study, we have synthesized several new methyl helianthrone derivatives and compared their phototoxicity with that of hypericin. In contrast to hypericin, methyl helianthrones are soluble in aqueous solutions and have a broad range of light absorbance, which allows the use of polychromatic light. Structural modifications of methyl helianthrone demonstrated that substitution of hydrogen atoms of methyl helianthrone at Positions 2 and 5 with Br atoms or methylation of its phenolic hydroxyls, significantly increases the corresponding singlet oxygen quantum yield and their phototoxicity toward alphaT3-1, M2R and LNCaP cells. The phototoxicity of some of these compounds was similar to that of hypericin. Methyl helianthrones, like hypericin, accumulated mainly in the perinuclear region as evident by confocal microscopy. Irradiation of cells pretreated with methyl helianthrone derivatives generates intracellular reactive oxygen species and lipid free radicals, as shown by a fluorescentic probe and electron paramagnetic resonance methods, respectively. The phototoxicity of these methyl helianthrones as well as their ability to oxidize membrane lipids were significantly decreased on addition of specific Type-II inhibitors, suggesting the involvement of singlet oxygen as the main oxidant.     

THE EFFECT OF LIPOSOMES'' MEMBRANE COMPOSITION ON THE BINDING OF THE PHOTOSENSITIZERS HPD AND PHOTOFRIN II

Abstract— Photofrin II (PF-II) is the commercial name of the active photosensitizer which is used in photodynamic therapy of cancer. The effect of the composition of lipid membranes on the binding of PF-II was studied and compared to hematoporphyrin derivative (Hpd), which is a complex mixture of porphyrins and from which PF-II is separated. We find that increasing the content of cholesterol in the bilayer decreases the partitioning of PF-II into the bilayer, similar to what we have found earlier with Hpd. However, inserting DMPC or DPPC into the membrane, which was shown to decrease the binding of Hpd, causes the opposite trend with PF-H. A membrane fluidizer such as benzyl alcohol also has different effects on the membrane binding of Hpd and PF-II. The rate of binding of PF-II to a lipid membrane is about 10 times lower than that of Hpd. These results as well as I^- quenching of the fluorescence of the two porphyrins indicate that PF-II is immersed less homogeneously and deeper in the bilayer than Hpd. The unique additive-dependent binding of PF-II to lipid membranes calls for care in using Hpd as a model photosensitizer.     

Effects of alkali metal halide salts on the hydrogen bond network of liquid water

Measurements of the oxygen K-edge X-ray absorption spectrum (XAS) of aqueous sodium halide solutions demonstrate that ions significantly perturb the electronic structure of adjacent water molecules. The addition of halide salts to water engenders an increase in the preedge intensity and a decrease in the postedge intensity of the XAS, analogous to those observed when increasing the temperature of pure water. The main-edge feature exhibits unique behavior and becomes more intense when salt is added. Density functional theory calculations of the XAS indicate that the observed red shift of the water transitions as a function of salt concentration arises from a strong, direct perturbation of the unoccupied molecular orbitals on water by anions, and does not require significant distortion of the hydrogen bond network beyond the first solvation shell. This contrasts the temperature-dependent spectral variations, which result primarily from intensity changes of specific transitions due to geometric rearrangement of the hydrogen bond network.     

Chemically modified "polar patch" mutants of subtilisin in peptide synthesis with remarkably broad substrate acceptance: designing combinatorial biocatalysts

A significant enhancement of the applicability of the serine protease subtilisin Bacillus lentus (SBL) in peptide synthesis was achieved by using the strategy of combined site-directed mutagenesis and chemical modification to create chemically modified mutant (CMM) enzymes. The introduction of polar and/or homochiral auxiliary substituents, such as X=oxazolidinones, alkylammonium groups, and carbohydrates at position 166 at the base of the primary specificity S(1) pocket created SBL CMMs S166C-S-X with strikingly broad structural substrate specificities. These CMMs are capable of catalyzing the coupling reactions of not only L-amino acid esters but also D-amino acid esters as acyl donors with glycinamide to give the corresponding dipeptides in good yields. These powerful enzymes are also applicable to the coupling of L-amino acid acyl donors with alpha-branched acyl acceptor, L-alaninamide. Typical increases in isolated yields of dipeptides of 60-80 % over SBL-WT (e.g. 0 % yield of Z-D-Glu-GlyNH(2) using SBL-WT-->74 % using S166C-S-(CH(2))(2) NMe(3) (+)) demonstrate the remarkable synthetic utility of this "polar patch" strategy. Such wide-ranging systems displaying broadened and therefore similarly high, balanced yields of products (e.g. 91 % Z-L-Ala-GlyNH(2) and 86 % yield of Z-D-Ala-GlyNH(2) using S166C-S-(3R,4S)-indenooxazolidinone) may now allow the use of biocatalysts in parallel library synthesis.     

Single-molecule fluorescence spectroscopy of TOTO on poly-AT and poly-GC DNA

Excited state lifetime and amplitude measurements were made on thiazole orange dimer (TOTO), a dimeric DNA-intercalating fluorophore, at single-molecule concentrations. As expected from previous study, the excited state lifetime of TOTO intercalated in DNA is dependent on the sequence of the double-stranded DNA, having values of 2.2 ns in poly-GC DNA and 1.8 ns in poly-AT DNA. The distribution of excited state lifetimes of single molecules of TOTO intercalated into oligonucleotides having varying proportions of poly-GC sequences relative to poly-AT sequences were analyzed as a function of the fraction of poly-GC. By using excited state lifetime distributions from the purely GC and purely AT oligonucleotides as a basis set, it was possible to estimate the GC content of oligonucleotides with intermediate GC composition to within a few percent error. This serves as a model for the analysis of equilibrium binding distributions in DNA using single-molecule methods.     

Evidence for ionic samarium(II) species in THF/HMPA solution and investigation of their electron-donating properties

The fundamental nature of samarium(II) complexes in THF/HMPA (HMPA = hexamethylphosphoramide) solutions containing SmI2 has been clarified by means of cyclic voltammetry, conductivity measurements, UV spectroscopy, and kinetic measurements. The principal species is not [SmI2(hmpa)4] as previously suggested, but either the ionic cluster [Sm(hmpa)4(thf)2+2I- if four equivalents of HMPA is present in the THF solution or [Sm(hmpa)6]2+ 2I- in the presence of at least 10 equivalents of HMPA. The formal potential of the [Sm(hmpa)4(thf)2]3+ 2I-/[Sm(hmpa)4(thf)2]2- 2I- redox couple determined by cyclic voltammetry was -1.79 +/- 0.08 V versus SCE. The order of reactivity of the samarium(II) complexes was found to be [Sm(hmpa)6]2+2I- > [Sm(hmpa)4(thf)2]2+2I- > SmI2 in their respective reactions with 1-iodobutane and with benzyl chloride. Very high rate enhancements, of the order of 1,000-15,000-fold, were observed upon addition of HMPA to the THF solution containing SmI2, Comparison of these rate constants with the corresponding rate constants for electron transfer (ET) reactions involving aromatic radical anions revealed that none of the reactions studied can be classified as outer-sphere ET processes and that the inner-sphere electron-donating abilities of the [Sm(hmpa)4(thf)2]2+ 2I- and SmI2 complexes are comparable. The inner-sphere ET character of the transition state increases on going from 1-iodobutane and benzyl bromide to benzyl chloride and acetophenone.     

The importance of ground-state vibronic mixing in molecular Raman scattering

We consider Albrecht's theory for Raman scattering of fundamentals in the far and pre-resonance regions. Destructive interferences inherent to the A and B terms augment the conventionally suppressed C term dramatically. Raman excitation profiles for the ν₁(a_1g) and ν₆(e_2g fundamentals in benzene can be well fitted with theoretical C-term profiles involving the ¹E_1u(π-π^*) state at 1800 A.