Direct measurement of the energy thresholds to conformational isomerization in tryptamine: experiment and theory

The methods of stimulated emission pumping-hole filling spectroscopy (SEP-HFS) and stimulated emission pumping population transfer spectroscopy (SEP-PTS) were applied to the conformation-specific study of conformational isomerization in tryptamine [TRA, 3-(2-aminoethyl)indole]. These experimental methods employ stimulated emission pumping to selectively excite a fraction of the population of a single conformation of TRA to well-defined ground-state vibrational levels. This produces single conformations with well-defined internal energy, tunable over a range of energies from near the zero-point level to well above the lowest barriers to conformational isomerization. When the SEP step overcomes a barrier to isomerization, a fraction of the excited population isomerizes to form that product. By carrying out SEP excitation early in a supersonic expansion, these product molecules are subsequently cooled to their zero-point vibrational levels, where they can be detected downstream with a third tunable laser that probes the ground-state population of a particular product conformer via a unique ultraviolet transition using laser-induced fluorescence. The population transfer spectra (recorded by tuning the SEP dump laser while holding the pump and probe lasers fixed) exhibit sharp onsets that directly determine the energy thresholds for conformational isomerization in a given reactant-product conformer pair. In the absence of tunneling effects, the first observed transition in a given X-Y PTS constitutes an upper bound to the energy barrier to conformational isomerization, while the last transition not observed constitutes a lower bound. The bounds for isomerizing conformer A of tryptamine to B(688-748 cm(-1)), C(1)(860-1000 cm(-1)), C(2)(1219-1316 cm(-1)), D(1219-1282 cm(-1)), E(1219-1316 cm(-1)), and F(688-748 cm(-1)) are determined. In addition, thresholds for isomerizing from B to A(<1562 cm(-1)), B to F(562-688 cm(-1)), and out of C(2) to B(<747 cm(-1)) are also determined. The A-->B and B-->A transitions are used to place bounds on the relative energies of minima B relative to A, with B lying at least 126 cm(-1) above A. The corresponding barriers have been computed using both density functional and second-order many-body perturbation theory methods in order to establish the level of theory needed to reproduce experimental results. While most of the computed barriers match experiment well, the barriers for the A-F and B-F transitions are too high by almost a factor of 2. Possible reasons for this discrepancy are discussed.     

Tandem mass spectrometry with online high-flow reversed-phase extraction and normal-phase chromatography on silica columns with aqueous-organic mobile phase for quantitation of polar compounds in biological fluids

In this work, high-flow online reversed-phase extraction was coupled with normal phase on silica columns with aqueous-organic mobile phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) to quantify drug candidates in biological fluids. The orthogonal separation effect obtained from this configuration considerably reduced matrix effects and increased sensitivity for highly polar compounds as detected by selected reaction monitoring. This approach also significantly improved the robustness and limit of detection of the assays. An evaluation of this system was performed using a mixture of albuterol and bamethan in rat plasma. Assay validation demonstrated acceptable accuracy (< 8% difference) and precision (< 6% CV) for these model compounds. The system has been used for the quantitation of polar ionic compounds in biological fluids in support of drug discovery programs. This assay was used to analyze samples for a BMS proprietary compound (A) in a rat pharmacokinetic study and is shown as an example to demonstrate the precision, accuracy, and sufficient sensitivity of this system.     

Total assignment of the proton and carbon NMR spectra of the alkaloid quindoline — utilization of HMQC-TOCSY to indirectly establish protonated carbon-protonated carbon connectivities

The reisolation of the indoloquinoline alkaloid quindoline (also known as norcryptolepine) from Cryptolepis sanguinolenta is reported. The structure was unequivocally confirmed by two-dimensional nmr methods; the proton and carbon spectra were assigned for the first time. Because of congestion in the proton spectrum HMQC-TOCSY was used as an alternative to the more familiar COSY experiment. In addition to establishing proton-proton connectivities, HMQC-TOCSY affords the added benefit of providing, in an indirect sense, connectivity information between protonated carbons.     

Interplay between cubic and hexagonal phases in block copolymer solutions

The phase behavior of a symmetric styrene-isoprene (SI) diblock copolymer in a styrene-selective solvent, diethylphthalate, was investigated by in situ small-angle X-ray scattering on isotropic and shear-oriented solutions and by rheology and birefringence. A remarkable new feature in this phase diagram is the coexistence of both body-centered cubic (bcc) and hexagonally close-packed (hcp) sphere phases, in a region between close-packed spheres (cps) and hexagonally packed cylinders (hex) over the concentration range phi approximately 0.33-0.45. By focusing on the transitions among these various ordered phases during heating and cooling cycles, we observed a strong hysteresis: supercooled cylinders persisted upon cooling. The stability of these supercooled cylinders is quite dependent on concentration, and for phi > or = 0.40, the supercooled cylinders do not revert to spheres even after quiescent annealing for 1 month. The spontaneous formation of spheres due to the dissociation of cylinders is kinetically hindered in this case, and the system is apparently not amenable to any pretransitional fluctuations of cylinders prior to the cylinder-to-sphere transition. This contrasts with the case of cylinders transforming to spheres upon heating in the melt. The application of large amplitude shear to the supercooled cylinders is effective in restoring the equilibrium sphere phases.     

Developmental Responses of Amphibians to Solar and Artificial UVB Sources: A Comparative Study

Abstract— Many amphibian species, in widely scattered locations, currently show population declines and/or reductions in range, but other amphibian species show no such declines. There is no known single cause for these declines. Differential sensitivity to UVB radiation among species might be one contributing factor. We have focused on amphibian eggs, potentially the most UVB-sensitive stage, and compared their resistance to UVB components of sunlight with their levels of photolyase, typically the most important enzyme for repair of the major UV photoproducts in DNA, cyclobutane pyrimidine dimers. Photolyase varied 100-fold among eggs/oocytes of 10 species. Among three species–Hyla regilla, Rana cascadae, and Bufo boreas–for which resistance of eggs to solar UVB irradiance in their natural locations was measured, hatching success correlated strongly with photolyase. Two additional species, Rana aurora and Ambystoma gracile, now show similar correlations. Among the low-egg-photolyase species, R. cascadae and B. boreas are showing declines, and the status of A. gracile is not known. Of the two high-photolyase species, populations of H. regilla remain robust, but populations of R. aurora are showing declines. To determine whether levels of photolyase or other repair activities are affected by solar exposures during amphibian development, we have initiated an extended study of H. regilla and R. cascadae, and of Xenopus laevis, laboratory-reared specimens of which previously showed very low photolyase levels. Hyla regilla and R. cascadae tadpoles are being reared to maturity in laboratories supplemented with modest levels of UV light or light filtered to remove UVB wavelengths. Young X. laevis females are being reared indoors and outdoors. Initial observations reveal severe effects of both UVA and UVB light on H. regilla and R. cascadae tadpoles and metamorphs, including developmental abnormalities and high mortalities. Assays of photolyase levels in the skins of young animals roughly parallel previous egg/oocyte photolyase measurements for all three species.     

Imaging of small molecules in tissue sections with a new intermediate-pressure MALDI linear ion trap mass spectrometer

We describe a new intermediate-pressure MALDI linear ion trap mass spectrometer and its capabilities for imaging mass spectrometry. The instrument design is described and is characterized in terms of four performance issues (1) MALDI performance at intermediate pressure; (2) analysis of samples on non-conductive and conductive glass slides; (3) critical importance of tandem mass spectrometry (both MS² and MS³) for identification of analyte species and imaging of isobaric species; (4) capability for repeated analysis of the same tissue section. Application of the new instrument to imaging phospholipids in rat brain sections is described in detail.     

Flow-injection determination of sugars with immobilized enzyme reactions and chemiluminescence detection

Glucose is determined by reaction with gluocose oxidase to produce hydrogen peroxide which is quantified via a chemiluminescence reaction with luminol. Sucrose, maltose, lactose and fructose are determined by enzymatic conversion to glucose (using invertase, amyloglucosidase, lactase. and glucose isomerase, respectively) and subsequent determination of the glucose, All enzymes are immobilized on controlled-pore glass and contained in flow-through reactors. For glucose, sucrose, and maltose the linear log-log working range 0.2 μM-1 mM, with a detection limit of 0.1 μM; for lactose and fructose the linear working range is 3 μM-1 mM with a detection limit of 1 μM. Assay time is 2 min.     

Determination of the distribution of light, optical properties, drug concentration, and tissue oxygenation in-vivo in human prostate during motexafin lutetium-mediated photodynamic therapy

It is desirable to quantify the distribution of the light fluence rate, the optical properties, the drug concentration, and the tissue oxygenation for photodynamic therapy (PDT) of prostate cancer. We have developed an integrated system to determine these quantities before and after PDT treatment using motorized probes. The optical properties (absorption (micro(a)), transport scattering (micro(s'), and effective attenuation (micro(eff)) coefficients) of cancerous human prostate were measured in-vivo using interstitial isotropic detectors. Measurements were made at 732 nm before and after motexafin lutetium (MLu) mediated PDT at different locations along each catheter. The light fluence rate distribution was also measured along the catheters during PDT. Diffuse absorption spectroscopy measurement using a white light source allows extrapolation of the distribution of oxygen saturation StO2, total blood volume ([Hb]t), and MLu concentration. The distribution of drug concentration was also studied using fluorescence from a single optical fiber, and was found to be in good agreement with the values determined by absorption spectroscopy. This study shows significant inter- and intra-prostatic variations in the tissue optical properties and MLu drug distribution, suggesting that a real-time dosimetry measurement and feedback system for monitoring these values during treatment should be considered in future PDT studies.     

Axial ligand substituted nonheme FeIV=O complexes: observation of near-UV LMCT bands and Fe=O Raman vibrations

Axial ligand substitution of a mononuclear nonheme oxoiron(IV) complex, [FeIV(O)(TMC)(NCCH3)]2+ (1) (TMC = 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane), leads to the formation of new FeIV=O species with relatively intense electronic absorption features in the near-UV region. The presence of these near-UV features allowed us to make the first observation of Fe=O vibrations of S = 1 mononuclear nonheme oxoiron(IV) complexes by resonance Raman spectroscopy. We have also demonstrated that the reactivity of nonheme oxoiron(IV) intermediates is markedly influenced by the axial ligands.     

Nature of vibrational coupling in helical peptides: an isotopic labeling study

Infrared (IR) and vibrational circular dichroism (VCD) spectra were measured for a series of isotopically ((13)C on two or more amide Cdouble bond]O) labeled, 25 residue, alpha-helical peptides of the sequence Ac-(AAAAK)(4)AAAAY-NH(2) that were also studied in the previous paper. Theoretical IR and VCD simulations were performed for correspondingly isotopically labeled Ac-A(24)-NHCH(3) constrained to an alpha-helical conformation by use of property tensor transfer from density functional theory (DFT) calculations on Ac-A(10)-NHCH(3). The simulations predicted and experiments confirmed that the vibrational coupling constants between i, i + 1 and i, i + 2 residues differ in sign, thus leading to a reversal of the (13)C VCD pattern and explaining the large shift in the (13)C amide I frequency as reported in the previous paper. The sign of the coupling constant remained consistent for larger label separation (with the exception of i, i + 4) and for more labels with uniform separation. Such effects confirm that the isotopically labeled group vibrations are essentially only coupled to each other and are effectively uncoupled from those of the unlabeled groups. This development confirms the utility of isotopic labels for site-specific structural studies with vibrational spectra. Observed spectral effects cannot be explained by considering only transition dipole coupling (TDC) between amide oscillators, particularly for smaller label separations, but the TDC and ab initio predicted couplings roughly converge at large separation.