A fluorescence-based method for direct measurement of submicrosecond intramolecular contact formation in biopolymers: an exploratory study with polypeptides.

A fluorescent amino acid derivative (Fmoc-DBO) has been synthesized, which contains 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) as a small, hydrophilic fluorophore with an extremely long fluorescence lifetime (325 ns in H2O and 505 ns in D2O under air). Polypeptides containing both the DBO residue and an efficient fluorescence quencher allow the measurement of rate constants for intramolecular end-to-end contact formation. Bimolecular quenching experiments indicated that Trp, Cys, Met, and Tyr are efficient quenchers of DBO (k(q) = 20, 5.1, 4.5, and 3.6 x 10(8) M(-1) x s(-1) in D2O), while the other amino acids are inefficient. The quenching by Trp, which was selected as an intrinsic quencher, is presumed to involve exciplex-induced deactivation. Flexible, structureless polypeptides, Trp-(Gly-Ser)n-DBO-NH2, were prepared by standard solid-phase synthesis, and the rates of contact formation were measured through the intramolecular fluorescence quenching of DBO by Trp with time-correlated single-photon counting, laser flash photolysis, and steady-state fluorometry. Rate constants of 4.1, 6.8, 4.9, 3.1, 2.0, and 1.1 x 10(7) s(-1) for n = 0, 1, 2, 4, 6, and 10 were obtained. Noteworthy was the relatively slow quenching for the shortest peptide (n = 0). The kinetic data are in agreement with recent transient absorption studies of triplet probes for related peptides, but the rate constants are significantly larger. In contrast to the flexible structureless Gly-Ser polypeptides, the polyproline Trp-Pro4-DBO-NH2 showed insignificant fluorescence quenching, suggesting that a high polypeptide flexibility and the possibility of probe-quencher contact is essential to induce quenching. Advantages of the new fluorescence-based method for measuring contact formation rates in biopolymers include high accuracy, fast time range (100 ps-1 micros), and the possibility to perform measurements in water under air.     

The He-LiH potential energy surface revisited. II. Rovibrational energy transfer on a three-dimensional surface

We use our rigid rotor He-LiH potential energy surface [B. K. Taylor and R. J. Hinde, J. Phys. Chem. 111, 973 (1999)] as a starting point to develop a three-dimensional potential surface that describes the interaction between He and a rotating and vibrating LiH molecule. We use a fully quantum treatment of the collision dynamics on the current potential surface to compute rovibrational state-to-state cross sections. We compute excitation and relaxation vibrational rate constants as a function of temperature by integrating these cross sections over a Maxwell-Boltzmann translational energy distribution and summing over Boltzmann-weighted initial rotational levels. The rate constants for vibrational excitation of LiH are very small for temperatures below 300 K. Rate constants for vibrational relaxation of excited LiH molecules, however, are several orders of magnitude larger and show very little temperature dependence, suggesting that the collisions that result in vibrational relaxation are governed by long-range attractive interactions.     

Use of a native affinity ligand for the detection of G proteins by capillary isoelectric focusing with laser-induced fluorescence detection

Affinity probe capillary isoelectric focusing (CIEF) with laser-induced fluorescence was explored for detection of Ras-like G proteins. In the assay, a fluorescent BODIPY FL GTP analogue (BGTPgammaS) and G protein were incubated resulting in formation of BGTPgammaS-G protein complex. Excess BGTPgammaS was separated from BGTPgammaS-G protein complex by CIEF using a 3-10 pH gradient and detected in whole-column imaging mode. In other cases, a single point detector was used to detect zones during the focusing step of CIEF using a 2.5-5 pH gradient. In this case, analyte peaks passed the detector in approximately 5 min at an electric field of 350 V/cm. Detection during focusing allowed for more reproducible assays at shorter times but with a sacrifice in sensitivity compared to detection during mobilization. Resolution was adequate to separate BGTPgammaS-Ras and BGTPgammaS-Rab3A complexes. Formation of specific complexes was confirmed by adding GTPgammaS to samples containing BGTPgammaS-G protein. GTPgammaS competed with BGTPgammaS for G protein binding sites resulting in decreased BGTPgammaS-G protein peak heights. The concentrating effect of CIEF enabled detection limits of 30 pM.     

Positron binding energies for alkali hydrides

Ab initio multireference single- and double-excitation configuration interaction (MRD-CI) calculations are carried out to study the interactions of positrons with the members of the alkali hydride class of molecules. A new computer program has been constructed for this purpose that makes use of the Table-Direct-CI method for construction of the required Hamiltonian matrixes and electronic/positronic wave functions. The calculations indicate that the binding energy (positron affinity PA) of a single positron to these systems increases by an increment of 0.2-0.3 eV as the atomic number of the alkali atom is increased. It is found that the positron prefers a location in the more electronegative regions of such molecules, similarly as has been found in earlier calculations for the urea and acetone molecules. The positron orbital itself possesses a diffuse charge distribution with relatively small expectation values of the kinetic energy in all four systems considered. Each of the four positronic molecules is stable with respect to formation of either positronium (Ps) or HPs according to the present calculations. Relatively large changes in the equilibrium bond distance of the hydrides occur as a result of the positron interaction. The importance of bond dipole moments in producing the binding of positrons to molecules is discussed, as well as the role that the electronegativity of the constituent atoms plays in determining the magnitude of the PA for a given system.     

Potential Mechanisms of Photodynamic Inactivation of Virus by Methylene BlueI. RNA–Protein Crosslinks and Other Oxidative Lesions in Qβ Bacteriophage

A spectrum of oxidative lesions was observed in a bacteriophage-based model system that is very sensitive to the photodynamic activity of selected dyes. When suspensions of the intact bacteriophage Qβ were exposed to methylene blue plus light (MB+L), inactivating events, or "hits" occurred that were oxygen-dependent and that were associated with the formation of several specific lesions: (1) carbonyl moieties on proteins, (2) 8-oxo-7,8-dihydroguanine (8-oxoGua), and (3) single-strand breaks (ssb) in the RNA genome and (4) RNA-protein crosslinks. Formation of carbonyl groups associated with protein in the Qβ phage preparation correlated positively with photoinactivation of the phage with increasing doses of either of the sensitizers MB or rose bengal. Strand breaks in the Qβ genomic RNA were observable at high MB concentrations but appeared not to be significant at the lower concentrations of MB, as full-length Qβ RNA was observable well beyond the 99% inactivation point in MB dosage. It was shown that the number of 8-oxoGua lesions were unlikely to be sufficient to account for the number of lethal events. Following exposure to MB+L, crosslink formation between Qβ RNA and protein was observed by virtue of the location of RNA at the interface of phenol-aqueous extractions of phage suspensions. A significant increase over background of RNA-protein complexes (including full-length Qβ RNA) was observed at the lowest concentration of MB tested (0.5 μ M ), which corresponded roughly to an average of 2 lethal hits per phage or approximately 13% survival compared to the zero MB control (100% survival). Due to its close correlation with Qβ inactivation and its expected lethality, RNA-protein crosslink formation may be important as an inactivating lesion in bacteriophage Qβ following MB+L exposure.     

Ion chromatographic analysis of cyanate in gold processing samples containing large concentrations of copper(I) and other metallocyanide complexes

An ion chromatographic (IC) method was developed for the determination of cyanate in gold cyanidation samples containing large concentrations of metallo-cyanide complexes. The analysis was performed on a Waters HC IC-Pak A anion-exchange column with an anthranilic acid eluent, with detection achieved using indirect UV at 355 nm. Two procedures were developed for removal of the metallo-cyanide complexes prior to the IC analysis. The first was a manual off-line method which used solid-phase extraction cartridges containing a strong anion-exchange resin to trap the complexes and to then enable determination of cyanate without interference. In the second approach, an automated on-line method was developed which used an anion-exchange guard column to trap the complexes and a column switching valve to allow backflushing of the cyanate from the guard column. This enabled the total analysis to be performed in a time of 10–14 min, depending on the sample composition. Finally, a comparison of results obtained by the standard Kjeldahl nitrogen method for cyanate and the IC method revealed an interference in the Kjeldahl method for samples containing large concentrations of Cu(I)-cyanide complexes.     

Nucleophile Ringöffnung von α-Nitrocyclopropancarbonsäure-arylestern mit sterisch geschützter,aber elektronisch wirksamer Carbonyl- und Nitro-Gruppe. Ein neues Prinzip der α-Aminosäure-Synthese (2-Aminobutansäure-a4-Synthon)

Nucleophilic Ring Opening of Aryl α-Nitrocyclopropanecarboxylates with Sterically Protected but Electronically Effective Carbonyl and Nitro Group. A New Principle of α-Amino Acid Synthesis (2-Aminobutanoic Acid a⁴-Synthon) The readily available 2,4,6-tri(tert-butyl)-and 2,6-di(tert-butyl)-4-methoxypahenol esters 2 of α-nitrocyclo-propanecarboxaylic acid ring opening with C-, N-, O-, and S-nucleophiles (cyanide, malonate, azide, anilines, alkoxides, phenoxides, thiolates) in DMF or alcohol solvents (80–95% yield). The products 6 – 14 are 2-nitrobutanoates with the newly introduced substituent in the 4-position. Reduction of the NO₂ group with Zn/AcOH/Ac₂O gives N-acetyl-α-amino acid esters 16 – 22 (40–90% yield). Subsequent oxidative cleavage (H₂O₂/HCOOH) of The p-methoxy-phenyl esters 18 and 20 produces free amino acids (65% 23 and 67% 24 , respectively). Thus, the nitro ester 2 corresponds to a 2-aminobutanoic-acid a⁴-synthon, it is a ‘homo-Michael acceptor’ producing γ-substituted α-amino acids.     

The bent RC  N linkage: An AB initio study of NH2CN,NF2CN and PF2CN

The ab initio energies, nuclear and electron repulsions and charge distributions have been calculated using moderately large basis sets as a function of the RC  N angle (R  NH₂, NF₂ or PF₂). The optimum RC  N angles were calculated to be 178.9°, 176.6°, and 175° for NH₂CN, NF₂CN, and PF₂CN, respectively. A rationalization of the differing bends is presented in terms of nuclear-nuclear and electron-electron repulsions.     

Stereospecific synthesis of dienone iron tricarbonyl complexes by friedelcrafts acylation

Acylation of diene Fe(CO)₃ complexes using the Perrier complexes RCOCl/AlCl₃ in methylene chloride at 0°C gives dienone complexes in high yield. Substitution occurs only at unsubstituted terminal carbons of the diene unit. Quenching the reaction mixtures in cold aqueous ammonia gives cis dienone complexes only. Trans dienone complexes are prepared by subsequent isomerization in methanolic sodium methoxide. Formylation of diene Fe(CO)₃ complexes proceeds in modest yield using dichloromethylmethyleter/AlCl₃ in methylene chloride to give trans-dienal complexes. Reduction of the dienone and dienal complexes as well as those of dienols and dienoic esters with 4 : 1 AlCl₃/LiAlH₄ results in complete removal of the oxygen function to give trans-diene complexes in good yield.     

Facilitated transport from ternary cation mixtures through water—chloroform—water membrane systems containing macrocyclic ligands

Cation fluxes were determined for various three-component, equimolar mixtures of alkali metal, alkaline earth, and Pb²⁺ cations in a H₂O---CHCl₃---H₂O liquid membrane system incorporating macrocyclic polyethers as carriers. Carrier ligands studied were 18-crown-6, dicyclohexano-18-crown-6, 1,10-diaza-18-crown-6, 21-crown-7, dibenzo-24-crown-8, and cryptand [2.2.2]. Correlations were found between transport and relative cation:polyether cavity radii, the type of substituents present on the polyether ring, and the type and number of donor atoms present. All the ligands studied transported Pb²⁺ at higher rates than the other Mⁿ⁺ in the mixtures. Transport behavior in these multi-cation systems can be predicted from Mⁿ⁺—polyether complex stability constant data in most cases.