Silylation-mediated oxidation of dihydropyrimidine bases and nucleosides

Vigorous trimethylsilylation of dihydropyrimidine and dihydro-s-triazine bases and nucleosides results in their dehydrogenation to the analogous aromatic derivatives. This transformation, which cannot be effected by conventional dehydrogenating reagents, has been studied in detail using gas chromatographic and mass spectrometric methods. For 5,6-dihydro-5-azacytidine (2a) the oxidation is quantitative, requires molecular oxygen as well as reagents capable of both N- and O-silylation, is accelerated by heat and selected free radiacal initiators, and is inhibited by diethyldithiocarbamate and galvinoxyl free radical. This anomalous silylation reaction occurs on both an analytical and preparative scale. Silylation-mediated oxidation also exhibits a substantial deuterium isotope effect with preferential retention of deuterium at the new site of unsaturation. The potential of this new reaction for the synthesis of aromatic systems and for the insertion of non-exchangeable isotopic labels in suitable reduced precursors is illustrated.     

Mechanism of photochemical oxidation of bacterioviridin]

The mechanism of bacterioviridin photochemical oxidation has been studied by the methods of ESR, flash-photolysis and low-temperature spectrophotometry. ESR spectrum of pigment cation-radical, a singlet line with H=11 G, g = 2.0027, has been recorded. The bands with maxima at 370, 470, 525, 590, 840 nm correspond to bacterioviridin cation -- radical in the absorption spectra. When -- benzoquinone is used as an electron acceptor with excitation light 640 nm the product of bacterioviridin irreversible oxidation is formed with the absorption band maximum 760 nm and absorption between 350 and 370 nm. It is suggested that this product is of double-oxidized non-radical nature and the mechanism of its formation through oxidation of the pigment cation-radical is discussed. The regeneration reaction of double-oxidized bacterioviridin up to cation-radical form in the presence of triphenylamine as a reducing agent has been carried out. The rate constants of cation-radical decay in the dark and desactivation of triplet state have the following values: K1=(1,64+/-0,15)-10(3) sec-1, K2=(13+/-2,0)-10(3) sec-1 correspondingly. The activation energy of the radical decay in the dark is Eact =(13,2-0,5) kcal/mole.     

Microsomal and photochemical oxidation and reduction of 1-piperidinoanthraquinone

In microsomes of control Wistar rats, the NADPH-dependent reduction of 1-piperidinoanthraquinone (1-PA) to the corresponding hydroquinone, in the absence of oxygen, has been observed. Two facts ((i) inhibition of the formation of 1-piperidinoanthrahydroquinone (1-PAH) by metyrapone and antibodies to cytochrome P-450, and (ii) increase in the rate of 1-PAH formation upon induction of rats by phenobarbital) indicate that cytochrome P-450 participates in the reduction of 1-PA. Since 1-PA is a substrate of cytochrome P-450 and is oxidized in microsomes to (N-anthraquinonyl-1)-δ-aminovaleric acid (AAV), model experiments have been conducted to examine whether the reduced forms of 1-PA are involved in its oxidation. During photochemical generation of 1-PAH and its subsequent oxidation (N-anthraquinonyl-1)-β-aminovaleric aldehyde (AAVal) is formed. However, this product is formed without participation of activated forms of the substrate and oxygen. AAVal) is formed. However, photochemical systems, apparently, is a precursor of AAV in microsomal oxidation of 1-PA. AAVal is a substrate of cytochrome P-450 (the Type I of binding) and is oxidized quantitatively in microsomal systems to yield AAV. The data obtained enable us to propose a possible mechanism of enzyme oxidation of 1-PA.     

Fast photochemical oxidation of proteins coupled with mass spectrometry

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical footprinting approach whereby radicals, produced by UV laser photolysis of hydrogen peroxide, induce oxidation of amino acid side-chains. Mass Spectrometry (MS) is employed to locate and quantify the resulting irreversible, covalent oxidations to use as a surrogate for side-chain solvent accessibility. Modulation of oxidation levels under different conditions allows for the characterisation of protein conformation, dynamics and binding epitopes. FPOP has been applied to structurally diverse and biopharmaceutically relevant systems from small, monomeric aggregation-prone proteins to proteome-wide analysis of whole organisms. This review evaluates the current state of FPOP, the progress needed to address data analysis bottlenecks, particularly for residue-level analysis, and highlights significant developments of the FPOP platform that have enabled its versatility and complementarity to other structural biology techniques.     

Periodate oxidation in chemistry of nucleic acid. Dialdehyde derivatives of nucleosides, nucleotides, and oligonucleotides

The employment of periodate oxidation in the chemistry of nucleic acids and their components is reviewed. The reaction mechanism, structural requirements to substrates, and synthesis of dialdehyde derivatives of nucleosides, nucleotides, and oligonucleotides are discussed in the first part. The second part involves chemical, physicochemical, and biological properties of the dialdehyde derivatives, as well as their use for the affinity modifications of proteins.     

A preparative method for the enzymatic 5'-monophosphorylation of nucleosides

A simple procedure to prepare a stable preparation of bovine liver adenosine kinase was developed. The adenosine kinase was used to phosphorylate a variety of purine nucleosides using ATP or [gamma-32P]ATP as the phosphoryl donor. A convenient scheme to purify the nucleotides was also developed.     

A blood analog for laser-induced photochemical anemometry

A transparent viscoelastic blood analog fluid was developed for use with Laser-Induced Photochemical Anemometry. To provide solubility of the photochemical tracer, 1′, 3′, 3′-trimethyl-6-nitroindoline-2-spiro-2-benzopyran (TNSB dye, Kodak Chemicals), the analog solvent needed to be nonpolar, thus currently available aqueous blood analogs were not suitable. An analog consisting of 0.04% ethylhydroxyethylcellulose dissolved in gamma-butyrolactone produced a pseudoplastic steady shear response with low elasticity in unsteady shear, while being compatible with the photochemical tracer.     

Model identification of the biochemical oxidation process

A fairly general model of the biochemical oxidation, which takes into account the activity of microorganisms, is presented. Parameters of the model have been determined by fitting the model to available experimental data through the use of a straightforward gradient technique.     

The oxidation rate of high-potential c-type cytochrome in the photochemical reaction centre is temperature-independent

The temperature dependence of laser-induced (694.3 nm, 30 ns, 10 mJ.cm-2) high-potential cytochrome c (Em=+290 mV) oxidation kinetics was studied in Ectothiorhodospira shaposhnikovii chromatophores. It was shown that the rate constant of this reaction is independent of temperature in the range of 300 K to 120 K.     

Modifications generated by fast photochemical oxidation of proteins reflect the native conformations of proteins

Hydroxyl radical footprinting (HRF) is a nonspecific protein footprinting method that has been increasingly used in recent years to analyze protein structure. The method oxidatively modifies solvent accessible sites in proteins, which changes upon alterations in the protein, such as ligand binding or a change in conformation. For HRF to provide accurate structural information, the method must probe the native structure of proteins. This requires careful experimental controls since an abundance of oxidative modifications can induce protein unfolding. Fast photochemical oxidation of proteins (FPOP) is a HRF method that generates hydroxyl radicals via photo‐dissociation of hydrogen peroxide using an excimer laser. The addition of a radical scavenger to the FPOP reaction reduces the lifetime of the radical, limiting the levels of protein oxidation. A direct assay is needed to ensure FPOP is probing the native conformation of the protein. Here, we report using enzymatic activity as a direct assay to validate that FPOP is probing the native structure of proteins. By measuring the catalytic activity of lysozyme and invertase after FPOP modification, we demonstrate that FPOP does not induce protein unfolding.     

Base-selective oxidation and cleavage of DNA by photochemical cosensitized electron transfer.

A photochemical mechanism for single-strand cleavage of DNA is proposed in which a photoexcited intercalator transfers an electron to an externally bound cosensitizer. Once formed, the oxidized intercalator oxidizes an adjacent base, creating a charge-separated complex from which reactions leading to cleavage of the sugar-phosphate backbone occur in competition with back electron transfer. Using ethidium bromide (EB) as the intercalator and methyl viologen (MV) as the externally bound cosensitizer, a 10-fold enhancement in the rate of single-strand break formation was found in pBR322 DNA over that for EB alone using 488-nm excitation. The rate of cleavage correlated with the amount of MV bound to DNA. In accord with the expected redox properties of the one-electron-oxidized EB and the DNA bases, cleavage occurs selectively at guanines. Although the reaction proceeds in nitrogen-purged solutions, the rate of cleavage in air-saturated solutions was enhanced 2-fold. Treatment of irradiated samples with alkali leads to a 2-fold increase in the yield of single-strand breaks. These results support a mechanism in which cleavage occurs by selective oxidation of guanines in DNA, initiated by photochemical cosensitized electron transfer from intercalated EB to externally bound MV, and may provide a basis for the development of light-activated base-selective DNA cleaving agents.     

N-Dithiasuccinoyl (Dts)-glycine: A novel oxidation reagent for the formation of intramolecular disulfide bridges under mild conditions

Summary Specific intramolecular cyclization to form disulfide bridges in peptides represents a major challenge to the synthetic art. The N-dithiasuccinoyl (Dts) function was originally proposed as an amino protecting group, removable under mild conditions by thiolysis [Barany, G. and Merrifield, R.B., J. Am. Chem. Soc., 99 (1977) 7363; 102 (1980) 3084]. We demonstrate here that this chemistry can be inverted, i.e., Dts-amines can be used as mild oxidation reagents that promote formation of intramolecular disulfide bridges. With oxytocin and deamino-oxytocin as models, and with Dts-glycine as the oxidant, we have shown the efficacy of this method under a variety of conditions: both components in solution; dithiol-peptide on a polymeric support and Dts-glycine in solution; and soluble dithiol-peptide with Dts-glycyl-resin. Kinetics have been determined under a range of conditions in mixtures of acetonitrile-phosphate buffer. The logarithm of the reaction rate (based on a simplified first-order assumption) varies linearly with pH; the slope of these correlations is 0.68±0.02. Under suboptimal conditions, by-products have been observed that include parallel and antiparallel dimers as well as trisulfide analogues. Optimized conditions give good yields and purities of the desired disulfides. Particular advantages of this approach include the lack of reactivity of Dts-glycine with all non-sulfhydryl side-chain functionalities found in peptides, and the fact that Dts-mediated oxidation is irreversible.This work was taken in part from the Ph.D. Thesis of L. Chen, University of Minnesota, Minneapolis, MN, U.S.A., January 1997 (in preparation). Preliminary reports of this work were made at the 14th American Peptide Symposium, Columbus, OH, U.S.A., June 18–23, 1995 (poster P-119), and at the Fourth International Symposium on Solid Phase Synthesis & Combinatorial Chemical Libraries, Edinburgh, Scotland, U.K., September 12–16, 1995 (Ref. 18).     

Effect of temperature modulations on TEMPO-mediated regioselective oxidation of unprotected carbohydrates and nucleosides

Regioselective oxidation of unprotected and partially protected oligosaccharides is a much sought-after goal. Herein, we report a notable improvement in the efficiency of TEMPO-catalyzed oxidation by modulating the temperature of the reaction. Mono-, di-, and tri-saccharides are oxidized regioselectively in yields of 75 to 92%. The present method is simple to implement and is also applicable for selective oxidations of other mono- and poly-hydroxy compounds including unprotected and partially protected nucleosides.