THE MECHANISM OF PHOTOCHEMICAL ADDITION OF CYSTEINE TO URACIL AND FORMATION OF DIHYDROURACIL

Abstract— A proposed mechanism for the photochemical addition of L -cysteine to uracil with the concurrent formation of dihydrouracil is shown to proceed through the triplet excited state of uracil which can abstract hydrogen atoms from cysteine to form dihydrouracil. This triplet state is the same one as that leading to photodimerization. The thiyl radicals generated add to ground state uracil molecules. The data permit a re-evaluation of the quantum yield for intersystem crossing of uracil in water which shows dimerization in aqueous solution to have a maximum efficiency of 56 per cent. The formation of the cross-adduct and dihydrouracil may be sensitized but the efficiency of the reaction is related to the ability of the sensitizer to be photoreduced and not to its triplet energy.     

A new sample preparation and separation combination for the precise,accurate, and simultaneous determination of uracil and dihydrouracil in human plasma by reversed‐phase HPLC

We have developed an efficient procedure and detection method using reversed‐phase high‐performance liquid chromatography for the simultaneous measurement of uracil and dihydrouracil in human plasma. The procedure, including chromatographic conditions and sample preparation, was optimized and validated. Optimization of the sample preparation included deproteinization, extraction, and cleanup. A new sample preparation method which resulted in an improved extraction yield of analytes and significantly reduced interference at low‐wavelength UV detection was developed. The developed method was validated for specificity, linearity, limits of detection and quantitation, precision, and accuracy. All calibration curves showed excellent linear regression (R ² > 0.9990) within the testing range. The limit of detection for uracil and dihydrouracil was 2.5 and 5.0 ng/mL, respectively. The extraction yields were >94% for uracil and 91% for dihydrouracil. Intra‐ and interassay precision and accuracy for uracil and dihydrouracil were lower than 8% at all tested concentrations. The proposed method was successfully applied to measure plasma concentrations of uracil and dihydrouracil in colorectal cancer patients scheduled to receive fluoropyrimidine‐based chemotherapy.     

PHOTOCHEMICAL REACTION BETWEEN PROTEIN AND NUCLEIC ACIDS: PHOTOADDITION OF TRYPTOPHAN TO PYRIMIDINE BASES*1

Abstract— The photochemical interactions between tryptophan and nucleic acid bases were studied. When aqueous solutions of tryptophan were irradiated (Λ > 260 nm) at neutral pH in the presence of each of the nucleic acid bases, pyrimidine bases but not purine bases were altered. Air was found to decrease the rate of reaction. Two classes of photoproducts were isolated by thin layer and ion-exchange chromatography and partially characterized. One was the dihydro-pyrimidine forms of the base (see Reeve and Hopkins, 1979) and the other consisted of tryptophan-pyrimidine photoadducts. Four tryptophan-uracil and two tryptophan-thymine adducts each with a 1:1 molecular stochiometry were found. Spectroscopic measurements and a positive reaction with Ehrlich's reagent indicate that the indole nucleus remained intact, but that the pyrimidine base was reduced at the 5, 6 double bond. The absence of a positive ninhydrin reaction and the effect of pH on the quantum yield of the photoadduct formation indicated that the ionized a-amino acid group of tryptophan was involved in photoadduct formation. Indole derivatives lacking an a-amino group were also found to form photoadducts with pyrimidine bases. The experimental results are consistent with a reaction mechanism involving tryptophan excitation to the first excited singlet state as the initial event. Radical scavenging experiments indicate that tryptophan free radicals, formed by electron dissociation from the excited state, react with the ground state pyrimidine.     

BIOLOGICAL DEACTIVATION AND SINGLE-STRAND BREAKAGE OF PLASMID DNA BY PHOTOSENSITIZATION USING TRIS(2,2''- BIPYRIDYL)RUTHENIUM(II) AND PEROXYDISULFATE

Abstract— Single-strand break formation and biological deactivation of plasmid pBR322 DNA in the presence of tris(2,2′-bipyridyl)-ruthenium(II), Ru(bpy)2/3;⁺, and K₂S₂O₈, upon irradiation with visible light(400–500 nm), were studied in aqueous solution at room temperature. Conditions of complete binding of Ru(bpy)2/3;⁺ to the strand were employed. The damage is initiated mainly by the SO^2/3;; radical anion. Under anoxic conditions at a ratio of nucleotide to sensitizer concentrations (N/S) of 18 and S₂O2/8^- concentrations of 0.5 mM the quantum yield of single-strand break (ssb) formation is φ_ssb= 8.4 times 10^-3 while that of biological deactivation (bd) is Ø_bd= 7.6 times 10^-3 (φ_ssb= 5.2 times 10^-36.4 times 10^-3, 6.0 times 10^-3 and φ_bd= 4.2 times 10^-3, 5.2 times 10^-3, 4.8 times 10^-3 at N/S=3, 6, and 9, respectively). The quantum yields are approximately 2.5 times smaller in air-saturated solutions. At N/S = 18 about 33 SO₄^-radical anions are required per one lethal event. φ_bd increases linearly with the S₂Oφ^- concentration (up to 0.5 mM). The damage to DNA is drastically reduced on addition of mono- or divalent salts (e.g. NaC10₄, MgCl₂). These additives cause the release of Ru(bpy)2⁺ from the strand. The observed damage to DNA is thus the result of a site specific reaction. When the phenanthroline analogue, Ru(phen)φ⁺, is used as sensitizer, φ_ssb and φ_bd are three times smaller.     

Photoreactions of tris(2,2'-bipyridyl)-ruthenium(II) with peroxydisulfate in deoxygenated aqueous solution in the presence of nucleic acid components, polynucleotides, and DNA

Abstract— The photobleaching of excited tris(2,2′-bipyridyl)-ruthenium(II), *Ru(bpy)₃²⁺, by peroxydis-ulfate in the presence of DNA and a series of polynucleotides, mononucleosides (uridine, cytidine, adenosine, guanosine), and purine or pyrimidine bases was studied in deoxygenated aqueous solution at room temperature. A reaction scheme is proposed which is confirmed by data obtained from mixing experiments with Ru(III) and bleaching measurements of Ru(II) using either continuous visible light or a nanosecond laser pulse (353 nm). The primary photobleaching step is the formation of Ru(bpy)₃²⁺ and the SO₄^- radical anion. In the presence of nucleic acids the two oxidizing species are formed in close proximity to the strand, since we used conditions where the Ru(bpy) ₃²⁺ ion is bound to the strand. Concerning the secondary reactions two clear cases (and several more complex cases) can be distinguished. On addition of uracil to the Ru(bpy)₃²⁺/S₂O₈^2- system the quantum yield for photobleaching is not significantly changed (φ^rel? 0.95), whereas it drops to virtually zero for guanosine-containing substrates, including DNA. The former result is explained by a reaction of SO₄^- with uracil leading to theN–1 radical which oxidizes Ru(II) to Ru(IIl). In contrast, the guanine moiety reacts with Ru(III) converting it into Ru(II). Therefore, in the presence of S₂O₈^2- and a substrate carrying a guanine moiety, Ru(bpy₃²⁺ acts as a photocatalyst.     

Ultraviolet inactivation of papain.

Abstract— Flash photolysis transient spectra (Λ > 250 nm) of aqueous papain show that the initial products are the neutral tryptophan radical Tip (Λ_max 510 nm), the tryptophan triplet state ³Trp (Λ_m., 460nm), the disulfide bridge electron adduct –?S^-— (Λ_max 420nm) and the hydrated electron e_aq^-. The –?S^-– yield was not altered by nitrous oxide or air, indicating that the formation of this product does not involve electrons in the external medium. The original papain preparation was activated by irradiating under nitrogen. The action spectrum supports previous work attributing the low initial activity to blocking of cysteinyl site 25 with a mixed disulfide. Flash lamp irradiation in nitrogen led to activation at low starting activities and inactivation at higher starting activities, while only inactivation at the same quantum yield was observed with air saturation. The results are consistent with photoionization of an essential tryptophyl residue as the key inactivating step.     

Reaction of sulphate radical anion (SO<Superscript>.</Superscript><Subscript>4</Subscript><Superscript>-</Superscript>) with hydroxy-and methyl-substituted pyrimidines: a pulse radiolysis study

Reactions of sulphate radical anion (SO·₄ ^-) with 4,6-dihydroxy-2-methyl pyrimidine (DHMP), 2,4-dimethyl-6-hydroxy pyrimidine (DMHP), 6-methyl uracil (MU) and 5,6-dimethyl uracil (DMU) have been studied by pulse radiolysis at pH 3 and at pH 10. The transient intermediate spectra were compared with those from the reaction of hydroxyl radical (·OH). It is proposed that SO·₄ ^- produces radical cations of these pyrimidines in the initial stage. These radical cations are short-lived except in the case of DMHP where a relatively longer lived radical cation is proposed to be formed. When there is a hydrogen atom attached to the N(1) or N(3) position, a deprotonation from these sites is highly favored. When there is no hydrogen attached to these sites, deprotonation from a substituted methyl group is favored. At acidic pH, deprotonation from nitrogen is observed for DHMP, MU and DMU. At basic pH, the radical cation reacts with OH^- leading to the formation of OH adducts.     

Uracil ring opening in the reaction of 5-formyl-2′-deoxyuridine with primary alkyl amines

Treatment of 5-formyl-2′-deoxyuridine (f⁵dU) with stoichiometric amounts of strongly nucleophilic, non-hindered primary alkyl amines led to fast and quantitative formation of the corresponding Schiff bases. In the presence of excess amines, novel nucleosides with ring opened pyrimidine bases were formed as a result of the Michael addition of a second amine to the pre-formed imines. In the reaction of f⁵dU with aromatic amines, the formation of Schiff base derivatives was slower and even under prolonged treatment with an excess of amine the uracil ring remained intact.     

Zur chemischen struktur der streptovirudine—I: Säurehydrolytischer abbau und konstitution der gebildeten fettsäuren

Streptovirudin is a complex of related nucleoside antibiotics. Eight components have been isolated in pure form. Both the antibiotic complex and the pure components were hydrolyzed by heating with acid. A sensitive method using mass spectrometry has been developed for the detection of the nucleoside bases uracil and dihydrouracil in the hydrolysates. Furthermore, the following fatty acids have been identified as degradation products on the basis of gaschromatographic and mass spectrometric studies: 10-methyl-2-undecenoic acid, 10-methyl-2-dodecenoic acid, 12-methyl-2-tridecenoic acid and 12-methyl-2-tetradecenoic acid.     

Functional monomers and polymers. CXXI. Methylation and interaction studies on adenine containing polymethacrylate derivatives

Studies of the methylation of polymethacrylate derivatives with adenine bases were made in comparison to those with uracil bases. The polymethacrylate derivatives with adenine bases were methylated by using methyl iodide in dimethyl sulfoxide solution to produce polymers that contained N¹-methyladenine and N¹, N⁶-dimethyladenine units. The products were identified by spectroscopic data and by preparing their model compounds. The methylated polymers obtained were further applied in a study of polymer complex formation with uracil-base polymers.     

Photodynamic oxidation of iodide ion and aromatic amino acids by eosin

Abstract— The photosensitized oxidation of I^-, tyrosine, and tryptophan by aqueous eosin has been investigated with a photostationary technique, in which the system is irradiated with sinusoidally modulated light and phase sensitive detection is used to measure the spectra of short-lived intermediates and their rate constants. It has been shown that the principal oxidizing agent for I^- is semi-oxidized eosin (X) produced by the reduction of oxygen by triplet state eosin. The same mechanism obtains when femcyanide ion is substitued for oxygen. A second oxidizing agent is effective in oxygenated solutions at high I^- concentrations, attributed to O₂^-. The X mechanism controls when tyrosine or tryptophan are irradiated at low substrate concentrations in the presence of oxygen or femcyanide. However, at high substrate concentrations, or in the absence of the electron accepting agents, the mechanism switches to the ‘dye-substrate’ process where triplet state eosin oxidizes the aromatic substrate and is reduced to the semiquinone. The conversion quantum yields agree with the predictions based on the measured quenching and reaction rate constants.     

Electrochemical reduction of uracil in dimethyl sulfoxide: Autoprotonation of the anion radical

The electrochemical reduction of uracil in dimethyl sulfoxide was investigated, using d.c.and a.c. polarography, cyclic voltammetry, and controlled potential electrolysis. Uracil is reduced in a one-electron step (E_1/2=?2.3 V); the apparent number of electrons transferred (n) decreases from one at infinite dilution to one-half at concentrations above 1mM. The concentration dependent n-value is due to proton transfer by the parent compound to the radical anion formed on reduction. Such a proton transfer, which has been observed for 2-hydroxypyrimidine, deactivates part of the uracil, which would otherwise be available for reduction, by formation of the more difficultly reducible conjugate base. The uracil anion forms insoluble mercury salts, producing two oxidation waves (E_1/2 of ?0.1 and ?0.3 V); the latter wave is due to formation of a passivating film on the electrode. Digital simulations indicate that the protonation rate exceeds 10⁵M^?1 s^?1 and that, at low uracil concentration, some of the free radical formed on protonation is further reduced. At concentrations exceeding 1 mM, all of the free radical dimerizes. The effect of added acids and base on the electrochemical behavior is described.     

Photoreaction of Ketoprofen with Tryptophan and Tyrosine in Phosphate Buffer Solution

Reaction of excited ketoprofen (KP) with tryptophan (Trp) and tyrosine (Tyr) in a phosphate buffer solution was studied by the transient absorption spectroscopy. Both amino acids, which would interact with KP in bovine serum albumin [Monti, S. [2009] Phys. Chem. Chem. Phys., 11, 9104–9113], accelerated the proton transfer reaction to yield 3‐ethylbenzophenone ketyl biradical (EBPH) from KP carbanion, which was produced by photoexcitation of KP^? through decarboxylation. By means of the actinometry method with benzophenone, the reaction quantum yield was successfully estimated to be fairly large, and Trp, Tyr, DOPA and 4‐methylphenol were found to be a good proton donor for the carbanion. The formation rate constants of EBPH by the amino acids (k_r) were also determined to be (2.7 ± 0.1) × 10⁹ M^?1s^?1 for Trp and (7.8 ± 0.4) × 10⁸ M^?1s^?1 for Tyr, which were larger than those by basic amino acids and dipeptides reported. The reason for the highly efficient proton transfer reaction with Trp and Tyr would be explained by difference of the activation energy for the reaction. These results suggest that the proton transfer should be a key process for an initial photoreaction of KP with a protein, causing photosensitization in vivo.     

Reactions of OH* and eaq − with uracil and thymine in the presence of Cu(II) ions in dilute aqueous solutions: A pulse radiolysis study

The reactions of OH^* and e_aq ^? adducts of uracil and thymine with Cu(II) ions in aqueous solutions were followed by pulse radiolysis. The transient absorption spectra of the OH^* adducts of uracil when followed in the presence of Cu(II) ions show growth in absorption at wavelengths 420 and 350 nm at 15 μs and 65 μs after the pulse respectively. Similar transient absorption spectra of thymine showed growth in absorption at wavelengths 390 and 320 nm at 38 μs and 65 μs after the pulse respectively. The rates of electron transfer from the OH^* adducts of uracil and thymine to various Cu(II) compounds when monitored at 360 nm lie between 10⁶ and 10⁸ mol^?1 dm³ s^?1 this implies that the electron transfer process is not an efficient process. Low rate constants coupled with the spectral changes suggest formation of a radical copper adduct which decays by water insertion to give cis-glycols as the major product. The electron transfer from the electron adducts of uracil and thymine to various copper(II) compounds takes place more efficiently (rate constants of the order of 10⁸ and 10⁹ mol^?1 dm³ s^?1) compared with that from the OH^* adducts. The t-butanol radicals formed on scavenging the OH^* radicals also produce adducts with Cu(I) ions which are formed on oxidation of the electron adducts by Cu(II) ions. This adduct has absorption around 400 nm both in the case of uracil and thymine.     

Photogeneration of superoxide and decarboxylated peptide radicals by carboquone, mitomycin C and streptonigrin. An electron spin resonance and spin trapping study

Visible light (405–615 nm) excitation of carboquone, mitomycin C, and streptonigrin dissolved in dimethylsulfoxide in the presence of oxygen generates superoxide anion radicals (O₂^?). The quantum yields for these reactions range from 4.2 times 10^?2 (carboquone, λ= 615 ± 10 nm) to 7.3 times 10^?6 (streptonigrin, λ=545 ± 10 nm). O₂^? radicals were spin trapped with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and identified by electron spin resonance (ESR). The efficiency of DMPO to spin trap O₂^? in dimethylsulfoxide was determined and indicated that 91% of the O₂^? present in dimethylsulfoxide is trapped by DMPO. The oxidation of the photoexcited drug molecules occurs via a direct electron transfer to dissolved oxygen in solution. Ultraviolet irradiation (λ= 313 ± 10 nm) of the aminoquinone drug solutions (80% H₂O, 20% dimethylsulfoxide) in the presence of peptides results in the decarboxylation of the peptides. In this case the photoexcited drugs are reduced, abstracting an electron from the C-terminal carboxyl group of the peptides. The reaction is specific to the C-terminal amino acid of the peptide. The decarboxylated peptide radicals were spin trapped with 2-methyl-2-nitrosopropane (MNP) and identified by ESR.     

Photochemical Hydrogen Abstraction and Electron Transfer Reactions of Tetrachlorobenzoquinone with Pyrimidine Nucleobases

Pentachlorophenol, a widespread environmental pollutant that is possibly carcinogenic to humans, is metabolically oxidized to tetrachloroquinone (TCBQ) which can result in DNA damage. We have investigated the photochemical reaction dynamics of TCBQ with two pyrimidine type nucleobases (thymine and uracil) upon UVA (355 nm) excitation using the technique of nanosecond time-resolved laser flash photolysis. It has been found that 355 nm excitation populates TCBQ molecules to their triplet state ³TCBQ^*, which are highly reactive towards thymine or uracil and undergo two parallel reactions, the hydrogen abstraction and electron transfer, leading to the observed photoproducts of TCBQH· and TCBQ·^- in transient absorption spectra. The concomitantly produced nucleobase radicals and radical cations are expected to induce a series of oxidative or strand cleavage damage to DNA afterwards. By characterizing the photochemical hydrogen abstraction and electron transfer reactions, our results provide potentially important molecular reaction mechanisms for understanding the carcinogenic effects of pentachlorophenol and its metabolites TCBQ.     

Design, synthesis and conformational analysis of turn inducer cyclopropane scaffolds: microwave assisted amidation of unactivated esters on catalytic solid support to obtain γ-turn mimic scaffolds

Novel constrained 1-aroyl-cyclopropane-2,3-cis-dicarboxylic acid bis-[(2-hydroxy-ethyl)-amides] (17a-e) with varied torsional angles have been synthesized in high yield from unactivated esters of 1-aroyl-2,3-cis-diethoxycarbonylcyclopropanes (15a-e) on a catalytic solid support with reduced reaction times by using the monomode-microwave irradiation; 15a-e were obtained by diastereoselective ethoxycarbonylmethylene transfer from a sulfur ylide to ethyl β-aroylacrylates (10a-e). Torsional angles and interatomic distance measurements on the energy minimized structures of the obtained molecules (17a-e, DFT, B3LYP/6-31G^∗ level) have established these molecules as valuable γ-turn mimic scaffolds.     

Assisted intramolecular proton transfer in (uracil)2Ca2+ complexes

The structure and relative stability of the complexes between uracil dimers and Ca²⁺, as well as the proton transfer (PT) processes within these dimers, have been investigated by the density functional theory methods. Although in uracil dimers PT occurs as an almost synchronous double PT processes that connect the diketo dimer with a keto-enol dimer, the process within the most stable (uracil)₂Ca²⁺ complexes is much more complicated, and the product of the reaction looks like the result of an intramolecular PT from one of the NH groups of one monomer to one of the carbonyl groups of the same monomer. An analysis of the force profile along the reaction coordinate shows that the intimate mechanism implies three elementary steps, two intermolecular PTs, and an in-plane displacement of one monomer with respect to the other. The result of this so-called assisted intramolecular proton transfer is the formation of a dimer in which only one monomer is a keto-enol derivative, the other monomer being apparently unchanged, although it suffers significant structural rearrangements along the reaction coordinate. Quite importantly, this dimer is significantly stabilized upon Ca²⁺ association; therefore, while the most stable uracil dimers correspond systematically to associations involving only the diketo forms, in (uracil)₂Ca²⁺ complexes the most stable structures correspond to those in which one of the monomers is a keto-enol uracil isomer.     

The addition of hydroxylamine to the halogenated uracils

Hydroxylamine adds reversibly to C-6 of uracil bases via a multistep mechanism. Equilibrium constants measured at several pH values, 25°, μ = 3.0 M are: uracil, 0.25 M^?1; 5-iodouracil, 0.30 M^?1, 5-bromouracil, 0.24 M^?1, 5-chlorouracil, 0.06 M^?1 and 5-fluorouracil, 5.11 M^?1. The kinetics of hydroxylamine addition to both 5-bromo-and 5-iodouracil are complex. At low hydroxylamine buffer concentrations, the rate constants are second-order with respect to hydroxylamine buffer but at higher concentration a first-order dependence is approached. Hydroxylamine elimination from 5-iodo-6-hydroxylamino-5,6-dihydrouracil is subject to general base catalysis by tris(hydroxy-methyl)aminomethane but at higher concentrations the rate constants are not proportional to the concentration of general base. This reaction is subject to solvent effects where increasing ethanol concentration depresses the rate when measured at constant pH. These kinetics can be rationalized in terms of a multistep reaction pathway in which hydroxylamine addition to C-6 of the halogenated uracil precedes general acid catalyzed proton transfer to C-5 yielding the final 6-hydroxylaminodihydropyrimidine product.     

Electrochemistry of ABTS at Glassy Carbon Electrodes

The electrochemical and the mass transport behavior of ABTS^2-/ABTS^·- (2,20-azinobis(3-ethylbenzothiazoline-6-sulfonate)) redox couple at glassy carbon electrode (GCE) in phos-phate buffer solution (PBS, pH=4.4) is studied in detail by cyclic voltammetry combined with rotating disk electrode system. From the i-E curves recorded at different electrode ro-tating rate, rate constant, and transfer coefficient for ABTS^2-?ABTS^·-+e reaction at GCE electrode and the diffusion coefficient of ABTS^2- in PBS are estimated to be 4.6×10^-3 cm/s, 0.28, and 4.4×10^-6 cm²/s, respectively. The transfer coefficient with a value of ca. 0.28 dif-fers largely from the value of 0.5 that is always assumed in the literature. The origins for the difference of the rate constant determined and the challenges for estimating the stan-dard rate constant are discussed. The performance for such ABTS^2- mediated bio-cathode toward oxygen reduction reaction is discussed according to the over-potential drop as well as current output limit associated with the charge transfer kinetics of ABTS^2-?ABTS^·-+eredox reaction and/or the mass transport effect.