PHOTOCHEMICAL COUPLING OF 5-BROMOURACIL TO TRYPTOPHAN, TYROSINE AND HISTIDINE, PEPTIDE-LIKE DERIVATIVES IN AQUEOUS FLUID SOLUTION

Direct irradiation of 5-bromouracil (BU) in aqueous fluid solution in the presence of tryptophan (trp), tyrosine (tyr) or histidine (his) derivatives using a XeCl excimer laser at 308 nm yielded photocoupling of BU to the aromatic ring of each amino acid. Irradiation of BU at 308 nm most likely results in excitation of the n-φ* transition, intersystem crossing to the triplet manifold, and coupling via electron transfer from the aromatic amino acid. The coupling observed was regiospecific between the 5-position of uracil (U) and the 2-position of the indole and phenol rings and the 5-position of the imidazole ring of the respective amino acids. Quantum yields of photocoupling to BU ranged from 1 × 10^-3 to 7 × 10^-3 and paralleled known rates of electron transfer and ionization potentials of the aromatic rings. The photocoupling between BU and some of the aromatic amino acid peptide-like derivatives possibly mimics photocrosslinking of BU-DNA to associated proteins, a potentially useful photoreaction for studying nucleic acid-protein interactions. Formation of crosslinks of the type proposed here might be detected by the characteristic fluorescence emission of the uracil amino acid adducts.     

Mechanistic Studies Relevant to Bromouridine-Enhanced Nucleoprotein Photocrosslinking: Possible Involvement of an Excited Tyrosine Residue of the Protein

The results of mechanistic studies on formation of uridine (U) and N-acetyl-in-(5-uridinyl)tyrosine N-ethylam-ide (2) from irradiation of aqueous, pH 7 solutions of bromouridine (BrU) and N-acetyltyrosine JV-ethylamide (1) are reported. Solutions were irradiated with monochromatic laser emission at 266, 308 and 325 nm. Quantum yield measurements as a function of excitation wavelength suggest that both products result from excitation of the tyrosine derivative followed by electron transfer to BrU, possibly with intermediacy of the hydrated electron. The BrU radical anion ejects bromide to form the uri-dinyl radical, which then abstracts a hydrogen atom from 1 or adds to the aromatic ring of 1. Formation of adduct 2 is a model for photocrosslinking of nucleic acids bearing the bromouracil chromophore to adjacent tyrosine residues of proteins in nucleoprotein complexes. The value of 325 nm excitation in photocrosslinking, where the tyrosine chromophore is more competitive for photons, was demonstrated with an RNA bound to the MS2 bacteriophage coat protein; more than a 60% increase in the yield of photocrosslinking relative to that obtained with 308 nm excitation was achieved.     

Stacking of the mutagenic DNA base analog 5-bromouracil

The potential energy surface of the stacked 5-bromouracil/uracil (BrU/U) dimer has been investigated in the gas phase and in solution (water and 1,4-dioxane), modeled by a continuum solvent using the polarizable continuum model. Minima and transition states were optimized using DFT (the M06-2X density functional and the 6-31+G(d) basis set). Six stacked gas-phase BrU/U minima were located: four in the face-to-back orientation and two face-to-face. The global minimum in the gas phase is a face-to-face structure with a twist angle of 60° and a zero-point energy-corrected interaction energy of ?10.7 kcal/mol. The BrU/U potential energy surface is geometrically and energetically similar to that of U/U (Hunter and Van Mourik in J Comput Chem 33:2161, 2012). Energy calculations were also performed on experimental geometries of stacked dimers (47 containing BrU stacking with either adenine, cytosine, guanine or thymine and 51 containing thymine also stacking with one of those four bases) taken from DNA structures in the Protein Data Bank. Single-point interaction energies were computed at different levels of theory including MP2, CCSD(T) and DFT using the mPW2PLYP-D double-hybrid functional augmented with an empirical dispersion term, using basis sets ranging from aug-cc-pVDZ to aug-cc-pVQZ. No strong evidence was found for the suggestion that the mutagenicity of BrU is due to enhanced stacking of BrU compared to the corresponding stacked dimers involving thymine.     

Unique charge transfer properties of the four-base pi-stacks in Z-DNA

To investigate the photoreactions of BrU in Z-DNA, the photoirradiation of 5'-d(C1G2C3G4BrU5G6C7G8)-3'/5'-d(C9mG10C11A12C13mG14C15G16)-3'(ODN 1-2) was investigated. In accord with previous observations, B-form ODN 1-2 with the 5'-GBrU sequence showed very weak photoreactivity. However, Z-form ODN 1-2 in 2 M NaCl underwent photoreaction to afford 5'-d(CGC)rGd(UGCG)-3' together with the formation of imidazolone (Iz) contained 5'-d(CIzCACmGCG)-3'. The results clearly indicate that structural changes caused by the B-Z transition dramatically increased the photoreactivity of ODN 1-2. Inspection of the molecular structure of Z-DNA suggests that there is unique four-base pi-stacks at the G4-BrU5-C11-mG10 in ODN 1-2. These results suggest that the intriguing possibility that the mG10 in a complementary strand located at the end of the four-base pi-stacks may act as an electron donor. To test the hypothesis of interstrand charge transfer from mG10 to BrU5 within the four-base pi-stacks in Z-DNA, ODN 1-3 samples in which the putative donor G10 residue was replaced with 8-methoxyguanine (moG) were prepared, since moG is known to trap cation radicals to yield Iz moieties in DNA. Photoirradiation of ODN 1-3 efficiently produced 5'-d(CGC)rGd(UGCG)-3' together with formation of 5'-d(CIzCACmGCG)-3'. These results clearly indicate that the interstrand charge transfer from mG10 to BrU5 initiates the photoreaction. In clear contrast, other replacements of G with moG did not enhance the photoreactivity. The present study revealed the presence of unique four-base pi-stacks in Z-DNA and photoirradition of BrU in Z-DNA causes efficient electron transfer from G within this cluster.     

The (5-4) and (6-4) adducts of 1-methylthymine and their Dewar valence isomers

Previous studies of the photochemical reaction of 1-methylthymine (MeT) in frozen aqueous solution have indicated that four cyclobutane type dimers are formed. We have restudied this system and have found that, in addition to cyclobutane dimers, both a (5-4) adduct and a (6-4) adduct of MeT are formed in significant amounts. Upon standing in aqueous solution, the (5-4) adduct is susceptible to reaction to form an isomeric form of the parent adduct, possibly via ring-opening and closure reactions at C-6 of the saturated pyrimidine ring component of the adduct. Irradiation of each of these three adducts with UVB light produces a pair of Dewar-type adducts. The nine products were individually characterized by mass spectrometry, proton NMR spectroscopy and UV spectroscopy. A less comprehensive study showed that irradiation of thymidine in frozen aqueous solution produces a diastereomeric pair of (5-4) adducts, along with the previously known diastereomeric pair of (6-4) adducts.     

The photochemistry of uracil: a reinvestigation

Results from a re-examination of the photochemical reactions undergone by uracil (Ura) are presented. Irradiation of Ura in frozen aqueous solution at -78.5°C produces two diastereomeric (6-4) products, namely the cis and trans isomers of 5-hydroxy-6-4'-(pyrimidin-2'-one)-5,6-dihydrouracil. Upon heating in 0.1m HCl, each of these compounds decomposes to form 6-4'-(pyrimidin-2'-one)uracil. In addition, evidence for production of a hydrate of a trimer of Ura is presented, Irradiation of this compound at 254nm forms Ura and a (6-4) adduct as products. The compounds 5-4'-(pyrimidin-2'-one)uracil and 6-hydroxy-5,6-dihydrouracil were also present after Ura was irradiated in frozen aqueous solution. Cyclobutane dimers (CBDs) are formed when Ura is irradiated in the frozen aqueous state, in fluid aqueous and acetonitrile solution and in the presence of photosensitizers (e.g. acetone). Published information, concerning the identity and relative quantitative importance of the four CBD isomers (cis-syn, cis-anti, trans-syn and trans-anti) formed in these photochemical systems, is incomplete and often in substantial disagreement. Using chemical methods in conjunction with HPLC, the identity and relative amounts of the four dimers have been determined in each of these systems. Consequently, a number of inconsistencies found in the literature concerning dimer product identity and quantitative distribution have been resolved.     

The cyclobutane dimers of 2'-deoxyuridine, 2'-deoxycytidine, 5-methyl-2'-deoxycytidine and 5-bromo-2'-deoxyuridine

Irradiation of DNA and RNA pyrimidine nucleosides with UV light in frozen aqueous solution or in solution with acetone often results in the formation of cyclobutane dimers (CBDs). Many of these photodimers have not been characterized. We present here the results of work designed to achieve the isolation, spectroscopic characterization and determination of the stereochemical nature of a number of little studied or previously unstudied CBDs of four 2'-deoxyribonuclesides. These nucleosides are 2'-deoxyuridine (dUrd), 2'-deoxycytidine (dCyd), 5-methyl-2'-deoxycytidine (5-MedCyd) and 5-bromo-2'-deoxyuridine (5-BrdUrd). In particular, we have isolated and characterized six dUrd CBDs, five dCyd CBDs, five 5-MedCyd CBDs and four 5-BrdUrd CBDs. Photoproducts were studied by UV spectroscopy, mass spectrometry, proton NMR spectroscopy and via chemical approaches. Also presented are results from less definitive studies of a number of (6-4) (or 5-4) photoadducts of these nucleosides. In addition, results from exploratory photochemical studies of other 2'-deoxyribonucleosides in frozen solution, as well as some mixtures of two nucleosides, are given. The latter results indicate that 5-iodo-2'-deoxyuridine (5-IdUrd), 5-bromo-2'-deoxycytidine and 5-iodo-2'-deoxycytidine each form putative CBDs and that 5-BrdUrd is capable of forming putative mixed CBDs and (6-4) and/or (5-4) adducts with thymidine (Thd); 5-IdUrd similarly forms a (6-4) (or (5-4)) adduct with Thd.     

Isomerization kinetics of panipenem in aqueous solution

The isomerization kinetics of panipenem (INN: (+)-(5R,6S)-3-[(S)-1-(acetimidoylpyrrolidin-3-yl)thio]-6-[(R)-hydroxyethyl]-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, CAS No. 87726-17-8) in aqueous solution were investigated. An equilibrium between the Z-form and E-form was observed, and it was found that the isomerization rates were affected by the pH of the solution. Under acidic conditions, the isomerization rates were small. However, the isomerization rates were increased with the pH value. This phenomenon resulted from the extent of proton dissociation from the acetimidoyl group.     

Reaktionen von N-alkoxycyclimoniumsalzen—II. Heterocyclen aus ringöffnungsreaktionen von N-methoxypyridiniumsalzen

Rearrangement of 3-substituted N-methoxypyridinium-salts (R= COOH, COOCH₃, CONH₂) in aqueous solution by alkali resp. ammonia yields 3 - methoxyiminomethylpyridone - (2). Under the same conditions, 5 - hydroxy - 3′- (2′- methoxyiminoethylidene) - pyrrolidone - (2) is formed from N - Methoxypyridinium - 4 - carbonitrile.     

Exploitation of reversed micelles as a medium in mimetic peroxidase-catalyzed fluorogenic reaction between hydrogen peroxide and l-tyrosine

The peroxidase activity of mimetic enzyme, iron tetrasulfonatophthalocyanine (FeTSPc), was characterized in reversed micelles of hexadecyltrimethylammonium bromide (CTAB) formed in n-heptane-n-pentanol solution (2:1, V:V). The assay is based on its catalytic effect on the oxidation reaction of l-tyrosine (l-tyr) by hydrogen peroxide. The influences of environmental factors, such as the water content, CTAB concentration and pH, on the peroxidase activity of FeTSPc were investigated. It was observed that the reaction rate was distinctly enhanced in CTAB reversed micelles as compared with the rate in aqueous solution. Under optimum conditions, application of the FeTSPc-catalyzed fluorescence system in reversed micelles to the determination of H(2)O(2) and FeTSPc led to a highly sensitive system compared with that in aqueous solution, permitting detection limits of 5x10(-9) mol l(-1) H(2)O(2) and 2.3x10(-9) mol l(-1) FeTSPc. The advantages and limitations of employing the reversed micellar media in such mimetic peroxidase-catalyzed fluorescent detection schemes were discussed.     

Room Temperature Phosphorescence from Tryptophan and Halogenated Tryptophan Analogs in Amorphous Sucrose

Abstract. Tryptophan phosphorescence lifetime and quantum yield are sensitive to the local environment. The phosphorescence from tryptophan analogs, however, has not been studied. We report here data on the room temperature phosphorescence of tryptophan, 4-, 5- and 6-fluoro-DL-tryptophan (4-F-trp, 5-F-trp and 6-F-trp) and 5-bromo-DL-tryptophan (5-Br-trp) embedded in glassy powders of freeze-dried sucrose. In aqueous solution, the absorption of the analogs was either blue-shifted (4-F-trp), red-shifted (5-F-trp and 5-Br-trp) or not shifted (6-F-trp) with respect to tryptophan. The phosphorescence emission spectra of all analogs were red-shifted compared to trp (442 nm) with maxima at 446 nm (5-F-trp), 451 mn (6-F-trp), 452 nm (5-Br-trp) and 469 nm (4-F-trp). The 5-F-trp and 6-F-trp analogs had emission intensities similar to tryptophan (relative quantum yields of 0.68 and 0.91, respectively, compared to tryptophan), while the intensities of the 4-F and 5-Br analogs were lower (relative quantum yields of 0.039 and 0.022, respectively). All analogs exhibited complex decay behavior requiring several exponentials for an adequate fit; the average lifetimes were all lower than that of trp (1039 ms). The average lifetimes of the fluorinated analogs (5-F, 721 ms; 6-F, 482 ms and 4-F, 35 ms) scaled approximately with the relative quantum yields while that of 5-Br (0.53 ms) was significantly lower. Analysis of the individual lifetimes suggested that the fluorinated analogs differ in their sensitivity to environmental interactions, with 5-F- and 6-F-trp quenched 1.5-2-fold and 4-F-trp about 23-fold more efficiently than tryptophan. The red-shifted 5-F-trypto-phan analog, which has been incorporated into proteins, may provide an alternative phosphorescence probe for selective phosphorescence detection of a specific protein in a complex mixture.     

Quantitative phosphorescence study of interactions of cytosine and cytidine and its nucleotides in frozen aqueous solution: evidence for anomalous heavy-atom effect

Phosphorescence vs. pH titration curves of cytosine, cytidine, cytidine-5'-mono-phosphate (CMP), -diphosphate (CDP) and -triphosphate (CTP) were obtained in methanol/water 10/90 v/v and in various aqueous sodium halide solutions frozen at 77 degrees K. As shown by the shape of the titration curve, molecular aggregates or "puddles" of cytosine and cytidine were shown to occur only in relatively concentrated frozen solution (10(-3)M), these aggregates being dissociated in dilute frozen solutions (相似文献   

Photoinduzierte 1, 3-dipolare Cycloaddition von 3-Phenyl-2H-azirinen an Azodicarbonsäure-diäthylester. 32. Mitteilung über Photoreaktionen

Irradiation of 2, 2-dimethyl-3-phenyl- ( 1a ), 2, 3-diphenyl-2H-azirine ( 1b ) or the azirine-precursors 1-azido-1-phenyl-propene ( 2a ) and 1-azido-1-phenyl-ethylene ( 2b ), respectively, in benzene in the presence of azodicarboxylic acid diethylester, yields the corresponding 1, 2-carbethoxy-3-phenyl-Δ³-1, 2, 4-triazolines 4a–d (Scheme 1). Refluxing 4 ( a, c or d ) in 0, 2–0, 4M aqueous ethanolic potassium hydroxide leads to the formation of the 1-carbethoxy-3-phenyl-Δ²-1, 2, 4-triazolines 6 ( a, c or d ). Under the same conditions 4b is converted to 3, 5-diphenyl-1, 2, 4-triazole ( 7b , Scheme 2). In 10M aqueous potassium hydroxide solution heating of either 4 ( c or d ) or 6 ( c or d ) yields the 3-phenyl-1, 2, 4-triazoles 7 ( c or d ). Photolysis of 1-carbethoxy-5, 5-dimethyl-3-phenyl-Δ²-1, 2, 4-triazoline ( 6a ) in benzene in the presence of oxygen and trifluoroacetic acid methylester gives the 5-methoxy-2, 2-dimethyl-4-phenyl-5-trifluoromethyl-3-oxazoline ( 13 , Scheme 5). 5, 5-Dimethyl-3-phenyl-1, 2, 4-triazole seems to be the intermediate, which on losing nitrogen gives the benzonitrile-isopropylide ( 3a ).     

PHOTOREACTIONS OF ALLOPURINOL AND HYPOXANTHINE WITH 2-PROPANOL

Abstract— Ultraviolet irradiation of allopurinol with 2-propanol, either in aqueous solution at 254 nm or in aqueous acetone (used as a photosensitizer) at > 290 nm, gave 2-(3'-hydroxy-2',3'-dimethylbutan-2'-yl)pyrazolo[3,4-d]pyrimidin-4(5H)-one (3) and 6-(2'-hydroxypropan-2'-yl)pyrazolo[3,4-d]pyrimidin-4(5H)-one (4) as major products. In addition, two minor photoproducts, which were not characterized, were detected by thin layer chromatography. Under similar irradiation conditions, hypoxanthine yielded 8-(2'-hydroxypropan-2'-yl)-2-(propan-2'-yl)hypoxanthine (5), 8-(3'-hydroxy-2',3'-dimethylbutan-2'-yl)hy-poxanthine (6) and 8-(2'-hydroxypropan-2'-yl)hypoxanthine (7) together with two unidentified minor photoproducts. Compounds 5 and 6 appear to be secondary photoproducts derived from 7. Initiation of the reaction by photodecomposition of di- t -butyl peroxide led to specific formation of 4 from allopurinol and of 7 from hypoxanthine. The allopurinol and hypoxanthine photoproducts were characterized by their ¹H-NMR, UV, and mass spectra.     

Hydrophilic molecularly imprinted polymers for bisphenol A prepared in aqueous solution

We have prepared a hydrophilic molecularly imprinted polymer (MIP) for the hydrophobic compound bisphenol A (BPA) in aqueous solution using 3-acrylamido-N,N,N-trimethylpropan-1-aminium chloride (AMTC) as the functional monomer. Under redox-polymerization conditions, BPA forms an ion-pair with AMTC, which was confirmed by ¹H-NMR titration. The imprinting effect in aqueous solution was evaluated by comparison of this material with the corresponding non-imprinted polymer (NIP) and with a control polymer (CP) bearing no AMTC. The MIP showed the highest activity among the three polymers, and the imprinting factors as calculated from the amount of BPA bound to the MIP divided by the amounts bound to NIP and CP, respectively, are 1.8 and 6.0. The MIP was selective for BPA in aqueous solution, while structurally related compounds are not recognized. Such a selectivity for a hydrophobic compound is rarely observed in aqueous medium because non-specific binding of BPA inevitably leads to hydrophobic interaction.

     

Aminocyclodextrins to facilitate the deprotonation of 4-tert-butyl-alpha-nitrotoluene

6A-Amino-6A-deoxy-beta-cyclodextrin enhances the rate of the deprotonation of 4-tert-butyl-alpha-nitrotoluene. The rate constants for reaction of the cyclodextrin-bound species, kinc = 4 x 10(-3), 9 x 10(-3) and 19 x 10(-3) s(-1), at pH 6.0, 6.5 and 7.0, respectively, in 0.1 mol dm(-3) aqueous phosphate buffer containing 1% methanol at 298 K. These rate constants correspond to a rate acceleration (kinc/kun) of ca. 10 times at each pH. Under the same conditions, 6A-dimethylamino-6A-deoxy-beta-cyclodextrin and 6A-(2-aminoethylamino)-6A-deoxy-beta-cyclodextrin are more effective; at pH 6.0, 6.5 and 7.0, for the former, kinc = 3 x 10(-2), 7 x 10(-2) and 12 x 10(-2) s(-1), whilst for the latter, kinc = 4 x 10(-2), 5 x 10(-2) and 9 x 10(-2) s(-1), respectively. Each cyclodextrin also decreases the pKa of the nitrotoluene, from 6.8 in free solution, to 6.2 when bound. The accelerated deprotonation by 6A-amino-6A-deoxy-beta-cyclodextrin is reflected in the enhanced rates of hydrogen-deuterium exchange of the nitrotoluene in deuterium oxide, and in the conjugate addition of the nitrotoluene to methyl vinyl ketone in aqueous solution.     

Mineralization of single flexible polyelectrolyte molecules

Conformation of a single flexible polyelectrolyte molecule with a hydrophobic backbone in aqueous solution is effected by the interplay of the short-range intramolecular attraction and the long-range Coulomb repulsion. The conformation can be frozen if the molecule is trapped by a solid substrate. With this approach, we prepared the range of single molecule templates from poly(2-vinylpyridine) (P2VP) deposited on the surface of Si-wafer or mica in different conformations from an elongated wormlike coil to compact globule. Pd(+2) was coordinated by P2VP via an ion exchange reaction exposing the samples to palladium acetate acidic aqueous solution. In the next step, Pd(+2) was reduced by dimethylamine borane. This route results in wire-shaped metallic nanoparticle assembles of about 2-5 nm in diameter and 50-700 nm in length. The conformation and size of the underlaying polyelectrolyte molecules determine the dimensions of nanoparticles.     

Synthesis of 3- and 5-amino-5-(3)-(pyrrol-2-yl)isoxazoles

5-Amino-3-(pyrrol-2-yl)isoxazoles were selectively prepared by the reaction of 2-(2,2-dicyano-1-ethylthioethenyl)pyrroles with hydroxylamine in methanol. Under analogous conditions, 2-(2-carbamoyl-2-cyano-1-ethylthioethenyl) pyrroles with hydroxylamine gave 5-aminoisoxazoles and their structural isomers, 3-aminoisoxazoles (3-5% yield). The latter were selectively prepared by reacting 2-(2-carbamoyl-2-cyano-1-ethylthioethenyl)pyrroles with hydroxylamine in the presence of aqueous NaOH and from the products of intramolecular cyclization of 2-(2-carbamoyl-2-cyano-1-ethylthioethenyl)pyrroles, 1-ethylthio-3-iminopyrrolizines and hydroxylamine.     

Transformations of dialkyl(4-hydroxy-2-butynyl)-(3-phenylallyl)ammonium bromides in an KOH aqueous solution or in the presence of powdered KOH

Under the action of a twofold excess of KOH and heating in aqueous solution, and also under the conditions of the Stevens rearrangement (with KOH powder and a small amount of methanol) dialkyl-(4-hydroxy-2-butynyl)(3-phenylallyl)ammonium bromides form dialkyl[4-(1-phenylallyl)-2,5-dihydro-2-furyl]amines. Rearrangement–cleavage reaction also occurs under the same conditions.     

Development of Zn(II) sensors based on the assisted transfer of metal ions by hydrophobic ligands through gel-supported microinterfaces

Square-wave voltammetry (SWV) has been used to study the transfer of zinc(II) ion under static conditions, assisted by 5,6-diphenyl-3-(2-pyridyl)-1,2,4-triazine (DPT), through gel-supported microinterfaces. Microhole arrays created by laser photoablation of thin polyester films were used to support an organic gel phase prepared by addition of 1,3:2,4-dibenzylidenesorbitol (DBS) to a solution of o-nitrophenyloctyl ether (o-NPOE) with the appropriate supporting electrolyte. The results show that SWV can be used with the gel-supported microinterfaces if a gelified aqueous reference is used for the organic phase. Under such conditions a preliminary estimate of the detection limit for the determination of Zn2+ is 5 x 10(-8) mol L(-1).