REGIOSELECTIVE 1-ALKYLATION OF 2′-DEOXYGUANOSINE

ABSTRACT

A method has been found for the regioselective alkylation of the nitrogen at the 1-position of 2′-deoxyguanosine. This consists in the reaction, in tetrahydrofuran solution, of a fully protected form of dG, namely the 3′5′-O-bis(tert-butyldimethylsilyl)-N ²-dimethylaminomethylene derivative, with an alkyl halide in the presence of cesium carbonate. The yields of these previously unavailable derivatives of 2′-deoxyguanosine range from good to excellent. Confirmation of the structure of these substances comes from a comparison of their spectroscopic properties with those of the known 1-methyl homologue. In particular, the UV spectra of these new derivatives and the known 1-methyl homologue are essentially identical.     

The alkylation of 2′-deoxyguanosine and of thymidine with diazoalkanes. Some observations on O-alkylation

The reaction in ether-methanol between 2'-deoxyguanosine and diazomethane or its ethyl or n-butyl homologue gives 1-, O(6)- and 7-alkyl-2'-deoxyguanosine. N(2),O(6)-Dimethyl-2'-deoxyguanosine was also detected. The hydrolysis of the methyl and the ethyl derivatives gives the corresponding alkylguanines: the O(6)-alkyl-2'-deoxyguanosines were sequentially hydrolysed, first to 2-amino-6-alkoxypurines, subsequently to guanine. The mass spectra of O(6)-alkyl-2'-deoxyguanosines (methyl and ethyl) and of the corresponding 2-amino-6-alkoxypurines were determined. The reaction of diazomethane with thymidine afforded O(4)-methylthymidine, in addition to the previously detected 3-methylthymidine.     

Improved synthesis of 2'-amino-2'-deoxyguanosine and its phosphoramidite

2'-Amino-2'-deoxynucleosides and oligonucleotides containing them have proven highly effective for an array of biochemical applications. The guanosine analogue and its phosphoramidite derivatives have been accessed previously from 2'-amino-2'-deoxyuridine by transglycosylation, but with limited overall efficiency and convenience. Using simple modifications of known reaction types, we have developed useful protocols to obtain 2'-amino-2'-deoxyguanosine and two of its phosphoramidite derivatives with greater convenience, fewer steps, and higher yields than reported previously. These phosphoramidites provide effective synthons for the incorporation of 2'-amino-2'-deoxyguanosine into oligonucleotides.     

Synthesis of 2,3-anhydro- and 3,4-anhydro-hexopyranosides having a methyl branch on the oxirane ring,and their reactions with some lithium methylcuprate reagents

Three 2,3-anhydroaldohexopyranosides having a 2-C-methyl or 3-C-methyl branch, as well as three 3,4-anhydroaldohexopyranosides having a 3-C-methyl (7) or 4-C-methyl branch, were newly synthesized. The reactions of these, together with those of a known 3-C-methyl epoxide (2), with three kinds of lithium methylcuprate were investigated. Except for 2 and 7, the vicinal monodeoxy di-C-methyl derivatives were obtained by attack of the cuprates at the sterically less-hindered site of the oxirane ring, irrespective of the stereoelectronic effect. Formation of a unique, acyclic 1-enitol derivative from 2, and of a 4-enolone derivative from 7, was ascertained. Differences in the reactivity among the cuprates was also observed.     

Pryogalloloestrogens -- a new group of oestrogen metabolites.

After incubation of radioactive catecholoestrogen monomethyl ethers with rat liver slices the following well known metabolic pathways were observed: 1) demethylation, 2) 16alpha-hydroxylation, 3) oxidoreduction at C-atom 17, and 4) conjugation with glutathione, sulphuric acid and glucuronic acid. In addition, for the first time a further aromatic ortho-hydroxylation, leading to pyrogalloloestrogen derivatives, was detected. Thus, the incubation of 2-methoxyoestrone yielded 2,4-dihydroxyoestrone 2-methyl ether as the main metabolite of the lipophile fraction. Under the same conditions, 4-methoxyoestrone was converted to 2,4-dihydroxyoestrone 4-methyl ether and 2,4-dihydroxyoestradiol-17beta 4-methyl ether; these compounds were the quantitatively most important metabolites not only in the lipophile but also in the sulphate and glucuronide fractions. The identity of these new metabolic products was established by chromatography, microchemical reactions and recrystallisation to constant specific radioactivity.     

A new, but old, nucleoside analog: the first synthesis of 1-deaza-2'-deoxyguanosine and its properties as a nucleoside and as oligodeoxynucleotides

The first synthesis of 5-amino-3-(2'-deoxy-beta-D-ribofuranosyl)imidazo[4,5-b]pyridin-7-one (1-deaza-2'-deoxyguanosine) is described. The compound was converted from the known AICA-deoxyriboside. The tautomeric structure of the base moiety was determined by theoretical calculation to be a hydroxyl form. Although the analog was found to be labile to acidic conditions, 1-deaza-2'-deoxyguanosine was successfully converted into a phosphoramidite derivative, which was incorporated into oligodeoxynucleotides by the standard phosphoramidite method. Thermal stabilities of oligodeoxynucleotides containing 1-deaza-2'-deoxyguanosine were investigated by thermal denaturing experiments. Also, a triphosphate analog of 1-deaza-2'-deoxyguanosine was synthesized for polymerase extension reactions. Single nucleotide insertion reactions using a template containing 1-deaza-2'-deoxyguanosine, as well as 1-deaza-2'-deoxyguanosine triphosphate, were performed using the Klenow fragment (exonuclease minus) polymerase and other polymerases. No hydrogen bonded base pairs, even a 1-deaza-2'-deoxyguanosine:cytidine base pair, were indicated by thermal denaturing studies. However, though less selective and less effective than the natural guanosine counterpart, the polymerase extension reactions suggested the formation of a base pair of 1-deaza-2'-deoxyguanosine with cytidine during the insertion reactions.     

Identification of the main oxidation products of 8-methoxy-2'-deoxyguanosine by singlet molecular oxygen

It is now well established that oxidation of 2'-deoxyguanosine (dGuo) in DNA by singlet molecular oxygen [O2 (1Delta(g))] produces 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), whereas the main degradation products of free dGuo in aqueous solution have been identified as the two diastereomers of spiroiminodihydantoin nucleoside. Interestingly, O2 (1Delta(g))-mediated oxidation of free 8-oxodGuo gives rise to a pattern of degradation products that is different from that observed when the nucleoside is inserted into DNA. The reasons for these differences and the mechanisms involved in the oxidation reactions are not yet completely understood for either dGuo or 8-oxodGuo, either free or within DNA. In the present work, we report a study of the reaction of O2 (1Delta(g)) toward a modified nucleoside, 8-methoxy-2'-deoxyguanosine (8-MeOdGuo), either free or incorporated into an oligonucleotide. The reason for the choice of 8-MeOdGuo as a chemical model to study in more detail the oxidation pathways of 8-oxodGuo or, more precisely, of the tautomeric 8-hydroxy-2'-deoxyguanosine was dictated by the fact that only the 7,8-enolic tautomer is present in the molecule. The thermolysis of an endoperoxide of a naphthalene derivative as a clean chemical source of 18O-labeled O2 (1Delta(g)) was used to oxidize 8-MeOdGuo. The main O2 (1Delta(g)) oxidation products that were separated and analyzed by HPLC coupled to tandem mass spectrometry were identified as the 2'-deoxyribonucleoside derivatives of 2,2,4-triamino-5-(2H)oxazolone, 2,5-diamino-4H-imidazol-4-one together with the methyl-substituted derivatives of spiroiminodihydantoin, oxidized iminoallantoin and urea. On the other hand, O2 (1Delta(g)) oxidation of 8-MeOdGuo-containing oligonucleotide generated imidazolone as the predominant degradation product. These results provided new mechanistic insights into the reactions of O2 (1Delta(g)) with purine nucleosides.     

Distinct reactions catalyzed by bacterial and yeast trans-aconitate methyltransferases

The trans-aconitate methyltransferase from the bacterium Escherichia coli catalyzes the monomethyl esterification of trans-aconitate and related compounds. Using two-dimensional (1)H/(13)C nuclear magnetic resonance spectroscopy, we show that the methylation is specific to one of the three carboxyl groups and further demonstrate that the product is the 6-methyl ester of trans-aconitate (E-3-carboxy-2-pentenedioate 6-methyl ester). A similar enzymatic activity is present in the yeast Saccharomyces cerevisiae. Although we find that yeast trans-aconitate methyltransferase also catalyzes the monomethyl esterification of trans-aconitate, we identify that the methylation product of yeast is the 5-methyl ester (E-3-carboxyl-2-pentenedioate 5-methyl ester). The difference in the reaction catalyzed by the two enzymes may explain why a close homologue of the E. coli methyltransferase gene is not found in the yeast genome and furthermore suggests that these two enzymes may play distinct roles. However, we demonstrate here that the conversion of trans-aconitate to each of these products can mitigate its inhibitory effect on aconitase, a key enzyme of the citric acid cycle, suggesting that these methyltransferases may achieve the same physiological function with distinct chemistries.     

Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine under anaerobic conditions by reductively activated nitro 5-deazaflavin derivatives.

Electrolytically reduced 6- and 8-nitro-5-deazaflavin derivatives have been found to interact to react specifically with guanine base by means of cyclic voltammetry. Electrolytic reductions of 6- and 8-nitro-5-deazaflavin derivatives in the presence of the 2'-deoxyguanosine under anaerobic conditions resulted in prominent formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine.     

Analysis of DNA-bound advanced glycation end-products by LC and mass spectrometry

Sugars and sugar degradation products readily react in vitro with guanine derivatives, resulting in the formation of DNA-bound advanced glycation end-products (DNA-AGEs). The two diastereomers of N(2)-(1-carboxyethyl)-2'-deoxyguanosine (CEdG(A,B)) and the cyclic adduct of methylglyoxal and 2'-deoxyguanosine (mdG) (N(2)-7-bis(1-hydroxy-2-oxopropyl)-2'-deoxyguanosine have also been detected in cultured cells and/or in vivo. LC-MS/MS methods have been developed to analyze sensitively DNA adducts in vitro and in vivo. In this paper, the chemical structures of possible DNA-AGEs and the application of LC-MS/MS to measure DNA-AGEs are reviewed.     

Native corrinoids from Clostridium cochlearium are adeninylcobamides: spectroscopic analysis and identification of pseudovitamin B(12) and factor A

The corrinoids from the obligate anaerobe Clostridium cochlearium were extracted as a mixture of Co(beta)-cyano derivatives. From 50 g of frozen cells, approximately 2 mg (1.5 micromol) of B(12) derivatives was obtained as a crystalline sample. Analysis of the corrinoid sample of C. cochlearium by a combination of high-pressure liquid chromatography and UV-Vis absorbance spectroscopy revealed the presence of three cyano corrinoids in a ratio of about 3:1:1. The spectroscopic data acquired for the sample indicated the main components to be pseudovitamin B(12) (Co(beta)-cyano-7"-adeninylcobamide) (60%) and factor A (Co(beta)-cyano-7"-[2-methyl]adeninylcobamide) (20%). Authentic pseudovitamin B(12) was prepared by guided biosynthesis from cobinamide and adenine. Both pseudovitamin B(12) and its homologue, factor A, were subjected to complete spectroscopic analysis by UV-Vis, circular dichroism, mass spectrometry, and by one- and two-dimensional (1)H, (13)C-, and (15)N nuclear magnetic resonance (NMR) spectroscopy. The third component was indicated by the mass spectra to be an isomer of factor A and is likely (according to NMR) to be 7"-[N(6)-methyl]-adeninylcobamide, a previously unknown corrinoid. C. cochlearium thus biosynthesizes as its native "complete" B(12) cofactors the 7"-adeninylcobamides and two homologous corrinoids, in which the nucleotide base is a methylated adenine.     

Synthesis and antimicrobial activity of meso-substituted polymethine cyanine dyes

The condensation reaction of equivalent amounts of 2-cyanomethyl benzooxazole or its derivatives with variously substituted aromatic aldehydes gave 2-cyano-styryl benzooxazole or its derivatives. The subsequent reaction of the 2-cyano-styryl benzooxazoles with 2(4)-methyl substituted heterocyclic quaternary salts afforded meso-substituted styryl-2(4)-polymethine cyanines. The condensation reaction of 2-cyanomethyl benzooxazole or its derivatives with alpha-nitroso-beta-naphthol followed by reaction with 2(4)-methyl substituted heterocyclic quaternary salts gave meso-substituted aza-2(4)-polymethine cyanines. The reaction of 2-cyanomethyl benzooxazole or its derivatives with N-methyl heterocyclic quaternary salts followed by the reaction with 2-methylquinolinium methiodide afforded the corresponding meso-substituted trimethine cyanine dyes. Elemental analyses, visible absorption, IR, (1)H NMR spectroscopy, and mass spectra established the structures of these compounds. The relationship between the structure and properties of these dyes has been studied and the solvatochromic behavior of some selected cyanine dyes in organic solvents is discussed. Finally, the antimicrobial activity of selected novel dyes was investigated in vitro using a wide spectrum of microbial strains.     

Hyaluronidase-catalyzed copolymerization for the single-step synthesis of functionalized hyaluronan derivatives

Hyaluronidase-catalyzed copolymerization was carried out with monomer combinations of 2-methyl (1a)/2-vinyl (1b), 2-methyl (1a)/2-ethyl (1c), 2-methyl (1a)/2-n-propyl (1d), and 2-vinyl (1b)/2-ethyl (1c) oxazoline derivatives of hyalobiuronate [GlcAbeta(1-->3)GlcN]. All copolymerization reactions proceeded successfully in a regio and stereoselective manner, giving rise to hyaluronan derivatives bearing different N-acyl groups at the C2 position of the glucosamine unit in the polymer chain. The composition of the N-acyl groups was controlled by varying the comonomer feed ratio. The copolymerization mechanism was also discussed.     

New pteridine substrates for dihydropteridine reductase and horseradish peroxidase.

The oxidation of 4,5-diaminopyrimidin-6(1H)-one, 5,6,7,8-tetrahydropteridin-4(3H)-one, its 6-methyl and cis-6,7-dimethyl derivatives, and 6-methyl- and cis-6-7-dimethyl-5,6,7,8-tetrahydropterins, by horseradish peroxidase/H2O2 is enzymic and follows Michaelis-Menten kinetics, and its Km and kcat. values were determined. This oxidation of 5,6,7,8-tetrahydropterins produces quinonoid dihydropterins of established structure, and they are known to be specific substrates for dihydropteridine reductase. By analogy the peroxidase/H2O2 oxidation of the 5,6,7,8-tetrahydropteridin-4(3H)-ones should produce similar quinonoid dihydro species. The quinonoid species derived from 5,6,7,8-tetrahydropteridin-4(3H)-one and its 6-methyl and cis-6,7-dimethyl derivatives are shown to be viable substrates for human brain dihydropteridine reductase, and apparent Km and Vmax. values are reported.     

Chemical synthesis and spontaneous glycosidic hydrolysis of 3-methyl-2''-deoxyguanosine and 2''-deoxywyosine [1]

3-methyl-2'-deoxyguanosine (1a) is obtained from 2'-deoxyguanosine by a reaction sequence involving conversion into tricyclic 1,N-2-isopropeno derivative (4-desmethyl-2'-deoxywyosine, 3a) followed by methylation which results in 2'-deoxywyosine (2a) and final removal of the 1,N-2 blocking system. Compounds 1a and 2a undergo spontaneous hydrolytic cleavage of their glycosidic bonds at pH 7, 37 degrees C, which makes them the most labile of all known nucleosides composed of structural units occurring in nature.     

Formation of a diimino-imidazole nucleoside from 2'-deoxyguanosine by singlet oxygen generated by methylene blue photooxidation

Singlet oxygen ((1)O(2)) is capable of inducing genotoxic, carcinogenic and mutagenic effects. It has previously been reported that the reaction of (1)O(2) with 2'-deoxyguanosine, which is a major target of (1)O(2) among the DNA constituents, leads to formation of various oxidized products including 8-oxo-7,8-dihydro-2'-deoxyguanosine and spiroiminodihydantoin, amino-imidazolone and diamino-oxazolone nucleosides. In addition to these products, we report that a novel diimino-imidazole nucleoside, 2,5-diimino-4-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-2H,5H-imidazole (dD), is formed by reaction of 2'-deoxyguanosine with (1)O(2) generated by irradiation with visible light in the presence of methylene blue under aerobic conditions. Its identification is based on identical chromatographic and spectroscopic data with an authentic compound, which we recently isolated and characterised from the reaction mixture of 2'-deoxyguanosine with reagent HOCl and a myeloperoxidase-H(2)O(2)-Cl(-) system. The yield of dD was increased by D(2)O and decreased by azide. dD was not generated from 8-oxo-7,8-dihydro-2'-deoxyguanosine. These results indicate that dD is generated by (1)O(2) directly from 2'-deoxyguanosine, but not via 8-oxo-7,8-dihydro-2'-deoxyguanosine. dD may play a role in the genotoxicity of singlet oxygen in cells.     

Reassignment of the guanine-binding mode of reduced mitomycin C

Mitomycin C (1) is a clinically used antitumor antibiotic that binds covalently to deoxyribonucleic acid under reductive or acidic catalysis. We have determined the structures of the adducts resulting from attack of reductively activated 1 on the dinucleoside phosphate d(GpC) to be N2-(2' beta, 7'-diaminomitosen-1'alpha-yl)-2'-deoxyguanosine (2) and its 1' beta-isomer (3). This represents a revision of the previously reported structures for these adducts in that the mitomycin residue is linked to the N2- rather than O6-position of 2'-deoxyguanosine. This revision is the result of applying to the mitomycin case a newly developed general method that leads to unambiguous assignment of the linkage position in complex alkylated guanosines. The method as described here takes advantage of the resolution enhancement gained by calculation of the second derivatives of absorbance Fourier transform infrared spectra. In addition, we present 1H NMR data that corroborate the assigned structures of 2 and 3 and that should serve as a useful reference for future investigations into the binding of mitomycin C to DNA. The convenient synthesis of adducts 2 and 3 from deoxyguanosine and mitomycin C reported here should facilitate such investigations as well. Furthermore, we demonstrate a useful acetylation procedure for adducts and metabolites of mitomycin C that furnishes spectroscopically superior chemical derivatives (e.g., triacetates 4 and 5, derived from acetylation of adducts 2 and 3).     

Methylation of thymine and uracil with free methyl cations formed due to beta-decay of tritiated methane: possible implication in mutagenesis and carcinogenesis

Exposure of solid thymine and uracil at room temperature to free methyl cations, produced due to beta-decay of tritiated methane, resulted in formation of their 1-, O2-, 3-, O4-, and 6-methyl derivatives. In addition, uracil formed a 5-methyl derivative (thymine); tritium-containing thymine and uracil were also detected. Both thymine and uracil formed predominantly unidentified products which resulted presumably from their oligomerization. Incubation at -195 degrees C did not markedly change the pattern of reaction products. Aqueous-ammonia solutions of these pyrimidines formed methylated derivatives and considerable amounts of methanol and tritiated water. The possible implication of these reactions in mutagenic and carcinogenic effects of tritium-substituted hydrocarbons is discussed.     

Antioxidant and cytotoxic flavonols from Calotropis procera

Phytochemical investigations of Calotropis procera leaves have led to the isolation of two new compounds: quercetagetin-6-methyl ether 3-O-beta-D-4C1-galacturonopyranoside (3) and (E)-3-(4-methoxyphenyl-2-O-beta-D-4C1 -glucopyranoside)-methyl propenoate (4), along with eleven known metabolites: nine flavonol and two cinnamic acid derivatives. All metabolites were isolated for the first time from the genus Calotropis, except for 1 isolated previously from Calotropis gigantea. The structures were determined by spectroscopic methods (UV, ESI-MS, 1H, 13C NMR, 1H-1H COSY, HSQC, and HMBC). The radical scavenging activity of the aqueous methanol extract and compounds 8-13 was measured by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. Cytotoxic screening of the same compounds was carried out on brine shrimps as well.     

Inhibition of 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) activity of the antimutagenic human MTH1 protein by nucleoside 5'-diphosphates

The hMTH1 protein, a human homologue of E. coli MutT protein, is an enzyme converting 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) to 8-oxo-2'-deoxyguanosine 5'-monophosphate (8-oxo-dGMP) and inorganic pyrophosphate. It is thought to play an antimutagenic role by preventing the incorporation of promutagenic 8-oxo-dGTP into DNA. As found in our previous investigations, 8-oxo-2'-deoxyguanosine 5'-diphosphate (8-oxo-dGDP) strongly inhibited 8-oxo-dGTPase activity of MTH1. Following this finding, in the present study we have tested the canonical ribo- and deoxyribonucleoside 5'-diphosphates (NDPs and dNDPs) for possible inhibition of 8-oxo-dGTP hydrolysis by hMTH1 extracted from CCRF-CEM cells (a human leukemia cell line). Among them, the strongest inhibitors appeared to be dGDP (Ki=74 microM), dADP (Ki=147 microM), and GDP (Ki=502 microM). Other dNDPs and NDPs, such as dCDP, dTDP, ADP, CDP, and UDP were much weaker inhibitors, with Ki in the millimolar range. Based on the present results and published data, we estimate that the strongest inhibitors, dGDP and dADP, at physiological concentrations not exceeding 5 microM and GDP at mean concentration of 30 microM, taken together, can decrease the cellular hMTH1 enzymatic activity vs. 8-oxo-dGTP (expected to remain below 500 pM) by up to 15%. The other five NDPs and dNDPs tested cannot markedly affect this activity.