The effect of bisulfite on the dehalogenation of 5-chloro-, 5-bromo-, and 5-iodouracil

The 5-chloro-, bromo-, and iodo-analogs of uracil are dehalogenated in the presence of sodium bisulfite to yield 5,6 dihydrouracil-6-sulfonate as the final product. Under similar conditions, 5-fluorouracil adds bisulfite to yield 5-fluoro-5,6 dihydrouracil-6-sulfonate but is not dehalogenated. Ultraviolet absorption spectra of 5-bromouracil and 5-iodouracil reacting under pseudo first-order conditions with bisulfite indicate that dehalogenation proceeds via a pathway which has 5-halo-5,6-dihydrouracil-6-sulfonate and uracil as intermediates. In the case of 5-chlorouracil, the rate of bisulfite attack on the 6-position of the chlorouracil ring system is very slow relative to the rate of bisulfite addition to uracil. Hence, although dechlorination does occur, ultraviolet absorption spectra of reaction mixtures containing bisulfite and 5-chlorouracil do not reveal the uracil absorption peak observed with both 5-iodouracil and 5-bromouracil. Fluorine and proton nmr spectra indicate that bisulfite addition to 5-fluorouracil is stereoselective as is the case of bisulfite addition to uracil.     

The role of bisulfite in the debromination of 5-bromouracil. The debromination of 5-bromo-5,6-dihydrouracil-6-sulfonate

5-Bromouracil is dehalogenated in the presence of bisulfite buffers to yield uracil which subsequently adds bisulfite to form 5,6-dihydrouracil-6-sulfonate. Presumably, 5-bromo-5,6-dihydrouracil-6-sulfonate is an intermediate in uracil formation. Kinetic data indicate that the disappearance of 5-bromouracil in the presence of bisulfite buffers is second order with respect to total bisulfite concentration, thus indicating the participation of 2 moles of either sulfite or bisulfite in the overall reaction, Iodometric titrations of total bisulfite combined with spectral analysis of the various pyrimidine and dihydropyrimidine species present indicate that, in addition to the total bisulfite required to form 5,6-dihydrouracil-6-sulfonate, an additional mole of sulfite is consumed per mole of 5-bromouracil dehalogenated. These data combined with the finding that sulfate is generated during dehalogenation are indicative of a pathway for the dehalogenation of the intermediate 5-bromo-5,6-dihydro-uracil-6-sulfonate which involves the attack of sulfite either directly or via an intervening molecule of water to yield uracil. Subsequent reactions of halogen-containing intermediates yield sulfate and bromide as final products of the reaction.     

Co-operative mutagenic actions of bisulfite and nitrogen nucleophiles.

The combined effect of bisulfite and a nitrogen nucleophile, i.e. semicarbazide, methoxyamine or hydroxylamine, to chemically modify cytosine and to cause mutation and inactivation of bacteriophage lambda was investigated. A rapid transamination of cytidine with each of the amines took place in the presence of bisulfite, and the reaction product was solely the N(4)-transaminated 5,6-dihydrocytidine-6-sulfonate. Modifications of cytidine with bisulfite alone and with the nitrogen nucleophile alone were much slower reactions than those using a combination of bisulfite and the nucleophile. Whereas the product of the modification with the bisulfite/semicarbazide, 5,6-dihydro-4-semicarbazido-2-ketopyrimidine ribofuranoside-6-sulfonate, is convertible to 4-semicarbazido-2-ketopyrimidine ribofuranoside by treatment with a phosphate buffer, the products of the modification with the bisulfite/methoxyamine and with the bisulfite/hydroxylamine, i.e. 4-methoxy-5,6-dihydrocytidine-6-sulfonate and 4-hydroxy-5,6-dihydrocytidine-6-sulfonate, were stable in phosphate buffer.Inactivation and the “clear” mutation of bacteriophage lambda were observed when the phage was treated with sodium bisulfite in the presence of semicarbazide, methoxyamine or hydroxylamine. Under the conditions used, only very small increases in the mutation frequency were obtained by treatment of the phage with bisulfite alone or with the base alone. It was concluded that the residues, 5,6-dihydro-4-semicarbazido-2-ketopyrimidine-6-sulfonate, 4-methoxy-5, 6-dihydrocytosine-6-sulfonate and 4-hydroxy-5,6-dihydrocytosine-6-sulfonate in DNA are the causes of the mutation.When phage that had been inactivated by the semicarbazide/bisulfite reagent was subsequently treated with a phosphate buffer, a reactivation took place. The rate of the reactivation increased as the concentration of phosphate in the buffer increased. This reactivation was not accompanied by change in the mutation frequency. No reactivation was observed after a similar incubation when the prior inactivation had been induced by either methoxyamine/bisulfite or hydroxylamine/bisulfite. These results indicate that the 4-semicarbazido-2-ketopyrimidine residue is also mutagenic but is less lethal than the corresponding 5,6-dihydro-6-sulfonate structure.These results offer the first clear example of the co-operative mutagenic action of two different reagents.     

Reaction of cytidine with semicarbazide in the presence of bisulfite. A rapid modification specific for single-stranded polynucleotide.

Semicarbazide reacted rapidly with 5,6-dihydrocytidine-6-sulfonate, which was formed from cytidine by addition of bisulfite across the 5,6-double bond. The transaminated product, 5,6-dihydro-4-semicarbazido-2-ketotopyrimidine-6-sulfonate ribofuranoside, was identified by comparison with that formed by treatment of 4-semicarbazido-2-ketopyrimidine ribofuranoside with bisulfite. The progress of the transamination was monitored spectrophotometrically by use of a strong absorbance of the product in alkali. The reaction between cytidine and the semicarbazide-bisulfite mixture was optimal at pH 4.5. Complete transformation of cytidine into the product required only 5 min with the use of 3M semicarbazide-1M sodium bisulfite, pH 5.0, at the reaction temperature 37 degrees C. The product was stable in unbuffered solution but in phosphate buffers it underwent elimination of bisulfite to give 4-semicarbazido-2-ketopyrimidine ribofuranoside. The rate of the elimination at pH 7.0 and 37 degrees C increased proportionally with the increase of the phosphate concentration. Complete elimination was obtained by treatment with 1 M sodium phosphate for 2 h. When heat-denatured calf-thymus DNA was treated with 3 M semicarbazide-1 M bisulfite at 37 degrees C and pH 5.0 the transamination of reactive cytosine residues was completed by 10 min of incubation. At 20 degrees C, it required 85 min of incubation. Cytosine residues in native DNA did not react at all even by prolonged incubations. The modified DNA samples were further treated with a phosphate buffer at pH 7, producing 4-semicarbazido-2-ketopyrimidine residues in the DNA. Analysis of the base compositions of these samples by perchloric acid hydrolysis showed that the modification was selective to cytosine, which had been expected from studies with monomers. It also showed that the reactive cytosine residues in the denatured DNA, constitute about 80% of the total cytosine, which was consistent with the view that heat-denatured DNA still contains a considerable amount of secondary structure. The semicarbazide-bisulfite modification is expected to be a sensitive method to locate cytosine residues in single-stranded regions of polynucleotides.     

The effect of thiols on the dehalogenation of 5-iodo and 5-bromouracil

The 5-iodo- and 5-bromo- analogs of uracil are dehalogenated in the presence of both cysteine and 2-mercaptoethanol to yield uracil. Presumably, the reaction involves the initial addition of the thiol group across the 5,6 double bond of the halopyrimidine to yield the corresponding 5-halo, 5,6-dihydrouracil-6-thioether which then dehalogenates to yield uracil. Under comparable conditions, cysteine causes more rapid dehalogenation of both halouracils than does 2-mercaptoethanol.Thiol containing compounds catalyze hydrogen-deuterium exchange at carbon five of uracil (1–3) and have been implicated as having a catalytic effect in the deamination of cytosine (4,5). Presumably, these reactions involve the reversible nucleophilic addition of the thiol group across the 5,6 double bond of the pyrimidine to yield the corresponding 5,6 dihydropyrimidine with a substituted thioether group on carbon six. This pathway is supported by comparable reactions involving the addition of bisulfite to the pyrimidine ring system (6–10). Different from the bisulfite addition compounds, the thioether containing dihydropyrimidine adducts have not been isolated and characterized; however, 5′-deoxy-5′,6-epithio-5,6-dihydro-2′,3′-0-isopropylideneuridine resulting from the intramolecular attack of the 5′ thiol group on carbon six of the uracil ring system of 5′-deoxy-5′-thio-2′,3′-0-isopropylideneuridine has been isolated and characterized (11).In a recent communication, we reported that bisulfite buffer systems catalyze the dehalogenation of 5-iodo-, 5-bromo-, and 5-chlorouracil (12). The object of this work is to demonstrate that cysteine and 2-mercaptoethanol, sulfur nucleophiles with more physiological importance than bisulfite, also cause halopyrimidine dehalogenation under nearly physiological conditions of temperature and pH.     

Reaction of bisulfite with the 5-hydroxymethyl group in pyrimidines and in phage DNAs.

5-Hydroxymethylcytosine reacted with bisulfite and, instead of undergoing usual deamination process, gave cytosine 5-methylenesulfonate as the product. The conversion was rapid and quantitative, and the optimum pH was 4.5. The product was isolated as crystals and characterized. Cytosine 5-methylenesulfonate was only very slowly deaminated by treatment with bisulfite. 5-Hydroxymethyl-2'-deoxycytidine 5'-phosphate reacted with bisulfite in the same way as 5-hydroxymethylcytosine. Residues of 5-hydroxymethylcytosine in native as well as denatured T2 DNA were convertible to those of cytosine 5-methylenesulfonate by treatment of the DNA with bisulfite. While it is known that the 5-hydroxy-methyl groups of T-even bacteriophage DNA can be enzymatically glucosylated, this observation offers chemical evidence that the 5-hydrozymethyl groups in DNA are situated in such a way that they can readily react with external agents. 5-Hydroxymethyluracil gave uracil 5-methylenesulfonate on treatment with bisulfite. This reaction was much slower than that of 5-hydroxymethylcytosine, and the optimum pH was between 6 and 7.     

A polymerase engineered for bisulfite sequencing

Bisulfite sequencing is a key methodology in epigenetics. However, the standard workflow of bisulfite sequencing involves heat and strongly basic conditions to convert the intermediary product 5,6-dihydrouridine-6-sulfonate (dhU6S) (generated by reaction of bisulfite with deoxycytidine (dC)) to uracil (dU). These harsh conditions generally lead to sample loss and DNA damage while milder conditions may result in incomplete conversion of intermediates to uracil. Both can lead to poor recovery of bisulfite-treated DNA by the polymerase chain reaction (PCR) as either damaged DNA and/or intermediates of bisulfite treatment are poor substrate for standard DNA polymerases. Here we describe an engineered DNA polymerase (5D4) with an enhanced ability to replicate and PCR amplify bisulfite-treated DNA due to an ability to bypass both DNA lesions and bisulfite intermediates, allowing significantly milder conversion conditions and increased sensitivity in the PCR amplification of bisulfite-treated DNA. Incorporation of the 5D4 DNA polymerase into the bisulfite sequencing workflow thus promises significant sensitivity and efficiency gains.     

Transformation of Polyoxins D,E, and F with Sodium Bisulfite

Polyoxins D, E, and F which possess 5-carboxyuracil as the nucleobase were reacted selectively with sodium bisulfite at pH 4.0 resulting in facile decarboxylation to afford corresponding 5,6-dihydrouracil-6-sulfonates and uracil type polyoxins (polyoxins L, M, and K) in good yield. The former compounds were also converted to the latter almost quantitatively with mild alkali treatment. Biological activities of the transformed compounds were described.     

Alteration of the kinetic parameters for aminoacylation of Escherichia coli formylmethionine transfer RNA by modification of an anticodon base.

Treatment of Escherichia coli formylmethionine tRNA with 2 M sodium bisulfite, pH 7.0, in 10 mM MgCl2 at 25 degrees results in formation of uridine/bisulfite adducts at U18 in the dihydrouridine loop, U37 in the anticodon, and U48 in the variable loop. Two products, corresponding to the two diastereoisomers of 5,6-dihydrouridine-6-sulfonate, are formed at each reactive site in the tRNA. Although none of the modifications cause complete loss of methionine acceptor activity, the modified tRNA is amino-acylated at a reduced rate and has a decreased affinity for E. coli methionyl-tRNA synthetase. Aminoacylation of [35S]bisulfite-labeled tRNAfMet with a limiting amount of purified enzyme followed by separation of the acylated and unacylated molecules and structural analysis has shown that the presence of a specific diastereoisomer of the uridine/bisulfite adduct in the anticodon base U37 alters the kinetic parameters for aminoacylation of tRNAfMet.     

Comparison of bisulfite modification of 5-methyldeoxycytidine and deoxycytidine residues.

Sodium bisulfite is a mutagen which can specifically deaminate more than 96% of the cytosine residues in single-stranded DNA via formation of a 5,6-dihydrocytosine-6-sulfonate intermediate. Under the same reaction conditions, only 2-3% of the 5-methylcytosine (m5Cyt) residues in single-stranded XP-12 DNA, which has 34 mole% m5Cyt, was converted to thymine (Thy) residues. In contrast, at the deoxynucleoside and free base levels, the same treatment with bisulfite and then alkali converted 51% and > 95%, respectively, of the m5Cyt to the corresponding Thy derivatives. However, the rate of reaction of m5Cyt and its deoxyribonucleoside was much slower than that of the analogous quantitative conversion of cytosine or deoxycytidine to uracil or deoxyuridine, respectively. The much lower reactivity of m5Cyt and its derivatives compared to that of the unmethylated analogs is primarily due to a decrease in the rate of formation of the sulfonate adduct.     

Recognition of a cytosine base lesion by a human damage-specific DNA binding protein.

Sodium bisulfite reacts with cytosine and 5-methylcytosine, forming the 5,6-dihydrosulfonate adducts which deaminate to the uracil and thymine adducts, respectively. At alkaline pH, the sulfonate groups are then released, generating uracil and thymine. In DNA, the resulting G:U and G:T base mismatches generated are potential sites of mutagenesis. Using a human damage-specific DNA binding protein as a probe, we have found protein-recognizable lesions in bisulfite-treated DNA and poly d(I-C), but not in treated poly d(A-T) or poly d(A-U). Although this suggests that the lesion recognized is cytosine-derived, there was no correlation between the number of uracils induced and the number of binding sites, suggesting that the protein-bound damage is not a uracil-containing mismatch. Modification of the treatment protocol to reduce elimination of the bisulfite from the base adducts increased the level of binding, suggesting that the protein recognizes a base-sulfonate adduct.     

Specific Deprotonation Reactions of the Pyrimidine Radical Cation Resulting from the Menadione Mediated Photosensitization of 2'-Deoxycytidine

Menadione(2-methyl-1, 4-naphthoquinone) was shown to sensitize 2'-deoxycytidine to near ultraviolet light according to two main mechanisms. Reaction of a water molecule with the initially photo-induced pyrimidine radical cation and subsequent addition of molecular oxygen leads to the preponderant formation of the four cis and trans diastereoisomers of 5,6-dihydroxy-5,6-dihydro-2'-deoxyuridine. Pyrimidine ring opening and rearrangement products are also generated through the intermediate 6-hydroxy-5,6-dihydro-2'-deoxyurid-5-yl radical. The competitive deprotonation reaction of the radical cation is likely to involve two sites. Loss of an amino group proton is the likely initial event to explain the formation of 2'-deoxyuridine which is resistant to further photooxidation. The second deprotonation reaction involves the osidic carbon C(1'). The resulting radical will further react with oxygen leading to the release of free cytosine with concomitant formation of 2-deoxy-D-ribono-1,4-lactone. This reaction which is not prevented by hydroxyl radical scavengers constitutes to our knowledge the first example of a pyrimidine radical which is able to initiate selective intramolecular reaction at position 1 within the sugar moiety.     

Reaction of Bromotrichloromethane Derived Free Radicals with Uracil in a Model System. Structures of Products Formed

Free radicals generated by benzoyl peroxide-mediated catalytic decomposition of bromotrichloromethane (eg. trichloromethyl) were allowed to react under nitrogen or under air with uracil. Under nitrogen two reaction products were formed, one was identified as 5-chlorouracil and the other as a 5-bromouracil. Under air, besides the above two products other nine were also formed: 5,6-dihydrouracil; 5-hydroxyuracil; a chlorohydroxy adduct of uracil; a bromohydroxy derivative of uracil having the 5,6 bond in the saturated form; other bromohydroxy derivative of uracil having the double bond intact; 5,6-dihydroxyuracil; two dihalogenated hydroxylated uracil derivatives and one peak we were not able to descipher its structure. No single reaction product formed had carbon centered radicals (eg. trichloromethyl) added from CBrCl₃ and consequently would be missed in 'in vivo' covalent binding studies where ^l4C haloalkane (CBrCl₃ or carbon tetrachloride) were employed. If similar reaction products resulted during interaction of CBrCl₃ reactive metabolites with uracil in RNAs, significant deleterious effects in their function would be expected. That possibility, however, remains to be established.     

Rapid,Stepwise Substitution of Fluorines In 5-Trifluoromethyl-2′-Deoxyuridine by Bisulfite

Abstract

On treatment with bisulfite at neutral pH, 5-trifluoromethyl-2′-deoxyuridine (CF3dUR) underwent rapid substitution of the fluorine atoms by bisulfite to give first the monosulfonate and then the disulfonate derivatives. It was shown that the monosulfonate product has reactivity to bisulfite with a potency half that of CF3dUR. These findings demonstrate the stepwise nature of the fluorine release from CF3dUR and constitute evidence that 5-exo-methylene type intermediates are involved in the nucleophile-mediated release of the fluorine from CF3dUR.     

Specific Deprotonation Reactions of the Pyrimidine Radical Cation Resulting from the Menadione Mediated Photosensitization of 2′-Deoxycytidine

Menadione(2-methyl-1, 4-naphthoquinone) was shown to sensitize 2′-deoxycytidine to near ultraviolet light according to two main mechanisms. Reaction of a water molecule with the initially photo-induced pyrimidine radical cation and subsequent addition of molecular oxygen leads to the preponderant formation of the four cis and trans diastereoisomers of 5,6-dihydroxy-5,6-dihydro-2′-deoxyuridine. Pyrimidine ring opening and rearrangement products are also generated through the intermediate 6-hydroxy-5,6-dihydro-2′-deoxyurid-5-yl radical. The competitive deprotonation reaction of the radical cation is likely to involve two sites. Loss of an amino group proton is the likely initial event to explain the formation of 2′-deoxyuridine which is resistant to further photooxidation. The second deprotonation reaction involves the osidic carbon C(1′). The resulting radical will further react with oxygen leading to the release of free cytosine with concomitant formation of 2-deoxy-D-ribono-1,4-lactone. This reaction which is not prevented by hydroxyl radical scavengers constitutes to our knowledge the first example of a pyrimidine radical which is able to initiate selective intramolecular reaction at position 1 within the sugar moiety.     

Bisulfite genomic sequencing: systematic investigation of critical experimental parameters

Bisulfite genomic sequencing is the method of choice for the generation of methylation maps with single-base resolution. The method is based on the selective deamination of cytosine to uracil by treatment with bisulfite and the sequencing of subsequently generated PCR products. In contrast to cytosine, 5-methylcytosine does not react with bisulfite and can therefore be distinguished. In order to investigate the potential for optimization of the method and to determine the critical experimental parameters, we determined the influence of incubation time and incubation temperature on the deamination efficiency and measured the degree of DNA degradation during the bisulfite treatment. We found that maximum conversion rates of cytosine occurred at 55°C (4–18 h) and 95°C (1 h). Under these conditions at least 84–96% of the DNA is degraded. To study the impact of primer selection, homologous DNA templates were constructed possessing cytosine-containing and cytosine-free primer binding sites, respectively. The recognition rates for cytosine (≥97%) and 5-methylcytosine (≥94%) were found to be identical for both templates.     

Cytogenetic damage induced in human lymphocytes by sodium bisulfite.

The frequencies of chromosomal aberrations (CA), sister-chromatid exchanges (SCE), and micronuclei (MN) in human blood lymphocytes exposed to sodium bisulfite (sulfur dioxide) at various concentrations ranging from 5 x 10(-5) M to 2 x 10(-3) M in vitro were studied. It was shown that sodium bisulfite (NaHSO3 and Na2SO3, 1:3 M/M) caused an increase in SCE and MN in human blood lymphocytes in a dose-dependent manner, and also induced mitotic delays and decreased mitotic index. For CA, our results indicated that sodium bisulfite induced an increase of chromatid-type aberrations in lymphocytes from three of four donors in a dose-dependent manner. The chemical at low concentrations induced chromatid-type aberrations, but not chromosome-type aberrations; high concentrations induced both chromatid- and chromosome-type aberrations. No cytogenetic damage in human lymphocytes was induced by sodium sulfate. The results have confirmed that sulfur dioxide is a clastogenic and genotoxic agent.     

The reaction of bisulfite with liposomal membranes

Bisulfite has been shown to induce leakage of encapsulated substances from liposomal vesicles. The bisulfite induced leakage of either DNP-tyrosine, potassium ferricyanide, or [³H]glycine was observed to be greater with lipsomes composed of phospholipids containing unsaturated fatty acids. The leakage of encapsulated substances from liposomes was found to be concentration dependent when incubated for a constant time interval and time dependent when incubated at a constant bisulfite concentration. In addition, bisulfite caused the leakage of approximately 5 times more [³H]glycine from unilamellar liposomes than from multilamellar liposomes. These findings are consistent with the interaction of bisulfite with liposomal membranes via reaction with sites of unsaturation.     

Structural investigation of the biosynthesis of alternative lower ligands for cobamides by nicotinate mononucleotide: 5,6-dimethylbenzimidazole phosphoribosyltransferase from Salmonella enterica.

Nicotinate mononucleotide (NaMN):5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella enterica plays a central role in the synthesis of alpha-ribazole, a key component of the lower ligand of cobalamin. Surprisingly, CobT can phosphoribosylate a wide range of aromatic substrates, giving rise to a wide variety of lower ligands in cobamides. To understand the molecular basis for this lack of substrate specificity, the x-ray structures of CobT complexed with adenine, 5-methylbenzimidazole, 5-methoxybenzimidazole, p-cresol, and phenol were determined. Furthermore, adenine, 5-methylbenzimidazole, 5-methoxybenzimidazole, and 2-hydroxypurine were observed to react with NaMN within the crystal lattice and undergo the phosphoribosyl transfer reaction to form product. Significantly, the stereochemistries of all products are identical to those found in vivo. Interestingly, p-cresol and phenol, which are the lower ligand in Sporomusa ovata, bound to CobT but did not react with NaMN. This study provides a structural explanation for how CobT can phosphoribosylate most of the commonly observed lower ligands found in cobamides with the exception of the phenolic lower ligands observed in S. ovata. This is accomplished with minor conformational changes in the side chains that constitute the 5,6-dimethylbenzimidazole binding site. These investigations are consistent with the implication that the nature of the lower ligand is controlled by metabolic factors rather by the specificity of the phosphoribosyltransferase.     

Biogenic Aldehydes in Brain: On Their Preparation and Reactions with Rat Brain Tissue

When 1 mM serotonin, dopamine, or norepinephrine was incubated with a monoamine oxidase preparation (mitochondrial membranes) in the presence of 4 mM sodium bisulfite, 85-95% of the amines were oxidized to the corresponding aldehydes. In the absence of bisulfite, the recoveries were only approximately 30%, and dark colored products were formed during the incubations. The aldehydes derived from tyramine, octopamine, methoxytyramine, and normetanephrine were also prepared by the use of this method. The bisulfite-aldehyde compounds were stable during storage at -20 degrees C. Bisulfite-free aldehyde solutions were made by diethylether extraction. When the aldehydes derived from dopamine or serotonin were incubated with rat brain homogenates, they were found to disappear in an aldehyde dehydrogenase- and aldehyde reductase-independent manner. The disappearance of the latter aldehyde was more pronounced, and the results indicated that this aldehyde may react with both proteins and phospholipids.