Frank A. Sedor

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

Abstract 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.

The dehalogenation of iodouracil by cysteine. Intramolecular general-acid catalysis of cysteine addition to 5-iodouracil☆

Abstract The rate-determining step of the cysteine-catalyzed deiodination of 5-iodouracil is the formation of 5-iodo-6-cysteinyl-5,6-dihydrouracil. The rate of the reaction depends upon the concentration of un-ionized 5-iodouracil and the following ionic species of cysteine; − OOC(NH 3 + )CHCH 2 S − . Unlike the reaction of 2-mercapto-ethanol with 5-iodouracil, the cysteine reaction is not subject to catalysis by imidazolium ion and tris(hydroxymethyl)aminomethane hydrochloride. When the rates of cysteine reacting with 5-iodouracil are measured in both H 2 O and D 2 O, a large kinetic isotope effect is observed ( k 2 H 20 k 2 D 20 = 4.10 ), thus implicating the protonated α amino group of cysteine as an intramolecular general acid catalyst for the reaction. These results and possible mechanisms for the actual dehalogenation of the intermediate 5-iodo-6-cysteinyl-5,6-dihydrouracil are discussed in terms of a possible mechanism for enzymatic halopyrimidine dehalogenation.

The addition of bisulfite to 5-fluorouracil. Evidence for a change in rate determining step

Abstract The kinetics of bisulfite addition to 5-fluorouracil were studied as a function of increasing concentrations of potential general acids. Values of k obsd [ SO 3 = ] measured at 25°C and ionic strength 1.0 M increased linearly and then became invariant with increasing concentrations of either HSO3− or (OHCH2CH2)2N+C(CH2OH)3 HCl (BisTris+HCl). A small kinetic hydrogen-deuterium isotope effect ( k HS k DS = 1.10 ) was observed for the general acid catalysed portion of the addition reaction. The kinetics of bisulfite elimination from 5-fluoro-5,6-dihydrouracil-6-sulfonate were studied in ethanolamine buffers. As previously observed with 1,3-dimethyl-5,6-dihydrouracil-6-sulfonate, this reaction is subject to general base catalysis and exhibits a large kinetic hydrogen-deuterium isotope effect ( k 2 H 2 O k 2 D 2 O = 3.8 ). The kinetic results for the addition reaction are consistent with a multistep reaction pathway involving the initial formation of an oxyanion sulfite addition intermediate (II) which subsequently adds a proton and undergoes tautomerization to yield the final 5-fluoro-5,6-dihydrouracil-6-sulfonate product. Thus the elimination of bisulfite from 5-fluoro-5,6-dihydrouracil-6-sulfonate probably proceeds by an ElcB mechanism which involves, at relatively low concentrations of general base, rate determining general base catalyzed proton abstraction from carbon 5 to yield intermediate II followed by the rapid elimination of sulfite to yield 5-fluorouracil. These results may be related to both the enzymatically catalyzed dehalogenation of bromoand iodouracil and the methylation of deoxyuridylate by thymidylate synthetase.

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

Abstract 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.

The dehalogenation of halocytosines by bisulfite buffers

Abstract Both 5-bromo- and 5-iodocytosine are rapidly dehalogenated in dilute bisulfite buffers to yield cytosine. With 5-bromocytosine, but not with 5-iodocytosine, extrapolation of semilogarithmic plots of extent reaction versus time indicates the bisulfite buffer concentration-dependent formation of an intermediate which subsequently reacts to control the rate of 5-bromocytosine dehalogenation. The disappearance of both halocytosines has a second-order dependence on bisulfite buffer concentration. Both imidazole and acetate buffers catalyze the reaction of 5-iodocytosine, but not that of 5-bromocytosine, with bisulfite. In the case of acetate buffer catalysis of the reaction of 5-iodocytosine with bisulfite, the dependence of the observed rate constants changes from first order to zero order as a function of increasing buffer concentration. The observed rate constants for 5-bromocytosine dehalogenation increase, reach a maximum at about 4.5, and then decrease as a function of pH. Iodometric titration of sulfite utilization coupled with spectrophotometric analysis of pyrimidine reactants and products indicates that 1 mole of sulfite is consumed per mole of halocytosine dehalogenated. The spectrophotometrically determined p K a values for the conjugate acids of 5-bromo- and 5-iodocytosine at 25°C and ionic strength 1.0 M are 3.25 and 3.56, respectively. These results are discussed in terms of a multistep reaction pathway which is analogous to the bisulfite-catalyzed dehalogenation of the 5-halouracils.

The dehalogenation of the 5-halo-5,6-dihydrouracils: Uracil formation from 5-iodo- and 5-bromo-5,6-dihydrouracil

Abstract The elimination of halide ion from either 5-bromo- or 5-iodo-5,6-dihydrouracil to yield uracil is a slow reaction which, in the case of 5-iodo-5,6-dihydrouracil, is 400 times slower than the enzymatic release of 125I− from 5-[125I]iodouracil. The elimination of HBr from 5-bromo-5,6-dihydrouracil is subject to general base catalysis by tris(hydroxymethyl)aminomethane (k2Tris base = 11 × 10−4 M−1 min−1, 37°C, ionic strength 1.0 M). At pH values near and above physiological, both the bromo- and iododihydropyrimidines are subject to hydrolysis of the dihydropyrimidine ring, a reaction which parallels halide elimination to yield uracil. The resulting 2-halo-3-ureidopropionate then cyclizes via intramolecular attack of the ureido oxygen atom to yield halide ion and 2-amino-2-oxazoline-5-carboxylic acid as final products. In dilute hydroxide ion, the kinetics of 5-bromo-5,6-dihydrouracil hydrolysis (25°C, ionic strength 1.0 M) show a change in rate-determining step as a function of increasing hydroxide ion concentration, a result which, as in the case of 5,6-dihydrouracil, can be explained in terms of the formation of a tetrahedral addition intermediate. The data are discussed relative to enzymatically catalyzed halopyrimidine dehalogenation.