J. Org. Chem., Vol. 43, No. 21, 1978
Deamination of Cytidine by Bisulfite
4197
Deamination of Cytidine by Bisulfite: Mechanism at Neutral pH S. Slae and R. Shapiro* Department of Chemistry, Graduate School of Arts and Sciences, New York University, New York, New York 10003 Receiued March 17.1978
The kinetics of cytidine deamination in the presence of bisulfite were studied at elevated temperatures at pH 6.55 and 7.30. The reaction was found to be second order in bisulfite ion. The activation enthalpy and entropy were determined. General base catalysis was observed in the presence of added bases. A mechanism is suggested which describes the kinetic properties of this reaction. By extrapolation of our data, the rate of deamination of cytidine M bisulfite within a cell, at 37 “ C , has been estimated at 4.0 X lo-’* s-l. by
-
The specific deamination of cytosine derivatives to those I VI, Scheme I) has of uracil by b i ~ u l f i t e(for ~ ~ example, ~,~ been used as a synthetic method in nucleoside chemistry and as a means for the chemical modification of nucleic acids.4 Treatment of viruses and bacteria by bisulfite induces mutations, which have been ascribed to cytosine deamination within DNA.5 In an earlier study, we described the principal mechanistic features of the reaction.6 Our data were primarily collected in acidic solution, in which the optimal rate of deamination is observed. One unresolved point of uncertainty was the nature of the buffer catalysis observed in the deamination step (IV V, Scheme I). In order to understand this step more fully, we have recently completed a study of the model compound, l-methyl-5,6-dihydro~ytosine.~ In our present work, we have conducted additional studies of the deamination of cytidine a t neutral pH. With this data, and the conclusions from our model study, we can describe the deamination mechanism fully, and extrapolate the rate to physiological conditions. This information is necessary for an accurate estimate of the possible hazard of bisulfite as an environmental mutagen.8
-
Results Kinetic Order of the Reaction. The kinetics of the bisulfite-catalyzed deamination of cytidine were measured at pH 6 . 5 5 , ~= 3.0 and 75 “C under pseudo-first-order conditions at several bisulfite concentrations. The results shown in Table I conform to a rate equation second order in bisulfite
where k’so3 = (2.08 f 0.1) X mo1-ls-l. General Base Catalysis. When general-base catalysts were added to solutions of cytidine and bisulfite ion at pH 6.55 and 7.30, j~ = 3.0, and 75 “C, an increase in the deamination rate was revealed, the overall reaction rate being described by the rate law
When this increase in observed reaction rate,
was combined with the known stoichiometric concentration of base, the catalytic effect of each general-base catalyst, h’g, was obtained. In solutions of biphosphate and bisulfite ions, the observed “biphosphate” or “sulfite” catalysis is due to the presence of two catalytic species, HzPO4- and HP042-, or HS03- and S032-.An adjustment can be made to the observed rate constants h g , for the minor catalytic effects of HzPO4- and HSOB-, so that a good estimate of the h’s for HP042- and S032-alone, ( k ’ g ) ~ ~can j , be made.l0 A further correction for the actual concentration of free base present under experimental conditionsll is made in Table 11, where the resulting values of COR are compiled. Activation Parameters. Rate constants for bisulfitecatalyzed deamination of cytidine were measured a t elevated temperatures at pH 6.55 ahd 7.30, and the results are summarized in Table 111.The activation parameters derived from
Scheme I. Mechanism of Bisulfite-Catalyzed Deamination of Cytidine
HSO,-(K’Add)
B, H , O , H’, -NH4+ ( k * )
I
R I1 -Ht (KHAdd)
1
R
I
-HSO,-
I
R IV R = 0-D-ribofuranosyl
R VI
4198
J.Org. Chem., Vol, 43, No. 21, 1978
Slae and Shapiro
--
Table I. Observed Catalytic Coefficients in Cytidine Deamination in the Presence of Bisulfitee
los x [ H S0 3-1 sr
[Bls~
kobsd,
s-l a
lo5 x
k'B,
M-2 S - b
0.75 0.93 1.0 0.75 1.0 1.0
pH 6.55 1.03 f 0.01 1.79 f 0.03 2.08f 0.02 0.56 M imidazole 1.40 f 0.02 0.25M H2P042.55 f 0.02 0.50M H ~ P O J - 3.15 f 0.17
1.0 1.0 1.0 1.0
pH 7.30 0.098f 0.01 0.40M CH:jCOz- 0.141 f 0.006 0.108f 0.006 0.50 M imidazole 0.134 k 0.005 0.072f 0.005 0.13 M HP04'0.109 f 0.005 0.086f 0.06
0.66 f 0.01 1.88f 0.02 2.14 f 0.10
Estimated uncertainties in values of k are the standard derivations. k ' =~ ( k & d - h,bsdS03)/([HS03-]s~[B]s~) as explained in the text. p = 3.0, 75 "C. 0
this data were: pH 6.55, AH* = 4.1 f 0.7kcal/mol, AS* = -69 f 2 eu; pH 7.30, AH* = 7.0 f 0.5 kcal/mol, AS* = -66 f 2 eu. Discussion p H Dependence of Reaction Rates. The cytidine deamination reaction in the presence of bisulfite was shown previously to proceed through either a protonated (IV) or neutral (11) intermediate (see Scheme I), depending on the experimental conditions.6 In the first case, a rapid equilibration between the species I, 11,111,and IV is followed by the ratedetermining reaction of the protonated intermediate, IV V. In the second case, the rate-determining reaction of the V, follows its rapid preequilibrium forintermediate, I1 11. Since either step IV --* V or I1 V may be mation, I catalyzed by a base such as sulfite ion, the observation of second-order rate dependence on the stoichiometric bisulfite concentration as well as general base catalysis is consistent with either case. The lack of nucleophilic catalysis is demonstrated by the small ratio of catalytic coefficients for imidazole (k/q),J compared to monohydrogen phosphate ( k / q ) ~ p o ~ '=- 3.42 a t pH 6.55. The observed rate constant for this reaction in the presence of bisulfite alone, kobstfSo3, as well as that for the increase in observed rate constant due to the presence of additional base catalysts, kobsdB. exhibit a calculable dependence on pH. The results of these calculations are compared with experimental measurements in Table IV (see the Supplementary Material for the derivation of these calculations). Under the experimental conditions described here, the mechanism involving the protonated intermediate (IV V) accurately predicts the
-
- -
-
-
pH dependence of hobsdS03 and kobsdB, while the alternative mechanism (I 11 V) is inconsistent with these results. Activation Parameters. The enthalpies and entropies of activation measured for the bisulfite-catalyzed cytidine deamination differ significantly from those determined for the deamination of l-methyl-5,6-dehydrocytosine,MDC7 (AH* = 20.7 f 0.8 kcal/mol, AS* = -11.0 f 2.6 eu for the reaction in acidic media). The mechanism proposed here for cytidine deamination can account for these differences. In the bisulfite-catalyzed cytidine deamination, intermediate IV is formed in a rapid preequilibrium, and it is this species that is the equivalent of the protonated MDC, the reacting species under the conditions studied for MDC (pH 0.4). In the mechanism proposed for cytidine deamination, where IV V is the slow step, the overall activation enthalpy (and entropy) is the sum of the enthalpies (and entropies) for steps I I1 (equilibrium),I11 IV (equilibrium), and IV V (kinetic). Based on earlier measurements6 of the temperature dependence of the cytidine sulfonate adduct formation, an approximate estimate of AH" = -9.3 f 4 kcal/mol, and AS" = -60 f 20 eu can be made for transformation I11 --* IV. The transformation equivalent to I I11 for cytidine18 has AH" = -4.4 kcal/mol and AS" = 5.0 eu. Therefore for the step IV V, the estimated parameters are AH* = 18 f 5 kcal/mol and AS* = -14 f 25 eu. When MDC deamination is compared with the kinetically equivalent step of cytidine deamination, the activation parameters are similar. Br#nsted Relationship. The relative rate constants of the general bases compared to sulfite ion can be shown to be equal to the equation
-
-
-
-
-
[HS03-]ST[B]ST) kobsdSo3/[HSO3-IST2
hobsdB/(
The relative catalytic coefficients for the rate-determin, be calculated from ing step of this mechanism, h g / h s ~can the relative observed rate constants (see the Supplementary Material for the derivation). Values so obtained are presented in Table 11. The Br9nsted relationship21for the relative rate constants of the rate-determining step at pH 6.55, = 3.0 and 75 "C yields2* log ( k l q ) = (0.35 f 0.07)[(pKa+ log ( p / q ) ] - (8.59f 0.47) (4) Studies of the general-base-catalyzed deamination of 1methyl-5,6-dihydro~ytosine~ (37"C,1.1 = L O ) , which proceeds through a similar rate-determining step, yielded a fl of 0.19 f 0.03for a series of bases including structural types similar to those used in the present study. The agreement between the values of p is good, considering the differences in experi-
Table 11. General-Base Catalvsis in Cvtidine Deamination in the Presence of 1 M Bisulfite Ione
pH 6.55 so32-
imid
HP042-
sop imid HP042-
CH3C02-
6.37 7.12 6.27
20.8 6.61 20.1
6.37 7.12 6.27 4.76
0.98 0.72 0.86 1.08
15.6 6.1 18.1
0.60 0.21 0.65
26.0 f 0.3 31.5 f 0.5 27.8 f 1.8
1.00 1.21 f 0.06 1.07f 0.08
pH 7.30
0.93 0.72 0.84 1.08
0.90 0.60 0.915 0.997
1.01 f 1 1.20 f 0.1 0.94 f 0.7 1.08 f 0.08
1.00 1.20 f 0.10 0.94f 0.23 1.08f 0.09
estimated error limits pK estimates for these experimental conditions based on extrapolations of the best literature are f 0.05. Adjustment made for the catalytic contribution of conjugate acids, HzP04- and HS03-, as explained in the text. ( k ' ~ ) ~ ~ ~ = ( k ' ~ ) , d j ( [ H + ] + KHB)/KHB. Average of two measurements, see Table I. e p = 3.0,75"C.
J. Org. Chem., Vol. 43, No. 21, 1978
Deamination of Cytidine by Bisulfite Table 111. Temperature Dependence of Cytidine Deamination Rate Constants in the Presence of 1 M Bisulfiten
io5 x temp, "C 55.5 64.5 75.0 86.0 a
Table IV, Dependence of Cytidine Deamination Rate Constants on pH in the Presence of 1 M Sulfite and Added Rases
io7 x kobsd, S-l (pH 7.30)
kobsd, S-l
(pH 6.55)
[(k'B)6.55/(k'B);.301
SO?imidazole HP04'-
= 3.0.
mental conditions and the uncertainties involved in the estimate of /3 for cytidine. Reaction Mechanism. T h e mechanism presented in Scheme I is consistent with the kinetic order of the reaction, rate dependence on pH, general-base catalysis, absence of nucleophilic catalysis, lack of general-acid catalysis? solvent isotope effect,6 and activation parameters. Although the details of transformation IV V have not been examined, this process probably occurs through a tetrahedral intermediate (2a) similar to the mechanism proposed
-
+ I
HO
R'
experimental ratio
base
5.29 f 0.30 6.55 f 0.06 9.80 0.60 14.1 f 0.30
1.31 f 0.07 1.49 f 0.04 2.08 0.09 2.42 f 0.22
4199
15.1 f 2.0 7.7 f 1.0 16.8 f 2.0
17.7 f 1.1
9.2 f 0.04 21.5 f 7.5
2.6 f 0.3 1.3 f 0.2 2.8 0.3
*
= k o b s d S 0 3 / [ H S 0 3 - ] ~ ~ 2 for s0s2- and kobsdB/ [HSO~-]ST[B]ST for imidazole and HP0d2-. These are the apparent third-order rate constants corrected for pH to reflect true concentrations of reactive forms; 6.55 and 7.30 are the pH values for the respective determination of k'obsd. * See Appendix in supplementary material for the basis of these calculations. Case 1 and 2 as described in the text. p = 3.0,75 "C.
(see the Supplementary Material for the derivation). Extrapolation from rate data at elevated temperatures yields kobsdSo3
-
calculated ratiobSc case 1 case 2
= 2.5
x
S-'
for 37 "C, p H 7.30, p = 3.0. In order to estimate the rate of deamination within a living cell, we must introduce into eq 5 the total concentration of [B~]sT.This has been estimated as bases within the cell, 0.16 M ( p = 0.161.23-25 Thus the physiological deamination rate of cytidine by bisulfite in vivo at M bisulfite can be estimated a t 4.0 X M bisulfite it would be 4.0 X s-l. 10-12 s-1, while at The significance of this data for questions of environmental mutagenesis will be discussed elsewhere.
R 2 0
Experimental Section "
3
a, R b, R
= =
P-D-ribofuranosyl; R' CH,; R' = H
= SO,-
-
-.
for the deamination of l-methyl-5,6-dihydrocytosine (lb 2b 3b) and similar amidines? The rate-determining step of this mechanism is t,hought to be either t h e formation of intermediate 2, or the reaction of the protonated form with which it is in equilibrium under the experimental conditions (4). These processes could occur through transition states VI1 +H,N N
OH
Y
&A R I R 4
or VIII, respectively.
I
NH2 1711
VI11
The Rate of Cytidine Deamination of Bisulfite u n d e r Physiological Conditions. It can be shown that in the presence of general base, the rate of cytidine deamination proceeding through intermediate IV can be approximately described by the following rate constant (eq 5 )
Materials and Apparatus. Schwartz Mann cytidine and commercially available reagent grade chemicals were used. Imidazole was recrystallized from benzene three times before use (mp 88.5-89.0"C; lit. 90-91 "C). Water was doubly distilled in Pyrex vessels and deoxygenated with nitrogen. For reactions in the presence of sulfite ion, 10-3 M hydroquinine was added as a radical inhibitor. Apparatus. Ultraviolet absorbance measurements were recorded on a Cary 15 recording spectrophotometer. pH measurements were made at various temperatures on a Radiometer Model 22 pH meter equipped with a glass electrode which had been precalibrated with pH standard solutions at the temperatures of measurement. pH values are f0.02. Kinetics of Cytidine Deamination. Ampules (2 mL) sealed with rubber septum caps were filled with reaction mixtures buffered to the desired pH at appropriate temperatures and kept in a constant temperature bath regulated to f0.1 "C. Temperatures were measured with a thermometer calibrated by the National Bureau of Standards. Samples (50 pL) were removed with a syringe and stored in pH 11.5 buffer solution of 0.2 M Na2HP04 for at least 24 h. The relative concentrations of the reactant (cytidine) and product (uridine) were calculated for each sample from the observed absorbances at 270 and 280 nm by means of the formulas, [Uridine] = (A270 - 1.21A2~)/3044; [Cytidine] = (2.39 A280 - A270)/8805. A plot of log xcrt against time was analyzed by the method of least squares on a Hewlett Packard 3000 computer. Estimated errors in k are the standard deviations of the slopes for these kinetic plots. Catalytic coefficients of general bases were obtained by rate measurements in aqueous buffers kept at constant pH. Ionic strength was held constant by the addition of sodium chloride.
Acknowledgment. This research was supported by Grant ES 01033 from the National Institutes of Health, U S . Public Health Service. R. Shapiro has been a Research Career Development Awardee (GM-50188) of the National Institutes of Health. Supplementary Material Available: An Appendix containing
Cook and Holman
4200 J. Org. C'hem., Val. 43, No. 21, 1978
( ~ S O ~ Z - [ S O ~ ~ - ] ) I ( ~ H S=~1.50 ~ - [(pH H S6.55), O ~ - ]9.0 ) (pH 7.30). The possible error introduced into the calculations by this estimate is not significant enough to modify the final conclusions. (11) Based on best p K estimates vailable from the for these R e g i s t r y No.--Cytidine, 65-46-3; bisulfite, 15181-46-1 conditions. (12) A. Albert and E. P. Serjeant, "Ionization Constants of Acids and Bases", Methuen, London, 1962. G. Kciturn, W. Vogel, and K. Andrussow, "Dissociation Constants of Organic (13) References a n d Notes Acids in Aqueous Solution", Butterworths, London, 1961. (1) R. Shapiro, R . E. Servis, and M. Welcher, J. Am. Chem. SOC. 92, 1422 (14) D. D. Perrin, "Dissociation Constants of Inorganic Acids and Bases In (1970). Aqueous Solutions", Butterworths, London, 1969. (2) H. Hayatsu. Y. Wataya, and K. Kai, J. Am. Chem. SOC.92, 724 (1970). (15) D. D. Perrin, "Dissociation Constants of Organic Bases in Aqueous Solu(3) H. Hayatsu, Y . Wataya, and K . Kai, Biochemistry, 9, 2858 (1970). tions", Butterworths, London, 1965. (4) H. Hayatsu, Prog. Nucleic AcidRes. Mol. Biol. 16, 75 (1976). (16) L. G. Sillen and A. E. Martell, "Stability Constants of Metal ion Complexes", (5) R. Shapiro, Mutation Res. 39, 149 (1977). 2nd ed, The Chemical Society, London, 1964. (6) R. Shapiro. V. DiFate, and M. Welcher, J. Am. Chem SOC. 96, 906 (17) L. G. Sillen and A. E. Martell, "Stability Constants of Metal-Ion Complexes, (1974). Supplement No. l " , The Chemical Society, London, 1971. (7) S. Slae and R. Shapiro, J. Org. Chem. 43, 1721 (1978). (18) H. A. Sober, ed., "Handbook of Biochemistry", Chemical Rubber Publishing (8) Science, 187, 502 (1975). Co.. Cleveland. Ohio. 1970. (19) Nucleophilic catalysis is characterized by high ratios of ( k / 9 ) , , / ( k / 9 ) ~ p o , (9) Symbolism used in this paper includes the following: fi is the ionic strength (in molarity): kobd indicates the observed rate constant; ST indicates the of about 1000.20 (20) H. S. Johnson, Adv. Phys. Org. Chem., 5, 237 (1967). stoichiometric concentration before correction for equilibration of the different species present; Sub indicates Substrate; "Bisulfite" describes (21) W. P. Jencks, "Catalysis In Chemistry and Enzymology", McGraw-Hill, New York. N.Y., 1969. the overall effects of a solution of bisulfite and sulfite ions without identifying the specific species causing that effect. (22) The Brgnsted relationship reported here should be considered only illus(IO) Assuming that the catalytic properties of general bases are similar for the trative, since bases of different structural types are compared. In addition, errors in the estimated values of KHBand the small change in catalyst well-characterized deamination of l-methyl-5,6dihydro~ytosine,~ and the strength (ApK = 0.85) studied and possible specific salt effects affect the ratedetermining step of cytidine deamination (See Reaction Mechanism), uncertainty in the Brgnsted relationship. then kson = 2kHS03; k ~ p o ~ z=-5k~,po,-. Under the experimental conditions, the relative concentrations of the basic species present can be calculated (23) J. L. Gamble, "Chemical Anatomy, Physiology, and Pathology of Extrato be: [S032-]/[HS03-] = 1.50 (pH 6.55), 9.00 (pH 7.30); [HP04*-]/ cellular Fluid", 6th ed,Howard University Press, Cambridge, Mass.. 1954, [H2P04-] = 1.86 (pH 6.55) 10.8 (pH 7.30). The relative catalytic contripp 5,9, 11. O 3.0 ~ - [ H S (24) ~ ~ - D. ] ) Jensen, "The Principles of Physiology", Appieton-Century-Crofts, New bution of each species becomes ( ~ S O ~ ~ - ~ S ~ ~ * - ] ) I ( ~ H S = York, N.Y., 1976, p 54. (PH 6.55). 18 (PH 7.30); ( k ~ p o ~ ~ - [ H P 0])/(kii2P04-[H2P04]) 4 = 9.3 (PH 6.55), 54 (pH 7.30). Should the rate constant ratio ( k ~ o ~ z - / k ~equal ~ o ~ - ) (25) A. C. Guyton, "Textbook of Medical Physiology". 4th ed, Saunders, Philone (a lower limit). the relative catalytic contribution would become adelphia, Pa., 1971, p 39
the derivations mentioned in the text (6 pages). Ordering information is given o n any current masthead page.
Fluorinated Pyrimidine Nucleosides. 2.' Reaction of Hydrochloride 2,2'-Anhydro-1 -~-D-arabinofuranosyl-5-fluorocytosine with Nitrogen and Sulfur Nucleophiles Alan F. Cook* and Michael J. Holman Chemical Research Department, Hoffmann-La Roche Inc., N u t l e y , N e w Jersey 071 10 Received M a r c h 15. 1978 Reaction o f 2,2'-anhydro-l-~-~-arabinofuranosyl-5-~uorocytosine hydrochloride (1, anhydro-ara-FC) w i t h a m chloride (2) by attack a t Cz o f t h e pyrimim o n i a gave l-B-~-arabinofuranosyl-2,4-diamino-5-fluoropyrimidinium dine ring. Reaction o f 1 w i t h methylamine gave t h e corresponding 2-methylamino derivative 3, w h i c h was r a p i d l y converted i n t o t h e 2,4-bis(methylamino)arabinoside4 by amine exchange a t C4. T r e a t m e n t o f 1 w i t h ethylamine or n-propylamine similarly produced t h e corresponding 2,4-bis(alkylamino) derivatives 14 a n d 15. Reaction o f 1, 2, o r 4 w i t h methylamine for a prolonged reaction period resulted in rearrangement w i t h loss o f t h e sugar m o i e t y to produce 2-amino-5-fluoro-l-methyl-4-methyliminopyrimidine hydrohalide (91, t h e structure o f w h i c h was conf i r m e d by X - r a y crystallography. T h e reaction o f 1 w i t h 15N-enriched ammonia was examined; in a d d i t i o n t o Cz attack a n d amine exchange a t C4, evidence was f o u n d for incorporation o f 16Ni n t o t h e p y r i m i d i n e ring. Reaction o f 1 w i t h sodium hydrosulfide or hydrogen sulfide induced defluorination w i t h o u t cleavage o f t h e anhydro b o n d (21); t h e oxazolidinethione 22 was also isolated as a b y p r o t o give 2,2'-anhydro-l-/3-~-arabinofuranosylcytosine duct. T r e a t m e n t of t h e corresponding sulfur- a n d nitrogen-bridged analogues 23 a n d 26 w i t h sodium hydrosulfide also produced t h e corresponding defluorinated anhydro nucleosides 25 a n d 27.
2,2'-Anhydro-l-~-~-arabinofuranosyl-5-fluorocytosine shown that the corresponding unfluorinated analogue 1-phydrochloride (1, anhydro-ara-FC; Scheme I), a compound D-arabinofuranosyl-2,4-diaminopyrimidiniumchloride was produced by the reaction of 2,2'-anhydro-l-/3-~-arabinofufirst synthesized by Fox et has been shown by Burchenal et aL3 to be a promising new agent for the treatment of acute ranosylcytosine with a r n m ~ n i aAlthough .~ difficulty was exmyeloblastic leukemia. As part of a synthetic program in the perienced by Doerr and Fox in the isolation of the unfluoarea of fluorinated pyrimidine nucleosides, we have employed rinated analogue due to the hygroscopic nature of the salt, anhydro-ara-FC as starting material for the preparation of together with its propensity for recrystallization to the 2,2'anhydro compound, the hydrochloride salt of 2, in contrast, some 5-fluoropyrimidine nucleosides with potential antitumor activity. The reactions of nitrogen and sulfur nucleophiles was found to be stable indefinitely a t room temperature. In aqueous solution, 2 was found to be much less stable; storage form the basis for this report. of a solution for 4 days at room temperature resulted in almost Reaction of anhydro-ara-FC (1) with methanolic ammonia yielded the highly crystalline 2,4-diamino-5-fluoropyrimidine complete conversion to arabinosyl-5-fluorocytosine. A small arabinoside 2 by reaction at Cz of the pyrimidine ring.4 This amount of a byproduct was isolated from the reaction of 1 with r e a c t i o n is to be expected since Doerr and Fox have previously ammonia; this was identified as 2-amino-p-D-arabinofu0022-3263/78/1943-4200$01.00/00 1978 American Chemical Society
