5826 the hydrolysis of derivatives of salicylic acid, the nucleophilic catalysis becomes relatively less favorable, and the pK,’s of nucleophile and leaving group are approximately equal at the borderline. But if the leaving group is lost, as it is from derivatives of dicarboxylic acids, nucleophilic catalysis is relatively more favorable than in intermolecular reactions, and leaving groups up to about 6.5 pK units more basic than the nucleophile can be displaced. Implications for Enzymic Catalysis. On the basis of the evidence and arguments presented in this paper we consider that the mechanism outlined in Scheme I11 is an important pathway for the hydrolysis of the anion Scheme I11
I
m
1
of 3,5-dinitroaspirin. This mechanism achieves a potentiation of intramolecular nucleophilic catalysis of the hydrolysis of this ester by a second, independent, intramolecular process in which the leaving group of the first step catalyzes the further reaction of the intermediate formed. Such tight control and integration of consecutive intramolecular processes is a characteristic generally associated with enzymic catalysis. For example, the formation of an acyl enzyme with a second potentially nucleophilic group involved in the active site is probably a not uncommon situation. 3,5-Dinitroaspirin is a model for such a system; the acyl group migrates rapidly and reversibly between the two nucleophilic centers, and is hydrolyzed slowly relative to this process by one of several possible routes involving general species catalysis of the attack of water on the acyl group by the free nucleophilic center. In this paper we have shown that in the hydrolysis of 3,5-dinitroaspirin both nucleophilic centers can act as general base catalysts in this way. In the following paper we present evidence that the addition of a proton to the system, which might be expected to inhibit the nucleophilic mechanism, actually enhances catalysis. Acknowledgment. We are grateful to Dr. W. P. Jencks for valuable exchanges of comment and unpublished information. We acknowledge also a maintenance grant from the Science Research Council of Great Britain, and a Studentship from Gonville and Caius College (to A. R. F.).
Intramolecular Nucleophilic Catalysis in the Hydrolysis of Substituted Aspirin Acids A. R. Fersht and A. J. Kirby Contribution f r o m the University Chemical Laboratory, Cambridge, England. Received January 16, 1968 Abstract: The hydrolysis of acetyl 3,5-dinitrosalicylic acid is faster than that of the anion, even though the
hydrolysis of the anion is already accelerated by intramolecular nucleophilic catalysis. The evidence does not support our earlier suggestion that this further acceleration is due to intramolecular general acid catalysis by the neighboring carboxyl group. Solvolysis of the acid in 50 aqueous methanol produces significant amounts of methyl 3,5-dinitrosalicylate, indicating that a mixed salicylic acetic anhydride is an intermediate, and therefore that intramolecular nucleophilic catalysis is involved in this case also. Catalysis is observed for the hydrolysis of aspirin itself, and for its monosubstituted derivatives, and is shown to assist the attack of other nucleophiles than water. Nucleophilic catalysis appears to be favored for the hydrolysis of the aspirin acids because of a more favorable equilibrium constant for the formation of the protonated form, rather than the anion, of the mixed anhydride intermediate.
W
e have shown that intramolecular catalysis of the hydrolysis of aspirin anion’ and its singly substituted derivatives2 involves the carboxylate group as a general base, and that the mechanism changes to nucleophilic catalysis for 3,5-dinitroa~pirin.~This catalysis is apparent in the case of aspirin and the singly substituted compounds from their characteristic (1) A. R. Fersht and A. 3. Kirby, J . Amer. Chem. SOC.,89, 4857 (1967).
(2) A. R. Fersht and A. J. Kirby, ibid., 89,4853 (1967). (3) A . R. Fersht and A. J. Kirby, ibid., 90,5818 (1968).
Journal of the American Chemical Society / 90.21
pH-rate profile^,^ which show that the aspirin anions are hydrolyzed more rapidly than the protonated forms. The pH-rate profile for the hydrolysis of 35dinitroaspirin (Figure 1) also shows a pH-independent region between pH 4 and 8, but in this case the free acid is considerably more reactive than the anion.5 We have established that the hydrolysis of the anion is accelerated (4) L. J. Edwards, Trans. Faraday SOC.,46, 723 (1950). (5) A. R. Fersht and A . J. Kirby, J . Amer. Chem. Soc., 89, 5961 (1967); preliminary communication.
October 9, 1968
5827
by about 50 times by intramolecular nucleophilic catalysis,3 so that the hydrolysis of the free acid, which is some 30 times faster still, must be more strongly catalyzed, presumably by the free carboxyl group. Furthermore, this catalysis is presumably available also to those aspirin derivatives showing the classical pH-rate profile for hydrolysis. We have therefore investigated the scope, as well as the mechanism, of this catalysis.
end of each kinetic run, were measured using a Vibron electrometer, fitted with a C-33B pH-measuring attachment. The results of the pK. measurements, a t 39" and ionic strength 1.0, were for aspirin 3.36 =t0.05, for 5-methoxyaspirin, 3.37 f 0.04, and for 4-methoxyaspirin, 3.89 f 0.04. Detailed hydrolysis data for 3,5-dinitroaspirin are given in Table 11. Table II. Hydrolysis Data for 3,5-Dinitroaspirin Acid at 39' (Ionic Strength l.Oa) Conditions
Experimental Section Materials. Inorganic salts were either purified reagent grade or analytical grade. The preparation of the substituted acetylsalicylic acids has been described p r e v i o u ~ l y . Aspirin ~~~ methyl ester was prepared from methyl salicylate and acetic anhydride containing a trace of concentrated HsSOd, and had mp 48.5-49" (lit.6 mp 49"). Solvents were purified as before. Kinetic Methods and Results. The hydrolysis rates of the substituted aspirins were measured spectrophotometrically, as previously d e ~ c r i b e d at , ~ 39" and ionic strength 1.0, using the wavelengths listed in Table I. Final absorbances for runs followed by initial rates were measured by tenfold dilution with the, correct buffer, maintained at ionic strength 1.0 with added KCl. The methyl esters were added from stock solutions in dioxane, so that the final reaction mixtures contained 3 % of dioxane. Table I. Rate Constants for the Hydrolysis of Substituted Aspirin Acids, at 39" (Ionic Strength 1.0) Substituted salicylic acida
khyd
k E + X lo3, M-1 min-1
298.Y 0.59 =k 0.02 298.Y 0.038 f 0.001 31lC 1.59 i. 0.18 319 1.50 i 0.16 330d 0 . 5 2 i 0.02 304c 2 . 1 f 0.25 302c 1.95 f 0.20 297d 0 . 7 9 f 0.01 347d 4.98 f 0.32 347d 32.5 i. 0 . 6 317d 23.2 i. 0 . 7 30@ 16.3 i 0 . 3 308 80.1 i 3 . 7
4.72 i. 0.05
Followed at (mp)
Aspirin Aspirin, methyl ester 5-Chloroaspirin 5-Bromoaspirin 5-Methoxyaspirin 4-Chloroaspirin 4-Bromoaspirin 4-Methoxyaspirin 4-Nitroaspirin 3-Nitroaspirin 5-Nitroaspirin 5-Nitroaspirin in DzO 5-Nitroaspirin at 49.2" 5-Nitroaspirin at 59.4"
X lo4, min-1
308 240 f 10
4 . 2 8 ' 2 0.05 4.32 i. 0.04 5.30 i 0 . 0 7 3.88 f 0 . 0 7 3.96 f 0.05 4.35 f 0.04 3.88 f 0.08 1.02 f 0 . 0 2 3.71 f 0.11 4 . 5 f 0.16 9.81 f 0.48 24.5 f 1.2
For 5-nitroaspirin, AH,,* = 23 f 0.6 kcal/mol; AS3,*= - 5 f 3 e u I a The iodo compounds were too insoluble to use. Isosbestic point for aspirin anion, acid, and methyl ester. "Absorption maximum for the substituted acid. d Wavelength of maximum difference in absorption between aspirin and salicylic acid produced. e k z for catalysis by Df. f Calculated from the rate constants given for 39, 49.2, and 59.4'.
The spontaneous rate constants for the hydrolysis of the aspirin acids were measured by extrapolation to zero acid concentration of the good straight lines obtained by plotting the observed hydrolysis rates against the concentration of HCl. In each case six runs a t HCl concentrations between 0.1 and 0.6 M gave the spontaneous and acid-catalyzed rate constants, listed in Table I. The values are corrected for residual contributions from hydrolysis of the anions. The accuracy of the low rate constants for aspirin and its 4- and 5-methoxy derivatives was improved by three extra runs in HCl solutions near pH 2. The large contributions to the observed rate from the hydrolysis of the anions at this pH were calculated from the known rate constants for anion hydrolysis,* using the Henderson-Hasselbach equation. The pK;s of these acids were measured under the experimental conditions by the spectrophotometric method.' pH's for these measurements, routinely a t the ( 6 ) H. Erdmann, Chem. Ber., 32, 3572 (1899).
DH
1.0 M HC1 1.O M HCl, ionic strength 3.0 (KC1) 2.9 M HCl, ionic strength 2.9 1.0 M DC1 in DzO 0.5 M HCI 0.5 M H C 1 at 32.1" 0.5 M HC1 at 24.9" 0.2 M HCl 0.1 M HCl 0.05 M HC1 0.033 M HCl 0.02 M HCl 0.01 M HCl 0.005 M HC1 Formate bufferb Acetate bufferb Phosphate bufferb Carbonate bufferb
1.39 1.57 1.75 2.06 2.35 2.59 2.87 3.51 4.89 5.60 6.44 8.75 9.68 10.10
khvd.
min-1
0.746 1.15 1.04 0.562 0.741 0.338 0.140 0.730 0.682 0.582 0.534 0.468 0.344 0.232 0.157 0.099 0.047 0.028 0.027 0.027 0.048 0.176 0.386
Corrected rate constant for free acid 0.746 =t0.001 2.68 f 0 . 0 2 x 10-2 Corrected rate constant for anion 3.6 f 0 . 1 x 10-3 Rate constant for hydrolysis of methyl esterC For free acid, AH,,* = 21.4 f 0.7 kcal/mol ASs9* = 1.4 f 3 eud 0 Followed at 307 mp for the acid form, and at 338 mp for the anion, as described previously. Extrapolated to zero buffer concentration. c Followed in phosphate buffers, pH 6.4, and extrapolated to zero buffer concentration. Based on first-order rate constants in 0.5 M HCl.
Hydrolysis in 50% Aqueous Methanol. A 4.2-mg sample of 3,5-dinitroaspirin was hydrolyzed for ten half-lives at 39" in 250 ml of a solvent composed of equal volumes of methanol and 2 M HC1. The hydrolysis mixture was then compared spectrophotometrically with solutions of authentic samples of 3,5-dinitrosalicylic acid and its methyl ester, in the same solvent (Table 111). Table III
Wavelength A, m i
Reaction product
3.5-Dinitrosalicylic acid
Methyl 3,5dinitrosalicylate
285 325 340 350
1.8206 1.6542 1.3034 1.oooO
1,6842 1,6083 1.2763 1.oooO
3.8192 2.4385 1.5962 1.oooO
The composition of the reaction mixture was calculated from these ratios, using the ratio of the absorbtivities of the two components a t 350 mp (Cacid/€ester = 2.055). This gave a n estimate for the composition of the reaction product of 12.6 =k 0.6% of methyl ester. Two further determinations at room temperature gave the similar figure of 12.2 =k 0.7 % methyl ester. When the experiment (7) A. Albert and E. J. Sergeant, "Ionization Constants of Acids and Bases," Methuen and Co., Ltd., London, 1962.
Fersht, Kirby / Aspirin Acids
5828 Table IV. Apparent Second-Order Rate Constants for the Reactions of Oxy Anions with Substituted Aspirins at 39" (Ionic Strength 1.0) of free base in buffer
Concn range
No. of runs
kz,' M-1 min-1
4 6 6 4 5 6 6 6 6 6 6
1 . 0 4 x 10-2 2 . 1 2 x 10-2 4.05 X 10-2 9 . 9 x 10-2 0.192 0.63 0.72 3.32 x 10-2 3.08 X 10-2 2.87 X 10-2 3.66 x 10-2
G0.9 0.1-1 .o
4 5
3.0 9.1
4-Methoxyaspirin 91 0.1-0.8 33 0.1-0.8
6 6
6 . 0 x 10-6 1 . 7 x 10-4
of free base, M
Oxy anion
PH
Methoxyacetate
6.4b 3.45 2.94 6.4b 3.57 2.83 2.49 6.45b 5.65 4.95 3.57
3,5-Dinitroaspirin 100 GO.8 50 0.1-0.8 25 0.05-0.4 100 0-0.8 50 0.1-0.7 16.7 0.02-0.16 9.1 0.02-0.16 100 0-0.8 91 0.1-0.8 67 0.1-0.8 9.1 0.02-0.16
Formate
6 . 3b 3.56
100 50
Acetate
5.7 4.4
Formate
6 . 3b 5.6d 3.54 3.24
Formate
Acetate
