general basic catalysis.pdf

7/22/2019 General Basic Catalysis.pdf

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1GX h I YRON L. BlTNDE R .INI) BYRONt7 .TURNQUEST \ . < I . 7 J[CONTRIBUTIONROM THE DEPARTMENTF CIIEMISTRY,LLINOISNSTITUTE1 TECIINOLOGY]

General Basic Catalysis of Ester Hydrolysis and Its Relationship to EnzymaticHydrolysisBYMYRON. BENDER ND BYRONW. T UR NQC E ST ~

RECEIVEDL-CUST30, 1956The hydrolyses of p-nitrophenyl acetate, 2,4-dinitrophenyl acetate, phenyl acetate and ethyl thiolacetate are catalyzedby a number of bases including pyridine, 3- and 4-picoline, trimethyl amine, imidazole, N-methylimidazole, quinoline andacetate ion. The hydrolysisof p-nitrophenyl acetate meets th e requirements of general basic catalysis since the rat e is pro-

portional to th e summation of catalytic constants times catalyst concentrations at constant p H and ionic strength. Thecatalytic rate cons tants are related to th e basicity of the catalyzing amines of constant steric requirement in a manner similarto the Brfinsted catalysis law. Th e slope of this linear free energy relationship is 1.62 , th e highest recorded for a generalbasic catalysis. Th e mechanism of this general basic catalysis, involving not a rate-determining proton transfer but rathe rthe addition of the base to the su bstrate producing an unstable intermediate, offers a n explanation fo r the deviation of theslope from its usual limitation. Imidazole catalysis of the hydrolysis of various esters demonstrates that the rate of hy-drolysis increases with th e acidity of th e alcoholic residue of the ester. I t appears th at general basic catalysis of est er hy-drolysis is of importance only for esters containing an alcohol th at is a reasonably stron g acid p K ,

acid, acetate ion, chloroacetic acid and bisulfateion on the hydrolysis of ethyl a ~ e t a t e . ~ owever,the reported catalytic rate constants are minute(the catalytic constant for acetate ion was reportedto be 0.25 X lo- l./mole sec. while that for hy-droxide ion is 1.08 l./mole sec.) and their realityis questionable. These data have been criticallydiscussed from the viewpoint of th e primary salteffect and th e effect of th e nature of the bufferon the rate.l0V1l General catalysis by acetic acidand acetate ion, although of small magnitude, hasbeen reported in the esterification of acetic acid i nmethanol.12A consideration of the mechanism of the hy-drolysis of carboxylic acid der ivatives by hydrogenand hydroxyl ions points to the direction in whichgeneral basic catalysis of ester hydrolysis may besought, 3 Wiberg14 stat es th at for th e reaction

L Y + Jthe initial addition depends on the nucleophilicityof ut th e partitioning of th e addition compoundto form reactant or product is related to th e relativestabilities of Y and X. One would then expectgeneral basic catalysis of ester hydrolysis when theanion formed from the ester is at least of compa-rable stability (basicity) to the attacking base.Accordingly, in order to demonstrate generalbasic catalysis, esters were chosen in which thealcoholic portion of th e ester was a much strongeracid than the ethanol in Dawsons original work.Among such esters are phenyl, negatively sub-stituted phenyl and thiol esters.

(9) H. M. awson and W. owson, J . Chem. Soc. 2444 (1927);H. &I . Dawson and W. Lowson, bid . , 393 (1929); H. iM. Dawson,E. R . Pycock and E. Spivey, i b i d . , 291 (1933).10) R. P. Bell, Acid-Base Catalysis, Oxford Univ. rem, Lon -don, 1941, . 80.(11) G. Gorin, personal communication.(12) A. C. Rolfe and C . N. Hinshelwood, T r a n s . Fa ra d a y Soc., 30(13) M. I Bender, THIS JOURNAL, 73, G26 (1951); 1.I. ,, Bender1 4 ) I


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April 5, 1957 BASIC CATALYSIS OF ESTERHYDROLYSIS 1657ExperimentalMaterials .-2,4-Dinitrophenyl acet ate was prepared from2,4-dinitrophenol and acetic anhydride.l6 Two recrystalliza-tions from Skellysolve B gave an almost colorless product,m.p. 71-71.5" (l it. m.p. 72"). The preparation of p-nitro-phenyl acetate was described previously.6 Ethyl thiolacetatewas prepared from ethanethiol and a ceyl chloride, b. p.115-115.5" (lit. b.p. 115').'6 Phenyl acetate was anEastman Kodak Co. white label product, n Z 0 ~.5025.Imidazole was described previously.6 In order to avoidturbidity (probably du e to hydrocarbon contamination) ofthe solution when 4-picoline was mixed with water, the

Eastman Kodak Co. white label product was distilled(b. p. 142-143'), converted to the hydrochloride and crys-tallized from ethanol-ether, reconverted to the free aminewith alkali and redistilled, b.p. 143-144', n Z o ~.5035(lit. b.p. 145", n z o ~.5029).'7 3-Picoline, a Reilly Coal Ta rCo. product, was fractionated through a Todd distillationassembly packed with a monel spiral; the middle fractionwas collected using a reflux ratio of seven-to-one, b.p.144.l0, @ 2 2 . 5 ~ 1.5049 (lit. b.p. 143.8", nZ6D 1.5038).17Fisher reagent grade pyridine was redistilled, b.p. 115:.Fisher reagent grade quinoline was redistilled, b.p. 236 .Eastman Kodak Co. white label trimethylamine (as thehydrochloride) was used without further purification. N-Methylimidazole was prepared from imidazole and methyliodide, b.p. 196-198' (li t. b.p. 19S0).'8 The neutra l equiva-lent was determined by t itration of 40-mg. samples with0.02 NH Cl using a Beckman model H pH meter. Titrationcurves were drawn an d the end-point taken a s the inflectionpoint of these curves; neut. equiv. found 80.7 and 81.0,calcd. 82.1. The ionization constant of N-methylimidazolewas determined potentiometrically in the above manner.The pH of the half-neutralization point of t he amine, usedas t he value of PK as found to be 7.00 and 6.98 for solu-tions 0.02 A4 in total amine. Dedichenlg has reported apK, of 7.34 for this amine a t ionic strengths of the sameorder of magnitude a s above. J . T . Baker reagent gradeacetic acid was redistilled, b.p. 118'. Commercial 1,4-dioxane was purified a s described previously.6Kinetic Determinations.-The general procedure for thespectrophotometric determination of the kinetics using aCary recording spectrophotometer has been given pre-viously.6 The hydrolysis of p-nitrophenyl aceta te wasfollowed by measurement of the appearance of p-nitrophe-nolate ion a t 400 mp; the hydrolysis of 2,4-dinitrophenylaceta te was followed by measurement of th e appearanceof 2,4 -dinitrophenolate ion a t 360 mp; the hydrolysis ofphenyl acetate was followed by measurement of t he appear-ance of phenol a t 272 mp and the hydrolysis of ethyl thiolace-ta te was followed by measurement of the disappearance ofester at 245 mr . All species were followed a t or very neartheir absorption maxima except in the case of ethyl thiolace-tate where the intense absorption of imidazole interferedwith measurements at t he absorption maximum of the ester.The specific rate constants were calculated from the spec-trophotometric da ta b y the method of Guggenheimso orby the use of the usual first-order equation. The averagearithmetic deviation from the Guggenheim plots varied fromless than 1 in most cases to 4 or 5 in the worst cases(with 3-picoline, 4-picoline and pyridine as catalysts-thiswas due to a slight decrease in rate after the reaction wasabout 70% complete). The rate constants for the imid-azole-catalyzed hydrolysis of e thyl thiolaceta te were alwayslarger in the first 15% of the reaction, reasonably constantthrough the middle range and smaller after about 60-70%completion.The a ttempted hydrolyses of ethyl ace tate were performedin the following manner . The ester and catalyst were mixeda t 25.04'. Aliquots of the mixTure were withdrawn andtitra ted in the indicated manner. m-Aminobenzoic acid,anthranilic acid, benzoic acid, pyridine, manganous chlorideand sodium thiosulfate solutions were titrated to p H 8 withstandard alkali using phenolphthalein or a Beckman modelH pH meter; nickelous chloride an d cupric chloride solutions(15) J. J. Blanksma, C h e m . Weekb l a d , 6 25 (1909).(16) P.N. ylander and D. S. Tarbell, THISJOURNAL 72 3021(17) T. Eauchi, Btrll . C h e m . SOC. a p a n , 3,228 1928).18) 0 Wallach, Ber . , 16, 645 (1882).

(19) G. edichen, i b i d . , 39, 1840 (1906).2 0 ) E. . Guzgenheim, P h i l . M a g . , 71 2, 538 1920).

(1950).

were titrated to pH 4.0 with standard acid using the PHmeter.The hydrolysis of acetylcholine bromide with imidazolewas followed by periodic examination of the infrared spec-trum in a Perkin-Elmer double beam infrared spectro-photometer. The solutions were prepared as follows: imid-azole was dissolved in a solution of DCI in deuterium oxide(99.8%) to give a solution containing equal amounts of freeamine and deuteronated amine; to this solution was added asolution of ester in deuterium oxide. The concentrationswere adjusted so th at the final solution was 0.10 M in esterand 0.43 M in each imidazole species. Calcium fluorideinfrared cells were used for the analysis of the deuteriumoxide solutions.The hydrolysis of methy l p-nitrobenzoate with imidazolemas followed by measurement of the r ate of appearance of p -nitrobenzoate ion with th e Cary recording spectrophotome-ter. The ester has a maximum a t 266 mp and p-nitrobenzo-at e ion has a maximum a t 272 mp . However, at 290 mpthe optical density of the latter is much greater than thatof the former so that the increase in optical density at thiswave length can be used as a measure of the hydrolysis re-action.Results

Hydrolysis of p-Nitrophenyl Acetate by TertiaryAmines.-In Table I are listed the catalytic rateTABLE

THEHYDROLYSISF p-NITROPHEKYLCETATEBY TERTIARYAMINES~ko X 102I./mole ki X lo'. f iK? ofCatalyst pH sec. s e c - 1 log ka amineb

Pyridine 63-Picoline 64-Picoline 6N-Methyl-

imidazole 7Imidazole 7Trimethyl-

amine 8Hydroxide ionImidazole 7

.35

.37

.08.05.15.35.85

0.0531 -3.275 5.23',261 -2.583 5.66d.795 0.52 -2.100 6.05"

31. 7 -0.499 7.00'46.9 1.4 5 -0.323 7.04'9.8 7 12.0 -1.006 9.72'

510 2.699 15. 73.64

5% dioxane-water, 26.2'. Aqueous solut ion, 25'.c A . Gero and J . J . Markham, J . Org. Chem., 16, 18.55(1951). d E . F. G. Herington, Disc. Furuday Soc. 9 , 26(1950). Determined in thi s investigation; Dedichenlg re-ported 7.34. f A . H. M. Kirby and A . Neuberger, Bio-chem. J . , 32, 1146 (1938). OH. S. Harned and R. -4.Robinson, THIS OURNAL,0 , 3154 (1928).constants, kc, for the hydrolysis of p-nitrophenylaceta te by several heterocyclic bases and onealiphatic tertiary amine. These constants wereobtained from the slopes of lines of a plot of kobsversus the free amine concentration, using the leastsquares method (Fig. 1). In Fig. 1 it is seen tha tfor six catalysts the observed hydrolytic rateconstants varied in a linear fashion with t he buffer(catalyst) concentration at constant hydrogen ionconcentration and a t constant ionic strength.It has already been pointed out that the rate ofhydrolysis of p-nitrophenyl acetate by imidazolewas independent of primary salt effects6; doublingthe ionic streng th of t he medium (from 0.117 to0.245 M ) n the hydrolysis of p-nitrophenyl acetatewith 4-picoline also had no effect on the specificrate constant.Th e rate of alkaline hydrolysis of 9-nitrophenylacetate was estimated from the intercepts of Fig. 1.If i t is assumed that k H ( H f ) = 0, the intercepts,Ki , are equal to ~ H , O+ k o H ( O H - ) . A plot of iversus (OH-) then yields K O H as the slope and


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165s M Y R O N L. BENDER ND BYRONTi. TURNQIJI?ST Vol. T )(Free amine), mole/l.

0.004 0.012 0.020

0.2 0.4 0.6 0.8 1.0(Fre e ami ne), moles/l.

Fig. 1.-Hydrolysis of P-nitrophenyl acetate with variousamines a t 26.2": A , imidazole; B, N-methylimidazole; C,trimethylamine; D, 4-picoline; E, 3-picoline; F, yridine.Top scaleA , B and C; bottom scale D, E and F.~ H , O s the intercept. The data in Table I wereused for the plot illustrated in Fig. 2. The second-order rate constant for hydroxide ion was estimated

0.5 1.0 1.5 2.0Hydroxide ion concentration X lo6,moles/l.

Fig. 2 -The alkalitie hydrolysis of p-nitrophenyl acetate in,5 dioxane-water at 26.2".

to be 510 I./mole sec. from least squared data ofTable I and K H ~ Owas estimated a t 5 X 10-5 sec.-I.Hydrolysis of 2,4-Dinitrophenyl Acetate, PhenylAcetate and Ethyl Thiolacetate by Various Bases.-The catalytic rate constants for the hydrolysis ofthese esters were obtained in the same manner asthose for p-nitrophenyl acetate. The constants arelisted in Table I1 and the plots from which theywere obtained are given in Fig. 3.(Free dniitie,, mole IOOO, ? O O l i 0015

1 I18.0

15.0

1

4x 9.0

12.00

6.0

3 O

I I0.1 0.2 0.3 0 4

(Fre e amine), mole/l.Fig. 3.-Hydrolysis of various esters with imidazole and

acet ate ion. Imidazole catalysis: A , 2,4-dinitrophenylacetate (to p and left scales); B, ethyl thiolacetate (bottomand right scales); C, phenyl acet ate (botto m and lef t scales).Acetate catalysis: D, 2,4-dinitrophenyl acetate (top scaleX l o * and left scale X lo-]).

Attempted Hydrolysis of Ethyl Acetate, Acetyl-choline and Methyl $-Nitrobenzoate with VariousAcids and Bases.-A study has been made of th ehydrolysis of ethyl acetate by various acids andbases. In all cases given in Table I11 there was nomeasurable amount of reaction a t the indicatedperiod of time.The extent of hydrolysis of acetylcholine bromideby imidazole in deuterium oxide solution was esti-mated from the diminution of the carbonyl peal;of the ester a t 1730 em.-'. In twenty days at 25the optical density fell from about 0.264 to 0.140indicating approximately 40y0 hydrolysis. It wasassumed that the deuterium oxide solution wasnear neutrality since pk', was 7.04 for imidazolein water at 25". The half-life calculated for thehydrolysis of acetylcholine in water a t pH 7.0 isabout 60 days ( k o ~.20 l.,/mole sec. in water a tIn view of the uncertainty in the trueconcentration of OD- and in the value for thehydrolysis constant by deuteroxide ion, catalysis by

( 21 ) J Batterworth, D n Bley a n d G S Stone , Biochein J , IS,3 0 (1953)


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April 5 , 1057 BASICCATALYSISF ESTER YDROLYSIS 1650TABLE1

THEHYDROLYSIS O F 2,4-DIXITROPHENYL ACETATE, PHENYL ACETATEAND ETHYL HIOLACETATEY VARIOUS BASk, X 102Este r Cataly st PH I./mole sec. log ko of alcohol log kOE

2,4-Dinitrophenyl acetate Imidazole 6.1 2 649 0.82 9 4 .0 2Quinoline 5.00 15.0dAcetate ion 5.3 0 0. O5gePhenyl acetate Imidazole 7.4 9 ,588' -2 .196 9.89 -0.240b

Ethy l thiolacetate Imidazole 7 .13 .0166' -3. 770 10.5 ,386*26.2'.p-Sitrophenyl acetate Imidazole -0. 323 7.1 6 0.906b

56% acetone-water, 25', ref. 22. c Aqueous solution, 25'. Determined a t only one concentration of quino-line, 67, dioxane-water. e 57, dioxane-water. Aqueous solution. 62Y0 acetone-water, J . R. Schaefgen, THISOURXAL,70 1308 (1948).TABLE11

ATTEMPTEDHYDROLYSISF ETHYL CETATE ITH VARIOUSACIDSA N D BASES^(Cata lyst ) , (Ester) , Time,Cata lyst M M months

m-ilminobenzoic acid 0.038 0.037 1 .0Anthranilic acid ,025 , 0 24 1 . 0Benzoic acid-sodium

benzoate .017 (0.017 ) .017 1. 0Pyridine-pyridinium ion .040 ( ,040) ,040 1 . 0Manganese chloride ,033 ,033 1.0Nckelous chloride .015 .Ol5 0.25Nickelous chloride" .010 .025 .25Sodium thiosulfate ,025 ,025 .25Cupric chlorideb ,015 ,013 . 5Cupric chloride' .025 ,025 .5

a pH 7.5, 0.060 M tris-(hydroxymethy1)-aminomethanePH 7.7, 0.050 Muffer.tris buffer. Aqueous solution, 25.04'.pI 6.5, 0.079 M tris buffer.imidazole must be considered to be unproven;if such hydrolysis does exist, i t must be quite small.The amount of hydrolysis of methyl p-nitro-benzoate in imidazole solution was determined bythe increase in the optical density a t 290 mp.For a solution 1.82 X M in ester, 0.601 Min total amine and 0.430 M in free amine at pH7.50 in 5 dioxane-water a t 25", the opticaldensity increased from 1.050 to 1.190 in six days;the increase found after this length of time repre-sents about 25y hydrolysis. The half-life of th ehydrolysis of this ester by hydroxide ion at 25was approximated to be about 17 days at pH 7.5by extrapolation of the known rate constant in 56%acetone-water to 5% dioxane-water.22 It there-fore appears tha t the small amount of hydrolysisfound for methyl p-nitrobenzoate in imidazolesolution is explainable on the basis of an alkalinereaction.

DiscussionMechanism of th e Reaction.-Catalysis of esterhydrolysis by bases other than hydroxide ion isbelieved to proceed according to the mechanismoutlined for imidazole-catalyzed hydrolysisa in-

volving attack of the ester by the base giving anacylammonium (or acid anhydride) intermediatewhich is subsequently hydrolyzed by water, yieldingcarboxylate ion and regenerating the catalyst.Only in t he imidazole case is there direct evidencefor intermediate for mat ioa 6 In none of the otherammonium ion intermediates is there the possibilityof stabilization of the intermediate by loss of a( 2 2 ) E . Tommila and C. N. Hinshelwood, J . Chcm. SOC., 1801

(19381

proton. Th e concentration of these intermediateswould, therefore, not be expected to attain anexperimentally measurable value. Catalysis ofhydrolysis by aceta te ion should give an anhydrider 09 -I

HzOO R 8 lk5 R'C-OH + B + ROH (1)

as intermediate by analogy with the acyl derivativesof the amines.23 The rate constant for the un-catalyzed hydrolysis of acetic anhydride is givenz4as 2.5 X sec.-l in aqueous solution at 25';the similarity between this value and the value forN-acetylimidazole indicates that these two inter-mediates are of comparable stability. I t is notsurprising th at the rates of hydrolytic cleavage areclose since the latter is the nitrogen analog ofthe former. It was not possible, however, to ob-tain physical evidence for the existence of theanhydride. Since good first-order rate cons tantswere obtained whether the intermediate is shortor long-lived, the concentration of the intermediatemust have no effect on the determination of theseconstants for our condit ions of excess catalyst .This result substantiates the earlier kinetic argu-ments.6General Basic Catalysis.-For general basiccatalysis to be operative, the rat e must be propor-tional to the summation of catalyt ic constantstimes catalyst concentrations at constant PH andionic strength. It has been demonstrated tha t thehydrolysis of p-nitrophenyl acetate fulfills thisrequirement. A corollary of general basic ca-talysis is th at a linear relationship exists between thelog of th e rate cons tant and the log of the equilib-rium constant of the catalyzing base, the Brgn-s ted re la t i~nship .~~he d ata from the hydrolysisof p-nitrophenyl aceta te with various tertiaryamines given in Table I was used to make such alinear free energy plot according to the method ofBr#nsted, in which log k, is plotted zersus P K a(Fig. 4).It can be seen that the relationship is followedover three powers of ten in the rate. Trimethyl-amine shows a large negative deviation which is

(23) Cf.L. P. Ham mett , "Physical Organic Chemistry," McGraw-(24) A. C. D.Rivet t and N. V. Sidnwick, J . Chem. Soc. , 97, 732(25) 5. N. rmsted and K. J. Pedetsea, Z . p h y s i k . Chem . , 108,185

Hill Book Co. nc.,New York,N. Y.. 940,p. 356(1910).(1924).


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b

I6 8 10

PKA .Fig. 4.-Linear free energy relationsh ip in the hydrolysis

of P-nitrophenyl acetate : a, yridine; B, 4-picoline; C,imidazole; D, S-methyliinid3zole; E, trimethylamine; F,3-picoline.expected because of t he much greater steric re-quirements of this amine compared to that of t heheterocyclic bases. Th e slope of the line deter-mined by th e method of least squares is 1.62;this is the largest slope recorded for such a linearfree energy relationship. 6 s 7Figure 4 gives information concerning theidentity of the catalyt ic nitrogen atom of imidazole.A pr ior i , one would expect the imino nitrogen to bemore basic (and more nucleophilic) since imidazoleis an analog of a vinylamine (containing nitrogenin place of carbon) and since vinylamines areprotonated on carbon atom two of t he doublebond. N-Methylimidazole shows no deviationfrom the linear free energy relationship; a deviationwould be expected if the attacking nitrogen con-tained a methyl group in place of hydrogen (com-pare trimethylamine). Th e nitrogen participatingin the catalytic attack must then be the iminonitrogen. Th e intermediate obtained from thisamine as catalyst, N-acetyl-N-methylimidazoliumion, should be much less stable than N-acetyliniid-azole since the former cannot stabilize itself by lossof a proton while the latter can. Th e ready ca-talysis by imidazole (and N-methylimidazole) aswell as their high basicities is due t o th e resonancestabilization of their respective quaternary salts.

Comparison of the rate constant for the alkalinehydrolysis of pn itr ophenyl acetate given in Table Iwith the da ta in Fig. 4 indicates th at the hydroxideion has an extremely large negative deviation in logk cn. 11powers of ten) . Th e same type of negativedeviation exists for the acetate ion-catalyzed hy-drolysis of 2,4-dinitrophenyl aceta te compared to( 2 6 ) Reference 2 8 , p ? Y O(27) K. P. Bell, Acid-Base C.~ ; i t n l y s i s , Onford Univ. Press . I.on

d o n , 1041, Chaps 5 . 7

th at of quinoline. These two bascs have pKvalues of 4.75 and 4.94, respectively; yet the ratioof their catalytic constants is 173. The presentresults appear to indicate that attack at a carbonylcarbon atom is governed in part by the charge 011the attacking species, with tincharged reagentsbeing more eflicient than negatively cliargcdreagents of th e same basicity.It has been shown by Pedersen for general basiccatalysis involving protoil transfer that if themeasured rate copstai1t, ,tis, is equal to the rateconstant for proton transfer, the slope, a , of theRr@iisted catalysis law plot must have the limits0 < cy < 1 .27 f26 It has been pointed out that i i isuch a situation as a approaches ullity, generdbasic catalysis cannot be detected due to the rniichgreater reactivi ty of hydroxyl ion as a catalyst.:The question might arise as to why general basiccatalysis is observed in the present instance whenthe slope is 1 G2. This phenomenon is explicable interms of the large negative deviation in log kof the hydroxide ion catalysis. If the hydroxideion catalysis were included in an approximatelinear relationship, the slope would be in the rangeof 0.37-0.47 which is the region in which generalbasic catalysis is often observed.* If the rela-tionship shown in Fig. 4 is a true Br@nsted ela-tionship, the slope of 1.62 indicates that in thisreaction kobs cannot he equal to the rate constantfor proton transfer. It has been postulated thatkc in these ester hydrolyses does not involve protontransfer and is equal to the complex rate constant,k l k 8 / k 2+ k3 (equation I . The complexity of kcmakes it difficult to assess the limits of a iu thepresent instance.It can be seen that these ester hydrolyses con-form to general basic catalysis if one extends theconcept of basic catalysis to include not only a rate-determining proton transfer but also a rate-deter-mining introduction of base into the subst rateto form an unstable addition compound, theaccepted mechanism for the hydroxide ion-cat-alyzed hydrolysis of esters and th e favored inecli-anism for the amine- and acetate-catalyzed hy-drolyses of esters.Th e Br@nsted relationship has been appliedin the past, for the most part , to reactions in whichproton transfer occurs in the catalytic step n soriginally set forth by Br@tistecl. There are tworeports in which this relationship has been appliedto reactions involving nucleophilic attack on carbon.One is the hydrolysis of anhydrides2g n which therat e was proportional to t he basicity of aniincsincluding pyridine, 3- and 4-picoline and isociEino-line with a slope of 0.925 but was not proportionalto th e basicity of sterically hindered amines suchas quinoline, 2,G-lutidine and 2-picoline. Thesecond example of such a relation is th e catalysis ofthe hydrolysis of chloroacetate by general bases.Th e rate was shown to follow the basicity of thecatalyst over a large range of basicity involving31 bases with a slope of 0 203 These exatnples

(28) K. J . Pedersen, J . P h y s . C h e w . 58 , 581 (1934).(29) V . Gold and IZ C.. jrfierson, .I C h e n i . Soc., 14014 ( 195 : { ) .30 ) 11 h l . D ~ w s o n , < I < , P \ w c k x u d >, IC Smith, ~ b ~ d ,1 731) :. I; S mi th , ; b i d . , 521 1043)(1943).


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April 5, 1057 BASICCATALYSIS OF ESTERHYDROLYSIS 1661together with the .present results bring up thequestion of whether general basic catalysis mustinvolve proton transfer in the rate-determining step.The function of a general basic catalys t appears toinclude not only a rate-determining proton transferbu t also the introduction of a base into the sub-strate to form an unstable i r ~t er me di at e, ~~ .~ ~hichis essentially the situation in the two cases citedabove as well as in t he present research.Correlation of the Rate of Hydrolysis with theAcidity of the Alcoholic Residue.-An at temp t wasmade to correlate the catalytic rate constants ofimidazole-catalyzed hydrolysis with the p K a Sof the alcoholic residue of the various esters. Thedat a in Table I1 were used for the plot illustratedin Fig. 5. Although a linear relationship does notexist between the rate constant and the acidity ofthe alcohol, it is evident that the rate increases withthe acid strength of the alcohol derived from theester in accord with the mechanistic argumentspresented earlier. For a series of esters consistingof alcoholic and acyl portions of cons tant stericrequirement, a linear free energy relationshipwould in all probability be applicable as has beenfound in the alkaline hydrolysis of substi tutedbenzyl acetates where the rates followed theHammett relation with a reaction constant of0.760.34 Figure 5 also shows a plot in whichcancellation of th e steric effects of the alcoholicgroup of t he ester is attempted by plotting the logof t he rate constants of the alkaline hydrolysisversus the log of the r ate constants for imidazolecatalysis. The da ta are given in Table 11. Forthree esters the logarithms of alkaline and imid-azole catalysis are linearly related. This relation-ship is similar to those obtained by Taft35 whofound that the linear free energy relationships inester hydrolysis were independent of solvent andattacking base (hydroxide and methoxide ions).The relationship between alkaline and imidazolecatalysis breaks down when an extension to methyl$-nitrobenzoate is attempted. The relationshippredicts catalysis of this ester by imidazole equiv-alent to tha t for phenyl acetate; experimentallythere is litt le if any imidazole-catalyzed hydrolysisof methyl p-nitrobenzoate. Changing steric re-

TABLEVFROM p-NITROPHENYLCETATE Y VARIOUS ATALYSTSRATECONSTANTS FOR THE FORMATION OF p-NITROPHENOL

Solvent, 5 Temp.,Catalyst k, . /mole see. aqueous s o h O C Ref.Insulin 0.5 8 Isopropyl alc. 25 36DFP-a-chymo-

trypsin 0.4 7 Isopropyl alc. 25 36a-Chymotrypsin >33 Isopropyl alc. 25 36a-Chymotrypsin 3 Isopropyl alc. 18 37Imidazole 0, 47 5 Dioxane 2 6 . 2Hydroxide ion 511 Dioxane 20.2

Sec.-. b This itivestigation.(32) J. W. Baker and E. Rothstein, Handbuch der Katalyse.(33) E. L King, Catalysis, Vol. 11, P. H. Emmett , ed ., Reinhold(34) Reference 23, p . 191.(35) R . W. Taft, J r . , TH IS O U R N A L , 4 , 2 7 2 9 (1952).

Val. 2 , G. M. Schwab, ed ., J . Springer, Vienna,1940, p. 51.Pub. Corp., New York,N . Y . 955,pp. 389-397.

4

3Jwz1

pK. of alcohol.4 6 8 10

I I 1

-1 0 1 2 3-log k O H .Fig. 5.-The imidazole-catalyzed hydrolysis of various

esters in 5% dioxane-water a t 26.2: curve I, botto m scale;curve 11, op scale; A , p-nitrophenyl acetate; B, phenyl ace-tat e; C, ethyl thiolacetat e; D, 2,4-dinitrophenyl acetate.quirements can explain part of this failure but i t isprobable tha t there are other factors.General Basic Catalysis of Ester Hydrolysis as aModel System for Enzymatic Hydrolysis.--Sinceit has been postulated th at th e active catalytic siteof a-chymotrypsin consists of a general base,namely, imidazole, it is interesting to compare thekinetics of the a-chymotrypsin-catalyzed andimidazole-catalyzed hydrolyses of p-nitrophenylacetate. These da ta together with other hydrolysesof p-nitrophenyl acetate are shown in Table IV.In discussing the d ata in Table IV , the enzyme-catalyzed hydrolysis of this ester will be assumedto proceed through the rn ec ha ni ~r n ~~ -~ ~

kakikz

II /ICHICOR + En -- CHICOR. . E l i ) ---+I1CH3CEn + OR- ( 2 )

(3)I1 kp ICH3CEn + H20 - CHaCOH + En + HThe fact that the reactions involving insulin andDFP-a-chymotrypsin have the same rate constant

(36) B . S. Hartley and B. A. Kilby, Biochem. J . , 66, 288 (1954).(37) H. Gutfreund. Disc . F a v e d a y Soc., 20 107 (1955); 11. Gut-freund, Advances in Catalysis, Val. IX, in press, Academic Press,

Inc., New York, 1956; H. Gutfreund and J. M. Sturtevant, Biochem.J. 63, 656 (1956). In the last paper serine, and not imidazole, ispostulated to participate in step k , , thus liberating p-nitrophenol.I. B.Wilson and F. Bergmann, J.Bioi. Chem. . 186 83 (1950).

(38) ADDEDIN PROOF.-From th e repor ted result s of G. H. Dixon,W. J. Dreyer and H. Neurath, THIS OURNAL,8, 4810 (1956), andof Gutfreund and Sturtevantn with respect to the pH dependence ofsteps ks and k d , it appears that imidazole participates in step k r andnot ka. This implies that step k , may consist of a two-step processinvolving an acyl-imidazole intermediate.


7/7

as imidazole indicates that the catalytic species ofthese non-enzymatic proteins is the imidazolering of a histidine residue.Even though p-nitrophenyl acetate does notpossess the structural requirements of a typicalsubs trate of a-chymotrypsin, the da ta of Table IVindicate that the ester is hydrolyzed by the cat-alytically active site of this enzyme since diiso-propyl fluorophosphate inhibits this reaction aswell as the reactions of typical substrates. Con-firmation of this hypothesis has come from theisolation of acetyl-cr-chymotrypsiii from the reac-tion of o-nitrophenyl acetate and a-chyniotrypsin.This conipound possesses one acetyl group perniolecule of a-chymotrypsin and is enzymaticallyiiiactive; the acetyl group is rapidly hydrolyzedi n neutral solution, regenerating the activec~11zy111e.~~.I large difference in rate exists between therate of formation of p-nitrophenol by a-chymo-trypsin on the one hand and by imidazole orDFP-a-chymotrypsin on the other hand.The large difference in rate between active andDFP-inhibited enzyme is most easily explained bythe reaction of the ester at the active site in theformer case and a t an inactive imidazole ring in t helatter. It is significant tha t there are two histidineresidues in a-chymotrypsin which can account forthese two sites,4o For the rate of t he former re-action, the rapid spectrophotometric dat a of Gut -freund are preferred t o those of Hartley and Kilbysince Gutfreund's rate constant applies to kaof reaction 2 and is presumably analogous to therate constant for the imidazole-catalyzed reaction.The equivalence of the rate constants for imid-azole and DFP-chymotrypsin catalysis and themuch larger rate constant for a-chymotrypsincatalysis indicates that if the active site of a-chymotrypsin involves an imidazole ring3' themechanism is certainly more complex than a simpledisplacement on ester by imidazole to give an acyl-enzyme intermediate followed by hydrolytic cleav-age of the intermediate. This conclusion isstrengthened by th e fact tha t esters of aliphaticalcohols are readily hydrolyzed by a-chymotrypsin,but there is little or no catalysis by imidazole.If the secondary imidazole in DFP-a-chymotrypsinis equivalent to imidazole alone and is thus notsterically hindered, the imidazole in a-chymotrypsinmust coiitain a unique cnr-ironnient to cause such alarge increase i n rate.In contrast to the large difference in the rate ofliberation of p-nitrophenol with a-chymotrypsinand imidazole, the rate of formation of acetatefrom the acetyl-enzyme and N-acetylimidazoleare reasonably close. Th e former was reportedas IO-? sec.-l , 6 whereas the latter is found to be

(:{ I) A . K . Balls and 11. S Wood, J . Bioi. Ch cm . . 219 245 (1956).GO J . FI. Northrup. 11.Kunitz and R . M. Herriott, "Crystalline

l inzymcs ," Columbia Utiiv. Press, New York, N. Y . 1948, p . 26.

1.3 X lop3 set.- . Siiice the. rate of foririationof acid is usually measured in enzymatic catalysis,differences in rate for various substrates wouldimply that specificity is important in the hydrolyticcleavage of t he intermediate as well as in previoussteps. I t should be pointed out that S-acetyl-imidazole, while a relatively reactive intermediate,is still quite selective since it will react preferen-tially with more powerful nucleophiles such asphosphate ion and thiols rather than with water.Catalysis of ester hydrolysis by imidazole by 110ineans explains all the phases of enzymatic liy-drolysis. However, there are some aspects ofthese reactions which appear to be siniilar i . e . ,formation of an acyl-catalys t intermediate and itsra te of hydrolysis) so tha t it is at least reasonable topostulate imidazole as a pa rt of the active centerin a-chymotrypsin and to use the catalysis byimidazole as a starting point for thc constructioriof a more complete iiiodel for cu-cliyiiiotryl)sili-catalyzed hydrolysis.Hydrolysis of esters by iriiidazole may possiblyserve as a model system or the enzyme papain.Smith4' has presented evidence that the action ofthis enzyme is due to formation of a thiol ester ofthe enzyme (transesterification of the ester withthe sulfhydryl group of the enzyme) and sub-sequent hydrolysis of the thiol ester. Since thehydrolysis of a thiol ester is more readily catalyzedby imidazole than the hydrolysis of the corre-sponding oxygenated ester, it is entirely feasiblethat papain catalyzes hydrolysis through such amechanism. I t should be pointed out t ha t whereasthe hydroxide ion-catalyzed hydrolysis of ethylthiolacetate is only slightly faster than that ofethyl acetate,'6 the iinidazole-catalyzed hydrolysisof t he former is apparently infinitely faster thanthat of th e latter.The use of thiol esters in biological reactions is byno nieans limited to hydrolytic reactions. Theimportant acyl transfer and oxidative decarboxyla-

tion reactions involve thiolesters such as acetyl-coenzyme and acetl-lgluta thione; it is possiblethat these reactions occur through acy1a:nineintermediates.ADDED N PRoon.-After submission of this and th e preced ing paper ,

it was found that many aspects of this research were carried out 111-dependently and simultaneously b y T. C. Rruice and G. 1, Schrnir,: i ~ r h . i o c h c n z . B i o p h y s . , 63, 84 (1 25ti), and Tms JOUIINAL, 79, 1tXG( l S 5 7 ) . Substantial agreement exists between the results of theseinvestigations. The authors gratefully acknowledge the wiiortun ityof reading the nlan uscript nf Bruice and Schmir prior to publication.

Acknowledgment.-The authors gratefully :tc-knowledge valuable discussions with Drs. FBergmann, G. Gorin, 3. Gutfreund, D. E. Kosh-land, Jr., N.Kilpatrick, F H. Westheimer ant1K. B. n'iberg.CHICAGO0, ILLINOIS

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