ACETYLTRANSFER FROM ACETYLIMIDAZOLE
s o v . 5, 1964 [ COXTRIBCTION No 308 FROM
4655
GRADUATE DEPARTMEXT OF BIOCHEMISTRY, BRAXDEIS UNIVERSITY, ~ V A L T H AMASSACHUSETTS Y, 021543
THE
Equilibria and Rates for Acetyl Transfer among Substituted Phenyl Acetates, Acetylimidazole, 0-Acylhydroxamic Acids, and Thiol Esters' BY JOSEPH GERSTEIN A N D WILLIAM P. JENCKS RECEIVED JCLY 6, 1964 The equilibrium and rate constants for acetyl transfer from acetylimidazole to a series of substituted phenols have been determined. The effects of polar substituents on the transition state of the reaction are intermediate between those oti the reactants and on the products for the equilibrium AcIniH
+
+ -0PhX
k2
rlcOPhX
+ Im.
k- 2
Determination of the equilibrium constants for acetyl transfer t o 9-methylacetohydroxamic acid and K-acetylB-mercaptoethylamine has made possible t h e calculation of the free energies of hydrolysis of these compounds, the phenyl acetates, acetylimidazole, and other "energy-rich" compounds for which t h e equilibrium constants for reactions with these compounds are known.
In order t o consider in detail the effect of structure on reactivity and the mechanism of acyl transfer reactions, it is desirable to know the rate constants in both directions and the equilibrium constants for a series of such reactions. The results of such determinations for a series of phenyl acetates and imidazole are reported here. In addition, some rate and equilibrium constants for acetyl transfers involving acetylimidazole, the ester group of N,O-diacetyl-N-methylhydroxylamine, and the 'thiol ester group of N,Sdiacetyl-p-mercaptoethylamine have been determined. By relating these values to the free energy of hydrolysis of thiol the results permit the calculation of the free energies of hydrolysis of these and related ' 'energy-rich' ' compounds,
Experimental Materials.-Commercially available phenols a n d imidazole were redistilled or recrystallized. Substituted phenyl acetates a n d S,O-diacetyl-S-methylhydroxylaminewere kindly supplied by D r . Jack Kirsch. S-Acetylimidazole, m . p . 101-103°, 245 mp, was prepared by the method of Boyer4 and showed A, E 2920; reported5 A, 245 mp, e 3000; the latter value was used for calculation of acetylimidazole concentration in these experiments. S,S-Diacetyl-&mercaptoethylamine6 ( S-2-mercaptoethylacetamide acetate) was prepared by t h e dropwise addition with vigorous stirring of 0.3 mole of acetic anhydride t o 0.1 mole of mercaptoethylamine hydrochloride in 30 ml. of water a t 0-3". T h e solution was maintained at p H 8.0 b y the simultaneous addition of 8 J1 potassium hydroxide. After standing for 20 min. a t rooin temperature, the mixture was extracted with ether and the ether extract was dried with sodium sulfate. After evaporation of solvent the product was distilled; b . p . 140142' (0.85 m m . ) , 1.5068, d 1.11. An aqueous solution S-acetyl-P-mercaptoethylamine (S-2-mercaptoethylacetof amide) was prepared by the reaction of 0.7 mmole of N,Sdiacetyl-8-mercaptoethylamine with 1.8 mmoles of potassium hydroxide in 3.2 ml. of water a t 25' for 40 min. followed by neutralization with a small excess of hydrochloric acid. Measurement of thiol appearance by the nitroprusside reaction' showed t h a t the reaction was complete in 15 min. under these conditions and ultraviolet spectroscopy indicated t h a t the solution did not (1) Supported by research grants f r o m the Xational Science Foundation and from the National Cancel- Institute of the Sational Institutes of Health (C.4-033975) and a training grant from the A-ational Institute of Neurological Diseases and Blindness (R-TI-NB-.5241). ( 2 ) IT P Jencks. S. Cordes, a n d J . Carriuolo, J . B i d C h e m . , 296, 3608 (19fiO) (3) W. P. Jencks and M. Gilchrist, J . A m Chrm. Soc., 86, 4651 (1964). (1) J . H Boyer. Biochem. P r e p n . , 4, 54 (1955). ( 5 ) E. R . S t a d t m a n . "The Mechanism of Enzyme Action," W. D. McElr o y and B. Glass, Ed., T h e Johns Hopkins Press, Baltimore, M d . , 1954. p. 581. ! 6 ) T. IVieland and E. Bokelmann, A ~ I M6 7 , ,6 , 20 (1952). ( 7 ) E . R . S t a d t m a n , "Methods in Enzymology," Vol. 111, S. P. Colonick and S 0 Kaplan, Ed , Academic Press, Inc , X e n York, N . Y . , 1957, p. 939.
contain detectable amounts of thiol ester or thiazoline. NMethylacetohydroxamic acid was prepared in a similar manner by the saponification of A-,0-diacetyl-N-methylhydroxylamine. I n a typical preparation 1.i mmoles of N,0-diacetyl-S-methylhydroxylamine was added to 4.0 mmoles of potassium hydroxide in 6 ml. of water a t 25' and the mixture was allowed t o stand for 1 h r . before neutralization with 0.75 ml. of 2 M hydrochloric acid. A control experiment in which the release of hydroxarnic acid was followed b y the addition of aliquots to ferric chloride solution8 showed t h a t the reaction was complete in 28 min. under these conditions. Determination of pK,' Values.-The pK,' of S-methylacetohydroxamic acid was found by titration t o be 8.79 i 0.02 a t 25" and ionic strength 1.0. Measurement of the p H of diluted samples gave a pK,' value of 8.85 a t ionic strengh 0.01. These values were confirmed b y measurements of the absorbance of the N-methylacetohydroxamate anion a t 260 mp. In a series of tris(hydroxymethy1)aminomethane (Tris) buffers S-methylacetohydroxamate anion exhibits an ultraviolet absorption maximum a t 227 m p , e 1.96 X lo4,in 0.01 M X a O H , and has an extinction coefficient of 1575 a t 260 mp. T h e corresponding acid shows only end absorption a t 227 m p ( E 8.9 X lo3 in 0.01 31 H C l ) and has negligible absorption a t 260 m p ( e ca. 7 ) . Acetohydroxamate anion exhibits a weaker absorption maximum a t 215 nip ( e 6.6 X IO3). T h e spectrophotometric method gave a pK,' value for S-methylacetohydroxamic acid a t 25" of 8.78 f 0.03 a t ionic strength 1.0 and 8.84 f 0.02 a t ionic strength 0.01. In 6.2% acetonitrile a t 25" and ionic strength 1 . 0 t h e p K , ' was found to be 8.97 i 0.03 by the same method. T h e pK,' of the thiol group of S-acetyl-a-mercaptoethylamine was found to be 9.38 f 0.04 a t 25' and ionic strength 1.0 by measurement of the absorption of the thiol anion a t 250 m p (e250 3120) a t a series of p H values between 9.2 and 9.7 and in 0.01 M S a O H . A slow oxidation of the thiol in the alkaline solutions necessitated a (small) extrapolation of the absorbance readings to zero time. T h e pK,' was found to be 9.47 f 0.05 a t ionic strength 0.01. The pK,' of p-chorophenol a t 25' and ionic strength 1.0 was found t o be 9.20 i 0.03 in water and 9.29 i 0.04 in 6.2r; acetonitrile by measurements of the absorption of the p-chlorophenolate anion a t 298 m p ; the absorption of p-chlorophenol is negligible a t this wave length. The pK,' of p-nitrophenol a t 25" and ionic strength 1.0 in 2C; acetonitrile was found to be 7.02 i 0.02 in a similar manner, utilizing the absorption of the p-nitrophenolate anon a t 401 mp. T h e pK,' values of other phenols9 and of acetylimidazolium ionlo were taken from the literature. Rate Measurements.-The rates of reactions of phenols with acetylimidazole were generally followed by observing the decrease in acetylimidazole absorption a t 245 m p (248 mp in the case of p-chlorophenol and 260 mp in the case of N-methylacetohpdroxamic acid). When the absorption of the phenol a t this wave length was large, the measurements were made against a blank cuvette containing all the constituents of the reaction misture except acetylimidazole. Spectrophotometric measure( 8 ) F 1,ipmann and 1,. C . Tuttle, J Bioi. Chrni., 169,21 (194.5). (9) M. h l . Fickling, A . Fischer, B. R M a n n . J . Packel-. and J I'aughan, J . A m . C h r n i . Soc.. 81, 4 2 2 6 (1969) ( I O ) W. P . Jencks and J Carriuolo, J Btoi C h e w . . 294, 1272, 1280 (1959)
4656
GERSTEIN A N D WILLIAM P
JOSEPH
0.8 I
I
I
Kea
0.6
A E
0 (0
N
w
y
0.4
U
m
a
309
I
0 v)
m
269
a
4
0.2
I
I
2
3
TIME ( MIN.). Fig. 1.-Determination of t h e equilibrium constant for acetyl transfer between S-methylacetoliydroxainic acid and imidazole by measurement of acetylimidazole absorption a t 260 mp. JI hydroxarnic Initial concentrations for curves 1-3: 9.0 X acid, 0 238 M imidazole a s t h e free base, 1.94, 3.24, a n d 4.53 X 10 - 3 J1 S.0-diacetyl-N-niethylhydroxylaniine, pH 7.2 ; curves 4-6: 1.5 X 10-3 31 liydroxamic acid, 0.192 di imidazole as tlie free base, 3.4-3.8 X lo-' 31 acetylimidazole, 0.65, 1.30, and M I;,O-diacetyI-N-riiethyl~iydroxylaniine, p H 7.0. 1.91 X Ionic strength maintained a t 1.0 with potassium chloride T h e calculated values of t h e equilibrium constant are shown above t h e curves. ments were made with a Zeiss PMQ I1 spectrophotometer equipped with a brass cuvette holder through which water a t 25 k 0.1' was circulated. Reactions were carried o u t with a large excess of phenol so t h a t t h e observed rates followed pseudofirst-order kinetics and were inuch faster than t h e rates of acetyliniidazole hydrolysis. For those reactions in which the absorption of tlie phenol was too high for re:idings in a 1 cm. path length cuvette, a quartz insert was inserted and the reaction was followed with a 0.2- or 0 . ~ 5 - c n ipath . length. Tlie reactioii of m-nitrophenol with acetylimidazole was followed wit.li acetj-limidazole present in excess b y measuring the disappearance of t h e absorption of t h e phenol a t 338 nip. First- and second-order rate constaiits were obtained as described previously.'" The observed pseudo-first-order rate constants were corrected for t h e rate of acetylimidazole hydrolq-sis, measured under t h e same experimental coiiditions; this correction was small in all cases. Direct Measurement of Equilibrium Constants.-The experimental conditions used for the direct determination of equilibriuin constants are given in Table 11. T h e Incasurenieiit of tlie equilibrium constant for acetyl transfer between imidazole and the hydroxyl group of S-rnetlij-IacetoIiydroxaiIiic acid i e q . 1'1 is described here in detail as an example of tlie methods used. T h e
0 XcIni
il
0
CH1 I
+ CHaC--NOH
Im
+
l
CH3 i
CH3C-XOXc
(1)
reaction was followed by measuring tlie change in absorption a t 260 m p attributed t o acetylimidazole (Fig. 1 ) . The initial concentrations of reactants were varied over tlie ranges shown in Table 11. A solution of acetylimidazole iii dilute imidazole buffer, pH 7.0, was prepared before each experiment and was kept a t 0'. Tlie concentration of acetylimidazole in this solution was determined b y measurement of the absorbance a t 2-15 mp of diluted aliquots. Solutions of N-0-diacetyl-S-inethylh>-droxylamine were prepared before each experiment and were shown t o be free of hydroxaiiiic acid b y t h e absence of color development upon addition of aliquots to acidic ferric chloride solution.i T h e rate of hydrolysis of this cornpound was shown
Vol 8(i
JENCKS
t o be negligible a t t h e pH values a t which equilibriurn rneasuremerits were made. T h e reactants were brought t o 25' and the reaction was initiated by the addition of acetylimidazole in imidazole buffer t o the other reactants in a 4-ml. cuvette. T h e contents was miled b y inversion and t h e absorption a t 260 nip was followed until the readings were stable over a period of several miti. (Fig. 1 ) . The p H of the mixture was then determined. Tlie rate of acetylimidazole hydrolysis under the same conditions of temperature, imidazole concentration, p H , and ionic strength was determined separately. Tlie amount of acetyliniidazole which had hydrolyzed during t h e time required t o reach equilibrium was estimated b y multiplying t h e average concentraion of acet)-limidazole b y t h e hydrolysis rate constant and t h e time. This correction was less than 1'L of t h e acetylimidazole conceritration in this experiment and was less t h a n 7C;; in all experiments. T h e measured absorbance a t 260 mp was corrected for tlie absorption of N-methylacetoliydroxamate anion, determined from the initial concentration of N-methylacetohydroxaniic acid, the p H , and the amount of this compound which was formed or utilized during t h e approach t o equilibrium. This correction was about L5ci of tlie observed absorbance in this experiment. The corresponding correction for the reaction with phenol was also 5 ? ; , b u t it was as much a s 30c; in the reactions with pniethoxyplienol, which necessitated the use of successive approximations t o make ail accurate correction in t h e latter reaction. The equilihriurii concentration of acetylimidazole 1810. Tlie equilibrium conwas then calculated, based on centrations of t h e other reactants were determined from tlie initial concentrations and tlie amount of reaction which had taken place. T h e measurements of equilibria involving phenyl acetates were carried o u t in a similar manner, except t h a t quartz inserts were used t o reduce t h e p a t h length of 1.0-cni. cuvettes t o 0.2 or 0.05 c m . T h e p a t h lengths were calibrated with solutions of known absorbance. T h e determination of t h e equilibrium constant for acetyl transfer between p-chlorophenol and Sniethylhydroxarriic acid presented technical difficulties because of t h e low solubility of p-chlorophenyl acetate in water and because it was not possible t o carry o u t t h e reaction under conditions in which the pH remained constant during the reaction. Consequently, experiments in which p-chlorophenyl acetate was added initially were carried o u t in 6.": acetonitrile; t h e results in this solvent shoi\-ed moderately good agreement with those obtained in water in tlie absence of added p-cliloroplienyl acet a t e ( T a b l e 11). T h e pH values of t h e reaction mixtures used for the spectrophotometric measurements were obtained by following the pH of identical reaction mixtures a s a function of time. T h e rate of hydrolysis of S,O-diacetyl-S-niethylliydroxylamine was determined by following tlie appearance of S-methylacetohytlroxamate a t 27% mp under t h e conditions of the equilibrium experiments and was fouiid t o proceed with a rate cons t a n t of 0.018 n i i n - i a t p H 8.53 and 0.013 miti.-' a t pH 8.49 in G.Z"-; acetonitrile. The value of for p-clilorophenoxide was found to be 2420 i 20 for two samples of recrystallized and one of resublimed p-chloroplienol; Spencer and Williams" report QUE
"00.
The reaction of acetylimidazole with p-nitrophenol was followed by measurement of tlie absorption of the p-nitrophenolate ion a t 401 nip. The concentration of p-nitrophenol was calculated from t h e measured p H and t h e pK,' of p-nitrophenol, wl~icli was measured separately under t h e same esperiiiiental conditions. Reactions of S-acetyl-@-tiiercaptoethylamine were carried d l ethyleneo u t in 0.05 Jf Tris buffer which contained diarnitietetraacetic acid. I t was shown in control experiments t h a t tlie thiol did not undergo significant oxidation and t h e thiol ester and S,O-diacetyl-S-methylhydroxylarnine did not undergo hydrolysis during the time interval required for t h e attainment of equilibrium. T h e reaction of S,S-diacetyl-~-iiiercaptoeth~~larni~~e with S rriethylacctoliydroxai~iicacid was followed by measurement of the concentration of free thiol b y the nitroprusside reaction.' The assay mixture contained 2.0 nil. of saturated sodium chloride, 0.4 inl. of 1.6 .l1 potassium carbonate t o which had been added 0.8 .lI hydrochloric acid, 0.4 ml. of 2.75; sodium nitroprusside, and 0.10 nil. of sample. All reagents were made up in ,\I ethylenediarninetetraacetic acid. Readings were taken 30 sec. after the addition of sample. (11) B. Spencer a n d R . T.Williams, Biochem
I., 48,
637 (19.71).
Nov. 5 , 1964
4657
ACETYLTRANSFER FROM ACETYLIMIDAZOLE
The concentrations of reactants and products a t equilibrium were calculated from absorbance measurements after the initial rapid change in absorbance had leveled off. In reaction mixtures in which the final readings were not perfectly stable, because of hydrolysis, the equilibrium position was determined by the graphical method of Stadtmanj as well as b y measurements at several time intervals after the initial rapid change in absorption; no significant differences were found between the results of these procedures.
Results Equilibrium constants were obtained from measurements of the rate of the reaction in both directions, from the relationship K,, = k l 'L1,or by direct measurement of the equilibrium concentrations of the reactants and products. In a few instances the results of the two methods were compared and internal consistency was demonstrated by showing that the same equilibrium constant was obtained by comparing two different pairs of reactions. A11 measurements were made a t 25' and a t an ionic strength maintained a t 1.0 with potassium chloride. Rate Measurements.-The rate constants, k - , , for the reaction of imidazole with a series of substituted phenyl acetates (eq. 2) were taken from a recently reported series of measurements which had been carried
o u t under the same experimental conditions.I2 T h e rate constants of the reverse reaction, k l , were determined by measuring the rate of acetylimidazole disappearance with the phenol present in large excess, except in the case of m-nitrophenol, for which reaction acetylimidazole was present in excess and the disappearance of phenol was measured spectrophotometrically. Pseudo-first-order kinetics were followed for a t least two half-times in each reaction. The results obtained for the reaction of p-chlorophenol with acetylimidazole are shown in Fig. 2 , as an example. T h e rate constant for acetylimidazole hydrolysis was determined under the same conditions and was subtracted from the observed rate constants if necessary, but in most instances this correction was negligible. T h e reactions were carried out in dilute imidazole buffer to avoid side reactions. Doubling the concentration of imidazole buffer did not affect the rate constant or extent of reaction. The same rate constant was observed if the p H was increased from 7.04 to 7.25. This demonstrates that the rate of the reaction is independent of p H in this p H region and follows the rate lawIfl rate
= kl
[AcIm][ R O H ]
(2a)
Since the mechanism of the reaction actually involves the attack of phenolate anion on the conjugate acid of acetylimidazole,lfl the rate may be described more appropriately, for mechanistic purposes, b y the kinetically indistinguishable rate law rate
=
kz [AcImH+][RO-]
(2b)
in which kl = k l K A c I m H + , " K R O H ; K R O Hand K A c I m H * are the acid dissociation constants for the phenol and the conjugate acid of acetylimidazole, respectively. T h e rate constants, k1 and k p , for the reactions of acetylimidazole with a series of phenols and N-methyl(12) J. F. Kirsch and W. P. Jencks, J . A m . Chem. SOL.,86, 837 (1964).
2 .o
.o
0.01
[ p-C
0.03 h lorophenol
1.
0.05 M
Fig. 2.-Rate of disappearance of acetylimidazole a s a function of p-chlorophenol concentration a t 25' and ionic strength 1.0: 0, p H 7.04, 0 007 M imidazole; E , p H 7 25, 0 01 ,?I imidazole; A , p H 7.05, 0 014 M imidazole
acetohydroxamic acid are summarized in Table I . T h e value of kl of 20.1 M-'m h - l for the reaction with phenol a t ionic strength 1.0 is in satisfactory agreement with the corresponding values of 16-19 Jf-' min.-' measured previously in the absence of added salt.Io For each reaction with a substituted phenol it was shown t h a t the rate constant is not altered by doubling the concentration of imidazole buffer, which demonstrates that the reactions proceed to completion and are not subject to significant catalysis by imidazole under the experimental conditions which were used For the reactions with phenol, p-chlorophenol, m-nitrophenol, and N-methylacetohydroxamic acid it was shown that the rate constants are independent of p H over a range of 0.2-0.3 p H unit, which demonstrates that the rate law of eq. 2a and 2b holds for these reactions. Since it was not possible to measure the rate constant for the reaction of acetylimidazole with pnitrophenol directly, for technical reasons, this rate constant was calculated from the rate constant for the reverse reaction and the directly measured equilibrium constant (see below). In contrast to the reactions with phenols, the observed rate constants for the reaction of N-methylacetohydroxamic acid with acetylimidazole increase with increasing imidazole buffer concentration a t constant p H (Fig. 3, upper curve). The second-order rate constant of 510 M-l min.-' for the reaction (Table I ) was obtained by dividing the observed pseudofirst-order rate constant for acetylimidazole disappearance, extrapolated to zero imidazole concentration, by the concentration of N-methylacetohydroxamic acid. T h e dependence of the rate upon imidazole concentration was shown to be caused partly by the fact t h a t the reaction does not proceed to completion a t the higher imidazole concentrations and partly by catalysis of the reaction by imidazole. Since the hydroxamic acid and imidazole are present in large excess, the observed reaction is pseudo first order in both directions and, in any given experiment, kobsd = kf k,, where kf and k , are the pseudo-first-order rate
+
4658
JOSEPH
GEKSTEIN A N D WILLIAM P.
Vol. 86
JENCKS
TABLE I RATESO F
Init. concn. of acetylimidazole,
M
ROH
25'
K E A C T I O X S \VITH L4CETYLIMIDAZOLE AT
Concn. of imidazole buffer ( a s free base), M
AXD I O S I C STRENGTH
1.0 kzb
x ?io. of detmn.
Concn. of K O H , M
ki>a pH
M
-1
min. -.I
10 -6,
\\'ave
M-1
length,
min. -1
mii
pKR' KOH
p-Methoxyphenol 2 . 8 x 10-3 0 015 0.02-0.10 5 7.2 25 3' 96 0 2Gd 10.20' ,015 p-Methylphenol 2 . 0 x 10-3 ,023-0.07 7 7.2 17 7c 69 0 24Sd 10 19" Phenol 2 7 x 10-3 ,015 034- 10 6 7 3' 20.1'.h 48 2 215d 9 99" p-Chlorophenol 7 . 8 x 10Y4 007 ,015- ,043 7 7.0' 43 3c 24.8 248' 9.38' m-Sitrophenol 0.016-0.062 ,004-0.02 0013 7 6 7' 66.5I.I 3 6 338' 8.35. k p-Xitrophenol . . . . . . . . 7 1 38.3 0.13 7 14" X-Methylacetohydroxamic acid 5 o x 10-4 .os ,0025-0.0095 11 6 9' 510' 87.5 260 8 85"' a For rate = k,[acetylimidazole] [ R O H ] . For rate = k2[AcImH+][RO-1. Doubling of the imidazole concentration resulted in no significant change in rate, d P a t h length of 0.5 mm. e Ref. 13. f Rates found t o change by less t h a n 7Yc upon increasing the k i = 17.9 J P Lmin.-' a t p H 7.3 a n d ji = 0.25. Path length of 2 m m . achieved by use of quartz insert. pH by 0 . 2 unit. Kef. 9. I Reaction shown to have gone to completion by production of same final absorbance a t 338 mji (owing to nz-nitrophenyl acetate) at all Calculated from the equilibrium constant (Table 11) and the rate constant of the reverse reacconcentrations of acetylimidazole. tion (ref, 1 2 . ) . I The catalytic constant for imidazole in this reaction (k,,, I",) is 800 I . * .\I-* min.-' (see Results). This work. ,
constants for the forward and reverse reactions, respectively, and Keq' = kf 'kr, where Keq' is the equilibrium ratio of N,O-diacetyl-N-methylhydroxylamine to acetylimidazole under the conditions of the experiment.I4 T h e value of Keq' was evaluated for each
creasing imidazole concentration to an extent which is much larger than the experimental error in the determination of kf. This indicates that the reaction is subject to general base catalysis by imidazole. The value of the catalytic constant of the reaction for the rate law
0 CH3
I1
rate
+
~
kl [AcImj [CH3C-NOH]
=
0 CH3
4.0
'I
II
' 1
k,[XcIm] [CHaC-NOH] [ I m ]
3.0
-
i
I
.-i 2.0 E
Y
r
I.o
0.1
0.3
0.5 M
[ IMIDAZOLE] b. Fig 3.-Effect of imidazole concentration on the rate of the M acetylimidazole with 3.5 X -M reaction of 5 X N-methylacetohydroxarnic acid a t 25' and ionic strength 1.0: 0 , k o b a d ; 0 , k t = kob.d([LkIm]r - [AcIm],,)/[AcIm]i, where [ X c I r n ] ~and [AcIm],, are the initial and equilibrium concentrations of acetylimidazole, respectively (see text).
experiment from the measured absorbance a t zero time, the absorbance after the initial reaction had proceeded to equilibrium, and the absorbance after the acetylimidazole and N,O-diacetyl-N-methylhydroxylamine had undergone complete hydrolysis. T h e values of kf calculated in this manner are shown as the lower curve in Fig. 3 . T h e values of kf increase with in(13) A . I . Biggs, 7ru:is F a r a d a y Soc , 5 2 , 35 (1956). (11) A . A . Frost and R . C . Peal-son, "Kinetics and blechanism." John Wiley and Sons, Inc, New Vork, N Y , 1956, p. 172.
(3)
is 800 J P min.-'. Based on the apparent second-order rate constant for the reverse reaction of 4.27 in the presence of 1 M imidazole12and on the fact t h a t t h e ratio of the catalyzed and uncatalyzed reactions must be the same in both directions, the rate constant for the uncatalyzed reaction of imidazole with N,O-diacetylN-methylhydroxylamine is 1.7 - 1 f - I niin.-' and the equilibrium constant, from the ratio of the rate constants in the two directions, is 5lO;'l.T = 300. Equilibrium Measurements.-In a number of instances it was possible to measure the equilibrium position of the reactions directly, because the transacylation reactions occur much faster than hydrolysis a t readily attainable concentrations of reactants. The concentration of acetylimidazole or phenol was measured directly in the reaction mixture by spectrophotometry, with the use of quartz inserts to obtain short path length in 1-cm. cuvettes. T h e concentration of free thiol in the reaction of N,O-diacetyl-N-methylhydroxylamine with N-acetylrnercaptoethylamine (eq. 4) was determined by the nitroprusside reaction in 0 CH30
II
~
11
H
I
+
O
/1
CH~COS-CCHI HSCH2CH2N-CCH3 0 H O Hac 0 I 11 I/ i_ It HOS-CCH3 CH3CSCHzCHgh-CCHa
+
(4)
buffered solutions. A typical experiment for the measurement of the acetyl-transfer reaction between imidazole and the hydroxyl group of N-methylacetohydroxamic acid (eq. 1) is described in detail in the Experimental section and is illustrated in Fig. 1. T h e experimental conditions and the results are summarized in Table IT. In each case equilibrium was approached
ACETYL
Nov. 5 , 1964
T R A N S F E R FROM
ACETYLIMIDAZOLE
4659
TABLE I1 FOR DIRECTDETERMINAT~OSS OF EQVILIBRILXCONSTASTS AT 25" CONDITIOXS
---
Acetylimidazole, 'M x 10%
I n i t . concn.---ROH, AcOR, Af x 103 .M x 103
Compound
Acetylimidazole 1 98d 1 .98d 1 .83d
p - Sitrophenol
20-80 20-60 30-50
p-Sitrophenol p - Sitrophenol
0- 1 G2.0 0.8-3.0
0
p-LVethoxyphenol Phenol Phenol S-Methylacetohydroxamic acid
11-66 10
+ AcS(Me)OH
+ imidazole
AND
No. of detn.
5
0.014
4Ole 401' 4Ole
0 2-0.35 0 234 0 234 0.24 0.19
245 245 245 260 260
6 7
p-CIPhOH
IONIC STRENGTH 1.O"
5 3
5 4 3
pH
7.12 7.2 7.12
7.6 7.5 7.5 7.2 7.0
0.9-1 . 1 1 . O-1 . 4 1.0-1.1 Mean 132-139 45.6-48.1 45.3-51.5 289-309 269-282 Mean
1.o 1.2 1.1 1.1 136 47 49f 298 278 287
+ ilcK(Me)OAc
AcN(Me)OH, M x 103
19-48 4 3-9 5
3 5 XcX(Me)OXc
HSEttiHAc, .M x 102
0 6-2 0
PCPA, .M x 101
3 9 1 5
AcN(Me)OAc, M x 102
7 5 10 10 1.9-4.5
0.9-1.4 0.45
p-CIPhOAc CIPhOH, M x 103
M
AcOR 0.014 0.014
0 74 0.74 0.94
18 30 30
0.36
AcK ( M e ) OAc, M x 10%
+ ROH
Imidazole,b
Wave length, mp
298 298
5 2
8 5' 8 5Q
9.3-13.3 8.3-10.5 Mean
11.6 9.4h 11.2
+ H S E t X H h c 1-AcN(Me)OH + AcSEtSHAc
AcS(Me)OH,
AcSEtNHAc, .lf x 102
M
Wave length
KO. of detn.
7
PH
Range
K1
Av.
0-1.5 0-1.6 0 1-0.2 1.1-1 9 1 8,23' 19 2-21 . o 20.1 -1s the free base. Maintained with KC1. For eq. 2, with all reactants in the uncharged form. Total p-nitrophenol concenIonic strength 0.25. 0.17 M Tris buffer. I n 6.2% acetonitrile. Concentration of R S H tration. e I n 2% acetonitrile. measured by the nitroprusside reaction (ref. 7). I 0.05 M Tris buffer, M ethylenediarninetetraacetate. ti
Q
from both directions and was attained from a range of different concentrations of starting materials. T h e equilibrium constants, K I , refer to the concentrations of nonionized reactants and products a t ionic strength 1.0 and 25'. Structure of the Anion of Acetohydroxamic Acid.Possible structures for the anion of unsubstituted hydroxamic acids are I , 11, and I11 (R = H). I t has recently been suggested, from the results of ultraviolet and infrared spectroscopic studies, t h a t the anion
8II
0
I1
RC-3-0 R
-
I
OR -
R C- S-OR I1
R&=X-O111
of benzohydroxarnic acid exists partly or entirely in forms I1 or I11 (R = H ) , in which a proton has been removed from the nitrogen atom.'j-16 T h e anion of Nmethylacetohydroxamic acid can exist only in form I (R = CH3). T h e difference in ultraviolet spectrum between the anions of acetohydroxamic acid (Ama, 21.5 mp, e 6.6 X lo3) and N-methylacetohydroxamic acid (Amax 227 mF, e 1.96 X IO4) is consistent with a difference in structure between these two anions. On the other hand, the fact that N-methylacetohydroxamic acid (pK, 8.8) is a stronger acid than acetohydroxaniic acid (pKa 9.4)'7a means t h a t dissociation of a hydroxamic acid to structure I occurs readily. Acetohydroxamic acid must, therefore, dissociate largely or entirely to structure I and the possibility should be considered t h a t the spectral differences represent only (15) R . E. Plapinger, J . Org. Chem., 24, 802 (1959). (16) 0. Exner and B. KakBE, Collection Czech. Chem. C o m m u n , 28, 1656 (1963), 0. Exner. i b i d . , 89, 1337 (1964). (17) (a) W. M. Wise and W. W. B r a n d t , J . A m . Chem. Soc , '77, 1058 (1955); (b) Steinberg and Swidler (personal communication) have determined t h e dissociation conqtants of K m e t h y l - and 0-methylbenzohydroxamic acid a n d oi cthylbenzohydroximic acid and conclude that the anion of benzohydroxamic acid exists in forms I a n d 11 in approximately equimolar concentration.
an effect of methyl substitution on the spectrum of I.17b Since the effect of methyl substitution on the dissociation constant is in the opposite direction from that expected from the inductive effect of the methyl group, it is probable that specific solvation of the N-H of acetohydroxamic acid provides some stabilization of this molecule; such solvation differences may also be expected t o affect the ultraviolet spectra. Discussion T h e equilibrium constants and free energies for acetyltransfer reactions which involve acetylimidazole, substituted phenyl acetates, a thiol ester, a n d the acetyl ester group of N,O-diacetyl-N-methylhydroxylamine are summarized in Table I11 according to two conventions. T h e values of K I refer to the nonionized species of reactants and products. T h e values of ICII refer to the ionic species of the reactants which actually undergo reaction and are related to K I by the ionization constants of the compounds. For nucleophilic reagents the reactive species is the anion and for acetylimidazole it is the acetylimidazolium cation. lo Where comparison is possible, there is moderately good agreement between the values of KI determined from the ratio of the rate constants in two directions and from direct measurement of equilibrium concentrations. For the reaction of acetylimidazole with p methoxyphenol, for example, the two methods give values of 129 and 136, respectively. After correction for catalysis of the reaction by imidazole, the rate measurements give an equilibrium constant of 300 for the reaction of acetylimidazole with N-methylacetohydroxamic acid, compared to the directly measured value of 287. T h e value of 37 from the rate measurements for the reaction of acetylimidazole with phenol is probably more accurate than the value of 48 from direct measurements, because of the high blanks attributed to phenol absorption in the latter measure-
I(Xi0
JOSEPH
GERSTEISANI) ~VILLLUIP.JEXCKS
1-01
nients. A further internal check of the consistency of the data can be made for the reactions involving acetylimidazole, p-chlorophenyl acetate, arid N,O-diacetyl-Smethylhydroxylamine. The equilibrium constant for acetyl transfer from acetylimidazole t o p-chlorophenol is 27 and t h a t for acetyl transfer from p-chlorophenyl TABLE III SUXXARY OF EQUILIBRIUM COSSTAYTS ASD FREE-ENERGY DIFFERENCES FOR ACETYLTRASSFER" KI
~
.
~
K , T f X 10
-
/! C F 3 C H 2 0 A c
