Amine-Catalyzed Hydrolyses of Cyclodextrin Cinnamates

Proc. Natl. Acad. Sci. USA Vol. 73, No. 9, pp. 2969-2972, September 1976 Chemistry

Amine-catalyzed hydrolyses of cyclodextrin cinnamates (cyclodextrin-catalyzed ester hydrolysis/acyl-cyclodextrin/acceleration of deacylation/enzyme model) MAKOTO KOMIYAMA AND MYRON L. BENDER Division of Biochemistry, Department of Chemistry, Northwestern University, Evanston, Illinois 60201 Contributed by Myron L. Bender, July 6,1976

ABSTRACT Hydrolyses of 6B-cyclodextrin cinnamate EXPERIMENTAL (#CDC) and a-cyclodextrin cinnamate were catalyzed by amines such as 1,4-diazabicyclo(2.2.2)octane, triethylamine, quinucli- Materials. #CDC and aCDC were kindly furnished by Y. dine, piperidine, diisobutylamine, and n-butylamine. The rate constant of hydrolyses of the #CDC-amine complexes follows Kurono. They were recrystallized before use. The purity of the order: 1,4-diazabicyclo(2.2.2)octane > n-butylamine > qui- fCDC and aCDC were found to be higher than 96% and-98%, nuclidine > piperidine > triethylamine >> diisobutylamine. The respectively, by absorption spectroscopy at 273 nm, which is ratio of the catalytic rate constant for the ,8CDC/1,4-diazabi- the isosbestic point between trans-cinnamic acid and (3CDC cyclo(2.2.2)octane complex to the spontaneous rate constant for or aCDC. Aza2bicOct was purified by recrystallization and had ,8CDC is about 6-fold and is almost independent of pH below a melting point of 158° see ref. Other pH 11.5; but, it then drastically increases with pH above pH 11.5, (159-160° reported, 5). up to 57-fold at pH 13.6 which is much higer than previous reagents were purchased from the Aldrich Chemical Co. attempts. The pH-rate constant profile and isotope effect with Kinetics. The hydrolysis of ,#CDC or aCDC was assayed by deuterium oxide solvent indicate that 1,4-diazabicyclo(2.2.2) absorption spectroscopy at 305 nm and 200. The ionic strength octane, included in #CDC, assists the catalytic nucleophilic was maintained at 1.0 M (KCI) unless otherwise noted. The rate attack by hydroxide ion toward the carbonyl carbon of #CDC. constants were determined by first-order kinetics, with at least Acceleration of deacylation of acyl-cyclodextrins, by amines, two half-lives. The change of pH in the sample solution, before has made the cyclodextrin-catalyzed hydrolysis of esters an even better model of hydrolytic enzyme reactions than those devel- and after the reaction was carried out, did not exceed +0.02 pH oped previously. units. When amine (B) forms a complex with ,BCDC or aCDC (S), It is well known that cyclodextrins catalyze the hydrolyses of BS, the observed rate constant (kobs) of the hydrolysis of ,BCDC phenyl esters (1). The cyclodextrin-catalyzed hydrolysis of esters or aCDC in the presence of B is expressed by Eq. 1: can be used as a model of serine esterase-catalyzed hydrolyses, because it proceeds through the pathway of binding, acylation, kobs[S] = ks[S] + kB[S][B] + kjS[BS] [1] and deacylation steps which is characteristic of enzymatic re- where ks and kgs are the first-order rate constants for S and BS, actions. Besides, cyclodextrin reactions exhibit many kinds of respectively; kB is the second-order rate constant for the hy- kinetic features shown by enzyme reactions, including stereo- drolysis catalyzed by B, which is not included in S. For the specificity, competitive inhibition, saturation, D-L-specificity, hydrolyses in the present paper, the second term in the right- etc. (2). However, a much smaller rate constant for the deacy- hand side of Eq. 1 is much smaller than the first and third terms, lation of acyl-cyclodextrins than that of the corresponding ac- because a plot of kob, versus [B] showed saturation at large values ylation resulted in the ineffective use of cyclodextrins as cata- of [B]. Thus, rearrangement of Eq. 1 using Eq. 2, under the lysts (2, 3). Thus, acceleration of deacylation of acyl-cyclo- condition that [B]o>> [S]o, gives Eq. 3 or Eq. 4: dextrins is quite important to make cyclodextrins an even better enzyme model. Recently, Kurono et al. (4) showed that the Kd = ([B]o- [BSX[S]O- [BS])/[BS] [2] hydrolyses of a-cyclodextrin cinnamate and f3-cyclodextrin = Kd/kms(l/[B]o) + cinnamate are accelerated by 3- and 2-fold, respectively, when 1/(kobs-ks) 1/kBp [3] 5-nitrobenzimidazole is included in the cyclodextrin cinna- kms = (kob. - ksXKd/[B]o + 1) [4] mates. This acceleration, however, is not sufficient for cyclodextrin to be a true enzyme model. where Kd is the dissociation constant of BS and the sub- In this report, accelerations of hydrolyses of a-cyclodextrin script 0 refers to the initial concentration. cinnamate (aCDC) and ,B-cyclodextrin cinnamate (I#CDC) by The values of kBS and Kd were determined by plotting 1/(kob, other amines, which come closer to a true enzyme model, are - ks) versus 1/[B]o in Eq. 3. When the value of Kd was deter- reported. The amines studied include 1,4-diazabicyclo(2.2.2)- mined by this procedure, kms could then be determined by the octane (DABCO; Aza2bicOct), triethylamine (Et3N), quinu- use of Eq. 4. clidine (QCD), piperidine (Pip), diisobutylamine (iBt2NH), and For D20 experiments, pD was determined by the equation: n-butylamine (BtNH2). pD = pH meter reading + 0.4 (6). The pH-rate constant profile and solvent isotope effect with deuterium oxide for Aza2bicOct on the hydrolysis of ,#CDC are shown. Furthermore, a mechanism for the amine-catalyzed RESULTS hydrolysis of aCDC or ,BCDC is proposed. The hydrolysis of flCDC or aCDc was accelerated by Abbreviations: aCDC, a-cyclodextrin cinnamate; fiCDC, /l-cyclo- Aza2bicOct, Et3N, QCD, Pip, and BtNH2, where the rate ac- dextrin cinnamate; Aza2bicOct, 1,4-diazabicyclo(2.2.2)octane; Et3N, celeration approached a maximum at high values of [B]o. On triethylamine; QCD, quinuclidine; Pip, piperidine; iBt2NH, diiso- the other hand, isobutylamine hardly affected the rate of hy- butylamine; BtNH2, n-butylamine. drolysis of ,BCDC. 2969 Downloaded by guest on September 30, 2021 2970 Chemistry: Komiyama and Bender Proc. Natl. Acad. Sci. USA 73 (1976)

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-5 i / [B]O M-l 9 10 I1 12 13 14 FIG. 1. Plots of 1/(kobs - ks) versus 1/[B]o in the hydrolysis of #CDC at 200 and pH 12.0; 0, Aza2bicOct; 0, Et3N. pH FiG. 2. The pH-rate constant profile at 200 for the hydrolysis of Fig. 1 shows the plot of 1/(kob, - ks) versus 1/[B]o, for the f3CDC catalyzed by Aza2bicOct; 0, kBS; *, kS. hydrolysis of f3CDC catalyzed by Aza2bicOct or Et3N. Table 1 lists the values of kg~s and Kd as well as the ratio of kBS increases the frequency factor for catalysis. This interpretation to ks. The dissociation constant, Kd, of the (BCDC-Aza2bicOct is supported by the fact that kgS for QCD is about % that for complex obtained at pH 12.0 is equal to that obtained at pH Aza2bicOct, though Kd for QCD is smaller than that for 10.0, within experimental error. The Kd value for the Aza2bicOct. QCD has a similar structure to Aza2bicOct, except aCDC-Aza2bicOct complex is about three times that for the that one of two nitrogen atoms in Aza2bicOct is replaced by a f3CDC-Aza2bicOct complex. This result is consistent with the carbon atom. finding by Kurono et al. (4) that the Kd value for the complex Fig. 2 shows the pH dependence of kgs for Aza2bicOct as well between aCDC and 5-nitrobenzimidazole is about 1.5 times as that of ks. A linear increase of KBS with pH and a slope of that for the complex between (#CDC and 5-nitrobenzimidazole. unity in the pH region investigated were noted. Almost all Besides, the ratio of kBs to ks in catalysis by Aza2bicOct is Aza2bicOct is in the neutral state in the pH range investigated. 1.7-fold larger for ,BCDC than for aCDC. Thus, the rate constant, kgs, can be expressed by Eq. 5: In the hydrolysis of ,iCDC, as for the kas values: Aza2bicOct > BtNH2 > QCD > Pip > Et3N >> iBt2NH, whereas for Kd: kBS = kOH- [OH- [5] BtNH2 > Et3N > Aza2bicOct > Pip > QCD. Thus, no rela- tionship was observed between catalytic activity of the-base and Below pH 12.5, the value of kgS was determined at [B]o = 0.435 its complex formation constant. Besides, the catalytic activity M by using Eq. 4, while above pH 12.5, it was determined is not linear with the pKa value of the bases. The geometry of at [Blo = 45 mM. The decrease of the value of [B]o at higher pH the complex between ,#CDC and the amine should govern the was necessary to keep the ionic strength at 1.0 M, since magnitude of kgs, in the same manner as the geometry of the Aza2bicOct functions as a buffer. An increase in the ionic complexes between cyclodextrins and phenyl esters governs strength from 1.0 M to 1.5 M, by the use of KCl, decreased the their hydrolyses (1). The high catalytic activity of Aza2bicOct kBs value by about 20%. The rather large dependence of the rate can be also ascribed to the presence of two basic centers, which constant upon the ionic strength is consistent with that for the alkaline hydrolysis of trifluoroethyl acetate (8). Table 1. Values of kBS and Kd at 200, pH 12, The rate constant for the f3CDC spontaneous hydrolysis, ks, and I = 1.0 M for the hydrolysis of,CDC increased linearly with pH up to pH 11.2, with a slope of unity. Above pH 11.2, however, the slope gradually decreased with kBS Kd kBS pKa of pH, which is attributable to an appreciable ionization of the 10-i sec-' 10-2 M base* hydroxyl groups of 3CDC. Catalyst kS Fig. 3. shows the dependence of the ratio (kBS/ks) upon pH Aza2bicOct 8.0 12 6.7 8.19 for the hydrolysis of ,BCDC catalyzed by Aza2bicOct. Up to pH 0.08t 11 5.0 8.19 11.5, the ratio (kBs/ks) remained rather constant at about 6. EtN 2.9 38 2.4 10.78 Above pH 11.5, however, the ratio drastically increased with QCD 5.6 9.2 4.7 10.95 pH. At pH 13.6, the ratio attained a value of 57, which is about Pip 3.9 9.4 3.3 11.28 28 times the acceleration by 5-nitrobenzimidazole (4). iBt2NH 0.0 - 0.0 10.91 The rate constant (koH-), based on the concentration of hy- BtNH2 6.0 43 5.0 10.28 droxide anion, for the hydrolysis of the ,3CDC-Aza2bicOct Aza2bicOctl 6.2 35 3.6 8.19 complex in D20 was found to be 1.41 + 0.02 times that in H20 at pH 10.0, 11.0, or 12.0: the ratio of the autoprotolysis constant For #CDC, ks = 1.2 X 10-3 sec-1 at pH 12 and 1.6 X 10-5 of H20 and D20 was taken as 6.5 (9). This result indicates nu- sec-I at pH 10. cleophilic attack by hydroxyl anion toward ,BCDC, because the * See ref. 7. tAtpH 10. nucleophilicity of deuteroxide anion is 20-40% higher than t This reaction used aCDC as a substrate; ks = 1.7 x 10-3 sec-1 hydroxide anion (10-15). Nucleophilic attack by Aza2bicOct at pH 12. toward f3CDC is ruled out by the D20 effect (16). The rather Downloaded by guest on September 30, 2021 Chemistry: Komiyama and Bender Proc. Nati. Acad. Sci. USA 73 (1976) 2971 Neither Aza2bicOct, Et3N, nor QCD can form a stable amide from I3CDC or aCDC, since they are tertiary amines. On the other hand, some part of the observed change of absorbance at 80 305 nm in the presence of Pip, iBt2NH, or BtNH2, may be due to the formation of amides as in ammonia-catalyzed ester cleavage (17). However, lack of acceleration of the hydrolysis 60 by iBt2NH implies that the important reaction in the presence of Pip, iBt2NH, or BtNH2 is hydrolysis of flCDC rather than the amide formation. 401 The value (1.6 X 10-5 sec-I at pH 10.0, 20°, I = 1.0 M) of the 0 rate constant, ks, for the spontaneous hydrolysis of I3CDC in the present paper is close to that (1.8 X 10-5 sec-1 at pH 10.0, 20°, I = 0.15 M) obtained by Kurono et al. (4). The latter value was 20 calculated from the value of ks (3.47 X 10-4 hr'1) at pH 7.35, 20 250, using the activation energy of the reaction (31 kcal/mol). The slightly smaller value in the present paper is due to the 10 I 12 13 14 larger ionic strength. pH The pH-rate constant profile in Fig. 2 and the D20 effect show that the hydrolysis of 3CDC by complexed Aza2bicOct FIG. 3. The ratio (kBS/kS) for the hydrolysis of flCDC catalyzed is associated with attack by hydroxide anion. The alkaline hy- by Aza2bicOct at 200 as a function of pH. drolysis of esters proceeds via the formation of a tetrahedral addition intermediate and the rate-determining step is nu- poor leaving group, ,B-cyclodextrin, in 3CDC, does not favor cleophilic attack by the hydroxide anion (18, 19). Aza2bicOct, nucleophilic catalysis by Aza2bicOct (17). which is in the neighborhood of the attacking hydroxide anion as the result of inclusion in ,8CDC, can assist attack by the hy- DISCUSSION droxide anion toward ,3CDC. The assistance by Aza2bicOct probably takes place through hydrogen bonding between the The present study showed that the deacylation of acyl-cyclo- hydrogen atom of the hydroxide anion and the nitrogen atom dextrins is much enhanced by amines such as Aza2bicOct, of Aza2bicOct. The hydrogen bonding can stabilize the tran- BtNH2, QCD, Pip, and Et3N. Small rate constants for the- sition state more than the ground state (14) and enhance the in state. deacylation of acyl-cyclodextrins are one of the defects of the nucleophilicity of the hydroxide anion the ground Both cyclodextrin-catalyzed hydrolysis of esters as a model of serine effects result in the rate enhancement. The catalysis by amines esterases, though cyclodextrin reactions exhibit many en- other than Aza2bicOct probably proceeds in the same way as zyme-like features (2). Thus, the present finding of acceleration that by Aza2bicOct. of the deacylation step by amines provides further potentiality In summary, we find that the deacylation of acyl-cyclodex- for cyclodextrins to be an even better enzyme model. For ex- trins is much enhanced by certain amines. This implies that ample, the observed rate constants for the acylation and the cyclodextrins can be even better model enzymes in the presence deacylation in the ,B-cyclodextrin-catalyzed hydrolysis of m- of thqse amines. chlorophenyl benzoate are 2.2 X 10-3 sec-1 and 2.7 X 10-4

sec 1, respectively, under the conditions that [IB-cyclodextrin]o = 10-2 M at pH = 10.6, 250, and I = 0.2 M (3). The ob- We thank Dr. Yukihisa Kurono for kindly providing samples of served rate constant for the acylation is four times (5.5 X 10-4 aCDC and ,6CDC. This research was supported by Grant MPS-72- sec-1) that for the alkaline hydrolysis of m-chlorophenyl ben- 05013 from the National Science Foundation. zoate. However, f,-cyclodextrin cannot be called a true catalyst here, because the rate constant for the deacylation of ,B-cyclodextrin benzoate is smaller than that for the alkaline hydrolysis 1. VanEtten, R. L., Sebastian, J. F., Clowes, G. A. & Bender, M. L. of m-chlorophenyl acetate. However, the rate constant for the (1967) J. Am. Chem. Soc. 89,3242-3253. deacylation of f3-cyclodextrin benzoate, catalyzed by complexed 2. Bender, M. L. & Komiyama, M. (1976) Bioorg. Chem., in Aza2bicOct, can be 1.8 X 10-3 sec-1 (which is 3.3-fold larger press. than that for the alkaline hydrolysis of m-chlorophenyl ben- 3. VanEtten, R. L., Clowes, G. A., Sebastian, J. F. & Bender, M. L. (1967), J. Am. Chem. Soc. 89,3253-3262. zoate), when the accelerated deacylation by complexed 4. Kurono, Y., Stamoudis, V. & Bender, M. L. (1976), Bioorg. Chem., Aza2bicOct is tentatively assumed to be the same (6.7 fold) as in press. that for the hydrolysis of ,BCDC. Thus, f3-cyclodextrin in the 5. Fieser, L. F. & Fieser, M. (1967) Reagentsfor Organic Synthesis presence of Aza2bicOct can be called a true catalyst for ester (John Wiley and Sons, Inc., New York), p. 1203. hydrolysis, and it is much better enzyme model than ,B-cyclo- 6. Glasoe, P. K. & Long, F. A. (1960) J. Phys. Chem. 64, 188- dextrin in the absence of Aza.bicOct. 190. The ratio of the rate constant for the complexed Aza2- 7. Perrin, D. D. (1965) Dissociation Constants of Organic Bases bicOct-catalyzed deacylation of fl-cyclodextrin benzoate to that in Aqueous Solution (Pure and Applied Chemistry, London). for the alkaline hydrolysis of m-chlorophenyl benzoate is 3.3 8. Kirsch, J. F. & Jencks, W. P. (1964) J. Am. Chem. Soc. 86, and is almost independent of pH because both rate constants 837-846. 9. Kingerley, R. W. & LaMer, V. K. (1941), J. Am. Chem. Soc. 63, increase proportionally with pH. In the absence of Aza2bicOct, 3256-3262. however, the ratio of the rate constant for the alkaline hydro- 10. Wiberg, K. B. (1955) Chem. Rev. 55,713-743. lyisis of m-chlorophenyl benzoate to that for the spontaneous 11. Jencks, W. P. & Carriuolo, J. (1960) J. Am. Chem. Soc. 82, deacylation of fl-cyclodextrin benzoate should increase with 675-681. pH, and attain the value of 16 at pH 13.6. 12. Bunton, C. A. & Shiner, V. J., Jr. (1961) J. Am. Chem. Soc. 83, Downloaded by guest on September 30, 2021 2972 Chemistry: Komiyama and Bender Proc. Nati. Acad. Sci. USA 73 (1976)

42-47, 3207-3214. 16. Bender, M. L., Pollock, E. J. & Neveu, M. C. (1962) J. Am. Chem. 13. Bunton, C. A. & Shiner, V. J., Jr. (1961) J. Am. Chem. Soc. 83, Soc. 84,595-599. 3207-3214. 17. Jencks, W. P. and Carriuolo, J. (1961) J. Am. Chem. Soc. 83, 14. Bruice, T. C. & Fife, T. H.- (1962) J. Am. Chem. Soc. 84, 1743-1750. 1973-1979. 18. Bender, M. L. (1960) Chem. Rev. 60,53-113. 15. Gruhn, W. B. & Bender, M. L. (1975) BMoorg. Chem. 4,219- 19. Jencks, W. P. & Gilchrist, M. (1968) J. Am. Chem. Soc. 90, 236. 2622-2637. Downloaded by guest on September 30, 2021