Distribution Statement Each transmittal of this document outside the agencies of the U.S. Government must have prior approval of the CO, Edgewood Arsenal, ATTN: SMUEA-TSTI-T, Edgewood Arsenal, Maryland 21010. Disclaimer The findings in this report are not to be construed as an official Department of the Army position unless so designated by other authorized documents. Disposition Destroy this report when no longer needed. Do not return it to the originator.

MECHANISM OF ENZYME ACTION Semiannual Report by C. G. Overberger H. Maki I. Cho M. Morimoto P. S. Yuen C. M. Shen J. C. Salamone March 1968 Each transmittal of this document outside the agencies of the U.S. Government must have prior approval of the CO, Edgewood Arsenal, ATTN: SMUEA-TSTI-T, Edgewood Arsenal, Maryland 21010 DEPARTMENT OF THE ARMY EDGEWOOD ARSENAL Research Laboratories Physical Research Laboratory Edgewood Arsenal, Maryland 21010 Contract DAAA-15-67-C-0567 Project 1BO14501B71A THE UNIVERSITY OF MICHIGAN Ann Arbor, Michigan

Foreword The work described in this report was authorized under Project 1B014501B71A, Life Sciences Basic Research in Support of Materiel (U). This work was started in March and completed in December 1967. Reproduction of this document in whole or in part is prohibited except with permission of CO, Edgewood Arsenal, ATTN: SMUEA-RPR, Edgewood Arsenal, Maryland, 21010; however, Defense Documentation Center is authorized to reproduce the document for U.S. Government purposes. The information in this document has not been cleared for release to the general public.

Digest Solvolytic rate measurements of esters catalyzed by synthetic, imidazolecontaining polymers have revealed many analogies to those of naturally occurring enzymic processes. In particular, the multifunctional interaction of catalytic groups at the active site of an enzyme with a substrate can also exist in synthetic, polymeric imidazoles. Reactions catalyzed by polymers with pendent imidazole or benzimidazole functions have revealed the existance of cooperative interactions between two imidazole (or benzimidazole) functions(1-3) the utilization of electrostatic attraction of substrate to a charged site on a polymer chain thereby accumulating the substrate in the vicinity of catalytically active groups, and cooperative interactions between imidazole and hydroxyl or phenol functions. In a continuation of the study of copolymers of 4(5)-vinylimidazole with acrylic acid, believed to be synthetic, macromolecular models for acetyl(4) cholinesterase, the solvolytic rates of neutral, negatively, and positively charged phenyl esters were determined as a function of the copolymer composition and sequence distribution. In this manner, a proper distribution of charged groups (carboxylate) and catalytically active functions (imidazole) was obtained which had optimal catalytic activity. Since the copolymers containing high contents of acrylic acid could behave principally as polyelectrolytes, the effect of anionic poly(acrylic acid) on the solvolytic rates of neutral, negatively, and positively charged substrates was also investigated. In this system a strong inhibition of the

solvolytic rate of the positively charged ester 3-acetoxy-N-trimethylanilinium iodide (ANTI) was observed. The dissociation constant of this system (polyanion-substrate) was found to be of the same order of magnitude as that of acetylcholinesterase with its substrate. The preparation of poly(3-vinyl-1,2,4-triazole), which was reported in the Final Report covering the Period March 4, 1964-March 4, 1967, Contract No. DA18-035-AMC-121(A), has been improved. Solvotic reactions employing 1,2,4-triazole and poly(3-vinyl-1,2,4-triazole) with neutral and negatively charged substrates are being investigated. Results appear to indicate that the nucleophilic reaction of neutral triazole with substrate does not contribute significantly to the reaction rate, while that of either anionic triazole or general base catalyzed triazole is most important in the solvolytic process. An investigation of the effects of solvent composition on the catalytic activities of imidazole-containing polymers is in progress. Alteration in solvent composition (alcohol-water) has shown marked differences in catalytic capabilities.(35) By varying the concentration of ethanol and water in the solvent system, it may be possible to correlate the effects of a tight polymer coil to enhanced catalytic action, since the imidazole groups would have a greater probability of being in juxtaposition and can act as multifunctional catalysts. Light scattering measurements will be employed to determine the end-to-end distance in the polymer. At present, the effect of varying solvent on apparent pK1 measurements have been performed, as well as the effect of solvent composition on polymer solubility (turbidity measurements).

It was recently reported that solvolytic reactions catalyzed by a copolymer of 4(5)-vinylimidazole and p-vinylphenol indicated the existence of cooperative, multifunctional interactions between pendent, neutral imidazole, and anionic phenol groups. In order to further elucidate this cooperative interaction a methylene compound with terminal imidazole and phenol groups was synthesized. Catalytic effects of this compound and of poly-4(5)vinylimidazole on the solvolytic rates of phosphoric esters are presently under investigation.

Table of Contents Page I. Effects of Copolymer Composition and Monomer Sequence on Catalytic Activity of Copolymers of 4(5)-Vinylimidazole and Acrylic Acid 8 II. Effects of Anionic Poly(acrylic acid) on Solvolytic Rates of Neutral, Negatively, and Positively Charged Esters —An Inhibitory Effect 13 III. Effects of Solvent Composition in Polymeric Imidazole Catalysis 24 IV. Catalytic Activities of 1,2,4-Triazole and Poly(3-vinyl1, 2,4-triazole) 29 V. Preparation of 4(5)-[9-(p-Hydroxyphenyl)nonyl]-imidazole 35 VI. Experimental 36 VII. Literature Cited 41 VIII. Distribution List 42

List of Illustrations Table Page I. Ratios of Catalytic Rate Constants to Fractions of Neutral, Isolated Imidazole Sequences as a Function of Copolymer Composition 12 II. First-Order Rate Constants for the Solvolyses of PNPA, NABA, and ANTI as a Function of Poly(acrylic acid) Concentration, pH 8.98 15 III. Differences in First-Order Rate Constants for the Solvolysis of ANTI in the Presence and Absence of Poly(acrylic acid) 22 IV. Homopolymerization and Copolymerization Conditions of 4(5)Vinylimidazole with Styrene 25 Figure 1. Catalytic rate constants for the solvolysis of ANTI at pH values of 8.38 (0), 8.98 (A), and 9. 94 (0 ), catalyzed by copolymers of 4(5)-vinylimidazole and acrylic acid, in 28.5% ethanol-water, ionic strength 0.02, temp. 26~C. 10 2. Fraction of isolated, neutral imidazole sequences as a function of the imidazole content in copolymers of 4(5)-vinylimidazole and acrylic acid, at pH values 8, 9, and 10. 11 3. Dependence of first-order observed rate constants for PNPA (0), NABA (A), and ANTI (0) solvolyses on poly(acrylic acid) concentration, 28.5% ethanol-water, ionic strength 0. 02, buffer concentration 0. 02 M, temp. 26~C. 16 4. Determination of a coefficient of repulsion. 19 5. Determination of the polyion-substrate dissociation constant and the reactivity of the bound substrate. 23 6. Effect of solvent composition (ethanol-water) on (apparent) pK1 values for imidazole (0), poly-4(5)-vinylimidazole [sample No. 2, (A)], and copoly [4(5)-vinylimidazole-styrene] [sample No. 5, (0)], ionic strength 0.05, temp. 26~C. 26

List of Illustrations (Concluded) Figure Page 7. Turbidity of poly-4(5)-vinylimidazole [samples No. 1 ([]) and 2 (A)] and copoly[4(5)-vinylimidazole-styrene] [sample No. 5 (0)] at pH 8.0 as a function of increasing water content in ethanol-water solvent systems, buffer (TRIS-HC1) concentration 0.1 M, ionic strength 0.1, temp. 260~C. 27 8. Turbidity of poly-4(5)-vinylimidazole [samples No. 1 ( 0) and 2 (A) and copoly[4(5)-vinylimidazole-styrene] [sample No. 5 (0)] at pH 9.0 as a function of increasing water content in ethanol-water solvent systems, buffer (TRIS-HC1) concentration 0.1 M, ionic strength 0 1, temp. 26~C. 28 9. Dependence of the solvolytic rates of PNPA (0,), NABA ( O,I), and NABS (A,A) on the concentrations of 1,2,4-triazole and poly-3-vinyl-l,2,4-triazole, respectively; in 28. 5% ethanolwater, pH 8.89, buffer concentration (TRIS-HC1) 0.02 M, ionic strength 0. 02, temp. 26~C. 31 10. pH-rate profiles for the solvolyses of PNPA (0,$), NABA (EO,), and NABS (A,A) catalyzed by 1,2,4-triazole and poly-3-vinyl1,2,4-triazole, respectively; 28.5% ethanol-water, ionic strength 0. 02, buffer (TRIS-HC1) concentration 0. 02 M, temp. 26~C. 32 11. Dependence of the catalytic rate constants for the solvolyses of PNPA (0), NAkBA (EO), and NABS (A) on the fraction of anionic 1,2,4-triazole functions, 28.5% ethanol-water, ionic strength 0. 02, buffer (TRIS-HC1) concentration 0. 02 M, temp. 26~C. 33 12. Dependence of the catalytic rate constants for the solvolyses of PNPA (0), NABA ([E), and NABS (A) on the fraction of anionic 1,2,4-triazole functions in poly-3-vinyl-1,2,4-triazole, 28.5% ethanol-water, ionic strength 0.02, buffer (TRIS-HC1) concentration 0.02 M, temp. 26~C. 34

Results and Discussion I. Effects of Copolymer Composition and Monomer Sequence on Catalytic Activity of Copolymers of 4(5)-Vinylimidazole and Acrylic Acid. In the last report(6) results of the solvolyses of the neutral ester p-nitrophenyl acetate (PNPA), the negatively charged ester 4acetoxy-3-nitrobenzoic acid (NABA) and the positively charged ester 3-acetoxy-N-trimethylanilinium iodide (ANTI), each catalyzed by a series of copolymers of 4(5)-vinylimidazole and acrylic acid at one pH value, were given. When the positively charged ester ANTI was employed, a bellshaped curve was obtained in a plot of the second-order catalytic rate constant vs. mole % imidazole in the copolymers. This curve showed that maximum catalytic activity occurred in the region of 46 mole % imidazole when the ionic strength was 0.02. However, when the ionic strength was increased to 0.50, no optimal effects were observedo These results and an analysis of the sequence distribution in the copolymers support the belief that the isolated neutral imidazole sequence (I) is primarily responsible for catalytic action. The leveling effect of increased ionic strength cdon / hioot strongly suggests that ion-pair formation occurs between the anionic carboxylate sites and the positively charged substrate0

In order to obtain additional information regarding this system, the effect of varying pH on the catalytic action of copolymers of 4(5)-vinylimidazole and acrylic acid with ANTI was studied. In Figure 1 is shown a plot of the second-order catalytic rate constant for solvolysis of ANTI at three pH values vs. mole % neutral imidazole functions, This figure reveals rate maxima in the composition region of 42-50% mole % imidazole for each pH value studied. If this data is compared to a graph of the fraction of isolated neutral imidazole sequences as a function of the mole % imidazole in the (6) copolymers shown in Figure 2 and the preceeding report, it is seen that good agreement exists between the bell-shaped curves with regard to shape and location of maxima. This further substantiates the isolated, neutral imidazole sequence (I) being the most catalytically active component,

10 60 50'E 40 3020 1010 20 30 40 50 60 70 80 90 100 COPOLYMER COMPOSITION ( mole % in 4 (5) - Vinylim i dazole) Figure 1. Catalytic rate constants for the solvolysis of ANTI at pH values of 8.38 (0), 8.98 (A) and 9.94 (0U), catalyzed by copolymers of 4(5)vinylimidazole and acrylic acid, in 28.5% ethanol-water, ionic strength 0.02, temp. 26~C.

11 U.I 1.0 80.9 z d0-8 0.8 0.7 pH 10 J 0.6,H0 6 0 /X _ pH 9 w 0.5 0.4.g~~~~ ~~pH8 0 0.3 C) o 0.2 z 0 0.1 I. 0 10 20 O30 40 50 60 70 80 90 100 COPOLYME R COMPOSITION (mole % in 4 (5) -Vinylimidozole') Figure 2. Fraction of isolated, neutral imidazole sequences as a function of the imidazole content in copolymers of 4(5)-vinylimidazole and acrylic acid, at pH values 8, 9, and 10.

12 If the ratios of the catalytic rate constants (kcat) to the fractions of neutral, isolated imidazole sequences (alfl) are compared for the different copolymer compositions (Table I), it is noted that the values reported for imidazole contents of 26.1 mole % and 79.4 mole % deviate considerably from the values found for imidazole contents of 31.9, 46.3 and 58.8 mole %. Table I(a) Ratios of Catalytic Rate Constants to Fractions of Neutral, Isolated Imidazole Sequences as a Function of Copolymer Composition. kcat/alfl mole % imidazole pH 8.38 pH 8.95 pH 9.90 26.1 18.7 1709 48.0 31.9 22.2 33o1 81.0 46.3 19.9 31.3 83.2 58.8 22,2 35.6 91.5 79 4 72.0 130 205 (a) In 28.5% ethanol-water at an ionic strength of 0.02, temp. 26~0c. These data are indicative of two factors involved in the solvolysis of ANTI catalyzed by the copolymers of 4(5)-vinylimidazole and acrylic acid. Firstly, at copolymers compositions greater than 50 mole % imidazole, contributions between imidazoleimidazole sequences could occur in accord to such effects previously observed in reactions catalyzed by poly-4(5)-vinylimidazole.(1'3)

The other factor could be the inhibition of the solvolytic rate of ANTI (presented in section II) caused by an increase in the amount of carboxylate-carboxylate sequences. (8) The effects of pH on the solvolytic rates of PNPA and NABA catalyzed by the above mentioned copolymers and a monomeric analog of the isolated sequences, y-4-imidazoylbutyric acid, are currently under investigation, II.onic Po(acrylic acid) on Solvolytic Rates of Neutral, Negatively, and Positively Charged Esters - An Inhibitory Effect In general, the effects of polyions (containing no catalytically active functions) on the reaction rates of two small charged reagents can be classified into two categories(7' 9) The large electrostatic potential which characterizes a polyelectrolyte solution can lead to corresponding fluctuations in the local concentration of the charged species. If a reaction involves two ionic species of the same charge, such as hydroxide ions and an anionic ester, a polyion of the opposite sign (polycation) will tend to concentrate the reactive reagents in its vicinity thereby increasing their collision frequency. In such a case the polyion will act as a catalyst. On the other hand, if two oppositely charged species are the reactive reagents, such as a cationic ester and hydroxide ions, a polyion of either charge will tend to concentrate one reagent in its vicinity and repel the other0 The polyion will

then act as an inhibitoro The above effects have been experimentally substantiated(7'8' 10,11) It has also been postulated that if two similarly charged species are the reactive reagents, a polyion of the same charge will accelerate the reaction since both species are repelled from the polymer chain and are in a higher concentration (lower volume) than would be realized in the absence of polyelectrolyte.(7'9) In order to further understand the behavior of the copolymers of 4(5)-vinylimidazole and acrylic acid towards neutral and charged esters, particularly when the mole % of acrylic acid is high, a study of the solvolytic rates of PNPA, NABA and ANTI was undertaken0 Data for the solvolyses of PNPA, NABA and ANTI solvolysed in the presence and absence of poly(acrylic acid) are listed in Table II. These measurements were obtained in 28~5% ethanol-water at pH 8o98, ionic strength 0.020 In Figure 3, plots of the firstorder observed rate constant (kobsd = kmead klank) s. ttal concentration of poly(acrylic acid) are presented0 This figure reveals several surprising effectso It is seen that the reaction with both PNPA and NABA is first-order in both substrate and polyion (hydroxide ion), an effect not previously observed for either a neutral substrate or an anionic substrate whose hydroxide ion catalysis occurs in the presence of a polyanion. Furthermore, the reaction with ANTI clearly reveals an inhibitory effect displaying saturation characteristics, an effect previously reported

15 by Morawetz and Shafer for the hydrolysis of singly and doubly charged cationic esters in the presence of partially neutralized poly(methacrylic acid).(7) Table II(a) First-order Rate Constants for the Svolyses of PNPA, NABA and ANTI as a Function of Poly(acrylic aci'd) Concentration, PH 8.98 - -1 10 min [Poly(AA)] ANTI NABA PNPA x 103 m kblank kmeasd kobsd kblank kmeasd kobsd kblank kmeasd kobsd 0.000 7o23 0 —- o.00 3,03. 0. o000 3.76. O ooo0 0,467 6.40 -0o83 3o.68 o68 4.23 0,47 0o934 | _ a 6.16 -1.07 4.33 1.30 4.54 o.68 3.000 5 56 -1.67 704 4o01 5.72 1.96 4.670 5 8 __ 2.60 (a) In 28o5% ethanol-water, ionic strength 0.02, temp. 260c. Regarding the solvolysis of NABA, the effect of enhanced solvolytic action occurring when two similarly charged species react in the presence of a similarly charged polyion has been theorized(7~'9) but never experimentally verified. This effect can be accounted for by the electrostatic repulsion of both reactive species (anionic ester and hydroxide ions) from the polyanion where they are then concentrated in a decreased volume of solutiono From this experiment a coefficient of repulsion can be calculatedo Consider the rate of solvolysis of substrate in the absence of polyion is given by the expression

~6~~~16 C 4E 0 2 -o [poly(AA)] x 10 (M) Figure 3. Dependence of first-order observed rate constants for PNPA (0), NABA (A), and ANTI (0) solvolyses on poly(acrylic acid) concentration, 28. 5% ethanol-water, ionic strength 0. 02, buffer concentration 0. 02 M, temp. 26 ~C.

17 Ms MOH (1) S][OH+ +][S kw + kbk [ k][1[i o ~-~ =V~ BkO' —'~ V where R0 and k0 are the rate and rate constant in the absence of polyion, [S] is the substrate concentration, Ms and MOH are the moles of substrate and hydroxide ions, respectively, V is the total volume of solution, kw is the rate constant for water catalysis, kB is the rate constant for buffer catalysis and B is the buffer concentration. In the presence of polyion, the reactive reagents will be excluded from the polyion by a certain factor (according to the Boltzman distribution), and the rate of solvoly3is will then be given by Rp = k [S]p [OH-]p + kw + kB [B] [S] Ms MOHp = ko p V -Vp + kw + kB [B] [S] Rp = kp [S] [OH] + kw + kB [B] [S] (2) where Rp and kp are the rate and rate constant in the presence of polyion, [S]p and [OH-]p are the concentrations of substrate and hydroxide ions resulting from exclusion from the polymer domain, and Vp is the volume from which substrate and hydroxide ions are excluded. This equation assumes that the concentration of reactive species in the vicinity of polyion is negligibleo The observed rate of reaction is then R1 Rp - Ro= o "s i |(\-,v |

18 2VpV - V 2 Rp Ro = ko Ms MOH v2 (V-v) v (2v-v )(3) Rp R0= k0 [S] [OH-] Since V~> Vp, 2V 2V Rp- RO k [S [O] -- ko ] [ ] v V (4) Vp can be defined as the summation of the excluded volume for each polyion (assuming no interactions of polymer chains), VP = Vpi =Kn (5) where K is the effective repulsive volume coefficient associated with one polymer molecule and np is the number of polymer molecules. Therefore, since np/V = [P] RD-O =.k = 2K[P] (6) Ro k0 Employing equation 6 for the base catalyzed solvolysis of NABA in the presenc.e of anionic poly(acrylic acid) (Figure 4), a straight line is obtained having a slope of 441 M. The constant K is then determined to be 220.5 M1. If the polymer is an extended cylinder in solution, the constant K would then indicate an excluded volume of substrate and hydroxide ions of approximately 240 A from a single monomer segment.

19 1.2 0,0 0.8 0.4 1 2 3 4 [poly(AA)] x 103 (M) Figure 4. Determination of a coefficient of repulsion.

20 Previous solvolytic reactions of neutral esters in the presence of polyions have revealed no rate alterations,(7'10) since the ester should neither be concentrated near the polyion nor in the bulk of solution. However, we observed a totally unexpected rate acceleration for the solvolysis of PNPA in the presence of anionic poly(acrylic acid) (Figure 3), In this case it appears that PNPA is excluded from the vicinity of the polyelectrolyte since if it were in the vicinity of the polyanion a rate depression would be expected Another factor involved could be rate acceleration caused by nucleophilic attack of anionic carboxylate groups on the acetyl functions, where PNPA could be attracted to the polyanion by either hydrophobic forces or ion-dipole interactions; however, if this effect were operative, a similar or even greater rate enhancement should occur with the positively charged ester ANTI which would be strongly attracted to the polyanion by electrostatic forces. As mentioned previously, the reaction of hydroxide ions with ANTI in the presence of anionic poly(acrylic acid) revealed a rate depression. This would therefore exclude the possible involvement of carboxylate functions in the solvolysiso At the present time the causes of this effect are not understood and further research is necessary to elucidate this phenomenon. From Figure 3 it is seen that a strong depression of the solvol ytic rate of ANTI is obtained when the reaction takes place in the presence of anionic poly(acrylic acid)~ Furthermore, at high polymer concentrations the reaction rate approaches a limiting value. This effect is analogous to that previously observed by Morawetz and

21 Shafer for the hydrolysis of singly and doubly charged cationic esters in the presence of anionic polymeric acids(7'8). It is accounted for by the electrostatic attraction of the positively charged polyanion, from which the catalyzing hydroxide ions are repelled. The results of Morawetz and Shafer indicated that a kinetic scheme similar to that obtained for an enzyme-substrate complexation could be obtained from such an inhibitory system, They were able to calculate the dissociation constant KD from the complex of polymer P with reagent R, ioeo, [P] [R] KD = PR] (7) they employed the equation ko= [R] + kb [PR] (8) LRI + [PRI where k0 is the rate constant for the uncomplexed substrate and kb the rate constant for the substrate bound to the polyion, while k is the observed rate constant. Combining equations 6 and 7 they obtained k = kb + KD (kO k (9) a relationship analogous to that determined by Lineweaver and Burk for enzyme-substrate complexationo They assumed k0 to be identical to the rate constant resulting from hydrolysis in the absence of polyion~ The linear relationship suggested by equation 9 can be used to determine the reactivity of the bound reagent and the dissociation

22 constant of the polyion-substrate. In table III data pertaining to the differences in first-order rate constants in the presence and absence of polyion, to be used in plotting equation 9, are presented. Table III(a) Differences in First-order Rate Constants for the Solvo]ysis of ANTI in the Presence and Absence of Poly(acrylic acid) k x 103 (min-) (k - kp)/[P] (min-1 M-1) 6.40 1.765 6.16 1.150 5056 0o551 5043 0.386 (a) In 28.5% ethanol-water, pH 8.96, ionic strength 0.02. Constants obtained from Table IIo These data are plotted in Figure 5. The value of kb is determined from the intercept to be 5.25 x 10-3 min-1, a value slightly less than the solvolytic rate for the free reagent (ko = 7~23 x 10 -l min, see Table II)o The dissociation constant KD for the polyionsubstrate complex is determined from the slope to be 7o14 x 10-4 M, a value of the same order of magnitude as the Michaelis constant Km for the reaction of acetylchlinesterase with acetylcholine (Km = 2.6 x 10o-). The latter reaction is in part due to the electrostatic attraction of the positively charged substrate to an anionic site on the enzymeo

8 6 x 4 I l l I 0.4 0.8 1.2 1.6 ko-k (min M') [poly (AA)] Figure 5. Determination of the polyion-substrate dissociation constant and the reactivity of the bound substrate.

III. Effect of Solvent Composition on Polymeric Imidazole Catalysis Poly-4(5)-vinylimidazole is soluble in the mixed solvent system of ethanol-water, insoluble in water and difficulty soluble in ethanol Since previous experiments have indicated a marked dependence of solvent composition on the solvolytic rates of esters,3'5) a study of the varying proportions of ethanol-water on the catalytic capabilities of poly-4(5)-vinylimidazole and a copolymer. of 4(5)-vinylimidazole with styrene (a copolymer containing inert hydrophobic groups) is under investigation. It can be expected that varying the solvent content will lead to alterations ii the polymer conformations, as well as in the apparent dissociation constants of the imidazole groups at half neutralization. Since the pendent imidazole functions in poly-4(5)vinylimidazole are believed to be involved in cooperative, multifunctional interactions, a rate enhancement should occur when the polymer is in a tight coil in solution, In this instance, the catalytic functions could be in juxtaposition for enhanced multifunctional interactions. The dimensions of the polymer will be determined by light scattering measurements. At the present time turbidimetric titrations have been performed on two copolymers of 4(5)-vinylimidazole amd a copolymer of 4(5)-vinylimidazole and styrene. The inert, hydrophobic styrene groups could lead to different conformations than that of poly4(5)-vinylimidazole in'ethanol-water solvent systems. In Figure 6 is shown a plot of the effect of ethanol content on the (apparent) pK1 values of the imidazole functions for imidazole,

for poly-4(5)-vinylimidazole, and for a copolymer of 4(5)-vinylimidazole and styrene. These data are tentative and subject to further investigation. The poly-4(5)-vinylimidazole employed in this investigation was prepared by free radical polymerization in benzene (sample No. 2, see Table IV) from which the growing polymer chain precipitates. This plot reveals that for poly-4(5)-vinylimidazole and (sample No. 5) copoly[4(5)-vinylimidazole-styrene] the apparent pK1 values for the imidazole groups show a minimum in the region of 50% ethanol-water, whereas the pK1 values for imidazole decrease with increasing ethanol content. In Figures 7 and 8 the turbidity of samples No. I and 2 (Table IV) of poly-4(5)-vinylimidazole and sample Noo 5 of copolymer of 4(5)-vinylimidazole and styrene are shown (for two pH values) vs. varying ethanol content. The conditions employed in polymerization for a series of homopolymers and are shown in Table IV. Table IVf Homo olymerization and Copolymerization Conditions otif 4(5)-Vinvy-'rmidazole wtkth S3tyrene Sample No,. V4(5)VIm1 [S1tyrenel. AiBN Sgo lvnt Tm. Tine - onv 1 0.011 m.. 002 g re thanol. 65: 2'4 h 33.1 2 0 w - 0. 02 L h 88.3 3 0,00:79 m 0.0025 m 0.02 g me t-an:" o5~ i in 0:.n 4 0.0071 m 0.0033 m 0.02 g methanol 65~ 40 min l o 5 0,0071 m 0.0033 > 0o02 g rmethanol 65~ 13.5 h 6907

7.0 6.8 6.6 A 6.4 6.2 6.0.. I I I I 20 40 60 80 0% ethanol (v/v) Figure 6. Effect of solvent composition (ethanol-water) or (apparent) pK1 values for imidazole ( O), poly-4(5)-vinylimidazole [sample No. 2, (A)], and copoly [4(5)-vinylimidazole-styrene] [sample No. 5, (0)], ionic strength 0.05, temp. 26~C.

40 30 20 10 20 40 60 80 0/0 H20 in ethanol- H20 Figure 7. Turbidity of poly-4(5)-vinylimidazole [samples No. 1 ( a ) and 2 (A)] and copoly [4(5)-vinylimidazole-styrene] [sample No. 5 (0)] at pH 8.0 as a function of increasing water content in ethanol-water solvent systems, buffer (TRIS-HC1) concentration 0.1 M, ionic strength 0.1, temp. 260C.

40 30 20 1.0 20 40 60 80 0/o H20 in ethanol- H20 2 Figure 8. Turbidity of poly-4(5)-vinylimidazole [samples No. 1 (0) and 2 (A)] and copoly[4(5)-vinylimidazole-styrene] [sample No. 5(0)] at pH 9. A as a function of increasing water content in ethanol-water solvent systems, buffer (TRIS-HC1) concentration 0.1 M, ionic strength 0.1, temp. 26~C.

29 From Figures 7 and 8 it is noted that the copolymer (sample No. 5) precipitates more easily from solution than either samples No. 1 or 2 with increasing water content, and that sample No. 2 (having a higher intrinsic viscosity than sample No. 1) precipitates before sample No. o1 From these results it should be possible to obtain an optimal catalytic activity in the regions where the polymers are approaching their minimum solubility, i.e., when the tightly coiled polymers will have an even higher "effective local concentration" of catalytically active functions, IV. Catalytic Activities of 1,2,4-Triazole and Poly-3-vinyl-1,2,4triazole The preparation of poly-3-vinyl-1,2,4-triazole from the thermal decomposition of 3-f-acetoxyethyl-1,2,4-triazole was previously re(6) ported(6) An improved synthesis has been obtained based on the dehydration of 3-P-hydroxethyl-1,2,4-triazole over molecular sieves 5A. This procedure gives the monomer in 15-20% yield, nmr and infrared spectra consistent with the vinyl structure. 3-Vinyl-l1,2,4-triazole polymerizes on standing as well as by free radical and thermal initiation. Solvolytic rates of PNPA, NABA, NABS and ANTI catalyzed by 1,2,4-triazole and poly-3-vinyl-1,2,4-triazole in several solvent systems are under investigation. At the present time some data have been completed for reactions in 28.5% ethanol-water. The concentration dependence of the first-order observed rate constant for the solvolyses of PNPA, NABA and NABS,.each catalyzed

by 1,2,4-triazole and by poly-3-vinyl-1,2,4-triazole, is shown in Figure 9. This figure reveals that the reaction is an overall second-order process, i.e, first-order in both catalyst and substrate. In Figure 10 the pH-rate profiles for PNPA, NABA and NABS solvolyses are presented. It is seen that 1,2,4-triazole is a catalyst superior to poly-3-vinyl-1,2,4-triazole in the pH region investigated. Although this is not an unusual occurrence, the marked enhancement of the monomeric triazole catalyzed reaction with increasing pH strongly suggests the involvement of anionic triazole functions which could arise from a general base catalysis. Potentiometric titrations of 1,2, 4-triazole and poly-3-vinyl1,2,4-triazole in 28.5% ethanol-water gave pK2 values of 10.1 and 11.0, respectively. The pK1 value for 1,2,4-triazole was determined to be 2.3. For poly-3-vinyl-1,2,4-triazole, the apparent pK1 value appears to be lower than its monomeric analog. Therefore, at intermediate pH most of the triazole functions are in the neutral form. In this region both polymeric and monomeric catalysis appear to be equally inefficient, suggesting that the nucleophilic reaction of triazole on substrate does not contribute significantly to the rate of reactiono Indeed, the participation of anionic functions is clearly revealed in Figures li and 12 where the second-order catalytic rate constant kcat is plotted vs. a2 (the fraction of anionic triazole functions) for monomeric and polymeric catalyses, respectively, It is interesting to note that if these figures are compared, the reaction of (anionic) poly-3-vinyl-1,2,4-triazole is

25 rf) 20 o i155 I 2 3 4 5 conc. x 104 (M) Figure 9. Dependence of the solvolytic rates of PNPA (0,0), NABA (S, ), and NABS (A,&) on the concentrations of 1,2,4-triazole and poly-3-vinyl-1,2, 4-triazole, respectively; in 28.5% ethanol-water, pH 8.89, buffer concentration (TRIS-HC1) 0.02 M, ionic strength 0. 02, temp. 26~C.

60.50 E,- 40 300 020 20 O 6.5 7.0 7.5 8.0 8.5 9.0 pH Figure 10. pH-rate profiles for the solvolyses of PNPA (0,0), NABA (OE, ), and NABS (A,A) catalyzed by 1,2,4-triazole and poly-3-vinyl-1,2,4-triazole, respectively; 28.5% ethanol-water, ionic strength 0.02, buffer (TRIS-HC1) concentration 0. 02 M, temp. 26~C.

33?80 60 E 400 20 20 40 60 80 a2 x 10~ Figure 11. Dependence of the catalytic rate constants for the solvolyses of PNPA (0), NABA (C), and NABS (A) on the fraction of anionic 1,2,4-triazole functions, 28.5% ethanol-water, ionic strength 0.02, buffer (TRIS-HC1) concentration 0. 02 M, temp. 26~C.

34 E 8E8 ) 4 2 4 6 I I 10 2 x 103 Figure 12. Dependence of the catalytic rate constants for the solvolyses of PNPA (0), NABA (0), and NABS (A) on the fraction of anionic 1,2,4-triazole functions in poly-3-vinyl-1,2,4-triazole, 28. 5% ethanol-water, ionic strength 0.02, buffer (TRIS-HC1) concentration 0.02 M, temp. 26~C.

approximately as efficient as (anionic) l,2,4-triazole in catalyzing the solvolysis of PNPAo On the other hand, the reaction of the polymer with NABS is considerably less efficient than that of its monomeric analog. This may indicate that anionic sites exist on the polymer chain which repel the anionic substrate. These results indicate that the rate expression for the solvolysis of the substrates studied is composed of at least three terms, viz. Rate = kl [Tz][S] + k2 [Tzl[OHIf[S] + k3 [OH-I[S] The existence of bifunctional catalysis (e.g., k4 [Tz]2[S]) has not yet been determined, We are currently investigating the effect of N-methyltriazole on the solvolytic rates of the above montioned substrates, and possibly the deuterium isotope effects In this manner we hope to substantiate this unexpected general base catalysis of esters containing good leaving groups. V0 Preparation of 4(5)-[ 9-(p-hydroxyphenyl)nonyl] imidazole A copolymer of 4(5)-vinylimidazole and p-vinylphenol has been found to be a very efficient catalyst for the solvolyses of neutral, negatively and positively charged substrates.5) The strong enhancement of this copolymer, relative to the solvolytic rates o obtained with imidazole, poly-4(5)-vinylimidazole, phenol, poly-pvinylphenol, and a copolymer of 4(5)-vinylimidazole-p-methoxystyrene, was rationalized by the cooperative interaction between imidazole and

36 anionic phenol participating in a multifunctional attack on substrate. The synthesis of the model compound 4(5)-[9-p-hydroxyphenyl)nonyl]imidazole (VIII) was undertaken to determine if such cooperative interactions can occur in small molecules by employing a proper spacing between the catalytic functions. The following synthetic has been successfully employed. Ac2O 0 0 CHO0 — HOOC-(CH2 )8-COOH 2 0.o -(CH2)8- 0 AiCi~ II CH H2NNH2 -(CH2)8 COOH. H30- - (CH2)9 COOH III IV S012 CH3- -(CH)9-cC2 H g - (CH) V VI H2NH-NH2OAc CH —-H )9 i> N~c~cl ~-r~-Vi.C-NH20 A > CH30- -(CH2 )9 VII H HO- -(CH2 )9 VIII H Solvolytic rate measurements are currently in progress. VI. Experimental 3-Vinyl-l, 2, 4-triazole. Into an activated 4" column of molecular sieves (dried at 300~ for 30 min., 0.4 mm) at room temperature

was placed 0.1 to 0.2 g of 3-6-hydroxyethyl-l, 2,4-triazoleo Mixing was effected by shaking the stoppered column. The column' was then connected to a dry-ice condenser and then immersed into an oil bath pre-heated to 280-3000, After 9-10 mino, vacuum (0.4 mm) was applied and both monomer and water condensed on the cold finger. Water was subsequently removed by lyophilizationo Yield 15-20%, mop. 38-40~ Anal. Calcdo for C4H5N3 C, 50.52; H, 5029; N, 44o17o Found: C, 50o34; H, 5,34; N, 44o24, Poly (sebacic acid anhydride) (II) (1 mole) was prepared by the procedure of Hill(l2) 9-(p-Methoxyphenyl-9-keto ) nonanoic Acid IIIo To 1 mole of poly(sebacic acid anhydride) in a mixture of 250 ml dry nitrobenzene and 1200 ml. dry tetrachloroethane was added 266 g (2 mole) of aluminium chloride with vigorous stirring at 00, To this mixture was then added 108 g (1 mole) of anisole dropwise, keeping temperature below 50, The violet colored solution was stirred for a further 4 h and then left to stand at 0-5~ for 3 days. The violet complex was destroyed with 250 ml. 37% hydrochloric acid, from which a white solid precipitated. The solvent was removed by steam distillation and a brown solid resulted. The solid was washed several times with benzene (1 lo )and the benzene solution evaporated in vacuo. The solid obtained was dissolved in 1 N sodium hydroxide solution and acidified with hydrochloric acid. The solid obtained was twice ex

tracted with 500 ml of hot water to remove any unreacted sebacic acid. After decolorization with Norit-A. in 50% ethanol-water, the product was obtained in 51% yield (74-3 g), m.p. 97-99~, Analytical sample recrystallized from cyclohexane yielded colorless needles, m.p. 98-98.5~. Anal. Calcd. for C H2404: C, 69.84; H, 8.27; 0, 21.89. Found: C, 69.83; H, 8,34; 0, 22.04. l0-(p-Methoxyphenyl) nonanoic Acid (IV). To 10.0 g (0.034 mole) 9 —(p-methoxyphenyl-9-keto) nonanoic acid in 80 ml of diethylene glycol was added 5.0 g of sodium hydroxide pellets and 5 ml of 85% hydrazine hydrate. The mixture was refluxed for 1 h. The water formed during the reaction was distilled off slowly until the reaction temperature reached 200~. The mJixture was refluxed for 3 h at 200~ and then cooled and added to 500 ml water. The pH was adjusted to 6 with hydrochloric acid. A light-yellow solid was obtained (9o75 g, 94% yield) which was recrystallized from heptane. Solution was decolorized with Norit-A. and colorless crystals were obtained, mop. 6405-66.5~. Further recrystallization from heptane gave mop. 66.5-67.5~. Anal. Clacd. for C17H2603: C, 73.35; H, 9. 41; 0, 17.24. Found: C, 73.27; H, 9.42; 0, 17.09.

39 l0-(_P-Methoxyphenyl) nonanoyl Chloride (V). To 24.5 g mole) of 10-(p-methoxyphenyl) nonanoic acid was added 100 ml thionyl chloride~ The mixture was refluxed for 4 h and the excess thionyl chloride was distilled off at atmospheric pressure. The residual oil was then distilled at 160-170~/0,05 mm yielding 14.5 g (55%) of a colorless liquid. Analo Calcdo for C17H2502CCl C, 68.79; H, 8)49; C1, 11.95. Found: C, 68.96; H, 8~53; C1, 1184. a-Chloromethyl-(9-p-methoxyphenyl) nonyl Ketone (VI)o To 14.5 g (0.050 mole) l0-(p-methoxyphenyl) nonanoyl chloride was added excess diazomethane in ether (prepared from N-nitroso-N-methylurea) at 0~ under a nitrogen atmosphere and anhydrous conditionso The solution was stirred for 45 min and dry hydrogen chloride was added. The mixture was left at 0~ for an additional 45 mino The ether solution was then washed with 3-100 ml portions of ice-water and once with 100 ml Ool N sodium bicarbonate solution. The solution was dried over anhydrous sodium sulfate and the ether evaporated giving 13,2 g (87% yield) of colorless crystals, mopo 59-61~o Analytical sample recrystallized from dry heptane, mopo 60o5-61~o Anal. Calcdo for C18H2702CC 1 C, 69.55; H, 8.76; C1, 11o41. Foundo C, 69.39; H, 8.64; C1, 11.46. 4(5)-[9-(p-hydroxyphenyl) nonyl] imidazoleo A solution of 1.2 g

40 of crude 4(5)-[9-(p-methoxyphenyl) nonyl] imidazole dissolved in 12 ml of glacial acetic acid was saturated with 6 g of dry hydrogen bromide gas. The reaction mixture was stood at room temperature for 24 h, at 50~ for 2.5 h, and 70~ for 1 h. The acetic acid was distilled off and the residue suspended in anhydrous ether. Ammonia gas was bubbled through the solution until pH 9 was reached. The ether was then evaporated under reduced pressure yielding a lightyellow solid. The product was crystallized from 50% ethanol-water, mop. 104-106.5~. Further recrystallization from 60% ethanol-water gave colorless needles, m.p. 106-108.5~, Anal. Calcd. for C18H26N20: C, 75.48; H, 9.15; N, 9.78. Found: C, 75.34; H, 9.03; N, 9.80. 4(5)-[9-(p-Methoxyphenyl) nonyl] imidazole (VII), In 250 ml of dry diethylene glycol was added 14.0 g (0.045 mole) a-chloromethyl(9-p-methoxyphenyl) nonyl ketone and 1803 g (0.175 mole) of dry formamidine acetate salto The mixture was heated for 50 h at 135~ under a nitrogen atmosphere during which time the solution became brown. The diethylene flycol was removed in vacuo and the residual oil was added to a Dowex 50 Wx8 ion exchange resin (acidic form). The desired compound was released by treatment with 0.1 N ammonium hydroxide in 90% ethanol-water, The brown solution was evaporated yielding a brown oil which was distilled at 180~/ 0.025 mm. A light-yellow waxy solid was obtained (5 g, 37% yield), m.p. 58-73~o Analytical sample recrystallized from diethyl malonate, m.p. 74-76~O Anal. Calcd. for C19gH28N20: C, 75.95; H, 9.39; N, 9.33. Found: C, 75.86; H, 9.24; N, 9.31.

41 VII. Literature Cited (1) C. G. Overberger, T. St. Pierre, N. Vorchheimer, J. Lee and S. Yaroslavsky, J. Am. Chem. Soc., 87, 296 (1965). (2) C. G. OVerberger, T. St. Pierre and S. Yaroslavsky, ibid., 87, 4310 (1961). (3) C. G. Overberger, T. St. Pierre and C. Yaroslavsky and S. Yaroslavsky, ibid., 88, 1184 (1966). (4) C. G. Overberger, R. Sitaramaiah, T. St. Pierre and S. Yaroslavsky, ibid., 7, 3270 (1965). (5) C. G. Overberger, J. C. Salamone and S. Yaroslavsky, ibid., in press. (6) Contract DA 18-035-AMC-121(A), Final Report Covering the Period March 4, 1964-March 4, 1967. (7) H. Morawetz and J. A. Shafer, J. Phys. Chem., 67, 1293 (1963). (8) H. Morawetz and J. A. Shafer, Biopolymers, 1, 71 (1963). (9) H. Morawetz, J. Polymer Sci., 42, 125 (1960). (10) H. Morawetz, C. G. Overberger, J. C. Salamone and S. Yaroslavsky, ibid., B4, 609 (1966). (11) C. L. Arcus, T. L. Howard and D. S. South, Chem. Ind. (London), 1756 (1964). (12) W. Hill, J. Am. Chem. Soc., 54, 4105 (1932).

42 VIII. Distribution List No. of Recipient Copies Defense Documentation Center 20 Cameron Station Alexandria, Virginia 22314 Commanding Officer 20 Edgewood Arsenal Attn: Dr. Joseph Epstein Research Laboratories Edgewood Arsenal, Maryland 21010 Record Copy 1 Contract Project Officer 4 Director, Research Laboratories 1 Chief, Library Branch 5 Technical Information Division Technical Support Directorate Chief, Technical Releases Branch 8 Technical Information Division Technical Support Director (for DOD clearance purposes) Alternate Project Officer 1

UNCLASSIFIED Security Classification DOCUMENT CONTROL DATA - R&D (Security claaeftication of title, body of abstract and indexing annotation must be entered when the overall report ie clesaified) 1. ORIGINATING ACTIVITY (Corporate author) 2. REPORT SECURITY C LASSIFICATION The University of Michigan I UNCLASSIFIED Ann Arbor, Michigan 2b. GROJP NA 3. REPORT TITLE MECHANISM OF ENZYME ACTION 4. DESCRIPTIVE NOTES (Type of report and tncusetve dater) Semiannual Report - March-December 1967 5. AUTHOR(S) (Leat name. fitrt name, initial) Overberger, C. G., Maki, H., Cho, I., Morimoto, M., Yuen, P. S., Shen, C. M., and Salamone, J. C. 6. REPO RT DATE 7-. TOTAL NO. OF PAGES 7b. NO. OF REFS February 1968 56 12 aa. CONTRACT OR GRANT NO. a. ORIGINATOR'S REPORT NUM-BER(S) DAAA-15-67 -C - 0567 b. PROJECT NO. SEMIANR 1B014501B71A C. O 6. O THoER R E PORT NO(S) (A ny other number. that may be aafgneod thie report) cd. 08979-1-T 1 0. AVA IL ABILITY/LIMITATION NOTICES Each transmittal of this document outside the agencies of the U.S. Government must have prior approval of the CO, Edgewood Arsenal, ATTN: TSTI-T, Edgewood Arsenal, Maryland 21010. 11. SUPPL EMENTARY NOTES 12. SPONSORING MILITARY ACTIVITY Life sciences basic research in Edgewood Arsenal Research Laboratories, fLife sciences basiei research in Edgewood Arsenal, Maryland 21010 support of materiel | (J. Epstein, Proj. 0., Ext. 25114) 13.ABSTRACT The objectives of the contract are to synthesize polymeric catalysts with structures similar to vinylimidazole and to determine the catalytic activities of these compounds against various phosphate esters and selected neutral and charged carbon esters. Copolymers of 4(5)-vinylimidazole with acrylic acid were evaluated for their effects on the solvolytic rates of neutral, negatively and positively charged phenyl esters as a function of the copolymer composition and sequence distribution. A distribution of charged groups (carboxylate) and catalytically active functions (imidazole) was obtained which had optimum catalytic activity. Also, the effect of anionic poly(acrylic acid) on the above substrates was investigated. A strong inhibition of the solvolytic rate of the positively charged ester 3-acetoxy-N-trimethylanilinium iodide was found. The dissociation constant of this system (polyanion-substrate) was found to be of the same order of magnitude as that of acetylcholinesterase with its substrate. D 1D JAN 64 1473 UNCLASSIFIED Security Classification

UNCLASSIFIED Security Classification 14. K LINK A LINK B LINK C KEY WORDS _._____________________ _ ~ROLE WT ROLE WT ROLE WT Acrylic acid Poly(3-vinyl-1,2,4-triazole) Charged esters Polymeric imidazole catalysis 1,2,4-Triazole 4(5)-[9-(c -Hydroxyphenyl)nonyl- Catalytic activity imidazole Solvolytic rates Inhibitory effect Monomer sequence Solvent composition Enzymes Mechanism of action Copolymer composition 4(5)-Vinylimidazole Anionic poly(acrylic acid) INSTRUCTIONS 1. ORIGINATING ACTIVITY: Enter the name and address imposed by security classification, using standard statements of the contractor, subcontractor, grantee, Department of De- such as: fense activity or other organization (corporate author) issuing (1) "Qualified requesters may obtain copies of this the report. report from DDC. " 2a. REPORT SEICUUTY CLASSIFICATION: Enter the. overt (2) "Foreign announcement and dissemination of this all security classification of the report. Indicate whether "Restricted Data" is included. 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UNIVERS OF MICHIGAN 3 9015 03695 1625