Distribution Statement This document is subject to special export controls and each transmittal to foreign governments or foreign nationals may be made only with prior approval of the CO, Edgewood Aresenal, 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 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 M. Morimoto P. S. Yuen C. M. Shen R. R. Deupree J. C. Salamone March 1969 This document is subject to special export controls and each transmittal to foreign governments or foreign nationals may be made only with 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 Laboratories Edgewood Arsenal, Maryland 21010 Contract DAAA-15-67-C- 0567 Project iB0145OiB7iA THE UNIVERSITY O0 MICHIGAN ANN ARBOR, MICH'IGAN

Foreword The work described in this report was authorized under Project 1B014501B71A, Life Sciences Basic Research in support of Material (U). This work was started in July and completed in December 1968. Reproduction of this document in whole or in part is prohibited except with permission of CO, Edgewood Arsenal, ATTN: SMUEA-RPRE, Edgewood Arsenal, Maryland, 21010; however, Defense Documentation Center is authorized to reproduce the document for the U. S. Government purposes. The information in this document has not been cleared for release to the general public.

Digest The reactions of imidazole and benzimidazole containing polymers with rea.ctive esters have revealed several analogies to those of enzyme catalyzed processes. Although the reactions of synthetic macromolecules have been considerably less efficient than those of natural macromolecules, the synthetic polymers have revealed a higher reactivity than their monomeric analogs,(1-3) a specificety- in their solvolytic reactions,(2-4) a competitive inhibition by substances similar to the reactive substrate,(495) a bifuactional interaction between. two catalytic functions and a substrate,(367) n and a complexation (saturatio.n) by high and low molecular weight esters.(4'8) In a further investigation of the catalytic interactions between pendent neutral and ani.onic imr dazole groups, mathematical evidence is presented which indicates that this reaction is dependent upon both monomeric and dimeric neut"ral imidazole interactions, Similarly, for poly-4-vinylpyridine catalyzed reactions, it is possible that a similar neutral-neutral interaction occurs. An investigation of the solvolysis of a paraffinic, anionic ester catalyzed by poly-4(5)-vinyl.imidazole has revealed several unusual catalytic effects which appear to be solvent dependent. When the solvent system contains a high water contents very rapid solvolyteic rates are obtained. These solvolytic rates appear to approach those of enzymic reactions, Under appropriate conditions iLt is also possible to obtain saturation phenomena which illustrate the contributton of hydrophobic interactions in polymer-substrate complexation. Since polymeric catalysts have revealed many analogies to hydrolytic enzymes, we are attempting to prepare a, new type of polymeric catalyst which can undergo oxidatlion- reductions reactions. We are also continuing our investigation of salt effeects in the hydrolysis of tri(choline chloride) phosphate acd the solvoly-ti effects of dimeric imidazole-phenol model systems.

Table of Contents Page LIST OF ILLUSTRATIONS 5 RESULTS AND DISCUSSION 6 I. Discussion of Recent Visit to Edgewood Arsenal 6 II. Bifunctional Interactions in Polymeric Imidazole and Pyridine Systems 6 III. Hydrophobic Bonding in Macromolecule-Substrate Complexation 11 IV. The Effect of Ionic Strength on the Inhibition of a Phosphate Ester 15 V. Esterolytic Behavior of Dimeric Analogs 17 VI. Oxidation-Reduction Polymers 18 VII. Significance of this Research 19 VIII. Glossary 19 IX. Literature Cited 20 DISTRIBUTION LIST 21

List of Illustrations Table Page I. Effect of Ethanol-Water Solvent Composition in Time for Half Solvolyses of NDBA Catalyzed Poly-4(5)-Vinylimidazole and Imidazole 13 II. Effect of Ionic Strength on the Inhibition of TCCP 17 III. Second-Order Rate Constants for the Solvolysis of PNPA Catalyzed by Imidazole, Phenol, and Imidazole-Phenol Dimer 18 Figure 1. k /e1l - cl profiles for the solvolyses of DNPA catalyzed by poly-4(5)-vinylimidazole in 28.5% ethanol-water and 10% methanolwater. 9 2. k /ac - al profile for the solvolyses of DNPA catalyzed by polt-4(5)-vinylimidazole catalyzed solvolysis of NDBA as a function of catalyst and substrate concentration. 11 3. The observed initial rates of the poly-4(5)-vinylimidazole catalyzed solvolysis of NDBA as a function of catalyst and substrate concentration. 14 4. Modified Lineweaver-Burk plots for [PVIm]/v obd. vs. [PVIm] and [CLBA/vd. vs. [ELBA]. 16 s~~~~b s~~~~~

MECHANISM OF ENZYME ACTION Results and Discussion I. DISCUSSION OF RECENT VISIT TO EDGEWOOD ARSENAL During a recent visit to the Research Laboratories, Physical Research Laboratory of Edgewood Arsenal, C. G. Overberger and J. C. Salamone presented a review of the effects of imidazole containing polymers on the solvolytic rates of carbon and phosphorous esters. Initially, C. G. Overberger discussed the types of cooperative interactions which have been found between two pendent imidazole groups and a substrate. He also discussed the cooperative interactions between imidazole and hydroxyl, imidazole and phenol, as well as imidazole and carboxylate groups. Such cooperative effects are in part the cause of the enhanced catalytic abilities of the polymers in relation to their low molecular weight analogs. The catalytic ability of 1,2,4-triazole and poly-1,2,4-triazole were also discussed. It was mentioned that this system was quite different from the imidazole system in that the triazole reactions were principally dependent upon the anionic functions. J. C. Salamone then discussed the effects of hydrophobic bonding which were under investigation using poly-4(5)-vinylimidazole as the catalyst and neutral and anionic, paraffinic esters as substrates. The largest catalytic effect was noted for the system which utilized both hydrophobic and electrostatic forces to effect complexation between substrate and polymer. The effects of ionic strength on the solvolytic rate of tri(choline chloride) phosphate in the presence of anionic polyacrylic acid were also discussed. The results of this subject are presented in Section IV. II. BIFUNCTIONAL INTERACTIONS IN POLYMERIC IMIDAZOLE AND PYRIDINE SYSTEMS In the catalytic solvolysis of PNPA by poly-4(5)-vinylimidazole, two types of cooperative mechanisms were proposed for the enhanced catalytic effect in 28.5% ethanol-water solution at pH values above 7.5(3). Interactions of anionic and neutral, pendent imidazole groups with the substrate were considered to be the most likely mechanism for the solvolysis of PNPA. The alternative cooperative interaction of two neutral pendent imidazole groups in the same concerted manner was also considered, particularly for the polymer in 10% methanol-water solution at intermediate pH values.(9) The proposed anionicneutral interaction mechanism was supposedly substantiated by a study of the activation parameters for the solvolysis of PNPA.(9)

The enhanced catalytic effect of poly-4(5)-vinylimidazole in the solvolysis of PNPA was observed to begin at pH - 7.5. Calculations of ao (the fraction of cationic functions) and al (the fraction of neutral functions) showed that at pH 8, o =- 0.1 and cl = 0.9, while c2 was estimated to 10-8. Since this system is on the cationic side of the isoelectric point, it would appear to be very unlikely that an anionic-neutral interaction could participate in the catalytic process. The argument that poly-N-vinylimidazole and poly-2-methyl-N-vinylimidazole are poor catalysts for the solvolysis of PNPA(3) because these catalysts contained no anionic sites and could not have any anionic-neutral interaction, supposedly substantiated the proposed anionic neutral imidazole interaction in the poly-4(5)-vinylimidazole catalyzed process. However, a closer examination of the poly-N-vinylimidazole and poly-2-methyl-N-vinylimi dazole systems appears to support further either the interaction of two neutral, pendent imidazole groups or individual neutral, pendent imidazole groups in the catalytic solvolysis of PNPA by poly-4(5)-vinylimidazole in 28.5% ethanol-water solution. The investigati.on of PNPA solvolysis catalyzed by poly-N-vinylimidazole was performed in the pH range of 7-9.(3) The apparent pK1 of the pendent imidazole groups in this polymer was determined to be 4.4. Therefore, under the conditions studied, the polymer was practically neutral. The increase in the fraction of the neutral, pendent groups was very small from pH 7 (al = 0.93) to pH 9 (l, = 0.97). It is not surprising to find that the catalytic rates remained almost constant, since there was no significant increase in al. For the N-alkylated polymers, the solvolytic mechanism would seem to imply either an. individual pendent, neutral imidazole reaction or a bifunctional pendent, neutral-neutral imidazole reaction. The lack of an enhanced catalytic effect for poly-2-methyl-N-vinylimidazole in the solvolysis of PNPA cannot be used as a substantiation of the anionicneutral pendent imidazole group interaction proposed for poly-4(5)-vinylimidazole. In this case the low apparent PKa (4.8) of poly-2-methyl-N-vinylimidazole and the added steric factor of the 2-methyl substitution are also involved. Therefore, whether poly-2-methyl-N-vinylimidazole is a good catalyst or not is irrelevant to the catalytic properties of poly-4( 5)-vinylimidazole. The second order catalytic rate constants for the poly-4(5)-vinylimidazole catalyzed solvolysis of PNPA in 28.5% ethanol-water solution obtained above pH 8,4 should be considered with reservation, since it was found that the precipitation range of this polymer was in the vicinity of pH 8.5. A heterogeneous system would obviously further complicate the process. It was in this region that such anionic-neutral interactions were proposed. It was previously reported for the momomeric imidazole catalyzed solvolysis.:f PNPA that there were no interfunctional interactions between two imidazole functions and the substrate.(l0,ll) Studies of the activation parameters of poly-4( 5)-vinylimidazole and poly-N-vinylimidazole catalyzed solvolyses of PNPA indicated that multifunctional interactions appeared to be the pathway that

lead to the enhanced effects observed for poly-4(5)-vinylimidazole in 28.5% ethanol-water and 10% methanol-water solutions, while no effects were possible for poly-N-vinylimidazole. A neutral-neutral interaction was proposed for poly4(5)-vinylimidazole and PNPA in 10% methanol-water, while an anionic neutral interaction was considered to be most likely for the 28.5% ethanol-water solution system. From a consideration of the facts that poly-4(5)-vinylimidazole was investigated in the cationic side of the isoelectric point and that the poly-N-vinylimida.zole and poly-2-methyl-N-vinylimida.zole reactions do not necessarily support an anionic-neutral reaction in the polcy-4(5)-vinylimidazole system, it is possible that the poly-4(5)-vinylimidazole solvolyses of PNPA in 10% methanol-water and 28.5% ethanol-water both involve neutral-neutral interactions. If the neutral-neutral interaction (oi) is the principle factor responsible for the observed enhanced catalytic effect of poly-4(5)-vinylimidazole in the solvolysis of PNPA, and if the neutral, pendent imidazole group (el) also participates to a lesser extent, then we would express kcat explicitly by the following equations: kc = kllc + klol (1) cat then k cat = klll + kl (2) Using equation (2), a linear plot of kcat/li versus al should be obtained if the proposed neutral-neutral pendent group interaction is operative in 28.5% ethanol-water, as well as in 10% methanol-water. The data, obtained for the catalyzed solvolyses of PNPA by poly-4(5)-vinylimidazole in both 28.5% ethanolwater and 10% methanol-water solutions(3,9) were treated graphically according to equation (2). Indeed, as revealed in Figure 1, a, linear relationship was obtained for each solvent system with intercepts at al = 0.25 and values of kll of 30.50 1/mole-man and 102.70 l/mol-min for 28.5% ethanol-water and 10% methanol-water, respectively. These results strongly indicate that neutralneutral interactions are very probable for both of these systems. A close examination of the rate al profiles for the catalytic solvolyses of NABS by poly-4(5)-vinylimidazole(7) and by poly-4-vinylpyridine(2) reveals that maximal catalytic rates for these polymers are found to be in the vicinity of a1 = 0.75. The intercept on the al axis at 0.25 for the catalytic solvolyses of PNPA by poly-k4(5)-vinylimidazole seems to be related to this value. This intercept suggests that at ol = 0.25 the polymer will have no catalytic activity and that at al values below 0.25, the polymer will either have no catalytic activity or will instead function as an inhibitor. There is no reason, however,

50 40 _J 0 30 20 I 0 2.4.6.8 a1 Figure 1. kcat/ai - al profiles for the solvolyses of PNPA catalyzed by poly4(5)-vinylimidazole in 28.5% ethanol-water (0), and 10% methanol-water (s); ionic strength 0.02, 260C. why the partially protonated polymer should function as a, competitive inhibitor toward a neutral substrate, although such an effect may occur with an oppositely charged ester.(l 2) At a,= 0.25, poly-4(5)-vinylimidazole is ca. 75% protonated. Two neutral, pendent imidazole groups will, on an average, be separated by three protonated, pendent imidazole groups. The polymer chain would be extended because of internal electrostatic repulsion between the prQtonated functions, and the polymer would be more rigid than a polymer having a piotonation of less than 75%. These effects coupled together would render difficult the interactions of neutralneutral pendent imidazole groups. Consequently, catalytic activity would not be detectable below ac = 0.25 if the neutral-neutral interactions are solely responsible for the catalytic effect in these solvolytic processes..=._~~~~~~~

Therefore, we may state that if neutral-neutral interactions are important in a catalyzed solvolytic process, then a separation of two pendent, neutral groups by a certain number of inert pendent groups would be expected to make such neutral-neutral interactions impossible to occur. Indeed, such an effect has previously been noted in solvolytic reactions catalyzed by a copolymer of 4(5)-vinylimidazole and p-methoxystrene. In this case, the catalytic imidazole functions, when diluted by the inert p-methoxyphenyl groups, were very inefficient catalysts.(7) For the case of poly-4(5)-vinylimidazole, this would mean that the neutral-neutral interactions could not extend beyond the fourth unit; therefore, a, = 0.25 may be considered as the threshold value for such an interaction to occur. The above statement is related to the observed maximum rate at al = 0.75 for the catalytic solvolysis of NABS by poly-4(5)-vinylimidazole. At al - 0.75 the polymer is about 25% protonated, that is, every protonated, pendent imidazole group is separated by three neutral, pendent imidazole groups. The observed maximum rate at ac 0.75 would mean that in the case of a cationic-neutral interaction, the three neutral pendent groups contribute to the maximum interaction upon the negative substrate which is bound to a, protonated, pendent group by electrostatic force. Therefore, the observed distorted bell-shape rate-ol profiles for the solvolyses of negatively charged substrates by poly-4(5)vinylimidazole and by poly-4-vinylpyridine can be interpreted by following the above reasoning. That is, if we assume the fourth neutral pendent group interacts insignificantly with the negative substrate bound to the protonated pendent group by electrostatic force, then the rapid decrease of the catalytic rates at values above 0.75 would be a direct result of the decrease of the cationic sites. The gradual decrease of the catalytic rates at al values less than 0.75 is probably a consequence of the gradual decrease of the neighboring neutral pendent group contribution. In the case of the poly-4-vinylpyridine catalyzed solvolysis of the neutral substrate 2,4-dinitrophenyl acetate (DNPA), both the nonlinear rate-a, profile and the inefficiency of the polymer in comparison to its monomeric analog 4picoline were attributed to the decreased nucleophilicity of the pendent pyridine groups.(2) This nonlinear relationship, however, can also be considered by the cooperative interaction of two pendent, neutral pyridine groups. If we assume that neutral-neutral interactions are also operative in the poly-4vinylpyridine system, then according to equation (2), a linear relationship would be expected to occur between kcat/al and o,. Indeed, such a linear relaticnship was obtained, and an intercept in the vicinity of al = 0.2 was found (Figure 2). In this case, it is obvious that no anionic-neutral interaction is possible, since the pyridine moiety cannot exist in the anionic form. It is interesting to again note that for a polymeric catalyst and a neutral substrate, there seems to be no catalytic effect at al values below 0.2. Although the poly-4-vinylpyridine system appears to be dependent on neutral-neutral interactions, such as described for poly-4(5)-vinylimidazole, it is unusual that the polymeric catalyst is less efficient than its monomeric analog. This effect may, however, be caused by the high content of ethanol in the solvent system 10

z 8 I _J o 6'0 4 lo 4.2.4.6. 8 aI Figure 2. kcat/al - al profile for the solvolyses of DNPA catalyzed by poly4-vinylpyridine in 50% ethanol-water, 0.04M in KC1, 36.80C. (50% ethanol-water). Analogous results have been reported for the decreased catalytic behavior of poly-4(5)-vinylimidazole in solvents of high alcohol contents. (13) Therefore, we may generalize that for any polymer catalyzed solvolysis of an active ester, where neutral-neutral pendent group interactions are important to the solvolytic process, there should be a threshold al value below which the polymer can either have no effect on the reaction rate or can inhibit the solvolytic reaction. For any polymer reactions involving cationic-neutra~ or anionic-neutral pendent group interactions, there should be a maximum catalytic rate at a certain al value. It would be interesting to investigate the solvolysis of a. neutral substrate by a synthetic polymeric catalyst at al values below 0.2, an area which has not been reported. III. HYDROPHOBIC BONDING IN MACROMOLECULE-SUBSTRATE COMPLEXATION The complexation of catalytically active, synthetic macromolecules with low and high molecular weight reagents has been investigated by several workers.(4,8,14,15) These association reactions, which are characteristic of 11

enzyme-substrate complexation, presumably involved an accumulation of the substrate in the vicinity of the polymer chain by either electrostatic or hydrophobic forces. Although the kinetically determined binding constants of these reactions were similar to those of enzymic reactions, the synthetic macromolecule catalyzed processes have been characterized by relatively low reactivities. It has been recently found that partially protonated poly-l4(5)-vinylimidazole was able to complex with the negatively charged ester sodium 3-nitro-4acetoxybenzenesulfonate (NABS) under conditions in which the substrate concentration was in excess of the polymeric catalyst concentration.(8) This saturation effect was not obtained when the neutral ester p-nitrophenyl acetate (PNPA) was employed. These results suggested that the saturation of poly-4(5)vinylimidazole with NABS was facilitated by electrostatic forces, whereas the lack of saturation with PNPA indicated that hydrophobic forces were insufficient to accumulate the neutral ester in the vicinity of the polymer chain. Reactions of poly-4(5)-vinylimidazole with neutral and charged substrates in alcohol-water solvent systems have indicated that cooperative, multifunctional interactions among pendent imidazole functions and a substrate were possible.(13) Such cooperative interactions appear to be in part responsible for the enhanced catalytic ability of the polymer relative to its monomeric analog, imidazole. In order to further improve the rate of the cooperative imidazole attack on a substrate, it was felt that a very rapid poly-4(5)-vinylimidazole catalyzed reaction could be attained if both hydrophobic and electrostatic forces contributed to the complexation of a substrate with the macromolecule. The substrate chosen for this investigation was 3-nitro-4-dodecanoyloxybenzoic acid (NDBA), a long araffinic chain analog of the anionic ester 3-nitro-4-acetoxybenzoic acid. 3) The solvolysis of NDBA by poly-4(5)-vinylimidazole in ethanol-water solvent systems was expected to involve predominantly hydrophobic interactions in solvents containing a high water content (higher polarity) and electrostatic interactions in solvents containing a high ethanol content (lower polarity). In alcohol-water solvent systems, we have previously observed that a high alcohol content apparently increased the electrostatic interaction between poly4(5)-vinylimidazole and an anionic ester (NABS) and decreased the apolar interaction with a neutral ester (PNPA).(7) These results are in contrast to those of similar reactions conducted at a lower alcohol concentration where the apolar interaction increases relative to the electrostatic interaction. In Table I are presented the times for half solvolyses of NDBA catalyzed by imidazole and by poly-4(5)-vinylimidazole for several ethanol-water solvent systems. In each case, the catalyst concentrations were greater than the substrate concentration such that a first order disappearance of NDBA could be expected.(3) The imidazole catalyzed reactions were found to be second-order processes in all solvent systems, and it is noted that the half-lives for NDBA solvolyses increased considerably with increased ethanol content. The poly4(5)-vinylimidazole catalyzed reaction was, however, more complex. At ethanol contents of 60, 80, and 90% the polymeric reaction was a second-order process and the polymer was more efficient than its monomeric analog by a factor of 3.5 12

at 60%, of 4.8 at 80%, and of 13 at 90%. This increase in polymeric reactivity could in part be caused by an increase in electrostatic forces as well as by a possible contribution of hydrophobic forces. Since poly-4(5)-vinylimidazole is insoluble in water or ethanol, it is conceivable that hydrophobic interactions could increase as the limit of solubility of the polymer is approached. TABLE I* EFFECT OF ETHANOL-WATER SOLVENT COMPOSITION ON TIME FOR HALF SOLVOLYSES OF NDBA CATALYZED BY POLY-4( 5)-VINYLIMIDAZOLE AND IMIDAZOLE Ethanol PVIm Imidazole (, volume) (min) ( min) 15 < 0.5 146 20 < 0.5 197 30 < 0.5 352 40 15 635 60 454 1,610 80 861 4,146 90 826 11,093 *pH 7.9, ionic strength 0.02; half solvolyses times were determined for catalyst concentrations of 1.90 x 104m and substrate concentration of 1.78 x 10-4m.(16) From Table I it is also noted that the reactivity of poly-4(5)-vinylimidazole relative to imidazole, increases dramatically at ethanol contents of 15% to 40%. In fact, at low ethanol contents these reactions were too fast to measure by our standard procedure.(3) These extremely rapid catalyses, which are currently under investigation by stopped-flow spectroscopy, are indicative of a strong hydrophobic reaction between the paraffinic substrate and the vinyl polymer. In the region of the 40% ethanol-water the polymer reactions were found to deviate from yielding a first-order solvolysis of NDBA. In Figure 3A the initial rates (vobsd) 8) for the solvolysis of a fixed concentration of NDBA as a function of increasing concentration of neutral polymer (PVIm) appear to be approaching a limiting value. Such an occurrence is indicative of macromoleculesubstrate complexation, although this is unexpected owing to the higher concentration of catalyst to substrate that was employed.(4) When the concentration of neutral poly-4(5)-vinylimidazole was fixed below a varying concentration of NDBA (Figure 3B), it was again found that the initial solvolysis rates were approaching a limiting value. 13

24 A 20 16 12 B r0 0 0 1 1 1 1 1I 0 2 4 6 8 10 [ PVIm] or [NDBA] x 104 (M) Figure 3. The observed initial rates of the poly-4(5)-vinylimidazole catalyzed solvolysis of NDBA as a function of catalyst and substrate concentration.

The latter kinetic data are characteristic of the Michaelis-Menten mechanism,(817) in which the catalyst (an enzyme) reacts with the substrate to form a catalyst-substrate complex, and this complex is decomposed giving back the free catalyst and the products of the reaction. If it is assumed that systems A and B of Figure 3 can be described by the Michaelis-Menten mechanism for complexation, and that there are no interactions between bound materials, which could affect this complexation, it is possible to determine the Michaelis constants (Km) for these reactions; by utilizing a modified Lineweaver-Burk plot,(8) [PVIm]/vobsd can be plotted versus [PVIm] when poly-4(5)-vinylimidazole concentration is in excess (Figure 4A) and [NDBA]/vobsd can be plotted versus [NDBA] when substrate concentration is in excess (Figure 4B). From a least squares treatment of these data, values of Km were determined to be (4.77 +0.57) x 10-4M for [P4VIm] > [NDBA] (A) and (4.53 +o.64) x 10-4M for [NDBA] > [P4VIm] (B). The first order rate constants for the decomposition of the macromoleculesubstrate complex were calculated(3) to be 0.032 +0.004 min-1 for A and 0.088 +0.013 min-1 for B. The novel similarity of the Km values would appear to suggest that the mechanisms of complexation for both systems are similar, regardless of which material is in excess concentration. Although the determined values of Km are the same order of magnitude as those of certain enzyme-substrate reactions, the efficiencies of these polymeric reactions in 43.7% ethanol-water leave much to be desired. We hope to report in the near future the results of the rapid solvolyses of NDBA at low ethanol contents, as well as the effects of temperature, pH and ionic strength on the solvolyses of a variety of neutral and anionic paraffinic substrates. IV. THE EFFECT OF IONIC STRENGTH ON THE INHIBITTON OF A PHOSPHATE ESTER In the last summary progress report(15) and at our recent visit to Edgewood Arsenal we discussed briefly the effect of ionic strength on the hydrolysis of tri(choline chloride) phosphate (TCCP) in the presence and absence of anionic polyacrylic acid. We have now completed our investigation of this system. The kinetics of the reaction + - + + [(CH3)3N CH2CH2013PO + OH + (CH3)3N CH2CH20H + [(CH3)3N CH2CH20]2PO2 DCCP were followed by means of a pH-stat at pH 9.0 in the presence and absence of polyacrylic acid, and at varying ionic strength. The influence of ionic strength (4) on the rate of the uncatalyzed reactions is as predicted by Laidler(l9) for reactions which involve charged species; i.e., the logarithm of the initial rate is linearly related to the square root of the ionic strength. This behavior arises from the change in the activity coefficient with a change in p as predicted by the Debye-Htckle theory —the equation is 15

600 B,1 300 E-, 200 /:100 l A.. -6 -4 -2 0 2 4 6 8 10 E PVIml or [ NDBAI x 104(M) Figure 4. Modified Lineweaver-Burk plots for [PVIm]/vobsd. vs. [PVIm] and [NDBA]/Vobsd. vs. [NDBA].

log K = log K~ + 1.02 Z ZB p where K is the rate at any l, K' is the rate at zero l, and ZA and ZB are the charges of A and B, respectively. For the blank reaction, 1.02 ZAZB is found to be -0.75. Since hydroxide ion has a charge of -1, the charge of phosphate center under attack would then have a charge of +0.75. This appears to be an unusual method for ascertaining the charge of phosphorous under conditions in which the ester is hydrolyzed. In the presence of polyacrylic acid the reaction was strongly inhibited, and this effect decreased with increasing ionic strength (Table II). The inhibition factor is defined by K~/K. TABLE II* EFFECT OF IONIC STRENGTH ON THE INHIBITION OF TCCP (M.min-') [KC1] 6 + log K 6 + log K K~/K 0.00 0.011 1.685 o.603 12.10 0.02 0.031 1.580 0.796 6.10 0.o4 0.051 1.515 o.940 3.76 o. o6 0.071 1.460 1.032 2.68 0.10 0.111 1.370 1.130 1.74 0.15 0.161 1.290 1.130 1.45 0.20 0.211 1.210 1.080 1.35 *pH 9.0, 10% ethanol-water; [polyacrylic acid] = 0.01 M, [TCCP] = 0.001 M. From Table II it can be seen that as the ionic strength is increased, the inhibition factor is markedly decreased. These data suggest that the increased salt content shields the triply charged TCCP from the anionic carboxylate of the polymer chain. V. ESTEROLYTIC BEHAVIOR OF DIMERIC ANALOGS In our last summary progress report(l8) the preparation of the dimeric imidazole-phenol dimer, 4(5)-(2-hydroxyphenyl)imidazole (I), was presented. In Table III are listed the second order catalytic rate constants for the solvolysis of p-nitrophenyl acetate (PNPA) catalyzed by imidazole, phenol, and 4(5)-(2-hydroxyphenyl)imidazole. The pK1 for the imidazole group of I was determined to be 6.o, while the pKa of the phenol group of I was determined to 17

OH (I) TABLE III* SECOND-ORDER RATE CONSTANTS FOR THE SOLVOLYSIS OF PNPA CATALYZED BY IMIDAZOLE, PHENOL, AND IMIDAZOLE-PHENYL DIMER pH Imidazole Phenol Dimer 7.32 8.4 - 2.2 8.13 11.5 - 2.3 9.22 11.8 - 3.3 9.81 10.1 - 13.6 10-34 11.3 7.25 32.7 *In 30% n-propanol-water, y = 0.02, 26~. be 11.0. Since the pKa of monomeric phenol is 10.4, it is conceivable that the enhanced action of I may be caused by a cooperative interaction of imidazole and phenoxide groups. A situation, such as this, has been reported for a copolymer of 4(5)-vinylimidazole and p-vinylphenol.(7) Further studies with I are in progress. VI. OXIDATION-REDUCTION POLYMERS Since polymeric catalysts have revealed several analogies to hydrolytic enzymes, we are attempting to prepare a synthetic, polymeric system which may reveal similarities to the redox enzymes. A system, which is amenable to an oxidation-reduction reaction and one which is of importance as a preparative organic procedure, is the oxidation of halides or tosylates to carbonyl compounds. Using the concept of bifunctional interactions between two pendent groups on a polymer and substrate, it is possible that copolymers of 4-vinylpyridine and 4-vinylpyridine-nN-oxide (II) or 4-vinylpyridine and vinylmethyl sulfoxide (III) would be very efficient oxidative agents. For each of these copolymers the halide or tosylate could be attacked quickly by the oxide. The tetrahedral intermediate thus formed could 18

then be decomposed by a nucleophilic attack from a neighboring neutral pyridine group, thereby giving the desired aldehyde or ketone. H2 H2 H2 CH2 \ CH CH CH Io CH3 6 (II) (III) These systems represent a type of synthetic polymer not yet investigated, since the oxidation-reduction process is dependent upon a cooperative attack between two functional groups and the substrate. The preparations of these copolymers are currently in progress. VII. SIGNIFICANCE OF THIS RESEARCH The research described in this report has indicated how polymeric reactions have revealed many analogies to the catalytic reactions of enzymes. Although these synthetic, polymeric catalysts have appeared to be considerably less efficient than enzymes in their hydrolytic reactions, recent evidence indicates that such rapid reactions are also possible using the principle of hydrophobic and electrostatic interactions between a substrate and a synthetic macromolecule. It is hoped that the reactions described above will lead to a more comprehensive understanding of the behaviors of naturally occurring macromolecules, as well as elucidating the behaviors of synthetic macromolecules with low molecular weight materials. VIII. GLOSSARY PNPA - p-nitrophenyl acetate a - fraction of neutral functions ao fraction of cationic functions a - fraction of anionic function DNPA - 2,4-dinitrophenyl acetate keat - second-order catalytic rate constant NABS - sodium 3-nitro-4-acetoxybenzenesulfonate PVIm - poly-4(5)-vinylimidazole NDBA - 3-nitro-4-dodecanoyloxybenzoic acid TCCP - tri(choline chloride) phosphate 19

IX. LITERATURE CITED 1. For a review see H. Morawetz, "Macromolecules in Solution, " (High Polymers, Vol. XXI), Interscience Publishers, New York, 1965, Chapter IX. 2. R. L. Letsinger and T. J. Savereide, J. Amer. Chem. Soc., 84, 3122 (1962). 3. C. G. Overberger, T. St. Pierre, N. Vorchheimer, J. Lee, and S. Yaroslavsky, ibid., 87, 296 (1965). 4. R. L. Letsinger and I. Klaus, ibid., 87, 3380 (1965). 5. C. G. Overberger, unpublished results. 6. C. G. Overberger, T. St. Pierre, and S. Yaroslavsky, J. Amer. Chem. Soc., 87, 4310 (1965). 7. C. G. Overberger, J C. Salamone, and S. Yaroslavsky, ibid., 89, 6231 (1968). 8. C. G. Overberger, R. Corett, J. C. Salamone, and S. Yaroslavsky, Macromolecules, 1, 331 (1968). 9. C. G. Overberger, T. St. Pierre, C. Yaroslavsky, and S. Yaroslavsky, J. Amer. Chem. Soc., ibid., 88, 1184 (1966). 10. T. C. Bruice and G. L. Schmir, ibid., 80, 148 (1958). 11. J. F. Krisch and W. P. Jencks, ibid., 86, 833 (1964). 12. H. Morawetz and J. A. Shafer, Biopolymers, 1, 77 (1963); j. Phys. Chem., 67, 1293 (1963). 13. C. G. Overberger, in preparation for Accounts Chem. Res. 14. Yu E. Kirsh, V. A. Kabanov, and V. A. Kargin, Dokl. Akad. Nauk (Eng. Trans.), 177, 976 (1967); Vysokomol. Soedin., A10, 349 (1968). 15. C. Aso, T. Kunitake, and F. Shimada, J. Polymer Sci., B6, 467 (1968). 16. This concentration is below the critical micelle concentration. 17. L. Michaelis and M. Menten, Biochem. Z., 49, 333 (1913). 18. Mechanism of Enzyme Action, Semiannual Report, covering the period January 1968 - June 1968. 19. K. Laidler, "Chemical Kinetics," 2nd Ed., McGraw-Hill, New York, 1965, p. 219. 20

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, Technical Information Division Technical Support Directorate - 5 Chief, Technical Releases Branch, Technical Information Division Technical Support Directorate (for DOD clearance purposes) - 8 Alternate Project Officer - 1 21

UNCLASSIFIED becunLY %-sa SIL |UtlV I DOCUMENT CONTROL DATA- R & D (Security classification of title, body of abstract and indexing annotation must be entered when the overall report Is classified) 1. ORIGINATING ACTIVITY (Corporate author) 12e. REPORT SECURITY CLASSIFICATION The University of Michigan UNCLASSIFIED Ann Arbor, Michigan 48104 2b. GROUP NA 3. REPORT TITLE Mechanism of Enzyme Action 4. DESCRIPTIVE NOTES (Type of report and inclusive dates) Semiannual Report - June-December 1968 5. AU THOR(S) (First name, middle initial, la t name) Overberger, C. G., Morimoto, M., Yuen, P. S., Shen, C. M., Deupree, R. R., and Salamone, J. C. 6. REPORT DATE 7a. TOTAL NO. OF PAGES 7b. NO. OF REFS March 1969 20 19 Sa. CONTRACT OR GRANT NO. 9a. ORIGINATOR'S REPORT NUMBER(S) DAAA-15-67-C- 0567 b. PROJECT NO. Seminar lBO14 5 OlB71A C. 9b. OTHER REPORT NO(S) (Any other numbers that may be assigned this report) d. 10. DISTRIBUTION STATEMENT This document is subject to special export controls and each transmittal to foreign governments or foreign nationals may be made only with prior approval of the CO, Edgewood Arsenal. ATTN: SMUEA-TSTI-T, Ed;ewood Arsenal, Maryland 21010. 11. SUPPLEMENTARY NOTES /12. SPONSORING MILITARY ACTIVITY Edgewood Arsenal Research Laboratories Life sciences basic research in support Edgewood Arsenal, Maryland 21010 of material j (J. Epstein, Proj. 0. Ext. 25114) 13. ABSTRACT In this report are presented the effects of cooperative interactions between two pendent imidazole functions on a polymer chain and a substrate. Such cooperative interaction lead to enhanced catalytic efficiencies of polymeric catalysts. The effect of hydrophobic and electrostatic binding in synthetic, polymeric catalysis is discussed. Reactions of this type appear to be as efficient as an enzymic process. Inhibition of the hydrolysis rate of the triply charged phosphate ester tri(choline chloride) phosphate in the presence of anionic polyacrylic acid is discussed. A new oxidation reduction system employing macromolecules and low molecular weight materials is presented DD PO,,1473 iO REPLACES DO FORM l47a. 1 JAN @,. WHICH IS wu "vy I > OSO~sLKTKO~S roYius. UNCLASSIFIED Security Classification

UNCLASSIFIED Security Classification 14. LINK A LINK S LINK C KEY WORDS ROLE WT ROLE WT ROLE WT Cooperative interaction Bifunctional interaction Imidazole Poly-4(5)-vinylimidazole Hydrophobic bonding Electrostatic bonding NDBA. Charged substrated Inhibition Hydrophobic interactions Enzyme- substrate complexation Saturation Phosphate ester Tri(choline chloride) phosphate Anionic polyacrylic acid Dimeric analogs UNCLASSIFIED Security Classification

UNIVERSITY OF MICHIGAN 111 11111111 1 I032I21 111III 746 3 9015 03022 7469