Distribution Statement This document is subject to special export cbntrols 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. 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 Terminal Report by C. G. Overberger R. R. Deupree R. C. Glowaky H. Maki M. Morimoto T. J. Pacansky C. J. Podsiadly J. C. Salamone C. M. Shen P. S. Yuen June 1967-June 1, 1970 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 DAAA15-67-C-0567 Proje ct lB061l102B71A The University of Michigan Ann Arbor, Michigan 48104

Foreword The work described in this report was authorized under Project lB0O61102B71A, Life Sciences Basic Research in Support of Materiel (U). This work was started in June 1967 and completed in May, 1970. Reproduction of this document in whole or in part is prohibited except with permission of CO, Edgewood Arsenal, Attn: SMUA-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.

TABLE OF CONTENTS Page I. Publications List 4 II. Objectives 8 III. Introduction g IV. Discussion 10 V. Future Research 14 VI. Glossary 16 VII. Literature Cited 17 Distribution List 19

MECHANISM OF ENZYME ACTION I. Publications List (1) Synthesis of 5(6)-Vinylbenzimidazole and of 2-Vinylbenzimidazole. C. G. Overberger, B. Kosters, and To. St. Pierre, J. Polymer Sci., Part A-i, Vol. 5, 1987-92 (1967)o The syntheses of 2- and 5(6)-vinylbenzimidazole are described. These compounds were characterized by appropriate chemical reactions and physical properties. Incidental to this work, 3,4-diaminostyrene was also prepared. (2) Cooperative Effects in the Esterolytic Action of Synthetic Macromolecules Containing Imidazole and Hydroxyl Functions. C. G. Overberger, J. C. Salamone, and S. Yaroslavsky, J. Amer. Chem. Soc., 89, 6231-6236 (1967). The imidazole group of histidine and the hydroxyl group of serine are the purported participants in the active site of a-chymotrypsin. Copolymers of 4(5)-vinylimidazole with vinyl alcohol and with p-vinylphenol were prepared in order to investigate whether interactions between two different functional groups within a vinyl polymer chain would occur in esterolytic catalyses. The substrates investigated were neutral and negatively and positively charged phenyl esters. Rates of solvolyses were determined in either 28. DO ethanolwater or in 80% methanol-water. The copolymer of 4(5)-vinylimidazole and pvinylphenol was found a highly superior catalyst at high pH values toward each substrate investigated than any of its monomeric or polymeric analogs. These rate enhancements are not associated with the apparent dissociation constants of the imidazole or phenol groups, but can be attributed to bifunctional catalysis by imidazole and anionic phenol. One probable mechanism is the nucleophilic attack of imidazole on the substrate, followed by reaction of anionic phenol acting as a general base on the tetrahedral intermediate. Considerable rate enhancements were also observed for reactions catalyzed by copolymers of 4(5)-vinylimidazole and vinyl alcohol. (3) Selective Catalytic Effects of Strongly Ionizing Polycations on Ester Solvolysis. C. Go Overberger, H. Morawetz, J. C. Salamone, and S. Yaroslavsky, Jo Amer. Chem. Soc., 90, 651 (1968). The cationic polyion, poly(l-vinyl-3-methylimidazolium iodide) (PVMI), was found to enhance the solvolytic rates of the negatively charged esters

4-acetoxy-3-nitrobenzoic acid and sodium 4-acetoxy-3-nitrobenzenesulfonate in 28. 0o ethanol-water solutions. These rates were unaffected by the monomeric analog of the polyion, l,3-dimethylimidazolium iodide. At high pH values, the enhanced solvolytic rates of the negatively charged esters may be accounted for by the high local concentration of hydroxyl counterions in the vicinity of the polymer chain. At intermediate pH values, the large catalytic effect is attributed to an enhanced susceptibility of the anionic ester to direct water attack in the vicinity of the polycation. The solvolyses of uncharged esters (p-nitrophenyl acetate and p-nitrophenyl hexanoate) were not accelerated- indicating that hydrophobic forces are insufficient to concentrate the esters in the neighborhood of the-polyion. A copolymer of 1-vinyl-3-methylimidazolium iodide (VMI) containing 86 mol % vinyl alcohol residues catalyzed the hydrolysis of anionic esters with an efficiency similar to that of the PVMI homopolymer, if the two were compared at equal stoichiometric concentrations of imidazolium groups. On the other hand, a copolymer of VMI containing 63 mol % p-vinylphenol residues was about twice as effective as PVMI. This is due to the fact that the polycation draws the anionic es-ter into a region with a high local concentration of the phenoxide nucleophile. (4) Macromolecule-Substrate Complexation. A Saturation Phenomenon Exhibited by Poly(4(5)-vinylimidazole) and an Anionic Ester. C. G. Overberger, R. Corett, J. C. Salamone, and S. Yaroslavsky, Macromolecules, Vol. 1, 331 (1968). The x~ates of solvolyses of the neutral ester p-nitrophenyl acetate (PNPA) and the negatively charged ester sodium 4-acetoxy3-nitrobenzenesulfonate (NABS), each catalyzed by poly (4(5)-vinylimidazole), were investigated under conditions in which the substrate concentration was in excess of the catalyst concentration. In contrast to the solvolytic behavior of the neutral ester PNPA, the reaction of the negatively charged ester NABS gave rise to a kinetic scheme similar to that of hydrolytic enzymes in that the initial solvolysis rate reached a limiting value at high substrate concentration. This would appear to indicate the saturation of the polymeric catalyst with anionic substrate. The lack of a saturation effect exhibited by PNPA in the concentration range and pH value'investigated can perhaps be accounted for by the inadequacy of hydrophobic forces to sufficiently concentrate the neutral ester in the vicinity of the polymer chain, whereas at intermediate pH the anionic substrate would be strongly attracted to the protonated imidazole sites on the polymer by electrostatic forces. Employing the basic Michaelis-Menten kinetic system, values of Km and k2 for the solvolysis of sodium 4-acetoxy-3-nitrobenzenesulfonate were found to be (3.8+0.2) x 10-4 M and 0.63+0.04 min-1, respectively. (5) Optically Active Imidazole-Containing Polymers. C. G. Overberger and Iwhan Cho, J. of Polymer Sci., Part A-I, 6, 2741 (1968).

In order to investigate the stereospecificity of enzyme-catalyzed reactions, an optically active copolymer of 4(5)-vinylimidazole and 2,5(S)-dimethyl.-lhepten-3-one was synthesized, and its effects on the solvolytic rates, in ethanolwater, of the p-nitrophenyl and 4-carboxy-2-nitrophenyl esters of 3(R)- and 3(S)-methylpentanoic acid and of the commercially available N-carbobenzoxy-(R)and (S)-phenylal.anine p-nitrophenyl esters were investigated. The optically active comonomer was prepared by thermal decomposition of solid (+)-l-piperidino2,5(S)-dimethylheptan-3-one hydrochloride, which was obtained from the reaction of 2(S) -methylbutyllithium with 3-piperidino-2-methylpropionitrile. The 3(R)methylpentanoic acid was prepared in 92% optical purity from L-alloisoleucine via diazotization in concentrated hydrobromic acid and subsequent reductive debromination with zinc amalgam in dilute hydrochloric acid, In the optically active copolymer-catalyzed solvolyses of the optically active esters performed at pH values of 6-8 no significant differences between the solvolytic rates of (R) and (S) isomers of substrates were observedo Poly-L-histidine was also employed as a, catalyst for the solvolyses of these substrateso At pH 6.0 in ethanol-water the latter catalyst also failed to exhibit specificity toward (R) and (S) substrates. (6) Esterolytic Catalyses by Triazoles. C. G. Overberger and PO S. Yuen, Jo Amer. Chemo Soc., in press. The catalytic effects of 1,2,4-triazole, 1,2,3-triazole and several of their respective derivatives in the solvolyses of neutral, negatively and positively charged phenyl esters were investigated. Similar studies were performed with poly- 3-vinyl-1,2, 4-triazole. For all. systems it was observed that their catalytic effect exhibited a linear dependence on the concentration of their respective anionic forms. A comparison of catalytic behavior of the triazole and imidazole systems was made. The difference of their respective catalytic behavior was shown to arise from their PKa values rather than the nature of the catalyzing species. An empirical relationship between catalytic ability and pKa value was obtained for these system, and can be expressed by the equation log ki npKi + log C, where n and log C were determined to be 0.612 and -2097, respectively. This equation could possibly be used to predict the catalytic effect of a closely related five-membered nitrogen heterocycleo (7) Cooperative Effects Involved in Esterolytic Reactions Catalyzed by Imidazole-Carboxylic Acid Copolymerso Co Go Overberger and H. Maki, Macromolecules, 3, 214 (1970)o In order to elucidate the occurrence of cooperative interactions between pendent imidazole and carboxylate groups in 4(5) -vinyl.imidazole-acryl.i.c acid copolymer-catalyzed esterolytic reactions, copolymers of 4(5)-vinylimidazole and vinylsulfonic acid were prepared and their catalytfic activiteies were investigated. The latter copolymers, rich in vinylsulfonic acid, had no catalytic activity, indicating the occurrence of cooperative interactions between pendent imidazole and carboxylate groups in the former copolymerso

(8) Esterolytic Catalyses by Copolymers Containing Imidazole and Carboxyl Functions. C. G. Overberger and H. Maki, Macromolecules, 3, 220 (1970). I The electrostatic interactions involved in the imidazole-carboxylic acid copolymer-catalyzed solvolyses of 3-acetoxy-N-trimethylanilinium iodide (ANTI), p-nitropohenyl acetate (PNPA) and 3-nitro-4-acetoxybenzoic acid (NABA) were studied and compared with the monomeric analog 7-4(5)-imidazolebutyric acid. The effects of copolymer composition of the 4(5)-vinylimidazole-acrylic acid copolymers on their catalytic activities were investigated in detail. These effects became apparent by inspecting the dependencies of their activities on the monomer sequence distributions, which were found to control the overall catalytic activities of the copolymers for the charged esters. The most catalytically active species toward ANTI is the carboxylate-imidazole-carboxylate triad. (9) Synthesis of (R)-3-Methylpentanoic Acid. C. G. Overberger and Iwhan Cho, J. Org. Chem., 33, 3321 (1968). (10) The Effect of Ethanol-Water Solvent Composition on Poly-4(5)Vinylimidazole Catalyzed Esterolytic Reactions. C. G. Overberger, M. Morimoto, I. Cho and J. C. Salamone, Macromolecules, 2, 553 (1969). (11) Esterolytic Action of Synthetic Macromolecules. C. G. Overberger and J. C. Salamone, Accounts of Chem. Res., 2, 217 (1969). (12) Esterolytic Activities of Copolymers Containing Imidazole Groups. C. G. Overberger, Jo Co Salamone, I. Cho, and H. Maki, Annals of the New York Academy of Sciences, Vol. 155, Article 2, pp. 431-446 (1969)0 (13) Specific Catalytic Action of Large Molecules Containing Imidazole Groups. C. G. Overberger, J. C. Salamone, and S. Yaroslavksy, Pure and Applied Chem., Vol. 15, 453-464 (1967). (14) Cooperative Effects in the Esterolytic Action of Synthetic Polymers Containing Pendent Imidazole Groups. C. G. Overberger, H. Maki, and J. C. Salamone, Svensk kemisk tidskrift, 80:5 (1968).

IIo Objectives The objectives of this work have been to synthesize new catalytically active, synthetic macromolecules and to study their activities toward carbon and phosphate esters. In these investigations it was hoped that a careful study of the reaction kinetics would allow certain analogies to enzyme behavior to be drawn. Further, it was believed the field of macromolecular reactions was in itself interesting and quite different from those reactions of low molecular weight materialso

III. Introduction During the past decade there has been a considerable advance in understanding the reactivities of catalytically active, synthetic macromolecules with low molecular weight substrates.(l) This area of macromolecular research has been of considerable importance because of possible analogies with enzyme catalyzed processes. Although the synthetic macromolecular catalysts lack the tertiary structure, and often the secondary structure, of naturally occurring proteins, it has been possible to achieve certain behavioral patterns of enzymes. For example, synthetic polymeric species can be characterized by (a) higher reactivities than corresponding monomeric systems,(2) (b) specificity of substrate hydrolysis (particularly if the substrate is of a charge opposite to that of the charged groups on the polymer),(2-4) competitive inhibition by substances similar to the reactive substrate,(5, ) (d) bifunctional catalysis involving the interactions of two pendent functional groups and substrate, (2,4,7-9) and (c) saturation (complexation) by high and low molecular weight reagents.(5 0-1 However, until recently, it has not been possible to achieve the high catalytic efficiency that is usually associated with an enzyme catalyzed reaction.

IVo Discussion In our previous reports we have described that the behaviors of synthetic polymers toward low molecular weight compounds are of two different types. First, if two low molecular weight ionic species are the reactive reagents, a polymer with charged groups will tend to concentrate and/or repel one or both low molecular weight reagents in its vicinity and, consequently, will function as either an inhibitor or an accelerator of the reaction. Secondly, if catalytically active functions are added to a polymer which contains charged groups, the polymer itself, and not its counterions, is able to react with either charged or neutral reagentso Several workers have investigated enhanced or inhibited catalytic actions of polyions that contain no catalytic functions (Type 1) on the reactivities of similarly and/or oppositely charged, low molecular weight reagents.(1113,l1518) This effect has been theoretically treated by considerations of the distribution of the electrostatic potential(ll, 13,5,1 7) and by investigation of the primary salt effect. (18) Reactions which involve polymers of the second type would apparently be more related to those of hydrolytic enzymes, since the charged groups can serve as electrostatic binding sites thereby accumulating a substrate in a, high local concentration of catalytically active substituentso This cooperative effect would lead to enhanced catalytic action. (25,19) Using a polymeric reaction system of Type I, we have recently reported(20) that anionic polyacrylic acid or polyvinylsulfonic acid could effectively inhibit the hydrolysis of the positively charged phosphate ester tris (choline chloride) phosphate (TCCP), an analog of the biologically important acetylcholineo Furthermore, from this investigation we were able to ascertain by a mathematical treatment the number of negative charges on the polymer backbone which are necessary for complete inhibition of the solvolytic rate and the binding constant of the substrate to the polymer. The implications of this work are that it may be passible to use synthetic macromolecules to either accumulate or inhibit the hydrolysis of a phosphate ester. Although the above example indicated how a negatively charged polyion could accumulate a positively charged phosphate and then inhibit its hydrolysis from the catalyzing hydroxyl ions (or any other hydrolyzing species) it should be possible to incorporate other groups on the polymer chain which could facilitate hydrophobic interactions with a neutral phosphate ester. By this technique, therefore, it is conceivable that a synthetic polymer could be utilized to accumulate and possibly hydrolyze a toxic agent, such as certain phosphotriesters, and thereby protect vital enzymes from becoming phosphorylatedo Unfortunately, in this investigation we were not able to find a synthetic oxygen or nitrogens-containing nucleophilic polymer which would attack and hydrolyze TCCP. This investigation should be continued, since if such a polymer

could be found, then the possiblity of attracting a phospotriester by either electrostatic or hydrophobic interactions, and then allowing certain nucleophilic groups to attack and hydrolyze the triester, would result in a novel and perhaps biologically important macromolecule. This type of macromolecule could be efficacious in ensuring the life process from certain toxicological agents. Another area which has been of considerable interest to us is in the possible elucidation of enzyme action through the use of synthetic macromolecules of typeII in which charged groups can electrostatically attract a substrate into the vicinity of catalytically active groups on the polymer chain. In this regard, we have been particularly interested in elucidating the effect of cooperative, multifunctional interactions between catalyst groups at the active site of an enzyme and a reactive substrate by using a synthetic macromolecule of the abovementioned type. Indeed, we have found that several types of bifunctional interactions can occur between pendent groups in either poly-4(5)vinylimidazole or poly-5(6)-vinylbenzimidazole.(1) Investigations of this type are of considerable importance owing to the fact that the high catalytic efficiency of enzyme reactions is in part dependent on such cooperative, multifunctional interactions. In our solvolytic work to date, we have determined that bifunctional interactions of the following types could occur(l): (1) cooperative interaction of one neutral and one anionic pendent benzimidazole group in the poly-5(6)-vinylbenzimidazole catalyzed solvolysis of the anionic esters sodium 3-acetoxy-4-nitrobenzenesulfonate (NABS) and 4-nitro-3-acetoxybenzoic acid (NABA), and the neutral ester p-nitrophenyl acetate (PNPA) at high pH. (2) cooperative interaction of two neutral pendent imidazole groups in the poly-4(5)-vinylimidazole catalyzed solvolysis of PNPA at high pH and of two neutral pendent benzimidazole groups in the solvolysis of NABS at intermediate pH. (3) cooperative interaction of one neutral and one protonated pendent imidazole group in the poly-4(5)-vinylimidazole catalyzed solvolyses of NABA and NABS at intermediate pH and in the poly5(6)-vinylbenzimidazole catalyzed solvolysis of NABS at low pH. (4) cooperative interaction of one neutral, pendent imidazole and one anionic, pendent phenol in the copolymer of 4(5)-vinylimidazole and p-vinylphenol catalyzed solvolyses of PNPA, NABA, NABS and the positively charged ester 3-acetoxy-N-trimethylanilinium iodide (ANTI). (5) cooperative interaction of one neutral, pendent imidazole and one anionic, pendent carboxylate in the copolymers at 4(5)-vinyllimidazole and acrylic acid catalyzed solvolysis of ANTI. 11

Although a variety of cooperative interactlions between pendent groups in the synthetic imidazole containing polymers have been elucidated, it had not been possible to achieve a high catalytic activity, such as that which would be characteristic of an enzymic processo Since all the above cooperative interactions were obtained for reactions that were second-order, ie., first-order in polymer and first-order in substrate, we attempted to better compare our synthetic system with an enzymic system by'"saturating"v poly-4(5)-vinylimidazole with a low molecular weight substrate. This effect of saturation had previously never been reported with a synthetic macromolecule and a low molecular weight substrate. When the neutral ester PNPA was employed, we were unable to saturate (or complex) poly-4(5)vinylimidazole. These results suggested that hydrophobic interactions were insufficient to accumulate PUPA in the vicinity of the catalytically active polymer~ However, when the negatively charged ester was employed with a partially protonated poly-4(5)-vinylimidazole, we did achieve saturation, In fact, by this reaction we were able to obtain a kinetic system of the MichaelisMenten type, which is used so often. in enzyme reactions, and we determined that the Michaelis constant (Kin) was the same order of magnitude as that of certain enzyme substrate reactionso(i4) Although this si.milarity, however, was of considerable interest, the turnover number for this process was very small, indicating that the overall catalytic process was not particularly efficient0 The above reaction did show that it was possible to complex a synth.etic, catalytically active macromolecule with a reactive substrate. The method of attraction in this case was primar.ily through electrostati- forces. Since enzyme-substrate complexation presumably involves more hydrophobic or nonpolar interactions than electrostatic interactions, we have begun an investigation of nonpolar interactions in poly-4(5)-vinylimidazole catalyzed reactions. When the long-chain, anionic ester 4-nit:ro-3=dodecanoyloxybenzoic acid (NDBA) was employed as a substrate and poly-4(5>)vinylimidazole was the catalyst, a remarkable catalytic efficiency was achieved when the solvent system had a low concentration of ethanolo In fact it was determined that the polymeric reaction was approximately 2000 times faster than that of the monomeric analog imidazole(2l) These extremely rapid rate constants, on the order of certain enzymic reactions, had to be determined by stopped-flow spectroscopy~ By this system we were able to achieve turnover numbers which would be characteristic of an enzyme catalyzed hydrolysis of a nonspecific substrate. This is the first reported example of a synthetic polymer which not only complexed with the substrate, but also achieved a remarkably high solvol.ytic C rate. Perhaps the most significant point of this investigation was the pronounced effect of the ethanol.water solvent composition on the solvolytic rate of NDBAo It was found that the maximum rate was achieved in 3W ethanol-water (v/v)0 In this ratio it has been reported that the ethanoLwater solvent system achieves a maximum Tstructurednesso =(2l) When the temperature of this reaction i.s varied, it is found that a maximum rate occurs near room temperatureo

This is a characteristic behavior of enzymic systems, and it is highly unusual to find this with a synthetic, macromolecular catalyst. These results suggest that if the structuredness of the solvent is important in synthetic systems, then a similar effect may occur in enzymic systems; i.e., an ordering of the water at the enzyme's active site may be necessary for high catalytic efficiency. 13

V. Future Research It is apparent that the reactions of synthetic polymers are extremely interesting, particularly with regard to the very unusual catalytic effects which can be achieved by a proper choice of reaction conditions. In this regard we wish to continue our investigation of nonpolar interactions in these synthetic, macromolecular reactions. For example, the studies with NDBA have all been performed below the critical micelle concentration (cmc) of this substrate. As one approaches the cmc, the apolar interactions between poly-4( 5) -vinylimidazole and substrate should. increase dramatically, possibly leading to high catalytic rates. It is also of interest to synthesize a positively charged, long chain phenyl ester and to study its solvolysis. It is known that the short-chain, positively charged ester ANTI is not solvolyzed very effectively by poly-4(5)-vinylimidazole, perhaps because of the electrostatic repulsion which is involved. It could be expected, however, that a long chain, positively charged. ester, would overcome this electrostatic barrier, since the apolar interactions with polymer would presumably outweigh any electrostatic effects. These studies will lead to a better understanding of apolar interactions between macromolecules (either natural or synthetic) and substrates, an area of extreme importance in reactions which involve complexation before a hydrolytic reacti on. We are also interested in utilizing poly-4( 5 )-vinylimidazole to help elucidate the role of ribonuclease (RNase) in the hydrolysis of RNA.(20) Since RNase is believed to contain two imidazole functions at its active site, poly-4(5)vinylimidazole might effectively cleave ribo-nucleotides in much the same fashion as RNase. This area would. add a new dimension to the reactions of catalytically active, synthetic polymers. Another new area in which catalytically active, synthetic macromolecules may find application is in the hydrolysis of amide bonds. Although a considerable amount of research has been performed on the hydrolyses of (carbon) esters, very little attention has been given to the studies of amide hydrolyses by macromolecular catalysts. Of particular interest i'n this regard is the fact that Bruice(22) has shown that the protonated imidazole function is a catalytically active species in an intramolecular amide catalyzed hydrolysis. This process strongly suggests that a partially protonated poly-4(5)-vinylimidazole may be a very effective catalyst for amide cleavage. Furthermore, should this reaction prove feasible, it also should be possible to study the hydrolysis of a prote in catalyzed by poly-4(5)-vinylimidazole.

As suggested before, with a proper choice of reaction conditions it should be possible to design'a synthetic polymer for a specific catalytic process. We now know that one could use electrostatic forces to accumulate a charged substrate to an oppositively charged polymer. It is also possible to incorporate hydrophobic sites on a polymer chain which would then allow a substrate to be attracted to the polymer by apolar interactions. Furthermore, the electrostatic interactions and/or the apolar interactions could be used to bind the substrate to the polymer, resulting in a polymer-substrate complex. We also know that multifunctional interactions can exist between pendent groups on the synthetic polymer in their interactions with substrate, thereby leading to enhanced catalytic action. The cooperative interactions involved could exist between one type of functional group or between two different functional groups. In addition, these interactions could be between a protonated and a neutral group, a neutral and a neutral group, or a neutral and an anionic group. From our recent studies on solvent effects, it seems clear that there is also a question of "structuredness" of the solvent medium which must also be considered. In conclusion, although synthetic macromolecules will undoubtedly never replace an enzyme for a specific hydrolysis reaction, it should be possible, based on the previously discussed effects, to design a hydrolytic system in which a synthetic polymer will function as a very efficient catalyst. 15

VI. Glossary TCCP tris (choline chloride) phosphate NABS sodium 4-acetoxy- 3-nitrobenzenesulfonate NABA 4-nitro- 3-acetoxybenzoic acid PNPA p-nitrophenyl acetate ANTI 3-acetoxy-N-trimethylanilinium iodide Km Michaelis constant NDBA 4-nitro- 3-dodecanoyloxybenzoic acid v/v volume/volume cmc critical micelle concentration RNase ribonuclease RNA ribonucleic acid 16

VII. Literature Cited 1. C. G. Overberger and J. C. Salamone, Accounts Chem. Res., 2, 217 (1969). 2. For a review see H. Morawetz, "Macromolecules in Solution," (High Polymers, Vol. XXI), Interscience Publishers, New York, 1965, Chapter IX. 3. R. L. Letsinger and T. J. Savereide, J. Amer. Chem. Soc., 84, 3122 (1962). 4. C. G. Overberger, T. St. Pierre, N. Vorchheimer, J. Lee, and S. Yaroslavsky, ibid., 87, 296 (1965). 5. R. L. Letsinger and I. Klaus, ibid., &T7 3380 (1965). 6. C. G. Overberger, unpublished results. 7. C. G. Overberger, T. St. Pierre, and S. Yaroslavsky, J. Amer. Chem. Soc., 87, 4310 (1965). 8. C. G. Overberger, J. C. Salamone, and S. Yaroslavsky, ibid., 89, 6231 (1968). 9. J. Hine, F. E. Rogers, and R. E. Notari, ibid., 90, 3279 ('1968). 10. Yu. E. Kirsh, V. A. Kabanov, and V. A. Kargin, Dokl. Akad. Nauk (English translation), 177, 976 (1967). 11. H. Morawetz and J. A. Shafer, J. Phys. Chem., 67, 1293 (1963). 12. S. Yoshikawa and 0. K. Kim, Bull. Chem. Soc. Jap., )9, 1729 (1966). 13. B. Vogel and H. Morawetz, J. Amer. Chem. Soc., 2, 1368 (1968). 14. C. G. Overberger, R. Corett, J. C. Salamone, and S. Yaroslavsky, Macromolecules, 1, 331 (1968). 15. H. Morawetz, J. Polymer Sci., 42, 125 (1960). 16. C. L. Arcus, T. L. Howard, and D. S. South, Chem. Ind. (London), 1756 (1964). 17. H. Morawetz, C. G. Overberger, J. C. Salamone, and S. Yaroslavsky, J. Amer. Chem. Soc., 90, 651 (1968). 18. N. Ise and F. Matusi, ibid.., 9, 4242 (1968). 17

19. C. G. Overberger, R. Sitaramaiah, T. St. Pierre, and S. Yaroslavsky, ibid., 87, 3270 (1965). 20. Contract CAAA-15-67-C-0567. Semiannual Reports, July 1968-December 1968 and January 1969-June 1969. 21. C. G. Overberger, M. Morimoto, I. Cho, and J. C. Salamone, Macromolecules, 2, 553 (1969). 22. For a review see T. C. Bruice in "Enzyme Models and Enzyme Structure," Brookhave Symposia in Biology, No. 15, Brookhaven National Laboratory, 1962. 18

Distribution List No. of Recipient Copies Defense Documentation Center Cameron Station Alexandria, Virginia 22314 20 Commanding Officer Edgewood Arsenal ATTN: Dr. Joseph Epstein Research Laboratories Edgewood Arsenal, Maryland 21010 20 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 19

Unclassified | ~i~ttt~.OCUN K T COT1ROW.. DATA. R & D _ ~ nq tn t#Iti od01almirwt md ietnt anted Wh ver e i. tl nEled': I. QRIGINA TINS tVIet (WOrporote.UfLh).. REPORaT oSECUitY CL)ASSIICAIION UNCLASSIFIED The University of Michigan Ann Arbor, Michigan 48104 NA MECHANISM OF ENZYME ACTION 4. Os9CrIPtIVR' O.1t2 (Tpte etfrpott and #nctuelve dates) Terminal Report- June 1967-June 1. 1970 S. AU'S H rtS, n..ame".I.dd.le., W at1as name). C. G. Overberger, R. R. Deupree, R. C. Glowakyr, H. Maki, M. Morimoto, T. J. Pacansky, C. J. Podsiadly, J. C. Salamone, C. M. Shen, and P. S. Yuen'. REPORT DA-t' a *. TOTAL NO. OF PAGES 7b. NO. O nF Rs May 7, -1970 19 22 b. PROJEfCT'r NO. TERN1B061102B71A C.!b. OTHER REPORT NO(S) (any other numbers that may be assigned thishl report) d. 08979-5-F I0. DIOTRItIUTION STATMENT This document is subject to special export controls and each transmittal to foreign governments or foreign governments or foreign nationals may be made only with prior approval of the CO, Edgewood Arsenal, Attn: SMUEA-TSTI-T, idgewood Arsenal... Marvland 21010.. _:............. -......... 11. SUPPLEMEWTARV NOTES I2. SPONSORING MILITARY ACTIVITY Edgewood Arsenal Life Sciences Basic Research Research Laboratories Materiel support Edgewood Arsenal, Maryland 21010 I3. AGSTRACT I (J. s P~j. C., X311) The objectives of this work have been to synthesize new catalytically active, synthetic macromolecules and to study their activities toward carbon and phosphate ester. In these investigations it was hoped that a careful study of the reaction kinetics would allow certain analogies to enzyme behavior to be drawn. Further, it was believed the field of macromolecular reactions wasin itself interesting and quite different from those reactions of low molecular weight materials. During the period of this contract we have found several extremely interesting catalytic effects displayed by synthetic imidazole containing polymers. Indeed, in one investigation it was found that very rapid solvolytic rates could be achieved, and that these rates of.reaction were comparable to those of certain enzymic reactions. In this report are also described several new areas where synthetic macromolecules may again reveal very unusual catalytic effects. DD iNO 173 Unclassified ecurity Classification

Unclassified__ security asscation LINK A LINK s L.INI-o KIVE WORD _ FOLa WX WTO L WO T-' Y * I~~~~~~~ I wrI 1rc t I~' ~ Bifunctional Catalysis Saturation Complexation Catalytic Activity Phosphotriesters Electrostatic Hydrophobic Interactions Nucleophilic Michaelis Constant Turnover Number Nonpolar Apolar Unclas sified recurity ClIrMulcifi:tln

UNIVERSITY OF MICHIGAN 3 9015 03695 1609