Tuning Substrate Selectivity of a Cationic Enzyme Using Cationic

Aug 2, 2006 - Noncovalent interactions between an artificial molecular scaffold and a protein are interesting due to the possibility of reversible mod...
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Langmuir 2006, 22, 7695-7700

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Tuning Substrate Selectivity of a Cationic Enzyme Using Cationic Polymers Raghunath Roy, Britto S. Sandanaraj, Akamol Klaikherd, and S. Thayumanavan* Department of Chemistry, UniVersity of Massachusetts, Amherst, Massachusetts 01003 ReceiVed February 20, 2006. In Final Form: May 17, 2006 Noncovalent interactions between an artificial molecular scaffold and a protein are interesting due to the possibility of reversible modulation of the activity of the protein. R-Chymotrypsin is a positively charged protein that has been shown to interact with negatively charged polymers. Here we show that positively charged polymers are also capable of electrostatically binding to this protein. The resulting experiments show that the ability of a polymer to bind a protein does not depend only on the pI of the protein. We also realized that the variations in charge density in the polymer backbone afford different selectivities of the enzyme toward charged substrates.

Introduction Enzymes with modified activity and high substrate selectivity are of great interest in biocatalysis,1-6 biosensors,7-9 and enzymerelated biotechnology. Modifying the enzymatic properties of a protein through noncovalent interactions with an artificial molecular scaffold is interesting because of the reversible nature of the interaction.10-15 We have recently reported that complementary electrostatic interactions between a polymer and a protein could be used to bring about a binding-induced substrate selectivity to the enzyme.15 In that case, the positively charged R-chymotrypsin (ChT; pI ) 8.8) exhibits a hyperactivity toward a positively charged substrate, while the negatively charged substrate exhibits inhibition. Such selectivity is also observed with negatively charged monolayer-protected metal nanoparticles complexed with the enzyme.16 It has been suggested in these cases that the negatively charged polymer scaffold acts as a mediator for recruiting and enhancing the local concentration of the substrate near the active site, thus enhancing the rate of the reaction relative to that of the unbound enzyme. Thus, the charge on the polymer dictates the substrate selectivity; the charge on the polymer is dictated by the pI of the protein due to the need for charge complementarity. Therefore, the pI of the protein limits the nature of substrate selectivity one could obtain. In the * To whom correspondence should be addressed. E-mail: thai@ chem.umass.edu. (1) Bornscheuer, U. T. Angew. Chem., Int. Ed. 2003, 42, 3336-3337. (2) Cao, L. Q.; van Langen, L.; Sheldon, R. A. Curr. Opin. Biotechnol. 2003, 14, 387-394. (3) Hult, K.; Berglund, P. Curr. Opin. Biotechnol. 2003, 14, 395-400. (4) Gross, R. A.; Kumar, A.; Kalra, B. Chem. ReV. 2001, 101, 2097-2124. (5) Zhu, G.; Wang, P. J. Am. Chem. Soc. 2004, 126, 11132-11133. (6) Novik, S. J.; Dordick, J. S. Biotechnol. Bioeng. 2006, 68, 665-667. (7) Bordusa, F. Chem. ReV. 2002, 102, 4817-4867. (8) Haupt, K.; Mosbach, K. Chem. ReV. 2000, 100, 2495-2504. (9) McQuade, T. D.; Pullen, E. A.; Swager, T. M. Chem. ReV. 2000, 100, 2537-2574. (10) Fischer, N. O.; Verma, A.; Goodman, C. M.; Simard, J. M.; Rotello, V. M. J. Am. Chem. Soc. 2003, 125, 13387-13391. (11) Verma, A.; Simard, J. M.; Rotello, V. M. Langmuir 2004, 20, 41784181. (12) Hamuro, Y.; Calama, M. C.; Park, H. S.; Hamilton, A. D. Angew. Chem., Int. Ed. Engl. 1997, 36, 2680-2683. (13) Park, H. S.; Lin, Q.; Hamilton, A. D. J. Am. Chem. Soc. 1999, 121, 8-13. (14) Lin, Q.; Park, H. S.; Hamuro, Y.; Lee, C. S.; Hamilton, A. D. Biopolymers 1998, 47, 285-298. (15) Sandanaraj, B. S.; Vutukuri, D. R.; Simard, J. M.; Klaikherd, A.; Hong, R.; Rotello, V. M.; Thayumanavan, S. J. Am. Chem. Soc. 2005, 127, 1069310698. (16) Hong, R.; Emrick, T.; Rotello, V. M. J. Am. Chem. Soc. 2004, 126, 13572-13573.

Figure 1. Front (A) and rear (B) views of R-chymotrypsin.

case of ChT, one could obtain hyperactivity toward a positively charged substrate using a complementary negatively charged polymer. However, is it possible to achieve a polymer scaffold that affords hyperactivity toward negatively charged substrates for a positively charged protein? In this paper, we report our approach to this issue using ChT. Our approach to this problem is a simple one. We asked whether we could design positively charged polymers that could bind to the positively charged chymotrypsin. While such an approach seems counterintuitive, it is important to remember that proteins are polyampholytes (i.e., proteins contain both positively and negatively charged residues). Although limited, there is literature precedence for such interactions.17-23 Upon carefully examining the structure of ChT, one realizes that the active site of the enzyme is surrounded mostly with cationic amino acids (15 cationic residues vs 5 anionic residues). However, the backside of the enzyme is dominated by anionic amino acids (9 anionic residues vs 5 cationic residues), as shown in Figure 1. Thus, we designed polymethacrylamides P1-P5 containing varying degrees of positively charged amino groups as the side chain functionality. In the present study, these polymers have been shown to be able to modulate the substrate selectivity of the enzyme.

Results and Discussion The amino side chain functionality of the polymer is based on lysine. This choice is based on the fact that lysine is one of (17) Veparelli, P.; Alfani, N.; Cantarella, M. J. Mol. Catal. B: Enzym. 2001, 15, 1-8. (18) Spreti, N.; Alfani, F.; Cantarella, M.; D’Amico, F.; Germani, R.; Savelli, G. J. Mol. Catal. B: Enzym. 1999, 6, 99-110. (19) Aluin, E.; Lissi, E.; Duarte, R. Langmuir 2003, 19, 5374-5477. (20) Spreti, N.; Profio, P. D.; Marte, L.; Bufali, S.; Brinchi, L.; Savelli, G. Eur. J. Biochem. 2001, 268, 6491-6497. (21) Renner, C.; Piehler, J.; Schrader, T. J. Am. Chem. Soc. 2006, 128, 620628. (22) Porcar, I.; Gareil, P.; Tribet, C. J. Phys. Chem. B 1998, 102, 7906-7909.

10.1021/la060496j CCC: $33.50 © 2006 American Chemical Society Published on Web 08/02/2006

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Scheme 1. Synthesis of Random Copolymers P1-P5 with Different Densities of Cationic Residues

Scheme 2. Synthesis of the Lysine Derivative 3

the most common cationic residues found at interfaces of proteins during protein-protein interactions.24-28 Structures of polymers P1-P5, with different densities of the lysine functionalities, are shown in Scheme 1. These polymers were synthesized through a postsynthetic modification of an active ester polymer based on N-hydroxysuccinimide (NHS), 2.29 We used atom transfer radical polymerization (ATRP)30-33 to polymerize the NHS ester of methacrylic acid (1) and obtain the corresponding polymer 2, poly(NHSMA), with narrow polydispersity (Mn ) 15300, DP ) 83, PDI ) 1.13).29 It is important that polymers P1-P5 are synthesized from a preformed polymer such as 2. This route avoids any possible differences in the effect of the polymerprotein interactions due to the difference in the number of repeat units within the polymer chain. This ensures that the number of total repeat units is the same in all polymers, the difference being only in the density of cationic residues within the polymer chain. To tune the percentage of lysine-based amino functionalities, polymer 2 was sequentially treated with the lysine derivative 3 (23) Tribet, C.; Porcar, I.; Bonnefont, P. A.; Audubert, R. J. Phys. Chem. B 1998, 102, 1327-1337. (24) Berg, T. Angew. Chem., Int. Ed. 2003, 42, 2462-2481. (25) DeLano, W. l.; Ultsch, M. H.; DeVos, A. M.; Wells, J. A. Science 2000, 287, 1279-1283. (26) Bogan, A. A.; Thomas, K. S. J. Mol. Biol. 1998, 280, 1-9. (27) Stites, W. E. Chem. ReV. 1997, 97, 1233-1250. (28) Scott, R. A.; Mauk, A. G. Cytochrome C: A Multidisciplinary Approach; University Science Books: Sausalito, CA, 1996. (29) Savariar, E. N.; Thayumanavan, S. J. Polym. Sci., Part A: Polym. Chem. 2004, 42, 6340-6345. (30) Matyjaszewski, K.; Xia, J. Chem. ReV. 2001, 101, 2921-2990. (31) Kamigatio, M.; Ando, T.; Sawamoto, M. Chem. ReV. 2001, 101, 36893745. (32) Tang, W.; Tsarevsky, N. V.; Matyjaszewski, K. J. Am. Chem. Soc. 2006, 128, 1598-1604. (33) Bontempo, D.; Heredia, K. L.; Fish. B. A.; Maynard, H. D. J. Am. Chem. Soc. 2004, 126, 15372-15373.

and isopropylamine in different ratios. This reaction afforded the precursor polymer represented by 4, as shown in Scheme 1. Deprotection of the benzyloxycarbonyl (Cbz) functionality using HBr/acetic acid liberated the side chain amino functionality of lysine to afford polymers P1-P5. The ratio of the lysine functionality to the isopropyl moiety was estimated by 1H NMR and was found to closely correspond to the feed ratio of the amine 3 and isopropylamine. The lysine derivative 3 was synthesized from the commercially available N-()-(carboxybenzyl)-N-(R)-Boc-lysine (5). The free carboxylic acid group of 5 was coupled with (isopropoxypropyl)amine under EDC coupling conditions to afford compound 6 in 81% yield. The R-amino Boc group was selectively deprotected using TFA in the presence of a benzyloxycarbonyl protecting group to achieve compound 3 in 88% yield, as shown in Scheme 2. The purpose of the (isopropoxypropyl)amine is simply to mask the negative charge of the carboxylic acid in lysine. The effect of polymer upon the enzymatic activity of ChT was monitored spectroscopically using catalytic hydrolysis of chromogenic substrates S1-S3. The assay studies were done by preincubating ChT (3.2 µM) with the polymer (5 µM) in 5 mM sodium phosphate buffer solution (pH 7.4) for 2 h followed by addition of 2 mM substrate. The rate of formation of the hydrolyzed product p-nitroaniline was monitored at 405 nm. The percentage of enzymatic activity was calculated by normalizing against control experiments for each substrate under identical conditions in the absence of any polymer. The relative activities of ChT under the influence of polymers P1-P5 with respect to substrates S1-S3 are shown in Figure 2. It is important to note the structural differences among substrates S1-S3. These substrates are identical in structure, except for the amino, hydroxyl, and carboxylate functionalities

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Figure 2. Modulation of enzymatic selectivity under the influence of polymers P1-P5. Scheme 3. Synthesis of Polymers P6 and P7

present in the side chain. Therefore, substrates S1-S3 are positively charged, neutral, and negatively charged substrates, respectively, for the enzyme, but otherwise identical in structure. The percent enzymatic activities shown in Figure 2 are normalized to the activity of native chymotrypsin with the particular substrate in the absence of the polymer. For example, the percent activity of the ChT/P1 mixture with respect to S1 is 163%, where the activity of ChT toward S1 in the absence of P1 is considered to be 100%. The observed trend becomes clear immediately when one examines the relative effects of P1 and P5 on the substrate selectivity. Polymer P1 has only 20% cationic functionalities in the side chain, whereas polymer P5 contains a cationic functionality in every repeat unit. In the presence of P1, the enzyme is more selective toward the positively charged substrate (163% toward S1, 96% toward S2, and 29% toward S3). In other words, the enzyme exhibits significant inhibition toward substrate S3, while exhibiting hyperactivity toward S1, in the presence of P1. On the other hand, the enzyme exhibits significant hyperactivity toward all substrates in the presence of polymer P5. The hyperactivity however is pronounced greatly with substrate S3 and least pronounced with substrate S1. The effect of all polymers with respect to the neutral substrate S2 falls between those of S1 and S3. Similarly, while P1 and P5 exhibit opposite substrate selectivities, polymers P2-P4 exhibit intermediary selectivities. There are two functionalities present in polymers P1-P4. One is based on the cationic (lysine derivative) side chain functionality, and the other one is based on N-isopropylmethacrylamide. It is necessary that we find out whether this neutral functionality by itself has any interaction or influence on the protein function. Accordingly, we synthesized poly(Nisopropylmethacrylamide) and tested the effect of this polymer on the activity of the enzyme. There were no discernible

differences in the activity of the enzyme, which suggests that the key residues in the polymer-protein interaction are indeed the cationic residues of the polymer. It is then interesting to identify whether there is any influence of the (isopropoxypropyl)amine tail of the lysine derivative in the modulation of ChT activity. To find this, commercially available poly-L-lysine was tested against ChT activity. Poly-L-lysine hyperactivates the ChT activity by 160% against S3 in a way similar to that of P5. This suggests that neither the (isopropoxypropyl)amine tail nor the polymethacrylate backbone of the polymer is crucial in the modulation of enzymatic activity. Considering these findings, we were interested in finding out whether any amine-based cationic polymer would be able to modify the enzymatic activity. To test this possibility, we synthesized poly[(2-aminoethyl)methacrylamide] (P6) and tested its effect on ChT activity (Scheme 3); we did not observe any change in activity. This polymer did not exert any modulation of enzymatic activity in any of the substrates. The distance between the amine functionality and the polymer backbone in polymers P1-P5 or poly-L-lysine is longer than that in P6. Therefore, we synthesized poly[(5-aminopentyl)methacrylamide] (P7) using the same procedure followed for the synthesis of P6 (Scheme 3). The enzymatic activity of ChT was about 124% against S3. This hyperactivity, combined with our observations with poly-L-lysine, suggests that the linker length between the amino moiety and the polymer backbone is one of the important factors in determining the observed variations in the substrate selectivity of ChT. The effect of P5 upon the activity of ChT can be explained on the basis of electrostatics, which is similar to the effects observed with the negatively charged polymers that we had previously reported.15 In our previous case, the negatively charged polymer that is bound to the protein is thought to recruit the positively charged substrates close to the active site of the protein

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Figure 3. (A) Circular dichroism and (B) fluorescence spectra of ChT in the presence of polymers P1-P5.

and thus enhance the activity of the enzyme toward the positively charged substrate S1. In the current case, since the polymer that is bound to the protein is positively charged, it is reasonable to expect that a similar mechanism would afford enhanced activity toward substrate S3. On the other hand, polymer P1 also seems to bind to the protein, since the activity of ChT is significantly modified, especially with substrates S1 and S3. The reasons for the enzyme’s hyperactivity toward S1 and inhibition toward S3 in the presence of P1 are not clear. One could not explain the observed selectivity on the basis of simple electrostatics. Therefore, it is clear that the binding event between the polymer and the protein has another effect on the activity of the enzyme, besides the electrostatic effect outlined above. This can be seen upon examining the activity of the polymer-protein complex toward substrate S2. This substrate is neutral, and therefore, the observed inhibition or hyperactivity could not be attributed to an electrostatic interaction between the polymer and the substrate. Thus, when one observes the percent activity of ChT with respect to S2, the activity of the enzyme increases with increasing density of the lysine functionalities. Therefore, there is an inherent enhancement in the enzymatic activity of ChT upon binding to the cationic residues of the polymer. The effect of this binding upon the selectivity of the enzyme is exactly opposite that expected from the electrostatic effect described above. This is clear from the difference in selectivity when one goes from P1 to P5, as shown in Figure 2. It is possible that the other effect of binding the polymer to the protein is based on an allosteric effect. It should be pointed out however that there is neither literature precedence nor clear evidence to support this supposition. To search for any evidence of allosteric interactions, we carried out circular dichroism (CD) experiments of ChT in the presence of the polymers. The results of these studies are shown in Figure 3A. The far-UV CD spectrum of ChT in sodium buffer solution (pH 7.4) shows the characteristic bands around 204 and 230 nm that are related to the β sheet and R helix in the secondary structure, respectively.34 The CD spectrum of a thermally denatured ChT does not have a characteristic band at 230 nm in addition to a blue shift of the 204 nm band. On the other hand, a red shift of the fluorescence maxima (∼20 nm) of ChT is considered to be an indication of loss of native structure due to exposure to a more polar solvent environment.35-37 In the presence of polymers P1-P5, we did not observe either of these, suggesting that there is no denaturation of the protein. This is additionally substantiated by the ionic strength based recovery of the enzymatic activity study (vide infra). (34) Provencher, S. W.; Glo¨ckner, J. Biochemistry 1981, 20, 33-37.

It should be noted however that the relative intensity of the two peaks changes when bound to the cationic polymers. While this could be taken to suggest that there is an allosteric conformational change in the protein, it is complicated by the fact that the lysine side chain of the polymer itself is chiral and nonracemic. Therefore, the change in intensity of the peak at 204 nm could be due to contributions from the polymer chain. To test for this, we investigated the CD spectrum of ChT in the presence of the achiral polymer P7. In this case, there is no change in either of the peaks in the CD spectrum. This observation supports our assumption that the features seen with polymers P1-P5 are likely due to the inherent chirality of the side chains. It is also important to recognize that a lack of change in the CD spectrum itself cannot be construed as a lack of subtle conformational changes in proteins since CD represents global changes in the conformation of ChT. Fluorescence spectroscopy also provides insight into the protein structure. ChT has eight tryptophan residues; four of them are distributed on the surface, and the rest of them are in the core.38 The λmax emission of native ChT is around 334 nm, while the emission maximum of the thermally denatured enzyme is red shifted to 356 nm. Fluorescent spectra of ChT35-37 in the presence of polymers also did not afford any significant spectral changes and thus provide no further information (Figure 3B). Therefore, the hypothesis regarding a possible allosteric effect explaining the substrate selectivities still remains provisional. The discussions so far suggest that the nature of the interaction between the polymer and the protein is likely to be based on electrostatics. If this were the case, then an increase in the ionic strength of the solution would screen out the interaction. To test this possibility, the activity of ChT in the presence of polymers P1-P5 was examined at different ionic strengths (solutions containing 0.01-0.3 M NaCl). The activity of ChT was monitored with respect to the hydrolysis of chromogenic substrate S3 at different time intervals. A control experiment was also done to normalize the ChT activity at these salt concentrations in the absence of the polymer. The difference in the activity of the enzyme in the presence and absence of the polymer becomes negligible with increasing concentrations of NaCl, as shown in Figure 4. These results suggest that it is reasonable to assume that the interaction between the polymers and ChT is primarily electrostatic. (35) Ladokhin, A. S. In Encyclopedia of Analytical Chemistry; Meyers, R. A., Ed.; John Wiley & Sons Ltd.: Chichester, U.K., 2000; pp 5762-5799. (36) Desie, G.; Boens, N.; De Schryver, F. C. Biochemistry 1986, 25, 83018308. (37) Dorovska-Taran, V.; Veeger, C.; Visser, A. J. Eur. J. Biochem. 1993, 218, 1013-1019. (38) Celej, M. S.; D’Andrea, M. G.; Campana, P. T.; Fidelio, G. D.; Bianconi, M. L. Biochem. J. 2004, 378, 1059-1066.

Tuning Substrate SelectiVity of a Cationic Enzyme

Figure 4. Effect of ionic strength on the ChT activity against substrate S3.

We also carried out several other control experiments to rule out some of the alternate possibilities. For example, is there an inherent selectivity of ChT toward substrates S1-S3, and therefore, are the observed selectivities significant? First, the difference in the activity of ChT toward these substrates is small, relative to the observed differences.16 We have also independently confirmed these results in the literature. Second, note that each percent enhancement or inhibition reported here is normalized with respect to the activity of ChT toward that particular substrate in the absence of the polymer. Therefore, the observed effects are true indicators of the supramolecular interaction between the polymer and the protein. Is it possible that the polymer does not bind to the protein but directly reacts with the substrate and therefore the observed selectivities might not have anything to do with polymer-protein interaction? We performed simple control experiments to address this possibility. None of the substrates were hydrolyzed to produce the p-nitroaniline product in the presence of polymers P1-P5 without the presence of ChT. Thus, these experiments also suggest that the observed substrate hydrolysis is indeed due to the enzyme influenced by the interaction between the polymer and the protein. In our previous study, we observed that a micellar assembly formed from the anionic amphiphilic polymer is able to modulate cationic ChT activity.15,39 To examine whether the micellar assembly is important for the above effect, we investigated whether polymers P1-P5 form micelle-type structures in water. We followed the evolution of emission spectra of pyrene in the presence of various concentrations of polymers (P1-P5). The I1/I3 values of 1.8 did not change with the concentration of the polymers, which suggests that these polymers do not form any micelle-type assembly. Therefore, it can be concluded that a micelle-type assembly is not required for modulation of ChT’s function.

Summary We have synthesized a set of lysine-based polymethacrylamides through an easy postmodification of poly(NHSMA). Upon studying the interaction of these polymers containing different amounts of the lysine-based cationic residues with the enzyme chymotrypsin, we have shown the following: (i) A positively charged polymer can be utilized to bind to a protein with a pI > 7, i.e., to a protein that is considered to be positively charged. (39) Basu, S.; Vutukuri, D. R.; Shyamroy, S.; Sandanaraj, B. S.; Thayumanavan, S. J. Am. Chem. Soc. 2004, 126, 9890-9891.

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(ii) The interaction is based on electrostatics, and this is attributed to the fact that proteins are polyampholytes and therefore should be capable of binding either of the charged polyelectrolytes with appropriate side chain functionality. (iii) Systematic variation of the percent cationic functionalities in the side chain of the polymer affords differences in substrate selectivities. While the selectivity from high-density cationic polymers is based on electrostatic interactions between the polymer and substrate, the selectivity arising from the lower charge density cationic polymer affords the opposite trend, and the reason for this is not clear to us at this time. (iv) Micellar assembly of the polymer is not required for modulation of protein activity. (v) The competition between the two interactions allows for tuning the substrate selectivity by tuning the charge density in the polymer chain. While the proposed mechanisms for selectivity are consistent with the observed experimental results, a deeper understanding of the interaction is needed. This is a subject of ongoing studies in our laboratories. Experimental Section Materials. N-()-(Carboxybenzyl)-N-(R)-Boc-lysine DCHA salt was purchased from Acros Chemicals and purified by washing with 5% HCl solution. N-Hydroxybenzotriazole (HOBt), trifluoroacetic acid (TFA), and poly-L-lysine (MW 21000, PDI ) 1.4) were also purchased from Acros Chemicals. N-[3-(Dimethylamino)propyl]N′-ethylcarbodiimide hydrochloride (EDC) was from Aldrich and 1,5-diaminopentane from Alfa Aesar. Three different chromogenic substrates (S1-S3) were synthesized following the reported procedure.16 R-Chymotrypsin from bovine pancreas (E.C. 3.4.21.1) was purchased from Sigma-Aldrich. All the above chemicals were used without further purification. Dichloromethane and other dry solvents were purified following the reported procedure.41 Synthesis of Compound 6. N-()-(Carboxybenzyl)-N-(R)-Boclysine (3.2 g, 8.9 mmol) and EDC (1.88 g, 9.8 mmol) in 75 mL of DCM were stirred in an ice bath (0 °C) for 10 min, and then HOBt (1.32 g, 9.8 mmol) and (3-isopropoxypropyl)amine (5.2 g, 45 mmol) were added under an argon atmosphere. The reaction mixture was allowed to stir for 12 h at room temperature. The reaction mixture was washed with water followed by 5% HCl and 10% NaHCO3 solutions. The dichloromethane layer was filtered through anhydrous Na2SO4 and evaporated to dryness to get the crude product, which was purified by silica gel column chromatography (20% ethyl acetate/ hexane): yield 3.3 g, 81%; 1H NMR (400 MHz, CDCl3) δ 7.32 (m, 5H), 6.71 (br s, 1H), 5.08 (m, 3H), 4.92 (br s, 1H) 4.10 (br s, 1H), 3.15-3.55 (m, 7H), 1.48-1.77 (m, 8H), 1.42 (s, 9H), 1.15(dd, 6H); 13C (100 MHz, CDCl ) δ 171.9, 156.8, 136.9, 128.8, 128.3, 80.1, 3 72.1, 67.4, 66.9, 54.7, 40.8, 38.7, 32.7, 29.7, 29.6, 28.6, 22.8, 22.4; FAB/MS m/z (M+) 479.9 (calcd 479.6). Synthesis of Compound 3. Compound 2 (3 g, 6.2 mmol) and 40 mL of 25% trifluoroacetic acid in dichloromethane were stirred at 0 °C for 2 h. After the reaction was over, trifluoroacetic acid and dichloromethane were evaporated and the crude solid reaction mixture was diluted with dichloromethane and washed with saturated NaHCO3 solution. The organic layer was filtered through anhydrous sodium sulfate and evaporated in a rotary evaporator to afford the product: yield 2.1 g, 88%; 1H NMR (400 MHz, CDCl3) δ 7.58 (s, 1H), 7.29 (m, 5H), 5.12 (s, 2H), 4.90 (br s, 1H) 3.47-3.57 (m, 3H) 3.32-3.37 (m, 3H) 3.16-3.21 (m, 2H), 1.47-1.81 (m, 10H), 1.15 (d, J ) 6 Hz, 6H); 13C (100 MHz, CDCl3) δ 175.05, 157.4, 136.9, 128.7, 128.8, 71.9, 67.1, 66.8, 55.4, 37.9, 34.9, 30.0, 29.8, 23.1, 22.4; FAB/MS m/z (M+) 379.2 (calcd 379.2). General Procedure for Functionalization of Poly(NHSMA). Poly(N- hydroxylsuccinimide methacrylate) (0.131 g, 0.72 mmol) and compound 3 (0.275 g, 0.72 mmol) were added to a dry round(40) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99, 20322039. (41) Perrin, D. D.; Armarego, W. L. F. Purification of Laboratory Chemicals, 3rd ed.; Pergamon Press: Oxford, U.K., 1988.

7700 Langmuir, Vol. 22, No. 18, 2006 bottom flask in an argon atmosphere. Dry DMSO (0.8 mL) and triethylamine (84 mg, 1.4 mmol) were added, and the reaction mixture was stirred at 60 °C for 5 h. The substitution reaction was monitored by FT-IR spectroscopy following the disappearance of the 1735 cm-1 peak.42 The polymer thus formed was precipitated in cold diethyl ether. The polymer was digested with DCM, precipitated in ether, filtered off, and dried. Incorporation of different amounts of lysine amine 3 into poly(NHSMA) was done by feeding the exact mole ratio of the amine, and the resulting mixture was allowed to react for 5 h followed by addition of excess isopropylamine into the same round-bottom flask to replace the remaining active ester from the polymer (yield 50-60%). General Procedure for Deprotection of the Carboxybenzyl Group from the Polymer. Carboxybenzyl-protected lysine polymer (300 mg) and 3 mL of 33% HBr in acetic acid were added to a dry round-bottom flask and stirred for 30 min at room temperature. The deprotected bromide salt of the polymer was washed several times with diethyl ether to remove acetic acid and HBr. It was then dried to get the pure product. The complete disappearance of 1H NMR signals at δ 7.33 and 5.08 confirmed full deprotection of the carboxybenzyl group. Activity Assay Studies. All experiments were performed in 5 mM sodium phosphate buffer at pH 7.4 with [ChT] ) 3.2 µM and [polymer] ) 5 µM. The enzymatic hydrolysis reaction was initiated by adding a substrate S1-S3 (26 µM) stock solution (16 µL) in DMSO/EtOH (1:9) to a preincubated (2 h) ChT-polymer solution (42) Godwin, A.; Hartenstein, M.; Muller, A.; Brocchini, S. Angew. Chem., Int. Ed. 2001, 40, 594-597.

Roy et al. (184 µL) to reach a final substrate concentration of 2 mM. Hydrolysis of substrates was monitored for 10-30 min at 405 nm. The assays were performed in triplicate, and the averages are reported. The standard deviation was usually less than 10%. The activity of native chymotrypsin (control) was taken to be 100%. From this value, the relative activity of polymer-bound ChT was calculated. For salt effect studies, all the parameters were kept unchanged except the NaCl salt concentrations. Fluorescence and CD Experiments. ChT (3.2 µM) was incubated with polymers P1-P5 (5 µM) in 5 mM sodium phosphate buffer (pH 7.4). ChT was excited at 295 nm, and the emission was recorded from 300 to 450 nm on a spectrofluorimeter, using a 10 mm quartz cuvette. CD experiments were performed using a quartz cuvette with a 1 mm path length. Three scans were taken for each sample from 190 to 250 nm at a rate of 20 nm min-1. All experiments were performed at a constant temperature of 25 °C. All fluorescence and CD experiments were performed under conditions identical with those of the activity assays (5 mM sodium phosphate buffer, pH 7.4, [ChT] ) 3.2 µM, [polymer] ) 5 µM).

Acknowledgment. Support from the U.S. Army Research Office is gratefully acknowledged. We are also grateful to the NSF-MRSEC at the University of Massachusetts for supporting this effort. Supporting Information Available: 1H NMR spectra of polymers P1-P5 and synthetic details of polymers P6 and P7. This material is available free of charge via the Internet at http://pubs.acs.org. LA060496J