Functionalized Semitelechelic Poly[N-(2-hydroxypropyl

Semitelechelic poly[N-(2-hydroxypropyl)methacrylamide]s (ST-PHPMA) with different ... The semitelechelic polymers have been characterized by end-group...
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Bioconjugate Chem. 1998, 9, 793−804

793

Functionalized Semitelechelic Poly[N-(2-hydroxypropyl)methacrylamide] for Protein Modification Zheng-Rong Lu,† Pavla Kopecˇkova´,†,‡ Zhuchun Wu,§ and Jindrˇich Kopecˇek*,†,‡ Departments of Pharmaceutics and Pharmaceutical Chemistry/CCCD and of Bioengineering, Mass Spectrometry Facility, University of Utah, Salt Lake City, Utah 84112. Received May 12, 1998; Revised Manuscript Received July 17, 1998

Semitelechelic poly[N-(2-hydroxypropyl)methacrylamide]s (ST-PHPMA) with different functional end groups, namely carboxyl, methyl ester, hydrazide, and amino groups, were prepared by chain transfer free-radical polymerization. 2,2′-Azobisisobutyronitrile (AIBN) was used as an initiator and 3-mercaptopropionic acid, methyl 3-mercaptopropionate, 3-mercaptopropionic hydrazide, and 2-mercaptoethylamine were used as chain-transfer agents. The semitelechelic polymers have been characterized by end-group analysis, size-exclusion chromatography (SEC), and matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS). The effects of the concentrations of the mercaptans and the initiator on the molecular weight of the polymers have been investigated. The higher the concentration of mercaptan, the lower the molecular weight of ST-PHPMA. The concentration of initiator did not have a significant effect on the molecular weight of the semitelechelic polymers. The end groups of the ST polymers can be readily transformed by polymeranalogous reactions. A model protein, R-chymotrypsin, has been modified with ST-PHPMA-CONHNH2 and STPHPMA-COOSu and the conjugates characterized by MALDI-TOF MS. The activity of modified chymotrypsins toward a high molecular weight substrate, P-Gly-Leu-Phe-NAp (where P is the HPMA copolymer backbone, and NAp is p-nitroanilide), was slightly lower than the activity of the native enzyme. The cleavage of a low molecular weight substrate, Z-Gly-Leu-Phe-NAp, by modified chymotrypsins was dependent on their structure. Whereas the activity of the amino group modified chymotrypsins was higher than that of the native enzyme, the activity of carboxyl-modified chymotrypsins was lower than that of the native enzyme. In summary, the data seem to indicate that ST-PHPMA is an effective protein-modifying agent.

INTRODUCTION 1

Semitelechelic (ST) polymers are linear macromolecules containing a functional group at one end of the polymer chain. The functional group can be used for conjugating or grafting the polymer chains to other molecular species or surfaces without causing cross* To whom correspondence should be addressed. Phone: (+801) 581 7211. Fax: (+801) 581 7848. E-mail: [email protected]. † Departments of Pharmaceutics and Pharmaceutical Chemistry/CCCD. ‡ Department of Bioengineering. § Mass Spectrometry Facility. 1 Abbreviations: AIBN, 2,2′-azobisisobutyronitrile; DCC, dicyclohexyl carbodiimide; DCU, dicyclohexyl urea; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DPn(end), number average degree of polymerization determined by end group analysis; HPMA, N-(2-hydroxypropyl)methacrylamide; IBN, radical formed by decomposition of AIBN; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Mn, number average molecular weight; Mn(end), number average molecular weight determined by end group analysis; Mw, weight average molecular weight; MWCO, molecular weight cut off; NAp, p-nitroanilide; HOSu, N-hydroxysuccinimide; ONp, p-nitrophenoxy; P, HPMA copolymer backbone; PBS, phosphate-buffered saline; PEG, poly(ethylene glycol); PHPMA, polyHPMA; SEC, size-exclusion chromatography; ST, semitelechelic; TNBS, trinitrobenzene sulfonic acid; Tris, 2-amino-2-(hydroxymethyl)-1,3-propanediol; Tris/CaCl2 buffer, 80 mM Tris + 20 mM CaCl2, pH 8.0; Z, benzyloxycarbonyl.

linking. For example, the covalent attachment of methoxy poly(ethylene glycol) (mPEG) to therapeutic proteins increases their resistance to proteolysis, reduces their antigenicity, and prolongs their intravascular half-life (1-3). The attachment of PEG to biomaterial surfaces results in protein repulsion as a result of PEG’s low interfacial free energy with water, hydrophilicity, high surface mobility, and steric stabilization effects (4). Covalent conjugation of PEG with hydrophobic anticancer drugs results in more soluble and more easily formulated and delivered drugs (5). Numerous ST polymers of different structures have been evaluated and many possess effects similar to PEG when conjugated to proteins. Structural characteristics of ST polymers, such as the molecular weight (6), linearity, or branching (7), chemical structure (8-15), and degree of substitution (16) are decisive for the properties of the conjugates. A suitable route for the synthesis of ST polymers is radical polymerization in the presence of chain-transfer agents. Okano et al. have shown that mercaptans are effective at introducing functional groups to the ends of macromolecules and regulating the molecular weight via chain-transfer reactions (12, 17). N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers are water-soluble biocompatible polymers used in anticancer drug delivery (reviewed in ref 18). HPMA copolymers containing reactive groups at side-chain termini were used for the modification of trypsin (19), chymotrypsin (19, 20), and acetylcholinesterase (21). The attachment via side-chain termini most probably resulted

10.1021/bc980058r CCC: $15.00 © 1998 American Chemical Society Published on Web 10/02/1998

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in multiple point attachment of the macromolecules to the protein. Nevertheless, a dramatic increase in the acetylcholinesterase survival in the blood stream of mice (21) and in the thermostability of modified enzymes (19, 21) was observed when compared to the native proteins. The multipoint attachment of the polymers to protein has the disadvantage that it may cause unexpected crosslinking. The synthesis of semitelechelic poly[N-(2-hydroxypropyl)methacrylamide] (ST-PHPMA) permits onepoint attachment of HPMA macromolecules to surfaces and proteins, and avoids the possible cross-linking caused by multipoint attachment. Its covalent attachment to nanospheres resulted in surfaces with decreased biorecognizability (10). It appears that ST-PHPMA has potential as a protein or biomaterial surface-modifying agent. Consequently, this study is devoted to the synthesis of ST-PHPMA containing different functional groups, namely amino, carboxyl, N-hydroxysuccinimide ester, methyl ester, and hydrazide groups. These groups were introduced at one end of the polymer chains using functionalized mercaptans as chain-transfer agents or by polymeranalogous reactions of semitelechelic intermediates. The functionality and the molecular weight of STPHPMA were characterized by end-group determination and size exclusion chromatography (SEC). Carboxyl and amino group directed modifications of a model protein, R-chymotrypsin, with ST-PHPMA were performed. The impact of chemical modification on the enzymatic activity of ST-PHPMA-substituted chymotrypsins was determined using low and high molecular weight substrates. The macromolecules and conjugates have been characterized by classical methods (SEC, UV, protein content, and functional group determination) and by matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) (22). EXPERIMENTAL PROCEDURES

Chemicals. Phe and Leu were of L-configuration. HPMA (23) and the substrates Z-Gly-Leu-Phe-NAp (16) and HPMA copolymer attached Gly-Leu-Phe-NAp, P-GlyLeu-Phe-NAp (16) were prepared as described. R-Chymotrypsin from bovine pancreas was from Sigma, St. Louis, MO. 2,2′-Azobisisobutyronitrile (AIBN) was purified by recrystallization from ethanol. Methyl 3-mercaptopropionate, 3-mercaptopropionic acid, and 2-mercaptoethylamine (Aldrich, Milwaukee, WI) were used without further purification. Solvents and all other reagents were of reagent grade or better. Synthesis of 3-Mercaptopropionic Hydrazide. Methyl 3-mercaptopropionate (10 g, 83 mmol) was added dropwise to an excess of hydrazine monohydrate (10 g, 200 mmol) dissolved in 30 mL of methanol, and the reaction mixture was stirred at rt for 72 h. Methanol was removed under vacuum and the product separated by chromatography on a silica gel column eluted with methanol/diethyl ether (1:4). The fraction containing the product was collected and the solvent removed by rotoevaporation. The oily product, 3-mercaptopropionic hydrazide, was dried under vacuum; yield 8.3 g, (83%); Rf ) 0.62 (methanol/acetonitrile, 1:4). 1H NMR (CDCl3, ppm): 1.57 (t, 1H, SH), 2.43 (t, 2H, COCH2), 2.76 (m, 2H, CH2SH), 3.96 (sh, 2H, NH2), 7.55 (sh, 1H, NH). MS (scan ES+, m/z): 121.6 (M+ + 1). Synthesis of Semitelechelic Polymers. Semitelechelic HPMA polymers were prepared by free-radical solution polymerization in methanol using functionalized mercaptans as chain-transfer agents (17) and AIBN as the initiator. A typical synthesis is described below using

Lu et al. Table 1. Synthesis and Characterization of ST-PHPMA-COOSua Polymers original ST-PHPMAyieldb of COOH ratio ratio yield of conversion Mn [DCC]/ [HOSu]/ polymer COOH to code (SEC)c Mn(end)d [COOH] [COOH] COOSu (%) 1 2 3 4 5

5400 5400 4580 4340 3440

5510 5510 3800 3740 2680

5 17 14 20 20

5 20 14 20 20

86 76 54 90 89

50e 77b 80e 94b 75e

a HOSu, N-hydroxysuccinimide. b Yield determined after hydrolysis from released N-hydroxysuccinimide. c Mn, the number average molecular weight determined from size-exclusion chromatography. d Mn, the number average molecular weight determined from titration of COOH end groups. e Yield determined after polymeranalogous reaction with glycyl p-nitroanilide using  ) 1.3 × 104 M-1 cm-1 in H2O.

as an example the synthesis of a ST-PHPMA-COOH polymer. All polymers were isolated by precipitation as described below, except the ST-PHPMA-CONHNH2 polymers which were dialyzed against water using a membrane with molecular weight cut off (MWCO) 500 and isolated by freeze-drying. Synthesis of ST-PHPMA-COOH. HPMA (1.0 g, 7.0 mmol), 3-mercaptopropionic acid (22 mg, 0.21 mmol), and AIBN (50 mg, 0.3 mmol) were dissolved in 10 mL of methanol. The solution was bubbled with nitrogen and sealed in an ampule. The reaction mixture was stirred at 50 °C for 24 h. After polymerization, the volume of the reaction mixture was reduced, the polymer was isolated by precipitation into diethyl ether, filtered, and washed with diethyl ether. The product was purified by reprecipitation three times. The weight-average (Mw) and number-average (Mn) molecular weights and polydispersity were determined by size-exclusion chromatography (SEC) on a Superdex 75 HR 10/30 column calibrated with ST-PHPMA samples of narrow polydispersity (Mw/Mn < 1.1; molecular weight of calibration samples were determined by MALDI-TOF MS) using a Pharmacia FPLC system (PBS buffer, pH 7.3); Mn ) 5410, Mw ) 7530, Mw/Mn ) 1.4. The content of end groups determined by titration was 0.181 mmol/g. The numberaverage molecular weight [Mn(end)] calculated from the content of end groups was 5510; the ratio of molecular weights obtained from SEC and end group determination, Mn/Mn(end) ) 0.98. Fractionation of ST-PHPMA Polymers. The fractionation of ST-PHPMA polymers was performed by SEC on a Superdex 75 HR 16/60 column (PBS buffer, pH 7.3). For example, ST-PHPMA-NH2 in PBS buffer was applied on the column, and the polymer fractions were collected at different elution time intervals. The polymer fractions were desalted by dialysis, lyophilized, and characterized by MALDI-TOF MS and SEC on a Superdex 75 HR 10/ 30 column (PBS buffer, pH 7.3) using a Pharmacia FPLC system equipped with a refractive index detector. Polymeranalogous Transformations. Synthesis of ST-PHPMA-COOSu. ST-PHPMA-COOSu polymers were synthesized by the reaction of ST-PHPMA-COOH and N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide (DCC) in anhydrous DMF. Polymers with different properties were obtained under different synthetic conditions, and the yield and degree of the modification are reported in Table 1. The procedure will be demonstrated by the synthesis of polymer 3. ST-PHPMACOOH [Mn(end) ) 3800; 280 mg, 0.073 mmol] and Nhydroxysuccinimide (120 mg, 1.0 mmol) were dissolved

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Table 2. Characterization of ST-PHPMA-CONHNH2 Prepared by Hydrazinolysis of ST-PHPMA-COOCH3 ST-PHPMA-CONHNH2 code 6 7 8 9

ST-PHPMA-COOCH3 Mn(end)a Mn Mw 2000 3000 2900 4200

2510 2890 3480 4690

3160 3780 6340 8830

mg

hydrazine (mmol)

yield of polymer (%)

Mn(end)b

Mn

Mw

Mw/Mnc

Mn/Mn(end)

500 500 700 700

10 10 15 10

46 40 58 65

2250 2800 3330 5100

3020 3610 4830 6070

4237 5270 7930 10700

1.47 1.46 1.64 1.74

1.34 1.29 1.45 1.19

a Number average molecular weight estimated from proton NMR. b Number average molecular weight determined by modified TNBS assay. c Data from size exclusion chromatography.

in 5 mL of anhydrous DMF and cooled in an ice-water bath. DCC (200 mg, 1.0 mmol) in 3 mL of DMF was added dropwise with stirring. The mixture was kept at 4 °C for 48 h, and at rt for an additional 12 h. The volume of the reaction mixture was reduced under vacuum, dicyclohexyl urea (DCU) was removed by filtration, and the polymer solution was precipitated by dropping it into an excess of diethyl ether. The product (3) was purified by reprecipitation three times (methanol solution to diethyl ether) and dried under vacuum. Yield: 150 mg, 54%. The amount of active ester groups was determined by detecting the released N-hydroxysuccinimide anion after addition of an excess of 1-amino2-propanol; 260 nm ) 8500 M-1 cm-1 (24). The yield of active ester formation was 80%, based on the COOH content determined by titration. Synthesis of ST-PHPMA-CONHNH2 by Hydrazinolysis of ST-PHPMA-COOCH3. An alternate route of STPHPMA-CONHNH2 synthesis was the hydrazinolysis of ST-PHPMA-COOCH3. The ST-PHPMA-COOCH3 polymers used for hydrazinolysis, the yield and properties of ST-PHPMA-CONHNH2 are reported in Table 2. The synthesis of polymer 8 (Table 2) is shown as an example. ST-PHPMA-COOCH3 (0.7 g, 0.2 mmol) was dissolved in 5 mL of methanol and added dropwise to a 20 mL methanol solution of hydrazine monohydrate (0.75 g, 15 mmol) with stirring. The reaction mixture was refluxed for 5 h and concentrated by rotoevaporation. The product was isolated by precipitation into an excess of diethyl ether, dialyzed against deionized water (tubing, MWCO 500), and lyophilized; yield 0.41 g, 58%. The weightaverage (Mw), number-average (Mn) molecular weights, and polydispersity were determined by SEC on a Superdex 75 column calibrated with fractions of PHPMA (PBS buffer, pH 7.3) using a Pharmacia FPLC system, Mn ) 4830, Mw ) 7930, Mw/Mn ) 1.64. The content of hydrazide end groups was determined by the modified TNBS assay (0.300 mmol/g) (25). The number-average molecular weight [Mn(end)] based on the content of end groups was 3330, Mn/Mn(end) ) 1.45. Methods for End Group Determination. Determination of COOH Groups. ST-PHPMA-COOH samples were titrated with sodium hydroxide standard solution using an ABU80 autoburet and PHM84 research pH meter as monitor. Determination of COOCH3 Groups by Hydrolysis. ST-PHPMA-COOCH3 samples ( 1.1) when compared to SEC. There were attempts to improve the analysis of MALDI-TOF MS spectra of polydisperse samples (32); however, the best agreement between data from MALDI-TOF MS and traditional methods was obtained for polymer samples with a narrow polydispersity (31, 34). The analyses of ST-PHPMA polymers here also show that there is a good agreement of the average molecular weights between MALDI-TOF MS and SEC for polymers with a narrow polydispersity (PD < 1.1), and the average molecular weight of the polymers with high polydispersity (PD > 1.2) determined by the MS was lower than those determined by SEC. Table 5 lists the average molecular weights determined by SEC, MALDI-TOF MS for ST-PHPMA polymer fractions A and B, polymer-chymotrypsin conjugate (III), and some ST-PHPMA polymers. For polymer fractions (A and B) with a narrow polydispersity (Mw/Mn < 1.1), which were fractionated by SEC from polymer 13 (Table 5), both the number-average and weight-average molecular weights from the MS were only slightly lower than those from SEC, and the difference between two methods

was less than 7%. The molecular weights of the chymotrypsin-polymer conjugates determined by MALDI-TOF MS also agreed well with those determined by SEC equipped with an on-line light-scattering detector (MiniDawn, Wyatt). The difference between the numberaverage molecular weights (3%) was smaller than the difference between the weight-average molecular weights (14%). However, for polymers 10-13 with higher polydispersity (PD > 1.2), the molecular weights from MALDITOF MS were considerably lower than those obtained from SEC (using a column calibrated with fractions), because MALDI-TOF MS underestimated the contribution from higher molecular weight polymer chains (32). The difference between the molecular weights obtained from the two methods increased with increasing polydispersity. Although MALDI-TOF MS could not give the true shape of the molecular weight distribution for STPHPMA polymers with high polydispersity (33), the accurate determination of the molecular weight of individual polymer chains may provide very important information on the chemical structure of the end groups and the composition of the individual macromolecules of semitelechelic polymers (29, 30). Figure 5 shows the MALDI-TOF mass spectrum of STPHPMA-CONHNH2 (10; Table 5). The mass spectrum clearly showed the presence of HPMA polymer chains with different end groups in the polymer mixture. The peak-to-peak increment for the polymer chains with identical end groups was 143.2, the molar mass of HPMA. The peak series a at m/z ) (n × 143.2) + 23.0 (Na+) + 68.1 + 1.0 (n is the number of repeat units; mass of initiator residue Me2[CN]C ) 68.1) corresponds to the polymer chains initiated by isobutyronitrile (IBN) radicals and then terminated by a proton from the chain transfer agent. The sodium ion was used to cationize the polymer chains. The peak series b at m/z ) (n × 143.2) + 23.0 (Na+) + 120.2 (molar mass of 3-mercaptopropionic hydrazide ) 120.2 Da) represents the semitelechelic polymer chains H-(HPMA)n-SCH2CH2CONHNH2. There was an unknown series c at m/z ) (n × 143.2) + 23.0 (Na+) + 146.0 in the spectrum of ST-PHPMA-CONHNH2, whose origin is not known. One might suspect that these macromolecules are products of side reactions during polymerization. Figure 6 shows the MALDI-TOF mass spectrum of STPHPMA-COOH (11; Table 5) which was used, after esterification to ST-PHPMA-COOSu, for the synthesis of conjugate III. The spectrum contains the peak series a which corresponds to the polymer chains initiated by IBN radicals and then terminated by a proton from the chaintransfer agent (similar to that shown in the spectrum in Figure 5). The peak series d at m/z ) (n × 143.2) + 23.0 (Na+) + 106.1 (molar mass of mercaptopropionic acid ) 106.1) and e at m/z ) (n × 143.2) + 23.0 (Na+) + 128.1

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Figure 5. The MALDI-TOF mass spectrum (A) of ST-PHPMA-CONHNH2 (10; Table 5) and its expanded spectrum (B) in the region of 1300-1800 Da. IBN is the isobutyronitrile radical, formed by decomposition of AIBN; Hyz is the hydrazide end group; X is an unknown end group.

(molar mass of sodium mercaptopropionate ) 128.1) represents the peaks of the semitelechelic polymer chains of H-(HPMA)n-SCH2CH2COOH and H-(HPMA)n-SCH2CH2COONa, respectively. The latter peak series is due to the replacement of the proton in the carboxyl group by Na+ during the analysis by MALDI-TOF MS. It appears that the polymerization of HPMA in the presence of potent chain-transfer agents permits the synthesis of semitelechelic macromolecules. The polymerization method is based on the assumption that the reaction of the chain-transfer agent occurs with primary radicals formed by the decomposition of AIBN. However, this takes place only when a high excess of the chaintransfer agent is present. The participation of the chaintransfer reaction with growing polymer chains results in macromolecules containing the initiator residues at one end of the macromolecule and a saturated chain end, formed by hydrogen atom transfer, at the other (see peak series a in Figures 5 and 6). Moreover, for a given

composition of the polymerization mixture, the probability of the reaction of chain-transfer agent with primary radicals will decrease with time (conversion). It is important to note, that from the point of view of protein modification, macromolecules having the structure a are nonreactive, inert components of ST-PHPMA. Modification of Chymotrypsin with ST-PHPMA Polymers. Both carboxyl and amino groups can be used for the modification of proteins with semitelechelic polymers. The selection of the modification mode may be important for the biological activity of the proteinpolymer conjugate. For example, Sakam and Pardridge (35) have shown that carboxyl-directed pegylation of brain-derived neurotrophic factor preserved the biological activity of the conjugate, whereas the amino group directed modification did not. Pettit et al. (36) have shown that amino group directed pegylation of interleukin-15 alters the biological activity of the conjugate. There are 17 carboxyl groups and 17 amino groups in

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Figure 6. The MALDI-TOF mass spectrum (A) of ST-PHPMA-COOH (11; Table 5) and its expanded spectrum (B) in the region of 1500-1900. IBN is the isobutyronitrile radical, formed by decomposition of AIBN.

R-chymotrypsin. ST-PHPMA-CONHNH2 and ST-PHPMACOOSu were used for the carboxyl and amino group directed modification of chymotrypsin, respectively. The amino-directed modification of proteins with polymers containing active ester groups is the most frequently used method. The carboxyl group directed modification with polymers containing hydrazo groups is a new method, which was first introduced by Zalipsky (3, 25, 37). The difference of the pKa of hydrazide (pKa ≈ 3.0) and the amino groups of proteins (pKa ) 6.8-8.0 for R-amino, 10.4-11.1 for -amino of lysine) makes it possible to react the hydrazo groups with the carboxyl groups of proteins in the presence of water soluble carbodiimide at mildly acidic pH, while the amino groups on the same proteins remain deactivated due to protonation. Conjugates I, II, III, and IV have been prepared by the reaction of the amino groups on R-chymotrypsin with ST-PHPMA-COOSu nos. 2, 4, 5, and 5 listed in Table 2,

respectively. This is a relatively easy reaction. To achieve a high yield of conjugation, a proper pH of the reaction mixture has to be chosen based on the pH dependence of the rate constants of hydrolysis and aminolysis. All the conjugates were characterized by MALDI-TOF MS, and Figure 1 shows the MALDI-TOF mass spectrum of conjugate III. The average molecular weights of the conjugates calculated from the MS are listed in Table 6. Conjugate V has been prepared by the reaction of chymotrypsin with a large excess of ST-PHPMA-CONHNH2; the molar ratio of chymotrypsin to polymer was about 1:120. The conjugation reaction was carried out at pH 4.5-5.0 in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and the polymer chains were attached to carboxyl groups of chymotrypsin via hydrazide bonds. Since carboxyl groups were consumed by conjugation, dilute HCl was added to

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Table 6. Enzymatic Activity of Polymer Modified r-Chymotrypsins. Kinetics of Cleavage of Low Molecular Weight and Polymeric p-Nitroanilides conjugate

polymer conjugate conjugate conjugation Mn(end)a Mnb Mwb degree (%)c

native chymotrypsin I

5510

44 900

46 600

21(22)

II

3740

49 400

51 000

38(43)

III

2680

40 900

41 700

35(34)

IV

2680

36 100

37 800

25(25)

V

2490

46 600

47 800

48

bond moded

kinetic parameters Z-GLF-NAp P-GLF-NAp

kcat (s-1) KM (M) kcat/KM (M-1 s-1) P-CONH-E kcat (s-1) KM (M) kcat/KM (M-1 s-1) P-CONH-E kcat (s-1) KM (M) kcat/KM (M-1 s-1) P-CONH-E kcat (s-1) KM (M) kcat/KM (M-1 s-1) P-CONH-E kcat (s-1) KM (M) kcat/KM (M-1 s-1) P-NHNHCO-E kcat (s-1) KM (M) kcat/KM (M-1 s-1)

0.83 3.9 × 10-4 2140 0.24 5.5 × 10-5 4400 0.27 6.5 × 10-5 4200 0.21 5.0 × 10-5 4200 0.18 4.5 × 10-5 4000 0.34 3.0 × 10-4 1100

0.94 1.3 × 10-3 720 0.91 2.2 × 10-3 410 0.34 5.7 × 10-4 600 0.65 1.75 × 10-4 370 0.55 1.1 × 10-3 480 0.64 1.6 × 10-3 410

a Number average molecular weight determined by end group analysis. b Determined by MALDI-TOF MS. c The conjugation degree was calculated from Mn of the conjugate and the Mn(end) of the polymers; the data in the parentheses were obtained from TNBS assay.d P represents polymer, and E the enzyme.

maintain the pH at 4.5-5.0. The MALDI-TOF mass spectrum of conjugate V (Figure 2) showed a broad peak of the conjugate because of the random conjugation of macromolecules with different molecular weights to the enzyme. The main peak around m/z ) 45 000 reflects the single charged conjugate, whereas the small peak around m/z ) 22 000 corresponds to the double charged conjugate. The average molecular weights calculated from the MS are listed in Table 6. Chymotrypsin was also modified with narrow fractions of ST-PHPMA-CONHNH2, and conjugates VI, VII, VIII, and IX were prepared by conjugation with the polymer fractions (Table 3). In the preparation of conjugates VI and IX, the polymer enzyme molar ratio used was lower than that used in the preparation of conjugates V, VII, and VIII, and a larger excess of the coupling agent EDC was used to achieve a high conjugation degree. The results indicated that the larger excess of EDC was helpful when the polymer enzyme molar ratio was lower. The molar ratio in the preparation of conjugate IX was only 23:1, but the conjugation degree obtained was the same as that of conjugate VIII, which was prepared with the molar ratio of 100:1. All the conjugates prepared with the polymer fractions were characterized by SEC and MALDI-TOF MS, and the mass spectrum of conjugate VI is shown in Figure 3. The calculated average molecular weights of the conjugates and the average number of polymer chains attached to each chymotrypsin molecule were calculated from the MS and reported in Table 3. As expected, conjugates prepared from the polymer fractions possessed a more uniform structure than those prepared from unfractionated semitelechelic macromolecules. The MALDI-TOF MS of the conjugates also indicated that there was no cross-linking of proteins under the mildly acidic reaction conditions used, and no peak of protein-protein conjugate was found in the spectrum (see Figure 3 as an example). The molecular mass of the protein-protein conjugate would be about 50 000 Da (molar mass of R-chymotrypsin is 25 240 Da) if there was cross-linking between protein molecules. The molecular mass of most of the conjugates is well below 50 000 Da. Even when a larger excess of EDC and a lower polymer enzyme molar ratio was used, no evidence of protein-protein cross-linking was shown in the SEC and MALDI-TOF MS of the conjugate IX. The number

average and weight average molecular weights of the conjugate were 45 600 and 46 200, lower than the molecular weight of a possible protein-protein conjugate. It seems that about 10 is the maximum number of STPHPMA polymer chains which can be attached to one chymotrypsin molecule. It appeared, however, that the molecular weight of the ST-PHPMA played a role. The results in Table 3 indicated that the molecular weight of the polymer fractions affected the conjugation degree of the conjugates. Lower molecular weight polymer fractions produced conjugates with a higher conjugation degree, because the smaller spherical size of the lower molecular weight fraction possessed a lower sterical exclusion effect. The influence of the properties of the ST-PHPMA on the distribution of molecular weights of the R-chymotrypsin conjugates was demonstrated by MALDI-TOF mass spectra (compare Figure 3A with Figures 5 and 6). The conjugation degree of all conjugates was calculated from the Mn of the conjugates and the Mn(end) of the polymers (Table 6). The conjugation degrees of the amino group directed conjugates were also determined by the TNBS assay. The conjugation degrees obtained from both methods were in good agreement. Depending on the conjugation conditions and on the structure of the STPHPMA, there were about 3-9 polymer chains attached to each enzyme molecule. The number of chains attached depended on the ratio of the enzyme to the polymer in the reaction mixture as well as on the molecular weight of the ST-PHPMA used. A higher concentration ratio of polymer to enzyme gave conjugates with a higher conjugation degree; see, for example, conjugates III and IV. For conjugates I and III, the same reaction conditions and the same molar ratios were used; ST-PHPMA 5, with a lower molecular weight, gave a higher conjugation degree in the conjugate III when compared to STPHPMA 2 and conjugate I. In conjugates VI and VII, the relatively high conjugation degree (∼50%) was achieved when a high molar ratio of polymer to enzyme, and a low molecular weight narrow fraction of STPHPMA-CONHNH2 was used in the conjugation. When the polymer enzyme molar ratio was low in the carboxyl directed modification, a much larger excess of coupling agent was necessary to achieve a relatively high conjugation degree.

Functionalized Semitelechelic PolyHPMA

The conjugation degree of ST-PHPMA with chymotrypsin was lower (about 9 vs 14 chains) than that of the chymotrypsin conjugates with PEG-SC (16). This might be attributed to the solution structure difference between the two polymers. PHPMA has a random coil structure in aqueous solution, while PEG possesses a more extended one. The coiled PHPMA may cover more surface of the enzyme, and the steric effect may prevent more PHPMA macromolecules from attaching to the enzyme. One may hypothesize that PHPMA may cover a larger area of the protein than the extended macromolecules per modifying chain of a particular molecular weight. The impact of different shapes of macromolecules on the protein repulsion characteristics of a modified biomolecule or biomaterial surface needs careful evaluation. Activity of ST-PHPMA-Chymotrypsin Conjugates. The activity of ST-PHPMA-chymotrypsin conjugates for enzymatically catalyzed hydrolysis of peptide substrates was investigated. Native R-chymotrypsin was used as control; Z-Gly-Leu-Phe-NAp and P-Gly-Leu-PheNAp were the substrates. The Michealis-Menten kinetic constants of the enzymatic cleavage reaction of the substrates were calculated from Lineweaver-Burk plots of the initial rates of the release of p-nitroaniline and are listed in Table 6. Both the chymotrypsin conjugates and the native chymotrypsin were active for the cleavage of the substrates. All conjugates and the native enzyme exhibited a high activity for Z-Gly-Leu-Phe-NAp and a lower activity for P-Gly-Leu-Phe-NAp. The steric hindrance of the polymer chain of P-Gly-Leu-Phe-NAp renders the formation of the enzyme-polymer substrate complex more difficult, resulting in lower turnover rates, in agreement with previously published data (20). For the cleavage of Z-Gly-Leu-Phe-NAp, amino group modified conjugates I, II, III, and IV showed higher reactivities than the native enzyme, similar to literature data (11, 16). Carboxyl group modified conjugate V showed a lower activity toward Z-Gly-Leu-Phe-NAp than the native enzyme. This indicated that the type of bond between the enzyme and the polymers affects the activity of the conjugates, at least for the low molecular weight substrate, as observed by modification of other proteins (35, 36). It is known that a carboxyl group of an aspartic acid was involved in the active site of chymotrypsin (38). In addition, the conjugation of a carboxyl group in the vicinity of the enzyme active site might cause the decrease of the activity of the carboxyl group-modified chymotrypsin conjugates. The activity of chymotrypsinST-PHPMA conjugates to catalyze the hydrolysis of the polymer-bound substrate, P-Gly-Leu-Phe-NAp, was similar and lower than the native enzyme with small variations. Conjugate II possessed a slightly higher activity than the average for all the conjugates. It appears that the conjugation degree and the molecular weight of the polymers did not have a pronounced effect on the activity of chymotrypsin-ST-PHPMA conjugates toward P-GlyLeu-Phe-NAp. These data seem to indicate that the sterical hindrance that occurs upon the formation of the enzyme-polymer substrate complex is the leading cause of the decreased potential for enzyme-substrate formation. ACKNOWLEDGMENT

The research was supported in part by Amgen, Inc., Thousand Oaks, CA.

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