Preparation and Characterization of Poly(ethylene glycol) Vinyl Sulfone

and Department of Chemistry, University of Alabama in Huntsville, Huntsville, Alabama 35899. Received September 30, 1995X. Synthesis of the vinyl sulf...
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Bioconjugate Chem. 1996, 7, 363−368

363

Preparation and Characterization of Poly(ethylene glycol) Vinyl Sulfone Margherita Morpurgo,† Francesco M. Veronese,† David Kachensky,‡ and J. Milton Harris*,§ Departmento di Scienze Farmaceutiche, Centro di Studio di Chimica del Farmaco e dei Prodotti Biologicamente Attivi del CNR, Universita di Padova, Padova, Italy, Synergen, Inc., 1885 33rd Street, Boulder, Colorado 80301, and Department of Chemistry, University of Alabama in Huntsville, Huntsville, Alabama 35899. Received September 30, 1995X

Synthesis of the vinyl sulfone and chloroethyl sulfone derivatives of poly(ethylene glycol) (PEG) is described. The chloroethyl sulfone (CES-PEG) is rapidly converted to the vinyl sulfone (VS-PEG) in the presence of base but is stable in water at neutral pH. Reactions with small molecules such as β-mercaptoethanol and NR-acetyllysine show that the vinyl sulfone derivative is highly selective for reaction with sulfhydryl groups relative to reaction with amino groups. Also, VS-PEG is stable in water. These properties indicate that VS-PEG should be useful for selective attachment of PEG to protein cysteine groups. This hypothesis was verified by reacting VS-PEG with cysteine groups of reduced ribonuclease (RNase); the reaction is rapid and selective at pH 7-9. Reaction at lysine sites of unreduced RNase occurs slowly at pH 9.3 and is essentially complete after 100 h. Amino acid residues other than lysine and cysteine are not reactive toward VS-PEG. The covalent linkage between VS-PEG and lysine or cysteine groups is shown to be stable.

INTRODUCTION

EXPERIMENTAL PROCEDURES

(PEG)1

Poly(ethylene glycol) derivatives have wide utility for a variety of biomedical and biotechnical applications (1-8), and interest continues in the synthesis of new PEG derivatives that possess novel properties (1, 9-21). Selective modification of cysteine groups is especially desirable since these groups can be introduced into specific locations in a protein, with the result that the PEG attachment site and extent of modification (i.e., “PEGylation”) are known and controlled (17). In the present work we describe the synthesis and characterization of vinyl sulfone PEG (VS-PEG) and its precursor chloroethyl sulfone PEG (CES-PEG). These derivatives are of interest because previous work has shown that small compounds such as methyl and ethyl vinyl sulfone are selective for reaction with sulfhydryl groups relative to reaction with amino groups (22, 23), and as noted above, there is much interest in selective coupling of PEG to cysteine groups of proteins (14-21). Thus, it is of interest to determine if VS-PEG can offer advantages over the available sulfhydryl-selective PEGs such as maleimide, dithiopyridine, and iodoacetamide. In the following we describe the synthesis of CES-PEG and VS-PEG, and we report rates for reaction of CESPEG and VS-PEG with small thiols, small amines, the cysteine-containing tripeptide glutathione, and native and reduced RNase. The stability of the reagents and the products in aqueous medium is also determined. * Author to whom correspondence should be addressed. † Universita di Padova. ‡ Synergen, Inc. § University of Alabama. X Abstract published in Advance ACS Abstracts, April 1, 1996. 1 Abbreviations: PEG, poly(ethylene glycol); VS-PEG, vinyl sulfone derivative of PEG; CES-PEG, chloroethyl sulfone derivative of PEG; MAL-PEG, maleimide derivative of PEG; RNase, bovine pancreatic ribonuclease; ALMe, NR-acetyl-L-lysine methyl ester; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); DTDP, 4,4′-dithiodipyridine; TEA, triethylamine; TNBS, trinitrobenzenesulfonic acid; mPEG, methoxypoly(ethylene glycol) (MW 5000).

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Bovine pancreatic ribonuclease (RNase), NR-acetyl-Llysine methyl ester (ALMe), N-acetyl-L-cysteine, 5,5′dithiobis(2-nitrobenzoic acid) (DTNB), fluorescamine, glutathione, methanesulfonyl chloride, and hydroin buffers were purchased from Sigma (St. Louis, MO). The maleimide derivative of PEG (MAL-PEG) was purchased from Shearwater Polymers (Huntsville, AL). β-Mercaptoethanol was obtained from Merck (Rahway, NJ). 4,4′Dithiodipyridine (DTDP), triethylamine (TEA), trinitrobenzenesulfonic acid (TNBS), methoxypoly(ethylene glycol) (MW 5000) (mPEG), and p-nitrophenyl chloroformate were purchased from Aldrich (Milwaukee, WI). Proton NMR spectroscopy was performed with a Bruker 200 MHz or a Varian Gemini 200 MHz instrument. Ultraviolet spectroscopy was perfomed with a HewlettPackard diode array or a Perkin-Elmer λ2 instrument, and fluorescence was measured on a Perkin-Elmer LS5B spectrometer. Ultrafiltration was performed with an Amicon system and a PM 10 membrane (cutoff 10 000 Da). Gel filtration chromatography was performed on a Pharmacia FPLC system using a Superose 12 TM column and a UV detector. Synthesis of mPEG Chloroethyl Sulfone (CESPEG). The synthesis involves four steps. First, mPEG mesylate was prepared by a modification of the method of Harris et al. (24). A toluene solution (150 mL) of mPEG 5000 (15 g) was dried by azeotropic distillation. After cooling, 50 mL of dry dichloromethane, 250 µL (1.06 equiv) of methanesulfonyl chloride, and 440 µL (1.06 equiv) of triethylamine were added while the solution was cooled in an ice bath. After 20 h, the solution was filtered, the volume reduced to 20 mL, and the product precipitated by addition of dry diethyl ether. The solid mesylate was dried in vacuo: 1H NMR (dimethyl sulfoxide-d6) 3.17 ppm (s, 3H, -SO2CH3), 3.23 ppm (s, 3H, CH3O-), 3.49 ppm (s, mPEG backbone, -OCH2-), 4.30 ppm (t, 2H, -CH2OSO2-). In the second step, the mesylate was reacted with β-mercaptoethanol to form mPEG hydroxyethyl sulfide. Mesylate (14 g) was dissolved in 150 mL of distilled water, and 770 µL (4 equiv) of β-mercaptoethanol and © 1996 American Chemical Society

364 Bioconjugate Chem., Vol. 7, No. 3, 1996

5.5 mL (4 equiv) of 2 N NaOH solution were added. The reaction was stirred at reflux for 3 h. After cooling, the product was extracted three times with dichloromethane, and the collected organic fractions were dried over anhydrous sodium sulfate. The volume was reduced to 20 mL, and the product was precipitated by addition to dry diethyl ether: 1H NMR (dimethyl sulfoxide-d6) 3.23 ppm (s, 3H, CH3O-), 2,57 ppm (t, 2H, -SCH2-), 2.65 ppm (t, 2H, -CH2OH), 3.49 ppm (s, mPEG backbone, -OCH2-), 4.76 ppm (t, 1H, -OH). In the third step, the sulfide was oxidized to produce mPEG hydroxyethyl sulfone. Thirteen grams of the sulfide was dissolved in 20 mL of 0.123 M, pH 5.6, tungstic acid solution, and 15 mL of distilled water was added. After cooling to 0 °C, 1.04 mL of 30% hydrogen peroxide was added. The reaction was carried out overnight, and the product was extracted three times with dichloromethane. The collected organic fractions were dried over anhydrous sodium sulfate, the volume reduced to 20 mL by rotary evaporation, and the product precipitated by addition to dry diethyl ether: 1H NMR (dimethyl sulfoxide-d6) 3.23 ppm (s, 3H, CH3O-), 3.25 ppm (t, 2H, -CH2SO2-), 3.37 ppm (t, 2H, -SO2CH2-), 3.49 ppm (s, mPEG backbone), 3.77 ppm (t, 2H, -CH2OH), 5.04 ppm (t, 1H, -OH). In the fourth step, the hydroxysulfone is converted to the chloride by reaction with thionyl chloride. Hydroxysulfone (13 g) was stirred overnight in 35 mL of freshly distilled (over quinoline) thionyl chloride. Excess thionyl chloride was removed by distillation. Two hundred milliliters of toluene was added and removed by distillation twice. The final product was obtained by solution in dry dichloromethane followed by precipitation upon addition to dry ethyl ether: 1H NMR (dimethyl sulfoxided6) 3.23 ppm (s, 3H, CH3O-), 3.49 ppm (s, mPEG backbone), 3.64 ppm (t, 2H, -CH2SO2-), 3.79 ppm (t, 2H, -SO2CH2-), 3.94 ppm (t, 2H, -CH2Cl); yield 90%; purity >95% from NMR. Synthesis of mPEG Vinyl Sulfone (VS-PEG). The chloroethyl sulfone (CES-PEG) is readily converted to the vinyl sulfone (VS-PEG) by reaction with a variety of bases. For example, solution of 2 g of CES-PEG 5000 in 25 mL of dichloromethane containing 2 equiv of triethylamine, followed by addition to 50 mL of dry diethyl ether, and vacuum drying, produced 1.8 g of VS-PEG: 1H NMR (dimethyl sulfoxide-d6) 3.23 ppm (s, 3H, CH3O-), 3.49 ppm (s, mPEG backbone), 3.74 ppm (t, 2H, -CH2SO2-), 6.21 ppm (m, 2H, dCH2), 6.97 ppm (m, 1H, -SO2CHd). The degree of end group conversion, as shown by NMR, was 100%. The extent of end groups converted to vinyl sulfone can also be estimated by reaction of VS-PEG with excess mercaptoethanol followed by titration of remaining mercaptoethanol with DTDP. However, it should be noted that a weakness of this method is that the basic conditions used in the analytical method could convert any remaining CES-PEG to VS-PEG. VS-PEG was added under stirring to a 2 mM solution of β-mercaptoethanol in 0.1 M borate buffer (pH 9.5) containing 5 mM EDTA to minimize disulfide formation. After 15 min of stirring to complete reaction, 2 mM DTDP was added and stirred for 5 min. The absorbance was measured at 324 nm. The excess β-mercaptoethanol can be calculated from the 4-thiopyridone extinction coefficient of 19 800 M-1 cm-1 (25). This method also shows that the degree of end group conversion in synthesis of VS-PEG is 100%. Kinetic Studies of VS-PEG and CES-PEG Reaction with Thiols. The reactivity of VS-PEG and CESPEG toward thiols was evaluated by monitoring thiol concentration with DTDP, as described in the preceding

Morpurgo et al. Table 1. Approximate Rate Constants for Reaction of CES-PEG with Model Thiols and Amines at 21 ( 1 °C substrate

pH k2 (M-1 min-1) k1 (min-1) t1/2 (min)

thiolsa β-mercaptoethanol 8.0 9.0 9.5 N-acetylcysteine 8.0 8.5 9.0 glutathione 8.0 8.5 9.0 reduced RNase 8.0 9.0 amined NR-acetyllysine 8.0 methyl ester 9.5

1.5 fastb very fastc 1.0 1.4 8.9 2.1 3.2 15 3.5 14

0.029 0.020 0.028 0.18 0.041 0.065 0.30 0.069 0.27

no reaction 0.76

24 ≈1 ,1 34 25 3.9 17 11 2.3 10 2.6 1 day

0.023

30

a

Error limits (3%. b Reaction over within 15 min. c Reaction over within 2 min. d Error limits (7%.

paragraph (25), or with Ellman’s assay (26). To obtain first-order kinetics, the mPEG derivative was used in 10 times the concentration of the thiol. The goal of these studies was to obtain qualitative kinetics, so the reactions were conducted under ambient conditions (21 ( 1 °C) rather than in a constant temperature bath. Four different thiols were studied: β-mercaptoethanol, Nacetyl-L-cysteine, glutathione, and reduced RNase. Rate constants for these reactions are given in Table 1. To measure the reactivity of VS-PEG and CES-PEG with β-mercaptoethanol, the PEG derivative was added, under stirring, to a 2 mM thiol solution in 0.1 M buffer containing 5 mM EDTA. At scheduled times 50 µL aliquots of the reaction mixture were removed and quenched by addition to 950 µL of pH 7.2 buffer (0.1 M phosphate, 5 mM EDTA), followed by addition to 1.0 mL of 2 mM DTDP. After 5 min, the absorbance at 324 nm was measured. The ln of absorbance was plotted versus reaction time, and second-order rate constants were calculated by dividing the slopes of these plots by the derivative concentration (20 mM). Kinetics of reaction with N-acetyl-L-cysteine and glutathione were monitored with Ellman’s assay. A solution of 20 mM PEG derivative and 2 mM thiol was prepared in 0.1 M buffer (containing 5 mM EDTA). Aliquots (30 µL) were withdrawn and quenched by addition to 920 µL of pH 7.27 buffer (0.1 M phosphate, 5 mM EDTA). Fifty microliters of DTNB was added, and after 5 min, the absorbance at 412 nm was measured. Second-order rate constants were obtained by dividing the slope of the ln A versus time plot by the concentration of the derivative. The reactivity of PEG derivatives with reduced RNase was measured as follows. Protein (MW 13 700, 14 mg) was dissolved in 300 µL of 25% β-mercaptoethanol in 4.5 M guanidinium chloride, and the solution was heated at 100 °C for 5 min. The reduced protein (four cystine disulfide bonds reduced to eight cysteine thiol groups) was isolated from excess β-mercaptoethanol by gel filtration chromatography using pH 4.3 buffer as eluent (0.05 M Tris/ethyl acetate, 0.2 M guanidinium chloride). The protein fractions were concentrated by ultrafiltration and the thiol content of the sample measured by Ellman’s assay. Kinetic measurements were made, as described in the previous paragraph, using 2 mM thiol and 20 mM PEG derivative. Kinetic Studies of VS-PEG and CES-PEG Reaction with Amines. The reactivity of VS-PEG and CESPEG toward amines was evaluated by monitoring amine concentration with fluorescamine assay (27-29) or TNBS

Bioconjugate Chem., Vol. 7, No. 3, 1996 365

Preparation of PEG Vinyl Sulfone Table 2. Degree of Lysine Substitution by Reaction of VS-PEG with Unreduced RNase (100 h, pH 9.3, Room Temperature) As Measured by TNBS Assay and Amino Acid Analysis ratio of protein NH2 to VS-PEGa

degree (%) of substitutionb by TNBS

degree (%) of substitutionb by amino acid analysis

1:1 1:6 1:10

25 75 89

28 68 76

a Amine concentration 0.59 M. b Eleven lysines are available for substitution.

Figure 1. Reaction between reduced RNase thiol groups and VS-PEG as evaluated from disappearance of free cysteines. The reaction was carried out at room temperature, pH 8.0 ([) and 9.0 (9). [SH] ) 0.81 mM; [VS-PEG] ) 8.1 mM.

groups of unreduced RNase was determined by monitoring amine concentration with the TNBS assay (Figure 2) (30); RNase has 11 lysine groups and the N-terminal amine with which to react (31). Several tubes were prepared, each containing 600 µL of RNase solution (1.35 mg/mL in 0.2 M, pH 9.3, borate buffer). Different aliquots (50, 300, and 500 µL) of CES-PEG solution (66.6 mg/mL in the pH 9.3 borate buffer) were added to obtain molar ratios between protein amine and VS-PEG (produced by reaction of CES-PEG with pH 9.3 buffer) of 1:1, 1:6, and 1:10, respectively. The final volume of each sample was then adjusted to 1.2 mL with buffer. A control tube was also prepared without CES-PEG addition. Aliquots were withdrawn at scheduled times and analyzed by using the TNBS assay. Degree of RNase Lysine Substitution. The extent of modification of RNase lysine groups by VS-PEG after approximately 100 h of reaction at pH 9.3 (as described in the preceding section) was determined both by TNBS assay (as described above) and by amino acid analysis (Table 2) (32). For amino acid analysis, equal volumes of 12 N HCl and phenol (final concentration, 0.5%) were added to each tube, and amino acid analysis was performed after 24 h of hydrolysis at 110 °C in closed vials. The amino acid composition of each modified sample was compared to that of the native one. Control experiments showed that VS-PEG attaches irreversibly to lysine groups, so the reduction in lysine content corresponds to modification extent. Stability of VS-PEG and CES-PEG in Aqueous Buffer. The lifetime of VS-PEG and CES-PEG at pH 7.0 and 9.0 was investigated by NMR examination using D2O as solvent. A 5% solution of CES-PEG in D2O was prepared, and the pH was adjusted by adding a small amount of the appropriate hydroin buffer powder. NMR experiments were carried out at scheduled times to follow the appearance of VS-PEG and mPEG hydroxysulfone. RESULTS AND DISCUSSION

Preparation of CES-PEG involves four steps: Figure 2. Reaction between native RNase and VS-PEG evaluated from disappearance of protein lysine groups. Lysine:VSPEG molar ratios 1:1 ([), 1:6 (9), 1:10 (2) were used. The reaction was carried out at room temperature and pH 9.3 ([NH2] ) 0.59 mM).

(30). To obtain first-order kinetics, the mPEG derivative was used in 10 times the concentration of the amine. Reactions were conducted under ambient conditions (21 ( 1 °C). Two different amines were studied: NR-acetylL-lysine methyl ester (ALMe) and native RNase. Rate constants for the small molecule ALMe are given in Table 1, and reactivity data for native RNase are give in Table 2 and Figure 2. Reactivity with ALMe was measured by following amine disappearance with the fluorescamine assay (2729). A 10-fold excess of PEG derivative was added under stirring to 0.3 mM ALMe solution in the appropriate buffer (0.1 M phosphate, pH 8, or 0.1 M borate, pH 9.5). A 50 µL aliquot was withdrawn and quenched at scheduled times by addition to 2.95 mL of 0.1 M, pH 8.0, phosphate buffer. Fluorescamine solution (1.0 mL, at a concentration of 0.3 mg/mL in acetone) was added with vigorous mixing. Fluorescence was measured after 10 min (λexc ) 390 nm, λem ) 475 nm). Second-order rate constants were obtained from the slope of the plot of ln fluorescence emission intensity versus time, divided by PEG derivative concentration. The reactivity of VS-PEG and CES-PEG with amino

Et3N mPEG-OH + CH3SO2Cl 98 mPEG-O3SCH3 (1) NaOH mPEG-O3SCH3 + HSCH2CH2OH 98 mPEG-SCH2CH2OH (2) mPEG-SCH2CH2OH + H2O2 f mPEG-SO2CH2CH2OH (3) mPEG-SO2CH2CH2OH + SOCl2 f mPEG-SO2CH2CH2Cl (4) The chlorosulfone is readily converted to VS-PEG by exposure to any of a variety of bases:

mPEG-SO2CH2CH2Cl + base f mPEG-SO2CHdCH2 (5) This final elimination step occurs rapidly at pH 9.0. A solution of CES-PEG in pH 9.0 buffer, followed within 2-3 min by NMR examination, showed only signals for VS-PEG. In contrast, a solution of CES-PEG in water at pH 7 showed only 4% VS-PEG after 48 h of incubation at room temperature; no hydroxysulfone was produced. VS-PEG was shown by these same NMR experiments to

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be stable indefinitely at pH 7 and 9. This water stability of VS-PEG is highly unusual for “activated” PEGs suitable for protein modification, and such stability is of great utility for large-scale preparations and for heterofunctional reagents, one of the functional groups of which must survive during coupling of the other functional group (1, 15, 21, 33, 34). Similarly, the stability of CESPEG at neutral pH provides a means of protecting the vinyl sulfone group. Isolation of VS-PEG is provided by solution of CESPEG in methylene chloride containing 2 equiv of triethylamine, followed by precipitation of the product by addition to diethyl ether. The efficiency of each of the above synthetic steps is readily monitored by NMR characterization of the products. It was found that all of the synthesis steps were very efficient, yielding almost complete functionalization and over 90% recovery. NMR examination of the final VS-PEG showed no other functional groups. Similarly, addition of excess mercaptoethanol, followed by titration of mercaptoethanol with dithiodipyridine, showed 100% vinyl sulfone. Experimental details are given for mPEG 5000, but we have found that the same chemistry works well for preparation of VS-PEG of molecular weights up to 20 000. Since VS-PEG and CES-PEG are intended for selective reaction with thiol groups relative to reaction with amino groups, we have measured rates of reaction, at room temperature and in aqueous solution, of these derivatives with thiols and primary amines (Table 1). The majority of the experiments were conducted by solution of CESPEG in basic solution, so that VS-PEG is formed in situ. As model thiols we used β-mercaptoethanol, N-acetyllcysteine, glutathione, and reduced RNase. NR-Acetyl-Llysine methyl ester and unreduced, native RNase were used as model amines. As can be seen from Table 1, reaction of VS-PEG with thiols at pH 8 takes place with half-lives of from 10 to 34 min. This rather large range of reactivities indicates that reaction of the large VS-PEG is sensitive to the steric environment of the thiol groups. Indeed, we have observed an even larger range of reactivity toward protein cysteine groups (35). An increase in pH to 9 gives an enhancement of VS-PEG reaction rate with thiols and reduces half-lives to 1-4 min (Table 1). At pH 9.5 reaction is too fast to measure. The extent of reaction with reduced RNase is illustrated in Figure 1. Here it is clear that cysteine modification is almost complete within 20 min at pH 9 and within 1 h at pH 8. Reaction of VS-PEG is also effective at pH 7. Although we did not measure rate constants at pH 7, qualitative experiments were performed. Equimolar amounts (0.01 M) of VS-PEG and N-acetyllcysteine were reacted at pH 7 and room temperature, and the reaction was monitored by the DTDP assay for free thiol. After 30 min, most thiol had reacted, and after 1.5 h, only slight amounts of thiol could be detected (data not shownsexperiment repeated three times). It is important to note that protein modification would normally be done with an excess of VS-PEG, so extensive reaction would occur within 30 min. The VS-PEG conjugate with N-acetylcysteine was isolated by extracting into methylene chloride, drying with magnesium sulfate, and precipitating by adding this solution to diethyl ether. Solution of the pure product in deuterated water, and subsequent NMR examination, showed the conjugate to be stable indefinitely in aqueous solution (data not shown). In contrast to thiol reactivity, reaction of VS-PEG with primary amine groups is very slow at pH 8, with no reaction detected for reaction of NR-acetyllysine methyl

Morpurgo et al.

ester after 24 h (Table 1). Reaction with amino groups is much faster at pH 9.5 but still is quite slow compared to reaction with thiols (half-life of 30 min for amine reaction versus a thiol reaction that is too fast to measure). Thus, VS-PEG is highly selective for reaction with thiols, and this selectivity, plus the water stability of VS-PEG, shows that this derivative is suitable for selective modification of protein cysteine groups. Despite the low reactivity of VS-PEG with amines, this derivative can be used for attachment to protein amino groups. Thus, we have found that VS-PEG gives 40% modification of lysine groups of unreduced RNase after 30 h if a 10 to 1 molar excess of VS-PEG to lysine groups is used at pH 9.3 (Figure 2). Reaction for approximately 100 h gives even more extensive modification (Table 2). Examination of the time course of this reaction, Figure 2, shows that the reaction is significantly slower than for reaction with the small molecule NR-acetyl-L-lysine methyl ester. Presumably this is a reflection of the bulk of VS-PEG and steric shielding of lysine groups in the protein. Although this low reactivity of VS-PEG with protein lysine groups might appear to preclude general utilization of this reaction for protein PEGylation, the reaction could be used for introduction of a small number of PEGs into a conjugate. Presumably the more reactive amino groups will be modified first, so the slow PEGylation provided by VS-PEG could possibly be used for selective modification of lysine groups. The extent of lysine modification was measured by two routes, the TNBS assay and amino acid analysis. The TNBS assay depends on colorimetric detection of reaction of TNBS with available lysine amino groups. Amino acid analysis is based on a reduction in the number of lysine groups that are found after protein hydrolysis. For this method to succeed, it is necessary that the lysine-ethyl sulfone linkage formed upon attachment of VS-PEG to amine be sufficiently durable to survive hydrolysis of the protein. Since the degrees of lysine modification as measured by TNBS and amino acid analysis are in reasonable agreement (Table 2), it is apparent that this linkage is very durable. Lower values for modification extent are found from amino acid analysis than from TNBS assay at high degrees of modification, but colorimetric methods such as the TNBS assay are known to overestimate the degree of PEGylation (32). For example, overestimation by colorimetric methods was shown when colorimetric methods were compared with the exact “norleucine-PEG method” (32). This latter method involves protein modification with a mPEG bearing a norleucine group in the polymer backbone. Norleucine is an unnatural amino acid, and direct estimation of the degree of protein PEGylation is obtained by PEGylating with the norleucine PEG followed by determining the concentration of norleucine in the hydrolyzed protein. The overestimation by colorimetric assay is consistent with reduced reactivity of TNBS toward lysine residues in the mPEG-modified protein (32). It is also noteworthy that amino acid analysis of PEGylated RNase showed complete recovery of all amino acids other than the modified lysines, especially since Masri and Friedman observed modification of histidine and tyrosine upon reaction of proteins with ethyl and methyl vinyl sulfones (22). Thus, it is apparent that VSPEG is unreactive toward nucleophilic amino acids other than lysine and cysteine, with reactivity toward the latter group being by far the greatest. One of the goals of our work is to compare the various thiol-selective PEG reagents. As noted above, VS-PEG is stable indefinitely in aqueous solution. It is known

Preparation of PEG Vinyl Sulfone

from the literature that maleimides undergo slow hydrolysis (36), and we have confirmed a similar degradation pathway for PEG maleimide. In our experiment PEG maleimide is dissolved in D2O and examined by NMR. Slow disappearance of the NMR vinyl absorptions at 7.01 ppm is observed. A half-life of 6.8 h at pH 7.5 was measured. In a continuation of the current work, which is to be published soon, we have found also that there appears to be slow hydrolysis of the amide linkages in PEG-protein conjugates prepared with MAL-PEG (35). As shown above, VS-PEG forms a stable linkage. A possible advantage of MAL-PEG is that it reacts more rapidly than VS-PEG. As described above, VS-PEG reacts completely with an equimolar solution of Nacetylcysteine at pH 7 and room temperature within 1.5 h. A companion experiment with MAL-PEG showed complete reaction in less than 1 h. Although we have not conducted a direct comparison of VS-PEG with PEG iodoacetamide and PEG dithiopyridine, some comments can be made. The dithiopyridine derivative forms a disulfide when coupled to thiols. An advantage of this linkage is that it can be cleaved, if desired, by exposure to reducing reagents, but it is also possible that this reversibility could become a problem in certain environments (16). Iodoacetamides are also selective for reaction with thiol groups, but reaction with other groups such as histidine has been noted, and slow hydrolysis is also observed (37). CONCLUSION

In summary, we have demonstrated that VS-PEG is readily prepared in high purity and that this PEG derivative is highly selective for reaction with thiol groups relative to amino groups. This selectivity extends to reaction with protein cysteine and lysine groups. Modification of lysines is slow but does occur at useful rates at pH 9.3. The linkages formed for reaction with both thiol and amino groups are stable. An additional useful property of VS-PEG is that the chloroethyl sulfone precursor serves as a protected form that can be deprotected by exposure to base. VS-PEG itself is stable in aqueous solution for days at pH values up to 9. The extent of protein PEGylation by VS-PEG can be monitored by colorimetric methods or by amino acid analysis. Future work concerns utilization of VS-PEG for PEGylation of therapeutically useful proteins (35). ACKNOWLEDGMENT

We gratefully acknowledge partial financial support of this work by the National Science Foundation and the Army Research Office (J.M.H.) and the CNR Finalized Project on Biotechnology and Bioinstrumentation (F.M.V.). We also acknowledge the technical assistance of Mr. Franco Schiavon. LITERATURE CITED (1) Harris, J. M., Ed. (1992) Poly(ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications; Plenum Press, New York. (2) Mabrouk, P. A. (1994) Effect of pegylation on the structure and function of horse cytochrome c. Bioconjugate Chem. 5, 236-241. (3) Alred, P. A., Tjerneld, F., Kozlowski, A., and Harris, J. M. (1992) Synthesis of dye conjugates of ethylene oxide-propylene oxide copolymers and application in temperature-induced phase partitioning. Bioseparation 2, 363-373. (4) Maruyama, K., Unezaki, S., Yuda, T., Ishida, O., Takahashi, N., Suginaka, A., Huang, L., and Iwatsuru, M. (1994)

Bioconjugate Chem., Vol. 7, No. 3, 1996 367 Enhanced delivery and antitumor effect of doxorubicin encapsulated in long-circulating liposomes. J. Liposome Res. 4, 143-165. (5) Shoichet, M. S., Winn, S. R., Harris, J. M., and Gentile, F. T. (1994) Poly(ethylene oxide)-grafted thermoplastic membranes for use as cellular hybrid bio-artificial organs in the central nervous system. Biotechnol. Bioeng. 43, 563-572. (6) Jones, D. S., Hachmann, J. P., Osgood, S. A., Hayag, M. S., Barstad, P. A., Iverson, G. M., and Coutts, S. M. (1994) Conjugates of double-stranded oligonucleotides with poly(ethylene glycol) and keyhole limpet hemocyanin: a model for treating systemic lupus erythematosus. Bioconjugate Chem. 5, 390-399. (7) Kwon, G., Suwa, S., Yokoyama, M., Okano, T., Sakurai, Y., and Kataoka, K. (1994) Enhanced tumor accumulation and prolonged circulation times of micelle-forming poly(ethylene oxide-aspartate) block copolymer-adriamycin conjugates. J. Controlled Release 29, 17-23. (8) Sawnhney, A. S., Pathak, C. P., van Rensburg, J. J., Dunn, R. C., and Hubbell, J. A. (1994) Optimization of photopolymerized bioerodible hydrogen properties for adhesion prevention. J. Biomed. Mater. Res. 28, 831-838. (9) Nathan, A., Zalipsky, S., Ertel, S. I., Agathos, S. N., Yarmush, M. L., and Kohn, J. (1993) Copolymers of lysine and polyethylene glycol: a new family of functionalized drug carriers. Bioconjugate Chem. 4, 54-62. (10) Chamow, S. M., Kogan, T. P., Venuti, M., Gadek, T., Harris, R. J., Peers, D. H., Mordenti, J., Shak, S., and Ashkenazi, A. (1994) Modification of CD4 immunoadhesin with monomethoxypoly(ethylene glycol) aldehyde via reductive amination. Biconjugate Chem. 5, 133-140. (11) Fuke, I., Hayashi, T., Tabata, Y., and Ikada, Y. (1994) Synthesis of poly(ethylene glycol) derivatives with different branchings and their use for protein modification. J. Controlled Release 30, 27-34. (12) Zalipsky, S., Seltzer, R., and Menon-Rudolph, S. (1992) Evaluation of a new reagent for covalent attachment of polyethylene glycol to proteins. Biotechnol. Appl. Biochem. 15, 100-114. (13) Miron, T., and Wilchek, M (1993) A simplified method for the preparation of succinimidyl carbonate polyethylene glycol for coupling to proteins. Bioconjugate Chem. 4, 568-569. (14) Kogan, T. P. (1992) The synthesis of substituted methoxypoly(ethylene glycol) derivatives suitable for selective protein modification. Synth. Commun. 22, 2417-2424. (15) Akerblom, E., Dohlsten, M., Bryno¨, C., Mastej, M., Steringer, I., Hedlund, G., Lando, P., and Kalland, T. (1993) Preparation and characterization of conjugates of monoclonal antibodies and staphylococcal enterotoxin A using a new hydrophilic cross-linker. Bionconjugate Chem. 4, 455-466. (16) Woghiren, C., Sharma, B., and Stein, S. (1993) Protected thiol-polyethylene glycol: a new activated polymer for reversible protein modification. Bioconjugate Chem. 4, 314-318. (17) Kuan, C.-T., Wang, Q.-C., and Pastan, I. (1994) Pseudomonas exotoxin A mutants. J. Biol. Chem. 269, 7610-7616. (18) Herman, S., Loccufier, J., and Schact, E. (1994) End-group modification of R-hydro-ω-methoxy-poly(oxyethylene). Macromol. Chem. Phys. 195, 203-209. (19) Herron, J. N., Gentry, C. A., Davies, S. S., Wei, A.-p., and Lin, J.-n. (1994) Antibodies as targeting moieties: affinity measurements, conjugation chemistry and applications in immunolipopsomes. J. Controlled Release 28, 155-166. (20) Goodson, R. J., and Katre, N. V. (1990) Site directed pegylation of recombinant Interleukin-2 at its glycolsylation site. Bio/Technology 8, 343-346. (21) Zalipsky, S. (1995) Functionalized polyethylene glycols for preparation of biologically relevant conjugates. Bioconjugate Chem. 6, 150-165. (22) Masri, M. S., and Friedman, M. (1988) Protein reactions with methyl and ethyl vinyl sulfones. J. Protein Chem. 7, 4954. (23) Dearfield, K. L., Harrington-Brock, D., Doerr, C. L., Rabinowitz, J. R., and Moore, M. M. (1991) Genotoxicity in mouse lymphoma cells of chemicals capable of Michael addition. Mutagenesis 6, 519-525.

368 Bioconjugate Chem., Vol. 7, No. 3, 1996 (24) Harris, J. M., Struck, E. C., Case, M. G., Paley, M. S., Yalpani, M., Van Alstine, J. M., and Brooks, D. E. (1984) Synthesis and characterization of poly(ethylene glycol) derivatives. J. Polym. Sci., Polym. Chem. Ed. 22, 341-352. (25) Grassetti, D. R., and Murray, J. F. (1967) Determination of sulfhydryl groups with 2,2′- or 4,4′-dithiopyridine. J. Appl. Biochem. Biotechnol. 119, 41-49. (26) Riddles, P. W., Blakeley, R. L., and Zerner, B. (1983) Reassessment of Ellman’s reagent. Methods Enzymol. 91, 4960. (27) Udenfriend, S., Stein, S., Bo¨hlen, P., Bairman, W., Leimgruber, W., and Weigele, M. (1972) Fluorescamine: a reagent for assay of amino acid, peptides, proteins and primary amines in the picomole range. Science 178, 871-872. (28) Stocks, S. J., Jones, A. J. M., Ramey, C. W., and Brooks, D. E. (1986) A fluorometric assay of the degree of modification of proteins with polyethylene glycol. Anal. Biochem. 154, 232236. (29) Karr, L. J., Donnelly, D. L., Kozlowski, A., and Harris, J. M. (1994) Use of poly(ethylene glycol)-modified antibody in cell extraction. Methods Enzymol. 228, 377-390. (30) Snyder, S. L., and Sobocinsky, P. Z. (1975) Improved 2,4,6trinitrobenzenesulfonic acid method for the determination of amines. Anal. Biochem. 64, 284-288. (31) DeaKyne, C. A., and Allen, L. C. (1979) Role of active-site residues in the catalytic mechanism of ribonuclease A. J. Am. Chem. Soc. 101, 3951-3959.

Morpurgo et al. (32) Sartore, L., Caliceti, P., Schiavon, O., Monfardini, C., and Veronese, F. M. (1991) Accurate evaluation of the polymer content in monomethoxy-poly(ethylene glycol)-modified proteins based on amino acid analysis. J. Appl. Biochem. Biotechnol. 31, 213-222. (33) Zalipsky, S., and Barany, G. (1986) Preparation of polyethylene glycol derivatives with two different functional groups at the termini. Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 27 (1), 1-2. (34) Harris, J. M., Herati, M. R. S., Sather, P. J., Brooks, D. E., and Fyles, T. M. (1992) Synthesis of new poly(ethylene glycol) derivatives. In ref 1, pp 371-381. (35) Kachensky, D., Kohno, T., Seely, J., Thompson, R. D., and Harris, J. M. (1996) Polyethylene glycol vinylsulfones: novel conjugation reagents for site-specific polypeptide modification. J. Controlled Release, in press. (36) Ishi, Y., and Lehrer, S. S. (1986) Effects of the state of the succinimido ring on the fluorescence and structural properties of pyrene maleimide-labeled RR-tropomyosin. Biophys. J. 50, 75-80. (37) Means, G. E., and Feney, R. E. (1990) Chemical modification of proteins: history and applications Bioconjugate Chem. 1, 2-12.

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