508
Bioconjugate Chem. 1093, 4, 568-569
TECHNICAL NOTES A Simplified Method for the Preparation of Succinimidyl Carbonate Polyethylene Glycol for Coupling to Proteins Talia Miron and Meir Wilchek’ Department of Biophysics, The Weizmann Institute of Science, Rehovot 76100, Israel. Received January 21, 1993’
An improved method for the activation of polyethylene glycol with commercially available succinimidyl carbonate is described. The activated polyethylene glycol was coupled to proteins in high yield.
INTRODUCTION Proteins modified with polyethylene glycol have become very important due to their numerous applications in diagnostics ( I ) , in medicine (2-4) and for the enzymatic synthesis in organic solvents (5, 6 ) . Recently, it has been shown that succinimidyl carbonate polyethylene glycol (SC-PEG)’ is the reagent of choice for covalent attachment to proteins (7).However, the described method for the preparation of this “activated PEG” is relatively time-consuming, laborious, and dangerous. In the following we describe a simple, rapid method for the preparation of SC-PEG in high yield by using N-succinimidyl chloroformate (8)or a commercially available reagent, namely N,N’-disuccinimidyl carbonate (9). These reagents have been used in our laboratory for the last 10 years for the activation of polyethylene glycol or monomethoxypolyethylene glycol, their coupling to proteins has been accomplished according to Scheme I and, the protocol is outlined below. EXPERIMENTAL PROCEDURES Reagents, all chemicals, solvents, polyethylene glycol (8000),and monomethoxypolyethylene glycol (2000 and 5000) were from Aldrich (Milwaukee, WI). Proteins were obtained from Sigma Chemical Co. (St. Louis, MO). N-Succinimidyl chloroformate was prepared according to the method of Gross and Bilk (IO). N,”-Disuccinimidyl carbonate and 44Dimethylamino)pyridine were from Fluka (Buchs, Switzerland). Prestained SDS-PAGE standards of low molecular weight was from Bio-Rad (Richmond, CA). Thin-layer chromatography (TLC) was carried out on aluminium sheets precoated with silica gel 60 F ~ from M Merck (Darmstadt, Germany). Preparation of SC-PEG. Polyethylene glycol or monomethoxypolyethylene glycol (1mmol of powder) was dissolved in 5-25 mL of dry (molecular sieve) dioxane by heating in a water bath. The clear solution was brought to room temperature, and either N-succinimidyl chloroformate or N,”-disuccinimidyl carbonate (5-7 mmol dissolved in 10 mL of dry acetone) was added. 4-(Dimethy1amino)pyridine(5-7 mmol in 10 mL of dry acetone) was added slowly under magnetic stirring. Activation proceeded for 2 h for N-succinimidyl chloroAbstract published in Advance ACS Abstracts, October 1, 1993. lAbbreviations used: PEG, polyethylene glycol; SC-PEG, succinimidyl carbonate polyethylene glycol. @
Scheme I. Preparation of Succinimidyl Carbonate Polyethylene Glycol Methyl Ether (SC-PEG) (A) and PEGProteins (B) 0
Ii
CI -C-0-N
s
0
(1) N-Hydroxysuccinimidyl chloroformate
(2) N, N’ Disuccinimidyl carbonate
(3) 4-Dimethylaminopyridine (DMAP)
(3) DMAP
CH3(OCH,CH,),OH + (1)OK (2)
Anhydrous condition
Poly(ethy1eneglycol) methyl ether mPEG
v SC-PEG
B. Co0
3
CHJ(OCH,CH~),,OCW-N
+ NH2-R
pH 7-8
*
d SC-PEG
Protein
PEG-Protein
formate and 6 h for N,”-disuccinimidyl carbonate a t room temperature. The activation mixture with N-succinimidyl chloroformate, contained a white precipitate of 4-(dimethylaminolpyridine hydrochloride, which was filtered through a fine sintered-glass funnel. The supernatant was collected, and the activated PEG was precipitated by diethyl ether until no more precipitation was observed (3-4 vol). In the activation mixture with N,”-disuccinimidyl carbonate, the activated PEG was directly precipitated from the clear solution by diethyl ether. Several
1043- 1a02193~2904-05~a~04.00/0 0 1993 American Chemical Society
Bioconjugate Chem., Vol. 4, No. 6, 1993 569
Technical Note
activated monomethoxypolyethylene glycol is shown in Figure 1, where hen egg white lysozyme solutions (mg/ mL) were modified with either SC-PEG-2000or SC-PEG-
5000.
27.5 325
18.5 -
%
Figure 1. SDS-PAGE (12%) of non-pegylated (Lane 0) and pegylated hen egg white lysozyme (lanes 1-6) carried out at 4 "C for 16 h. Pegylation was done either with SC-PEG-2000 (lanes 1-3) or with SC-PEG-5000 (lanes 4-6). SC-PEG was used in ratio to lysine residues of 0.3:l (lanes 1,4), in excess of 2:l (lanes 2,5),and 8:l (lanes 3,6). M is prestained SDS-PAGE molecular weight markers [from top to bottom, in kDa]: phosphorylase B (106), bovine serum albumin (80), ovalbumin (49.5), carbonic anhydrase (32.5),soybean trypsin inhibitor (27.5)lysozyme (18.5). On each lane (0-6) 20 pg was loaded. The gel was stained with coomassie briliant blue G-250.
cycles of redissolving of the product in acetone and precipitation were carried out in both cases. Removal of the starting materials in the activated PEG were followed by chromatography on TLC in chloroform-methanol (5: 1) system. The activated PEG was stored dry and desiccated a t 4 "C and is stable for a t least 1year. The yield was over 95% as determined spectrophotometrically according to a previously published procedure (II). When the reactions were performed with triethylamine instead of 4-(dimethylamino)pyridine,the yield of the activated PEG was only around 40%. No efforts were made to remove unreacted PEG a t this stage, since it does not disturb subsequent reaction with proteins. Coupling of SC-PEG.Amino-containingligands and proteins were derivatized with SC-PEG in 10-100 mM phosphate buffer, pH 6.5-7.5, a t 4 O C for 4-16 h. Nonreacting SC-PEG was removed by dialysis. An example for modulated modification of protein with
LITERATURE CITED (1) Sharp, K. A,, Yalpani, M., Howard, S. J., and Brooks, D. E. (1986) Synthesis and application of a poly(ethy1ene glycol)antibody affinity ligand for cell separation in aqueous polymer two-phase systems. Anal. Biochem. 154, 110-117. (2) Abuchowski, A., Kazo, G. M., Verhoest, C. R., Jr., Van Es, T., Kafkewitz, D., Nucci, M. L., Viau, A. T., and Davis, F. F. (1984) Cancer therapy with chemically modified enzymes. I. Antitumor properties of polyethylene glycol asparaginase conjugates. Cancer Biochem. Biophys. 7,175-186. (3) Berger, H., Jr., and Pizzo, S. V. (1988) Preparation of polyethylene glycol-tissueplasminogen activator adducts that retain functionalactivity: characteristicsand behavior in three animal species. Blood 71, 1641-1647. (4) Katre, N. V., Knauf, M. J., and Laird, W. J. (1987) Chemical modification of recombinant interleukin 2 by polyethylene glycol increases its potency in the murine Meth A sarcoma model. R o c . Natl. Acad. Sci. U.S.A. 84, 1487-1491. (5) Pina, C., Clark, D., and Blanch, H. (1989) The activity of PEG-modified chymotrypsin in aqueous and organic media. Biotech. Tech. 3, 333-338. (6) Gaertner, H., Watanabe, T., Sinisterra,J. V., and Puigserver, A. (1991) Peptide synthesis catalyzed by modified a-chymotrypsin in low-water organic media. J. Org. Chem. 56, 31493153. (7) Zalipsky, S., Seltzer, R., and Menon-Rudolph, S. (1992) Evaluation of a new reagent for covalent attachment of polyethylene glycol to proteins. Biotech. Appl. Biochem. 15, 100-114. (8) Wilchek, M., and Miron, T. (1982)Immobilizationof enzymes
and affinity ligands onto agarose via stable and uncharged carbamate linkages. Biochem. Internat. 4,629-635. (9) Wilchek, M., and Miron, T. (1985) Activation of Sepharose with NJV-disuccinimidyl carbonate. Appl. Biochem. Biotech. 11, 191-193. (10) Gross, H., and Bilk, L. (1967). Angew. Chem. 79,532-533. (11) Miron, T., and Wilchek, M. (1982) A spectrophotometric
assay for soluble and immobilized N-hydroxysuccinimide esters. Anal. Biochem. 126,433-435.
