Bioconjugate Chem. 1994, 5, 491-492
491
TECHNICAL NOTES Improved Method for Preparing N-Hydroxysuccinimide Ester-Containing Polymers for Affinity Chromatography M. Wilchek,* K. L. Knudsen, a n d T. Miron Department of Membrane Research and Biophysics, The Weizmann Institute of Science, Rehovot 76100, Israel. Received May 10, 1994@
N,N,","-Tetramethyl(succinimid0) uronium tetrafluoroborate is proposed as a reagent of choice for the activation of carboxyl groups and formation of N-hydroxysuccinimide esters on polymers. Unlike conventional methods which generate unstable gels, the reaction is appropriate for hydroxy-containing resins like Sepharose, cellulose, and dextran. The yields of activation and subsequent coupling capacity for ligands and proteins are very high. The respective columns can be used for affinity chromatography and immobilization of proteins.
INTRODUCTION
N-Hydroxysuccinimide (NHS) esters are widely used as coupling agents for protein modification (1,2) and as a means to immobilize ligands containing amino groups (e.g., proteins) onto solid supports for affinity chromatography ( 3 , 4). NHS-activated polymers are commercially available and commonly used. Several years ago (51, we demonstrated that the standard procedure to prepare NHS esters (namely N-hydroxysuccinimide and carbodiimides) leads to the formation of unstable immobilized compounds on polymers that also contain hydroxyl groups. This phenomenon is due to the formation of a p-alanine derivative which binds to the hydroxy-containing polymer, resulting in an unstable bond (5). In the latter study, we suggested alternative approaches to overcome this problem, including a two-step procedure and the use of trifluoroacetylNHS (6). In all cases, however, reduced yields of NHS ester were obtained. N,N,","-Tetramethyl(succinimido)uronium tetrafluoroborate (TSTU) was recently introduced as a reagent to produce NHS esters for carboxamide formation (7,8) without accompanying side reactions. In the present study we adapted TSTU for the rapid activation in high yields of carboxyl groups on Sepharose and other carriers, which also contain hydroxyl groups. The resultant columns are appropriate for use in affinity chromatography and protein immobilization. The reaction proceeds without the side reactions which characterize classical methods. EXPERIMENTAL PROCEDURES
Materials. N,N,","-Tetramethyl( succinimido)uronium tetrafluoroborate (TSTU) and 4-(dimethylamino)pyridine (DMAF') were obtained from Fluka (Buchs, Switzerland). N,N-Dimethylformamide (DMF) and 1,4Abstract published in Advance ACS Abstracts, August 1, 1994. @
dioxane were from Merck (Darmstadt, Germany). C1Sepharose 4B-+amino caproic acid was prepared as described previously (5). Activation Procedure. C1-Sepharose 4B-+aminocaproic acid was washed with 0.3 N HC1 and water and dehydrated gradually by increasing concentrations of dioxane (25%, 50%, and 100%). The filtered gel was found to contain about 50 pmol of carboxyl groups per gram, as determined by amino acid analysis (€-aminocaproic acid) after total hydrolysis. The dehydrated gel (1g) was suspended in 0.1 M TSTU in DMF (0.5 mL). DMAP was added dropwise (0.1 M in DMF, 0.5 mL), and about 2 mL of additional solvent (DMF or dioxane) was then added. The reaction mixture was stirred for 1 h a t room temperature, filtered, and washed successively with DMF, methanol, and isopropanol. The gel was stored in 2-propanol a t 4 "C. The yield of activation was quantitative (49-55 pmoll g) as determined by spectroscopic titration (9). After total hydrolysis, the sole amino acid observed was €-aminocaproic acid; no P-alanine could be detected. Coupling of Proteins. A suspension of the activated gel (containing about 1g of filtered gel) was washed with cold water to remove the 2-propanol. The gel was then added immediately to a solution of protein (between 1-10 mg in 0.1 M phosphate buffer, pH 7.0) and shaken overnight a t 4 "C. The conjugate was filtered and washed extensively with phosphate buffer. The amount of coupled protein was determined either by amino acid analysis or spectroscopically by assessing the amount of uncoupled protein after acidification of the resin. The yields were very high-usually over 90%. For example, 5-7 mg of avidin could be coupled per g of wet gel. Depending on their solubility, low-molecular-weight ligands were coupled either in organic solvents or in buffer. RESULTS AND DISCUSSION
The structure of TSTU and its interaction with a carboxyl group for peptide synthesis was originally
1043-1802/94/2905-0491$04.50/0 0 1994 American Chemical Society
492 Bioconjugafe Chem., Vol. 5, No. 5, 1994
Wilchek et al.
Scheme 1. Activation of Carboxyl Group by TSTU (A) and Its Use in Coupling of Proteins (B) A. Activation 8 . Cowing
described by Knorr et al. (7,8). The reaction is depicted in Scheme 1. In order to apply TSTU on polymeric carriers for immobilization purposes, experiments were performed using different reaction conditions and different concentrations of reagents. Optimization experiments demonstrated that a 1-h reaction period a t room temperature, using a ratio of carboxy1:TSTU:base of l:l:l, gives quantitative yields of NHS ester. We recommend using DMAP as base, since diisopropylethylamine or triethylamine caused extensive discoloration of carriers such as Sepharose. After coupling of protein or ligand, a small amount of black residue was left in solution. On the other hand, when the base was DMAP, white (colorless) gels were obtained. Recently, it was shown that NHS esters can also be prepared in a mixed aqueouslorganic solvent system using TSTU (8). This makes the system even more attractive since the solvents do not have to be dehydrated. The method was also applied to prepare NHS esters in high yields on other polymers which contain carboxyl and hydroxyl groups, such as (carboxymethy1)cellulose and (carboxymethy1)dextran. NHS esters of poly(ethy1ene glycols), which contain carboxyl groups, were also prepared by this method, but we found that for isolation purposes, the conventional approach using N-hydroxysuccinimide and diisopropylcarbodiimide is preferable in this case. Due to the high yield of activation, efficient coupling of proteins and ligands was obtained. When reacted with
1-5 mg of protein per g (wet-weight) of gel, coupling yields of about 90% for proteins were easily obtained with near-complete retention of biological activity. LITERATURE CITED (1) Bauminger, S., and Wilchek, M. (1980) The use of carbodiimide in the preparation of immunizing conjugates. Methods Enzymol. 70, 151-159. (2) Becker, J. M., and Wilchek, M. (1972) Inactivation by avidin of biotin-modified bacteriophage. Biochim. Biophys. Acta 264, 165-170. (3) Cuatrecasas, P., and Parikh, I. (1972)Adsorbents for affinity chromatography. Use of N-hydroxysuccinimide esters of agarose. Biochemistry 11, 2291-2299. (4) Wilchek, M., Miron, T., and Kohn, J. (1984) Affinity chromatography. Methods Enzymol. 104, 3-55. (5) Wilchek, M., and Miron, T. (1987) Limitations of Nhydroxysuccinimide esters in affinity chromatography and protein immobilization. Biochemistry 26, 2155-2161. (6) Sakakibara, S., and Inukai, N. (1965) The trifluoroacetate method of peptide synthesis. I. The synthesis and use of trifluoroacetate reagents. Bull. Chem. SOC.Jpn. 38, 19791984. (7) Knorr, R., Trzeciak, A., Bannwarth, W., and Gillessen, D. (1989) New coupling reagents in peptide chemistry. Tetrahedron Lett. 30, 1927-1930. ( 8 ) Bannwarth, W., and Knorr, R. (1991) Formation of carboxamides with N,N,N”’-tetramethyl(succinimido)uronium tetrafluoroborate in aqueous/organic solvent systems. Tetrahedron Lett. 32, 1157-1160. (9) Miron, T., and Wilchek, M. (1982) A spectrophotometric assay for soluble and immobilized N-hydroxysuccinimide esters. Anal. Biochem. 126, 433-435.
