Bioconjugate Chem. 1992, 3, 200-202
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TECHNICAL NOTE Activating Hydroxyl Groups of Polymeric Carriers Using 4 -Fluorobenzenesulf onyl Chloride Yu-An Chang,' Adrian Gee, Alan Smith, and William Lake Baxter Healthcare Corporation, Fenwal Division, 3015 South Daimler Street, Santa Ana, California 92705. Received November 15, 1991
4-Fluorobenzenesulfonyl chloride (fosyl chloride), due to the strong electron-withdrawing property of its fluoride atom, is found to be an excellent activating agent for the covalent attachment of biologicals to a variety of solid supports (e.g. functionalized polystyrene microspheres, Sepharose beads, or cellulose rods and hollow fibers). This reagent reacts rapidly with primary or secondary hydroxyl groups, a t ambient temperature and pressure, to form 4-fluorobenzenesulfonate leaving groups. The activated solid support can be used immediately or preserved for several months without loss of activity by freeze-drying or by storage at 4 OC in aqueous solution a t pH 5. Enzymes, antibodies, avidin, and other biologicals can be covalently attached to the activated solid phase with excellent retention of biological function. Potential therapeutic applications of the fosyl chloride chemistry for bioselective separation of human lymphocyte subsets from whole blood and tumor cells from bone marrow are presented.
INTRODUCTION
Bioselective-separation technology can be used to perform separations of a specific target population of cells, proteins, or antibodies from a heterogenous solution without the need of tedious and extensive chemical or physical separation techniques. This technolgoy uses affinity supports which are typically formed from hydroxylbearing polymeric carriers, in the form of columns, gels, or polymeric beads. A biologically active ligand is chemically bound to the carrier to provide a selective affinity for binding to the desired target population, i.e. cells, proteins, enzymes, antibodies, and antigens from solutions. Various methods have been used for chemically coupling biologically active ligands to hydroxyl-bearing polymeric carriers ( I ) . Cyanogen bromide (CNBr) (2)is one of the earliest and most widely used methods. Present methods using CNBr and similar reagents (3, 4 ) have several limitations. The activated solid supports are unstable, side reactions occur during activation and coupling, and the linkages formed between supports and ligands are labile (4). In addition, CNBr is a noxious, lachrymatory, and poisonous chemical which requires special handling. Mukaiyama et al. (6) first reported the use of l-methyl2-fluoropyridinium p-toluenesulfonate (FMP) for the preparation of various sulfides and amines from alcohols. Ngo (7) used this reagent to activate hydroxyl groups on polysaccharides such as agarose and Sepharose. A positively charged amine group on the FMP-activated polymeric carrier makes displacement of this group by similarly charged ligands difficult and inefficient. Mosbach and Nilsson (8a,b) have activated hydroxyl groups using p-toluenesulfonyl chloride (tosyl chloride) and 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride). Due to the electron-donatingcharacter of the methyl group on the p-toluenesulfonate, reaction with the nucleophilic groups on the biological often requires 16-24 h under 'A preliminary poster presentation has been given a t the 200th American Chemical Society national meeting (Division of Medicinal Chemistry), Washington, DC, August 26-31, 1990. 1043-1802/92/2903-0200$03.00/0
conditions of pH 8.0 or higher. Such harsh conditions can denature biologicals and may decrease the usefulness of the ligand-coupled carrier. Tresyl chloride is toxic, highly reactive, and volatile. The volatility and high reactivity of tresyl chloride requires that activation with this chemical be performed under strictly anhydrous conditions, making use difficult. In this study we describe a coupling chemistry which overcomes many of these disadvantages. 4-Fluorobenzenesulfonyl chloride (fosyl chloride) has a fluorine atom at the para position, providing a strong electron-withdrawing group to increase the reactivity of the sulfonate. Fosyl chloride is a solid of low volatility and requires no special handling conditions. Use of this reagent to activate hydroxyl carriers yields stable activated supports with minimal side reactions. In this study, we investigated fosyl chloride activation of different solid supports and the resultant biological activity of bound ligands. EXPERIMENTAL PROCEDURES
4-Fluorobenzenesulfonylchloride, p-toluenesulfonylchle ride, cyanogen bromide (CNBr), pyridine, triethylamine, and acetonitrile were purchased from Aldrich Chemical Co. (Milwaukee,WI). 4-(Dimethylamino)pyridine(DMAP) was purchased from Fluka (Ronkonkoma, NY). Tween20 and bovine serum albumin (BSA)were purchased from Sigma, St. Louis, MO. BCA micro protein assay reagents were purchased from Pierce (Rockford, IL). BoltonHunter reagent was purchased from Du Pont Co. NEN Research Products (Boston, MA). Dynal M-450 beads were purchased from Dynal Inc. (Great Neck, NY). Goat anti-mouse IgG was purchased from Jackson Immunoresearch Lab. (West Grove, PA). Anti-Leu 1 (CD 31, antiLeu 3a (CD 4), and anti-Leu 4 (CD 5) antibodies were purchased from Becton Dickinson (Mountain View, CA). General Reaction for Fosyl Chloride Activation of Hydroxyl Groups. The activation of hydroxylated carriers was performed in the presence of a base such as triethylamine or pyridine. DMAP was used as a catalyst in a dry polar aprotic organic solvent such as acetonitrile. 0 1992 American Chemical Society
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Technlpl Note
Scheme I. Fosyl Chloride Activation
CARRIER m HYDROXYL CPOUPS
T P a n CHLORIDE
ACINAmC*PIIIES
Scheme 11. Coupling of Fosyl Chloride Activated Carrier
r
PIIOuXilcNa
mL/100 mg/l mL; 7 mL) and fosyl chloride (2.03 g) was added to the beads. The mixture was rotated at ambient temperature for 1 h. The reagents were removed by filtration under reduced pressure, and then the beads were washed with acetonitrile (7 mL) and deionized water (7 mL). Deionized water (10 mL) and 3 drops of 1N HC1 were added to the beads to decrease the solution pH to 5 and the activated beads were sealed in the test tube with Parafilm and stored at 4 "C for future coupling of biologicals. (3)Activation of Cellulose Rods and Coupling of lZ6ILabeled GAM. Celluloserods (3mg; 1cm long) were added to each of two glass test tubes. To the tubes was added 4 mL of dry acetonitrile along with the quantity of reagents as follows: sample no.
The fosyl chloride reacted rapidly with the hydroxyl groups under ambient temperature and pressure to form 4-fluorobenzenesulfonate groups. This reactive derivative can be used immediately, preserved by freeze-drying, or stored at 4 "C and pH 5 in aqueous solution for future use. The unreacted reagents can be removed by washing with organic solvents (e.g. acetonitrile and acetone) followed by deionizedwater. The carrier can be made of polystyrene with copolymerswhich contain hydroxyl groups or polysaccharides such as cellulose or Sepharose. The carriers can be in the form of microspheres, beads, fibers, or rods. The activation of carriers and couplingof ligands are illustrated in Scheme 1. General Coupling Procedure of Biologicals to Activated Solid Supports. Biologicals containing amino or sulfhydryl groups are suitable for use as ligands to displace the 4-fluorobenzenesulfonate groups. The coupling reaction can be carried out under various conditions, e.g. temperature, pH, and solvents. The unreacted 4-fluorobenzenesulfonate groups can be easily removed by incubation with 0.05 M Tris buffer (pH 8). Scheme I1 illustrates the coupling of various biologicals to activated solid supportsand the following examplesillustrate specific coupling procedures. ( I ) Activation of PolystyreneParamagnetic Beads and Coupling of Goat Anti-Mouse (GAM) IgG t o Activated Magnetic Polystyrene Beads. Dynal M 450 polystyrene paramagnetic beads (5 mL) were washed with deionized water (3 mL) three times followed by acetonitrile (3 mL) three times. The beads were then resuspended in 6 mL of acetonitrile, and pyridine (0.08 mL), DMAP (160 mg), and fosyl chloride (600 mg) were added to the solution. The sample was rotated at ambient temperature for 6 h and then the beads were washed four times with acetonitrile (3 mL) and then washed four times with deionized water (3 mL). Part of the sample was freeze-dried and stored at 4 "C; the remaining sample was used directly for coupling to biologicals. Borate buffer (20 mL, 0.05 M, pH 9) was added to fosyl chloride activated beads (1.24 X lo9 beads) and 18.6 mg of a goat anti-mouse IgG preparation was added to the beads. The beads were rotated at ambient temperature overnight and then washed with Dulbecco's phosphate-buffered saline (DPBS) and stored at 4 "C. These GAM Ab coated beads were used for cell separation experiments (see below). (2) Activation of Sepharose Beads. Sepharose beads (2.04 g, Sigma, CL-4B-200) were filtered under reduced pressure and washed once with 15 mL of deionized water and then with 15 mL of acetonitrile three times to remove water. A solution of acetonitrile/DMAP/triethylamine(9
1 2
reagents DMAP pyridine fosyl chloride DMAP pyridine tresyl chloride
quantity 300 mg 150 pL 500 mg 300 mg 150 p L
450 mg
The samples were incubated at ambient temperature for 6 h and then washed with 0.05 M borate buffer (pH 9.5, 2 mL) two times, acetonitrile (2 mL) three times, and borate buffer (2 mL) three times. The samples were resuspended in the borate buffer (3 mL). 1251-LabeledGAM (1mg/mL, specific activity = 1998cpm/mg) was prepared using Bolton-Hunter reagent as per the manufacturer's procedure. A 90-pL sample of lZ5I-labeledGAM was added to each sample and incubated at room temperature overnight. Samples were then counted in the y counter for total counts to determine the quantity of antibody bound in each sample. Each sample was washed three times with 0.05% Tween/PBS and then resuspended in 1.0 mL of 0.05% Tween/PBS solution and transferred to new tubes. Tubes used for the original incubation were washed with 0.5 mL of buffer, and the wash was added to the new tubes. The tubes were then counted for the radioactivity of each sample by a y counter. RESULTS AND DISCUSSION
(1) Comparison of Fosyl Chloride to Other Activation Agents. ( A ) Comparison of Fosyl Chloride and Tresyl Chloride Activation of Cellulose Rods and Their Binding of 125I-Labeled GAM Antibody. Cellulose rods activated by fosyl chloride and tresyl chloride under the same conditions were incubated with lZ5I-labeledGAM Ab, and then samples were washed and transferred to new tubes. The tubes were counted for radioactivity and the quantity of the 1251-labeledGAM bound to the cellulose rod samples was thus determined (See Figure 1). ( B )Cell Depletion by Fosyl Chloride vs Tosyl Chloride Activated Polystyrene Beads. Goat anti-mouse Ab coupled polystyrene beads activated by fosyl chloride and tosyl chloride under the same conditions were used for separation of lymphocyte subsets, such as CD 3, CD 4, and CD 5 positive cells,2following the reported procedure (9). Comparable results were obtained (Table I). 2Lymphocyte subsets used in these experiments are human T-lymphocytes which express different surface antigens such as CD 3, CD 4, and CD 5. Consequently, these cells can be identified and separated using specific antibodies against these surface antigens.
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Bioconjugate Chem., Vol. 3, No. 2, 1992
Foiyl
Cl
Troayl CI
Actlvated Colluloss Rods
Figure 1. 1251-Labeled GAM Ab bound to fosyl or tresyl chloride activated cellulose rods. Table I. Comparison of Percent Cell Depletion by Fosyl Chloride and Tosyl Chloride Activated GAM Ab Coupled Beads lymphocyte % cell depletion subset fosyl chloride tosyl chloride 90.1 82.2 CD 3
?!
"
99.9
99.9 93.5
CD 4 CD 5
88.0
10 -I
P
1
I
8.60
1.15
time can be as short as 30 min and the activated solid support can be stored at 4 "C for several weeks without loss of binding activity. In summary, fosyl chloride was found to be a versatile reagent for the activation of a variety of solid supports which have a wide range of applications. Examples of other biologicals which have been successfully coupled to fosyl chloride activated solid supports include (1)avidin (a protein which has high affinity for biotinylated biologicals), (2) the enzyme catalase, and (3) human factor VIII, an anticoagulant protein (unpublished results available upon request). Potential therapeutic applications of the 4-fluorobenzenesulfonylchloride chemistry for bioselective separation of human lymphocyte subsets from whole blood, tumor cells from bone marrow, and allergenic antibodies from whole blood are currently under investigation. LITERATURE CITED (1) Scouten, W. (1987)A Survey of Enzyme Coupling Techniques. In Methods in Enzymology (K. Mosbach, Ed.) Vol. 135, pp 30-64, Academic Press, New York. (2) Porath, J., and Axen, R. (1976) Immobilization of Enzymes
to Agar, Agarose, and Sephadex Supports. In Methods in Enzymology (K. Mosbach, Ed.) Vol. 44, pp 19-45, Academic Press, New York. (3) Kohn, J., and Wilcheck, M. (1982) A new approach (cyanotransfer) for cyanogen bromide activation of Sepharose a t neutral pH, which yields activated resins free of interfering nitrogen derivatives. Biochem. Biophys. Res. Commun. 107, 878-884. (4) Kohn, J., and Wilcheck, M. (1983) New approaches for the
FOayt CI FOiyl
CNBr
CI 6 C N l r ACIlvntod Sophirow Bold.
Figure 2. Fosyl chloride vs CNBr activation.
(C)Binding of BSA to Fosyl Chloride and CNBr Actiuated Sepharose Beads. Sepharose beads (Sigma, CL-4B-200)were activated by CNBr (2)and fosyl chloride, respectively, and the activated samples were incubated with BSA under the same conditions. The quantities of the BSA bound to the samples were determined by BCA micro protein assay (Figure 2). The amount of BSA coupled was similar with both activation chemistries. The p-fluoro substitution imparts a strong electronwithdrawing effect, making the 4-fluorobenzenesulfonate an excellent leaving group. Consequently, substitution of ligand for the 4-fluorobenzenesulfonate on the solid supports can be completed faster than that with p-toluenesulfonate. The reaction time is thus reduced while biological activity is conserved. 4-Fluorobenzenesulfonylchloride can be used to activate a broad spectrum of solid supports, e.g. cross-linked agarose beads, cellulose fiber, cellulose rod, maleated cellulose rod, and polystyrene paramagnetic beads. The activation
use of cyanogen bromide and related cyanylating agents for the preparation of activated polysaccharide resins. In Affinity Chromatography and Biological Recognition (I. M. Chaiken, M. Wilcheck, and I. Parikh, Eds.) pp 197-207, Academic Press, New York. (5) (a) Tesser, G. I., Fisch, H. U., and Schwyzer, R. (1974) Limitations of affinity chromatography: Solvolytic detachment of ligands frompolymericsupports. Helu. Chim. Acta 57,17181730. (b) Kohn, J., and Wilcheck, M. (1983) Activation of polysaccharide resins by CNBr. In Solid Phase Biochemistry (W. H. Scouten, Ed.) pp 599-630, Wiley, New York. (6) Mukaiyama, T., Ikeda, S., and Kobayashi, S. (1975) A novel method for the preparation of various 2-pyridyl sulfides from alcohols. Chem. Lett. 1159-1162. ( 7 ) Ngo, T. T. (1986) Method of Activating Hydroxyl Groups of a Polymeric Carrier Using 2-Fluoro-1-methylpyridiniumToluene-4-sulfonate. US. Patent # 4,582,875. (8) (a) Mosbach, K. H., and Nilsson, K. G. I. (1983) Method of Covalently Binding Biologically Active Organic Substances to Polymeric Substances. U.S.Patent # 4,415,665. (b) Nilsson, K., and Mosbach, K. (1984) Immobilization of Ligands with Organic Sulfonyl Chlorides. In Methods in Enzymology (W. B. Jakoby, Ed.) Vol. 104,pp 56-69, Academic Press, New York. (9) Gee, A. P., Mansour, V., and Weiler, M. (1989) T-cell Depletion of Human Bone Marrow J. Zmmunogenet. 16,103115. Registry No. DMAP, 1122-58-3; CNBr, 506-68-3; fosyl chloride, 349-88-2;catalase, 9001-05-2;human factor VIII, 900127-8; Dynal M 450 polystyrene paramagnetic beads, 114451-546; Sepharose beads CL-4B-200,61970-08-9;cellulose, 9004-34-6; pyridine, 110-86-1;tresyl chloride, 1648-99-3.
