- Email: [email protected]
[20] Use of glycosylamines in preparation of oligosaccharide polyacrylamide copolymers
[20]
USE OF GLYCOSYLAMINES
22l
[20] Use of G l y c o s y l a m i n e s in P r e p a r a t i o n of Oligosaccharide Polyacrylamide Copolymers
By
ELISABET KALLIN
Introduction Reducing oligosaccharides can be converted to neoglycoconjugates through their glycosylamines. The oligosaccharides are transformed to N-acylated glycosylamine derivatives to obtain derivatives suitable for further conjugaton. In this chapter the N-acryloylation of glycosylamines and subsequent polymerization of the glycosylamides into polyacrylamide copolymers are described. Polyacrylamide copolymers are nontoxic, high molecular weight, water-soluble conjugates where the degree of incorporation of oligosaccharide can be chosen simply by altering the proportions of the reactants. These polyvalent structures have been successfully used as coating antigens in immunoassays. An advantage of these types of conjugates, over protein conjugates, is that they have a simple and well-characterized structure that reduces the risk for undesired immunological reactions. By using a combination of neoglycoproteins and oligosaccharide polyacrylamide copolymers in immunization and screening, antibodies directed to parts of an antigen other than the sugar epitope can be excluded. Glycosylamines can be obtained through treatment of a reducing saccharide with saturated ammonia in methanol.l This procedure, however, is generally not applicable to complex oligosaccharides for several reasons. First, the reaction is low-yielding and time-consuming. Second, the reaction with methanolic ammonia produces both the a and/3 forms of the glycosylamines, causing separation problems. Third, and most importantly, larger oligosaccharides are nearly insoluble in methanol, and the addition of water causes the reaction to proceed even slower. A better way to obtain pure/3-glycosylamines has been described for N-acetylglucosamine and N,N'-diacetylchitobiose.2 The method consists of treatment of the free saccharide with saturated aqueous ammonium bicarbonate for a period of 6 days at 30°. Pure /3-glycosylamines are 1 H. Paulsen and K. W. Pflughaupt, in " T h e C a r b o h y d r a t e s " (W. Pigman and D. Horton, eds.), Vol. 1B, p. 881. A c a d e m i c Press, N e w York, 1980; H. S. Isbell and H. L. F r u s h , Methods Carbohydr. Chem. 8, 255 (1980). 2 L. M. L i k h o s h e r s t o v , O. S. N o v i k o v a , V. A. Derevitskaja, and N. K. K o c h e t k o v , Carbohydr. Res. 146, cl (1986).
METHODS IN ENZYMOLOGY,VOL. 242
Copyright © 1994by AcademicPress, Inc. All rights of reproduction in any form reserved.
222
SYNTHETIC POLYMERS
[20]
o RO ~ ~ ~ , . ~
. RO
OH
NH~
c/O-NH + I
o
~._ /o RO~ , ~ ~ . j
o
NH-- C,,,~
ii
RO
~ ~ ~ . , ~ NH2
SCHEME 1. Synthesis of glycosylamines and N-acryloylglycosylamines. Conditions: i, NH4HCO3 ; ii, acryloyl chloride/Na2CO3 ; iii, cation exchange.
obtained after repeated evaporations and ion-exchange chromatography. This method has been shown to be extendable to sugars other than those terminating with N-acetylglucosamine. Several mono-, di-, and oligosaccharides, including fucosylated and sialylated structures, gave fl-glycosides in good yields. 3,4 The rate of the reactions is highly dependent on temperature, and on repeated additions of solid ammonium bicarbonate to compensate for evaporated ammonia and carbon dioxide. The possibility for liberated gases to pass out freely is important for a successful reaction. 2 Later results have shown, however, that the pH is the important factor. By adjusting the pH to 9 through the addition of a few drops of concentrated ammonia, the vessel could be closed and the reaction time lowered. Nuclear magnetic resonance (NMR) experiments have shown that the formation of glycosylamine occurs via the ammonium carbamate form of the amine (Scheme 1).3 Such carbamates are known to be stable in alkaline solution or as solid salts, but they decarboxylate rapidly on acidification, and this was also shown to be the case during the cation-exchange workup procedure. This procedure, performed both to obtain the free primary glycosylamine and to purify it from salt and reducing oligosaccharide, implies subjecting the glycosylamine to acidic conditions. It has to be performed with great care and might explain the lower yields previously reported. 2 Omitting the cation-exchange step in the workup procedure gave the oligosaccharide fl-carbamate derivatives in good yields. The amount of reducing sugar in the products was less than 10%, according to NMR. Contamination by free ammonium bicarbonate was minimized by three 3 E. Kallin, H. L6nn, T. Norberg, and M. Elofsson, J. Carbohydr. Chem. 8, 597 (1989). 4 R. Roy and C. Laferrier, J. Chem. Soc., Chem. Commun. 1709 (1990).
[20]
USE OF GLYCOSYLAMINES
o\
223
,o
a
o
0
SCHEME 2. Copolymerization of N-acryloylglycosylamines with acrylamide.
successive evaporations of highly diluted aqueous solutions, followed by lyophilization. Whether free or in the form of carbamates, the glycosylamines are stable in aqueous solutions above pH 8 for several days or weeks, and as lyophilized powders for long periods. They hydrolyze rapidly around pH 5 but are stable again at low pH (aqueous HCI) owing to the stabilizing effect caused by protonation of the amino group. Acylation of glycosylamines with acryloyl chloride proceeds smoothly in water-containing media provided a sufficient amount of base is added to prevent acidification of the reaction mixture. It is convenient to use a water-soluble salt as base. 5 Besides the advantage of giving a buffering effect to the system, the water-soluble salt is easily removed by a solidphase extraction procedure. Thus, treatment of/3-glycosylamines with acryloyl chloride in aqueous solution in the presence of sodium carbonate gives N-acryloylated/3-glycosylamines(Scheme 1). 3,4 Acylation of glycosyl-N-carbamates is done as easily as when using the free glycosylamines.3 The N-acryloylated glycosylamines are copolymerized with acrylamide to form high molecular weight, linear copolymers (Scheme 2). 6 By adding a cross-linking reagent to the reaction mixture insoluble gels can 5 p. H. Weigel, R. L. Schnaar, S. Roseman, and Y. C. Lee, this series, Vol. 83, p. 294. 6 V. Ho~'ejgf, P. Smolek, and J. Kocourek, Biochim. Biophys. Acta 538, 293 (1978).
224
SYNTHETIC POLYMERS
[20]
be prepared. 5,7 The degree of oligosaccharide incorporation in the copolymers is about 70% of the theoretical value. This is higher than what is the case when, for example, allyl glycosides are copolymerized with acrylamide. Then the lower reactivity of the allyl group compared to the acryloyl function necessitates the use of a larger amount of glycoside. 8 The molecular weight distribution of the copolymers is usually in the range of 100,000-500,000, centered around 300,000 although molecular weights of over 1,000,000 can be obtained. The molecular weight increases with decreasing concentrations of initiator and increasing concentrations of monomers. However, too high a concentration of monomers may result in an insoluble product. The dominant factors influencing the molecular weight distribution are the purity and concentration of the monomers and maintenance of an oxygen-free reaction mixture. General Methods
All reactions except the preparation of.glycosylamines are performed under nitrogen. Concentrations are performed at a bath temperature below 30°. Optical rotations are recorded at 21 ° with a Perkin-Elmer (Norwalk, CT) 241 polarimeter. The NMR spectra are recorded in D:O with a Bruker AM 500 instrument. Acrylamide (enzyme grade, Eastman Kodak Co., Rochester, NY) is used without further purification. Gel-permeation chromatography is performed on Fractogel TSK HW-55(F) (Merck, Darmstadt, Germany) with water as eluent. Dextran standards are from Pharmacosmos (Viby, Denmark). To prevent self-polymerization 2,6-di-tertbutyl-4-methylphenol (0.5%, w/v) in tetrahydrofuran is used as inhibitor solution. Organic solvents are of analytical grade. Other methods are the same as described before. 9
Preparation of Glycosylamines Solid ammonium bicarbonate is added until saturation to a solution of oligosaccharide (50 mg) in water (2.5 ml). Concentrated aqueous ammonia is added to bring the pH to 9. The mixture is stirred at room temperature for 2-5 days. Ammonium bicarbonate is added at intervals, assuring saturation by keeping a portion of solid salt constantly present in the mixture. When thin-layer chromatography (TLC) indicates no further conversion, 7 V. Hoi'ej~f and J. Kocourek, Biochim. Biophys. Acta 297, 346 (1973); V. Ho~ej~f and J. Kocourek, this series, Vol. 34, p. 361. s A. Y. Chernyak, K. V. Antonov, N. D. Kochetkov, L. N. Padyukov, and N. V. Tsvetkova, Carbohydr. Res. 141, 199 (1985). 9 E. Kallin, this volume [12].
[00]
USE OF GLYCOSYLAMINES
225
the mixture is diluted with water (100 ml) and concentrated to half the volume. This procedure is repeated twice, and the residue is lyophilized. The obtained crude glycosylamine, present in carbamate form, is used without further purification. It should be noted that TLC analysis often gives a false picture of how the reactions proceed, owing to the lability of the product under the chromatographic conditions.
Preparation of N-Acryloylated Glycosylamines Sodium carbonate (100 rag) and methanol (1.0 ml) are added to a solution of the crude glycosylamine (0.14 retool) in water (1.0 ml). The mixture is stirred at 0° while acryloyl chloride (60 ml, 0.74 mmol) in tetrahydrofuran (0.5 ml) is added dropwise. After 10 rain, the solution is diluted with water (3 ml) and concentrated to 2 ml. The solution is again diluted with water (2 ml), inhibitor solution (0.2 ml) is added, and the solution is concentrated to 1-2 ml and applied to a Bond Elut Cl8 cartridge (5 g gel), equilibrated in water. Elution with water gives salts, unreacted glycosylamine, and reducing sugar in the first fractions, and the desired product in the later fractions. In some cases, elution of the product is preferably speeded up by adding methanol to the eluant. Product-containing fractions are combined, mixed with a few drops of inhibitor solution, and concentrated to 2 ml. This solution is purified by gel filtration on a BioGel P-2 (Bio-Rad, Richmond, CA) column. Appropriate fractions are combined and lyophilized. The N-acryloylated glycosylamines are, as predicted, much more stable toward hydrolysis than the glycosylamines. However, the presence of the acryloyl group introduces a tendency to self-polymerization; therefore, addition of small amounts of inhibitor solution is necessary during some operations.
Preparation of Polyacrylamide Copolymers A solution of the N-acryloylglycosylamine (52 mmol) and acrylamide (210 mmol, 15 rag) in distilled water (0.4 ml) is deaerated by flushing with nitrogen for 20 min. The solution is then stirred at 0°, and N,N,N',N'tetramethylethylenediamine (TEMED, 0.002 ml) and ammonium persulfate (I.0 mg) are added. The mixture is slowly stirred at 0° for 2 hr, then at room temperature overnight. The viscous solution is diluted with water (1 ml) and fractionated by gel filtration on Fractogel HW 55(F). Fractions containing polymer are combined and lyophilized. The polyacrylamide copolymers are characterized by optical rotation and NMR. 1H NMR spectroscopy at 50° allows a good estimation of the
226
SYNTHETICPOLYMERS
[21]
degree of substitution of the polymer, through integration of the sugar anomeric signals and the CH and CH 2 groups in the polymer backbone. The molecular weight distribution of the copolymer is determined by gel filtration on Fractogel HW 55(F), using dextran standards for calibration.
[21] S y n t h e s i s of Poly(N-acetyl-fl-lactosaminide-carrying Acrylamide): C h e m i c a l - E n z y m a t i c H y b r i d Process B y KAZUKIYO KOBAYASHI, TOSHIHIRO AKAIKE,
and TAICH1 USUI
Introduction I n c r e a s i n g a t t e n t i o n is b e i n g p a i d to g l y c o p o l y m e r s 1w h i c h a r e s y n t h e t i c p o l y m e r s s u b s t i t u t e d w i t h p e n d a n t c a r b o h y d r a t e m o i e t i e s . S e v e r a l differe n t t y p e s o f g l y c o p o l y m e r s h a v e b e e n u s e d as b i o m e d i c a l m a t e r i a l s s u c h a s c e l l - s p e c i f i c c u l t u r e s u b s t r a t a , 2-6 artificial a n t i g e n s , 7'8 a n d t a r g e t e d d r u g d e l i v e r y a g e n t s . 9 T h e y a r e a l s o u s e f u l as t o o l s for i n v e s t i g a t i n g b i o l o g i c a l r e c o g n i t i o n p h e n o m e n a 1°-~7 u s i n g l e c t i n s a n d a n t i c a r b o h y d r a t e m o n o c l o n a l 1 R. Roy, F. D. Tropper, and A. Romanowska, Bioconjugate Chem. 3, 256 (1992). 2 p. H. Weigel, R. L. Schnaar, M. S. Kuhlenschmidt, E. Schmell, R. T. Lee, Y. C. Lee, and S. Roseman, J. Biol. Chem. 254, 10830 (1979). 3 A. Kobayashi, T. Akaike, K. Kobayashi, and H. Sumitomo, Makromol. Chem., Rapid Commun. 7, 645 (1986), 4 S. Tobe, Y. Takei, K. Kobayashi, and T. Akaike, Biochem. Biophys. Res. Commun. 184, 225 (1992). 5 A. Kobayashi, K. Kobayashi, S. Tobe, and T. Akaike, J. Biomater. Sci. Polym. Ed. 3, 499 (1992). 6 K. Kobayashi, A. Kobayashi, S. Tobe, and T. Akaike, in "Neoglycoconjugates" (Y. C. Lee and R. T. Lee, eds.) p. 261. Academic Press, San Diego, California, 1994. 7 N. K. Kochetkov, Pure Appl. Chem. 56, 923 (1984). 8 A. Rozalski, L. Brade, H.-M. Kuhn, J. Brade, P. Kosma, B. J. Appelmek, S. Kusumoto, and H. Paulsen, Carbohydr. Res. 193, 257 (1989). 9 R. Duncan, P. Kopeckova-Rojmanova, J. Strohalm, I. Hume, H. C. Cable, J. Pohl, J. B. Lloyd, and J. Kopecek, Br. J. Cancer 55, 165 (1987). 10 L. A. Carpino, H. Ringsdorf, and H. Ritter, Makromol. Chem. 177, 1631 (1976). II R. Roy and F. D. Tropper, J. Chem. Soc., Chem. Commun., 1058 (1988). 12R. Roy, F. D. Tropper, and A. Romanowska, J. Chem. Soc., Chem. Commun., 1611 (1992). 13R. Roy, F. D. Tropper, T. Morrison, and J. Boratynski, J. Chem. Soc., Chem. Commun., 536 (1991). 14S. Nishimura, K. Matsuoka, and K. Kurita, Macromolecules 23, 4182 (1990). ~5S. Nishimura, K. Matsuoka, T. Furuike, S. Ishii, K. Kurita, and K. Nishimura, Macromolecules 24, 4236 (1991).
METHODS IN ENZYMOLOGY, VOL. 242
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
USE OF GLYCOSYLAMINES
22l
[20] Use of G l y c o s y l a m i n e s in P r e p a r a t i o n of Oligosaccharide Polyacrylamide Copolymers
By
ELISABET KALLIN
Introduction Reducing oligosaccharides can be converted to neoglycoconjugates through their glycosylamines. The oligosaccharides are transformed to N-acylated glycosylamine derivatives to obtain derivatives suitable for further conjugaton. In this chapter the N-acryloylation of glycosylamines and subsequent polymerization of the glycosylamides into polyacrylamide copolymers are described. Polyacrylamide copolymers are nontoxic, high molecular weight, water-soluble conjugates where the degree of incorporation of oligosaccharide can be chosen simply by altering the proportions of the reactants. These polyvalent structures have been successfully used as coating antigens in immunoassays. An advantage of these types of conjugates, over protein conjugates, is that they have a simple and well-characterized structure that reduces the risk for undesired immunological reactions. By using a combination of neoglycoproteins and oligosaccharide polyacrylamide copolymers in immunization and screening, antibodies directed to parts of an antigen other than the sugar epitope can be excluded. Glycosylamines can be obtained through treatment of a reducing saccharide with saturated ammonia in methanol.l This procedure, however, is generally not applicable to complex oligosaccharides for several reasons. First, the reaction is low-yielding and time-consuming. Second, the reaction with methanolic ammonia produces both the a and/3 forms of the glycosylamines, causing separation problems. Third, and most importantly, larger oligosaccharides are nearly insoluble in methanol, and the addition of water causes the reaction to proceed even slower. A better way to obtain pure/3-glycosylamines has been described for N-acetylglucosamine and N,N'-diacetylchitobiose.2 The method consists of treatment of the free saccharide with saturated aqueous ammonium bicarbonate for a period of 6 days at 30°. Pure /3-glycosylamines are 1 H. Paulsen and K. W. Pflughaupt, in " T h e C a r b o h y d r a t e s " (W. Pigman and D. Horton, eds.), Vol. 1B, p. 881. A c a d e m i c Press, N e w York, 1980; H. S. Isbell and H. L. F r u s h , Methods Carbohydr. Chem. 8, 255 (1980). 2 L. M. L i k h o s h e r s t o v , O. S. N o v i k o v a , V. A. Derevitskaja, and N. K. K o c h e t k o v , Carbohydr. Res. 146, cl (1986).
METHODS IN ENZYMOLOGY,VOL. 242
Copyright © 1994by AcademicPress, Inc. All rights of reproduction in any form reserved.
222
SYNTHETIC POLYMERS
[20]
o RO ~ ~ ~ , . ~
. RO
OH
NH~
c/O-NH + I
o
~._ /o RO~ , ~ ~ . j
o
NH-- C,,,~
ii
RO
~ ~ ~ . , ~ NH2
SCHEME 1. Synthesis of glycosylamines and N-acryloylglycosylamines. Conditions: i, NH4HCO3 ; ii, acryloyl chloride/Na2CO3 ; iii, cation exchange.
obtained after repeated evaporations and ion-exchange chromatography. This method has been shown to be extendable to sugars other than those terminating with N-acetylglucosamine. Several mono-, di-, and oligosaccharides, including fucosylated and sialylated structures, gave fl-glycosides in good yields. 3,4 The rate of the reactions is highly dependent on temperature, and on repeated additions of solid ammonium bicarbonate to compensate for evaporated ammonia and carbon dioxide. The possibility for liberated gases to pass out freely is important for a successful reaction. 2 Later results have shown, however, that the pH is the important factor. By adjusting the pH to 9 through the addition of a few drops of concentrated ammonia, the vessel could be closed and the reaction time lowered. Nuclear magnetic resonance (NMR) experiments have shown that the formation of glycosylamine occurs via the ammonium carbamate form of the amine (Scheme 1).3 Such carbamates are known to be stable in alkaline solution or as solid salts, but they decarboxylate rapidly on acidification, and this was also shown to be the case during the cation-exchange workup procedure. This procedure, performed both to obtain the free primary glycosylamine and to purify it from salt and reducing oligosaccharide, implies subjecting the glycosylamine to acidic conditions. It has to be performed with great care and might explain the lower yields previously reported. 2 Omitting the cation-exchange step in the workup procedure gave the oligosaccharide fl-carbamate derivatives in good yields. The amount of reducing sugar in the products was less than 10%, according to NMR. Contamination by free ammonium bicarbonate was minimized by three 3 E. Kallin, H. L6nn, T. Norberg, and M. Elofsson, J. Carbohydr. Chem. 8, 597 (1989). 4 R. Roy and C. Laferrier, J. Chem. Soc., Chem. Commun. 1709 (1990).
[20]
USE OF GLYCOSYLAMINES
o\
223
,o
a
o
0
SCHEME 2. Copolymerization of N-acryloylglycosylamines with acrylamide.
successive evaporations of highly diluted aqueous solutions, followed by lyophilization. Whether free or in the form of carbamates, the glycosylamines are stable in aqueous solutions above pH 8 for several days or weeks, and as lyophilized powders for long periods. They hydrolyze rapidly around pH 5 but are stable again at low pH (aqueous HCI) owing to the stabilizing effect caused by protonation of the amino group. Acylation of glycosylamines with acryloyl chloride proceeds smoothly in water-containing media provided a sufficient amount of base is added to prevent acidification of the reaction mixture. It is convenient to use a water-soluble salt as base. 5 Besides the advantage of giving a buffering effect to the system, the water-soluble salt is easily removed by a solidphase extraction procedure. Thus, treatment of/3-glycosylamines with acryloyl chloride in aqueous solution in the presence of sodium carbonate gives N-acryloylated/3-glycosylamines(Scheme 1). 3,4 Acylation of glycosyl-N-carbamates is done as easily as when using the free glycosylamines.3 The N-acryloylated glycosylamines are copolymerized with acrylamide to form high molecular weight, linear copolymers (Scheme 2). 6 By adding a cross-linking reagent to the reaction mixture insoluble gels can 5 p. H. Weigel, R. L. Schnaar, S. Roseman, and Y. C. Lee, this series, Vol. 83, p. 294. 6 V. Ho~'ejgf, P. Smolek, and J. Kocourek, Biochim. Biophys. Acta 538, 293 (1978).
224
SYNTHETIC POLYMERS
[20]
be prepared. 5,7 The degree of oligosaccharide incorporation in the copolymers is about 70% of the theoretical value. This is higher than what is the case when, for example, allyl glycosides are copolymerized with acrylamide. Then the lower reactivity of the allyl group compared to the acryloyl function necessitates the use of a larger amount of glycoside. 8 The molecular weight distribution of the copolymers is usually in the range of 100,000-500,000, centered around 300,000 although molecular weights of over 1,000,000 can be obtained. The molecular weight increases with decreasing concentrations of initiator and increasing concentrations of monomers. However, too high a concentration of monomers may result in an insoluble product. The dominant factors influencing the molecular weight distribution are the purity and concentration of the monomers and maintenance of an oxygen-free reaction mixture. General Methods
All reactions except the preparation of.glycosylamines are performed under nitrogen. Concentrations are performed at a bath temperature below 30°. Optical rotations are recorded at 21 ° with a Perkin-Elmer (Norwalk, CT) 241 polarimeter. The NMR spectra are recorded in D:O with a Bruker AM 500 instrument. Acrylamide (enzyme grade, Eastman Kodak Co., Rochester, NY) is used without further purification. Gel-permeation chromatography is performed on Fractogel TSK HW-55(F) (Merck, Darmstadt, Germany) with water as eluent. Dextran standards are from Pharmacosmos (Viby, Denmark). To prevent self-polymerization 2,6-di-tertbutyl-4-methylphenol (0.5%, w/v) in tetrahydrofuran is used as inhibitor solution. Organic solvents are of analytical grade. Other methods are the same as described before. 9
Preparation of Glycosylamines Solid ammonium bicarbonate is added until saturation to a solution of oligosaccharide (50 mg) in water (2.5 ml). Concentrated aqueous ammonia is added to bring the pH to 9. The mixture is stirred at room temperature for 2-5 days. Ammonium bicarbonate is added at intervals, assuring saturation by keeping a portion of solid salt constantly present in the mixture. When thin-layer chromatography (TLC) indicates no further conversion, 7 V. Hoi'ej~f and J. Kocourek, Biochim. Biophys. Acta 297, 346 (1973); V. Ho~ej~f and J. Kocourek, this series, Vol. 34, p. 361. s A. Y. Chernyak, K. V. Antonov, N. D. Kochetkov, L. N. Padyukov, and N. V. Tsvetkova, Carbohydr. Res. 141, 199 (1985). 9 E. Kallin, this volume [12].
[00]
USE OF GLYCOSYLAMINES
225
the mixture is diluted with water (100 ml) and concentrated to half the volume. This procedure is repeated twice, and the residue is lyophilized. The obtained crude glycosylamine, present in carbamate form, is used without further purification. It should be noted that TLC analysis often gives a false picture of how the reactions proceed, owing to the lability of the product under the chromatographic conditions.
Preparation of N-Acryloylated Glycosylamines Sodium carbonate (100 rag) and methanol (1.0 ml) are added to a solution of the crude glycosylamine (0.14 retool) in water (1.0 ml). The mixture is stirred at 0° while acryloyl chloride (60 ml, 0.74 mmol) in tetrahydrofuran (0.5 ml) is added dropwise. After 10 rain, the solution is diluted with water (3 ml) and concentrated to 2 ml. The solution is again diluted with water (2 ml), inhibitor solution (0.2 ml) is added, and the solution is concentrated to 1-2 ml and applied to a Bond Elut Cl8 cartridge (5 g gel), equilibrated in water. Elution with water gives salts, unreacted glycosylamine, and reducing sugar in the first fractions, and the desired product in the later fractions. In some cases, elution of the product is preferably speeded up by adding methanol to the eluant. Product-containing fractions are combined, mixed with a few drops of inhibitor solution, and concentrated to 2 ml. This solution is purified by gel filtration on a BioGel P-2 (Bio-Rad, Richmond, CA) column. Appropriate fractions are combined and lyophilized. The N-acryloylated glycosylamines are, as predicted, much more stable toward hydrolysis than the glycosylamines. However, the presence of the acryloyl group introduces a tendency to self-polymerization; therefore, addition of small amounts of inhibitor solution is necessary during some operations.
Preparation of Polyacrylamide Copolymers A solution of the N-acryloylglycosylamine (52 mmol) and acrylamide (210 mmol, 15 rag) in distilled water (0.4 ml) is deaerated by flushing with nitrogen for 20 min. The solution is then stirred at 0°, and N,N,N',N'tetramethylethylenediamine (TEMED, 0.002 ml) and ammonium persulfate (I.0 mg) are added. The mixture is slowly stirred at 0° for 2 hr, then at room temperature overnight. The viscous solution is diluted with water (1 ml) and fractionated by gel filtration on Fractogel HW 55(F). Fractions containing polymer are combined and lyophilized. The polyacrylamide copolymers are characterized by optical rotation and NMR. 1H NMR spectroscopy at 50° allows a good estimation of the
226
SYNTHETICPOLYMERS
[21]
degree of substitution of the polymer, through integration of the sugar anomeric signals and the CH and CH 2 groups in the polymer backbone. The molecular weight distribution of the copolymer is determined by gel filtration on Fractogel HW 55(F), using dextran standards for calibration.
[21] S y n t h e s i s of Poly(N-acetyl-fl-lactosaminide-carrying Acrylamide): C h e m i c a l - E n z y m a t i c H y b r i d Process B y KAZUKIYO KOBAYASHI, TOSHIHIRO AKAIKE,
and TAICH1 USUI
Introduction I n c r e a s i n g a t t e n t i o n is b e i n g p a i d to g l y c o p o l y m e r s 1w h i c h a r e s y n t h e t i c p o l y m e r s s u b s t i t u t e d w i t h p e n d a n t c a r b o h y d r a t e m o i e t i e s . S e v e r a l differe n t t y p e s o f g l y c o p o l y m e r s h a v e b e e n u s e d as b i o m e d i c a l m a t e r i a l s s u c h a s c e l l - s p e c i f i c c u l t u r e s u b s t r a t a , 2-6 artificial a n t i g e n s , 7'8 a n d t a r g e t e d d r u g d e l i v e r y a g e n t s . 9 T h e y a r e a l s o u s e f u l as t o o l s for i n v e s t i g a t i n g b i o l o g i c a l r e c o g n i t i o n p h e n o m e n a 1°-~7 u s i n g l e c t i n s a n d a n t i c a r b o h y d r a t e m o n o c l o n a l 1 R. Roy, F. D. Tropper, and A. Romanowska, Bioconjugate Chem. 3, 256 (1992). 2 p. H. Weigel, R. L. Schnaar, M. S. Kuhlenschmidt, E. Schmell, R. T. Lee, Y. C. Lee, and S. Roseman, J. Biol. Chem. 254, 10830 (1979). 3 A. Kobayashi, T. Akaike, K. Kobayashi, and H. Sumitomo, Makromol. Chem., Rapid Commun. 7, 645 (1986), 4 S. Tobe, Y. Takei, K. Kobayashi, and T. Akaike, Biochem. Biophys. Res. Commun. 184, 225 (1992). 5 A. Kobayashi, K. Kobayashi, S. Tobe, and T. Akaike, J. Biomater. Sci. Polym. Ed. 3, 499 (1992). 6 K. Kobayashi, A. Kobayashi, S. Tobe, and T. Akaike, in "Neoglycoconjugates" (Y. C. Lee and R. T. Lee, eds.) p. 261. Academic Press, San Diego, California, 1994. 7 N. K. Kochetkov, Pure Appl. Chem. 56, 923 (1984). 8 A. Rozalski, L. Brade, H.-M. Kuhn, J. Brade, P. Kosma, B. J. Appelmek, S. Kusumoto, and H. Paulsen, Carbohydr. Res. 193, 257 (1989). 9 R. Duncan, P. Kopeckova-Rojmanova, J. Strohalm, I. Hume, H. C. Cable, J. Pohl, J. B. Lloyd, and J. Kopecek, Br. J. Cancer 55, 165 (1987). 10 L. A. Carpino, H. Ringsdorf, and H. Ritter, Makromol. Chem. 177, 1631 (1976). II R. Roy and F. D. Tropper, J. Chem. Soc., Chem. Commun., 1058 (1988). 12R. Roy, F. D. Tropper, and A. Romanowska, J. Chem. Soc., Chem. Commun., 1611 (1992). 13R. Roy, F. D. Tropper, T. Morrison, and J. Boratynski, J. Chem. Soc., Chem. Commun., 536 (1991). 14S. Nishimura, K. Matsuoka, and K. Kurita, Macromolecules 23, 4182 (1990). ~5S. Nishimura, K. Matsuoka, T. Furuike, S. Ishii, K. Kurita, and K. Nishimura, Macromolecules 24, 4236 (1991).
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