ANALYTICAL
BIOCHEMISTRY
A Simple
Method for the Preparation Containing Thioglycoside
REIKO Department
95, 260-269(1979)
T. LEE, STEPHANIE of Biology
and McCollum-Pratt Baltimore,
CASCIO,
of Polyacrylamide LigandW AND YUAN
Institute, The Johns Maryland 21218
CHUAN Hopkins
Gels
LEES University,
Received November 13, 1978 A simple method was developed for the preparation of acrylamide derivatives containing thioglycosides. The synthetic scheme involves the preparation of an O-acetylated I-thiosugar via a pseudothiourea derivative, the controlled addition of the thiosugar to N.N’-bismethyleneacrylamide to form a monoaddition product in which one of the acrylamide groups remains unchanged, and finally de-O-acetylation. Similar reaction schemes using bisacrylamido derivatives of 1,2-diaminoethane and 1,6-diaminohexane lead to analogous compounds with longer aglycon chain lengths. The thioglycoside derivatives of acrylamide thus prepared could be copolymerized with acrylamide to form polymers or gels containing thioglycosides as ligands. These gels were successfully used as affinity materials for the purification of peanut lectin and in cell-adhesion studies.
The elucidation of the roles of carbohydrates in biological reactions has been probed with carbohydrates of well-defined structure. Frequently, immobilization of such carbohydrates proves to be an invaluable aid to biological studies (1,2). We have chosen polyacrylamide gel as the matrix most suitable for such purposes, because of its flexibility and chemical and biological inertness. Earlier publications from our laboratory described two different methods for the preparation of polyacrylamide gels containing o-aminoalkyl glycosides or their l-thio analogs. In the first method, N-succinimidyl acrylate is copolymerized with acrylamide, and the active esters are subsequently replaced with aminoalkyl glycosides (3,4). The second method involved acryloylation of aminoalkyl glycosides and
its copolymerization with acrylamide (5). The carbohydrate-containing gels prepared by these methods have been effectively utilized for the isolation of lectins (3,6) and in studies of cell adhesion (2,7). We have now developed a simple method for the preparation of thioglycoside derivatives of acrylamide, which in turn can be used to produce carbohydrate-containing acrylamide polymers. The underlying prin-
1 This paper is dedicated to the memory of Dr. Alvin Nason. 2 Contribution No. 993 from the McCollum-Pratt Institute, The Johns Hopkins University. Supported by USPHS NIH Research Grants AM9970 and CA21901. 3 To whom all correspondence should be addressed. 0003-2697/79/070260-10$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
SCHEME
260
1
ACRYLAMIDE
GELS WITH THIOGLYCOSIDES
CH,=CHCONH(CH,),NHCOCH=CH, Compound No.
n 1
1 10 13
2 6
R-SCH,CH,CONH(CH,),NHCOCH=CH, Compound No.
n -
R
2
1
3 4
I
5
1
6
1
7 8
1 1
9
1
11
2
12 14
2 6
15
6
1
2,3,4,6,-Tetra-O-acetyl+?-D-galactopyranosyl @&alactopyranosyl 2,3,4,6-Tetra-O-acetyl-@-glucopyranosyl P-D-Glucopyranosyl 2,3,4,6-Tetra-O-acetyl-a-D-mannopyranosyl a-D-Mannopyranosyl 2-Acetamido-3,4,6-tri-0-acetyl-2deoxy-P-D-glucopyranosyl 2-Acetamido-2-deoxy-@r+ucopyranosyl 2,3,4,6-Tetra-O-acetyl-/3-D-galactopyranosyl P-D-Galactopyranosyl 2,3,4,6-Tetra-O-acetyI+-D-galactopyranosyl b-o-Calactopyranosyl SCHEME
2
ciple of this synthesis is illustrated in Scheme 1. Since the starting materials for this reaction can be either commercially purchased or easily prepared by simple procedures from available materials, the present method offers great advantages over the other methods. The gels prepared by the method described here have been satisfactorily used for lectin isolation and cell-adhesion studies. EXPERIMENTAL Materials and methods. Acrylamide from J. T. Baker Chemical Co. and N,N’-methylenebisacrylamide (l), acryloyl chloride, 1,Zdiaminoethane, and 1,6-diaminohexane from Aldrich Chemical Co. were used without purification. Partially purified peanut
261
lectin (“precolumn lectin”) was prepared as reported earlier (6). Pseudothiourea derivatives of 2,3,4,6tetra-0-acetyl-P-D-galactopyranose, D-Dglucopyranose (8), a-D-mannopyranose (9), and 2-acetamido-2-deoxy-3,4,6-tri-o-acetyl/3-o-glucopyranose (10) were prepared from the corresponding 1-bromo or 1-chloro sugars as described. All evaporations were performed under reduced pressure at 25-40°C with a rotatory evaporator. Melting points (uncorrected) were measured with a Fisher- Johns apparatus. Proton magnetic resonance spectra were recorded with a JEOL NMH- 100 spectrometer. Optical rotations were measured with a Cary 60 spectropolarimeter. Elemental analyses were performed by Galbraith Laboratories (Knoxville, Tenn.). Thin-layer chromatography (tlc)4 was performed with silica gel F-254 layers precoated on aluminum sheets (Merck), using the following solvent systems: (A) 4:l (v/v) ethyl acetate-acetone, (B) 9:4:2 (v/v/v) ethyl acetate-isopropyl alcohol-water. Acrylamido compounds were detected by fluorescence quenching. For detection of carbohydrates, tic sheets were sprayed with 15% sulfuric acid in 50% ethanol and heated for a few minutes at 140°C. Whenever necessary, chromatographic purification of O-acetylated sugar derivatives was achieved with a column of Sephadex LH-20 (4 x 190 cm) in 95% ethanol collecting l&ml fractions. Neutral sugars were determined by a modified version (11) of the phenol- sulfuric acid method, and thiol groups by Ellman’s method (12). The glycoside monomers were characterized by hydrolyzing a known dry weight with mercuric acetate (13), then analyzing it on an automated neutral sugar analyzer (14). Protein concentrations were determined by measuring absorbance at 280 nm. 4 Abbreviations used: tic, thin-layer chromatography: pmr, proton magnetic resonance: DMSO. dimethyl sulfoxide; TEMED, tetramethylethylenediamine.
262
LEE, CASCIO, AND LEE
The hemagglutination assay for peanut lectin was performed as described by Sutoh et al. (6). Polyacrylamide gel electrophoresis was performed according to the method of Reisfeld et al. (15), and proteins were stained with Coomassie brilliant blue. Preparation of I-thioaldopyranose tates. The pseudothiourea derivatives
ace-
100 2 eo 4 g 60 5 F’ ‘E Lz : 20
were converted into the corresponding 1-thio de0 rivatives in the following way: To a solution 0 I 2 3 4 5 22 of pseudothiourea derivative, 10 mmol(4.87 Time(h) g for neutral sugar derivatives and 4.42 g for FIG. 1. Rate of decrease of the thiol group in the the amino sugar derivative) in 40 ml of water, mixture containing I-thiogalactose and the were added potassium carbonate (1.66 g, 12 reaction following bisacrylamido compounds: (A). 1, (B) 10, mmol) and sodium bisulfite (1.04 g, 10 (0) 13, (0) 1 + 10, (0) 1 + 13. mmol), followed immediately by 40 ml of chloroform. The biphasic reaction mixture was stirred vigorously in an enclosed vessel. in solvent A showed three components with Within a few minutes, all the salts were very similar RF values. The three compodissolved in the aqueous layer. The appearnents were, in order of increasing RF value, ance of I-thiosugar in the chloroform layer a strong, uv-absorbing spot of remaining 1, was analyzed by Ellman’s method. The con- a uv-absorbing sugar derivative (2), and a small amount of non-uv-absorbing sugar centration of I-thiosugars reached a plateau derivative. This last material was obtained within 15-30 min, but stirring was usually in pure crystalline form after gel filtration continued for a total of 45 min, after which time the two layers were separated. The on a column of Sephadex LH-20 (see below) aqueous layer was extracted once with 20 ml and was identified as a bis-addition prodof chloroform. The chloroform solutions uct [N,N’-methylenebis(aminocarbonylethy1 were combined, washed once with 40 ml of 2,3,4,6-tetra-O-acetyl-l-thio-p-D-galactowater, and then evaporated to a syrup. pyranoside)]. This reaction mixture was one (N - Acrylamidomethyl)aminocarbonyl - routinely evaporated to approximately half of the original volume and then partiethyl 2,3,4,6-tetra-O-acetyl-l-thio-@D-gationed between 75 ml each of water and lactopyranoside (2). The syrupy I-thiosugar derivative obtained from 10 mmol of 2-S- chloroform. The aqueous phase was extracted again with 50 ml of chloroform. The (2,3,4,6-tetra-O-/3-D-galactopyranosyl)-2pseudothiourea hydrobromide was stirred combined chloroform layers were then with 10 ml of 50% ethanol, and a solution of washed with water (4 x 50 ml) to remove excess 1. The chloroform layer was dried l(3.08 g, 20 mmol) in 50 ml of 50% ethanol was added to it. Stirring briefly produced a with anhydrous sodium sulfate, filtered, and clear solution, to which were added 1 ml evaporated. The resulting syrup contained mainly the product 2 and a small amount pyridine and a small amount of hydroquioof the bis-addition by-product. These two none (5-6 mg). The progress of the reaction compounds could be separated completely was followed by measuring the disappearusing the Sephadex LH-20 column. Fracance of thiol group. The reaction of O-acetytions containing 2 were combined and lated thiosugar with 1 was complete within 24 h in most cases. The kinetics of these evaporated to a small volume, which, upon reactions are shown in Fig. 1. After over- standing in the cold, produced crystals; mp, 122- 123°C. The pmr data (CDCl& indicated night at room temperature, tic of the mixture
ACRYLAMIDE
GELS
WITH
the correct ratio of CH,CO- (6 2.0-2.1, m, 12) to -W&X,(6 2.7 and 2.95, m, 4) to -CH,protons (6 4.3, s, 2) to -NHCOprotons (6 7.65, broad, 2). Acryloyl proton signals were in the region of 5.7 to 6.35 ppm. For routine preparation of 3, however, one can proceed to the next step (de-O-acetylation) without the Sephadex LH-20 gel filtration. (N - Acrylamidomethyl)aminocarbonyl ethyl I-thio-PDgalactopyranoside (3). The
THIOGLYCOSIDES
263
tose derivative, 2. The yield of 4 was 80%. Pure, crystalline 4 obtained from Sephadex LH-20 chromatography melted at 168169°C. De-0-acetylation of 4 by sodium methoxide produced 5 in 82% yield: Rf in solvent B, 0.32; mp, 165- 167°C; [(~],~~-24.6 (c 8.8, water). The pmr spectrum was similar to that of 3. Anal. Calcd. for C,,H,,N,O,S (350.40): C, 44.56; H, 6.33; N, 8.00; S, 9.15. Found: C, 44.47; H, 6.37: N, 8.01; S, 8.93.
syrup containing mainly 2 and small amounts (N - Acrylamidomethyl)aminocarbonyl of by-products (see above) was de-o-acetyethyl 2,3,4,6-tetra-O-acetyl-1-thio-a-D-manlated in 20 ml dry methanol containing 0.01 nopyranoside (6) and (N-acrylamidomethyl)M sodium methoxide for 2 h at room tem- aminocarbonylethyl I-thio-cw-Dmannopyraperature. Crystals of 3 which formed over- noside (7). The mannose derivatives were night at 4°C were filtered and washed with prepared exactly as described for the galacmethanol (yield: 2.3 g, 6.54 mmol). The ad- tose derivatives 2 and 3. However, the addition of benzene and petroleum ether (bp, dition reaction mixture contained, in addi35-60°C) to the filtrate produced an addi- tion to the two expected products, a contional 0.14 g (0.4 mmol) of crystalline 3. The taminant which was not observed in the total yield of crystalline 3 from the pseudopreparation of either the galactose or glucose thiourea derivative was 69.4%. The product derivative. The syrup obtained upon evapthus obtained was usually pure enough to be oration of the washed chloroform solution was dissolved in 15 ml of 95% ethanol and used in polymer formation (see below). However, the complete removal of impuriapplied to the column of Sephadex LH-20. ties could be accomplished by passing the The elution profile is shown in Fig. 2. The aqueous solution of the product through a largest peak contained mainly 6. Fractions 98 - 107 were combined and evaporated to a Sephadex G-15 column (2 x 145 cm) equilibrated with 0.1 M acetic acid. Fractions small volume; however, compound 6 could containing 3 were evaporated to a small not be obtained in crystalline form. It was volume, to which 95% ethanol was added. dehydrated by the addition and evaporation Upon standing in the cold, pure 3 crystalof absolute ethanol and toluene, and then lized: R, in solvent B, 0.23: mp, 193°C; and de-0-acetylated as described for 3. Benzene [a],,25-5.10 (c 5.3, water). The spectrum of and petroleum ether (35-60°C) were added ?H-exchanged sample in dimethyl sulfoxide to the deacetylation mixture to produce two (DMSO)-dG showed the presence of both crops of crystals. The yield of crystalline 7 ethylene and acryloyl groups. The anomeric from 9 mmol of the pseudothiourea derivaproton signal was at 4.30 (d, 1, J = 7 Hz). tive was 2.86 mmol(32%). Recrystallization Anal. Calcd. for C,,H,,N,O,S (350.40): from absolute ethanol-benzene-petroleum C, 44.56; H, 6.33; N, 8.00; S, 9.15. Found: ether (35-60”(Z) produced pure, crystalline C, 44.49; H, 6.45; N, 8.07; S, 9.06. 7 having mp, 166-167°C: [c~],,~“+117.3~ (c. (IV - Acrylamidomethyl)aminocarbonyl - 8.9, water); and R, in solvent B of 0.38. ethyl 2,3.4,6-terra-O-acetyl-I-thio-pDgluAnalysis for mannose of a mercuric hydrolycopyranoside (4) and (N-acrylamidomethyl) sate of this material yielded 102% of the aminocarbonylethyl 1-thio-p-o-glucopyratheoretical amount of mannose on the drynoside (5) The glucose derivative (4) was weight basis. prepared exactly as described for the galac(N - Ac~lamidomethyl)aminocarbonyl -
264
LEE,
CASCIO,
ethyl 2-acetamido-3,4,6-tri-0-acetyl-2-deoxyI-thio-@Dglucopyranoside (8). The generation of the thiosugar from the pseudothiourea derivative was carried out exactly as described for 2. The thiosugar obtained from 11.05 g (25 mmol) of the pseudothiourea derivative was dissolved in 200 ml of 50% ethanol, to which was added 1 (7.71 g, 50 mmol), hydroquinone (20 mg), and pyridine (2 ml). Within 3 h, product 8 began to crystallize. After overnight at room temperature, the crystals were filtered, washed with 50% ethanol, and dried in a vacuum desiccator, yielding 9.15 g (17.7 mmol) of 8. The filtrate was concentrated to about 150 ml. To this was added about 25 ml of 95% ethanol, and the solution was left in the cold, thus producing an additional 1.12 g (2.2 mmol) of 8. The total yield of 8 was 79.5%. These crystals were not readily soluble in either acetone or chloroform and did not move in tic in solvent A or B. The dried crystals were pulverized, resuspended in 50% ethanol, and stirred several hours at room temperature. These washed crystals appeared as
0.6
\ t
I
LH-20(4 in
x 150cm) 95% EtOH
AND
LEE
single, immobile spots on tic solvent A; mp, 276-277°C. Anal. Calcd for C21H31N3010S (517.55): S, 6.20. Found: S, 6.42. (N - Acrylamidomethyl)aminocarbonyl ethyl 2-acetamido-2-deoxy I-thio-P-Dglucopyranoside (9). Washed crystalline 8 (1.18 g, 2.3 mmol) was stirred in 200 ml of dry methanol to form a fine suspension, which was then made 5 mM in sodium methoxide and stirred overnight at room temperature. During the reactions, particles of 8 disappeared, but a new type of solid was observed to form. This new solid dissolved readily upon addition of an equal volume of water. The tic (solvent B) of this solution indicated the presence of only one sugar derivative, suggesting that de-Oacetylation was complete. This suspension was kept in the cold overnight, filtered, washed with dry methanol, and dried in a vacuum desiccator, yielding 0.78 g (2.1 mmol) of 9 (91%): RF in solvent B, 0.23; mp, 232-234°C; [CX]D 25-20.6”(c 3.7, water). The pmr spectrum of 9 in DMSO-ds after D,O exchange gave the methyl signal at 1.83 ppm, and the anomeric proton signal at 4.42 ppm. Other features of the spectrum were similar to that of 3. For analytical purposes, 9 was further washed as a fine suspension in 95% ethanol overnight at room temperature, filtered, and dried. Anal. Calcd. for C15HZ5N307S * Hz0 (409.46): C, 44.00; H, 6.65; N, 10.26; S, 7.83. Found: C, 43.93; H, 6.89; N, 10.29;
S, 7.87.
1
60
-470
FRACTION
I 80
,1, 90
100
110
NUMBER
FIG. 2. Purification of 6 on a Sephadex LH-20 umn. An aliquot from each fraction (18 ml) analyzed by phenol-sulfuric acid method.
colwas
N, N’-Diacryloyl-1,2-diaminoethane (10). Acryloyl chloride (8 ml, 98 mmol) was added dropwise to a solution of ethylenediamine (10 ml, 149 mmol) in 40 ml of l,Cdioxane, which was chilled in an ice bath. This mixture was stirred for l/2 h in the cold, then for 1 h at room temperature. The mixture was stirred with 50 ml of chloroform and filtered, and the precipitate was washed with 50 ml of 1,Cdioxane. The filtrate and washings were combined, mixed with 50 mg of hydroquinone, and evaporated to dry-
ACRYLAMIDE
GELS
WITH
tress. The residue was dissolved in 95% ethanol, and the insoluble gelatinous material was removed by filtration with the aid of Celite. The filtrate was concentrated to a syrup, from which crystals of 10 were obtained in three crops after addition of anhydrous ether. The total yield was 52%. The product was pure by tic in solvent A; mp, 125-126°C [lit. 141.51435°C; Hirose et al. (16)]. (N - Acrylamidoethyljaminocarbonylethyl 2,3,4,6-O-acetyl-I-thio-@Dgalactopyranoside (11) and (N-acrylamidoethyljaminocarbonylethyl I-thio-/SD-galactopyranoside (12). Preparation of 11 from 10 (10 mmol)
and the pseudothiourea derivative of galactose (5 mmol) was carried out in essentially the same way as for 2, except that the reaction mixture was kept at room temperature for 7 days, at which time there was still a small amount of unreacted thiol. The mixture was evaporated to a syrup, dissolved in 30 ml of 95% ethanol, and fractionated in two batches on the column of Sephadex LH-20. The elution profile was similar to the one shown in Fig. 2, except that the position of all three peaks were two or three fractions earlier than those shown in Fig. 2. Unreacted 10, which was located by tic (solvent A) in fractions 107- 117, was recovered by evaporation, yielding 1.21 g (7.2 mmol). The major peak (fractions 95101) contained 11. These fractions were combined and evaporated to yield a syrup. This syrup was dissolved in absolute ethanol and toluene and evaporated. The syrupy 11 was deacetylated as described for 3. Addition of benzene and petroleum ether (3560°C) produced a brown, amorphous precipitate at first. Addition of more petroleum ether to the filtrate produced crystalline 12; the yield was 0.38 g, 1.04 mmol (21%). 12 was recrystallized from ethanol-benzenepetroleum ether; mp, 148- 150°C; [a]~*~ +4.6” (c 6.2, water); RF in solvent B, 0.17. A portion of this material was hydrolyzed by mercury and analyzed by the automated sugar analyzer, yielding 98% of the theo-
THIOGLYCOSIDES
265
retical value of galactose based on dry weight. Anal. Calcd. for C14H24N207S1 (364.42): C, 46.14; H, 6.64: N, 7.69; S, 8.80. Found: C, 45.68; H, 6.83, N, 7.58; S, 8.48. N,N’
- Diacryloyl
- 1,6 - diaminohexane
(13). Acryloyl chloride (16 ml, 176 mmol) was added dropwise to a solution of 1,6diaminohexane (5.8 g, 50 mmol) and sodium carbonate (31.3 g, 250 mmol) in 250 ml of water while being chilled in an ice bath. After 15 min, the precipitate was filtered, washed with cold water, and crystallized from ethanol-ether. Two crops of crystalline 13 were obtained in 51.8% (5.8 g) and 13.8% (1.54 g) yield, respectively. The melting point of the first crop was 140141°C [lit. 145~6°C; Hirose et al. (16)]. (N-Acrylamidohexyl)aminocarbonylethyl 2,3,4,6-tetra-O-acetyl-l-thio-~-n-galactopyranoside (14) and (N-acrylamidohexyl) aminocarbonylethyl I-thio-&bgalactopyranoside (15). This reaction was carried out
in half the scale as the preparation of 2, substituting 13 for 1. The reaction mixture was left standing at room temperature for 7 days, at which time about 8% of the thiol group had still not reacted. The mixture was concentrated to a syrup, dissolved in 30 ml of 95% ethanol, and fractionated, in two batches, on the column of Sephadex LH-20. The elution was similar to that shown in Fig. 2, except that all three peaks were eluted earlier than the corresponding peaks in Fig. 2. The main peak was located in fractions 88-95, and unreacted 13 was eluted in fractions 97- 107. About 1.76 g (7.85 mmol) of 13 was recovered from the column effluent, indicating that the yield of 14 was no greater than 43%. The fractions of the major carbohydrate peak from two column runs were combined and evaporated to syrupy 14. This syrup was dried by dissolving it in absolute ethanol and toluene and evaporating off the solvents. The residue was deacetylated as described for 3. Addition of benzene to the deacetylation mixture produced a small amount of a brown, amorphous
266
LEE,
CASCIO,
precipitate, which was removed by filtration. Addition of absolute ethanol, benzene, and petroleum ether (35-60°C) and storage in the cold produced three crops of crystalline 15. The combined yield was 0.58 g (1.4 mmol), 28%; mp, 138-140°C; [alDz5 -2.90” (c 2.3, water); RF in solvent B, 0.31. A portion of this material, hydrolyzed by mercury and analyzed by the automated sugar analyzer, yielded 100% of the theoretical quantity of galactose based on dry weight. Anal. Calcd. for ClsH32NZ0,S, (420.53): C, 51.41; H, 7.67; N, 6.66; S, 7.63. Found: C, 51.32; H, 7.95; N, 6.68; S, 7.86. Comparison of I-thiogalactopyranose addition to 1, 10 and 13. The addition of the thiol group of 1-thiogalactopyranose to the N,N’-bisalkaneacylamide compounds (1,lO and 13) was compared under the following conditions and using the same preparation of l-thiogalactopyranose. The reaction mixture consisted of 0.25 mmol of the thiosugar; 0.5 mmol of either 1, 10, or 13; and 50 ~1 of pyridine in 2.8 ml of 50% methanol. In addition, two reaction mixtures were prepared in which two kinds of bisacrylamide compounds were present (1, and 10 in one, 1 and 13 in the other). The thiol content of the mixture was measured at various times. Figure 1 shows the results of these experiments. Obviously the addition of lthiogalactopyranose to 1 is much faster than to 10, which in turn is slightly faster than to 13. The rates of decrease of thiol group in the reactions containing two bisacrylamide compounds were the sum of the rates of the individual bisacrylamide compounds. Therefore it appears that the presence of one bisacrylamide compound did not stimulate or inhibit addition of thiol group to the other bisacrylamide compound. Purification of peanut lectin by af$nity chromatography. Two types of acrylamide gels containing 1-thiogalactopyranosides were prepared. In the first type, soluble, linear copolymers of acrylamide and 3 in 1: 1 molar ratio were prepared, which were then trapped in a 12% acrylamide gel with
AND
LEE
10% crosslinking. The second type was a 12% acrylamide gel containing 2% (molar) of 3 as monomer with 10% crosslinking. The exact procedures for the preparation of gels are as follows: Type I: To a solution of acrylamide (71 mg, 1 mmol) and 3 (350 mg, 1 mmol) in 10 ml of water were added 0.5 ml of 2.5% N,N,N’,N’-tetramethylethylenediamine (TEMED) and 0.5 ml of 0.5 hi ammonium persulfate. The reaction mixture was kept at room temperature for a few hours. The size distribution of polymers prepared in this manner was determined previously by passing them through a column of Bio-Gel A-5M (1.25 x 35 cm) using 5 mM NaCl as eluant. It was found that 80% of the carbohydrate (phenol-sulfuric acid color) was contained in polymers with molecular weights larger than 500,000. A thick solution of polymer was diluted with 5 ml of water and added to a solution containing 3 g of acrylamide and 0.3 g of 1 in 10 ml of water. Polymerization was initiated by the addition of 5 ~1 of TEMED and 0.5 ml of 5% ammonium persulfate. After polymerization overnight at room temperature, the gel was broken up in a Virtis homogenizer at about 500 r-pm for 1 h. The ground gel was washed and defined by repeated decantation in water. Type II: Acrylamide (3 g, 42.2 mmol), 1 (0.3 g), and 3 (0.3 g, 0.86 mmol) were dissolved in 25 ml of water. Polymerization was initiated by the addition of 5 ~1 of TEMED and 0.5 ml of 5% ammonium persulfate. The gel thus formed was ground and defined as described for type I. The efficiency of incorporation of 3 into the two types of gel was determined in the following way: A known volume of gel was washed with water (3x) in a graduated centrifuge tube by centrifugation, and removal of the supernate was accomplished by suction. The gel was dehydrated by washing with 95% ethanol (2x) and acetone (2x), then dried in a vacuum desiccator, and weighed. A volume of 1.1 ml of packed gel was equivalent to 90 mg dry weight. Another
ACRYLAMIDE
GELS WITH THIOGLYCOSIDES
l4-
1.2Yz g tow 8 083 B % 0.6s 04-
,.?
02-
20
40
EFFLUENT
60
80
100
120
( ml I
FIG. 3. Affinity chromatography of “precolumn fraction” of peanut lectin. Elution procedure is described in the text. Arrow indicates the introduction of 0.05 M galactose.
portion of packed, water-washed (but not dehydrated) gel (0.1 ml) was treated with mercuric acetate (0.1 M in 0.02 M acetic acid, 1.5 ml) at 55°C for 1 h to hydrolyze the lthioglycosidic linkage and release the sugar from the gel (13). The supernate of the hydrolysate and two water washes of the gel were passed through a column of Dowex 50-X8 (H+-form, 0.8 g packed into a Pasteur pipet), which was washed four times with 0.5 ml of 0.02 M acetic acid. The eluate was made made up to 4 ml, and a sample therefrom was analyzed by the phenol-sulfuric acid method. A sample of 2-(6-aminohexanamido) ethyl l-thio-P-D-galactopyranoside (17) was subjected to mercuric acetate hydrolysis (at room temperature), chromatographed on the Dowex 50 column, and used as standard in the analysis. Incorporation of galactose into the type I gel was found to be only 37%, while incorporation into the type II gel was 90% of the galactose derivative (3) used in the gel formation. The concentrations of galactose in types I and II gels was 6.2 and 15.3 PmoYml of packed gel, respectively.
267
Ground gel (20 ml) was packed into a column (2.5 x 4 cm), and washed with 0.15 M NaCl. The effluent contained a negligible amount of sugar. As freeze-dried “precolumn lectin” fraction (6) of peanut lectin (0.27 g) was stirred in 10 ml of 0.15 M NaCl, passed through a column of packed glass wool to remove insoluble matter, and then applied to the acrylamide-gel column of either types I or II in the cold. Elution was performed as described by Sutoh et ~11.(6), first with 0.15 M NaCl until the absorbance at 280 nm returned to the background level and then with 0.05 M galactose. As shown in Fig. 3, elution with NaCl produced one, large protein peak with no hemagglutination activity, while elution with galactose produced a single, sharp protein peak. The latter peak was dialyzed in the cold against 0.15 M NaCl in 0.01 M Tris-HCl buffer, pH 7.2. The dialyzed material ran as a single band in polyacrylamide gel electrophoresis at pH 7.5 as shown in Fig. 4, and had the same specific activity as the peanut lectin purified by a different method (6). These acrylamide derivatives of l-thioglycosides were also used to prepare polyacrylamide gels for cell-adhesion studies (18). The gels prepared with these derivatives yielded essentially the same results obtained earlier with the gels described in the introduction (2); namely, chicken hepatocytes adhered specifically to gels containing 2-acetamido-2-deoxyl-thio+-D-glucopyranoside, while rat hepatocytes adhered specifically to gels containing 1-thio-P-Dgalactopyranoside. DISCUSSION
Polyacrylamide gels are suitable matrices for the immobilization of molecules for biological studies. The carbohydrate-containing polyacrylamide gels described here can be readily prepared and are suitable for a wide range of biological applications. In particular, the use of the w-acryloyl thioglycoside has many advantages. First of all,
268
LEE, CASCIO, AND LEE
6 FIG. 4. Polyacrylamlcie gel electrophoresis of protein fractions obtained in the affinity chromatography. Right: lectin peak, eluted with galactose; left: the protein peak not absorbed to the affinity column.
the incorporation of the preformed ligand plus a “spacer” arm into the gel obviates the problems of chemical heterogeneity created by derivatizing a preformed gel. In addition, the o-acryloyl thioglycosides can be prepared in good yield by simple methods from readily available reagents. N,N’-Methylenebisacrylamide and its analogs with longer alkyl chains are commercially available, and, if necessary, can be prepared from acryloyl chloride and diaminoalkane in a single step. Some Ithiosugars are commercially available, and they are also easily prepared from commercially available acetylated glycosyl halides via their pseudothiurea derivative (9,19). This synthetic scheme also provides for flexibility in varying the sugar and the
chain length of the “spacer” arm. The presence of the S-glycosidic linkage is an advantage working with biological systems since it is highly resistant to enzymatic cleavage yet is readily and specifically hydrolyzed by mercury salts, which facilitates analysis (13). Thioglycosides have already been shown to behave similarly to O-glycosides in affinity chromatography and cell-adhesion studies (18). The addition reaction was carried out using twofold molar excess of the bisacrylamide (equivalent to fourfold excess of the acrylamide groups) to reduce formation of the bis-addition product. The use of an even larger excess of the bisacrylamide resulted in only slight reduction in the formation of the bis-addition product and caused subsequent purification to be more cumbersome. The reaction of 1-thio-P-o-galactose, -/3D-glucose, and 2-acetamido-2-deoxy-/3-Dglucose with 1 proceeded rapidly to completion with very little formation of bisadduct. After the addition reaction was complete, unreacted 1 could be removed easily by partitioning between chloroform and water. The desired products remained in chloroform, while 1 was extracted into water. This procedure gave sufficient purification so that upon de-O-acetylation products could be obtained as crystals. On the other hand the reaction of I-thio-a-r>mannose with 1 was considerably slower after 1 day than the other thiosugars, and did not proceed to completion. It was necessary to fractionate this reaction mixture on a column of Sephadex LH-20. The yield of the a-D-mannose derivative (7) was only -30% compared to yields of 70-90% for the derivatives of p-o-galactose, P-o-glucose, and 2-acetamido-2-deoxy-&D-glucose. These differences in the reaction rates and the yields may be attributed to steric hindrance of the axial-thiol group in the (Y-Dmannose derivative. Reactions of I-thiogalactose with 10 and 13 instead of 1 also proceed very slowly. The fact that neither a stimulatory nor an
ACRYLAMIDE
269
GELS WITH THIOGLYCOSIDES
inhibitory effect was exhibited when two bisacrylamido compounds were present in the same reaction mixture suggests that the reaction rate is an intrinsic characteristic of each bisacrylamide compound. The addition reactions using 10 and 13 did not go to completion under the conditions described. It was necessary, therefore, to purify the reaction products on a column of Sephadex LH-20. This gel filtration also served the purpose of separating and recovering unreacted bisacrylamido compounds (10 and 13), which, unlike 1, were not easily extracted into water from chloroform. Although the procedure described here used O-acetylated I-thiosugars, the corresponding de-0-acetylated sugars can also be used for monoaddition to bisacrylamido derivatives. However, the overall yields in the latter case are generally much lower than those obtained with the 0-acetylated sugars. Polysaccharides entrapped in polyacrylamide have been shown to be effective for affinity chromatography of lectins (6,20). In this study, both the conventional method of gel formation (type II) and the entrapment of preformed linear acrylamide polymers which contained thioglycosides in crosslinked polyacrylamide gel (type I) were tested. The type II gel was more effective in terms of incorporation of the thioglycoside monomer into the gel than the type I gel. In the latter case, it appears that a considerable amount of the preformed linear polymer was too small to be trapped in the network of crosslinked polyacrylamide under the conditions described here. In spite of the difference in the total lthiogalactoside content, the two types of gels behaved nearly identically during affinity chromatography. It may be reasoned, therefore, that performance of ligandcontaining polyacrylamide gels is not only a
function of total ligand concentration, but is a function of available ligand concentration. REFERENCES I. Chipowsky, S., Lee, Y. C., and Roseman, S. (1973) Proc. Nat. Acud. Sci. USA 70, 23092312. 2. Schnaar. R. L., Weigel, P. H., Kuhlenschmidt, M., Lee, Y. C., and Roseman, S. ( 1978) J. Bio!. Chem.
253.
7940.
3. Schnaar, R. L., and Lee, Y. C. (1975) Biochemistry 14, 153.5. 4. Schnaar, R., Weigel, P. H., Roseman, S., and Lee, Y.
C.
(1979)
Methods
Carbohydr.
Chem.
8,
in press. 5. Weigel, P. H., Schnaar, R., Roseman. S., and Lee, Y. C. (1979) Methods Carhohydr. Chem. 8, in press. 6. Sutoh, K., Rosenfeld, L., and Lee, Y. C. (1977) Anal.
B&hem.
79,
329.
7. Weigel, P. H., Schmell, E., Lee, Y. C., and Roseman, S. (1978) J. Biol. Chem. 253, 330-333. 8. Horton, D. (t963) Methods Curbohydr. Chem. 2, 433. 9. Lee, Y. C., Stowell, C. P., and Krantz, M. J. (1976)
15, 3956.
Biochemistry
10. Horton, D., and Wolfrom, Chem.
21,
M. L. (1%2) J. Org.
1794.
11. McKelvy, J. F.. and Lee, Y. them.
Biophys.
132,
C. (1969)
12. Ellman, G. L. (1959) Arch. Biochcm. 82, 70. 13. Krantz, M. J.. and Lee, Y. C. (1976) them.
Arch.
Bio-
99. Biophys. Anal.
Bio-
71, 318.
14. Lee, Y. C. (1972) in Methods in Enzymology (Ginsburg, V.. ed.), Vol. 28. p. 63, Academic Press, New York. 1s. Reisfeld, R. A., Lewis, U. J., and Williams, D. E. ( 1%2) Nuture (Landon) 195, 281. 16. Hirose, K., Ihashi, Y ., Taguchi, S., and Yoshizawa, M. (1966) Kogyokagaku Zasshi (J~~pan) 69, 240-244. 17. Lee, R. T., and Lee, Y. C. (1974) Curbohydr. Has. 34, 151. 18. Weigel, P. H., Schnadr. R., Schmell, E., KuhlenSchmidt. M. S.. Lee, R. T.. Lee. Y. C.. and Roseman, S.. in press. 19. Chipowsky, S.. and Lee, Y. C. ( 1973) C’arh,thvdr. Res.
31, 339.
20. Horisberger,
M.
(1977)
Carbohydr.
Re$.
53,
231.
BIOCHEMISTRY
A Simple
Method for the Preparation Containing Thioglycoside
REIKO Department
95, 260-269(1979)
T. LEE, STEPHANIE of Biology
and McCollum-Pratt Baltimore,
CASCIO,
of Polyacrylamide LigandW AND YUAN
Institute, The Johns Maryland 21218
CHUAN Hopkins
Gels
LEES University,
Received November 13, 1978 A simple method was developed for the preparation of acrylamide derivatives containing thioglycosides. The synthetic scheme involves the preparation of an O-acetylated I-thiosugar via a pseudothiourea derivative, the controlled addition of the thiosugar to N.N’-bismethyleneacrylamide to form a monoaddition product in which one of the acrylamide groups remains unchanged, and finally de-O-acetylation. Similar reaction schemes using bisacrylamido derivatives of 1,2-diaminoethane and 1,6-diaminohexane lead to analogous compounds with longer aglycon chain lengths. The thioglycoside derivatives of acrylamide thus prepared could be copolymerized with acrylamide to form polymers or gels containing thioglycosides as ligands. These gels were successfully used as affinity materials for the purification of peanut lectin and in cell-adhesion studies.
The elucidation of the roles of carbohydrates in biological reactions has been probed with carbohydrates of well-defined structure. Frequently, immobilization of such carbohydrates proves to be an invaluable aid to biological studies (1,2). We have chosen polyacrylamide gel as the matrix most suitable for such purposes, because of its flexibility and chemical and biological inertness. Earlier publications from our laboratory described two different methods for the preparation of polyacrylamide gels containing o-aminoalkyl glycosides or their l-thio analogs. In the first method, N-succinimidyl acrylate is copolymerized with acrylamide, and the active esters are subsequently replaced with aminoalkyl glycosides (3,4). The second method involved acryloylation of aminoalkyl glycosides and
its copolymerization with acrylamide (5). The carbohydrate-containing gels prepared by these methods have been effectively utilized for the isolation of lectins (3,6) and in studies of cell adhesion (2,7). We have now developed a simple method for the preparation of thioglycoside derivatives of acrylamide, which in turn can be used to produce carbohydrate-containing acrylamide polymers. The underlying prin-
1 This paper is dedicated to the memory of Dr. Alvin Nason. 2 Contribution No. 993 from the McCollum-Pratt Institute, The Johns Hopkins University. Supported by USPHS NIH Research Grants AM9970 and CA21901. 3 To whom all correspondence should be addressed. 0003-2697/79/070260-10$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
SCHEME
260
1
ACRYLAMIDE
GELS WITH THIOGLYCOSIDES
CH,=CHCONH(CH,),NHCOCH=CH, Compound No.
n 1
1 10 13
2 6
R-SCH,CH,CONH(CH,),NHCOCH=CH, Compound No.
n -
R
2
1
3 4
I
5
1
6
1
7 8
1 1
9
1
11
2
12 14
2 6
15
6
1
2,3,4,6,-Tetra-O-acetyl+?-D-galactopyranosyl @&alactopyranosyl 2,3,4,6-Tetra-O-acetyl-@-glucopyranosyl P-D-Glucopyranosyl 2,3,4,6-Tetra-O-acetyl-a-D-mannopyranosyl a-D-Mannopyranosyl 2-Acetamido-3,4,6-tri-0-acetyl-2deoxy-P-D-glucopyranosyl 2-Acetamido-2-deoxy-@r+ucopyranosyl 2,3,4,6-Tetra-O-acetyl-/3-D-galactopyranosyl P-D-Galactopyranosyl 2,3,4,6-Tetra-O-acetyI+-D-galactopyranosyl b-o-Calactopyranosyl SCHEME
2
ciple of this synthesis is illustrated in Scheme 1. Since the starting materials for this reaction can be either commercially purchased or easily prepared by simple procedures from available materials, the present method offers great advantages over the other methods. The gels prepared by the method described here have been satisfactorily used for lectin isolation and cell-adhesion studies. EXPERIMENTAL Materials and methods. Acrylamide from J. T. Baker Chemical Co. and N,N’-methylenebisacrylamide (l), acryloyl chloride, 1,Zdiaminoethane, and 1,6-diaminohexane from Aldrich Chemical Co. were used without purification. Partially purified peanut
261
lectin (“precolumn lectin”) was prepared as reported earlier (6). Pseudothiourea derivatives of 2,3,4,6tetra-0-acetyl-P-D-galactopyranose, D-Dglucopyranose (8), a-D-mannopyranose (9), and 2-acetamido-2-deoxy-3,4,6-tri-o-acetyl/3-o-glucopyranose (10) were prepared from the corresponding 1-bromo or 1-chloro sugars as described. All evaporations were performed under reduced pressure at 25-40°C with a rotatory evaporator. Melting points (uncorrected) were measured with a Fisher- Johns apparatus. Proton magnetic resonance spectra were recorded with a JEOL NMH- 100 spectrometer. Optical rotations were measured with a Cary 60 spectropolarimeter. Elemental analyses were performed by Galbraith Laboratories (Knoxville, Tenn.). Thin-layer chromatography (tlc)4 was performed with silica gel F-254 layers precoated on aluminum sheets (Merck), using the following solvent systems: (A) 4:l (v/v) ethyl acetate-acetone, (B) 9:4:2 (v/v/v) ethyl acetate-isopropyl alcohol-water. Acrylamido compounds were detected by fluorescence quenching. For detection of carbohydrates, tic sheets were sprayed with 15% sulfuric acid in 50% ethanol and heated for a few minutes at 140°C. Whenever necessary, chromatographic purification of O-acetylated sugar derivatives was achieved with a column of Sephadex LH-20 (4 x 190 cm) in 95% ethanol collecting l&ml fractions. Neutral sugars were determined by a modified version (11) of the phenol- sulfuric acid method, and thiol groups by Ellman’s method (12). The glycoside monomers were characterized by hydrolyzing a known dry weight with mercuric acetate (13), then analyzing it on an automated neutral sugar analyzer (14). Protein concentrations were determined by measuring absorbance at 280 nm. 4 Abbreviations used: tic, thin-layer chromatography: pmr, proton magnetic resonance: DMSO. dimethyl sulfoxide; TEMED, tetramethylethylenediamine.
262
LEE, CASCIO, AND LEE
The hemagglutination assay for peanut lectin was performed as described by Sutoh et al. (6). Polyacrylamide gel electrophoresis was performed according to the method of Reisfeld et al. (15), and proteins were stained with Coomassie brilliant blue. Preparation of I-thioaldopyranose tates. The pseudothiourea derivatives
ace-
100 2 eo 4 g 60 5 F’ ‘E Lz : 20
were converted into the corresponding 1-thio de0 rivatives in the following way: To a solution 0 I 2 3 4 5 22 of pseudothiourea derivative, 10 mmol(4.87 Time(h) g for neutral sugar derivatives and 4.42 g for FIG. 1. Rate of decrease of the thiol group in the the amino sugar derivative) in 40 ml of water, mixture containing I-thiogalactose and the were added potassium carbonate (1.66 g, 12 reaction following bisacrylamido compounds: (A). 1, (B) 10, mmol) and sodium bisulfite (1.04 g, 10 (0) 13, (0) 1 + 10, (0) 1 + 13. mmol), followed immediately by 40 ml of chloroform. The biphasic reaction mixture was stirred vigorously in an enclosed vessel. in solvent A showed three components with Within a few minutes, all the salts were very similar RF values. The three compodissolved in the aqueous layer. The appearnents were, in order of increasing RF value, ance of I-thiosugar in the chloroform layer a strong, uv-absorbing spot of remaining 1, was analyzed by Ellman’s method. The con- a uv-absorbing sugar derivative (2), and a small amount of non-uv-absorbing sugar centration of I-thiosugars reached a plateau derivative. This last material was obtained within 15-30 min, but stirring was usually in pure crystalline form after gel filtration continued for a total of 45 min, after which time the two layers were separated. The on a column of Sephadex LH-20 (see below) aqueous layer was extracted once with 20 ml and was identified as a bis-addition prodof chloroform. The chloroform solutions uct [N,N’-methylenebis(aminocarbonylethy1 were combined, washed once with 40 ml of 2,3,4,6-tetra-O-acetyl-l-thio-p-D-galactowater, and then evaporated to a syrup. pyranoside)]. This reaction mixture was one (N - Acrylamidomethyl)aminocarbonyl - routinely evaporated to approximately half of the original volume and then partiethyl 2,3,4,6-tetra-O-acetyl-l-thio-@D-gationed between 75 ml each of water and lactopyranoside (2). The syrupy I-thiosugar derivative obtained from 10 mmol of 2-S- chloroform. The aqueous phase was extracted again with 50 ml of chloroform. The (2,3,4,6-tetra-O-/3-D-galactopyranosyl)-2pseudothiourea hydrobromide was stirred combined chloroform layers were then with 10 ml of 50% ethanol, and a solution of washed with water (4 x 50 ml) to remove excess 1. The chloroform layer was dried l(3.08 g, 20 mmol) in 50 ml of 50% ethanol was added to it. Stirring briefly produced a with anhydrous sodium sulfate, filtered, and clear solution, to which were added 1 ml evaporated. The resulting syrup contained mainly the product 2 and a small amount pyridine and a small amount of hydroquioof the bis-addition by-product. These two none (5-6 mg). The progress of the reaction compounds could be separated completely was followed by measuring the disappearusing the Sephadex LH-20 column. Fracance of thiol group. The reaction of O-acetytions containing 2 were combined and lated thiosugar with 1 was complete within 24 h in most cases. The kinetics of these evaporated to a small volume, which, upon reactions are shown in Fig. 1. After over- standing in the cold, produced crystals; mp, 122- 123°C. The pmr data (CDCl& indicated night at room temperature, tic of the mixture
ACRYLAMIDE
GELS
WITH
the correct ratio of CH,CO- (6 2.0-2.1, m, 12) to -W&X,(6 2.7 and 2.95, m, 4) to -CH,protons (6 4.3, s, 2) to -NHCOprotons (6 7.65, broad, 2). Acryloyl proton signals were in the region of 5.7 to 6.35 ppm. For routine preparation of 3, however, one can proceed to the next step (de-O-acetylation) without the Sephadex LH-20 gel filtration. (N - Acrylamidomethyl)aminocarbonyl ethyl I-thio-PDgalactopyranoside (3). The
THIOGLYCOSIDES
263
tose derivative, 2. The yield of 4 was 80%. Pure, crystalline 4 obtained from Sephadex LH-20 chromatography melted at 168169°C. De-0-acetylation of 4 by sodium methoxide produced 5 in 82% yield: Rf in solvent B, 0.32; mp, 165- 167°C; [(~],~~-24.6 (c 8.8, water). The pmr spectrum was similar to that of 3. Anal. Calcd. for C,,H,,N,O,S (350.40): C, 44.56; H, 6.33; N, 8.00; S, 9.15. Found: C, 44.47; H, 6.37: N, 8.01; S, 8.93.
syrup containing mainly 2 and small amounts (N - Acrylamidomethyl)aminocarbonyl of by-products (see above) was de-o-acetyethyl 2,3,4,6-tetra-O-acetyl-1-thio-a-D-manlated in 20 ml dry methanol containing 0.01 nopyranoside (6) and (N-acrylamidomethyl)M sodium methoxide for 2 h at room tem- aminocarbonylethyl I-thio-cw-Dmannopyraperature. Crystals of 3 which formed over- noside (7). The mannose derivatives were night at 4°C were filtered and washed with prepared exactly as described for the galacmethanol (yield: 2.3 g, 6.54 mmol). The ad- tose derivatives 2 and 3. However, the addition of benzene and petroleum ether (bp, dition reaction mixture contained, in addi35-60°C) to the filtrate produced an addi- tion to the two expected products, a contional 0.14 g (0.4 mmol) of crystalline 3. The taminant which was not observed in the total yield of crystalline 3 from the pseudopreparation of either the galactose or glucose thiourea derivative was 69.4%. The product derivative. The syrup obtained upon evapthus obtained was usually pure enough to be oration of the washed chloroform solution was dissolved in 15 ml of 95% ethanol and used in polymer formation (see below). However, the complete removal of impuriapplied to the column of Sephadex LH-20. ties could be accomplished by passing the The elution profile is shown in Fig. 2. The aqueous solution of the product through a largest peak contained mainly 6. Fractions 98 - 107 were combined and evaporated to a Sephadex G-15 column (2 x 145 cm) equilibrated with 0.1 M acetic acid. Fractions small volume; however, compound 6 could containing 3 were evaporated to a small not be obtained in crystalline form. It was volume, to which 95% ethanol was added. dehydrated by the addition and evaporation Upon standing in the cold, pure 3 crystalof absolute ethanol and toluene, and then lized: R, in solvent B, 0.23: mp, 193°C; and de-0-acetylated as described for 3. Benzene [a],,25-5.10 (c 5.3, water). The spectrum of and petroleum ether (35-60°C) were added ?H-exchanged sample in dimethyl sulfoxide to the deacetylation mixture to produce two (DMSO)-dG showed the presence of both crops of crystals. The yield of crystalline 7 ethylene and acryloyl groups. The anomeric from 9 mmol of the pseudothiourea derivaproton signal was at 4.30 (d, 1, J = 7 Hz). tive was 2.86 mmol(32%). Recrystallization Anal. Calcd. for C,,H,,N,O,S (350.40): from absolute ethanol-benzene-petroleum C, 44.56; H, 6.33; N, 8.00; S, 9.15. Found: ether (35-60”(Z) produced pure, crystalline C, 44.49; H, 6.45; N, 8.07; S, 9.06. 7 having mp, 166-167°C: [c~],,~“+117.3~ (c. (IV - Acrylamidomethyl)aminocarbonyl - 8.9, water); and R, in solvent B of 0.38. ethyl 2,3.4,6-terra-O-acetyl-I-thio-pDgluAnalysis for mannose of a mercuric hydrolycopyranoside (4) and (N-acrylamidomethyl) sate of this material yielded 102% of the aminocarbonylethyl 1-thio-p-o-glucopyratheoretical amount of mannose on the drynoside (5) The glucose derivative (4) was weight basis. prepared exactly as described for the galac(N - Ac~lamidomethyl)aminocarbonyl -
264
LEE,
CASCIO,
ethyl 2-acetamido-3,4,6-tri-0-acetyl-2-deoxyI-thio-@Dglucopyranoside (8). The generation of the thiosugar from the pseudothiourea derivative was carried out exactly as described for 2. The thiosugar obtained from 11.05 g (25 mmol) of the pseudothiourea derivative was dissolved in 200 ml of 50% ethanol, to which was added 1 (7.71 g, 50 mmol), hydroquinone (20 mg), and pyridine (2 ml). Within 3 h, product 8 began to crystallize. After overnight at room temperature, the crystals were filtered, washed with 50% ethanol, and dried in a vacuum desiccator, yielding 9.15 g (17.7 mmol) of 8. The filtrate was concentrated to about 150 ml. To this was added about 25 ml of 95% ethanol, and the solution was left in the cold, thus producing an additional 1.12 g (2.2 mmol) of 8. The total yield of 8 was 79.5%. These crystals were not readily soluble in either acetone or chloroform and did not move in tic in solvent A or B. The dried crystals were pulverized, resuspended in 50% ethanol, and stirred several hours at room temperature. These washed crystals appeared as
0.6
\ t
I
LH-20(4 in
x 150cm) 95% EtOH
AND
LEE
single, immobile spots on tic solvent A; mp, 276-277°C. Anal. Calcd for C21H31N3010S (517.55): S, 6.20. Found: S, 6.42. (N - Acrylamidomethyl)aminocarbonyl ethyl 2-acetamido-2-deoxy I-thio-P-Dglucopyranoside (9). Washed crystalline 8 (1.18 g, 2.3 mmol) was stirred in 200 ml of dry methanol to form a fine suspension, which was then made 5 mM in sodium methoxide and stirred overnight at room temperature. During the reactions, particles of 8 disappeared, but a new type of solid was observed to form. This new solid dissolved readily upon addition of an equal volume of water. The tic (solvent B) of this solution indicated the presence of only one sugar derivative, suggesting that de-Oacetylation was complete. This suspension was kept in the cold overnight, filtered, washed with dry methanol, and dried in a vacuum desiccator, yielding 0.78 g (2.1 mmol) of 9 (91%): RF in solvent B, 0.23; mp, 232-234°C; [CX]D 25-20.6”(c 3.7, water). The pmr spectrum of 9 in DMSO-ds after D,O exchange gave the methyl signal at 1.83 ppm, and the anomeric proton signal at 4.42 ppm. Other features of the spectrum were similar to that of 3. For analytical purposes, 9 was further washed as a fine suspension in 95% ethanol overnight at room temperature, filtered, and dried. Anal. Calcd. for C15HZ5N307S * Hz0 (409.46): C, 44.00; H, 6.65; N, 10.26; S, 7.83. Found: C, 43.93; H, 6.89; N, 10.29;
S, 7.87.
1
60
-470
FRACTION
I 80
,1, 90
100
110
NUMBER
FIG. 2. Purification of 6 on a Sephadex LH-20 umn. An aliquot from each fraction (18 ml) analyzed by phenol-sulfuric acid method.
colwas
N, N’-Diacryloyl-1,2-diaminoethane (10). Acryloyl chloride (8 ml, 98 mmol) was added dropwise to a solution of ethylenediamine (10 ml, 149 mmol) in 40 ml of l,Cdioxane, which was chilled in an ice bath. This mixture was stirred for l/2 h in the cold, then for 1 h at room temperature. The mixture was stirred with 50 ml of chloroform and filtered, and the precipitate was washed with 50 ml of 1,Cdioxane. The filtrate and washings were combined, mixed with 50 mg of hydroquinone, and evaporated to dry-
ACRYLAMIDE
GELS
WITH
tress. The residue was dissolved in 95% ethanol, and the insoluble gelatinous material was removed by filtration with the aid of Celite. The filtrate was concentrated to a syrup, from which crystals of 10 were obtained in three crops after addition of anhydrous ether. The total yield was 52%. The product was pure by tic in solvent A; mp, 125-126°C [lit. 141.51435°C; Hirose et al. (16)]. (N - Acrylamidoethyljaminocarbonylethyl 2,3,4,6-O-acetyl-I-thio-@Dgalactopyranoside (11) and (N-acrylamidoethyljaminocarbonylethyl I-thio-/SD-galactopyranoside (12). Preparation of 11 from 10 (10 mmol)
and the pseudothiourea derivative of galactose (5 mmol) was carried out in essentially the same way as for 2, except that the reaction mixture was kept at room temperature for 7 days, at which time there was still a small amount of unreacted thiol. The mixture was evaporated to a syrup, dissolved in 30 ml of 95% ethanol, and fractionated in two batches on the column of Sephadex LH-20. The elution profile was similar to the one shown in Fig. 2, except that the position of all three peaks were two or three fractions earlier than those shown in Fig. 2. Unreacted 10, which was located by tic (solvent A) in fractions 107- 117, was recovered by evaporation, yielding 1.21 g (7.2 mmol). The major peak (fractions 95101) contained 11. These fractions were combined and evaporated to yield a syrup. This syrup was dissolved in absolute ethanol and toluene and evaporated. The syrupy 11 was deacetylated as described for 3. Addition of benzene and petroleum ether (3560°C) produced a brown, amorphous precipitate at first. Addition of more petroleum ether to the filtrate produced crystalline 12; the yield was 0.38 g, 1.04 mmol (21%). 12 was recrystallized from ethanol-benzenepetroleum ether; mp, 148- 150°C; [a]~*~ +4.6” (c 6.2, water); RF in solvent B, 0.17. A portion of this material was hydrolyzed by mercury and analyzed by the automated sugar analyzer, yielding 98% of the theo-
THIOGLYCOSIDES
265
retical value of galactose based on dry weight. Anal. Calcd. for C14H24N207S1 (364.42): C, 46.14; H, 6.64: N, 7.69; S, 8.80. Found: C, 45.68; H, 6.83, N, 7.58; S, 8.48. N,N’
- Diacryloyl
- 1,6 - diaminohexane
(13). Acryloyl chloride (16 ml, 176 mmol) was added dropwise to a solution of 1,6diaminohexane (5.8 g, 50 mmol) and sodium carbonate (31.3 g, 250 mmol) in 250 ml of water while being chilled in an ice bath. After 15 min, the precipitate was filtered, washed with cold water, and crystallized from ethanol-ether. Two crops of crystalline 13 were obtained in 51.8% (5.8 g) and 13.8% (1.54 g) yield, respectively. The melting point of the first crop was 140141°C [lit. 145~6°C; Hirose et al. (16)]. (N-Acrylamidohexyl)aminocarbonylethyl 2,3,4,6-tetra-O-acetyl-l-thio-~-n-galactopyranoside (14) and (N-acrylamidohexyl) aminocarbonylethyl I-thio-&bgalactopyranoside (15). This reaction was carried out
in half the scale as the preparation of 2, substituting 13 for 1. The reaction mixture was left standing at room temperature for 7 days, at which time about 8% of the thiol group had still not reacted. The mixture was concentrated to a syrup, dissolved in 30 ml of 95% ethanol, and fractionated, in two batches, on the column of Sephadex LH-20. The elution was similar to that shown in Fig. 2, except that all three peaks were eluted earlier than the corresponding peaks in Fig. 2. The main peak was located in fractions 88-95, and unreacted 13 was eluted in fractions 97- 107. About 1.76 g (7.85 mmol) of 13 was recovered from the column effluent, indicating that the yield of 14 was no greater than 43%. The fractions of the major carbohydrate peak from two column runs were combined and evaporated to syrupy 14. This syrup was dried by dissolving it in absolute ethanol and toluene and evaporating off the solvents. The residue was deacetylated as described for 3. Addition of benzene to the deacetylation mixture produced a small amount of a brown, amorphous
266
LEE,
CASCIO,
precipitate, which was removed by filtration. Addition of absolute ethanol, benzene, and petroleum ether (35-60°C) and storage in the cold produced three crops of crystalline 15. The combined yield was 0.58 g (1.4 mmol), 28%; mp, 138-140°C; [alDz5 -2.90” (c 2.3, water); RF in solvent B, 0.31. A portion of this material, hydrolyzed by mercury and analyzed by the automated sugar analyzer, yielded 100% of the theoretical quantity of galactose based on dry weight. Anal. Calcd. for ClsH32NZ0,S, (420.53): C, 51.41; H, 7.67; N, 6.66; S, 7.63. Found: C, 51.32; H, 7.95; N, 6.68; S, 7.86. Comparison of I-thiogalactopyranose addition to 1, 10 and 13. The addition of the thiol group of 1-thiogalactopyranose to the N,N’-bisalkaneacylamide compounds (1,lO and 13) was compared under the following conditions and using the same preparation of l-thiogalactopyranose. The reaction mixture consisted of 0.25 mmol of the thiosugar; 0.5 mmol of either 1, 10, or 13; and 50 ~1 of pyridine in 2.8 ml of 50% methanol. In addition, two reaction mixtures were prepared in which two kinds of bisacrylamide compounds were present (1, and 10 in one, 1 and 13 in the other). The thiol content of the mixture was measured at various times. Figure 1 shows the results of these experiments. Obviously the addition of lthiogalactopyranose to 1 is much faster than to 10, which in turn is slightly faster than to 13. The rates of decrease of thiol group in the reactions containing two bisacrylamide compounds were the sum of the rates of the individual bisacrylamide compounds. Therefore it appears that the presence of one bisacrylamide compound did not stimulate or inhibit addition of thiol group to the other bisacrylamide compound. Purification of peanut lectin by af$nity chromatography. Two types of acrylamide gels containing 1-thiogalactopyranosides were prepared. In the first type, soluble, linear copolymers of acrylamide and 3 in 1: 1 molar ratio were prepared, which were then trapped in a 12% acrylamide gel with
AND
LEE
10% crosslinking. The second type was a 12% acrylamide gel containing 2% (molar) of 3 as monomer with 10% crosslinking. The exact procedures for the preparation of gels are as follows: Type I: To a solution of acrylamide (71 mg, 1 mmol) and 3 (350 mg, 1 mmol) in 10 ml of water were added 0.5 ml of 2.5% N,N,N’,N’-tetramethylethylenediamine (TEMED) and 0.5 ml of 0.5 hi ammonium persulfate. The reaction mixture was kept at room temperature for a few hours. The size distribution of polymers prepared in this manner was determined previously by passing them through a column of Bio-Gel A-5M (1.25 x 35 cm) using 5 mM NaCl as eluant. It was found that 80% of the carbohydrate (phenol-sulfuric acid color) was contained in polymers with molecular weights larger than 500,000. A thick solution of polymer was diluted with 5 ml of water and added to a solution containing 3 g of acrylamide and 0.3 g of 1 in 10 ml of water. Polymerization was initiated by the addition of 5 ~1 of TEMED and 0.5 ml of 5% ammonium persulfate. After polymerization overnight at room temperature, the gel was broken up in a Virtis homogenizer at about 500 r-pm for 1 h. The ground gel was washed and defined by repeated decantation in water. Type II: Acrylamide (3 g, 42.2 mmol), 1 (0.3 g), and 3 (0.3 g, 0.86 mmol) were dissolved in 25 ml of water. Polymerization was initiated by the addition of 5 ~1 of TEMED and 0.5 ml of 5% ammonium persulfate. The gel thus formed was ground and defined as described for type I. The efficiency of incorporation of 3 into the two types of gel was determined in the following way: A known volume of gel was washed with water (3x) in a graduated centrifuge tube by centrifugation, and removal of the supernate was accomplished by suction. The gel was dehydrated by washing with 95% ethanol (2x) and acetone (2x), then dried in a vacuum desiccator, and weighed. A volume of 1.1 ml of packed gel was equivalent to 90 mg dry weight. Another
ACRYLAMIDE
GELS WITH THIOGLYCOSIDES
l4-
1.2Yz g tow 8 083 B % 0.6s 04-
,.?
02-
20
40
EFFLUENT
60
80
100
120
( ml I
FIG. 3. Affinity chromatography of “precolumn fraction” of peanut lectin. Elution procedure is described in the text. Arrow indicates the introduction of 0.05 M galactose.
portion of packed, water-washed (but not dehydrated) gel (0.1 ml) was treated with mercuric acetate (0.1 M in 0.02 M acetic acid, 1.5 ml) at 55°C for 1 h to hydrolyze the lthioglycosidic linkage and release the sugar from the gel (13). The supernate of the hydrolysate and two water washes of the gel were passed through a column of Dowex 50-X8 (H+-form, 0.8 g packed into a Pasteur pipet), which was washed four times with 0.5 ml of 0.02 M acetic acid. The eluate was made made up to 4 ml, and a sample therefrom was analyzed by the phenol-sulfuric acid method. A sample of 2-(6-aminohexanamido) ethyl l-thio-P-D-galactopyranoside (17) was subjected to mercuric acetate hydrolysis (at room temperature), chromatographed on the Dowex 50 column, and used as standard in the analysis. Incorporation of galactose into the type I gel was found to be only 37%, while incorporation into the type II gel was 90% of the galactose derivative (3) used in the gel formation. The concentrations of galactose in types I and II gels was 6.2 and 15.3 PmoYml of packed gel, respectively.
267
Ground gel (20 ml) was packed into a column (2.5 x 4 cm), and washed with 0.15 M NaCl. The effluent contained a negligible amount of sugar. As freeze-dried “precolumn lectin” fraction (6) of peanut lectin (0.27 g) was stirred in 10 ml of 0.15 M NaCl, passed through a column of packed glass wool to remove insoluble matter, and then applied to the acrylamide-gel column of either types I or II in the cold. Elution was performed as described by Sutoh et ~11.(6), first with 0.15 M NaCl until the absorbance at 280 nm returned to the background level and then with 0.05 M galactose. As shown in Fig. 3, elution with NaCl produced one, large protein peak with no hemagglutination activity, while elution with galactose produced a single, sharp protein peak. The latter peak was dialyzed in the cold against 0.15 M NaCl in 0.01 M Tris-HCl buffer, pH 7.2. The dialyzed material ran as a single band in polyacrylamide gel electrophoresis at pH 7.5 as shown in Fig. 4, and had the same specific activity as the peanut lectin purified by a different method (6). These acrylamide derivatives of l-thioglycosides were also used to prepare polyacrylamide gels for cell-adhesion studies (18). The gels prepared with these derivatives yielded essentially the same results obtained earlier with the gels described in the introduction (2); namely, chicken hepatocytes adhered specifically to gels containing 2-acetamido-2-deoxyl-thio+-D-glucopyranoside, while rat hepatocytes adhered specifically to gels containing 1-thio-P-Dgalactopyranoside. DISCUSSION
Polyacrylamide gels are suitable matrices for the immobilization of molecules for biological studies. The carbohydrate-containing polyacrylamide gels described here can be readily prepared and are suitable for a wide range of biological applications. In particular, the use of the w-acryloyl thioglycoside has many advantages. First of all,
268
LEE, CASCIO, AND LEE
6 FIG. 4. Polyacrylamlcie gel electrophoresis of protein fractions obtained in the affinity chromatography. Right: lectin peak, eluted with galactose; left: the protein peak not absorbed to the affinity column.
the incorporation of the preformed ligand plus a “spacer” arm into the gel obviates the problems of chemical heterogeneity created by derivatizing a preformed gel. In addition, the o-acryloyl thioglycosides can be prepared in good yield by simple methods from readily available reagents. N,N’-Methylenebisacrylamide and its analogs with longer alkyl chains are commercially available, and, if necessary, can be prepared from acryloyl chloride and diaminoalkane in a single step. Some Ithiosugars are commercially available, and they are also easily prepared from commercially available acetylated glycosyl halides via their pseudothiurea derivative (9,19). This synthetic scheme also provides for flexibility in varying the sugar and the
chain length of the “spacer” arm. The presence of the S-glycosidic linkage is an advantage working with biological systems since it is highly resistant to enzymatic cleavage yet is readily and specifically hydrolyzed by mercury salts, which facilitates analysis (13). Thioglycosides have already been shown to behave similarly to O-glycosides in affinity chromatography and cell-adhesion studies (18). The addition reaction was carried out using twofold molar excess of the bisacrylamide (equivalent to fourfold excess of the acrylamide groups) to reduce formation of the bis-addition product. The use of an even larger excess of the bisacrylamide resulted in only slight reduction in the formation of the bis-addition product and caused subsequent purification to be more cumbersome. The reaction of 1-thio-P-o-galactose, -/3D-glucose, and 2-acetamido-2-deoxy-/3-Dglucose with 1 proceeded rapidly to completion with very little formation of bisadduct. After the addition reaction was complete, unreacted 1 could be removed easily by partitioning between chloroform and water. The desired products remained in chloroform, while 1 was extracted into water. This procedure gave sufficient purification so that upon de-O-acetylation products could be obtained as crystals. On the other hand the reaction of I-thio-a-r>mannose with 1 was considerably slower after 1 day than the other thiosugars, and did not proceed to completion. It was necessary to fractionate this reaction mixture on a column of Sephadex LH-20. The yield of the a-D-mannose derivative (7) was only -30% compared to yields of 70-90% for the derivatives of p-o-galactose, P-o-glucose, and 2-acetamido-2-deoxy-&D-glucose. These differences in the reaction rates and the yields may be attributed to steric hindrance of the axial-thiol group in the (Y-Dmannose derivative. Reactions of I-thiogalactose with 10 and 13 instead of 1 also proceed very slowly. The fact that neither a stimulatory nor an
ACRYLAMIDE
269
GELS WITH THIOGLYCOSIDES
inhibitory effect was exhibited when two bisacrylamido compounds were present in the same reaction mixture suggests that the reaction rate is an intrinsic characteristic of each bisacrylamide compound. The addition reactions using 10 and 13 did not go to completion under the conditions described. It was necessary, therefore, to purify the reaction products on a column of Sephadex LH-20. This gel filtration also served the purpose of separating and recovering unreacted bisacrylamido compounds (10 and 13), which, unlike 1, were not easily extracted into water from chloroform. Although the procedure described here used O-acetylated I-thiosugars, the corresponding de-0-acetylated sugars can also be used for monoaddition to bisacrylamido derivatives. However, the overall yields in the latter case are generally much lower than those obtained with the 0-acetylated sugars. Polysaccharides entrapped in polyacrylamide have been shown to be effective for affinity chromatography of lectins (6,20). In this study, both the conventional method of gel formation (type II) and the entrapment of preformed linear acrylamide polymers which contained thioglycosides in crosslinked polyacrylamide gel (type I) were tested. The type II gel was more effective in terms of incorporation of the thioglycoside monomer into the gel than the type I gel. In the latter case, it appears that a considerable amount of the preformed linear polymer was too small to be trapped in the network of crosslinked polyacrylamide under the conditions described here. In spite of the difference in the total lthiogalactoside content, the two types of gels behaved nearly identically during affinity chromatography. It may be reasoned, therefore, that performance of ligandcontaining polyacrylamide gels is not only a
function of total ligand concentration, but is a function of available ligand concentration. REFERENCES I. Chipowsky, S., Lee, Y. C., and Roseman, S. (1973) Proc. Nat. Acud. Sci. USA 70, 23092312. 2. Schnaar. R. L., Weigel, P. H., Kuhlenschmidt, M., Lee, Y. C., and Roseman, S. ( 1978) J. Bio!. Chem.
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3. Schnaar, R. L., and Lee, Y. C. (1975) Biochemistry 14, 153.5. 4. Schnaar, R., Weigel, P. H., Roseman, S., and Lee, Y.
C.
(1979)
Methods
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in press. 5. Weigel, P. H., Schnaar, R., Roseman. S., and Lee, Y. C. (1979) Methods Carhohydr. Chem. 8, in press. 6. Sutoh, K., Rosenfeld, L., and Lee, Y. C. (1977) Anal.
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7. Weigel, P. H., Schmell, E., Lee, Y. C., and Roseman, S. (1978) J. Biol. Chem. 253, 330-333. 8. Horton, D. (t963) Methods Curbohydr. Chem. 2, 433. 9. Lee, Y. C., Stowell, C. P., and Krantz, M. J. (1976)
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12. Ellman, G. L. (1959) Arch. Biochcm. 82, 70. 13. Krantz, M. J.. and Lee, Y. C. (1976) them.
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14. Lee, Y. C. (1972) in Methods in Enzymology (Ginsburg, V.. ed.), Vol. 28. p. 63, Academic Press, New York. 1s. Reisfeld, R. A., Lewis, U. J., and Williams, D. E. ( 1%2) Nuture (Landon) 195, 281. 16. Hirose, K., Ihashi, Y ., Taguchi, S., and Yoshizawa, M. (1966) Kogyokagaku Zasshi (J~~pan) 69, 240-244. 17. Lee, R. T., and Lee, Y. C. (1974) Curbohydr. Has. 34, 151. 18. Weigel, P. H., Schnadr. R., Schmell, E., KuhlenSchmidt. M. S.. Lee, R. T.. Lee. Y. C.. and Roseman, S.. in press. 19. Chipowsky, S.. and Lee, Y. C. ( 1973) C’arh,thvdr. Res.
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