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Chemoenzymic synthesis of the GM3-oligosaccharide [NeuNAcα(2–3)Galβ(1–4)Glc] on a water-soluble polymer support
Reactive & Functional Polymers 39 (1999) 147–153
Chemoenzymic synthesis of the G M3 -oligosaccharide [NeuNAca(2–3)Galb(1–4)Glc] on a water-soluble polymer support Alexandra Tuchinsky, Uri Zehavi* Institute of Biochemistry, Food Sciences and Nutrition, Faculty of Agricultural, Food and Environmental Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel Received 13 October 1997; accepted 23 November 1997
Abstract A water-soluble polymer, carrying 0.16 meq of lactose / g was prepared following the copolymerization of 2-nitro-4(carboxyhydrazido-N-acryloyl)benzyl 2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl)-b-D-glucopyranoside and acrylamide. Subsequently, employing a sialyltransferase–phosphatase system a NeuNAc-residue was transferred regio- and stereoselectively to the acceptor polymer, to yield, following photochemical release, the free G M3 -oligosaccharide [NeuNAca(2–3)Galb(1–4)Glc]. 1999 Elsevier Science B.V. All rights reserved. Keywords: G M3 -Oligosaccharide; Modified polyacrylamide; Sialyltransferase; Photolysis; Polymer support; Oligosaccharide synthesis; NeuNAca(2–3)Galb(1–4)Glc
1. Introduction The oligosaccharide moieties of glycosphingolipids and glycoproteins are involved in a variety of biological processes [1]. Sequences containing NeuNAc-residues are present, for instance, in antigenic determinants, receptors for proteins, viruses, or bacteria, are important in cell–cell interaction, the circulation half-life time of glycoproteins, and change during cellular differentiation [2]. As the consequence, partial saccharides structures of glycosphingolipids and of glycoproteins, including G M3 *Corresponding author. Tel.: 1972-8-9481914; fax: 1972-89476189; e-mail: [email protected]
oligosaccharides, are useful as heptenes in immunological reaction and may interfere specifically in carbohydrate–lectin or carbohydrate–carbohydrate interaction [3]. Numerous chemical syntheses of the G M3 oligosaccharide were reported, the first one being [4]. Enzymic syntheses of the compound were published as well [5–10]. Enzymic oligosaccharide synthesis based on glycosyltransferases is an attractive alternative to classical chemical synthesis largely due to high stereo- and regioselectivity of glycosyltransferases in regard to the glycoside bond formation, no tedious protection / deprotection steps are required, fewer reaction steps and milder reaction conditions are employed [11]. On the
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other hand, water-soluble, saccharide carrying polymers may serve as high-performance acceptors for enzymic synthesis of oligosaccharides and glycoconjugates providing advantages as the use of a large excess of reagents and the facile separation of polymeric products. Furthermore, saccharide-carrying polymers may serve in affinity chromatography of lectins, enzymes, etc. Herein we describe the synthesis of the G M3 oligosaccharide [NeuNAca(2–3)Galb(1–4)Glc] on a water-soluble, light-sensitive polyacrylamide, employing Galb-1,4-GlcNAca-2,3(N)-sialyltransferase and calf intestinal alkaline phosphatase. Following the enzymic transfer, the product saccharide was, released from the polymer support by photolysis in a very high yield (85.6%). 2. Experimental
2.1. General methods Procedures were as previously described in Ref. [12]. CMP-[ 14 C]NeuNAc (11.3 Gbq / mmol) was purchased from New England Nuclear Research Products (Boston, USA). CMPNeuNAc, Triton X-100 and bovine a-lactalbumin were from Sigma Chemical Co. (St. Louis, MO 63178, USA). G M3 -Oligosaccharide [NeuNAca(2–3)Galb(1–4)Glc] was purchased from Accurate Chemical Company (NY, USA). Calf intestinal alkaline phosphatase (EC 3.13.1) was obtained from Boehringer Menaheim. Recombinant rat liver Galb-1,4-GlcNAca-2,3-(N)sialyltransferase (EC 2.4.99.6) was a gift from Dr James C. Paulson (Cytel Corp., La Jolla, CA, USA).
2.2. 4 -Carboxymethyl-2 -nitrobenzyl 2,3,6 -triO-acetyl-4 -O-(2,3,4,6 -tetra-O-acetyl-b -Dgalactopyranosyl)-b -D-glucopyranoside (1) This compound was prepared in analogy to a literature procedure for a related gluco-deriva-
tive [12] with the following amounts: 2,3,6-triO-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-D-galacbromide topyranosyl)-a-D-glucopyranosyl (acetobromo lactose) [13] (12 g, 16.9 mmol), methyl 4-hydroxymethyl-3-nitrobenzoate [14] (3 g, 14.3 mmol), calcium sulphate (1.26 g, 9.9 mmol), calcium carbonate (2.8 g, 17.7 mmol), nitromethane (120 ml) and silver perchlorate (3 g, 14.2 mmol). Following filtration through a Celite filter and evaporation, the residue was applied to a silica gel column (500 g, 5 cm in diameter), which was eluted with the following mixtures of ethyl acetate–petroleum ether 2:8, 1 l; 3:7, 1 l affording unreacted methyl 4-hydroxymethyl-3-nitrobenzoate (0.4 g); 2:3, 1 l; 1:1, 0.25 l; 3:2, 0.5 l giving compound 1 (7.4 g, 62.8%), m.p. 97–998C, a 31 D 5.861.98C (c 2.4, methanol), R f 0.27 (1:1, ethyl acetate–petroleum ether), 1 H NMR (CDCl 3 ): d 8.74 (d, 1H, J30,50 1.7 Hz, H-30, aryl), 8.29 (dd, 1H, J50,60 8.1 Hz, H-50, aryl), 7.81 (d, 1H, H-60, aryl), 5.36 (dd, 1H, J39,49 3.3 Hz, J49,59 0.9 Hz, H-49), 5.29 (d, 1H, J 15.6 Hz, benzylic CH 2 ), 5.25 (t, 1H, H-3), 5.13 (t, 1H, J29,39 10.4 Hz, H-29), 5.08 (d, 1H, benzylic CH 2 ), 4.97 (dd, 1H, J39,49 3.3 Hz, H-39), 4.67 (d, 1H, J1,2 7.9 Hz, H-1), 4.49 (d, 1H, J19,29 8.1 Hz, H-19), 4.94 (m, 1H, H-6b), 3.98 (s, 3H, OCH 3 ), 3.89 (t, 1H, J4,5 7.8 Hz, H-4), 3.84 (t, 1H, J5,6a 9.2 Hz, H-6a), 3.68 (m, 1H, H-5), 2.17, 2.12, 2.06, 1.98 (each s, each 3H, acetyl), 2.09–2.07 (m, 9H, 3 acetyls), lit. J1,2 7.8 Hz [14]. Anal. Calcd. for total sugar: 41.7%. Found: 42.3%. MS: Calcd. 829.3 (M). Found: 828.5 (M 2 H)2 .
2.3. 4 -Carboxy-2 -nitrobenzyl 4 -O-b -Dgalactopyranosyl-b -D-glucopyranoside (2) Compound 1 (6.56 g, 7.9 mmol) was treated with sodium methoxide (0.1 M, 70 ml) in methanol (530 ml) and dichlormethane (53 ml) for 25 min. The solution was neutralised with a cation-exchange resin (Amberlite IR-120, H 1 form), filtered and concentrated [15]. The re-
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sulting syrup was suspended in water (100 ml) and treated with sodium hydroxide (1 M, 70 ml) for 20 min. The solution was neutralised again with a cation-exchange resin, the suspension was filtered and the solution was freeze dried giving 2 (3.88 g, 94%), m.p. 163–1658C, a 31 D 2 7.561.98C (c 1.2, water), R f 0.15 (1:1, methanol–chloroform), 1 H NMR (D 2 O): d 8.61 (d, 1H, J30,50 1.2 Hz, H-30, aryl), 8.24 (dd, 1H, J50,60 8.0 Hz, H-50, aryl), 7.93 (d, 1H, H-60, aryl), 5.32 and 5.22 (2d, each 1H, J 15.0 Hz, benzylic CH 2 ), 3.95–3.21 (m, 13H, saccharide protons). Anal. Calcd. for total sugar: 64.5%. Found: 64.8%. MS: Calcd. 521.1 (M). Found 522.1 (M–H)1 .
2.4. 4 -Carboxy-2 -nitrobenzyl 2,3,6 -tri-Oacetyl-4 -O-(2,3,4,6 -tetra-O-acetyl-b -Dgalactopyranosyl)-b -D-glucopyranoside (3) Compound (2) (3.68 g, 6.6 mmol) was suspended in acetic anhydride (8 ml) and pyridine (16 ml) and was kept in an ice-water bath for 1 h. Stirring was continued overnight at room temperature, ice was added and the mixture was stirred for 2 h at 08C. The reaction mixture was extracted with ethyl acetate (2 3 20 ml), the extracts were combined and washed with 5% HCl and water, dried over Na 2 SO 4 , filtered, and evaporated in vacuo to yield the chromatographically pure product 3 31 (5.15 g, 89%), m.p. 86–898C, a D 2 10.361.98C (c 2.0, methanol), R f 0.1 (2:1, ethyl acetate–petroleum ether) and 0.6 (2:1, 1 methanol–chloroform), H NMR (CDCl 3 ): d 8.78 (d, 1H, J30,50 1.4 Hz, H-30, aryl), 8.33 (dd, 1H, J50,60 8.2 Hz, H-50, aryl), 7.85 (d, 1H, H-60, aryl), 5.36 (d, 1H, J39,49 2.8 Hz, H-49), 5.29 (d, 1H, J 15.7 Hz, benzylic CH 2 ), 5.26 (t, 1H, J3,4 9.3 Hz, H-3), 5.12 (t, 1H, J29,39 10.4 Hz, H-29), 5.08 (d, 1H, benzylic CH 2 ), 4.97 (dd, 1H, H-39), 4.68 (d, 1H, J1,2 7.9 Hz, H-1), 4.50 (d, 1H, J19,29 8.1 Hz, H-19), 4.52 (m, 1H, H-6b), 3.89 (t, 1H, J4,5 8.3 Hz, H-4), 3.85 (t, 1H, J5,6a 9.2 Hz, H-6a), 3.68 (m, 1H, H-5), 2.16, 2.12, 2.06, 1.97
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(each s, each 3H, acetyl), 2.07 (m, 9 H, 3 acetyls). Anal. Calcd. for total sugar: 42.4%. Found: 41.8%. MS: Calcd. 816.0 (M). Found: 814.9 (M 2 H)2 .
2.5. 2 -Nitro-4 -carboxyhydrazido-N9 -(tertbutyloxycarbonyl)-benzyl 2,3,6 -tri-O-acetyl-4 O-(2,3,4,6 -tetra-O-acetyl-b -Dgalactopyranosyl)-b -D-glucopyranoside (4) A suspension of compound 3 (4.31 g, 5.3 mmol) and tert-butyloxycarbonylhydrazine (0.71 g, 5.3 mmol) in methanol (38 ml) was cooled in an ice-water bath while dicyclohexylcarbodiimide (1.23 g, 5.3 mmol) was added in several portions with stirring. The stirring was continued for 1 h at 08C and at room temperature overnight and the solvent was removed in vacuo. The residue was applied to a silica gel column (150 g, 5 cm in diameter) which was eluted (flash) with the following mixtures of ethyl acetate–petroleum ether 4:6, 0.5 l; 1:1, 0.5 l; 6:4, 1 l and 7:3, 0.5 l giving compound 4 (3.7 g, 75%), m.p. 120–1238C, a 31 D 2 3.761.98C (c 1.3, methanol), R f 0.18 (1:1, ethyl acetate– petroleum ether), 1 H NMR (CDCl 3 ): d 8.68 (s, 1H, hydrazido NH), 8.50 (d, 1H, J30,50 1.7 Hz, H-30, aryl), 8.07 (dd, 1H, J50,60 7.8 Hz, H-50, aryl), 7.80 (d, 1H, H-60, aryl), 6.81 (s, 1H, hydrazido NH), 5.35 (dd, 1H, J39,49 3.4 Hz, J49,59 0.9 Hz, H-49), 5.25 (d, 2H, J 15.2 Hz, benzylic CH 2 ), 5.24 (t, 1H, J3,4 9.3 Hz, H-3), 5.14-5.08 (m, 2H, H-2, H-29), 5.06 (d, 1H, benzylic CH 2 ), 4.96 (dd, 1H, H-39), 4.66 (d, 1H, J1,2 7.9 Hz, H-1), 4.51 (m, 1H, H-6b), 4.49 (d, 1H, J19,29 7.9 Hz, H-19), 3.89 (t, 1H, J5,6a 7.2 Hz, H-6a), 3.85 (t, 1H, J4,5 9.2 Hz, H-4), 3.67 (m, 1H, H-5), 2.16, 2.11, 2.06, 2.05, 1.97 (each s, each 3 H, acetyl), 2.07 (s, 6H, 2 acetyls), 1.51 (s, 9H, tert-butyl). Anal. Calcd. for total sugar: 37.3%. Found: 36.7%. MS: Calcd. 929.2 (M). Found: 830.1 2 (M 2 H-t-BOC) .
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2.6. 2 -Nitro-4 -(carboxyhydrazido-N9 acryloyl)-benzyl2,3,6 -tri-O-acetyl-4 -O(2,3,4,6 -tetra-O-acetyl-b -D-galactopyranosyl)b -D-glucopyranoside (5) Compound 4 (3.07 g, 3.3 mmol) was dissolved in a mixture of trifluoroacetic acid–dichlorometane 1:1 (4.5 ml), the reaction a mixture was stirred at 08C for 15 min and at room temperature for additional 15 min. Dichlorometane (25 ml) was added and the solvent was removed in vacuo. This procedure was repeated four times, yielding the unprotected hydrazide that was further used without isolation. The unprotected hydrazide, 2,6-di-tert-butyl-4methylphenol (15 mg) and triethylamine (0.38 g) were dissolved in chloroform (20 ml) at 08C and acryloyl chloride (0.35 g, 3.5 mmol) was added dropwise over 10 min to the stirred reaction mixture. Stirring was continued for an additional hour at 08C and at room temperature overnight. The solution was washed with icecold water (25 ml portions) and saturated brine, dried with MgSO 4 , and filtered. 2,6-Di-tertbutyl-4-methylphenol (10 mg) was added to the chloroform solution that was removed in vacuo. Residue was applied to a silica gel column (100 g, 5 cm in diameter) which was successively eluted (flash) with the following mixtures of ethyl acetate–petroleum ether 4:6, 0.5 l; 1:1, 0.5 l and 6:4, 1 l giving compound 5 (1.48 g, 51.5%), m.p. 190–1948C (dec.), a 31 D 2 11.961.98C (c 1.9, methanol), R f 0.51 (ethyl acetate), 1 H NMR (CDCl 3 ): d 11.72 and 9.65 (2s, each 1H, hydrazido NH), 8.54 (d, 1H, J30,50 1.3 Hz, H-30, aryl), 8.14 (dd, 1H, J50,60 8.1 Hz, H-50, aryl), 7.76 (d, 1H, H-60, aryl), 6.44–6.29 (m, 3H, CH=CH 2 , acryloyl), 5.34 (dd, 1H, J39,49 3.3 Hz, J49,59 0.8 Hz, H-49), 5.22 (d, 1H, J 14.08 Hz, benzylic CH 2 ), 5.23 (t, 1H, J3,4 9.3 Hz, H-3), 5.06 (d, 1H, benzylic CH 2 ), 5.09–5.00 (m, 1H, H-2,29), 4.95 (dd, 1H, H-39), 4.64 (d, 1H, J1,2 7.8 Hz, H-1), 4.49–4.45 (m, 1H, H-6b), 4.48 (d, 1H, J19,29 7.8 Hz, H-19), 4.14–4.06 (m, 1H, H-6a), 3.87 (t, 1H, J4,5 7.1 Hz, H-4), 3.66 (m, 1H, H-5), 2.14, 2.10, 2.03, 1.95 (each s,
each 3H, acetyl), 2.07–2.05 (m, 9H, 3 acetyls); assignment was supported by COSY and deuterium exchange experiments. Anal. Calcd. for total sugar: 39.2%. Found: 38.8%. MS: Calcd. 883.2 (M). Found: 882.2 (M 2 H)2 .
2.7. 2 -Nitro-4 -( N9 - -carboxyhydrazido)benzyl2,3,6 -tri-O-acetyl-4 -O-(2,3,4,6 -tetra-Oacetyl-b -D-galactopyranosyl)-b -Dglucopyranoside (6) Polymer 6 was prepared in analogy to a literature procedure [16] (small scale) with the following amounts: compound 5 (0.81 g, 0.92 mmol), acrylamide (4.19 g, 58.1 mmol), 2,29azo-bis-isobutyronitrile (AIBN initiator of polymerisation) (25 mg and additionally 50 mg), tetrahydrofuran (50 ml). The polymerisation was carried out at 508C additional portion of AIBN (25 mg) in THF (4 ml) were introduced daily into the tube through a cannula, and the reaction was continued for 3 days. Purification gave the acetylated lacto– polymer 6 (3.1 g, 62%), 0.17 meq of lactose / g, IR: 3398 (wide, OH, NH), 3200, 2928, 1756 21 (CO), 1662 (CO), 1618, 1452 and 1419 cm .
2.8. 2 -Nitro-4( N9 - -carboxyhydrazido)benzyl 4 -O-b -D-galactopyranosyl-b -D-glucopyranoside (7) Polymer 6 (500 mg) was suspended in 9:1 methanol–dichlorometane (40 ml) and treated with sodium methoxide in methanol (0.1 M, 6 ml). The mixture was stirred overnight at room temperature and the resulting solution was dialysed employing a Diaflo UM2 membrane, lyophilised and gave of 390 mg of polymer 7 (0.16 meq of lactose / g), IR: almost identical with 7, only the absorbance at 1756 cm 21 has disappeared. Molecular weights were determined as 7000 (2 / 3 of total) and 12 000. Compound 7 (20 mg in 10 ml of water) was irradiated followed by ultrafiltration through a Diaflo UM2 membrane. Determination of total
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sugar in the filtrate showed the release of 0.12 meq of lactose / g.
2.9. 2 -Nitro-4 -( N9 - -carboxyhydrazido)benzyl [3 -O-(5 -Acetamido-3,5 -dideoxy-D-glycero-a -Dgalacto-2 -nonulopyranosylonic acid)-(4 -O-b -Dgalactopyranosyl)] -b -D-glucopyranoside (8) The incubation mixture (100 ml) contained polymer 7 (1 mg, 0.16 meq Lac / g), CMPNeuNAc (100 mg, 162 nmol) including CMP[ 14 C]NeuNAc (212, 205 d.p.m. / mmol, 345,611 d.p.m. / mg), a-lactalbumin (60 mg), 50 mM sodium cacodilate (pH 6.5), Triton X-100 (0.5%), calf intestinal alkaline phosphatase (3 U), and Galb-1,4-GlcNAca-2,3-(N)-sialyltransferase (100 mU). The mixture was vortexes and placed in an incubator at 378C. The same amounts of CMP-NeuNAc and calf intestinal alkaline phosphatase were added at regular intervals of 24 h. After 3 days of incubation the polymer was isolated by ultrafiltration, washed extensively with water [until only very little radioactivity (46 d.p.m. / ml) emerged in the eluants] and lyophilised. Polymer 8 (11 222 400 d.p.m. / g, 0.05 meq G M3 -oligosaccharide / g, represented a 33.1% incorporation yield of NeuNAc.
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enzyme specificity with a minimum of synthetic steps and mild reaction conditions ([11], and references cited therein). Our previous work [12] dealt with the preparation of an improved water-soluble acceptor polymer; in difference from prior acceptor polymer preparations, the present polymer afforded higher transfer yield and acceptor loads. The acceptor polymer (in the current work, polymer 6) was made by incorporating a suitable acryloyl saccharide derivative (5) already during the copolymerisation with acrylamide and the saccharide load was determined by the proportion of the two starting materials. The light-sensitive nitrobenzyl glycoside (1) was obtained in 62.8% yield from 2,3,6-tri-Oacetyl - 4 - O-(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl)-a-D-glucopyranosyl bromide and methyl 4-hydroxymethyl-3-nitrobenzoate in a Koeigs–Knorr reaction with silver perchlorate as a promoter. Deblocking gave compound 2 that was acetylated to compound 3 and condensed with tert-butyloxycarbonylhydrazine (DCC), to yield compound 4. The t-BOC group was removed by TFA in CH 2 Cl 2 and condensed with acryloyl chloride to yield compound 5.
2.10. Release of GM 3 -oligosaccharide [ NeuNAca(2 – 3)Galb(1 – 4)Glc] (9) from polymer 8 The photolysis (.350 nm) of polymer 8 [17], following ultrafiltration, the filtrate was lyophilised, subjected to TLC and counting affording G M3 -oligosaccharide (9) at a 85.6% yield (9 549 231 d.p.m. / g) and, and polymer 10 in the nondialysable residue. 3. Results and discussion Enzymic oligosaccharide synthesis on polymer supports was pioneered and intensively studied by us and recently also by others, leading to useful preparative results based on
Scheme 1. Intermediates in the chemoenzymic synthesis of G M3 oligosaccharide.
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Copolymerisation with acrylamide in the presence of AIBN gave compound 6 and removing of the protecting groups yielded a water-soluble polymer 7 (0.16 mmol Lac / g) (Scheme 1). Polymer 7 was an acceptor in the sialyltrans-
Fig. 1. TLC (Merk plate, n-butanol / acetic acid / water, 20 / 10 / 15 by vol) separation of radioactive products released by irradiation of compound 8. The marker G M3 -oligosaccharide, [NeuNAca(2– 3)Galb(1–4)Glc], was run analogised the product (9) and was detected by spraying with sulfuric acid and heating.
ferase reaction, using radioactively labelled CMP-NeuNAc. It has been previously shown, that lactose is a poor acceptor for sialyltransferase [18]. The reaction efficiency was increased in three ways: conditions that minimise the hydrolysis of the sugar nucleotide, excess of both CMP-NeuNAc and addition of calf intestinal alkaline phosphatase to hydrolyse the released by product of the sialylation, CMP [19] which is a strong inhibitor of sialyltransterase; the transfer yield was 33.1%, a yield comparable with transglycosylation [20] and definitely with chemical sialylation. Interestingly, enzymic (chymotrypsin) removal of the product from polymer that was introduced by us [21] was recently applied in the context of the synthesis of G M3 oligosaccharide [22]. Finally, the last step of the present synthesis constituted of photolysis, that was utilised by us very frequently in the past, releasing G M3 -oligosaccharide, (5-acetamido-3,5-dideoxy-D-glycero-a-D-galacto-2-nonulopyranosylonic acid)-(2–3)-b-Dgalactopyranosyl-(1–4)-b-D-glucopyranose, Ne-
Scheme 2. Photochemical release of G M3 -oligosaccharide (9).
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uNAca(2–3)Galb(1–4)Glc, (9) from the product polymer 8 chromatographically identical with a marker, proceeded at a very high yield (85.6%, Fig. 1, Scheme 2). Acknowledgements The authors are grateful to Dr James C. Paulson at the Cytel Corp. (La Jolla, CA, USA) for providing us with a generous supply of recombinant rat liver Galb-1,4-GlcNAca-2,3(N)-sialyltransferase and to Mr Ariel Lustig for determination of molecular weight. This work was supported by the German–Israeli Foundation for Scientific Research and Development (G.I.F.), by the Ministry of Science, Niedersachsen, and by the Volkswagen Stiftung. References [1] Y. Ichikawa, G.C. Look, C.-H. Wong, Anal. Biochem. 202 (1992) 215 (and literature cited therein). [2] S. Hakomori, J. Biol. Chem. 265 (1990) 18713. [3] S. Hakomori, Biochem. Soc. Trans. 21 (1993) 583. [4] A.Y. Khorlin, I.M. Prialova, I.B. Bystrova, Carbohydr. Res. 19 (1971) 272.
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[5] S. Sabesan, J.C. Paulson, J. Am. Chem. Soc. 108 (1986) 2068. [6] H.T. de Heij, M. Kloosterman, P.L. Koppen, J.H. van Boom, D.H. van der Eijnden, Carbohydr. Chem. 7 (1988) 209. [7] K.G.I. Nilsson, Carbohydr. Res. 188 (1989) 9. [8] T. Ichiawa, G.J. Shen, C.H. Wong, J. Am. Chem. Soc. 113 (1991) 4698. [9] A.K. Ray, U. Nilsson, G. Magnusson, J. Am. Chem. Soc. 114 (1992) 2256. [10] V. Kren, J. Thiem, Angew. Chem. Int. Ed. Engl. 34 (1995) 893. [11] U. Zehavi, J. Thiem, in: M.D. Bednarsky, E.S. Simon (Eds.), ACS Symposium Series, vol. 466, ch. 7, pp. 90–98, American Chemical Society, Washington, DC, 1991, and literature cited therein. [12] A. Tuchinsky, U. Zehavi, React. Funct. Polym. 31 (1996) 11. [13] F.J. Reyther, R.G. Young, J. Am. Chem. Soc. 74 (1952) 4210. [14] U. Zehavi, S. Sadeh, M. Herchman, Carbohydr. Res. 124 (1983) 23. ¨ [15] S. Kopper, U. Zehavi, Carbohydr. Res. 193 (1989) 296. [16] A. Pollak, H. Blumenfeld, M. Wax, R.L. Baughn, G.M. Whitesides, J. Am. Chem. Soc. 102 (1980) 6324. [17] U. Zehavi, M. Herchman, Carbohydr. Res. 128 (1984) 160. [18] J.C. Paulson, J.I. Rearick, R.L. Hill, J. Biol. Chem. 252 (1977) 2363. [19] C. Unverzagt, H. Kunz, J.C. Paulson, J. Am. Chem. Soc. 112 (1990) 9308. [20] S.-I. Nishimura, K.B. Lee, K. Matsuoka, Y.C. Lee, Biochem. Biophys. Res. Commun. 199 (1994) 249. [21] U. Zehavi, M. Herchman, Carbohydr. Res. 133 (1984) 339. [22] K. Yamada, S.-I. Nishimura, Tetrahedron Lett. 36 (1995) 9493.
Chemoenzymic synthesis of the G M3 -oligosaccharide [NeuNAca(2–3)Galb(1–4)Glc] on a water-soluble polymer support Alexandra Tuchinsky, Uri Zehavi* Institute of Biochemistry, Food Sciences and Nutrition, Faculty of Agricultural, Food and Environmental Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel Received 13 October 1997; accepted 23 November 1997
Abstract A water-soluble polymer, carrying 0.16 meq of lactose / g was prepared following the copolymerization of 2-nitro-4(carboxyhydrazido-N-acryloyl)benzyl 2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl)-b-D-glucopyranoside and acrylamide. Subsequently, employing a sialyltransferase–phosphatase system a NeuNAc-residue was transferred regio- and stereoselectively to the acceptor polymer, to yield, following photochemical release, the free G M3 -oligosaccharide [NeuNAca(2–3)Galb(1–4)Glc]. 1999 Elsevier Science B.V. All rights reserved. Keywords: G M3 -Oligosaccharide; Modified polyacrylamide; Sialyltransferase; Photolysis; Polymer support; Oligosaccharide synthesis; NeuNAca(2–3)Galb(1–4)Glc
1. Introduction The oligosaccharide moieties of glycosphingolipids and glycoproteins are involved in a variety of biological processes [1]. Sequences containing NeuNAc-residues are present, for instance, in antigenic determinants, receptors for proteins, viruses, or bacteria, are important in cell–cell interaction, the circulation half-life time of glycoproteins, and change during cellular differentiation [2]. As the consequence, partial saccharides structures of glycosphingolipids and of glycoproteins, including G M3 *Corresponding author. Tel.: 1972-8-9481914; fax: 1972-89476189; e-mail: [email protected]
oligosaccharides, are useful as heptenes in immunological reaction and may interfere specifically in carbohydrate–lectin or carbohydrate–carbohydrate interaction [3]. Numerous chemical syntheses of the G M3 oligosaccharide were reported, the first one being [4]. Enzymic syntheses of the compound were published as well [5–10]. Enzymic oligosaccharide synthesis based on glycosyltransferases is an attractive alternative to classical chemical synthesis largely due to high stereo- and regioselectivity of glycosyltransferases in regard to the glycoside bond formation, no tedious protection / deprotection steps are required, fewer reaction steps and milder reaction conditions are employed [11]. On the
1381-5148 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S1381-5148( 98 )00011-X
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other hand, water-soluble, saccharide carrying polymers may serve as high-performance acceptors for enzymic synthesis of oligosaccharides and glycoconjugates providing advantages as the use of a large excess of reagents and the facile separation of polymeric products. Furthermore, saccharide-carrying polymers may serve in affinity chromatography of lectins, enzymes, etc. Herein we describe the synthesis of the G M3 oligosaccharide [NeuNAca(2–3)Galb(1–4)Glc] on a water-soluble, light-sensitive polyacrylamide, employing Galb-1,4-GlcNAca-2,3(N)-sialyltransferase and calf intestinal alkaline phosphatase. Following the enzymic transfer, the product saccharide was, released from the polymer support by photolysis in a very high yield (85.6%). 2. Experimental
2.1. General methods Procedures were as previously described in Ref. [12]. CMP-[ 14 C]NeuNAc (11.3 Gbq / mmol) was purchased from New England Nuclear Research Products (Boston, USA). CMPNeuNAc, Triton X-100 and bovine a-lactalbumin were from Sigma Chemical Co. (St. Louis, MO 63178, USA). G M3 -Oligosaccharide [NeuNAca(2–3)Galb(1–4)Glc] was purchased from Accurate Chemical Company (NY, USA). Calf intestinal alkaline phosphatase (EC 3.13.1) was obtained from Boehringer Menaheim. Recombinant rat liver Galb-1,4-GlcNAca-2,3-(N)sialyltransferase (EC 2.4.99.6) was a gift from Dr James C. Paulson (Cytel Corp., La Jolla, CA, USA).
2.2. 4 -Carboxymethyl-2 -nitrobenzyl 2,3,6 -triO-acetyl-4 -O-(2,3,4,6 -tetra-O-acetyl-b -Dgalactopyranosyl)-b -D-glucopyranoside (1) This compound was prepared in analogy to a literature procedure for a related gluco-deriva-
tive [12] with the following amounts: 2,3,6-triO-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-D-galacbromide topyranosyl)-a-D-glucopyranosyl (acetobromo lactose) [13] (12 g, 16.9 mmol), methyl 4-hydroxymethyl-3-nitrobenzoate [14] (3 g, 14.3 mmol), calcium sulphate (1.26 g, 9.9 mmol), calcium carbonate (2.8 g, 17.7 mmol), nitromethane (120 ml) and silver perchlorate (3 g, 14.2 mmol). Following filtration through a Celite filter and evaporation, the residue was applied to a silica gel column (500 g, 5 cm in diameter), which was eluted with the following mixtures of ethyl acetate–petroleum ether 2:8, 1 l; 3:7, 1 l affording unreacted methyl 4-hydroxymethyl-3-nitrobenzoate (0.4 g); 2:3, 1 l; 1:1, 0.25 l; 3:2, 0.5 l giving compound 1 (7.4 g, 62.8%), m.p. 97–998C, a 31 D 5.861.98C (c 2.4, methanol), R f 0.27 (1:1, ethyl acetate–petroleum ether), 1 H NMR (CDCl 3 ): d 8.74 (d, 1H, J30,50 1.7 Hz, H-30, aryl), 8.29 (dd, 1H, J50,60 8.1 Hz, H-50, aryl), 7.81 (d, 1H, H-60, aryl), 5.36 (dd, 1H, J39,49 3.3 Hz, J49,59 0.9 Hz, H-49), 5.29 (d, 1H, J 15.6 Hz, benzylic CH 2 ), 5.25 (t, 1H, H-3), 5.13 (t, 1H, J29,39 10.4 Hz, H-29), 5.08 (d, 1H, benzylic CH 2 ), 4.97 (dd, 1H, J39,49 3.3 Hz, H-39), 4.67 (d, 1H, J1,2 7.9 Hz, H-1), 4.49 (d, 1H, J19,29 8.1 Hz, H-19), 4.94 (m, 1H, H-6b), 3.98 (s, 3H, OCH 3 ), 3.89 (t, 1H, J4,5 7.8 Hz, H-4), 3.84 (t, 1H, J5,6a 9.2 Hz, H-6a), 3.68 (m, 1H, H-5), 2.17, 2.12, 2.06, 1.98 (each s, each 3H, acetyl), 2.09–2.07 (m, 9H, 3 acetyls), lit. J1,2 7.8 Hz [14]. Anal. Calcd. for total sugar: 41.7%. Found: 42.3%. MS: Calcd. 829.3 (M). Found: 828.5 (M 2 H)2 .
2.3. 4 -Carboxy-2 -nitrobenzyl 4 -O-b -Dgalactopyranosyl-b -D-glucopyranoside (2) Compound 1 (6.56 g, 7.9 mmol) was treated with sodium methoxide (0.1 M, 70 ml) in methanol (530 ml) and dichlormethane (53 ml) for 25 min. The solution was neutralised with a cation-exchange resin (Amberlite IR-120, H 1 form), filtered and concentrated [15]. The re-
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sulting syrup was suspended in water (100 ml) and treated with sodium hydroxide (1 M, 70 ml) for 20 min. The solution was neutralised again with a cation-exchange resin, the suspension was filtered and the solution was freeze dried giving 2 (3.88 g, 94%), m.p. 163–1658C, a 31 D 2 7.561.98C (c 1.2, water), R f 0.15 (1:1, methanol–chloroform), 1 H NMR (D 2 O): d 8.61 (d, 1H, J30,50 1.2 Hz, H-30, aryl), 8.24 (dd, 1H, J50,60 8.0 Hz, H-50, aryl), 7.93 (d, 1H, H-60, aryl), 5.32 and 5.22 (2d, each 1H, J 15.0 Hz, benzylic CH 2 ), 3.95–3.21 (m, 13H, saccharide protons). Anal. Calcd. for total sugar: 64.5%. Found: 64.8%. MS: Calcd. 521.1 (M). Found 522.1 (M–H)1 .
2.4. 4 -Carboxy-2 -nitrobenzyl 2,3,6 -tri-Oacetyl-4 -O-(2,3,4,6 -tetra-O-acetyl-b -Dgalactopyranosyl)-b -D-glucopyranoside (3) Compound (2) (3.68 g, 6.6 mmol) was suspended in acetic anhydride (8 ml) and pyridine (16 ml) and was kept in an ice-water bath for 1 h. Stirring was continued overnight at room temperature, ice was added and the mixture was stirred for 2 h at 08C. The reaction mixture was extracted with ethyl acetate (2 3 20 ml), the extracts were combined and washed with 5% HCl and water, dried over Na 2 SO 4 , filtered, and evaporated in vacuo to yield the chromatographically pure product 3 31 (5.15 g, 89%), m.p. 86–898C, a D 2 10.361.98C (c 2.0, methanol), R f 0.1 (2:1, ethyl acetate–petroleum ether) and 0.6 (2:1, 1 methanol–chloroform), H NMR (CDCl 3 ): d 8.78 (d, 1H, J30,50 1.4 Hz, H-30, aryl), 8.33 (dd, 1H, J50,60 8.2 Hz, H-50, aryl), 7.85 (d, 1H, H-60, aryl), 5.36 (d, 1H, J39,49 2.8 Hz, H-49), 5.29 (d, 1H, J 15.7 Hz, benzylic CH 2 ), 5.26 (t, 1H, J3,4 9.3 Hz, H-3), 5.12 (t, 1H, J29,39 10.4 Hz, H-29), 5.08 (d, 1H, benzylic CH 2 ), 4.97 (dd, 1H, H-39), 4.68 (d, 1H, J1,2 7.9 Hz, H-1), 4.50 (d, 1H, J19,29 8.1 Hz, H-19), 4.52 (m, 1H, H-6b), 3.89 (t, 1H, J4,5 8.3 Hz, H-4), 3.85 (t, 1H, J5,6a 9.2 Hz, H-6a), 3.68 (m, 1H, H-5), 2.16, 2.12, 2.06, 1.97
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(each s, each 3H, acetyl), 2.07 (m, 9 H, 3 acetyls). Anal. Calcd. for total sugar: 42.4%. Found: 41.8%. MS: Calcd. 816.0 (M). Found: 814.9 (M 2 H)2 .
2.5. 2 -Nitro-4 -carboxyhydrazido-N9 -(tertbutyloxycarbonyl)-benzyl 2,3,6 -tri-O-acetyl-4 O-(2,3,4,6 -tetra-O-acetyl-b -Dgalactopyranosyl)-b -D-glucopyranoside (4) A suspension of compound 3 (4.31 g, 5.3 mmol) and tert-butyloxycarbonylhydrazine (0.71 g, 5.3 mmol) in methanol (38 ml) was cooled in an ice-water bath while dicyclohexylcarbodiimide (1.23 g, 5.3 mmol) was added in several portions with stirring. The stirring was continued for 1 h at 08C and at room temperature overnight and the solvent was removed in vacuo. The residue was applied to a silica gel column (150 g, 5 cm in diameter) which was eluted (flash) with the following mixtures of ethyl acetate–petroleum ether 4:6, 0.5 l; 1:1, 0.5 l; 6:4, 1 l and 7:3, 0.5 l giving compound 4 (3.7 g, 75%), m.p. 120–1238C, a 31 D 2 3.761.98C (c 1.3, methanol), R f 0.18 (1:1, ethyl acetate– petroleum ether), 1 H NMR (CDCl 3 ): d 8.68 (s, 1H, hydrazido NH), 8.50 (d, 1H, J30,50 1.7 Hz, H-30, aryl), 8.07 (dd, 1H, J50,60 7.8 Hz, H-50, aryl), 7.80 (d, 1H, H-60, aryl), 6.81 (s, 1H, hydrazido NH), 5.35 (dd, 1H, J39,49 3.4 Hz, J49,59 0.9 Hz, H-49), 5.25 (d, 2H, J 15.2 Hz, benzylic CH 2 ), 5.24 (t, 1H, J3,4 9.3 Hz, H-3), 5.14-5.08 (m, 2H, H-2, H-29), 5.06 (d, 1H, benzylic CH 2 ), 4.96 (dd, 1H, H-39), 4.66 (d, 1H, J1,2 7.9 Hz, H-1), 4.51 (m, 1H, H-6b), 4.49 (d, 1H, J19,29 7.9 Hz, H-19), 3.89 (t, 1H, J5,6a 7.2 Hz, H-6a), 3.85 (t, 1H, J4,5 9.2 Hz, H-4), 3.67 (m, 1H, H-5), 2.16, 2.11, 2.06, 2.05, 1.97 (each s, each 3 H, acetyl), 2.07 (s, 6H, 2 acetyls), 1.51 (s, 9H, tert-butyl). Anal. Calcd. for total sugar: 37.3%. Found: 36.7%. MS: Calcd. 929.2 (M). Found: 830.1 2 (M 2 H-t-BOC) .
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2.6. 2 -Nitro-4 -(carboxyhydrazido-N9 acryloyl)-benzyl2,3,6 -tri-O-acetyl-4 -O(2,3,4,6 -tetra-O-acetyl-b -D-galactopyranosyl)b -D-glucopyranoside (5) Compound 4 (3.07 g, 3.3 mmol) was dissolved in a mixture of trifluoroacetic acid–dichlorometane 1:1 (4.5 ml), the reaction a mixture was stirred at 08C for 15 min and at room temperature for additional 15 min. Dichlorometane (25 ml) was added and the solvent was removed in vacuo. This procedure was repeated four times, yielding the unprotected hydrazide that was further used without isolation. The unprotected hydrazide, 2,6-di-tert-butyl-4methylphenol (15 mg) and triethylamine (0.38 g) were dissolved in chloroform (20 ml) at 08C and acryloyl chloride (0.35 g, 3.5 mmol) was added dropwise over 10 min to the stirred reaction mixture. Stirring was continued for an additional hour at 08C and at room temperature overnight. The solution was washed with icecold water (25 ml portions) and saturated brine, dried with MgSO 4 , and filtered. 2,6-Di-tertbutyl-4-methylphenol (10 mg) was added to the chloroform solution that was removed in vacuo. Residue was applied to a silica gel column (100 g, 5 cm in diameter) which was successively eluted (flash) with the following mixtures of ethyl acetate–petroleum ether 4:6, 0.5 l; 1:1, 0.5 l and 6:4, 1 l giving compound 5 (1.48 g, 51.5%), m.p. 190–1948C (dec.), a 31 D 2 11.961.98C (c 1.9, methanol), R f 0.51 (ethyl acetate), 1 H NMR (CDCl 3 ): d 11.72 and 9.65 (2s, each 1H, hydrazido NH), 8.54 (d, 1H, J30,50 1.3 Hz, H-30, aryl), 8.14 (dd, 1H, J50,60 8.1 Hz, H-50, aryl), 7.76 (d, 1H, H-60, aryl), 6.44–6.29 (m, 3H, CH=CH 2 , acryloyl), 5.34 (dd, 1H, J39,49 3.3 Hz, J49,59 0.8 Hz, H-49), 5.22 (d, 1H, J 14.08 Hz, benzylic CH 2 ), 5.23 (t, 1H, J3,4 9.3 Hz, H-3), 5.06 (d, 1H, benzylic CH 2 ), 5.09–5.00 (m, 1H, H-2,29), 4.95 (dd, 1H, H-39), 4.64 (d, 1H, J1,2 7.8 Hz, H-1), 4.49–4.45 (m, 1H, H-6b), 4.48 (d, 1H, J19,29 7.8 Hz, H-19), 4.14–4.06 (m, 1H, H-6a), 3.87 (t, 1H, J4,5 7.1 Hz, H-4), 3.66 (m, 1H, H-5), 2.14, 2.10, 2.03, 1.95 (each s,
each 3H, acetyl), 2.07–2.05 (m, 9H, 3 acetyls); assignment was supported by COSY and deuterium exchange experiments. Anal. Calcd. for total sugar: 39.2%. Found: 38.8%. MS: Calcd. 883.2 (M). Found: 882.2 (M 2 H)2 .
2.7. 2 -Nitro-4 -( N9 - -carboxyhydrazido)benzyl2,3,6 -tri-O-acetyl-4 -O-(2,3,4,6 -tetra-Oacetyl-b -D-galactopyranosyl)-b -Dglucopyranoside (6) Polymer 6 was prepared in analogy to a literature procedure [16] (small scale) with the following amounts: compound 5 (0.81 g, 0.92 mmol), acrylamide (4.19 g, 58.1 mmol), 2,29azo-bis-isobutyronitrile (AIBN initiator of polymerisation) (25 mg and additionally 50 mg), tetrahydrofuran (50 ml). The polymerisation was carried out at 508C additional portion of AIBN (25 mg) in THF (4 ml) were introduced daily into the tube through a cannula, and the reaction was continued for 3 days. Purification gave the acetylated lacto– polymer 6 (3.1 g, 62%), 0.17 meq of lactose / g, IR: 3398 (wide, OH, NH), 3200, 2928, 1756 21 (CO), 1662 (CO), 1618, 1452 and 1419 cm .
2.8. 2 -Nitro-4( N9 - -carboxyhydrazido)benzyl 4 -O-b -D-galactopyranosyl-b -D-glucopyranoside (7) Polymer 6 (500 mg) was suspended in 9:1 methanol–dichlorometane (40 ml) and treated with sodium methoxide in methanol (0.1 M, 6 ml). The mixture was stirred overnight at room temperature and the resulting solution was dialysed employing a Diaflo UM2 membrane, lyophilised and gave of 390 mg of polymer 7 (0.16 meq of lactose / g), IR: almost identical with 7, only the absorbance at 1756 cm 21 has disappeared. Molecular weights were determined as 7000 (2 / 3 of total) and 12 000. Compound 7 (20 mg in 10 ml of water) was irradiated followed by ultrafiltration through a Diaflo UM2 membrane. Determination of total
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sugar in the filtrate showed the release of 0.12 meq of lactose / g.
2.9. 2 -Nitro-4 -( N9 - -carboxyhydrazido)benzyl [3 -O-(5 -Acetamido-3,5 -dideoxy-D-glycero-a -Dgalacto-2 -nonulopyranosylonic acid)-(4 -O-b -Dgalactopyranosyl)] -b -D-glucopyranoside (8) The incubation mixture (100 ml) contained polymer 7 (1 mg, 0.16 meq Lac / g), CMPNeuNAc (100 mg, 162 nmol) including CMP[ 14 C]NeuNAc (212, 205 d.p.m. / mmol, 345,611 d.p.m. / mg), a-lactalbumin (60 mg), 50 mM sodium cacodilate (pH 6.5), Triton X-100 (0.5%), calf intestinal alkaline phosphatase (3 U), and Galb-1,4-GlcNAca-2,3-(N)-sialyltransferase (100 mU). The mixture was vortexes and placed in an incubator at 378C. The same amounts of CMP-NeuNAc and calf intestinal alkaline phosphatase were added at regular intervals of 24 h. After 3 days of incubation the polymer was isolated by ultrafiltration, washed extensively with water [until only very little radioactivity (46 d.p.m. / ml) emerged in the eluants] and lyophilised. Polymer 8 (11 222 400 d.p.m. / g, 0.05 meq G M3 -oligosaccharide / g, represented a 33.1% incorporation yield of NeuNAc.
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enzyme specificity with a minimum of synthetic steps and mild reaction conditions ([11], and references cited therein). Our previous work [12] dealt with the preparation of an improved water-soluble acceptor polymer; in difference from prior acceptor polymer preparations, the present polymer afforded higher transfer yield and acceptor loads. The acceptor polymer (in the current work, polymer 6) was made by incorporating a suitable acryloyl saccharide derivative (5) already during the copolymerisation with acrylamide and the saccharide load was determined by the proportion of the two starting materials. The light-sensitive nitrobenzyl glycoside (1) was obtained in 62.8% yield from 2,3,6-tri-Oacetyl - 4 - O-(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl)-a-D-glucopyranosyl bromide and methyl 4-hydroxymethyl-3-nitrobenzoate in a Koeigs–Knorr reaction with silver perchlorate as a promoter. Deblocking gave compound 2 that was acetylated to compound 3 and condensed with tert-butyloxycarbonylhydrazine (DCC), to yield compound 4. The t-BOC group was removed by TFA in CH 2 Cl 2 and condensed with acryloyl chloride to yield compound 5.
2.10. Release of GM 3 -oligosaccharide [ NeuNAca(2 – 3)Galb(1 – 4)Glc] (9) from polymer 8 The photolysis (.350 nm) of polymer 8 [17], following ultrafiltration, the filtrate was lyophilised, subjected to TLC and counting affording G M3 -oligosaccharide (9) at a 85.6% yield (9 549 231 d.p.m. / g) and, and polymer 10 in the nondialysable residue. 3. Results and discussion Enzymic oligosaccharide synthesis on polymer supports was pioneered and intensively studied by us and recently also by others, leading to useful preparative results based on
Scheme 1. Intermediates in the chemoenzymic synthesis of G M3 oligosaccharide.
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Copolymerisation with acrylamide in the presence of AIBN gave compound 6 and removing of the protecting groups yielded a water-soluble polymer 7 (0.16 mmol Lac / g) (Scheme 1). Polymer 7 was an acceptor in the sialyltrans-
Fig. 1. TLC (Merk plate, n-butanol / acetic acid / water, 20 / 10 / 15 by vol) separation of radioactive products released by irradiation of compound 8. The marker G M3 -oligosaccharide, [NeuNAca(2– 3)Galb(1–4)Glc], was run analogised the product (9) and was detected by spraying with sulfuric acid and heating.
ferase reaction, using radioactively labelled CMP-NeuNAc. It has been previously shown, that lactose is a poor acceptor for sialyltransferase [18]. The reaction efficiency was increased in three ways: conditions that minimise the hydrolysis of the sugar nucleotide, excess of both CMP-NeuNAc and addition of calf intestinal alkaline phosphatase to hydrolyse the released by product of the sialylation, CMP [19] which is a strong inhibitor of sialyltransterase; the transfer yield was 33.1%, a yield comparable with transglycosylation [20] and definitely with chemical sialylation. Interestingly, enzymic (chymotrypsin) removal of the product from polymer that was introduced by us [21] was recently applied in the context of the synthesis of G M3 oligosaccharide [22]. Finally, the last step of the present synthesis constituted of photolysis, that was utilised by us very frequently in the past, releasing G M3 -oligosaccharide, (5-acetamido-3,5-dideoxy-D-glycero-a-D-galacto-2-nonulopyranosylonic acid)-(2–3)-b-Dgalactopyranosyl-(1–4)-b-D-glucopyranose, Ne-
Scheme 2. Photochemical release of G M3 -oligosaccharide (9).
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uNAca(2–3)Galb(1–4)Glc, (9) from the product polymer 8 chromatographically identical with a marker, proceeded at a very high yield (85.6%, Fig. 1, Scheme 2). Acknowledgements The authors are grateful to Dr James C. Paulson at the Cytel Corp. (La Jolla, CA, USA) for providing us with a generous supply of recombinant rat liver Galb-1,4-GlcNAca-2,3(N)-sialyltransferase and to Mr Ariel Lustig for determination of molecular weight. This work was supported by the German–Israeli Foundation for Scientific Research and Development (G.I.F.), by the Ministry of Science, Niedersachsen, and by the Volkswagen Stiftung. References [1] Y. Ichikawa, G.C. Look, C.-H. Wong, Anal. Biochem. 202 (1992) 215 (and literature cited therein). [2] S. Hakomori, J. Biol. Chem. 265 (1990) 18713. [3] S. Hakomori, Biochem. Soc. Trans. 21 (1993) 583. [4] A.Y. Khorlin, I.M. Prialova, I.B. Bystrova, Carbohydr. Res. 19 (1971) 272.
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[5] S. Sabesan, J.C. Paulson, J. Am. Chem. Soc. 108 (1986) 2068. [6] H.T. de Heij, M. Kloosterman, P.L. Koppen, J.H. van Boom, D.H. van der Eijnden, Carbohydr. Chem. 7 (1988) 209. [7] K.G.I. Nilsson, Carbohydr. Res. 188 (1989) 9. [8] T. Ichiawa, G.J. Shen, C.H. Wong, J. Am. Chem. Soc. 113 (1991) 4698. [9] A.K. Ray, U. Nilsson, G. Magnusson, J. Am. Chem. Soc. 114 (1992) 2256. [10] V. Kren, J. Thiem, Angew. Chem. Int. Ed. Engl. 34 (1995) 893. [11] U. Zehavi, J. Thiem, in: M.D. Bednarsky, E.S. Simon (Eds.), ACS Symposium Series, vol. 466, ch. 7, pp. 90–98, American Chemical Society, Washington, DC, 1991, and literature cited therein. [12] A. Tuchinsky, U. Zehavi, React. Funct. Polym. 31 (1996) 11. [13] F.J. Reyther, R.G. Young, J. Am. Chem. Soc. 74 (1952) 4210. [14] U. Zehavi, S. Sadeh, M. Herchman, Carbohydr. Res. 124 (1983) 23. ¨ [15] S. Kopper, U. Zehavi, Carbohydr. Res. 193 (1989) 296. [16] A. Pollak, H. Blumenfeld, M. Wax, R.L. Baughn, G.M. Whitesides, J. Am. Chem. Soc. 102 (1980) 6324. [17] U. Zehavi, M. Herchman, Carbohydr. Res. 128 (1984) 160. [18] J.C. Paulson, J.I. Rearick, R.L. Hill, J. Biol. Chem. 252 (1977) 2363. [19] C. Unverzagt, H. Kunz, J.C. Paulson, J. Am. Chem. Soc. 112 (1990) 9308. [20] S.-I. Nishimura, K.B. Lee, K. Matsuoka, Y.C. Lee, Biochem. Biophys. Res. Commun. 199 (1994) 249. [21] U. Zehavi, M. Herchman, Carbohydr. Res. 133 (1984) 339. [22] K. Yamada, S.-I. Nishimura, Tetrahedron Lett. 36 (1995) 9493.