Enzymatic Synthesis of Chondroitin 4-Sulfate with Well-Defined

Synthesis of chondroitin sulfate (ChS) with well-defined structure was achieved for the first time by hyaluronidase-catalyzed polymerization...
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Biomacromolecules 2005, 6, 2935-2942

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Enzymatic Synthesis of Chondroitin 4-Sulfate with Well-Defined Structure Shun-ichi Fujikawa, Masashi Ohmae, and Shiro Kobayashi*,† Department of Materials Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan Received May 29, 2005; Revised Manuscript Received August 23, 2005

Synthesis of chondroitin sulfate (ChS) with well-defined structure was achieved for the first time by hyaluronidase-catalyzed polymerization. N-Acetylchondrosine (GlcAβ(1f3)GalNAc) oxazoline derivatives sulfated at C4 (1a), C6 (1b), and both C4 and C6 (1c) in the GalNAc unit were synthesized as transition state analogue substrate monomers for hyaluronidase (HAase) catalysis. Compound 1a was effectively polymerized by the enzyme, giving rise to synthetic ChS sulfated perfectly at the C4 position in all N-acetylgalactosamine units (Ch4S, 2a) in good yields. Molecular weights (Mn) of 2a ranged from 4000 to 18 400, which were controlled by varying reaction conditions. Compounds 1b and 1c were not catalyzed by the enzyme, affording the corresponding disaccharides through the oxazoline ring-opening without formation of polysaccharides. Introduction Chondroitin sulfate (ChS) belongs to a family of glycosaminoglycans (GAGs) including hyaluronan (HA), chondroitin (Ch), dermatan sulfate (DS), heparan sulfate/heparin, and keratan sulfate, which are widely distributed in extracellular matrixes and on cell surfaces.1 ChS has a β(1f4)linked repeating unit of β-D-glucuronyl-(1f3)-N-acetyl-Dgalactosamine (GlcAβ(1f3)GalNAc) disaccharide, whose hydroxyl groups are randomly sulfated.1 Molecular weights of naturally occurring ChS range from 0.5 to 35 × 104. Biosynthesis of ChS is performed by catalysis of the specific glycosyltransferases with uridine 5′-diphospho (UDP)-GlcA and UDP-GalNAc as substrates1,2 and the sulfotransferases with 3′-phosphoadenosine 5′-phosphosulfate;3 however, the synthesis mechanism of ChS is not fully understood. Recently, important biological activities of ChS in living systems has been increasingly reported.1 Particularly, it is essential for the development of the central nervous system, which is closely associated with the sulfation patterns.1b,4 Naturally occurring ChS is further classified into five groups according to the major component in the molecule, i.e., ChS-A mainly containing GlcAβ(1f3)GalNAc4S unit (sulfated at C4 in GalNAc; A unit), ChS-C (GlcAβ(1f3)GalNAc6S; C unit), ChS-D (GlcA2Sβ(1f3)GalNAc6S; D unit), ChS-E (GlcAβ(1f3)GalNAc4,6S; E unit), and ChS-K (GlcA3Sβ(1f3)GalNAc4S; K unit).1b,4 These natural ChS variants contain other units in various proportions, for example, ChS-A from whale cartilage incorporates a C unit in about 20% as a minor unit, and in shark cartilage ChS-C has a D unit in about 10%.5 Thus, incorporation of minor units causes substantial structural diversity of ChS molecules, * To whom correspondence should be addressed. Tel: +81-75-3832459. Fax: +81-75-383-2461. E-mail: [email protected]. † Present address: R&D Center for Bio-based Materials, Kyoto Institute of Technology, Kyoto 606-8585, Japan. Tel/Fax: +81-75-724-7688. E-mail: [email protected].

Scheme 1. Enzymatic Synthesis of ChS via HAase-catalyzed Polymerization

which makes the results of studies on ChS functions indistinct. Synthesis of ChS with a uniform structure has challenged many scientists; up to now, hexasaccharides consisting of structural units A,6 C,6 and D7 have been synthesized as the longest ChS variants via conventional chemical methods. In vitro synthesis of ChS via biosynthetic pathways has not been achieved due to difficulties in isolation and characterization of the enzymes involved in ChS biosynthesis. The investigation of the biological activities of naturally occurring ChS at a molecular level requires a structurally well-defined ChS polymer. Enzymatic polymerization utilizing a glycoside hydrolase as catalyst has been demonstrated to be effective as a singlestep polysaccharide synthesis.8 Several synthetic polysaccharides of natural and unnatural types were successfully produced with the total structure control by enzymatic polymerization of substrate monomers activated at the anomeric carbon atoms.8-12 These observations motivated us to explore the synthesis of structurally well-defined ChS bearing regio-selective sulfate groups. In the present paper, hyaluronidase (HAase)-catalyzed synthesis of ChS consisting of a single structural unit is reported (Scheme 1). Such ChS will become not only a key substance to study the bioactivity

10.1021/bm050364p CCC: $30.25 © 2005 American Chemical Society Published on Web 09/22/2005

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associated with sulfate groups, but also a probe for investigation of substrate specificity of the particular sulfotransferases and an epitope for preparation of antibodies recognizing microstructures in natural ChS molecules. Experimental Section Measurements. NMR spectra were recorded with a Bruker DPX-400 spectrometer. FABMS spectra were obtained on a JEOL JMS HX-110A spectrometer using 2,4dinitrobenzyl alcohol or dithiothreitol/thioglycerol (1/1, v/v) as the matrix. Optical rotations were measured with a JASCO P-1010 polarimeter. Melting points were determined with a YAMATO MP-21. Yields and molecular weights of the products given after the reactions were determined by size exclusion chromatography (SEC) measurements (GPC-8020 system; TOSOH) with Shodex Ohpak SB-804HQ column (8.0 × 300 mm) eluting with 0.1 M aqueous sodium nitrate (flow rate; 0.5 mL/min, 40 °C) calibrated by hyaluronan standards (Mn ) 800, 2000, 4000, and 50 000). Materials. Hyaluronidases from ovine testes (OTH) and from bovine testes (BTH) were purchased from Sigma (OTH, Type V, Lot No. 122k1378, 3720 units/mg, and BTH, type IV-S, lot no. 38H7026, 1010 units/mg). HAase of bee venom was purchased from Sigma (lot no. 79H1017). All enzymes were used without further purification. Chondroitin sulfate A from whale cartilage was purchased from Seikagaku Co. (lot no. S03101, Mn ) 28 800). Triethylammonium benzyl 2-acetamido-6-O-acetyl2-deoxy-3-O-(methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate)-4-O-sulfonato-β-D-galactopyranoside (5a). Benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-O-(methyl 2,3,4tri-O-acetyl-β-D-glucopyranosyluronate)-β-D-galactopyranoside (3)12 (696 mg, 0.972 mmol) in an acetic acid (16.0 mL)water (4.0 mL) mixture was stirred at 80 °C for 1 h. The reaction mixture was then evaporated to dryness, and the residue was subjected to silica gel column chromatography (chloroform-methanol 20:1-15:1, v/v) to give benzyl 2-acetamido-2-deoxy-3-O-(methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate)-β-D-galactopyranoside (4) (505 mg, 0.806 mmol, 87%). To a solution of compound 4 (505 mg, 0.806 mmol) in pyridine (10.0 mL) was added acetyl chloride (70 µL, 0.967 mmol) dropwise at -40 °C under argon atmosphere. The mixture was stirred at -40 °C for 1.5 h followed by further addition of acetyl chloride (35 µL, 0.484 mmol). After stirring for 1.5 h, methanol (2.0 mL) was added at -40 °C for quenching of excess reagent, and the mixture was evaporated to dryness. The residue was subjected to silica gel column chromatography (n-hexanes-ethyl acetate 1:3-0:1, v/v). Fractions containing the product were combined and evaporated to dryness. To a solution of the residue in dimethylformamide (10.0 mL) was added sulfurtrioxide trimethylamine complex (895 mg, 6.432 mmol), and then the reaction mixture was stirred at 50 °C under argon atmosphere overnight. Methanol (2.0 mL) was added to the solution for quenching of excess reagent, and the mixture was evaporated to dryness. The residue was subjected to silica gel chromatography (chloroform-methanol 20:1-15: 1, v/v, containing 0.5% triethylamine) to give 5a (669 mg,

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0.785 mmol, 80%) as white amorphous. [R]22D -26° (c 1.0, CHCl3). 1H NMR (400 MHz, CDCl3, TMS) δ 9.50 (1H, bs, SO3H‚N(CH2CH3)3), 7.35-7.28 (5H, m, aromatic), 6.20 (1H, bs, NHCOCH3), 5.23 (1H, t, H-3′, J2′,3′ ) J3′,4′ ) 9.03 Hz), 5.16 (1H, t, H-4′, J3′,4′ ) J4′,5′ ) 9.03 Hz), 5.01-4.98 (2H, m, H-2′, H-1′), 4.83 (1H, d, PhCH2, J ) 11.54 Hz), 4.79 (1H, s, H-4), 4.70 (1H, d, H-1, J1,2 ) 7.53 Hz), 4.59 (1H, d, PhCH2, J ) 11.54 Hz), 4.49-4.43 (2H, m, H-6), 4.27 (1H, d, H-3, J2,3 ) 9.54 Hz), 4.14 (1H, d, H-5′, J4′,5′ ) 9.54 Hz), 3.91-3.87 (2H, m, H-2, H-5), 3.73 (3H, s, COOCH3), 3.18 (6H, q, SO3H‚N(CH2CH3)3, J ) 7.03 Hz), 2.08-1.94 (15H, m, CH3CO), 1.35 (9H, t, SO3H‚N(CH2CH3)3, J ) 7.03 Hz). HRMS (FAB); found 851.3112 m/z (-1.0 ppm) (calcd. for C36H55N2O19S [M+H]+ 851.3120). 2-Methyl-4,5-dihydro-[triethylammonium 6-O-acetyl1,2-di-doxy-3-O-(methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate)-4-O-sufonato-r-D-galactopyranoso][2,1d]-1,3-oxazole (6a). A mixture of 5a (213 mg, 0.250 mmol) and 20% palladium hydroxide on charcoal (133 mg) in methanol (20.0 mL) was stirred at room temperature under hydrogen atmosphere for 2 h. The reaction mixture was then filtered through diatomaceous earth (Celite) and evaporated to dryness. The residue was dissolved in anhydrous dichloromethane (10.0 mL) followed by the successive addition of triethylamine (60 µL, 0.230 mmol), 4-(dimethylamino)pyridine (30 mg, 0.138 mmol) and p-toluenesulfonyl chloride (94 mg, 0.276 mmol) at room temperature. The mixture was stirred at room temperature under argon atmosphere for 9 h, and then triethylamine (60 µL, 0.230 mmol), 4-(dimethylamino)pyridine (30 mg, 0.138 mmol), and p-toluenesulfonyl chloride (94 mg, 0.276 mmol) were further added. After an additional 12 h, the reaction mixture was evaporated to dryness, and the residue was subjected to silica gel column chromatography (chloroform-methanol 20:1, v/v, containing 0.5% triethylamine), size exclusion chromatography (SEC) by Sephadex LH-20 (eluent; methanol containing 1% triethylamine), and silica gel chromatography (chloroformmethanol 20:1, v/v, containing 0.5% triethylamine) to afford 6a (100 mg, 0.135 mmol, 54%) as white amorphous. [R]24D +4.6° (c 0.68, CHCl3). 1H NMR (400 MHz, CDCl3, TMS) δ 9.45 (1H, bs, SO3H‚N(CH2CH3)3), 5.93 (1H, d, H-1, J1,2 ) 6.53 Hz), 5.27 (1H, t, H-3′, J2′,3′ ) J3′,4′ ) 9.03 Hz), 5.20 (1H, t, H-4′, J3′,4′ ) J4′,5′ ) 9.03 Hz), 5.15 (1H, d, H-1′, J1′,2′ ) 7.53 Hz), 4.97 (H, dd, H-2′, J1′,2′ ) 7.53 Hz, J2′,3′ ) 9.03 Hz), 4.81 (1H, dd, H-4, J3,4 ) 4.52 Hz, J4,5 ) 3.01 Hz), 4.48 (1H, dd, H-6a, J5,6a ) 3.51 Hz, J6a,6b ) 12.55 Hz), 4.39 (1H, dd, H-6b, J5,6b ) 9.04 Hz, J6a,6b ) 12.55 Hz), 4.24 (1H, ddd, H-5, J4,5 ) 3.01 Hz, J5,6a ) 3.51 Hz, J5,6b ) 9.04 Hz), 4.20 (1H, dd, H-3, J2,3 ) 5.52 Hz, J3,4 ) 4.52 Hz), 4.11 (1H, d, H-5′, J4′,5′ ) 9.03 Hz), 4.07 (1H, dd, H-2, J1,2 ) 6.53 Hz, J2,3 ) 5.52 Hz), 3.75 (3H, s, COOCH3), 3.19 (6H, q, SO3H‚N(CH2CH3)3, J ) 7.03 Hz), 2.07-2.00 (15H, m, CH3CO), 1.38 (9H, t, SO3H‚N(CH2CH3)3, J ) 7.03 Hz). HRMS (FAB); found 743.2529 m/z (-2.1 ppm) (calcd. for C29H47N2O18S [M+H]+ 743.2544). 2-Methyl-4,5-dihydro-[sodium 1,2-di-deoxy-3-O-(sodium β-D-glucopyranosyluronate)-4-O-sulfonato-r-D-galactopyranoso][2,1-d]-1,3-oxazole (1a). To a solution of 6a (75 mg, 0.101 mmol) in methanol (3.0 mL) was added 1 M

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aqueous sodium hydroxide (303 µL, 0.303 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 1 h and then evaporated and lyophilized. The residue was dissolved in water, and the pH of the solution was adjusted to 8.0 by DOWEX 50W-X4 resin. After filtration of the mixture through cotton, the filtrate was lyophilized to give 1a (41 mg, purity 92%). 1H NMR (400 MHz, D2O, acetone) δ 5.89 (1H, d, H-1, J1,2 ) 6.02 Hz), 4.53 (1H, d, H-1′, J1′,2′ ) 8.03 Hz), 3.92-3.88 (3H, m, H-3, H-5, H-6a), 3.65-3.57 (2H, m, H-2, H-6b), 3.50 (1H, d, H-5′, J4′,5′ ) 9.04 Hz), 3.363.30 (2H, m, H-4′, H-3′), 3.19 (1H, dd, H-2′, J1′,2′ ) 8.03 Hz, J2′,3′ ) 8.53 Hz), 1.84 (3H, s, CH3C of oxazole). HRMS (FAB); found 526.0228 m/z (+1.6 ppm) (calcd. for C14H19NNa3O14S [M+Na]+ 526.0219). Triethylammonium benzyl 2-acetamido-4-O-acetyl-2deoxy-3-O-(methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate)-6-O-sulfonato-β-D-galactopyranoside (5b). To a solution of compound 4 (323 mg, 0.515 mmol) in dimethylformamide (5.0 mL) was added sulfurtrioxide trimethylamine complex (215 mg, 1.54 mmol). The reaction mixture was stirred at 50 °C under argon atmosphere overnight, and then methanol (1.0 mL) was added for quenching of excess reagent. The mixture was evaporated to dryness, and the residue was subjected to silica gel column chromatography (chloroform-methanol 10:1-7:1, v/v, containing 0.5% triethylamine). Fractions containing the product were collected and combined, followed by evaporation. Acetic anhydride (282 µL, 2.89 mmol) was then added dropwise to the residue dissolved in pyridine (5.0 mL) at 0 °C under dry atmosphere. The mixture was stirred at room temperature for 24 h followed by evaporation to dryness. The residue was purified by silica gel column chromatography (chloroform-methanol 20:1-10:1, v/v, containing 0.5% triethylamine) to provide 5b (334 mg, 0.393 mmol, 76%) as white amorphous. [R]26D -21° (c 0.89, CHCl3). 1H NMR (400 MHz, CDCl3, TMS) δ 9.46 (1H, bs, SO3H‚N(CH2CH3)3), 7.30-7.29 (5H, m, aromatic), 6.06 (1H, bs, NHCOCH3), 5.44 (1H, d, H-4, J3,4 ) 3.01 Hz), 5.20 (1H, t, H-3′, J2′,3′ ) J3′,4′ ) 9.53 Hz), 5.14 (1H, t, H-4′, J3′,4′ ) J4′,5′ ) 9.53 Hz), 4.92 (1H, dd, H-2′, J1′,2′ ) 8.03 Hz, J2′,3′ ) 9.53 Hz), 4.89 (1H, d, PhCH2, J ) 12.05 Hz), 4.85 (1H, d, H-1, J1,2 ) 8.54 Hz), 4.72 (1H, d, H-1′, J1′,2′ ) 8.03 Hz), 4.58 (1H, d, PhCH2, J ) 12.05 Hz), 4.48 (1H, dd, H-3, J2,3 ) 7.53 Hz, J3,4 ) 3.01 Hz), 4.153.98 (4H, m, H-6, H-5, H-5′), 3.81 (1H, dd, H-2, J1,2 ) 8.54 Hz, J2,3 ) 7.53 Hz), 3.73 (3H, s, COOCH3), 3.14 (6H, q, SO3H‚N(CH2CH3)3, J ) 7.53 Hz), 2.08-1.92 (15H, m, CH3CO), 1.35 (9H, t, SO3H‚N(CH2CH3)3, J ) 7.53 Hz). HRMS (FAB); found 851.3115 m/z (-0.6 ppm) (calcd. for C36H55N2O19S [M+H]+ 851.3120). 2-Methyl-4,5-dihydro-[triethylammonium 4-O-acetyl1,2-di-doxy-3-O-(methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate)-6-O-sufonato-r-D-galactopyranoso][2,1d]-1,3-oxazole (6b). A mixture of compound 5b (89.0 mg, 0.104 mmol) and 20% palladium hydroxide on charcoal (100 mg) in methanol (10.0 mL) and triethylamine (100 µL) was stirred at room temperature under hydrogen atmosphere for 1 h. The reaction mixture was then filtered through Celite, and the filtrate was evaporated to dryness. The residue was dissolved in anhydrous dichloromethane (5.0 mL), followed

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by the addition of triethylamine (32 µL, 0.124 mmol), 4-(dimethylamino)pyridine (16.0 mg, 0.0741 mmol), and p-toluenesulfonyl chloride (50.6 mg, 0.148 mmol) at room temperature. The reaction mixture was stirred at room temperature under argon atmosphere for 24 h, and then triethylamine (32 µL, 0.124 mmol), 4-dimethylaminopyridine (16.0 mg, 0.0741 mmol), and p-toluenesulfonyl chloride (50.6 mg, 0.148 mmol) were also added. After an additional 24 h, the mixture was evaporated to dryness, and the residue was subjected to silica gel column chromatography (chloroform-methanol 20:1-10:1, v/v, containing 0.5% triethylamine), SEC by Sephadex LH-20 (eluent; methanol containing 1% trietylamine), and silica gel column chromatography (chloroform-methanol 20:1, v/v, containing 0.5% triethylamine) to give 6b (50 mg, 0.0674 mmol, 65%) as white amorphous. [R]24D +4.6° (c 0.68, CHCl3). 1H NMR (400 MHz, CDCl3, TMS) δ 9.57 (1H, bs, SO3H‚N(CH2CH3)3), 5.93 (1H, d, H-1, J1,2 ) 7.02 Hz), 5.46 (1H, dd, H-4, J3,4 ) 3.51 Hz, J4,5 ) 2.01 Hz), 5.28 (1H, t, H-3′, J2′,3′ ) J3′,4′ ) 9.04 Hz), 5.20 (1H, t, H-4′, J3′,4′ ) J4′,5′ ) 9.04 Hz), 5.07 (1H, d, H-1′, J1′,2′ ) 7.53 Hz), 4.97 (H, dd, H-2′, J1′,2′ ) 7.53 Hz, J2′,3′ ) 9.04 Hz), 4.25 (1H, dt, H-5, J4,5 ) J5,6b ) 2.01 Hz, J5,6a ) 6.52 Hz), 4.16 (1H, dd, H-6a, J5,6a ) 6.52 Hz, J6a,6b ) 11.05 Hz), 4.07-4.02 (1H, m, H-5′, H-6b), 3.89 (1H, dd, H-3, J2,3 ) 7.03 Hz, J3,4 ) 3.51 Hz), 3.773.76 (4H, m, H-2, COOCH3), 3.14 (6H, q, SO3H‚N(CH2CH3)3, J ) 7.03 Hz), 2.06-2.00 (15H, m, CH3CO), 1.36 (9H, t, SO3H‚N(CH2CH3)3, J ) 7.03 Hz). HRMS (FAB); found 743.2543 m/z (-0.3 ppm) (calcd. for C29H47N2O18S [M+H]+ 743.2544). 2-Methyl-4,5-dihydro-[sodium 1,2-di-deoxy-3-O-(sodium β-D-glucopyranosyluronate)-6-O-sulfonato-r-D-galactopyranoso][2,1-d]-1,3-oxazole (1b). To a solution of compound 6b (138 mg, 0.186 mmol) in methanol (5.0 mL) was added 1 M aqueous sodium hydroxide (560 µL, 0.557 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 1 h and then concentrated and lyophilized. The residue was dissolved in water, and the pH of the solution was adjusted to 8.0 by DOWEX 50W-X4 resin. After removal of the resin by filtration through cotton, the filtrate was lyophilized to afford 1b (91 mg, purity 93%). 1H NMR (400 MHz, D2O, acetone) δ 5.88 (1H, d, H-1, J1,2 ) 7.03 Hz), 4.44 (1H, d, H-1′, J1′,2′ ) 7.53 Hz), 4.03-3.95 (4H, m, H-4, H-5, H-6), 3.76 (1H, t, H-2, J1,2 ) J2,3 ) 7.03 Hz), 3.64 (1H, dd, H-3, J2,3 ) 7.03 Hz, J3,4 ) 3.51 Hz), 3.50 (1H, d, H-5′, J4′,5′ ) 7.02 Hz), 3.31-3.28 (2H, m, H-4′, H-3′), 3.18 (1H, d, H-2′, J1′,2′ ) J2′,3′ ) 7.53 Hz), 1.81 (3H, s, CH3C of oxazole). HRMS (FAB); found 526.0228 m/z (+1.7 ppm) (calcd. for C14H19NNa3O14S [M+Na]+ 526.0219). Triethylammonium benzyl 2-acetamido-2-deoxy-3-O(methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate)4,6-di-O-sulfonato-β-D-galactopyranoside (5c). To compound 4 (260 mg, 0.372 mmol) dissolved in dimethylformamide (10.0 mL) was added sulfurtrioxide trimethylamine complex (1.03 g, 7.432 mmol). The reaction mixture was stirred at 50 °C under argon atmosphere for 48 h, and then methanol (5.0 mL) was added to quench excess reagent followed by evaporation to dryness. The resulting residue was subjected to silica gel column chromatography (chlo-

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roform-methanol 10:1-3:1, v/v, containing 0.5% triethylamine) and SEC by Sephadex LH-20 (eluent; methanol) to produce 5c (346 mg, 0.350 mmol, 94%) as white amorphous. [R]25D -23° (c 1.0, CHCl3). 1H NMR (400 MHz, CDCl3, TMS) δ 9.10 (1H, bs, SO3H‚N(CH2CH3)3), 7.23-7.16 (5H, m, aromatic), 5.25-5.18 (2H, m, H-3′, H-4′), 5.07 (1H, d, H-1′, J1′,2′ ) 8.03 Hz), 4.99 (H, dd, H-2′, J1′,2′ ) 8.03 Hz, J2′,3′ ) 9.54 Hz), 4.82-4.80 (2H, m, PhCH2, H-4), 4.69 (1H, d, H-1, J1,2 ) 8.04 Hz), 4.50 (1H, d, PhCH2, J ) 11.55 Hz), 4.45 (1H, dd, H-6a, J5,6a ) 5.02 Hz, J6a,6b ) 10.54 Hz), 4.374.33 (2H, m, H-6b, H-5′), 4.28-4.17 (2H, m, H-2, H-3), 3.86 (1H, t, H-5, J5,6a ) 5.02 Hz), 3.73 (3H, s, COOCH3), 3.19 (6H, q, SO3H‚N(CH2CH3)3, J ) 7.03 Hz), 2.03-1.99 (12H, m, CH3CO), 1.33 (9H, t, SO3H‚N(CH2CH3)3, J ) 7.03 Hz). HRMS (FAB); found 1012.3607 m/z (+0.0 ppm) (calcd. for C40H67N3O21S2Na [M+Na]+ 1012.3607). 2-Methyl-4,5-dihydro-[triethylammonium 1,2-di-deoxy-3-O-(methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate)-4,6-di-O-sufonato-r-D-galactopyranoso][2,1-d]-1,3oxazole (6c). A mixture of compound 5c (282 mg, 0.285 mmol) and 20% palladium hydroxide on charcoal (250 mg) in methanol (25.0 mL) and triethylamine (250 µL) was stirred at room temperature under hydrogen atmosphere for 2 h. The reaction mixture was then filtered through Celite, and the filtrate was evaporated to dryness. The residue was dissolved in anhydrous dichloromethane (25.0 mL) followed by the successive addition of triethylamine (76 µL, 0.288 mmol), 4-(dimethylamino)pyridine (37.0 mg, 0.173 mmol), and p-toluenesulfonyl chloride (119 mg, 0.345 mmol). The reaction mixture was stirred at room temperature under argon atmosphere for 18 h and then evaporated to dryness. The residue was subjected to silica gel column chromatography (chloroform-methanol 10:1-5:1, v/v, containing 0.5% triethylamine) and SEC by Sephadex LH-20 (eluent; methanol containing 1% triethylamine) to give 6c (232 mg, 0.263 mmol, 92%) as white amorphous. [R]26D +4.7° (c 1.0, CHCl3). 1H NMR (400 MHz, CDCl3, TMS) δ 10.07 (1H, bs, SO3H‚N(CH2CH3)3), 5.90 (1H, d, H-1, J1,2 ) 6.53 Hz), 5.27 (1H, t, H-3′, J2′,3′ ) J3′,4′ ) 8.53 Hz), 5.19-5.15 (2H, m, H-4′, H-1′), 4.95 (1H, t, H-2′, J1′,2′ ) J2′,3′ ) 8.53 Hz), 4.74 (1H, t, H-4, J4,5 ) 3.01 Hz), 4.37 (1H, t, H-6a, J5,6a ) J6a,6b ) 8.04 Hz), 4.27-4.15 (4H, m, H-3, H-5, H-6b, H-5′), 4.01 (1H, t, H-2, J1,2 ) J2,3 ) 6.53 Hz), 3.74 (3H, s, COOCH3), 3.17 (6H, q, SO3H‚N(CH2CH3)3, J ) 7.03 Hz), 2.08-1.99 (12H, m, CH3CO), 1.37 (9H, t, SO3H‚N(CH2CH3)3, J ) 7.03 Hz). HRMS (FAB); found 882.3194 m/z (-2.0 ppm) (calcd. for C33H60N3O20S2 [M+H]+ 882.3212). 2-Methyl-4,5-dihydro-[sodium 1,2-di-deoxy-3-O-(sodium β-D-glucopyranosyluronate)-4,6-di-O-sulfonato-rD-galactopyranoso][2,1-d]-1,3-oxazole (1c). To a solution of compound 6c (232 mg, 0.263 mmol) in methanol (8.0 mL) was added 1 M aqueous sodium hydroxide (789 µL, 0.789 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 1 h and then concentrated and lyophilized. The residue was dissolved in water, and the pH of the solution was adjusted to 8.0 by DOWEX 50W-X4 resin. After filtration of the resin through cotton, the filtrate was lyophilized to give 1c (152 mg, purity 80%). 1H NMR (400 MHz, D2O, acetone) δ 5.90 (1H, d, H-1, J1,2 ) 5.52 Hz), 4.67 (1H, d,

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H-4, J4,5 ) 3.51 Hz), 4.55 (1H, d, H-1′, J1′,2′ ) 8.03 Hz), 4.18 (1H, dt, H-5, J4,5 ) 3.51 Hz, J5,6a ) J5,6b ) 6.02 Hz), 4.08-4.06 (2H, m, H-6a, H-6b), 3.95-3.93 (2H, m, H-2, H-3), 3.51 (1H, d, H-5′, J4′,5′ ) 8.04 Hz), 3.34-3.29 (2H, m, H-3′, H-4′), 3.20 (1H, t, H-2′, J1′,2′ ) J2′,3′ ) 8.53 Hz), 1.84 (3H, s, CH3C of oxazole). HRMS (FAB); found 627.9609 m/z (+0.4 ppm) (calcd. for C14H19NNa3O14S [M+Na]+ 627.9607). Typical Polymerization Procedure for 1a. Monomer 1a (7.0 mg, 13.9 µmol) in a phosphate buffer (50 mM, pH 7.5, 139 µL) was incubated with OTH (0.7 mg) at 30 °C. Monomer consumption was monitored by TLC (solvent: acetonitrile/methanol ) 2/1, v/v). A spot derived from monomer was observed clearly on a TLC trace with Rf ) 0.2. After 1.5 h, the mixture was heated at 90 °C for 3 min to inactivate the enzyme. A small portion of the mixture was analyzed by SEC measurements (Mn ) 11 700, Mw ) 21 000). Fractions containing the product 2a were collected, combined, and desalted through Sephadex G-10 column chromatography eluting with distilled water followed by lyophilization to give 2a (5.0 mg, 71% isolated yield). 1H NMR (400 MHz, D2O, acetone) δ 4.56 (1H, s, H-4), 4.37 (1H, d, H-1, J1,2 ) 7.52 Hz), 4.27 (1H, d, H-1′, J1′,2′ ) 7.04 Hz), 3.85-3.83 (2H, m, H-2, H-3), 3.63-3.57 (4H, m, H-6a, H-6b, H-5, H-4′), 3.47 (1H, d, H-5′, J4′,5′ ) 9.04 Hz), 3.39 (1H, t, H-3′, J2′,3′ ) J3′,4′ ) 7.04 Hz), 3.18 (1H, t, H-2′, J1′,2′ ) J2′,3′ ) 7.04 Hz), 1.84 (3H, m, CH3CO). 13C NMR (100 MHz, D2O, acetone) δ 174.58 (C-6′), 174.07 (CH3CO), 103.35 (C-1′), 100.49 (C-1), 79.98 (C-4′), 76.28 (C-5′), 76.16 (C-4), 75.21 (C-3), 74.14 (C-5), 73.09 (C-3′), 71.82 (C-2′), 60.58 (C-6), 51.06 (C-2), 22.09 (CH3CO). Results and Discussion Synthesis of Sugar Oxazoline Monomers Having Sulfate Groups. On the basis of our new concept “transitionstate analogue substrate” (TSAS),10-12 regio-selectively sulfated sugar oxazoline monomers (1a-1c) were designed for HAase catalysis.11-13 HAase employed normally is active for the hydrolysis of ChS as well as HA, Ch, and DS.14 Compounds 1a, 1b, and 1c having 4-, 6-, and 4,6-di-sulfate groups, respectively, were synthesized starting from compound (3)12 (Scheme 2). The 4,6-O-benzylidene group of 3 was hydrolyzed by 80% aqueous acetic acid to afford compound (4). The 6-hydroxyl group of 4 was acetylated by acetyl chloride, and the 4-hydroxyl group was sulfonated by sulfurtrioxide trimethylamine complex to provide 4-Osulfo derivative (5a). Compounds (5b) and (5c) were prepared from 4 by treatment with sulfurtrioxide trimethylamine complex. The 1-O-benzyl group of 5 was removed by hydrogenation using palladium(II) hydroxide-charcoal under hydrogen atmosphere followed by treatment with p-toluenesulfonyl chloride in dichloromethane containing triethylamine and 4-(dimethyamino)pyridine, giving rise to the corresponding oxazoline derivatives (6). Finally, all of the O-acetyl protecting groups and methyl ester were hydrolyzed by aqueous sodium hydroxide-methanol mixed solution to provide sulfated oxazolines 1a-1c. Reactions of Sugar Oxazoline Monomers 1a-1c with HAase. Figure 1 illustrates the time-dependence of the

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a (i) 80% AqAcOH, 70 °C; (ii) AcCl/pyridine, then Me N‚SO /DMF, 54%; (iii) Me N‚SO /DMF, then Ac O, DMAP/pyridine, 74%; (iv) Me N‚SO /DMF, 3 3 3 3 2 3 3 quant.; (v) Pd(OH)2-C, H2/MeOH, then TsCl, DMAP/CH2Cl2, Et3N, (6a) 54%, (6b) 53%, (6c) 80%; (vi) 1 M aq. NaOH-MeOH (1:10, v/v), (1a) 84%, (1b) 93%, (1c) 80%

Table 1. Enzymatic Polymerization of the Sugar Oxazolines 1a-1c under Various Conditions polymerizationa

polymer

entry

oxazoline

enzyme

enzyme amount/ wt % for oxazoline

1 2 3 4 5

1a 1b 1c 1a 1a

OTH OTH OTH BTH bee venom

10 10 10 10 10

timeb/h

structure

yieldc/%

Mnd

Mwd

1.5 18 35 48 48

2a 2b 2c 2a 2a

75 0 0 20 0

11 700

21 000

8500

11 600

a In a phosphate buffer (50 mM, pH 7.5) at 30 °C. Initial concentration of sugar oxazoline was 0.10 M. b Indicating the time for complete consumption of sugar oxazoline monitored by TLC (acetonitrile/methanol ) 2/1, v/v). c Determined by HPLC containing products with molecular weight more than tetrasaccharide. d Determined by SEC calibrated with hyaluronan standards.

concentration of the sugar oxazolines with HAase from ovine testes (OTH; 3720 units/mg; black symbols) and without enzyme (white symbols). The concentration change of the oxazoline was monitored at 30 °C by 1H NMR spectroscopy measuring the integration of the H-1 proton. With enzyme catalyst, it is possible for two kinds of reactions to occur; enzymatic polymerization of 1a-1c leading to the production of the corresponding polysaccharides 2a-2c, enzymatic and nonenzymatic hydrolysis of the sugar oxazolines through the oxazoline ring-openening, affording β-D-glucopyranosyluronate-(1f3)-2-acetamido-2-deoxy-4-O-sulfo-D-galactopyranose from 1a, β-D-glucopyranosyluronate-(1f3)-2-acetamido2-deoxy-6-O-sulfo-D-galactopyranose from 1b, and β-Dglucopyranosyluronate-(1f3)-2-acetamido-2-deoxy-4,6-diO-sulfo-D-galactopyranose from 1c. Notably, the consumption rate of 1a was very fast by OTH (b); 1a disappeared within 1 h, whereas it remained in 97% without enzyme (O; Figure

1a). The sugar oxazoline is a high-energy compound, which is activated at the anomeric carbon. Therefore, 1a was gradually decomposed through the oxazoline ring-opening in aqueous media without enzyme, affording to the corresponding disaccharides. Significant differences were observed between 1b (Figure 1b) and 1c (Figure 1c), which were completely consumed within 18 h (1b) and 35 h (1c) with the enzyme, and they remained in 37 and 15% after the respective period without enzyme. These results indicate that 1a is most reactive with the enzymatic catalysis, whereas 1b and 1c are much less reactive. Table 1 indicates the polymerization results with varying reaction conditions. Compound 1a produced the corresponding polymer 2a having Mn ) 11 700 in a 75% yield through OTH-catalyzed polymerization in 1.5 h under total control of regio-selectivity and stereochemistry (entry 1). This Mn value corresponds to that of naturally occurring ChS. Under

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Figure 2. (A) 1H NMR spectra of 2a (synthetic Ch4S) and (B) naturally occurring ChS-A from whale cartilage.

Figure 1. Reaction-time courses of the sugar oxazolines (a) 1a, (b) 1b, and (c) 1c with OTH (black symbols) and without enzyme (white symbols). Reaction conditions: In a phosphate buffered D2O (50 mM, pD 7.5); reaction temperature, 30 °C; initial concentration of each sugar oxazoline, 0.1 M; amount of OTH, 10 wt % for sugar oxazoline.

similar polymerization conditions, the corresponding nonsulfated monomer took 25 h for the complete monomer consumption.12 However, the HAase used was from ICN Biochemicals Inc.12 and the present HAase is from Sigma. When we polymerized the nonsulfated monomer with the present HAase, it took about 2 h for the complete monomer consumption, which is to be compared in terms of the catalytic activity of enzymes from different sources. Compounds 1b and 1c were exclusively hydrolyzed by the enzyme without formation of the corresponding polymers 2b and 2c (entries 2 and 3). Considering the polymerization mechanism of the oxazoline monomers as we proposed previously,11,12 these results clearly show that the sulfate group at the C6 position on GalNAc in the sugar oxazolines prevents the polymerization; compounds 1b and 1c were probably not recognized at the acceptor site in the enzyme for the chain elongation but recognized and activated at the donor site, leading to the hydrolysis products through the oxazoline ring-opening.13 Thus, the sugar oxazoline 1a acts

as a TSAS monomer for HAase catalysis. Polymerization of monomer 1a was further investigated with HAase from bovine testes (BTH, 1010 units/mg) and HAase from bee venom. BTH also catalyzed the polymerization (entry 4); however, the bee venom enzyme did not catalyze the polymerization (entry 5). OTH was more effective than BTH and led to rapid production of 2a with higher molecular weight, and hence, OTH was mainly used in the following experiments. Figure 2 shows 1H NMR spectra of 2a and natural ChS-A from whale cartilage. The signals at δ 4.02 and 3.99 derived from H-4 and H-6 of GalNAc6S (C unit) were observed in 80 (A unit): 20 (C unit), respectively on the spectrum of naturally occurring ChS-A from whale cartilage (Figure 2B). However, the spectrum of 2a (synthetic Ch4S) showed the signals derived only from A unit, definitely supporting the structural uniformity. 13C NMR spectrum also reveals that the structure of 2a is composed of only the A unit (Figure 3A), whereas natural ChS-A incorporates the C unit; the broad signal observed at δ 67.0 is derived from C6 of GalNAc6S (Figure 3B). Enzymatic Polymerization of 1a Under Various Conditions. To find the optimal conditions for the polymerization of 1a with OTH, reaction parameters such as pH, enzyme amount, concentration of 1a, and reaction temperature were examined (Table 2). The reaction proceeded at pH ranging from 6.0 to 9.0 (entry 1 in Table 1, and entries 6-10). However, no catalytic activities were observed at pH 9.5 or higher (entry 11). Yield of 2a reached 75% at pH 7.5 (entry 1), and the Mn value marked 18 000 at pH 8.0 (entry 8). Thus, the product polymer 2a was effectively provided at pH 7.5-8.0. The amount of the enzyme strongly affected reaction time and yield of 2a (entries 1, 12-14), indicating that 5-10 wt % of OTH produced polymer 2a in good yields with Mn values higher than 10 000. The monomer concentration influenced the yield and Mn value of 2a (entries 15 and 16); 0.05 and 0.2 M of 1a gave 2a in 75% yield with Mn ) 9100 and 68% yield with Mn ) 18 400, respectively. It is

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are to be compared with those (∼ 4600 and ∼6800) of natural (and also synthetic) Ch,12 and natural ChS-A (28 800 and 34 800) from whale cartilage. Mn of 2a corresponds to 72-74 saccharide units. Conclusion

Figure 3. (A) 13C NMR spectra of 2a (synthetic Ch4S) and (B) naturally occurring ChS-A from whale cartilage. Table 2. Enzymatic Polymerization of 1a under Various Conditions polymerization of 1aa

polymer 2a

OTH/ initial wt % conc./ temp./ timeb/ yieldc/ entry for 1a M pH °C h %

Mnd

Mwd

4000 8200 18 000 13 000 11 900

5600 13 700 32 400 18 500 15 800

10 000 15 500 8200 9100 18 400 12 300 13 000 12 700 11 100 6500

15 300 28 500 14 200 16 100 36 500 21 600 23 700 24 300 20 800 9400

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

10 10 10 10 10 10 1 5 20 10 10 10 10 10 10 10 10

0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.05 0.20 0.10 0.10 0.10 0.10 0.10 0.10

6.0 7.0 8.0 8.5 9.0 9.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5

30 30 30 30 30 30 30 30 30 30 30 0 10 20 40 50 60

0.5 0.5 5.0 72 72 168 48 6.0 0.5 1.5 2.5 3.0 2.5 2.0 1.0 5.0 4.0

66 74 58 14 12 0 6 68 74 75 68 79 77 79 60 16 0

a In a phosphate buffer; 50 mM. b Indicating the time for complete consumption of 1a monitored by TLC (acetonitrile/methanol ) 2/1, v/v). c Determined by HPLC containing 2a with molecular weight more than tetrasaccharide. d Determined by SEC calibrated with hyaluronan standards.

surprising that the polymerization proceeded at the temperatures ranging widely from 0 to 50 °C (entries 1 and 1721); at 40 °C or lower, the polymer 2a having a higher Mn was rapidly produced in good yields. At 20 °C, the polymer 2a was obtained in the highest yield of 79% with Mn ) 12 700 (entry 19). At 60 °C or higher, the polymerization did not occur probably due to inactivation of the enzyme, affording only the hydrolyzed compound from 1a (entry 22). The highest Mn and Mw values (18 400 and 36 500) of 2a

Monomer 1a was highly reactive with HAase catalysis, giving rise to ChS having sulfate group exclusively at C4 position on all GalNAc residues (Ch4S) with high molecular weight. Furthermore, this method enabled the regulation of molecular weight of the product Ch4S. Because of the difficulty to obtain pure Ch4S from nature and by other synthetic methods, it will become a potent tool not only for investigating the biological functions of ChS but also as a basic substance in the development of biomaterials, medicines, and food supplements. The present study will provide with important information for many fields of sciences, for instance, enzymology and biochemistry for investigation of substrate specificity of the enzyme, medical science for development of carbohydrate drugs, and polymer chemistry and carbohydrate chemistry for utilization of hydrolase as synthesis catalyst. Acknowledgment. The authors thank Drs. T. Miyoshi and T. Morikawa of DENKA Co. (Tokyo, Japan) for their gift of hyaluronan samples for SEC calibration standards. This study was partially supported by the 21st COE program for a United Approach to New Materials Science in Kyoto University. References and Notes (1) (a) DeAngelis, P. Anat. Rec. A 2002, 268, 317-326. (b) Sugahara, K.; Mikami, T.; Uyama, T.; Mizuguchi, S.; Nomura, K.; Kitagawa, H. Curr. Opin. Struct. Biol. 2003, 13, 612-620. (c) Sellec, S. B. Trends Genet. 2000, 16, 206-212. (2) (a) Yada, T.; Sato, T.; Kaseyama, H.; Gotoh, M.; Iwasaki, H.; Kikuchi, N.; Kwon, Y.-D.; Togayachi, A.; Kudo, T.; Watanabe, H.; Narimatsu, H.; Kimata, K. J. Biol. Chem. 2003, 278, 39711-39725. (b) Uyama, T.; Kitagawa, H.; Tanaka, J.; Tamura, J.; Ogawa, T.; Sugahara, K. J. Biol. Chem. 2003, 278, 3072-3078. (3) (a) Kusche-Gullberg, M.; Kjelle´n, L. Curr. Opin. Struct. Biol. 2003, 13, 605-611. (b) Chapman, E.; Best, M. D.; Hanson, S. R.; Wong, C.-H. Angew. Chem., Int. Ed. 2004, 43, 3526-3548. (4) (a) Sugahara, K.; Yamada, S. Trends Glycosci. Glycotechnol. 2000, 12, 321-349. (b) Laabs, T.; Carulli, D.; Geller, H. M.; Fawcett, J. W. Curr. Opin. Neurobiol. 2005, 15, 116-120. (5) Mathews, M. B. Clin. Orthop. Relat. Res. 1966, 48, 267-283. (6) (a) Jacquinet, J.-C. Abstract of Papers, XXth International Carbohydrate Symposium, Hamburg, August 2000, B-118, p 117. (b) Belot, F.; Jacquinet, J.-C. Carbohydr. Res. 2000, 326, 88-97. (7) Karst, N.; Jacquinet, J.-C. Eur. J. Org. Chem. 2002, 815-825. (8) (a) Kobayashi, S.; Uyama, H.; Kimura, S. Chem. ReV. 2001, 101, 3793-3818. (b) Kobayashi, S.; Uyama, H.; Ohmae, M. Bull. Chem. Soc. Jpn. 2001, 74, 613-635. (c) Kobayashi, S.; Sakamoto, J.; Kimura, S. Prog. Polym. Sci. 2001, 26, 1525-1560. (d) Kobayashi, S. J. Polym. Sci., Polym. Chem. Ed. 1999, 37, 3041-3056. (e) Kobayashi, S.; Shoda, S.; Uyama, H. In Catalysis in Precision Polymerization; Kobayashi, S., Ed.; John Wiley & Sons: Chichester, U.K., 1997; Chapter 8. (f) Kobayashi, S.; Shoda, S.; Uyama, H. AdV. Polym. Sci. 1995, 121, 1-30. (9) (a) Kobayashi, S.; Kashiwa, K.; Kawasaki, T.; Shoda, S. J. Am. Chem. Soc. 1991, 113, 3079-3804. (b) Kobayashi, S.; Hobson, L. J.; Sakamoto, J.; Kimura, S.; Sugiyama, J.; Imai, T.; Itoh, T. Biomacromolecules 2000, 1, 168-173. (10) (a) Kobayashi, S.; Kiyosada, T.; Shoda, S. J. Am. Chem. Soc. 1996, 118, 13113-13114. (b) Sakamoto, J.; Sugiyama, J.; Kimura, S.; Imai, T.; Ito, T.; Watanabe, T.; Kobayashi, S. Macromolecules 2000, 33, 4155-4160, 4982.

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(11) (a) Kobayashi, S.; Morii, H.; Itoh, R.; Kimura, S.; Ohmae, M. J. Am. Chem. Soc. 2001, 123, 11825-11826. (b) Ochiai, H.; Ohmae, M.; Mori, T.; Kobayashi, S. Biomacromolecules 2005, 6, 10681084. (12) Kobayashi, S.; Fujikawa, S.; Ohmae, M. J. Am. Chem. Soc. 2003, 125, 14357-14369.

Communications (13) Markovic-Housley, Z.; Miglierini, G.; Soldatova, L.; Rizkallah, P. J.; Mu¨ller, U.; Schirmer, T. Structure 2000, 8, 1025-1035. (14) Frost, G.-I.; Cso´ka, T.; Stern, R. Trends Glycosci. Glycotechnol. 1996, 8, 419-434.

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