Enzymatic Synthesis of Chondroitin and Its Derivatives Catalyzed by Hyaluronidase

DOI: 10.1021/ja036584x

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Journal of the American Chemical Society p. 14357 - 14369 (2003)

Update date:2022-08-04

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Authors:

Kobayashi, Shiro

Fujikawa, Shun-Ichi

Ohmae, Masashi

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Article abstract of DOI:10.1021/ja036584x

The enzymatic polymerization to provide synthetic chondroitin and its derivatives is reported here, the first example of such in vitro synthesis to date. N-Acetylchondrosine (GlcAβ(1→3)GalNAc) oxazoline (1a) and its derivatives (1b-1F) were designed and s

Full text of DOI:10.1021/ja036584x

Published on Web 11/04/2003

Enzymatic Synthesis of Chondroitin and Its Derivatives

Catalyzed by Hyaluronidase

Shiro Kobayashi,* Shun-ichi Fujikawa, and Masashi Ohmae

Contribution from the Department of Materials Chemistry, Graduate School of Engineering,

Kyoto UniVersity, Kyoto 615-8510, Japan

Received June 10, 2003; E-mail: kobayasi@mat.polym.kyoto-u.ac.jp

Abstract: The enzymatic polymerization to provide synthetic chondroitin and its derivatives is reported

here, the first example of such in vitro synthesis to date. N-Acetylchondrosine (GlcAâ(1f3)GalNAc) oxazoline

(1a) and its derivatives (1b-1f) were designed and synthesized as novel transition state analogue substrate

monomers for catalysis by hyaluronidase. Hyaluronidase is a hydrolysis enzyme of chondroitin that also

catalyzes the formation of repeated glycosidic bonds in in vitro synthesis, rather than in the catabolic direction.

Monomers of 2-methyl (1a), 2-ethyl (1b), and 2-vinyl (1f) oxazoline derivatives were polymerized using

this enzyme, via ring-opening polyaddition with total control of regioselectivity and stereochemistry. These

reactions provided the corresponding synthetic chondroitin (natural type; N-acetyl, 2a) and the derivatives

(unnatural type) with N-propionyl (2b) and N-acryloyl (2f) functional groups at the C2 position of all the

galactosamine units, in good yields. Monomers of 2-n-propyl (1c) and 2-isopropyl (1d) oxazoline derivatives

were polymerized to produce 2c and 2d in low yield. The 2-phenyl oxazoline derivative (1e) did not afford

any enzyme-catalyzed products. M_nvalues of 2a and 2b reached 4800 and 4000, respectively. The M_n

value of 2a corresponds to that of the naturally occurring chondroitin. Thus, hyaluronidase catalysis allows

the in vitro production of not only natural type but also the formation of unnatural type chondroitins.

Introduction

tide, chondroitin synthase, with the potential to synthesize Ch

has been cloned. This possesses the two glycosyl transferase

Chondroitin (Ch) and chondroitin sulfate (ChS) are naturally

occurring heteropolysaccharides belonging to the family of

glycosaminoglycans (GAGs), which also includes hyaluronan

(HA), heparin/heparan sulfate, and dermatan sulfate (DS). Ch

is a nonsulfated derivative of ChS, consisting of a â-D-

glucuronyl-(1f3)-N-acetyl-D-galactosamine (N-acetylchon-

drosine, GlcAâ(1f3)GalNAc) disaccharide repeating unit con-

nected through â(1f4) glycosidic linkages. ChS exists

predominantly as polysaccharide side chains in proteoglycans

(PGs) in extracellular matrixes (ECMs), where it plays an

important role in the bioactivities of living systems,¹such as

controlling morphogenesis by regulation of topology and

function.²Ch is widely used as a therapeutic material for the

prevention or alleviation of symptoms of diseases, such as

rheumatoid arthritis³and in food supplements.⁴

activities required for chain polymerization.⁶Some microbial

Ch synthases have been identified⁷and have been found to

produce Ch as capsular polysaccharides surrounding microbes,

which contain no sulfate groups or core proteins. In mammalian

cells, Ch is regioselectively sulfated at C4, C6, and/or C2′ of

the N-acetylchondrosine repeating unit, by the action of specific

sulfotransferases⁸during chain elongation of Ch backbone. This

process results in the structural diversity of ChS.⁹

A number of reports have been published which describe the

functions of ChS at a molecular level.¹⁰It has been found to

play a critical role in the brain matrix, promoting neurite cell

growth¹¹and neural cell migration.¹²Nematodes are animals

(5) (a) Sato, T.; Gotoh, M.; Kiyohara, K.; Akashima, T.; Iwasaki, H.;

Kameyama, A.; Mochizuki, H.; Yada, T.; Inaba, N.; Togayachi, A.; Kudo,

T.; Asada, M.; Watanabe, H.; Imamura, T.; Kimata, K.; Narimatsu, H. J.

Biol. Chem. 2003, 31, 3063-3071. (b) Uyama, T.; Kitagawa, H.; Tanaka,

J.; Tamura, J.; Ogawa, T.; Sugahara, K. J. Biol. Chem. 2003, 31, 3072-

3078. (c) Prydz, K.; Dalen, K. T. J. Cell Sci. 2000, 113, 193-205.

(6) Kitagawa, H.; Uyama, T.; Sugahara, K. J. Biol. Chem. 2001, 276, 38721-

38726.

Biosynthesis of Ch is catalyzed by glycosyl transferases with

UDP-GlcA and UDP-GlcNAc as glycosyl donors in the Golgi

apparatus.⁵Recently, human cDNA encoding a single polypep-

(7) (a) DeAngelis, P. L.; Padgett-McCue, A. J. J. Biol. Chem. 2000, 275,

24124-24129. (b) Ninomiya, T.; Sugiura, N.; Tawada, A.; Sugimoto, K.;

Watanabe, H.; Kimata, K. J. Biol. Chem. 2002, 277, 21567-21575.

(8) Habuchi, O. Biochim. Biophys. Acta 2000, 1474, 115-127.

(9) Sugahara, K.; Yamada, S. Trends Glycosci. Glycotechnol. 2000, 12, 321-

349.

(10) (a) Watanabe, H.; Kimata, K.; Line, S.; Strong, D.; Gao, L.; Christine, A.

K.; Yamada, Y. Nat. Genet. 1994, 7, 154-157. (b) Zhuo, L.; Yoneda, M.;

Zhao, M.; Yingsung, W.; Yoshida, N.; Kitagawa, Y.; Kawamura, K.;

Suzuki, T.; Kimata, K. J. Biol. Chem. 2001, 276, 7693-7696.

(11) (a) Faissner, A.; Clement, A. M.; Lochter, A.; Streit, A.; Mandl, C.;

Schachner, M. J. Cell Biol. 1994, 126, 783-799. (b) Clement, A. M.;

Nadanaka, S.; Masayama, K.; Mandl, C.; Sugahara, K.; Faissner, A. J.

Biol. Chem. 1998, 273, 28444-28453.

(1) (a) Iozzo, R. V. Annu. ReV. Biochem. 1998, 67, 609-652. (b) Sugahara,

K.; Yamada, S. Trends Glycosci. Glycotechnol. 2000, 12, 321-349.

(2) (a) Bullock, S. L.; Fletcher, J. M.; Beddington, R. S. P.; Wilson, V. A.

Genes DeV. 1998, 12, 1894-1906. (b) Perrimon, N.; Bernfield, M. Nature

2000, 404, 725-728. (c) Lin, X.; Wei, G.; Shi, Z.; Dryer, L.; Esko, J. D.;

Wells, D. E.; Matzuk, M. M. DeV. Biol. 2000, 224, 299-311. (d) Lander,

A. D.; Selleck, S. B. J. Cell Biol. 2000, 148, 227-232. (e) Chudo, H.;

Toyoda, H. Seikagaku 2001, 73, 449-457. (f) Habuchi, O. Cell Technol.

2001, 20, 204-210. (g) Maeda, N. Cell Technol. 2001, 20, 1074-1083.

(3) Johnson, K. A.; Hulse, D. A.; Hart, R. C.; Kochevar, D.; Chu, Q.

Osteoarthritis Cart. 2001, 9, 14-21.

(4) McAlindon, T. E.; LaValley, M. P.; Gulin, J. P.; Felson, D. T. JAMA 2000,

283, 1469-1475.

J. AM. CHEM. SOC. 2003, 125, 14357-14369

14357

A R T I C L E S

Kobayashi et al.

Scheme 1. Enzymatic Polymerization to Synthetic Ch and Its

which produce nonsulfated Ch;¹³however, their function has

not yet been identified.

Derivatives

Structurally defined polysaccharide samples are essential for

the investigation of the function of Ch and ChS at a molecular

level. Such an approach has been achieved over the last two

decades using synthetic chemistry.¹⁴The synthesis of ChS has

proved challenging, with hexasaccharides containing a 4- or 6-O-

sulfate group on GalNAc units being the longest derivatives

synthesized to date.¹⁵Therefore, a facile and efficient method

for the synthesis of Ch and ChS is urgently required.

Enzymatic polymerization catalyzed by a glycosyl hydrolase

has been demonstrated as an effective nonbiosynthetic approach

for constructing structurally well-defined oligo- and polysac-

charides.¹⁶This methodology has the advantage that the

polymeric products are generated by a single-step polymerization

reaction of designed activated substrate monomers in a regio-

and stereoselective manner. For example, not only natural

homopolysaccharides of cellulose,¹⁷xylan,¹⁸and chitin,¹⁹but

also unnatural heteropolysaccharides of alternatingly 6-O-

methyl-cellulose²⁰and a cellulose-xylan hybrid polysaccha-

ride²¹have been synthesized by this method. A mutant hydrolase

was also effective for synthesis of â(1f4)-oligo- and polysac-

charides.²²Furthermore, we have recently achieved enzymatic

polymerization to form synthetic HA which has a number

average molecular weight of (M_n) ∼2 × 10⁴catalyzed by

hyaluronidases (HAases; endo-â-N-acetylhexosaminidases²³). In

this case, N-acetylhyalobiuronate (GlcAâ(1f3)GlcNAc) di-

saccharide oxazoline derivative, an activated GlcNAc form of

the repeating unit in HA, was used as a transition state analogue

substrate monomer.^24,25In these reactions, the hydrolase enzyme

which catalyzes the glycosidic bond cleavage of the polysac-

charides by hydrolysis in living systems, catalyzed repeated

glycosidic bond formation by transglycosylation of the monomer

in vitro. Two of the above enzymatic polymerizations utilize

hydrolysis enzymes as catalysts, Chitinase (family 18) for chitin

synthesis and HAases (family 56) for HA synthesis. Both

reactions involve the glycosidic bond cleavage of the (1f4)-

â-N-acetyl-D-glucosaminide linkage.^19,24,26

Ch, ChS, and DS are known to be hydrolyzed at their (1f4)-

â-N-acetyl-D-galactosaminide linkage with HAase catalysis.²³

Here, we report a facile and efficient synthesis of synthetic Ch

(natural type) by enzymatic ring-opening polyaddition. The

reaction is catalyzed by the HAases with an N-acetylchondrosine

oxazoline derivative, a repeating disaccharide form of Ch with

an activated GalNAc, which has the potential to serve as a

transition state analogue substrate monomer for the enzyme.

The polymerization proceeded in a perfect regio-selective and

stereo-controlled fashion. This reaction has been extended to

the synthesis of Ch derivatives (unnatural type) with N-

propionyl, N-butyryl, N-isobutyryl, N-benzoyl, and N-acryloyl

groups in place of the N-acetyl group in natural Ch, by using

the corresponding 2-ethyl, 2-n-propyl, 2-isopropyl, 2-phenyl,

and 2-vinyl oxazoline derivatives as novel monomers for the

enzyme (Scheme 1). The present reactions provide the first

successful synthesis of natural type Ch and its unnatural type

derivatives by nonbiosynthetic pathways. These polymeric

products may serve as a novel class of substrates for use in

various scientific fields such as medicinal chemistry, organic

chemistry, biochemistry, enzymology, and polymer chemistry.

(12) (a) Maeda, N.; Nishiwaki, T.; Shintani, T.; Hamanaka, H.; Noda, M. J.

Biol. Chem. 1996, 271, 21446-21452. (b) Maeda, N.; Noda, M. J. Cell

Biol. 1998, 142, 203-216.

(13) (a) Bulik, D. A.; Wei, G.; Toyoda, H.; Kinoshita-Toyoda, A.; Waldrip, W.

R.; Esko, J. D.; Robbins, P. W.; Selleck, S. B. Proc. Natl. Acad. Sci. U.S.A.

2000, 97, 10838-10843. (b) Berninsone, P.; Hwang, H.-Y.; Zemtseva, I.;

Horvitz, H. R.; Hirschberg, C. B. Proc. Natl. Acad. Sci. U.S.A. 2001, 98,

3738-3743.

(14) For example, see the following reviews: (a) Tamura, J. Trends Glycosci.

Glycotechnol. 1994, 6, 29-50. (b) Tamura, J. Trends Glycosci. Glycote-

chnol. 2001, 13, 65-88.

(15) Karst, N.; Jaquinet, J.-C. Eur. J. Org. Chem. 2002, 815-825.

(16) (a) Kobayashi, S.; Shoda, S.; Uyama, H. AdV. Polym. Sci. 1995, 121, 1-30.

(b) Kobayashi, S.; Shoda, S.; Uyama, H. In Catalysis in Precision

Polymerization; Kobayashi, S., Ed.; John Wiley & Sons: Chichester, 1997;

Chapter 8. (c) Kobayashi, S. J. Polym. Sci., Polym. Chem. Ed. 1999, 37,

3041-3056. (d) Kobayashi, S.; Uyama, H.; Kimura, S. Chem. ReV. 2001,

101, 3793-3818. (e) Kobayashi, S.; Sakamoto, J.; Kimura, S. Prog. Polym.

Sci. 2001, 26, 1525-1560. (f) Kobayashi, S.; Uyama, H.; Ohmae, M. Bull.

Chem. Soc. Jpn. 2001, 74, 613-635.

Results and Discussion

(17) Kobayashi, S.; Kashiwa, K.; Kawasaki, T.; Shoda, S. J. Am. Chem. Soc.

1991, 113, 3079-3804.

(18) Kobayashi, S.; Wen, X.; Shoda, S. Macromolecules 1996, 29, 2698-2700.

(19) (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.

Monomer Design. It is generally accepted that an enzyme-

catalyzed reaction proceeds at an accelerated rate relative to

that of the uncatalyzed reaction by the reduction of the activation

energy. This is achieved by stabilization of the reaction transition

state, which occurs on formation of the enzyme-substrate

complex.²⁷Furthermore, all enzymatic reactions are reversible,

including complex formation.²⁸These observations imply that

the formation of a desired enzyme-substrate complex in a

(20) Okamoto, E.; Kiyosada, T.; Shoda, S.; Kobayashi, S. Cellulose 1997, 4,

161-172.

(21) Fujita, M.; Shoda, S.; Kobayashi, S. J. Am. Chem. Soc. 1998, 120, 6411-

6412.

(22) Fort, S.; Boyer, V.; Greffe, L.; Davis, G. J.; Moroz, O.; Christiansen, L.;

Schu¨lein, M.; Cottaz, S.; Driguez, H. J. Am Chem. Soc. 2000, 122, 5429-

5437.

(23) Frost, G. I.; Cso´ka, T.; Stern, R. Trends Glycosci. Glycotechnol. 1996, 8,

419-434.

(26) Williams, S. J.; Mark, B. L.; Vocadlo, D. J.; James, M. N. G.; Withers, S.

G. J. Biol. Chem. 2002, 277, 40055-40065.

(27) (a) Haldane, J. In Enzymes; Longmans, Green, Co.; MIT Press: New York,

1930; p 182. (b) Pauling, L. Chem. Eng. News 1946, 24, 1375. (c) Kollman,

P. A.; Kuhn, B.; Donini, O.; Perakyla, M.; Stanton, R.; Bakowies, D. Acc.

Chem. Res. 2001, 34, 72-79.

(24) Kobayashi, S.; Morii, H.; Itoh, R.; Kimura, S.; Ohmae, M. J. Am. Chem.

Soc. 2001, 123, 11825-11826.

(25) Markovic-Housley, Z.; Miglierini, G.; Soldatova, L.; Rizkallah, P. J.; Mu¨ller,

U.; Schirmer, T. Structure 2000, 8, 1025-1035.

14358 J. AM. CHEM. SOC. VOL. 125, NO. 47, 2003

Enzymatic Synthesis of Chondroitin

A R T I C L E S

Scheme 2. Syntheses of Substrate Monomers 1a-1f

Figure 1. Two possibilities of the monomer design for chondroitin

synthesis.

reaction pathway can be controlled whether proceeding in the

forward or reverse direction. Therefore, an appropriate combina-

tion of monomer and enzyme is crucial for controlling the

pathway, so that the enzyme-monomer complex may form,

leading irreversibly to the desired product. Hydrolase enzymes

have been used as catalysts for polymerization in the synthesis

of various polysaccharides, where glycosidic bond formation

is repeatedly induced by the hydrolysis enzyme.^16-21,24In these

reactions, the enzyme that inherently catalyzes the glycosidic

bond cleavage (the forward direction), also catalyzes in vitro

bond formation (the reverse direction). These results strongly

suggest that the enzyme and designed substrate monomer formed

the complex expected, which is considered very close in

structure for both hydrolysis and polymerization, and thus was

able to produce the target polysaccharide. To achieve such a

complex, the monomer substrate must be recognized by the

enzyme. Therefore, a monomer was designed with a structure

thought to be similar to that involved in the transition state of

the enzyme catalyzed hydrolysis. We named the monomers

designed as “transition state analogue substrate monomers”.^16-21,24

We have designed a monomer for Ch synthesis, based on

the concept of enzymatic polymerization with hydrolase ca-

talysis.There are two possibilities for monomer design (Figure

1); an oxazoline-type disaccharide monomer (A) designed for

HAase (endo-â-N-acetylhexosaminidase; EC3.2.1.35) cataly-

sis^23,24cleaving the (1f4)-â-N-acetyl-D-galactosaminide link-

age, and a fluoride-type disaccharide monomer (B) designed

for endo-â-glucuronidase catalysis cleaving the (1f3)-â-D-

glucuronide linkage.²⁹The oxazoline structure of monomer (A)

is analogous to that used in the syntheses of chitin¹⁹and

hyaluronan.²⁴These natural polysaccharides contain the N-acetyl

group at the C2 position of the D-glucosamine and D-galac-

tosamine units, respectively, and their hydrolysis by Chitinase

and hyaluronidase is considered to involve an oxazolinium

transition state. Combination of the former monomer (A) and

enzyme is feasible since HAases are commercially available;

(i) TMSOTf, MS4A/CH₂Cl₂, 91%, (ii) AcSH, 86%, (iii) 80% aqAcOH,

∆, (iv) Ac₂O/pyridine, 81% (2 steps), (v) Pd(OH)₂-C, H₂/MeOH, (vi) Ac₂O/

pyridine, quant. (2 steps), (vii) 80% aqAcOH, ∆, (viii) Ac₂O/pyridine, 87%

(2 steps), (ix) Pd(OH)₂-C, H₂/MeOH, (x) (R¹CO)₂O (b, d) or R¹COCl (c,

e, f), Et₃N/MeOH, (xi) Ac₂O/pyridine, (b) 38%, (c) 57%, (d) 41%, (e) 58%,

(f) 38% (3 steps), (xii) TMSOTf/CH₂Cl₂, (a) 89%, (b) 82%, (c) 64%, (d)

76%, (e) 64%, (f) 77%, (xiii) MeONa/MeOH, (xiv) carbonate buffer (50

mM, pH 10.6), (a) 82%, (b) 93%, (c) 86%, (d) 83%, (e) 87%, (f) 86% (2

steps).

however, the latter (B) is not possible due to the difficulty in

obtaining a supply of the enzyme. The specific endo-â-

glucuronidase for Ch degradation has been identified in the rat

liver only²⁹and is not commercially available. Therefore, in

the present study, we selected the combination of the A

monomer and the enzyme for the syntheses of natural type Ch

and also unnatural type Chs.

Synthesis of Oxazoline Monomers. All substrate monomers

(1a-1e) were successfully synthesized according to the reactions

outlined in Scheme 2. The key intermediate (5) was readily

prepared through a glycosylation reaction of 2-azidogalactoside

derivative (4) with trichloroacetimidate of peracetylated methyl

(28) Lehninger, A. L. In Biochemistry; Worth Publishing: New York, 1970;

chapter 8.

(29) (a) Takagaki, K.; Nakamura, T.; Majima, M.; Endo, M. FEBS Lett. 1985,

181, 271-274. (b) Takagaki, K.; Nakamura, T.; Majima, M.; Endo, M. J.

Biol. Chem. 1988, 263, 7000-7006.

J. AM. CHEM. SOC. VOL. 125, NO. 47, 2003 14359

A R T I C L E S

Kobayashi et al.

Figure 2. Reaction-time courses of 1a with H-OTH (2) and without the

enzyme (b).

glucuronate (3). For the synthesis of monomer 1a for natural

type Ch, the 2-azido group of 5 was converted to acetamido

group by treatment with thioacetic acid. The 4,6-O-benzylidene

group of 6 was removed by acetolysis followed by acetylation

with acetic anhydride in pyridine to give compound 7. The 1-O-

benzyl group was removed by hydrogenation followed by

acetylation to afford 9a.

Figure 3. (A) H and (B) ¹³C NMR spectra of synthetic Ch.

For the syntheses of monomers 1b-1e, for unnatural Chs,

compound 5 was also treated with aqueous acetic acid to remove

the 4,6-O-benzylidene group, followed by acetylation with acetic

anhydride in pyridine to provide 8. Both the 2-azido and 1-O-

benzyl groups of 8 were hydrogenated to amino and hydroxyl

groups, respectively. Compounds 9b-9f were prepared by

modification of the amino group to various amido groups

through the reaction with the corresponding acid anhydrides (9b,

9d) or acid chlorides (9c, 9e, 9f) in methanol prior to acetylation

of the anomeric hydroxyl group.

Formation of the oxazoline ring in 9a-9f was performed

using trimethylsilyl triflate. All O-acetyl groups of 10a-10f

were then removed by sodium methoxide in methanol followed

by hydrolysis of the methyl ester in carbonate buffer (pH 10.6)

which afforded the substrate monomers (1a-1f).

Table 1. ¹³C NMR Chemical Shift Values^aof Synthetic Ch and

Natural Ch

GalNAc

CdO

synthetic Ch^b100.38 50.55 79.91 67.23 74.52 60.61 174.17 22.04

natural Ch^c

100.36 50.50 79.82 67.24 74.49 60.61 174.14 22.02

GlcA

synthetic Ch

natural Ch

103.87

103.83

72.01

71.97

73.24

73.19

79.23

79.17

75.90

75.88

174.54

174.51

^aAll chemical shift values are indicated by ppm from the methyl carbon

of acetone as an internal standard (at 29.8 ppm in D₂O). ^bObtained in this

study. ^cPurchased from Seikagaku Co.

measured at 23 °C, became two characteristic doublet signals

at δ 4.37 and 4.30 with the same coupling constant of 7.53 Hz,

on measurement at 70 °C. The former signal was assigned to

the anomeric proton derived from GlcA, and the latter from

GalNAc, showing the existence of glycosidic linkages with the

â-orientation only. In B, the specific signals derived from the

anomeric carbon atoms of GlcA and GalNAc were observed at

δ 103.87 and 100.38, respectively. In addition, the signals at δ

79.91 and 79.23 were assigned to C3 of GalNAc and C4 of

GlcA by C-H correlation spectroscopy. These results indicate

the formation of a GlcAâ(1f3)GalNAcâ(1f4) repeating

structure, natural type (synthetic) Ch 2a. All of the chemical

shift values were closely similar to those of the natural Ch

sample (Table 1). The yield and molecular weight of synthetic

Ch were 50% and M_n2100, respectively (M_w2500; mainly

decasaccharide) as determined by SEC measurements.

Synthesis of Ch (natural type) via Enzymatic Polymeri-

zation of 1a. Substrate monomer 1a designed for natural type

Ch was polymerized with the ovine testicular HAase (H-

OTH: hydrolysis activity, 2160 units/mg) in phosphate buffer

(pH 7.5). Figure 2 illustrates the reaction-time courses of 1a

with H-OTH (2) and without the enzyme (b). Without the

enzyme, 1a was gradually consumed due to nonenzymatic

hydrolysis to produce a disaccharide, GlcAâ(1f3)GalNAc.³⁰

On addition of H-OTH, 1a was consumed more rapidly due

to the enzyme-catalyzed reaction as well as the nonenzymatic

hydrolysis. On reaction completion, the enzyme was thermally

inactivated and the resulting mixture purified by Sephadex G-10

size exclusion chromatography (SEC) to afford the polymeric

product.

Figure 3 shows the H (A) and ¹³C (B) NMR spectra of the

product. An overlapping broad signal around δ 4.33 in A

(corresponding to two anomeric protons of GlcA and GalNAc),

Enzymatic Polymerization of 1a under Various Reaction

Conditions. Polymerization of 1a was performed under varying

pH conditions, in phosphate buffer, for different reaction times

using H-OTH as the catalyst (entries 1-7, Table 2). The

(30) Structure was identified by HPLC and MALDI-TOF/MS measurements;

m/z 396.96 [M-H]^-.

14360 J. AM. CHEM. SOC. VOL. 125, NO. 47, 2003

Enzymatic Synthesis of Chondroitin

A R T I C L E S

Table 2. Enzymatic Polymerization of Monomer 1a under Various Reaction Conditions

polymerization^a

polymer (2a)

monomer

concentration/M

enzyme amount/

wt % for 1a

temp

/°C

time

yield

entry

enzyme^b

/ %

M ^d

H-OTH

OTH

H-BTH

BTH

bee venom

H-OTH

6.0

7.0

7.5

8.0

8.5

9.0

7.5

7.5^e

7.5^f

0.10

0.05

0.10

0.20

0.10

1600

1900

2100

4600

2200

4600

1700

2200

2500

6700

2700

6500

4100

4600

4800

4400

4300

2500

2600

2800

6000

6800

7100

7000

6600

6400

6300

6600

3200

3400

3600

∼1

1700

1800

2100

^aIn a phosphate buffer: 50 mM. ^bOTH: ovine testicular hyaluronidase (560 units/mg), BTH: bovine testicular hyaluronidase (330 units/mg), H-OTH:

ovine testicular hyaluronidase (2160 units/mg from ICN Biochemicals, Inc.), H-BTH: bovine testicular hyaluronidase (1010 units/mg from SIGMA).

^cDetermined by HPLC containing products with molecular weight higher than tetrasaccharides. ^dDetermined by SEC calibrated with hyaluronan standards.

^eIn a phosphate buffer: 500 mM. ^fIn a phosphate buffer (50 mM) containing sodium chloride (500 mM).

monomer consumption became slower when increasing the pH

from 6.0 to 9.0. In these reactions, monomer consumption was

complete within the respective reaction times except for entries

4 and 6. These observations are in agreement with the reported

phenomena that the optimal pH range for hydrolysis of natural

Ch is between 4 and 6;²³the hydrolysis activity of the enzyme

is notably higher in the regions with relatively low pH. Recent

reports have investigated the hydrolysis activities of hyalu-

ronidase enzymes in detail, in terms of pH dependency versus

enzyme preparation and their structure.³¹

The optimum polymer yield was obtained at pH 7.5,

indicating that at this pH the polymerization proceeded ef-

fectively prior to the formation of the hydrolysis product. The

M_nof synthetic Ch (2a) reached 4600 (22-24 saccharides) (the

M_nvalue of which is similar to that of naturally occurring Ch)³²

for shorter reaction times of 6 h at pH 8.0 and 8.5 (entries 4

and 6). At pH 6.0, the monomer was consumed completely via

enzymatic polymerization as well as nonenzymatic hydrolysis

within 1 h, giving rise to a 28% yield of synthetic Ch. This

indicates that the polymerization and hydrolysis of 1a was fast,

as was the hydrolysis of the product Ch. At pH 9.0 synthetic

Ch was not obtained, which was most likely to be due to a

complete suppression of the glycosylation reaction leading to

synthetic Ch.

temperature was also investigated for the same enzyme at 20

°C for 3 h, 30 °C for 2 h, and 40 °C for 1 h (entries 13, 9, and

14, respectively). It was found that temperature did not greatly

effect the yield and molecular weight for reaction times less

than 3 h. However, it is notable that synthetic Ch 2a with a

higher molecular weight was obtained with shorter reaction

times in the pH range of 7.5 to 8.5 (entries 4, 6, 8-14).

Catalytic activity was examined for other enzymes, OTH

(hydrolysis activity, 560 units/mg), bovine testicular HAase

(BTH: 330 units/mg, H-BTH: 1010 units/mg), and bee venom

containing HAase, at pH 7.5. OTH and BTH had a hydrolysis

activity lower than H-OTH or H-BTH, and although polym-

erization proceeded, synthetic Ch was produced in lower yields

(entries 3 vs 16 and 17 vs 18, respectively). Shorter reaction

times conferred a yield reduction of 2a but a higher molecular

weight was observed (entry 15). Bee venom showed a reduced

catalytic activity for the polymerization reaction (entry 19).

Increasing the buffer concentration or the addition of NaCl

to the solution resulted in a decrease in both the yield and

molecular weight (entries 20 and 21) The effect of organic

solvents such as methanol, acetonitrile, acetone, and tetrahy-

drofuran were investigated. Monomer 1a was polymerized in

phosphate buffer (pH 7.0)/methanol (2:1 v/v) using OTH at 30

°C and afforded the tetra- and hexasaccharides in low yield, as

determined by MALDI-TOF/MS. Similar results were obtained

with the three other organic solvents, suggesting that the use of

an organic cosolvent drastically reduces the catalytic activity.

Polymerization Mechanism of 1a. Figure 4 illustrates the

postulated reaction mechanism catalyzed by a hyaluronidase of

family 56. Hydrolysis of Ch occurs by the following steps as

shown in A and B.^24,25Protonation of the oxygen atom in the

â(1f4) glycosidic linkage occurs after the recognition of the

substrate Ch by the enzyme. Subsequently, the carbonyl oxygen

atom of GalNAc at the donor site attacks its own anomeric

carbon atom from the R-side to assist in cleavage of the

As H-OTH catalysis was optimal at pH 7.5, polymerization

reactions for the investigation of other reaction parameters were

performed at this pH. Monomer concentration did not signifi-

cantly affect the yield and molecular weight of polymer 2a with

relatively short reaction times of 2 or 3 h (entries 8-10).

Varying the amount of H-OTH enzyme (entries 11, 9, and 12)

did not confer a large effect, with reaction times of 2 h. Reaction

(31) (a) Oettl, M, Hoechstetter, J.; Asen, I.; Bernhardt, G.; Buschauer, A. Eur.

J. Pharm. Sci. 2003, 18, 267-277. (b) Frost, G. I.; Stern, R. Anal. Biochem.

1997, 251, 263-269.

(32) Poole, A. R. Biochem. J. 1986, 236, 1-14.

J. AM. CHEM. SOC. VOL. 125, NO. 47, 2003 14361

A R T I C L E S

Kobayashi et al.

Figure 5. Reaction-time courses of monomers 1b-1f with H-OTH (2)

and without the enzyme (b).

Enzymatic Polymerization to Ch Derivatives (unnatural

type) with Various Amido Groups. Newly designed monomers

1b-1f for the synthesis of Ch derivatives were subjected to

enzymatic polymerization. The reaction with H-OTH was

performed under the previously identified optimal conditions

of pH 7.5 and 30 °C. Figure 5 shows the reaction-time courses

for five substrate monomers. In all reactions performed in the

absence of the enzyme (control), the disaccharide was produced

via hydrolysis from the corresponding monomers 1b-1f.

Interestingly, monomers of 1b, 1c, 1d, and 1f for unnatural type

Ch were significantly catalyzed by the enzyme, as transition

state analogue substrates, resulting in ring-opening polyaddition

of the oxazoline monomers. Notably, the 2-ethyl oxazoline

monomer (1b) was consumed at an almost identical rate as the

2-methyl derivative (1a). However, no notable difference was

observed for compound 1e in monomer consumption in the

presence or absence of the enzyme. The catalysis of these

oxazoline derivatives at the donor site of the HAase is strongly

dependent on the nature of the 2-substituent in the oxazoline-

ring.³³

Figure 4. Postulated reaction mechanisms of HAase catalysis for the

hydrolysis of Ch (A and B) and for the polymerization of monomer 1a (C

and D).

glycosidic bond, which results in a high-energy oxazolinium

ion species in B. Water nucleophilically attacks the oxazolinium

anomeric carbon to open the oxazolinium ring resulting in the

formation of the hydrolysis products of natural Ch, GlcAâ-

(1f3)GalNAc and/or Ch chains with GlcA and GalNAc end

structures.

In the polymerization, monomer 1a is readily recognized by

the enzyme and is activated by protonation at the donor site in

C, because the protonated monomer structure in C is identical

to that of the oxazolinium transition state in B. Therefore, 1a

can be regarded as a transition state analogue substrate monomer

in an activated form. The structure of 1a facilitates recognition

and further activation by the enzyme, with lowering the

activation energy for the subsequent reactions. The 4-hydroxyl

group of GlcA in another molecule of 1a, or in the nonreducing

end of the growing chain placed in the acceptor site, regio-

selectively adds to the anomeric carbon of the oxazolinium ion

of 1a from the â-side with ring-opening to form a â(1f4)

glycosidic linkage between GalNAc and GlcA, as shown in D.

Repetition of this regio- and stereoselective glycosylation, ring-

opening polyaddition reaction of 1a is catalyzed by the enzyme,

giving rise to synthetic Ch; thus, the monomer formula of 1a is

the same as that of the product Ch.^19,24

Table 3 illustrates the results of the polymerization reactions

of substrates 1b-1f. Unnatural N-propionyl (2b) and N-acryloyl

(2f) derivatives of Ch were obtained from 1b and 1f in a 46%

yield with M_nof 2700 (mainly 12-14 saccharides) and in a

(33) From the time-conversion courses of these monomers in Figures 2 and 5,

reaction rate constants for the control experiments (without enzyme) could

be evaluated. Rate constants of the pseudo-first order (k₁at 30 °C in a

phosphate buffer at pH 7.5) were as follows; 1.26 × 10^-5s^-1(1a), 4.98 ×

10^-6s^-1(1b), 2.41 × 10^-6s^-1(1c), 3.26 × 10^-6s^-1(1d), 6.59 × 10^-7s^-1

(1e), and 2.86 × 10^-5s^-1(1f).

14362 J. AM. CHEM. SOC. VOL. 125, NO. 47, 2003

Enzymatic Synthesis of Chondroitin

A R T I C L E S

Table 3. Enzymatic Polymerization of Monomers 1b-1f

polymerization^a

polymer

yield^d

time

entry

monomer

enzyme

structure

/ %

M ^e

H-OTH^b

H-OTH^c

122

168

239

∼1

2700

3600

3400

4600

^aIn a phosphate buffer at pH 7.5: 50 mM, monomer concentration: 0.1

M, amount of enzyme:10 wt % for monomer, reaction temperature: 30

°C. ^bH-OTH (2502 unit/mg) from ICN Biochemicals, Inc. ^cH-OTH

(1870 unit/mg) from SIGMA. ^dDetermined by HPLC containing products

with molecular weight higher than tetrasaccharides. ^eDetermined by SEC

calibrated with hyaluronan standards.

19% yield with M_n3400 (mainly 16-18 saccharides) respec-

tively (entries 1 and 5 in Table 3). Ring-opening polyaddition

of 1b and 1f proceeded in a regio- and stereoselective fashion

to produce polymers 2b and 2f. Their structures were confirmed

by H and ¹³C NMR as having a â-D-glucuronyl-(1f3)-â-N-

propionyl-D-galactosaminyl-(1f4) repeating unit and a â-D-

glucuronyl-(1f3)-â-N-acryloyl-D-galactosaminyl-(1f4) repeat-

ing unit for 2b and 2f, respectively. Monomers 1c and 1d

provided oligomers of the N-butyryl (2c) and N-isobutyryl (2d)

derivatives of Ch up to decasaccharide and octasaccharide

respectively, as determined by MALDI-TOF/MS (entries 2 and

3). No polymeric or oligomeric products were formed through

the reaction of 1e with the enzyme (entry 4).

It should be emphasized that catalysis by the natural enzyme

allowed production of not only the natural type Ch but also

unnatural type Ch such as the N-propionyl (2b) and N-acryloyl

(2f) derivatives. The substrate oxazoline structure is important

for monomer design; 2-substituents in the oxazoline-ring can

be varied in the approximate order 2-methyl > 2-ethyl > 2-vinyl

. 2-n-propyl > 2-isopropyl. 2-Phenyl oxazoline monomer 1e

was not recognized by the enzyme.

Figure 6. Polymerization behavior of 1a (A) and 1b (B); monomer

consumption (b), polymer yields (9) and molecular weight (M_n) (2).

Conclusion

Synthetic Ch 2a (natural type) was successfully prepared for

the first time via enzymatic ring-opening polyaddition of

N-acetylchondrosine oxazoline derivative 1a, a novel transition

state analogue substrate monomer, using HAase as catalyst. The

polymerization reactions catalyzed by both BTH and OTH

proceeded smoothly at 30 °C in the pH range of 6.0 to 8.5 with

total control of regioselectivity and stereochemistry. M_nvalues

of 2a ranged from 1600 (octasaccharide) to 4800 (24 saccharide)

(M_w, 1700-7100). The higher M_nvalue of 2a corresponds to

that of naturally occurring Ch. Thus, synthetic Ch 2a provides

a natural type Ch sample with a well-defined structure for use

in the present study. Unnatural type Chs with N-propionyl (2b)

and N-acryloyl (2f) groups were also prepared by utilizing

natural HAase as a catalyst for polymerization of the novel

substrate monomers of 2-ethyl (1b) and 2-vinyl (1f) oxazoline

derivatives, respectively. These unnatural Chs cannot be pro-

duced through biosynthetic pathways and are therefore expected

to be potential materials for use in various scientific fields, such

as polymeric drugs, biomaterials, tissue engineering, and gly-

cotechnology. Furthermore, the fact that the vinyl oxazoline

monomer 1f is a good substrate for the HAase implies possible

applications for the preparation of functionalized Ch utilizing

the reactive vinyl group. This would permit syntheses of

structurally defined new macromonomers, telechelics and gels,

which have natural or unnatural Ch chains. Fundamental work

Polymerization Behaviors of Monomers 1a and 1b. Cor-

relation among consumption of monomers, yields of polymeric

products and M_nwas examined in more detail for 1a and 1b

(Figure 6, A and B). The yield of product 2a of natural type

Ch increased gradually with monomer consumption and then

reached a plateau at around 50% after about 10 h, as shown in

A. It is notable that the molecular weight value (M_n) of 2a at 1

h was 4700, larger than the value following reaction completion.

It had already been observed in Table 2 that shorter reaction

times produced 2a with higher M_nvalues. The product Ch is

an intact substrate for the catalyst HAase, therefore the M_nvalue

of 2a was gradually reduced to 2100 due to enzymatic

hydrolysis. In B, the yield of the product 2b gradually increased

with a decrease in monomer concentration and the M_nof 2b

after 1 h was 4000. Polymer 2b with a N-propionyl group in

the galactosamine unit is an unnatural substrate for the enzyme

HAase, however, the value of M_ngradually decreased to 2700.

The rate of decrease of M_nfor 2a, was less than that of 2b,

indicating that 2b was hydrolyzed more slowly by the enzyme.

The natural enzyme HAase served as a catalyst for both

polymerization of monomer 1b and hydrolysis of the unnatural

substrate 2b. These observations suggest that the M_nvalue can

be controlled to some extent by selecting reaction conditions.

J. AM. CHEM. SOC. VOL. 125, NO. 47, 2003 14363

A R T I C L E S

Kobayashi et al.

gel chromatography (toluene-ethyl acetate 1:0-1:3) followed by

crystallization from ethanol to give pure 6 (220 mg, 0.307 mmol, 86%)

as a white crystal. [R]_D-9.0° (c 1.0, CHCl₃); mp 230 °C (decomposi-

tion); ¹H NMR (400 MHz, CDCl₃, TMS) δ 7.57-7.52 (2H, m,

aromatic), 7.37-7.30 (8H, m, aromatic), 5.75 (1H, d, NH, J_NH,2) 7.03

Hz), 5.57 (1H, s, PhCH), 5.23-5.19 (3H, m, H-4′, H-3′, H-1), 5.01

(1H, t, H-2′, J_1′,2′) J_2′,3′) 8.29 Hz), 4.96-4.91 (2H, m, PhCH₂, H-1′),

4.79 (1H, dd, H-3, J_2,3) 11.5 Hz, J_3,4) 3.53 Hz), 4.56 (1H, d, PhCH₂,

J ) 12.10 Hz), 4.38-4.35 (2H, m, H-6a, H-4), 4.10 (1H, dd, H-6b,

J_5,6b) 1.51 Hz, J_6a,6b) 11.80 Hz), 4.01 (1H, d, H-5′, J_4′,5′) 9.54 Hz),

3.69 (3H, s, COOCH₃), 3.52-3.46 (2H, m, H-2, H-5), 2.02-2.01 (9H,

m, CH₃CO), 1.92 (3H, s, CH₃CONH); HRMS (FAB) calcd. for C₃₅H₄₂-

NO₁₅[M+H]⁺716.2554, found 716.2554.

Benzyl 2-acetamido-4,6-di-O-acetyl-2-deoxy-3-O-(methyl 2,3,4-

tri-O-acetyl-â-^D-glucopyranosyluronate)-â-D-galactopyranoside (7).

Compound 6 (960 mg, 1.34 mmol) in 80% aqueous acetic acid (15.0

mL) was stirred at 80 °C for 1 h to remove benzylidene acetal. The

reaction mixture was then evaporated to dryness, and the residue was

subjected to silica gel chromatography (chloroform-methanol 10:1) to

give benzyl 2-acetamido-2-deoxy-3-O-(methyl 2,3,4-tri-O-acetyl-â-^D-

glucopyranosyluronate)-â-^D-galactopyranoside (760 mg, 1.21 mmol,

90%). Acetic anhydride (470 µL, 4.84 mmol) was then added dropwise

to the solution of this compound (760 mg, 1.21 mmol) in pyridine (10.0

mL) at 0 °C. The mixture was stirred for 3 h at room temperature under

dry atmosphere, and then evaporated. The residue was diluted with

chloroform, washed with 4% (w/v) aqueous potassium hydrogen sulfate,

saturated aqueous hydrogen carbonate, and saturated aqueous sodium

chloride. The organic layer was dried over anhydrous magnesium

sulfate, filtered through diatomaceous earth, and evaporated. The residue

was purified by silica gel chromatography (n-hexane-ethyl acetate 1:2-

1:4) to afford pure 7 (700 mg, 0.984 mmol, 81%) as a white crystal

(from ether). [R]_D-20° (c 1.0, CHCl₃); mp 173-174 °C;¹H NMR (400

MHz, CDCl₃, TMS) δ 7.37-7.29 (5H, m, aromatic), 5.64 (1H, d, NH,

J_NH,2) 7.03 Hz), 5.39 (1H, d, H-4, J_3,4) 3.01 Hz), 5.22-5.14 (2H,

m, H-4′, H-3′), 5.02 (1H, d, H-1, J_1,2) 8.03 Hz), 4.97 (1H, t, H-2′,

J_1′,2′) J_2′,3′) 8.53 Hz), 4.89 (1H, d, PhCH₂, J ) 11.80 Hz), 4.70-

4.65 (2H, m, H-1′, H-3), 4.58 (1H, d, PhCH₂, J ) 11.80 Hz), 4.17

(1H, dd, H-6a, J_5,6a) 6.28 Hz, J_6a,6b) 11.29 Hz), 4.08 (1H, dd, H-6a,

J_5,6a) 6.28 Hz, J_6a,6b) 11.29 Hz), 3.98 (1H, d, H-5′, J_4′,5′) 9.54 Hz),

3.87 (1H, t, H-5, J_5,6a) J_5,6b) 6.28 Hz), 3.75 (3H, s, COOCH₃), 3.46

(1H, ddd, H-2, J_1,2) 8.03 Hz, J_NH,2) 7.03 Hz, J_2,3) 9.28 Hz), 2.09-

2.00 (15H, m, CH₃CO), 1.89 (3H, s, CH₃CONH); HRMS (FAB) calcd.

for C₃₂H₄₂NO₁₇712.2453 [M+H]⁺, found 712.2453.

toward these applications, including the preparation of sulfated

derivatives of ChS and DS is now in progress in our laboratory.

Experimental Section

Measurements. NMR spectra were recorded with a Bruker DPX-

400 spectrometer. FABMS spectra were obtained on a JEOL XPS

spectrometer using 2,4-dinitrobenzyl alcohol or dithiothreitol/thioglyc-

erol (1/1, v/v) as matrix. Optical rotations were measured with a JASCO

P-1010 polarimeter. Melting points were determined with a YAMATO

MP-21. Concentrations of oxazoline monomers in the reaction mixtures

were calculated by HPLC measurements (LC8020 system; TOSOH)

with Shodex Asahipak NH2P-50 4E column (4.6 × 250 mm) eluting

with phosphate buffer (10 mM, pH 7.0)-acetonitrile mixed solution (30:

70 (v/v), flow rate; 0.5 mL/min, 30 °C). 3 µL of the reaction mixture

was sampled, then injected to HPLC. Yields and molecular weight

values of the products given after the reactions were determined by

SEC measurements (GPC-8020 system; TOSOH) with Shodex Ohpak

SB-803HQ 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 (M_n800, 2000, 4000) and chondroitin sodium salt (M_n4000,

Seikagaku Co.). MALDI-TOF/MS analysis of the product was per-

formed with a JEOL JMS-ELITE spectrometer by using 2,5-dihy-

droxybenzoic acid as a matrix on Nafion-coated plate³⁴under negative

ion mode.

Materials. Hyaluronidases from ovine testes were purchased from

ICN Biochemicals Inc. (Lot No. 9303B, OTH, 560 units/mg; 6830B,

H-OTH, 2160 units/mg). Hyaluronidases from bovine testes were

purchased from SIGMA (Lot No. 30K7049, BTH, 330 units/mg;

38H7026, H-BTH, 1010 units/mg). Bee venom was purchased from

SIGMA (Lot No. 79H1017). All enzymes were used without further

purification.

Benzyl 2-azido-4,6-O-benzylidene-2-deoxy-3-O-(methyl 2,3,4-tri-

O-acetyl-â-^D-glucopyranosyluronate)-â-D-galactopyranoside (5). A

mixture of benzyl 2-azido-4,6-O-benzylidene-2-deoxy-â-^D-galactopy-

ranoside (4) (334 mg, 0.871 mmol) and methyl (2,3,4-tri-O-acetyl-R-

D-glucopyranosyl trichloroacetimidate)uronate (3) (500 mg, 1.05 mmol)

in anhydrous dichloromethane (9.0 mL) was stirred at -20 °C under

argon atmosphere in the presence of molecular sieves 4A (MS4A; 1.80

g). After 30 min, trimethylsilyl trifluoromethanesulfonate (TMSOTf;

190 µL, 1.05 mmol) in anhydrous dichloromethane (1.0 mL) was added

dropwise. After 1 h, the reaction mixture was quenched with triethyl-

amine (0.1 mL), filtered through diatomaceous earth (Celite), diluted

with chloroform, washed with saturated aqueous hydrogen carbonate

and saturated aqueous sodium chloride. The organic layer was dried

over anhydrous magnesium sulfate, then filtered through diatomaceous

earth, and concentrated. The residue was purified by silica gel

chromatography (toluene-ethyl acetate 4:1-2:1) followed by crystal-

lization from diethyl ether to give pure 5 (555 mg, 0.793 mmol, 91%)

as a white crystal. [R]_D-10° (c 1.0, CHCl₃); mp 232 °C (decomposi-

tion); ¹H NMR (400 MHz, CDCl₃, TMS) δ 7.54-7.52 (2H, m,

aromatic), 7.38-7.31 (8H, m, aromatic), 5.56 (1H, s, PhCH), 5.26-

2-Acetamido-4,6-di-O-acetyl-2-deoxy-3-O-(methyl 2,3,4-tri-O-

acetyl-â-^D-glucopyranosyluronate)-â-D-galactopyranosyl acetate (9a).

A mixture of 7 (700 mg, 0.984 mmol) and 20% palladium hydroxide

on activated carbon (200 mg) in methanol (20.0 mL) was stirred at

room temperature under hydrogen atmosphere for 3 h. The reaction

mixture was then filtered through diatomaceous earth and evaporated.

The residue was dried by vacuum pump for overnight. Then, to a

solution of this compound in pyridine (10.0 mL) was added acetic

anhydride (320 µL, 3.39 mmol) at 0 °C. The reaction mixture was stirred

at room temperature under dry atmosphere for 3 h, and then evaporated.

The residue was diluted with chloroform, washed with 4% (w/v)

aqueous potassium hydrogen sulfate, saturated aqueous hydrogen

carbonate and saturated aqueous sodium chloride. The organic layer

was dried over anhydrous magnesium sulfate. The solution was then

filtered through diatomaceous earth, and evaporated. The residue was

purified by silica gel chromatography (ethyl acetate) to give 9a (680

5.22 (2H, m, H-3′, H-4′), 5.06 (1H, dd, H-2′, J_1′,2′) 7.52 Hz, J_2′,3′

)

8.52 Hz), 4.99 (1H, d, PhCH₂, J ) 11.60 Hz), 4.92 (1H, d, H-1′, J_1′,2′

) 7.52 Hz), 4.69 (1H, d, PhCH₂, J ) 11.60 Hz), 4.37-4.29 (3H, m,

H-6a, H-1, H-4), 4.09-4.02 (2H, m, H-6b, H-5′), 3.88 (1H, dd, H-2,

J_1,2) 8.04 Hz, J_2,3) 10.5 Hz), 3.72 (3H, s, COOCH₃), 3.49 (1H, dd,

H-3, J_2,3) 10.50 Hz, J_3,4) 3.52 Hz), 3.37 (1H, s, H-5), 2.06-2.01

(9H, m, Ac); HRMS (FAB) calcd. for C₃₃H₃₈N₃O₁₄[M+H]⁺700.2354,

found 700.2357.

Benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-O-(methyl 2,3,4-

tri-O-acetyl-â-^D-glucopyranosyluronate)-â-D-galactopyranoside (6).

Compound 5 (250 mg, 0.358 mmol) in thioacetic acid (2.5 mL) was

stirred at room temperature under dry atmosphere for 24 h. The reaction

mixture was then evaporated, and the residue was purified by silica

mg, 1.03 mmol, quant., R / â ) 1/1) as white amorphous. H NMR

(400 MHz, CDCl₃, TMS) δ 6.34-6.32 (2H, m, H-1R, NHR), 5.89 (1H,

d, H-1â, J_1,2) 8.53 Hz), 5.78 (1H, d, NHâ, J_2,NH) 8.03 Hz), 5.49

(1H, d, H-4â, J_3,4) 3.01 Hz), 5.29-5.13 (7H, m, H-4R, H-3′Râ,

H-4′Râ, H-2′R), 5.02 (1H, dd, H-2′â, J_1′,2′) 8.03 Hz, J_2′,3′) 8.53

Hz), 4.86 (1H, d, H-1′R, J_1′,2′) 8.03 Hz), 4.77 (1H, d, H-1′â, J_1′,2′

8.03 Hz), 4.55 (1H, ddd, H-2R, J_1,2) 3.52 Hz, J_2,NH) 7.03 Hz, J_2,3

)

(34) Jacobs, A.; Dahlman, O. Anal. Chem. 2001, 73, 405-410.

14364 J. AM. CHEM. SOC. VOL. 125, NO. 47, 2003

Enzymatic Synthesis of Chondroitin

A R T I C L E S

8.53 Hz), 4.38 (1H, dd, H-3â, J_2,3) 10.79 Hz, J_3,4) 3.01 Hz), 4.24-

3.97 (11H, m, H-3R, H-6Râ, H-5′ Râ, H-2â, H-5Râ), 3.76 (6H, s,

COOCH₃), 2.19-2.02 (36H, m, CH₃CO), 1.93-1.90 (6H, d, CH₃-

CONH); HRMS (FAB) calcd. for C₂₇H₃₈NO₁₈[M+H]⁺664.2089, found

664.2092.

CH₃CO); HRMS (FAB) calcd. for C₃₀H₃₈N₃O₁₆[M+H]⁺696.2252,

found 696.2249.

4,6-Di-O-acetyl-2-deoxy-3-O-(methyl 2,3,4-tri-O-acetyl-â-^D-glu-

copyranosyluronate)-2-propanamide-â-^D-galactopyranosyl acetate

(9b). A mixture of 8 (100 mg, 0.144 mmol) and 20% palladium

hydroxide on activated carbon (50.0 mg) in methanol (10.0 mL) was

stirred at room temperature under hydrogen atmosphere for 4 h. The

reaction mixture was filtered through diatomaceous earth and evapo-

rated. To a solution of the residue in methanol (10.0 mL) was added

triethylamine (1.0 mL) and propionic anhydride (180 µL, 1.44 mmol)

at 0 °C under dry atmosphere. The reaction mixture was stirred at room

temperature for 2 h, quenched with pyridine (1.00 mL), and evaporated.

The residue was concentrated by vacuum pump for overnight. To a

solution of the residue in pyridine (10.0 mL) was added acetic anhydride

(84 µL, 0.863 mmol) at 0 °C. The reaction mixture was stirred at room

temperature under dry atmosphere for 3 h, and then evaporated. The

residue was diluted with chloroform, washed with 4% (w/v) aqueous

potassium hydrogen sulfate, saturated aqueous hydrogen carbonate and

saturated aqueous sodium chloride. The organic layer was dried over

anhydrous magnesium sulfate, filtered through diatomaceous earth, and

evaporated. The residue was purified by silica gel chromatography (n-

hexane-ethyl acetate 1:4) to give 9b (37.0 mg, 0.0546 mmol, 38%, R

2-Methyl-[4,6-di-O-acetyl-1,2-di-deoxy-3-O-(methyl 2,3,4-tri-O-

acetyl-â-^D-glucopyranosyluronate)-r-D-galactopyrano]-[2,1-d]-2-ox-

azoline (10a). To a solution of 9a (200 mg, 0.301 mmol) in anhydrous

dichloromethane (10.0 mL) was added dropwise TMSOTf (110 µL,

0.603 mmol) at 0 °C under argon atmosphere. The reaction mixture

was stirred at room temperature for 12 h, quenched with triethylamine

(200 µL) at 0 °C, and evaporated. The residue was purified by silica

gel chromatography (n-hexane-ethyl acetate 1:2-2:5, containing 0.5%

(v/v) triethylamine) to give pure 10a (161 mg, 0.268 mmol, 89%) as

white amorphous. [R]_D+34° (c 1.0, CHCl₃); ¹H NMR (400 MHz,

CDCl₃, TMS) δ 5.93 (1H, d, H-1, J_1,2) 6.53 Hz), 5.40 (1H, d, H-4,

J_3,4) 3.51 Hz), 5.30-5.20 (2H, m, H-3′, H-4′), 5.07 (1H, d, H-1′,

J_1′,2′) 8.03 Hz), 4.99 (1H, t, H-2′, J_1′,2′) J_2′,3′) 8.03 Hz), 4.18-4.12

(3H, m, H-6, H-5), 4.08 (1H, d, H-5′, J_4′,5′) 9.53 Hz), 3.93 (1H, dd,

H-3, J_2,3) 6.53 Hz, J_3,4) 3.51 Hz), 3.83 (1H, t, H-2, J_1,2) J_2,3

)

6.53 Hz), 3.76 (3H, s, COOCH₃), 2.08-2.02 (18H,, m, CH₃CO, CH₃C

of oxazoline); HRMS (FAB) calcd. for C₂₅H₃₄NO₁₆[M+H]⁺604.1877,

found 604.1879.

/ â ) 5/1) as white amorphous. H NMR (400 MHz, CDCl₃, TMS) δ

6.34 (1H, d, H-1R, J_1,2) 3.51 Hz), 6.16 (1H, d, NHR, J_1,2) 8.03 Hz),

5.90 (1H, d, H-1â, J_1,2) 8.52 Hz), 5.62 (1H, d, NHâ, J_2,NH) 8.52

Hz), 5.27 (1H, s, H-4R), 5.25-5.13 (3H, m, H-3′R, H-4′R, H-2′R),

4.85 (1H, d, H-1′R, J_1′,2′) 8.04 Hz), 4.57 (1H, ddd, H-2R, J_1,2) 3.51

Hz, J_2,NH) 8.03 Hz, J_2,3) 8.53 Hz), 4.23-4.19 (2H, m, H-3R, H-6aR),

4.11-4.07 (2H, m, H-5′R, H-6bR), 4.00 (1H, dd, H-5R, J_5,6a) 6.53

Hz, J_5,6b) 11.54 Hz), 3.76 (3H, s, COOCH₃), 2.19-2.01 (20H, m,

CH₃CO, CH₃CH₂CONH), 1.09 (3H, t, CH₃CH₂CONH, J ) 8.03 Hz);

HRMS (FAB) calcd. for C₂₈H₄₀NO₁₈[M+H]⁺678.2245, found

678.2248.

2-Methyl-[1,2-di-deoxy-3-O-(sodium â-^D-glucopyranosyluronate)-

r-^D-galactopyrano]-[2,1-d]-2-oxazoline (1a). To a solution of 10a (240

mg, 0.398 mmol) in methanol (1.0 mL) was added about 28% (w/v)

sodium methoxide in methanol (420 mg) at 0 °C under argon

atmosphere. The reaction mixture was stirred at room temperature under

argon atmosphere for 30 min, and evaporated. The residue was dissolved

with carbonate buffer (50 mM, pH 10.6, 4.0 mL) and stirred at room

temperature for 1 h. The reaction mixture was then lyophilized to give

1a (160 mg, purity 82%). H NMR (400 MHz, D₂O, acetone) δ 5.92

(1H, d, H-1, J_1,2) 7.03 Hz), 4.47 (1H, d, H-1′, J_1′,2′) 8.03 Hz), 4.00

(1H, d, H-4, J_3,4) 3.52 Hz), 3.80-3.73 (2H, m, H-2, H-5), 3.64-3.52

(4H, m, H-3, H-6, H-5′), 3.34-3.32 (2H, m, H-4′, H-3′), 3.21 (1H, dd,

H-2′, J_1′,2′) 8.03 Hz, J_2′,3′) 9.03 Hz), 1.84 (3H, s, CH₃C of oxazoline);

HRMS (FAB) calcd. for C₁₄H₂₀NNaO₁₁[M+H]⁺402.1012, found

402.1018.

2-Ethyl-[4,6-di-O-acetyl-1,2-di-deoxy-3-O-(methyl 2,3,4-tri-O-

acetyl-â-^D-glucopyranosyluronate)-r-D-galactopyrano]-[2,1-d]-2-ox-

azoline (10b). To a solution of 9b (100 mg, 0.148 mmol) in anhydrous

dichloromethane (5.0 mL) was added TMSOTf (40 µL, 0.220 mmol)

at 0 °C under argon atmosphere. The reaction mixture was stirred at

room temperature for 18 h, quenched with triethylamine (200 µL) at 0

°C, and evaporated. The residue was purified by silica gel chromatog-

raphy (n-hexane-ethyl acetate 1:2-2:5, containing 0.5% (v/v) triethyl-

amine) to give pure 10b (75.0 mg, 0.121 mmol, 82%) as white

Benzyl 4,6-di-O-acetyl-2-azido-2-deoxy-3-O-(methyl 2,3,4-tri-O-

acetyl-â-^D-glucopyranosyluronate)-â-D-galactopyranoside (8). A

solution of 5 (555 mg, 0.793 mmol) in 80% aqueous acetic acid (15.0

mL) was stirred at 80 °C for 1 h. The reaction mixture was then

evaporated, and the residue was purified by silica gel chromatography

(n-hexane-ethyl acetate 1:2-1:4) to give benzyl 2-azido-2-deoxy-3-

O-(methyl 2,3,4-tri-O-acetyl-â-^D-glucopyranosyluronate)-â-D-galacto-

pyranoside (453 mg, 0.741 mmol, 93%). Then, to a solution of this

compound (453 mg, 0.741 mmol) in dry pyridine (10.0 mL) was added

dropwise acetic anhydride (430 µL, 4.46 mmol) at 0 °C. The reaction

mixture was stirred at room temperature under dry atmosphere for 4 h

followed by evaporation. The residue was diluted with chloroform,

washed with 4% (w/v) aqueous potassium hydrogen sulfate, saturated

aqueous hydrogen carbonate and saturated aqueous sodium chloride.

The organic layer was dried over anhydrous magnesium sulfate, filtered

through diatomaceous earth, and evaporated. The residue was purified

by silica gel chromatography (n-hexane-ethyl acetate 1:1) followed

by crystallization from diethyl ether to give pure 8 (478 mg, 0.687

mmol, 93%) as a white crystal. [R]_D+0.12° (c 1.0, CHCl₃); mp 144-

145 °C;¹H NMR (400 MHz, CDCl₃, TMS) δ 7.39-7.31 (5H, m,

aromatic), 5.33 (1H, d, H-4, J_3,4) 3.01 Hz), 5.23-5.21 (2H, m, H-4′,

H-3′), 4.99-4.93 (2H, m, H-2′, PhCH₂), 4.80 (1H, d, H-1′, J_1′,2′) 8.03

Hz), 4.70 (1H, d, PhCH₂, J ) 12.05 Hz), 4.29 (1H, d, H-1, J_1,2) 8.03

Hz), 4.17 (1H, dd, H-6a, J_5,6a) 5.52 Hz, J_6a,6b) 11.54 Hz), 4.08 (1H,

amorphous. [R]_D+20° (c 0.75, CHCl₃); H NMR (400 MHz, CDCl₃,

TMS) δ 5.93 (1H, d, H-1, J_1,2) 7.02 Hz), 5.38 (1H, d, H-4, J_3,4

)

3.51 Hz, J_4,5) 2.00 Hz), 5.31-5.20 (2H, m, H-3′, H-4′), 5.09 (1H, d,

H-1′, J_1′,2′) 7.52 Hz), 5.00 (1H, dd, H-2′, J_1′,2′) 7.52 Hz, J_2′,3′) 8.53

Hz), 4.16-4.11 (3H, m, H-6, H-5), 4.05 (1H, d, H-5′, J_4′,5′) 9.53

Hz), 3.94 (1H, dd, H-3, J_2,3) 7.02 Hz, J_3,4) 3.51 Hz), 3.83 (1H, t,

H-2, J_1,2) J_2,3) 7.02 Hz), 3.76 (3H, s, COOCH₃), 2.32 (2H, q,

CH₃CH₂of oxazoline, J ) 7.53 Hz), 2.08-2.02 (15H, m, CH₃CO),

1.18 (3H, t, CH₃CH₂of oxazoline, J ) 7.53 Hz); HRMS (FAB) calcd.

for C₂₆H₃₆NO₁₆[M+H]⁺618.2034, found 618.2035.

2-Ethyl-[1,2-di-deoxy-3-O-(sodium â-^D-glucopyranosyluronate)-

r-^D-galactopyrano]-[2,1-d]-2-oxazoline (1b). To a solution of 10b

(75.0 mg, 0.121 mmol) in methanol (1.21 mL) added about 28% (w/v)

sodium methoxide in methanol (2.3 mg) at 0 °C under argon

atmosphere. The reaction mixture was stirred at 0 °C for 30 min then

at room temperature for 30 min followed by evaporation. The residue

was dissolved with carbonate buffer (50 mM, pH 10.6, 1.2 mL) and

stirred for 1 h at room temperature. The mixture was freeze-dried to

give 1b (56.0 mg, purity 93%). H NMR (400 MHz, D₂O, acetone) δ

5.91 (1H, d, H-1, J_1,2) 7.53 Hz), 4.47 (1H, d, H-1′, J_1′,2′) 8.03 Hz),

4.00 (1H, s, H-4), 3.78-3.73 (2H, m, H-2, H-5), 3.63-3.52 (4H, m,

H-3, H-6, H-5′), 3.34-3.32 (2H, m, H-4′, H-3′), 3.22 (1H, t, H-2′,

J_1′,2′) J_2′,3′) 8.03 Hz), 2.16 (2H, q, CH₃CH₂of oxazoline, J ) 7.53

dd, H-6a, J_5,6a) 7.02 Hz, J_6a,6b) 11.54 Hz), 3.98 (1H, d, H-5′, J_4′,5′

)

10.04 Hz), 3.75 (3H, s, COOCH₃), 3.73-3.64 (2H, m, H-5, H-2), 3.53

(1H, dd, H-3, J_2,3) 10.54 Hz, J_3,4) 3.01 Hz), 2.11-2.01 (15H, m,

J. AM. CHEM. SOC. VOL. 125, NO. 47, 2003 14365

A R T I C L E S

Kobayashi et al.

Hz), 0.96 (3H, t, CH₃CH₂of oxazoline, J ) 7.53 Hz); HRMS (FAB)

calcd. for C₁₅H₂₃NO₁₁Na [M+H]⁺416.1169, found 416.1161.

CH₃CH₂CH₂CONH, J ) 7.53 Hz); HRMS (FAB) calcd for C₁₆H₂₄-

NNa₂O₁₁[M+Na]⁺452.1145, found 452.1141.

4,6-Di-O-acetyl-2-butanamide-2-deoxy-3-O-(methyl 2,3,4-tri-O-

acetyl-â-^D-glucopyranosyluronate)-â-D-galactopyranosyl acetate (9c).

A mixture of 8 (200 mg, 0.288 mmol) and 20% palladium hydroxide

on activated carbon (100 mg) in methanol (20.0 mL) was stirred at

room temperature under hydrogen atmosphere for 3.5 h. The reaction

mixture was filtered through diatomaceous earth and evaporated. To a

solution of the residue in methanol (20.0 mL) was added triethylamine

(1.0 mL) and butyryl chloride (60 µL, 0.575 mmol) at 0 °C under dry

atmosphere. The reaction mixture was stirred at 0 °C for 2 h, quenched

with pyridine (1.0 mL), and evaporated. The residue was dried by

vacuum pump for overnight. Then, to a solution of this residue in

pyridine (20.0 mL) was added acetic anhydride (168 µL, 1.73 mmol)

at 0 °C. The mixture was stirred at room temperature under dry

atmosphere for 12 h, and then evaporated. The residue was diluted

with chloroform, washed with 4% (w/v) aqueous potassium hydrogen

sulfate, saturated aqueous hydrogen carbonate and saturated aqueous

sodium chloride. The organic layer was dried over anhydrous magne-

sium sulfate, then filtered through diatomaceous earth, and evaporated.

The residue was purified by silica gel chromatography (n-hexane-

ethyl acetate 1:1-1:3) to give pure 9c (113 mg, 0.163 mmol, 57%, R

/ â ) 50/9) as white amorphous. ¹H NMR (400 MHz, CDCl₃, TMS) δ

6.34 (1H, d, H-1R, J_1,2) 3.52 Hz), 6.23 (1H, d, NHR, J_1,2) 7.53 Hz),

5.89 (1H, d, H-1â, J_1,2) 8.53 Hz), 5.27-5.13 (4H, m, H-4R, H-3′R,

H-4′R, H-2′R), 4.85 (1H, d, H-1′R, J_1′,2′) 8.53 Hz), 4.56 (1H, dt, H-2R,

J_1,2) 3.52 Hz, J_2,NH) J_2,3) 7.53 Hz), 4.25-4.11 (2H, m, H-3R,

H-6aR), 4.09-4.06 (2H, m, H-5′R, H-6bR), 4.00 (1H, dd, H-5a, J_5,6a

) 6.53 Hz, J_5,6b) 11.04 Hz), 3.76 (3H, s, COOCH₃), 2.19-2.01 (20H,

m, CH₃CO, CH₃CH₂CH₂CONH), 1.62-1.59 (2H, m, CH₃CH₂CH₂-

CONH), 0.883 (3H, t, CH₃CH₂CH₂CONH, J ) 7.03 Hz); HRMS (FAB)

calcd for C₂₉H₄₂NO₁₈[M+H]⁺692.2402, found 692.2402.

4,6-Di-O-acetyl-2-deoxy-3-O-(methyl 2,3,4-tri-O-acetyl-â-^D-glu-

copyranosyluronate)-2-(2-methylpropanamido)-â-^D-galactopyrano-

syl acetate (9d). A mixture of 8 (315 mg, 0.453 mmol) and 20%

palladium hydroxide on activated carbon (150 mg) in methanol (30.0

mL) was stirred at room temperature under hydrogen atmosphere for

3.5 h. The reaction mixture was filtered through diatomaceous earth

and evaporated. To a solution of the residue in methanol (30.0 mL)

was added triethylamine (1.0 mL) and isobutyric anhydride (240 µL,

1.44 mmol) at 0 °C under dry atmosphere. The mixture was stirred at

room temperature for 2 h, then quenched with pyridine (3.0 mL), and

evaporated. The residue was dried by vacuum pump for overnight. Then,

to a solution of the residue in pyridine (30.0 mL) was added acetic

anhydride (265 µL, 2.72 mmol) at 0 °C. The mixture was stirred at

room temperature under dry atmosphere for 6 h, then evaporated. The

residue was diluted with chloroform, washed with 4% (w/v) aqueous

potassium hydrogen sulfate, saturated aqueous hydrogen carbonate and

saturated aqueous sodium chloride. The organic layer was dried over

anhydrous magnesium sulfate, then filtered through diatomaceous earth

and evaporated. The residue was purified by silica gel chromatography

(n-hexane-ethyl acetate 1:2-1:3) to give pure 9d (129 mg, 0.187

mmol, 41%, R / â ) 20/3) as white amorphous. H NMR (400 MHz,

CDCl₃, TMS) δ 6.35 (1H, d, H-1R, J_1,2) 3.51 Hz), 6.10 (1H, d, NHR,

J_1,2) 7.53 Hz), 5.90 (1H, d, H-1â, J_1,2) 8.54 Hz), 5.27 (1H, d, H-4R,

J_3,4) 2.00 Hz), 5.26-5.13 (3H, m, H-3′R, H-4′R, H-2′R), 4.84 (1H,

d, H-1′R, J_1′,2′) 8.53 Hz), 4.55 (1H, ddd, H-2R, J_1,2) 3.51 Hz, J_2,NH

) 7.53 Hz, J_2,3) 8.53 Hz), 4.25-4.20 (2H, m, H-3R, H-6aR), 4.11-

4.07 (2H, m, H-5′R, H-6bR), 4.00 (1H, dd, H-5a, J_5,6a) 6.52 Hz, J_5,6b

) 11.04 Hz), 3.76 (3H, s, COOCH₃), 2.27 (1H, m, (CH₃)₂CHCONH),

2.19-1.95 (18H, m, CH₃CO), 1.10-1.04 (6H, m, (CH₃)₂CHCONH);

HRMS (FAB) calcd. for C₂₉H₄₂NO₁₈[M+H]⁺692.2402, found

692.2402.

2-Propyl-[4,6-di-O-acetyl-1,2-di-deoxy-3-O-(methyl 2,3,4-tri-O-

acetyl-â-^D-glucopyranosyluronate)-r-D-galactopyrano]-[2,1-d]-2-ox-

azoline (10c). To a solution of 9c (113 mg, 0.163 mmol) in anhydrous

dichloromethane (5.0 mL) was added TMSOTf (45 µL, 0.245 mmol)

at 0 °C under argon atmosphere. The reaction mixture was stirred at

room temperature for 6 h, then quenched with triethylamine (0.5 mL)

at 0 °C, and evaporated. The residue was purified by silica gel

chromatography (n-hexane-ethyl acetate 1:1, containing 0.5% (v/v)

triethylamine) to give pure 10c (66.0 mg, 0.105 mmol, 64%) as white

2-Isopropyl-[4,6-di-O-acetyl-1,2-di-deoxy-3-O-(methyl 2,3,4-tri-

O-acetyl-â-^D-glucopyranosyluronate)-r-D-galactopyrano]-[2,1-d]-2-

oxazoline (10d). To a solution of 9d (118 mg, 0.171 mmol) in

anhydrous dichloromethane (6.0 mL) was added TMSOTf (47 µL, 0.256

mmol) at 0 °C under argon atmosphere. The reaction mixture was stirred

at room temperature for 15 h, then quenched with triethylamine (100

µL) at 0 °C followed by concentration in vacuo. The residue was

purified by silica gel chromatography (n-hexane-ethyl acetate 1:1,

containing 0.5% (v/v) triethylamine) to give pure 10d (82.0 mg, 0.130

mmol, 76%) as white amorphous. [R]_D+17° (c 0.82, CHCl₃); ¹H NMR

(400 MHz, CDCl₃, TMS) δ 5.91 (1H, d, H-1, J_1,2) 7.03 Hz), 5.36

(1H, t, H-4, J_3,4) J_4,5) 2.51 Hz), 5.31-5.19 (2H, m, H-3′, H-4′),

amorphous. [R]_D+25° (c 0.66, CHCl₃); H NMR (400 MHz, CDCl₃,

TMS) δ 5.93 (1H, d, H-1, J_1,2) 7.03 Hz), 5.38 (1H, t, H-4, J_3,4) J_4,5

) 3.51 Hz), 5.29 (1H, t, H-3′, J_2′,3′) J_3′,4′) 9.03 Hz), 5.22 (1H, m,

H-4′, J_3′,4′) J_4′,5′) 9.03 Hz), 5.10 (1H, d, H-1′, J_1′,2′) 8.03 Hz), 5.00

(1H, dd, H-2′, J_1′,2′) 8.03 Hz, J_2′,3′) 9.03 Hz), 4.13 (3H, m, H-6,

5.09 (1H, d, H-1′, J_1′,2′) 8.53 Hz), 5.00 (1H, t, H-2′, J_1′,2′) J_2′,3′

8.53 Hz), 4.15-4.09 (3H, m, H-6, H-5), 4.03 (1H, d, H-5′, J_4′,5′

)

H-5), 4.05 (1H, d, H-5′, J_4′,5′) 9.54 Hz), 3.93 (1H, dd, H-3, J_2,3

)

7.03 Hz, J_3,4) 3.51 Hz), 3.82 (1H, t, H-2, J_1,2) J_2,3) 7.03 Hz), 3.75

(3H, s, COOCH₃), 2.28 (2H, t, CH₃CH₂CH₂CONH, J ) 7.03 Hz),

2.08-2.02 (15H, m, CH₃CO), 1.70-1.62 (2H, m, CH₃CH₂CH₂CONH),

0.973 (3H, t, CH₃CH₂CH₂CONH, J ) 7.53 Hz); HRMS (FAB) calcd

for C₂₇H₃₈NO₁₆[M+H]⁺632.2190, found 632.2191

9.54 Hz), 3.94 (1H, dd, H-3, J_2,3) 7.03 Hz, J_3,4) 2.51 Hz), 3.83 (1H,

t, H-2, J_1,2) J_2,3) 7.03 Hz), 3.75 (3H, s, COOCH₃), 2.58 (1H, m,

(CH₃)₂CHCONH), 2.08-2.02 (15H, m, CH₃CO), 1.20-1.18 (6H, m,

(CH₃)₂CHCONH). HRMS (FAB) calcd. for C₂₇H₃₈NO₁₆[M+H]⁺

632.2190, found 632.2186.

2-Propyl-[1,2-di-deoxy-3-O-(sodium â-^D-glucopyranosyluronate)-

r-^D-galactopyrano]-[2,1-d]-2-oxazoline (1c). To a solution of 10c (66.0

mg, 0.105 mmol) in methanol (1.1 mL) was added about 28% (w/v)

sodium methoxide in methanol (2.0 mg) at 0 °C under argon

atmosphere. The reaction mixture was stirred at 0 °C for 20 min then

at room temperature for 10 min followed by concentration in vacuo.

The residue was dissolved with carbonate buffer (50 mM, pH 10.6,

1.1 mL) and stirred at room temperature for 1 h, then freeze-dried to

2-Isopropyl-[1,2-di-deoxy-3-O-(sodium â-^D-glucopyranosyluronate)-

r-^D-galactopyrano]-[2,1-d]-2-oxazoline (1d). To a solution of 10d

(82.0 mg, 0.130 mmol) in methanol (1.3 mL) added about 28% (w/v)

sodium methoxide in methanol (2.5 mg) at 0 °C under argon

atmosphere. The reaction mixture was stirred at 0 °C for 1 h then at

room temperature for 1 h. To the mixture was added about 28% (w/v)

sodium methoxide in methanol (2.5 mg) and stirred for 1 h followed

by concentration in vacuo. The residue was dissolved with carbonate

buffer (50 mM, pH 10.6, 1.3 mL) at room temperature and stirred for

give 1c (46.0 mg, purity 86%). H NMR (400 MHz, D₂O, acetone) δ

5.98 (1H, d, H-1, J_1,2) 7.03 Hz), 4.52 (1H, d, H-1′, J_1′,2′) 7.53 Hz),

4.06 (1H, d, H-4, J_3,4) 2.51 Hz), 3.84-3.79 (2H, m, H-2, H-5), 3.71-

3.57 (4H, m, H-3, H-6, H-5′), 3.40-3.38 (2H, m, H-4′, H-3′), 3.28

(1H, t, H-2′, J_1′,2′) J_2′,3′) 7.53 Hz), 2.21 (2H, t, CH₃CH₂CH₂CONH,

J ) 7.53 Hz), 1.56-1.47 (2H, m, CH₃CH₂CH₂CONH), 0.813 (3H, t,

1 h. The reaction mixture was lyophilized to give 1d (65.0 mg, purity

83%). H NMR (400 MHz, D₂O, acetone) δ 5.91 (1H, d, H-1, J_1,2

7.03 Hz), 4.46 (1H, d, H-1′, J_1′,2′) 8.03 Hz), 4.01 (1H, d, H-4, J_3,4

)

2.01 Hz), 3.77-3.73 (2H, m, H-2, H-5), 3.65-3.52 (4H, m, H-3, H-6,

H-5′), 3.36-3.30 (2H, m, H-4′, H-3′), 3.23 (1H, t, H-2′, J_1′,2′) J_2′,3′

)

14366 J. AM. CHEM. SOC. VOL. 125, NO. 47, 2003

Enzymatic Synthesis of Chondroitin

A R T I C L E S

8.03 Hz), 2.45 (1H, m, (CH₃)₂CHCONH), 1.01-0.97 (6H, m, (CH₃)₂-

CHCONH); HRMS (FAB) calcd. for C₁₆H₂₅NO₁₁Na [M+H]⁺430.1325,

found 430.1323.

(3H, m, H-6, H-5′), 3.45-3.32 (2H, m, H-4′, H-3′), 3.27 (1H, t, H-2′,

J_1′,2′) J_2′,3′) 7.53 Hz); HRMS (FAB) calcd. for C₁₉H₂₃NO₁₁Na

[M+H]⁺464.1169, found 464.1173.

4,6-Di-O-acetyl-2-benzamido-2-deoxy-3-O-(methyl 2,3,4-tri-O-

acetyl-â-^D-glucopyranosyluronate)-D-galactopyranosyl acetate (9e).

A mixture of 8 (300 mg, 0.431 mmol) and 20% palladium hydroxide

on activated carbon (150 mg) in methanol (30.0 mL) was stirred at

room temperature under hydrogen atmosphere for 3.5 h. The reaction

mixture was then filtered through diatomaceous earth and evaporated.

To a solution of the residue in methanol (30.0 mL) was added

triethylamine (1.0 mL) and benzoyl chloride (100 µL, 0.863 mmol) at

0 °C under dry atmosphere. The mixture was stirred at 0 °C for 1 h,

quenched with pyridine (3.0 mL), and evaporated. The residue was

dried by vacuum pump for overnight. Then, to a solution of the residue

in pyridine (30.0 mL) was added acetic anhydride (252 µL, 2.59 mmol)

at 0 °C. The reaction mixture was kept stirring at room temperature

under dry atmosphere for 3 h, then evaporated. The residue was diluted

with chloroform, washed with 4% (w/v) aqueous potassium hydrogen

sulfate, saturated aqueous hydrogen carbonate and saturated aqueous

sodium chloride. The organic layer was dried over anhydrous magne-

sium sulfate, then filtered through diatomaceous earth, and evaporated.

The residue was purified by silica gel chromatography (n-hexane-

ethyl acetate 1:1-1:2) to give pure 9e (180 mg, 0.248 mmol, 58%, R

4,6-Di-O-acetyl-2-acrylamido-2-deoxy-3-O-(methyl 2,3,4-tri-O-

acetyl-â-^D-glucopyranosyluronate)-â-D-galactopyranosyl acetate (9f).

A mixture of 8 (502 mg, 0.722 mmol) and 20% palladium hydroxide

on activated carbon (250 mg) in methanol (50.0 mL) was stirred at

room temperature under hydrogen atmosphere for 3.5 h. The reaction

mixture was filtered through diatomaceous earth and evaporated. To a

solution of the residue in methanol (50.0 mL) was added triethylamine

(1.0 mL) and acryloyl chloride (292 µL, 3.61 mmol) at 0 °C under dry

atmosphere. The mixture was kept stirring at 0 °C for 1 h, then added

acryloyl chloride (292 µL, 3.61 mmol) additionally. After 1 h, the

reaction mixture was quenched with pyridine (2.0 mL) and concentrated

in vacuo. The residue was dried under diminished pressure for

overnight. Then, to a solution of the residue in pyridine (20.0 mL)

was added acetic anhydride (422 µL, 4.33 mmol) at 0 °C. The reaction

mixture was stirred at room temperature under dry atmosphere for 4 h,

and then evaporated. The residue was diluted with chloroform, washed

with 4% (w/v) aqueous potassium hydrogen sulfate, saturated aqueous

hydrogen carbonate and saturated aqueous sodium chloride. The organic

layer was dried over anhydrous magnesium sulfate, then filtered through

diatomaceous earth, and concentrated in vacuo. The residue was purified

by silica gel chromatography (n-hexane-ethyl acetate 1:1-1:3) to give

pure 9f (183 mg, 0.271 mmol, 38%, R / â ) 4/1) as white amorphous.

¹H NMR (400 MHz, CDCl₃, TMS) δ 6.42 (1H, d, NHR, J_NH,2) 7.52

Hz), 6.38 (1H, d, H-1R, J_1,2) 3.52 Hz), 6.32 (1H, dd, CH_AH_BdCH_C,

/ â ) 5/2) as white amorphous. H NMR (400 MHz, CDCl₃, TMS) δ

7.72-7.37 (5H, m, aromatic), 6.55 (1H, d, NHR, J_1,2) 7.53 Hz), 6.44

(1H, d, H-1R, J_1,2) 3.01 Hz), 6.16 (1H, d, H-1â, J_1,2) 9.04 Hz), 5.37

(1H, d, H-4R, J_3,4) 2.00 Hz), 5.23-5.10 (3H, m, H-3′R, H-4′R, H-2′R),

4.87 (1H, d, H-1′R, J_1′,2′) 8.03 Hz), 4.83 (1H, ddd, H-2R, J_1,2) 3.01

Hz, J_2,NH) 7.53 Hz, J_2,3) 8.54 Hz), 4.37 (1H, dd, H-3R, J_2,3) 8.54

J_A,B) 1.51 Hz, J_A,C) 17.06 Hz), 6.07 (1H, dd, CH_AH_BdCH_C, J_B,C

)

10.04 Hz, J_A,C) 17.06 Hz), 5.96 (1H, d, H-1â, J_1,2) 9.04 Hz), 5.61

(1H, dd, CH_AH_BdCH_C, J_A,B) 1.51 Hz, J_B,C) 10.04 Hz), 5.27-5.14

(4H, m, H-4R, H-3′R, H-4′R, H-2′R), 4.84 (1H, d, H-1′R, J_1′,2′) 8.06

Hz), 4.64 (1H, ddd, H-2R, J_1,2) 3.52 Hz, J_2,NH) 7.52 Hz, J_2,3) 7.54

Hz), 4.28-4.21 (2H, m, H-3R, H-6aR), 4.12-4.08 (2H, m, H-5′R,

H-6bR), 4.01 (1H, dd, H-5a, J_5,6a) 6.53 Hz, J_5,6b) 9.04 Hz), 3.78

(3H, s, COOCH₃), 2.18-2.00 (18H, m, CH₃CO); HRMS (FAB) calcd

for C₂₈H₃₈NO₁₈[M+H]⁺676.2089, found 676.2090.

2-Vinyl-[4,6-di-O-acetyl-1,2-di-deoxy-3-O-(methyl 2,3,4-tri-O-

acetyl-â-^D-glucopyranosyluronate)-r-D-galactopyrano]-[2,1-d]-2-ox-

azoline (10f). To a solution of 9f (100 mg, 0.148 mmol) in anhydrous

dichloromethane (5.0 mL) was added TMSOTf (41 µL, 0.222 mmol)

at 0 °C under argon atmosphere. The reaction mixture was stirred at

room temperature for 3 h, then quenched with triethylamine (0.5 mL)

at 0 °C, and evaporated. The residue was purified by silica gel

chromatography (n-hexane-ethyl acetate 2:1-1:1, triethylamine 0.5%)

to give pure 10f (70.0 mg, 0.114 mmol, 77%) as white amorphous.

[R]_D+42° (c 0.70, CHCl₃); ¹H NMR (400 MHz, CDCl₃, TMS) δ 6.24

(1H, dd, CH_AH_BdCH_C, J_B,C) 10.04 Hz, J_A,C) 17.57 Hz), 6.16 (1H,

dd, CH_AH_BdCH_C, J_A,B) 2.01 Hz, J_A,C) 17.57 Hz), 6.01 (1H, d, H-1,

Hz, J_3,4) 2.00 Hz), 4.28 (1H, dd, H-6aR, J_5,6a) 5.52 Hz, J_6a,6b

)

11.04 Hz), 4.14 (1H, dd,, H-6bR, J_5,6b) 7.03 Hz, J_6a,6b) 11.04 Hz),

4.06-4.00 (2H, dd, H-5′R, H-5R), 3.62 (3H, s, COOCH₃), 2.20-1.91

(18H, m, CH₃CO); HRMS (FAB) calcd. for C₃₂H₄₀NO₁₈[M+H]⁺

726.2245, found 726.2248.

2-Phenyl-[4,6-di-O-acetyl-1,2-di-deoxy-3-O-(methyl 2,3,4-tri-O-

acetyl-â-^D-glucopyranosyluronate)-r-D-galactopyrano]-[2,1-d]-2-ox-

azoline (10e). To a solution of 9e (180 mg, 0.248 mmol) in anhydrous

dichloromethane (9.0 mL) was added TMSOTf (68 µL, 0.372 mmol)

at 0 °C under argon atmosphere. The reaction mixture was stirred at

room temperature for 12 h, then quenched with triethylamine (200 µL)

at 0 °C, and evaporated. The residue was purified by silica gel

chromatography (toluene-ethyl acetate 3:1-2:1, containing 0.5% (v/

v) triethylamine) to give pure 10e (105 mg, 0.158 mmol, 64%) as white

amorphous. [R]_D+30° (c 1.0, CHCl₃); H NMR (400 MHz, CDCl₃,

TMS) δ 7.96-7.43 (5H, m, aromatic), 6.15 (1H, d, H-1, J_1,2) 6.02

Hz), 5.42 (1H, s, H-4), 5.33 (1H, t, H-3′, J_2′,3′) J_3′,4′) 8.29 Hz),

5.27-5.19 (2H, m, H-4′, H-1′), 5.04 (1H, t, H-2′, J_1′,2′) J_2′,3′) 8.29

Hz), 4.17 (3H, m, H-6, H-5), 4.11-4.02 (3H, m, H-5′, H-2, H-3), 3.72

(3H, s, COOCH₃), 2.10-2.04 (15H, m, CH₃CO); HRMS (FAB) calcd.

for C₃₀H₃₆NO₁₆[M+H]⁺666.2034, found 666.2043.

2-Phenyl-[1,2-di-deoxy-3-O-(sodium â-^D-glucopyranosyluronate)-

r-^D-galactopyrano]-[2,1-d]-2-oxazoline (1e). To a solution of 10e (105

mg, 0.158 mmol) in methanol (1.6 mL) was added about 28% (w/v)

sodium methoxide in methanol (3.0 mg) at 0 °C under argon

atmosphere. The reaction mixture was stirred at 0 °C for 0.5 h, then at

room temperature for 1 h. To the mixture was added additional sodium

methoxide in methanol (3.0 mg) at room temperature, and stirred for

30 min followed by evaporation. The residue was dissolved with

carbonate buffer (50 mM, pH 10.6, 1.6 mL) at room temperature, then

stirred for 1 h followed by lyophilization to give 1e (60.0 mg, purity

87%). ¹H NMR (400 MHz, D₂O, acetone) δ 7.77-7.34 (5H, m,

J_1,2) 6.52 Hz), 5.79 (1H, dd, CH_AH_BdCH_C, J_A,B) 2.01 Hz, J_B,C

)

10.04 Hz), 5.41 (1H, s, H-4), 5.29 (1H, t, H-3′, J_2′,3′) J_3′,4′) 9.03

Hz), 5.22 (1H, t, H-4′, J_3′,4′) J_4′,5′) 9.03 Hz), 5.09 (1H, d, H-1′, J_1′,2′

) 7.53 Hz), 5.00 (1H, dd, H-2′, J_1′,2′) 7.53 Hz, J_2′,3′) 9.03 Hz), 4.15

(3H, m, H-6, H-5), 4.07 (1H, d, H-5′, J_4′,5′) 9.03 Hz), 3.95-3.94

(2H, m, H-3, H-2), 3.76 (3H, s, COOCH₃), 2.09-2.02 (15H, m, CH₃-

CO); HRMS (FAB) calcd for C₂₆H₃₃NO₁₆[M+H]⁺616.1877, found

616.1893.

2-Vinyl-[1,2-di-deoxy-3-O-(sodium â-^D-glucopyranosyluronate)-

r-^D-galactopyrano]-[2,1-d]-2-oxazoline (1f). To a solution of 10f (70.0

mg, 0.114 mmol) in methanol (1.2 mL) was added about 28% (w/v)

sodium methoxide in methanol (2.2 mg) at 0 °C under argon

atmosphere. The reaction mixture was stirred at 0 °C for 0.5 h, then at

room temperature for 0.5 h. To the mixture was added again sodium

methoxide in methanol (2.2 mg). After 0.5 h, the reaction mixture was

concentrated in vacuo. The residue was dissolved with carbonate buffer

(50 mM, pH 10.6, 1.2 mL) at room temperature and kept stirring for

aromatic), 6.13 (1H, d, H-1, J_1,2) 7.03 Hz), 4.53 (1H, d, H-1′, J_1′,2′

7.53 Hz), 4.06 (1H, d, H-4, J_3,4) 2.51 Hz), 4.00 (1H, dd, H-2, J_1,2

7.03 Hz, J_2,3) 7.53 Hz,), 3.83 (1H, dd, H-5, J_5,6a) 7.53 Hz, J_5,6b

)

11.54 Hz), 3.74 (1H, dd, H-3, J_2,3) 7.53, J_3,4) 7.53 Hz), 3.68-3.52

J. AM. CHEM. SOC. VOL. 125, NO. 47, 2003 14367

A R T I C L E S

Kobayashi et al.

1 h followed by lyophilization to afford 1f (72 mg, purity 86%). H

NMR (400 MHz, D₂O, acetone) δ 6.14-6.02 (3H, m, CH_AH_BdCH_C,

CH_AH_BdCH_C, H-1), 5.78 (1H, dd, CH_AH_BdCH_C, J_A,B) 1.51 Hz, J_B,C

) 10.04 Hz), 4.54 (1H, d, H-1′, J_1′,2′) 7.53 Hz), 4.08 (1H, d, H-4, J_3,4

) 2.00 Hz), 3.95 (1H, t, H-2, J_1,2) 7.03 Hz), 3.84 (1H, dd, H-5, J_5,6a

) 7.03 Hz, J_5,6b) 10.54 Hz), 3.73-3.58 (4H, m, H-3, H-6, H-5′),

3.40-3.38 (2H, m, H-4′, H-3′), 3.28 (1H, t, H-2′, J_1′,2′) J_2′,3′) 7.53

Hz); HRMS (FAB) calcd for C₁₅H₂₁NNaO₁₁[M+H]⁺414.1012, found

414.1004.

distilled water. The resulting solution was applied to MALDI-TOF/

MS analysis, and the spectrum showed peaks of oligomers of 2c.

MALDI-TOF/MS; m/z 830.78 [M-H]^-(n ) 2, tetrasaccharide),

1238.04 [M-H]^-(n ) 3, hexasaccharide), 1645.52 [M-H]^-(n ) 4,

octasaccharide), 2052.56 [M-H]^-(n ) 5, decasaccharide). In the

control experiment (without the enzyme), the only product was the

disaccharide derived from hydrolysis of 1c, 2-butanamido-2-deoxy-3-

O-(sodium â-^D-glucopyranosyluronate)-D-galactopyranose: MALDI-

TOF/MS; m/z 424.21 [M-H]^-.

A solution of 1d (5.0 mg, 11.6 µmol) in a phosphate buffer (50

mM, pH 7.5, 116 µL) was incubated with H-OTH (0.5 mg) at 30 °C.

After 168 h, the resulting suspension was heated at 90 °C for 3 min

and poured into excess amount of tetrahydrofuran to inactivate the

enzyme. The precipitate was collected by centrifugation, dissolved in

water, and analyzed by SEC measurement, but no polymeric products

were detected by SEC measurement. The reaction mixture was purified

through Sephadex G-10 column eluting with 0.1 M aqueous sodium

chloride, then desalted through Sephadex G-10 column eluting with

distilled water. The resulting solution was applied to MALDI-TOF/

MS analysis, and the spectrum showed peaks of oligomers of 2d.

MALDI-TOF/MS; m/z 830.90 [M-H]^-(n ) 2, tetrasaccharide),

1237.17 [M-H]^-(n ) 3, hexasaccharide), 1644.54 [M-H]^-(n ) 4,

octasaccharide). In the control experiment (without the enzyme), the

only product was the disaccharide derived from hydrolysis of 1d,

2-deoxy-2-(2-methylpropanamido)-3-O-(sodium â-^D-glucopyranosylu-

ronate)-^D-galactopyranose: MALDI-TOF/MS; m/z 424.87 [M-H]^-.

A solution of 1e (5.0 mg, 10.8 µmol) in a phosphate buffer (50 mM,

pH 7.5, 108 µL) was incubated with H-OTH (0.5 mg) at 30 °C. After

239 h, the resulting mixture was heated at 90 °C for 3 min and poured

into excess amount of tetrahydrofuran to inactivate the enzyme. The

precipitate was collected by centrifugation, dissolved in water, and

analyzed by SEC measurement and MALDI-TOF/MS, but neither

polymeric products nor oligomers were detected. In the control

experiment (without the enzyme), the only product was the disaccharide

derived from hydrolysis of 1e, 2-benzamido-2-deoxy-3-O-(sodium â-^D-

glucopyranosyluronate)-^D-galactopyranose: MALDI-TOF/MS; m/z 458.55

[M-H]^-.

A solution of 1f (25.0 mg, 60.5 µmol), in a phosphate buffer (50

mM, pH 7.5, 605 µL) was incubated with H-OTH (2.5 mg) at 30 °C.

After 24 h, the resulting suspension was heated at 90 °C for 3 min and

poured into excess amount of tetrahydrofuran to inactivate enzyme.

The precipitate was collected by centrifugation, dissolved in water, and

analyzed by SEC measurement (yield 19%, M_n3400, M_w4600) and

MALDI-TOF/MS. The reaction mixture was purified through Sephadex

G-10 column eluting with 0.1 M aqueous sodium chloride. Fractions

containing the products were collected and subjected to SEC column

(Shodex ohpak SB-803HQ, 0.1M sodium nitrate), then desalted through

Sephadex G-10 column eluting with distilled water to give 2f (3.0 mg,

12%). ¹H NMR (400 MHz, D₂O, acetone) δ 6.11-5.98 (2H, m,

CH_AH_BdCH_C, CH_AH_BdCH_C), 5.59 (1H, d, CH_AH_BdCH_C, J_B,C) 8.03

Hz), 4.34 (1H, d, H-1, J_1,2) 7.03 Hz), 4.25 (1H, d, H-1′, J_1′,2′) 8.53

Hz), 3.93-3.89 (2H, m, H-4, H-2), 3.64-3.44 (6H, m, H-3, H-4′, H-5′,

H-6, H-5), 3.33 (1H, t, H-3′, J_2′,3′) 9.03 Hz), 3.14 (1H, t, H-2′, J_1′,2′

) 8.53 Hz). ¹³C NMR (100 MHz, D₂O, acetone) δ 174.25 (C-6′), 168.92

(CH₃CH₂CO), 129.78 (CH₂dCH), 126.74 (CH₂dCH), 103.84 (C-1′),

100.05 (C-1), 80.11 (C-3), 78.62 (C-4′), 75.75 (C-5′), 74.56 (C-5), 73.09

(C-3′), 72.02 (C-2′), 67.30 (C-4), 60.67 (C-6), 50.60 (C-2). In the control

experiment (without the enzyme), the only product was the disaccharide

derived from hydrolysis of 1f, 2-acrylamido-2-deoxy-3-O-(sodium â-^D-

glucopyranosyluronate)-^D-galactopyranose: MALDI-TOF/MS; m/z 408.06

[M-H]^-.

Enzymatic Polymerization of 1a. A typical polymerization proce-

dure of 1a is given as follow: 1a (15.0 mg, 37.5 µmol) in a phosphate

buffer (50 mM, pH 7.5, 375 µL) was incubated with H-OTH (1.5

mg) at 30 °C. After 23 h, the mixture was heated at 90 °C for 3 min

to inactivate the enzyme and poured into excess amount of tetrahy-

drofuran. The precipitate was collected by centrifugation. A small part

of the precipitate was dissolved in water for SEC measurements (yield

50%, M_n2100, M_w2500) and MALDI TOF/MS. The residual

precipitate was dissolved in water and subjected to Sephadex G-10

size exclusion chromatography eluting with 0.1 M aqueous sodium

chloride to remove hydrolyzed disaccharide fraction, then desalted

through Sephadex G-10 column eluting with distilled water. The

fractions containing polysaccharides were combined and lyophilized

to afford 2a as a white solid (6.6 mg, 44%). ¹H NMR (400 MHz, D₂O,

acetone) δ 4.30 (2H, bs, H-1, H-1′), 3.93 (1H, bs, H-4), 3.80 (1H, bt,

H-2), 3.61-3.50 (6H, m, H-3, H-4, H-5′, H-6, H-5), 3.38 (1H, bt, H-3′),

3.16 (1H, bt, H-2′), 1.83 (1H, s, CH₃CO). Measurement at 70 °C; 4.37

(1H, d, H-1, J_1,2) 7.53 Hz), 4.30 (1H, d, H-1′, J_1′,2′) 7.53 Hz); ¹³C

NMR (100 MHz, D₂O, acetone) δ 174.54 (C-6′), 174.17 (CH₃CO),

103.87 (C-1′), 100.38 (C-1), 79.91 (C-3), 79.23 (C-4′), 75.90 (C-5′),

74.52 (C-5), 73.24 (C-3′), 72.01 (C-2′), 67.23 (C-4), 60.61 (C-6), 50.55

(C-2), 22.04 (CH₃CO).

Enzymatic polymerizations of 1b-1f. A solution of 1b (9.0 mg,

21.6 µmol) in a phosphate buffer (50 mM, pH 7.5, 216 µL) was

incubated with H-OTH (0.9 mg) at 30 °C. After 35 h, the resulting

suspension was heated at 90 °C for 3 min and poured into excess

amount of tetrahydrofuran to inactivate enzyme. The precipitate was

collected by centrifugation, dissolved in water, and analyzed by SEC

measurement (yield 46%, M_n2700, M_w3600) and MALDI-TOF/MS.

The reaction mixture was purified by Sephadex G-10 size exclusion

chromatography eluting with 0.1 M aqueous sodium chloride, then

desalted through Sephadex G-10 column eluting with distilled water

to give 2b as a white solid (3.0 mg, 33%). H NMR (400 MHz, D₂O,

acetone) δ 4.33 (1H, d, H-1, J_1,2) 8.53 Hz), 4.28 (1H, d, H-1′, J_1′,2′

)

8.03 Hz), 3.92 (1H, s, H-4), 3.82 (1H, t, H-2, J_1,2) 8.53 Hz), 3.63-

3.46 (6H, m, H-3, H-4′, H-5′, H-6, H-5), 3.37 (1H, t, H-3′, J_2′,3′) 8.03

Hz), 3.16 (1H, t, H-2′, J_1′,2′) 8.03 Hz), 2.10 (2H, q, CH₃CH₂CO, J )

7.53 Hz), 0.91 (3H, t, CH₃CH₂CO, J ) 7.53 Hz). ¹³C NMR (100 MHz,

D₂O, acetone) δ 178.34 (C-6′), 174.15 (CH₃CH₂CO), 103.82 (C-1′),

100.21 (C-1), 79.66 (C-3), 78.86 (C-4′), 75.97 (C-5′), 74.49 (C-5), 73.18

(C-3′), 72.06 (C-2′), 67.40 (C-4), 60.65 (C-6), 50.40 (C-2), 28.97

(CH₃CH₂CO), 8.76 (CH₃CH₂CO). In the control experiment (without

the enzyme), the only product was the disaccharide derived from

hydrolysis of 1b, 2-deoxy-2-propanamido-3-O-(sodium â-^D-glucopy-

ranosyluronate)-^D-galactopyranose: MALDI-TOF/MS; m/z 410.62

[M-H]^-.

A solution of 1c (5.0 mg, 11.6 µmol) in a phosphate buffer (50 mM,

pH 7.5, 116 µL) was incubated with H-OTH (0.5 mg) at 30 °C. After

122 h, the reaction mixture was heated at 90 °C for 3 min and poured

into excess amount of tetrahydrofuran to inactivate the enzyme. The

resulting precipitate was collected by centrifugation, dissolved in water,

and its small part was analyzed by SEC measurement, but no polymeric

products were detected. The residual solution was subjected to Sephadex

G-10 column chromatography eluting with 0.1 M aqueous sodium

chloride, then desalted through Sephadex G-10 column eluting with

Enzymatic Polymerization of 1a in an Organic Cosolvent. A

typical polymerization procedure of 1a in methanol as an organic

cosolvent is given as follow: 1a (5.0 mg, 12.5 µmol) in a phosphate

buffer (50 mM, pH 7.0, 83 µL) including methanol (42 µL) was

14368 J. AM. CHEM. SOC. VOL. 125, NO. 47, 2003

Enzymatic Synthesis of Chondroitin

A R T I C L E S

incubated with OTH (0.5 mg) at 30 °C. After 26 h, a small amount of

the mixture was subjected to SEC measurement, but no polymeric

products were detected. The reaction mixture was then subjected to

Acknowledgment. We thank Drs. T. Miyoshi and T. Morika-

wa of Denka Co. (Tokyo, Japan) for supplying hyaluronan

samples for calibration standards. This work was partially

supported by the Mitsubishi Foundation and by the 21st COE

program for a United Approach to New Materials Science in

Kyoto University.

Dowex 50W-X8 (NH₄form) to remove sodium ion. The resulting

solution was applied to MALDI-TOF/MS analysis, and the spectrum

showed peaks of oligomers of 2a. MALDI-TOF/MS; m/z 775.35

[M-H]^-(n ) 2, tetrasaccharide), 1154.21 [M-H]^-(n ) 3, hexa-

saccharide).

JA036584X

J. AM. CHEM. SOC. VOL. 125, NO. 47, 2003 14369

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