Bottom-Up Synthesis of Hyaluronan and Its Derivatives via Enzymatic

Biomacromolecules 2005, 6, 1068-1084

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Bottom-Up Synthesis of Hyaluronan and Its Derivatives via Enzymatic Polymerization: Direct Incorporation of an Amido Functional Group Hirofumi Ochiai, Masashi Ohmae, Tomonori Mori,† and Shiro Kobayashi* Department of Materials Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan Received November 13, 2004; Revised Manuscript Received December 29, 2004

This paper reports the synthesis of hyaluronan (HA) and its derivatives via the hyaluronidase-catalyzed polymerization of 2-substituted oxazoline derivative monomers designed as “transition-state analogue substrates”. Polymerization of 2-methyl oxazoline monomer from N-acetylhyalobiuronate (GlcAβ(1f3)GlcNAc) effectively proceeded at pH 7.5 and 30 °C, giving rise to synthetic HA (natural type) in an optimal yield of 78% via ring-opening polyaddition under total control of regioselectivity and stereochemistry. Hyaluronidase catalysis enabled the polymerization of 2-ethyl, 2-n-propyl, and 2-vinyl monomers, affording the corresponding HA derivatives (unnatural type) with N-propionyl, N-butyryl, and N-acryloyl functional groups, respectively, at the C2 position of all glucosamine units in good yields. The 2-isopropyl oxazoline derivative provided the N-isobutyryl derivative of HA in low yields. Monomers of 2-phenyl and 2-isopropenyl oxazoline derivatives were not polymerized. The mechanism of the polymerization is discussed. Introduction In 1934, the first report of hyaluronan (hyaluronic acid; HA) by Meyer and Palmer described HA to be a high molecular weight biopolysaccharide in the vitreous humor of cattle eyes.1 The chemical structure of HA was determined as having a repeating unit of β-D-glucuronyl-(1f3)-N-acetylD-glucosamine (GlcAβ(1f3)GlcNAc, N-acetylhyalobiuronate), which is linked in a β(1f4) fashion.2 The molecular weight value of naturally occurring HA often reaches one million dalton. Biosynthesis of HA is performed by the catalysis of HA synthase existing in plasma membrane with uridine 5′-diphospho-GlcNAc (UDP-GlcNAc) and UDP-GlcA as substrates.3 HA belongs to the group of glycosaminoglycans (GAGs) including chondroitin (Ch), Ch sulfate, dermatan sulfate, heparin/heparan sulfate, and keratan sulfate, all of which are biomacromolecular hetero-polysaccharides.4 HA exists in extracellular matrixes (ECMs) as a scaffold for other ECM molecules such as collagens, fibronectins, and proteoglycans having GAG side chains.5 Interactions of HA with ECM molecules construct a strong network of viscoelastic ECMs. HA exhibits a number of crucial bioactivities, e.g., maintaining cartilage elasticity,6 regulation of cell differentiation, proliferation, and migration leading to tissue morphogenesis and remodeling,7,8 and matrix formation around cumulus cells during ovulation and fertilization.9 Recently, intracellular HA was found in many kinds of * To whom correspondence should be addressed. E-mail: kobayasi@ mat.polym.kyoto-u.ac.jp. Tel: +81-75-383-2459. Fax: +81-75-383-2461. † Present address: Department of Functional Materials Science, Faculty of Engineering, Saitama University, Saitama 338-8570, Japan.

proliferating cells such as smooth muscle cells,10 fibroblasts,10 and keratinocytes.11 Investigation of its more detailed functions is very actively ongoing; it is likely that HA exerts some influences on the cell mitosis.10 Such multifunctions of naturally occurring HA have been utilized in medical and pharmaceutical fields.12-19 Particularly in the cataract surgery and in the treatment of arthritis, various kinds of HA products are actually used.12 In addition, chemically modified HA samples were developed for artificial ECMs with using cross-linked HA.20 Mainly aiming at the clinical applications, cross-linked HA derivatives were prepared by the reaction of HA hydroxyl groups and the protein amino or imino groups with formaldehyde21 or other chemicals.22-25 Photocurable HA derivatives were also synthesized by introduction of photoreactive chromophores.26 Noncurable HA derivatives find useful medical and pharmaceutical applications,27-35 in particular for the drug delivery system.36 Synthesis of such HA derivatives is achieved by modification of natural HA. Precise control of the modification is normally difficult due to low reactivity of functional groups on a high molecular weight HA molecule. Further, unexpected side reactions such as bond cleavage of the HA chain causing reduction of the molecular weight often occur during the modification under severe reaction conditions.37 HA derivatives with a perfectly controlled structure bear the potentials to regulate their biological activities as well as their chemical and physical properties. Therefore, their precise production is challenging in synthetic chemistry. Enzymatic polymerization utilizing a glycoside hydrolase as a catalyst has been demonstrated to be effective for a single-step synthesis of oligo- and polysaccharides with

10.1021/bm049280r CCC: $30.25 © 2005 American Chemical Society Published on Web 02/09/2005

Enzymatic Synthesis of Hyaluronan and Its Derivatives

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Scheme 1. Enzymatic Polymerization of Monomers (1) to Synthetic HA and Its Derivatives (2)

perfectly controlled structure via nonbiosynthetic pathways.38 Synthetic polysaccharides were prepared by enzymecatalyzed repeated regio-selective and stereo-controlled glycosidations of sugar monomers activated at the anomeric position such as glycosyl fluorides and sugar oxazolines. For example, cellulose39 was prepared for the first time by polymerization catalyzed with cellulase from β-D-cellobiosyl fluoride as a substrate monomer, xylan40 with cellulase from β-D-xylobiosyl fluoride, amylose oligomers41 with amylase from R-D-maltosyl fluoride, and chitin42 with chitinase from a N,N′-diacetylchitobiose oxazoline derivative. All reactions led to natural-type homopoly- and homo-oligosaccharides. Mutant enzymes were also shown to be effective for the synthesis of β(1f4)-poly- and oligosaccharides.43 In addition, unnatural-type polysaccharides, i.e., an alternating 6-O-methylcellulose44 and a cellulose-xylan hybrid polymer,45 were synthesized by cellulase catalysis using 6-O-methyl-β-D-cellobiosyl fluoride monomer and by xylanase using β-D-xylosyl-(1f4)-β-D-glucosyl fluoride, respectively. Recently, the synthesis of natural-type heteropolysaccharides of HA46 and Ch47 was achieved by the hyaluronidase (HAase)-catalyzed regio-selective and stereo-controlled ring-opening polyaddition of N-acetylhyalobiuronate (GlcAβ(1f3)GlcNAc) oxazoline and N-acetylchondrosine (GlcAβ(1f3)GalNAc) oxazoline derivatives as substrate monomers, respectively. All the substrate monomers above were designed and synthesized according to our new concept of “transition-state analogue substrate”.38,46,47 Thus, the methodology of enzymatic polymerization utilizing glycoside hydrolases is a “bottom-up” synthesis of polysaccharides from molecularly designed sugar monomers through enzyme-catalyzed glycosidation. This situation allows us to construct various natural and unnatural polysaccharides.38 The polysaccharides produced by enzymatic polymerization possess perfectly controlled structures, which provide good samples for the examination of biological activities as well as chemical and physical properties at a molecular level. In the present study, we report the “bottom-up” synthesis of HA and its derivatives bearing various amido functional groups via an enzymatic ring-opening polyaddition (Scheme 1). The 2-methyl oxazoline monomer (1a) was preliminarily

Figure 1. Possible monomer designs of (A) an oxazoline-type monomer for HAase and (B) a fluoride-type monomer for hyaluronoglucuronidase.

reported to be an effective monomer leading to natural-type HA;46 in addition, the reaction conditions of 1a with HAase as well as the synthesis method of 1a have been refined. 2-Ethyl (1b), 2-n-propyl (1c), 2-isopropyl (1d), 2-phenyl (1e), 2-vinyl (1f), and 2-isopropenyl (1g) oxazoline monomers were newly prepared as substrate monomers for HAase to generate HA derivatives bearing the corresponding N-propionyl, N-butyryl, N-isobutyryl, N-benzoyl, N-acryloyl, and N-methacryloyl functional groups in place of the N-acetyl group in natural-type HA. In particular, monomer 1f led to a reactive unnatural HA derivative having olefinic groups in the N-acyl group. These results will provide a novel insight for synthesis of HA derivatives toward the new materials architecture in various fields of science. Results and Discussion Monomer Design. On the basis of the concept of “transition-state analogue substrate” in the enzymatic polymerization,38,46,47 two types of possible monomers are considered from the chemical structure of HA (Figure 1). One is an oxazoline-type monomer derived from GlcAβ(1f3)GlcNAc (1a; Figure 1A), and the other is a β-fluoride-type monomer from GlcNAcβ(1f4)GlcA (Figure 1B). Both of them are activated substrate monomers; compound 1a is ready to form the corresponding oxazolinium ion via protonation of the oxazoline, and GlcNAcβ(1f4)GlcAβ-fluoride is expected to give an oxocarbenium ion by releasing fluorine anion from the monomer. The former is developed for HAase catalysis46,47,48 (EC 3.2.1.35; hyaluronoglucosaminidase), responsible for the catabolism of β(1f4)-N-acetyl-D-glucosaminide linkages in HA,49 and the latter for hyaluronoglucuronidase catalysis48a,50 (EC 3.2.1.-; endo-β-glucuronidase), normally catalyzing the bond cleavage of β(1f3)-D-glucuronide linkages in HA. HAase is a commercially available enzyme; therefore, the combination of monomer 1a and HAase is feasible for synthesis of natural-type HA. In addition, the synthesis of 1a can be extended to that of other monomers (1b-1g), enabling the synthesis of unnatural-type HA derivatives, i.e., 2-substitu-

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Scheme 2. Synthesis of Substrate Monomer 1aa

Ochiai et al. Scheme 3. Synthesis of Substrate Monomers 1b-1ga

a Key: (i) BF3‚OEt2, MS4A/CH2Cl2, 78%; (ii) 80% aqAcOH; (iii) Pd(OH)2-C, H2/MeOH; (iv) Ac2O/pyridine, 98% (3 steps); (v) TMSOTf/ CH2ClCH2Cl, 90%; (vi) NaOMe/MeOH; (vii) carbonate buffer (50 mM, pH 12), 84% (2 steps).

tions of the oxazoline part lead to corresponding HA derivatives through oxazoline ring opening. On the other hand, it is inconvenient to employ GlcNAcβ(1f4)GlcAβfluoride as a monomer because hyaluronoglucuronidase is not a commercial enzyme. Some eliminases (EC 4.2.2.1; hyaluronan lyases) are known to cleave the (1f4)-β-Nacetyl-D-glucosaminide linkages in HA. However, their catalytic mechanism is completely different from the enzymes mentioned above: They produce the 4,5-unsaturated structure at the nonreducing terminal of the sugar residue.48a Thus, we selected the route using HAase and oxazoline-type monomers for the synthesis of HA and its derivatives. Synthesis of Monomers. In our previous paper,46 the yield of 1a was not satisfactory for the large-scale synthesis. Therefore, we searched for a better route to 1a. Previously, the glycosidation reaction to a disaccharide derivative (5) has been performed with methyl (2,3,4-tri-O-acetyl-R-Dglucopyranosyl bromide)uronate51 and benzyl 2-acetamido2-deoxy-4,6-O-isopropylidene-β-D-glucopyranoside (4)52 as a glycosyl donor and a glycosyl acceptor, respectively. The reaction provided the corresponding disaccharide (5) in a low yield of 29% due to a lower reactivity of the glycosyl donor bringing about multiple unknown byproducts. In the present study, methyl(2,3,4-tri-O-acetyl-R-D-glucopyranosyl trichloroacetimidate)uronate (3)53 was employed as a more effective glycosyl donor using boron trifluoride diethyl ether complex (BF3‚OEt2) as a promoter, giving rise to 5 in a much better yield of 78% (Scheme 2). The 4,6-O-isopropylidene group of 5 was removed by acid hydrolysis, then the O-benzyl group at the anomeric position was hydrogenated by palladium(II) hydroxide-charcoal under hydrogen atmosphere. A fully O-acetyl protected derivative (6) was produced in a 98% yield after 3 steps. Formation of the oxazoline ring (7) was achieved in a 90% yield by reaction with trimethylsilyl triflate (TMSOTf). All the O-acetyl protecting groups of 7 were removed with a sodium methoxide-methanol mixture followed by alkaline hydrolysis of the methyl ester in a carbonate buffer to give 1a in an 84% yield. Total yield of 1a through the present route

a Key: (i) BF ‚OEt , BnOH, MS4A/toluene, -40 °C, 6 h, 85%; 3 2 (ii) NaOMe/MeOH-CHCl3, rt, 3 h, quant.; (iii) (CH3)2C(OCH3)2, CSA, Drierite/MeCN, 60 °C, 24 h, 71%; (iv) BF3‚OEt2, MS4A/CH2Cl2, 71%; (v) 80% aqAcOH, 78%; (vi) Pd(OH)2-C, H2/MeOH; (vii) RCOCl, Et3N/ MeOH; (viii) Ac2O/pyridine, (b) 77%, (c) 63%, (d) 56%, (e) 68%, (f) 54%, (g) 31% (3 steps); (ix) TMSOTf/CH2ClCH2Cl, (b) 74%, (c) 77%, (d) 48%, (e) 52%, (f) 78%, (g) 56%; (x) NaOMe/MeOH; (xi) carbonate buffer (50 mM, pH 12), (b) 81%, (c) 85%, (d) 79%, (e) 84%, (f) 87%, (g) 83% (2 steps).

has been much improved as 58%, which is to be compared with that of the previous one (19%). Monomers (1b-1g), all new compounds, for the synthesis of HA derivatives (unnatural-type) were prepared according to the reactions outlined in Scheme 3. To facilitate the production of HA derivatives bearing various amido functional groups, an azido group was introduced at the C-2 position of the glucopyranose unit in place of the acetamido group. A readily accessible compound (8)54 was reacted with benzyl alcohol to produce a 1-O-benzyl-protected derivative (9). The O-acetyl groups of 9 were all removed by sodium methoxide to afford benzyl 2-azido-2-deoxy-β-D-glucopyranoside (10). The 4- and 6-hydroxyl groups of 10 were protected by isopropylidene group, giving rise to a glycosyl acceptor (11). Then, compound 11 was glycosylated with a glycosyl donor 3 using BF3‚OEt2 as a promoter to afford the disaccharide derivative (12) in a 71% yield. The 4,6-Oisopropylidene group and the 1-O-benzyl group of 12 were successively removed by acid hydrolysis to a glycol (13) in an 80% aqueous acetic acid, followed by hydrogenation with palladium(II) hydroxide-charcoal under a hydrogen atmo-


Enzymatic Synthesis of Hyaluronan and Its Derivatives Table 1. Enzymatic Polymerization of 1a under Various Conditions polymerization of 1aa entry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20g 21h 22 23 24 25 a

polymer (2a)

pH

concentration of 1a/M

enzyme amount / wt % for 1a

temp/ °C

OTH OTH OTH OTH

6.0 6.5 7.0 7.5

0.10 0.10 0.10 0.10

10 10 10 10

30 30 30 30

8 14 48 52

OTH OTH OTH OTH OTH OTH OTH OTH OTH OTH OTH OTH OTH OTH OTH OTH OTH H-OTH BTH

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 7.5 7.5 7.5 7.5

0.10 0.10 0.10 0.10 0.01 0.02 0.05 0.20 0.50 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10

10 10 10 10 10 10 10 10 10 1 3 5 20 10 10 10 10 10 10

30 30 30 30 30 30 30 30 30 30 30 30 30 20 40 30 30 30 30

60 60 72 168i 48 48 48 52 60 72 48 48 24 144 24 48 48 24 60

bee venom H-OTH

7.5 7.5

0.10 0.10

10 10

30 30

72 6i

enzymeb

timec/ h

yieldd/%

Mne

Mwe

19 56 72 78 53f 73 67 65 0 32 39 51 64 61 13 39 60 73 70 56 23 25 80 53 34f trace 69

1300 2100 4400 5500 13300 5700 5400 4600

1400 2500 9800 13800 22000 14900 13500 11300

2600 3400 5100 5600 5500 3900 5800 5500 4600 4700 4900 3200 3600 4300 7800 17700

5000 7700 13000 14400 13800 10900 14500 12900 12900 10700 12400 8200 9000 11600 17600 25000

6800

19500

b

In a carbonate buffer (50 mM). OTH (560 units/mg from ICN Biochemicals, Inc.); BTH (500 units/mg from SIGMA); H-OTH (3720 units/mg from SIGMA). c Indicating the time for complete consumption of 1a except for entries 8 and 25. d Determined by HPLC containing products with molecular weight higher than tetrasaccharides unless otherwise indicated. e Determined by SEC calibrated with hyaluronan standards. f Isolated yields after purification. g In a carbonate buffer (500 mM). h In a carbonate buffer (50 mM) with the addition of NaCl (500 mM). i Reaction was terminated at the indicated time.

sphere. The amino group converted from the azido group during the reduction was reacted with propionyl, butyryl, isobutyryl, benzoyl, acryloyl, or methacryloyl chloride, followed by acetylation of the hydroxyl groups to produce the corresponding N-propionyl (14b), N-butyryl (14c), N-isobutyryl (14d), N-benzoyl (14e), N-acryloyl (14f), or N-methacryloyl (14g) derivatives. Oxazoline ring formation of these N-acyl derivatives was achieved by treatment with TMSOTf, giving rise to the corresponding 2-substituted oxazolines (2-ethyl (15b), 2-n-propyl (15c), 2-isopropyl (15d), 2-phenyl (15e), 2-vinyl (15f), and 2-isopropenyl (15g)). Finally, all O-acetyl groups were removed by sodium methoxide in methanol followed by alkaline hydrolysis of the methyl ester in a carbonate buffer to produce the corresponding monomers 1b-1g in high yields. Enzymatic Polymerization of 1a. To find the optimal conditions in the polymerization of 1a with HAase, reaction parameters of pH, concentration of 1a, amount of HAase, reaction temperature, buffer concentration, and NaCl addition,48c,55 reaction time and type of enzyme were examined (Table 1). In all cases, three kinds of reaction, polymerization of 1a to product polymer (2a), enzymatic, and nonenzymatic hydrolyses of 1a resulting in formation of N-acetylhyalobiuronate (16a),56 explain the reaction behaviors of 1a (Scheme 4). In entries 1-8, the polymerization was performed with ovine testicular HAase (OTH) under varying pH conditions

Scheme 4. Three Kinds of Reaction Occur during the Polymerization of 1a

at 30 °C. Monomer 1a was completely consumed within 8 h at pH 6.0, giving rise to 2a with molecular weight (Mn, determined by size-exclusion chromatography, SEC) of 1300 (approximately 6 saccharides) in a 19% yield (entry 1). At pH 6.5, the reaction provided 2a having Mn 2100 (approximately 10 saccharides) in an improved yield (56%) within 14 h (entry 2). It is known that the optimal pH range for the hydrolysis of HA by HAase is from 4 to 6;49 therefore, these results indicate that at pH 6 or 6.5 the polymerization of 1a was catalyzed rapidly to produce 2a, but the enzyme also catalyzed the hydrolysis of product 2a to reduce the yield and molecular weight of 2a. At a pH ranging from 7.0 to 9.0 (entries 3-7), the yields and Mn values of 2a became higher; 2a having Mn 5500 (approximately 28 saccharides) was produced in a 78% yield at pH 7.5 after 52 h (entry 4), and a Mn value of 2a reached 5700 (approximately 30

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Figure 2. Effects of pH on the reaction rate in a qualitative sketch: (A) polymerization of monomer 1a and (B) hydrolysis of HA.

saccharides) at pH 8.0, which was obtained in a 73% yield (entry 5). Both the yield and Mn value became gradually smaller at a pH range from 8.0 to 8.5. At pH 9.5, however, the reaction did not afford 2a but only 16a, the hydrolysis product of 1a (87%), and 13% of 1a remained unchanged after 168 h (entry 8). From these results, the polymerization of 1a proceeded prior to the hydrolysis under neutral or weak alkaline conditions, giving rise to 2a having Mn over 5000 in good yields. However, at a higher pH of 9.5 the polymer 2a was not formed, which was due to a complete suppression of the glycosidation reaction leading to synthetic HA. Thus, the pH of the reaction drastically affects the reaction time for complete consumption of 1a, yields, and molecular weight values of the product polymer 2a. On the basis of these results, the pH effects on the reaction rate are illustrated in Figure 2, which qualitatively explains the consumption behaviors of monomer 1a as well as the hydrolysis of HA (2a). The polymerization of 1a, therefore, is to be carried out at a pH where the difference in rate of the polymerization of 1a and the hydrolysis of product HA is maximal, e.g., pH 7.5. Without the enzyme at pH 7.5, 1a was gradually consumed, affording 16a quantitatively after 96 h (data not shown). The structure of 2a was unambiguously determined.46 Since the catalysis of OTH seems optimal at pH 7.5, particularly in terms of the yield of 2a (entry 4), other reaction parameters were examined at this pH. Concentration effects of monomer 1a were studied at 30 °C by using 10 wt % of OTH (entries 4 and 9-13). The consumption rate of 1a became slightly slower while raising the concentration of 1a. At a lower concentration, the yield and Mn value of 2a were lower; at a high concentration, the Mn value of 2a reached 5600 (approximately 28 saccharides, entry 12). The enzyme amount also affected the yield and Mn value of 2a (entries 14-17). A small amount of the enzyme caused the yield reduction of 2a. An appropriate concentration of the enzyme was around 3-10 wt % for 1a for the higher Mn values. A higher concentration (20 wt %, entry 17) accelerated the consumption rate of 1a but slightly lowered the Mn value, which is probably due to the increased number of initiated HA chains from 1a, compared with entry 4. Next, the reaction temperature was varied (entries 4, 18, and 19). At temperatures of 20, 30, and 40 °C, the higher the

Figure 3. Time dependence of concentration of 1a (4), yields of 2a (0) and Mn value of 2a (O) with (A) OTH and (B) H-OTH. Reaction conditions: concentration of 1a, 0.1 M; enzyme, 10 wt % for 1a; carbonate buffer (50 mM, pH 7.5); reaction temperature, 30 °C.

temperature, the faster the reaction rate; however, the yield and Mn value of 2a were little affected. A higher buffer (NaCO3) concentration (entry 20) and the addition of NaCl (entry 21) slightly promoted the consumption of 1a but greatly reduced the yield and Mn value of the product 2a. Previous papers described acceleration of HAasecatalyzed hydrolysis of HA by the addition of NaCl.48c,55 Therefore, these results can be explained; in a higher concentration of NaCO3 or NaCl, 1a was slightly more readily recognized by the enzyme to cause the polymerization to 2a and/or the hydrolysis to 16a, and further product 2a was also hydrolyzed in a much faster rate, resulting in a lower yield of 2a. In entries 22-24, the polymerization was performed by using an additional three types of HAase. H-OTH, which has higher hydrolysis activity units than OTH, exhibited a higher polymerization rate for 1a, giving rise to 2a having an Mn value of 4300 in an 80% yield for a shorter reaction time of 24 h (entry 22). Bovine testicular HAase (BTH) was also effective for the polymerization, producing 2a having a slightly higher Mn value of 7800 in a 53% yield within 60 h (entry 23). Bee venom containing HAase showed little catalytic activity (entry 24). As expected from the above, at around pH 7.5 the consumption of 1a to product 2a is the fastest among three reactions (Scheme 4). This allows us to anticipate a higher Mn value of 2a in the initial stage of polymerization of 1a in a higher concentration prior to hydrolysis of 2a, which was actually observed in a shorter reaction time of 6 h (entry 25). Figure 3 shows time dependence of concentration of monomer 1a, the yield, and Mn value of polymer 2a in the


polymerization by using OTH (A) and H-OTH (B). With OTH catalysis, the yield of 2a gradually increased when 1a completely disappeared (52 h). The Mn value in A reached 5900 at 20 h and then gradually decreased to 5500. H-OTH exhibited a higher activity for the consumption of 1a, giving rise to 2a in relatively good yields. Concentration of 1a was drastically reduced to 0.02 M, accompanying a steep rise of the yield of 2a to almost 70% within 4 h. Notably, the Mn value of 2a reached 6900 at 2 h, then it became smaller to 4500 at 24 h. These results are in accord with those of entry 25 (Table 1). Thus, the polymerization of 1a proceeded most effectively in a 50 mM carbonate buffer containing 0.1 M of 1a by using 10 wt % OTH as a catalyst at pH 7.5 and 30 °C. Isolated yields of 2a through the reactions with OTH were 53% (Mn 13300, approximately 66 saccharides, entry 4) and with BTH 34% (Mn 17700, approximately 88 saccharides, entry 23), respectively, in the present study. These data agree well with those reported previously (52% and Mn 13500 by OTH; 39% and Mn 17400 by BTH).46 It is to be noted that the reactions with organic cosolvents such as methanol, acetonitrile, and tetrahydrofuran did not proceed by using OTH catalysis at 30 °C (concentration of 1a, 0.1 M; carbonate buffer (50 mM, pH 7.5)/organic solvent, 2/1 (v/v)). Enzymatic Polymerization of Monomers 1b-1g. Catalysis behaviors of monomers 1b-1g were examined with OTH under similar conditions optimized above: In a carbonate buffered D2O (50 mM, pD 7.5); at 30 °C; concentration of monomers, 0.1 M; OTH, 10 wt % for monomers. Consumption of the monomers was monitored by 1H NMR spectroscopy with measuring the anomeric proton of the monomers, which enables the direct observation of the oxazoline ring-opening reaction of the monomer. Figure 4 illustrates the reaction-time courses of monomers 1b-1g along with 1a, with and without the enzyme. Monomer 1a was most rapidly consumed by the enzyme catalysis within 20 h. 2-Ethyl oxazoline monomer 1b was effectively consumed and disappeared within 26 h. Consumption of 1c (2-n-propyl) with the catalysis was a little faster compared to that without the enzyme. A very small difference in rate was observed in the reaction of 1d (2-isopropyl), suggesting that it is less catalyzed by OTH than 1c due to the longer reaction time for complete consumption of 1d than 1c. No significant differences were observed in the reaction of 1e (2-phenyl); it was not catalyzed by OTH at all, but only hydrolysis of 1e took place. Notably, 2-vinyl monomer 1f was consumed with effective catalysis by the enzyme, whereas the consumption was quite slow without the enzyme. 2-Isopropenyl monomer 1g was hardly catalyzed by the enzyme, similar to the case of monomer 1e. Table 2 shows the polymerization results of monomers 1b-1g using OTH and BTH as catalyst. In the enzymatic reactions three kinds of reaction, polymerization of the monomers, enzymatic, and nonenzymatic hydrolyses of the monomers to N-acylhyalobiuronate (16b-16g),56 are possible to occur, similar to the reaction of monomer 1a (Scheme 4). Both OTH and BTH catalyzed the reaction of 1b, giving


Figure 4. Reaction-time courses of monomers (A) 1a, (B) 1b, (C) 1c, (D) 1d, (E) 1e, (F) 1f, and (G) 1g with OTH (4) and without the enzyme (O) in D2O.

rise to the corresponding polymer 2b in 52% (Mn 9700, approximately 46 saccharides, entry 1) and 30% (Mn 6900, entry 2) yields, respectively, through a perfect regio-selective and stereo-controlled ring-opening polyaddition. In entries 3 and 4, OTH catalyzed the polymerization of 1c with higher activity than BTH to give 2c (Mn 8000, approximately 38 saccharides). However, 2-isopropyl oxazoline monomer 1d was difficult to polymerize by both enzymes (entries 5 and 6). Oligomers 2d (4-14 saccharides by OTH, 4-12 saccharides by BTH) were formed in a trace amount. Monomer 1e was not catalyzed by the enzymes (entries 7 and 8), affording the hydrolyzed compound 16e (69%) and unchanged 1e (31%) after 168 h. Polymerization of 1f smoothly proceeded by OTH, resulting in the formation of 2f having Mn 9100 (approximately 44 saccharides) in a 41% yield (entry 9). Both the yield and Mn value of 2f were drastically reduced by using BTH as catalyst (entry 10). Neither the polymer 2g nor its oligomers were formed from 1g (entries 11 and 12), although a small difference was observed in its catalytic behavior between the enzymatic reaction and the

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Table 2. Enzymatic Polymerization of Monomers 1b-1g polymerizationa

polymer

entry monomer enzymeb timec/h structure yieldd,e/% 1

1b

OTH

48

2b

2

1b

BTH

60

2b

3

1c

OTH

60

2c

4

1c

BTH

72

2c

5 6 7 8 9

1d 1d 1e 1e 1f

OTH BTH OTH BTH OTH

96 96 168g 168g 48

2d 2d 2e 2e 2f

10

1f

BTH

60

2f

OTH BTH

168g

2g 2g

11 12

1g 1g

168g

65d 52e 41d 30e 47d 40e 21d 13e trace trace 0 0 50d 41e 21d 14e 0 0

M nf

Mwf

6900 9700 4700 6900 4500 8000 1600 1800

17500 18500 10900 11800 13500 16900 1800 2900

5900 16800 9100 19500 3100 5900 3700 6700

a In a carbonate buffer at pH 7.5, 50 mM; monomer concentration, 0.10 M; amount of enzyme, 10 wt % for monomer; reaction at 30 °C. b OTH (560 unit/mg), BTH (500 unit/mg). c Reaction time for the complete consumption of the monomer unless otherwise indicated. d Determined by HPLC containing products with molecular weight higher than tetrasaccharides. e Isolated yields after purification. f Determined by SEC measurements calibrated with hyaluronan standards. g Reaction was terminated at the indicated time.

Figure 5. (A) 1H and (B)

13C

NMR spectra of 2f.

nonenzymatic one in Figure 4. The hydrolyzed compound 16g (84%) and unchanged 1g (16%) were confirmed after 168 h. Thus, monomers 1b, 1c, and 1f were polymerized by the catalysis of HAase, giving rise to the corresponding polymers 2b, 2c, and 2f in good yields. All 1H and 13C NMR data of 2b, 2c, and 2f (Figure 5) confirmed their structures, having the hyaluronan main chain with a different amido group.

All these behaviors of the monomers from Figure 4 and Table 2 suggest the reactivity order of HAase-catalyzed reaction to be: 1a (2-methyl) > 1b (2-ethyl) ≈ 1f (2-vinyl) > 1c (2-n-propyl) . 1d (2-isopropyl) > 1g (2-isopropenyl) ≈ 1e (2-phenyl). These can be understood primarily in terms of a steric factor of the substituents. In addition, it is surprising that the monomer consumption was even a little faster in D2O (Figure 4) than in H2O (Tables 1 and 2) under similar reaction conditions, in particular for the reactions to produce the polymers.57 Polymerization Mechanism. The present ring-opening polyaddition of the oxazoline monomers 1 utilizes the catalyst function of hydrolysis enzyme HAase. Figure 6A shows the possible transition states (or intermediates) in the hydrolysis of natural HA with HAase, which is generally accepted. In the hydrolase enzyme, there are donor and acceptor sites and two carboxylic acid groups are involved in the catalysis.38 A key step for the hydrolysis is protonation onto the oxygen atom of the glycosidic bond as seen in stage a. Then, the acetyl oxygen atom attacks from the R direction onto the anomeric C1 carbon and at the same time cleaves the C-O bond to form an oxazolinium ion via substrate-assisted stabilization as given in stage a to b. A water molecule attacks nucleophilically the C1 of the oxazolinium to open the ring, giving rise to the hydrolysis product from stage b to c. On the other hand, as shown in Figure 6B, polymerization employs the oxazoline monomer which is easily recognized at the donor site. There is no need to protonate the less basic oxygen atom but only to protonate the more basic oxazoline nitrogen atom. Therefore, the polymerization lacks stage a and starts from stage b. The oxazolinium ion readily formed from the monomer is attacked from the β direction by the 4-OH group of GlcA of the other monomer or of the growing chain located at the acceptor site, inducing the ring opening of the oxazolinium ion to give a β(1f4) glycosidic bond as well as the N-acetylglucosamine unit from stage b to c. Repetition of stages b and c eventually produces HA or HA derivatives under total control of stereo- and regio-selectivities. It is to be noted that the structures of a transition state (intermediate) of b in both parts A and B of Figure 6 are very close to each other; both involve a protonated oxazolinium ion in common. The difference is a nucleophile, water in the hydrolysis and the 4-OH group of GlcA in the polymerization. From these observations, we proposed a “transition-state analogue substrate” concept,38,42,46,47 because enzymatic catalysis is due to the stabilization of a transition state, lowering the activation energy of the reaction.58 This difference in catalysis function is pointed out in Figure 2. Hydrolysis of HA needs a little lower pH than polymerization of monomers 1. The polymerization undergoes even at a higher pH, which means that the oxazolinium formation is easier from the monomer than from the hydrolysis of HA demanding the protonation on the glycosidic oxygen atom. A similar observation was already reported, for the hydrolysis of chitin with chitinase, two carboxylic acid groups were required but for the glycosidation of oxazoline type monomers only one carboxylic



Figure 6. Comparison of mechanism in the hyaluronidase catalysis. (A) Hydrolysis of natural HA and (B) polymerization of the oxazoline monomers to HA and its derivatives.

group basically showed activity, demonstrated by using a mutant chitinase.43b Since the present monomers are designed and prepared in such a structure, they are readily recognized and activated by HAase (thus “activated monomer mechanism”) to lead to HA and HA derivatives with perfectly regulating these complicated structures. It is also noteworthy that present catalysis allowed the production of natural HA as well as three unnatural HAs having N-propionyl, N-butyryl, and N-acryloyl amido groups. This suggests that natural HAase possesses a space at the active site enough for these sterically larger substituents compared with N-acetyl group or that HAase is dynamic during polymerization catalysis as a host for the larger group guests. Isopropyl, phenyl, and isopropenyl groups on the monomer were sterically too large for the HAase catalysis. Circular Dichroism (CD). Further, circular dichroism was measured for six polymer samples (Figure 7). For naturaltype HA (Figure 7A), the dichroism was observed at 207 nm due to the amido group, which is the same as reported before.59 The higher the molecular weight, the deeper the dichroism. With three unnatural-type HA derivatives (Figure 7B), the chroism was observed for polymers of 2b (N-propionyl) and 2c (N-butyryl) at 209 nm and the polymer of 2f (N-acryloyl) at 205 nm. Conjugated amido group affected the dichroism in the 220-270-nm region as seen in part f. Conclusion

Figure 7. Circular dichroism: (A) (a) natural HA (Mn ) 2.0 × 103), (b) synthetic HA (Mn ) 1.4 × 104), and (c) natural HA (Mn > 1.0 × 106). (B) HA derivatives, (d) 2b (Mn ) 6.6 × 103), (e) 2c (Mn ) 3.1 × 103), and (f) 2f (Mn ) 5.1 × 103). The concentration of the sample was 1.00 mM.

The present study demonstrated the “bottom-up” synthesis of HA derivatives as well as synthetic HA by HAasecatalyzed ring-opening polyaddition of sugar monomers bearing various 2-substitutions at the oxazoline ring. The synthesis method of N-acetylhyalobiuronate oxazoline (1a) was improved, and synthetic HA (2a) was produced in good yields under optimal conditions at pH 7.5 and 30 °C by the catalysis of HAase with total control of regioselectivity and stereochemistry. Novel 2-ethyl (1b), 2-n-propyl (1c), and

2-vinyl (1f) oxazoline monomers were also polymerized by the enzyme catalysis under the similar reaction conditions, giving rise to the corresponding HA derivatives (unnatural HA) bearing N-propionyl (2b), N-butyryl (2c), or N-acryloyl (2f) group in every respective glucosamine unit. The present method enables a tailored production of HA derivatives with perfectly controlled structure under mild reaction conditions without using toxic chemicals. In particular, 2f contains a reactive vinyl group and can lead to

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macromonomers, telechelics, and graft copolymers; it has a potential to serve as a new HA-related biomaterial. Thus, the enzyme-catalyzed “bottom-up” synthesis will become a potent method for construction of HA derivatives applicable in various fields of science such as macromolecular chemistry, medicinal chemistry, pharmacology, physiology, biochemistry, and enzymology. Experimental Section Measurements. NMR spectra were recorded on a Bruker DPX-400 spectrometer. For solutions in D2O, acetone served as a reference 2.22 ppm (1H) and 30.89 ppm (13C). All assignments were based on correlation spectrsocopy, DEPT, and HMQC experiments. Optical rotations were measured with a JASCO P-1010 polarimeter. Melting points were determined with a YAMATO MP-21. High-performance liquid chromatography (HPLC) analysis was performed using a TOSOH LC8020 system equipped with refractive index (RI) and UV detectors. SEC analysis was carried out on a TOSOH GPC-8020 system equipped with a RI detector with a Shodex OHpak SB-803HQ column (8.0 × 300 mm), eluting with 0.1 M aqueous NaNO3 (flow rate, 0.5 mL/min) at 40 °C. The calibration curves were obtained by using hyaluronan as the standard. Fast-atom bombardment (FAB) mass spectra were obtained on a JMS-HX110A spectrometer using 2,4-dinitrobenzyl alcohol or dithiothreitol/thioglycerol (1/1, v/v) as a matrix. Matrix-assisted laser desoprtion ionization time-of-flight (MALDI-TOF) mass spectrometry analysis of the product was performed with a JEOL JMSELITE spectrometer by using 2,5-dihydroxybenzoic acid as a matrix on a Nafion-coated plate60 under negative ion mode. CD spectra were recorded in a H2O solution at 20 °C on a CD spectrometer (J-600, JASCO) using an optical cell of 0.1 cm path length. Materials. OTH (Lot No. 9303B, 560 units/mg) was purchased from ICN Biochemicals Inc. H-OTH (Lot No. 122K1378, 3720 units/mg), BTH (Lot No. 81K7042, 500 units/mg), and bee venom from Apis mellifera (Lot No. 79H1017) were purchased from SIGMA. All enzymes were used without further purification. 1,2-Dichloroethane was distilled from P2O5 and stored over activated 4-Å molecular sieves (MS4A) under argon atmosphere before use. Other chemicals were of reagent grade and used without further purification. Purification by column chromatography was carried out by elution of a column (15 × 150, 30 × 150, 45 × 150 mm2) of silica gel (Silica Gel 60, 75 µm average particle size; Nacalai Tesque, Kyoto, Japan) using a stepwise gradient-elution procedure. Monomer purities were determined by 1H NMR subtracting the amount of buffer salts. Benzyl (Methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate)-(1f3)-2-acetamido-2-deoxy-4,6-O-isopropylideneβ-D-glucopyranoside (5). To a solution of methyl (2,3,4tri-O-acetyl-R-D-glucopyranosyl trichloroacetimidate) uronate (3)53 (1.34 g, 2.79 mmol) and benzyl 2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranoside (4)52 (700 mg, 1.99 mmol) in dry CH2Cl2 (20 mL) in the presence of activated MS4A (2.4 g) was added a solution of BF3‚ OEt2 (0.35 mL, 2.76 mmol) in dry CH2Cl2 (1.4 mL) at 0 °C. The reaction mixture was stirred at room temperature

Ochiai et al.

under argon atmosphere overnight. The reaction was stopped by the addition of Et3N (0.7 mL). The mixture was filtered through diatomaceous earth (Celite), poured into saturated aqueous NaHCO3, and extracted with CHCl3. The organic layer was washed with saturated aqueous NaCl, dried over MgSO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (2:1 to 1:2 n-hexanesEtOAc) to give 5 (1.04 g, 1.56 mmol, 78%) as a white solid: 1H NMR (400 MHz, CDCl3, TMS) δ 7.36-7.26 (m, 5H, aromatic), 5.70 (d, 1H, J2,NH ) 7.03 Hz, NH), 5.24-5.15 (m, 3H, H-1, H-3′, H-4′), 4.95 (t, 1H, J1′,2′ ) J2′,3′ ) 8.53 Hz, H-2′), 4.86-4.83 (m, 2H, H-1′, CH2Ph), 4.55-4.49 (m, 2H, H-3, CH2Ph), 3.96-3.92 (m, 2H, H-6, H-5′), 3.82-3.71 (m, 5H, H-4, H-6, COOCH3), 3.42 (m, 1H, H-5), 3.04 (m, 1H, H-2), 2.03-1.87 (4s, 12H, CH3CO), 1.59, 1.41 (2s, 6H, (CH3)2C). (Methyl 2,3,4-Tri-O-acetyl-β-D-glucopyranosyluronate)(1f3)-2-acetamido-1,4,6-tri-O-acetyl-2-deoxy-D-glucopyranose (6). Compound 5 (495 mg, 0.741 mmol) was dissolved in 80% aqueous AcOH (10 mL). The reaction mixture was stirred at 80 °C for 2 h and then concentrated to dryness. The residue was dissolved in MeOH (50 mL), and 20% palladium(II) hydroxide on activated carbon (50 mg) was added. The mixture was vigorously stirred at room temperature under hydrogen atmosphere for 6 h followed by filtration through Celite and concentrated to dryness. To the residue was added pyridine (15 mL) and Ac2O (10 mL) at 0 °C, and the mixture was stirred at roomtemperature overnight. The reaction was terminated by the addition of MeOH (10 mL) at 0 °C. The mixture was diluted with CHCl3 and washed sequentially with 1 M aqueous HCl, saturated aqueous NaHCO3, and saturated aqueous NaCl. The organic layer was dried over MgSO4 and then filtered through Celite and concentrated. The residue was subjected to silica gel column chromatography (2:1 to 1:1 n-hexanes-EtOAc, then EtOAc) to give 6 (480 mg, 0.723 mmol, 98%, R/β ) 74/26) as a white solid. 1H NMR data were consistent with the previous values.46 2-Methyl-4,5-dihydro-[4,6-di-O-acetyl-1,2-dideoxy-3-O(methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate)-RD-glucopyranoso][2,1-d]-1,3-oxazole (7). To a solution of 6 (540 mg, 0.814 mmol) in dry 1,2-dichloroethane (4.9 mL) was added trimethylsilyl triflate (TMSOTf, 0.22 mL, 1.22 mmol) in dry 1,2-dichloroethane (0.51 mL) at 50 °C under argon atmosphere. After the mixture was stirred for 8 h, the reaction was stopped by the addition of Et3N (0.5 mL) at 0 °C. The mixture was concentrated, and the residue was purified by silica gel column chromatography (2:1 to 1:4 n-hexanes-EtOAc) and further purified by Sephadex LH20 column chromatography (MeOH) to give 7 (442 mg, 0.732 mmol, 90%) as a white amorphous powder. 1H NMR data were consistent with the previous values.46 2-Methyl-4,5-dihydro-[1,2-dideoxy-3-O-(sodium β-Dglucopyranosyluronate)-r-D-glucopyranoso][2,1-d]-1,3-oxazole (1a). To a solution of 7 (361 mg, 0.598 mmol) in dry MeOH (5 mL) was added NaOMe in MeOH (0.1 M, 1.5 mL, 0.150 mmol). After stirring at room temperature for 1 h, the reaction mixture was concentrated to dryness. The residue was dissolved in a carbonate buffer (50mM, pH 12,


6.0 mL). After stirring at room temperature for 2 h, the solution was lyophilized to give 1a (264 mg, purity 84%), which was characterized.46 Benzyl 3,4,6-Tri-O-acetyl-2-azido-2-deoxy-β-D-glucopyranoside (9). To a solution of 3,4,6-tri-O-acetyl-2-azido-2deoxy-R-D-glucopyranosyl trichloroacetimidate (8)54 (1.64 g, 2.79 mmol) and benzyl alcohol (0.72 mL, 6.92 mmol) in dry toluene (20 mL) in the presence of activated MS4A (2.6 g) was added a solution of BF3‚OEt2 (87 µL, 0.687 mmol) in dry CH2Cl2 (0.91 mL) at -40 °C. After the mixture was stirred for 6 h, the mixture was filtered through Celite, poured into saturated aqueous NaHCO3, and extracted with CHCl3. The organic layer was washed with saturated aqueous NaCl, dried over MgSO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (10:1 to 4:1 n-hexanes-EtOAc) to give 9 (1.23 g, 2.93 mmol, 85%) as a white solid: Rf 0.61 (1:1 n-hexanes-EtOAc); [R]26D -3.1° (c 1.0, CHCl3); mp 130-131 °C (from EtOH); 1H NMR (400 MHz, CDCl , TMS) δ 7.39-7.32 (m, 5H, 3 aromatic), 5.05-4.96 (m, 2H, H-3, H-4), 4.95 (d, 1H, J ) 12.04 Hz, CH2Ph), 4.71 (d, 1H, J ) 12.05 Hz, CH2Ph), 4.44 (d, 1H, J1,2 ) 8.03 Hz, H-1), 4.29 (dd, 1H, J5,6a ) 4.52 Hz, J6a,6b ) 12.05 Hz, H-6a), 4.15 (dd, 1H, J5,6b ) 2.26 Hz, J6a,6b ) 12.30 Hz, H-6b), 3.64 (ddd, 1H, J4,5 ) 9.66 Hz, J5,6a ) 4.64 Hz, J5,6b ) 2.01 Hz, H-5), 3.57 (dd, 1H, J1,2 ) 8.03 Hz, J2,3 ) 10.04 Hz, H-2), 2.11, 2.08, 2.01 (3s, 9H, CH3CO); 13C NMR (100 MHz, CDCl3) δ 170.56, 169.90, 169.52 (CH3CO), 135.96-128.07 (aromatic), 100.14 (C-1), 72.36 (C-3), 71.68 (C-5), 71.17 (CH2Ph), 68.29 (C-4), 63.56 (C-2), 61.80 (C-6), 20.65, 20.58, 20.48 (CH3CO); highresolution mass spectrometry (HRMS) (FAB) m/z calcd for C19H24N3O8 [M + H]+ 422.1563, found 422.1564. Benzyl 2-Azido-2-deoxy-β-D-glucopyranoside (10). Compound 9 (1.23 g, 2.93 mmol) was dissolved in 10:1 (v/v) MeOH-CH2Cl2 (33 mL), and then NaOMe in MeOH (28 wt %, 0.2 mL) was added dropwise. After the mixture was stirred at room temperature for 3 h, it was neutralized with Dowex 50W-X4 (H+ form). The resulting solution was filtered through cotton and concentrated to dryness to provide 10 (862 mg, 2.92 mmol, quant.) as a white amorphous powder: Rf 0.23 (EtOAc); [R]26D 15.7° (c 1.0, CHCl3); 1H NMR (400 MHz, CD3OD, TMS) δ 7.41-7.27 (m, 5H, aromatic), 4.94 (d, 1H, J ) 11.54 Hz, CH2Ph), 4.69 (d, 1H, J ) 12.05 Hz, CH2Ph), 4.41 (d, 1H, J ) 7.53 Hz, H-1), 3.89 (dd, 1H, J5,6b ) 2.01 Hz, J6a,6b ) 11.55 Hz, H-6a), 3.69 (dd, 1H, J5,6b ) 5.52 Hz, J6a,6b ) 12.05 Hz, H-6b), 3.353.18 (m, 4H, H-2, H-3, H-4, H-5); 13C NMR (100 MHz, CD3OD) δ 138.49-128.75 (aromatic), 101.74 (C-1), 77.86 (C-3), 76.19 (C-5), 71.72 (CH2Ph), 71.41 (C-4), 68.07 (C-2), 62.46 (C-6); HRMS (FAB) m/z calcd for C13H18N3O5 [M + H]+ 296.1246, found 296.1256. Benzyl 2-Azido-2-deoxy-4,6-O-isopropylidene-β-Dglucopyranoside (11). Compound 10 (500 mg, 1.69 mmol) was dissolved in MeCN (5 mL) followed by the addition of 2,2-dimethoxypropane (1.0 mL, 8.13 mmol), Drierite (500 mg), and (()-camphor-10-sulfonic acid (79 mg, 0.340 mmol). The reaction mixture was stirred at 60 °C for 24 h. The mixture was concentrated and subjected to silica gel column chromatography (10:1 n-hexanes-EtOAc) to afford


11 (400 mg, 1.19 mmol, 71%) as a colorless syrup: Rf 0.56 (1:1 n-hexanes-EtOAc); [R]25D -11.9° (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3, TMS) δ 7.38-7.31 (m, 5H, aromatic), 4.92 (d, 1H, J ) 11.55 Hz, CH2Ph), 4.68 (d, 1H, J ) 11.54 Hz, CH2Ph), 4.46 (d, 1H, J1,2 ) 8.03 Hz, H-1), 3.95 (dd, 1H, J5,6a ) 5.27 Hz, J6a,6b ) 10.79 Hz, H-6a), 3.82 (t, 1H, J6a,6b ) 10.54 Hz, H-6b), 3.60 (t, 1H, J3,4 ) J4,5 ) 9.04 Hz, H-4), 3.50 (dt, 1H, J3,3-OH ) 2.01 Hz, J2,3 ) J3,4 ) 9.29 Hz, H-3), 3.42 (dd, 1H, J1,2 ) 7.76 Hz, J2,3 ) 9.28 Hz, H-2), 3.42 (dt, 1H, J4,5 ) 9.91 Hz, J5,6a ) 5.18 Hz, H-5), 2.53 (d, 1H, J3,3-OH ) 2.01 Hz, 3-OH), 1.52, 1.43 (2s, 6H, (CH3)2C); 13C NMR (100 MHz, CDCl3) δ 136.31-128.05 (aromatic), 100.99 (C-1), 99.94 ((CH3)2C), 73.33 (C-4), 72.31 (C-3), 71.34 (CH2Ph), 67.05 (C-5), 66.57 (C-2), 61.82 (C-6), 28.91, 18.99 ((CH3)2C); HRMS (FAB) m/z calcd for C16H22N3O5 [M + H]+ 336.1563, found 336.1559. Benzyl (Methyl 2,3,4-Tri-O-acetyl-β-D-glucopyranosyluronate)-(1f3)-2-azido-2-deoxy-4,6-O-isopropylideneβ-D-glucopyranoside (12). To a solution of the donor 353 (809 mg, 1.69 mmol) and the acceptor 11 (405 mg, 1.21 mmol) in dry CH2Cl2 (12 mL) in the presence of activated MS4A (1.46 g) was added a solution of BF3‚OEt2 (0.2 mL, 1.58 mmol) in dry CH2Cl2 (0.6 mL) at -20 °C. After the mixture was stirred for 30 min under argon atmosphere, the reaction was terminated by the addition of Et3N (0.5 mL). The mixture was filtered through Celite, poured into saturated aqueous NaHCO3, and extracted with CHCl3. The organic layer was washed with saturated aqueous NaCl, dried over MgSO4, filtered, and concentrated. Silica gel column chromatography (8:1 to 3:1 n-hexanes-EtOAc) of the residue afforded 12 (564 mg, 0.865 mmol, 71%) as a white solid: Rf 0.40 (1:1 n-hexanes-EtOAc); [R]25D -33.3° (c 1.0, CHCl3); mp 144-145 °C (from EtOH); 1H NMR (400 MHz, CDCl3, TMS) δ 7.37-7.31 (m, 5H, aromatic), 5.27-5.18 (m, 2H, H-3′, H-4′), 5.01 (t, 1H, J1′,2′ ) J2′,3′ ) 8.78 Hz, H-2′), 4.90 (d, 1H, J ) 11.54 Hz, CH2Ph), 4.72 (d, 1H, J1′,2′ ) 8.03 Hz, H-1′), 4.66 (d, 1H, J ) 11.54 Hz, CH2Ph), 4.38 (m, 1H, H-1), 3.95 (d, 1H, J4′,5′ ) 9.54 Hz, H-5′), 3.92 (m, 1H, H-6a), 3.80 (t, 1H, J ) 10.54 Hz, H-6b), 3.75 (s, 3H, COOCH3), 3.70 (m, 1H, H-4), 3.42-3.40 (m, 2H, H-2, H-3), 3.17 (dt, 1H, J4,5 ) 10.04 Hz, J5,6a ) 5.52 Hz, H-5), 2.07, 2.02, 2.02 (3s, 9H, CH3CO), 1.43, 1.36 (2s, 6H, (CH3)2C); 13C NMR (100 MHz, CDCl3) δ 170.11, 169.32, 169.19 (CH3CO), 167.04 (C-6′), 136.23-127.96 (aromatic), 101.04 (C-1′), 100.93 (C-1), 99.66 ((CH3)2C), 80.91 (C-3), 72.87 (C-5′), 72.72 (C-4), 72.04 (C-3′), 71.31 (C-2′), 71.27 (CH2Ph), 69.48 (C-4′), 66.91 (C-5), 65.47 (C-2), 61.81 (C-6), 52.73 (CH3O), 28.80 ((CH3)2C), 20.55, 20.45, 20.42 (CH3CO), 18.43 ((CH3)2C); HRMS (FAB) m/z calcd for C29H38N3O14 [M + H]+ 652.2354, found 652.2357. Benzyl (Methyl 2,3,4-Tri-O-acetyl-β-D-glucopyranosyluronate)-(1f3)-2-azido-2-deoxy-β-D-glucopyranoside (13). Compound 12 (2.00 g, 3.06 mmol) was dissolved in 80% aqueous AcOH (10 mL), and the reaction mixture was stirred at 80 °C for 3 h. The mixture was diluted with CHCl3, washed with saturated aqueous NaHCO3, and saturated aqueous NaCl. The organic layer was dried over MgSO4, filtered through Celite, and concentrated. The residue was purified by silica gel column chromatography (1:1 to 1:4

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n-hexanes-EtOAc) to give 13 (1.46 g, 2.39 mmol, 78%) as a white solid: Rf 0.31 (1:4 n-hexanes-EtOAc); [R]26D -27.5° (c 1.0, CHCl3); mp 95-96 °C (from EtOH); 1H NMR (400 MHz, CDCl3, TMS) δ 7.38-7.31 (m, 5H, aromatic), 5.30 (t, 1H, J2′,3′ ) J3′,4′ ) 9.54 Hz, H-3′), 5.20 (t, 1H, J3′,4′ ) J4′,5′ ) 9.54 Hz, H-4′), 5.05 (dd, 1H, J1′,2′ ) 8.03 Hz, J2′,3′ ) 9.53 Hz, H-2′), 4.91 (d, 1H, J ) 12.05 Hz, CH2Ph), 4.72 (d, 1H, J ) 12.05 Hz, CH2Ph), 4.68 (d, 1H, J1′,2′ ) 8.03 Hz, H-1′), 4.39 (d, 1H, J1,2 ) 8.03 Hz, H-1), 4.07 (d, 1H, J4′,5′ ) 10.04 Hz, H-5′), 3.94 (m, 1H, H-6a), 3.90 (s, 1H, 4-OH), 3.78 (m, 1H, H-6b), 3.75 (s, 3H, COOCH3), 3.56 (t, 1H, J3,4 ) J4,5 ) 9.04 Hz, H-4), 3.38 (dd, 1H, J1,2 ) 8.03 Hz, J2,3 ) 9.54 Hz, H-2), 3.30 (m, 1H, H-5), 3.23 (t, 1H, J2,3 ) J3,4 ) 9.29 Hz, H-3), 2.09, 2.04, 2.03 (3s, 9H, CH3CO); 13C NMR (100 MHz, CDCl3) δ 169.96, 169.43, 169.31 (CH3CO), 166.72 (C-6′), 136.52-127.98 (aromatic), 101.28 (C-1′), 100.71 (C-1), 85.94 (C-3), 75.32 (C-5), 71.69 (C-3′), 71.52 (C-5′), 71.43 (CH2Ph), 70.91 (C-2′), 69.47 (C-4), 68.71 (C-4′), 65.06 (C-2), 62.73 (C-6), 53.24 (CH3O), 20.56, 20.49, 20.43 (CH3CO); HRMS (FAB) m/z calcd for C26H33N3O14 [M + H]+ 612.2041, found 612.2042. (Methyl 2,3,4-Tri-O-acetyl-β-D-glucopyranosyluronate)(1f3)-1,4,6-tri-O-acetyl-2-deoxy-2-propanamido-D-glucopyranose (14b). To a solution of 13 (200 mg, 0.327 mmol) in MeOH (7 mL) was added 20% palladium(II) hydroxide on activated carbon (200 mg). The reaction mixture was vigorously stirred at room temperature under hydrogen atmosphere for 10 h. The mixture was filtered through Celite and rinsed with MeOH (10 mL). Triethylamine (0.1 mL) and propionyl chloride (58 µL, 0.664 mmol) was added, and the mixture was stirred at 0 °C for 12 h. After addition of pyridine (5 mL), the mixture was concentrated. Pyridine (5 mL) and Ac2O (5 mL) was added, and the mixture was stirred at 0 °C for 1 h, then at room temperature for 4 h. The reaction was stopped by the addition of MeOH (5 mL) at 0 °C. The mixture was concentrated, diluted with CHCl3, and washed sequentially with 1 M aqueous HCl, saturated aqueous NaHCO3, and saturated aqueous NaCl. The organic layer was dried over MgSO4, then concentrated. The residue was purified by silica gel column chromatography (3:1 to 1:2 n-hexanes-EtOAc) to yield 14b (170 mg, 0.251 mmol, 77%, R/β ) 78/22) as a white solid: Rf 0.41 (EtOAc); 1H NMR for R form (400 MHz, CDCl3, TMS) δ 6.05 (d, 1H, J1,2 ) 3.51 Hz, H-1), 5.24 (d, 1H, J2,NH ) 10.04 Hz, NH), 5.22 (t, 1H, J2′,3′ ) J3′,4′ ) 9.29 Hz, H-3′), 5.14 (t, 1H, J3′,4′ ) J4′,5′ ) 9.79 Hz, H-4′), 5.08 (t, 1H, J3,4 ) J4,5 ) 9.79 Hz, H-4), 4.84 (t, 1H, J1′,2′ ) J2′,3′ ) 8.53 Hz, H-2′), 4.68 (d, 1H, J1′,2′ ) 7.53 Hz, H-1′), 4.57 (dt, 1H, J1,2 ) 3.52 Hz, J2,3 ) 10.29 Hz, H-2), 4.26 (dd, 1H, J5,6a ) 4.01 Hz, J6a,6b ) 12.01 Hz, H-6a), 4.07 (dd, 1H, J5,6b ) 2.01 Hz, J6a,6b ) 12.55 Hz, H-6b), 4.02 (d, 1H, J4′,5′ ) 10.04 Hz, H-5′), 3.98 (m, 1H, H-5), 3.82 (t, 1H, J2,3 ) J3,4 ) 10.04 Hz, H-3), 3.75 (s, 3H, COOCH3), 2.24 (q, 2H, J ) 4.18 Hz, CH3CH2), 2.20-2.01 (18H, m, CH3CO), 1.17 (t, 3H, J ) 7.53 Hz, CH3CH2); 13C NMR for R form (100 MHz, CDCl3) δ 173.16, 170.70, 170.56, 169.58, 169.41, 169.28, 168.68 (CH3CO), 166.94 (C-6′), 100.23 (C-1′), 91.16 (C-1), 76.17 (C-3), 72.38 (C-5′), 71.69 (C-3′), 71.10 (C-2′), 69.62 (C-5), 69.42 (C-4′), 67.55 (C-4), 61.67 (C-6), 52.77 (CH3O), 50.93

Ochiai et al.

(C-2), 29.45 (CH3CH2), 20.89, 20.68, 20.48, 20.46, 20.43, 20.36 (CH3CO), 9.42 (CH3CH2); HRMS (FAB) m/z calcd for C28H40O18N [M + H]+ 678.2245, found 678.2236. 2-Ethyl-4,5-dihydro-[4,6-di-O-acetyl-1,2-dideoxy-3-O(methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate)-rD-glucopyranoso][2,1-d]-1,3-oxazole (15b). Compound 14b (170 mg, 0.251 mmol) was dissolved in dry 1,2-dichloroethane (1.1 mL) followed by the addition of TMSOTf (68 µL, 0.376 mmol) as described in the preparation of compound 7. The mixture was concentrated, and the residue was purified by silica gel column chromatography (3:1 to 1:2 n-hexanes-EtOAc), then Sephadex LH-20 column chromatography (MeOH) to give 15b (115 mg, 0.187 mmol, 74%) as a white amorphous powder: Rf 0.46 (20:1 CHCl3MeOH); [R]28D -46° (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3, TMS) δ 5.96 (d, 1H, J1,2 ) 7.52 Hz, H-1), 5.305.19 (m, 3H, H-4, H-3′, H-4′), 5.01 (t, 1H, J1′,2′ ) J2′,3′ ) 8.53 Hz, H-2′), 4.92 (d, 1H, J1′,2′ ) 8.04 Hz, H-1′), 4.184.07 (m, 5H, H-2, H-3, H-6a, H-6b, H-5′), 3.75 (s, 3H, COOCH3), 3.61 (m, 1H, H-5), 2.39 (q, 2H, J ) 7.36 Hz, CH3CH2), 2.11-2.01 (m, 15H, CH3CO), 1.23 (t, 3H, J ) 7.53 Hz, CH3CH2); 13C NMR (100 MHz, CDCl3) δ 170.78, 170.54, 170.09, 169.91, 169.36, 169.13 (CH3CO, OCdN), 166.86 (C-6′), 100.83 (C-1′), 99.34 (C-1), 78.08 (C-3), 72.21 (C-5′), 72.13 (C-3′), 71.05 (C-2′), 69.05 (C-4′), 67.75 (C-4), 67.25 (C-5), 65.05 (C-2), 63.71 (C-6), 52.79 (CH3O), 21.47 (CH3CH2), 20.91, 20.70, 20.58, 20.57, 20.48 (CH3CO), 9.95 (CH3CH2); HRMS (FAB) m/z calcd for C26H36O16N [M + H]+ 618.2034, found 618.2037. 2-Ethyl-4,5-dihydro-[1,2-dideoxy-3-O-(sodium β-Dglucopyranosyluronate)-r-D-glucopyranoso][2,1-d]-1,3oxazole (1b). Compound 15b (154 mg, 0.250 mmol) was treated with NaOMe in MeOH (0.1 M, 0.66 mL, 0.066 mmol) as described in the synthesis of compound 1a. The remaining methyl ester was hydrolyzed in a carbonate buffer (50 mM, pH 12, 2.5 mL) to afford 1b (114 mg, purity 81%): 1H NMR (400 MHz, D2O, acetone) δ 6.10 (d, 1H, J1,2 ) 7.03 Hz, H-1), 4.66 (d, 1H, J1′,2′ ) 8.04 Hz, H-1′), 4.31 (m, 1H, H-2), 4.15 (m, 1H, H-3), 3.87-3.75 (m, 3H, H-4, H-6a, H-5′), 3.67 (dd, 1H, J5,6b ) 6.28 Hz, J6a,6b ) 12.30 Hz, H-6b), 3.53-3.51 (m, 2H, H-3′, H-4′), 3.36-3.29 (m, 2H, H-5, H-2′), 2.40 (q, 2H, J ) 7.19 Hz, CH3CH2), 1.16 (t, 3H, J ) 7.28 Hz, CH3CH2); 13C NMR (100 MHz, D2O, acetone) δ 176.48 (C-6′), 172.88 (OCdN), 102.52 (C-1′), 100.62 (C-1), 79.74 (C-3), 76.44 (C-5′), 76.02 (C-3′), 73.41 (C-2′), 72.84 (C-5), 72.40 (C-4′), 68.48 (C-4), 64.52 (C-2), 62.25 (C-6), 21.66 (CH3CH2), 9.84 (CH3CH2); HRMS (FAB) m/z calcd for C15H23O11NNa [M + H]+ 416.1169, found 416.1167. (Methyl 2,3,4-Tri-O-acetyl-β-D-glucopyranosyluronate)(1f3)-1,4,6-tri-O-acetyl-2-but anamido-2-deoxy-D-glucopyranose (14c). A solution of compound 13 (314 mg, 0.513 mmol) in MeOH (9 mL) was hydrogenolysed in the presence of 20% palladium(II) hydroxide on activated carbon (300 mg) for 24 h as described for the preparation of compound 14b. The mixture was filtered through Celite and rinsed with MeOH (15 mL), and Et3N (0.3 mL) and butyryl chloride (82 µL, 0.785 mmol) were added at 0 °C. After the mixture was stirred at 0 °C for 3 h, pyridine (4 mL) was


added, and the mixture was concentrated. Acetylation with pyridine (2 mL) and Ac2O (3 mL) was carried out as described in the preparation of compound 14b. The residue was purified by silica gel column chromatography (1:1 n-hexanes-EtOAc, then EtOAc) to yield 14c (222 mg, 0.321 mmol, 63%, R/β ) 76/24) as a white solid: Rf 0.53 (EtOAc); 1H NMR for R form (400 MHz, CDCl3, TMS) δ 6.03 (d, 1H, J1,2 ) 4.02 Hz, H-1), 5.42 (d, 1H, J2,NH ) 9.53 Hz, NH), 5.20 (t, 1H, J2′,3′ ) J3′,4′ ) 9.29 Hz, H-3′), 5.11 (t, 1H, J3′,4′ ) J4′,5′ ) 9.79 Hz, H-4′), 5.05 (t, 1H, J3,4 ) J4,5 ) 9.54 Hz, H-4), 4.81 (t, 1H, J1′,2′ ) J2′,3′ ) 8.53 Hz, H-2′), 4.69 (d, 1H, J1′,2′ ) 7.53 Hz, H-1′), 4.51 (dt, 1H, J1,2 ) 3.68 Hz, J2,3 ) 10.17 Hz, H-2), 4.17 (dd, 1H, J5,6a ) 4.27 Hz, J6a,6b ) 12.30 Hz, H-6a), 4.05-3.89 (m, 4H, H-3, H-5, H-6b, H-5′), 3.72 (s, 3H, COOCH3), 2.16-1.98 (m, 20H, CH3CO, CH3CH2CH2), 1.68-1.57 (m, 2H, CH3CH2CH2), 0.94 (t, 3H, J ) 7.28 Hz, CH3CH2CH2); 13C NMR for R form (100 MHz, CDCl3) δ 172.45, 170.80, 170.08, 169.56, 169.37, 169.29, 168.61 (CH3CO), 166.95 (C-6′), 100.18 (C-1′), 91.14 (C-1), 76.07 (C-3), 72.45 (C-5′), 71.69 (C-3′), 71.22 (C-2′), 69.68 (C-5), 69.43 (C-4′), 67.59 (C-4), 61.67 (C-6), 52.80 (CH3O), 50.98 (C-2), 38.54 (CH3CH2CH2), 20.90, 20.70, 20.51, 20.50, 20.46, 20.39 (CH3CO), 18.81 (CH3CH2CH2), 13.70 (CH3CH2CH2); HRMS (FAB) m/z calcd for C29H42NO18 [M + H]+ 692.2402, found 692.2405. 2-Propyl-4,5-dihydro-[4,6-di-O-acetyl-1,2-dideoxy-3-O(methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate)-RD-glucopyranoso][2,1-d]-1,3-oxazole (15c). Compound 14c (222 mg, 0.321 mmol) in dry 1,2-dichloroethane (4 mL) was treated with TMSOTf (87 µL, 0.481 mmol) as described in the production of compound 7. The residue was purified by silica gel column chromatography eluting with 4:1 to 1:2 n-hexanes-EtOAc and then by Sephadex LH-20 column chromatography eluting with MeOH to give 15c (155 mg, 0.246 mmol, 77%) as a white amorphous powder: Rf 0.47 (20:1 CHCl3-MeOH); [R]28D -4.0° (c 0.1, CHCl3); 1H NMR (400 MHz, CDCl3, TMS) δ 5.96 (d, 1H, J1,2 ) 7.53 Hz, H-1), 5.28 (t, 1H, J3′,4′ ) 9.29 Hz, H-3′), 5.22 (t, 1H, J4′,5′ ) 9.29 Hz, H-4′), 5.20 (m, 1H, H-4), 5.01 (t, 1H, J ) 8.54 Hz, H-2′), 4.92 (d, 1H, J1′,2′ ) 7.53 Hz, H-1′), 4.19-4.10 (m, 4H, H-3, H-6, H-6, H-5′), 4.08 (m, 1H, H-2), 3.75 (s, 3H, COOCH3), 3.67 (m, 1H, H-5), 2.34 (t, 2H, J ) 7.28 Hz, CH3CH2CH2), 2.10-2.02 (m, 15H, CH3CO), 1.74-1.65 (m, 2H, CH3CH2CH2), 1.02 (t, 3H, J ) 7.53 Hz, CH3CH2CH2); 13C NMR (100 MHz, CDCl3) δ 170.80, 170.11, 169.94, 169.56, 169.39, 169.17 (CH3CO, OC)N), 166.89 (C-6′), 100.86 (C-1′), 99.25 (C-1), 78.12 (C-3), 72.24 (C-5′), 72.17 (C-3′), 71.10 (C-2′), 69.09 (C-4′), 67.76 (C-4), 67.27 (C-5), 65.07 (C-2), 63.74 (C-6), 52.81 (CH3O), 29.86 (CH3CH2CH2), 20.91, 20.72, 20.59, 20.58, 20.50 (CH3CO), 19.00 (CH3CH2CH2), 13.64 (CH3CH2CH2); HRMS (FAB) m/z calcd for C27H38NO16 [M + H]+ 632.2191, found 632.2190. 2-Propyl-4,5-dihydro-[1,2-dideoxy-3-O-(sodium β-Dglucopyranosyluronate)-r-D-glucopyranoso][2,1-d]-1,3oxazole (1c). Compound 15c (155 mg, 0.246 mmol) was treated with NaOMe in MeOH (0.1 M, 0.62 mL, 0.062 mmol) as described for the synthesis of compound 1a. The methyl ester was hydrolyzed in a carbonate buffer (50 mM,


pH 12, 1.23 mL) to provide 1c (115 mg, purity 85%): 1H NMR (400 MHz, D2O, acetone) δ 6.11 (d, 1H, J1,2 ) 7.02 Hz, H-1), 4.66 (d, 1H, J1′,2′ ) 8.03 Hz, H-1′), 4.29 (m, 1H, H-2), 4.15 (t, 1H, J2,3 ) J3,4 ) 2.76 Hz, H-3), 3.87-3.75 (m, 3H, H-4, H-6a, H-5′), 3.68 (dd, 1H, J5,6a ) 6.28 Hz, J6a,6b ) 12.30 Hz, H-6b), 3.56-3.49 (m, 2H, H-3′, H-4′), 3.38-3.30 (m, 2H, H-5, H-2′), 2.37 (t, 2H, J ) 7.28 Hz, CH3CH2CH2), 1.67-1.60 (m, 2H, CH3CH2CH2), 0.95 (t, 3H, J ) 7.53 Hz, CH3CH2CH2); 13C NMR (100 MHz, D2O, acetone) δ 176.44 (C-6′), 171.70 (OCdN), 102.38 (C-1′), 100.68 (C-1), 79.91 (C-3), 76.37 (C-5′), 76.00 (C-3′), 73.38 (C-2′), 72.90 (C-5), 72.37 (C-4′), 68.37 (C-4), 64.60 (C-2), 62.14 (C-6), 29.90 (CH3CH2CH2), 19.17 (CH3CH2CH2), 13.58 (CH3CH2CH2); HRMS (FAB) m/z calcd for C16H25NO11Na [M + H]+ 430.1325, found 430.1332. (Methyl 2,3,4-Tri-O-acetyl-β-D-glucopyranosyluronate)(1f3)-1,4,6-tri-O-acetyl-2-deoxy-2-(2-methylpropanamido)D-glucopyranose (14d). Compound 13 (317 mg, 0.519 mmol) in MeOH (9 mL) containing 20% palladium(II) hydroxide on activated carbon (300 mg) was treated as described for the preparation of 14b. The mixture was filtered through Celite and rinsed with MeOH (17 mL), and then Et3N (0.3 mL) and isobutyryl chloride (82 µL, 0.777 mmol) were added. The mixture was stirred at 0 °C for 3 h. After the addition of pyridine (4 mL), the mixture was concentrated. The crude product was treated with Ac2O (3 mL) and pyridine (4 mL) as described for the preparation of 14b. Silica gel column chromatography of the residue (1:1 n-hexanes-EtOAc, then EtOAc) gave 14d (200 mg, 0.288 mmol, 56%, R/β ) 88/12) as a solid: Rf 0.57 (EtOAc); 1H NMR for R form (400 MHz, CDCl , TMS) δ 6.05 3 (d, 1H, J1,2 ) 3.52 Hz, H-1), 5.41 (d, 1H, J2,NH ) 9.53 Hz, NH), 5.21-5.12 (m, 2H, H-3′, H-4′), 5.06 (t, 1H, J3,4 ) J4,5 ) 9.79 Hz, H-4), 4.88 (t, 1H, J1′,2′ ) J2′,3′ ) 8.53 Hz, H-2′), 4.68 (d, 1H, J1′,2′ ) 7.53 Hz, H-1′), 4.52 (dt, 1H, J1,2 ) 3.69 Hz, J2,3 ) J2,NH ) 10.17 Hz, H-2), 4.19 (dd, 1H, J5,6a ) 4.52 Hz, J6a,6b ) 12.55 Hz, H-6a), 4.08 (dd, 1H, J5,6b ) 2.01 Hz, J6a,6b ) 12.55 Hz, H-6a), 4.03 (d, 1H, J4′,5′ ) 9.04 Hz, H-5′), 3.96 (t, 1H, J2,3 ) J3,4 ) 9.54 Hz, H-3), 3.76-3.74 (m, 4H, H-5, COOCH3), 2.38 (m, 1H, (CH3)2CH), 2.202.00 (18H, m, CH3CO), 1.20-1.15 (m, 6H, (CH3)2CH); 13C NMR for R form (100 MHz, CDCl3) δ 176.17, 170.77, 170.01, 169.51, 169.34, 169.25, 168.58 (CH3CO), 166.94 (C-6′), 100.21 (C-1′), 91.05 (C-1), 75.99 (C-3), 72.52 (C-5′), 71.72 (C-3′), 70.88 (C-2′), 69.62 (C-5), 69.41 (C-4′), 67.61 (C-4), 61.71 (C-6), 52.79 (CH3O), 50.95 (C-2), 35.50 ((CH3)2CH), 20.85, 20.69, 20.49, 20.47, 20.37 (CH3CO), 19.49, 19.35 ((CH3)2CH); HRMS (FAB) m/z calcd for C29H42NO18 [M + H]+ 692.2402, found 692.2396. 2-Isopropyl-4,5-dihydro-[4,6-di-O-acetyl-1,2-dideoxy-3O-(methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate)r-D-glucopyranoso][2,1-d]-1,3-oxazole (15d). Compound 14d (199.5 mg, 0.288 mmol) in dry 1,2-dichloroethane (3 mL) was treated with TMSOTf (78 µL, 0.433 mmol) as described in the production of compound 7. Compound 15d (88.2 mg, 0.140 mmol, 48%) was isolated through silica gel column chromatography (4:1 to 1:2 n-hexanes-EtOAc) then Sephadex LH-20 column chromatography (MeOH) as a white amorphous powder: Rf 0.73 (20:1 CHCl3-MeOH);

1080


[R]28D -5.0° (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3, TMS) δ 5.97 (d, 1H, J1,2 ) 7.53 Hz, H-1), 5.30-5.18 (m, 3H, H-4, H-3′, H-4′), 5.01 (t, 1H, J1′,2′ ) J2′,3′ ) 8.53 Hz, H-2′), 4.92 (d, 1H, J1′,2′ ) 8.03 Hz, H-1′), 4.20-4.08 (m, 5H, H-2, H-3, H-6a, H-6b, H-5′), 3.75 (s, 3H, COOCH3), 3.59 (m, 1H, H-5), 2.66 (m, 1H, (CH3)2CH), 2.10-2.02 (m, 15H, CH3CO), 1.24 (d, 3H, J ) 7.03 Hz, (CH3)2CH), 1.23 (d, 3H, J ) 6.53 Hz, (CH3)2CH); 13C NMR (100 MHz, CDCl3) δ 173.53, 170.77, 170.10, 169.91, 169.36, 169.11 (CH3CO, OCdN), 166.84 (C-6′), 100.91 (C-1′), 99.17 (C-1), 77.87 (C-3), 72.19 (C-5′), 72.14 (C-3′), 71.03 (C-2′), 69.04 (C-4′), 67.79 (C-4), 67.20 (C-5), 64.80 (C-2), 63.88 (C-6), 52.80 (CH3O), 28.24 ((CH3)2CH), 20.92, 20.69, 20.58, 20.57, 20.49 (CH3CO), 19.29, 19.22 ((CH3)2CH); HRMS (FAB) m/z calcd for C27H38O16N [M+H]+ 632.2191, found 632.2189. 2-Isopropyl-4,5-dihydro-[1,2-dideoxy-3-O-(sodium β-Dglucopyranosyluronate)-r-D-glucopyranoso][2,1-d]-1,3oxazole (1d). Compound 15d (88.2 mg, 0.140 mmol) was treated with NaOMe in MeOH (0.1 M, 0.35 mL, 0.035 mmol) as described in the synthesis of compound 1a. The remaining methyl ester was hydrolyzed in a carbonate buffer (50 mM, pH 12, 698 µL) to afford 1d (66.0 mg, purity 79%): 1H NMR (400 MHz, D2O, acetone) δ 6.10 (d, 1H, J1,2 ) 7.03 Hz, H-1), 4.66 (d, 1H, J1′,2′ ) 7.53 Hz, H-1′), 4.30 (m, 1H, H-2), 4.15 (m, 1H, H-3), 3.87-3.75 (m, 3H, H-4, H-6a, H-5′), 3.66 (dd, 1H, J5,6a ) 6.27 Hz, J6a,6b ) 12.30 Hz, H-6b), 3.56-3.49 (m, 2H, H-3′, H-4′), 3.35-3.27 (m, 2H, H-5, H-2′), 2.69 (m, 1H, (CH3)2CH), 1.21-1.18 (m, 6H, (CH3)2CH); 13C NMR (100 MHz, D2O, acetone) δ 176.39 (C-6′), 175.59 (OCdN), 102.48 (C-1′), 100.47 (C-1), 79.77 (C-3), 76.27 (C-5′), 75.98 (C-3′), 73.37 (C-2′), 72.84 (C-5), 72.34 (C-4′), 68.49 (C-4), 64.50 (C-2), 62.20 (C-6), 28.61 ((CH3)2CH), 19.19, 19.01 ((CH3)2CH); HRMS (FAB) m/z calcd for C16H25NO11Na [M + H]+ 430.1325, found 430.1328. (Methyl 2,3,4-Tri-O-acetyl-β-D-glucopyranosyluronate)(1f3)-1,4,6-tri-O-acetyl-2-benzamido-2-deoxy-D-glucopyranose (14e). A solution of compound 13 (103 mg, 0.168 mmol) in MeOH (6 mL) was hydrogenolysed in the presence of 20% palladium(II) hydroxide on activated carbon (100 mg) for 24 h as described for the preparation of compound 14b. The mixture was filtered through Celite and rinsed with MeOH (12 mL), and then Et3N (0.1 mL) and benzoyl chloride (30 µL, 0.258 mmol) were added at 0 °C. After the mixture was stirred at 0 °C for 3 h, pyridine (4 mL) was poured into the mixture, then concentrated. The crude product was acetylated with Ac2O (3 mL) and pyridine (4 mL) as described for the preparation of compound 14b. The residue was purified by silica gel column chromatography (1:1 n-hexanes-EtOAc, then EtOAc) to give 14e (82.6 mg, 0.114 mmol, 68%, R/β ) 84/16) as a white solid: Rf 0.64 (EtOAc); 1H NMR for R form (400 MHz, CDCl3, TMS) δ 7.74-7.47 (m, 5H, aromatic), 6.33 (d, 1H, J2,NH ) 9.53 Hz, NH), 6.17 (d, 1H, J1,2 ) 3.01 Hz, H-1), 5.19-5.09 (m, 3H, H-4, H-3′, H-4′), 4.90 (t, 1H, J1′,2′ ) J2′,3′ ) 8.29 Hz, H-2′), 4.80 (d, 1H, J1′,2′ ) 8.03 Hz, H-1′), 4.73 (dt, 1H, J1,2 ) 3.35 Hz, J2,NH ) J2,3 ) 10.04 Hz, H-2), 4.22 (dd, 1H, J5,6a ) 3.76 Hz, J6a,6b ) 12.30 Hz, H-6a), 4.16-4.02

Ochiai et al.

(m, 4H, H-3, H-5, H-6b, H-5′), 3.74 (s, 3H, COOCH3), 2.161.94 (m, 18H, CH3CO); 13C NMR for R form (100 MHz, CDCl3) δ 170.77, 169.85, 169.39, 169.18, 168.59, 167.31 (CH3CO), 166.82 (C-6′), 133.60, 132.04, 128.69, 126.88 (aromatic), 100.49 (C-1′), 91.01 (C-1), 76.40 (C-3), 72.31 (C-5′), 71.82 (C-3′), 70.84 (C-2′), 69.68 (C-5), 69.27 (C-4′), 67.62 (C-4), 61.67 (C-6), 52.69 (CH3O), 51.65 (C-2), 20.78, 20.64, 20.60, 20.42, 20.33, 20.26 (CH3CO); HRMS (FAB) m/z calcd for C32H40NO18 [M + H]+ 726.2245, found 726.2250. 2-Phenyl-4,5-dihydro-[4,6-di-O-acetyl-1,2-dideoxy-3-O(methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate)-rD-glucopyranoso][2,1-d]-1,3-oxazole (15e). Compound 14e (43.2 mg, 0.0595 mmol) in dry 1,2-dichloroethane (2 mL) was treated with TMSOTf (16 µL, 0.0884 mmol) as described in the preparation of compound 7. The mixture was concentrated and purified by silica gel column chromatography (4:1 to 1:2 n-hexanes-EtOAc), then Sephadex LH20 column chromatography (MeOH), to afford 15e (20.7 mg, 0.031 mmol, 52%) as a white amorphous powder: Rf 0.66 (EtOAc); [R]28D +17° (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3, TMS) δ 8.00-7.45 (m, 5H, aromatic), 6.18 (d, 1H, J1,2 ) 7.03 Hz, H-1), 5.31 (1H, t, J2′,3′ ) J3′,4′ ) 9.04 Hz, H-3′), 5.27-5.22 (m, 2H, H-4, H-4′), 5.04 (t, 1H, J1′,2′ ) J2′,3′ ) 8.54 Hz, H-2′), 4.98 (d, 1H, J1′,2′ ) 8.03 Hz, H-1′), 4.31-4.30 (m, 2H, H-2, H-3), 4.19-4.16 (m, 3H, H-6a, H-6b, H-5′), 3.75 (s, 3H, COOCH3), 3.70-3.66 (m, 1H, H-5), 2.09-2.03 (m, 15H, CH3CO); 13C NMR (100 MHz, CDCl3) δ 170.73, 170.10, 169.89, 169.35, 169.16 (CH3CO), 166.85 (C-6′), 165.06 (OCdN), 132.19, 128.55, 128.34, 126.40 (aromatic), 100.88 (C-1′), 99.91 (C-1), 78.28 (C-3), 72.28 (C-5′), 72.17 (C-3′), 71.10 (C-2′), 69.11 (C-4′), 67.75 (C-4), 67.60 (C-5), 65.67 (C-2), 63.53 (C-6), 52.79 (CH3O), 20.84, 20.69, 20.60, 20.58, 20.48 (CH3CO); HRMS (FAB) m/z calcd for C30H36O16N [M + H]+ 666.2034, found 666.2034. 2-Phenyl-4,5-dihydro-[1,2-dideoxy-3-O-(sodium β-Dglucopyranosyluronate)-r-D-glucopyranoso][2,1-d]-1,3oxazole (1e). Compound 15e (20.7 mg, 0.0311 mmol) was treated with NaOMe in MeOH (0.1 M, 78 µL, 0.0078 mmol) as described for the synthesis of compound 1a. The methyl ester was hydrolyzed in a carbonate buffer (50 mM, pH 12, 155.5 µL) to provide 1e (15.8 mg, purity 84%): 1H NMR (400 MHz, D2O, acetone) δ 7.92-7.50 (m, 5H, aromatic), 6.27 (d, 1H, J1,2 ) 7.03 Hz, H-1), 4.73 (1H, d, J1′,2′ ) 7.53 Hz, H-1′), 4.47 (m, 1H, H-2), 4.26 (m, 1H, H-3), 3.93 (m, 1H, H-4), 3.82-3.79 (m, 2H, H-6a, H-5′), 3.70 (dd, 1H, J5,6b ) 6.02 Hz, J6a,6b ) 12.05 Hz, H-6b), 3.61-3.54 (m, 2H, H-3′, H-4′), 3.41-3.31 (m, 2H, H-5, H-2′); 13C NMR (100 MHz, D2O, acetone) δ 176.47 (C-6′), 166.69 (OCd N), 133.51, 129.49, 128.84, 126.08 (aromatic), 102.55 (C-1′), 101.12 (C-1), 80.01 (C-3), 76.45 (C-5′), 76.05 (C-3′), 73.43 (C-2′), 73.02 (C-5), 72.42 (C-4′), 68.43 (C-4), 65.17 (C-2), 62.07 (C-6); HRMS (FAB) m/z calcd for C19H23NO11Na [M + H]+ 464.1169, found 464.1166. (Methyl 2,3,4-Tri-O-acetyl-β-D-glucopyranosyluronate)(1f3)-1,4,6-tri-O-acetyl-2-acrylamido-2-deoxy-D-glucopyranose (14f). Compound 13 (250 mg, 0.409 mmol) in MeOH (8 mL) containing 20% palladium(II) hydroxide on activated


carbon (250 mg) was treated as described for the preparation of compound 14b. The mixture was filtered through Celite and rinsed with MeOH (16 mL), and then Et3N (0.16 mL) and acryloyl chloride (66 µL, 0.813 mmol) were added at 0 °C. The mixture was stirred at room temperature for 12 h. After the addition of pyridine (2 mL), the mixture was concentrated. The crude product was treated with Ac2O (5 mL) and pyridine (5 mL) as described for the preparation of compound 14b. The residue was purified by silica gel column chromatography (2:1 to 1:2 n-hexanes-EtOAc) to yield 14f (149 mg, 0.220 mmol, 54%, R/β ) 86/24) as a white solid: Rf 0.47 (EtOAc); 1H NMR for R form (400 MHz, CDCl3, TMS) δ 6.33 (dd, 1H, JA,B ) 1.25 Hz, JA,C ) 16.81 Hz, CHAHB)CHC), 6.11 (dd, 1H, JB,C ) 10.04, JA,C ) 17.07 Hz, CHAHB)CHC), 6.09 (d, 1H, J1,2 ) 3.51 Hz, H-1), 5.77 (dd, 1H, JA,B ) 1.25 Hz, JB,C ) 10.29 Hz, CHAHB)CHC), 5.57 (d, 1H, J2,NH ) 9.54 Hz, NH), 5.22 (t, 1H, J2′,3′ ) J3′,4′ ) 9.54 Hz, H-3′), 5.17-5.08 (m, 2H, H-4, H-4′), 4.84 (dd, 1H, J1′,2′ ) 7.78 Hz, J2′,3′ ) 9.29 Hz, H-2′), 4.70 (d, 1H, J1′,2′ ) 8.04 Hz, H-1′), 4.62 (dt, 1H, J1,2 ) 3.85 Hz, J2,3 ) 10.04 Hz, H-2), 4.22 (dd, 1H, J5,6a ) 4.27 Hz, J6a,6a ) 12.30 Hz, H-6a), 4.07 (dd, 1H, J5,6b ) 2.01 Hz, J6a,6a ) 12.55 Hz, H-6b), 4.03 (d, 1H, J4′,5′ ) 9.54 Hz, H-5′), 4.01-3.96 (m, 2H, H-3, H-5), 3.75 (s, 3H, COOCH3), 2.191.99 (m, 18H, CH3CO); 13C NMR for R form (100 MHz, CDCl3) δ 170.74, 169.84, 169.63, 169.55, 169.20, 168.84 (CH3CO), 166.87 (C-6′), 129.85 (CH2dCH), 128.05 (CH2d CH), 100.23 (C-1′), 90.87 (C-1), 76.13 (C-3), 72.09 (C-5′), 71.66 (C-3′), 70.96 (C-2′), 69.56 (C-5), 69.33 (C-4′), 67.60 (C-4), 61.70 (C-6), 52.63 (CH3O), 51.19 (C-2), 20.70, 20.56, 20.39, 20.30, 20.22, 20.12 (CH3CO); HRMS (FAB) m/z calcd for C28H38NO18 [M + H]+ 676.2089, found 676.2090. 2-Vinyl-4,5-dihydro-[4,6-di-O-acetyl-1,2-dideoxy-3-O(methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate)-rD-glucopyranoso][2,1-d]-1,3-oxazole (15f). A solution of compound 14f (151.0 mg, 0.224 mmol) in dry 1,2-dichloroethane (3 mL) was treated with TMSOTf (60 µL, 0.332 mmol) as described in the preparation of compound 7. The residue was purified through silica gel column chromatography (2:1 to 1:2 n-hexanes-EtOAc) then Sephadex LH-20 column chromatography (MeOH as eluent) to give 15f (107.5 mg, 0.175 mmol, 78%) as a white amorphous powder: Rf 0.57 (15:1 CHCl3-MeOH); [R]29D +38° (c 0.95, CHCl3); 1H NMR (400 MHz, CDCl , TMS) δ 6.27-6.24 (m, 2H, 3 CHAHBdCHC, CHAHBdCHC), 6.04 (m, 1H, H-1), 5.84 (dd, 1H, JA,B ) 3.27 Hz, JB,C ) 8.78 Hz, CHAHBdCHC), 5.28 (t, 1H, J2′,3′ ) J3′,4′ ) 9.04 Hz, H-3′), 5.25-5.20 (m, 2H, H-4, H-4′), 5.01 (t, 1H, J1′,2′ ) J2′,3′ ) 8.53 Hz, H-2′), 4.94 (d, 1H, J1′,2′ ) 8.03 Hz, H-1′), 4.19-4.14 (m, 5H, H-2, H-3, H-6a, H-6b, H-5′), 3.75 (s, 3H, COOCH3), 3.61 (m, 1H, H-5), 2.10-2.01 (m, 15H, CH3CO); 13C NMR (100 MHz, CDCl3) δ 170.72, 170.06, 169.88, 169.33, 169.13 (CH3CO), 166.86 (C-6′), 164.33 (OCdN), 128.18 (CH2d CH), 123.72 (CH2dCH), 100.77 (C-1′), 99.51 (C-1), 78.35 (C-3), 72.29 (C-5′), 72.14 (C-3′), 71.12 (C-2′), 69.10 (C-4′), 67.71 (C-4), 67.56 (C-5), 65.66 (C-2), 63.44 (C-6), 52.77 (CH3O), 20.85, 20.67, 20.57, 20.54, 20.45 (CH3CO); HRMS (FAB) m/z calcd for C26H33NO16 [M + H]+ 616.1877, found 616.1873.


2-Vinyl-4,5-dihydro-[1,2-dideoxy-3-O-(sodium β-D-glucopyranosyluronate)-r-D-glucopyranoso][2,1-d]-1,3-oxazole (1f). Compound 15f (43.9 mg, 0.0713 mmol) was treated with NaOMe in MeOH (0.1 M, 0.18 mL, 0.018 mmol) as described in the synthesis of compound 1a. The remaining methyl ester was hydrolyzed in a carbonate buffer (50 mM, pH 12, 357 µL) to afford 1f (33.9 mg, purity 87%): 1H NMR (400 MHz, D2O, acetone) δ 6.25-6.16 (m, 2H, CHAHBdCHC, CHAHBdCHC), 5.93 (m, 1H, H-1), 5.97 (m, 1H, CHAHBdCHC), 4.66 (d, 1H, J1′,2′ ) 7.53 Hz, H-1′), 4.42 (m, 1H, H-2), 4.18 (m, 1H, H-3), 3.87-3.67 (m, 4H, H-4, H-6a, H-6b, H-5′), 3.60-3.46 (m, 2H, H-3′, H-4′), 3.42-3.26 (m, 2H, H-5, H-2′); 13C NMR (100 MHz, D2O, acetone) δ 176.43 (C-6′), 166.00 (OCdN), 130.00 (CH2dCH), 123.22 (CH2dCH), 102.43 (C-1′), 100.73 (C1), 79.72 (C-3), 76.42 (C-5′), 75.99 (C-3′), 73.36 (C-2′), 72.91 (C-5), 72.34 (C-4′), 68.33 (C-4), 64.82 (C-2), 62.11 (C-6); HRMS (FAB) m/z calcd for C15H21NNaO11 [M + H]+ 414.1012, found 414.1008. (Methyl 2,3,4-Tri-O-acetyl-β-D-glucopyranosyluronate)(1f3)-1,4,6-tri-O-acetyl-2-deoxy-2-methacrylamido-Dglucopyranose (14 g). Compound 13 (139 mg, 0.227 mmol) in MeOH (14 mL) containing 20% palladium(II) hydroxide on activated carbon (70 mg) was treated as described for the preparation of 14b. The mixture was filtered through Celite and rinsed with MeOH (12 mL), and then Et3N (0.3 mL) and methacryloyl chloride (82 µL, 0.785 mmol) were added at 0 °C. After the mixture was stirred at room temperature for 4 h, pyridine (4 mL) was added and the resulting mixture was concentrated. The crude product was acetylated with Ac2O (10 mL) and pyridine (10 mL) as described for the preparation of 14b. The residue was purified by silica gel column chromatography (4:1 to 1:2 n-hexanesEtOAc) to provide 14g (48 mg, 0.0696 mmol, 31%, R/β ) 86/14) as a white solid: Rf 0.28 (1:3 n-hexanesEtOAc); 1H NMR for R form (400 MHz, CDCl3, TMS) δ 6.08 (d, 1H, J1,2 ) 3.52 Hz, H-1), 5.66-5.63 (m, 2H, NH, CHAHBdC(CH3)), 5.44 (m, 1H, CHAHBdC(CH3)), 5.205.12 (m, 2H, H-3′, H-4′), 5.10 (t, 1H, J3,4 ) J4,5 ) 9.79 Hz, H-4), 4.89 (dd, 1H, J1′,2′ ) 7.78 Hz, J2′,3′ ) 9.29 Hz, H-2′), 4.63 (d, 1H, J1′,2′ ) 7.53 Hz, H-1′), 4.59 (dt, 1H, J1,2 ) 3.68 Hz, J2,3 ) 10.16 Hz, H-2), 4.21 (dd, 1H, J5,6a ) 4.52 Hz, J6a,6b ) 12.55 Hz, H-6a), 4.08 (dd, 1H, J5,6b ) 2.26 Hz, J6a,6b ) 12.29 Hz, H-6b), 4.03 (d, 1H, J4′,5′ ) 9.53 Hz, H-5′), 4.013.95 (m, 2H, H-3, H-5), 3.75 (s, 3H, COOCH3), 2.19-1.98 (m, 21H, CH3CO, CHAHBdC(CH3)); 13C NMR for R form (100 MHz, CDCl3) δ 170.80, 170.11, 169.50, 169.35, 169.28, 168.59, 167.75, 166.91, 166.86 (CH3CO, C-6′), 139.71 (CH2dC), 128.05 (CH2dC), 100.61 (C-1′), 91.16 (C-1), 76.51 (C-3), 72.57 (C-5′), 71.91 (C-3′), 70.91 (C-2′), 69.79 (C-5), 69.42 (C-4′), 67.56 (C-4), 61.66 (C-6), 52.82 (CH3O), 51.18 (C-2), 20.92, 20.73, 20.51, 20.41, 20.35 (CH3CO), 18.71 (C(CH3)dCH2); HRMS (FAB) m/z calcd for C27H36NO16 [M + H]+ 630.2034, found 630.2030. 2-Isopropenyl-4,5-dihydro-[4,6-di-O-acetyl-1,2-dideoxy3-O-(methyl 2,3,4-Tri-O-acetyl-β-D-glucopyranosyluronate)r-D-glucopyranoso][2,1-d]-1,3-oxazole (15g). Compound 14g (68.7 mg, 0.0996 mmol) was dissolved in dry 1,2dichloroethane (2 mL), followed by the addition of TMSOTf

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(26 µL, 0.144 mmol) as described in the preparation of compound 7. The mixture was concentrated, and the residue was purified by silica gel column chromatography (4:1 to 1:2 n-hexanes-EtOAc), then Sephadex LH-20 column chromatography (MeOH), to give 15g (35.3 mg, 0.056 mmol, 56%) as a white amorphous powder: Rf 0.57 (15:1 CHCl3-MeOH); [R]27D +24° (c 0.7, CHCl3); 1H NMR (400 MHz, CDCl3, TMS) δ 6.04 (d, 1H, J1,2 ) 7.03, H-1), 5.99 (s, 1H, CHAHBdC(CH3)), 5.58 (s, 1H, CHAHBd C(CH3)), 5.28 (t, 1H, J2′,3′ ) J3′,4′ ) 9.04 Hz, H-3′), 5.255.20 (m, 2H, H-4, H-4′), 5.01 (t, 1H, J1′,2′ ) J2′,3′ ) 8.53 Hz, H-2′), 4.94 (d, 1H, J1′,2′ ) 8.03, H-1′), 4.25-4.10 (m, 5H, H-2, H-3, H-6a, H-6b, H-5′), 3.75 (s, 3H, COOCH3), 3.633.58 (m, 1H, H-5), 2.10-2.01 (m, 18H, CH3CO, CHAHBd C(CH3)); 13C NMR (100 MHz, CDCl3) δ 170.74, 170.08, 169.83, 169.33, 169.11 (CH3CO), 166.82 (C-6′), 165.93 (OCdN), 131.35 (CH2dC(CH3)), 123.95 (CH2dC(CH3)), 100.84 (C-1′), 99.45 (C-1), 77.90 (C-3), 72.21 (C-5′), 72.13 (C-3′), 71.03 (C-2′), 69.06 (C-4′), 67.73 (C-4), 67.39 (C-5), 65.57 (C-2), 63.62 (C-6), 52.77 (CH3O), 20.83, 20.66, 20.54, 20.44 (CH3CO), 18.82 (C(CH3)dCH2); HRMS (FAB) m/z calcd for C27H36O16N [M + H]+ 630.2034, found 630.2030. 2-Isopropenyl-4,5-dihydro-[1,2-dideoxy-3-O-(sodium β-Dglucopyranosyluronate)-r-D-glucopyranoso][2,1-d]-1,3oxazole (1g). Compound 15g (34.2 mg, 0.0543 mmol) was treated with NaOMe in MeOH (0.1 M, 0.14 mL, 0.014 mmol) as described in the synthesis of compound 1a. The remaining methyl ester was hydrolyzed in a carbonate buffer (50 mM, pH 12, 272 µL) to afford 1g (25.5 mg, purity 83%): 1H NMR (400 MHz, D2O, acetone) δ 6.15 (d, 1H, J1,2 ) 7.03 Hz, H-1), 5.97 (s, 1H, CHAHBdC(CH3)), 5.58 (s, 1H, CHAHBdC(CH3)), 4.67 (d, 1H, J1′,2′ ) 7.53 Hz, H-1′), 4.43 (m, 1H, H-2), 4.19 (s, 1H, H-3), 3.89-3.73 (m, 3H, H-4, H-6a, H-5′), 3.68 (m, 1H, H-6b), 3.52-3.50 (m, 2H, H-3′, H-4′), 3.40-3.27 (m, 2H, H-5, H-2′), 1.95 (s, 3H, CHAHBdC(CH3)); 13C NMR (100 MHz, D2O, acetone) δ 176.44 (C-6′), 167.43 (OCdN), 131.65 (CH2dC(CH3)), 125.39 (CH2dC(CH3)), 102.54 (C-1′), 100.60 (C-1), 79.80 (C-3), 76.40 (C-5′), 75.98 (C-3′), 73.37 (C-2′), 72.82 (C-5), 72.36 (C-4′), 68.42 (C-4), 65.19 (C-2), 62.12 (C-6), 18.71 (CH2dC(CH3)); HRMS (FAB) m/z calcd for C16H23NO11Na [M + H]+ 428.1169, found 428.1167. Enzymatic Polymerization of 1a. A typical polymerization procedure of 1a (entry 4, Table 1) is given as follows: 1a (10.0 mg, 24.9 µmol) in a carbonate buffer (50 mM, pH 7.5, 249 µL) was incubated with OTH (1.0 mg) at 30 °C. Consumption of 1a in the reaction mixture was monitored by HPLC measurements with Shodex Asahipak NH2P-50 4E column eluting with phosphate buffer (10 mM, pH 7.0)/ MeCN mixed solution (30:70 (v/v), flow rate of 0.5 mL/ min, 30 °C). After 48 h, the reaction was terminated by thermal inactivation of the enzyme at 90 °C for 5 min. The mixture was analyzed by SEC measurement (yields 78%) and MALDI-TOF/MS. The polymeric product was separated by HPLC through a Shodex OHpak SB-803HQ column using 0.1 M aqueous NaNO3 as eluent. The combined fractions were desalted by dialysis against distilled water using a Spectra/Por CE dialysis membrane (molecular weight cutoff of 1000) to afford 2a (5.3 mg, yields 53%): 1H NMR

Ochiai et al.

(400 MHz, D2O, acetone) δ 4.54 (m, 1H, H-1), 4.46 (m, 1H, H-1′), 3.95-3.65 (m, 6H, H-2, H-3, H-6a, H-6b, H-4′, H-5′), 3.64-3.40 (m, 3H, H-4, H-5, H-3′), 3.34 (m, 1H, H-2′), 2.01 (s, 3H, CH3CO). In the control experiment (without the enzyme), the only product was the disaccharide derived from hydrolysis of 1a, (sodium β-Dglucopyranosyluronate)-(1f3)-2-acetamido-2-deoxy-4-O-Dglucopyranose (16a): MALDI-TOF/MS m/z 397.15 [M - H]-. Enzymatic Polymerization of 1a in an Organic Cosolvent. A typical polymerization procedure of 1a in methanol as an organic cosolvent is given as follows: 1a (2.9 mg, 7.2 µmol) in a carbonate buffer (50 mM, pH 7.5, 48 µL) including methanol (24 µL) was incubated with OTH (0.3 mg) at 30 °C. After 96 h, a small amount of the mixture was subjected to SEC measurement, but no polymeric products were detected. The resulting solution was applied to MALDI-TOF/MS analysis, and the spectrum showed peaks of oligomers of 2a. MALDI-TOF/MS m/z 775.23 [M - H]- (n ) 2, tetrasaccharide). Consumption of Monomers 1a-1g with OTH Catalysis. A typical procedure for monitoring the monomer consumption is given as follows: Compound 1a (8.0 mg, 20.0 µmol) was dissolved in a carbonate buffered D2O solution (50 mM, pD 7.5, 100 µL), and the solution was divided into two parts (50 µL each). To the control solution was added only carbonate buffered D2O solution (50 mM, pD 7.5, 50 µL). To the other solution was added OTH (0.4 mg) in a carbonate buffered D2O solution (50 mM, pD 7.5, 50 µL). These two samples were kept standing at 30 °C in NMR probe tubes. Concentration of 1a was calculated from the integration values of the signals from the H-1 proton and the methyl protons by 1H NMR spectroscopy. Enzymatic Polymerizations of 1b-1f. A polymerization procedure of entry 1 (Table 2) was as follows. A solution of 1b (10.0 mg, 24.1 µmol) in a carbonate buffer (50 mM, pH 7.5, 241 µL) was incubated with OTH (1.0 mg) at 30 °C as described in the polymerization of 1a. After 48 h, the resulting suspension was heated at 90 °C for 5 min to inactivate the enzyme. The residue was analyzed by SEC measurement (yields 65%). The polymeric product was separated by HPLC through a Shodex OHpak SB-803HQ column using 0.1 M aqueous NaNO3 as eluent. The combined fractions were desalted by dialysis against distilled water using a Spectra/Por CE dialysis membrane (molecular weight cutoff of 1000) to give 2b (5.2 mg, yields 52%): 1H NMR (400 MHz, D2O, acetone) δ 4.57 (d, 1H, J1,2 ) 7.02 Hz, H-1), 4.45 (d, 1H, J1′,2′ ) 6.52 Hz, H-1′), 3.91-3.65 (m, 6H, H-2, H-3, H-6a, H-6b, H-4′, H-5′), 3.58-3.44 (m, 3H, H-4, H-5, H-3′), 3.34 (m, 1H, H-2′), 2.30 (m, 2H, CH3CH2), 0.91 (t, 3H, J ) 7.53 Hz, CH3CH2); 13C NMR (100 MHz, D2O, acetone) δ 179.18 (C-6′), 174.69 (NHCO), 103.51 (C-1′), 100.84 (C-1), 82.60 (C-3), 80.01 (C-4′), 76.85 (C-5′), 75.91 (C-5), 74.05 (C-3′), 73.01 (C-2′), 69.00 (C-4), 61.11 (C-6), 54.79 (C-2), 29.90 (CH3CH2), 9.68 (CH3CH2). In the control experiment (without the enzyme), the only product was the disaccharide derived from hydrolysis of 1b, (sodium β-D-glucopyranosyluronate)-(1f3)-2-deoxy-2-pro-


panamido-D-glucopyranose (16b): MALDI-TOF/MS m/z 410.66 [M - H]-. To a solution of compound 1c (10.0 mg, 23.3 µmol) in a carbonate buffer (50 mM, pH 7.5, 233 µL) was added OTH (1.0 mg), and the mixture was incubated at 30 °C for 60 h (entry 3, Table 2) as described in the polymerization of 1a. The reaction mixture was heated at 90 °C for 5 min to inactivate the enzyme. The mixture was analyzed by SEC measurement (yields 47%). The polymeric product was separated by HPLC through a Shodex OHpak SB-803HQ column using 0.1 M aqueous NaNO3 as eluent. The combined fractions were dialyzed using Spectra/Por CE dialysis membrane (molecular weight cutoff of 1000) to remove salts, giving rise to 2c (4.0 mg, yields 40%) after lyophilization: 1H NMR (400 MHz, D2O, acetone) δ 4.59 (m, 1H, H-1), 4.49 (m, 1H, H-1′), 3.95-3.68 (m, 6H, H-2, H-3, H-6a, H-6b, H-4′, H-5′), 3.59-3.45 (m, 3H, H-4, H-5, H-3′), 3.35 (m, 1H, H-2′), 2.20 (m, 2H, CH3CH2CH2), 1.61 (m, 2H, CH3CH2CH2), 0.93 (m, 3H, CH3CH2CH2); 13C NMR (100 MHz, D2O, acetone) δ 178.56 (C-6′), 174.84 (NHCO), 103.47 (C-1′), 100.50 (C-1), 82.54 (C-3), 79.41 (C-4′), 76.97 (C-5′), 76.04 (C-5), 74.17 (C-3′), 73.11 (C-2′), 69.16 (C-4), 61.17 (C-6), 54.94 (C-2), 38.73 (CH3CH2CH2), 19.31 (CH3CH2CH2), 13.87 (CH3CH2CH2). In the control experiment (without the enzyme), the only product was the disaccharide derived from hydrolysis of 1c, (sodium β-Dglucopyranosyluronate)-(1f3)-2-butanamido-2-deoxy-D-glucopyranose (16c): MALDI-TOF/MS m/z 425.31 [M - H]-. A solution of compound 1d (5.6 mg, 13.0 µmol) in a carbonate buffer (50 mM, pH 7.5, 130 µL) was incubated with OTH (0.56 mg) at 30 °C as described in the polymerization of 1a. After 96 h, the reaction mixture was heated at 90 °C for 5 min to inactivate the enzyme then analyzed by SEC measurement. The mixture was subjected to Sephadex G-10 column chromatography eluting with distilled water. The fractions containing carbohydrates were combined, then analyzed by MALDI-TOF/MS: MALDI-TOF/MS m/z 829.17 [M - H]- (n ) 2, tetrasaccharide), 1235.11 [M - H](n ) 3, hexasaccharide), 1641.66 [M - H]- (n ) 4, octasaccharide), 2046.82 [M - H]- (n ) 5, decasaccharide), 2453.10 [M - H]- (n ) 6, dodecasaccharide), 2860.14 [M - H]- (n ) 7, tetradecasaccharide). In the control experiment (without the enzyme), the disaccharide derived from hydrolysis of 1d, (sodium β-D-glucopyranosyluronate)(1f3)-2-deoxy-2-methylpropanamido-D-glucopyrano se was the sole product (16d): MALDI-TOF/MS m/z 423.944 [M - H]-. To a solution of compound 1e (5.0 mg, 10.8 µmol) in a carbonate buffer (50 mM, pH 7.5, 108 µL) was added OTH (0.5 mg), and the mixture was incubated at 30 °C for 168 h as described in the polymerization of 1a. The mixture was heated at 90 °C for 5 min to inactivate the enzyme. The residue was analyzed by SEC measurement and MALDITOF/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, (sodium β-D-glucopyranosyluronate)-(1f3)-2benzamido-2-deoxy-D-glucopyranose (16e): MALDI-TOF/ MS m/z 458.11 [M - H]-.


A solution of 1f (10.0 mg, 24.2 µmol) in a carbonate buffer (50 mM, pH 7.5, 242 µL) was incubated with OTH (1.0 mg) at 30 °C (entry 9, Table 2) as described in the polymerization of 1a. After 48 h, the reaction mixture was heated at 90 °C for 5 min to inactivate the enzyme. The mixture was analyzed by SEC measurement (yields 50%). HPLC purification with a Shodex OHpak SB-803HQ column using 0.1 M aqueous NaNO3 as eluent followed by dialysis against distilled water using a Spectra/Por CE dialysis membrane (molecular weight cut off: 1000) afforded 2f (4.1 mg, yields 41%): 1H NMR (400 MHz, D2O, acetone) δ 6.31-6.07 (m, 2H, CHAHBd CHC, CHAHBdCHC), 5.75 (m, 1H, CHAHBdCHC), 4.55 (m, 1H, H-1), 4.38 (m, 1H, H-1′), 3.98-3.58 (m, 6H, H-2, H-3, H-6a, H-6b, H-2′, H-4′, H-5′), 3.56-3.38 (m, 3H, H-4, H-5, H-3′), 3.29 (m, 1H, H-2′); 13C NMR (100 MHz, CDCl3) δ 174.85 (C-6′), 169.88 (NHCO), 130.76 (CH2dCH), 128.49 (CH2dCH), 103.76 (C-1′), 100.86 (C-1), 83.31 (C-3), 79.93 (C-4′), 76.76 (C-5′), 76.02 (C-5), 74.07 (C-3′), 73.14 (C-2′), 69.08 (C-4), 61.17 (C-6), 54.98 (C-2). The disaccharide derived from hydrolysis of 1f, (sodium β-D-glucopyranosyluronate)-(1f3)-2-acrylamido-2-deoxy-D-glucopyranose (16f) was the only product in the control experiment (without the enzyme): MALDI-TOF/MS m/z 408.09 [M - H]-. A solution of 1g (3.0 mg, 7.1 µmol) in a carbonate buffer (50 mM, pH 7.5, 71 µL) was incubated with OTH (0.30 mg) at 30 °C as described in the polymerization of 1a. After 168 h, the enzyme was thermally inactivated at 90 °C for 5 min. The residue was analyzed by SEC measurement and MALDITOF/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 1g, (sodium β-D-glucopyranosyluronate)-(1f3)-2deoxy-2-methacrylamido-D-glucopyranose (16g): MALDITOF/MS m/z 422.20 [M - H]-. 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 and by Program for Promotion of Basic Research Activities for Innovative Bioscience (BRAIN). References and Notes (1) Meyer, K.; Palmer, J. W. J. Biol. Chem. 1934, 107, 629-634. (2) Weissmann, B.; Meyer, K. J. Am. Chem. Soc. 1954, 76, 1753-1757. (3) (a) Itano, N.; Kimata, K. IUBMB Life 2002, 54, 195-199. (b) Weigel, P. H. IUBMB Life 2002, 54, 201-211. (4) Ernst, S.; Langer, R.; Cooney, C. L.; Sasisekharan, R. Crit. ReV. Biochem. Mol. Biol. 1995, 30, 387-444. (5) (a) Selleck S. B. Trend Genet. 2000, 16, 206-212. (b) Iozzo, R. V. Annu. ReV. Biochem. 1998, 67, 609-652. (6) (a) Watanabe, H.; Yamada, Y. Nat. Genet. 1999, 21, 225-229. (b) Watanabe, H.; Kimata, K.; Line, S.; Strong, D.; Gao, L. Y.; Kozak, C. A.; Yamada, Y. Nat. Genet. 1994, 7, 154-157. (c) Hardingham, T. E.; Muir, H. Biochim. Biophys. Acta 1972, 279, 401-405. (7) (a) Toole, B. P. Cell DeV. Biol. 2001, 12, 79-87. (b) Sherman, L.; Sleeman, J.; Herrlich, P.; Ponta, H. Curr. Opin. Cell Biol. 1994, 6, 726-733. (c) Toole, B. P. Proteoglycans and hyaluronan in morphogenesis and differentiation. In Cell Biology of Extracellular Matrix, 2nd ed.; Hay, E., Ed.; Plenum Press: New York, 1991; pp 305341. (d) Toole, B. P. Glycosaminoglycans in morphogenesis. In Cell Biology of Extracellular Matrix; Hay, E., Ed.; Plenum Press: New York, 1981; pp 259-294.

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