Formal synthesis of a disaccharide repeating unit (IdoA–GlcN) of heparin and heparan sulfate

Abstract

A concise route to access the key disaccharide repeating unit (IdoA–GlcN) of heparan sulfate is described. The synthesis was accomplished by commercially available diacetone α-D-glucose to functional group transformations, which led to the formation of a L-iduronate donor. This L-iduronate donor was subsequently coupled with a glucosyl acceptor to form the corresponding key disaccharide repeating unit (IdoA–GlcN) of heparan sulfate in good overall yield.

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Introduction

heparan sulfate (HS) is a member of the glycosaminoglycans (GAGs) family, which perform a variety of crucial biological functions and have been broadly employed as therapeutic agents.¹ It is a complex polysaccharide that has shown influential biological activities by mediating the action of numerous proteins, such as growth factors, cytokines, chemokines, viral proteins, and coagulation factors.² It also mediates various physiologically important processes, such as viral and bacterial infection, angiogenesis, tumor metastasis, cell adhesion, and lipid metabolism.³ Owing to wide range of biological applications, the heparan sulfate structural framework has attracted significant interest in the development of new medicines. Recently, a possible role of HS in Alzheimer's disease and Parkinson's disease has been also found.⁴ Moreover, heparin was discovered in 1916 and has been used as a drug for the treatment of thrombotic disorders for nearly a century.⁵ The discovery of heparin has contributed extensively towards the development of numerous advanced medical and surgical procedures.⁶ Heparin, a specialized highly sulfated form of HS is not only widely used as an anticoagulant but also in the prevention and treatment of arterial and venous thrombosis.^1b,7

Heparin (HP) and HS has similar disaccharide repeating units. HS consists of a disaccharide repeating unit of either iduronic acid (IdoA) or glucuronic acid (GlcA), and glucosamine (GlcN) residues, each of them are capable of carrying sulfate groups (Fig. 1). However, nearly 90% of the disaccharide units in HP contain IdoA, while only 20% of the disaccharide units in HS contain IdoA. HS can be isolated from many cell types, whereas heparin is an exclusive product of mast cells.⁸ Owing to the versatile nature of heparin and heparan sulfate, the syntheses of these molecules have attracted considerable attention in recent years.⁹ Recently, Hung et al. developed facile methodologies for the synthesis of heparin/HS-like oligosaccharides and then using the same strategy also synthesized the heparin based anticoagulant drug fondaparinux in the acquisition of L-iduronate from diacetone D-glucose.¹⁰ Moreover, the availability of the L-iduronate donor (IdoA) is rare and commercially it is expensive.

Fig. 1 Structure of heparan sulfate 1 and its disaccharide repeating unit (IdoA–GlcN) 2.

Therefore, development of an efficient process to synthesize L-iduronate donor (IdoA) is needed. However, among the monosaccharide units of HS, the analogue that represents IdoA and GlcN requires particular attention. Herein, we report a facile protocol for the synthesis of L-iduronate donor using diacetone α-D-glucose and its application towards the formal synthesis of the disaccharide repeating unit (IdoA–GlcN) of heparan sulfate by glycosylation with a suitable glucosyl acceptor.

Results and discussion

Accordingly, first we proposed a retrosynthetic strategy for the synthesis of the disaccharide repeating unit (IdoA–GlcN) 3, as shown in Scheme 1. The disaccharide repeating unit (IdoA–GlcN) 3 could be obtained from the glycosylation reaction between imidate 4 and glucosyl acceptor 5. The adduct imidate 4 could be prepared from the L-idose derivative 6, which would be inverted from diacetone D-glucose 7.

Scheme 1 Retrosynthetic plan for disaccharide repeating unit (IdoA–GlcN) 3.

While designing the chemical synthesis of the L-iduronate donor (IdoA), a choice was made as to whether the carboxyl function of the uronic acid units would be generated before or after chain assembly. In general, carboxylate group reduces the reactivity at the anomeric center during glycosylation and also renders the C5 position more susceptible to unwanted epimerization, especially when protected as an ester. Several groups reported that uronic acids can function as effective glycosyl donors.¹¹ The IdoA residue is a crucial part of most protein binding sites in HP and HS.¹² Numerous synthetic efforts for its acquisition have been put forward.¹³ A common approach involves the chemical manipulation of the more abundant D-glucose-based compounds, which differ from L-idose only by the C5 stereochemistry. The transformation has been achieved through S_N2 substitution of alkyl sulfonate groups,¹⁴ stereoselective hydro-boration of exo-glucals,¹⁵ and hydride mediated C5 inversion of the uronate derivative.¹⁶ Alternatively, D-xylose¹⁷ and the D-xylodi-aldose derived from D-glucose¹⁸ were extended stereoselectively at C5, generating several IdoA derivatives.

Initially, we prepared L-iduranyl triol 6 starting from diacetone D-glucose 7 according to the reaction sequence shown in Scheme 2. The 3–OH group of diacetone D-glucose 7 was initially protected by the benzyl group in the presence of sodium hydride in DMF, followed by the usual aqueous workup, and column chromatography provided the resultant product 8 in quantitative yield with the expected purity.^18a 5,6-O-Isopropylidene group of diacetone α-D-glucose 8 was then hydrolyzed regioselectively using 75% acetic acid to provide the diol 9 in 88% yield. The oxidative cleavage of diol 9 by sodium periodate in water furnished aldehyde 10 in quantitative yield, which was pure enough to use for the sequential step without further purification.

Scheme 2 Preparation of L-idopyranose derivative 6.

To generate the pyranose ring of L-iduronic acid, we followed the Bonnaffé et al.¹⁹ procedure where a bulky trisphenylthiomethane group was installed at the C-5 position to afford compound 11. In this regard, first we treated n-BuLi with trisphenylthiomethane. The in situ generated trisphenylthiomethyl lithium was then treated with aldehyde 10 at −30 °C to afford compound 11 in 62% yield. The synthesis of 11 was necessary for achieving the exact configuration of the L-iduronyl sugar. Although for this inversion reaction of aldehyde 10 to compound 11, we tried several reaction conditions while changing the temperature, relative equivalent and time, the best result was obtained at higher temperature (−30 °C), which provided 11 in 62% yield, which is different from the literature report (−78 °C, 92% yield).¹⁹ The cleavage of thioether functionality using CuO with CuCl₂ in a mixture of MeOH–H₂O–DCM as the solvent provided compound 12 in 86% yield by retaining the methyl ester group at the C-6 position. Finally, acid hydrolysis of the 1,2-O-isopropylidene of 12 provided the L-iduranyl ester 6 in quantitative yield.

To obtain the disaccharide repeating unit 3 from the L-iduranyl ester 6 various synthetic steps were carried out, as shown in Scheme 3. Initially, the acetylation of L-idopyranose 6 was accomplished by treating acetyl chloride in the presence of pyridine and catalytic amount of DMAP at −40 °C, which afforded the β-form of triacetate 13 in 87% yield. The preparation of orthoester 14 was achieved through the one-pot bromination and cyclization of triacetate 13.²⁰ In an attempt for the bromination of 13, various brominating reagents, such as TMSBr and TiBr₄, were used. When TMSBr was used, several spots were observed on TLC. We then subjected the crude to cyclization using freshly distilled 2,4,6-collidine in a methanol solution but could not obtain the expected orthoester 14. However, when TiBr₄ was used, the anomeric bromination of triacetate 13 was provided as sole product according to TLC (R_f = 0.4, EtOAc–hexane: 1/2), which upon subsequent treatment with freshly distilled 2,4,6-collidine in methanol provided orthoester 14 in overall 64% yield for two steps. The de-acetylation of orthoester 14 was achieved using 0.5 N NaOMe in MeOH at 0 °C which delivered the 4-hydroxy compound 15 in 51% yield.

Scheme 3 Formal synthesis of disaccharide repeating unit (IdoA–GlcN) 3 of heparan sulfate.

Our efforts to enhance the yield of 15 were unsuccessful, even after using different reaction conditions. The lower yield was attributed to the formation of an olefin as a side product (R_f = 0.4, EtOAc–hexane: 1/2) via removal of the acidic C-5 proton followed by removal of the C-4 hydroxyl group. Owing to the requirement of free C-4 OH group in L-iduronate of disaccharide 3 for further elongation of the chain assembly,⁹ we installed the temporary protecting chloroacetyl group at C-4 OH of the L-iduronate donor. However, deprotection of the chloroacetyl group could be achieved under weak basic conditions without disturbing the other acetates in the disaccharide 3.²¹ Therefore, compound 15 when masked with a chloroacetyl chloride group at C-4 OH in the presence of pyridine, provided fully protected iduronyl compound 16 in 89% yield. Next, we cleaved the orthoester group under acidic conditions to furnish a hemiacetal compound 17 in 94% yield. The L-iduronate imidate 4 was generated by treating the hemiacetal 17 with trichloroacetonitrile under basic conditions. This well-developed concise strategy was then applied successfully for preparation of the disaccharide repeating unit (IdoA–GlcN) 3 of heparan sulfate. However, L-iduronate imidate donor 4 glycosylated with the glucosyl acceptor 5 (ref. 22) in the presence of TMSOTf, provided the key disaccharide repeating unit (IdoA–GlcN) 3 of heparan sulfate in an α/β mixture ratio of 1/3 in reasonable yield.

Conclusion

We accomplished the formal synthesis of the disaccharide repeating unit (IdoA–GlcN) 3 of heparan sulfate starting from diacetone α-D-glucose with a glucosyl acceptor through a simple synthetic route. The use of a chloroacetyl group at C-4 OH of L-iduronate moiety could provide direct access to the chain elongation on disaccharide 3 to furnish the trisaccharide moiety by coupling with the appropriate donor. This strategy disclosed the simple route for the synthesis of the key disaccharide repeating unit (IdoA–GlcN) of heparan sulfate and it is expected to provide access to other structurally related analogues for exploring their biological activities.

Experimental section

General information

Some reactions were conducted in flame-dried glassware under a nitrogen atmosphere. Dichloromethane, tetrahydrofuran, toluene, methanol, and N,N-dimethylformamide were purified and dried using a safe purification system containing activated Al₂O₃. All reagents obtained from commercial sources were used without purification, unless mentioned otherwise. Flash column chromatography was carried out on Silica Gel 60 (230–400 mesh, E. Merck). TLC was performed on pre-coated glass plates of Silica Gel 60 F254 (0.25 mm, E. Merck); detection was performed by spraying with a solution of Ce(NH₄)₂(NO₃)₆ (0.5 g), (NH₄)₆Mo₇O₂₄ (24 g) and H₂SO₄ (28 mL) in water (500 mL) and subsequent heating on a hot plate. The optical rotations were measured at 589 nm (Na) at ∼27 °C. The ¹H, ¹³C NMR, DEPT, ¹H–¹H COSY, ¹H–¹³C COSY, and ¹H–¹H NOESY spectra were recorded with 400 and 600 MHz instruments. The chemical shifts are in ppm from Me₄Si, generated from the CDCl₃ lock signal at δ 7.26 ppm. The IR spectra were taken with a FT-IR spectrometer using KBr plates. The mass spectra were analyzed on a Finnigan LTQ-OrbitrapxL instrument with an ESI† source.

3-O-Benzyl-1,2:5,6-O-di-isopropylidene-α-D-glucofuranose (8). Commercial available diacetone D-glucose 7 (15 g, 57.69 mmol) was dissolved in DMF (144 mL). Benzyl bromide (10.09 mL, 86.53 mmol) was then added to the reaction mixture and stirred for 5 min. The reaction mixture was cooled to 0 °C. NaH (3.75 g, 95.18 mmol) was added in portions (1.25 g × 3) to the cooled solution (0 °C) and the reaction mixture was stirred for 2 hours at room temperature (24 °C). The reaction was quenched with IPA (6 mL) at 0 °C followed by the slow addition of H₂O (115 mL) at 0 °C. The resulting mixture was extracted with EtOAc (100 mL × 3). The organic phase was concentrated under reduced pressure, dried over MgSO₄, filtered, concentrated, and purified by flash column chromatography (EtOAc–hexane, 1 : 3 to 1 : 1) to afford 8 (20.21 g) in quantitative yield as a colourless oil. R_f = 0.6 (EtOAc–hexane = 1/2); [α]²⁴_D = −0.3 (c 1.0, CH₂Cl₂); IR (KBr) ν 3050, 3018, 1467, 1394 cm⁻¹; ¹H NMR (600 MHz, CDCl₃) δ 7.35–7.27 (m, 5H, Ph), 5.89 (d, J = 4.2 Hz, 1H, H-1), 4.68 (d, J = 11.4 Hz, 1H, CH₂Ph), 4.64 (d, J = 12 Hz, 1H, CH₂Ph), 4.87 (d, J = 4.2 Hz, 1H, H-2), 4.38–4.35 (m, 1H, H-4), 4.15 (dd, J = 7.8, 3.0 Hz, 1H, H-5), 4.12 (dd, J = 8.4, 6.0 Hz, 1H, CH₂), 4.02 (d, J = 3.0 Hz, 1H, H-3), 4.01 (dd, J = 8.4, 6.0 Hz, 1H, CH₂), 1.49 (s, 3H, CH₃), 1.42 (s, 3H, CH₃), 1.37 (s, 3H, CH₃), 1.31 (s, 3H, CH₃) ppm; ¹³C NMR (150 MHz, CDCl₃) δ 137.6 (CH), 128.3 (CH × 2), 127.8 (CH), 127.6 (CH × 2), 11.7 (C), 108.9 (C), 105.2 (CH), 82.6 (CH), 81.6 (CH), 81.2 (CH), 72.5 (CH), 72.3 (CH₂), 67.3 (CH₂), 26.8 (CH₃), 26.7 (CH₃), 26.2 (CH₃), 25.4 (CH₃) ppm. HRMS (M + Na⁺) calcd for C₁₉H₂₆O₆Na 373.1622, found: 373.1620.

3-O-Benzyl-1,2-O-isopropylidene-α-D-glucofuranose (9). Acetic acid (70 mL) and water (30 mL) were added to the 8 (10 g, 28.23 mmol) in a round bottom flask. After the reaction mixture was stirred at 55 °C for 3 hours, the combined layers were concentrated under reduced pressure. The residue was purified by column chromatography (EtOAc–hexane, 1 : 3 to 1 : 1) to afford 9 (7.83 g, 88%) as a colourless oil. R_f = 0.2 (EtOAc–hexane = 1/2); [α]²⁴_D = −1.6 (c 1.0, CH₂Cl₂); IR (KBr) ν 3510, 3079, 1487, 1444 cm⁻¹; ¹H NMR (600 MHz, CDCl₃) δ 7.35–7.28 (m, 5H, Ph), 5.90 (d, J = 3.6 Hz, 1H, H-1), 4.69 (d, J = 12 Hz, 1H, H-6a), 4.60 (d, J = 3.6 Hz, 1H, H-2), 4.56 (d, J = 11.4 Hz, 1H, H-6b), 4.11–4.08 (m, 2H, H-4, H-5), 4.02–3.99 (m, 1H, H-3), 3.78 (dd, J = 11.4, 3.0 Hz, 1H, CH₂Ph), 3.67 (dd, J = 11.4, 5.4 Hz, 1H, CH₂Ph), 2.92 (m, 2H, 2-OH), 1.46 (s, 3H, CH₃), 1.29 (s, 3H, CH₃) ppm; ¹³C NMR (150 MHz, CDCl₃) δ 137.2 (C), 128.3 (CH × 2), 127.6 (CH), 127.5 (CH × 2), 111.4 (C), 104.8 (CH), 81.9 (CH), 81.6 (CH), 79.7 (CH), 71.9 (CH₂), 68.7 (CH), 64.0 (CH₂), 26.4 (CH₃), 25.9 (CH₃) ppm; HRMS (M + Na⁺) calcd for C₁₆H₂₂O₆Na 333.1309, found: 333.1308.

3-O-Benzyl-1,2-O-isopropylidene-α-D-xylo-dialdose (10). Compound 9 (0.43 g) was dissolved in water (3 mL), DCM (3 mL) and NaIO₄ (0.63 g, 2.13 mmol) were added to the reaction mixture portion wise over 20 min. Stirring was continued at 30 °C for 1.5 h and EtOH (5 mL) was added. The salts were filtered off, washed with water, and the filtrate was extracted with DCM (10 mL × 3) and dissolved in Et₂O. The resulting residue was washed with H₂O, dried over MgSO₄, filtered, and concentrated to give 10 as a colourless syrup (0.36 g, 95%). The ¹H and ¹³C spectra were identical to those previously reported.^18a This was used in the next step without further purification.

Tris(thiophenyl)-3-O-benzyl-1,2-O-isopropylidene-β-L-orthoido-furanuronate (11). A solution of tris(phenylthio)methane (289 mg, 0.85 mmol) in anhydrous THF (2 mL) was cooled to −30 °C and n-BuLi (531 μL, 0.85 mmol) was added slowly. The mixture was kept stirring at −30 °C for 1 h. A solution of aldehyde 10 (200 mg, 0.71 mmol) in anhydrous THF (2 mL) was then injected into the reaction mixture at −30 °C. The resulting mixture was stirred at −30 °C for 2 h and the reaction was quenched with a NH₄Cl solution (2 mL). The aqueous phase was extracted with EtOAc (10 mL × 3); the combined organic phases were washed with brine (2 mL), dried over anhydrous MgSO₄, filtered, concentrated, and purified by column chromatography (EtOAc–hex, 1 : 3 to 1 : 1) to give product 11 (270 mg) in 62% yield as a white solid. R_f = 0.4 (EtOAc–hexane = 1/2); mp = 101–103 °C; IR (KBr) ν 3525, 3059, 1735, 1472 cm⁻¹; ¹H NMR (600 MHz, CDCl₃) δ 7.69–7.67 (m, 6H, Ph), 7.35–7.24 (m, 12H, Ph), 7.08–7.07 (m, 2H, Ph), 6.00 (d, J = 3.6 Hz, 1H, H-1), 4.82 (t, J = 2.4 Hz, 1H, H-4), 4.53 (d, J = 4.2 Hz, 1H, H-2), 4.50 (d, J = 12.0 Hz, 1H, CH₂Ph), 4.23 (d, J = 12 Hz, 1H, CH₂Ph), 4.21 (d, J = 2.4 Hz, 1H, H-5), 3.62 (d, J = 3.6 Hz, 1H, H-3), 3.25 (s, 1H, OH), 1.50 (s, 3H, CH₃), 1.33 (s, 3H, CH₃) ppm; ¹³C NMR (150 MHz, CDCl₃) 136.9 (C), 136.6 (CH × 6), 131.3 (C × 2), 129.1 (CH × 4), 128.43 (CH × 3), 128.39 (CH × 5), 127.8 (CH), 127.6 (CH × 2), 112.2 (C), 104.9 (C), 83.1 (CH), 81.9 (CH), 79.9 (C), 77.7 (CH), 76.7 (CH), 72.8 (CH), 71.7 (CH₂), 27.2 (CH₃), 26.6 (CH₃) ppm. HRMS (M + Na⁺) calcd for C₃₄H₃₄O₅NaS₃ 641.14606, found: 641.14616.

Methyl 3-O-benzyl-1,2-O-isopropylidene-α-L-idofuranosyluronate (12). Methanol (114 mL), CuO (730 mg, 9.19 mmol), CuCl₂ (2.76 g, 20.55 mmol), and water (10 mL) were added sequentially to a solution of 11 (3.34 g, 5.41 mmol) in CH₂Cl₂ (10 mL). The reaction mixture was shaken vigorously for 2 h, filtered through Celite 545 and concentrated without warming above 30 °C. The residue was dissolved in CH₂Cl₂ (50 mL), and water (50 mL) was added, giving a Cu salt precipitate that was eliminated by filtration through a Celite 545. After decantation, the aqueous layer was extracted with CH₂Cl₂ (50 mL × 2). The combined organic layers were washed with a sat. aqueous NaHCO₃ solution (20 mL) and water (20 mL), filtered through a silicon-treated filter, and concentrated. Flash chromatography of the residue (EtOAc–Hexane, 1 : 3 to 1 : 1) gave 12 as a colourless oil (1.56 g, 86%). R_f = 0.3 (EtOAc–hexane = 1/3); [α]²⁸_D = 24.3 (c 1.0, CH₂Cl₂); IR (KBr) ν 3525, 3062, 1735, 1497, 1376 cm⁻¹; ¹H NMR (600 MHz, CDCl₃) δ 7.36–7.28 (m, 5H, Ph), 6.00 (d, J = 3.6 Hz, 1H, H-1), 4.71 (d, J = 11.4 Hz, 1H, CH₂Ph), 4.67 (d, J = 4.2 Hz, 1H, H-2), 4.54–4.50 (m, 3H, H-5, H-4, CH₂Ph), 4.18 (d, J = 4.2 Hz, 1H, H-3), 3.72 (s, 3H, COOCH₃), 3.45 (s, 1H, 5-OH), 1.47 (s, 3H, CH₃), 1.33 (s, 3H, CH₃) ppm; ¹³C NMR (150 MHz, CDCl₃) δ 171.7 (C), 136.6 (C), 128.3 (CH × 2), 127.9 (CH), 127.7 (CH × 2), 112.2 (C), 105.0 (CH), 82.8 (CH), 80.1 (CH), 72.1 (CH₂), 69.6 (CH), 53.3 (CH), 52.4 (CH), 26.8 (CH₃), 26.4 (CH₃) ppm. HRMS (M + Na⁺) calcd for C₁₇H₂₂O₇Na 361.1258, found: 361.1255.

Methyl 3-O-benzyl-L-idopyranosyluronate (6). Compound 12 (1.2 g, 3.54 mmol) was dissolved in a mixture of trifluoroacetic acid (6.66 mL) and water (720 μL). After 20 min stirring at room temperature (24 °C) the solvents were evaporated and the resulting solution was coevaporated with water (5 mL × 3). The residue was crystallized from EtOAc, to which the minimum pyridine necessary to reach neutrality was added, giving a quantitative yield of 6 (1.05 g) as white solid. R_f = 0.1 (EtOAc–hexane = 1/1); mp = 111–112 °C. IR (KBr) ν 3573, 3033, 2954, 1742, 1445 cm⁻¹; ¹H NMR (600 MHz, CDCl₃) δ 7.37–7.28 (m, 5H, Ph), 5.07 (s, 1H, H-1), 4.62 (s, 2H, CH₂Ph), 4.56 (s, 1H, H-5), 4.02 (s, 1H, H-4), 3.96 (t, J = 3.0 Hz, 1H, H-3), 3.90 (s, 1H, H-2), 3.76 (s, 3H, COOCH₃) ppm; ¹³C NMR (150 MHz, CDCl₃) δ 170.4 (C), 137.2 (CH), 128.4 (CH × 2), 128.0 (CH), 127.5 (CH × 2), 93.1 (CH), 75.5 (CH), 74.3 (CH), 72.2 (CH₂), 68.1 (CH), 67.5 (CH), 52.6 (CH₃) ppm. HRMS (M + Na⁺) calcd for C₁₄H₁₈O₇Na 321.0945, found: 321.0948.

Methyl 1,2,4-tri-O-acetyl-3-O-benzyl-β-L-idofuranoate (13). N,N-Dimethylaminopyridine (156 mg, 1.28 mmol), pyridine (10.34 mL, 128.5 mmol) and acetyl chloride (5.48 mL, 77.1 mmol) were added to a cooled (−40 °C) suspension of crystalline 6 (3.83 g, 12.85 mmol) in CH₂Cl₂ (80 mL). After 10 h stirring at this temperature, the mixture was diluted with dichloromethane (150 mL) and the resulting organic phase was washed with a saturated NaHCO₃ solution (50 mL × 3), water (50 mL × 2), 10% H₂SO₄ (50 mL × 3), and water (50 mL × 3), filtered through a phase separator filter, and concentrated. The residue was purified by flash chromatography (EtOAc–hex, 1 : 3 to 1 : 1) to give 13 (4.78 g, 87%) as a white solid. R_f = 0.5 (EtOAc–hexane = 1/2); mp = 111–113 °C; IR (KBr) ν 3692, 3570, 2955, 1750, 1443, 1373, 1227, 1145 cm⁻¹; ¹H NMR (600 MHz, CDCl₃) δ 7.38–7.30 (m, 5H, Ph), 6.07 (d, J = 1.8 Hz, 1H, H-1), 5.15–5.14 (m, 1H, H-4), 5.02–5.02 (m, 1H, H-2), 4.78 (d, J = 2.4 Hz, 1H, H-5), 4.75 (d, J = 11.4 Hz, 1H, CH₂Ph), 4.72 (d, J = 11.4 Hz, 1H, CH₂Ph), 3.95 (t, J = 3.0 Hz, 1H, H-3), 3.76 (s, 3H, CO₂Me), 2.10 (s, 3H, Ac), 2.09 (s, 3H, Ac), 2.02 (s, 3H, Ac) ppm; ¹³C NMR (150 MHz, CDCl₃) 169.7 (C), 169.6 (C), 168.4 (C), 167.2 (C), 136.5 (C), 128.5 (CH × 2), 128.2 (CH), 127.8 (CH × 2), 89.8 (CH), 73.2 (CH), 72.9 (CH₂), 72.7 (CH), 67.0 (CH), 65.0 (CH), 52.5 (CH), 20.7 (CH₃), 20.7 (CH₃), 20.5 (CH₃) ppm. HRMS (M + Na⁺) calcd for C₂₀H₂₄O₁₀Na 447.1262, found: 447.1276.

Methyl 4-O-acetyl-3-O-benzyl-β-L-idopyranuronate-1,2-(methyl-orthoacetate) (14). TiBr₄ (106 mg, 0.23 mmol) was added to a solution of 13 (100 mg, 0.18 mmol) in CH₂Cl₂ (2 mL). The resulting mixture was stirred at room temperature (30 °C) for 2 h and then diluted with CH₂Cl₂ (10 mL), and washed with ice-cold water (5 mL). The organic layer was filtered through a Celite 545 pad, and the filtrate was filtered through a phase silicon-treated filtered, concentrated, giving methyl 1-bromo-2,4-di-O-acetyl-3-O-benzyl-L-idofuranoate. A solution of methyl 1-bromo-2,4-di-O-acetyl-3-O-benzyl-L-idofuranoate in anhydrous DCM (2 mL) containing freshly distilled 2,4,6-trimethyl pyridine (150 μL) and methyl alcohol (140 μL) was then stirred for 2 days at room temperature (30 °C). The mixture was diluted with dichloromethane (10 mL) and the resulting organic phase was washed with a saturated NaHCO₃ solution (2 mL), filtered, and concentrated. The residue was purified by flash chromatography (EtOAc–hexane, 1 : 3 to 1 : 1) to give 14 (58 mg, 64% in two steps) as light yellow oil. R_f = 0.5 (EtOAc–hexane = 1/2); [α]²⁸_D = −2.0 (c 1.0, CH₂Cl₂); IR (KBr) ν 3552, 3031, 1763, 1472, 1505, 1440 cm⁻¹; ¹H NMR (600 MHz, CDCl₃) δ 7.37–7.30 (m, 5H, Ph), 5.55 (d, J = 3.0 Hz, 1H, H-1), 5.18–5.17 (m, 1H, H-4), 4.79 (d, J = 12 Hz, 1H, CH₂Ph), 4.67 (d, J = 11.4 Hz, 1H, CH₂Ph), 4.54 (d, J = 1.2 Hz, 1H, H-5), 4.12 (t, J = 2.4 Hz, 1H, H-3), 4.07–4.06 (m, 1H, H-2), 3.77 (s, 3H, CO₂CH₃), 3.23 (s, 3H, OCH₃), 2.02 (s, 3H, OCOCH₃), 1.72 (s, 3H, CH₃) ppm; ¹³C NMR (150 MHz, CDCl₃) δ 170.0 (C), 168.0 (C), 136.7 (C), 128.5 (CH × 2), 128.2 (CH), 127.9 (CH × 2), 124.1 (C), 96.5 (CH), 76.0 (CH), 72.8 (CH₂), 71.2 (CH), 69.5 (CH), 66.7 (CH), 52.5 (CH₃), 49.0 (CH₃), 24.9 (CH₃), 20.6 (CH₃) ppm. HRMS (M + Na⁺) calcd for C₁₉H₂₄O₉Na 419.1313, found: 419.1316.

Methyl 3-O-benzyl-β-L-idopyranuronate 1,2-(methylorthoacetate) (15). Compound 14 (1.59 g, 0.55 mmol) was dissolved in methanol (20 mL) and cooled to −0 °C. A 0.5 M solution NaOMe (6.40 mL) was added, and the reaction mixture was stirred at 0 °C for 2 h and at 5 °C overnight. The solution was diluted with CH₂Cl₂ (20 mL) at 5 °C, quenched with aqueous NaHCO₃ and H₂O (10 mL each), and then extracted with (25 mL × 3). The organic fractions were dried over MgSO₄ and concentrated under reduced pressure. Purification by silica gel flash chromatography [EtOAc–hexanes (1 : 4 to 1 : 1) + 1% Et₃N] yielded 15 (971 mg, 68%) as a colourless oil. R_f = 0.3 (EtOAc–hexane = 1/2); [α]²⁸_D = −0.5 (c 1.0, CH₂Cl₂); IR (KBr) ν 3570, 3030, 1762, 1737, 1511, 1441 cm⁻¹; ¹H NMR (600 MHz, CDCl₃) δ 7.39–7.31 (m, 5H, Ph), 5.50 (d, J = 1.8 Hz, 1H, H-1), 4.71 (d, J = 11.4 Hz, 1H, CH₂Ph), 4.64 (d, J = 11.4 Hz, 1H, CH₂Ph), 4.48 (s, 1H, H-4), 4.15–4.08 (m, 2H, H-2, H-5), 3.81 (s, 3H, CO₂CH₃), 3.29 (s, 3H, OCH₃), 2.78 (d, J = 11.4 Hz, 1H, H-3), 1.75 (s, 3H, CH₃) ppm; ¹³C NMR (150 MHz, CDCl₃) δ 168.8 (C), 136.7 (C), 128.6 (CH × 2), 128.3 (CH), 127.8 (CH × 2), 123.4 (CH), 96.7 (CH), 75.7 (CH), 72.9 (CH), 72.8 (CH₂), 71.7 (CH), 66.9 (CH), 52.4 (CH₃), 50.2 (CH₃), 24.3 (CH₃) ppm. HRMS (M + Na⁺) calcd for C₁₇H₂₂O₈Na 377.1207, found: 377.1211.

Methyl 3-O-benzyl-4-O-chloroacetyl-β-L-idopyranuronate-1,2-(methylorthoacetate) (16). DCM (15 mL) and compound 15 (374 mg, 1.05 mmol) were charged into a round bottom flask under a nitrogen atmosphere then cooled to 0 °C. Pyridine (422 μL, 5.25 mmol) was subsequently added to the reaction mixture and then cooled to −20 °C. A solution of chloroacetyl chloride (333 μL, 4.2 mmol) was slowly charged into the reaction mixture at −20 °C. After stirring the reaction mixture for 12 h, the reaction mass was diluted with DCM (10 mL) and quenched into cold water (10 mL). The organic and aqueous layers were separated and the organic layer was washed with a NaHCO₃ solution and dried over magnesium sulfate. After evaporation, the residue was purified in a silica gel column using the solvent system 20 : 80 : 1 (EtOAc–Hexane–Et_3N) to afford 16 as a faint yellow solid (401 mg, 89%). R_f = 0.4 (EtOAc–hexane = 1/2); mp = 120–121 °C; IR (KBr) ν 3607, 3089, 3002, 1752, 1497, 1329 cm⁻¹; ¹H NMR (600 MHz, CDCl₃) δ 7.39–7.32 (m, 5H, Ph), 5.56 (d, J = 2.4 Hz, 1H, H-1), 5.24 (t, J = 1.2 Hz, 1H, H-4), 4.81 (d, J = 12.0 Hz, 1H, CH₂Ph), 4.67 (d, J = 11.4 Hz, 1H, CH₂Ph), 4.57 (d, J = 1.2 Hz, 1H, H-5), 4.15 (t, J = 2.4 Hz, 1H, H-3), 4.07 (s, 1H, H-2), 4.05 (d, J = 15.0, 1H, ClCH₂CO), 4.00 (d, J = 15.0, 1H, ClCH₂CO), 3.79 (s, 3H, COOMe), 3.24 (s, 3H, OCH₃), 1.71 (s, 3H, CH₃) ppm; ¹³C NMR (150 MHz, CDCl₃) δ 167.6 (C), 166.6 (C), 136.5 (C), 128.6 (CH × 2), 128.4 (CH), 128.0 (CH × 2), 124.1 (CH), 96.5 (CH), 75.8 (CH), 73.0 (CH₂), 71.0 (CH), 69.3 (CH), 68.3 (CH), 52.7 (CH₃), 49.2 (CH₃), 40.4 (CH₂), 24.9 (CH₃) ppm. HRMS (M + Na⁺) calcd for C₁₉H₂₃O₉ClNa 453.0923, found: 453.0941.

Methyl 2-O-acetyl-3-O-benzyl-4-O-(chloroacetyl)-2-O-acetyl-L-idopyranuronate (17). Compound 16 (100 mg) was dissolved in a solution of AcOH–H₂O (2 mL, 9/1) and stirred for 30 min at 28 °C. After evaporation, the residue was purified in a silica gel column using the solvent system 1 : 3 (EtOAc–toluene) to afford product 17 (90 mg, 94%) as a colourless oil (β/α mixture). R_f = 0.2 (EtOAc–hexane = 1/2). IR (KBr) ν 3607, 3089, 3002, 1752, 1497, 1329 cm⁻¹; ¹H NMR (600 MHz, CDCl₃) δ 7.39–7.31 (m, 5H, Ph), 5.31 (d, J = 7.2 Hz, 1H, H-1β), 5.27–5.26 (m, 1H, H-2α), 5.18–5.17 (m, 1H, H-1α), 5.03 (d, J = 1.8 Hz, 1H, H-4α), 4.92–4.91 (m, 0.6H, H-5α), 4.83–4.82 (m, 1H, H-5β), 4.77 (s, 1H, CH₂Ph), 4.74 (s, 0.6H, H-4β), 4.39 (d, J = 8.4 Hz, 1H, H-2β), 4.03–3.99 (m, 2H, H-3β, CH₂Cl), 3.97 (d, J = 1.8 Hz, 1H, CH₂Cl), 3.95 (d, J = 1.2 Hz, 1H, 0.6H, H-3β), 3.93 (dt, J = 3.0, 1.8 Hz, 1-H, H-3α), 3.77–3.76 (m, 3H, 2 × CH₃OMe), 2.11 (s, 3H, CH₃OAc), 2.05 (s, 3H, CH₃OAc); ¹³C NMR (150 MHz, CDCl₃) δ 170.1 (C), 169.6 (C), 168.2 (C), 167.3 (C), 166.3 (C), 136.4 (C), 128.6 (CH × 2), 128.5 (CH), 128.4 (CH), 128.0 (CH × 2), 127.8 (CH), 92.8 (CH), 91.9 (CH), 73.4 (CH₂), 73.1 (CH₂), 72.6 (CH), 72.2 (CH), 71.7 (CH), 68.5 (CH), 68.5 (CH), 67.6 (CH), 66.5 (CH), 65.3 (CH), 52.6 (CH), 40.2 (CH₃), 40.2 (CH₃) ppm. HRMS (M + Na⁺) calcd for C₁₈H₂₁O₉NaCl 439.0766, found: 439.0783.

Methyl 2-O-acetyl-3-O-benzyl-4-O-(chloroacetyl)-2-O-acetyl-L-idopyranuronate trichloroacetimidate (4). Trichloroacetonitrile (173 μL, 1.72 mmol) and K₂CO₃ (166 mg, 1.15 mmol) were added to a solution of 17 (96 mg, 0.23 mmol) in CH₂Cl₂ (4 mL). After stirring for 12 hours at room temperature (22 °C), the reaction mixture was quenched with water and NaHCO₃, dried over MgSO₄, filtered, and concentrated to give the crude residue, which was purified by column chromatography [EtOAc–hexane (1 : 3 to 1 : 1) + 1% NEt₃] to afford 4 as a 1 : 1 α/β mixture (80 mg, 62%). R_f 0.6 (EtOAc–hexane = 1/2). The imidate 19 were used directly in the next step.

Methoxy-[methyl-2-O-acetyl-3-O-benzyl-(4-chloroacetyl)-L-idofuranuranuate]-(1 → 4)-O-6-O-acetyl-2-benzylformate-2-deoxy-α,β-D-glucopyranoside (3). The imidate 4 (80 mg, 0.14 mmol) and acceptor 5 (ref. 22) (59 mg, 0.12 mmol) were azeotropically dried with toluene and dissolved in DCM (2 mL). A 4 Å molecular sieve (100 mg) was added, and the mixture was then stirred for 30 min at 21 °C. TMSOTf (8 μL, 0.045 mmol) was then added and stirred for 5 minutes. The mixture was stirred continuously for another 1 hour. The residue was purified by column chromatography with (EtOAc–toluene, 1 : 4) to afford the colourless oil 3α (11 mg) and white solid 3β (33 mg) in 40% yield. 3α-form: R_f = 0.3 (EtOAc–hexane = 1/1.5); [α]²⁴_D = 0.5 (c 1.0, CH₂Cl₂); IR (KBr) ν 3796, 3034, 2362, 1734, 1409, 1370, 1237 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.35–7.24 (m, 15H, Ph), 5.66 (d, J = 7.6 Hz, 1H, H-5′), 5.32 (s, 1H, H-1′), 5.22 (s, 1H, H-2′), 5.09–5.03 (m, 2H, CH₂Ph), 4.82–4.80 (m, 2H, N-H & CH₂Ph), 4.68 (d, J = 9.6 Hz, 1H, CH₂Ph), 4.65–4.63 (m, 1H, H-4′), 4.62 (d, J = 3.6 Hz, 1H, H-1), 4.54 (d, J = 11.2 Hz, 1H, CH₂Ph), 4.41 (d, J = 11.2 Hz, 1H, CH₂Ph), 4.32–4.23 (m, 2H, 6-Ha′, 6-Hb′), 4.12–4.10 (m, 1H, H-3′), 4.07–3.90 (m, 3H, OCH₂Cl, CH₂Ph), 3.77–3.63 (m, 3H, H-3, H-4, H-5), 3.61 (s, 3H, COOMe), 3.32 (s, 3H, OMe), 2.10 (s, 3H, CH₃OAc), 2.05 (s, 3H, CH₃OAc) ppm; ¹³C NMR (150 MHz, CDCl₃, ppm): δ 170.8 (C), 169.5 (C), 167.6 (C), 166.1 (C), 155.7 (C), 138.0 (C), 136.7 (C), 136.2 (C), 128.4 (CH), 128.4 (CH × 3), 128.3 (CH × 3), 128.2 (CH × 2), 128.2 (CH × 4), 128.0 (CH), 127.6 (CH), 107.8 (C), 98.8 (CH), 80.7 (CH), 79.6 (CH), 79.4 (CH), 78.9 (CH), 76.2 (CH), 74.7 (CH₂), 72.6 (CH₂), 68.6 (CH), 66.9 (CH₂), 62.4 (CH₂), 55.1 (CH₃), 54.6 (CH), 52.5 (CH₃), 40.5 (CH₂), 20.8 (CH₃), 20.7 (CH₃) ppm. HRMS (M + Na⁺) calcd for C₄₂H₄₈O₁₆NClNa 880.2554, found: 880.2530. 3β-form: R_f = 0.2 (EtOAc–hexane = 1/1.4); mp = 116–118 °C; IR (KBr) ν 3851, 3589, 2953, 1736, 1646, 1521, 1368, 1240 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.38–7.12 (m, 15H, Ph), 5.16–5.05 (m, 3H, CH₂Ph, H-1′, H-5), 4.99 (d, J = 2.4 Hz, 1H, NH), 4.97 (d, J = 2.4 Hz, 1H, CH₂), 4.86–4.83 (m, 2H, H-4, H-5′), 4.76–4.71 (m, 2H, H-2, CH₂Ph), 4.65 (t, J = 3.6 Hz, 1H, H-1), 4.61 (s, 1H, CH₂), 4.49 (d, J = 11.2 Hz, 2H, CH₂Ph), 4.21 (dd, J = 12.4, 3.6 Hz, 1H, 6Ha′), 4.07–4.05 (dt, J = 10.8, 3.6 Hz, 1H, 6Hb′), 3.96 (d, J = 7.2 Hz, 2H, CH₂Cl), 3.92–3.90 (m, 1H, H-3′), 3.82 (t, J = 3.2 Hz, 1H, H-2′), 3.57–3.48 (m, 2H, H-4′, CH₂Ph), 3.40 (s, 3H, COOMe), 3.35 (s, 3H, OMe), 2.12 (s, 3H, CH₃OAc), 2.03 (s, 3H, CH₃OAc) ppm; ¹³C NMR (150 MHz, CDCl₃) δ 170.7 (C), 169.6 (C), 168.2 (C), 166.4 (C), 155.7 (C), 137.8 (C), 137.0 (C), 136.0 (C), 128.5 (CH), 128.4 (CH × 3), 128.1 (CH × 3), 128.1 (CH × 2), 128.1 (CH × 3), 127.9 (CH), 127.2 (CH), 98.8 (C), 97.5 (CH), 78.9 (CH), 74.8 (CH), 74.3 (CH), 72.8 (CH₂), 72.4 (CH₂), 69.4 (CH), 69.2 (CH₂), 67.1 (CH), 66.9 (CH), 66.4 (CH), 63.0 (CH₂), 62.1 (CH₂), 55.2 (CH₃), 54.5 (CH), 52.2 (CH₃), 40.3 (CH₂), 29.3 (CH₂), 20.9 (CH₃), 20.8 (CH₃) ppm. HRMS (M + Na⁺) calcd for C₄₂H₄₈O₁₆NClNa 880.2554, found: 880.2536.

Acknowledgements

The authors wish to thank the Ministry of Science and Technology (MOST) in Taiwan (103-2113-M-005-010) and National Chung Hsing University for their financial support.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra17050d

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