Synthesis of 1 , 3-divalent glycoconjugates with diverse structures and their functionalization

A series of novel 1,3-difunctionalized glycoconjugates were synthesized using a sequence of regioselective functionalization and stereoselective glycosidation of D-glucose and D-GlcNAc. Regioselective C-3 functionalization of sugar molecules was achieved by chemical functionalization of isopropylidene or oxazoline protected sugar derivatives. The structural diversity at the anomeric carbon was explored by stereoselective chemical glycosidation. The oxazoline protected D-GlcNAc derivative gave either pyranose or furanose derivatives on glycosidation depending on the amount of Lewis acid used. The diversely functionalized glycoconjugates with azide or alkyne groups are potentially useful for the synthesis of multifunctionalized complex glycoconjugates via click reactions.


INTRODUCTION
Chemical synthesis of diversely functionalized biomolecules with targeted medical applications is a rapidly growing area of research in synthetic organic chemistry and chemical biology.Diversity oriented chemical synthesis of hybrid biomolecules not only generates a large number of compounds within a shorter period, but also produces a series of diversely functionalized molecules from the common starting material and by a common methodology.2][3] Carbohydrates, being the most abundant biomolecule with unparalleled structural cipation of the C-2 acetamido group in addition to the scope for the formation of hexofuranose and hexopyranose rings as products (Fig. 1).

General Information
All the solvents were used after distillation and dry solvents were prepared using standard methods.All reagents purchased from commercial sources were used without any purification. 1H-and 13 C-NMR spectra were recorded using 400 and 500 MHz NMR spectrometer.All mass spectra were recorded in Q-TOF electrospray ionization spectrometer.Column chromatography was performed over 100-200 mesh silica with ethyl acetate and hexane as the eluent.
Analytical and spectral data of the synthesized compounds are given in Supplementary material to this paper.

Syntheses
Synthesis of 1,2,4,6-tetra-O-acetyl-3-O-propargyl-D-glucopyranose (4).A solution of sodium hydride (240 mg, 10 mmol) in dry DMF (10 mL) in a 100-mL round bottom flask was cooled to 0 °C and a solution of diacetone glucose (1.3 g, 5.0 mmol in 10 mL DMF) was added.The reaction was continued at that temperature for 30 min and then propargyl bromide (10 mmol) was added under stirring.The reaction mixture was allowed to come to room temperature and the reaction continued for 24 h at room temperature.After completion of the reaction, as indicated by TLC, ethyl acetate (50 mL) was added followed by water (50 mL) and the two layers were separated.The organic layer was washed with distilled water (3×30 mL) followed by brine solution (30 mL), dried over anhydrous sodium sulphate and concentrated to dryness.The crude product was dissolved in a mixture of THF and water (9:1).To this solution, Dowex H + (30 wt.%) resin was added.The reaction mixture was stirred for 24 h at room temperature.After disappearance of the starting material, confirmed by TLC analysis, the reaction mixture was filtered through filter paper.After filtration of the resin, the filtrate was then concentrated and repeatedly washed with ethyl acetate to give a syrup.Acetylation of the hydroxyl groups of the syrupy product was realised using acetyl chloride along with sodium acetate as base to obtain 1,2,4,6-tetra-O-acetyl-3-O-propargyl-Dglucopyranose. 28The crude product was purified by column chromatography to give an overall yield of 80 %.

Synthesis of methyl 2,4,6-tri-O-acetyl-3-O-propargyl-α-D-glucopyranoside (7).
3-O-Propargyl-D-glucopyranose (3, 1 mmol) was dissolved in dry methanol.To this, IR 120 H + resin (0.5 g) was added and the reaction mixture was stirred at room temperature.After completion of the reaction, as indicated by TLC, the crude product was filtered and concentrated to dryness to give a syrup.The syrupy product was per-O-acetylated using acetyl chloride along with sodium acetate as base in acetonitrile at 60 °C to give the methyl 2,4,6-tri-O-acetyl-3-O--propargyl-α-D-glucopyranoside in 90 % yield after column purification using a mixture of ethyl acetate and hexane (1:2) as eluent.
Synthesis of 1,3-difunctionalized GlcNAc derivatives 10 and 11. 2-Acetamido-2-deoxy--D-glucopyranose (5 mmol) was converted to the corresponding oxazoline derivative 8 following a literature procedure using dry acetone and ferric chloride. 22odium hydride (96 mg, 4.0 mmol) dissolved in dry DMF (5 mL) was cooled to 0 °C.To this, oxazoline 8 (2 mmol) dissolved in DMF (5 mL) was added dropwise under stirring.After 30 min, propargyl bromide (2 mmol) was added to the reaction mixture and the reaction allowed to come to room temperature and continued until the starting material had been consumed.The reaction mixture was diluted with ethyl acetate (50 mL) and the organic layer was repeatedly washed with water, dried over sodium sulphate and concentrated to dryness to obtain the crude product 9, which was used for further reactions without any purification.The crude product 9 (1 mmol) obtained in the previous step was dissolved in anhydrous propargyl alcohol (1 mL).To this, p-TSA (0.5 equiv.) was added under nitrogen.The reaction was continued until disappearance of the starting material.The reaction mixture was dried under vacuum.Acetylation of hydroxyl groups was realised using acetic anhydride and pyridine (1:1, 2 mL).The crude product was purified by column chromatography using a mixture of ethyl acetate and hexane (1:1) to afford compound 10.
Compound 11 was prepared from 9 using the same methodology as that for 10 except n-decanol (1 mL for 1 mmol) was used in place of propargyl alcohol.
Synthesis of 1,3-difunctionalized GlcNAc derivatives 13 and 14.A solution of oxazoline 8 (2 mmol) in dry DMF (5 mL) was added dropwise to a mixture of sodium hydride (96 mg, 4 mmol) in dry DMF (5 mL) under stirring at 0 °C.After 30 min, n-decyl bromide (2 mmol) was added to the reaction mixture and stirring was continued at room temperature until the starting material had disappeared.The reaction mixture was diluted with ethyl acetate (50 mL) and the organic layer was repeatedly washed with water, dried over sodium sulphate and concentrated to dryness to obtain the crude product 12.
Crude product 12 (1 mmol) was dissolved in anhydrous n-decanol (1 mL) and p-TSA (0.2 equiv.) was added under nitrogen.The reaction mixture was dried under vacuum after disappearance of the starting material.Purification of the product using column chromatography with ethyl acetate and hexane (1:1) as the eluent resulted in compound 13.
Compound 14 was prepared from 12 using the similar methodology to that used for 13 except 1 equiv. of p-TSA (in place of 0.2 equiv.used in synthesis of compound 13) and propargyl alcohol (1 mL for 1 mmol) in place of n-decanol, followed by overnight reaction with a mixture of acetic anhydride and pyridine (1:1, 2 mL).

RESULTS AND DISCUSSION
The synthesis of 1,3-difunctionalized glycoconjugates derived from D-glucose was initiated by the alkylation of the C-3 hydroxyl group of 1,2:5,6-di-O--isopropylidene-α-D-glucofuranose (1) with propargyl bromide using sodium hydride as the base in dry DMF to furnish the corresponding C-3 O-propargylated derivative 2. The two acetonide protecting groups were removed under acidic condition using Dowex H + resin in aqueous THF to obtain C-3 O-propargylated glucopyranose 3. Per-O-acetylation of compound 3 was realised using acetyl chloride and sodium acetate in acetonitrile.The crude product was purified by column chromatography using a mixture of ethyl acetate and hexane as eluent to furnish 1,2,4,6-tetra-O-acetyl-3-O-propargyl-D-glucopyranose (4) in 80 % overall yield over three steps (Scheme 1). 27In the 1 H-NMR (500 MHz, CDCl 3 ) spectrum of 5, the anomeric proton appeared as a doublet at 4.52 ppm with a coupling constant of 9.0 Hz, confirming the β-linkage.The formation of the compound was further confirmed by 1 H-1 H COSY, 13 C-NMR and ESI-MS HRMS spectral data.4) was chosen as the glycosyl donor.Glycosidation was realised by taking n-dodecanol and SnCl 4 in dry DCM as catalyst to furnish the 3-O-propargylated glycolipid 6 as the α-isomer (Scheme 3).The formation of the α-isomer was confirmed by 1 H-NMR where the anomeric proton appeared as a doublet at 5.06 ppm with a coupling constant of 3.6 Hz.The formation of the compound was further confirmed by 1 H-1 H COSY, 13 C-NMR and the presence of the molecular ion peak in the ESI-MS HRMS spectroscopic data.In addition to the C-3 propargylated per-O-acetylated derivative 4, the 3-O--propargylated free sugar derivative 3 was also used in the glycosidation reaction under acidic conditions.The reaction of compound 3 with methanol in the presence of a catalytic amount of IR120 H + resin followed by per-O-acetylation of the hydroxyl groups using acetyl chloride in presence of sodium acetate resulted in the formation of the 3-O-propargylated per-O-acetylated methyl glycoside 7 (Scheme 4).The anomeric proton had a coupling constant of 2.8 Hz with the H-2 proton in the 1 H-NMR spectrum, which suggested the formation of the isomer as the only product.Formation of the compound was further confirmed by other spectroscopic techniques, i.e., 1 H-1 H COSY, 13 C-NMR and presence of the molecular ion peak in the ESI-MS HRMS spectroscopic data.After synthesizing different 1,3-difunctionalized glycoconjugates derived from D-glucose, several 1,3-difuctionalized glycoconjugates were synthesized from 2-acetamido-2-deoxy-D-glucopyranose (D-GlcNAc).For the synthesis of D-GlcNAc derivatives, a novel oxazoline intermediate 8 was synthesized following a literature procedure, by reacting 2-acetamido-2-deoxy-D-glucopyranose with acetone catalyzed by ferric chloride.The 1,2-oxazoline ring of sugars has advantages over 1,2-isopropylidene protections, not only for its facile opening but also for selective formation of a β-glycosidic bond. 22xazoline 8 was reacted with propargyl bromide using sodium hydride as base to synthesize the 3-O-propargylated oxazoline derivative 9. Compound 9 was used for glycosidation using different alcohols, i.e., propargyl alcohol and n-decanol, using p-TSA (0.5 mol %) as catalyst followed by per-O-acetylation using acetic anhydride and pyridine to furnish the 1,3-dipropargylated GlcNAc derivative 10 and 3-O-propargylated n-decyl glucoside 11, respectively (Scheme 5).Formation of both the compounds was confirmed by NMR and ESI-MS HRMS spectroscopic data.The 1 H-NMR spectrum of compound 10 revealed the formation of furanose glycoside where the anomeric proton appeared as a multiplet in the range 5.09--5.03ppm along with the H-4 proton.The exocyclic H-5 proton appeared as a multiplet in the 3.95-3.91ppm range.Two alkyne protons appeared as multiplets in the 2.51-2.49ppm range.In case of compound 11, the anomeric proton appeared as a doublet at 4.85 ppm with a coupling constant of 2.4 Hz, confirming the formation of the furanose ring.The Lewis acid (0.5 mol % p-TSA) used in these reactions for glycosidation also catalyzed the deprotection of the 5,6-isopropylidene group.
After synthesizing the 3-O-propargylated glycosides, attempts were made towards the synthesis of the corresponding C-3 decyl glycosides.For the synthesis of these derivatives, oxazoline 8 was used as the synthon and was reacted with n-decyl bromide using sodium hydride as the base to obtain the C-3 O-decyl derivative 12, which was used for the glycosidation reaction (Scheme 6).Compound 12 was reacted with two different alcohols in presence of p-TSA in different amounts.When it was reacted with n-decanol using 0.2 mol % of p-TSA, the resultant product, compound 13, was found to be a furanose glycoside without affecting the acetonide protection.In the 1 H-NMR spectrum of compound 13, the anomeric proton appeared as a singlet at 4.88 ppm.Two singlets appeared at 1.43 and 1.35 ppm, indicating the presence of acetonide protection.The formation of the compound was further confirmed by ESI-MS HRMS.When compound 12 was reacted with propargyl alcohol with 1 mol % of p-TSA followed by per-O-acetylation using acetic anhydride and pyridine, the resulting glycoconjugate 14 was found to be in the hexopyranose form, for which the anomeric proton was found to be a doublet at 4.85 ppm with a coupling constant of 8.0 Hz.This experiment showed the scope of the reaction for the synthesis of glycosides with different conformations (hexopyranose or hexofuranose) using different amounts of the Lewis acid.Scheme 6. Synthesis of C-3 decyl functionalized GlcNAc derivatives.

CONCLUSIONS
In summary, seven 1,3-difunctionalized glycoconjugates were synthesized from oxazoline or isopropylidene protected carbohydrate derivatives that yielded either pyranose or furanose derivatives depending on the amount of Lewis acid used in glycosidation.This is a unique example of substrate specific regio and stereoselectivity synthesis of furanose and pyranose ring containing glycolipids with one or two long chain alkyl groups or even propargyl groups.The propargyl functionalized glycolipids are useful synthetic biomolecules that can be utilized as clickable chemical ligating agent for the synthesis of hybrid glycolipids by a Cu(I)-catalyzed click reaction with azide functionalized biomolecules.2,4,6-Tri-O-acetyl-3-O-propargyl-β-D-glucopyranosyl azide could be used for the synthesis of complex glycoconjugates or glycopolymers by a [3+2] cycloaddition reaction.The concept of selective protection and deprotection of the hydroxyl groups is potentially useful for the functionalization of the carbohydrate derivatives at different positions leading to biologically important glycoconjugates.This methodology for formation of hexopyranose or hexofuranose ring containing glycosides derived from D-glucose or D-GlcNAc could be explored for the synthesis of other diversely functionalized glycoconjugates.

Scheme 2 .
Scheme 2. Synthesis of 2,4,6-tri-O-acetyl-3-O-propargyl-β-D-glucopyranosyl azide 5.The methodology of synthesising 1,3-difunctionalized glycoconjugates was further extended to the preparation of other glycoconjugates, such as the α-linked C-3 O-propargylated glycolipid.For the synthesis of selectively functionalized glycolipids, 1,2,4,6-tetra-O-acetyl-3-O-propargyl-D-glucopyranose (4) was chosen as the glycosyl donor.Glycosidation was realised by taking n-dodecanol and SnCl 4 in dry DCM as catalyst to furnish the 3-O-propargylated glycolipid 6 as the α-isomer (Scheme 3).The formation of the α-isomer was confirmed by 1 H-NMR where the anomeric proton appeared as a doublet at 5.06 ppm with a coupling constant of 3.6 Hz.The formation of the compound was further confirmed by 1 H-1 H COSY,13 C-NMR and the presence of the molecular ion peak in the ESI-MS HRMS spectroscopic data.