Expeditious synthesis of the tetrasaccharide cap domain of the Leishmania donovani lipophosphoglycan using one-pot glycosylation reactions

Abstract

Expeditious syntheses of the tetrasaccharide cap related to the lipophosphoglycan of Leishmania donovani from two monosaccharides and a disaccharide building block were achieved by one-pot sequential glycosylation reactions. The monosaccharide building blocks were synthesized from D-mannose, and the disaccharide building block was prepared from lactose by C2-epimerisation of its glucose unit through C2 hydroxy oxidation–reduction.

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Introduction

Leishmaniasis, a vector-borne parasitic disease caused by obligate intracellular protozoa, is endemic in more than 88 countries affecting over 12 million people worldwide, causing the deaths of 59 000 people, and leading to almost 1.5 to 2 million new cases each year.¹ Unfortunately, there is no effective vaccine for the treatment of leishmaniasis and the approved drugs suffer from a series of disadvantages like complicated side effects, high cost and ineffectiveness to patients residing at many corners of the world.² The visceral form of leishmaniasis (VL) is also known as kala-azar (Hindi for black sickness or fever) and causes swelling of the liver and spleen. It is often lethal without treatment. Shockingly, about 500 000 new cases of VL occur each year, and approximately 50 percent of the reported VL infections occur in the Asian subcontinent.³ Although kala-azar is most threatening in this subcontinent, cases have also been diagnosed in overseas travellers and US Gulf War veterans.⁴ Unfortunately both human immunodeficiency virus (HIV) and VL exert a synergistic detrimental effect as they target similar immune cells.⁵

Direct interactions via the carbohydrate sites of lipophosphoglycans (LPG), which are present abundantly on the promastigote, cause receptor mediated phagocytosis by macrophages and binding of the parasite to the epithelial cells of the sandfly midgut.⁶ It has been reported that LPG is antigenic and also a virulent factor for survival of the parasite and its infectivity.^6,7 LPG deficient mutant Leishmania can neither survive in vector sandfly nor transform to their amastigote form but insertion of exogenous LPG into the plasma membrane can restore both of these functions and can cause risky infection.^7b,8 An earlier structural analysis of Leishmania donovani LPG⁹ showed that heterogeneous LPG consists of four distinct domains:¹⁰ (i) a neutral oligosaccharide cap at the terminal non reducing end, (ii) a variable phosphoglycan repeating unit, (iii) a conserved phosphosaccharide and (iv) a glycophosphatidylinositol (GPI) anchor. The unique features of the structure include a 1,4-β linkage between galactose and mannose in the cryptic tetrasaccharide cap, and phosphoglycan repeats. Each of the domains has been studied and shown to display important structure–activity relationships.¹¹ The neutral oligosaccharide cap contains a signal for termination of phosphoglycan assembly⁶ and an epitope for recognition by macrophage receptors.¹² Attachment of the parasite to the digestive tract of the sandfly and human macrophages is also mediated by this tetrasaccharide cap.

The role of LPG in host–parasite interactions led to major interest from synthetic organic chemists who worked towards its total synthesis and its evaluation for chemotherapeutic vaccine design. The first synthesis of the tetrasaccharide cap was reported by Fraser-Reid.¹³ Although thereafter a few other research groups synthesized the cap tetrasaccharide,^14–17 the introduction of an economic one-pot total synthesis is still necessary and requires the refinement of the entire synthetic protocol.

In a continuation of our work on oligosaccharide syntheses,¹⁸ we report herein the synthesis of the saccharide cap A (Fig. 1), related to the repeating unit present in the LPG of Leishmania donovani, as its 3-(N-benzyloxycarbonyl)propyl glycoside (1, Fig. 2) by sequential one-pot protocols, from the corresponding monosaccharide and disaccharide building blocks.

Fig. 1 Tetrasaccharide cap domain of Leishmania donovani.

Fig. 2 Retro-synthetic analysis of 1.

Result and discussion

For one-pot total synthesis of the tetrasaccharide cap of Leishmania donovani two different pathways have been envisioned. The first requires sequential glycosylation based on a trichloroacetimidate donor and the second is based on a hydroxy donor. For each pathway, retro-synthetic analysis of the fully protected tetrasaccharide 2 led to three building blocks: two orthogonal donors, D-mannose-hydroxy 6 or -trichloroacetimidate 7; thiomannoside 5; and a disaccharide acceptor 3 (Fig. 2). The first glycosyl donor 6 or 7 is a super armed donor with 2-O-acetyl and 3,4,6-tri-O-benzyl protection so that glycosylation through acetoxonium ions produces only α-disaccharides via neighbouring group participation. The second mannose component, phenyl 3,4,6-tri-O-benzyl-1-thio-α-mannopyranoside 5, was chosen to serve a dual purpose during the course of the reaction. At the first step of the sequential glycosylation, it would serve as a glycosyl acceptor with the 2-OH group and then at the second step as a thioglycosyl donor. We thought to prepare the reducing end disaccharide acceptor, 3-(N-benzyloxycarbonyl)propyl glycoside 3, with C-2 axial free OH from lactose (Gal-β 1→4-Glc) via a suitable protecting group manipulation and gluco–manno conversion through inversion of the C-2 centre to give Gal-β 1→4-Man.

Thus, phenyl 2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl-(1→4)-2-O-acetyl-3,6-di-O-benzyl-1-thio-β-D-glucopyranoside 9 ¹⁹ was prepared from commercially available D-lactose via the 1,2-orthoester 8 ¹⁹ (Scheme 1). The reported yield for this thiolation of the 1,2-orthoester 8 was very poor at only 22%. After several trials we modified the reaction conditions by increasing the thiophenol equivalency to 5 and decreasing the reaction time to 1.5 hour. These modifications generated the desired product 9 in 78% yield. Glycosylation of thioglycoside 9 with benzyl-N-(3-hydroxy propyl)carbamate using trichloroisocyanuric acid (TCCA) and TMSOTf²⁰ produced 3-(N-benzyloxycarbonyl)propyl 2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl-(1→4)-2-O-acetyl-3,6-di-O-benzyl-β-D-glucopyranoside 10 in 96% yield. Zemplén deacetylation²¹ of compound 10 furnished 3-(N-benzyloxycarbonyl)propyl 2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl-(1→4)-3,6-di-O-benzyl-β-D-glucopyranoside 11 in almost quantitative yield. To prepare the projected acceptor we needed to invert the equatorial C2 hydroxy group of 11. Initially its conversion to the corresponding triflate followed by an S_N2 inversion at the C2 was envisioned by us, but this sequence of reactions did not result in the desired outcome. Thus we opted for an alternate route viz., C2 oxidation-stereoselective reduction,^17,22 to achieve the desired galactose-(1→4)-mannose acceptor. With this end in view, compound 11 was oxidised using Dess–Martin periodinane (DMPI)²³ in dry CH₂Cl₂ in the presence of catalytic pyridine to produce the corresponding 2-keto compound. Direct reduction of that ketone with sodium borohydride in methanol produced the desired acceptor 3-(N-benzyloxycarbonyl)propyl 2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl-(1→4)-3,6-di-O-benzyl-β-D-galactopyranosyl-(1→4)-3,6-di-O-benzyl-β-D-mannopyranoside 3. The change in substrate polarity (checked by TLC) and spectral analysis confirmed this inversion via oxidation–reduction with 76% yield.

Scheme 1 Synthesis of the disaccharide acceptor 3. Reagents and conditions: (a) I₂, Ac₂O, l h, quantitative. (b) AcOH, 33% HBr–AcOH, 5 h. (c) 2,6-Leutidine, MeOH : DCM 3 : 1, TBAB, 72 h, 81%. (d) BnCl, KOH, THF, 60 °C, 8 h, 88%. (e) PhSH, HgBr₂, MeCN, 60 °C, 1.5 h, 78%. (f) OH(CH₂)₃NHCbZ, TCCA, TMSOTf, 0 °C, DCM, 10 min, 96%. (g) NaOMe, MeOH : DCM (1 : 1), 3 h, quantitative. (h), (i) DMPI, DCM, Py, 2 h. (ii) NaBH₄, MeOH, overnight, 76% over 2 steps.

The carbohydrate moiety 14 ²⁴ was prepared via the 1,2-orthoester 13 ^24a,b of commercially available D-mannose using reported methods (Scheme 2). Zemplén deacetylation²¹ of compound 14 furnished phenyl 3,4,6-tri-O-benzyl-1-thio-D-mannopyranoside 5 ^24a in quantitative yield. Thioglycoside hydrolysis²⁵ of compound 14 was then effected using TCCA in wet acetone to afford 2-O-acetyl-3,4,6-tri-O-benzyl-1-thio-D-mannopyranose 6 in 96% yield. Treatment of 6 with trichloroacetonitrile and DBU in dry CH₂Cl₂ produced the acetimidate donor 7 ^24b in 91% yield.

Scheme 2 Synthesis of D-mannose based donors and acceptor. Reagents and conditions: (a) Ac₂O, I₂, rt, 15 min, quantitative. (b) AcOH, 33% HBr–AcOH, 5 h. (c) 2,6-Leutidine, DCM : MeOH 3 : 1, 8 h, 92%. (d) BnCl, KOH, 60 °C, 8 h, 87%. (e) PhSH, HgBr₂, MeCN, 60 °C, 2 h, 92%. (f) NaOMe, MeOH, rt, 3 h, quantitative. (g) TCCA, (CH₃)₂CO–H₂O, 30 min, 96%. (h) CCl₃CN, DBU, CH₂Cl₂, 91%.

After being equipped with the appropriately tailored building blocks, the one-pot sequential glycosylation reactions leading to the protected tetrasaccharide 2 were started from the reducing end of the compound. Trichloroacetimidate donor 7 and thioglycoside acceptor 5 were allowed to couple using 20 mol% TMSOTf²⁶ in dry CH₂Cl₂ at 0 °C (Scheme 3). After 20 minutes, the consumption of both of the starting materials (as indicated by TLC) resulted in a clean conversion to the desired disaccharide 4. Into the same reaction vessel, the non reducing acceptor 3 was added along with 1 equivalent of NIS. The reaction temperature was cooled to −30 °C and again 20 mol% TMSOTf was added to it. After 30 minutes, TLC showed full conversion to the fully protected tetrasaccharide, and the reaction was quenched by the addition of NEt₃ at that temperature. The crude product was purified by column chromatography providing an 87% yield of the pure desired tetrasaccharide derivative 2.

Scheme 3 Sequential one-pot synthesis of the tetrasaccharide derivative 2.

After achieving the final protected tetrasaccharide via trichloroacetimidate donor activation, we tried to rebuild the same tetrasaccharide in a more economical way, i.e. via sequential one-pot glycosylation based on the initial 1-hydroxy donor.²⁷ For that purpose, to a mixture of 1-hydroxy donor 6, Ph₂SO and TTBP in CH₂Cl₂ at −60 °C was added Tf₂O. The temperature was raised to −40 °C and maintained for 1 hour. Then acceptor 5 was added, and the reaction mixture was allowed to reach room temperature slowly during 3 hours. After a clean conversion to the disaccharide (as indicated by TLC) the reaction mixture was again cooled to −60 °C, and additional Tf₂O was added. After 10 minutes at that temperature, acceptor 3 was added, and the resulting mixture was allowed to reach 0 °C. TLC was used to check the reaction's progress after 1 hour, and the reaction was quenched by addition of NEt₃. The crude product was purified by column chromatography giving an 82% yield of the pure tetrasaccharide derivative 2; this yield was comparable with that based on the previous approach.

NMR and HRMS analyses of compound analyses of compound 2 unambiguously confirmed its formation and structure. Finally deacetylation under Zemplén conditions, followed by global debenzylation using hydrogen and palladium-charcoal in a mixture of water and methanol, afforded the desired tetrasaccharide 1 (Scheme 4). Compound 1 was characterised by NMR techniques (¹H-, ¹³C-, COSY and HSQC) and also by HRMS. The anomeric protons of compound 1 appear at δ 5.44 (b, H₁′′′), 4.87 (bs, H₁), 4.76 (bs, H₁′′) and 4.44 (d, J 8 Hz, H₁′) ppm and the corresponding carbons appear at 99.9, 100.8, 100.5 and 103.1 ppm, respectively.

Scheme 4 Deprotection of the tetrasaccharide derivative 2.

Conclusion

In conclusion, the syntheses of the tetrasaccharide cap of Leishmania donovani via one-pot sequential glycosylation techniques were successfully achieved in excellent yields. A number of notable features are: (a) inversion at C2 of the D-glucosyl moiety of lactose by applying an oxidation-stereoselective reduction approach; (b) step-by-step economic synthesis of the glycosyl donors and acceptors as well as the glycosylated products; and (c) [1 + 1 + 2] one-pot sequential glycosylations. All the reactions described are clean, stereoselective and high yielding.

Experimental

NMR spectra were recorded on a Bruker DPX 300 NMR spectrometer operating at 300 and 500 MHz for ¹H-NMR, and at 75, 100 and 125 MHz for ¹³C-NMR in CDCl₃ and D₂O. HRMS data were recorded on a Q-tof-Micro mass spectrometer using the electron spray ionization method. Specific rotations were measured on a Jasco J-815 spectrometer.

Phenyl 2-O-acetyl-3,4,6-tri-O-benzyl-1-thio-α-D-mannopyranoside (14)²⁴

To a solution of 3,4,6-tri-O-benzyl-β-D-mannopyranosyl-1,2-(methyl orthoacetate) (13, 2.88 g, 5.70 mmol), in dry CH₃CN (30 mL) containing 4 g of molecular sieves (4 Å), thiophenol (5.5 mL, 28.5 mmol, 5 equiv.) and HgBr₂ (205 mg, 0.57 mmol, 0.1 equiv.) were added at room temperature. This mixture was heated with stirring at 60 °C under an argon atmosphere for 2 hours, and then cooled to room temperature. The reaction mixture was filtered through a Celite bed, and the residue was washed with CH₂Cl₂ (3 × 5 mL). The combined filtrate and washings were washed subsequently with cold 5% aqueous NaOH (250 mL) followed by water (2 × 100 mL). The organic layer was dried over anhydrous Na₂SO₄ and concentrated. The residue was purified by column chromatography on silica gel (PE/EtOAc, 9 : 1) to afford 14 as a white solid (3.02 g, 92%); m.p. 64–66 °C; [α]²⁴_D +105.0 (c 1.2, CHCl₃); lit²⁴ m.p. 62–63 °C; [α]¹⁹_D +106.0 (c 1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 2.16 (s, 3H, COCH₃), 3.73 (dd, 1H, J = 1.7, 10.8 Hz), 3.87 (dd, 1H, J = 4.4, 10.8 Hz), 3.93–4.02 (m, 2H), 4.34 (m, 1H), 4.46–4.60 (m, 3H), 4.69 (d, 1H, J = 19.6 Hz), 4.73 (d, 1H, J = 18.8 Hz), 4.90 (d, 1H, J = 10.7 Hz), 5.55 (s, 1H), 5.62 (d, 1H, J = 1.5 Hz), 7.19–7.36 (m, 18H, ArH), 7.47–7.50 (m, 2H, ArH). ¹³C NMR (75 MHz, CDCl₃): δ 21.1, 68.9, 70.4, 72.0, 72.5, 73.4, 74.6, 75.3, 78.5, 86.3, 126.6, 127.0, 127.6, 127.69, 127.71, 127.8, 127.9, 128.2, 128.3, 128.4, 128.5, 129.1, 131.9, 133.5, 133.7, 137.6, 138.2, 138.3, 170.3. HRMS (TOF): calc. for (M + Na)⁺ C₃₅H₃₆O₆SNa 607.2131, found 607.2128.

Phenyl 3,4,6-tri-O-benzyl-1-thio-α-D-mannopyranoside (5)^24a

To a solution of 14 (1.0 g, 1.6 mmol) in dry methanol (10 mL), 1 M methanolic NaOMe (0.2 mL) solution was added, and the reaction mixture was stirred for 3 hours at room temperature. The reaction mixture was then neutralized with Dowex-50W cation exchange resin (H⁺) and filtered. The resin was washed with methanol, and the combined filtrate and washings were concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAc, 5 : 1) to give glassy syrupy product 5 (0.92 g, 99%); [α]²⁷_D +135.9 (c 2.52, CHCl₃); lit²⁴ [α_D] +182.5 (c 1.26, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 3.69 (m, 1H), 3.81 (m, 1H), 3.86–3.98 (m, 2H), 4.26–4.33 (m, 2H), 4.47 (d, 1H, J = 12.0 Hz), 4.54 (d, 1H, J = 10.8 Hz), 4.63 (d, 1H, J = 12.0 Hz), 4.73 (s, 2H), 4.85 (d, 1H, J = 10.8 Hz), 5.62 (s, 1H), 7.22–7.38 (m, 18H, ArH), 7.46–7.48 (m, 2H, ArH). ¹³C NMR (75 MHz, CDCl₃): δ 68.9, 70.0, 72.2, 72.4, 73.5, 74.6, 75.3, 77.3, 80.4, 87.5, 127.5, 127.6, 127.8, 127.9, 128.0, 128.08, 128.14, 126.17, 128.3, 128.4, 128.5, 128.7, 129.1, 131.7, 133.9, 137.7, 138.3, 138.4. HRMS (TOF): calc. for (M + Na)⁺ C₃₅H₃₄O₄SNa 565.2025, found 565.2024.

2-O-Acetyl-3,4,6-tri-O-benzyl-1-thio-D-mannopyranose (6)^24b

A suspension of 14 (1.0 g, 1.6 mmol) and TCCA (372 mg, 1 equiv.) in (CH₃)₂CO–H₂O (4 : 1) was stirred at 0 °C for 30 minutes. After completion of the reaction (as indicated by TLC) (CH₃)₂CO was evaporated in vacuo, and the resulting mixture was diluted with CH₂Cl₂ (15 mL). The solution was washed subsequently with saturated aqueous NaHCO₃ (200 mL) and water. The organic layer was dried over anhydrous Na₂SO₄ and concentrated. The residue was filtered through a column of silica gel (eluent: PE/EtOAc, 4 : 1) to afford 2-O-acetyl-3,4,6-tri-O-benzyl-1-thio-D-mannopyranose 6 as a white foam (808 mg, 96%). ¹H NMR (300 MHz, CDCl₃): δ 2.16 (s, 3H, COCH₃), 3.59–3.78 (m, 5H), 4.03–4.11 (m, 2H), 4.47–4.66 (m, 4H), 4.73 (m, 1H), 4.86 (m, 1H), 5.22 (t, 1H, J = 1.5 Hz), 5.38 (m, 1H), 7.15–7.18 (m, 2H, ArH), 7.27–7.37 (m, 15H, ArH). HRMS (TOF): calc. for (M + Na)⁺ C₂₉H₃₂O₇Na 515.2046, found 515.2040.

2-O-Acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl trichloroacetimidate (7)^24b

To a suspension of 6 (800 mg, 1.5 mmol) in dry CH₂Cl₂ (10 mL), trichloroacetonitrile (0.23 mL, 2.25 mmol, 1 equiv.) and DBU (0.04 mL, 0.45 mmol, 0.3 equiv.) were added dropwise at −5 °C. After completion of the reaction, CH₂Cl₂ was removed under rotary evaporation. The sticky brown mass was purified by chromatography on a 60–120 mesh silica gel column (PE/EtOAc, 9 : 1) to give pure compound 7 as a colourless syrup (920 mg, 91%). [α]²⁴_D +30.1 (c 1.2, CHCl₃); lit^24b [α]¹⁹_D +36.3 (c 0.9, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 2.20 (s, 3H, COCH₃), 3.72 (m, 1H), 3.85 (m, 1H), 4.00–4.06 (m, 3H), 4.51 (d, 1H, J = 8.1 Hz), 4.54 (d, 1H, J = 6.6 Hz), 4.58 (d, 1H, J = 11.2 Hz), 4.69 (d, 1H, J = 12.1 Hz), 4.74 (d, 1H, J = 11.2 Hz), 4.88 (d, 1H, J = 10.6 Hz), 5.5 (m, 1H), 6.31 (d, 1H, J = 1.7 Hz), 7.17–7.20 (m, 2H, ArH), 7.29–7.37 (m, 13H, ArH), 8.69 (s, NH). HRMS (TOF): calc. for (M + Na)⁺ C₃₁H₃₂Cl₃NO₇Na 659.1142, found 659.1148.

Phenyl 2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl-(1→4)-2-O-acetyl-3,6-di-O-benzyl-1-thio-β-D-glucopyranoside (9)²⁰

To a solution of 2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl-(1→4)-3,6-di-O-benzyl-α-D-glucopyranose-1,2-(methylorthoacetate) (8, 2.2 g, 2.34 mmol), in dry CH₃CN (30 mL) containing 4 g of molecular sieves (4 Å), thiophenol (1.15 mL, 11.7 mmol, 5 equiv.) and HgBr₂ (84 mg, 0.23 mmol, 0.1 equiv.) were added at room temperature. This mixture was heated under stirring at 60 °C under an argon atmosphere for 1.5 hours, and then cooled to room temperature. The reaction mixture was filtered through a Celite bed, and the residue was washed with CH₂Cl₂ (3 × 5 mL). The combined filtrate and washings were subsequently with cold 5% aqueous NaOH (250 mL) followed by water (2 × 100 mL). The organic layer was dried over anhydrous Na₂SO₄ and concentrated. The residue was purified by column chromatography on silica gel (PE/EtOAc, 9 : 1) to afford 9 as a white solid. It was crystallized from methanol (1.85 g, 78%); m.p. 94–96 °C; [α]²⁴_D −5.9 (c 1.1, CHCl₃); lit²⁰ m.p. 96 °C [EtOH]; [α]²⁵_D −5.1 (c 0.7, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 2.05 (s, 3H, COCH₃), 3.33–3.52 (m, 5H), 3.63 (t, 1H, J = 8.9 Hz), 3.78–3.83 (m, 3H), 3.94–4.01 (m, 2H), 4.22–4.35 (m 2H), 4.39–4.49 (m 3H), 4.53–4.67 (m, 3H), 4.74–4.79 (m, 2H), 4.83–4.88 (m, 2H), 4.97–5.07 (m, 3H), 7.22–7.36 (m, 33H, ArH), 7.52–7.55 (m, 2H, ArH). ¹³C NMR (75 MHz, CDCl₃): δ 21.1, 68.3, 68.4, 71.4, 72.7, 73.2, 73.5, 73.7, 74.7, 75.4, 79.8, 80.0, 82.3, 82.6, 86.0 (C₁), 103.0 (C₁′), 127.05, 127.2, 127.42, 127.48, 127.52, 127.58, 127.6, 127.7, 127.8, 127.9, 128.1, 128.2, 128.3, 128.4, 128.5, 128.9, 132.3, 133.2, 138.47, 138.51, 138.8, 138.9, 139.1, 169.5 (C=O). HRMS (TOF): calc. for (M + Na)⁺ C₆₂H₆₄O₁₁SNa 1039.4067, found 1039.4068.

3-(N-Benzyloxycarbonyl)propyl 2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl-(1→4)-2-O-acetyl-3,6-di-O-benzyl-β-D-glucopyranoside (10)

To a mixture of phenyl 2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl-(1→4)-2-O-acetyl-3,6-di-O-benzyl-1-thio-β-D-glucopyranoside 9 (1.8 g, 1.77 mmol) and benzyl-N-(3-hydroxy propyl)carbamate (445 mg, 2.13 mmol, 1.2 equiv.) in dry CH₂Cl₂ (25 mL), 3 g of flame activated molecular sieves (4 Å) were added. The mixture was stirred at room temperature under an argon atmosphere. After 40 minutes the mixture was cooled to 0 °C, and TCCA (410 mg, 1.77 mmol, 1 equiv.) was added to it. Then TMSOTf (0.096 mL, 0.53 mmol, 0.3 equiv.) was added via a micro-syringe. After 10 minutes, the acceptor was consumed completely (as checked by TLC), the reaction mixture was filtered off through a Celite bed, and the bed was washed with CH₂Cl₂. The combined filtrate and washings were washed subsequently with a saturated NaHCO₃ solution and water. The organic layer was dried over anhydrous Na₂SO₄ and concentrated to afford the glycosylated product. The crude mass was purified by silica gel column chromatography (60–120 mesh) (PE/EtOAc 3 : 1) to get pure product 10 (1.89 g) as a colourless syrup in 96% yield. [α]²⁹_D −2.57 (c 7.0, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 1.68–1.76 (bs, 2H), 1.97 (s, 3H, COCH₃), 3.24 (m, 1H), 3.33–3.37 (m, 3H), 3.38–3.43 (m, 2H), 3.46 (m, 1H), 3.55–3.59 (m, 2H), 3.69 (d, 1H, J = 10.0 Hz), 3.74–3.77 (m, 2H), 3.85 (m, 1H), 3.9 (d, 1H, J = 2.5 Hz), 3.95 (t, 1H, J = 9.0 Hz), 4.23 (d, 1H, J = 12.0 Hz), 4.29–4.34 (apparent t, 2H, J = 10.5, 11.5 Hz), 4.36–4.38 (dd, 2H, J = 1.5, 9 Hz), 4.48 (d, 1H, J = 12.0 Hz), 4.53–4.59 (dd, 2H, J = 11.5, 19.0 Hz), 4.67–4.77 (m, 3H), 4.82 (d, 1H, J = 11.0 Hz), 4.94–4.98 (m, 3H), 5.08 (bs, 2H), 5.29 (bs, 1H), 7.19–7.36 (m, 35H, ArH). ¹³C NMR (75 MHz, CDCl₃): δ 20.9, 29.5, 38.2, 66.5, 66.7, 68.1, 68.3, 72.5, 72.6, 73.1, 73.5, 73.7, 74.3, 74.7, 75.3, 79.9, 80.8, 82.5, 100.8 (C₁), 102.9 (C₁′), 127.2, 127.4, 127.5, 127.57, 127.62, 127.7, 127.9, 128.0, 128.1, 128.2, 128.30, 128.33, 128.4, 128.5, 136.9, 138.1, 138.2, 138.5, 138.8, 139.0, 139.1, 156.6 (C=O), 169.6 (C=O). HRMS (TOF): calc. for (M + Na)⁺ C₆₇H₇₃NO₁₄Na 1138.4929, found 1138.4932.

3-(N-Benzyloxycarbonyl)propyl 2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl-(1→4)-3,6-di-O-benzyl-β-D-glucopyranoside (11)

To a solution of 10 (1.85 g, 1.66 mmol) in a mixture of dry CH₂Cl₂ (10 mL) and dry methanol (8 mL) 1 M methanolic NaOMe (2 mL) solution was added, and the reaction mixture was stirred for 3 hours at room temperature. The reaction mixture was then neutralized with Dowex-50W cation exchange resin (H⁺) and filtered. The resin was washed with methanol, and the combined filtrate and washings were concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAc, 3 : 1) to give glassy syrupy product 11 (1.75 g, 99%); [α]²⁷_D −1.14 (c 5.1, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 1.79–1.81 (bs, 2H), 3.26 (m, 1H), 3.34–3.43 (m, 4H), 3.45–3.49 (m, 2H), 3.55 (t, 1H, J = 8.5 Hz), 3.64–3.69 (m, 2H), 3.74–3.78 (m, 2H), 3.89–3.96 (m, 3H), 4.27–4.32 (m, 3H), 4.36–4.41 (m, 2H), 4.48 (d, 1H, J = 12.0 Hz), 4.55 (d, 1H, J = 11.5 Hz), 4.68–4.71 (m, 2H), 4.75 (m, 2H), 4.81 (d, 1H, J = 11.0 Hz), 4.96 (d, 1H, J = 11.0 Hz), 5.06–5.08 (m, 3H), 5.51 (bs, 1H), 7.17–7.35 (m, 35H). ¹³C NMR (75 MHz, CDCl₃): δ 29.4, 38.2, 66.6, 67.4, 68.3, 72.6, 73.1, 73.5, 73.6, 74.7, 75.27, 75.32, 76.3, 79.9, 82.5, 82.8, 102.7 (C₁), 102.8 (C₁′), 127.37, 127.48, 127.5, 127.71, 127.75, 127.75, 127.9, 128.0, 128.1, 128.2, 128.27, 128.3, 128.40, 128.42, 128.49, 138.5, 136.8, 138.1, 138.2, 138.8, 139.0, 156.7 (C=O). HRMS (TOF): calc. for (M + Na)⁺ C₆₅H₇₁NO₁₃Na 1096.4823, found 1096.4825.

3-(N-Benzyloxycarbonyl)propyl 2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl-(1→4)-3,6-di-O-benzyl-β-D-mannopyranoside (3)

To a solution of 11 (1.70 g, 1.58 mmol) in a mixture of anhydrous CH₂Cl₂ (10 mL) and dry pyridine (0.25 mL, 3.16 mmol, 2 equiv.) was added DMPI (1.4 g, 3.16 mmol, 2 equiv.), and the reaction mixture was allowed to stir at room temperature for 2 hours. The reaction mixture was diluted with CH₂Cl₂ (75 mL), and the organic layer was successively washed with saturated NaHCO₃ solution and water, then dried over anhydrous Na₂SO₄ and finally concentrated under reduced pressure. To the solution of this crude keto product in distilled methanol (30 mL), was added NaBH₄ (895 mg, 23.7 mmol, 15 equiv.) and the reaction mixture was allowed to stir overnight at room temperature. The solvent was removed under reduced pressure, and the crude mass was dissolved in CH₂Cl₂ (100 mL). The organic layer was successively washed with saturated NaHCO₃ solution and water, dried over anhydrous Na₂SO₄ and concentrated under vacuum. The crude mass was purified by silica gel column chromatography (60–120 mesh) (PE/EtOAc 3 : 2) to get pure product 3 (1.29 g) as a colourless syrup in 76% yield. [α]²⁸_D −6.54 (c 2.8, CHCl₃); ¹H (500 MHz, CDCl₃): δ 1.79 (bs, 2H, CH₂), 3.29–3.32 (m, 2H), 3.39–3.43 (m, 2H), 3.45–3.48 (m, 2H), 3.53–3.63 (m, 3H), 3.69–3.72 (m, 2H), 3.76 (d, 1H, J = 9.5 Hz), 3.89 (bs, 2H), 4.02–4.05 (m, 2H), 4.29–4.32 (m, 2H), 4.36–4.43 (m, 4H), 4.57 (d, 1H, J = 11.5 Hz), 4.65–4.71 (m, 4H), 4.74–4.78 (m, 2H), 4.94 (d, 1H, J = 11.5 Hz), 5.06–5.09 (bs, 2H), 5.44 (bs, 1H), 7.17–7.31 (m 35H, ArH). ¹³C NMR (75 MHz, CDCl₃): δ 29.6, 38.3, 66.5, 67.3, 68.5, 68.9, 72.3, 72.7, 73.1, 73.5, 74.4, 74.6, 75.0, 75.1, 77.3, 79.1, 79.8, 82.6, 99.8 (C₁), 103.2 (C₁′), 127.4, 127.5, 127.6, 127.7, 127.8, 127.9, 128.0, 128.1, 128.18, 128.24, 128.3, 128.38, 128.42, 128.5, 136.8, 137.0, 138.2, 138.4, 138.7, 138.9, 156.6 (C=O). HRMS (TOF): calc. for (M + Na)⁺ C₆₅H₇₁NO₁₃Na 1096.4823, found 1096.4826.

3-(N-Benzyloxycarbonyl)propyl (2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1→2)-[(2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl)-(1→4)]-3,6-di-O-benzyl-β-D-mannopyranoside (2)

To a solution of 7 (175.0 mg, 0.26 mmol) and 5 (136.6 mg, 0.24 mmol) in dry CH₂Cl₂ (60 mL), 8 g of activated molecular sieves (4 Å) were added, and the reaction mixture was stirred under an argon atmosphere for 45 minutes. Then the reaction vessel was placed in a 0 °C cold bath, and TMSOTf (10 μL, 0.05 mmol, 0.2 equiv.) was added to it via a micro-syringe. After 10 min, complete consumption of both the starting materials was observed. Acceptor 3 (223.6 mg, 0.21 mmol) and NIS (58.5 mg, 0.26 mmol, 1 equiv.) were then added to the same vessel. After the addition, the reaction temperature was cooled to −30 °C and another 20 mol% of TMSOTf (10 μL, 0.02 mmol) was added to it. The second step of the reaction was completed after 30 minutes (as indicated by TLC). The reaction mixture was quenched by Et₃N, filtered through a Celite bed, and the bed was washed with CH₂Cl₂ (3 × 15 mL). The combined filtrate and washings were washed subsequently with saturated sodium thiosulphate, aqueous NaHCO₃ (2 × 50 mL) and water (2 × 50 mL). The organic layer was dried over anhydrous Na₂SO₄ and concentrated to furnish a syrupy compound. The crude product was purified by flash column chromatography (eluent: PE/EtOAc, 3 : 1) to afford the desired fully protected tetrasaccharide 2 as a white foam (359.15 mg, 87%); [α]²⁵_D²⁵ −14.93 (c 1.97, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 1.75 (bs, 2H, CH₂), 2.07 (s, 3H, COCH₃), 3.11–3.20 (m, 3H, NCH, H₃′′, H₅′), 3.34–3.38 (m, 3H, NCH, H₆, H₆′′), 3.47 (apparent t, 1H, J = 7.0, 8.0 Hz, H₅′′), 3.55–3.67 (m, 8H, OCH, H₂′, H₅, H₆, H₆′, H₆′′, H₆′′′), 3.75–3.81 (m, 5H, OCH, H₂′′, H₄′′, H₃′, H₆′), 3.91–3.98 (m, 2H, H₄′′′, H₆′′′), 4.20–4.28 (m, 7H, H₄, H₃′′, 5BnH), 4.33–4.53 (m, 12H, H₃, H₅′′′, H₁′, H₁′′, 8BnH), 4.57–4.66 (m, 5H, H₂, 4BnH), 4.74–4.80 (apparent t, 2H, J = 13.0 Hz, 2BnH), 4.84–4.90 (apparent t, 3H, J = 12.5, 13.5 Hz, H₁, 2BnH), 5.00–5.10 (2d, 3H, J = 11.5, 12.0 Hz, 3BnH), 5.46 (s, 1H, H₁′′′), 5.54 (bs, 1H, NH), 5.66 (bs, 1H, H₂′′′), 7.07–7.11 (m, 12H, ArH), 7.15–7.32 (m, 51H, ArH), 7.55 (d, 2H, J = 7.0 Hz, ArH). ¹³C NMR (125 MHz, CDCl₃): δ 21.2 (CH₃), 29.9 (CH₂), 37.8 (NCH₂), 66.4 (BnC), 66.6 (C₆/C₆′), 66.9 (BnC), 68.5 (C₆′′), 69.2 (C₂′′′), 69.6 (BnC), 70.1(OCH₂), 70.3 (C₆/C₆′), 70.5 (C₆′′′), 71.5 (BnC), 71.8 (BnC), 72.4 (BnC), 72.44, 72.6 (BnC), 72.7, 73.2 (BnC), 73.3 (BnC), 74.3 (C₄′′′), 74.5 (BnC), 74.6 (BnC), 74.8, 74.9 (BnC), 75.0, 75.1, 75.3, 75.4 (C₅′′′), 75.7, 75.9, 78.7, 79.9, 82.7 (C₃′′), 83.0 (C₅′), 83.4, 97.8 (C₁′′′), 99.9 (C₁), 101.2 (C₁′′), 102.8 (C₁′), 127.26, 127.33, 127.4, 127.5, 127.63, 127.66, 127.7, 127.81, 127.86, 127.9, 128.1, 128.2, 128.3, 128.4, 128.5, 128.6, 137.0, 138.6, 139.2, 156.9 (C=O), 170.6 (C=O). HRMS (TOF): calc. for (M + Na)⁺ C₁₂₁H₁₂₉₁NO₂₄Na 2002.8803, found 2002.8810.

3-(N-Benzyloxycarbonyl)propyl (2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1→2)-[(2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl)-(1→4)]-3,6-di-O-benzyl-β-D-mannopyranoside (2)

2-O-Acetyl-3,4,6-tri-O-benzyl-1-thio-D-mannopyranose 6 (74.4 mg, 0.152 mmol), Ph₂SO (67.8 mg, 0.335 mmol, 2.2 equiv.), and TTBP (56.8 mg, 0.228 mmol, 1.5 equiv.) were dissolved in CH₂Cl₂ (20 mL), and the reaction was cooled to −60 °C. Tf₂O (0.03 mL, 0.183 mmol, 1.2 equiv.) was added, and the reaction mixture was slowly brought to −40 °C in 1 h, after which phenyl 3,4,6-tri-O-benzyl-1-thio-α-D-mannopyranoside 5 (74.4 mg, 0.137 mmol, 0.9 equiv.) in 1 mL dry CH₂Cl₂ was added. The reaction mixture was slowly warmed to room temperature. After 1 hour at room temperature the mixture was cooled to −60 °C, and Tf₂O (0.03 mL, 0.183 mmol, 1.2 equiv.) was added. After the reaction had been kept at −60 °C for 10 minutes, 3-(N-benzyloxycarbonyl)propyl 2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl-(1→4)-3,6-di-O-benzyl-β-D-mannopyranoside 3 (134 mg, 0.122 mmol, 0.8 equiv.) was added. The reaction mixture was warmed to 0 °C and stirred for 1 hour, after which it was quenched with by Et₃N, filtered through a Celite bed, and the bed was washed with CH₂Cl₂ (3 × 15 mL). The combined filtrate and washings were washed subsequently with saturated aqueous NaHCO₃ (3 × 50 mL) and water (3 × 50 mL). The organic layer was dried over anhydrous Na₂SO₄ and concentrated to furnish a syrupy compound. The crude product was purified by flash column chromatography (eluent: PE/EtOAc, 3 : 1) to afford the desired fully protected tetrasaccharide 2 as a white foam (198.17 mg, 82%); the spectral data matched with that for the substrate synthesized previously.

3-(N-Benzyloxycarbonyl)propyl α-D-mannopyranosyl-(1→2)-(α-D-mannopyranosyl)-(1→2)-[(β-D-galactopyranosyl)-(1→4)]-β-D-mannopyranoside (1)

Protected tetrasaccharide 2 (75 mg, 0.038 mmol) was dissolved in CH₂Cl₂ and methanol (1 : 1; 8 mL), and NaOMe (1 M in methanol, 0.5 mL) was added. The mixture was stirred for 2 hours and then the reaction was quenched with Dowex-50W cation exchange resin (H⁺). The resin was filtered off and then washed with methanol (4 × 5 mL). The combined filtrate and washings were evaporated under reduced pressure. A mixture of the resulting mass and 10% Pd–C (70 mg) was taken in methanol (3 mL) and H₂O (1 mL) and stirring was continued under an H₂ atmosphere for 24 h. The catalyst was filtered through a Celite bed, and the bed was washed with methanol (3 × 5 mL). The combined filtrate and washings were concentrated under reduced pressure. The product was passed through a 0.45 μm Millipore membrane, and lyophilized to afford 1 as a white foam (22.89 mg, 84%); ¹H NMR (500 MHz, D₂O): δ 2.03 (m, 2H, CH₂), 3.13–3.16 (t, 2H, J = 7.0 Hz, NCH₂), 3.38–3.45 (m, 2H), 3.54–3.62 (m, 2H, H₄, H₂′), 3.65–3.69 (m, 4H, H₃′), 3.73–3.86 (m, 10H, OCH, H₅, H₃′′), 3.94–4.01 (m, 5H, OCH, H₄′), 4.05–4.13 (m, 3H, H₃, H₂′′), 4.31 (d, 1H, J = 2.5 Hz, H₂), 4.36 (s, 1H H₂′′), 4.44 (d, 1H, J = 8.0 Hz, H₁′), 4.76 (s, 1H, H₁′′), 4.88 (s, 1H, H₁), 5.44 (s, 1H, H₁′′′). ¹³C NMR (125 MHz, D₂O): 27.0 (CH₂), 37.3 (NCH₂), 60.8, 61.3, 61.8, 66.6, 66.9 (OCH₂), 67.2, 68.8, 70.2, 70.4, 70.9, 71.1, 72.7, 72.9 (C₂′′), 73.2, 74.5, 75.4, 76.0, 77.0, 78.1 (C₂), 99.9 (C₁′′′), 100.5 (C₁′′), 100.8 (C₁), 103.1 (C₁′). HRMS (TOF): calc. for (M + Na)⁺ C₂₇H₄₉NO₂₁Na 746.2695, found 746.2687.

Acknowledgements

Financial support from CSIR [02(0186)/14/EMR-II] to RG and from CAS-UGC and FIST-DST, India, to the Department of Chemistry, Jadavpur University are acknowledged. MMM (SRF) and NB (RA, CSIR project) thank UGC and CSIR, India, respectively, for their fellowships.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra03856e

‡ At present: Laboratory of Bioorganic Chemistry, Graduate School of Information Science, Nagoya University, Japan.

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