Synthetic routes toward the trisaccharide related to the lipopolysaccharide of Burkholderia sp. HKI-402 (B4)

Abstract

A stepwise and a three component one-pot sequential glycosylation reaction has been used for the synthesis of trisaccharide related to the LPS of Burkholderia sp. HKI-402 (B4) employing trichloroacetimidate and thio donors.

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Introduction

The interactions between bacterial symbionts and their hosts (higher organisms) can either be beneficial or detrimental to the host.¹ In many cases, living organisms harbour endosymbiotic bacteria for providing improved defence mechanisms, survival, and even acquired virulence.² The fungus Rhizopus microsporus offers endosymbiotic bacteria protection in exchange for the production of the causal agent of rice seedling blight, rhizoxin. It has been proved that rhizoxin is not biosynthesized by the fungus itself, but by endosymbiotic bacteria of the genus Burkholderia sp. residing in it.³ Rhizoxin, a lactone antibiotic, is a phytotoxin which exhibits strong antimitotic activity in most eukaryotic cells, including various human cancer cell lines. Owing to this, rhizoxin has attracted considerable interest as a potential antitumor drug.⁴ Similarly there are innumerable antimitotic agents with different potential uses produced by several types of bacteria and that could have deadly effects, such as chronic or acute toxic damage of internal organs.

Lipopolysaccharides (LPSs) are one of the major components of the bacterial cell wall and are crucial for every kind of host-bacteria interaction. Consequently, it is of immense importance to identify and synthetically mimic the bacterial cell wall LPSs in order to understand and manipulate the survival and virulence of bacteria for vaccination.

The synthesis of the trisaccharide related to the repeating unit (Fig. 1) present in the O-antigen LPS portion of Burkholderia sp. HKI-402 (B4),⁵ by stepwise and one-pot sequential glycosylation reactions will be discussed. To the best of our knowledge, this is the first synthesis of this trisaccharide.

Fig. 1 Trisaccharide repeating unit present in the LPS of Burkholderia sp. HKI-402 (B4).

Stepwise oligosaccharide syntheses⁶ are generally expensive and tedious procedures because they demand extensive protecting group manipulation and purification after each step. In contrast, one-pot sequential oligosaccharide syntheses are more cost effective, fast, and environmentally friendly. Owing to these advantages, many complex oligosaccharides have been synthesized using one-pot protocols, such as Globo-H hexasaccharide, heparin pentasaccharide, Le^y, α-Gal epitopes and Gb₃ saccharides.⁷ Continuing our work on oligosaccharide syntheses,⁸ we report herein the synthesis of the trisaccharide related to the repeating unit present in the LPS of Burkholderia sp. HKI-402 (B4) from the corresponding monosaccharide building blocks as 3-(N-benzyloxycarbonyl) propyl glycoside by stepwise and sequential one-pot protocols.

Results and discussion

Retrosynthetic analysis of the fully protected trisaccharide 2 led to five monomeric sugar units, two orthogonal donors, L-rhamnose trichloroacetimidate 3 and thioglycoside 4 along with donors 5a and 5b, and glucosamine acceptor 6 for the stepwise and sequential one-pot synthetic approaches (Fig. 2).

Fig. 2 Retrosynthetic analysis of 1.

Treatment of tetra-acetylated L-rhamnose (7)⁹ with thiophenol and BF₃·Et₂O in dry CH₂Cl₂ furnished phenyl 2,3,4-tri-O-acetyl-1-thio-α-D-rhamnopyranoside (8)⁹ in 91% yield after purification by column chromatography. Thioglycoside hydrolysis of 8 was carried out using TCCA¹⁰ in aqueous (CH₃)₂CO to give 2,3,4-tri-O-acetyl-L-rhamnopyranoside (9)⁹ in 88% yield. Treatment of 9 with trichloroacetonitrile and DBU in dry CH₂Cl₂ provided trichloroacetimidate donor (3) ⁹ in 89% yield. Zemplén deacetylation¹¹ of 8 resulted in quantitative formation of phenyl 1-thio-α-D-rhamnopyranoside (10).⁹ 2,3-O-Isopropylidenation of 10 with 2,2-dimethoxypropane and catalytic camphorsulfonic acid in dry CH₃CN followed by O-benzylation of the resulting crude phenyl 2,3-O-isopropyl-1-thio-α-D-rhamnopyranoside (11)¹² with benzyl bromide and sodium hydride in dry DMF furnished phenyl 4-O-benzyl-2,3-O-isopropyl-1-thio-α-D-rhamnopyranoside (12).¹² 60% AcOH was used for 2,3-O-isopropylidene removal in the next step. After column chromatography, 71% phenyl 4-O-benzyl-1-thio-α-D-rhamnopyranoside (13)¹² was obtained over 3 steps from 10. Next, 2,3-O-stannylene acetal formation of 13 was carried out with dibutyltin oxide in dry toluene. Then, benzyl bromide was added to the same reaction vessel to furnish phenyl 3,4-O-benzyl-1-thio-α-D-rhamnopyranoside (4).¹³ Thus, L-rhamnose monosaccharide units 3 and 4 were obtained. Next the 2-OH group of 4 was protected with various methods to furnish its O-benzyl (5a),^13a and O-naphthylmethyl (5b)^13b derivatives (Scheme 1).

Scheme 1 Synthesis of L-rhamnopyranose donor and acceptor.

Glucosamine acceptor 6 was synthesized in six steps from D-glucosamine hydrochloride (Scheme 2). D-glucosamine hydrochloride (14) was converted to N-phth-protected tetra-O-acetyl-D-glucosamine (16) ¹⁴ as an anomeric mixture following a reported method. Thereafter, thiolation of 16 with thiophenol in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) or BF₃·Et₂O in CH₂Cl₂ furnished 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-1-thio-β-D-glucopyranoside (17)¹² in comparable yields (∼85%). Glycosylation was then carried out with 17 as the donor and 3-(N-benzyloxycarbonyl) propanol as the acceptor, using the TCCA/TMSOTf¹⁵ activator system to provide (18) ^15,16 in 86% yield. It is to be noted that this method¹⁵ furnished a better yield (86%) compared with the reported method¹⁶ (65%). Zemplén deacetylation of 18 generated 19 in quantitative yield. Benzylidenation of the deacetylated product was carried out using benzaldehyde dimethylacetal and catalytic camphorsulfonic acid in dry CH₃CN.

Scheme 2 Synthesis of D-glucosamine acceptor.

With all the appropriately protected monomeric intermediates in hand, the glycosylation leading to the protected trisaccharide (2) started from the non-reducing end of the compound. For this purpose, N-phthalimido acceptor 6 was reacted with phenyl 2-O-benzyl or 2-O-naphthylmethyl-3,4-O-benzyl-1-thio-α-D-rhamnopyranosides in the presence of NIS/TMSOTf.¹⁷ Both rhamnoside donors 5a and 5b underwent successful glycosylations in 92% and 89% yields, respectively. However, deacylation following Zémplen's method or with NEt₃/MeOH/H₂O failed to give the corresponding desired product. Surprisingly both reactions proceeded with unusual decomposition. To explore another route, 21 was denaphthylmethylated with DDQ, which progressed smoothly, yielding the desired disaccharide acceptor (22) in 87% yield. In contrast, when thiorhamnoside donor 8 was allowed to react with 22 in the presence of N-(p-methylphenylthio)-ε-caprolactam (NMPTC)-TMSOTf¹⁸ or NIS/TMSOTf,¹⁷ both of these reactions failed to produce the fully protected trisaccharide, even after exploring different temperature controls. Using trichloroacetimidate donor 3 instead of thiorhamnoside solved the problem and yielded desired trisaccharide 2 in 73% yield (Scheme 3).

Scheme 3 Stepwise synthesis of the desired trisaccharide.

After achieving the final protected trisaccharide via multistep synthesis, we formulated a reverse synthetic route starting from the reducing end and working towards the desired product by a one-pot sequential glycosylation reaction. Compound 3 and 4 were coupled by TMSOTf¹⁹ in dry CH₂Cl₂ at −30 °C (Scheme 4). After completion of the initial reaction (indicated by TLC), acceptor 6 and NIS were added to the reaction mixture in the same vessel. The reaction temperature was then gradually increased to room temperature. After completion, the crude product was purified by flash chromatography to give pure trisaccharide derivative (2) in 81% yield. A comparison of the one-pot synthesis (Scheme 4) of the protected trisaccharide (2) with the multistep synthesis (Scheme 3) clearly indicates the efficacy of the one-pot protocol (overall 81%) over the sequential one (overall 67.4%) in terms of reaction yield, atom economy and environmental impact.

Scheme 4 One-pot synthesis of the target trisaccharide.

The structure of 2 was confirmed by ¹H- and ¹³C-NMR, COSY, HMBC, HSQC and HRMS. Compound 2 showed three consecutive anomeric protons, listed from the non-reducing end at δ 3.87 (s), 4.56 (brs) and 5.17 (d, J 8.5 Hz), and the corresponding anomeric carbons at δ 98.7 (¹J_CH 173.8 Hz), 99.7 (¹J_CH 164.7 Hz) and 99.6 (¹J_CH 173.6 Hz), respectively. The observed NMR spectral data indicate the presence of two α-linked rhamnopyranose and one β-linked glucopyranose residues in 2.

The trisaccharide derivative was deprotected in a stepwise reaction scheme, starting with N-phth deprotection by ethylene diamine in butanol. Then acetylation using pyridine and acetic anhydride, followed by selective de-O-acetylation under Zémplen conditions, and a final global debenzylation using hydrogen and palladium-charcoal in a mixture of acetic acid, water and methanol, ultimately produced the desired product (1) in overall 72% yield (Scheme 4). Compound 1 was characterized by ¹H- and ¹³C-NMR, DEPT, COSY, HSQC and HRMS. The three consecutive anomeric protons of 1 from the non-reducing end appeared at δ 4.85 (s), 5.07 (s) and 4.58 (d, J = 8.5 Hz).

Conclusion

In summary, we successfully obtained the target trisaccharide via a stepwise glycosylation approach with chain extension from the non-reducing end as well as exploiting a sequential one-pot glycosylation route. Our fast, high yielding stereoselective one-pot protocol for obtaining the desired trisaccharide unit similar to the repeating unit of the LPS of Burkholderia sp. HKI-402 (B4) is particularly useful. The NMR data for the synthetic target are in good agreement with the reported data, with slight deviations due to the incorporation of the 3-(N-benzyloxycarbonyl) propyl as the aglycon moiety.

Experimental

NMR spectra were recorded on a Bruker DPX 200, 300 and 500 spectrometers for ¹H- and ¹³C-NMR, respectively, in CDCl₃. HRMS data were recorded on a Q-tof-Micro mass spectrometer by electron spray ionization. Specific rotations were measured on a Jasco J-815 spectrometer.

2,3,4-Tri-O-acetyl-L-rhamnopyranose (9)⁹

To a solution of compound 8 (2 g, 5.24 mmol) in aqueous (CH₃)₂CO (4 : 1), TCCA (1.2 g, 5.24 mmol) was added at 0 °C and stirred for 40 min. Then the white precipitate was filtered, and the bed was washed with CH₂Cl₂ (3 × 5 mL). The combined filtrate and washings were evaporated, and the resulting residue was dissolved again in CH₂Cl₂. The organic layer was washed with saturated NaHCO₃ solution (200 mL) and water (200 mL). The organic layer was dried over anhydrous Na₂SO₄ and evaporated under vacuum to furnish compound 9. Column filtration of the crude product furnished the pure compound as a white solid (9, 1.34 g, 88%). ¹H NMR (500 MHz, CDCl₃): δ 1.24 (d, J = 6.0 Hz, 3H), 1.99 (s, 3H, COCH₃), 2.06 (s, 3H, COCH₃), 2.16 (s, 3H, COCH₃), 3.19 (d, J = 3.0 Hz, 1H), 4.13 (m, 1H), 5.08 (t, J = 10.0 Hz, 1H), 5.17 (s, 1H), 5.28 (m, 1H), 5.36 (m, 1H).

2,3,4-Tri-O-acetyl-L-rhamnopyranosyl trichloroacetimidate (3)⁹

To a solution of compound 9 (1 g, 3.45 mmol) and CCl₃CN (0.52 mL, 5.18 mmol) in dry CH₂Cl₂ (15 mL), DBU (0.1 mL, 0.69 mmol) was added at −5 °C, and the reaction was stirred at that temperature. After 5 h, excess solvent was removed, and the resulting residue was purified by flash column chromatography (PE/EtOAc, 3 : 1) to furnish pure compound 3 as a colorless syrup (1.33 g, 89%). ¹H (200 MHz, CDCl₃): δ 1.26 (s, J = 6.2 Hz, 3H, CH₃), 2.00 (s, 3H, COCH₃), 2.06 (s, 3H, COCH₃), 2.18 (s, 3H, COCH₃), 4.08 (m, 1H), 5.17 (t, J = 10.0 Hz, 1H), 5.36 (dd, J = 3.4, 10.2 Hz, 1H), 5.45 (m, 1H), 6.19 (d, J = 1.5 Hz, 1H, C-1), 8.72 (s, 1H, NH).

Phenyl-1-thio-α-L-rhamnopyranoside (10)⁹

A suspension of compound 8 (2 g, 5.24 mmol) in 0.1 M NaOMe in dry MeOH (10 mL) was stirred overnight at room temperature. After completion of the reaction (indicated by TLC), Dowex 50 (H⁺) resin was poured into the clear solution, and it was swirled occasionally. After 30 min the resin was filtered, and the bed was washed with distilled MeOH (3 × 5 mL). The combined filtrate and washings was evaporated to dryness to furnish compound 10 in quantitative yield. It was dried thoroughly and directly used without characterization for the next step.

Synthesis of phenyl 4-O-benzyl-1-thio-α-L-rhamnopyranoside (13)¹² from 10

To a solution of compound 10 (1 g, 3.9 mmol) and 2,2-dimethoxypropane (0.72 mL, 5.85 mmol) in dry CH₃CN, CSA (271.8 mg, 1.17 mmol) was added at 0 °C. The reaction mixture was stirred for 5 h. After completion (indicated by TLC), the reaction was quenched by the addition of Et₃N. Excess solvent was removed under vacuum, and the resulting residue was dissolved in CH₂Cl₂. The organic solution was washed with saturated NaHCO₃ solution (100 mL) and water (100 mL). The organic layer was dried over anhydrous Na₂SO₄ and evaporated under vacuum to furnish crude product 11. This was used for the next step without purification. To crude 11, 60% NaH in oil (234.0 mg, 5.85 mmol) and BnBr (0.7 mL, 5.85 mmol) were added in dry DMF at 0 °C. After completion (indicated by TLC), the reaction was quenched by MeOH, and the excess solvent was removed under vacuum. The resulting white paste was dissolved in CH₂Cl₂ and washed with water (100 mL). The organic layer was dried over anhydrous Na₂SO₄ and evaporated under vacuum to furnish crude product 12. Compound 12 was treated with 80% aqueous AcOH (15 mL) at 60 °C for 6 h. Then excess AcOH was co-evaporated with toluene, furnishing a thick brown syrup. This was dissolved in CH₂Cl₂ and washed with saturated NaHCO₃ solution (100 mL) and water (100 mL). The organic layer was dried over anhydrous Na₂SO₄ and evaporated under vacuum to furnish crude product 13. Purification of 13 by silica gel column chromatography (PE/EtOAc, 2 : 1) yielded phenyl 4-O-benzyl-1-thio-α-L-rhamnopyranoside as a white solid (13, 0.96 g, 71% over 3 steps). M.p. 110–112 °C. [α]²⁵_D −187.5 (c 1.5, CHCl₃). Lit¹² m.p. 111–113 °C. [α]_D −201 (c 0.9, CHCl₃). ¹H (500 MHz, CDCl₃): δ 1.36 (d, J = 6.0Hz, 3H, CH₃), 3.41 (t, J = 9.5 Hz, 1H), 3.94 (dd, J = 3.0, 9.0 Hz, 1H), 4.19 (m, 1H), 4.22 (m, 1H), 4.76 (s, 2H), 5.47 (d, J = 1.0 Hz, 1H, C-1), 7.24–7.34 (m, 4H, ArH), 7.36–7.38 (m, 4H, ArH), 7.45–7.47 (m, 2H, ArH). ¹³C (125 MHz, CDCl₃): δ 18.0, 68.7, 71.9, 72.6, 75.1, 81.8, 87.4, 127.4, 128.0, 128.1, 128.7, 129.1, 131.4, 134.2, 138.1.

Phenyl 3,4-di-O-benzyl-1-thio-α-L-rhamnopyranoside (4)¹³

A suspension of compound 13 (0.5 g, 1.44 mmol) and Bu₂SnO (430.6 mg, 1.73 mmol) in dry toluene (10 mL) was refluxed in Dean–Stark apparatus for 5 h. Then, BnBr (0.26 mL, 2.16 mmol) and tetrabutylammonium bromide (232.1 mg, 0.72 mmol) were added to the resulting clear solution. It was stirred at 80 °C until completion of the reaction (indicated by TLC). Excess toluene was removed under vacuum, and the resulting crude product was directly used for column chromatography on silica gel. Column elution with PE/EtOAc (3 : 1) furnished pure compound 4 as a colorless syrup (0.57 g, 90%). [α]²⁵_D −181.5 (c 1.5, CHCl₃); lit^13a [α]²⁵_D −196.1 (c 1.2, CHCl₃); ¹H (300 MHz, CDCl₃): δ 1.23 (d, 3H, J = 6.0 Hz, CH₃), 2.61 (s, 1H), 3.45 (t, 1H, J = 9.3 Hz), 3.78 (dd, 1H, J = 3.3, 10.0 Hz), 4.12 (m, 1H), 4.16 (s, 1H), 4.57 (d, 1H, J = 11.1 Hz), 4.64 (s, 2H), 4.81 (d, 1H, J = 10.8 Hz), 5.44 (s, 1H), 7.14–7.37 (m, 15H, ArH).

Phenyl 2-O-benzoyl-3,4-di-O-benzyl-1-thio-α-L-rhamnopyranoside (5a)^13a

To a solution of compound 4 (524 mg, 1.2 mmol) and dry pyridine, BzCl (0.21 mL, 1.8 mmol) was added. It was stirred at ambient temperature until completion of the reaction (indicated by TLC). Excess pyridine was removed under vacuum, and the organic layer was washed with brine solution. The organic layer was dried over anhydrous Na₂SO₄ and concentrated to afford the benzoylated product. Column elution by PE/EtOAc, 4 : 1 furnished pure compound 5a as a white foam (616 mg, 95%). ¹H (500 MHz, CDCl₃): δ 1.41 (d, J = 6.0 Hz, 3H), 3.65 (t, J = 9.0 Hz, 1H), 4.05 (dd, J = 2.5, 9.0 Hz, 1H), 4.32 (m, 1H), 4.62 (d, J = 11.5 Hz, 1H), 4.68 (d, J = 11.0 Hz, 1H), 4.81 (d, J = 11.5 Hz, 1H), 4.96 (d, J = 11.0 Hz, 1H), 5.57 (s, 1H, PhCH), 5.86 (d, J = 1.5 Hz, 1H, H-1), 7.28–7.38 (m, 13H, ArH), 7.47–7.50 (m, 4H, ArH), 7.59–7.62 (m, 1H, ArH), 8.08–8.10 (d, J = 7.5 Hz, 2H, ArH). ¹³C (125 MHz, CDCl₃): δ 18.1, 69.2, 71.1, 71.7, 75.5, 78.5, 80.2, 86.2, 127.7, 127.8, 128.1, 128.2, 128.4, 128.5, 129.1, 129.9, 131.8, 133.3, 134.0, 137.7, 138.3, 165.7.

Phenyl 3,4-di-O-benzyl-2-O-(2-naphthylmethyl)-1-thio-α-L-rhamnopyranoside (5b)^13b

To a solution of compound 4 (100 mg, 0.23 mmol) and dry DMF, 60% NaH in oil (9 mg, 0.35 mmol) and NapBr (77.4 mg, 0.35 mmol) were added. The mixture was stirred at ambient temperature until completion of the reaction (indicated by TLC). Excess NaH was quenched with MeOH, DMF was removed under vacuum, and was washed with brine solution. The organic layer was dried over anhydrous Na₂SO₄ and concentrated to afford the naphthylmethylated product. Column elution by PE/EtOAc, 5 : 1 furnished pure compound 5b as a white solid (121.5 mg, 92%). ¹H (300 MHz, CDCl₃): δ 1.43 (d, J = 6.3 Hz, 3H), 3.79 (t, J = 9.4 Hz, 1H), 3.92 (dd, J = 9.4, 3.1 Hz, 1H), 4.09 (dd, J = 3.0, 1.7 Hz, 1H), 4.18–4.26 (m, 1H), 4.65 (d, J = 11.7 Hz, 1H), 4.69 (d, J = 11.7 Hz, 1H), 4.73 (d, J = 10.8 Hz, 1H), 4.86 (d, J = 12.5 Hz, 1H), 4.93 (d, J = 12.6 Hz, 1H), 5.05 (d, J = 10.8 Hz, 1H), 5.56 (d, J = 1.5 Hz, 1H), 7.23–7.30 (m, 3H), 7.31–7.44 (m, 12H), 7.50–7.59 (m, 3H), 7.77–7.90 (m, 4H). ¹³C (75 MHz, CDCl₃) δ: 18.0, 69.5, 72.3, 72.4, 75.6, 76.6, 80.1, 80.6, 86.0, 126.05, 126.14, 126.2, 127.0, 127.3, 127.8, 127.9, 128.0, 128.1, 128.3, 128.5, 129.1, 133.1, 133.3, 134.7, 135.4, 138.3, 138.6.

3-(N-benzyloxycarbonyl) propyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranoside (18)^15,16

To a mixture of 17 (150.0 mg, 0.285 mmol) and 3-(N-benzyloxycarbonyl) propanol (50.0 mg, 0.238 mmol) in dry CH₂Cl₂ (5 mL), flame activated molecular sieves (4 Å) were added. The mixture was stirred at room temperature under argon. After 40 min the mixture was cooled to −5 °C and TCCA (55.3 mg, 0.238 mmol) was added. Then TMSOTf (12.9 μL, 0.071 mmol) was added via a micro syringe. After the acceptor was consumed completely (checked by TLC) the reaction was quenched by Et₃N (130.0 μL). The reaction mixture was filtered off through a Celite bed. The filtrate was diluted with CH₂Cl₂ and washed with saturated NaHCO₃ solution and water. The organic layer was dried over anhydrous Na₂SO₄ and concentrated to afford the glycosylated product. The crude product was purified by column chromatography on silica gel (60–120 mesh) (PE : EtOAc 4 : 1) to afford 8 (128.7 mg) in 86% yield as a white foam. [α]²⁵_D +10.9 (c 1.0, CHCl₃); lit.^15,16 [α]_D +18.1 (c 1.1, CHCl₃). ¹H (300 MHz, CDCl₃): δ 1.68–1.70 (m, 2H), 1.85 (s, 3H, COCH₃), 2.03 (s, 3H, COCH₃), 2.07 (s, 3H, COCH₃), 3.04–3.16 (m, 2H), 3.55 (m, 1H), 3.82–3.89 (m, 2H), 4.19 (dd, J = 2.1, 12.2 Hz, 1H), 4.22–4.34 (m, 2H), 4.95 (m, 1H), 5.01 (s, 2H), 5.16 (t, J = 9.6 Hz, 1H), 5.38 (d, J = 8.5 Hz, 1H), 5.75 (dd, J = 9.1, 10.7 Hz, 1H), 7.30–7.35 (m, 5H, ArH), 7.70–7.73 (m, 2H, ArH), 7.81–7.83 (m, 2H, ArH). The spectral data were consistent with those in the literature.¹⁵

3-(N-benzyloxycarbonyl) propyl-2-deoxy-2-phthalimido-β-D-glucopyranoside (19)

A suspension of compound 18 (1 g, 2.08 mmol) in 0.1 M NaOMe in dry MeOH (10 mL) was stirred for 45 min at room temperature. After completion of the reaction (indicated by TLC), Dowex 50 (H⁺) resin was poured into the clear solution, and it was swirled occasionally. After 30 min the resin was filtered, and the bed was washed with distilled MeOH (3 × 5 mL). The combined filtrate was evaporated to dryness to furnish title compound 19 in quantitative yield. It was dried thoroughly and used directly without characterization for the next step.

3-(N-benzyloxycarbonyl) propyl-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-glucopyranoside (6)

To a suspension of 19 (0.5 g, 1.41 mmol) and benzaldehyde dimethylacetal (0.32 mL, 2.12 mmol) in dry CH₃CN (25 mL), anhydrous FeCl₃ (68.6 mg, 0.42 mmol) was added, and it was stirred at ambient temperature. After completion of the reaction (indicated by TLC) the solvent was evaporated under reduced pressure. The solid residue was dissolved in CH₂Cl₂ and washed with saturated NaHCO₃ solution (200 mL) and water (200 mL). The organic layer was dried over anhydrous Na₂SO₄ and evaporated under vacuum to furnish the crude product. It was purified by column chromatography on silica gel (eluent: PE/EtOAc, 1 : 1) to give pure compound 5 as a white foam (0.5 g, 81%). [α]²⁵_D −39.8 (c 1.48, CHCl₃). ¹H (300 MHz, CDCl₃): δ 1.69 (m, 1H), 2.54 (m, 1H), 3.09–3.13 (m, 1H), 3.49–3.68 (m, 3H), 3.77–3.90 (m, 2H), 4.24 (dd, J = 8.4, 10.4 Hz, 1H), 4.38 (m, 1H), 4.62 (m, 1H), 4.90 (bs, 1H), 5.02 (s, 1H), 5.27 (d, 1H, J = 8.6 Hz), 5.30 (s, 1H), 5.55 (s, 1H, PhCH), 7.29–7.39 (m, 8H, ArH), 7.48–7.51 (m, 2H, ArH), 7.69–7.72 (m, 2H), 7.81–7.84 (m, 2H). ¹³C (75 MHz, CDCl₃): δ 29.4, 37.8, 56.7, 66.3, 66.5, 68.6, 71.7, 82.2, 98.9, 101.9, 123.5, 126.3, 128.0, 128.4, 128.5, 129.0, 129.3, 129.8, 131.58, 131.6, 131.63, 134.1, 134.2, 134.5, 136.4, 136.7, 137.0, 156.4, 168.2, 168.5, 192.6. HRMS m/z for (C₆₄H₇₀N₂O₂₀Na⁺) calcd: 1209.4420, found: 1209.4421.

3-(N-benzyloxycarbonyl) propyl 2-O-benzoyl-3,4-di-O-benzyl-α-L-rhamnopyranosyl-(1 → 3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-glucopyranoside (20)

To a solution of 5a (160 mg, 0.30 mmol) and 6 (145 mg, 0.25 mmol) in dry CH₂Cl₂ (12 mL) activated molecular sieves (4 Å) were added, and the reaction was stirred under argon for 45 min. NIS (67 mg, 0.30 mmol) and TMSOTf (16 μL, 0.08 mmol) were then added to the reaction vessel a micro syringe keeping the temperature at −5 °C. After the addition, the reaction temperature was raised gradually to room temperature. The reaction was complete after 15 min (indicated by TLC). The reaction mixture was then filtered through a Celite bed, and the bed was washed with CH₂Cl₂ (3 × 5 mL). The combined filtrate was washed with saturated aqueous NaHCO₃ (1 × 100 mL), saturated aqueous Na₂S₂O₃ (1 × 100 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 column chromatography (eluent: PE/EtOAc, 2 : 1) to afford disaccharide 20 as a white foam (230 mg, 92%). ¹H NMR (500 MHz, CDCl₃): δ 0.80 (d, J = 6.0 Hz, 3H), 1.67 (m, 2H), 3.08–3.12 (m,2H). 3.35 (t, J = 9.5 Hz, 1H), 3.53 (m, 2H), 3.65–3.68 (m, 2H), 3.81 (t, J = 10.0 Hz, 1H), 3.83–3.86 (m, 2H), 3.93 (m, 1H), 4.31 (dd, J = 8.5, 10.0 Hz, 1H), 4.38 (m, 1H), 4.44 (d, J = 11.5 Hz, 1H), 4.52 (d, J = 11.0 Hz, 1H), 4.56 (d, J = 11.5 Hz, 1H), 4.61–4.65 (m, 2H), 4.81 (d, J = 11.0 Hz, 1H), 4.88 (brs,1H), 5.04–5.07 (m,3H), 5.25 (d, J = 8.5 Hz, 1H), 5.54 (s, 1H,PhCH), 7.22 (brs, 8H, ArH), 7.28–7.38 (m, 12H, ArH), 7.47–7.51 (m, 3H, ArH), 7.66–7.71 (m, 3H, ArH), 7.86 (bs, 2H, ArH). HRMS m/z for (C₅₉H₅₈N₂O₁₄Na⁺) calcd: 1041.3786, found: 1041.3785.

3-(N-benzyloxycarbonyl) propyl 3,4-di-O-benzyl-2-O-(2-naphthylmethyl)-α-L-rhamnopyranosyl-(1 → 3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-glucopyranoside (21)

To a solution of 5b (88 mg, 0.15 mmol) and 6 (75 mg, 0.13 mmol) in dry CH₂Cl₂ (8 mL) activated molecular sieves (4 Å) were added, and the reaction was stirred under argon for 45 min. NIS (34 mg, 0.15 mmol) and TMSOTf (8.3 μL, 0.05 mmol) were then added to the reaction vessel via a micro syringe keeping the temperature at −5 °C. After the addition, the reaction temperature was raised gradually to room temperature. The reaction was complete after 15 min (indicated by TLC). The reaction mixture was then filtered through a Celite bed, and the bed was washed with CH₂Cl₂ (3 × 5 mL). The combined filtrate was washed with saturated aqueous NaHCO₃ (1 × 50 mL), saturated aqueous Na₂S₂O₃ (1 × 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 column chromatography (eluent: PE/EtOAc, 4 : 1) to afford disaccharide 21 as a syrup (119.5 mg, 89%). ¹H NMR (400 MHz, CDCl₃): δ 0.87 (d, J = 6.0 Hz, 3H), 1.69 (m, 1H), 3.10–3.14 (m, 2H), 3.42–3.56 (m, 3H), 3.64–3.69 (m, 2H), 3.76–3.93 (m, 4H), 4.20 (s, 2H), 4.29 (t, J = 9.5 Hz, 1H), 4.40 (d, J = 11.0 Hz, 2H), 4.50 (d, J = 10.8 Hz, 1H), 4.52 (d, J = 9.2 Hz, 1H), 4.64 (t, J = 9.2 Hz, 1H), 4.75 (s, 1H), 4.83 (d, J = 10.8 Hz, 1H), 4.91 (bs, 1H), 5.03 (s, 2H), 5.29 (d, J = 8.4 Hz, 1H), 5.54 (s, 1H, PhCH), 7.06 (d, J = 8.4 Hz, 1H, ArH), 7.24–7.36 (m, 18H, ArH), 7.43–7.78 (m, 12H, ArH), 8.67 (bs, 1H, NH). ¹³C NMR (125 MHz, CDCl₃): δ 17.5, 29.6, 29.8, 38.1, 56.7, 66.6, 66.7, 67.6, 68.5, 68.8, 72.0, 72.6, 74.5, 75.1, 76.3, 77.4, 79.7, 80.4, 80.8, 98.6, 98.9, 102.0, 123.7, 125.4, 125.88, 125.92, 126.1, 126.5, 127.3, 127.49, 127.54, 127.6, 127.8, 127.9, 128.0, 128.1, 128.2, 128.3, 128.36, 128.38, 128.6, 129.2, 131.3, 133.0, 133.3, 134.6, 135.5, 136.8, 137.1, 138.7, 138.9, 156.4. HRMS m/z for (C₆₃H₆₂N₂O₁₃Na⁺) calcd: 1077.4150, found: 1077.4152.

3-(N-benzyloxycarbonyl) propyl 3,4-di-O-benzyl-α-L-rhamnopyranosyl-(1 → 3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-glucopyranoside (22)

To a solution of 22 (64 mg, 0.06 mmol) in CH₂Cl₂/H₂O (5 mL, 19 : 1) DDQ (17 mg, 0.08 mmol) was added, and the reaction was stirred at room temperature for 2 h. The reaction mixture was washed with water (2 × 100 mL) and the organic layer was dried over anhydrous Na₂SO₄ and concentrated to furnish a syrupy compound. The crude product was purified by column chromatography (eluent: PE/EtOAc, 2 : 1) to afford disaccharide acceptor 22 as a syrup (48 mg, 87%). ¹H NMR (500 MHz, CDCl₃): δ 0.70 (d, J = 6.3 Hz, 3H), 1.59 (m, 1H), 2.94–3.05 (m, 2H), 3.15 (t, J = 9.0 Hz, 1H), 3.43 (m, 1H), 3.47–3.74 (m, 5H), 3.75–3.89 (m, 3H), 4.18 (dd, J = 8.7, 10.2 Hz, 1H), 4.31 (dd, J = 5.6, 13.6 Hz, 1H), 4.39–4.46 (m, 3H), 4.49–4.55 (m, 3H), 4.58–4.67 (m, 2H), 4.76 (d, J = 8.0 Hz, 1H), 4.80 (bs, 1H), 4.95 (bs, 2H), 5.18 (d, J = 8.7 Hz, 1H), 5.44 (s, 1H, PhCH), 7.12–7.30 (m, 18H, ArH), 7.41–7.44 (m, 2H, ArH), 7.63–7.66 (m, 2H, ArH), 7.75–7.78 (m, 2H, ArH). ¹³C NMR (125 MHz, CDCl₃): δ 17.4, 29.8, 38.1, 56.8, 66.6, 67.6, 67.8, 68.7, 68.8, 72.0, 74.3, 75.2, 77.3, 79.8, 80.0, 80.9, 98.9, 99.6, 102.1, 123.9, 126.6, 127.7, 127.9, 128.0, 128.2, 128.3, 128.4, 128.60, 128.62, 129.2, 131.4, 134.7, 137.1, 138.0, 138.6, 156.4. HRMS m/z for (C₅₂H₅₄N₂O₁₃Na⁺) calcd: 937.3524, found: 937.3523.

Stepwise synthesis of 3-(N-benzyloxycarbonyl) propyl 2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1 → 2)-3,4-di-O-benzyl-α-L-rhamnopyranosyl-(1 → 3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-glucopyranoside (2)

To a solution of 3 (22 mg, 0.05 mmol) and 22 (40 mg, 0.04 mmol) in dry CH₂Cl₂ (5 mL), activated molecular sieves (4 Å) were added, and the reaction was stirred under argon for 45 min. The reaction vessel was placed in a −15 °C cold bath, and NIS (11.0 mg, 0.05 mmol) and TMSOTf (4 μL, 0.02 mmol) were added via a micro syringe. After 10 min, complete consumption of both the starting materials was observed. The reaction mixture was then filtered through a Celite bed, and the bed was washed with CH₂Cl₂ (3 × 5 mL). The combined filtrate was washed with saturated aqueous NaHCO₃ (1 × 50 mL), saturated aqueous Na₂S₂O₃ (1 × 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, 1 : 1) to afford the desired fully protected trisaccharide (2) as a white foam (40.5 mg, 78%).

One-pot sequential synthesis of 3-(N-benzyloxycarbonyl) propyl 2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1 → 2)-3,4-di-O-benzyl-α-L-rhamnopyranosyl-(1 → 3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-glucopyranoside (2)

To a solution of 3 (50.0 mg, 0.11 mmol) and 4 (43.6 mg, 0.10 mmol) in dry CH₂Cl₂ (8 mL) activated molecular sieves (4 Å) were added, and the reaction was stirred under argon for 45 min. The reaction vessel was placed in a −30 °C cold bath, and TMSOTf (5.4 μL, 0.03 mmol) was added to it via a micro syringe. After 10 min, complete consumption of both the starting materials was observed. Acceptor 6 (53.0 mg, 0.09 mmol) and NIS (30.0 mg, 0.13 mmol) were then added to the same vessel. After the addition, the reaction temperature was raised gradually to room temperature. The second step of the reaction was complete after 30 min (indicated by TLC). The reaction mixture was then filtered through a Celite bed, and the bed was washed with CH₂Cl₂ (3 × 5 mL). The combined filtrate was washed with saturated 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, 1 : 1) to afford the desired fully protected trisaccharide (2) as a white foam (86.0 mg, 81%). [α]²⁵_D −34.2 (c 1.30, CHCl₃). ¹H NMR (500 MHz, CDCl₃): δ 0.72–0.76 (2d, J = 6.5 Hz, 6H, 2 × CH₃), 1.55 (m, 1H, CH₂), 1.85 (s, 3H, COCH₃), 1.91 (s, 3H, COCH₃), 1.95 (s, 3H, COCH₃), 2.98–3.03 (m, 2H, NCH₂), 3.20–3.24 (apparent t, 1H, J = 9.5 Hz, H-4′), 3.34 (br s, 1H, H-5′), 3.43 (m, 1H, CH₂), 3.51–3.60 (m, 3H, H-4, H-6a, PhCH₂), 3.65–3.79 (m, 4H, H-5, H-2′, H-3′, CH₂), 3.87 (s, 1H, H-1′), 4.15 (apparent t, J = 9.0, 10.0 Hz, 1H, H-2), 4.30 (dd, J = 4.5, 10.5 Hz, 1H, H-6), 4.35 (d, J = 12.0 Hz, 1H, PhCH₂), 4.42–4.48 (m, 3H, H-3, H-5′′, PhCH₂), 4.56 (br s, 1H, H-1′′), 4.69–4.73 (m, 2H, H-4′′, PhCH₂), 4.77 (m, 1H, H-2′′), 4.95 (br s, 2H, PhCH₂), 5.02–5.03 (m, 2H, H-3′, PhCH₂), 5.17 (d, J = 8.5 Hz, 1H, H-1), 5.44 (s, 1H, PhCH), 7.19–7.36 (m, 18H, ArH), 7.47–7.51 (m, 2H, ArH), 7.69–7.80 (m, 2H, ArH); ¹³C NMR (125 MHz, CDCl₃): δ 17.1 (CH₃), 17.3 (CH₃), 20.7 (COCH₃), 20.79 (COCH₃), 20.84 (COCH₃), 29.5, 38.0, 56.6 (C-2), 66.4 (C-4), 66.6 (PhCH₂, C-6a), 68.3 (OCH₂), 68.7 (C-5), 68.8 (C-2′, C-6, PhCH₂), 69.8 (C-3′′), 71.0 (C-4′′), 71.9 (2 × PhCH₂), 75.2 (2 × PhCH₂), 75.5 (C-2′′, C-5′′, C-3), 78.1 (C-5′), 79.0 (C-3′), 79.9 (C-4′), 80.7 (C-4), 98.7 (C-1), 99.6 (C-1′), 99.7 (C-1′′), 102.0 (PhCH), 126.4, 127.4, 127.47, 127.54, 128.0, 128.1, 128.2, 128.3, 128.4, 128.5, 129.1, 131.4. 134.2, 137.0, 138.5, 138.7, 156.3, 169.4, 169.8, 170.1; HRMS m/z for (C₆₄H₇₀N₂O₂₀Na⁺) calcd: 1209.4420, found: 1209.4421. The NMR spectra of compound 2 obtained by both protocols were identical.

3-(N-benzyloxycarbonyl) propyl α-L-rhamnopyranosyl-(1 → 2)-α-L-rhamnopyranosyl-(1 → 3)-2-deoxy-2-acetamido-β-D-glucopyranoside (1)

Protected trisaccharide 2 (60 mg, 0.034 mmol) was refluxed in butanol (6 mL) and ethylene diamine (1.5 mL) for 8 h and then excess solvent was removed under reduced pressure. Residual water was co-evaporated with toluene (2 × 5 mL) and the resulting compound was treated with dry pyridine (5 mL) and Ac₂O (2 mL) for 12 h at room temperature. After complete conversion, excess solvent was removed under vacuum. The crude residue was column filtered (PE/EtOAc, 3 : 2) and the pure product was dissolved in MeOH (7 mL) and NaOMe (1 M in MeOH, 0.5 mL) was added. The mixture was stirred for 10 h and then the reaction was quenched with Dowex-50W cation exchange resin (H⁺). The resin was filtered off and then washed with MeOH (4 × 5 mL). The combined filtrate and washings was evaporated under reduced pressure. The resulting residue and 10% Pd–C (70 mg) was dissolved in AcOH (1 mL), MeOH (3 mL), and H₂O (1 mL) and was stirred under H₂ for 24 h. The catalyst was filtered through a Celite bed, and the bed was washed with MeOH (3 × 5 mL). The combined filtrate and washings were concentrated under reduced pressure. It was passed through a 0.45 μm Millipore membrane, and lyophilized to afford 1 as a white foam (30.0 mg, 90%); ¹H NMR (500 MHz, D₂O): δ 1.25–1.30 (2d, 6H, J = 6.0 Hz, 2 × CH₃), 1.87–1.90 (m, 2H), 2.10 (s, 3H, COCH₃), 2.93–2.99 (m, 2H), 3.43–3.54 (m, 4H), 3.59 (apparent t, J = 8.0, 9.5 Hz, 1H), 3.71–3.86 (m, 7H), 3.94–4.05 (m, 4H), 4.58 (d, J = 8.5 Hz, 1H), 4.85 (s, 1H), 5.07 (s, 1H). ¹³C (75 MHz, D₂O): δ 16.5, 16.9, 22.5, 23.1, 26.8, 37.5, 55.6, 60.7, 67.7, 68.6, 68.9, 69.1, 69.8, 70.1, 72.1, 75.9, 80.4, 81.4, 99.7, 100.5, 102.7, 174.6. HRMS (ESI-TOF) Calcd for C₂₃H₄₂N₂O₁₄Na [M + Na]+ 593.2534, found 593.2531.

Acknowledgements

Financial support from DST-SERB, India (Scheme no. SR/S1/OC-61/2012) to RG and from CAS-UGC and FIST-DST, India to the Department of Chemistry, Jadavpur University are acknowledged. NB (SRF) and MMM (SRF) thank CSIR and UGC, India respectively, for their fellowships.

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Footnote

† Electronic supplementary information (ESI) available: General experimental procedure, copies of ¹H-, ¹³C-, COSY- HSQC-NMR spectra of compounds 1, 2 and HMBC-NMR spectra of 2. See DOI: 10.1039/c4ra03954h

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