Chemical synthesis of the hexasaccharide related to the repeating unit of the capsular polysaccharide from carbapenem resistant Klebsiella pneumoniae 2796 and 3264

Abstract

The total synthesis of the hexasaccharide repeating unit of the CPS from carbapenem resistant K. pneumoniae 2796 and 3264 is reported using a sequential glycosylations approach. The total synthesis has been accomplished by glycosylation of rationally protected monosaccharide synthons derived from the commercially available sugars. The required uronic acid on the galactose moiety was successfully installed by a TEMPO-mediated late stage oxidation. The glycosylations were performed by the NIS-mediated activation of thioglycosides using H₂SO₄-silica as the promoter. Chloroacetate group was extensively used as a temporary protecting group to facilitate stereoselective glycosylations.

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Introduction

Bacterial capsular polysaccharides (CPS) and lipopolysaccharides (LPS) are responsible for their virulence factor. CPS and LPS are made up of oligosaccharide repeating units with varied sugar residues. As they remain exposed on the outer surface of the bacterial cell wall, they play pivotal role in infection. Due to the presence of different sugar residues in the oligosaccharide repeats, CPS and LPS demonstrate diverse character and act as the elicitor of innate immune responses. Literature reports suggest that there are potential scope with these bacterial O-antigens as vital candidates for anti-microbial agents and vaccine-targets.^1–3 However, it require tedious isolation and purification processes to harness these complex oligosaccharides in adequate quantity. Thus the chemical synthesis of the required oligosaccharides remains the only way to explore their vivid biological roles and potential as vaccine targets.

Klebsiella pneumoniae (K. pneumoniae) is an opportunistic pathogen that is responsible for community or hospital acquired infections. It mostly affects the urinary and respiratory tracts. Both CPS and LPS of the K. pneumonia are found to be responsible for the virulence. The CPS antigens are used for K-typing of K. pneumoniae whereas LPS antigens are used for O-typing.⁴ The capsular antigens are protective against capsular pathogens such as H. influenza type b, meningococci and pneumococci.⁵ Among large number of K. pneumoniae K-antigens only a few are associated with human disease.⁶ This is a limiting factor for the development of suitable vaccine against this pathogen. Particularly the carbapenem resistant K-antigens (CRKP) are rarely known in the literature. Only recently, Kubler-Kielb has reported the structures of the CRKP CPS and LPS from clinical isolates collected from the infected patients of a CRKP outbreak in the US.⁷ Herein, we report the total synthesis of the hexasaccharide repeating unit of the CPS from K. pneumoniae 2796 and 3264 in the form of its p-methoxyphenyl glycoside (Fig. 1). The particular aglycon in the reducing end will enable us to form further glycoconjugates after the selective removal of the same from the per-O-acetylated derivative of the target oligosaccharide.

Fig. 1 Structure of the target hexasaccharide in the form of its p-methoxyphenyl glycoside.

Results and discussion

Judicious retro-synthetic analysis indicated that a sequential glycosylations strategy would be the best fit for the successful synthesis of the target hexasaccharide 1. Thus, three rhamnose moieties were planned to be stitched by using the same synthon 5 with the reducing end galactose moiety 6. The chloroacetate group was thought to be the perfect choice as a temporary protecting group as it can ensure the required 1,2-trans glycosylations as well as can be de-protected selectively to pave the path for introduction of the next sugar unit. Next, a suitably protected galactose synthon 14 having a non-participating group at 2-position thought to be ideal for the required 1,2-cis linkage. A per-O-acylated rhamnose derivative 17 will complete the target hexasaccharide in its protected form. Finally, a TEMPO mediated oxidation of the primary hydroxyl group of the non-reducing end galactose moiety followed by global de-protection would furnish the required molecule (Fig. 2).

Fig. 2 Retrosynthetic analysis for the total synthesis of the target hexasaccharide 1.

Therefore, the we started our synthesis with known p-tolyl 4-O-benzoyl-2,3-O-isopropylidene-1-thio-α-L-rhamnopyranoside (2).⁸ Hydrolysis of the isopropylidene group using 80% AcOH at 80 °C (ref. 9) gave the diol 3 in 91% yield. Next, selective benzylation at the equatorial 3-OH was accomplished by following stannylene chemistry¹⁰ to give the derivative 4 in 85% yield. Finally, protection of the sole 2-OH with chloroacetate¹¹ group afforded the required donor 5 in 89% yield. Donor 5 was coupled with the known acceptor 6 (ref. 12) by the activation of the thiotolyl using NIS in the presence of H₂SO₄-silica¹³ at 0 °C to afford the disaccharide 7 in 91% yield. It is worth noting that the use of H₂SO₄-silica as the promoter for NIS-mediated activation of the thioglycoside donor found to be beneficial compared to the use of toxic, fuming and hygroscopic TfOH or TMSOTf. Further, selective de-protection of the chloroacetate group using thiourea¹⁴ gave the disaccharide acceptor 8 in 87% isolated yield. Subsequently, glycosylations with the same donor 5 followed by de-protection of the chloroacetate group using the same reagent combination and condition was iterated twice to obtain the tetrasaccharide acceptor 12 (Scheme 1). The yields of the individual steps involved are mentioned in the Scheme 1.

Scheme 1 Synthesis of the tetrasaccharide acceptor 12.

In a separate experiment, known p-tolyl 2,3-di-O-benzyl-6-O-(4-methoxybenzyl)-1-thio-β-D-galactopyranoside (13)¹⁵ was treated with chloroacetic anhydride in the presence of dry pyridine to give the completely protected donor (14) in 88% yield. Next, glycosylations of the donor 14 with the tetrasaccharide acceptor 12 using NIS in the presence of H₂SO₄-silica at −50 °C gave the protected pentasaccharide 15 in 82% yield. Presence of the non-participating benzyl group at the 2-position of the galactosyl donor 14 and the reaction at very low temperature assured the formation of the desired 1,2-glycoside as the sole isolated product. Further, selective de-protection of the chloroacetate group using thiourea afforded the pentasaccharide acceptor 16 in 85% yield. Finally, glycosylations of 16 with the known donor 17 (ref. 16) using the same NIS/H₂SO₄-silica at 0 °C furnished the protected hexasaccharide 18 in 84% yield. At this stage, the strategically placed 4-methoxybenzyl group was selectively de-protected by oxidative cleavage using DDQ¹⁷ to afford the hexasaccharide derivative 19 in 78% isolated yield. Oxidation of the primary hydroxyl group using TEMPO in the presence of bis-acetoxy iodobenzene (BAIB)¹⁸ followed by catalytic hydrogenolysis and Zemplen de-O-acetylation¹⁹ gave the target hexasaccharide 1 in 64% yield over three steps (Scheme 2). The amorphous white powder of compound 1 was triturated with CH₂Cl₂ and filtered to remove aromatic impurities.

Scheme 2 Synthesis of the target hexasaccharide 1.

Conclusions

In conclusion, we have successfully accomplished the total synthesis of the hexasaccharide repeating unit of the CPS from K. pneumoniae 2796 and 3264 in the form of its p-methoxyphenyl glycoside. The practical synthetic strategy used the minimum protecting group manipulations and the chloroacetate group was used extensively as the temporary protecting group to ensure stereoselective glycosylations using rhamnose synthons. A TEMPO-mediated late stage oxidation was used successfully to generate the desired uronic acid moiety. The synthetic will definitely enhance the scope for further biological evaluation of the target oligosaccharide related to the carbopenem resistant K. pneumoniae strains and pave the path for a potential vaccine target.

Experimental section

General methods

All solvents and reagents were dried prior to use according to standardized methods.²⁰ The commercially purchased reagents were used without any further purification unless mentioned otherwise. All reactions were monitored by Thin Layer Chromatography (TLC) on Silica-Gel 60-F₂₅₄ with detection by fluorescence followed by charring after immersion in 10% ethanolic solution of H₂SO₄. Flash chromatography was performed with Silica Gel 230–400 mesh. ¹H and ¹³C NMR spectra were recorded on Bruker Avance 500 MHz spectrometer (¹H NMR at 500 MHz and ¹³C NMR at 125 MHz). HRMS analysis was performed with Micromass Q-TOF micro (Waters Corporation) instrument by +ve mode electro-spray ionization.

p-Tolyl-4-O-benzoyl-1-thio-α-L-rhamnopyranoside (3). Compound 2 (6 g, 14.5 mmol) was dissolved in AcOH–H₂O (8 : 1, 36 mL) and the solution was stirred at 80 °C for 2 h until the starting material was completely converted to a slower moving spot as suggested by TLC (n-hexane–EtOAc; 3 : 1). The solvents were evaporated and co-evaporated twice with toluene followed by purification of the crude product by flash chromatography using (n-hexane–EtOAc; 3.5 : 1) to afford pure compound 3 (4.9 g, 91%) as colourless syrup. [α]²⁵_D +103° (c 1.0, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 8.09–7.15 (m, 9H, ArH), 5.51 (d, 1H, J_1,2 1.5 Hz, H-1), 5.09 (t, 1H, J_3,4, J_4,5 9.5 Hz, H-4), 4.50 (m, 1H, H-5), 4.26 (bd, 1H, J_1,2 1.5 Hz, H-2), 4.07 (dd, 1H, J_2,3 3.0 Hz, J_3,4 9.5 Hz, H-3), 3.31 (bs, 1H, OH), 2.79 (bs, 1H, OH), 2.34 (s, 3H, SC₆H₄CH₃), 1.31 (d, 3H, J_5,6 6.0 Hz). ¹³C NMR (CDCl₃, 125 MHz) δ: 167.7 (COC₆H₅), 137.9, 133.6 (2), 132.1 (2), 129.9 (4), 129.3, 128.5 (2) (ArC), 87.6 (C-1), 76.6, 72.3, 70.9, 67.0, 21.1 (SC₆H₄CH₃), 17.5 (C–CH₃). HRMS calcd for C₂₀H₂₂O₅SNa (M + Na)⁺: 397.1086, found: 397.1084.

p-Tolyl-4-O-benzoyl-3-O-benzyl-1-thio-α-L-rhamnopyranoside (4). A mixture of compound 3 (4.8 g, 13 mmol) and Bu₂SnO (4.3 g, 17 mmol) was refluxed for 3 h at 80 °C in dry MeOH (30 mL). The resulting solution was concentrated in vacuo. The crude residue was dried under vacuum for 1 h. The residue was then dissolved in dry DMF (20 mL) followed by addition of Bu₄NI (5.3 g, 14.5 mmol) and stirred at room temperature for 10 min. BnBr (2.0 mL, 17 mmol) was added to the mixture and the solution was stirred for 10 h. After evaporating the solvents in vacuo the residue was dissolved in CH₂Cl₂ (40 mL) and washed successively with H₂O (50 mL), Na₂SO₃ (50 mL) and brine (50 mL). The organic layer was collected, dried (Na₂SO₄) and evaporated in vacuo. The crude product was purified by flash chromatography using n-hexane–EtOAc (6 : 1) as eluent to give the pure compound 4 (5.0 g, 85%). [α]²⁵_D +96° (c 1.0, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 8.03–7.10 (m, 14H, ArH), 5.45 (d, 1H, J_1,2 < 1.0 Hz, H-1), 5.42 (t, 1H, J_3,4, J_4,5 9.5 Hz, H-4), 4.65, 4.51 (ABq, 2H, J_A–B 12.0 Hz, CH₂Ph), 4.37 (m, 1H, H-5), 4.32 (dd, 1H, J_1,2 < 1.0 Hz, J_2,3 3.0 Hz, H-2), 3.90 (dd, 1H, J_2,3 3.0 Hz, J_3,4 9.5 Hz, H-3), 3.08 (bs, 1H, OH), 2.31 (s, 3H, S–C₆H₄–CH₃), 1.24 (d, 3H, J_5,6 6.5 Hz). ¹³C NMR (CDCl₃, 125 MHz) δ: 165.9 (COC₆H₅), 137.9, 137.3, 133.3, 132.1 (2), 130.0 (3), 129.9 (2), 128.6 (2), 128.5 (2), 128.1 (4) (ArC), 87.4 (C-1), 77.5, 73.2, 71.7, 69.8, 67.7, 21.2 (SC₆H₄CH₃), 17.5 (C–CH₃). HRMS calcd for C₂₇H₂₂O₅SNa (M + Na)⁺: 487.1555, found: 487.1553.

p-Tolyl-4-O-benzoyl-3-O-benzyl-2-O-chloroacetyl-1-thio-α-L-rhamnopyranoside (5). Compound 4 (5.0 g, 11 mmol) and chloroacetic acetic anhydride (4.3 g, 25 mmol) were taken in 20 mL dry CH₂Cl₂ and cooled to 0 °C. Pyridine (3.6 mL, 44 mmol) was then added and after 2 h TLC (n-hexane–EtOAc; 7 : 1) showed complete conversion of the reactant to a faster moving spot, the mixture was evaporated in vacuo and co-evaporated twice with toluene to obtain a syrupy residue, which was then purified by flash chromatography using n-hexane–EtOAc (8 : 1) as eluent to give the pure compound 5 (5.2 g, 89%) as light yellow syrup. [α]²⁵_D +113° (c 0.8, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 8.04–7.15 (m, 14H, ArH), 5.73 (dd, 1H, J_1,2, 1.5 Hz, J_2,3 3.0 Hz, H-2), 5.45 (d, 1H, J_1,2 1.5, H-1), 5.36 (t, 1H, J_3,4, J_4,5 10.0 Hz, H-4), 4.66, 4.45 (ABq, 2H, J_A–B 12.5 Hz, CH₂Ph), 4.36 (m, 1H, H-5), 4.25, 4.17 (ABq, 2H, J_A–B 15.5 Hz, COCH₂Cl), 3.98 (dd, 1H, J_2,3 3.0 Hz, J_3,4 10.0 Hz, H-3), 2.35 (s, 3H, S–C₆H₄–CH₃), 1.29 (d, 3H, J_5,6 6.5 Hz). ¹³C NMR (CDCl₃, 125 MHz) δ: 166.7 (COCH₂Cl), 165.5 (COC₆H₅), 138.3, 136.8, 133.2, 132.4 (2), 129.9 (2), 129.8 (2), 129.5, 129.1, 128.3 (2), 128.2 (2), 128.0 (2), 127.8 (ArC), 86.1 (C-1), 74.2, 72.6, 71.8, 71.3, 67.9, 74.2, 72.6, 71.8, 71.3, 67.9, 40.8 (COCH₂Cl), 21.0 (SC₆H₄CH₃), 17.2 (C–CH₃). HRMS calcd for C₂₉H₂₉ClO₆SNa (M + Na)⁺: 563.1271, found: 563.1269.

p-Methoxyphenyl 4-O-benzoyl-3-O-benzyl-2-O-chloroacetyl-α-L-rhamnopyranosyl-(1→3)-4-O-acetyl-2,6,-di-O-benzyl-β-D-galactopyranoside (7). A mixture of donor 5 (1.9 g, 3.6 mmol), known acceptor 6 (1.4 g, 2.8 mmol) and MS (4 Å) (1.5 g) in dry CH₂Cl₂ (15 mL) was stirred under nitrogen atmosphere for 30 min. NIS (1 g, 4.4 mmol) was added and the mixture was cooled in ice-water bath (∼5 °C) before adding H₂SO₄-silica (75 mg). The mixture was stirred at the same temperature for 30 min when TLC (n-hexane–EtOAc; 3 : 1) indicated complete consumption of the donor. The reaction mixture was neutralized with Et₃N and the mixture was filtered through a pad of Celite. The filtrate was washed successively with aq. Na₂S₂O₃ (2 × 30 mL), aq. NaHCO₃ (2 × 30 mL) and brine (30 mL). Organic layer was separated, dried over Na₂SO₄ and evaporated in vacuo. The syrupy crude product thus obtained was purified by flash chromatography using n-hexane–EtOAc (4 : 1) as eluent to afford pure disaccharide 7 (2.3 g, 91%) as white foam. [α]²⁵_D +86° (c 0.9, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 8.07–7.12 (m, 20H, ArH), 7.05, 6.82 (2d, 4H, C₆H₄OCH₃), 5.52 (dd, 1H, J_2′,3′ 1.5 Hz, J_1′,2′ < 1.0 Hz, H-2′), 5.39 (bd, 1H, J_3,4 1.5 Hz, H-4), 5.23 (d, 1H, J_1′,2′ < 1.0 Hz, H-1′), 5.22 (t, 1H, J_3′,4′, J_4′,5′ 10.0 Hz, H-4′), 5.06, 4.75 (ABq, 2H, J_A–B 11.0 Hz, CH₂Ph), 4.92 (d, 1H, J_1,2 7.0 Hz, H-1), 4.61, 4.42 (ABq, 2H, J_A–B 12.5 Hz, CH₂Ph), 4.55, 4.48 (ABq, 2H, J_A–B 12.0 Hz, CH₂Ph), 4.21, 4.12 (ABq, 2H, J_A–B 15.5 Hz, CH₂Cl), 4.13 (m, 1H, H-5′), 3.93 (m, 2H, H-2, H-3), 3.88 (t, 1H, J_4,5, J_5,6a, J_5,6b 6.5 Hz, H-5), 3.83 (dd, 1H, J_2′,3′ 1.5 Hz, J_3′,4′ 10.0 Hz, H-3′), 3.78 (s, 3H, OC₆H₄OCH₃), 3.58 (m, 2H, H-6a, H-6b), 1.97 (s, 3H, COCH₃), 1.25 (d, 3H, J_5′,6′ 6.5 Hz). ¹³C NMR (CDCl₃, 125 MHz) δ: 169.9 (COCH₃), 166.6 (COCH₂Cl), 165.6 (COC₆H₅), 155.4, 151.1, 137.6, 137.5, 137.3, 133.1, 129.9 (2), 129.7, 128.5 (4), 128.3 (4), 128.2 (2), 127.8 (4), 127.7 (2), 127.6, 118.2 (2), 114.6 (2) (ArC), 102.9 (C-1), 98.5 (C-1′), 79.0, 75.1, 74.7, 73.6, 73.5, 72.7, 72.5, 71.1, 69.9, 69.1, 68.2, 67.3, 55.6 (OC₆H₄OCH₃), 40.8 (COCH₂Cl), 20.6 (COCH₃), 17.6 (C–CH₃). HRMS calcd for C₅₁H₅₃ClO₁₄Na (M + Na)⁺: 947.3022, found: 947.3019.

p-Methoxyphenyl 4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→3)-4-O-acetyl-2,6,-di-O-benzyl-β-D-galactopyranoside (8). A mixture of disaccharide 7 (2.2 g, 2.4 mmol), thiourea (900 mg, 12 mmol) and 2,4,6-collidine (1.6 mL, 12 mmol) were refluxed in 20 mL CH₂Cl₂–MeOH (2 : 3) for 10 hours when TLC (n-hexane–EtOAc; 2.5 : 1) confirmed the complete conversion of the starting material to a slower moving spot. The solvents were evaporated in vacuo, the solid residue was dissolved in CH₂Cl₂ and washed with 1 (N) HCl (2 × 30 mL). The organic layer was collected, filtered, dried (Na₂SO₄) and evaporated in vacuo. The crude residue thus obtained was further subjected to flash chromatography using n-hexane–EtOAc (3 : 1) as eluent to give the pure disaccharide acceptor 8 (1.8 g, 87%). [α]²⁵_D +121° (c 0.8, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 8.09–7.17 (m, 20H, ArH), 7.07, 6.83 (2d, 4H, C₆H₄OCH₃), 5.42 (m, 1H, H-4), 5.34 (t, 1H, J_3′,4′, J_4′,5′ 9.5 Hz, H-4′), 5.30 (d, 1H, J_1′,2′ < 1 Hz, H-1′), 5.04, 4.78 (ABq, 2H, J_A–B 11.0 Hz, CH₂Ph), 4.93 (d, 1H, J_1,2 7.5 Hz, H-1), 4.61, 4.50 (ABq, 2H, J_A–B 12.5 Hz, CH₂Ph), 4.56, 4.49 (ABq, 2H, J_A–B 11.5 Hz, CH₂Ph), 4.10 (m, 1H, H-5′), 3.97–3.89 (m, 4H, H-2, H-3, H-5, H-2′), 3.78 (s, 3H, OC₆H₄OCH₃), 3.73 (dd, 1H, J_2′,3′ 2.0 Hz, J_3′,4′ 9.5 Hz, H-3′), 3.60 (m, 2H, H-6a, H-6b), 2.60 (bs, 1H, OH), 2.03 (s, 3H, COCH₃), 1.26 (d, 3H, J_5′,6′ 6.5 Hz). ¹³C NMR (CDCl₃, 125 MHz) δ: 169.8 (COCH₃), 165.7 (COC₆H₅), 155.4, 151.3, 137.8, 137.7, 137.4, 133.0, 129.9, 129.8, 128.4 (2), 128.3 (4), 128.3 (3), 128.2 (2), 127.8, 127.7 (2), 127.7 (2), 127.6 (2), 118.3 (2), 114.5 (2) (ArC), 102.9 (C-1), 100.5 (C-1′), 79.0, 75.8, 75.4, 75.0, 73.6, 72.9, 72.8, 71.3, 69.9, 68.4, 68.2, 66.9, 55.5 (OC₆H₄OCH₃), 20.6 (COCH₃), 17.5 (C–CH₃). HRMS calcd for C₄₉H₅₂O₁₃Na (M + Na)⁺: 871.3306, found: 871.3304.

p-Methoxyphenyl 4-O-benzoyl-3-O-benzyl-2-O-chloroacetyl-α-L-rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→3)-4-O-acetyl-2,6,-di-O-benzyl-β-D-galactopyranoside (9). A mixture of disaccharide acceptor 8 (1.6 g, 1.9 mmol), donor 5 (1.3 g, 2.5 mmol) and 4 Å MS (2 g) in dry CH₂Cl₂ (20 mL) was stirred under nitrogen atmosphere for 30 min. NIS (716 mg, 3.2 mmol) was added and cooled to 0 °C. After 15 min H₂SO₄-silica (75 mg) was added and stirring continued for 15 min till TLC (n-hexane–EtOAc; 2.5 : 1) confirmed the complete consumption of the donor. Then the reaction mixture was filtered through a pad of Celite. The filtrate was washed successively with aq. Na₂S₂O₃ (2 × 30 mL), aq. NaHCO₃ (2 × 30 mL) and brine (30 mL). Organic layer was separated, dried (Na₂SO₄) and evaporated in vacuo. The crude product thus obtained was subjected to flash chromatography using n-hexane–EtOAc (3 : 1) as the eluent to obtain the pure trisaccharide 9 (2.1 g, 90%) as white foam. [α]²⁵_D +87° (c 0.8, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 8.14–7.12 (m, 30H, ArH), 7.01, 6.79 (2d, 4H, C₆H₄OCH₃), 5.64 (dd, 1H, J_{2′′,3′′} 2.5 Hz, J_{1′′,2′′} < 1.0 Hz, H-2′′), 5.41 (m, 1H, H-4), 5.36 (t, 1H, J_3′,4′, J_4′,5′ 9.5 Hz, H-4′), 5.22 (d, 1H, J_1′,2′ < 1.0 Hz, H-1′), 5.21 (t, 1H, J_{3′′,4′′}, J_{4′′,5′′} 10.0 Hz, H-4′′), 4.98, 4.82 (ABq, 2H, J_A–B 11.5 Hz, CH₂Ph), 4.89 (m, 2H, H-1, H-1′′), 4.65–4.47 (m, 6H, 3 × CH₂Ph), 4.20, 4.12 (ABq, 2H, J_A–B 15.0 Hz, COCH₂Cl), 4.08 (m, 1H, H-5′), 4.02 (dd, 1H, J_{2′′,3′′} 2.5 Hz, J_{3′′,4′′} 10.0 Hz, H-3′′), 3.97 (m, 1H, H-5′′), 3.89–3.86 (m, 4H, H-2, H-2′, H-3, H-5), 3.81 (dd, 1H, J_2′,3′ 2.5 Hz, J_3′,4′ 9.5 Hz, H-3′), 3.77 (s, 3H, OC₆H₄OCH₃), 3.58 (m, 2H, H-6a, H-6b), 2.03 (s, 3H, COCH₃), 1.30 (d, 3H, J_5′,6′ 7.0 Hz, C–CH₃), 1.07 (d, 3H, J_{5′′,6′′} 6.0 Hz, C–CH₃). ¹³C NMR (CDCl₃, 125 MHz) δ: 170.0 (COCH₃), 166.4 (COCH₂Cl), 165.7 (COC₆H₅), 165.6 (COC₆H₅), 155.4, 151.2, 137.8, 137.7, 137.3, 133.1, 130.1, 129.9 (2), 129.8, 128.5 (4), 128.4 (5), 128.3 (5), 128.2 (2), 128.1 (2), 127.8 (4), 127.7, 127.6 (3), 118.3 (3), 114.6 (3) (ArC), 103.0 (C-1), 100.4 (C-1′), 99.4 (C-1′′), 78.3, 76.5, 76.0, 75.5, 74.6, 74.7, 73.5, 73.3, 72.9, 72.5, 71.0, 71.2, 70.3, 69.6, 68.4, 67.6, 67.4, 55.6 (OC₆H₄OCH₃), 40.9 (COCH₂Cl), 20.7 (COCH₃), 17.8, 17.4 (2 × C–CH₃). HRMS calcd for C₇₁H₇₃ClO₁₉Na (M + Na)⁺: 1287.4332, found: 1287.4330.

p-Methoxyphenyl 4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→3)-4-O-acetyl-2,6,-di-O-benzyl-β-D-galactopyranoside (10). Pure trisaccharide 9 (2 g), thiourea (600 mg, 7.9 mmol) and collidine (1 mL, 7.9 mmol) were dissolved in 20 mL CH₂Cl₂–MeOH (2 : 3). The mixture was refluxed for 10 h when the complete conversion of the starting material to a slower moving spot was judged by the TLC (n-hexane–EtOAc; 2 : 1). The mixture was evaporated in vacuo. The solid residue thus obtained was dissolved in CH₂Cl₂ and washed with 1 (N) HCl (2 × 30 mL) and brine (2 × 30 mL). The organic layer was separated, dried (Na₂SO₄) and evaporated in vacuo to get the crude product. It was purified by flash chromatography using n-hexane–EtOAc (5 : 2) as the eluent to get the pure compound 10 (1.7 g, 88%) as white powder. [α]²⁵_D +78° (c 0.7, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 8.08–7.10 (m, 30H, ArH), 6.96, 6.74 (2d, 4H, C₆H₄OCH₃), 5.35 (m, 1H, H-4), 5.33 (t, 1H, J_3′,4′, J_4′,5′ 10.0 Hz, H-4′), 5.30 (t, 1H, J_{3′′,4′′}, J_{4′′,5′′} 10.0 Hz, H-4′′), 5.19 (d, 1H, J_1′,2′ 2.0 Hz, H-1′), 5.09 (d, 1H, J_{1′′,2′′} 2.0 Hz, H-1′′), 4.91, 4.82 (ABq, 2H, J_A–B 12.5 Hz, CH₂Ph), 4.86 (d, 1H, J_1,2 7.5 Hz, H-1), 4.64, 4.52 (ABq, 2H, J_A–B 12.0 Hz, CH₂Ph), 4.56–4.42 (m, 4H, 2 × CH₂Ph), 4.31 (dd, 1H, J_{1′′,2′′} 2.0 Hz, J_{2′′,3′′} 3.0 Hz, H-2′′), 4.03 (m, 1H, H-5′), 3.97 (dd, 1H, J_1′,2′ 2.0 Hz, J_2′,3′ 3.0 Hz, H-2′), 3.93 (m, 1H, H-5′′), 3.91 (dd, 1H, J_{2′′,3′′} 3.0 Hz, J_{3′′,4′′} 10.0 Hz, H-3′′), 3.85–3.82 (m, 3H, H-2, H-3, H-5), 3.75 (dd, 1H, J_2′,3′ 3.0 Hz, J_3′,4′ 10.0 Hz, H-3′), 3.69 (s, 3H, OC₆H₄OCH₃), 3.53 (m, 2H, H-6a, H-6b), 2.62 (bs, 1H, OH), 1.93 (s, 3H, COCH₃), 1.23 (d, 3H, J_5′,6′ 6.5 Hz, C–CH₃), 1.03 (d, 3H, J_{5′′,6′′} 6.5 Hz, C–CH₃). ¹³C NMR (CDCl₃, 125 MHz) δ: 169.9 (COCH₃), 165.7, 165.6 (2 × COC₆H₅), 155.3, 151.1, 137.7 (3), 137.3, 133.0, 132.9, 130.0, 129.9, 129.8 (2), 129.7 (2), 128.4 (4), 128.3 (6), 128.2 (4), 128.1 (2), 127.8, 127.7 (4), 127.6, 127.5 (2), 118.2 (2), 114.5 (2) (ArC), 103.0 (C-1), 101.0 (C-1′′), 100.6 (C-1′), 77.8, 75.9 (2), 75.6, 75.2, 74.4, 73.6, 73.2, 72.8 (2), 71.6, 71.3, 69.6, 68.4, 68.0, 67.5, 66.9, 55.5, 20.6 (COCH₃), 17.7, 17.3 (2 × C–CH₃). HRMS calcd for C₆₉H₇₂O₁₈Na (M + Na)⁺: 1211.4616, found: 1211.4613.

p-Methoxyphenyl 4-O-benzoyl-3-O-benzyl-2-O-chloroacetyl-α-L-rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→3)-4-O-acetyl-2,6,-di-O-benzyl-β-D-galactopyranoside (11). A mixture of trisaccharide acceptor 10 (1.5 g, 1.3 mmol), donor 5 (890 mg, 1.6 mmol) and 4 Å MS (1.5 g) in dry CH₂Cl₂ (15 mL) was stirred under nitrogen atmosphere for 30 min. NIS (444 mg, 1.9 mmol) was then added and the mixture was cooled to 0 °C. After that H₂SO₄-silica (50 mg) was added to it and allowed to stir for 30 min when TLC (n-hexane–EtOAc; 2 : 1) showed complete consumption of the donor. The mixture was filtered through a Celite pad and the filtrate was successively washed with aq. Na₂S₂O₃ (2 × 30 mL), aq. NaHCO₃ (2 × 30 mL) and brine (30 mL). The organic layer was collected, dried (Na₂SO₄) and evaporated in vacuo. Crude product thus obtained was purified by flash chromatography using n-hexane–EtOAc (2 : 1) as the eluent. The pure tetrasaccharide 11 (1.8 g, 89%) was obtained as white foam. [α]²⁵_D +142° (c 0.8, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 8.10–7.10 (m, 40H, ArH), 6.99, 6.78 (2d, 4H, C₆H₄OCH₃), 5.65 (dd, 1H, J_{1′′′,2′′′} < 1.0 Hz, J_{2′′′,3′′′} 3.0 Hz, H-2′′′), 5.39–5.34 (m, 3H, H-4, H-4′, H-4′′), 5.22 (d, 1H, J_1′,2′ 1.5 Hz, H-1′), 5.21 (t, 1H, J_{3′′′,4′′′}, J_{4′′′,5′′′} 10.0 Hz, H-4′′′), 5.14 (d, 1H, J_{1′′,2′′} 1.5 Hz, H-1′′), 4.96, 4.82 (ABq, 2H, J_A–B 12.5 Hz, CH₂Ph), 4.94 (d, 1H, J_{1′′′,2′′′} < 1.0 Hz, H-1′′′), 4.88 (d, 1H, J_1,2 7.0 Hz, H-1), 4.70–4.42 (m, 8H, 4 × CH₂Ph), 4.29 (dd, 1H, J_{1′′,2′′} 1.5 Hz, J_{2′′,3′′} < 1.0 Hz, H-2′′), 4.16, 4.09 (ABq, 2H, J_A–B 15.5 Hz, COCH₂Cl), 4.07–4.01 (m, 4H, H-3′′, H-3′′′, H-5′, H-5′′), 3.96 (m, 1H, H-5′′′), 3.95 (dd, 1H, J_1′,2′ 1.5 Hz, J_2′,3′ 2.5 Hz, H-2′), 3.90–3.84 (m, 3H, H-2, H-3, H-5), 3.78 (dd, 1H, J_2′,3′ 2.5 Hz, J_3′,4′ 9.5 Hz, H-3′), 3.76 (s, 3H, OC₆H₄OCH₃), 3.57 (m, 2H, H-6a, H-6b), 1.97 (s, 3H, COCH₃), 1.29 (d, 3H, J_5′,6′ 6.5 Hz, C–CH₃), 1.14 (d, 3H, J_{5′′,6′′} 6.5 Hz, C–CH₃), 1.09 (d, 3H, J_{5′′′,6′′′} 6.5 Hz, C–CH₃). ¹³C NMR (CDCl₃, 125 MHz) δ: 169.9 (COCH₃), 166.4 (COCH₂Cl), 165.8, 165.7, 165.6 (3 × COC₆H₅), 155.5, 151.3, 137.8, 137.7, 137.4, 133.2, 133.1 (2), 130.0 (2), 129.9 (2), 129.8 (2), 128.4 (14), 128.3 (8), 128.2 (2), 128.1 (2), 127.8 (6), 127.7 (2), 127.5 (2), 118.3 (2), 114.6 (2) (ArC), 103.1 (C-1), 101.1 (C-1′′), 100.7 (C-1′), 99.2 (C-1′′′), 78.2, 76.1, 75.7, 75.4, 74.6, 73.9, 73.7 (2), 73.3, 73.1, 72.9, 72.7, 71.8 (2), 71.3, 70.2, 69.6, 68.4, 67.7, 67.6, 67.3, 55.6, 40.9 (COCH₂Cl), 20.6 (COCH₃), 17.8, 17.7, 17.5 (3 × C–CH₃). HRMS calcd for C₉₁H₉₃ClO₂₄Na (M + Na)⁺: 1627.5643, found: 1627.5641.

p-Methoxyphenyl 4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→3)-4-O-acetyl-2,6,-di-O-benzyl-β-D-galactopyranoside (12). To a solution of the tetrasaccharide 11 (1.7 g, 1.1 mmol) and thiourea (400 mg, 5.3 mmol) in 20 mL CH₂Cl₂–MeOH (2 : 3), collidine (0.7 mL, 5.3 mmol) was added and the mixture was refluxed for 12 h till TLC (n-hexane–EtOAc; 2 : 1) indicated the complete conversion of the starting material to a slower moving spot. The reaction mixture was evaporated in vacuo and the residue was dissolved in CH₂Cl₂. It was further washed with 1 (N) HCl (2 × 30 mL) and brine (2 × 30 mL). Resulting organic layer was collected, dried (Na₂SO₄) and evaporated in vacuo to obtain the crude product. It was further purified by flash chromatography using n-hexane–EtOAc (2 : 1) as the eluent to obtain the pure tetrasaccharide acceptor 12 (1.5 g, 90%) as white foam. [α]²⁵_D +102° (c 0.9, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 8.11–7.12 (m, 40H, ArH), 6.99, 6.79 (2d, 4H, C₆H₄OCH₃), 5.41–5.33 (m, 4H, H-4, H-4′, H-4′′, H-4′′′), 5.23 (d, 1H, J_1′,2′ 1.5 Hz, H-1′), 5.19 (d, 1H, J_{1′′′,2′′′′} 1.5 Hz, H-1′′′), 5.12 (d, 1H, J_{1′′,2′′} 1.5 Hz, H-1′′), 4.95, 4.82 (ABq, 2H, J_A–B 12.5 Hz, CH₂Ph), 4.88 (d, 1H, J_1,2 7.0 Hz, H-1), 4.72–4.44 (m, 8H, 4 × CH₂Ph), 4.37 (dd, 1H, J_{1′′′,2′′′} 1.5 Hz, J_{2′′′,3′′′} < 1.0 Hz, H-2′′′), 4.30 (dd, 1H, J_{1′′,2′′} 1.5 Hz, J_{2′′,3′′} < 1.0 Hz, H-2′′), 4.08–4.02 (m, 4H, H-3′′, H-3′′′, H-5′, H-5′′), 4.01–3.96 (m, 2H, H-2′, H-5′′′), 3.88–3.84 (m, 3H, H-2, H-3, H-5), 3.79 (dd, 1H, J_2′,3′ 3.5 Hz, J_3′,4′ 6.5 Hz, H-3′), 3.77 (s, 3H, OC₆H₄OCH₃), 3.58 (m, 2H, H-6a, H-6b), 2.57 (bs, 1H, OH), 1.96 (s, 3H, COCH₃), 1.28 (d, 3H, J_5′,6′ 6.0 Hz, C–CH₃), 1.16 (d, 3H, J_{5′′,6′′} 6.0 Hz, C–CH₃), 1.11 (d, 3H, J_{5′′′,6′′′} 6.0 Hz, C–CH₃). ¹³C NMR (CDCl₃, 125 MHz) δ: 169.4 (COCH₃), 165.9, 165.8, 165.6 (3 × COC₆H₅), 155.4, 155.2, 127.8, 137.7 (4), 137.5, 133.1 (2), 133.0 (2), 130.6 (6), 129.8 (2), 128.4 (6), 128.3 (4), 128.2 (2), 128.0 (4), 127.8 (6), 127.7 (4), 127.6 (4), 118.3 (2), 114.5 (2) (ArC), 103.0 (C-1), 101.2 (C-1′′′), 100.9 (C-1′′), 100.6 (C-1′), 78.1, 76.4, 76.2, 76.0, 75.4 (2), 74.8, 74.6, 73.7, 73.2, 73.1, 73.0, 72.9, 71.7, 71.6, 71.5, 69.6, 68.5, 68.2, 67.6 (2), 66.9, 55.6, 20.6 (COCH₃), 17.8, 17.7, 17.4 (3 × CH₃). HRMS calcd for C₈₉H₉₂O₂₃Na (M + Na)⁺: 1551.5927, found: 1551.5925.

4-Tolyl 2,3-di-O-benzyl-4-O-chloroacetyl-6-O-(4-methoxybenzyl)-1-thio-β-D-galactopyranoside (14). The compound 15 (1.3 g, 2.2 mmol) was dissolved in dry CH₂Cl₂ (20 mL) in presence of chloroacetic anhydride (770 mg, 4.5 mmol) and kept at 0 °C for 10 min. Pyridine (0.7 mL, 9.0 mmol) was then added to the reaction mixture and stirred for 2 hours when TLC (n-hexane–EtOAc; 4 : 1) showed complete conversion of the starting material to a faster moving spot. The reaction mixture was evaporated in vacuo and co-evaporated with toluene. The crude product thus obtained was further subjected to purification by flash chromatography using n-hexane–EtOAc (6 : 1) as eluent to get the pure compound 14 (1.3 g, 88%). [α]²⁵_D +139° (c 1.0, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 7.54–6.96 (m, 18H, ArH), 5.74 (dd, 1H, J_3,4 3.0 Hz, J_4,5 < 1.0 Hz, H-4), 4.83–4.53 (m, 4H, 2 × CH₂Ph), 4.67 (d, 1H, J_1,2 9.0 Hz, H-1), 4.55, 4.42 (ABq, 2H, J_A–B 12.5 Hz, CH₂PhOMe), 4.10, 4.03 (ABq, 2H, J_A–B 15.5 Hz, CH₂Cl), 3.85 (s, 3H, OCH₂C₆H₄OCH₃), 3.79 (dd, 1H, J_5,6a 6.5 Hz, J_6a,6b 9.5 Hz, H-6a), 3.71 (dd, 1H, J_2,3 9 Hz, J_3,4 3 Hz, H-3), 3.65 (t, 1H, J_1,2, J_2,3 9.0 Hz, H-2), 3.64 (m, 1H, H-5), 3.55 (dd, 1H, J_5,6b 7.5 Hz, J_6a,6b 9.5 Hz, H-6b), 2.37 (s, 3H, S–C₆H₄–CH₃). ¹³C NMR (CDCl₃, 125 MHz) δ: 166.6 (COCH₂Cl), 159.3, 138.0, 137.6, 137.3, 132.6 (2), 129.7 (2), 129.5 (2), 129.4, 129.3, 128.3 (2), 128.2 (4), 128.0 (2), 127.8, 127.7, 113.7 (2) (ArC), 87.8 (C-1), 80.9, 76.5, 75.6, 75.2, 73.1, 72.0, 68.7, 67.0, 55.1, 40.7 (COCH₂Cl), 21.0 (SC₆H₄CH₃). HRMS calcd for C₃₇H₃₉ClO₇Na (M + Na)⁺: 685.2001, found: 685.1998.

p-Methoxyphenyl 2,3-di-O-benzyl-4-O-chloroacetyl-6-O-p-methoxybenzyl-α-D-galactopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→3)-4-O-acetyl-2,6,-di-O-benzyl-β-D-galactopyranoside (15). A mixture of tetrasaccharide acceptor 12 (1.3 g, 0.85 mmol), donor 14 (740 mg, 1.1 mmol) and MS 4 Å (2.0 g) in dry CH₂Cl₂ (20 mL) was stirred under nitrogen atmosphere for 30 min. NIS (322 mg, 1.4 mmol) was added and the mixture was cooled to −50 °C followed by the addition of H₂SO₄-silica (50 mg) and the mixture was allowed to stir at the same temperature for 15 minutes. As TLC (n-hexane–EtOAc; 5 : 2) suggested the full consumption of the donor, the reaction was quenched by Et₃N. It was then followed by filtration of the mixture through a Celite pad. Resulting solution was then successively washed with aq. Na₂S₂O₃ (2 × 30 mL), aq. NaHCO₃ (2 × 30 mL) and brine (30 mL). It was then dried (Na₂SO₄) and evaporated in vacuo. The crude product was then purified by flash chromatography using n-hexane–EtOAc (3 : 1) as the eluent. Thus the pure pentasaccharide 15 (1.4 g, 82%) was furnished as white foam. [α]²⁵_D +68° (c 0.8, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 8.10–6.99 (m, 55H, ArH), 6.81, 6.79 (2d, 4H, C₆H₄OCH₃), 5.69 (bd, 1H, J_3,4 1.5 Hz, H-4), 5.42–5.37 (m, 4H, H-4, H-4′, H-4′′, H-4′′′), 5.21 (d, 1H, J_1′,2′ 1.0 Hz, H-1′), 5.15 (d, 1H, J_{1′′′,2′′′′} < 1.0 Hz, H-1′′′), 5.11 (d, 1H, J_{1′′,2′′} 1.0 Hz, H-1′′), 4.96, 4.83 (ABq, 2H, J_A–B 12.5 Hz, CH₂Ph), 4.88 (d, 1H, J_1,2 7.0 Hz, H-1), 4.83, 4.76 (ABq, 2H, J_A–B 10.5 Hz, CH₂Ph), 4.79 (d, 1H, J_{1′′′′,2′′′′} 3.5 Hz, H-1′′′′), 4.70–4.15 (m, 12H, 6 × CH₂Ph), 4.53 (m, 1H, H-5′′′′), 4.35 (m, 2H, H-2′′, H-2′′′), 4.12 (m, 1H, H-3′′′′), 4.08–4.01 (m, 3H, H-5′, H-3′′, H-3′′′), 4.01, 4.90 (ABq, 2H, J_A–B 15.0 Hz, COCH₂Cl), 3.99–3.94 (m, 3H, H-2′, H-5′′, H-5′′′), 3.87–3.86 (m, 2H, H-3, H-5), 3.81–3.78 (m, 2H, H-2, H-3′), 3.77 (s, 3H, OC₆H₄OCH₃), 3.73 (s, 3H, OCH₂C₆H₄OCH₃), 3.64 (dd, 1H, J_{1′′′′,2′′′′} 3.5 Hz, J_{2′′′′,3′′′′} 6.5 Hz, H-2′′′′), 3.58 (m, 2H, H-6a, H-6b), 3.27 (m, 2H, H-6a′′′′, H-6b′′′′), 1.95 (s, 3H, COCH₃), 1.28 (d, 3H, J_5′,6′ 6.0 Hz, C–CH₃), 1.16 (d, 3H, J_{5′′,6′′} 6.0 Hz, C–CH₃), 1.09 (d, 3H, J_{5′′′,6′′′} 6.0 Hz, C–CH₃). ¹³C NMR (CDCl₃, 125 MHz) δ: 169.9 (COCH₃), 166.7 (COCH₂Cl), 165.8, 165.6 (2) (3 × COC₆H₅), 159.1, 155.4, 151.2, 138.8, 138.0, 137.9, 137.7 (2), 137.5, 133.1, 132.9, 130.1, 130.0, 129.9 (2), 129.8, 129.7 (2), 128.4 (10), 128.2 (10), 128.2 (10), 128.1 (4), 127.8 (4), 127.7 (2), 127.6, 127.5 (2), 127.4 (4), 127.3, 118.3 (2), 114.5 (2), 113.6 (2) (ArC), 103.0 (C-1), 101.3 (C-1′′′), 100.6 (C-1′), 99.1 (C-1′′), 97.2 (C-1′′′′), 78.1, 76.0, 75.9, 75.7, 75.5, 75.2, 75.0, 74.7, 74.6, 74.3, 73.7 (2), 73.2, 73.1, 73.0, 72.9, 72.1 (2), 71.6, 71.0, 70.3, 70.0, 68.5, 67.6 (2), 67.4, 67.2, 55.6, 55.1, 41.0 (COCH₂Cl), 20.6 (COCH₃), 17.8, 17.6 (2) (3 × CH₃). HRMS calcd for C₁₁₉H₁₂₃ClO₃₀Na (M + Na)⁺: 2089.7685, found: 2089.7683.

p-Methoxyphenyl 2,3-di-O-benzyl-6-O-p-methoxybenzyl-α-D-galactopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→3)-4-O-acetyl-2,6,-di-O-benzyl-β-D-galactopyranoside (16). A solution of the pentasaccharide 15 (1.3 g, 0.6 mmol), thiourea (240 mg, 3.2 mmol) and 2,4,6-collidine (0.4 mL, 3.2 mmol) in 20 mL CH₂Cl₂–MeOH (2 : 3) was refluxed for 10 h. When the TLC (n-hexane–EtOAc; 2 : 1) suggested the complete conversion of the starting material to a slower moving spot, the reaction mixture was evaporated in vacuo. It was then dissolved in CH₂Cl₂ and washed with brine (2 × 30 mL). The organic layer was collected, dried (Na₂SO₄) and evaporated. The crude product thus obtained was purified by flash chromatography using n-hexane–EtOAc (2 : 1) to give the pure pentasaccharide acceptor 16 (1.1 g, 85%) as white foam. [α]²⁵_D +108° (c 0.7, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 8.07–6.98 (m, 54H, ArH), 6.78, 6.76 (2d, 4H, C₆H₄OCH₃), 5.44–5.34 (m, 4H, H-4, H-4′, H-4′′, H-4′′′), 5.21 (d, 1H, J_1′,2′ 1.0 Hz, H-1′), 5.15 (d, 1H, J_{1′′′,2′′′} < 1.0 Hz, H-1′′′), 5.11 (d, 1H, J_{1′′,2′′} 1.0 Hz, H-1′′), 4.95, 4.81 (ABq, 2H, J_A–B 11.5 Hz, CH₂Ph), 4.87 (d, 1H, J_1,2 7.0 Hz, H-1), 4.82 (d, 1H, J_{1′′′′,2′′′′} 3.0 Hz, H-1′′′′), 4.70–4.40 (m, 14H, 7 × CH₂Ph), 4.37–4.36 (m, 3H, H-2′′, H-2′′′, H-4′′′′), 4.13 (m, 1H, H-5′′′′), 4.06–3.90 (m, 8H, H-2′, H-3′′, H-3′′′, H-5′, H-5′′, H-5′′′, H-6a′′′′, H-6b′′′′), 3.80–3.79 (m, 4H, H-2, H-3, H-3′, H-5), 3.77 (s, 3H, OC₆H₄OCH₃), 3.70 (s, 3H, OCH₂C₆H₄OCH₃), 3.58 (m, 3H, H-6a, H-6b, H-2′′′′), 3.64 (m, 1H, H-3′′′′), 2.82 (br s, 1H, OH), 1.94 (s, 3H, COCH₃), 1.27 (d, 3H, J_5′,6′ 6.0 Hz, C–CH₃), 1.16 (d, 3H, J_{5′′,6′′} 6.0 Hz, C–CH₃), 1.09 (d, 3H, J_{5′′′,6′′′} 6.0 Hz, C–CH₃). ¹³C NMR (CDCl₃, 125 MHz) δ: 169.9 (COCH₃), 165.7 (2), 165.6 (3 × COC₆H₅), 159.1, 155.5, 151.2, 139.0, 138.0, 137.8 (2), 137.7, 137.6, 133.1, 132.8, 130.2, 130.1 (2), 129.9 (2), 129.8 (4), 129.4 (2), 128.4 (10), 128.3 (4), 128.2 (8), 128.1 (6), 127.9 (4), 127.8 (4), 127.7 (2), 127.6 (2), 127.5 (2), 127.3 (2), 118.3 (2), 114.6 (2), 113.7 (2) (ArC), 103.0 (C-1), 101.3 (C-1′′′), 100.7 (C-1′), 99.2 (C-1′′), 96.8 (C-1′′′′), 78.1, 76.0, 75.5, 74.6, 73.7 (2), 73.3 (4), 73.2 (2), 73.0, 72.7, 72.4, 72.2, 71.6, 70.8, 69.6, 69.4, 68.5, 68.3, 68.2 (2), 67.7, 67.6 (2), 55.6, 55.1, 20.6 (COCH₃), 17.8, 17.7, 17.6 (3 × CH₃). HRMS calcd for C₁₁₇H₁₂₂O₂₉Na (M + Na)⁺: 2013.7969, found: 2013.7967.

p-Methoxyphenyl 2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→4)-2,3-di-O-benzyl-6-O-p-methoxybenzyl-α-D-galactopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→3)-4-O-acetyl-2,6,-di-O-benzyl-β-D-galactopyranoside (18). Pentasaccharide acceptor 16 (1.0 g, 0.5 mmol), known donor 17 (260 mg, 0.7 mmol) and 4 Å MS (2 g) were taken in 20 mL of dry CH₂Cl₂. The mixture was stirred under nitrogen for 45 min followed by the addition of NIS (190 mg, 0.8 mmol). After cooling the reaction mixture to 0 °C H₂SO₄-silica (75 mg) was added to it. Reaction was continued at the same temperature. After 30 minutes when the TLC (n-hexane–EtOAc; 2 : 1) suggested that the whole of the acceptor was consumed, the mixture was neutralised with Et₃N and filtered through a Celite pad. The filtrate was successively washed with aq. Na₂S₂O₃ (2 × 30 mL), aq. NaHCO₃ (2 × 30 mL) and brine (30 mL). The organic layer was separated, dried (Na₂SO₄) and evaporated in vacuo to get the crude product. It was purified by flash chromatography using n-hexane–EtOAc (3 : 2) as eluent to obtain the pure hexasaccharide 18 (960 mg, 84%) as the white foam. [α]²⁵_D +132° (c 0.7, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 8.11–7.00 (m, 54H, ArH), 6.99, 6.79 (2d, 4H, C₆H₄OCH₃), 5.50 (s, 1H, H-2′′′′′), 5.44–5.39 (m, 4H, H-4, H-4′, H-4′′, H-4′′′), 5.37–5.34 (m, 2H, H-3′′′′′, H-5′′′′′), 5.21 (m, 2H, H-1′, H-1′′′′′), 5.09 (m, 2H, H-1′′, H-1′′′), 5.03 (t, 1H, J_{3′′′′′,4′′′′′}, J_{4′′′′′,5′′′′′} 10.0 Hz, H-4′′′′′), 4.96, 4.83 (ABq, 2H, J_A–B 11.5 Hz, CH₂Ph), 4.89 (d, 1H, J_1,2 6.5 Hz, H-1), 4.78–4.28 (m, 14H, 7 × CH₂Ph), 4.70 (d, 1H, J_{1′′′′,2′′′′} 3.0 Hz, H-1′′′′), 4.36–4.34 (m, 2H, H-2′′′, H-4′′′′), 4.21 (s, 1H, H-2′′), 4.08–3.93 (m, 8H, H-2′, H-3′′, H-3′′′, H-5′, H-5′′, H-5′′′′, H-6a′′′′, H-6b′′′′), 3.88–3.78 (m, 5H, H-2, H-3, H-3′, H-5, H-5′′′), 3.77 (s, 3H, OC₆H₄OCH₃), 3.68 (s, 3H, OCH₂C₆H₄OCH₃), 3.59–3.53 (m, 3H, H-6a, H-6b, H-2′′′′), 3.48 (m, 1H, H-3′′′′), 2.09, 2.06, 2.01, 1.96 (4s, 12H, 4 × COCH₃), 1.29 (d, 3H, J_5′,6′ 6.0 Hz, C–CH₃), 1.16 (d, 3H, J_{5′′,6′′} 6.0 Hz, C–CH₃), 1.13 (d, 3H, J_{5′′′′′,6′′′′′} 6.0 Hz, C–CH₃), 1.09 (d, 3H, J_{5′′′,6′′′} 6.0 Hz, C–CH₃). ¹³C NMR (CDCl₃, 125 MHz) δ: 170.0, 169.9, 169.7, 169.5 (4 × COCH₃), 165.7, 165.6, 165.5 (3 × COC₆H₅), 158.9, 155.4, 151.1, 139.0, 138.5, 137.9, 137.7 (2), 137.6, 137.5, 133.0 (2), 132.8, 131.0, 130.3, 130.1, 130.0 (2), 129.8 (4), 129.2 (2), 128.4 (4), 128.3 (8), 128.2 (8), 128.1 (4), 128.1 (4), 127.8, 127.7 (2), 127.6 (2), 127.5 (4), 127.2 (4), 127.1, 118.2 (2), 114.5 (2), 113.5 (2) (ArC), 102.9 (C-1), 101.3 (C-1′′′), 100.6 (C-1′), 99.2 (C-1′′), 98.6 (C-1′′′′′), 96.6 (C-1′′′′), 81.3, 78.1, 76.1, 76.0 (2), 75.4, 75.1, 74.7, 74.6, 74.2, 73.9 (2), 73.6, 73.2 (2), 73.1, 73.0, 72.9, 72.7, 72.5, 72.2, 71.6, 71.5, 71.1, 70.4, 69.7, 69.5, 69.4, 68.5, 68.1, 67.6, 67.5, 67.5, 66.7, 64.7, 55.5, 55.0, 20.8 (2), 20.7, 20.6 (2) (4 × COCH₃), 17.7, 17.6 (2), 17.4 (4 × CH₃). HRMS calcd for C₁₂₉H₁₃₈O₃₆Na (M + Na)⁺: 2285.8866, found: 2285.8863.

p-Methoxyphenyl 2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→4)-2,3-di-O-benzyl-α-D-galactopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→3)-4-O-acetyl-2,6,-di-O-benzyl-β-D-galactopyranoside (19). To a solution of the pure hexasaccharide 18 (950 mg, 0.4 mmol) in CH₂Cl₂ (24 mL) water (5 mL) and DDQ (190 mg, 0.8 mmol) were consecutively added and vigorously stirred for 3 h when the TLC (n-hexane–EtOAc; 2 : 1) suggested complete conversion of the starting material to a slower moving spot. The reaction mixture was washed successively with H₂O and brine. Organic layer was collected, dried (Na₂SO₄) and evaporated in vacuo. The crude product thus obtained was purified by flash chromatography using n-hexane–EtOAc (3 : 1) to afford the pure compound 19 (702 mg, 78%) as foam. [α]²⁵_D +172° (c 0.7, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 8.11–7.11 (m, 50H, ArH), 6.99, 6.79 (2d, 4H, C₆H₄OCH₃), 5.46–5.38 (m, 6H, H-2′′′′′, H-4, H-4′, H-4′′, H-4′′′′, H-5′′′′′), 5.28 (dd, 1H, J_{2′′′′′,3′′′′′} 3.5 Hz, J_{3′′′′′,4′′′′′} 6.5 Hz, H-3′′′′′), 5.24 (d, 1H, J_1′,2′ < 1.0 Hz, H-1′), 5.16 (d, 1H, J_{1′′′′′,2′′′′′} < 1.0 Hz, H-1′′′′′), 5.11 (d, 1H, J_{1′′,2′′} < 1.0 Hz, H-1′′), 5.07 (d, 1H, J_{1′′′,2′′′} < 1.0 Hz, H-1′′′), 5.02–4.42 (m, 12H, 6 × CH₂Ph), 4.99 (m, 1H, H-4′′′′′), 4.90 (d, 1H, J_1,2 6.5 Hz, H-1), 4.43 (m, 1H, H-2′′′), 4.37 (s, 1H, H-2′′), 4.18 (t, 1H, J_{3′′′,4′′′}, J_{4′′′,5′′′} 6.0 Hz, H-4′′′), 4.05–3.95 (m, 8H, H-2′, H-3′′, H-3′′′, H-5′, H-5′′, H-5′′′′, H-6a′′′′, H-6b′′′′), 3.91–3.86 (m, 3H, H-2, H-3, H-3′, H-5, H-5′′′), 3.81 (dd, 1H, J_2′,3′ 7.5 Hz, J_3′,4′ 2.5 Hz, H-3′), 3.77 (s, 3H, OC₆H₄OCH₃), 3.71–3.58 (m, 3H, H-6a, H-6b, H-2′′′′), 3.47 (m, 1H, H-3′′′′), 2.06, 1.99, 1.98 (3s, 12H, 4 × COCH₃), 1.29 (d, 3H, J_5′,6′ 6.0 Hz, C–CH₃), 1.21 (d, 3H, J_{5′′,6′′} 6.0 Hz, C–CH₃), 1.17 (d, 3H, J_{5′′′′′,6′′′′′} 6.0 Hz, C–CH₃), 1.09 (d, 3H, J_{5′′′,6′′′} 6.5 Hz, C–CH₃). ¹³C NMR (CDCl₃, 125 MHz) δ: 170.0, 169.9, 169.7, 169.5 (4 × COCH₃), 166.0, 165.8, 165.6 (3 × COC₆H₅), 155.4, 151.2, 138.9, 138.5, 138.0, 137.8 (2), 137.7, 137.2, 133.3, 133.1, 132.9, 130.1, 130.0 (2), 128.5 (2), 128.4 (8), 128.3 (6), 128.2 (6). 128.1 (8), 128.0, 127.8 (2), 127.7 (2), 127.6 (2), 127.5 (2), 127.4, 127.3, 127.2, 118.3 (2), 114.5 (2) (ArC), 103.3 (C-1), 101.1 (C-1′′′), 100.6 (C-1′), 99.2 (C-1′′), 98.8 (C-1′′′′′), 95.8 (C-1′′′′), 78.2, 77.7, 76.3, 76.1, 76.0, 75.4, 75.3, 75.0, 74.9, 74.6, 73.7, 73.4, 73.2, 72.9, 72.5, 72.2, 71.7, 71.7, 71.0, 70.9, 70.8, 70.7, 70.3, 69.6, 68.9, 68.6, 68.4, 67.7, 67.6, 67.5, 67.0, 62.4, 55.6, 20.8 (2), 20.7, 20.6 (4 × COCH₃), 17.8, 17.6 (2), 17.4 (4 × CH₃). HRMS calcd for C₁₂₁H₁₃₀O₃₅Na (M + Na)⁺: 2165.8290, found: 2165.8287.

p-Methoxyphenyl-α-L-rhamnopyranosyl-(1→4)-α-D-galactopyranosyl uronic acid-(1→2)-α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-β-D-galactopyranoside (1). Compound 19 (702 mg, 0.3 mmol) was dissolved in CH₂Cl₂–H₂O (1.5 : 1; 20 mL). TEMPO (30 mg, 0.2 mmol) was added followed by iodosobenzene diacetate (480 mg, 1.6 mmol) and the mixture was stirred at 5 °C for 7 h. Aq. saturated Na₂S₂O₃ (5 mL) was added to stop the reaction. After diluting the reaction mixture with CH₂Cl₂, it was washed with brine (2 × 30 mL). The organic layer was separated, dried (Na₂SO₄), filtered and evaporated to a syrupy compound. It was then dissolved in MeOH (50 mL) and passed through a 10% Pd–C cartridge in a ThalesNano flow hydrogenation assembly under continuous flow of H₂ at atmospheric pressure. The hydrogenolysis of the benzyl groups were complete after 3 such cycles as evident from mass spectroscopy. NaOMe in MeOH (0.5 M, 1 mL) was added to the solution and it was stirred at room temperature for 12 h. The solution was neutralized by DOWEX 50W H⁺ resin, filtered and evaporated in vacuo to afford the final hexasaccharide 1 (257 mg, 75%) as white amorphous mass. [α]²⁵_D +54° (c 0.5, MeOH). HRMS calcd for C₄₃H₆₆O₂₉Na (M + Na)⁺: 1069.3587, found: 1069.3585. ¹H NMR (MeOD, 500 MHz) δ: 6.73, 6.70 (2d, 4H, C₆H₄OCH₃), 5.26 (d, 1H, J_1′,2′ ≤ 1.0 Hz, H-1′), 5.24 (d, 1H, J_{1′′′′′,2′′′′′} ≤ 1.0 Hz, H-1′′′′′), 5.21 (d, 1H, J_{1′′′′,2′′′′} ≤ 1.0 Hz, H-1′′′′), 5.11 (d, 1H, J_{1′′,2′′} ≤ 1.0 Hz, H-1′′), 5.06 (d, 1H, J_{1′′′,2′′′} ≤ 1.0 Hz, H-1′′′), 4.77 (d, 1H, J_1,2 8.0 Hz, H-1), 3.70 (s, 3H, C₆H₄OCH₃), 1.28–1.24 (m, 12H, 4 × CH₃). ¹³C NMR (125 MHz, MeOD) δ: 173.4 (COOH), 156.5, 152.1, 116.7 (2), 115.7 (2) (aromatic C), 103.3 (C-1), 102.7 (C-1′), 102.6 (C-1′′′), 102.4 (C-1′′), 102.3 (C-1′′′′′), 101.2 (C-1′′′′), 80.0, 79.8, 78.2, 77.7, 76.6, 76.4, 74.9, 74.4, 74.2, 73.9, 73.7, 73.5, 72.3, 72.2, 72.1, 72.0, 71.9, 71.8, 70.5, 70.4, 70.3, 70.2, 69.5, 63.9, 63.2, 56.2, 18.1, 18.0, 17.9 (2) (4 × C–CH₃).

Acknowledgements

VS is thankful to IISER Kolkata for Senior Research Fellowship. SERB, DST, India is gratefully acknowledged for funding through grant SB/S1/OC-48/2013 to BM.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Copies of ¹H and ¹³C NMR spectra of all new compounds. See DOI: 10.1039/c6ra07351d

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