Block Synthesis of Tetra- and Hexasaccharides (β-D-glycero-D-manno-Hepp-(1→4)-[α-L-Rhap-(1→3)-β-D-glycero-D-manno-Hepp-(1→4)]_n-α-L-Rhap-OMe (n = 1 and 2) Corresponding to Multiple Repeat Units of the Glycan from the Surface-Layer Glycoprotein from Bacillus thermoaerophilus

Abstract

A fully stereocontrolled block synthesis of the title tetra- and hexasaccharides has been achieved taking advantage of the ability of the 4,6-O-benzylidene acetal to control the stereochemistry of the β-d-glycero-d-mannoheptopyranoside unit, and of a 2,3-O-diphenylmethylene acetal to install the α-l-rhamnopyranosidic linkages. Comparison of the spectral data for the hexasaccharide with that of the natural confirms the structure of this very unusual and structurally challenging glycan.

Introduction

In 1995 Kosma and coworkers described the characterization of the surface-layer glycoprotein from Bacillus thermoaerophilus, and proposed the very unusual disaccharide motif →4)-[α-l-rhamnopyranosyl-(1→3)-β-d-glycero-d-mannoheptopyranosyl-(1→ as the repeating unit of the glycan chain, with the stereochemistry of the β-d-glycero-d-manno-heptopyranoside assigned on the basis of comparison of spectral data with synthetic methyl β-l-glycero-d-manno-heptopyranoside.¹ Having recently described the first stereocontrolled route to β-d-glycero-d-manno-heptopyranosides and their 6-deoxy analogs,² and applied this chemistry to a linear synthesis of methyl α-l-rhamnopyranosyl-(1→3)-β-d-glycero-d-mannoheptopyranosyl-(1→3)-6-deoxy-β-glycero-d-manno-heptopyranosyl-(1→4)-α-l-rhamnopyranoside, a tetrasaccharide subunit from the Plesimonas shigelloides lipopolysaccharide,³ we selected this oligomer as a proving ground for a block synthesis approach to glycans containing the unusual β-d-glycero-d-manno-heptoside structure.^4,5

Results and Discussion

Although our previous approach to the synthesis of the key d-glycero-d-manno-thioheptopyranoside donor was successful, and has subsequently been adopted by others,⁶ it suffered from a low yield at the level of the homologation step in which a six-carbon thiomannopyranoside was extended to a seven carbon system by Swern oxidation and Wittig olefination owing to competing elimination of a benzyloxy group from the 4-position in the course of the Wittig reaction.^2,3 We began, therefore, by seeking to improve the homologation step and were attracted to the use of the Ley-bisacetal function⁷ for protection of the 3,4-diol functionality on the grounds that elimination would be retarded by the confinement of the putative alkoxide leaving group within the a fused bicyclic ring system. Thus, treatment of the bisacetal 1⁸ with sodium hydride and benzyl bromide in DMF from −40 to 10 °C, according to the method described by Ley and others,⁹ afforded the 2-O-benzyl derivative 2 with good selectivity alongside a minor amount of the 2,6-di-O-benzyl system 3.¹⁰ Swern oxidation followed by standard Wittig homologation then afforded alkene 4 in 84% yield along with a minor amount of a methylthioalkene 5,¹¹ representing a distinct improvement over the earlier protocol (Scheme 1).

Scheme 1.

Improved homologation procedure

Catalytic osmoylation of 4 took place with excellent yield but poor selectivity for the formation of 6 and 8,¹² whatever the conditions employed, in keeping with our previous observations³ and those of other workers ¹³ with closely related compounds (Table 1). Some minor over-oxidation to the corresponding sulfones 7 and 9 was also observed in these reactions.¹⁴

Table 1.

Osmoylation of Alkene 4

Entry	Oxidant/ligand	Solvent	T/°C	6/8 (yield)	7/9 (yield)
1	OsO4/NMO	Acetone/H2O	22	2.2/1 (86%)	1.2/1 (8.4%)
2	AD- β-mix	t-BuOH/H2O	22	1/1.2 (97%)	not detected
3	AD-α-mix	t-BuOH/H2O	0	1.2/1 (93%)	not detected
4	K3Fe(CN)6/OsO4/(DHQ)2Pry	t-BuOH/H2O	22	1.5/1 (96%)	not detected
5	K3Fe(CN)6/OsO4/(DHQ)2Pry	t-BuOH/H2O/PhCH3	0	2.5/1 (96%)	not detected

Selective monobenzylation with dibutyltin oxide and benzyl bromide ¹⁵ of 6 and 8 afforded the corresponding 7-O-benzyl-6-ols 10 and 11 in excellent yield. Attempted inversion of the unwanted l,d-alcohol 11 to the desired d,d-isomer 10 by the Mitsunobu protocol¹⁶ was stymied by poor yields but the desired effect was achieved by recourse to displacement of the 6-O-triflate with potassium nitrite with in suit hydrolysis of the corresponding nitroso ester.¹⁷ Hydrolysis of the biacetal function in 10 to give a triol was followed by selective benzylidene acetal formation across the 1,3-diol functionality and introduction of a naphthylmethyl ether¹⁸ onto the remaining alcohol, thereby providing the known glycosyl donor 13 and, incidentally, confirming the d,d-stereochemistry of 6 (Scheme 2).

Scheme 2.

Synthesis of Heptosyl Donor 13

Two l-rhamnopyranosyl acceptors 16 and 17 were prepared by protection of the corresponding triols as the benzophenone acetals¹⁹ in moderate yield (Scheme 3). Somewhat better yields of 16 and 17 have been reported in the literature for alkylation of 14 and 15 with dichlorodiphenylmethane in pyridine. ^19a

Scheme 3.

Rhamnosyl Acceptor Preparation

A first glycosidic bond forming reaction involved activation of donor with 4-nitrophenylsulfenyl chloride and silver triflate in dichloromethane at −78 °C to give the corresponding glycosyl triflate followed by addition of the acceptor 16, when disaccharide 18 was obtained in 81% yield and exquisite β-selectivity in keeping with our previous observations in the benzylidene protected d-glycero-d-manno-heptoside series. The 4-nitrophenylsulfenyl chloride/silver triflate protocol²⁰ is a convenient variant on benzenesulfenyl triflate activation protocol²¹ that employs a commercial, stable sulfenyl chloride. The anomeric stereochemistry of the newly assigned glycosidic bond is readily assigned on the basis of the shield, upfield nature of the heptoside H5 resonance (δ 2.63), something that is highly characteristic of 4,6-O-benzylidene protected β-mannosides and which carries over to corresponding benzylidene acetals of the β-d-glycero-d-manno-heptosides. Confirmation of this anomeric stereochemistry derives from the anomeric 159.7 Hz ¹J_H1,C1 coupling constant.²² Removal of the naphthylmethyl ether with dichlorodicyanoquinone in wet dichloromethane gave the glycosyl acceptor 19 in good yield (Scheme 4).

Scheme 4.

Upstream Disaccharide Synthesis

An analogous protocol was then applied to the coupling of donor 13 with the thioglycoside-containing acceptor 17 to give the thiodisaccharide 20, again with exquisite β-selectivity (for the heptopyranoside: δ_H5 2.63, ¹J_H1,C1 159.0 Hz). Unfortunately, yield were lower with acceptor 17 than with the corresponding methyl glycoside 16, maximizing at 54% when 1.2 eq of the sulfenyl chloride was employed. However, the 1-benzenesulfinyl piperidine (BSP)/trifluoromethanesulfonic anhydride protocol proved superior when the activation was conducted in the presence of 1-octene as a sacrificial olefin, resulting in a 73% yield of the desired disaccharide (Scheme 5).

Scheme 5.

Disaccharide Donor Synthesis.

The tetrasaccharide 21, comprising two iterations of the Bacillus thermoaerophilus glycan repeating unit was then assembled by coupling of the disaccharide acceptor with the disaccharide donor 20 on activation with N-iodosuccinimide and silver triflate in excellent yield and α-selectivity. The 4-nitrophenylsulfenyl chloride/silver triflate protocol was also attempted but only gave the target compound in 26% yield. In addition to the two anomeric resonances characterizing the pre-installed β-mannoheptoside linkages, the ¹³C NMR spectrum of the tetrasaccharide contained resonances at δ 93.9 and 98.1 with ¹J_CH of 168.7 and 172.3 Hz indicative of the α-nature for the two rhamnoside linkages. Finally, global deprotection of 21 was achieved cleanly by hydrogenolysis over palladium on charcoal leading to the isolation of the target tetrasaccharide 22 in 85% yield (Scheme 6).

Scheme 6.

Tetrasaccharide Synthesis

The synthesis of the hexasaccharide 25 began with removal of the naphthylmethyl protecting group from tetrasaccharide 21 to give the acceptor 23, and was followed by NIS/AgOTf mediated coupling with the disaccharide donor 20 to give the hexasaccharide 24 in 64% yield as a single anomer. The α-nature of the rhamnopyranosidic linkage formed in this glycosylation again rested on the one bond CH coupling constant at the anomeric center of the newly formed glycosidic bond (167 Hz). Global deprotection was again achieved by hydrogenolysis, affording the target hexasaccharide in 60% yield (Scheme 7).

Scheme 7.

Hexasaccharide Synthesis

The spectral data for the central residues in hexasaccharide 25 were compared to those reported for the natural isolate and found to show a very high degree of concordance (supporting information), thereby confirming the structure proposed by Kosma and coworkers.

Conclusion

A block synthesis of the target hexasaccharide has been achieved and confirms the very rare β-dglycero-d-mannoheptospyranoside linkage at the heart of this glycan.

Experimental Section

General Experimental

Peak assignments in the NMR spectra were made in the usual manner with the assistance of phase sensitive COSY-45 and phase sensitive NOESY experiments.

Phenyl 2-O-benzyl-3,4-O-(2',3'-dimethoxybutane-2',3'-diyl)-1-thia-α-d-mannopyranoside (2) and Phenyl 2,6-di-O-benzyl-3,4-O-(2',3'-dimethoxybutane-2',3'-diyl)-1-thia-α-d-mannopyranoside (3)

To a cooled (−40 °C) solution of diol 1⁸ (12.24 g, 31.67 mmol) in dry DMF (140 mL) was added benzyl bromide (4.20 mL, 35.4 mmol) followed by 60% NaH in mineral oil (2.57 g, 64.3 mmol). The resultant mixture was stirred for 1 h at −40 °C, then for 16 h at − 10 °C. The reaction mixture was poured into water and extracted with CH₂Cl₂. The collected organic phase was further washed with brine, dried over anhydrous Na₂SO₄, and concentrated. The residue was purified by silica gel chromatography (AcOEt/hexane = 1/4 to 1/3, then AcOEt/ CH₂Cl₂ = 3/1 to 2/1) to afford the monobenzylated ether 2 (12.21 g, 25.6 mmol, 81%). [α]_D²⁴ = + 221.3 (c 1.2, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.43–7.41 (m, 4 H), 7.35-7.26 (m, 6 H), 5.48 (s, 1 H), 4.93 (d, 1 H, J = 11.5 Hz), 4.68 (d, 1 H, J = 11.5 Hz), 4.29 (t, 1 H, J = 10.0 Hz), 4.25-4.22 (m, 1 H), 4.08 (dd, 1 H, J = 2.5, 9.5 Hz), 3.99 (br. s, 1 H), 3.85-3.77 (m, 2 H), 3.33 (s, 3 H), 3.30 (s, 3 H), 1.90 (br. s, 1 H), 1.37 (s, 3 H), 1.34 (s, 3 H); ¹³C NMR (125 MHz, CDCl₃) δ 138.6, 134.2, 132.0, 129.3, 128.5, 128.2, 127.8, 100.3, 99.9, 87.8, 77.7, 73.4, 72.2, 69.6, 64.1, 61.7, 48.3, 48.1, 18.0; HRMS m/z: calcd for C₂₅H₃₂O₇NaS, 499.1766 [M+Na⁺]; Found C₂₅H₃₂O₇NaS, 499.1761; and 3⁸ (1.51 g, 3.90 mmol, 12%). [α]_D²³ = +172.2 (c 1.0, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.51-7.46 (m, 4 H), 7.36-7.24 (m, 11 H), 5.59 (s, 1 H), 4.94 (d, 1 H, J = 12.5 Hz), 4.74 (d, 1 H, J = 12.0 Hz), 4.66 (d, 1 H, J = 11.5 Hz), 4.55 (d, 1 H, J = 11.5 Hz), 4.50-4.48 (m, 1 H), 4.36-4.32 (m, 1 H), 4.13-4.10 (m, 1 H), 4.03 (s, 1 H), 3.88-3.84 (m, 2 H), 3.58 (s, 3 H), 3.25 (s, 3 H), 1.40 (s, 3 H), 1.36 (s, 3 H); ¹³C NMR (125 MHz, CDCl₃) δ 138.83, 138.81, 134.6, 132.0, 129.2, 128.5, 128.4, 128.0, 127.74, 127.71, 127.67, 127.5, 100.3, 99.9, 87.6, 77.9, 73.6, 73.0, 72.0, 69.8, 69.0, 64.2, 48.3, 48.2, 18.12, 18.09; HRMS m/z: calcd for C₃₂H₄₂NO₇NaS, 584.2682 [M+NH₄⁺]; C₃₂H₄₂NO₇NaS, 584.2674; calcd for C₃₂H₃₈O₇NaS, 589.2236 [M+Na⁺], Found C₃₂H₃₈O₇NaS, 589.2238.

Phenyl 2-O-benzyl-6,7-dideoxy-3,4-O-(2',3'-dimethoxybutane-2',3'-diyl)-1-thia-α-d-manno-hepto-6-enopyranoside (4) and Phenyl 2-O-benzyl-6,7-dideoxy-3,4-O-(2',3'-dimethoxybutane-2',3'-diyl)-1-thia-α-d-manno-hepto-(E)-7-methylthio-6-enopyranoside (5)

To a cooled (−75 °C) solution of oxalyl chloride (1.07 mL, 12.46 mmol) in dry CH₂Cl₂ (30 mL) was added a solution of DMSO (1.80 10 mL, 25.34 mmol) in dry CH₂Cl₂ (3 mL). After stirring for 10 min. at −75 °C, a solution of alcohol 2 (2.95 g, 6.19 mmol) in CH₂Cl₂ (10 mL + 1 mL rinse) was added dropwise. The mixture was stirred for 15 min. at −75 °C, then for 45 min at to −60 °C, and then for 45 min at −45 °C. At this stage Et₃N (6.80 mL, 48.92 mmol) was added dropwise, after which stirring was continued for 45 min. at −45 °C, before the reaction mixture was allowed to warm to −0 °C over 2 h. The reaction mixture was diluted with CH₂Cl₂, washed with saturated aqueous NaHCO₃ and brine. The organic layer was dried over MgSO₄, filtered, and concentrated, and the residue was dried for 12 hours at −20 °C in vacuum before it was taken up in THF (9 mL + 2 mL rinse) and added to a −40 °C stirred solution of the Wittig reagent formed by addition of n-BuLi in hexane (1.80 M, 7.5 mL, 13.50 mmol) at 0 °C to a suspension of Ph₃PCH₃Br (5.00 g, 14.00 mmol) in THF (30 mL) and cooling to −40 °C. The resulting mixture was stirred for 1.5 at −40 °C, then warmed to 10 °C over 2.5 h before it was poured into water and extracted with CH₂Cl₂. The collected organic phase was further washed with brine, dried over anhydrous Na₂SO₄, and concentrated. The residue was purified by silica gel chromatography (AcOEt/hexane = 1/15) to furnish alkene 4 (2.45 g, 5.17 mmol, 84%). [α]_D²⁴ = + 251.7 (c 1.3, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.45-7.41 (m, 4 H), 7.35-7.25 (m, 6 H), 6.02-5.95 (m, 1 H), 5.52 (s, 1 H), 5.45-5.42 (m, 1 H), 5.27-5.24 (m, 1 H), 4.91 (d, 1 H, J = 12.0 Hz), 4.71 (d, 1 H, J = 12.0 Hz), 4.63 (t, 1 H, J = 7.50 Hz), 4.09-4.03 (m, 2 H), 3.99 (m, 1 H), 3.33 (s, 3 H), 3.26 (s, 3 H), 1.38 (s, 3 H), 1.34 (s, 3 H); ¹³C NMR (125 MHz, CDCl₃) δ 138.7, 134.8, 134.0, 131.6, 129.2, 128.5, 128.2, 127.8, 127.6, 118.1, 100.2, 100.0, 87.6, 77.8, 73.2, 72.3, 69.8, 67.8, 48.2, 48.1, 18.1, 18.0; HR-ESI m/z: calcd for C₂₆H₃₂O₆NaS, 495.1817 [M+Na⁺], Found C₂₆H₃₂O₆NaS, 495.1826; and alkene 5 (58.1 mg, 112 µmol, 1.8%). [α]_d²³ = + 218.3 (c 0.97, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.43-7.38 (m, 4 H), 7.34-7.24 (m, 6 H), 6.45 (dd, 1 H, J = 1.0, 15.0 Hz), 5.47 (s, 1 H), 5.45 (dd, 1 H, J = 6.5, 15.5 Hz), 4.89 (d, 1 H, J = 12.5 Hz), 4.70 (d, 1 H, J = 12.0 Hz), 4.65 (t, 1 H, J = 7.5 Hz), 4.05-4.00 (m, 2 H), 3.96 (m, 1 H), 3.32 (s, 3 H), 3.25 (s, 3 H), 2.24 (s, 3 H), 1.36 (s, 3 H), 1.33 (s, 3 H); ¹³C NMR (125 MHz, CDCl₃) δ 138.6, 134.8, 131.6, 129.8, 129.3, 128.5, 128.2, 127.8, 127.6, 120.4, 100.2, 100.0, 87.7, 77.8, 73.3, 72.4, 69.7, 68.0, 48.2, 48.0, 18.1, 18.0, 14.6; HR-ESI m/z: calcd for C₂₇H₃₄O₆NaS₂, 541.1695 [M+Na⁺], Found C₂₇H₃₄O₆NaS, 541.1675.

Phenyl 2-O-benzyl-3,4-O-(2',3'-dimethoxybutane-2',3'-diyl)-d-glycero-1-thia-α-d-manno-heptopyranoside (6), Phenyl 2-O-benzyl-3,4-O-(2',3'-dimethoxybutane-2',3'-diyl)-d-glycero-1-sulfonyl-α-d-manno-heptopyranoside (7), Phenyl 2-O-benzyl-3,4-O-(2',3'-dimethoxybutane-2',3'-diyl)-l-glycero-1-thia-α-d-manno-heptopyranoside (8), and Phenyl 2-O-benzyl-3,4-O-(2',3'-dimethoxybutane-2',3'-diyl)-l-glycero-1-sulfonyl-α-d-manno-heptopyranoside (9)

Method 1

To a solution of alkene 4 (345 mg, 730 µmol) in 8/1 acetone/H₂O (9 mL), chilled in an ice-water bath, was added NMO (139 mg, 1.2 mmol) and OsO₄ (13.0 mg, 5 µmol). After stirring for 7 h, the reaction was quenched with a solution of sodium sulfite and extracted with AcOEt. The collected organic phase was further washed with brine, dried over Na₂SO₄, and concentrated. The residue was subjected to silica gel chromatography (AcOEt/CH₂Cl₂ = 1/4, then AcOEt/hexane = 1/1 and AcOEt/CH₂Cl₂ = 2/5) to afford 6 (219.3 mg, 433 µmol, 59%), [α]_D²⁴ = + 240.3 (c 0.94, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.43-7.40 (m, 4 H), 7.35-7.26 (m, 6 H), 5.43 (s, 1 H), 4.91 (d, 1 H, J = 12.0 Hz), 4.67 (d, 1 H, J = 12.0 Hz), 4.37 (t, 1 H, J = 10.0 Hz), 4.26 (dd, 1 H, J = 6.0, 10.0 Hz), 4.07 (dd, 1 H, J = 3.0, 10.0 Hz), 3.96-3.93 (m, 2 H), 3.70-3.63 (m, 2 H), 3.33 (s, 3 H), 3.31 (s, 3 H), 3.16 (d, 1 H, OH, J = 3.0 Hz), 2.20 (t, 1 H, OH, J = 6.5 Hz), 1.36 (s, 3 H), 1.33 (s, 3 H); ¹³C NMR (125 MHz, CDCl₃) δ 138.5, 133.7, 132.2, 129.4, 128.5, 128.2, 128.1, 127.9, 100.3, 100.2, 87.8, 77.5, 73.5, 73.4, 71.2, 69.4, 67.1, 63.2, 48.6, 48.3, 18.1, 18.0; HR-ESI m/z: calcd for C₂₆H₃₄O₈NaS, 529.1872 [M+Na⁺], Found C₂₆H₃₄O₈NaS, 529.1859; 7 (18.3 mg, 34 µmol, 4.6%). [α]_D²³ = + 148.7 (c 0.78, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.93 (d, 2 H, J = 7.5 Hz), 7.69 (t, 1 H, J = 7.5 Hz), 7.58 (t, 2 H, J = 7.5 Hz), 7.44 (d, 2 H, J = 7.0 Hz), 7.37-7.31 (m, 3 H), 5.00 (d, 1 H, J = 11.5 Hz), 4.72 (s, 1 H), 4.67 (d, 1 H, J = 11.0 Hz), 4.61 (d, 1 H, J= 3.0 Hz), 4.52-4.49 (m, 2 H), 4.35 (t, 1 H, J = 9.0 Hz), 3.79-3.75 (m, 1 H), 3.57-3.52 (m, 2 H), 3.35 (s, 3 H), 3.33 (s, 3 H), 3.27 (br s, 1 H, OH), 2.23 (br. s, 1 H, OH), 1.37 (s, 3 H), 1.35 (s, 3 H); ¹³C NMR (125 MHz, CDCl₃) δ138.1, 136.7, 134.6, 129.5, 129.3, 128.6, 128.4, 128.1, 100.3, 100.1, 93.0, 74.7, 74.1, 73.9, 71.9, 69.1, 66.1, 62.4, 48.6, 48.4, 18.0, 17.9; HR-ESI m/z: calcd for C₂₆H₃₄O₁₀NaS, 561.1770 [M+Na⁺], Found C₂₆H₃₄O₁₀NaS, 561.1748; 8 (101.0 mg, 199 µmol, 27%), [α]_D²⁴ = + 232.2 (c 1.1, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.43 (d, 1 H, J = 7.5 Hz), 7.38-7.28 (m, 8 H), 5.52 (s, 1 H), 4.95 (d, 1 H, J = 12.0 Hz), 4.67 (d, 1 H, J = 11.5 Hz), 4.42 (t, 1 H, J = 10.0 Hz), 4.14 (dd, 1 H, J = 2.0, 10.0 Hz), 4.08 (dd, 1 H, J = 3.0, 10.0 Hz), 3.97-3.95 (m, 1 H), 3.94-3.90 (m, 1 H), 3.50 (br. s, 2 H), 3.33 (s, 3 H), 3.32 (s, 3 H), 2.66 (d, 1 H, OH, J = 6.0 Hz), 1.85 (br s, 1 H, OH), 1.36 (s, 3 H), 1.33 (s, 3 H); ¹³C NMR (125 MHz, CDCl₃) δ 138.5, 133.3, 132.1, 129.5, 128.6, 128.2, 127.9, 100.3, 100.0, 87.6, 77.3, 73.5, 72.9, 69.7, 68.8, 65.1, 63.4, 48.3, 18.1, 18.0; HR-ESI m/z: calcd for C₂₆H₃₄O₈NaS, 529.1872 [M+Na⁺], Found C₂₆H₃₄O₁₀NaS, 529.1874; 9 (15.1 mg, 28 µmol, 3.8%). [α]_D ²³ = + 135.8 (c 0.67, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.85 (d, 2 H, J = 8.0 Hz), 7.70 (t, 1 H, J = 7.5 Hz), 7.59 (t, 2 H, J = 8.0 Hz), 7.44 (d, 2 H, J = 7.0 Hz), 7.37-7.30 (m, 3 H), 5.03 (d, 1 H, J = 11.0 Hz), 4.81 (s, 1 H), 4.67 (d, 1 H, J = 11.0 Hz), 4.60 (d, 1 H, J= 3.0 Hz), 4.49-4.45 (m, 1 H), 4.41-4.37(m, 2 H), 3.89 (m, 1 H), 3.51-3.44 (m, 2 H), 3.35 (s, 3 H), 3.32 (s, 3 H), 2.10 (br. s, 1 H, OH), 1.72 (br. s, 1 H, OH), 1.37 (s, 3 H), 1.33 (s, 3 H); ¹³C NMR (125 MHz, CDCl₃) δ 138.1, 136.9, 134.7, 129.6, 128.9, 128.6, 128.5, 128.1, 100.1, 100.0, 93.2, 76.8, 74.7, 71.9, 69.2, 68.9, 64.6, 62.0, 48.4, 48.3, 17.9; HR-ESI m/z: calcd for C₂₆H₃₄O₁₀NaS, 561.1770 [M+Na⁺], Found C₂₆H₃₄O₁₀NaS, 561.1746.

Method 2

To a mixture of alkene 4 (1.52 g, 3.2 mmol) in 1/1 t-BuOH/H₂O (32 mL) was added toluene (2.5 mL), K₂CO₃ (1.31 g, 9.7 mmol), and K₃Fe(CN)₆ (3.29 g, 10.0 mmol). After stirring for 10 min, the mixture was cooled to 0 °C followed by addition of (DHQ)₂Pry (29.1 mg, 0.033 mmol), and a solution of OsO₄ in t-BuOH (2.5 wt%, 161 µL). After the mixture was stirred for 24 h, additional t -BuOH/H₂O (8 mL, 1/1), K₂CO₃ (344 mg, 2.5 mmol), and K₃Fe(CN)₆ (832 mg, 2.5 mmol) were added. Stirring was continued for another 24 h before the reaction mixture was diluted with CH₂Cl₂, washed with sat. aqueous NaSO₃ and brine. The organic phase was dried over Na₂SO₄, filtered and concentrated. The residue was subjected to silica gel chromatography (AcOEt/CH₂Cl₂ = 1/4) to give 6 (1.12 g, 2.2 mmol, 68%) and 8 (455 mg, 0.9 mmol, 28%).

Phenyl 2,7-di-O-benzyl-3,4-O-(2',3'-dimethoxybutane-2',3'-diyl)-d-glycero-1-thia-α-d-manno-heptopyranoside (10)

To a solution of diol 6 (1.69 g, 3.33 mmol) in anhydrous toluene (80 mL) was added Bu₂SnO (957 mg, 3.84 mmol) after which the reaction mixture was heated to refluxed in a Dean-Stark apparatus. After 2 h, the resulting clear solution was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in DMF (30 mL) under N₂, and benzyl bromide (0.48 mL, 4.04 mmol) and CsF (1.07 g, 7.06 mmol) were added. The resulting mixture was stirred for 16 h at room temperature under N₂, then was diluted with CH₂Cl₂, washed with aqueous KF and brine. The organic phase was collected, dried over Na₂SO₄, filtered and concentrated. The residue was purified by silica gel chromatography (AcOEt/Hexane = 1/6) to yield ether 10 (1.90 g, 3.19 mmol, 96%). [α]_D²⁴ = + 186.5 (c 1.2, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.41-7.36 (m, 4 H), 7.34-7.23 (m, 11 H), 5.47 (s, 1 H), 4.89 (d, 1 H, J = 12.5 Hz), 4.67 (d, 1 H, J = 12.0 Hz), 4.53 (d, 1 H, J = 12.0 Hz), 4.48 (d, 1 H, J = 12.5 Hz), 4.36 (t, 1 H, J = 10.0 Hz), 4.20 (dd, 1 H, J = 5.0, 10.0 Hz), 4.13-4.10 (m, 1 H), 4.03 (dd, 1 H, J = 3.0, 10.5 Hz), 3.93-3.92 (m, 1 H), 3.57 (d, 2 H, J = 5.0 Hz), 3.30 (s, 3 H), 3.23 (s, 3 H), 2.84 (d, 1 H, OH, J = 2.5 Hz), 1.33 (s, 3 H), 1.28 (s, 3 H); ¹³C NMR (125 MHz, CDCl₃) δ 138.6, 138.5, 134.3, 131.6, 129.3, 128.6, 128.2, 128.1, 127.9, 127.8, 127.7, 100.3, 100.0, 87.5, 77.5, 73.6, 73.3, 72.8, 71.0, 69.6, 65.9, 48.5, 48.3, 18.1, 18.0; HR-ESI m/z: calcd for C₃₃H₄₀O₈NaS, 619.2342 [M+Na⁺], Found C₃₃H₄₀O₈NaS, 619.2323.

Phenyl 2,7-di-O-benzyl-3,4-O-(2',3'-dimethoxybutane-2',3'-diyl)-l-glycero-1-thia-α-d-manno-heptopyranoside (11)

Following the protocol for 10, diol 8 (1.16 g, 2.29 mmol) gave rise to ether 11 (1.24 g, 2.08 mmol, 91%). [α]_D²⁴ = + 198.7 (c 0.97, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.43 (d, 2 H, J = 7.5 Hz), 7.36-7.22 (m, 13 H), 5.56 (s, 1 H), 4.92 (d, 1 H, J = 12.0 Hz), 4.69 (d, 1 H, J = 11.5 Hz), 4.48-4.40 (m, 3 H), 4.15-4.13 (m, 2 H), 4.06 (dd, 1 H, J = 2.5, 10.0 Hz), 3.95 (br. s, 1 H), 3.38 (t, 1 H, J = 10.5 Hz), 3.31-3.29 (m, 7 H), 2.15 (br s, 1 H, OH), 1.36 (s, 3 H), 1.31 (s, 3 H); ¹³C NMR (125 MHz, CDCl₃) δ 138.6, 138.3, 134.1, 131.5, 129.2, 128.6, 128.5, 128.2, 127.9, 127.8, 127.6, 100.3, 99.9, 87.3, 77.3, 73.5, 73.3, 71.8, 71.3, 69.9, 67.6, 63.1, 48.2, 18.0; HR-ESI m/z: calcd for C₃₃H₄₀O₈NaS, 619.2342 [M+Na⁺], Found C₃₃H₄₀O₈NaS, 619.2333.

Formation of Phenyl 2,7-di-O-benzyl-3,4-O-(2',3'-dimethoxybutane-2',3'-diyl)-d-glycero-1-thia-α-d-manno-heptopyranoside (10) by Stereochemical Inversion of 11

Method 1

To a solution of alcohol 11 (267.9 mg, 450 µmol), 4-nitrobenzoic acid (155 mg, 924 µmol), and triphenylphosphine (238 mg, 910 µmol), cooled at 0 °C, in THF (125 mL) was added diisopropyl azodicarboxylate (177 µL, 899 µmol). The reaction mixture was allowed to ambient temperature and was stirred for 12 h before it was diluted with CH₂Cl₂, washed with saturated aqueous NaHCO₃ and brine. The residue was subjected to silica gel chromatography to give ester (73.1 mg, 98 µmol, 22%), which was treated with K₂CO₃ (45.8 mg, 331 µmol) in 2/1 MeOH/THF (3 mL). The resulting mixture was stirred for 16 h at ambient temperature under N₂, then concentrated under reduced pressure. The solid was diluted with CH₂Cl₂, washed with saturated aqueous NaHCO₃ and brine. The organic phase was dried over Na₂SO₄, filtered, concentrated, and purified by silica gel chromatography (AcOEt/hexane = 1/5) to afford 10 (55.4 mg, 93 µmol, 95%).

Method 2

To a chilled (−40 °C) solution of alcohol 11 (478.7 mg, 802 µmol) in CH₂Cl₂ (12 mL) was added anhydrous pyridine (163 µL, 2.0 mmol) and Tf₂O (240 µL, 1.45 mmol, 1.8 eq) after which the reaction mixture was stirred for 10 min at −40 °C, then warmed to −5 °C over 80 min, then was poured into cold 0.2 N HCl and extracted with CH₂Cl₂. The collected organic phase was further washed with cold aqueous NaHCO₃ and brine. The organic phase was dried over Na₂SO₄, filtered and concentrated. The resulting residue was dissolved in dry DMF (10 mL), 18-C-6 (57 mg, 0.22 mmol) and KNO₂ (460 mg, 5.4 mmol) were added. After stirring for 13 hours, more 18-c-6 (256.3 mg, 0.97 mmol) and KNO₂ (324 mg, 3.8 mmol) were added. Stirring was continued for 71 h before the reaction mixture was poured into 1 N aqueous K₂CO₃, and stirred for 2 h, and extracted with CH₂Cl₂. The organic phase was washed with brine, dried (Na₂SO₄), and concentrated. The residue was purified by silica gel chromatography (AcOEt/hexane = 1/5) to afford alcohol 10 (331 mg, 556 µmol, 69%).

Phenyl 2,7-di-O-benzyl-4,6-O-benzylindene-d-glycero-1-thia-α-d-manno-heptopyranoside (12)

To a solution of 1,2-diacetal 10 (2.21 g, 3.7 mmol) in CH₂Cl₂ (30 mL) was added a 10/1 mixture of TFA/H₂O (15 mL). The resulting mixture was stirred for 40 min at ambient temperature under N₂ before the volatiles were removed under reduced pressure and residue purified by silica gel chromatography (AcOEt/CH₂Cl₂ = 1/1 to 3/2) to give triol (1.600 g, 3.3 mmol, 89%), which was dissolved in dry CH₂Cl₂ (36 mL) and treated with camphorsulfonic acid (52.2 mg, 0.23 mmol) and benzaldehyde dimethyl acetal (2.4 mL, 16 mmol). After stirring for 14 h at room temperature under N₂, the mixture was diluted with CH₂Cl₂, washed with saturated aqueous NaHCO₃ and brine. The organic phase was dried over Na₂SO₄, filtered and concentrated. The residue was subjected to flash chromatography on silica gel to afford benzylidene 12 (1.641 g, 2.9 mmol, 87%). [α]_D²³ = + 144.3 (c 1.0, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.54-7.53 (m, 2 H), 7.41-7.24 (m, 18 H), 5.68 (s, 1 H), 5.62 (s, 1 H), 4.77 (d, 1 H, J = 11.5 Hz), 4.66 (d, 1 H, J = 11.5 Hz), 4.53 (d, 1 H, J = 12.5 Hz), 4.48 (d, 1 H, J = 12.0 Hz), 4.14-4.01 (m, 5 H), 3.58-3.51 (m, 2 H), 2.44 (d, 1 H, OH, J = 7.5 Hz); ¹³C NMR (125 MHz, CDCl₃) δ 138.4, 137.5, 137.4, 133.7, 131.9, 129.3, 128.9, 128.5, 128.4, 128.3, 127.8, 127.7, 126.7, 101.9, 86.0, 79.9, 78.8, 78.7, 73.7, 73.5, 69.4, 65.7; HRMS m/z: calcd for C₃₄H₃₄O₆NaS, 593.1974 [M+Na⁺]; Found C₃₄H₃₄O₆NaS, 593.1962.

Phenyl 2,7-di-O-benzyl-4,6-O-benzylindene-3-O-(2-naphthalenylmethyl)-d-glycero-1-thia-α-d-manno-heptopyranoside (13)

To a solution of alcohol 12 (1.60 g, 2.8 mmol) in dry DMF (24 mL), cooled in an ice-water bath, was added 60% NaH in mineral oil (230 mg, 5.8 mmol). After stirring for 20 min, tetrabutylammonium iodide (106 mg, 0.29 mmol) and 2-(bromomethyl)naphthalylene (843 mg, 3.7 mmol) were added and theresulting mixture was stirred for 12.5 h in the dark at room temperature under N₂. At this point MeOH (2.0 mL) was added, followed by neutralization with acidic resin to pH 6.0. The solid was filtered off and the filtrate was concentrated under vacuum. The residue was dissolved in CH₂Cl₂, and washed with aqueous NaHCO₃ and brine. The organic phase was dried over Na₂SO₄, filtered and concentrated. The residue was purified by silica gel chromatography (AcOEt/hexane = 1/8 to 1/6) to yield ether 13³ (1.941 g, 2.7 mmol, 97%). [α]_D²⁴ = + 110.0 (c 0.80, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.87-7.82 (m, 3 H), 7.76-7.74 (m, 1 H), 7.62-7.60 (m, 2 H), 7.49-7.46 (m, 3 H), 7.44-7.22 (m, 18 H), 5.80 (s, 1 H), 5.60 (s, 1 H), 4.98 (d, 1 H, J = 12.0 Hz), 4.84 (d, 1 H, J = 12.5 Hz), 4.80 (d, 1 H, J = 12.5 Hz), 4.76 (d, 1 H, J = 12.5 Hz), 4.57 (d, 1 H, J = 12.5 Hz), 4.51 (d, 1 H, J = 13.0 Hz), 4.42 (t, 1 H, J = 8.5 Hz), 4.19-4.12 (m, 2 H), 4.09-4.04 (m, 2 H), 3.63 (d, 1 H, J = 11.0 Hz), 3.57 (dd, 1 H, J = 5.5, 12.0 Hz); ¹³C NMR (125 MHz, CDCl₃) δ 138.6, 137.9, 136.1, 133.8, 133.6, 133.2, 131.8, 129.3, 129.1, 128.7, 128.5, 128.4, 128.2, 127.9, 127.8, 127.7, 126.6, 126.3, 126.1, 125.9, 101.4, 86.8, 78.8, 78.4, 77.9, 76.7, 73.7, 73.4, 73.3, 69.5, 66.4; HR-ESI m/z: calcd for C₄₅H₄₂O₆NaS, 733.2600 [M+Na⁺]; Found C₄₅H₄₂O₆NaS, 733.2561.

Methyl 2,3-O-diphenylmethylene-α-l-rhamnopyranoside (16)

Triol 14²³ (1.426 g, 8.0 mmol), benzophenone dimethyl acetal²⁴ (3.649 g, 16.0 mmol), and camphorsulfonic acid (308.4 mg, 1.39 mmol) were dissolved in dry DMF (20 mL). The mixture was stirred in a 50 °C water bath in a rotary evaporator for 8 h under the reduced pressure (27 mm Hg), to remove methanol formed during the reaction, then was poured into a saturated solution of NaHCO₃ in H₂O, and extracted with AcOEt. The organic phase was washed with brine, dried over Na₂SO₄, and concentrated. The residue was purified by flash chromatography on silica gel (AcOEt/hexane = 1/5to1/4) to afford 16^19a (1.29 g, 3.8 mmol, 47%). [α]_D²⁴ = − 71.6 (c 0.65, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.53-7.51 (m, 4 H), 7.34-7.27 (m, 6 H), 5.02 (s, 1 H), 4.28 (t, 1 H, J = 6.5 Hz), 4.06 (d, 1 H, J = 5.5 Hz), 3.71-3.66 (m, 1 H), 3.42-3.39 (m, 1 H), 3.38 (s, 3 H), 2.28 (br s, 1 H, OH), 1.27 (d, 3 H, J = 6.0 Hz); ¹³C NMR (125 MHz, CDCl₃) δ 143.1, 142.7, 128.4, 128.3, 126.3, 126.2, 109.8, 98.3, 79.1, 76.1, 74.1, 66.2, 55.2, 17.3; HR-ESI m/z: calcd for C₂₀H₂₂O₅Na, 365.1365 [M+Na⁺]; Found C₂₀H₂₂O₅Na, 365.1353.

Phenyl 2,3-O-diphenylmethylene-1-thia-α-l-rhamnopyranoside (17)

Following the protocol for 16, treatment of thioglycoside 15²⁵ (1.60 g, 6.2 mmol) with benzophenone dimethyl acetal²⁴ (2.84 g, 12.5 mmol) and camphorsulfonic acid (81 mg, 0.35 mmol) furnished alcohol 17^19a (1.03 g, 2.4 mmol, 39%). [α]_D²⁴ = − 191.0 (c 1.2, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.54-7.47 (m, 6 H), 7.46-7.25 (m, 9 H), 5.91 (s, 1 H), 4.34 (dd, 1 H, J = 5.5, 7.0 Hz), 4.29 (d, 1 H, J = 6.0 Hz), 4.14-4.09 (m, 1 H), 3.45-3.40 (m, 1 H), 2.14 (br s, 1 H), 1.19 (d, 3 H, J = 6.5 Hz); ¹³C NMR (125 MHz, CDCl₃) δ 143.2, 142.9, 133.6, 132.3, 129.3, 128.5, 128.4, 127.9, 126.2, 126.0, 109.8, 83.8, 79.3, 77.1, 74.9, 67.2, 17.3; HR-ESI m/z: calcd for C₂₅H₂₄O₄NaS, 443.1293 [M+Na⁺]; Found C₂₅H₂₄O₄NaS, 443.1277.

Methyl 2,7-di-O-benzyl-4,6-O-benzylindene-3-O-(2-naphthalenylmethyl)-d-glycero-β-d-manno-heptopyranosyl-(1→4)-2,3-O-(diphenylmethylene)-α-l-rhamnopyranoside (18)

A flask, charged with thioheptoside 13 (206.4 mg, 290 µmol), TTBP (240.1 mg, 967 µmol), silver triflate (232 mg, 903 µmol) and 4 Å molecular sieves (500 mg), was dried under vacuum for 1 h with the exclusion of light. Then dry CH₂Cl₂ (7 mL) was added, and the resulting mixture was stirred for 0.5 h at −78 °C before 4-nitrobenzenesulfenyl chloride (112.0 mg, 591 µmol) was added in one portion as a solid. After stirring for 10 min at −78 °C, the mixture was warmed to −45 °C, and stirring continued for 15 min before the reaction mixture was recooled to −78 °C. A solution of methyl rhamnoside 16 (130 mg, 380 µmol) in CH₂Cl₂ (1 mL + 0.5 mL rinse) then was added dropwise. The reaction mixture was stirred for 30 min at −78 °C, then warmed to −45 °C and stirring continued for 3 h. Saturated aqueous NaHCO₃ was added, and after filtration of the solid, the filtrate was further washed with aqueous NaHCO₃ and brine. The organic phase was dried over Na₂SO₄, and concentrated. The residue was purified by flash chromatography on silica gel (AcOEt/PhCH₃ = 1/40 to 1/30) to afford disaccharide 18 (221.1 mg, 234 µmol, 81%) as a white foam. [α]_D²⁴ = − 59.5 (c 0.63, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.80-7.78 (m, 3 H), 7.68-7.56 (m, 1 H), 7.52-7.27 (m, 25 H), 7.07-7.05 (m, 3 H), 5.71 (s, 1 H), 5.02 (s, 1 H), 4.97 (d, 1 H, J = 12.5 Hz), 4.88 (d, 1 H, J = 12.0 Hz), 4.85 (d, 1 H, J = 13.0 Hz), 4.78 (d, 1 H, J = 13.0 Hz), 4.54 (d, 1 H, J = 12.5 Hz), 4.50 (d, 1 H, J = 12.0 Hz), 4.38 (s, 1 H), 4.23 (dd, 1 H, J = 6.0, 7.5 Hz), 4.18 (t, 1 H, J = 9.5 Hz), 4.10 (t, 1 H, J = 8.0 Hz), 4.03-4.02 (m, 2 H), 3.65-3.60 (m, 1 H), 3.53-3.50 (m, 2 H), 3.39 (s, 3 H), 3.34 (dd, 1 H, J = 7.5, 11.5 Hz), 3.27 (dd, 1 H, J = 7.5, 10.0 Hz), 2.63 (t, 1 H, J = 9.5 Hz), 1.19 (d, 3 H, J = 6.0 Hz); ¹³C NMR (125 MHz, CDCl₃) δ 143.2, 143.1, 138.8, 138.5, 137.9, 133.5, 133.2, 129.0, 128.9, 128.7, 128.6, 128.4, 128.1, 127.9, 127.8, 126.6, 126.4, 126.3, 126.2, 126.1, 125.8, 109.4, 102.3 (J_C1–H1 = 159.7 Hz), 101.2, 98.0 (J_C1–H1 = 174.6 Hz), 80.8, 79.1, 78.9, 78.0, 77.8, 76.5, 76.2, 75.0, 73.6, 72.5, 69.5, 68.7, 64.6, 55.2, 17.8; HR-ESI m/z: calcd for C₅₉H₅₈O₁₁Na, 965.3877 [M+Na⁺]; Found C₅₉H₅₈O₁₁Na, 965.3904.

Methyl 2,7-di-O-benzyl-4,6-O-benzylindene-d-glycero-β-d-manno-heptopyranosyl-(1→4)-2,3-O-(diphenylmethylene)-α-l-rhamnopyranoside (19)

To a heterogenerous mixture of 18 (252.2 mg, 267 µmol) in 9/1 CH₂Cl₂/H₂O (6.6 mL) was added DDQ (96.0 mg, 423 µmol). The reaction mixture was stirred for 3 h at ambient temperature under N₂, then was diluted with CH₂Cl₂, washed with aqueous NaHCO₃ and brine. The organic phase was dried over Na₂SO₄, and concentrated. The residue was purified by flash chromatography on silica gel (AcOEt/hexane = 1/4 to 1/3) to furnish alcohol 19 (185.2 mg, 231 µmol, 86%) as a white foam. [α]_D²⁴ = − 88.1 (c 0.1.1, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.52-7.48 (m, 6 H), 7.41-7.22 (m, 18 H), 7.15 (t, 1 H, J = 7.5 Hz), 5.60 (s, 1 H), 5.05 (s, 1 H), 5.02 (d, 1 H, J = 11.5 Hz), 4.68 (d, 1 H, J = 12.0 Hz), 4.58 (s, 1 H), 4.55 (d, 1 H, J = 12.5 Hz), 4.52 (d, 1 H, J = 12.5 Hz), 4.35 (dd, 1 H, J = 6.0, 7.5 Hz), 4.06-3.99 (m, 3 H), 3.77 (t, 1 H, J = 9.5 Hz), 3.68-3.62 (m, 2 H), 3.49 (dd, 1 H, J = 1.5, 11.5 Hz), 3.39 (s, 3 H), 3.72-3.32 (m, 2 H), 2.60 (t, 1 H, J = 9.0 Hz), 2.37 (br. s, 1 H, OH), 1.21 (d, 3 H, J = 6.0Hz); ¹³C NMR (125 MHz, CDCl₃) δ 143.2, 138.5, 138.4, 137.5, 129.2, 128.7, 128.6, 128.5, 128.4, 128.2, 127.9, 126.6, 126.2, 126.1, 125.9, 109.5, 102.1, 101.6, 98.0, 80.6, 79.2, 79.0, 78.6, 78.5, 76.5, 75.8, 73.6, 71.1, 69.5, 68.3, 64.5, 55.2, 17.9; HR-ESI m/z: calcd for C₄₈H₅₀O₁₁Na, 825.3251 [M+Na⁺]; Found C₄₈H₅₀O₁₁Na, 825.3207.

Phenyl 2,7-di-O-benzyl-4,6-O-benzylindene-3-O-(2-naphthalenylmethyl)-d-glycero-β-d-manno-heptopyranosyl-(1→4)-2,3-O-diphenylmethylene-1-thia-α-l-rhamnopyranoside (20)

Method 1

A mixture of thioheptoside 13 (90.1 mg, 127 µmol), TTBP (70.9 mg, 285 µmol), and AgOTf (81.6 mg, 318 µmol) in CH₂Cl₂ (3 mL) in the presence of 4 Å molecular sieves (290 mg) was stirred for 0.5 h at −78 °C before 4-nitrobenzenesulfenyl chloride (29.6 mg, 156 µmol) was added as a solid. After stirring for 10 min, the reaction mixture was warmed to −45 °C, and stirred for 10 min before it was recooled to −78 °C followed by the addition of a solution of thiorhamnoside 17 (64.8 mg, 154 µmol) in CH₂Cl₂ (1 mL + 0.5 mL rinse). The resulting mixture was stirred for 4 h at −78 °C and then saturated aqueous NaHCO₃ was added to quench the reaction, and the solid was filtered off. The filtrate was washed with saturated aqueous NaHCO₃ and brine. The organic phase was dried over Na₂SO₄, filtered and concentrated. The residue was purified by silica gel chromatography (AcOEt/PhCH₃ = 1/40) to afford disaccharide 20 (70.1 mg, 69 µmol, 54%). [α]_D²³ = − 115.5 (c 0.74, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.83-7.80 (m, 3 H), 7.73-7.70 (m, 1 H), 7.54-7.28 (m, 30 H), 7.06-7.04 (m, 3 H), 5.89 (s, 1 H), 5.72 (s, 1 H), 4.99 (d, 1 H, J = 12.5 Hz), 4.94 (d, 1 H, J = 12.5 Hz), 4.90 (d, 1 H, J = 13.0 Hz), 4.83 (d, 1 H, J = 12.5 Hz), 4.54 (d, 1 H, J = 12.0 Hz), 4.50 (d, 1 H, J = 12.5 Hz), 4.31 (s, 1 H), 4.26 (d, 1 H, J = 5.5 Hz), 4.22-4.18 (m, 2 H), 4.11-4.04 (m, 2 H), 4.04 (d, 1 H, J = 2.5 Hz), 3.52 (dd, 1 H, J = 3.0, 10.0 Hz), 3.49 (d, 1 H, J = 10.0 Hz), 3.59-3.29 (m, 2 H), 2.60 (t, 1 H, J = 10.0 Hz), 1.14 (d, 3 H, J = 6.0 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 143.12, 143.07, 138.8, 138.5, 137.9, 136.1, 133.5, 133.2, 132.3, 129.4, 129.1, 128.6, 128.5, 128.4, 128.13, 128.07, 127.96, 127.90, 127.86, 126.6, 126.5, 126.4, 126.1, 125.8, 125.6, 109.4, 102.3 (J_C1–H1 = 159.0 Hz), 101.2, 83.9 (J_C1–H1 = 167.2 Hz), 81.2, 78.9, 78.8, 78.0, 77.9, 77.0, 76.1, 75.0, 73.6, 72.6, 69.5, 68.6, 66.3, 17.6; HR-ESI m/z: calcd for C₆₄H₆₀O₁₀NaS, 1043.3805 [M+Na⁺]; Found C₆₄H₆₀O₁₀NaS, 1043.3755.

Method 2

A mixture of thioheptoside 13 (535.6 mg, 753 µmol), TTBP (298.1 mg, 1.2 mmol), and BSP (168.5 mg, 805 µmol) in CH₂Cl₂ (15 mL) in the presence of 4 Å molecular sieves (500 mg) was stirred for 0.5 h at ambient temperature. Then the mixture was cooled to −60 °C, and Tf₂O (156 µL, 940 µmol) was added dropwise. After stirring for 6 min at −78 °C, 1-octene (2.0 mL, 12.7 mmol) was added, and stirring continued for 5 min. The reaction mixture was cooled to −78 °C followed by the addition of a solution of thiorhamnoside 17 (480.0 mg, 1.1 mmol) in CH₂Cl₂ (3 mL + 0.5 mL rinse). The resulting mixture was stirred for 1 h at −78 °C before saturated aqueous NaHCO₃ was added to quench the reaction, and the solid was filtered off. The filtrate was washed with saturated aqueous NaHCO₃ and brine. The organic phase was dried over Na₂SO₄, filtered and concentrated. The residue was purified by silica gel chromatography (AcOEt/PhCH₃ = 1/50) to afford disaccharide 20 (561.0 mg, 549 µmol, 73%).

Methyl 2,7-di-O-benzyl-4,6-O-benzylindene-3-O-(2-naphthaleneylmethyl)-d-glycero-β-d-manno-heptopyranosyl-(1→4)-2,3-O-(diphenylmethylene)-α-l-rhamnopyranosyl-(1→3)-2,7-di-O-benzyl-4,6-O-benzylindene-d-glycero-β-d-manno-heptopyranosyl-(1→4)-2,3-O-(diphenylmethylene)-α-l-rhamnopyranoside (21)

A mixture of alcohol 19 (90.0 mg, 112 µmol), and thioheptoside 20 (135.3 mg, 132 µmol) in CH₂Cl₂ (6 mL) in the presence of 4 Å molecular sieves (320 mg) was stirred for 50 min at ambient temperature under N₂ atmosphere. Then the mixture was cooled to −15 °C, and NIS (38.4 mg, 170 µmol) and AgOTf (16.9 mg, 66 µmol) was subsequently added. The reaction mixture was stirred for 2.5 h while the reaction temperature was increased to 5 °C. The solids were filtered off, and the filtrate was washed with saturated aqueous Na₂S₂O₃ containing NaHCO₃ and brine. The organic phase was dried over Na₂SO₄, filtered and concentrated. The residue was purified by flash chromatography on silica gel (AcOEt/PhCH₃ = 1/20 to 1/15) to afford tetrasaccharide 21 (164.2 mg, 96 µmol, 85%). [α]_D²³ = − 86.6 (c 1.2, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.80-7.76 (m, 3 H), 7.68-7.66 (m, 1 H), 7.57-7.19 (m, 46 H), 7.15-7.01 (m, 7 H), 5.70 (s, 1 H), 5.60 (s, 1 H), 5.07 (s, 1 H), 5.01 (s, 1 H), 4.85 (d, 1 H, J = 12.0 Hz), 4.83 (d, 1 H, J = 12.0 Hz), 4.77-4.64 (m, 4 H), 4.56-4.48 (m, 5 H), 4.34 (dd, 1 H, J = 5.5, 7.0 Hz), 4.30 (t, 2 H, J = 6.5 Hz), 4.14 (t, 1 H, J = 9.5 Hz), 4.11-4.04 (m, 3 H), 4.02-3.92 (m, 3 H), 3.91-3.86 (m, 1 H), 3.83 (dd, 1 H, J = 6.0, 9.5 Hz), 3.70-3.66 (m, 1 H), 3.52 (d, 1 H, J = 9.5 Hz), 3.47 (d, 1 H, J = 10.0 Hz), 3.43-3.41 (m, 4 H), 3.37-3.33 (m, 1 H), 3.28 (dd, 1 H, J = 7.5, 11.0 Hz), 3.23 (dd, 1 H, J = 7.5, 10.0 Hz), 2.67 (t, 1 H, J = 9.5 Hz), 2.46 (t, 1 H, J = 9.5 Hz), 1.23 (d, 1 H, J = 6.5 Hz), 1.03 (d, 1 H, J = 6.5 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 143.19, 143.15, 143.11, 143.04, 143.07, 138.5, 138.4, 138.1, 138.0, 137.6, 136.1, 133.5, 133.1, 129.2, 129.0, 128.66, 128.63, 128.6, 128.5, 128.45, 128.41, 128.39, 128.35, 128.2, 128.12, 128.09, 127.98, 127.90, 127.8, 126.6, 126.44, 126.38, 126.3, 126.0, 125.9, 125.82, 125.77, 109.6, 109.3, 102.5 (J_C1–H1 = 161.8 Hz), 102.4 (J_C1–H1 = 160.5 Hz), 101.6, 101.1, 98.1 (J_C1–H1 = 172.3 Hz), 93.9 (J_C1–H1 = 168.7 Hz) 81.0, 80.3, 79.2, 79.1, 78.96, 78.92, 77.9, 77.7, 76.5, 76.4, 75.6, 75.1, 74.9, 74.5, 74.0, 73.9, 73.5, 72.2, 69.6, 69.4, 69.1, 68.7, 65.0, 64.6, 55.2, 17.9, 17.5; ESI-HRMS m/z: calcd for C₁₀₆H₁₀₄O₂₁Na, 1735.6968 [M+Na⁺]; Found 1735.7017.

Methyl d-glycero-β-d-manno-heptopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→3)-d-glycero-β-d-manno-heptopyranosyl-(1→4)-α-l-rhamnopyranoside (22)

To a solution of tetrasaccharide 21 (37.5 mg, 22 µmol) in 4/4/1 MeOH/THF/H₂O (4.5 mL) was added AcOH (13.0 µL, 227 µmol) and 10% Pd/C (84.6 mg). The mixture was purged with H₂ six times and stirred for 84 h at ambient temperature under 1 atmosphere of H₂. The catalyst was filtered off, and the filtrate was concentrated. The residue was dissolved in H₂O, and washed with AcOEt. The aqueous layer was concentrated to give tetrasaccharide 22 (13.2 mg, 19 µmol, 85%). [α]_D ²³ = − 104.0 (c 0.45, MeOH); ¹H NMR (500 MHz, D₂O) δ 4.83 (d, 1 H, J = 1.5 Hz), 4.72 (s, 1 H), 4.71 (s, 1 H), 4.55 (d, 1 H, J = 1.5 Hz), 4.13 (d, 1 H, J = 1.8 Hz), 3.94-3.83 (m, 5 H), 3.78 (dd, 1 H, J = 2.0, 4.0 Hz), 3.71-3.47 (m, 13 H), 3.30 (dd, 1 H, J = 3.0, 9.5 Hz), 3.26-3.24 (m, 4 H), 1.22 (d, 3 H, J = 6.0 Hz), 1.18 (d, 3 H, J = 6.0 Hz); ¹³C NMR (125 MHz, D₂O) δ 100.98 (J_C1-H1 = 172.1 Hz), 100.86 (J_C1–H1 = 161.5 Hz), 100.78 (J_C1–H1 = 161.3 Hz), 96.3 (J_C1–H1 = 173.0 Hz), , 79.5, 79.2, 77.1, 76.2, 76.1, 73.4, 72.5, 70.8, 70.6, 70.5, 70.4, 68.3, 67.7, 67.3, 66.4, 66.2, 62.2, 54.9, 17.1; ESI-HRMS m/z: calcd for C₂₇H₄₈O₂₁Na, 731.2586 [M+Na⁺]; Found C₂₇H₄₈O₂₁Na, 731.2585.

Methyl 2,7-di-O-benzyl-4,6-O-benzylindene-d-glycero-β-d-manno-heptopyranosyl-(1→4)-2,3-O-(diphenylmethylene)-α-l-rhamnopyranosyl-(1→3)-2,7-di-O-benzyl-4,6-O-benzylindene-d-glycero-β-d-manno-heptopyranosyl-(1→4)-2,3-O-(diphenylmethylene)-α-l-rhamnopyranoside (23)

To a mixture of tetrasaccharide 21 (180.1 mg, 106 µmol) in 15/3/5 CH₂Cl₂/H₂O/phosphate buffer (pH = 7.4) (15/3/5) (9.2 mL) was added DDQ (38.6 mg, 170 µmol). The mixture was stirred for 2.5 h at ambient temperature under N₂, before another portion of DDQ (34.5 mg, 152 µmol) was added. After the mixture was stirred for another 2 h, it was diluted with CH₂Cl₂, and washed with saturated aqueous NaHCO₃ and brine. The collected organic phase was dried over Na₂SO₄, filtered and concentrated. The residue was subjected to silica gel chromatography to furnish alcohol 23 (123.6 mg, 79 µmol, 74%) as white foam. [α]_D²³ = − 96.5 (c 0.71, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.53-7.45 (m, 10 H), 7.40-7.02 (m, 40 H), 5.59 (s, 1 H), 5.58 (s, 1 H), 5.07 (s, 1 H), 5.02 (s, 1 H), 4.85 (d, 1 H, J = 12.0 Hz), 4.84 (d, 1 H, J = 12.0 Hz), 4.68 (d, 1 H, J = 12.0 Hz), 4.53-4.44 (m, 7 H), 4.41 (dd, 1 H, J = 5.5, 7.5 Hz), 4.35 (dd, 1 H, J = 5.5, 7.0 Hz), 4.09-3.99 (m, 5 H), 3.95 (t, 1 H, J = 10.0 Hz), 3.92-3.88 (m, 2 H), 3.83 (dd, 1 H, J = 3.5, 10.5 Hz), 3.81-3.64 (m, 4 H), 3.60-3.56 (m, 2 H), 3.52 (d, 1 H, J = 9.0), 3.44 (dd, 1 H, J = 1.5, 11.0 Hz), 3.41 (s, 3 H), 3.37-3.26 (m, 4 H), 2.67 (t, 1 H, J = 9.5 Hz), 2.44 (t, 1 H, 9.5 Hz), 2.30 (d, 1 H, J = 9.0 Hz, OH), 1.23 (d, 1 H, J = 6.5 Hz), 1.03 (d, 1 H, J = 6.5 Hz); ¹³C NMR (125 MHz, CDCl₃) δ 143.23, 143.19, 143.15, 143.13, 138.5, 138.4, 138.3, 138.1, 137.6, 129.2, 129.1, 128.8, 128.7, 128.62, 128.59, 128.56, 128.52, 128.49, 128.45, 128.41, 128.2, 128.1, 128.0, 127.94, 27.89, 126.7, 126.5, 126.4, 126.3, 126.0, 109.6, 109.4, 102.6, 102.2, 101.6, 98.1, 93.9, 81.1, 80.2, 79.2, 79.1, 79.03, 78.96, 78.6, 78.0, 76.6, 76.4, 75.6, 75.3, 74.9, 73.9, 73.8, 73.64, 73.60, 71.0, 69.7, 69.4, 69.1, 68.4, 64.9, 64.6, 55.3, 17.9, 17.6; ESI-HRMS m/z: calcd for C₉₅H₉₆O₂₁Na, 1595.6342 [M+Na⁺]; Found 1595.6427.

Methyl 2,7-di-O-benzyl-4,6-O-benzylindene-3-O-naphthalenylmethyl)-d-glycero-β-d-manno-heptopyranosyl-(1→4)-2,3-O-(diphenylmethylene)-α-l-rhamnopyranosyl-(1→3)-2,7-di-O-benzyl-4,6-O-benzylindene-d-glycero-β-d-manno-heptopyranosyl-(1→4)-2,3-O-(diphenylmethylene)-α-l-rhamnopyranosyl-(1→3)-2,7-di-O-benzyl-4,6-O-benzylindene-d-glycero-β-d-manno-heptopyranosyl-(1→4)-2,3-O-(diphenylmethylene)-α-l-rhamnopyranoside (24)

A solution of thioglycoside 20 (106.2 mg, 104 µmol) and alcohol 23 (121.6 mg, 77 µmol) in CH₂Cl₂ (8 mL) was stirred for 30 min in the presence of 4 Å molecular sieves at ambient temperature under N₂ before it was cooled to −15 °C, and treated with NIS (29.9 mg, 133 µmol) and AgOTf (27.4 mg, 106 µmol). The resulting mixture was stirred for 1.5 h while the temperature was increased to 10 °C. The solid was filtered off, and the filtrate was washed with saturated aqueous Na₂S₂O₃ containing NaHCO₃ and brine. The organic phase was dried over Na₂SO₄, filtered and concentrated. The residue was purified by flash chromatography on silica gel (AcOEt/PhCH₃ = 1/18) to afford hexasaccharide 24 (124.2 mg, 50 µmol, 64%). [α]_D²³ = − 92.0 (c 0.44, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.80-7.75 (m, 3 H), 7.68-7.66 (m, 1 H), 7.57-7.09 (m, 76 H), 6.93 (t, 2 H, J = 8.0 Hz), 5.69 (s, 1 H), 5.58 (s, 1 H), 5.07 (s, 1 H), 5.01 (s, 1 H), 4.88-4.81 (m, 3 H), 4.76-4.62 (m, 5 H), 4.55-4.42 (m, 8 H), 4.38-4.34 (m 3 H), 4.30-4.26 (m, 3 H), 4.15-4.01 (m, 7 H), 3.98-3.82 (m, 8 H), 3.74-3.65 (m, 2 H), 3.51-3.33 (m, 9 H), 3.30-3.25 (m, 3 H), 3.18 (dd, 1 H, J = 7.5, 10.0 Hz), 2.67 (t, 1 H, J = 9.5 Hz), 2.48 (t, 1 H, J = 9.0 Hz), 2.42 (t, 1 H, J = 9.5 Hz), 1.23 (d, 3 H, J = 6.5 Hz), 1.04 (d, 3 H, J = 6.5 Hz), 1.00 (d, 3 H, J = 6.5 Hz); ¹³C NMR (125 MHz, CDCl₃) δ 143.22, 143.18, 143.12, 143.08, 138.6, 138.5, 138.4, 138.06, 137.98, 137.8, 137.63, 137.60, 136.1, 133.5, 133.2, 129.2, 129.1, 129.0, 128.7, 128.65, 128.60, 128.53, 128.5, 128.45, 128.41, 128.4, 128.33, 128.30, 128.27, 128.2, 128.1, 128.0, 127.9, 127.86, 127.84, 126.6, 126.5, 126.4, 126.3, 126.04, 125.98, 125.83, 125.79, 109.6, 109.5, 109.3, 102.6, 102.5, 102.4, 101.7, 101.6, 101.1, 98.1 (J_C1–H1 = 169.5 Hz), 93.86 (J_C1–H1 = 168.5 Hz), 93.79 (J_C1–H1 = 167.0 Hz), 81.2, 80.5, 80.3, 79.2, 79.1, 79.0, 78.9, 77.9, 77.7, 76.6, 76.5, 76.4, 75.6, 75.1, 74.9, 74.5, 74.3, 73.9, 73.8, 73.7, 73.65, 73.62, 73.55, 72.9, 72.2, 69.7, 69.6, 69.4, 69.2, 69.1, 68.8, 64.9, 64.6, 55.3, 17.9, 17.5; ESI-HRMS m/z: calcd for C₁₅₃H₁₅₀O₃₁Na, 2506.0059 [M+Na⁺]; Found 2506.0273.

Methyl d-glycero-β-d-manno-heptopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→3)-d-glycero-β-d-manno-heptopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→3)-d-glycero-β-d-manno-heptopyranosyl-(1→4)-α-l-rhamnopyranoside (25)

To a solution of hexasaccharide 24 (58.3 mg, 23 µmol) in 3/1 MeOH/THF (6 mL) was added AcOH (100 µL, 1.7 mmol) and 10% Pd/C (100 mg). The mixture was purged with H₂ six times, and stirred for 24 h under H₂ at 50 psi. The solid was filtered off, and the filtrate was concentrated to dryness. The resulting residue was dissolved in water, and washed with CHCl₃ (5 mL × 3). The aqueous phase was concentrated under reduced pressure, and dried under vacuum to afford hexamer 25 (14.9 mg, 14 µmol, 60%). [α]_D²³ = −84.6 (c 0.71, H₂O); ¹H NMR (500 MHz, D₂O, 330 K, referenced to an external standard sodium 3-trimethylsilyl-(2,2,3,3-²H) propionate (δ = 0.00) in D₂O) δ 4.96 (s, 2 H), 4.84 (s, 2 H), 4.83 (s, 1 H), 4.68 (s, 1 H), 4.24 (br. s, 2 H), 4.05-3.96 (m, 8 H), 3.92-3.90 (m, 1 H), 3.83-3.60 (m, 19 H), 3.44-3.41 (m, 2 H), 3.38-3.35 (m, 4 H), 1.33 (d, 3 H, J = 6.5 Hz), 1.31 (d, 6 H, J = 6.5 Hz); ¹³C NMR (125 MHz, D₂O, 330 K, referenced to an external standard 1,4-dioxane (δ = 67.40) in D₂O) δ 101.63 (J_C1–H1 = 174.5 Hz), 101.48 (J_C1–H1 = 160.5 Hz), 101.36 (J_C1–H1 = 161.4 Hz), 97.09 (J_C1–H1 = 170.2 Hz), 80.27, 80.10, 77.99, 76.84, 76.71, 74.08, 73.29, 73.25, 71.52, 71.28, 71.17, 69.10, 68.34, 67.92, 67.29, 67.14, 63.03, 62.98, 55.59, 17.78; ESI-HRMS m/z: calcd for C₄₀H₇₀O₃₁Na, 1069.3799 [M+Na⁺]; Found 1069.3762; calcd for C₄₀H₇₀O₃₁Na₂, 546.1848 [M+2Na²⁺], Found, 546.1837.