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Publicly Available Published by De Gruyter July 12, 2019

Synthesis and biological evaluation of S-simplexides and other analogues of simplexide

  • Amélie Roux , Stefania Loffredo EMAIL logo , Anne Lise Ferrara and Paul V. Murphy EMAIL logo

Abstract

Simplexides are natural glycolipids isolated from the marine sponge Plakortis simplex, and contain alkyl 4-O-(α-D-glucopyranosyl)-β-D-galactopyranoside. Simplexides can release of cytokines (IL-6) and chemokines (CXCL-8) from human monocytes and cause the expansion of natural killer T-cells (iNKTs) in vitro, with iNKTs contributing to the sustenance of immune homeostasis. Herein, the stereoselective syntheses of S-glycosidic analogues, i.e. S-simplexides, are described. The routes included Lewis acid promoted anomerisation of glycosyl thiols and thioglycolipids, as well as anomeric S-alkylation. Synthesis of O-glycosidic analogues are included. Heptadecanyl O- and S-glycosides as well as the 17-tritriacontyl 4-O-(α-D-glucopyranosyl)-β-D-galactopyranoside, a component of the natural simplexide isolate, all induced IL-6 and CXCL-8 production at both 10 and 30 μg/mL concentrations from PBMCs whereas the two S-simplexides were inactive. It is speculated that the lack of activity for the S-disaccharide analogue could be due to inhibition of cellular α-glucosidase, preventing degradation of the simplex disaccharide to a simpler galactopyranoside, whereas lack of activity for the S-galactolipid analogue could be due to increased conformational flexibility of S-glycosides. On the other hand, simpler unbranched O- and S-glycolipid analogues were active. Natural simplexide, and a synthetic simplexide, the 18-pentatriacontanyl 4-O-(α-D-glucopyranosyl)-β-D-galactopyranoside, were more potent than the new compounds tested.

Introduction

Simplexides are glycolipids, which were originally isolated from the marine organism Plakortis simplex [1]. The lipid component of these marine natural products is distinctive and the disaccharide, where glucopyranoside is α-1→4 linked to β-galactopyranoside, is not found commonly. While five different alkyl chain types were observed in the isolated fraction of simplexide, it has been estimated that up to 25 different molecular species may be produced naturally.

Researchers in Naples have previous demonstrated that both the natural isolated simplexide fraction and synthetic simplexide 1c (Scheme 1, R=R′=CH2CH3) induced the release of cytokines (IL-6) and chemokines (CXCL-8) from human monocytes and the expansion of natural killer T-cells (iNKT) in vitro [2]. These observations suggested that simplexides could exert both proinflammatory and immunoregulatory activities by their interaction with CD1d. The activity of simplexides may, thus, relate to that of the α-GalCer family of glycosphingolipids [3]. α-Galactosyl ceramide (KRN-7000, α-GalCer) enters monocytes and binds to CD1d, an antigen-presenting molecule. CD1d presents α-GalCer to the T cell receptor (TCR), which recognizes it with very high affinity [4]. This leads to the activation of NKT cells, which subsequently release chemokines and cytokines. The latter have an important role influencing both immune and pathogenic processes. While there have been various syntheses and biological studies of α-GalCer and analogues [5], [6], [7], [8], there has been limited structure activity studies on simplexides to date. A few syntheses of components of the natural isolate and a fluorescent simplexide have been reported [9], [10], [11].

Scheme 1: Structure of natural simplexide glycolipids 1a–1e and simplexide analogues 2–6.
Scheme 1:

Structure of natural simplexide glycolipids 1a–1e and simplexide analogues 2–6.

In this manuscript we describe a synthesis of simplexide 1a (where R=R′=CH2CH3) as well as the syntheses of mimetics of 1a, S-simplexides, where sulfur replaces glycosidic oxygen atoms of the natural product. Glycomimetics [12], including S-glycosides, are of interest in drug and development or as probes for basic research. The synthesis of S-glycoside analogues [13] of natural bioactive O-glycosides is of interest because they are more stable in vivo than the corresponding O-glycosides, being resistant to hydrolysis by glycosidases [14]. However, there are conformational implications in replacing an oxygen with sulfur as thioglycosides can be more flexible [15]. In addition to S-glycosides, analogues which have the α-O-anomeric configuration between the galactose and the lipid residue are included in this study. Lewis acid promoted anomerisation has found application during the synthetic work. Biological studies on new compounds generated are included, and some simpler S and O-glycolipids show ability to stimulate release of IL-6 and CXCL-8 from PBMCs.

Results and discussion

Synthesis of glycolipids and mimetics

The preparation of 13 (Scheme 2) was achieved via the α-thiol 12 [16] which had been generated in situ by TiCl4 induced anomerisation. Thiol 12, prepared on a multi-gram scale herein, was converted to thioacetate 9, facilitate long term storage of a more stable precursor to 12. The thioacetate 9 was obtained as an 89:11 mixture of anomers on a multigram scale, a stereoselectivity lower than previously described for thiol 12 [11]. The unstable triflate 10 was prepared from known glucopyranoside 11 [17], [18] (Scheme 2). De-S-acetylation of 9 and reaction of the generated thiolate in the presence of 10, via anomeric S-alkylation, gave the S-disaccharide 13. The thioglycoside 13 was isolated as an 83:17 mixture of α:β anomers after the alkylation and subsequent chromatography. The methoxyphenyl glycoside 13 was then hydrolysed using cermic ammonium nitrate (CAN) in the presence of water-acetonitrile and the hemiacetal that was generated was converted into the trichloroacetimidate donor 14 (83:17 mixture of α-S and β-S-glucopyranosides) in two steps. Glycosidation with the known alcohol 15 [19] and subsequent protecting group removal gave the S-simplexide 5 as a single isomer.

Scheme 2: Synthesis of S-simplexide.
Scheme 2:

Synthesis of S-simplexide.

The disaccharide 18 was next used to synthesise naturally occurring 1a. Thus 18 was obtained from the glycosylation reaction using the thioglycoside 16 and glycosyl acceptor 17 using N-iodosuccinimide and TMSOTf as activator followed by reaction of the disaccharide product with N-bromosuccinimide in the presence of water and acetone. The glycosidation promoted by NIS-TfOH had a selectivity ~70:30 in favour of the α-anomer, with the β-glucopyranoside also formed; these diastereoisomers were not separated. A related glycosidation reaction was reported previously by Li and co-workers,6c who used a 6-O-TBDPS protected thiogalactopyranoside donor as part of a one pot strategy towards simplexide, in diethyl ether and dichloromethane; the formation of the β-glucopyranoside was not reported in their earlier report. It appears the absence of the TBDPS group and/or the use of dichloromethane only as solvent leads to a reduction in the stereoselectivity of the glycosylation reaction. The thioglycoside of 17 is less reactive than 16 due to its acyl groups, and was not activated with NIS at −78°C. Hemiacetal 18 (70:30 mixture of glucopyranosides) was converted into its trichloroacetimidate and subsequent glycosylation of alcohols 15 and 19 gave, respectively, β-galactopyranosides 20 and 21, with each being an 85:15 mixture of α:β D-glucopyranosides. Debenzoylation followed by de-O-benzylation using the Birch conditions gave 1a, which after chromatography was a 90:10 mixture of α and β-D-glucopyranosides (Scheme 3). The 1H- and 13C-NMR data for 1a (α-glucopyranoside component) showed very good agreement with all the data reported by Li and co-workers, with one exception. They report a signal at δ 4.61 (dd, J=11.6, 2.4 Hz, 1H) for one of the glucopyranoside H-6 protons, whereas we recorded a signal for this proton at δ 4.48 (dd, J=11.5, 2.5 Hz, 1H). Reaction of 21 gave 2 which was isolated as an 84:16 mixture of glucopyranosides with the α-anomer being the major constituent.

Scheme 3: Synthesis of simplexide 1a and analogues.
Scheme 3:

Synthesis of simplexide 1a and analogues.

The disaccharide 18 was used for the synthesis of thiogalactopyranosides 3, 4 and 6. Its reaction with Lawesson’s reagent (Scheme 3) gave a mixture of the galactopyranosyl thiols 22, where the β-anomer was the major component (β:α=74:26). The reaction of 1-iodoheptadecane 24 prepared from 19 (Scheme 4), with 22β in the presence of sodium hydride at 0°C in DMF followed by protecting group removal (NaOMe-MeOH, followed by Na-NH3) gave 3 via 27 (33% over 3 steps, shown in Scheme 5); the final product 3 contained a 64:36 mixture of α and β-glucopyranosides. The reaction of tritriacontan-17-ol with triphenylphosphine in the presence of imidazole gave the iodide 23 (Scheme 4). When 23 was reacted with a mixture of thiols 22β and 22α, it gave a mixture of thioglycosides 25β and 25α. Separation of the thioglycosides and removal of protecting groups from both anomers gave 4 and 6, both of which contained some β-glucopyranoside as indicated in Scheme 4. The preparation of α-anomer 4 was also shown to be possible via 26α, which was obtained from 25β after treatment with TiCl4. Thus, this Lewis acid was able to promote anomerisation of the β-S-glycolipid to the α-S-glycolipid. In earlier work from our group, glycosphingolipid anomerisation required the presence of a galacturonic acid residue rather than galactopyranoside. Here, this was not the case as the anomerisation proceeded for the S-galactopyranoside derivative. Interestingly, the TiCl4 was also found to remove, in a regioselective manner, two of the benzyl protecting groups, at C-2 and C-3, of the glucopyranoside residue. Remarkably, the TiCl4 did not remove the benzyl group at the glucopyranoside C-6, which might have been predicted to be more reactive. In terms of the anomerisation reaction promoted by TiCl4, it is more likely that it involves an endocyclic cleavage pathway as opposed to exocyclic cleavage. In the former, the Lewis acid coordinates to the pyranose ring oxygen atom and isomerisation is facilitated by breaking and reforming the bond between this oxygen and the anomeric carbon atom, with C-1 to C-2 bond rotation enabling the generation of the axial anomer. On the other hand, the exocyclic cleavage pathway involves breaking and reforming the bond between the anomeric carbon and sulfur. An earlier study showed that rates of S-galactospyranoside anomerisation to be faster than that of the O-galactopyranoside and it was argued that this is consistent with endocyclic cleavage rather than the alternative pathway [20]. Anomerisation of other types of glycosides with additional cyclic constraints are also believed to go via endocyclic cleavage as discussed by Manabe, Ito and their co-workers [21]. Recent research on this reaction has recently been reviewed [22].

Scheme 4: Synthesis of S-simplexides.
Scheme 4:

Synthesis of S-simplexides.

Scheme 5: Synthesis of simplexide analogue 3.
Scheme 5:

Synthesis of simplexide analogue 3.

Biological evaluation of the new glycolipids

The effects of simplexide 1a and mimetics (compounds 2–5) on cytokine and chemokine production by human PBMCs were examined and these were compared to those of the natural and synthetic simplexide 1c (R=R′=CH2CH2CH3), prepared in Naples as described previously [2]. Cells were incubated with increasing concentrations (3–30 μg/mL) of compounds and the release of cytokines and chemokines (IL-6, TNF-α and CXCL-8) was determined. The simpler O-glycosidic analogue 2 and its S-glycoside mimetic 3, as well as the synthetic 1a, all induced both IL-6 (Fig. 1) and CXCL-8 (Fig. 2) production at both 10 and 30 μg/mL concentrations from PBMCs whereas compounds 4–6 had no effect (Figs. 14). By contrast, none of tested new compounds induced the TNF-α release (Fig. 3). Natural and synthetic simplexide 1c, with the longer lipid chains, were more potent in inducing the release of IL-6 (Fig. 4a) and CXCL-8 (Fig. 4b) compared to the tested sample of 1a, and the mimetics 2 and 3.

Fig. 1: Effect of increasing concentrations of synthetic compounds on IL-6 release from PBMCs. Polystyrene plates were coated with indicated concentrations of compound 1a (panel A), 2 (panel B), 3 (panel C), 4 (panel D), 5 (panel E), 6 (panel F), natural and synthetic (panel A–F, compound 1c) simplexide glycolipids dissolved in methanol. Solvent was dried under nitrogen immediately before the addition of cells. Cells were then added and after 24 h of incubation, the supernatant was collected and centrifuged (1000×g, 4°C, 5 min). IL-6 concentrations were determined by ELISA. The values are expressed as pg of IL-6 per 106 cells. Data are the mean±SEM of six experiments.
Fig. 1:

Effect of increasing concentrations of synthetic compounds on IL-6 release from PBMCs. Polystyrene plates were coated with indicated concentrations of compound 1a (panel A), 2 (panel B), 3 (panel C), 4 (panel D), 5 (panel E), 6 (panel F), natural and synthetic (panel A–F, compound 1c) simplexide glycolipids dissolved in methanol. Solvent was dried under nitrogen immediately before the addition of cells. Cells were then added and after 24 h of incubation, the supernatant was collected and centrifuged (1000×g, 4°C, 5 min). IL-6 concentrations were determined by ELISA. The values are expressed as pg of IL-6 per 106 cells. Data are the mean±SEM of six experiments.

Fig. 2: Effect of increasing concentrations of synthesised compounds on CXCL-8 release from PBMC. Polystyrene plates were coated with indicated concentrations of compound 1a (panel A), 2 (panel B), 3 (panel C), 4 (panel D), 5 (panel E), 6 (panel F), natural and synthetic (panel A–F, compound 1c) simplexide glycolipids dissolved in methanol. Solvent was dried under nitrogen immediately before the addition of cells. Cells were then added and after 24 h of incubation, the supernatant was collected and centrifuged (1000×g, 4°C, 5 min). CXCL-8 levels were determined by ELISA. The values are expressed as pg of CXCL-8 per 106 cells. Data are the mean ± SEM of six experiments.
Fig. 2:

Effect of increasing concentrations of synthesised compounds on CXCL-8 release from PBMC. Polystyrene plates were coated with indicated concentrations of compound 1a (panel A), 2 (panel B), 3 (panel C), 4 (panel D), 5 (panel E), 6 (panel F), natural and synthetic (panel A–F, compound 1c) simplexide glycolipids dissolved in methanol. Solvent was dried under nitrogen immediately before the addition of cells. Cells were then added and after 24 h of incubation, the supernatant was collected and centrifuged (1000×g, 4°C, 5 min). CXCL-8 levels were determined by ELISA. The values are expressed as pg of CXCL-8 per 106 cells. Data are the mean ± SEM of six experiments.

Fig. 3: Effect of increasing concentrations of synthetic compounds on TNF-α release from PBMC. Polystyrene plates were coated with indicated concentrations of compound 1a (panel A), 2 (panel B), 3 (panel C), 4 (panel D), 5 (panel E), 6 (panel F), natural and synthetic (panel A–F) simplexide glycolipids dissolved in methanol. Solvent was dried under nitrogen immediately before the addiction of cells. Cells were then added and after 24 h of incubation, the supernatant was collected and centrifuged (1000×g, 4°C, 5 min). TNF-α were determined by ELISA. The values are expressed as pg of TNF-α per 106 cells. Data are the mean±SEM of six experiments.
Fig. 3:

Effect of increasing concentrations of synthetic compounds on TNF-α release from PBMC. Polystyrene plates were coated with indicated concentrations of compound 1a (panel A), 2 (panel B), 3 (panel C), 4 (panel D), 5 (panel E), 6 (panel F), natural and synthetic (panel A–F) simplexide glycolipids dissolved in methanol. Solvent was dried under nitrogen immediately before the addiction of cells. Cells were then added and after 24 h of incubation, the supernatant was collected and centrifuged (1000×g, 4°C, 5 min). TNF-α were determined by ELISA. The values are expressed as pg of TNF-α per 106 cells. Data are the mean±SEM of six experiments.

Fig. 4: Effect of 1a and mimetics 2–6 on IL-6 (panel A), CXCL-8 (panel B) and TNF-α (panel C) production from PBMC. Positive controls were natural and synthetic simplexide. Polystyrene plates were coated with indicated concentrations of glycolipids dissolved in methanol. Solvent was dried under nitrogen immediately before the addiction of cells. Cells were then added and after 24 h of incubation, the supernatant was collected and centrifuged (1000×g, 4°C, 5 min). Cytokines and chemokines were determined by ELISA. The values are expressed as percent increase versus MeOH control. Data are the mean±SEM of six experiments. *p<0.05 vs. control.
Fig. 4:

Effect of 1a and mimetics 2–6 on IL-6 (panel A), CXCL-8 (panel B) and TNF-α (panel C) production from PBMC. Positive controls were natural and synthetic simplexide. Polystyrene plates were coated with indicated concentrations of glycolipids dissolved in methanol. Solvent was dried under nitrogen immediately before the addiction of cells. Cells were then added and after 24 h of incubation, the supernatant was collected and centrifuged (1000×g, 4°C, 5 min). Cytokines and chemokines were determined by ELISA. The values are expressed as percent increase versus MeOH control. Data are the mean±SEM of six experiments. *p<0.05 vs. control.

It is known that S-glycosides can act as glycosidase inhibitors. A reviewer of this manuscript has pointed out that most glycosylceramides, composed of oligosaccharides rather than a monosaccharide, require lysosomal processing to monoglycosylceramides, before they can cause NKT cell stimulation. In this scenario, cellular glycosidases degrade the lipids presenting oligosaccharides to simpler lipids presenting monosaccharides, with the latter being required to stimulate cytokine release [23]. It is therefore possible that the S-glycosidic linkage in 5 prevents degradation to the simpler active galactolipid, and that this would explain the lack of activity found for 5 in this study. This suggests that it would be relevant to synthesise and evaluate the simpler galactopyranoside derivatives of simplexides, work which as far as we are aware, has not been carried out to date. The lack of activity displayed by 6, on the other hand, could be explained by different conformational preferences between it and the simplexides, as S-glycosides are known to have increased flexibility compared to the O-glycosides. There is no clear proposal as to why 4 may not be active and synthesis of the α–O-simplexide analogue would clarify the relationship between anomeric configuration and bioactivity for the simplexides. It is interesting that the simpler unbranched glycolipids 2 and 3 were active and could be investigated more in future research.

Summary and conclusions

The synthesis of S-simplexides were achieved herein, enabling a contribution to be made to the structure activity relationship of glycolipids of immunological interest. The synthesis of S-simplexides include application of Lewis acid promoted anomerisation of glycosyl thiols as well as anomerisation of a thioglycolipid. In previous work, successful anomerisation of glycosphingolipids, to give the axial glycosides, with TiCl4 has required the saccharide to be a galacturonic acid residue, with the anomerisation of galactosphingolipids being unsuccessful [24], [25], [26], [27], [28]. On the other hand, the thiogalactopyranoside 25β glycolipid is structurally the most complex glycolipid amenable to anomerisation to date, indicating the reaction may have other applications in this field. From a biological perspective, unbranched heptadecanyl O- and S-glycosidic analogues as well as the 17-tritriacontyl 4-O-(α-D-glucopyranosyl)-β-D-galactopyranoside, a component of the natural simplexide isolate, all induced IL-6 and CXCL-8 production at both 10 and 30 μg/mL concentrations from PBMCs whereas the S-simplexides were inactive. Natural simplexide, and a synthetic analogue, the 18-pentatriacontanyl 4-O-(α-D-glucopyranosyl)-β-D-galactopyranoside, were most potent of those samples tested. Hence it would appear that the conformational flexibility induced by having an S-glycoside as opposed to an O-glycoside leads to a loss of bioactivity or that inhibition of cellular α-glucosidase promoted degradation of the simplexide to simpler galactopyranosides could be a contributing factor. Simpler lipids such as that found in 2 and even the S-glycoside 3 displayed activity. The research suggests that simpler galactopyranoside analogues or of more components of the simplexide isolate and analogues may be worthwhile for the provision of new improved immunomodulatory glycolipids in due course.

Experimental section

Reagents and general synthesis and analytical methods

NMR spectra (see Supplementary information section) were recorded with a 500 MHz or 600 MHz Varian spectrometer. Chemical shifts are referenced to the internal solvent signal. All NMR assignments were assigned with the aid of COSY, HSQC and HMBC experiments. Coupling constants J are reported in Hertz (Hz). Unless otherwise stated 1H NMR and/or 13C NMR spectroscopic data for previously reported compounds, cited herein, were in good agreement with previously published data. The IR spectra were recorded as thin films using a PerkinElmer Spectrum 100 FT-IR Spectrometer with an ATR attachment. High resolution mass spectra were recorded using a Waters LCT Premier XE (ESI-TOF instrument). Silica gel (pore size 60 Å, particle size 40–60 μm 230–400 mesh particle size) was purchased from Sigma-Aldrich. Dichloromethane, THF and DMF reaction solvents were used as obtained from a Pure Solv™ Solvent Purification System. The following were purchased for biological experiments: L-glutamine, antibiotic-antimycotic solution (10 000 IU/mL penicillin, 10 mg/mL streptomycin, and 25 μg/mL amphotericin B), MEM non-essential amino acids solutions and Histopaque®-1077 (Sigma-Aldrich, St. Louis, MO, USA); RPMI and fetal calf serum (FCS) (MP Biomedicals Europe, Illkirch, France). All other reagents for bioassays were purchased from Carlo Erba (Milan, Italy).

Benzoylation

The reactant was dissolved in pyridine (10 mL per 1 mmol) and the solution was cooled down to 0°C. DMAP (0.05 equiv. per OH group) and BzCl (1.2 equiv. per OH group) were then added, and the solution was stirred at room temperature overnight. The mixture was then diluted with EtOAc. The organic phase was washed with HCl until the aqueous phase was acidic, then water and brine. It was then dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. Chromatography gave the product.

Debenzoylation

The reactant was dissolved in methanol (10 mL per mmol). Solid sodium prewashed with petroleum ether (0.2 equiv.) was then added to the solution. The mixture was stirred at room temperature overnight. It was then acidified to pH=6 with Amberlite (IR-120, strongly acidic, hydrogen form), filtered, and the solvent was removed under reduced pressure to give the product.

De-O-benzylation

The reactant was dissolved in THF (20 mL per mmol) and solid sodium prewashed with petroleum ether (20 equiv.) was added. The round bottom flask was cooled to −78°C and gaseous NH3 (excess) was bubbled into the mixture via a needle. The reaction stopped bubbling and was stirred vigorously for 16 h, while being allowed to attain room temperature. Water was then added, and the mixture was extracted with EtOAc three times. The organic phase was dried over Na2SO4, filtered and the solvent removed under reduced pressure to give the product after chromatography.

p-Methoxyphenyl 2,3-di-O-benzoyl-4-S-(2,3,4,6-tetra-O-benzoyl-α-D-glucopyranosyl)-4-thio-6-tert-butyldiphenylsilyl-β-D-galactopyranoside (13)

Compound 11 [29] (13.2 g, 27 mmol) was dissolved in pyridine (100 mL) and the solution was cooled to 0°C. Imidazole (5.4 g, 3 equiv.) and TBDPSCl (10 mL, 1.5 equiv.) were then added, and the mixture was stirred at 0°C for 3 h. It was then diluted with CH2Cl2, washed with HCl (5%), a satd NaHCO3 and water, dried over Na2SO4, filtered and the solvent was removed under reduced pressure. Chromatography (cyclohexane-EtOAc, 4:1) gave the intermediate (17.0 g, 87%) as a white solid; 1H NMR (500 MHz, CDCl3) δ 7.99 (overlapping signals, 4H, aromatic H), 7.71 (overlapping signals, 4H, aromatic H), 7.52 (overlapping signals, 2H, aromatic H), 7.40 (overlapping signals, 10H, aromatic H of TBDPS), 6.94 (overlapping signals, 2H, aromatic H), 6.72 (overlapping signals, 2H, aromatic H), 5.65 (dd, J=9.8, 7.9 Hz, 1H, H-2), 5.49 (t, J=9.5 Hz, 1H, H-3), 5.14 (d, J=7.9 Hz, 1H, H-1), 4.06 (overlapping signals, 2H, H-4, H-6a), 4.01 (dd, J=11.0, 5.3 Hz, 1H, H-6b), 3.73 (overlapping signals, 4H, H-5, OCH3), 3.20 (s, 1H, OH), 1.08 (s, 9H, Si-C(CH3)3). 13C NMR (126 MHz, CDCl3) δ 167.4 (C=O), 165.3 (C=O), 155.5 (C), 151.3 (C), 136.3 (C), 135.7 (C), 135.6 (C), 133.5 (C), 133.2 (C), 132.7 (C), 130.0 (CH), 129.8 (CH), 129.7 (CH), 128.4 (CH), 128.4 (CH), 127.8 (CH), 126.4 (CH), 126.3 (CH), 118.8 (CH), 114.5 (CH), 110.0 (CH), 100.7 (C-1), 77.2 (C-2), 76.1 (C-5), 71.4 (C-3), 70.7 (C-4), 64.0 (C-6), 55.6 (O-CH3), 26.8 (TBDPS), 19.2 (TBDPS). IR (film) cm−1: 2982, 1722, 1601, 1451, 1263, 1178, 1090, 1069, 1026, 707, 687. This intermediate (5 g, 6.8 mmol) was dissolved in CH2Cl2 (43 mL) and the solution was cooled to 0°C. Pyridine (1.7 mL, 3 equiv.) and triflic anhydride (1.6 mL, 1.4 equiv.) were then added, and the mixture was stirred at room temperature for 1 h. It was then diluted with CH2Cl2, washed with HCl 1 mol/L, water and brine, dried over Na2SO4, filtered and the solvent was removed under reduced pressure. Chromatography (cyclohexane-EtOAc, 8:1) gave unstable triflate 10 (3.1 g, 53%). Thiol 811 (5 g, 8.2 mmol) was dissolved in CH2Cl2 (75 mL) and pyridine (330 μL, 0.5 equiv., 4.1 mmol) was then added, followed by TiCl4 (1 M in CH2Cl2, 24.5 mL, 3 equiv.). The mixture was stirred at room temperature for 16 h and then diluted in EtOAc and washed with a saturated solution of NH4Cl. The aqueous phase was extracted with EtOAc, and the combined organic phases were washed with satd NaHCO3, brine, and then dried over Na2SO4 and filtered. After removal of the solvent under reduced pressure, the unpurified α-thiol 12 (5 g, 8.2 mmol) was suspended in pyridine (50 mL) and acetic anhydride (7.7 mL, 10 equiv.), and the resulting mixture was stirred at room temperature for 16 h. The mixture was then cooled to 0°C and methanol was then added followed by EtOAc. The organic phase was washed with water, then 1 M HCl until the pH of the aqueous phase was acidic, water and brine. It was then dried over Na2SO4, filtered and the solvent evaporated under reduced pressure. Chromatography (cyclohexane-EtOAc, 7:2) gave 9 (3.63 g, 68%) as a white solid, which was an 89:11 mixture of anomers. Analytical data for α-anomer : 1H NMR (500 MHz, CDCl3) δ 8.02 (overlapping signals, 2H, aromatic H), 7.93 (overlapping signals, 4H, aromatic H), 7.87 (overlapping signals, 2H, aromatic H), 7.52 (overlapping signals, 4H, aromatic H), 7.39 (overlapping signals, 6H, aromatic H), 7.30 (overlapping signals, 2H, aromatic H), 6.55 (d, J=5.5 Hz, 1H, H-1), 5.87 (t, J=9.8 Hz, 1H, H-3), 5.71 (overlapping signals, 2H, H-2, H-4), 4.59 (dd, J=11.9, 2.6 Hz, 1H, H-6a), 4.47 (overlapping signals, 2H, H-5, H-6b), 2.37 (s, 3H, CH3). 13C NMR (126 MHz, CDCl3) δ 191.2 (S-C=O), 166.1 (C=O), 165.7 (C=O), 165.1 (C=O), 165.0 (C=O), 133.6 (C), 133.5 (C), 133.3 (C), 133.1 (C), 129.9 (CH), 129.8 (CH), 129.8 (CH), 129.8 (CH), 129.7 (CH), 129.7 (CH), 129.6 (CH), 128.8 (CH), 128.7 (CH), 128.6 (CH), 128.5 (CH), 128.4 (CH), 128.4 (CH), 128.3 (CH), 128.3 (CH), 80.8 (C-1), 71.7 (C-5), 71.6 (C-3), 70.1 (C-4), 68.9 (C-2), 62.7 (C-6), 31.5 (CH3). IR (film) cm−1: 2982, 1722, 1601, 1451, 1263, 1178, 1090, 1069, 1026, 707, 687. Thioacetate 9 (1 g, 1.5 mmol) and freshly prepared triflate 10 (5.28 g, 4 equiv.) were then dissolved in DMF (66 mL) and the solution was cooled to 0°C. Diethylamine (3.2 mL, 20 equiv.) was then added and the mixture was stirred at 0°C for 5 h. It was then washed with water and extracted with EtOAc. The organic phase was dried over Na2SO4, filtered and the solvent removed under reduced pressure. Chromatography (cyclohexane-EtOAc, 5:1) gave 13 (1.26 g, 62%) as an 83:17 mixture of the α-S- and β-S-glucopyranoside (white solid). Analytical data for the α-anomer 13α: 1H NMR (500 MHz, CDCl3) δ 8.15 (overlapping signals, 2H, aromatic H), 8.00 (overlapping signals, 6H, aromatic H), 7.89 (overlapping signals, 2H, aromatic H), 7.80 (overlapping signals, 2H, aromatic H), 7.69 (overlapping signals, 5H, aromatic H), 7.44 (overlapping signals, 3H, aromatic H), 7.41 (overlapping signals, 11H, aromatic H), 7.21 (overlapping signals, 5H, aromatic H), 6.81 (overlapping signals, 2H, aromatic H), 6.67 (overlapping signals, 2H, aromatic H), 6.31 (d, J=5.6 Hz, 1H, H-1′), 6.11 (t, J=9.9 Hz, 1H, H-3′), 5.71 (overlapping signals, 2H, H-2, H-4′), 5.60 (dd, J=10.5, 4.7 Hz, 1H, H-3), 5.53 (dd, J=10.3, 5.9 Hz, 1H, H-2′), 4.95 (d, J=7.8 Hz, 1H, H-1), 4.60 (dt, J=10.1, 2.9 Hz, 1H, H-5′), 4.11 (m, 1H, H-4), 4.05 (dd, J=10.7, 7.8 Hz, 1H, H-6a), 3.98 (dd, J=12.6, 2.7 Hz, 1H, H-6′a), 3.88 (dd, J=10.7, 5.7 Hz, 1H, H-6b), 3.76 (t, J=6.9 Hz, 1H, H-5), 3.69 (s, 3H, OCH3), 3.57 (dd, J=12.7, 3.0 Hz, 1H, H-6′b), 1.07 (s, 9H, Si-C(CH3)3). 13C NMR (126 MHz, CDCl3) δ 165.8 (C=O), 165.6 (C=O), 165.5 (C=O), 165.3 (C=O), 165.2 (C=O), 164.8 (C=O), 155.4 (C), 151.1 (C), 135.6 (C), 135.6 (C), 133.7 (C), 133.6 (C), 133.3 (C), 133.2 (C), 133.2 (C), 133.1 (C), 133.0 (CH), 132.7 (CH), 130.1 (CH), 130.0 (CH), 129.9 (CH), 129.9 (CH), 129.9 (CH), 129.7 (CH), 129.7 (CH), 129.3 (CH), 129.1 (CH), 128.8 (CH), 128.8 (CH), 128.7 (CH), 128.6 (CH), 128.6 (CH), 128.4 (CH), 128.3 (CH), 128.3 (CH), 128.3 (CH), 128.3 (CH), 127.9 (CH), 127.8 (CH), 118.4 (CH), 114.4 (CH), 101.1 (C-1), 82.3 (C-1′), 74.6 (C-5), 72.7 (C-3), 71.5 (C-2′), 70.8 (C-3′), 70.6 (C-2), 68.7 (C-4′), 68.0 (C-5′), 62.6 (C-6), 61.7 (C-6′), 55.6 (O-CH3), 46.3 (C-4), 26.8 (TBDPS), 19.0 (TBDPS). IR (film) cm−1: 2935, 2858, 1725, 1602, 1507, 1451, 1259, 1178, 1090, 1067, 1026, 825, 801, 743, 703, 687. ESI-HRMS calcd for C77H70O17SSiNa 1349.4001, found m/z 1349.4005 [M+Na]+.

2,3-Di-O-benzoyl-4-S-(2,3,4,6-tetra-O-benzoyl-α-D-glucopyranosyl)-4-thio-6-tert-butyldiphenylsilyl-β-D-galactopyranosyl trichloroacetimidate (14)

Compound 13 (1.26 g, 0.95 mmol) was dissolved in a mixture of H2O-CH3CN (3:17, 20 mL) and ammonium cerium nitrate (2.6 g, 4.75 mmol) was added. The mixture was stirred at room temperature for 1 h. The solution was diluted in EtOAc, washed with water, a saturated solution of NaHCO3, dried over Na2SO4, filtered and the solvent removed under reduced pressure. Chromatography (cyclohexane-EtOAc, 4:1) gave the hemiacetal (0.59 g, 51%) as a white solid. The hemiacetal precursor (0.59 g, 0.48 mmol) was then dissolved in CH2Cl2 (15 mL). Trichloroacetonitrile (1 mL, 20 equiv.) and DBU (0.1 mL, 1.2 equiv.) were then added to the solution, which was stirred at room temperature for 1 h. The solvent was then removed under reduced pressure. Chromatography (cyclohexane-EtOAc, 6:1) gave 14 (0.49 g, 74%) as a mixture of glucopyranosides (83:17 α-S anomer: β-S anomer, white solid); 1H NMR (α-S anomer, 500 MHz, CDCl3) δ 8.47 (s, 1H, NH), 8.18 (overlapping signals, 2H, aromatic H), 8.07 (overlapping signals, 2H, aromatic H), 8.02 (overlapping signals, 3H, aromatic H), 8.98 (overlapping signals, 3H, aromatic H), 7.91 (overlapping signals, 2H, aromatic H), 7.79 (overlapping signals, 3H, aromatic H), 7.66 (overlapping signals, 5H, aromatic H), 7.41 (overlapping signals, 31H, aromatic H), 6.63 (d, J=3.8 Hz, 1H, H-1), 6.58 (d, J=5.7 Hz, 1H, H-1′), 6.17 (t, J=9.9 Hz, 1H, H-3′), 6.10 (dd, J=10.9, 4.5 Hz, 1H, H-3), 5.70 (overlapping signals, 2H, H-2, H-4′), 5.60 (m, 1H, H-2′), 4.65 (dt, J=10.1, 3.0 Hz, 1H, H-5′), 4.39 (overlapping signals, 2H, H-4, H-5), 4.04 (m, 1H, H-6′a), 4.00 (m, 1H, H-6a), 3.78 (dd, J=10.6, 5.3 Hz, 1H, H-6b), 3.64 (dd, J=12.6, 3.4 Hz, 1H, H-6′b), 1.04 (s, 9H, Si-C(CH3)3). 13C NMR (126 MHz, CDCl3) δ 165.8 (C=O), 165.6 (C=O), 165.4 (C=O), 165.4 (C=O), 165.3 (C=O), 164.8 (C=O), 160.5 (C=NH), 135.7 (C), 135.6 (C), 133.6 (C), 133.5 (C), 133.4 (C), 133.2 (C), 133.0 (C), 132.9 (CH), 132.6 (CH), 130.0 (CH), 130.0 (CH), 129.9 (CH), 129.8 (CH), 129.8 (CH), 129.7 (CH), 129.7 (CH), 129.1 (CH), 129.0 (CH), 128.9 (CH), 128.8 (CH), 128.7 (CH), 128.7 (CH), 128.6 (CH), 128.4 (CH), 128.3 (CH), 128.3 (CH), 128.3 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH), 93.5 (C-1), 90.7 (CCl3), 81.5 (C-1′), 71.8 (C-5), 71.5 (C-2′), 70.8 (C-3′), 69.4 (C-3), 69.0 (C-2), 68.8 (C-4′), 68.0 (C-5′), 62.0 (C-6), 61.7 (C-6′), 45.9 (C-4), 26.7 (TBDPS), 19.1 (TBDPS). IR (film) cm−1: 2931, 1726, 1452, 1264, 1106, 1093, 1070, 796, 735, 707. ESI-HRMS calcd for C72H64Cl3NO16SSiNa 1386.2678, found m/z 1386.2642 [M+Na]+. Selected 1H-NMR data for β-S-anomer: δ 5.12 (d, J=9.9 Hz, 1H, H-1′).

17-Tritriacontyl 4-S-(α-D-glucopyranosyl)-4-thio-β-D-galactopyranoside (5)

Compound 14 (490 mg, 0.36 mmol) and 15 (345 mg, 2 equiv.) were dissolved in CH2Cl2 (18 mL) in a round bottom flask containing molecular sieves 4 Å. A 0.1 mol/L solution of TMSOTf in CH2Cl2 (0.720 mL, 0.2 equiv.) was then added, and the mixture was stirred at room temperature for 3 h. The reaction was quenched with triethylamine, filtered, and the solvent removed under reduced pressure. Chromatography (cyclohexane-EtOAc, 8:1) gave the fully protected glycolipid (326 mg, 54%) as a white solid. 1H NMR (500 MHz, CDCl3) δ 8.09 (overlapping signals, 2H aromatic H), 8.02 (overlapping signals, 2H aromatic H), 7.95 (overlapping signals, 4H aromatic H), 7.85 (overlapping signals, 2H aromatic H), 7.77 (overlapping signals, 2H aromatic H), 7.66 (overlapping signals, 2H aromatic H), 7.60 (overlapping signals, 2H aromatic H), 7.53 (overlapping signals, 3H aromatic H), 7.37 (overlapping signals, 16H aromatic H), 7.25 (overlapping signals, 6H aromatic H), 6.25 (d, J=5.7 Hz, 1H, H-1′), 6.04 (t, J=9.9 Hz, 1H, H-3′), 5.62 (t, J=9.8 Hz, 1H, H-4′), 5.47 (overlapping signals, 3H, H-2′, H-2, H-3), 4.56 (dt, J=10.1, 2.9 Hz, 1H, H-5′), 4.44 (d, J=7.4 Hz, 1H, H-1), 4.02 (dd, J=4.5, 1.3 Hz, 1H, H-4), 3.94 (dd, J=10.6, 8.4 Hz, 1H, H-6a), 3.89 (dd, J=12.6, 2.8 Hz, 1H, H-6′a), 3.72 (dd, J=10.6, 5.4 Hz, 1H, H-6b), 3.58 (dd, J=8.1, 6.0 Hz, 1H, H-5), 3.50 (dd, J=12.6, 3.0 Hz, 1H, H-6′b), 3.40 (m, 1H, CH-(C16H33)2), 1.60 (s, 4H, lipid), 1.47–1.04 (overlapping signals, 54H, lipid), 1.00 (s, 9H, Si-C(CH3)3), 0.89 (m, 6H, lipid). 13C NMR (126 MHz, CDCl3) δ 165.8 (C=O), 165.6 (C=O), 165.4 (C=O), 165.3 (C=O), 165.0 (C=O), 164.8 (C=O), 135.6 (C), 135.5 (C), 133.5 (C), 133.4 (C), 133.3 (C), 133.2 (C), 133.0 (C), 133.0 (C), 132.9 (CH), 132.8 (CH), 130.1 (CH), 129.9 (CH), 129.9 (CH), 129.8 (CH), 129.7 (CH), 129.7 (CH), 129.7 (CH), 129.6 (CH), 129.1 (CH), 129.0 (CH), 128.9 (CH), 128.7 (CH), 128.6 (CH), 128.5 (CH), 128.2 (CH), 128.2 (CH), 128.2 (CH), 127.8 (CH), 127.7 (CH), 101.2 (C-1), 82.1 (C-1′), 81.2 (CH-(C16H33)2), 73.9 (C-5), 73.0 (C-3), 71.3 (C-2′), 71.2 (C-2), 70.8 (C-3′), 68.7 (C-4′), 67.8 (C-5′), 62.4 (C-6), 61.7 (C-6′), 46.5 (C-4), 34.6, 33.9, 31.9, 29.8, 29.7, 29.7, 29.6, 29.6, 29.6, 29.6, 29.5, 29.4, 29.4 (all lipid carbons), 26.7 (CH3, TBDPS), 25.0 (lipid), 24.8 (lipid), 22.7 (lipid), 19.0 (C, TBDPS), 14.1 (lipid). IR (film) cm−1: 3057, 2964, 1697, 1597, 1573, 1528, 1395, 1293, 1260, 1234, 1088, 992, 875, 794, 742, 695. ESI-HRMS calcd for C103H130O16SSiNa 1705.8747, found m/z 1705.8724 [M+Na]+. The disaccharide (326 mg, 0.19 mmol) was then dissolved in THF (5 mL) and acetic acid (0.05 mL) and the solution was cooled to 0°C. TBAF (1 mol/L in THF, 0.4 mL, 2 equiv.) was then added and the mixture was stirred at room temperature overnight. The solution was diluted in CH2Cl2 and the organic phase was washed with water, a saturated solution of NaHCO3, water and brine, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. After chromatography, debenzoylation gave 5 (114 mg, 39% over three steps) as a white solid. 1H NMR (500 MHz, CD3OD) δ 5.33 (d, J=5.4 Hz, 1H, H-1′), 4.23 (d, J=7.5 Hz, 1H, H-1), 4.14 (ddd, J=10.0, 6.0, 2.4 Hz, 1H, H-5′), 3.95 (dd, J=10.6, 7.8 Hz, 1H, H-6a), 3.84 (dd, J=11.8, 2.4 Hz, 1H, H-6′a), 3.74 (overlapping signals, 2H, H-3, H-6b), 3.68 (overlapping signals, 3H, H-5, H-2′, H-6′b), 3.61 (m, 1H, CH-(C16H33)2), 3.55 (dd, J=9.8, 8.8 Hz, 1H, H-3′), 3.38 (dd, J=4.6, 1.4 Hz, 1H, H-4), 3.26 (overlapping signals, 2H, H-2, H-4′), 1.49 (overlapping signals, 4H, lipid), 1.26 (overlapping signals, 56H, lipid), 0.88 (t, J=6.9 Hz, 6H, lipid). 13C NMR (126 MHz, CD3OD) δ 102.9 (C-1), 87.3 (C-1′), 79.3 (CH-(C16H33)2), 74.2 (C-3′), 74.2 (C-5), 73.3 (C-3), 73.1 (C-5′), 72.8 (C-2), 72.1 (C-2′), 70.4 (C-4′), 61.3 (C-6′), 61.0 (C-6), 51.7 (C-4), 34.4, 33.7, 31.7, 29.6, 29.4, 29.4, 29.3, 29.1, 24.8, 24.7, 22.4, 13.3 (all lipid carbons). IR (film) cm−1: 3351, 2981, 2915, 2850, 1737.5, 1471, 1462, 1381, 1251, 1152, 1070, 955, 828, 717. ESI-HRMS calcd for C45H88O10SCl 855.5787, found m/z 855.5795 [M+Cl].

17-Tritriacontyl 4-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-2,3,6-tri-O-benzoyl-1-thio-β-D-galactopyranoside (20)

Compound 17 (4.5 g, 7.5 mmol) and 16 (14.6 g, 22 mmol) were dissolved in CH2Cl2 (100 mL) in a round bottom flask containing molecular sieves 4 Å. This solution was stirred at room temperature for 15 min before being cooled to −78°C. N-Iodosuccinimide (1.7 g, 1.3 equiv.) TMSOTf (340 μL, 0.25 equiv.) were then added and the temperature was let to go to room temperature and stirred for 4 h. It was then diluted with CH2Cl2 and the organic phase was washed with a saturated solution of NaHCO3, water and brine, dried over Na2SO4, filtered and the solvent removed under reduced pressure. Chromatography (cyclohexane-EtOAc, 4:1) gave a disaccharide (4.97 g, 59%; 5:2 mixture of α-and β-anomers). ESI-HRMS calcd for C68H64O13SNa 1143.3965, found m/z 1143.4008 [M+Na]+. The mixture of anomers (3.20 g, 2.8 mmol) was dissolved in acetone-H2O (9:1, 12 mL). N-Bromosuccinimide (1.5 g, 3 equiv.) was then added and the solution was stirred at room temperature for 4 h. The mixture was then diluted with EtOAc, and the organic phase was washed with a saturated solution of NaHCO3, water and brine, dried over Na2SO4, filtered and the solvent removed under reduced pressure. Chromatography (cyclohexane-EtOAc, 3:1) gave intermediate 18 (1.80 g, 62%) as a yellow oil. This intermediate 18 (1 g, 0.98 mmol) was dissolved in CH2Cl2 (30 mL) and Cl3CCN (2 mL, 20 equiv.) and DBU (0.2 mL, 1.2 equiv.) were then added and the mixture was stirred for 1 h. The solvent was then removed under reduced pressure and chromatography (cyclohexane-EtOAc, 5:1) gave the trichloroacetimidate intermediate (0.80 g, 70%) as a white solid. This precursor (400 mg, 0.34 mmol) and 15 (664 mg, 4 equiv.) were then dissolved in CH2Cl2 (17 mL) in a round bottom flask containing molecular sieves 4 Å, and the solution was cooled to 0°C. TMSOTf (0.1 M in CH2Cl2, 0.9 mL, 0.09 mmol) was added, and the mixture was stirred at room temperature for 3 h. Triethylamine was added and after filtration, the solvent was removed under reduced pressure. Chromatography (cyclohexane-EtOAc, 9:1) gave 20 (219 mg, 43%) as a white solid. 1H NMR (500 MHz, CDCl3) δ 8.01 (overlapping signals, 2H, aromatic H), 7.94 (overlapping signals, 4H, aromatic H), 7.57 (overlapping signals, 2H, aromatic H), 7.45 (overlapping signals, 6H, aromatic H), 7.34 (overlapping signals, 4H, aromatic H), 7.27 (overlapping signals, 12H, aromatic H), 7.15 (overlapping signals, 5H, aromatic H), 5.69 (t, J=9.3 Hz, 1H, H-2), 5.25 (d, J=10.6 Hz, 1H, H-3), 5.00 (d, J=11.0 Hz, 1H, benzyl CH), 4.92 (m, 1H, benzyl CH), 4.79 (overlapping signals, 2H, benzyl CH x2), 4.71 (overlapping signals, 3H, H-6a, H-6b, benzyl CH), 4.41 (overlapping signals, 2H, H-4, benzyl CH), 4.34 (d, J=12.0 Hz, 1H, benzyl CH), 4.17 (t, J=9.6 Hz, 1H, H-3′), 4.09 (t, J=11.7 Hz, 1H, H-5′), 4.03 (overlapping signals, 2H, H-5, benzyl CH), 3.71 (t, J=9.6 Hz, 1H, H-4′), 3.55 (overlapping signals, 3H, H-2′, O-CH), 3.34 (d, J=11.0 Hz, 1H, H-6′a), 2.94 (d, J=10.9 Hz, 1H, H-6′b), 1.66 (overlapping signals, 4H, lipid), 1.23 (overlapping signals, 56H, lipid), 0.88 (t, J=7.0 Hz, 6H, lipid); 13C NMR (126 MHz, CDCl3) δ 166.4, 166.2, 165.3 (each C=O), 139.0 (C), 138.7 (C), 138.2 (C), 138.0 (C), 133.4 (C), 133.3 (C), 133.1 (C), 130.1 (CH), 130.0 (CH), 129.9 (CH), 129.8 (CH), 129.8 (CH), 129.5 (CH), 129.0 (CH), 128.6 (CH), 128.6 (CH), 128.5 (CH), 128.4 (CH), 128.4 (CH), 128.4 (CH), 128.4 (CH), 128.3 (CH), 128.3 (CH), 128.1 (CH), 128.0 (CH), 127.8 (CH), 127.8 (CH), 127.7 (CH), 127.6 (CH), 127.6 (CH), 127.6 (CH), 127.5 (CH), 127.5 (CH), 101.8 (C-1), 100.6 (C-1′), 82.4 (CH-O), 81.8 (C-3′), 79.9 (C-2′), 77.7 (C-4′), 76.2 (C-4), 75.7 (benzyl CH2), 74.9 (benzyl CH2), 74.3 (C-3), 73.9 (benzyl CH2), 73.4 (benzyl CH2), 72.8 (C-5), 71.5 (C-5′), 70.2 (C-2), 67.9 (C-6′), 63.0 (C-6), 35.1, 34.3, 32.1, 30.0, 29.9, 29.9, 29.8, 29.8, 29.5, 29.5, 25.5, 25.2, 22.8, 14.3 (all lipid carbons). IR (film) cm−1: 2923, 2852, 1725, 1651, 1601, 1452, 1271, 1095, 1070, 1028, 709. ESI-HRMS calcd for C94H128O14N 1494.9335, found m/z 1494.9321 [M+NH4]+.

1-Heptadecanyl 4-O-(2,3,4,6-tetra-O-benzyl-D-glucopyranosyl)-2,3,6-tri-O-benzoyl-β-D-galactopyranoside (21)

The trichloroacetimidate precursor (400 mg, 0.34 mmol), prepared from 18 as described above in the preparation of 20, and 19 (0.35 g, 4 equiv.) were dissolved in CH2Cl2 (17 mL) in a round bottom flask containing 4 Å molecular sieves, and the solution was cooled to 0°C. TMSOTf (0.1 M in CH2Cl2, 0.9 mL, 0.09 mmol) was then added, and the mixture was stirred at room temperature for 3 h. Triethylamine was added to quench the reaction. After filtration, the solvent was removed under reduced pressure. Chromatography (cyclohexane-EtOAc, 9:1) gave 21 (203 mg, 47%) as a white solid and which was a mixture of glucosides (85α:15β). 1H NMR (α-glucopyranoside, 500 MHz, CDCl3): δ 8.03 (overlapping signals, 2H, aromatic H), 7.95 (overlapping signals, 4H, aromatic H), 7.57 (m, 1H, aromatic H), 7.45 (overlapping signals, 5H, aromatic H), 7.35 (overlapping signals, 4H, aromatic H), 7.27 (overlapping signals, 13H, aromatic H), 7.15 (overlapping signals, 6H, aromatic H), 5.70 (dd, J=10.6, 7.7 Hz, 1H, H-2), 5.28 (dd, J=10.6, 3.1 Hz, 1H, H-3), 5.00 (d, J=10.9 Hz, 1H, benzyl CH), 4.93 (dd, J=7.2, 3.8 Hz, 1H, benzyl CH), 4.79 (overlapping signals, 4H, H-6a, H-6b, Bn CH x2), 4.69 (m, 1H, benzyl CH), 4.42 (overlapping signals, 2H, H-4, benzyl CH), 4.35 (d, J=12.1 Hz, 1H, benzyl CH), 4.16 (t, J=9.5 Hz, 1H, H-3′), 4.10 (m, 1H, H-5′), 4.05 (overlapping signals, 2H, H-5, benzyl CH), 3.94 (dt, J=9.9, 6.2 Hz, 1H, butyl CH(H)O), 3.72 (t, J=9.6 Hz, 1H, H-4′), 3.55 (m, 1H, H-2′), 3.51 (m, 1H, butyl CH(H)O), 3.34 (dd, J=11.0, 2.1 Hz, 1H, H-6′a), 2.93 (dd, J=11.0, 2.0 Hz, 1H, H-6′b), 1.53 (m, 2H, lipid), 1.34 (m, 28H, lipid), 0.88 (m, 3H, lipid). 13C NMR (126 MHz, CDCl3) δ 166.2, 166.1, 165.3 (each C=O), 138.9 (C), 138.6 (C), 138.0 (C), 137.9 (C), 133.3 (C), 133.2 (C), 133.0 (C), 130.2 (CH), 130.0 (CH), 129.8 (CH), 129.7 (CH), 129.7 (CH), 129.6 (CH), 129.3 (CH), 128.7 (CH), 128.5 (CH), 128.4 (CH), 128.3 (CH), 128.3 (CH), 128.2 (CH), 128.2 (CH), 128.0 (CH), 127.9 (CH), 127.9 (CH), 127.7 (CH), 127.6 (CH), 127.5 (CH), 127.4 (CH), 127.4 (CH), 101.6 (C-1), 100.6 (C-1′), 81.7 (C-3′), 79.7 (C-2′), 77.6 (C-4′), 75.9 (C-4), 75.6 (benzyl CH2), 74.7 (benzyl CH2), 73.9 (C-3), 73.9 (benzyl CH2), 73.3 (benzyl CH2), 72.7 (C-5), 71.4 (C-5′), 70.2(CH2-O), 69.9 (C-2), 67.7 (C-6′), 62.5 (C-6), 32.8, 31.9, 29.7, 29.7, 29.7, 29.6, 29.6, 29.6, 29.6, 29.5, 29.5, 29.4, 29.4, 29.3, 26.9, 25.8, 25.8, 22.7, 14.1 (all lipid). ESI-HRMS calcd for C78H96O14N 1270.6831, found m/z 1270.6843 [M+NH4]+. Selected 1H-NMR data for β-glucopyranoside: δ 5.55 (dd, J=10.4, 3.3 Hz, H-3), 5.86 (dd, J=10.5, 8.0 Hz, H-2).

17-Tritriacontyl 4-O-(D-glucopyranosyl)-β-D-galactopyranoside (1a)

Compound 20 (219 mg, 0.15 mmol) was reacted as described in the general procedures for debenzoylation and O-debenzylation successively to give 1a (97 mg, 82% over two steps, 90:10 mixture of α & β-glucopyranosides); 1H NMR (α-glucopyranoside, 600 MHz, pyridine-d5) δ 5.81 (d, J=3.8 Hz, 1H, H-1′ α), 4.93 (ddd, J=10.1, 5.7, 2.5 Hz, 1H, H-5′), 4.86 (d, J=7.5 Hz, 1H, H-1), 4.75 (d, J=3.3 Hz, 1H, H-4), 4.70 (t, J=9.6 Hz, 1H, H-6a), 4.58 (t, J=9.3 Hz, 1H, H-3′), 4.48 (dd, J=11.5, 2.5 Hz, 1H, H-6′a), 4.38 (dd, J=9.9, 7.6 Hz, 1H, H-2), 4.33 (overlapping signals, 2H, H-6b, H-6′b), 4.20 (overlapping signals, 3H, H-3, H-2′, H-4′), 4.16 (m, 1H, H-5), 3.98 (m, 1H, O-CH), 1.76 (overlapping signals, 4H, lipid), 1.52 (overlapping signals, 2H, lipid), 1.29 (overlapping signals, 54H), 0.88 (t, J=6.9 Hz, 6H, lipid). 13C NMR (151 MHz, Pyridine-d5) δ 105.1 (C-1), 103.1 (C-1′), 80.4 (CH-O), 80.1 (C-4), 76.0 (C-5), 75.6 (C-3), 75.4 (C-3′), 75.2 (C-5′), 74.6 (C-2′), 73.4 (C-2), 72.5 (C-4′), 62.9 (C-6′), 60.9 (C-6), 35.9, 35.0, 32.5, 30.7, 30.5, 30.4, 30.4, 30.4, 30.3, 30.3, 30.0, 26.1, 25.8, 23.3, 14.7 (all lipid carbons). IR (film) cm−1: 3400, 2955, 2915, 2850, 1470, 1111, 1042, 1024, 992, 793, 718, 660, 668. ESI-HRMS calcd for C45H88O11Cl 839.6015, found m/z 839.6030 [M+Cl]. Selected 1H-NMR spectroscopic data for the β-glucopyranoside: δ 5.27 (d, J=7.9 Hz, H-1′ β).

1-Heptadecanyl 4-O-(α-D-glucopyranosyl)-β-D-galactopyranoside (2)

Compound 21 (203 mg, 0.16 mmol) was reacted as described in the general procedures for de-O-benzoylation and de-O-benzylation successively to give 2 (75 mg, 80% over two steps) as a mixture of glucopyranosides. 1H NMR (α-D-glucopyranoside, 500 MHz, CDCl3) δ 4.98 (d, J=3.8 Hz, 1H, H-1′), 4.28 (d, J=7.4 Hz, 1H, H-1), 4.03 (overlapping signals, 2H, H-4, H-4′), 3.86 (overlapping signals, 3H, H-6a, H6′a, O-CH-lipid), 3.76 (dd, J=11.1, 5.7 Hz, 1H, H-6b), 3.70 (overlapping signals, 2H, H-3′, H-6′b), 3.64 (m, 1H, H-5), 3.56 (overlapping signals, 2H, H-3, O-CH-lipid), 3.50 (dd, J=10.1, 7.4 Hz, 1H, H-2), 3.46 (dd, J=9.8, 3.7 Hz, 1H, H-2′), 3.30 (dd, J=10.0, 9.0 Hz, 1H, H-5′), 1.64 (overlapping signals, 2H, lipid), 1.27 (s, 28H, lipid), 0.90 (m, 3H, lipid). 13C NMR (126 MHz, CDCl3) δ 104.2 (C-1), 101.5 (C-1′), 78.9 (C-4), 74.8 (C-5), 74.1 (C-3), 74.0 (C-3′), 73.5 (C-4′), 73.1 (C-2′), 72.1 (C-2), 71.2 (C-5′), 71.0 (O-CH2-lipid), 62.2 (C-6′), 60.1 (C-6), 32.4, 30.2, 30.1, 29.9, 26.4, 23.2, 14.3 (all lipid carbons). IR (film) cm−1: 3358, 2915, 2850, 1653, 1471, 1376, 1262, 1099, 1047, 1020, 794, 717. ESI-HRMS calcd for C29H56O11Cl 615.3511, found m/z 615.3499 [M+Cl]. ESI-HRMS calcd for C29H56O11Na 603.3720, found m/z 603.3743 [M+Na]+. Selected 1H-NMR data for β-glucopyranoside: δ 4.52 (d, J=7.7 Hz, H-1′), 4.22 (d, J=7.2 Hz, H-1).

4-O-(2,3,4,6-Tetra-O-benzyl-α-D-glucopyranosyl)-2,3,6-tri-O-benzoyl-1-thio-D-galactopyranose (22)

Intermediate 18 (700 mg, 0.69 mmol), prepared as described above in preparation of 20, was dissolved in dioxane (17 mL). Lawesson’s reagent (0.4 g, 1.5 equiv.) was then added, and the mixture was stirred at 100°C overnight. The solvent was then removed under reduced pressure. Chromatography (cyclohexane-EtOAc, 7:2) gave 22β (270 mg, 38%) and 22α (96 mg, 14%). Analytical data for 22β1H NMR (α-glucopyranoside, 500 MHz, CDCl3) δ 8.08 (overlapping signals, 3H, aromatic H), 8.97 (overlapping signals, 5H, aromatic H), 7.60 (overlapping signals, 16H, aromatic H), 7.49 (overlapping signals, 6H, aromatic H), 7.27 (overlapping signals, 28H, aromatic H), 5.68 (t, J=9.9 Hz, 1H, H-2), 5.32 (dd, J=10.3, 2.9 Hz, 1H, H-3), 5.03 (d, J=11.0 Hz, 1H, benzyl CH), 4.97 (overlapping signals, 2H, H-1′, benzyl CH), 4.83 (overlapping signals, 2H, benzyl CH x2), 4.78 (overlapping signals, 2H, H-1, H6a), 4.71 (overlapping signals, 2H, H-6b, benzyl CH), 4.47 (d, J=3.4 Hz, 1H, H-4), 4.41 (overlapping signals, 2H, benzyl CH x2), 4.16 (t, J=9.6 Hz, 1H, H-3′), 4.10 (overlapping signals, 3H, H-5, H-5′, benzyl CH), 3.76 (t, J=9.5 Hz, 1H, H-4′), 3.57 (dd, J=9.9, 3.5 Hz, 1H, H-2′), 3.43 (dd, J=11.0, 2.1 Hz, 1H, H-6′a), 3.04 (dd, J=11.0, 2.1 Hz, 1H, H-6′b), 2.51 (d, J=14.5 Hz, 1H, SH). 13C NMR (126 MHz, CDCl3) δ 166.1 (C=O), 166.1 (C=O), 165.5 (C=O), 138.8 (C), 138.5 (C), 137.9 (C), 137.9 (C), 133.4 (C), 133.3 (C), 133.2 (C), 130.0 (CH), 129.8 (CH), 129.7 (CH), 129.7 (CH), 129.3 (CH), 129.2 (CH), 128.5 (CH), 128.5 (CH), 128.4 (CH), 128.4 (CH), 128.3 (CH), 128.1 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH), 127.6 (CH), 127.5 (CH), 127.5 (CH), 100.5 (C-1′), 81.7 (C-3′), 79.8 (C-2′), 79.4 (C-1), 77.6 (C-4′), 77.3 (C-5), 76.2 (C-4), 75.6 (benzyl CH2), 74.9 (benzyl CH2), 74.7 (C-3), 74.0 (benzyl CH2), 73.4 (benzyl CH2), 72.1 (C-2), 71.5 (C-5′), 67.8 (C-6′), 62.9 (C-6). ESI-HRMS calcd for C61H58O13SNa 1053.3496, found m/z 1053.3496 [M+Na]+.

Analytical data for 22α:1H NMR (α-glucopyranoside, 500 MHz, CDCl3) δ 8.07 (overlapping signals, 3H, aromatic H), 8.95 (overlapping signals, 5H, aromatic H), 7.59 (m, 1H, aromatic H), 7.48 (overlapping signals, 5H, aromatic H), 7.19 (overlapping signals, 28H, aromatic H), 6.22 (t, J=5.3 Hz, 1H, H-1), 5.79 (dd, J=10.8, 5.5 Hz, 1H, H-2), 5.61 (dd, J=10.8, 3.0 Hz, 1H, H-3), 5.02 (m, 1H, benzyl CH), 4.93 (overlapping signals, 2H, benzyl CH, H-1′), 4.73 (overlapping signals, 6H, benzyl CH x3, H5, H6a, H6b), 4.47 (d, J=3.1 Hz, 1H, H-4), 4.39 (overlapping signals, 2H, benzyl CH x2), 4.10 (overlapping signals, 3H, H-3′, H-5′, benzyl CH), 3.70 (t, J=9.5 Hz, 1H, H-4′), 3.55 (dd, J=9.9, 3.4 Hz, 1H, H-2′), 3.28 (dd, J=11.0, 2.5 Hz, 1H, H-6′a), 2.91 (dd, J=11.0, 2.0 Hz, 1H, H-6′b), 1.88 (d, J=4.9 Hz, 1H, SH). 13C NMR (126 MHz, CDCl3) δ 166.0 (C=O), 166.0 (C=O), 165.3 (C=O), 138.8 (C), 138.3 (C), 137.8 (C), 137.7 (C), 133.4 (C), 133.4 (C), 133.2 (C), 129.9 (CH), 129.8 (CH), 129.8 (CH), 129.7 (CH), 129.3 (CH), 129.0 (CH), 128.5 (CH), 128.4 (CH), 128.4 (CH), 128.4 (CH), 128.4 (CH), 128.2 (CH), 128.2 (CH), 128.2 (CH), 128.1 (CH), 127.9 (CH), 127.9 (CH), 127.9 (CH), 127.9 (CH), 127.8 (CH), 127.8 (CH), 127.7 (CH), 127.7 (CH), 127.7 (CH), 127.6 (CH), 127.5 (CH), 127.5 (CH), 127.3 (CH) (aromatic carbons), 100.1 (C-1′), 81.8 (C-3′), 79.6 (C-2′), 78.2 (C-1), 77.6 (C-4′), 76.0 (C-4), 75.6 (benzyl CH2), 74.9 (benzyl CH2), 74.2 (benzyl CH2), 73.4 (benzyl CH2), 71.3 (C-5′), 70.5 (C-3), 69.6 (C-5), 68.4 (C-2), 67.7 (C-6′), 62.5 (C-6). ESI-HRMS calcd for C61H58O13SNa 1053.3496, found m/z 1053.3503 [M+Na]+.

17-Tritriacontyl 4-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-2,3,6-tri-O-benzoyl-1-thio-β-D-galactopyranoside (25β)

Compound 15 (2 g, 4.2 mmol) was dissolved in THF (50 mL). To this suspension were added imidazole (850 mg, 3 equiv.), triphenylphosphine (3.3 g, 3 equiv.) and iodine (3.2 g, 3 equiv.), and the resulting mixture was stirred at 45°C overnight. Methanol was then added to the solution, and the solvent was removed under reduced pressure. Chromatography (100% of cyclohexane) gave 23 (2.11 g, 86%) as a white solid; 1H NMR (500 MHz, CDCl3) δ 4.12 (tt, J=8.8, 4.6 Hz, 1H, I-CH), 1.85 (overlapping signals, 2H), 1.68 (overlapping signals, 2H), 1.51 (overlapping signals, 2H), 1.26 (overlapping signals, 54H), 0.88 (t, J=6.8 Hz, 6H). Compound 22β (100 mg, 0.27 mmol) was dissolved in DMF (5 mL) and cooled to 0°C. NaH (5 mg, 1.2 equiv) was then added and the reaction was stirred for 5 min, and a solution of 23 (115 mg, 2 equiv.) in DMF (5 mL) was added. The mixture was stirred at room temperature for 3 h. It was then diluted with Et2O, and the organic phase was washed with water and brine, dried over Na2SO4, filtered and the solvent removed under reduced pressure. Chromatography (cyclohexane-EtOAc, 15:1) gave 25β (88 mg, 61%, 80:20 mixture of α and β-glucopyranosides, white solid; 1H NMR (α-glucopyranoside, 500 MHz, CDCl3): δ 8.02 (overlapping signals, 2H, aromatic H), 7.93 (overlapping signals, 5H, aromatic H), 7.57 (overlapping signals, 2H, aromatic H), 7.45 (overlapping signals, 6H, aromatic H), 7.29 (overlapping signals, 26H, aromatic H), 7.15 (overlapping signals, 8H, aromatic H), 5.70 (t, J=10.0 Hz, 1H, H-2), 5.32 (dd, J=10.2, 3.0 Hz, 1H, H-3), 5.00 (d, J=10.9 Hz, 1H, benzyl CH), 4.92 (d, J=11.0 Hz, 1H, benzyl CH), 4.89 (d, J=3.5 Hz, 1H, H-1′), 4.81 (overlapping signals, 2H, benzyl CH x2), 4.76 (m, 1H, H-1), 4.73 (overlapping signals, 2H, H-6a, H-6b), 4.69 (t, J=11.7 Hz, 1H, benzyl CH), 4.44 (overlapping signals, 2H, H-4, benzyl CH), 4.37 (d, J=12.1 Hz, 1H, benzyl CH), 4.14 (t, J=9.5 Hz, 1H, H-3′), 4.08 (overlapping signals, 3H, H-5, H-5′, benzyl CH), 3.73 (t, J=9.6 Hz, 1H, H-4′), 3.53 (m, 1H, H-2′), 3.39 (dd, J=11.1, 2.1 Hz, 1H, H-6′a), 3.01 (dd, J=11.1, 1.9 Hz, 1H, H-6′b), 2.85 (m, 1H, S-CH lipid), 1.53 (overlapping signals, 7H, lipid), 1.20 (overlapping signals, 72H, lipid), 0.88 (t, J=6.8 Hz, 6H, lipid); 13C NMR (126 MHz, CDCl3) δ 165.1 (C=O), 165.0 (C=O), 164.2 (C=O), 137.8 (C), 137.6 (C), 137.0 (C), 136.9 (C), 132.2 (C), 132.1 (C), 132.0 (C), 129.2 (CH), 129.0 (CH), 128.8 (CH), 128.7 (CH), 128.6 (CH), 128.6 (CH), 128.3 (CH), 127.6 (CH), 127.4 (CH), 127.4 (CH), 127.3 (CH), 127.3 (CH), 127.3 (CH), 127.2 (CH), 127.2 (CH), 127.1 (CH), 127.0 (CH), 126.9 (CH), 126.9 (CH), 126.9 (CH), 126.7 (CH), 126.6 (CH), 126.6 (CH), 126.6 (CH), 126.5 (CH), 126.4 (CH), 126.4 (CH), 126.3 (CH), 99.5 (C-1′), 83.3 (C-1), 80.7 (C-3′), 78.7 (C-2′), 76.5 (C-4′), 75.6 (C-5), 75.3 (C-4), 74.6 (benzyl CH2), 74.2 (C-3), 73.7 (benzyl CH2), 72.8 (benzyl CH2), 72.3 (benzyl CH2), 70.5 (C-5′), 67.6 (C-2), 66.8 (C-6′), 62.1 (C-6), 45.8 (CH-S), 34.3, 34.2, 30.9, 28.7, 28.7, 28.6, 28.6, 28.6, 28.6, 28.6, 28.5, 28.5, 28.5, 28.3, 25.8, 25.3, 21.7, 13.1 (all lipid carbons); ESI-HRMS calcd for C94H124O13SNa 1515.8660, found m/z 1515.8699 [M+Na]+. Selected 1H-NMR data for β-glucopyranoside: δ 5.85 (t, J=10.0 Hz, H-2), 5.58 (dd, J=10.1, 3.2 Hz, H-3).

17-Tritriacontyl 4-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-2,3,6-tri-O-benzoyl-1-thio-α-D-galactopyranoside (25α)

Compound 22α (100 mg, 0.097 mmol) was dissolved in DMF (5 mL) and cooled to 0°C. NaH (5 mg, 1.2 equiv.) was then added and the reaction was stirred for 5 min, and a solution of 24 (115 mg, 2 equiv.) in DMF (5 mL) was then added. The mixture was stirred at room temperature for 3 h. It was then diluted with Et2O, and the organic phase was washed with water and brine, dried over Na2SO4, filtered and the solvent removed under reduced pressure. Chromatography (cyclohexane-EtOAc, 15:1) gave 25α (88 mg, 61%) as a white solid. 1H NMR (500 MHz, CDCl3) δ 8.04 (overlapping signals, 2H, aromatic H), 7.95 (overlapping signals, 4H, aromatic H), 7.57 (m, 1H, aromatic H), 7.46 (overlapping signals, 5H, aromatic H), 7.31 (overlapping signals, 19H, aromatic H), 7.20 (overlapping signals, 5H, aromatic H), 7.11 (overlapping signals, 6H, aromatic H), 5.96 (d, J=5.7 Hz, 1H, H-1), 5.75 (dd, J=11.0, 5.7 Hz, 1H, H-2), 5.56 (dd, J=11.0, 2.9 Hz, 1H, H-3), 5.03 (d, J=11.0 Hz, 1H, benzyl CH), 4.92 (d, J=10.9 Hz, 1H, benzyl CH), 4.89 (d, J=3.2 Hz, 1H, H-1′), 4.80 (overlapping signals, 2H, benzyl CH x2), 4.70 (overlapping signals, 4H, H-5, H-6a, H-6b, benzyl CH), 4.46 (d, J=3.0 Hz, 1H, H-4), 4.37 (overlapping signals, 2H, benzyl CH x2), 4.12 (t, J=9.5 Hz, 1H, H-3′), 4.05 (overlapping signals, 2H, H-5′, benzyl CH), 3.69 (t, J=9.7 Hz, 1H, C-4′), 3.54 (dd, J=9.6, 3.4 Hz, 1H, C-2′), 3.22 (dd, J=11.0, 2.3 Hz, 1H, H-6′a), 2.83 (d, J=10.8 Hz, 1H, H-6′b), 2.73 (m, 1H, S-CH lipid), 1.46 (overlapping signals, 4H, lipid), 1.36–1.03 (overlapping signals, 56H, lipid), 0.88 (t, J=6.7 Hz, 6H, lipid). 13C NMR (126 MHz, CDCl3) δ 166.0 (C=O), 165.9 (C=O), 165.6 (C=O), 138.9 (C), 138.4 (C), 137.8 (C), 137.7 (C), 133.2 (C), 133.2 (C), 133.1 (C), 129.9 (CH), 129.8 (CH), 129.8 (CH), 129.8 (CH), 129.7 (CH), 129.6 (CH), 129.4 (CH), 128.5 (CH), 128.4 (CH), 128.4 (CH), 128.4 (CH), 128.4 (CH), 128.3 (CH), 128.3 (CH), 128.3 (CH), 128.3 (CH), 128.2 (CH), 128.0 (CH), 127.9 (CH), 127.9 (CH), 127.9 (CH), 127.8 (CH), 127.8 (CH), 127.8 (CH), 127.7 (CH), 127.6 (CH), 127.5 (CH), 100.2 (C-1′), 81.9 (C-1), 81.8 (C-3′), 79.5 (C-2′), 77.6 (C-4′), 76.4 (C-4), 75.6 (benzyl CH2), 74.9 (benzyl CH2), 74.2 (benzyl CH2), 73.3 (benzyl CH2), 71.3 (C-3), 71.2 (C-5′), 69.0 (C-2), 69.0 (C-5), 67.7 (C-6′), 62.8 (C-6), 45.2 (CH-S), 35.8, 35.1, 34.6, 31.9, 29.7, 29.7, 29.7, 29.6, 29.6, 29.6, 29.5, 29.4, 27.1, 26.4, 22.7, 14.1 (all lipid carbons). ESI-HRMS calcd for C94H124O13SNa 1515.8660, found m/z 1515.8619 [M+Na]+. Selected 1H-NMR data for β-glucopyranoside: δ 6.01 (d, J=5.6 Hz, H-1), 5.85 (dd, J=10.8, 5.5 Hz, H-2), 5.79 (dd, J=10.8, 3.2 Hz, H-3).

17-Tritriacontyl 4-O-(D-glucopyranosyl)-1-thio-β-D-galactopyranoside (6)

Compound 25β (25 mg, 0.017 mmol) was de-O-benzoylated and de-O-benzylated successively. Chromatography (CH2Cl2-MeOH 9:1) gave 6 (11 mg, 80% over two steps, 77:23 mixture of α and β-glucopyranosides, white solid); 1H NMR (α-glucopyranoside, 500 MHz, CDCl3-CD3OD, 1:1) δ 4.97 (d, J=3.8 Hz, 1H, H-1′), 4.40 (d, J=9.5 Hz, 1H, H-1), 4.08 (d, J=2.9 Hz, 1H, H-4), 4.01 (ddd, J=9.8, 6.6, 2.5 Hz, 1H, H-5′), 3.90 (m, 1H, H-6′a), 3.83 (dd, J=11.0, 8.4 Hz, 1H, H-6a), 3.69 (overlapping signals, 4H, H-5, H-6b, H-3′, H-6′b), 3.55 (dd, J=9.6, 2.8 Hz, 1H, H-3), 3.47 (overlapping signals, 2H, H-2, H-2′), 3.29 (t, J=9.5 Hz, 1H, H-4), 2.90 (m, 1H, S-CH), 1.62 (overlapping signals, 4H, lipid), 1.42 (overlapping signals, 4H, lipid), 1.27 (overlapping signals, 52H, lipid), 0.89 (t, J=6.8 Hz, 6H, lipid). 13C NMR (126 MHz, CDCl3-CD3OD, 1:1) δ 101.5 (C-1′), 86.8 (C-1), 79.2 (C-4), 78.2 (C-5), 75.2 (C-3), 73.9 (C-3′), 73.3 (C-5′), 72.9 (C-2), 71.1 (C-2′), 71.0 (C-4), 62.2 (C-6′), 59.8 (C-6), 46.7 (S-CH), 35.3, 35.3, 32.3, 30.0, 29.9, 29.7, 26.8, 23.0, 14.2 (all lipid carbons). IR (film) cm−1: 3338, 2916, 2850, 1470, 1260, 1018, 797, 717. ESI-HRMS calcd for C45H87O10S 819.6020, found m/z 819.6049 [M−H]. Selected 1H-NMR data for β-glucopyranoside: δ 4.51 (d, J=7.4 Hz, H-1′), 4.35 (d, J=9.1 Hz, H-1).

17-Tritriacontyl 4-O-(α-D-glucopyranosyl)-1-thio-β-D-galactopyranoside (4)

Compound 25α (23 mg, 0.015 mmol) was de-O-benzoylated and de-O-benzylated successively. Chromatography (CH2Cl2-MeOH 9:1) gave 4 (11 mg, 83% over two steps, 90:10 mixture of α and β-glucopyranosides, white solid); 1H-NMR (500 MHz, CDCl3-CD3OD 1:1): δ 5.41 (d, J=5.4 Hz, 1H, H-1), 4.98 (d, J=3.7 Hz, 1H, H-1′), 4.32 (dd, J=8.0, 5.2 Hz, 1H, H-5), 4.09 (m, 1H, H-4), 4.04 (dd, J=10.5, 5.4 Hz, 1H, H-2), 3.93 (dd, J=10.0, 2.7 Hz, 1H, H-5′), 3.90 (d, J=11.0 Hz, 1H, H-6′a), 3.86 (dd, J=12.1, 8.8 Hz, 1H, H-6a), 3.68 (overlapping signals, 4H, H-3, H-3′, H-6b, H-6′b), 3.50 (dd, J=9.8, 3.8 Hz, 1H, H-2′), 3.29 (t, J=9.5 Hz, 1H, H-4′), 2.76 (p, J=6.5 Hz, 1H, CH-lipid), 1.61 (overlapping signals, 4H, lipid), 1.44 (overlapping signals, 9H), 1.29 (overlapping signals, 60H, lipid), 0.89 (t, J=6.8 Hz, 7H). 13C NMR (126 MHz, CDCl3-CD3OD 1:1) δ 102.0 (C-1′), 86.5 (C-1), 81.7 (C-4), 74.0 (C-5′), 74.0 (C-3′), 72.8 (C-2′), 71.8 (C-3), 71.1 (C-4′), 70.8 (C-5), 69.6 (C-2), 62.3 (C-6′), 60.7 (C-6), 46.2 (CH-lipid), 35.9, 35.3, 32.4, 30.2, 30.1, 30.1, 30.1, 29.8, 27.2, 23.1, 14.3 (all lipid carbons). IR (film) cm−1: 3385, 2913, 2849, 1471, 1260, 1081, 1019, 799, 716. ESI-HRMS calcd for C45H87O10S 819.6020, found m/z 819.6038 [M−H]. Selected 1H-NMR data for β-glucopyranoside: δ 5.38 (d, J=5.9 Hz), 4.50 (d, J=7.6 Hz, 1H).

1-Heptadecanyl 4-O-(2,3,4,6-tetra-O-benzyl-D-glucopyranosyl)-2,3,6-tri-O-benzoyl-1-thio-β-D-galactopyranoside (27)

Compound 19 (1 g, 3.9 mmol) was suspended in THF (50 mL) and to this was added imidazole (0.8 g, 3 equiv.), triphenylphosphine (3 g, 3 equiv.) and iodine (3 g, 3 equiv.), and the resulting mixture was stirred at 45°C overnight. Methanol was then added to the solution, and the solvent was removed under reduced pressure. Chromatography (cyclohexane) gave intermediate 24 (1.13 g, 79%) as a white solid. Analytical data for 24 was in agreement with that reported previously [30]. Compound 22β (100 mg, 0.097 mmol) was dissolved in DMF (5 mL) and cooled to 0°C. NaH (5 mg, 1.2 equiv.) was then added and the reaction was stirred for 5 min, and 24 (71 mg, 2 equiv.) in DMF (5 mL) was then added. The mixture was stirred at room temperature for 3 h. It was then diluted with Et2O, and the organic phase was washed with water and brine, dried over Na2SO4, filtered and the solvent removed under reduced pressure. Chromatography (cyclohexane-EtOAc, 14:1) gave 27 (53 mg, 43%, 81α:19β mixture of glucopyranosides, white solid); 1H NMR (α-glucopyranoside, 500 MHz, CDCl3): δ 8.03 (overlapping signals, 2H, aromatic H), 7.94 (overlapping signals, 4H, aromatic H), 7.59 (overlapping signals, 2H, aromatic H), 7.47 (overlapping signals, 5H, aromatic H), 7.29 (overlapping signals, 18H, aromatic H), 7.16 (overlapping signals, 4H, aromatic H), 5.80 (t, J=10.0 Hz, 1H, H-2), 5.34 (dd, J=10.2, 2.9 Hz, 1H, H-3), 4.98 (d, J=10.9 Hz, 1H, benzyl CH), 4.93 (overlapping signals, 2H, H-1′, benzyl CH), 4.80 (overlapping signals, 2H, benzyl CH x2), 4.75 (overlapping signals, 2H, H-6a, H-6b), 4.71 (overlapping signals, 2H, H-1, benzyl CH), 4.46 (overlapping signals, 2H, H-4, benzyl CH), 4.37 (d, J=12.1 Hz, 1H, benzyl CH), 4.10 (overlapping signals, 4H, H-5, H-3′, H-5′, benzyl CH), 3.73 (t, J=9.6 Hz, 1H, H-4′), 3.56 (dt, J=9.9, 3.0 Hz, 1H, H-2′), 3.34 (dd, J=10.9, 2.1 Hz, 1H, H-6′a), 2.95 (dd, J=11.0, 1.9 Hz, 1H, H-6′b), 2.82 (ddd, J=12.5, 8.3, 6.3 Hz, 1H, butyl CH(H)O), 2.72 (dt, J=12.5, 7.6 Hz, 1H, butyl CH(H)O), 1.61 (m, 2H, lipid), 1.25 (overlapping signals, 28H, lipid), 0.89 (m, 3H, lipid); 13C NMR (126 MHz, CDCl3) δ 166.1, 166.0, 165.3 (each C=O), 138.8 (C), 138.6 (C), 138.0 (C), 137.9 (C), 133.3 (C), 133.2 (C), 133.1 (C), 130.0 (CH), 129.7 (CH), 129.7 (CH), 129.5 (CH), 129.2 (CH), 128.6 (CH), 128.5 (CH), 128.4 (CH), 128.4 (CH), 128.4 (CH), 128.4 (CH), 128.3 (CH), 128.3 (CH), 128.3 (CH), 128.3 (CH), 128.2 (CH), 128.2 (CH), 128.1 (CH), 128.0 (CH), 127.9 (CH), 127.9 (CH), 127.7 (CH), 127.7 (CH), 127.6 (CH), 127.5 (CH), 127.5 (CH), 127.3 (CH), 100.2 (C-1′), 83.9 (C-1), 81.9 (C-3′), 79.7 (C-2′), 77.5 (C-4′), 76.5 (C-5), 75.7 (C-4), 75.6 (benzyl CH2), 75.0 (C-3), 74.7 (benzyl CH2), 73.9 (benzyl CH2), 73.3 (benzyl CH2), 71.5 (C-5′), 68.2 (C-2), 67.7 (C-6′), 62.7 (C-6), 29.8 (O-CH2), 29.8, 29.7, 29.7, 29.7, 29.6, 29.6, 29.5, 29.5, 29.4, 29.2, 28.9, 26.9, 22.7, 14.1 (all lipid carbons). ESI-HRMS calcd for C78H92O13SNa 1286.6602, found m/z 1286.6595 [M+Na]+. Selected 1H-NMR data for β-glucopyranoside: δ 5.90 (t, J=10.1 Hz, H-2), 5.59 (dd, J=10.1, 3.2 Hz, H-3).

1-Heptadecanyl 4-O-(α-D-glucopyranosyl)-1-thio-β-D-galactopyranoside (3)

Compound 27 (30 mg, 0.024 mmol) was reacted as described in the general procedures for debenzoylation and O-debenzylation successively. Chromatography (CH2Cl2-MeOH 9:1) gave 3 (11 mg, 77% over two steps, 64α:36β mixture of glucopyranosides, white solid); 1H NMR (600 MHz, CDCl3) δ 4.86 (d, J=3.9 Hz, 1H, H-1′), 4.28 (d, J=9.3 Hz, 1H, H-1), 3.99 (overlapping signals, 2H, H-2, H-4), 3.91 (ddd, J=12.6, 5.7, 2.5 Hz, 1H, H-5′), 3.80 (m, 1H, H-6a), 3.75 (overlapping signals, 2H, H-6′a, H-6′b), 3.70 (t, J=6.5 Hz, 1H, H-5), 3.58 (overlapping signals, 3H, H-3, H-3′, H-6b), 3.37 (dd, J=9.9, 3.8 Hz, 1H, H-2′), 3.20 (t, J=9.5 Hz, 1H, H-4′), 3.10 (m, 2H, O-CH2-lipid), 1.73 (m, 2H, lipid), 1.45 (overlapping signals, 6H, lipid), 1.18 (overlapping signals, 28H, lipid), 0.79 (t, J=7.0 Hz, 3H). 13C NMR (151 MHz, CDCl3) δ 100.4 (C-1′), 89.5 (C-1), 78.7 (C-5), 77.6 (C-4), 73.5 (C-3), 72.7 (C-3′), 72.3 (C-5′), 71.8 (C-2), 69.9 (C-4′), 65.9 (C-2), 60.9 (C-6′), 59.2 (C-6), 50.3 (S-CH2), 31.1, 28.9, 28.8, 28.8, 28.7, 28.6, 28.5, 28.3, 28.0, 27.8, 27.7, 24.1, 21.9, 20.3, 12.9 (all lipid carbons). Selected 1H-NMR data for β-glucopyranoside: δ 4.51 (d, J=7.8 Hz, H-1′), 4.30 (d, J=9.5 Hz, H-1).

17-Tritriacontyl 4-O-(4,6-di-O-benzyl-α-D-glucopyranosyl)-2,3,6-tri-O-benzoyl-1-thio-α-D-galactopyranoside (26α)

Compound 25β (50 mg, 0.033) was dissolved in CH2Cl2 (0.75 mL). TiCl4 (1 M in CH2Cl2, 90 μL, 2.5 equiv.) was then added, and the mixture was stirred at room temperature for 15 h. It was then diluted in EtOAc and washed with a saturated solution of NH4Cl. The aqueous phase was extracted with EtOAc, and the combined organic phases were washed with satd NaHCO3, brine, and then dried over Na2SO4 and filtered. After removal of the solvent under reduced pressure, chromatography (cyclohexane-EtOAc, 6:1) gave 26α (18 mg, 41%) as a white solid. 1H NMR (500 MHz, CDCl3) δ 8.05 (overlapping signals, 3H, aromatic H), 7.94 (overlapping signals, 4H, aromatic H), 7.58 (m, 1H, aromatic H), 7.47 (overlapping signals, 5H, aromatic H), 7.29 (overlapping signals, 13H, aromatic H), 7.15 (m 2H, aromatic H), 7.10 (overlapping signals, 3H, aromatic H), 5.94 (d, J=6.0 Hz, 1H, H-1), 5.72 (dd, J=11.1, 5.9 Hz, 1H, H-2), 5.57 (d, J=11.0 Hz, 1H, H-3), 5.02 (d, J=3.9 Hz, 1H, H-1′), 4.79 (m, 1H, H-5), 4.67 (overlapping signals, 3H, H-6a, H-6b, benzyl CH), 4.54 (d, J=2.8 Hz, 1H, H-4), 4.42 (d, J=11.5 Hz, 1H, benzyl CH), 4.35 (d, J=12.1 Hz, 1H, benzyl CH), 4.03 (m, 1H, benzyl CH), 3.97 (overlapping signals, 2H, H-3′, H-5′), 3.60 (s, 1H, H-2′), 3.55 (t, J=9.8 Hz, 1H, H-4′), 2.99 (d, J=11.3 Hz, 1H, H-6′a), 2.74 (m, 1H, S-CH), 2.63 (d, J=11.2 Hz, 1H, H-6′b), 2.51 (s, 1H, OH), 2.47 (d, J=8.1 Hz, 1H, OH), 1.63 (overlapping signals, 4H, lipid), 1.23 (overlapping signals, 54H, lipid), 0.88 (t, J=6.9 Hz, 6H, lipid). 13C NMR (126 MHz, CDCl3) δ 165.9, 165.8, 165.6 (each C=O), 138.4 (C), 137.5 (C), 133.3 (C), 133.3 (C), 133.3 (C), 129.9 (CH), 129.8 (CH), 129.8 (CH), 129.5 (CH), 129.3 (CH), 129.2 (CH), 128.5 (CH), 128.4 (CH), 128.4 (CH), 128.2 (CH), 128.0 (CH), 127.9 (CH), 127.7 (CH), 127.7 (CH), 99.8 (C-1′), 82.1 (C-1), 76.9 (C-4′), 74.7 (C-4), 74.4 (benzyl CH2), 74.3 (C-3′), 73.4 (benzyl CH2), 72.6 (C-2), 71.0 (C-3), 70.9 (C-5′), 68.7 (C-2, C-5 (overlapping signals)), 67.3 (C-6′), 62.0 (C-6), 45.4 (CH-S), 35.0, 34.6, 31.9, 29.7, 29.7, 29.7, 29.6, 29.6, 29.6, 29.4, 27.1, 26.3, 22.7, 14.1 (all lipid carbons). IR (film) cm−1: 3459, 2923, 2853, 1724, 1452, 1269, 1095, 1069, 1046, 1026, 708. ESI-HRMS calcd for C80H112O13SNa 1335.7721, found m/z 1335.7678 [M+Na]+.

Isolation and characterization of natural and synthetic simplexide 1c

Simplexide was isolated from Plakortis simplex at the Department of Pharmacy, University of Naples Federico II as described previously [1], [31]. The structure and purity of the isolated glycolipids was confirmed by 1H-NMR spectroscopy and mass spectrometry as described earlier [1]. As with most glycolipids from sponges, each of the obtained glycolipids was an inseparable mixture of homologues, which are identical in the polar part of the molecule but slightly different in the length and branching of the lipophilic chains. The relative amounts of branched and unbranched chains were evaluated by 1H-NMR, while their length was determined by mass spectrometry. Both resulted to be very close to those reported earlier [15], [32]. Stock solutions of glycolipid were prepared and stored in MeOH at a concentration of 3 mM unless otherwise specified and diluted to working concentration in RPMI immediately before the experiment.

Synthetic simplexide 1c was prepared as reported earlier from 1-octadecanol, 1-bromoheptadecane, methyl α-d-glucopyranose, and methyl β-d-galactopyranose and final purification of 1c was achieved using reverse-phase HPLC using an RP-18 column and MeOH as eluent as previously described.

Cell isolation and purification

The study protocol involving the use of human blood cells was approved by the Ethical Committee of the University of Naples Federico II (N°301/2018), and written informed consent was obtained from blood donors in according to the principles expressed in the Declaration of Helsinki. Peripheral blood mononuclear cells (PBMCs) were purified from buffy coats of healthy donors (HCV, HBsAg, HIV) obtained from the Leukapheresis Unit. PBMCs were obtained by dextran stratification and centrifugation over Histopaque®-1077.

Cell incubations

PBMCs were incubated (37°C, 24 h) in RMPI supplemented with 2 mM L-glutamine and 1% antibiotic solution and various concentrations of synthesised compounds (from 1a to 6) (3–30 μg/mL), or natural and synthetic simplexide (10 μg/mL). All compounds were routinely checked for LPS contamination (Limulus amebocyte test, MP Biomedicals) and discarded if the LPS concentration was above the detection limit of the assay (0.125 EU/mL). The experiments to compare the effects of synthetic and natural compounds were done in polystyrene plates coated with various concentrations of glycolipids dissolved in methanol. Solvent was dried under nitrogen immediately before the addiction of cells for 24 h at 37°C. At the end of the experiment, the supernatant was removed, centrifuged (1000 g, 4°C, 5 min) and stored at –80°C for subsequent determination of IL-6, CXCL-8 and TNF-α release.

Cytokine assay

The release of IL-6, CXCL-8 and TNF-α in the culture supernatant was measured in duplicate determinations by commercially available ELISA kits (R&D, Minneapolis, MN, USA) according to the manufacturer’s instructions. The results were normalized as pg/mL of proteins for 106 cells.

Statistical analysis

Data were analyzed with the GraphPad Prism 5 software package. The data are expressed as mean values±SE of the indicated number of experiments. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Dunnett’s test (when comparison was made against a control) or Bonferroni’s test (when comparison was made between each pair of groups) A p value of 0.05 or lower was considered to be significant.


Article note

A collection of invited papers based on presentations at the 29th International Carbohydrate Symposium (ICS-29), held in the University of Lisbon, Portugal, 14–19 July 2018.


Award Identifier / Grant number: 12/IA/1398

Award Identifier / Grant number: 14/SP/2710

Funding statement: The authors thank Alfonso Mangoni for kindly providing natural and synthetic simplexide 1c and also the reviewers for critical comments. This work was supported in part by grants from Regione Campania CISI-Lab Project, CRèME Project, and the TIMING Project. This publication has partly emanated from research supported by Science Foundation Ireland (SFI, grant number 12/IA/1398) and was co-funded under the European Regional Development Fund, Funder Id: http://dx.doi.org/10.13039/501100008530, under Grant Number 14/SP/2710.

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Supplementary Material

The online version of this article offers supplementary material (https://doi.org/10.1515/pac-2019-0218).


Published Online: 2019-07-12
Published in Print: 2019-07-26

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