HETEROCYCLES

Paper | Special issue | Vol. 84, No. 2, 2012, pp. 1067-1079
Received, 19th July, 2011, Accepted, 29th August, 2011, Published online, 2nd September, 2011.
DOI: 10.3987/COM-11-S(P)84

■ Synthesis of Novel Oligosaccharides Based on 1,4-Dioxanyloxy 3-Oxasugars

Margaret Morozova, Sonali Wickremasinghe, and Mark A. Rizzacasa*

School of Chemistry, The Bio21 Institute, The University of Melbourne, Building 102, 30 Flemington Road
Melbourne, Victoria 3010, Australia

Abstract

The synthesis of a new family of di and tri-3-oxaoligosaccharides based on the 1,4-dioxanyloxy or 3-oxapseudosugar moiety is described. The approach involved the glycosylation of trichloroacetamidate donors with acceptor alcohols to provide 3-oxadi- and trisaccharides. In all cases, the glycosylation was highly stereoselective providing the 1α anomers exclusively.

INTRODUCTION
Uncommon sugars are frequently found in secondary metabolites and these moieties often play crucial roles in determining the biological activities of these compounds.1 The potent anticancer metabolites silvestrol (1) and episilvestrol (2) (Figure 1) were isolated from several species of Aglaia2,3 and contain a common cyclopenta[b]benzofuran with five contiguous stereogenic centres as well as a novel 1,4-dioxanyloxy or 3-oxapseudosugar4 substituent (highlighted). More recently, two other isomers, 2”’-episilvestrol (3) and 2”’,5”’-diepisilvestrol (4) were isolated from Aglaia folveolata Pannell (Meliaceae).5 Both silvestrol (1) and episilvestrol (2) have potency against lung, breast, and prostate cancer cell lines in vitro, with LC50 in the nanomolar range.2,3

Silvestrol was also found to be active against the iv P388 murine leukemia model and possessed significant preclinical activity against B-cell malignancies with selectivity against B cell.6 Interestingly, other Aglaia metabolites, containing only the parent cyclopenta[b]benzofuran core, such as aglafoline (methyl rocaglate) and rocaglamide (Figure 1), have been found to be significantly less active than 1, suggesting that the presence of the novel dioxanyloxy group is critical to the activity of 1 and 2. So far, two total syntheses of silvestrol (1) have been reported.7,8
We have suggested a biosynthetic origin of the dioxylanoxy 3-oxapseudosugar found in episilvestrol (2) which begins with a O-aryl-D-glucopyranoside I (Scheme 1).4 Selective oxidative cleavage of the 2,3 diol in I gives the dialdehyde II which would rapidly undergo intramolecular acetal formation to give a stable acetal III. Reduction of the aldehyde and methylation of the acetal would then give the 1,4-dioxylanoxy group found in episilvestrol 2. An inversion at C5”’ would then give silvestrol (1).

Oligosaccharides are composed of two to ten monosaccharide residues linked together by glycoside bonds. They act as recognition sites for bacteria, viruses, toxins, antibodies and hormones and provide modulation of important biological processes such as cell-cell recognition and adhesion and viral or bacterial adhesion to host cells.9 Oligosaccharide mimics of glycoproteins and glycolipids have been utilized to study the structural basis of protein-carbohydrate interactions. For example, C-linked oligosaccharides have been used to show the absence of any hydrogen bond involvement by the intersaccharidic oxygen in the binding area of immunoglobulin confirming the nature of binding is identical to O-linked oligosaccharides.10 With the possibility of interesting biological applications in mind, we elected to investigate the synthesis of simple 1,6-linked-1,4-dioxylanoxy or 3-oxaoligosaccharides. Herein, we report the first stereoselective synthesis of di- and tri-3-oxasaccharides based on the 1,4-dioxanyloxy or 3-oxapseudosugar motif as found in episilvestrol (2).

RESULTS AND DISCUSSION
We envisaged that 3-oxaoligosaccharides based on the 1,4-dioxylanoxy unit could be synthesized from the protected precursor 5, utilized for our total synthesis of episilvestrol (2),7 and the C2 epimer 67b (Scheme 2). Removal of the PMB protecting group in dioxane 5 and appropriate activation would give a glycosyl donor 7 whilst desilylation of dioxane 6 would provide the 2β11 acceptor 8.

The stereochemistry of the glycosylation should follow from the analysis shown in Scheme 2 whereby the oxonium ion generated from 7 reacts with alcohols such as 8 on the same least hindered face in both half chair conformers A and B, however A may be the preferred conformation as the R group is in a psuedoequatorial orientation and the methoxy group is in the anomerically preferred axial position. This would afford a 1’,2’-diaxial linked or 1’α, 2’β 3-oxadisaccharide 9 which could be then be converted into a glycosyl acceptor by silyl group removal.

The synthesis of the donor and acceptor common intermediates 5 and 6 is shown in Scheme 3. The route follows our published synthesis of 57b and begins with Koenigs-Knorr glycosylation12 of bromide 10 with PMB-OH followed by deacetylation and benzylidene formation to afford 11. Selective benzylidene cleavage was achieved with BH3•THF in the presence of Cu(OTf)213 to give the alcohol 12 in 83% yield. The diol in compound 12 was then oxidatively cleaved with NaIO414 to give 1,4-dioxane aldehyde 13 as a ~3:1 mixture of anomers. Reduction of 13 with DiBALH afforded the diol 14 which was selectively silylated to yield the TBS ether 15. Methylation of the lactols 15 was achieved with NaHMDS followed by treatment with MeOTf15 to afford the 2β ketal 5 and 2α ketal 6 in a 2.2:1 ratio in 89% overall yield. This ratio was lower than our reported procedure7b using nBuLi as base but supplied both methyl ketal anomers 5 and 6 in high overall yield. The 2β isomer 5 displayed singlet at 4.29 ppm for H2 in its 1H NMR spectrum while in the 2α methylketal 6, the corresponding signal resonates as a doublet at 4.39 ppm (J = 1.8 Hz).

With the two glycosylation precursors in hand we next trialed several standard glycosylation methods. The procedure we utilized for the synthesis of both 1 and 2 involved a modified Mitsunobu coupling which was not applicable in this case.7 Amongst the other glycosylation protocols available,16 we found the trichloroacetamidate method pioneered by Schmidt17 to be the most adaptable and the synthesis of the first example of a 1’α, 2α, 2’β oxadisaccharide 9 is shown in Scheme 4. Desilylation of 6 gave the glycosyl acceptor 8 in high yield whilst the glycosyl donor 16 was prepared from acetal 5 by oxidative deprotection of the PMB ether and conversion of the intermediate lactols into the trichloroacetamidate 16.18 Glycosylation of acceptor 8 with the donor 16 was achieved using TMSOTf as the promoter to afford disaccharide 9 as a single α’-anomer, in a low yield (36%) based on the donor 16. When the reaction was conducted with BF3•OEt2 as the Lewis acid, the yield increased to 62%. The presence of powdered 4Å molecular sieves is critical for the success of the reaction. In the absence of sieves, considerable hydrolysis of the trichloroacetamidate was observed. Removal of the TBS group gave alcohol 17 followed by hydrogenolysis of the benzyl ethers provided triol 18. Confirmation of the stereochemistry of the new glycosyl linkage arose from NMR analysis of 18. The coupling constant for H1-H2 was 1.8 Hz, indicative of an eq-ax relationship whilst the coupling between H1’-H2’ was closer to 0 Hz, showing these protons are in a eq-eq orientation. This result is in accord with our prediction that axial approach of the donor on the intermediate oxonium ion to form the 1’α-anomer is preferred.
The synthesis of the alternative 1’α, 2β, 2’β-isomer is shown in Scheme 5. The 2β isomer 5 was desilylated to give acceptor 19 in good yield. Glycosylation using the donor 16 then afforded the 1’α-anomer 20 in good yield as the only detectable diastereoisomer. Removal of the TBS group afforded the alcohol 21 and debenzylation then gave triol 22. Again, 1H NMR couplings supported the assigned 1’α, 2β, 2’β stereochemistry of the glycosylation product 20 (3JH1,H2 = 3JH1’,H2’ = 0 Hz).

To test the effect of the C2 stereochemistry on the glycosylation reaction, the 2α glycosyl donor was synthesized from compound 5 (Scheme 6). Removal of the PMB group gave the lactols as a mixture of anomers which were converted in the trichloroacetamidate 23 under the standard conditions. Glycosylation using the acceptor 8 afforded the 1’α, 2α, 2’α 3-oxadisaccharide 24, again as the sole diastereoisomer. The coupling constants for H1-H2 and H1’-H2’ were also consistent with the assigned stereochemistry.

We next explored the synthesis of an example of a 3-oxatrisaccharide as shown in Scheme 7. Glycosylation of 17 with trichloroacetamidate 16 afforded the 3-oxatrisaccharide 25 as a single anomer. Removal of the TBS group gave the alcohol 26 in good yield. Again, the selectivity was high for the glycosylation reaction providing the 1”α anomer as confirmed by the H1"-H2" coupling constant of 0 Hz.

CONCLUSION
The synthesis of a new family of oligosaccharides has been achieved based on the 1,4-dioxanyloxy or 3-oxasugar moiety. The glycosylation reactions were based on the Schmidt trichloroacetamidate method and were highly stereoselective with only the 1α isomers formed in all cases, regardless of the C2 stereochemistry. The stereochemical outcomes could easily be determined by 1H NMR analysis. Examples of 3-oxadisaccharides and a trisaccharide were synthesized and this method could easily provide more extended 3-oxaoligosaccharides. Studies directed towards the biological activities of these novel sugars are currently underway.

EXPERIMENTAL

General
Proton nuclear magnetic resonance spectra (1H NMR, 400 MHz and 500 MHz) and proton decoupled carbon nuclear magnetic resonance spectra (13C NMR, 100 MHz and 125 MHz) were obtained in deuterochloroform with residual chloroform as internal standard. Chemical shifts are followed by multiplicity, coupling constant(s) (J, Hz), integration and assignments where possible. Optical rotations were recorded in a 10 cm microcell for a 1 mL solution and units are deg.cm2g-1. Flash chromatography was carried out on silica gel 60. Analytical thin layer chromatography (t.l.c.) was conducted on aluminium-backed 2mm thick silica gel 60 GF254 and chromatograms were visualized with 20% w/w phosphomolybdic acid in ethanol. High resolution mass spectra (HRMS) were obtained by ionizing samples via electron spray ionization (ESI). Anhydrous THF and CH2Cl2 were used from a solvent cartridge system. Dry methanol was distilled from magnesium methoxide. All other solvents were purified by standard methods. Petrol used refers to petroleum ether 40-60 ºC boiling range. All other commercially available reagents were used as received. The usual workup refers to extraction with particular solvent (3x), washing with water and brine, drying with MgSO4 and concentrating under reduced pressure.

Methyl ketals 5 and 6
To a solution of the lactols 157b (623.6 mg, 1.23 mmol) in THF (16 mL) at –78 ºC, was added a solution of LiHMDS (1.0M, 1.6 mmol) drop wise followed by MeOTf (234 µL, 1.82 mmol). The solution was stirred for 20 mins at -78 ºC and sat. aq. NaHCO3 and Et2O were added. The usual workup with Et2O and purification by flash chromatography with 10% EtOAc/petrol as eluent gave the 2β methyl ketal 57b (206 mg, 61%) as a colourless oil. 1H NMR (500 MHz) δ 7.19-7.27 (m, 7H), 6.80 (d, J = 8.7 Hz, 2H), 4.57 (ABq, J = 11.6 Hz, 2H), 4.55 (ABq, J = 11.2 Hz, 2H), 4.47 (s, 1H), 4.29 (s, 1H), 4.20-4.25 (m, 1H), 3.82 (dd, J = 11, 3.2 Hz, 1H), 3.77 (t, J = 11.2 Hz, 1H), 3.73 (s, 3H), 3.70 (dd, J = 11.2, 5.2 Hz, 1H), 3.63 (dd, J = 11.2, 2.8 Hz, 1H), 3.39-3.42 (m, 1H), 3.31 (s, 3H), 0.84 (s, 9H), 0.011 (s, 3H), 0.003 (s, 3H). Further elution gave the 2α methyl ketal 67b (96.6 mg, 28%) as a colourless oil. 1H NMR (500 MHz) δ 7.28-7.34 (m, 7H), 6.83 (d, J = 9 Hz, 2H), 4.74 (ABq, J = 11.8 Hz, 2H), 4.65 (ABq, J = 12 Hz, 2H), 4.56 (d, J = 1.8 Hz, 1H), 4.39 (d, J = 1.8 Hz, 1H), 4.14-4.18 (m, 1H), 4.09 (dd, J = 12, 2.9 Hz, 1H), 3.78 (s, 3H), 3.75 (dd, J = 11, 4.1 Hz, 1H), 3.67 (dd, J = 11, 5.3 Hz, 1H), 3.66 (t, J = 10 Hz, 1H), 3.50 (s, 3H), 3.40-3.46 (m, 1H), 0.89 (s, 9H), 0.065 (s, 3H), 0.061 (s, 3H).

2α Acceptor 8
The methyl ketal 6 (305.8 mg, 0.605 mmol) was dissolved in THF (4 mL) and the solution was cooled to 0 ˚C. TBAF (560 mg, 1.78 mmol) was then added and the solution was stirred for 1.5 h. The reaction was quenched with 0.2M citric acid. The usual workup with CH2Cl2 and purification by flash chromatography (40% EtOAc/petrol) gave the glycosyl acceptor 8 (304 mg, 90%) as a pale yellow oil: [α]24D –42.8 (c 5.01, CH2Cl2); IR (film) υmax: 3507, 2930, 1612, 1514, 1458, 1303, 1246, 1214, 1173, 1097, 1056; 1H NMR (500 MHz) δ 7.28-7.34 (m, 7H), 6.85 (d, J = 8.4 Hz, 2H), 4.67 (ABq, J = 11.2 Hz, 2H), 4.59 (ABq, J = 12 Hz, 2H), 4.58 (s, 1H), 4.39 (d, J = 1.8 Hz, 1H), 4.14-4.19 (m, 1H), 4.13 (dd, J = 11.5, 2.9 Hz, 1H), 3.78 (s, 3H), 3.73-3.76 (m, 1H), 3.64-3.69 (m, 1H), 3.59 (t, J = 10 Hz, 1H), 3.50 (s, 3H), 3.39-3.42 (m, 1H); 13C NMR (125 MHz) δ 159.4, 137.8, 130.0, 128.6, 128.0, 113.8, 99.2, 93.3, 78.7, 77.4, 77.2, 76.9, 72.5, 70.0, 67.3, 66.4, 56.9, 55.3; HRMS (ESI) calc. for C22H28O7 [M+Na]+: 427.1727; found 427.1727.

2β Donor 16
To a solution of the methyl ketal 5 (60 mg, 0.12 mmol) in CH2Cl2 (5 mL) and pH buffer (0.4 mL) was added DDQ (50 mg) at 0 ºC and the reaction mixture stirred at rt for 17 h. The mixture was filtered through celite and the filtrate was concentrated. The crude residue was purified by flash chromatography with 15% EtOAc/petrol as eluent to give the mixture of lactols (34.6 mg, 75%) as a colourless oil. To a solution of lactols (29.6 mg, 0.07 mmol) in CH2Cl2 (0.4 mL), was added DBU (2.2 µL, 0.015 mmol) and trichloroacetonitrile (33.7 µL, 0.34 mmol) at 0 ˚C under argon. The solution was stirred at 0 ˚C over 3 h and most of the solvent was removed under reduced pressure. The residue was purified by flash chromatography (1% NEt3, 10% EtOAc/petrol) to give the trichloroacetamidate 16 as a mixture of α and β isomers (20:1 by 1H-NMR) (36.1 mg, 91%), as a pale yellow oil: [α]25D –45.8o (c 0.80, CH2Cl­); IR (film) υmax: 2929, 2857, 1671, 1463, 1257, 1173, 1069, 970, 925, 836, 798, 779, 735; 1H NMR (500 MHz) δ 8.57 (s,1H, minor), 8.55 (s, 1H, major), 7.32-7.34 (m, 5H), 5.97 (s, 1H, major), 5.90 (d, J = 1.79 Hz, 1H, minor), 4.67 (ABq, J = 11.5 Hz, 4H, major and minor), 4.70 (d, J = 1.79 Hz, 1H, minor), 4.55 (s, 1H, major), 4.35-4.39 (m, 2H, major and minor), 4.01 (t, J = 11.5 Hz, 2H, major and minor), 3.66 (dd, J = 10.9 Hz, 5.4 Hz, 2H, major and minor), 3.71 (m, 2H, major and minor), 3.70 (dd, J = 10.5 Hz, 5.4 Hz, 2H, major and minor), 3.47 (s, 3H, minor), 0.87 (s, 18H, major and minor), 3.44 (s, 3H, major), 0.029 (d, 12H, major and minor); 13C NMR (125 MHz) δ 128.4, 128.0, 127.8, 94.3, 93.7, 79.7, 73.2, 68.7, 62.5, 59.5, 26.1, 18.4, -5.0s; HRMS (ESI) calc. for C22H34Cl3N1O6Si [M+Na]+: 564.11132; found 564.11132.

1’α, 2α, 2'β’-L-3-Oxadisaccharide 9
BF3•OEt2 (130 µL, 0.0120 mmol, 0.15M in CH2Cl2) was added to a stirred solution of trichloroacetimidate donor 16 (130 mg, 0.239 mmol), alcohol acceptor 8 (98.2 mg, 0.243 mmol) and freshly activated powdered 4Å molecular sieves (90 mg) in dry CH2Cl2 (5 mL) at -50 ˚C under a N2 atmosphere. The mixture was stirred for 4 h, quenched with sat. aq. NaHCO3 (2 mL) and filtered through a layer of celite. Usual workup with CH2Cl2 and purification by flash chromatography (20-30% EtOAc/petrol) afforded the disaccharide 9 (116 mg, 62%) as a colourless oil. [α]25D –60.7 (c 0.58, CH2Cl2); IR (film) υmax: 2928, 1613, 1514, 1455, 1327, 1249, 12216, 1249, 1216, 1159, 1114, 1064; 1H NMR (500 MHz) δ 7.28-7.35 (m, 12), 6.83 (d, J = 8.7 Hz, 2H), 4.71-4.74 (m, 3H), 4.56-4.60 (m, 5H), 4.38 (d, J = 4.38 Hz, 1H), 4.36 (s, 1H), 4.10-4.22 (m, 4H), 3.78-3.87 (m, 2H), 3.77 (s, 3H), 3.71 (dd, J = 10.9, 5.4 Hz, 1H), 3.65 (dd, J = 11.3, 3.3 Hz, 1H), 3.57-3.65 (m, 2H), 3.52 (m, 1H), 3.49 (s, 3H), 3.46 (m, 1H), 3.39 (s, 3H), 0.89 (s, 9H), 0.043 (s, 6H); 13C NMR (125 MHz) δ 138.6, 138.1, 130.0, 128.5, 128.4, 128.1, 127.9, 127.7, 113.8, 99.3, 96.0, 95.7, 93.4, 80.1, 77.9, s72.8, 68.9, 67.4, 66.2, 66.1, 62.7, 60.1, 56.9, 55.3, 54.9, 29.8 , 26.0, 18.4; HRMS (ESI) calc. for C42H60O12Si [M+Na]+: 807.3795; found 807.3795.

1’α, 2α, 2'β 3-Oxadisaccharide acceptor 17
TBAF (264 mg, 0.8 mmol) was added to a solution of the disaccharide 9 (220 mg, 0.28 mmol) in THF (2 mL) at 0 ˚C. The reaction was quenched with 0.2M citric acid after 4 h. The Usual workup with CH2Cl2 and purification by flash chromatography (50% EtOAc/petrol) yielded the 3-oxadisaccharide alcohol 17 (176 mg, 94%) as a pale yellow oil. [α]25D –78.3 (c 1.035, CH2Cl2); IR (film) υmax: 2925, 1514, 1455, 1248, 1159, 1117, 1064; 1H NMR (500 MHz) δ 7.28-7.35 (m, 12H), 6.83 (d, J = 8.7 Hz, 2H), 4.72 (ABq, J = 11.5Hz, 2H), 4.54-4.62 (m, 6H), 4.38 (d, J = 4.38 Hz, 1H), 4.36 (s, 1H), 4.10-4.22 (m, 4H), 3.78-3.87 (m, 3H), 3.78 (s, 3H), 3.71 (dd, J = 10.9, 5.4 Hz, 1H), 3.66 (dd, J = 11.3, 3.3 Hz, 1H), 3.58-3.63 (m, 2H), 3.58-3.63 (m, 2H), 3.51-3.53 (m, 1H), 3.49 (s, 3H), 3.42-3.44 (m, 1H), 3.41 (s, 3H); 13C NMR (125 MHz) δ 159.0, 138.1, 137.9, 130.1, 129.6, 128.7, 128.6, 128.2, 128.17, 128.11, 128.0, 113.9, 99.4, 95.9, 95.7, 93.7, 79.1, 77.7, 73.0, 72.4, 69.2, 67.2, 66.5, 66.3, 65.9, 60.6, 60.4, 57.0, 55.4, 55.0; HRMS (ESI) calc. for C36H46O12Si [M+Na]+: 693.2881; found 693.2882.

1’α, 2α, 2'β Triol 18
To a solution of the oxadisaccharide 17 (58.2 mg, 0.0741 mmol) in MeOH (3 mL) was added Pd(OH)2 (10.9 mg, 77.8 µmol) and the mixture was stirred under a H2 atmosphere for 18 h. The mixture was filtered through celite, washed with EtOAc and concentrated to give the triol 18 as a pale yellow oil (42 mg, 84%). [α]25D –156.9 (c 0.2, CH2Cl2); IR (film) υmax: 3465, 2930, 1612, 1515, 1457, 1249, 1159, 1115, 1060 cm-1. 1H NMR (500 MHz) δ 7.31 (d, J = 8.7 Hz, 2H), 6.90 (d, J = 8.7 Hz, 2H), 4.70-4.65 (m, 3H), 4.51 (s, 1H), 4.41 (d, J = 1.8 Hz, 1H), 4.39 (s, 1H), 4.11-4.17 (m, 2H), 4.08 (dd, J = 11.6 Hz, 2.9 Hz, 1H), 3.91-3.95 (m, 3H), 3.89 (t, J = 11 Hz, 1H), 3.80 (s, 3H), 3.64-3.73 (m, 4H), 3.59 (dd, J = 10.7, 3.0 Hz, 1H), 3.53-3.56 (m, 2H), 3.51 (s, 3H), 3.43 (s, 3H); 13C (125 MHz) δ 159.7, 129.8, 113.8, 99.1, 95.8, 93.8, 71.3, 70.5, 69.6, 68.7, 67.1, 66.2, 62.0, 56.9, 56.8, 55.3, 55.0, 54.9; HRMS (ESI) calc. for C22H34O12 [M+Na]+: 513.1943; found 513.1943.

2β Acceptor 19
The methyl ketal 5 (78.6 mg, 0.155 mmol) was dissolved in THF (1 mL) and the solution was cooled to 0 ˚C. TBAF (143.5 mg, 0.46 mmol) was then added and the solution was stirred for 1.5 h. The reaction was quenched with 0.2M citric acid. The usual workup with CH2Cl2 and purification by flash chromatography (40% EtOAc/petrol) gave the glycosyl acceptor 19 (56.1 mg, 91%); [α]24D –124.9 (c 0.8750, CH2Cl2); IR (film) υmax: 3503, 2924, 2837, 1612, 1586, 1514, 1454, 1400, 1303, 1246, 1211, 1196, 1175, 1175, 1154, 1108, 1027, 1058, 957, 892, 878, 851, 820, 738, 698; 1H NMR (500 MHz) δ 7.28-7.34 (m, 7H), 6.85 (d, J = 8.4 Hz, 2H), 4.73 (ABq, J = 11.5 Hz, 2H), 4.66 (ABq, J = 11.5 Hz, 2H), 4.56 (s, 1H), 4.56 (s, 1H), 4.32-4.28 (m, 1H), 4.13 (dd, J = 11.5, 2.9 Hz, 1H), 3.79 (s, 3H), 3.73-3.76 (m, 1H), 3.84-3.69 (m, 1H), 3.59 (t, J = 10 Hz, 1H), 3.39 (s, 3H), 3.47-3.42 (m, 1H); 13C NMR (125 MHz) δ 159.7, 138.0, 130.3, 129.4, 128.9, 128.4, 128.3, 114.2, 96.1, 94.3, 94. 3, 79.3, 72.7, 68.7, 66.0, 60.9, 60.8, 55.6, 55.2; HRMS (ESI) calc. for C22H28O7 [M+Na]+: 427.1727; found: 427.1727.

1’α, 2β, 2'β 3-Oxadisaccharide 20
BF3˙OEt2 (400 µL, 0.060 mmol, 0.15M in CH2Cl2) was added to a stirred solution of trichloroacetamidate donor 16 (57.2 mg, 0.12 mmol), alcohol acceptor 19 (56.3 mg, 0.13 mmol) and freshly activated powdered 4Å molecular sieves (90 mg) in dry CH2Cl2 (2 mL) at -60 ˚C under a Ar atmosphere. The mixture was stirred for 8 h, quenched with saturated NaHCO3 (0.5 mL) and filtered through a layer of celite. Usual workup with CH2Cl2 and purification by flash chromatography (20-30% EtOAc/petrol) afforded the disaccharide 20 (41.4 mg, 51%) as a colourless oil. [α]25D –89.0 (c. 1.85, CH2Cl2); IR (film) υmax: 2930, 1732, 1612, 1515, 1455, 1250, 1157, 1110, 1064; 1H NMR (500 MHz) δ 7.26-7.35 (m, 12H), 6.83 (d, J = 8.5 Hz, 2H), 4.75 (dd,J = 5.5Hz, 11.5 Hz, 2H), 4.73(s, 1H), 4.63 (s, 1H), 4.57 (dd, J = 5Hz, 11.5 Hz, 2H), 4.56 (s, 1H), 4.48 (d, J = 11.5 Hz), 4.36 (s, 2H), 4.33-4.27 (m, 1H), 4.22-4.18 (m, 1H), 3.78 (s, 3H), 3.71 (dd, J = 11.5, 4 Hz 1H), 3.68 (dd, J = 11.5, 2 Hz, 1H), 3.74-3.69 (m, 2H), 3.55-3.52 (m, 1H), 3.39 (s, 3H), 3.46-3.42 (m, 1H), 3.38 (s, 3H), 0.89 (s, 9H), 0.043 (s, 6H); 13C NMR (125 MHz) δ 159.7, 158.7, 138.8, 130.2, 128.7, 128.6, 128.3, 128.1, 127.9, 114.2, 96.2, 96.1, 96.1, 95.9, 94.4, 80.3, 78.8, 77.6, 77.4, 77.1, 73.1, 72.8, 68.7, 66.2, 65.7, 65.2, 62.9, 60.8, 60.4, 55.2, 55.1, 26.2, 18.6, -5.1; HRMS (ESI) calc. for C42H60O12Si [M+Na]+: 807.3746; found 807.3751.

1’α, 2β, 2'β 3-Oxadisaccharide acceptor 21
TBAF (50 mg, 0.19 mmol) was added to a solution of the disaccharide 20 (26 mg, 0.03 mmol) and THF (2 mL) at 0 ˚C. The reaction was quenched with 0.2M citric acid after 4 h. The Usual workup with CH2Cl2 and purification by flash chromatography (50% EtOAc/petrol) yielded the alcohol 21 (16 mg, 72%) as a pale yellow oil. [α]25D –75.2 (c 1.20, CH2Cl2); IR (film) υmax: 3496, 2923, 1613, 1586, 1514, 1454, 1303, 1248, 1196, 1111, 1061; 1H NMR (500 MHz): 7.34 (m, 12H), 6.86 (d, 8.5), 4.75 (dd, 4.5Hz, 12Hz, 2H), 4.61 (ABq, J = 11.5Hz, 2H), 4.59-4.55 (m, 4H), 4.50 (d, J = 11.5 Hz, 1H), 4.38 (s, 1H), 4.36 (s, 1H), 4.30-4.23 (m, 2H), 3.89 (dd, J =5.5 Hz, 11.5 Hz, 1H), 3.82-3.69 (m, 5H), 3.82 (s, 3H), 3.65 (dd, J = 3, 11.5 Hz, 1H), 3.52-3.49 (m, 1H), 3.43-3.78 (m, 1H), 3.44 (s, 3H), 3.42 (s, 3H); 13C NMR (125 MHz) δ 159.7, 138.3, 138.1, 130.2, 128.8, 128.7, 128.4, 128.3, 128.2, 114.2, 96.2, 96.1, 95.9, 94.5, 79.3, 78.5, 77.6, 77.4, 77.1, 73.05, 72.6, 68.8, 66.2, 66.1, 65.8, 60.9, 60.7, 60.6, 55.6, 55.19, 55.17; C36H46O12 [M+Na]+: 693.2881; found 693.2883.

1’α, 2β, 2'β 3-oxodisaccharide triol 22
To a solution of disaccharide 21 (12.4 mg, 0.018 mmol) in distilled MeOH (1 mL) was added Pd(OH)2 (2.5 mg, 0.018 mmol) and the mixture was stirred under a H2 atmosphere for 18 h. The mixture was filtered through celite, washed with EtOAc and concentrated and the crude product was purified via flash chromatography (EtOAc:MeOH:H2O 7:2:1) to afford the triol 22 (6.2 mg, 70%) as a colourless oil. [α]25D –103.9 (c 0.255, CH2Cl2); IR (film) υmax: 3414, 2924, 2853, 2017, 1728, 1613, 1586, 1515, 1457, 1378, 1249, 1197, 1156, 1111, 1060; 1H NMR (500 MHz) δ 7.29 (d, J = 8.5 Hz, 2H), 6.89 (d, J = 8 Hz, 2H ), 4.69 (d, J = 11.5 Hz, 1H), 4.56 (s, 1H), 4.53 (s, 1H), 4.51 (d, J = 12 Hz, 1H), 4.39 (s, 1H), 4.37 (s, 1H), 4.25-4.06 (m, 3H), 3.92-3.89 (m, 4H), 3.80 (s, 3H), 3.71-3.63 (m, 6H), 3.58-3.56 (m, 1H), 3.43 (s, 3H), 3.40 (s, 3H); 13C NMR (125 MHz) δ 160.4, 130.24, 114.3, 111.1, 96.1, 96.8, 111.1, 77.6, 77.0, 72.1, 71.2, 69.2, 67.4, 64.3, 63.5, 62.5, 60.04, 59.8, 55.6, 55.2; C22H34O12 [M+Na]+: 513.1942; found 513.1943.

2α Donor 23
To a solution of the methyl ketal 6 (120 mg, 0.24 mmol) in CH2Cl2 (5 mL) and pH buffer (0.8 mL) was added DDQ (100 mg) at 0 ºC and the reaction mixture stirred at rt for 2h. The mixture was filtered through celite and the filtrate was concentrated. The crude residue was purified by flash chromatography with 15% EtOAc/petrol as eluent to give the mixture of lactols (72 mg, 75%) as a colourless oil. To a solution of the lactols (63.8 mg, 0.16 mmol) in CH2Cl2 (2 mL), was added DBU (5 µL, 0.03 mmol) and trichloroacetonitrile (81 µL, 0.81 mmol) at 0 ˚C under argon. The solution was stirred at 0 ˚C over 2.5 h and most of the solvent was removed under reduced pressure. The residue was purified by flash chromatography (1% NEt3, 10% EtOAc/petrol) to give the trichloroacetamidate 23 as a mixture of 1α and β anomers (10:1 - 1H NMR), (77.6 mg, 89%) as a pale yellow oil. IR (film) υmax: 2954, 2928, 2856, 1734, 1669, 1497, 1471, 1455, 1463, 1388, 1361, 1332, 1316, 1284, 1254, 1197, 1216, 1173, 1073, 1028; 1H NMR (500 MHz) δ 8.55 (s, 1H, minor), 8.50 (s, 1H, major), 7.32-7.34 (m, 5H), 6.13(s, 1H, minor), 5.8 (d, J = 1.5 Hz, 1H, major), 4.67 (ABq, J = 12 Hz, 4H, major and minor), 4.59 (d, J = 1.5 Hz, 1H, minor), 4.55 (d, J = 1.5 Hz ,1H, major), 4.32-4.28 (m, 2H, minor), 4.18 (dd, J = 13 Hz, 4.5 Hz, 2H, major and minor), 4.06-4.03 (m, 12H, major), 3.92 (dd, J = 13 Hz, 4.5 Hz, 2H, major and minor), 3.80 (dd, J = 6 Hz, 11 Hz, 2H, major and minor), 3.74 (dd, J = 5.5 Hz, 11 Hz, 1H, major and minor), 3.68 (dd, J = 5.5 Hz, 11 Hz, 1H, major and minor), 3.54 (s, 3H, minor), 3.48 (s, 3H, major), 0.87 (s, 18H, major and minor), 3.44 (s, 3H, major), 0.02 & 0.03 (s, 12H, major and minor); 13C NMR (100 MHz) δ 128.7, 128.9, 127.8, 96.0, 94.5, 77.6, 73.1, 70.5, 62.5, 55.8, 30.05, 26.3; HRMS (ESI) calc. for C22H34Cl3NO6Si [M+Na]+: 564.11132; found 564.11133.

1’α, 2α, 2'α 3-Oxadisaccharide 24
BF3˙OEt2 (40 µL, 4 µmol, 0.15M in CH2Cl2) was added to a stirred solution of trichloroacetimidate donor 23 (45 mg, 80 µmol), alcohol acceptor 7 (35 mg, 90 µmol) and freshly activated powdered 4Å molecular sieves (50 mg) in dry CH2Cl2 (2 mL) at 60 ˚C under an Ar atmosphere. The mixture was stirred for 8 h, quenched with saturated NaHCO3 (0.5 mL) and filtered through a layer of celite. The usual workup with CH2Cl2 and purification by flash chromatography (50% EtOAc/petrol) afforded the 3-oxadisaccharide 21 (36 mg, 59%) as a colourless oil. [α]23D –61.6 (c 0.21, CH2Cl2); IR (film) υmax: 2927, 2855, 1613, 1586, 1514, 1497, 1454, 1388, 1359, 1328, 1303, 1248, 1215, 1172, 1099, 1060, 1028; 1H NMR (500 MHz) δ 7.34-7.27 (m, 12H), 6.85 (d, J = 9 Hz, 2H), 4.8 (d, J = 11.5 Hz, 1H), 4.72 (dd, J = 12 Hz, 6.5 Hz, 1H), 4.65 (d, J = 2 Hz, 1H), 4.59 (d, J = 1.5 Hz, 1H), 4.62-4.54 (m, 3H), 4.41 (d, J = 1.5 Hz, 1H). 4.38 (d, J = 2 Hz, 1H), 4.13 -4.05 (m, 4H), 3.78 (s, 3H), 3.79-3.61 (m, 8H), 3.51 (s, 3H), 3.48 (s, 3H), 3.48-3.45 (m, 1H), 0.90 (s, 9H), 0.03 (s, 6H); 13C NMR (125 MHz) δ 159.6,138.9, 138.7, 130.2, 129.8, 128.6, 128.4, 128.0, 128.0, 127.9, 114.1, 99.6, 99.6, 95.3, 93.6, 77.8, 76.9, 73.5, 73.3, 69.1, 68.1, 67.3, 67.1, 66.0, 63.5, 57.1, 55.6, 26.2, 18.6, -5.0; HRMS (ESI) calc. for C42H60O12Si [M+Na]+: 807.3746; found 807.3747.

1’α, 1”α, 2α, 2'β, 2'β 3-Oxatrisaccharide 25
A 0.15M solution of BF3•OEt2 in CH2Cl2 (34 µL, 0.005 mmol) was added to a stirred solution of donor 16 (20 mg, 0.03 mmol), alcohol acceptor 17 (24 mg, 0.04 mmol) and freshly activated powdered 4Å molecular sieves (90 mg) in dry CH2Cl2 (1 mL) at -60 ˚C under Ar atmosphere. The mixture was stirred for 8 h, quenched with saturated NaHCO3 (2 mL) and filtered through a layer of celite. Usual workup with CH2Cl2 and purification by flash chromatography (20-30% EtOAc/petrol) afforded the 3-oxatrisaccharide 26 (16 mg, 51%) as a colourless oil. [α]27D –53.6 (c 0.36, CH2Cl2); IR (film) υmax: 2929, 2326, 2342, 1722, 1514, 1455, 1251, 1158, 1115, 1066; 1H NMR (500 MHz) δ 7.33-7.27 (m, 17H), 6.84 (d, 12H), 4.75-4.68 (m, 3H), 4.67 (s, 1H), 4.61 (s, 2H), 4.59-4.55 (s, 6H), 4.53 (s, 2H), 4.38 (d, J = 1.5 Hz, 1H), 4.37 (s, 1H) , 4.36 (s, 1H), 4.22-4.1 (m, 4H), 3.9-3.77 (m, 4H), 3.76 (s, 3H), 3.74-3.6 (m, 5H), 3.59-3.52 (m, 3H), 3.49 (s, 3H), 3.84 (s, 6H), 0.89 (s, 9H), 0.043 (s, 6H); 13C NMR (125 MHz) δ 159, 137.5, 129.8, 127.4, 117.0, 114.0, 112.1, 85.6, 82.6, 70.4, 65.7, 55.3 54.9, 29.8, 26.0, 18.4, -5.0; HRMS (ESI) calc. for C56H78O17Si [M+Na]+: 1073.4905; found 1073.4902.

1’α, 1”α, 2β, 2’β, 2”β 3-Oxatrisaccharide alcohol 26
TBAF (8 mg, 20.0 µmol) was added to a solution of the trisaccharide 25 (7.6 mg, 7.0 µmol) in THF (1 mL) at 0 ˚C and the solution was stirred for 5 h. The reaction was quenched with 0.2M citric acid and the usual workup with CH2Cl2 followed by purification by flash chromatography (50% EtOAc/petrol) afforded trisaccharide alcohol 26 (5.1 mg, 77%); [α]25D –103.4 (c 0.73, CH2Cl2); IR (film) υmax: 2927, 1514, 1158, 1117, 1064 cm-1. 1H NMR (500 MHz) δ 7.28-7.35 (m, 17H), 6.83 (d, J = 8.8 Hz, 2H), 4.69-4.74 (m, 3H), 4.50-4.61 (m, 8H), 4.38 (d, J = 1.8 Hz, 1H), 4.36 (s, 1H), 4.31 (s, 1H), 4.12-4.24 (m, 4H), 3.78-3.87 (m, 3H), 3.76 (s, 3H), 3.50-3.75 (m, 11H), 3.49 (s, 3H), 3.39 (s, 3H), 3.34 (s, 3H), 2.17 (s, 1H); 13C NMR (125 MHz) δ 159.0, 130.0, 129.6, 128.6, 128.2, 128.0, 127.9, 120.6, 118.9, 113.8, 112.0, 109.0, 99.5, 99.4, 96.0, 95.8, 93.5, 85.4, 79.1, 78.4, 77.9, 77.4, 76.8, 72.9, 72.4, 69.8, 69.0, 67.3, 66.3, 66.1, 65.8, 60.5, 60.4, 60.3, 57.9, 54.9; HRMS (ESI) calc. for C50H64O17 [M+Na]+: 959.4045; found 959.4035.

ACKNOWLEDGEMENTS
We thank the Australian Research Council Discovery Grants Program for funding.

References

1. W. Chen, G. Zhang, L. Zhu, F. Fang, X. Cao, J. Kedenburg, J. Shen, D. Sun, and P. W. Wang, Frontiers in Modern Carbohydrate Chemistry, 2007, American Chemical Society, Chapter 2, p. 15.
2. B. M. Meurer-Grimes, J. Yu, and G. L. Vairo, U.S patent, 6710075 B2, 2004.
3. B. Y. Hwang, B.-N. Su, H. M. Chai, L. B. S. Kardono, J. J. Afriastini, S. Riswan, B. D. Santarsiero, A. D. Mesecar, R. Wild, C. R. Fairchild, G. D. Vite, W. C. Rose, N. R. Farnsworth, G. A. Cordell, J. M. Pezzuto, S. M. Swanson, and A. D. Kinghorn, J. Org. Chem., 2004, 69, 6156. CrossRef
4. M. A. Rizzacasa and M. El Sous, Tetrahedron Lett., 2005, 46, 293. CrossRef
5. L. Pan, L. B. S. Kardono, S. Riswan, H. Chai, E. J. C. Blanco, C. M. Pannell, D. D. Soejarto, T. G. McCloud, D. J. Newman, and A. D. Kinghorn, J. Nat. Prod., 2010, 73, 1873. CrossRef
6. D. M. Lucas, R. B. Edwards, G. Lozanski, D. A. West, J. D. Shin, M. A. Vargo, M. E. Davis, D. M. Rozewski, A. J. Johnson, B.-N. Su, V. M. Goettl, N. A. Heerema, T. S. Lin, A. Lehman, X. Zhang, D. Jarjoura, D. J. Newman, J. C. Byrd, A. D. Kinghorn, and M. R. Grever, Blood, 2009, 113, 4656. CrossRef
7. (a) M. El Sous, M. L. Khoo, G. Holloway, D. Owen, P. J. Scammells, and M. A. Rizzacasa, Angew. Chem. Int. Ed., 2007, 76, 7835; CrossRef (b) T. E. Adams, M. El Sous, B. C. Hawkins, S. Hirner, G. Holloway, M. L. Khoo, D. J. Owen, G. P. Savage, P. J. Scammells, and M. A. Rizzacasa, J. Am. Chem. Soc., 2009, 131, 1607. CrossRef
8. B. Gerard, R. Cencic, J. Pelletier, and J. A. Porco Jr., Angew. Chem. Int. Ed., 2007, 46, 7831. CrossRef
9. H. Osborn and T. H. Khan, Oligosaccharides: Their Synthesis and Biological Role, 2000, Oxford Chemistry Masters, p. 112.
10. J. Wang, P. Kovác, P. Sinaÿ, and C. P. J. Glaudemans, Carbohydrate Res., 1998, 308, 191. CrossRef
11. We have used the following nomenclature for the stereochemistry at both acetal carbons (C1 and C2) according to standard carbohydrate IUPAC rules. Groups cis to the C4 ring oxygen are denoted α and trans are β as shown below (see: Nomenclature of Carbohydrates Pure & Appl. Chem., 1996, 68, 1919.).
12. H. Paulsen, Angew. Chem., Int. Ed. Engl., 1982, 21, 155. CrossRef
13. C.-R. Shie, Z.-H. Tzeg, S. S. Kulkarni, B.-J. Uang, C.-Y. Hsu, and S.-C. Hung, Angew. Chem. Int. Ed., 2005, 44, 1665. CrossRef
14. T. Heidelberg and J. J. Thiem, Prakt. Chem./Chem.-Ztg., 1998, 340, 223. CrossRef
15. R. R. Schmidt, M. Reichrath, and U. Moering, Carbohydr. Chem., 1984, 3, 67. CrossRef
16. R. V. Stick and S. J. Williams in Carbohydrates: The Essential Molecules of Life, Elsevier, The Netherlands, 2009, Chapter 4, p. 133.
17. R. R. Schmidt and J. Michel, Angew. Chem., Int. Ed. Engl., 1980, 19, 731.
18. I. Ohashi, M. J. Lear, F. Yoshimura, and M. Hirama, Org. Lett., 2004, 6, 719. CrossRef