Carbohydrate Research 345 (2010) 2282–2286

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Carbohydrate Research

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Synthesis of a core disaccharide from the Streptococcus pneumoniae type23F capsular polysaccharide antigen

Somnath Dasgupta, Mark Nitz ⇑Department of Chemistry, 80 St. George Str. Toronto, ON, Canada

a r t i c l e i n f o

Article history:Received 11 June 2010Received in revised form 5 August 2010Accepted 6 August 2010Available online 10 August 2010

Keywords:PhosphoglycanStreptococcus pneumoniaeH-PhosphonatePhosphoramidite

0008-6215/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.carres.2010.08.002

⇑ Corresponding author. Tel.: +1 416 946 0640.E-mail address: mnitz@chem.utoronto.ca (M. Nitz

a b s t r a c t

The synthesis of methyl a-L-rhamnopyranosyl-(1?2)-b-D-galactopyranoside and methyl a-L-rhamnopyr-anosyl-(1?2)-3-(glycer-2-yl-phosphate)-b-D-galactopyranoside disaccharides from the Streptococcuspneumoniae type 23F capsular polysaccharide is reported. A simple protecting group strategy was fol-lowed using commercially available monosaccharides and phosphorylating reagents. H-Phosphonateand phosphoramidite coupling chemistries were explored for introducing the phosphodiester. Hydrazinehydrate was found to be a mild and efficient deacetylating agent, which was required to avoid phosphatemigration during the deprotection of the phosphodiester functionalized disaccharide.

� 2010 Elsevier Ltd. All rights reserved.

Streptococcus pneumoniae remains an important pathogen and amajor cause of human fatalities despite the impressive advances invaccine and antibiotic therapies against this organism.1 To under-stand the immune response against S. pneumoniae, antibodiesagainst the S. pneumoniae serotype 23F have been studied in detail.These studies have revealed the oligoclonal response against the23F polysaccharide antigen and the limited distribution of heavy(H) and light (L) variable gene usage in antibody production.2 Tocomplement the genetically detailed view of the immune responseagainst this pathogen, we have developed a synthesis for a portionof the serotype 23F antigen. The compounds generated in this re-port are required to develop further understanding of the struc-tural biology that governs polysaccharide recognition byantibodies against the 23F polysaccharide antigen.

The structure of the 23F antigen is shown in Figure 1; it consistsof a tetrasaccharide repeating unit with a phosphoglycerol sidechain.3 The recognition epitope of the 23F antigen has been shownto minimally consist of a rhamnose residue; however, the impor-tance of the flanking residues and the phosphoglycerol side chainhas not been determined.4,5 To aid in the elucidation of the keyintermolecular contacts in monoclonal antibodies against the 23Fpolysaccharide we report here the synthesis of the a-L-Rha(1?2)-b-D-Gal-OMe disaccharide (1) and the disaccharidefunctionalized with the phosphoglycerol side chain (2).

The complete tetrasaccharide repeating unit of the 23F polysac-charide has been synthesized previously by Vliegenthart and co-workers.6 In this synthesis difficulties were encountered in the

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).

introduction of the phosphoglycerol side chain and the final tetra-saccharide product required HPLC purification. Here we evaluatedtwo routes for the addition of the phosphoglycerol side chain usingphosphoramidite coupling and H-phosphonate coupling. UltimatelyH-phosphonate coupling proved to be more reproducible, easier tohandle and led to good yields of the desired phosphodiester.

Synthesis of the disaccharide 1 began by using methyl 6-O-tert-butyldiphenylsilyl-3,4-O-isopropylidene-b-D-galactopyranoside 37

as the acceptor and ethyl 2,3,4-tri-O-acetyl-1-thio-a-L-rhamnopyr-anoside 4 as the donor. The glycosylation was carried out using NISin the presence of TMSOTf to afford disaccharide 5 in 87% yield.Subsequently, the TBDPS group was selectively removed using

Figure 1. Structure of the S. pneumoniae type 23F capsular polysaccharide (top) andstructures synthesized (bottom).

Scheme 1. Synthesis of a-L-Rhap-(1?2)-b-D-Galp-OMe disaccharide.

Scheme 3. Glycero-phophodiester synthesis via phosphoramidate 9.

S. Dasgupta, M. Nitz / Carbohydrate Research 345 (2010) 2282–2286 2283

TBAF in THF to afford compound 6 in 81% isolated yield. The iso-propylidene group was selectively cleaved using 80% AcOH at80 �C and the acetyl groups were removed with NaOMe in MeOHto give the target disaccharide 1 in 78% overall yield (Scheme 1).

For the synthesis of disaccharide 2, containing the phosphoglyc-erol side chain, compound 6 was acetylated with pyridine and ace-tic anhydride, followed by removal of the isopropylidene groupusing 80% AcOH to furnish compound 7 in 96% overall yield. A3,4-orthoester was installed on galactose using trimethyl orthoac-etate and CSA.8 Upon treatment with 80% acetic acid the orthoesterrearranged to give the axial acetate revealing the equatorial OH atC3 of galactose in disaccharide 8 (Scheme 2).

Introduction of the phosphoglycerol side chain into disaccha-ride 8 was first explored with the benzylidene-protected phosph-itylating agent 9. The production of 9 was problematic. Treatingcis-1,3-O-benzylidene glycerol with N,N-diisopropylchlorophosph-oramidite in the presence of DIPEA gave the impure phosphorami-dite 9.9 Purification of the phosphoramidite (9) was attemptedusing silica gel chromatography but compound 9 was found tobe highly labile and, with utmost effort, could only be isolated ina 30% yield.10 Once the phosphoramidite 9 was obtained couplingbetween the disaccharide acceptor 8 and 9 proceeded smoothly inthe presence of tetrazole.11 Prior to purification, oxidation of thephosphite diester was carried out using tert-butylhydroperoxideto afford the phosphate triester 10 as a diastereomeric mixturein 91% yield. The base labile cyanoethyl group was removed withDBU to afford the desired fully protected phosphate diester 11 asa single isomer in 90% yield (Scheme 3).

Having found the phosphoramidite 9 to be extremely labile andchallenging to work with alternative approaches for installing thephosphoglycerol side chain were explored. The H-phosphonatemethod was chosen as it has the advantage that the intermediatemono and diesters can be purified easily by silica gel chromatogra-

Scheme 2. Synthesis of selectively protected disaccharide acceptor 8.

phy.12–14 The H-phosphonate monoester was prepared by reactingcompound 8 with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-onein pyridine. The phosphonic diester linkage was achieved by cou-pling the triethylammonium salt of the protected disaccharidephosphonate 12 with cis-1,3-O-benzylidene glycerol in the pres-ence of pivaloyl chloride and pyridine. Subsequent oxidation inthe presence of iodine afforded pure compound 11 after silica gelchromatography in 70% yield (Scheme 4).

Conditions for the global deprotection of the protected phosphatediester 11 were carefully chosen to reduce the possibility of phos-phate migration and/or ester cleavage. Phosphate diester migrationis facile between cis-diols, especially in ribofuranose systems, andhas also been documented in the synthesis of mannose-3-phosphateglycosides.15–17 The acetyl-protecting groups of disaccharide 11were first removed to avoid the migration from secondary to pri-mary alcohols on the glycerol substituent. Migration was observedbetween the O-3 and O-4 positions of galactose under basic deacet-ylation conditions (NaOMe or NH3). Neutral-nucleophilic acetolysiswith hydrazine hydrate in MeOH successfully avoided phosphatemigration. In the 1H NMR spectrum of the product disaccharide

Scheme 4. Glycero-phophodiester synthesis via H-phosphonate 12.

2284 S. Dasgupta, M. Nitz / Carbohydrate Research 345 (2010) 2282–2286

(13) only a single resonance was observed in the 31P NMR. The finalbenzylidene protecting group was removed by hydrogenation to af-ford pure compound 2 in 95% yield (Scheme 4).

Further evidence that the phosphate diester had not quantita-tively migrated was provided by comparison of the relative chem-ical shifts of the protons at H-3 and H-4 of galactose in compounds1 and 2. It was observed that H-3 (4.11 ppm) was downfield of H-4(4.03 ppm) in compound 2 as would be expected if the phosphodi-ester formed through O-3. In compound 1, lacking the phospho-glycerol group, the relative positions of the H-3 (3.53 ppm) andH-4 (3.75 ppm) resonances are reversed. Similar trends were ob-served in the 13C NMR spectra for the C-3 and C-4 resonances ofthe galactose ring between compounds 1 and 2.

In conclusion, we have provided an efficient synthesis of theinternal disaccharide a-L-Rha(1?2)-b-D-Gal-OMe as well as thephosphoglycerol functionalized disaccharide of the S. pneumoniaetype 23F capsular polysaccharide. As has been noted previouslythe H-phosphonate phosphodiester synthetic approach proved tobe robust.18 The acetolysis with hydrazine monohydrate in thepresence of migration-prone phosphodiesters may also prove use-ful in future synthesis. Report of the interactions of compounds 1and 2 with monoclonal antibodies against the 23F capsular poly-saccharide will be communicated in due course.

1. Experimental

1.1. General methods

Proton nuclear magnetic resonance spectra (1H NMR) and car-bon nuclear magnetic resonance spectra (13C NMR) were recordedon a Varian Mercury 400 or a Varian Mercury 300 NMR spectrom-eter. Chemical shifts for protons are reported in parts per million (dscale) downfield from tetramethylsilane and are referenced toresidual protium in the NMR solvents (CHCl3: d 7.27, CD2HOD: d3.31). Chemical shifts for carbon resonances are reported in partsper million (d scale) downfield from tetramethylsilane and are ref-erenced to the carbon resonances of the solvents (CDCl3: d 77.0,CD3OD: d 49.05). Data are represented as follows: chemical shift,multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet), integra-tion, coupling constant and assignment. High resolution massspectra were obtained from an ABI/Sciex Qstar mass spectrometerwith an ESI source. Silica column chromatography was performedusing silica gel (230–400 mesh) from Silicycle.

1.2. Methyl 2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl-(1?2)-6-O-tert-butyl-diphenylsilyl-3,4-O-isopropylidene-b-D-galactopyranoside (5)

A mixture of compounds 3 (1 g, 2.1 mmol), 4 (0.92 g,2.75 mmol) and MS 4 Å (1.5 g) in dry CH2Cl2 (20 mL) was stirredunder nitrogen for 1 h. After addition of NIS (0.8 g, 3.57 mmol),the mixture was cooled to 0 �C followed by addition of TMSOTf(0.05 mL) and the mixture was allowed to stir at 0 �C for 1 h. Whenthe acceptor 3 was completely consumed (Rf 0.3, n-pentane–EtOAc5:1), the mixture was filtered through a pad of Celite and the fil-trate was washed successively with Na2S2O3 (2 � 30 mL), NaHCO3

(2 � 30 mL) and brine (30 mL). The organic layer was collected,dried (Na2SO4) and concentrated in vacuo. The crude productwas purified by flash chromatography using 6:1 n-pentane–EtOAcas eluent to afford the disaccharide 5 (1.37 g, 87%) as a white foam:Rf 0.3 (n-pentane–EtOAc 5:1); 1H NMR (CDCl3, 400 MHz) d: 7.70–7.35 (m, 10H, ArH), 5.32 (dd, 1H, J = 4 Hz, 10 Hz), 5.24 (d, 1H,J = 4 Hz), 5.19 (s, 1H), 5.06 (t, 1H, J = 10 Hz), 4.23 (d, 1H, J = 4 Hz,),4.17–4.14 (m, 3H), 3.95–3.93 (m, 2H), 3.82 (m, 1H), 3.67 (t, 1H,J = 8.2 Hz), 3.51 (s, 3H), 2.12 (s, 3H), 2.04 (s, 3H), 1.97 (s, 3H),

1.49 (s, 3H), 1.31 (s, 3H), 1.18 (d, 3H, J = 6.2 Hz), 1.06 (s, 9H); 13CNMR (CDCl3) d: 170.0, 169.9 (2), 135.5 (2), 135.4 (2), 133.3,133.1, 129.6 (2), 127.6 (2), 127.5 (2), 110.1, 101.8, 96.2, 79.6,76.1, 73.2, 73.1, 70.9, 69.6, 69.2, 66.2, 62.5, 56.6, 27.9, 26.6, 26.2,20.9, 20.8, 20.7, 19.1, 17.1. ESIMS: m/z calcd for C38H52O13SiNa(M+Na)+: 767.3075; found 767.3078.

1.3. Methyl 2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl-(1?2)-3,4-O-isopropylidene-b-D-galactopyranoside (6)

To a stirred solution of compound 5 (1.3 g, 2.6 mmol) in dry THF(20 mL) at 0 �C was added AcOH (0.17 mL, 3.1 mmol) followed byn-Bu4NF (1 M in THF, 7.5 mL) and the solution was allowed to stirat room temperature for 12 h; after this time the starting materialhad been completely converted into a slower moving spot. The sol-vents were evaporated at temperatures below 30 �C in vacuo. Thecrude product was purified by flash chromatography using n-pen-tane–EtOAc (1:1) as eluent to afford pure compound 6 (0.71 g, 81%)as a colourless oil; Rf 0.1, (n-pentane–EtOAc 2:1); 1H NMR (CDCl3,300 MHz) d: 5.29 (dd, 1H, J = 10 Hz, 4.1 Hz), 5.23 (d, 1H, J = 4.2 Hz),5.17 (s, 1H), 5.05 (t, 1H, J = 10 Hz,), 4.23–4.19 (m, 2H), 4.14–4.12(m, 2H), 3.98 (m, 1H), 3.84 (m, 2H), 3.67 (t, 1H, J = 7.8 Hz,), 3.55(s, 3H), 2.14 (s, 3H), 2.04 (s, 3H), 1.97 (s, 3H), 1.49 (s, 3H), 1.31(s,3H), 1.19 (d, 3H, J = 6.2 Hz); 13C NMR (CDCl3) d: 170.2, 170 (2),170.2, 101.6, 101.5, 96.3, 79.7, 76.0, 74.1, 73.1, 71.0, 69.6, 69.3,66.4, 62.5, 56.9, 27.9, 26.3, 20.9, 20.8, 20.7, 17.1. ESIMS: m/z calcdfor C22H34O13Na (M+Na)+: 529.1897; found 529.1894.

1.4. Methyl a-L-rhamnopyranosyl-(1?2)-b-D-galactopyranoside(1)

A solution of compound 6 (100 mg, 0.19 mmol) in AcOH–H2O(9:1, 10 mL) was stirred at 80 �C for 2 h after which time the start-ing material was completely converted into a slower moving com-ponent. The solvents were evaporated and then co-evaporatedwith toluene. The residue thus obtained was dissolved in MeOH(5 mL) followed by the addition of NaOMe (0.5 M in MeOH,0.1 mL) at which time pH paper indicated that the solution wasalkaline. The reaction mixture was stirred at room temperaturefor 6 h. The solution was then neutralized with Amberlite IR 120H+ resin and filtered. The filtrate was evaporated to afford purecompound 1 (52 mg, 78%) as an amorphous white solid: Rf 0.3(CH2Cl2–MeOH 9:1); 1H NMR (D2O, 400 MHz) d: 4.84 (s, 1H, H-10), 4.25 (d, 1H, J1,2 = 7.8 Hz, H-1), 3.88 (d, 1H, J2,3 = 3.5 Hz, H-20),3.76 (m, 2H, H-4, H-50), 3.64–3.59 (m, 4H, H-3, H-6a, H-6b, H-30),3.53 (m, 1H, H-5), 3.43 (s, 3H, OCH3), 3.37 (dd, 1H, J1,2 = 7.8 Hz,J2,3 = 9.8 Hz, H-2), 3.29 (t, 1H, J3,4 = J4,5 = 9.7 Hz, H-40). 1.13 (d, 3H,J = 6.2 Hz, C–CH3); 13C NMR (D2O) d: 102.8, 101.6, 78.3, 75.1,73.4, 72.1, 70.3 (2), 69.1, 68.9, 61.1, 57.3, 16.6. ESIMS: calcd form/z C13H24O10Na (M+Na)+: 363.1297; found 363.1271.

1.5. Methyl 2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl-(1?2)-6-O-acetyl-b-D-galactopyranoside (7)

A solution of compound 6 (600 mg, 1.18 mmol) in pyridine(5 mL) and acetic anhydride (2 mL) was stirred at room tempera-ture for 1 h, when TLC showed complete conversion of the startingmaterial into a faster moving spot (Rf 0.2, n-pentane–EtOAc 2:1).The solvents were evaporated, and then co-evaporated with tolu-ene to remove any traces of pyridine and acetic anhydride. The res-idue was dissolved in AcOH–H2O (9:1, 10 mL) and stirred at 80 �Cfor 2 h at which time the starting material had been completelyconverted into a slower moving compound by TLC. The solventswere evaporated, co-evaporated with toluene and the crude prod-uct was purified by flash chromatography using n-pentane–EtOAc(1:2) as eluent to afford compound 7 (575 mg, 96%) as a white

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powder: 1H NMR (CDCl3, 300 MHz) d: 5.29 (dd, 1H, J = 10 Hz,4.1 Hz), 5.23 (d, 1H, J = 4.2 Hz), 5.17 (s, 1H), 5.06 (t, 1H,J = 9.9 Hz), 4.23–4.19 (m, 2H), 4.14–4.12 (m, 2H), 3.82 (m, 1H),3.69–3.64 (m, 3H), 3.52 (s, 3H), 2.13 (s, 3H), 2.09 (s, 3H), 2.05 (s,3H), 1.98 (s, 3H), 1.19 (d, 3H, J = 6.2 Hz). 13C NMR (CDCl3) d:171.1, 170.4, 170.2, 169.9, 102.6, 98.1, 74.0, 71.8, 70.9, 69.7, 69.4,68.9, 66.6, 62.4, 56.8, 20.9, 20.8 (2), 20.8, 17.1. ESIMS: calcd m/zfor C21H32O14Na (M+Na)+: 531.1690; found 531.1693.

1.6. Methyl 2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl-(1?2)-4,6-di-O-acetyl-b-D-galactopyranoside (8)

To a solution of compound 7 (500 mg, 0.98 mmol) in dry CH3CN(10 mL) was added trimethyl orthoacetate (0.17 mL, 1.42 mmol)and CSA (15 mg). The solution was stirred at room temperaturefor 1 h. The solution was then neutralized with triethylamine(100 lL) and the solvents were evaporated in vacuo. The residuewas dissolved in AcOH (80% aq, 10 mL) and stirred at room temper-ature for 45 min. The solvents were evaporated and co-evaporatedwith toluene and the resulting crude product was purified by flashchromatography using n-pentane–EtOAc 2:1 to furnish the disac-charide acceptor 8 (460 mg, 85%) as a white foam: Rf 0.3 (n-pen-tane–EtOAc 2:1); 1H NMR (CDCl3, 300 MHz) d: 5.31 (d, 1H,J = 4 Hz), 5.24 (m, 2H), 5.14 (s, 1H), 5.07 (t, 1H, J = 9.8 Hz), 4.30(d, 1H, J = 7.7 Hz), 4.16 (m, 3H), 3.92 (dd, 1H, J = 3.5 Hz, 9.1 Hz),3.80 (m, 1H), 3.70 (t, 1H, J = 9.4 Hz), 3.56 (s, 3H), 2.14 (s, 3H),2.12 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H), 1.99 (s, 3H), 1.21 (d, 3H,J = 6.2 Hz); 13C NMR (CDCl3) d: 171.4, 170.4, 170.2, 170.1, 169.9,102.5, 98.2, 76.0, 73.2, 70.9, 70.6, 70, 69.7, 69.3, 66.6, 61.7, 57.0,20.9, 21.8, 20.8 (3), 20.7, 17.0. ESIMS: calcd for m/z C23H34O15Na(M+Na)+: 573.1795; found 573.1791.

1.7. Methyl 2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl-(1?2)-4,6-di-O-acetyl-3-(cis-1,3-O-benzylidene glycer-2-yl-phosphate)-b-D-galactopyranoside (11)

To a solution of cis-1,3-O-benzylidene glycerol (200 mg,1.1 mmol), in dry CH2Cl2 (10 mL), under argon were addedDIPEA (0.25 mL, 1.66 mmol) and 2-cyanoethyl N,N-dii-sopropylchlorophosphoramidite (524 mg, 2.22 mmol). The reac-tion mixture was stirred at room temperature for 1 h at whichtime the TLC indicated that the reaction was complete. The reac-tion mixture was quenched with MeOH (1 mL) and stirred for5 min. After removing the solvents, the residue was passed througha silica column, eluted with n-pentane–EtOAc 5:1 (Rf 0.6) contain-ing 0.5% triethylamine to give the corresponding phosphoramidite9 (122 mg, 30%) as a colourless oil. The phosphoramidite obtainedwas used immediately in the next reaction. Compound 9 (122 mg,0.33 mmol) and compound 8 (63 mg, 0.11 mmol) were dissolved in3:1 CH2Cl2–CH3CN (12 mL), and to the solution was added 1-H tet-razole (0.33 mmol, 0.75 mL of 0.45 M solution in CH3CN). The reac-tion mixture was stirred for 6 h and then tert-butyl hydroperoxide(0.66 mmol, 0.13 ml of 5 M in decane) was added. The oxidationreaction was allowed to proceed for a further 1 h at which timethe TLC showed complete conversion of starting material into aslower moving compound. The solvents were evaporated and thecrude material was passed through silica column eluting with50:1 CH2Cl2–MeOH to give compound 10 (80 mg, 91%) as a diaste-reomeric mixture. The 31P NMR spectrum contains two peaks at d:0.35 and 3.49. The mixture of diastereomers (10) was dissolved inCH2Cl2 (5 ml) and to this solution was added DBU (70 lL,0.47 mmol). The reaction was allowed to proceed for 12 h at whichtime the TLC showed complete conversion of the starting materialinto a slower moving spot. Dichloromethane was evaporated andthe residue was passed through a small silica column eluting with(5:1 EtOAc–MeOH and 0.1% Et3N). Compound 11 was thus isolated

as a single isomer (67 mg, 90%) as a colourless oil. The spectra wereidentical to those described below.

Compound 12 (95 mg, 0.13 mmol) and cis-1,3-O-benzylideneglycerol (24 mg, 0.13 mmol) were co-concentrated in pyridine(2 � 5 mL), and the residue was dissolved in dry pyridine (4 mL).Pivaloyl chloride (48 lL, 0.39 mmol) was added, the mixture wasstirred for 16 h, at which time TLC showed complete conversionof the starting material into a faster moving spot. A solution of0.5 M iodine in 2:1 water–pyridine (0.2 mL) was added to the reac-tion. After 3 h, TLC indicated the total disappearance of the phos-phonate and a slower moving spot was formed. The excessiodine was destroyed with aqueous 5% sodium thiosulfate(10 mL), and the mixture was diluted with CH2Cl2 (50 mL), washedwith 1 M triethylammonium hydrogen carbonate (2 � 10 ml),dried (MgSO4), filtered and concentrated. Column chromatographywas done using (5:1 EtOAc–MeOH with 0.1% triethylamine) to givepure compound 11 (72 mg, 70%) as syrup. 1H NMR (CD3OD,400 MHz) d:7.48–7.33 (m, 5H), 5.62 (d, 1H, J = 3.6 Hz), 5.53 (s,1H), 5.46 (s, 1H), 5.21 (d, 1H, J = 4 Hz), 5.18 (dd, 1H, J = 4 Hz,10 Hz), 5.00 (t, 1H, J = 10 Hz), 4.45 (m, 1H), 4.37–4.25 (m, 4H),4.18 (d, 1H, J = 7.8 Hz), 4.11–4.06 (m, 3H), 4.04–3.91 (m, 2H),3.76 (t, 1H, J = 9.4 Hz), 3.49 (s, 3H), 2.12 (s, 3H), 2.10 (s, 3H), 2.04(s, 3H), 2.03 (s, 3H), 1.90 (s, 3H), 1.16 (d, 3H, J = 6.2 Hz). 31P NMR(CD3OD): d: �2.38. 13C NMR (CD3OD) d: 172.5, 172.3, 172.2,172.1, 171.9, 140, 129.9, 129.1 (2), 127.5 (2), 103.6, 102.3, 99.5,76.8, 72.2, 72, 71.7, 71.4, 71.3, 70.9, 69.2, 67.8, 63.6, 57.1, 48.4,21.1, 21.0, 20.7 (2), 20.5, 17.5. ESIMS: calcd for m/z C33H45O20

PNa(M+Na)+: 815.2140; found 815.2144.

1.8. Methyl 2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl-(1?2)-4,6-di-O-acetyl-3-triethyl ammonium phosphonate-b-D-galactopyranoside (12)

To a solution of 8 (100 mg, 0.18 mmol) in CH3CN (15 mL) wereadded pyridine (5 mL) and 2-chloro-4H-1,3,2-benzodioxaphospho-rin-4-one (65 mg, 0.35 mmol). After 1 h, when TLC showed disap-pearance of the starting material, water (3 mL) was added andafter 5 min the solvents were evaporated. The residue was dilutedwith CH2Cl2–Et3N 9.9:0.1 (20 mL) and washed with 1 M triethyl-ammonium hydrogen carbonate (2 � 10 mL), dried (MgSO4), fil-tered and concentrated. Column chromatography was done usingCH2Cl2–MeOH–Et3N (92:8:0.1) to give pure compound 12 as a syr-up (103 mg, 78%): Rf 0.3 (92:8:0.1 CH2Cl2–MeOH–Et3N); 1H NMR(CD3OD, 300 MHz) d: 5.48 (s, 1H), 5.41 (d, 1H, J = 3.2 Hz), 5.29(m, 1H), 5.16 (dd 1H, J = 3.4 Hz, 9.1 Hz), 5.05 (s, 1H), 4.99 (t, 1H,J = 10 Hz), 4.45 (m, 2H), 4.30–3.95 (m, 4H), 3.69 (t, 1H, J = 9.5 Hz),3.53 (s, 3H), 3.23 (m, 2H), 2.11 (s, 6H), 2.04 (s, 3H), 2.02 (s, 3H),1.93 (s, 3H), 1.31 (t, 3H, J = 7 Hz), 1.16 (d, 3H, J = 6.2 Hz); 31PNMR (CD3OD): d: 2.53; 13C NMR (CD3OD) d: 172.2, 172, 171.7(2),171.6, 170.2, 103.7, 99.6, 75.8, 72.1, 72, 71.5, 71.1, 70.8, 67.9,63.1, 57.2, 47.9, 20.7 (2), 20.6 (2), 17.5, 9.3. ESIMS: calcd for m/zC30H54NO7PNa (M+Na)+: 754.3027; found 754.3024.

1.9. Methyl a-L-rhamnopyranosyl-(1?2)-3-(1,3-O-benzylideneglycer-2-yl-phosphate)-b-D-galactopyranoside (13)

Compound 11 (40 mg, 0.05 mmol) was dissolved in MeOH(5 mL), and to the solution hydrazine hydrate (18 lL, 0.37 mmol)was added, and then the reaction was stirred at room temp for16 h. The reaction was then neutralized using Amberlite IR120 H+

resin. The solution was filtered and the filtrate was evaporated.The residue thus obtained was dissolved in minimum volume ofwater and loaded onto a C-18 Silica plug (500 mg) eluting first withwater and then with MeOH–water 99:1 to furnish pure compound13 (18 mg, 64%) as a white powder: 1H NMR (D2O, 300 MHz) d:7.41–7.35 (m, 5H), 5.65 (s, 1H), 4.86 (s, 1H), 4.32 (d, 1H,

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J = 7.8 Hz), 4.21–4.14 (m, 4H), 4.13 (dd, 1H, J = 4.1 Hz, 9 Hz), 4.09(d, 1H, J = 4 Hz), 4.01 (s, 1H), 3.80 (m, 1H), 3.65 (dd, 1H, J = 4 Hz,10 Hz), 3.60–3.54 (m, 3H), 3.53 (dd, 1H, J = 7.8 Hz, 8.1 Hz), 3.45(s, 3H), 3.28 (t, 1H, J = 9.6 Hz), 1.13 (d, 3H, J = 6.2 Hz); 31P NMR(D2O): d: 0.08; 13C NMR (CD3OD) d: 139.9, 130, 129.1 (2), 127.4(2), 104.5, 103, 102.6, 80.9, 76.3, 74.1, 72.2 (2), 71.9, 71.2, 69.8,69.3, 69.1, 62.9, 57.1, 17.8. ESIMS: calcd for m/z C23H35O15PNa(M+Na)+: 605.1611; found 605.1615.

1.10. Methyl a-L-rhamnopyranosyl-(1?2)-3-(glycer-2-yl-phosphate)-b-D-galactopyranoside (2)

To a solution of 13 (15 mg, 0.025 mmol) in MeOH (3 mL) wasadded 5% Pd–C (5 mg). Hydrogenolysis was performed for 12 h,the mixture was filtered through a Celite bed and concentrated. Itwas lyophilized from water to give compound 2 (12 mg, 95%) as awhite powder: 1H NMR (D2O, 300 MHz) d: 4.83 (s, 1H, H-10), 4.33(d, 1H, J1,2 = 7.8 Hz, H-1), 4.11 (m, 2H, H-3, H-200), 4.03 (d, 1H,J = 3.2 Hz, H-4), 3.97 (d, 1H, J2,3 = 3.1 Hz, H-20), 3.78 (m, 1H, H-50),3.65 (dd, J3,2 = 4 Hz, J3,4 = 10 Hz, H-30), 3.62–3.59 (m, 5H, H-1a00, H-1b00, H-3a00, H-3b00, H-6a), 3.58–3.52 (m, 2H, H-5, H-6b), 3.51 (dd,1H, J2,1 = 7.8 Hz, J2,3 = 8.1 Hz, H-2), 3.45 (s, 3H, OCH3), 3.30 (t, 1H,J3,4 = J4,5 = 9.7 Hz, H-40), 1.14 (d, 3H, J = 6.2 Hz, C–CH3); 31P NMR(D2O): d: 0.25; 13C NMR (D2O) d: 102.6, 101.7, 78.1, 77.2, 76.4,74.7, 72.1, 70.3, 70.2, 68.9, 67.9, 61.6, 61.1, 57.3, 16.5. ESIMS: calcdfor m/z C16H31O15PNa (M+Na)+: 517.1298; found 517.1295.

Acknowledgements

This work was supported by the Natural Science & EngineeringResearch Council (NSERC) of Canada. The authors also thank SteveBryson and Emil Pai for helpful discussions.

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.carres.2010.08.002.

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