Synthesis of a core disaccharide from the Streptococcus pneumoniae type 23F capsular polysaccharide antigen

Article abstract of DOI:10.1016/j.carres.2010.08.002

The synthesis of methyl α-l-rhamnopyranosyl-(1→2)-β-d- galactopyranoside and methyl α-l-rhamnopyranosyl-(1→2)-3-(glycer-2- yl-phosphate)-β-d-galactopyranoside disaccharides from the Streptococcus pneumoniae type 23F capsular polysaccharide is reported. A simple protecting group strategy was followed using commercially available monosaccharides and phosphorylating reagents. H-Phosphonate and phosphoramidite coupling chemistries were explored for introducing the phosphodiester. Hydrazine hydrate was found to be a mild and efficient deacetylating agent, which was required to avoid phosphate migration during the deprotection of the phosphodiester functionalized disaccharide.

Full text of DOI:10.1016/j.carres.2010.08.002

Carbohydrate Research 345 (2010) 2282–2286
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Synthesis of a core disaccharide from the Streptococcus pneumoniae type
23F 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
a b s t r a c t
Article history:
Received 11 June 2010
Received in revised form 5 August 2010
Accepted 6 August 2010
Available online 10 August 2010
The synthesis of methyl
anosyl-(1?2)-3-(glycer-2-yl-phosphate)-b-
a
-
L
-rhamnopyranosyl-(1?2)-b-
D-galactopyranoside and methyl a-L-rhamnopyr-
D
-galactopyranoside disaccharides from the Streptococcus
pneumoniae type 23F capsular polysaccharide is reported. A simple protecting group strategy was fol-
lowed using commercially available monosaccharides and phosphorylating reagents. H-Phosphonate
and phosphoramidite coupling chemistries were explored for introducing the phosphodiester. Hydrazine
hydrate was found to be a mild and efficient deacetylating agent, which was required to avoid phosphate
migration during the deprotection of the phosphodiester functionalized disaccharide.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Phosphoglycan
Streptococcus pneumoniae
H-Phosphonate
Phosphoramidite
Streptococcus pneumoniae remains an important pathogen and a
major cause of human fatalities despite the impressive advances in
vaccine and antibiotic therapies against this organism.1 To under-
stand the immune response against S. pneumoniae, antibodies
against the S. pneumoniae serotype 23F have been studied in detail.
These studies have revealed the oligoclonal response against the
23F polysaccharide antigen and the limited distribution of heavy
(H) and light (L) variable gene usage in antibody production.2 To
complement the genetically detailed view of the immune response
against this pathogen, we have developed a synthesis for a portion
of 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 by
antibodies against the 23F polysaccharide antigen.
introduction of the phosphoglycerol side chain and the final tetra-
saccharide product required HPLC purification. Here we evaluated
two routes for the addition of the phosphoglycerol side chain using
phosphoramidite coupling and H-phosphonate coupling. Ultimately
H-phosphonate coupling proved to be more reproducible, easier to
handle 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
-rhamnopyr-
as the acceptor and ethyl 2,3,4-tri-O-acetyl-1-thio-
a-L
anoside 4 as the donor. The glycosylation was carried out using NIS
in the presence of TMSOTf to afford disaccharide 5 in 87% yield.
Subsequently, the TBDPS group was selectively removed using
The structure of the 23F antigen is shown in Figure 1; it consists
of a tetrasaccharide repeating unit with a phosphoglycerol side
chain.3 The recognition epitope of the 23F antigen has been shown
to minimally consist of a rhamnose residue; however, the impor-
tance of the flanking residues and the phosphoglycerol side chain
has not been determined.4,5 To aid in the elucidation of the key
intermolecular contacts in monoclonal antibodies against the 23F
polysaccharide we report here the synthesis of the
Rha(1?2)-b- -Gal-OMe disaccharide (1) and the disaccharide
functionalized with the phosphoglycerol side chain (2).
a-L-
D
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
⇑
Corresponding author. Tel.: +1 416 946 0640.
E-mail address: mnitz@chem.utoronto.ca (M. Nitz).
Figure 1. Structure of the S. pneumoniae type 23F capsular polysaccharide (top) and
structures synthesized (bottom).
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.08.002

S. Dasgupta, M. Nitz / Carbohydrate Research 345 (2010) 2282–2286
2283
Scheme 1. Synthesis of
a-
L
-Rhap-(1?2)-b-
D-Galp-OMe disaccharide.
Scheme 3. Glycero-phophodiester synthesis via phosphoramidate 9.
TBAF in THF to afford compound 6 in 81% isolated yield. The iso-
propylidene group was selectively cleaved using 80% AcOH at
80 °C and the acetyl groups were removed with NaOMe in MeOH
to 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 group
using 80% AcOH to furnish compound 7 in 96% overall yield. A
3,4-orthoester was installed on galactose using trimethyl orthoac-
etate and CSA.8 Upon treatment with 80% acetic acid the orthoester
rearranged to give the axial acetate revealing the equatorial OH at
C3 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. Treating
cis-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 attempted
using silica gel chromatography but compound 9 was found to
be highly labile and, with utmost effort, could only be isolated in
a 30% yield.10 Once the phosphoramidite 9 was obtained coupling
between the disaccharide acceptor 8 and 9 proceeded smoothly in
the presence of tetrazole.11 Prior to purification, oxidation of the
phosphite diester was carried out using tert-butylhydroperoxide
to afford the phosphate triester 10 as a diastereomeric mixture
in 91% yield. The base labile cyanoethyl group was removed with
DBU to afford the desired fully protected phosphate diester 11 as
a single isomer in 90% yield (Scheme 3).
phy.12–14 The H-phosphonate monoester was prepared by reacting
compound 8 with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one
in pyridine. The phosphonic diester linkage was achieved by cou-
pling the triethylammonium salt of the protected disaccharide
phosphonate 12 with cis-1,3-O-benzylidene glycerol in the pres-
ence of pivaloyl chloride and pyridine. Subsequent oxidation in
the presence of iodine afforded pure compound 11 after silica gel
chromatography in 70% yield (Scheme 4).
Conditions fortheglobaldeprotectionof theprotectedphosphate
diester 11 were carefully chosen to reduce the possibility of phos-
phate migration and/or ester cleavage. Phosphate diester migration
is facile between cis-diols, especially in ribofuranose systems, and
has also been documented in the synthesis of mannose-3-phosphate
glycosides.15–17 The acetyl-protecting groups of disaccharide 11
were first removed to avoid the migration from secondary to pri-
mary alcohols on the glycerol substituent. Migration was observed
between the O-3 and O-4 positions of galactose under basic deacet-
ylation conditions (NaOMe or NH3). Neutral-nucleophilic acetolysis
with hydrazine hydrate in MeOH successfully avoided phosphate
migration. In the 1H NMR spectrum of the product disaccharide
Having found the phosphoramidite 9 to be extremely labile and
challenging to work with alternative approaches for installing the
phosphoglycerol side chain were explored. The H-phosphonate
method was chosen as it has the advantage that the intermediate
mono and diesters can be purified easily by silica gel chromatogra-
Scheme 2. Synthesis of selectively protected disaccharide acceptor 8.
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 final
benzylidene protecting group was removed by hydrogenation to af-
ford pure compound 2 in 95% yield (Scheme 4).
1.49 (s, 3H), 1.31 (s, 3H), 1.18 (d, 3H, J = 6.2 Hz), 1.06 (s, 9H); 13C
NMR (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.
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 compounds
1 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) and
H-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 of
the galactose ring between compounds 1 and 2.
1.3. Methyl 2,3,4-tri-O-acetyl-
a-L-rhamnopyranosyl-(1?2)-3,4-
O-isopropylidene-b- -galactopyranoside (6)
D
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 by
n-Bu4NF (1 M in THF, 7.5 mL) and the solution was allowed to stir
at room temperature for 12 h; after this time the starting material
had been completely converted into a slower moving spot. The sol-
vents were evaporated at temperatures below 30 °C in vacuo. The
crude 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 calcd
for C22H34O13Na (M+Na)+: 529.1897; found 529.1894.
In conclusion, we have provided an efficient synthesis of the
internal disaccharide
a-L-Rha(1?2)-b-D-Gal-OMe as well as the
phosphoglycerol functionalized disaccharide of the S. pneumoniae
type 23F capsular polysaccharide. As has been noted previously
the H-phosphonate phosphodiester synthetic approach proved to
be robust.18 The acetolysis with hydrazine monohydrate in the
presence of migration-prone phosphodiesters may also prove use-
ful in future synthesis. Report of the interactions of compounds 1
and 2 with monoclonal antibodies against the 23F capsular poly-
saccharide will be communicated in due course.
1. Experimental
1.1. General methods
1.4. Methyl
(1)
a-L-rhamnopyranosyl-(1?2)-b-D-galactopyranoside
Proton nuclear magnetic resonance spectra (1H NMR) and car-
bon nuclear magnetic resonance spectra (13C NMR) were recorded
on a Varian Mercury 400 or a Varian Mercury 300 NMR spectrom-
eter. Chemical shifts for protons are reported in parts per million (d
scale) downfield from tetramethylsilane and are referenced to
residual protium in the NMR solvents (CHCl3: d 7.27, CD2HOD: d
3.31). Chemical shifts for carbon resonances are reported in parts
per 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 mass
spectra were obtained from an ABI/Sciex Qstar mass spectrometer
with an ESI source. Silica column chromatography was performed
using silica gel (230–400 mesh) from Silicycle.
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-evaporated
with 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 was
alkaline. The reaction mixture was stirred at room temperature
for 6 h. The solution was then neutralized with Amberlite IR 120
H+ resin and filtered. The filtrate was evaporated to afford pure
compound 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 for
m/z C13H24O10Na (M+Na)+: 363.1297; found 363.1271.
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 stirred
under 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. When
the acceptor 3 was completely consumed (Rf 0.3, n-pentane–EtOAc
5:1), 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 product
was purified by flash chromatography using 6:1 n-pentane–EtOAc
as 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),
1.5. Methyl 2,3,4-tri-O-acetyl-
a-L-rhamnopyranosyl-(1?2)-6-O-
acetyl-b- -galactopyranoside (7)
D
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 starting
material 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 °C
for 2 h at which time the starting material had been completely
converted into a slower moving compound by TLC. The solvents
were 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

S. Dasgupta, M. Nitz / Carbohydrate Research 345 (2010) 2282–2286
2285
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/z
for C21H32O14Na (M+Na)+: 531.1690; found 531.1693.
as a single isomer (67 mg, 90%) as a colourless oil. The spectra were
identical to those described below.
Compound 12 (95 mg, 0.13 mmol) and cis-1,3-O-benzylidene
glycerol (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 was
stirred for 16 h, at which time TLC showed complete conversion
of the starting material into a faster moving spot. A solution of
0.5 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 excess
iodine was destroyed with aqueous 5% sodium thiosulfate
(10 mL), and the mixture was diluted with CH2Cl2 (50 mL), washed
with 1 M triethylammonium hydrogen carbonate (2 ꢀ 10 ml),
dried (MgSO4), filtered and concentrated. Column chromatography
was done using (5:1 EtOAc–MeOH with 0.1% triethylamine) to give
pure 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.
1.6. Methyl 2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl-(1?2)-4,6-
di-O-acetyl-b- -galactopyranoside (8)
D
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 temperature
for 1 h. The solution was then neutralized with triethylamine
(100 lL) and the solvents were evaporated in vacuo. The residue
was dissolved in AcOH (80% aq, 10 mL) and stirred at room temper-
ature for 45 min. The solvents were evaporated and co-evaporated
with toluene and the resulting crude product was purified by flash
chromatography 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.
1.8. 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)
1.7. Methyl 2,3,4-tri-O-acetyl-
di-O-acetyl-3-(cis-1,3-O-benzylidene glycer-2-yl-phosphate)-b-
-galactopyranoside (11)
a-L-rhamnopyranosyl-(1?2)-4,6-
To a solution of 8 (100 mg, 0.18 mmol) in CH3CN (15 mL) were
added 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 and
after 5 min the solvents were evaporated. The residue was diluted
with 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 using
CH2Cl2–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); 31P
NMR (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/z
D
To
a solution of cis-1,3-O-benzylidene glycerol (200 mg,
1.1 mmol), in dry CH2Cl2 (10 mL), under argon were added
DIPEA (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 which
time the TLC indicated that the reaction was complete. The reac-
tion mixture was quenched with MeOH (1 mL) and stirred for
5 min. After removing the solvents, the residue was passed through
a silica column, eluted with n-pentane–EtOAc 5:1 (Rf 0.6) contain-
ing 0.5% triethylamine to give the corresponding phosphoramidite
9 (122 mg, 30%) as a colourless oil. The phosphoramidite obtained
was used immediately in the next reaction. Compound 9 (122 mg,
0.33 mmol) and compound 8 (63 mg, 0.11 mmol) were dissolved in
3:1 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 oxidation
reaction was allowed to proceed for a further 1 h at which time
the TLC showed complete conversion of starting material into a
slower moving compound. The solvents were evaporated and the
crude material was passed through silica column eluting with
50:1 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 in
C
30H54NO7PNa (M+Na)+: 754.3027; found 754.3024.
1.9. Methyl
glycer-2-yl-phosphate)-b-
a
-
L
-rhamnopyranosyl-(1?2)-3-(1,3-O-benzylidene
-galactopyranoside (13)
D
Compound 11 (40 mg, 0.05 mmol) was dissolved in MeOH
(5 mL), and to the solution hydrazine hydrate (18 L, 0.37 mmol)
l
was added, and then the reaction was stirred at room temp for
16 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 of
water and loaded onto a C-18 Silica plug (500 mg) eluting first with
water and then with MeOH–water 99:1 to furnish pure compound
13 (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,
CH2Cl2 (5 ml) and to this solution was added DBU (70 lL,
0.47 mmol). The reaction was allowed to proceed for 12 h at which
time the TLC showed complete conversion of the starting material
into a slower moving spot. Dichloromethane was evaporated and
the residue was passed through a small silica column eluting with
(5:1 EtOAc–MeOH and 0.1% Et3N). Compound 11 was thus isolated

2286
S. Dasgupta, M. Nitz / Carbohydrate Research 345 (2010) 2282–2286
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.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2010.08.002.
References
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1
white powder: H 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,
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3.65 (dd, J3,2 = 4 Hz, J3,4 = 10 Hz, H-30), 3.62–3.59 (m, 5H, H-1a00, H-
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This work was supported by the Natural Science & Engineering
Research Council (NSERC) of Canada. The authors also thank Steve
Bryson and Emil Pai for helpful discussions.