Synthesis of a novel pentasaccharide core component from the lipooligosaccharide of Moraxella catarrhalis

Article abstract of DOI:10.1016/j.carres.2011.10.002

The novel pentasaccharide [p-(trifluoroacetamido)phenyl]ethyl 3-O-β-d-glucopyranosyl-4-O-β-d-glucopyranosyl-6-O-[2-O-(α-d- glucopyranosyl)-β-d-glucopyranosyl]-α-d-glucopyranoside (1), which includes a linker moiety to enable facile coupling to an antigeni

Full text of DOI:10.1016/j.carres.2011.10.002

Carbohydrate Research 346 (2011) 2805–2811
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Synthesis of a novel pentasaccharide core component from
the lipooligosaccharide of Moraxella catarrhalis
b,
Andrew G. Pearson a, Ian R. Peak b, Jennifer C. Wilson b, , I. Darren Grice
⇑
⇑
a School of Medical Science, Griffith University Gold Coast Campus, Queensland 4222, Australia
b Institute for Glycomics, Griffith University Gold Coast Campus, Queensland 4222, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
The novel pentasaccharide [p-(trifluoroacetamido)phenyl]ethyl 3-O-b-
D-glucopyranosyl-4-O-b-D-gluco-
Received 21 October 2010
Received in revised form 29 September
2011
Accepted 3 October 2011
Available online 17 October 2011
pyranosyl-6-O-[2-O-( -glucopyranosyl)-b- -glucopyranosyl]- -glucopyranoside (1), which includes
a
-
D
D
a-D
a linker moiety to enable facile coupling to an antigenic protein, was synthesised as a component of a
potential vaccine candidate against the Gram-negative bacterium Moraxella catarrhalis. This microorgan-
ism is one of three principal causative agents of otitis media in children. The pentasaccharide represents a
common cross-serotype (A, B and C) structure from the lipooligosaccharides of Moraxella catarrhalis.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Moraxella catarrhalis
Pentasaccharide
Linker
Lipooligosaccharide
Moraxella catarrhalis is a Gram-negative pathogen that, along
with Streptococcus pneumoniae and nontypeable Haemophilus influ-
enzae, is responsible for 15–25% of cases of otitis media (middle ear
infection) in children.1 There is a high incidence of otitis media
cases in infants in developed countries with at least 80% contract-
ing the disease before the age of 3 years. M. catarrhalis also contrib-
utes to a range of respiratory tract infections in adults and
exacerbates serious conditions such as chronic obstructive pul-
monary disease (COPD), a leading cause of mortality in the US.2
Currently, there are no licensed M. catarrhalis vaccines available
for prevention of otitis media.3
The major surface lipid of Gram-negative bacteria is lipopoly-
saccharide. M. catarrhalis lacks the O-antigenic repeating compo-
nent of lipopolysaccharide and therefore its bacterial outer
membrane is covered in lipooligosaccharide (LOS), containing core
oligosaccharide and lipid A components. Three major serotypes of
M. catarrhalis have been identified—A, B and C.4 As shown in Figure
the production of LOS-specific antibodies.6 Several studies using
serotype-specific LOS derivatives have shown serotype specific
protection on subsequent bacterial challenge.7 These studies high-
light the requirement to include LOS from all serotypes to provide
effective protection against all serotypes of M. catarrhalis.
A major limitation of including full length oligosaccharide in a
potential vaccine candidate is that a conserved region of the LOS
structure in all serotypes (see Fig. 1) is identical to the PK antigen
found on human erythrocyte cells.8 If included in a vaccine, this
structure could potentially stimulate an auto immune response.
Excluding the structure of the PK antigen reveals a less specific con-
served structure common to all M. catarrhalis LOS serotypes A, B
and C highlighted in Figure 1.
In an attempt to synthesise a truncated OS common to all
M. catarrhalis serotypes but excluding the structure of the PK anti-
gen that could be linked to an antigenic protein as a vaccine candi-
date, we embarked on the synthesis of the novel pentasaccharide 1
and the tetrasaccharide 2,9 as shown in Scheme 1. While a synthe-
sis of 2 has been previously reported, we employed a different ap-
proach in part for the synthesis of 2, to give us ready access to both
2 and the novel target compound 1 (see Fig. 2). In addition, full
NMR assignment of 2 is provided. The design of 1 and 2 includes
[p-trifluoroacetamido)phenyl]ethyl linker moiety, which Ekelöf
and Oscarson9 have also employed previously, that facilitates the
convenient attachment of an antigenic protein. Herein the synthe-
sis and full NMR characterisation of 1 and 2 are presented. It is ex-
pected that when linked to a carrier protein such as diphtheria
toxin, 1 will elicit an immune response that protects mice against
1, it is the heterogeneity in the central b-D-Glc-(1,4)-linked chain of
the core oligosaccharide that determines the specific serotype. De-
tails of the composition and biosynthesis of the core oligosaccha-
ride structures of the serotypes have been studied.5
Previous studies into the utility of LOS as a vaccine candidate
have demonstrated that exposure to M. catarrhalis LOS results in
⇑
Corresponding authors. Tel.: +61
7
5552 8077; fax: +61
5552 8098 (I.D.G.).
7 5552 8098
(J.C.W.); tel.: +61
7
5552 7027; fax: +61 7
E-mail addresses: jennifer.wilson@griffith.edu.au (J.C. Wilson), d.grice@griffith.
edu.au (I.D. Grice).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.10.002

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A. G. Pearson et al. / Carbohydrate Research 346 (2011) 2805–2811
Figure 1. Moraxella catarrhalis serotypes A, B and C oligosaccharide structures (top), along with a conserved-oligosaccharide core minus the terminal a-D-Galp-(1,4)-b-D-Galp
of the PK antigen (bottom).
Figure 2. The structure of the pentasaccharide-linker (1) and tetrasaccharide-linker (2).
all serotypes of M. catarrhalis. Although 2 is a reduced common
core in relation to 1, it also could be a component of a potential
vaccine candidate.
diethyl ether. Conditions were optimised to obtain the a-glycoside
and afford the fully protected novel pentasaccharide 9 in 53% yield.
Full NMR assignment of 6, 7 and 9 including labelling of the sugar
residues is provided in Table 1.
The chosen route for the synthesis of compound 1 is shown in
Scheme 1. The known trisaccharide derivative 3,10 which contained
Protected tetrasaccharide 7 and pentasaccharide 9 were then
deprotected by treatment with methoxide, followed by hydrogen-
olysis to afford deprotected compounds 2 and 1, respectively. For 9,
the initial methoxide treatment failed to remove the benzoate, and
the subsequent hydrogenolysis was slow to remove the benzyl
ethers (4 days). However, upon successful hydrogenolysis, the sin-
gle benzoate was easily removed by standard methoxide
treatment.
the
a-[p-(trifluoroacetamido)phenyl]ethyl glycosidic linkage, was
chosen as the key intermediate and was synthesised through 11
steps. The silyl protecting group TBDMS was removed from 3 to af-
ford the novel trisaccharide 4 in 75% yield. Trisaccharide 4 then
underwent a silver-triflate-promoted coupling in dichloromethane
with the chloroacetyl glucose derivative 510 (synthesised in 7 steps
from D-glucose) which had first been converted to the bromosugar,
to afford the novel tetrasaccharide 6 in 82% yield. The chloroacetyl
group installed to direct b-glycosylation was removed from 6 by
treatment with thiourea and NaHCO3 in methanol to afford 7 in
72% yield. The introduction of the benzylated glucosyl bromide do-
nor 811 was achieved by silver triflate-promoted glycosidation in
Full NMR assignment of 1 and 2 including labelling of the sugar
residues is provided in Table 2. Characterisation of 2 was achieved
by NMR analysis. Examination of the 1H 1D spectrum revealed only
three anomeric signals at 4.88, 4.56 and 4.41 ppm. The signals at
3
4.56 and 4.41 ppm were due to b-Glc residues with JH1H2 of 7.86

A. G. Pearson et al. / Carbohydrate Research 346 (2011) 2805–2811
2807
Scheme 1. Reagents and conditions: (a) aq AcOH, CH3CN, 75%; (b) (i) Br2, CH2Cl2, 0 °C; (ii) AgOTf, CH2Cl2, À35 °C, 82%; (c) thiourea, NaHCO3, CH3OH, 72%; (d) (i) NaOMe,
MeOH, (ii) H2, Pd/C; (e) 7, AgOTf, Et2O, À35 °C, 53%.
and 7.94 Hz, respectively. The signal at 4.88 ppm on integration
residue C and D were confirmed as the b-Glc-(1?4)-
a-Glc and b-
was deemed to be due to two anomeric signals, an
a
-Glc and the
Glc-(1?6)- -Glc by the presence of a correlation between H1 of
a
remaining b-Glc overlapped in the 1H dimension but well resolved
in the 13C dimension.
residue C to H4 of residue A and H1 of residue D to H6 of residue
A, respectively. These HMBC correlations were also confirmed with
a series of selective NOE experiments.
1H–13C-HSQC-TOCSY spectra with a 250 ms and 15 ms spin lock
pulse were acquired. The 15 ms 1H–13C-HSQC-TOCSY spectrum re-
vealed the H2/C2 and H3/C3 resonances for each of the glucose res-
idues. Additionally, the 250 ms 1H–13C-HSQC-TOCSY spectrum
revealed that the triplet at 4.01/77.1 ppm well resolved from the
Assignments of the ring protons were achieved using selective
experiments and low mixing time 1H–13C-HSQC-TOCSYs in combi-
nation with edited 1H–13C-HSQC and the 250 ms 1H–13C-HSQC-
TOCSY spectra. For instance for the clearly distinct anomeric pro-
tons of C1 and D1 selective TOCSY (180 ms) experiments were used
to locate the 1H resonances of their ring protons. There was overlap
in the 1H dimension of the anomeric protons of residues A and B
however, which complicated the assignment of residue B. This
was not an issue for residue A where the H3 proton at 4.01 ppm
was well resolved from other ring protons and could be used for
selective experiments to locate other ring protons within residue
A. Fortunately the excellent shift dispersion of the ring protons of
residue A made assignment of residue B resonances possible in a
selective TOCSY experiment with irradiation at 4.88 ppm. The
methylene protons of the ethylene bridge of the linker were as-
signed from the edited 1H–13C-HSQC, as well as the HMBC and
ring protons was associated with the
a-Glc residue A. This peak
had two weak correlations at 72.3 and 74.3 ppm. The peak at
72.3 ppm had already been identified as C2 and therefore the peak
at 4.01/77.1 ppm had to be C3 that would be expected to correlate
to C2 (d 72.3) and C4 (d 74.3) at a 15 ms spin lock time. Selective
irradiation of the H3 (4.01 ppm) in a selective COSY experiment
gave the 1H chemical shift of H2 (3.73 ppm) and H4 (3.85 ppm).
Examination of an overlapped edited 1H–13C-HSQC and the
250 ms 1H–13C-HSQC-TOCSY spectra revealed H5/C5 at 3.32/
70.4 ppm, respectively, by elimination since all other ring reso-
nances had been accounted for. The remaining methylene H6/C6
resonances were easily identified from the edited 1H–13C-HSQC
at 3.87/3.81 and 67.6 ppm, respectively.
NOE spectra. The central
a-Glc A H1 showed a correlation to the
With the assignment of the central
a
-Glc A in hand, the identi-
O-CH2 linker at 3.89/69.8 ppm. The methylene directly bonded to
the aromatic ring was clearly seen in the edited 1H–13C-HSQC spec-
trum at 2.96/35.4 ppm. Characteristic para-disubstituted aromatic
resonances were noted in the 1H spectrum and gave correlations in
the edited 1H–13C-HSQC at 7.49/123.0 and 7.39/130.6 ppm.
fication of the terminal b-Glc residues B, C and D was facilitated by
examination of selective NOE and 1H–13C-HMBC spectra. Residue B
was confirmed as the b-Glc-(1?3)-a-Glc by the presence of a cor-
relation between H1 of residue B to H3 of residue A. Likewise,

Table 1
1H and 13C chemical shifts (ppm) for protected compounds 6, 7, and 9 in CDCl3 at 298 K on a Bruker Avance spectrometer operating at 600 and 150 MHz, respectively

A. G. Pearson et al. / Carbohydrate Research 346 (2011) 2805–2811
2809
Table 2
1H and 13C chemical shifts (ppm) for target compounds 1 and 2 in D2O referenced to t-butanol at 298 K on a Bruker Avance spectrometer operating at 600 and 150 MHz,
respectively
1
H1
H2
H3
H4
H5
H6a
H6b
C1
C2
C3
C4
C5
C6
J1,2
A
B
C
D
E
4.91
3.7 Hz
4.95
8.0 Hz
4.56
7.9 Hz
4.56
3.80
3.34
3.31
3.41
3.41
4.14
3.51
3.45
3.55
3.71
3.78
3.69
4.23
3.94
3.81
3.90
3.77
3.72
3.74
3.70
3.69
3.66
98.6
72.5
74.0
73.7
76.3
72.2
76.4
76.4
76.4
75.2
73.3
74.7
70.2
70.2
70.2
70.2
70.5
70.2
70.2
70.2
70.2
69.2
61.2
61.3
61.3
61.3
3.41
3.38
3.47
3.38
102.1
101.1
103.4
97.9
ꢀ3.4
3.32
ꢀ3.4
3.32
7.9 Hz
5.34
3.8 Hz
1H d/13C d OCH2CH2Ar 3.88, 3.78/
OCH2CH2Ar 2.98, 2.88 (t, 2 H,
6.7 Hz)/34.6
COCF3 156.7 (q,
37 Hz)
COCF3 115.8 (q,
284 Hz)
Ar 7.42 (8.5 Hz)/122.9, 7.30/130.6,
quaternary: 138.5, 133.9
68.8
2
A
B
C
D
H1
J1,2
4.88
overlap
4.88
7.42 Hz
4.56
7.86 Hz
4.41
H2
H3
H4
H5
3.32
H6a
3.87
3.93
3.86
3.91
H6b
3.81
3.75
3.69
3.72
C1
98.4
C2
C3
C4
C5
C6
3.73
3.36
3.29
3.26
4.01
3.52
3.45
3.48
3.85
72.3
73.8
73.8
73.8
77.1
76.4
76.5
76.5
74.3
70.3
70.2
70.4
70.4
70.3
70.2
70.4
67.6
61.4
61.4
61.4
3.42
3.36
3.37
3.46
3.36
3.41
102.0
101.8
102.8
7.94 Hz
1H d/13C d OCH2CH2Ar 3.89/69.8
OCH2CH2Ar 2.96/35.4
COCF3 157.7 (q,
35 Hz)
COCF3 116.7 (q,
284 Hz)
Ar 7.49 (8.5 Hz)/123.0, 7.39/130.6,
quaternary: 139.0, 133.8
In a similar manner the chemical shift assignments of 1 were
determined. Compound 1 contains a central -Glc (A) with an ano-
meric resonance at 4.91 ppm with JH1H2 of 3.7 Hz and a terminal
Crucial for confirming the identity and connectivity of each of
the sugar rings was a series of selective NOE and a HMBC
experiments. HMBC correlations were confirmed for A1 to the
methylene linker O-CH2, B1 to A3, C1 to A4 and D1 to A6 protons
confirming the correct connectivity of the assigned sugar
residues.
a
3
a-Glc (E) at 5.34 ppm. Interestingly, the H6 protons of the central
-Glc A were located at 4.23 and 3.72 ppm from an edited
a
1H–13C-HSQC spectrum, well resolved from other H6/C6 reso-
nances from the other sugar rings. In addition, the
a-Glc A H3/C3
In summary, utilising D-glucose as the starting substrate we
signals were well resolved from other ring resonances at 4.14/
76.4 ppm. A 15 ms 1H–13C-HSQC-TOCSY experiment readily identi-
fied the 13C shift of the C2 and C4 resonances from the C3 reso-
nance. In the same way this spectrum also revealed the H2/C2
and H3/C3 positions from the H1/C1 line.
have described the 23-step synthesis of a novel pentasaccharide-
linker 1. The target pentasaccharide-linker represents a common-
core oligosaccharide from the LOS of M. catarrhalis serotypes A, B
and C. Full NMR characterisation of the key novel compounds have
been provided. In future studies the target pentasaccharide-linker
1 and the tetrasaccharide-linker 2 will be coupled to a carrier pro-
tein for investigation as potential therapeutic agents protecting
against otitis media.
In this spectrum, the central
a-Glc A was resolved from the b-
Glc residues B. The b-Glc residue B anomeric resonance was at
4.95 ppm. The terminal b-Glc residues C and D were overlapped
in the 1H dimension with anomeric signals at 4.56 ppm, however
their C1 resonances were resolved allowing unambiguous assign-
ment. Identification of the ring 1H resonances for each of the Glc
residues was greatly facilitated by acquiring a series of selective
TOCSY experiments irradiated at each anomeric proton position
with spin lock times starting at 15 ms and increasing in 15 ms
increments to 120 ms. With increasing mixing time successive
protons signals starting from H2 were readily identified. The
13C signals were identified in a similar manner using 1H–13C-
HSQC-TOCSY experiments also successively increasing the spin
lock time by 15 ms, starting at 15–120 ms.
1. Experimental
1.1. General methods
Solvents and reagents used were of analytical grade or distilled
prior to use. Reactions were monitored by thin layer chromatogra-
phy (TLC) using Merck pre-coated aluminium-backed silica plates
(60 F254). Plates were visualized after charring using ethanol/
H2SO4. Flash column chromatography was performed on Silica
Gel 60 (Merck).

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A. G. Pearson et al. / Carbohydrate Research 346 (2011) 2805–2811
1H and 13C NMR spectra for compound 4 was recorded on a
1.4. [p-(Trifluoroacetamido)phenyl]ethyl 2-O-benzyl-3-O-(2,3,4,
6-tetra-O-benzyl-b- -glucopyranosyl)-4-O-(2-O-benzoyl-3,4,6-
tri-O-benzyl-b- -glucopyranosyl)-6-O-(3,4,6-tri-O-benzyl-b-
glucopyranosyl)- -glucopyranoside (7)
Bruker Avance 300 MHz (300 and 75 MHz, respectively) at 298 K.
1H and 13C NMR spectra for the protected compounds 6, 7 and 9
were recorded on a Bruker Avance 600 MHz (600 and 150 MHz,
respectively) at 298 K. Signals are reported in chemical shifts (d in
ppm) relative to CDCl3 (d 1H 7.26 ppm; d 13C 77.0 ppm). Coupling
constants (J) are reported in Hz.
D
D
D-
a
-D
To a solution of 6 (169 mg, 82 mmol) in CH3OH (2 mL) and
CH2Cl2 (2 mL) was added thiourea (63 mg, 820 mmol) and NaHCO3
(69 mg, 820 mmol). The mixture was stirred at 55 °C until TLC re-
vealed complete reaction (10 h). The mixture was cooled to rt, di-
luted with CHCl3 (30 mL), and washed with saturated aq NaHCO3
(25 mL). Purification of the residue by silica gel chromatography
(hexanes/EtOAc 3:1) afforded the title compound (117 mg, 72%).
Full 1H and 13C NMR analysis are provided in Table 1. HRMS calcu-
lated for C118H120F3NO23Na [M+Na]+: 1998.8095. Found:
1998.8035.
The deprotected target compounds 1 and 2 (2.5 mg) were each
dissolved in 99.99% D2O (200 lL) and placed in 5 mm Shigemi
tubes. NMR spectra were acquired on a Bruker Avance 600 MHz
spectrometer equipped with a triple resonance (1H, 13C,15N) cryo-
probe operating at
a
probe temperature of 298 K. The 1H
(600.13 MHz) and 13C (150 MHz) spectra acquired included a 1H
1D, DEPTQ, edited 1H–13C-HSQC, 1H–13C-HSQC-TOCSY (15 ms,
30 ms and 160 ms spinlock pulses), 1H–13C-HMBC, gradient COSY,
selective gradient TOCSY (15, 30, 45, 60, 75, 90 and 160 ms spin-
lock pulses), and selective gradient NOESY (200 ms mixing time)
experiments. 1H and 13C chemical shifts were referenced to the
methyl resonances of t-butanol in D2O (d 1H 1.24 ppm; d 13C
30.29 ppm).
1.5. [p-(Trifluoroacetamido)phenyl]ethyl 3-O-(b-
syl)-4-O-(b- -glucopyranosyl)-6-O-(b- -glucopyranosyl)-
copyranoside (2)
D
-glucopyrano
D
D
a-D
-glu
Mass spectra were obtained on a Bruker Esquire 3000 electro-
spray ionization-ion trap mass spectrometer (LR-MS) and a Bruker
Daltonics Apex III 4.7e Fourier Transform spectrometer fitted with
an Apollo API source (HR-MS).
To a solution of 7 (88 mg, 44 mmol) in CH2Cl2 (1 mL) and
CH3OH (5 mL) at 0 °C was added Na metal (10 mg, 400 mmol).
After 3 h the solution was neutralised with Dowex 50 (H+) resin,
filtered and concentrated. The residue was dissolved in EtOAc/
MeOH/AcOH (2:2:1, 5 mL) and hydrogenolysed over 10% Pd/C at
40 psi for 20 h. The mixture was filtered, concentrated, and puri-
fied by HPLC (Synergi Hydro-RP Phenomenex column,
250 Â 10 mm, 3 mL/min, 16% CH3CN in H2O, eluted at 13.6 min)
to afford the title compound (13 mg, 33%). Full 1H NMR and 13C
NMR analyses are provided in Table 2. 13C NMR is consistent with
the limited assignments that were previously reported.9 ESIMS (m/
z): 904.3 [M+Na]+. HRMS calculated for C34H49F3NO22 [MÀH]À:
880.2704. Found: 880.2743.
1.2. [p-(Trifluoroacetamido)phenyl]ethyl 2-O-benzyl-3-O-(2,3,4,
6-tetra-O-benzyl-b-
D
-glucopyranosyl)-4-O-(2-O-benzoyl-3,4,6-
-glucopyranoside (4)
tri-O-benzyl-b- -glucopyranosyl)-a-D
D
To a solution of 310 (798 mg, 481 mmol) in CH3CN (5 mL) was
added AcOH (80% aqueous, 12 mL). The solution was stirred at
40 °C for 18 h, concentrated, and then purified by silica gel chroma-
tography (hexanes/EtOAc 3:1) to afford the title compound
(559 mg, 75%). 1H NMR (300 MHz, CDCl3) d 8.07–8.05 (m, 3 H);
7.53–7.00 (m, 47 H); 5.25–5.21 (m, 2H); 5.11 (d, J1,2 7.91 Hz, 1 H,
H-1 B); 4.98 (d, J 11.06 Hz, 1 H); 4.85–4.75 (m, 3 H); 4.72–4.40
(m, 14 H); 4.23 (d, J 11.5 Hz, 1 H); 3.81–3.27 (m, 18 H); 2.79 (t, J
7.11 Hz, 2 H). 13C NMR (75 MHz, CDCl3) d 165.5 (PhCOO); 154.8
(q, 37 Hz, COCF3); 138.9–120.5 (Ar); 115.9 (q, 287 Hz, COCF3);
102.3 (C-1 B); 97.3 (C-1 C); 96.1 (C-1 A); 84.9, 82.8, 82.4, 82.2,
78.5, 77.9, 74.8, 74.6, 74.6, 74.4, 72.4, 71.1 (12 Â C-2, C-3, C-4, C-
5); 75.6, 75.1, 74.8, 74.7, 74.5, 73.5, 73.3, 73.2 (8 Â PhCH2O);
69.4, 69.1, 68.1 (2 Â C-6 OBn, PhCH2C); 61.8 (C-6 A); 35.2 (PhCH2C).
HRMS calculated for C91H92F3NO18Na [M+Na]+: 1566.6159. Found:
1566.6233.
1.6. [p-(Trifluoroacetamido)phenyl]ethyl 2-O-benzyl-3-O-(2,3,4,
6-tetra-O-benzyl-b-
tri-O-benzyl-b- -glucopyranosyl)-6-O-(3,4,6-tri-O-benzyl-2-O-
{2,3,4,6-tetra-O-benzyl- -glucopyranosyl}-b- -glucopyrano
syl)- -glucopyranoside (9)
D-glucopyranosyl)-4-O-(2-O-benzoyl-3,4,6-
D
a
-D
D
a-D
To a solution of 7 (110 mg, 55.6 mmol) in diethyl ether (5 mL)
containing 4 Å molecular sieves was added a solution of 811
(250 mg, 412 mmol) in diethyl ether (5 mL). The mixture was stir-
red under argon at rt for 30 min. The reaction vessel was cooled to
À35 °C, and silver triflate (106 mg, 414 mmol) was added. After
50 min, the reaction vessel was allowed to warm to rt, triethyl-
amine was added and the stirring was continued for 20 min. The
mixture was diluted with CH2Cl2, filtered through Celite, and con-
centrated. Purification of the residue by silica gel chromatography
(hexanes/EtOAc 4:1) afforded the title compound (74 mg, 53%).
Full 1H and 13C NMR analyses are provided in Table 1. ESIMS (m/
1.3. [p-(Trifluoroacetamido)phenyl]ethyl 2-O-benzyl-3-O-(2,3,4,
6-tetra-O-benzyl-b-
tri-O-benzyl-b- -glucopyranosyl)-6-O-(3,4,6-tri-O-benzyl-2-O-
chloroacetyl-b- -glucopyranosyl)- -glucopyranoside (6)
D-glucopyranosyl)-4-O-(2-O-benzoyl-3,4,6-
D
D
a-D
To a solution of 510 (292 mg, 511 mmol) in CH2Cl2 (5 mL) at 0 °C
was added bromine (2.8 mL, 545 mmol). After 10 min, toluene was
added, the solution was concentrated, and the residue was co-
evaporated twice with toluene. The residue in CH2Cl2 (10 mL)
was added to a solution of 4 (526 mg, 341 mmol) in CH2Cl2 con-
taining 4 Å molecular sieves. The mixture was stirred under argon
for 30 min at rt; the temperature was then lowered to À35 °C and
silver triflate (175 mg, 681 mmol) was added. The mixture was
stirred at À35 °C for 45 min, at rt for 30 min, then triethylamine
was added, and the stirring was continued for 20 min. The mixture
was diluted with CH2Cl2, filtered through Celite and concentrated.
Purification of the residue by silica gel chromatography (hexanes/
EtOAc 4:1) afforded the title compound (576 mg, 82%). Full 1H and
13C NMR analyses are provided in Table 1. HRMS calculated for
z): 2497.8 [MÀH]À. HRMS calculated for
C152H154F3NO28Na
[M+Na]+: 2521.0502. Found: 2521.0524.
1.7. [p-(Trifluoroacetamido)phenyl]ethyl 3-O-(b-
osyl)-4-O-(b- -glucopyranosyl)-6-O-(2-O-{ -glucopyranosyl}-
b- -glucopyranosyl)- -glucopyranoside (1)
D-glucopyran
D
a-D
D
a-D
To a solution of 9 (63 mg, 25.2 mmol) in CH2Cl2 (1 mL) and
CH3OH (5 mL) Na metal (10 mg, 400 mmol) was added at 0 °C. After
3 h the solution was neutralised with Dowex 50 (H+) resin, filtered
and concentrated. The residue was dissolved in EtOAc/MeOH/AcOH
(2:2:1, 5 mL) and hydrogenated over 10% Pd/C at 40 psi for 4 d. The
mixture was filtered, concentrated, and purified by HPLC (Synergi
Hydro-RP Phenomenex column, 250 Â 10 mm, 3 mL/min, 16%
CH3CN in H2O, eluted at 14.8 min) to afford the de-O-benzylated
C
120H121ClF3NO24Na [M+Na]+: 2074.7811. Found: 2074.7720.

A. G. Pearson et al. / Carbohydrate Research 346 (2011) 2805–2811
2811
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compound that still contained the single benzoate. This was treated
with Na metal in anhydrous CH3OH at 0 °C for 2 h, neutralised with
Dowex 50 (H+) resin, filtered, and concentrated to afford the title
compound (8.5 mg, 32%). Full 1H NMR and 13C NMR analyses are
provided in Table 2. ESIMS (m/z): 1042.4 [MÀH]À. HRMS calculated
for C40H60F3NO27Na [M+Na]+: 1066.3203. Found: 1066.3239.
Acknowledgements
The authors acknowledge Queensland NMR network (QNN) and
Institute for Glycomics for access to facilities and funding.
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