Synthesis of phosphorylated fragments of Streptococcus pneumoniae type 19F capsular polysaccharide

Article abstract of DOI:10.1039/b205684d

Serotype 19F is one of the most virulent of the Streptococcus pneumoniae serotypes, causing meningitis, otitis media, septicemia and pneumonia. Effective protection against infection is mediated by antibodies directed against its capsular polysaccharide (CPS), composed of the trisaccharide repeating units (→4-β-D-ManpNAc-(1→4)-α-D-Glcp-(1→2)-α-L- Rhap-1→) bridged by phosphate diesters. Unfortunately, CPSs are typical T-independent type 2 immunogens. Therefore, the conjugation of putative saccharide haptens to a protein carrier, which invokes T-cell involvement, has proved to be extremely effective in enhancing the immune response. In a project aimed at ascertaining the role played by the different structural factors in the immunogenicity of new vaccines against Streptococcus pneumoniae type 19F, we planned the synthesis of a family of trisaccharide repeating unit derivatives where the phosphate group is a) absent; b) linked only to the rhamnose unit; or c) linked only to the N-acetylmannosamine unit. Compound of type a) and c) also bear an appropriate spacer for conjugation to the protein carrier. Moreover, the synthesis of a dimer of the CPS repeating unit, in which a phosphate diester bridges two repeating units, was carried out.

Full text of DOI:10.1039/b205684d

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Synthesis of phosphorylated fragments of Streptococcus
pneumoniae type 19F capsular polysaccharide
Giuseppina Soliveri,a Anna Bertolotti,a Luigi Panza,b Laura Poletti,a Christopher Jonesc and
Luigi Lay*a
1
a Department of Organic and Industrial Chemistry, University of Milan, Milan, Italy
b Department of Medical Science, University of Eastern Piemonte, Novara, Italy
c National Institute of Biological Standards and Control, South Mimms, Potters Bar, Herts,
UK EN6 3QG
Received (in Cambridge, UK) 12th June 2002, Accepted 12th July 2002
First published as an Advance Article on the web 29th August 2002
Serotype 19F is one of the most virulent of the Streptococcus pneumoniae serotypes, causing meningitis, otitis
media, septicemia and pneumonia. Effective protection against infection is mediated by antibodies directed against
its capsular polysaccharide (CPS), composed of the trisaccharide repeating units ( 4-β--ManpNAc-(1 4)-α--
Glcp-(1 2)-α--Rhap-1 ) bridged by phosphate diesters. Unfortunately, CPSs are typical T-independent type
2 immunogens. Therefore, the conjugation of putative saccharide haptens to a protein carrier, which invokes
T-cell involvement, has proved to be extremely effective in enhancing the immune response. In a project aimed
at ascertaining the role played by the different structural factors in the immunogenicity of new vaccines against
Streptococcus pneumoniae type 19F, we planned the synthesis of a family of trisaccharide repeating unit derivatives
where the phosphate group is a) absent; b) linked only to the rhamnose unit; or c) linked only to the N-acetylmanno-
samine unit. Compound of type a) and c) also bear an appropriate spacer for conjugation to the protein carrier.
Moreover, the synthesis of a dimer of the CPS repeating unit, in which a phosphate diester bridges two repeating
units, was carried out.
responses induced by glycoconjugate vaccines can be tailored to
optimize protection and immunogenicity, no systematic study
Introduction
Vaccination is the most cost-effective means to protect popu-
lations against infectious diseases caused by pathogenic
bacteria. Amongst these Streptococcus pneumoniae1 is one of
the major causes of death and disability throughout the world,2
causing meningitis, otitis, septicemia and pneumonia, both in
developed and in developing countries.3 Streptococcus pneu-
moniae belongs to the class of encapsulated bacteria that are
enveloped by a carbohydrate coat (capsule). This exerts a pro-
tective function against the host’s immune defense. The cap-
sular polysaccharides (CPSs) of the bacteria, which define the
different serotypes, are the key determinants of virulence, and
the resistance to infection from encapsulated bacteria is medi-
ated by the presence of antibodies directed against their CPSs.
Ninety serotypes4 of S. pneumoniae have been defined, associ-
ated with different diseases, age groups and geographical areas.
Therefore, the design of a “universal” vaccine is extremely
complex. Nevertheless, five polyvalent vaccines against these
bacteria, based on a mixture of some of their CPSs, have been
successfully developed.3a Unfortunately, as CPSs are typical
T-independent type 2 immunogens, the antibody response
induced by their administration drops in a few years in adults,
and children under two years of age are unresponsive. The
immune response can be improved by chemical conjugation of
the saccharide vaccines to a protein carrier (typically, CRM197,
a variant of diphtheria toxin), which invokes T-cell involve-
ment.5 Glycoconjugate vaccines of this kind have proved to
be extremely effective against Haemophilus influenzae type B
infections.
has been carried out to elucidate the principles on which this
would be based. Such knowledge would greatly aid the rapid
and rational development of novel vaccines against important
diseases and the optimization of the immunogenicity of
existing vaccines.
We are currently involved in a project aimed at a rational
development of glycoconjugate vaccines by elucidation of their
mechanism of action and the development of standardized
synthetic methodologies The whole project can contribute to
determining the role played by different structural factors in the
activity of new glycoconjugate vaccines. In particular, synthetic
oligoglycoconjugates can be useful in establishing the chain
length, the number of incorporated carbohydrate residues and
other structural features that are required for optimal immuno-
logical activity. Furthermore, they can overcome the problems
of product heterogeneity and biological contamination associ-
ated with the use of native carbohydrate material. Finally, they
allow a full characterisation and immunological evaluation of
the synthesised compounds. This will aid the development of
new vaccines and their acceptance onto the market by licensing
authorities.
The general target is a glycoconjugate molecule in which a
saccharidic portion is covalently linked to a carrier protein
through a spacer. We focused our attention on serotype 19F of
S. pneumoniae, whose CPS is composed of trisaccharide repeat-
ing units
(
4-β--ManpNAc-(1 4)-α--Glcp-(1 2)-α-L-
Rhap-1 ) linked through phosphodiester bridges. Syntheses
of the saccharidic part of the 19F repeating unit have already
been reported by us7 and others.8
In order to optimize the design of the saccharidic part we
needed to study the role of the phosphate group and the linkage
between the saccharidic part and the carrier protein. An
important issue to be addressed is the role of the phosphate
Rational development of glycoconjugate vaccines is ham-
pered by the lack of understanding of their fundamental
science—including the molecular mechanisms by which T-cell
dependent immune responses against the saccharide are
induced. Despite suggestions in the literature6 that the immune
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J. Chem. Soc., Perkin Trans. 1, 2002, 2174–2181
This journal is © The Royal Society of Chemistry 2002
DOI: 10.1039/b205684d

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group, as charged groups are often important epitopes for
pneumococcal PSs.9 In the 19F CPS the phosphate groups
are linked to the rhamnose of one unit and to the N-
acetylmannosamine of the subsequent unit, and thus it is of
interest to assess the structure–activity relationship when the
phosphate is a) absent; b) linked only to the rhamnose unit; c)
linked only to the N-acetylmannosamine unit; d) bridging two
repeating units.
A second question involves evaluation of the influence of the
configuration of the linkage between the saccharidic part and
the carrier protein, as this could affect the presentation of the
antigen.
To look into these problems, in the present paper we describe
the synthesis of a family of trisaccharides (4, 11ꢀ, 11ꢁ, 12ꢀ,
12ꢁ, 15) covering all of the above aspects. In compound 4 the
phosphate group is α-linked to the anomeric position of
the rhamnose unit. Compounds 11ꢀ and 11ꢁ bear a 4Љ-O-
phosphate group and have a linker at the anomeric position of
the rhamnose, which is α-linked in 11ꢀ, and β-linked in 11ꢁ.
Compounds 12ꢀ and 12ꢁ correspond, respectively, to 11ꢀ and
11ꢁ in which the phosphate group is absent. Finally, in dimer
15, bearing only the α-oriented spacer arm, the phosphate
group is linked both to the rhamnose and to the N-acetyl-
mannosamine units.
It is planned to test the above compounds as inhibitors in an
ELISA experiment in order to assess their ability to inhibit the
binding of the CPS to anti-Pn19F antiserum. This will indicate
which residues are most important for oligosaccharide–
antibody binding, although not those factors which may be
predictive of the induction of productive immunity by the final
oligosaccharide conjugate.
Scheme 1 Reagents and conditions: (a) EtOH, Wilkinson’s catalyst,
DBU, quantitative; (b) NIS, dibenzyl phosphate, CH2Cl2–Et2O, ms 4 Å,
72%; (c) H2, Pd(OH)2, EtOAc–MeOH–H2O ϩ HOAc, then Dowex
50W-X8 (Naϩ form), 95%.
Results and discussion
The strategically protected trisaccharide 1, corresponding to
the repeating unit of the capsular polysaccharide of Strepto-
coccus pneumoniae type 19F, has been obtained on a multigram
scale via a procedure previously described by us7 and employed
for the synthesis of compounds 4, 11ꢀ, 11ꢁ, 12ꢀ, 12ꢁ, 15.
The 1-O-allyl group of compound 1 was isomerized10 into
the corresponding 1-O-propenyl glycoside 2, which was directly
used as a glycosyl donor11 with dibenzyl phosphate as a nucleo-
phile. The glycosylation was promoted by NIS and TMSOTf
and afforded the glycosyl 1-O-dibenzyl phosphate 3 as the pure
α anomer (72%). Removal of the protecting groups was
achieved by hydrogenolysis over Pd(OH)2 and gave trisacchar-
ide 4 as its disodium salt (Scheme 1).
On the other hand, complete removal of the allyl group from
1 allowed the introduction of the N-trifluoroacetamidopropyl
group as an N-protected spacer arm for conjugation to protein
carriers. Thus, hydrolysis of 2 with iodine in moist tetrahydro-
furan12 afforded the known7b trisaccharide 5, which was
converted into the trichloroacetimidate 613 by treatment with
trichloroacetonitrile in CH2Cl2 in the presence of a catalytic
amount of DBU (Scheme 2). Despite many attempts, performed
employing different Lewis acids as well as different reaction
conditions, the glycosylation with 3-(trifluoroacetamido)-
propan-1-ol (purchased by Fluka) invariably led to α–β mix-
tures of the glycoside 7. The best result was achieved in dry
Et2O at room temperature using BF3ؒOEt2 as the acidic pro-
moter (98% yield of a ca. 5 : 2, α : β mixture). Nevertheless, we
took advantage of this result to explore the influence on the
antigenic presentation of the configuration of the linkage
joining the saccharidic moiety to the protein carrier. The two
anomers of glycoside 7 were carefully separated by medium
pressure chromatography giving pure 7ꢀ and 7ꢁ in 69 and 27%
yield, respectively. The benzylidene acetal of 7ꢀ was hydrolysed
with 90% aq. trifluoroacetic acid in CH2Cl2 and the resulting
diol 8ꢀ was regioselectively 6c-O-benzylated via the correspond-
Scheme 2 Reagents and conditions: (a) I2, THF–H2O,11 quantitative;
(b) Cl3CCN, DBU, CH2Cl2, 96%; (c) HO(CH2)3NHCOCF3, BF3ؒOEt2,
Et2O, rt, 98%, α : β = 5 : 2.
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Scheme 3 Reagents and conditions: (a) 90% aq. CF3COOH, CH2Cl2, 0 ЊC, quantitative; (b) Bu2SnO, toluene, then BnBr, tetrabutylammonium
iodide, 9ꢀ: 78%, 9ꢁ: 76%; (c) diphenyl phosphorochloridate, DIPEA, DMAP, CH2Cl2, 10ꢀ: 49%, 10ꢁ: 61%; (d) H2, Pd(OH)2, MeOH–H2O; (e) H2
(5 bar), PtO2, H2O, then Dowex 50W-X8 (Naϩ form), 11ꢀ: 71%, 11ꢁ: 81%.
ing stannylene acetal to afford compound 9ꢀ in 78% overall
yield (Scheme 3). 4c-O-Phosphorylation of 9ꢀ was carried out
with diphenyl phosphorochloridate in CH2Cl2 in the presence
of diisopropylethylamine and 4-N,N-dimethylaminopyridine
(DMAP)14 providing trisaccharide 10ꢀ in 49% yield. Complete
removal of the protecting groups was accomplished in two
steps. First, the benzyl ethers were cleaved by hydrogenolysis
over Pd(OH)2 in a methanol–water mixture. Thereafter, the
phenyl esters were removed by hydrogenation (5 bar) over PtO2
in water. After filtration over a Celite pad, the compound was
eluted with water through a column filled with Dowex 50W-X8
resin (Naϩ form) and lyophilised, giving 4c-O-phosphoryl-
ated trisaccharide 11ꢀ in 71% overall yield. Likewise, through
the same reaction sequence applied for the synthesis of 11ꢀ,
trisaccharide 11ꢁ was obtained from 7ꢁ (Scheme 3).
5). After work up and purification by filtration over a short
column of silica gel, 13 was recovered as its triethylammonium
salt in almost quantitative yield. The coupling of 13 with 5 was
carried out using pivaloyl chloride as a condensing agent in
pyridine and followed by in situ oxidation with iodine in
pyridine–water18 to give phosphodiester-linked hexasaccharide
14 its triethylammonium salt in 61% overall yield (Scheme 5).
The 1H NMR spectrum of compound 14 showed extensive
overlap of the signals, which did not allow its complete spectro-
scopic characterization. Only after removal of the protecting
groups by hydrogenation over Pd(OH)2 on charcoal, followed
by filtration over Dowex 50W-X8 resin (Naϩ form), was hexa-
saccharide 15 recovered as a white glass and fully characterized
by NMR spectroscopy. Moreover, HPLC analysis showed the
presence of a 9 : 1, α : β mixture of diastereoisomers of 15,
differing in the configuration of the anomeric linkage of the
inner rhamnose unit (unit d), as deduced from the NMR
spectra.
Moreover, to evaluate the influence on the immunological
activity of the 4c-O-phosphorylation 4c-OH trisaccharides 12ꢀ
and 12ꢁ were synthesised by hydrogenation over Pd(OH)2 of 7ꢀ
and 7ꢁ, respectively (Scheme 4).
Conclusions
We have described the synthesis of a new family of Strepto-
coccus pneumoniae 19F CPS fragments, differing in the position
of the phosphate group or in the anomeric configuration of a
spacer suitable for conjugation to a carrier protein. These com-
pounds allow the assessment of the role of the phosphate group
in the immunological activity of the fragments. Moreover,
the influence of the configuration of the linkage between the
saccharidic part and the carrier protein linkage in presenting
the antigen can be studied starting from compounds 12ꢀ,ꢁ.
Experimental
Scheme 4 Reagents and conditions: (a) H2, Pd(OH)2, MeOH–H2O,
95%.
TLC was carried out on Merck silica-gel 60 F254 plates (0.25
mm thickness), and spots were visualized by spraying with
a solution containing H2SO4 (31 cm3), ammonium molybdate
(21 g) and Ce(SO4)2 (1 g) in 500 cm3 of water, followed by
heating at 110 ЊC for 5 min. Column chromatography was
performed by the flash procedure using Merck silica-gel 60
(230–400 mesh). Melting points were determined with Büchi
apparatus and are not corrected. Optical rotations were meas-
ured at room temperature (25 ЊC) with a Perkin-Elmer 241
polarimeter and [α]D values are given in units of in 10Ϫ1 deg cm2
Finally, the synthesis of the hexasaccharide 15 was
attempted. From the other methods available for the synthesis
of glycosylphosphodiester bridges,15 we chose the H-phosphon-
ate approach.16 Within this context, in principle two different
procedures can be adopted, the first one consisting of the
synthesis of the anomeric H-phosphonate monoester16,17 of
trisaccharide 5 followed by coupling with compound 9ꢀ. Dis-
appointingly, we invariably obtained a ca. 7 : 3, α : β mixture
of anomers, using both the in situ formed triimidazolyl-
phosphine16,18 and 2-chloro-4H-1,3,2-benzodioxaphosphinin-
4-one19 in the formation of the H-phosphonate monoester on
5. Gratifyingly, the formation of the H-phosphonate monoester
on trisaccharide 9ꢀ,20 followed by the coupling with compound
5, allowed better stereocontrol—even if not absolute, as it
turned out at the end of our attempts. Hydrogen phosphonate
13 was obtained by the treatment of 9ꢀ with a slight excess of
2-chloro-4H-1,3,2-benzodioxaphosphinin-4-one in a mixture
of dry acetonitrile and pyridine at room temperature (Scheme
g
Ϫ1. NMR spectra were recorded at 30 ЊC on Bruker AC 300,
Varian Unity 500 and Varian XL 200 Gemini spectrometers.
In the description of the NMR spectra a, b, c, d, e and f refer to
the monosaccharide units in the oligosaccharides (a = reducing
end), and coupling constants are given in Hz. Signals corre-
sponding to aromatic carbons were omitted from the 13C
spectra. Analytical HPLCs were carried out on a system con-
sisting of a SpectraPhysics P2000 gradient pump, a Spectra-
Physics 100 UV/vis detector and a SpectraPhysics SP4270 inte-
grator, with a 20 cm × 4.6 mm HyperCarb column (5 µm)
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J. Chem. Soc., Perkin Trans. 1, 2002, 2174–2181

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Scheme 5 Reagents and conditions: (a) 2-chloro-4H-1,3,2-benzodioxaphosphinin-4-one, py, CH3CN, rt; (b) 5, PivCl, py, rt; (c) I2, py–H2O 19 : 1, rt,
61% from 9ꢀ; (d) H2, Pd(OH)2, MeOH–H2O, then Dowex 50W-X8 (Naϩ form), 96%.
(where not indicated). Elemental analyses were performed using
the Carlo Erba elemental analyzer 1108.
H-2a, H-3a, H-5a, H-3b, H-4b, H-4c, H-6c), 4.26 (1 H, d,
J 11.9, CHHPh), 4.45–5.04 (18 H, m, 7 × CH2Ph, CHHPh,
H-1b, H-1c, H-2c), 5.45 (1 H, s, CHPh), 5.55 (1 H, d, J 9.3,
NHAc), 5.68 (1 H, br d, J1a,2a 5.9, H-1a) and 7.05–7.50 (45 H,
m, 9 × Ph); δC(75.46 MHz; CDCl3, Me4Si) 17.8 (q, C-6a), 23.1
(q, CH3CO), 50.5 (d, C-3c), 67.9, 68.6, 69.4, 71.1, 72.1, 72.7,
73.3, 74.6, 75.2 (9 × t, C-6b, C-6c, 8 × CH2Ph), 67.1, 69.9, 70.4,
74.4, 75.6, 75.9, 77.7, 78.4, 79.3, 80.3 (10 × d, C-2a, C-3a, C-4a,
C-5a, C-2b, C-3b, C-4b, C-5b, C-3c, C-4c, C-5c), 96.1 (d, JC,P
8.7, C-1a), 96.9, 99.8 (2 × d, C-1b, C-1c), 101.6 (d, CHPh) and
177.7 (s, CONH); δP(200 MHz; CDCl3, D2O–H3PO4–H2O =
90 : 8.5 : 1.5) Ϫ2.39.
Prop-1-enyl (2-acetamido-3-O-benzyl-4,6-O-benzylidene-2-
deoxy-ꢁ-D-mannopyranosyl)-(1 4)-(2,3,6-tri-O-benzyl-ꢀ-D-
glucopyranosyl)-(1 2)-3,4-di-O-benzyl-ꢀ-L-rhamno-
pyranoside 2
To a solution of 1 (200 mg, 0.17 mmol) in dry THF (30 cm3)
was added a catalytic amount of (cycloocta-1,5-diene)bis-
(methyldiphenylphosphine)iridium hexafluorophosphate. The
stirred solution was degassed by freezing the solvent and allow-
ing it to warm to room temperature under vacuum and then
placed under H2 to activate the catalyst (the slightly red sus-
pension became pale yellow). After 2 min, the solution was
degassed once more and stirred at room temperature under N2.
After 2 h, TLC analysis showed a complete conversion of the
allyl ether into the 1-O-propenyl ether. The reaction mixture
was concentrated to dryness and the resulting yellow oil was
filtered on a short column of silica gel (petroleum ether–EtOAc
7 : 3) to remove the catalyst. Crude 2 (180 mg) was used directly
in the next step without further characterization.
(2-Acetamido-2-deoxy-ꢁ-D-mannopyranosyl)-(1 4)-(ꢀ-D-
glucopyranosyl)-(1 2)-ꢀ-L-rhamnopyranosyl dihydrogen
phosphate disodium salt 4
Trisaccharide 3 (32 mg, 0.022 mmol) was dissolved in EtOAc–
MeOH–H2O 1 : 1 : 1 (6 cm3, containing 1 drop of HOAc) and
hydrogenolyzed at 10 bar over Pd(OH)2 (40 mg) for 16 h. The
mixture was filtered over a Celite pad and the filtrate was con-
centrated, then eluted with water through a column filled with
Dowex 50W-X8 resin (Naϩ form). Lyophilization gave tri-
saccharide 4 (13 mg, 95%) as a white foam (Found: C, 36.85; H,
5.25; N, 2.10. C20H34NO18PNa2 requires C, 36.80; H, 5.24; N,
2.14%); [α]D ϩ18.6 (c 0.5 in H2O); the NMR spectrum of
compound 4 is reported in Table 1.
(2-Acetamido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-ꢁ-D-
mannopyranosyl)-(1 4)-(2,3,6-tri-O-benzyl-ꢀ-D-glucopyrano-
syl)-(1 2)-3,4-di-O-benzyl-ꢀ-L-rhamnopyranosyl dibenzyl
phosphate 3
Propenyl glycoside 2 (170 mg, 0.14 mmol) was dissolved in
CH2Cl2–Et2O 2 : 1 (6 cm3) containing 4 Å molecular sieves
(100 mg). The suspension was stirred under Ar for 1 h, then
cooled at Ϫ40 ЊC. NIS (150 mg, 0.6 mmol) and dibenzyl phos-
phate (160 mg, 0.57 mmol) were added, followed by a 0.1 M
solution of TMSOTf in CH2Cl2 (14 mm3, 0.0014 mmol). After
30 min the reaction mixture was neutralised with Et3N, filtered
over a Celite pad and the solvent evaporated. The crude residue
was first purified by flash chromatography (petroleum ether–
EtOAc 6 : 4). Final purification was achieved by medium pres-
sure chromatography (same eluent) to give 3 (143 mg, 72%) as a
white solid (Found: C, 72.5; H, 5.7; N, 9.1. C93H88NO18P
requires C, 72.6; H, 5.75; N, 9.1%); mp 115–116 ЊC; [α]D ϩ6.7
(c 1 in CHCl3); δH(300 MHz; CDCl3, Me4Si) 1.31 (3 H, d, J6a,5a
6.0, H-6a), 1.80 (3 H, s, CH3CO), 3.01–3.10 (1 H, m, H-5c), 3.18
(1 H, br d, J6b,6Јb 10.9, H-6b), 3.29–3.34 (2 H, m, H-6Јb, H-3c),
3.49–3.63 (4 H, m, H-4a, H-2b, H-5b, H-6c), 3.76–4.08 (7 H, m,
O-(2-Acetamido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-ꢁ-D-
mannopyranosyl)-(1 4)-O-(2,3,6-tri-O-benzyl-ꢀ-D-gluco-
pyranosyl)-(1 2)-3,4-di-O-benzyl-ꢀ,ꢁ-L-rhamnopyranosyl
trichloroacetimidate 6
Compound 5 (872 mg, 0.753 mmol), obtained from trisacchar-
ide 2 as previously described,7b was dissolved in dry CH2Cl2
(30 cm3) under a nitrogen atmosphere. After cooling to 0 ЊC,
CCl3CN (529 mm3, 5.27 mmol) and DBU (11 mm3, 0.075
mmol) were added under N2 and the mixture was stirred for 15
min at 0 ЊC. The mixture was concentrated and the residue was
purified by flash chromatography (petroleum ether–EtOAc 7 : 3
ϩ 1% Et3N) to give 6 (only α anomer, 942 mg, 96%) isolated as
a glass (Found: C, 65.40; H, 5.90; N, 2.10. C71H75N2O15Cl3
requires C, 65.46; H, 5.80; N, 2.15%); [α]D ϩ3.0 (c 1 in CHCl3);
δH(300 MHz; CDCl3, Me4Si) 1.39 (3 H, d, J6a,5a 6.0, H-6a), 1.79
(3 H, s, CH3CO), 3.06 (1 H, dt, J5c,6c 9.8, J5c,4c 9.8, J5c,6Јc 4.9,
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Table 1 1H NMR (D2O, 500 MHz, 303 K) and 13C NMR (D2O, 125.72 MHz, 303 K) of compound 4a
H-1
C-1
H-2
C-2
H-3
C-3
H-4
C-4
H-5
C-5
H-6
C-6
Residue
H-6Ј
NAc
a
b
c
5.37
93.8
5.04
98.8
7.87
100.2
3.97
70.2
3.56
72.2
4.53
54.2
[3.95]
78.6
3.87
72.2
3.80
73.0
3.45
73.1
3.67
79.7
3.51
67.5
[3.90]
70.3
4.07
71.3
3.43
77.5
1.27
17.8
3.74
60.8
3.91
61.3
3.74
3.80
2.05
23.0
a Chemical shifts referenced to highfield N-acetyl at 2.050 and 23.00 ppm. [ ] = uncertain assignment.
H-5c), 3.29 (1 H, br d, J6Јb,6b 10.8, H-6Јb), 3.34 (1 H, dd, J2c,3c
4.2, J3c,4c 9.8, H-3c), 3.44 (1 H, br d, J6b,6Јb 10.8, H-6b), 3.50–
3.69 (5 H, m, H-3a, H-4a, H-2b, H-5b, H-4c), 3.83–4.10 (5 H,
m, H-5a, H-3b, H-4b, H-6c, H-6Јc), 4.13 (1 H, br s, H-2a), 4.30
(1 H, d, J 11.9, CHHPh), 4.48 (1 H, d, J 12.9, CHHPh), 4.50
(1 H, br s, H-1c), 4.58–4.74 (8 H, m, H-2c, 3 × CHHPh,
2 × CH2Ph), 4.81 (1 H, d, J 11.8, CHHPh), 4.96–5.01 (3 H, m,
2 × CHHPh, H-1b), 5.47 (1 H, s, CHPh), 5.51 (1 H, d, J 9.6,
NHAc), 6.28 (1 H, br s, H-1a), 7.05–7.55 (35 H, m, 7 × Ph) and
8.53 (1 H, s, NH).
H-3c, H-4c, H-6Јc, O(CH2)2CH2NHCOCF3, OCH2(CH2)2-
NHCOCF3), 3.76–4.11 (7 H, m, H-2a, H-4a, H-5a, H-3b, H-4b,
H-5b, H-6c), 4.26 (1 H, d, J 11.9, CHHPh), 4.48 (1 H, br s,
H-1c), 4.50 (1 H, d, J 12.0, CHHPh), 4.57–4.76 (9 H, m,
3 × CHHPh, 2 × CH2Ph, H-1a, H-2c), 4.83 (1 H, d, CHHPh),
4.94 (1 H, d, J1b,2b 3.8, H-1b), 4.96 (1 H, d, J 11.0, CHHPh),
4.98 (1 H, d, J 11.8, CHHPh), 5.48 (1 H, s, CHPh), 5.50 (1 H,
d, J 9.7, NHAc), 6.75 (1 H, br s, NHCOCF3) and 7.05–7.60
(35 H, m, 7 × Ph); δC(75.46 MHz; CDCl3, Me4Si) 18.0 (q, C-6a),
23.1 (q, CH3CO), 28.3 (t, OCH2CH2CH2NHCOCF3), 38.3 (t,
O(CH2)2CH2NHCOCF3), 50.4 (d, C-2c), 65.8, 67.9, 68.6, 71.2,
72.2, 72.9, 73.5, 74.7, 75.3 (9 × t, C-6b, C-6c, 6 × CH2Ph,
OCH2(CH2)2NHCOCF3), 67.1, 68.6, 69.9, 74.9, 75.6, 75.9,
78.5, 79.0, 79.6, 80.0, 80.3 (11 × d, C-2a, C-3a, C-4a, C-5a,
C-2b, C-3b, C-4b, C-5b, C-3c, C-4c, C-5c), 97.1, 97.7, 99.6 (3 ×
d, C-1a, C-1b, C-1c), 101.7 (d, CHPh) and 170.4 (s, CONH).
3-Trifluoroacetamidopropyl (2-acetamido-3-O-benzyl-4,6-O-
benzylidene-2-deoxy-ꢁ-D-mannopyranosyl)-(1 4)-(2,3,6-tri-O-
benzyl-ꢀ-D-glucopyranosyl)-(1 2)-3,4-di-O-benzyl-ꢀ,ꢁ-L-
rhamnopyranoside 7ꢀ, 7ꢁ
Compound 6 (343 mg, 0.26 mmol) and 3-(trifluoroacetamido)-
propan-1-ol (608 mg, 3.55 mmol) were dissolved in dry Et2O at
rt, then BF3ؒEt2O (66 mm3, 0.52 mmol) was added dropwise
under N2 with stirring for 10 min. The reaction mixture was
quenched with Et3N and concentrated under reduced pressure.
The residue was purified by flash chromatography (petroleum
ether–EtOAc 6 : 4) to give 7 as a mixture of anomers (338 mg,
98%). The anomers were separated by medium pressure
chromatography (petroleum ether–EtOAc 65 : 35). First, the
β anomer 7ꢁ (94 mg) was obtained as a glass (Found: C,
67.70; H, 6.25; N, 2.10. C74H81N2O16F3 requires C, 67.77; H,
6.23; N, 2.14%); [α]D ϩ30.6 (c 1 in CHCl3); δH(300 MHz;
CDCl3, Me4Si) 1.38 (3 H, d, J6a,5a 6.0, H-6a), 1.72 (3 H, s,
CH3CO), 1.70–1.80 (2 H, m, OCH2CH2CH2NHCOCF3),
1.99–3.14 (2 H, m, H-5c, O(CH2)2CHHNHCOCF3), 3.23–3.64
(11 H, m, H-3a, H-4a, H-5a, H-2b, H-6b, H-6Јb, H-3c, H-4c,
H-6Јc, O(CH2)2CHHNHCOCF3, OCHH(CH2)2NHCOCF3),
3.74–3.82 (1 H, m, OCHH (CH2)2NHCOCF3), 3.92 (1 H, t,
J 9.2, H-4b or H-3b), 3.99 (1 H, t, J 9.2, H-4b or H-3b), 4.07
(1 H, dd, J6c,6Јc 10.4, J6c,5c 4.9, H-6c), 4.21–4.26 (3 H, m,
H-2a, H-5b, CHHPh), 4.39 (1 H, s, H-1a), 4.45 (1 H, br s,
H-1c), 4.47–4.74 (8 H, m, 7 × CHHPh, H-2c), 4.80 (1 H, d, J
11.7, CHHPh), 4.83 (1 H, d, J 11.7, CHHPh), 4.97 (1 H, d, J
11.7, CHHPh), 4.99 (1 H, d, J 10.9, CHHPh), 5.43 (1 H, d, J1b,2b
3.7, H-1b), 5.47 (1 H, s, CHPh), 5.49 (1 H, d, J 9.7, NHAc),
6.99 (1 H, br s, NHCOCF3) and 7.08–7.52 (35 H, m, 7 × Ph);
δC(75.46 MHz; CDCl3, Me4Si) 17.8 (q, C-6a), 23.2 (q, CH3CO),
28.6 (t, OCH2CH2CH2NHCOCF3), 37.2 (t, O(CH2)2CH2-
NHCOCF3), 50.6 (d, C-2c), 67.9, 68.1, 68.7, 71.2, 71.8,
72.2, 73.4, 74.4, 75.4 (9 × t, C-6b, C-6c, 6 × CH2Ph, OCH2-
(CH2)2NHCOCF3), 67.2, 69.6, 72.3, 73.8, 75.8, 75.9,
78.5, 79.2, 79.6, 80.0, 81.2 (11 × d, C-2a, C-3a, C-4a, C-5a,
C-2b, C-3b, C-4b, C-5b, C-3c, C-4c, C-5c), 97.6, 99.7, 101.6
(3 × d, C-1a, C-1b, C-1c), 101.7 (d, CHPh) and 170.3 (s,
CONH).
3-Trifluoroacetamidopropyl (2-acetamido-3,6-di-O-benzyl-2-
deoxy-ꢁ-D-mannopyranosyl)-(1 4)-(2,3,6-tri-O-benzyl-ꢀ-D-
glucopyranosyl)-(1 2)-3,4-di-O-benzyl-ꢀ-L-rhamnopyranoside
9ꢀ and its anomer 9ꢁ
A solution of 7ꢀ (240 mg, 0.183 mmol) in CH2Cl2 (10 cm3) was
cooled to 0 ЊC, then 90% aq. CF3COOH (2.5 cm3) was added
and the mixture was stirred for 30 min. The reaction was
quenched with sat. NaHCO3 and, after extraction with CH2Cl2,
the organic layer was washed with sat. NaHCO3 and water,
dried (Na2SO4), filtered and concentrated. The crude diol 8ꢀ
was dissolved in toluene (30 cm3) and Bu2SnO (68 mg, 0.275
mmol) was added. The mixture was refluxed for 3 h, then con-
centrated to 1/3 of its volume and allowed to cool to 50–60 ЊC.
Tetrabutylammonium iodide (102 mg, 0.275 mmol) and BnBr
(65 mm3, 0.549 mmol) were added and the mixture was refluxed
overnight. The reaction was quenched with MeOH and concen-
trated. The residue was purified by flash chromatography (pet-
roleum ether–EtOAc 6 : 4) to give 9ꢀ (187 mg, 78%) isolated as
a yellow glass (Found: C, 67.70; H, 6.40; N, 2.10. C74H83-
N2O16F3 requires C, 67.67; H, 6.37; N, 2.13%); [α]D ϩ19.0 (c 1 in
CHCl3); δH(300 MHz; CDCl3, Me4Si) 1.38 (3 H, d, J6a,5a 6.5,
H-6a), 1.75 (3 H, s, CH3CO), 1.84 (2 H, m, OCH2CH2CH2-
NHCOCF3), 2.50 (1 H, br s, OH), 3.06–3.15 (2 H, m, H-5c,
O(CH2)2CHHNHCOCF3), 3.24–3.66 (11 H, m, H-3a, H-2b,
H-6b, H-6Јb, H-3c, H-4c, H-6c, H-6Јc, O(CH2)2CHHNH-
COCF3, OCH2(CH2)2NHCOCF3), 3.77–3.90 (2 H, m, H-4a,
H-5a), 3.93–4.13 (4 H, m, H-2a, H-3b, H-4b, H-5b), 4.23 (1 H,
d, J 10.9, CHHPh), 4.37 (1H, d, J 11.9 CHHPh), 4.49–4.81
(12 H, m, 9 × CHHPh, H-1a, H-1c, H-2c), 4.92–4.97 (4 H, m,
H-1b, 3 × CHHPh), 5.73 (1 H, d, J 9.4, NHAc), 6.81 (1 H, br s,
NHCOCF3) and 7.15–7.50 (35 H, m, 7 × Ph); δC(75.46 MHz;
CDCl3, Me4Si) 18.1 (q, C-6a), 23.2 (q, CH3CO), 28.4 (t,
OCH2CH2CH2NHCOCF3), 38.3 (t, O(CH2)2CH2NHCOCF3),
49.4 (d, C-2c), 65.9, 68.2, 69.3, 71.0, 72.1, 72.8, 73.3, 73.6, 74.8,
75.3 (10 × t, C-6b, C-6c, 7 × CH2Ph, OCH2(CH2)2NHCOCF3),
67.3, 68.6, 69.9, 75.2, 75.8, 79.1, 79.8, 80.0, 80.2, 80.7, 81.4
(11 × d, C-2a, C-3a, C-4a, C-5a, C-2b, C-3b, C-4b, C-5b, C-3c,
C-4c, C-5c), 97.0, 97.7, 99.7 (3 × d, C-1a, C-1b, C-1c), 116.8 (q,
JC,F 287.4, COCF3), 157.0 (q, JC,F 38.6, COCF3) and 170.4 (s,
CONH).
Further elution gave the α anomer 7ꢀ (238 mg) as a glass
(Found: C, 67.70; H, 6.25; N, 2.10. C74H81N2O16F3 requires C,
67.77; H, 6.23; N, 2.14%); [α]D ϩ18.2 (c 1 in CHCl3); δH(300
MHz; CDCl3, Me4Si) 1.38 (3 H, d, J6a,5a 6.0, H-6a), 1.77 (3 H, s,
CH3CO), 1.80–1.90 (2 H, m, OCH2CH2CH2NHCOCF3), 3.07
(1 H, dt, J5c,6c 10.0, J5c,4c 10.0, J5c,6Јc 5.0, H-5c), 3.21 (1 H,
br d, J6b,6Јb 10.9, H6Јb), 3.31–3.70 (10 H, m, H-3a, H-2b, H-6b,
2178
J. Chem. Soc., Perkin Trans. 1, 2002, 2174–2181

View Article Online
The same reaction sequence was applied to compound 7ꢁ
(345 mg, 0.263 mmol) giving 9ꢁ (272 mg, 76%) as a glass
(Found: C, 67.70; H, 6.40; N, 2.10. C74H83N2O16F3 requires C,
67.67; H, 6.37; N, 2.13%); [α]D ϩ39.4 (c 1 in CHCl3); δH(300
MHz; CDCl3, Me4Si) 1.40 (3 H, d, J6a,5a 6.2, H-6a), 1.72 (3 H, s,
CH3CO), 2.40 (1 H, br s, OH), 3.05–3.68 (14 H, m, H-3a, H-4a,
H-5a, H-2b, H-6b, H6Јb, H-3c, H-4c, H-5c, H-6c, H-6Јc,
OCHH(CH2)2NHCOCF3, O(CH2)2CH2NHCOCF3), 3.72–3.81
(1 H, m, OCHH(CH2)2NHCOCF3), 3.95–4.05 (2 H, m, H-3b,
H-4b), 4.20–4.29 (2 H, m, H-2a, H-5b), 3.37–4.83 (14 H, m,
H-1a, H-1c, H-2c, 11 × CHHPh), 4.88–5.02 (3 H, m, 3 ×
CHHPh), 5.47 (1 H, d, J 3.7, H-1b), 5.62 (1 H, d, J 9.5, NHAc),
6.88 (1 H, br s, NHCOCF3) and 7.10–7.50 (35 H, m, 7 × Ph);
δC(75.46 MHz; CDCl3, Me4Si) 17.8 (q, C-6a), 23.1 (q, CH3CO),
28.7 (t, OCH2CH2CH2NHCOCF3), 37.2 (t, O(CH2)2CH2NH-
COCF3), 49.4 (d, C-2c), 67.9, 68.4, 69.3, 71.0, 71.5, 72.1, 73.2,
73.5, 74.4, 75.4 (10 × t, C-6b, C-6c, 7 × CH2Ph, OCH2-
(CH2)2NHCOCF3), 67.4, 69.5, 72.3, 73.4, 75.1, 76.0, 79.4, 79.6,
80.2, 81.2 (11 × d, C-2a, C-3a, C-4a, C-5a, C-2b, C-3b, C-4b,
C-5b, C-3c, C-4c, C-5c), 97.5, 99.8, 101.8 (3 × d, C-1a, C-1b,
C-1c), 115.9 (q, JC,F 287.4, COCF3), 157.1 (q, JC,F 38.6, COCF3)
and 170.3 (s, CONH).
H-4a, H-2b, OCHH(CH2)2NHCOCF3), 5.72–3.79 (1 H, m,
OCHH(CH2)2NHCOCF3), 3.93–4.02 (2 H, m, H-4b, H-3b),
4.23–4.41 (6 H, m, H-1a, H-2a, H-5b, 3 × CHHPh), 4.55–4.81
(11 H, m, H-1c, H-2c, H-4c, 8 × CHHPh), 4.90 (2 H, s, CH2Ph),
4.97 (1 H, d, J 11.1, CHHPh), 5.47 (1 H, d, J 3.7, H-1b), 5.71
(1 H, d, J 9.6, NHAc), 6.85 (1 H, br s, NHCOCF3) and 7.01–
7.45 (45 H, m, 9 × Ph); δC(75.46 MHz; CDCl3, Me4Si) 17.8 (q,
C-6a), 23.1 (q, CH3CO), 28.7 (t, OCH2CH2CH2NHCOCF3),
37.2 (t, O(CH2)2CH2NHCOCF3), 49.4 (d, C-2c), 67.9, 68.0,
68.4, 70.6, 71.4, 72.1, 73.2, 73.4, 74.4, 75.3 (10 × t, C-6b, C-6c, 7
× CH2Ph, OCH2(CH2)2NHCOCF3), 69.5, 72.3, 74.4, 74.6,
76.3, 77.9, 79.4, 79.6, 80.1, 81.2 (11 × d, C-2a, C-3a, C-4a, C-5a,
C-2b, C-3b, C-4b, C-5b, C-3c, C-4c, C-5c), 97.4, 99.6, 101.8
(3 × d, C-1a, C-1b, C-1c) and 170.4 (s, CONH); δP(200 MHz;
CDCl3, D2O–H3PO4–H2O = 90 : 8.5 : 1.5) Ϫ12.92.
3-Trifluoroacetamidopropyl (2-acetamido-2-deoxy-4-phospho-ꢁ-
D-mannopyranosyl)-(1 4)-(ꢀ-D-glucopyranosyl)-(1 2)-ꢀ-L-
rhamnopyranoside disodium salt 11ꢀ and its anomer 11ꢁ
Trisaccharide 10ꢀ (40 mg, 0.026 mmol) was dissolved in
MeOH–H2O–EtOAc 3 : 2 : 2 (7 cm3) and hydrogenolyzed at
atmospheric pressure over Pd(OH)2 (44 mg) for 16 h, then addi-
tional catalyst (45 mg) was added and the suspension stirred for
another 40 h. The mixture was filtered over a Celite pad and the
filtrate concentrated to dryness. The crude debenzylated prod-
uct was dissolved in H2O (2 cm3) and further hydrogenolyzed
over PtO2 (15 mg) at 5 bar for 16 h. The reaction mixture was
filtered over a Celite pad and the filtrate concentrated and
eluted with water through a column filled with Dowex 50W-X8
resin (Naϩ form). Lyophilization gave 4c-O-phosphorylated tri-
saccharide 11ꢀ (15 mg, 71%) as a white foam (Found: C, 37.20;
H, 5.00; N, 3.55. C25H40N2O19PF3Na2 requires C, 37.23; H,
5.00; N, 3.47%); [α]D ϩ12.0 (c 1 in H2O).
The same reaction sequence was applied to compound 10ꢁ
(36 mg, 0.023 mmol) affording trisaccharide 11ꢁ (15 mg,
81%) as a white foam (Found: C, 37.20; H, 5.00; N, 3.55.
C25H40N2O19PF3Na2 requires C, 37.23; H, 5.00; N, 3.47%); [α]D
ϩ25.0 (c 1 in H2O). NMR spectral data of compounds 11ꢀ and
11ꢁ are reported in Table 2.
3-Trifluoroacetamidopropyl (2-acetamido-3,6-di-O-benzyl-4-
diphenylphospho-2-deoxy-ꢁ-D-mannopyranosyl)-(1 4)-(2,3,6-
tri-O-benzyl-ꢀ-D-glucopyranosyl)-(1 2)-3,4-di-O-benzyl-ꢀ-L-
rhamnopyranoside 10ꢀ and its anomer 10ꢁ
To a solution of 9ꢀ (70 mg, 0.053 mmol) in dry CH2Cl2 (5 cm3)
were added Et(i-Pr)2N (18 mm3, 0.106 mmol) and DMAP (15
mg, 0.122 mmol) under N2. The mixture was cooled to 0 ЊC,
then diphenyl phosphorochloridate (25 mm3, 0.122 mmol) was
added. The reaction was stirred overnight at rt. Further por-
tions of Et(i-Pr)2N (18 mm3, 0.106 mmol) and diphenyl phos-
phorochloridate (22 mm3, 0.106 mmol) were added. After 30 h,
the mixture was diluted with CH2Cl2, washed with ice–water,
cold 0.5 M aq HCl, then with sat. NaHCO3, dried (Na2SO4),
filtered and concentrated. The residue was purified by flash
chromatography (petroleum ether–EtOAc 7 : 3) to give 10ꢀ
(40 mg, 49%) as a syrup (Found: C, 66.95; H, 6.05; N, 1.90.
C86H92N2O19PF3 requires C, 66.82; H, 6.00; N, 1.81%); [α]D
ϩ26.4 (c 1 in CHCl3); δH(300 MHz; CDCl3, Me4Si) 1.35 (3 H, d,
J6a,5a 6.5, H-6a), 1.72 (3 H, s, CH3CO), 1.74–1.86 (2 H, m,
OCH2CH2CH2NHCOCF3), 3.09 (1 H, m, H-5c), 3.21–3.69
(11 H, m, H-3a, H-2b, H-6b, H6Јb, H-3c, H-6c, H-6Јc, OCH2-
(CH2)2NHCOCF3, O(CH2)2CH2NHCOCF3), 3.74–3.81 (2 H,
m, H-4a, H-5a), 3.88–4.08 (4 H, m, H-2a, H-3b, H-4b, H-5b),
4.26 (1 H, d, J 11.0, CHHPh), 4.28 (1 H, d, J 11.7, CHHPh),
4.33 (1 H, d, J 12.2, CHHPh), 4.41 (1 H, d, J 11.7, CHHPh),
4.53–4.77 (12 H, m, H-1a, H-1c, H-2c, H-4c, 8 × CHHPh),
4.90–4.95 (3 H, m, H-1b, 2 × CHHPh), 5.65 (1 H, d, J 9.7,
NHAc), 6.75 (1 H, br s, NHCOCF3) and 7.02–7.42 (45 H, m,
9 × Ph); δC(75.46 MHz; CDCl3, Me4Si) 18.0 (q, C-6a), 23.1 (q,
CH3CO), 28.3 (t, OCH2CH2CH2NHCOCF3), 38.4 (t, O(CH2)2-
CH2NHCOCF3), 49.4 (d, C-2c), 65.9, 68.0, 68.2, 70.6, 72.1,
72.8, 73.3, 73.4, 75.3 (10 × t, C-6b, C-6c, 7 × CH2Ph, OCH2-
(CH2)2NHCOCF3), 68.6, 69.9, 74.4, 74.6, 74.7, 76.1, 77.8, 79.0,
79.8, 80.0, 80.5 (11 × d, C-2a, C-3a, C-4a, C-5a, C-2b, C-3b,
C-4b, C-5b, C-3c, C-4c, C-5c), 97.0, 97.7, 99.5 (3 × d, C-1a, C-
1b, C-1c) and 170.5 (s, CONH); δP(200 MHz; CDCl3, D2O–
H3PO4–H2O = 90 : 8.5 : 1.5) Ϫ12.43.
3-Trifluoroacetamidopropyl (2-acetamido-2-deoxy-ꢁ-D-manno-
pyranosyl)-(1 4)-(ꢀ-D-glucopyranosyl)-(1 2)-ꢀ-L-rhamno-
pyranoside 12ꢀ and its anomer 12ꢁ
Trisaccharide 7ꢀ (31 mg, 0.023 mmol) was dissolved in EtOAc–
MeOH–H2O 2 : 1 : 1 (8 cm3) and hydrogenolyzed at atmos-
pheric pressure over Pd(OH)2 (30 mg) for 16 h, then additional
catalyst (30 mg) was added and the suspension stirred for
another 36 h. The mixture was filtered over a Celite pad and the
filtrate lyophilised giving trisaccharide 12ꢀ (14 mg, 89%) as a
white foam (Found: C, 44.05; H, 6.00; N, 4.10. C25H41N2O19F3
requires C, 43.99; H, 6.05; N, 4.10%); [α]D = Ϫ 9.4 (c 1 in H2O).
Trisaccharide 7ꢁ (48 mg, 0.037 mmol) was hydrogenolyzed as
described for its α anomer, affording trisaccharide 12ꢁ (24 mg,
95%) as a white foam (Found: C, 44.05; H, 6.00; N, 4.10.
C25H41N2O16F3 requires C, 43.99; H, 6.05; N, 4.10%); [α]D ϩ17.9
(c 1 in H2O); the NMR spectral data of compounds 12ꢀ and
12ꢁ are reported in Table 3.
3-Trifluoroacetamidopropyl (2-acetamido-3,6-di-O-benzyl-2-
deoxy-4-O-hydrogen phosphonate-ꢁ-D-mannopyranosyl)-(1 4)-
(2,3,6-tri-O-benzyl-ꢀ-D-glucopyranosyl)-(1 2)-3,4-di-O-
benzyl-ꢀ-L-rhamnopyranoside triethylammonium salt 13
The same procedure was applied to compound 9ꢁ (50 mg,
0.038 mmol) giving 10ꢁ (36 mg, 61%) as a syrup (Found: C,
66.95; H, 6.05; N, 1.90. C86H92N2O19PF3 requires C, 66.82; H,
6.00; N, 1.81%); [α]D ϩ45.5 (c 1 in CHCl3); δH(300 MHz;
CDCl3, Me4Si) 1.38 (3 H, d, J6a,5a 6.1, H-6a), 1.71 (3 H, s,
CH3CO), 1.60–1.80 (2 H, m, OCH2CH2CH2NHCOCF3), 3.02–
3.15 (1 H, m, O(CH2)2CHHNHCOCF3), 3.23 (1 H, br d, J5c,4c
9.0, H-5c), 3.28–3.48 (8 H, m, H-3a, H-5a, H-6b, H6Јb, H-3c,
H-6c, H-6Јc, O(CH2)2CHHNHCOCF3), 3.54–3.62 (3 H, m,
To a stirred solution of 9ꢀ (88 mg, 0.067 mmol) in dry CH3CN
(500 mm3) and pyridine (300 mm3) was added a 0.4 M solution
of 2-chloro-4H-1,3,2-benzodioxaphosphinin-4-one in CH3CN
(206 mm3, 0.082 mmol) at rt. The mixture was stirred for
45 min, then py–H2O 1 : 1 (500 mm3) was added. The mix-
ture was diluted with CH2Cl2, washed with H2O and 1 M aq.
J. Chem. Soc., Perkin Trans. 1, 2002, 2174–2181
2179

View Article Online
Table 2 1H NMR (D2O, 500 MHz, 303 K) and 13C NMR (D2O, 125.72 MHz, 303 K) of compounds 11ꢀ and 11ꢁa
H-1
C-1
H-2
C-2
H-3
C-3
H-4
C-4
H-5
C-5
H-6
C-6
Residue
H-6Ј
NAc
11ꢀ
11ꢁ
a
4.89
98.6
5.00
98.7
4.89
100.8
3.79
66.5
4.70
101.6
5.19
101.1
4.91
100.6
3.91
69.1
3.95
77.6
3.6
3.86
3.51
73.1
3.69
80.1
3.99
70.8
3.35
38.2
3.42
73.2
3.69
79.9
4.02
71.8
3.38
37.9
3.69
70.1
4.08
71.5
3.49
77.4
3.35
1.30
17.7
3.91
61.0
3.90
61.7
[70.9]
3.92
72.5
3.97
73.4
1.91
28.8
3.65
73.2
3.91
72.8
3.99
73.1
1.88
29.1
b
3.77
3.90
72.3
c
4.56
53.6
3.56
2.08
23.2
anchor
a
4.08
79.2
3.52
73.1
4.58
53.9
3.64
3.42
73.7
4.15
70.5
3.52
77.1
3.49
1.33
17.7
3.77
61.00
3.90
61.6
b
3.77
3.90
c
2.08
23.2
anchor
a Chemical shifts referenced to the N-acetyl resonance at 2.083 and 23.23 ppm (compound 11ꢀ) and to 3-trimethylsilyl-2,2,3,3-tetra-
deuteriopropanoic acid Na salt (TSP-d4) at zero ppm and Ϫ1.8 ppm (compound 11ꢁ). [ ] = uncertain assignment.
Table 3 1H NMR (D2O, 500 MHz, 303 K) and 13C- NMR (D2O, 125.72 MHz, 303 K) of compounds 12ꢀ and 12ꢁa
H-1
C-1
H-2
C-2
H-3
C-3
H-4
C-4
H-5
C-5
H-6
C-6
Residue
H-6Ј
NAc
12ꢀ
12ꢁ
a
4.89
98.7
5.00
99.0
4.90
100.7
3.80
64.9
4.71
101.6
5.19
101.1
4.90
100.6
3.90
68.2
3.96
77.7
3.58
72.4
4.56
54.7
3.56
3.85
71.0
3.91
72.6
3.84
72.3
1.91
27.1
3.66
73.3
3.91
72.8
3.82
72.3
1.89
29.1
3.50
73.3
3.70
80.0
3.54
67.9
3.45
36.6
3.43
73.7
3.70
79.9
3.53
67.8
3.40
37.9
3.67
70.2
4.09
71.6
3.47
77.8
3.45
1.30
17.7
3.77
60.9
3.94
61.7
b
3.79
3.82
c
2.08
23.2
anchor
a
4.08
79.3
3.53
73.1
4.56
54.6
3.64
3.41
73.3
4.15
71.5
3.46
77.8
3.50
1.33
17.7
3.77
61.0
3.94
61.6
b
3.77
3.83
c
2.08
23.2
anchor
a Chemical shifts referenced to the N-acetyl resonance at 2.083 and 23.23 ppm.
triethylammonium hydrogen carbonate (TEAB) solution, dried
(Na2SO4), filtered and concentrated under reduced pressure.
The residue was purified on a short column of silica gel
(CH2Cl2–MeOH 9 : 1 ϩ 1% Et3N) to give 13 (97 mg) as a syrup
(Found: C, 65.00; H, 6.75; N, 2.80. C80H99N3O18PF3 requires C,
64.98; H, 6.75; N, 2.84%). The formation of the H-phosphonate
intermediate was ascertained by 1H NMR analysis, which
showed the diagnostic doublet at δ 6.95 (JH,P = 635 Hz). Com-
pound 13 was used directly in the following coupling step with-
out further characterization.
The organic layer was washed with 1 M aq. Na2S2O3 and 0.5 M
aq. TEAB, dried (Na2SO4), filtered and concentrated under
reduced pressure. The residue was purified by flash chrom-
atography (CH2Cl2–MeOH–Et3N 97 : 2 : 1); the fractions
containing the product were pooled and washed with 1 M aq.
TEAB, and compound 14 (98 mg, 61%) was recovered as a glass
(Found: C, 67.95; H, 6.60; N, 2.15. C149H172N4O33PF3 requires
C, 67.91; H, 6.58; N, 2.13%). 13C and 31P NMR analysis of
compound 14 showed a mixture of α and β anomers at C-1d,
1
causing extensive overlap of the signals in the H NMR spec-
trum. Therefore, full 1H NMR characterisation and optical
rotation measurements were performed on deprotected dimer
15. δC(75.46 MHz; CDCl3, Me4Si) 8.4 (q, NCH2CH3), 18.0,
18.1 (2 × q, C-6a, C-6d), 23.2 (q, CH3CO), 28.3 (t, OCH2CH2-
CH2NHCOCF3), 38.3 (t, O(CH2)2CH2NHCOCF3), 45.3 (t,
NCH2CH3), 50.5 (d, C-2c, C-2f ), 65.8, 68.1, 68.7, 70.7, 71.2,
71.7, 71.9, 72.8, 73.4, 74.5, 74.6, 74.8, 74.9, 75.2 (14 × t, C-6b,
C-6c, C-6e, C-6f, 13 × CH2Ph, OCH2(CH2)2NHCOCF3), 67.1,
68.5, 69.3, 69.6, 70.4, 71.4, 74.3, 75.5, 76.0, 76.2, 78.6, 78.8,
79.4, 79.8, 80.0, 80.3, 80.7, 81.1, 81.3, 81.5 (20 × d, C-2a, C-2b,
C-2d, C-2e, C-3a, C-3b, C-3c, C-3d, C-3e, C-3f, C-4a, C-4b,
C-4c, C-4d, C-4e, C-4f, C-5a, C-5b, C-5c, C-5d, C-5e, C-5f ),
94.0, 96.2, 96.7, 97.6, 98.9, 99.6, 101.6 (7 × d, C-1a, C-1b, C-1c,
C-1d, C-1e, C-1f, CHPh) and 170.1, 170.4 (2 × s, 2 × CONH);
δP(200 MHz; CDCl3, D2O–H3PO4–H2O = 90 : 8.5 : 1.5) Ϫ2.46
(major isomer), Ϫ1.09 (minor isomer).
3-Trifluoroacetamidopropyl (2-acetamido-3,6-di-O-benzyl-2-
deoxy-ꢁ-D-mannopyranosyl)-(1 4)-(2,3,6-tri-O-benzyl-ꢀ-D-
glucopyranosyl)-(1 2)-3,4-di-O-benzyl-ꢀ-L-rhamnopyranosyl
4c-[(2-acetamido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-ꢁ-D-
mannopyranosyl)-(1 4)-(2,3,6-tri-O-benzyl-ꢀ-D-gluco-
pyranosyl)-(1 2)-3,4-di-O-benzyl-ꢀ-L-rhamnopyranosyl
phosphate] triethylammonium salt 14
A mixture of 13 (97 mg, 0.066 mmol) and 5 (70 mg, 0.061
mmol) was dried by co-evaporation with pyridine (3 × 1 cm3).
The residue was dissolved in the same solvent (1 cm3) and PivCl
(19 mm3, 0.153 mmol) was added. The mixture was stirred for
40 min at rt, then a freshly prepared solution of I2 (31 mg, 0.122
mmol) in py–H2O 19 : 1 (1 cm3) was added. After 15 min the
mixture was diluted with CHCl3 and the phases were separated.
2180
J. Chem. Soc., Perkin Trans. 1, 2002, 2174–2181

View Article Online
Table 4 1H NMR (D2O, 500 MHz, 303 K) and 13C NMR (D2O, 125.72 MHz, 303 K) of compounds 15ꢀa
H-1
C-1
H-2
C-2
H-3
C-3
H-4
C-4
H-5
C-5
H-6
C-6
Residue
H-6Ј
NAc
a
4.86
98.3
5.00
98.7
4.89
100.2
5.48/5.49
94.8
4.96
98.5
4.87
100.2
3.925
77.3
3.56
72.1
4.57
54.2
4.01
77.8
3.55
72.1
4.53
54.3
1.88
1.88
3.82
70.6
3.87
3.48
72.8
3.68
79.7
4.05
73.1
3.49
72.8
3.67
79.7
3.51
67.5
[3.88]
1.27
17.5
3.75
60.7
3.83
61.3
1.29
17.6
3.75
60.7
3.81
61.3
b
4.07
71.3
3.54
76.5
3.87
[3.75]
[3.91]
c
3.98
72.2
3.92
70.0
[2.06]
[23.0]
d
e
3.88
4.05
71.3
3.44
77.5
[3.75]
[3.91]
f
3.81
73.1
3.42
[2.06]
[23.0]
anchor
3.76
3.53
a Chemical shifts referenced to the N-acetyl resonance at 2.083 and 23.23 ppm. [ ] = uncertain assignment.
New York, 1983, vol. 41, p. 155; (b) C. T. Bishop and H. J. Jennings,
in The Polysaccharides, ed. G. O. Aspinall, Academic Press,
New York, 1982, vol. 16, p. 292; (c) B. Arndt and M. Porro, in
Immunobiology of Proteins and Peptides VI, ed. M. Z. Atassi,
Plenum Press, New York, 1991, p. 129.
3-Trifluoroacetamidopropyl (2-acetamido-2-deoxy-ꢁ-D-manno-
pyranosyl-(1 4)-ꢀ-D-glucopyranosyl-(1 2)-O-ꢀ-L-rhamno-
pyranosyl 4c-[2-acetamido-2-deoxy-ꢁ-D-mannopyranosyl-
(1 4)-ꢀ-D-glucopyranosyl-(1 2)-ꢀ-L-rhamnopyranosyl
phosphate] triethylammonium salt 15
6 (a) P. W. Anderson, M. E. Pichichero, E. C. Stein, S. Porcelli,
R. F. Betts, D. M. Connuck, D. Korones, R. A. Insel, J. M.
Zahradnik and R. Eby, J. Immunol., 1989, 142(7), 2464; (b) C. A.
Laferriere, R. K. Sood, J. M. de Muys, F. Michon and H. J. Jennings,
Infect. Immun., 1998, 66(6), 2441; (c) C. A. Laferriere, R. K. Sood,
J. M. de Muys, F. Michon and H. J. Jennings, Vaccine, 1997, 15(2),
179; (d ) V. Pozsgay, C. Chu, L. Pannell, J. Wolfe, J. B. Robbins and
R. Schneerson, Proc. Natl. Acad. Sci. U. S. A., 1999, 96(9), 5194.
7 (a) L. Panza, F. Ronchetti, G. Russo and L. Toma, J. Chem. Soc.,
Perkin Trans. 1, 1987, 2745; (b) E. Bousquet, M. Khitri, L. Lay,
F. Nicotra, L. Panza and G. Russo, Carbohydr. Res., 1998, 311, 171.
8 (a) T. Sugawara and K. Igarashi, Carbohydr. Res., 1988, 172, 195;
(b) H. Paulsen, B. Helpap and J. P. Lorentzen, Carbohydr. Res., 1988,
179, 173; (c) E. Kaji, F. W. Lichtenthaler, Y. Osa and S. Zen, Bull.
Chem. Soc. Jpn., 1995, 68, 2401.
9 S. C. Szu, C. J. Lee, J. C. Parke, G. Schifman, J. Henrichsen,
R. Austrian, S. C. Rastogi and J. B. Robbins, Infect. Immun., 1982,
35(3), 777.
10 J. J. Oltvoort, C. A. van Boeckel, J. H. De Koning and J. H. van
Boon, Synthesis, 1981, 305.
11 G.-J. Boons, A. Burton and P. Wyatt, Synlett, 1996, 310.
12 M. A. Nashed and L. Anderson, J. Chem. Soc., Chem. Commun.,
1982, 1274.
Compound 14 (41 mg, 0.015 mmol) was hydrogenolyzed over
Pd(OH)2 (40 mg) in MeOH–H2O 1 : 1 (3 cm3) for 4 days at rt
(addition of a further 20 mg of catalyst was required to drive
the reaction to completion). The mixture was filtered over a
Celite pad and the filtrate was concentrated, then eluted with
water through a column filled with Dowex 50W-X8 resin (Naϩ
form). The fractions containing the dimer were pooled and
lyophilized to give 15 (18 mg, 96%) as a white solid (Found: C,
41.75; H, 5.70; N, 3.25. C45H74N3O33PF3Na requires C, 41.70;
H, 5.75, N, 3.24%). The diastereomeric ratio of α : β anomers
of 15 was determined to be 9 : 1 by HPLC analysis, performed
with a column of porous graphitised carbon (PGC “Hyper-
carb®”, Hypersil, Runcorn, UK; 4.6 × 250 mm, 7 µ)21 eluted
with an acetonitrile gradient (2 to 25%) in purified water (Elga)
containing 0.05% TFA at 0.6 cm3 minϪ1. Elution was monitored
by UV at 206 nm. [α]D ϩ24.0 (c 1 in H2O); the NMR spectral
data of compound 15ꢀ are reported in Table 4.
13 R. R. Schmidt, Angew. Chem., Int. Ed. Engl., 1986, 25, 212.
14 S. Sabesan and S. Neira, Carbohydr. Res., 1992, 223, 169.
15 (a) J. Thiem and M. Franzkowiak, J. Carbohydr. Chem., 1989, 8, 1
and references therein; (b) J. E. Marugg, M. Tromp, E.
Kuyl-Yeheskiely, G. A. van der Marel and J. H. van Boom,
Tetrahedron Lett., 1986, 27, 2661; (c) P. Westerduin, G. H.
Veeneman, J. E. Marugg, J. A. van der Marel and J. H. van Boom,
Tetrahedron Lett., 1986, 27, 1211; (d ) G. H. Veeneman, L. J. F.
Gomes and J. H. van Boom, Tetrahedron, 1989, 45, 7433.
16 (a) M. Lindberg, T. Norberg and S. Oscarson, J. Carbohydr. Chem.,
1992, 11, 243; (b) A. V. Nikolaev, J. A. Chudek and M. A. J.
Ferguson, Carbohydr. Res., 1995, 272, 179.
17 (a) P. Westerduin, G. H. Veeneman, G. A. van der Marel and
J. H. van Boom, Tetrahedron Lett., 1986, 27, 6271; (b) M. Nilsson
and T. Norberg, J. Chem. Soc., Perkin Trans. 1, 1998, 1699.
18 A. V. Nikolaev, I. A. Ivanova and V. N. Shibaev, Carbohydr. Res.,
1993, 242, 91.
Acknowledgements
This work was supported by EU (VACNET project, Contr.
BIO CT96 0158), MURST and CNR (Istituto di Scienze e
Tecnologie Molecolari).
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2181