Synthesis of a spacer-containing nonasaccharide fragment of Streptococcus pneumoniae 19F capsular polysaccharide

Article abstract of DOI:10.1039/a709293h

The nonasaccharide 1, representing three repeating units of the capsular polysaccharide from Streptococcus pneumoniae 19F, has been synthesized. The synthetic strategy is to first synthesize, by an AB + C route, trisaccharide derivative 14, having O-benzy

Full text of DOI:10.1039/a709293h

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Synthesis of a spacer-containing nonasaccharide fragment of
Streptococcus pneumoniae 19F capsular polysaccharide
Magnus Nilsson and Thomas Norberg
Department of Chemistry, Swedish University of Agricultural Sciences, PO Box 7015,
S-750 07 Uppsala, Sweden
The nonasaccharide 1, representing three repeating units of the capsular polysaccharide from
Streptococcus pneumoniae 19F, has been synthesized. The synthetic strategy is to first synthesize, by
an AB ؉ C route, trisaccharide derivative 14, having O-benzyl groups as persistent, and 1-O-allyl and
4Љ-O-acetyl groups as temporary, protecting groups. Then, specific replacement of the 1-O-allyl group
with an á-H-phosphonate monoester gives trisaccharide 17, which is coupled to a spacer. The obtained
derivative 18 is then extended from the 4Љ end in a stepwise fashion to give compound 20, using
trisaccharide 17 and solution-phase H-phosphonate chemistry. Finally, removal of the persistent
O-protecting groups gives the nonasaccharide 1.
Introduction
O
Bacterial capsular polysaccharides are important bacterial
surface components that have been studied for nearly a cen-
O
P
OH
tury by immunologists and chemists. Chemical structures
are now known for a great number of bacterial species,1 and
their immunological properties have been well characterized.
Immunologically, bacterial capsular polysaccharides give rise
to highly specific immune responses. Because of this, they
have been used in vaccines, replacing less efficient whole-cell
vaccines. Such polysaccharide vaccines, especially the modern
varieties consisting of polysaccharides conjugated to immuno-
genic proteins (conjugate vaccines2), have proved to be very
efficient in preventing human disease. For example, conjugate
vaccines against Haemophilus influenzae type b (HiB) are today
used widely as paediatric vaccines. Their use has dramatic-
ally reduced the incidence of childhood meningitis, a life-
threatening disease caused by the HiB organism. Spurred by the
success of HiB conjugate vaccines, intense efforts are now being
made in both industry and academia to further develop the
conjugate vaccine concept.
H3C
HO
O
HO
O
HO
NHAc
O
HO
O
HO
HO
O
O
OH
n
Streptococcus pneumoniae 19F polysaccharide
O
O
P
O
OH
H3C
HO
O
NH2
HO
O
HO
NHAc
O
HO
H
O
HO
In most previous work, the source of the bacterial capsular
polysaccharides has been bacterial cell cultures. After culturing
and harvesting of the bacterial cells, the polysaccharides are
isolated from the cells by centrifugation, precipitation and
chromatography, and then typically subjected to some kind of
chemical modification that makes possible conjugation to a
protein. These processes, culture, isolation and chemical modi-
fication, are not always trivial, and the whole process can be
hampered by difficulties in each of the steps.
An alternative source of bacterial polysaccharide structures
is chemical synthesis. It is today possible, using multistep
organic synthesis, to synthesize fragments of bacterial poly-
saccharides several repeating units in length. Although these
synthetic structures are of lower relative molecular mass than
the natural polysaccharides, they are often long enough to
function very well as immunogenic components of conjugate
vaccines.3 The advantage of a synthetic product over an isolated
one is its more defined structure and definite lack of bacterial
contaminants, and, in some cases, its economy of production.
Only very small amounts of synthetic material are required
in a vaccine, typically less than 10 micrograms per dose. This
means that a synthetic production facility can operate on a
quite modest scale, even though the synthetic process will
involve numerous chemical steps.
HO
O
O
OH
3
1
aimed at preparation of bacterial polysaccharide fragments.
Our own efforts in this regard include synthesis of penta-
and deca-meric fragments4,5 and a pentameric analogue6 of
H. influenzae type B capsular polysaccharide. We now report
synthesis of the nonasaccharide fragment 1 of the capsular
polysaccharide from Streptococcus pneumoniae 19F (native
structure, see above). Since this organism is an important
pathogen (it is a common cause of respiratory infections in
children), it is not surprising that synthetic efforts have been
reported before. The repeating-unit trisaccharide has been
synthesized by several groups.7–10 However, the crucial joining
together of several repeating units by phosphodiester linkages
to form larger structures, presumably required for a reliable
immune response, has, to our knowledge, not been reported.
Results and discussion
General strategy
Our general strategy for the synthesis of compound 1 was the
following: first, trisaccharide derivative 14 was synthesized,
having O-benzyl groups as persistent, and 1-O-allyl and 4Љ-O-
Several research groups have initiated synthetic programmes
J. Chem. Soc., Perkin Trans. 1, 1998
1699

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OAll
O
acetyl groups as temporary protecting groups. Then, specific
replacement of the 1-O-allyl group with an α-H-phosphonate
monoester gave a trisaccharide 17 which could be coupled
to a spacer (useful later in the conjugation to proteins). The
obtained derivative 18 was then extended from the 4Љ end in a
stepwise fashion to give nonasaccharide 20, using substrate 17
and solution-phase H-phosphonate chemistry. Finally, removal
of the persistent O-protecting groups gave the nonasaccharide
1.
H3C
BnO
OAll
BnO
BnO
O
H3C
BnO
O
BnO
AcO
BnO
N3
O
BnO
BnO
O
OH
O
OBn
12
13 α/β
(1) Synthesis of trisaccharide 14. As noted in the Introduction
section, several syntheses have been described of the S.
pneumoniae 19F trisaccharide repeating unit, using either
A ϩ BC7 or AB ϩ C8–10 strategies. To make synthesis of larger
structures possible, a protected trisaccharide repeating unit
should contain selectively removable temporary protecting
groups in the 1- and 4Љ-position. Our approach was to syn-
thesize such a trisaccharide 14, using thioglycoside blocks in an
AB ϩ C strategy. Many of the intermediates were crystalline,
and the chromatographic purifications necessary were not of
the most difficult type, which allowed for the production of
gram amounts of compound 14. The following steps were
carried out: Donor 4, suitably protected for later manipulation
at the 2- and 4-position, was prepared from substrate 211 by
selective benzylation at the 3-position as described12 (minor
modifications), giving compound 3 in 55% yield. Benzoylation
of compound 3 then gave compound 4 in 88% crystalline yield.
The conversion 13α/β → 14/15 was accomplished by,
first, reaction of azide 13α/β with triphenylphosphine, then
hydrolysis of the formed imine, and finally acetylation of the
amine by using pyridine–acetic anhydride to give the desired
compounds 14 (α-form) and 15 (β-form) in 58 and 28% yield,
respectively (from 13α/β). Deallylation of trisaccharide 14
using, first, Wilkinson’s catalyst and then 80% acetic acid gave
compound 16 in 80% yield.
OR
H3C
BnO
O
BnO
BnO
O
BnO
AcO
BnO
NHAc
O
BnO
O
O
OBn
OBn
O
Ph
O
O
O
HO
BnO
14 R = All
R2O
SMe
BnO
O
SMe
16 R = H (α/β-mixture)
OR1
OBn
OBn
BnO
AcO
BnO
2 R1 = R2 = H
3 R1 = H, R2 = Bn
4 R1 = Bz, R2 = Bn
5
OAll
CH3
BnO
NHAc
O
BnO
O
O
OBn
O
Conversion of fully protected monosaccharide 4 into the
bromo sugar followed by silver triflate (AgOTf)-promoted
coupling with compound 513 gave disaccharide 6 in 66% yield.
Debenzoylation of compound 6 using methanolic sodium
methoxide in 1,4-dioxane gave mono-ol 7 in 96% yield, with
a free 2Ј-OH group. Reaction of compound 7 with triflic
anhydride in dichloromethane–pyridine and subsequent reac-
tion of the product 8 with sodium azide in DMF gave azide 9
in 75% yield (from mono-ol 7). The manno configuration in
product 9 was evident from the 1H NMR coupling pattern.
15
Treatment of compound 16 with phosphorous acid and an
excess of 2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphorin-
ane in pyridine gave compound 17 in 61% yield. The α con-
figuration of product 17 was indicated by JC-1,H-1 (180 Hz) in the
NMR spectrum. Very little β product was detected.
O
O
P
O– Et3NH+
H
OBn
OR
H3C
BnO
O
6 R = Bz
7 R = H
8 R = Tf
O
BnO
O
O
BnO
BnO
BnO
SMe
O
O
OBn
Ph
O
BnO
AcO
BnO
NHAc
O
BnO
OBn
O
9 R1 = R2 = benzylidene
10 R1 = H, R2 = Bn
11 R1 = Ac, R2 = Bn
O
OBn
O
BnO
O
R1O
BnO
SMe
O
OBn
N3
17
R2O
Regioselective opening of the benzylidene acetal14 in com-
pound 9 gave alcohol 10 in 81% yield, which was 4Ј-O-
acetylated to give donor 11 in 94% yield. Methyl triflate-
promoted coupling of fully protected compound 11 with
acceptor 1215 in dichloromethane gave a 2:1 α/β-mixture of
trisaccharides (13 α/β) in 87% overall yield. Efforts to improve
the α:β ratio in this coupling included both change of solvent
(diethyl ether or diethyl ether–dichloromethane mixtures)
and change of coupling method (silver triflate- or halide-ion-
promoted coupling with the bromide derived from compound
11). No significant improvement was observed.
(2) Nonasaccharide synthesis. The attachment of
a
phosphate-linked spacer group at the reducing end of com-
pound 17 and subsequent stepwise oligomerization to give a
protected phosphodiester-linked nonasaccharide 20 was carried
out in solution (with chromatographic purification after each
coupling cycle) using a common H-phosphonate protocol
(coupling with pivaloyl chloride in pyridine, then oxidation
with iodine in water–pyridine). Thus, compound 17 was first
coupled with Cbz-ethanolamine16 and the product was then
deacetylated using methanolic sodium methoxide to give the
spacer-containing trisaccharide acceptor 18 in 91% yield.
Using compound 17, the H-phosphonate coupling/
deprotection cycle was performed twice on phosphate 18,
giving, first, the hexasaccharide 19 and then the nonasaccharide
20 in 87 and 83% yield, respectively. The deprotection steps
The obtained α/β mixture was difficult to separate by chrom-
atography, but after conversion of the azide group into an
acetamido group, the corresponding mixture of trisaccharides
was easily separated.
1700
J. Chem. Soc., Perkin Trans. 1, 1998

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Table 1 1H/13C Chemical shift (J/Hz in parentheses)a of compound 3
1
2
3
4
5
6a
3.77 (10.2)
6b
Glc
4.37 (9.4)
86.6
3.60 (8.6)
72.5
3.65–3.73
81.7*
3.65–3.73
81.4*
3.50
70.9
4.36 (4.8)
68.7
a Further 1H/13C chemical shifts are 2.21/12.1 (s, 3 H, SCH3), 5.57/101.4 (s, 1 H, PhCHO).
Table 2 1H/13C Chemical shift (J/Hz in parentheses)a of compound 4
1
2
3
4
5
6a
6b
Glc
4.52 (9.9)
83.9
5.36 (8.9)
71.2
3.91 (9.4)
79.3
3.83 (9.1)
81.8
3.58
70.8
3.82 (10.5)
68.7
4.41 (5.1)
a Further 1H/13C chemical shifts are 2.19/11.4 (s, 3 H, SCH3), 5.61/101.3 (s, 1 H, PhCHO) and 165.2 (C᎐O).
᎐
were monitored by MALDI-TOF MS† and FAB-MS,‡ since
TLC monitoring was found to be difficult.
Hydrogenolysis of compound 20 (Pd/C in ethanol–acetic
acid–water; atmospheric pressure) gave the deprotected nona-
saccharide 1 in 73% yield.
reversed. Coupling constants J are given within brackets, and
are measured in Hz. FAB-MS spectra were recorded with a
JEOL JMS-SX/SX-102A instrument. Ions were produced by
a beam of Xe atoms (6 keV), using a matrix of glycerol or
m-nitrobenzyl alcohol. For HRMS, poly(ethylene glycol)
(PEG) was used as an internal standard. For MALDI-TOF
MS, an LDI-1700XP instrument was used (negative-ion
mode), using trihydroxyacetophenone as a matrix; optionally
the sample was mixed with aq. 0.1  ammonium hydrogen
citrate, TLC was performed on Silica Gel F254 (Merck, Darm-
stadt, Germany) with detection by UV light and by staining
with 5% sulfuric acid in ethanol or 0.5% ninhydrin in butan-1-
ol. Column chromatography was performed on Matrex silica
gel 60 Å (35–70 µm, Amicon). Molecular sieves (powdered 3 Å
and 4 Å) were dried at 280 ЊC/0.5 mmHg overnight. Dichloro-
methane was distilled from P2O5 when necessary. Pyridine was
of pro analysi quality and, when used for H-phosphonate
coupling, was first distilled from ninhydrin and then fraction-
ally distilled from anhydrous barium oxide.
O
NHCbz
O
P
O
OH
H3C
BnO
O
BnO
18 n = 1
19 n = 2
20 n = 3
O
BnO
BnO
NHAc
O
H
O
BnO
BnO
O
O
OBn
n
1
The H NMR spectrum of compound 1 was well resolved
and showed doubling of the signals of the three anomeric pro-
tons in the ratio 2:1, which could be explained by assuming
that the terminating trisaccharide, C (the terminating trisac-
charide refers to the one with unsubstituted OH-4 of ManNAc),
differs slightly in chemical shift from the other two units, A and
B, or that the signals from ManNAc-C, Rha-A and Glc-A
give slightly different chemical shifts from the rest of the
residues. Chemical shifts of compound 1 are presented accord-
ing to the first assumption. Nuclear Overhauser Enhancement
Spectroscopy (NOESY) experiments showed NOE correlation
between the H-1s of rhamnose and the H-1s of glucose, indicat-
ing α anomeric configurations of the rhamnose units. Also, a
coupled 2D heteronuclear multiple quantum-filtered coherence
(HMQC) spectrum showed rhamnose JH-1,C-1 couplings indicat-
ing α anomeric configurations (174 Hz). Finally, the structure
of compound 1 was verified by the agreement between the
measured (HRMS, 1833.5186 Da) and calculated (1833.5142
Da) relative molecular mass of the M Ϫ H ion.
Methyl
3-O-benzyl-4,6-O-benzylidene-1-thio-â-D-gluco-
pyranoside 3. A mixture of methyl 4,6-O-benzylidene-1-thio-β-
-glucopyranoside 211 (2.00 g, 6.7 mmol) and sodium hydride
(80% in mineral oil; 0.50 g, 16.8 mmol) in DMF (16 ml) was
stirred at room temperature (rt) for 2 h. Copper() chloride
(0.90 g, 6.7 mmol) was added and the mixture was stirred for
15 min, then benzyl bromide (1.2 ml, 10.0 mmol) was added
and the mixture was stirred for 18 h. Additional benzyl bromide
(1 ml, 8.4 mmol) was then added and stirring was continued for
another 24 h. Methanol (1.5 ml) was added to destroy excess
of reagent. The mixture was diluted with ethyl acetate, washed
successively with 5% aq. ammonium hydroxide and brine, dried
(Na2SO4), filtered and concentrated. Column chromatography
(dichloromethane–acetone 95:5) of the residue gave the title
compound (1.43 g, 55%) as a solid, which could be recrystal-
lized from ethyl acetate–petroleum spirit (65–70 ЊC) to give
1
needles, mp 173–174 ЊC; [α]D ϩ49; H/13C NMR data (CDCl3)
are shown in Table 1 [HRMS: Calc. for C21H25O5S: 389.1423.
Found: 389.1427 (M ϩ Hϩ)].
Experimental
Methyl 2-O-benzoyl-3-O-benzyl-4,6-O-benzylidene-1-thio-â-
D-glucopyranoside 4. To a cooled (0 ЊC) solution of compound 3
(2.82 g, 7.25 mmol) in pyridine (40 ml) was added benzoyl
chloride (1.3 ml, 11 mmol) and the solution was stirred for 3 h.
Water (1.5 ml) was added and the solution was then diluted
with dichloromethane, washed successively with 2  sulfuric
acid and 1  aq. sodium hydrogen carbonate, dried (Na2SO4),
filtered and concentrated. The residue was recrystallized from
ethanol to give the title compound (3.16 g, 88%) as needles, mp
General methods
Concentrations were performed at reduced pressure (<40 ЊC
bath temperature, unless otherwise stated). Weighed yields are
calculated from residues dried in vacuo (0.5 mmHg) overnight.
Optical rotations were measured at 23 ЊC (c 1.0, CHCl3), using a
Perkin-Elmer 341 polarimeter; [α]D-values are given in 10Ϫ1 deg
cm2 gϪ1. NMR spectra were recorded for solutions in CDCl3
(internal Me4Si, δ 0.00) or D2O (internal acetone, δH 2.225, δC
31.0) at 300 K unless otherwise stated with Bruker DRX 400 or
DRX 600 spectrometers. Only selected NMR data are reported.
Assignments were corroborated by appropriate 2-D experi-
ments. Pairwise assignments marked with an asterisk could be
1
134–136 ЊC; [α]D ϩ26. H/13C NMR data (CDCl3) are shown
in Table 2 [HRMS: Calc. for C28H29O6S: 493.1685. Found:
493.1713 (M ϩ Hϩ)].
Methyl 4-O-(2-O-benzoyl-3-O-benzyl-4,6-O-benzylidene-â-D-
glucopyranosyl)-2,3,6-tri-O-benzyl-1-thio-â-D-glucopyranoside
6. To a cooled (0 ЊC) mixture of compound 4 (1.13 g, 2.3 mmol)
and molecular sieves (4 Å) in dichloromethane (25 ml) was
† Matrix-assisted laser-desorption time-of-flight mass spectrometry.
‡ Fast-atom bombardment mass spectrometry.
J. Chem. Soc., Perkin Trans. 1, 1998
1701

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Table 3 1H/13C Chemical shift (J/Hz in parentheses)a of compound 6
1
2
3
4
5
6a
6b
Glc
4.22 (9.7)
85.2
4.73 (8.7)
100.7
3.40 (9.2)
80.4
5.22 (8.7)
73.9
3.56 (9.2)
84.5
3.68 (9.2)
77.9
3.99 (9.7)
76.5
3.73 (9.2)
81.9
3.15
78.6
3.25
66.1
3.47
67.7
3.47
68.6
3.61 (2.6, 11.3)
4.21 (5.1, 10.5)
GlcЈ
a Further 1H/13C chemical shifts are 2.14/12.7 (s, 3 H, SCH3), 5.51/101.2 (s, 1 H, PhCHO) and 164.8 (C᎐O).
᎐
Table 4 1H/13C Chemical shift (J/Hz in parentheses)a of compound 9
1
2
3
4
5
6a
6b
Glc
4.35 (9.7)
85.5
4.67 (1.0)
100.3
3.46 (nd)
80.6
3.83 (3.6)
63.6
3.67 (9.2)
84.9
3.48 (9.7)
76.7*
3.99 (9.2)
77.6
3.90 (9.2)
78.5
3.47
3.75
68.8
3.51
3.75
78.6*
3.02
67.3
Man
3.99 (5.1, 10.7)
68.4
a Further 1H/13C chemical shifts are 2.29/12.8 (s, 3 H, SCH3) and 5.49/101.6 (s, 1 H, PhCHO).
Table 5 1H/13C Chemical shift (J/Hz in parentheses)a of compound 11
1
2
3
4
5
6a
6b
Glc
4.34 (9.4)
85.5
4.65 (nd)
99.4
3.38 (9.1)
80.8
3.85 (3.5)
61.7
3.68 (9.1)
84.8
3.34 (9.7)
78.2
3.96 (9.7)
76.8
5.09 (9.7)
68.8
3.48
78.4
3.27
74.4
3.75
69.0
3.31
70.2
3.79 (11.7, 3.2)
3.99 (4.1, 10.8)
Man
a Further 1H/13C chemical shifts are 1.86/20.9 (s, 3 H, OCOCH3) and 2.20/12.3 (s, 3 H, SCH3).
added bromine (0.12 ml, 2.3 mmol). The mixture was stirred for
18 min, then cyclohexene (0.53 ml, 5.2 mmol) was added and
the mixture was stirred at 0 ЊC for 10 min. After cooling of the
mixture to Ϫ40 ЊC, monosaccharide 513 (1.00 g, 2.1 mmol) and
2,6-di-tert-butyl-4-methylpyridine (0.427 g, 2.1 mmol) were
added and stirring was continued for another 30 min. A solu-
tion of silver triflate (0.617 g, 2.4 mmol) in toluene (25 ml) was
added dropwise to the mixture during 30 min at Ϫ40 to Ϫ50 ЊC,
and, after complete addition, the mixture was stirred for
another 40 min. Pyridine (1.3 ml) was added, the mixture
was filtered through Celite, and the filtrate was diluted with
dichloromethane, washed with 10% aq. sodium thiosulfate,
dried (Na2SO4), filtered and concentrated. Column chrom-
atography of the residue (stepwise gradient elution, toluene–
ethyl acetate 9:1–1:1) gave compound 6 as a solid (1.27 g,
66%), which could be recrystallized from ethyl acetate–ethanol
as needles, mp 187–191 ЊC; [α]D ϩ23; 1H/13C NMR data
(CDCl3) are shown in Table 3 [HRMS: Calc. for C55H56NaO11S:
947.3441. Found: 947.3457 (M ϩ Naϩ)].
Methyl 4-O-(2-azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-
â-D-mannopyranosyl)-2,3,6-tri-O-benzyl-1-thio-â-D-glucopyr-
anoside 9. To a stirred solution of benzoate 6 (2.41 g, 2.60
mmol) in 1,4-dioxane (20 ml) was added 0.5  methanolic
sodium methoxide (20 ml). The solution was heated to 40 ЊC
and stirred for 2 h 45 min. The solution was diluted with 1,4-
dioxane (25 ml), neutralized with Dowex 50 (Hϩ) resin, filtered
and concentrated. Column chromatography of the residue
(toluene–ethyl acetate 7:1) gave the free mono-ol 7 as a solid
(2.05 g, 96%).
was stirred at 70 ЊC for 4 h 30 min, then diluted with ethyl
acetate, washed with water, dried (Na2SO4), filtered and concen-
trated. Column chromatography of the residue with ethyl acet-
ate in petroleum spirit (stepwise gradient elution, 25–33%) gave
the title compound 9 as a syrup (1.54 g, 75%), [α]D Ϫ18; 1H/13C
NMR data (CDCl3) are shown in Table 4 [HRMS: Calc. for
C48H52N3O9S: 846.3424. Found: 846.3476 (M ϩ Hϩ)].
Methyl 4-O-(4-O-acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-â-D-
mannopyranosyl)-2,3,6-tri-O-benzyl-1-thio-â-D-glucopyranoside
11. To a cooled (ice) mixture of compound 9 (1.44 g, 1.70
mmol), sodium cyanoborohydride (0.60 g, 8.50 mmol) and
molecular sieves (3 Å) in THF (20 ml) was added dropwise
fluoroboric acid (54% in diethyl ether; 1.2 ml, 8.5 mmol). Add-
itional fluoroboric acid was added after 30 min (0.3 ml, 2.2
mmol), and again after 90 min. After 110 min, the reaction
mixture was filtered, diluted with ethyl acetate, washed with 1 
aq. sodium hydrogen carbonate, dried (Na2SO4), filtered and
concentrated. Column chromatography of the residue with
ethyl acetate in petroleum spirit (1:2) gave mono-ol 10 as a
syrup (1.18 g, 81%), which then was treated with pyridine–
acetic anhydride (2:1; 15 ml) for 14 h. Concentration and co-
concentration with toluene of the reaction mixture gave a
residue, which was purified by column chromatography with
petroleum spirit–ethyl acetate (2:1) as eluent. The appropriate
fractions were collected and concentrated to give compound 11
as an amorphous compound (1.16 g, 94%), [α]D ϩ20 (c 0.2);
1H/13C NMR data (CDCl3) are shown in Table 5 [HRMS: Calc.
for C50H55N3NaO10S: 912.3506. Found: 912.3535 (M ϩ Naϩ)].
Allyl O-[2-acetamido-4-O-acetyl-3,6-di-O-benzyl-2-deoxy-â-
D-mannopyranosyl)-(1→4)-O-(2,3,6-tri-O-benzyl-á-D-gluco-
pyranosyl]-(1→2)-3,4-di-O-benzyl-á-L-rhamnopyranoside 14 and
allyl O-[2-acetamido-4-O-acetyl-3,6-di-O-benzyl-2-deoxy-â-D-
mannopyranosyl)-(1→4)-O-(2,3,6-tri-O-benzyl-â-D-gluco-
A solution of the latter compound (2.00 g, 2.44 mmol) in
dichloromethane–pyridine (2:1; 30 ml) was stirred and cooled
(0 ЊC) while triflic anhydride (1 ml, 5.95 mmol) was added. The
solution was kept at 0 ЊC, and additional triflic anhydride was
added after 2 h 45 min (1 ml, 5.95 mmol), and after 10 h (0.5 ml,
2.98 mmol). After 11 h the reaction mixture was diluted with
dichloromethane, washed successively with 2  sulfuric acid,
1  aq. sodium hydrogen carbonate and water, dried (Na2SO4),
filtered and concentrated.
pyranosyl]-(1→2)-3,4-di-O-benzyl-á-L-rhamnopyranoside 15. A
mixture of disaccharide 11 (1.17 g, 1.32 mmol), rhamnoside
1215 (0.66 g, 1.71 mmol) and molecular sieves (4 Å) in dichloro-
methane (30 ml) was stirred at rt for 30 min. Then methyl tri-
flate (0.43 ml, 3.95 mmol) was added and stirring was continued
at rt for 24 h. Triethylamine (1.65 ml, 11.8 mmol) was added to
destroy excess of methyl triflate, and the mixture was filtered
A mixture of the crude residue containing triflate 8 (yellow
foam) and sodium azide (0.80 g, 12.3 mmol) in DMF (40 ml)
1702
J. Chem. Soc., Perkin Trans. 1, 1998

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Table 6 1H/13C Chemical shift (J/Hz in parentheses)a of compound 14
1
2
3
4
5
6a
6b
Rha
Glc
4.82 (1.5)
96.6
4.90 (3.5)
97.0
4.52 (nd)
99.4
4.02 (3.2)
75.1
3.52 (9.1)
79.7
4.58 (nd)
49.5
3.88 (9.4)
79.1
3.92 (9.1)
80.6
3.17 (9.7)
77.2
3.58 (9.4)
80.3
3.97 (9.7)
75.9
4.99 (nd)
68.0
3.73 (6.1)
68.3
4.08
69.7
3.17
73.8
1.36
18.0
3.22
68.0
3.33 (4.1)
69.0
3.36 (2.3, 10.8)
3.44 (3.5, 10.8)
Man
1
a Further H/13C chemical shifts are 1.73/23.2 (NHCOCH3), 1.93/20.8 (OCOCH3), 3.90 and 4.14/67.7 (CH2CH᎐CH2), 5.20/117.2 (CH2CH᎐CH2),
᎐
᎐
5.72/170.9 (NHAc) and 5.86/134.0 (CH2CH᎐CH2).
᎐
Table 7 1H/13C Chemical shift (J/Hz in parentheses)a of compound 17
1
2
3
4
5
6a
6b
Rha
Glc
5.67 (1.5)
4.07 (2.5)
75.2
3.49 (9.4)
79.4
4.57 (4.4)
49.4
3.92 (9.2)
79.1
3.87 (9.1)
80.6
3.15 (9.5)
77.2
3.59 (9.2)
79.6
3.95 (nd)
75.8
4.98 (9.7)
68.0
3.94 (6.2)
70.2
4.06
69.9
3.16
73.9
1.33
17.9
3.18
68.0
3.33 (4.1)
69.0
94.3 (180)
4.89 (3.8)
97.2 (171)
4.50 (nd)
99.4 (163)
3.32 (11.7)
Man
3.42 (3.2, 10.5)
a Further 1H/13C chemical shifts are 1.35/8.6 [(CH3CH2)3N], 1.71/22.8 (NHCOOCH3), 1.93/20.5 (OCOCH3), 3.06/45.8 [(CH3CH2)3N], 94.3 (C-1, JC,P
5.2) and 6.85 (d, JH,P 684, HPO3Ϫ).
through Celite, diluted with dichloromethane, washed with
water, dried (Na2SO4), filtered and concentrated. Column
chromatography of the residue (stepwise gradient elution
toluene–ethyl acetate 9:1–1:1) gave the anomeric mixture of
trisaccharides (13á/â) as a syrup (1.40 g, 87%, α:β-ratio 68:32)
which was not separated.
mixture was stirred for 54 h at rt. Additional 2-chloro-5,5-
dimethyl-2-oxo-1,3,2-dioxaphosphorinane (0.25 g, 1.35 mmol)
was added, the reaction mixture was heated to 40 ЊC, and stir-
ring was continued for 17 h. Then 1  aq. triethylammonium
hydrogen carbonate (2 ml) was added to destroy excess of
reagent, and the mixture was diluted with dichloromethane,
washed with 0.5  aq. triethylammonium hydrogen carbonate,
dried (Na2SO4), filtered and concentrated. The residue was
purified by column chromatography (ethyl acetate–acetic acid–
methanol–water 40:3:3:2), then was dissolved in dichloro-
methane and washed with 0.25  aq. triethylammonium
hydrogen carbonate. The organic layer was separated, concen-
trated and co-concentrated from dichloromethane (5 × 10 ml),
which gave, after drying in vacuo, the title compound 17 as a
foam (0.427 g, 61%), [α]D ϩ21 (c 0.2). 1H/13C NMR data
(CDCl3) are shown in Table 7 [HRMS: Calc. for C71H79NO18P:
1264.5034. Found: 1264.4949 (M Ϫ Hϩ)].
Preparation of deprotected nonasaccharide 1. To a stirred
solution of compound 17 (49.7 mg, 36.3 µmol) and Cbz-
ethanolamine16 (14.0 mg, 71.7 µmol) in pyridine (0.7 ml) under
N2 at rt was added pivaloyl chloride (13 µl, 109 µmol). After 20
min the formed H-phosphonate diester was oxidized during 15
min by addition of water (50 µl) and iodine (28 mg, 110 µmol).
Then the reaction mixture was diluted with dichloromethane,
washed successively with 10% aq. sodium thiosulfate, 2  aq.
sulfuric acid and 1  aq. triethylammonium hydrogen carbon-
ate and the dichloromethane layer was evaporated under a
stream of N2. The residue was dissolved in 0.2  methanolic
sodium methoxide (3.0 ml) and stirred overnight at rt. An
excess of acetic acid was added and the mixture was concen-
trated using a Rotavapor (bath temp. 30 ЊC). Column chrom-
atography of the residue (stepwise gradient elution, ethyl
acetate–methanol–acetic acid–water 100:3:3:2 and 40:3:3:2),
followed by concentration of appropriate fractions, gave a res-
idue, which was dissolved in dichloromethane and washed with
1  aq. triethylammonium hydrogen carbonate. The dichloro-
methane layer was evaporated with a stream of N2 and the
residue was dried in vacuo to give compound 18 as a foam (50.3
mg, 91%) [HRMS: Calc. for C79H88N2O20P: 1415.5667. Found:
1415.5669 (M Ϫ Hϩ)].
This mixture (1.39 g, 1.13 mmol) and triphenylphosphine
(0.43 g, 1.64 mmol) in dichloromethane (25 ml) was stirred at
37 ЊC for 23 h. Then the formed imine was hydrolysed by add-
ition of water (25 ml), and stirring was continued for 10 h at
37 ЊC. The organic layer and a dichloromethane wash were
combined, dried (Na2SO4), filtered and concentrated. The resi-
due was stirred overnight at rt with pyridine–acetic anhydride
(2:1; 15 ml), concentrated, and co-concentrated twice from
toluene. Column chromatography of the residue (stepwise
gradient elution, petroleum spirit–ethyl acetate 3:2–1:2)
gave first compound 14 as a foam (0.81 g, 58% from 13á/â),
[α]D ϩ18 and then compound 15 as a syrup (0.388 g, 28%
from 13á/â), [α]D Ϫ11; 1H/13C NMR data (CDCl3) of 14 are in
Table 6 [HRMS: Calc. for C74H83NNaO16: 1264.5609. Found:
1264.5774 (M ϩ Naϩ)]. 1H/13C NMR data (CDCl3) of 15:
δ 4.65/104.9 (1 H, d, J1Ј,2Ј 8.3, H-1Ј), 4.73/99.6 (m, H-1Љ) and
4.96/98.8 (d, J1,2 1.5, H-1) [HRMS: Calc. for C74H83NNaO16:
1264.5609. Found: 1264.5731 (M ϩ Naϩ)].
Triethylammonium
O-(2-acetamido-4-O-acetyl-3,6-di-O-
benzyl-2-deoxy-â-D-mannopyranosyl)-(1→4)-O-(2,3,6-tri-O-
benzyl-á-D-glucopyranosyl)-(1→2)-3,4-di-O-benzyl-á-L-rhamno-
pyranosyl hydrogen phosphonate 17. Trisaccharide 14 (0.80 g,
0.64 mmol) and tris(triphenylphosphine)rhodium() chloride
(0.06 g, 64 µmol) in ethanol (12 ml), toluene (4.8 ml) and water
(1.5 ml) was refluxed for 4 h. The reaction mixture was diluted
with water and extracted with diethyl ether. The combined
etheral layers were washed with saturated aq. potassium
chloride, dried (Na2SO4), filtered and concentrated. The residue
was stirred with 80% aq. acetic acid (40 ml) overnight at 80 ЊC,
then diluted with ethyl acetate, washed with 1  aq. sodium
hydrogen carbonate, dried (Na2SO4), filtered and concentrated.
Column chromatography of the residue (stepwise gradient
elution, toluene–ethyl acetate 3:1–3:2) gave compound 16 as
an amorphous product (0.61 g, 80%).
Compound 16 (0.614 g, 0.51 mmol) as a solution in pyridine
(1.9 ml) was mixed with a 2  solution of phosphorous acid
in pyridine (2.6 ml), then 2-chloro-5,5-dimethyl-2-oxo-1,3,2-
dioxaphosphorinane (0.47 g, 2.55 mmol) was added and the
To a stirred suspension of compounds 17 (43.1 mg, 31.4
µmol) and 18 (47.9 mg, 31.5 µmol) in pyridine (0.8 ml) under N2
at rt was added pivaloyl chloride (8 µl, 65.3 µmol). Additional
pivaloyl chloride (5 µl, 40.8 µmol) was added after 20 min, and
J. Chem. Soc., Perkin Trans. 1, 1998
1703

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Table 8 1H/13C Chemical shift (J/Hz in parentheses)a of compound 1
1
2
3
4
5
6a
1.31
J6,5 6.2
17.4
6b
Rha AB
Rha C
5.50
JH,P 7.0
94.5
JH,C 174
5.47
JH,P 7.5
94.4
JH,C 174
5.02
J1,2 3.7
98.4
JH,C 172
5.03
J1,2 3.7
98.4
4.03
77.5
4.00
77.6
3.58
71.8
3.59
(ol)
4.58
3.94
3.51
72.4
3.53
(ol)
3.70
3.89
69.7
(ol)
(ol)
71.2*
(ol)
(ol)
(ol)
(ol)
Glc AB
Glc C
3.89
4.08
71.0*
(ol)
3.72–3.80
60.5
(ol)
3.72–3.80
(ol)
70.9*
(ol)
79.4
(ol)
(ol)
(ol)
(ol)
(ol)
JH,C 172
4.91
Man AB
Man C
4.00
72.0*
3.82
72.6
4.07
3.56
76.4
3.45
77.3
3.85
61.2
3.82
(ol)
3.95
J2,3 4.0
53.9
100.0
JH,C 165
4.89
72.9*
3.52
4.55
J2,3 4.0
54.1
3.93
100.0
67.2
JH,C 165
a Further 1H/13C chemical shifts are 2.07/22.8 (CCOCH3), 2.08/22.8 (AB, COCH3), 3.28/40.8 (OCH2CH2NH2), 4.12/62.8 (OCH2CH2NH2) and 176.2
(C᎐O).
᎐
after 50 min water (50 µl) and iodine (24 mg, 94.5 µmol)
were added, and stirring was continued for 20 min. Work-up,
deacetylation of the obtained material, and isolation of com-
pound 19 were performed as described for the previous step,
giving product 19 as a foam (77.5 mg, 87%) (FABMS: Calc. for
C148H164N3O37P2: 2637.05. Found: m/z, 2637.044).
To a stirred suspension of compounds 17 (36.5 mg, 26.7
µmol) and 19 (75.8 mg, 26.7 µmol) in pyridine (0.9 ml) under
N2 at rt was added pivaloyl chloride (10 µl, 81.7 µmol). Add-
itional pivaloyl chloride (3 × 5 µl, 40.8 µmol) was added after
20, 40 and 60 min. Water (50 µl) and iodine (20 mg, 80 µmol)
were added after 90 min and then, after 115 min, work-up,
deacetylation of the obtained material, and isolation of prod-
uct 20 were performed as described for the previous step, giving
compound 20 as a foam (92.6 mg, 83%).
References
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Paper 7/09293H
Received 24th December 1997
Accepted 2nd March 1998
1704
J. Chem. Soc., Perkin Trans. 1, 1998