Synthesis of the branched trisaccharide L-glycero-α-D-manno- heptopyranosyl-(1 → 3)- [β-D-glucopyranosyl-(1 → 4)]-L-glycero-α-D-manno-heptopyranose, protected to allow flexible access to Neisseria and Haemophilus LPS inner core structures

Article abstract of DOI:10.1081/CAR-200044580

An efficient synthesis of the protected branched trisaccharide (2′S,3′S)-(7-O-benzyl-6-O-chloroacetyl-3,4-O-(2′, 3′-dimethoxybutane-2′,3′-diyl)-2-O-p-methoxybenzyl-L-glycero- α-D-manno-heptopyranosyl)-(1 → 3)-[(2,3,4,6-tetra-O-benzoyl-β-D- glucopyranosyl)

Full text of DOI:10.1081/CAR-200044580

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Synthesis of the Branched Trisaccharide
L‐Glycero‐α‐D‐manno‐heptopyranosyl‐(1 → 3
[β‐D‐glucopyranosyl‐(1 → 4)]‐L‐glycero‐α‐D‐m
Protected to Allow Flexible Access to
Neisseria and Haemophilus LPS Inner
Core Structures
Eva Segerstedt a , Karin Mannerstedt a , Mikael Johansson a & Stefan
Oscarson a
a Department of Organic Chemistry, Arrhenius Laboratory ,
Stockholm University , Floor 6, S‐106 91, Stockholm, Sweden
Published online: 09 Aug 2006.
To cite this article: Eva Segerstedt , Karin Mannerstedt , Mikael Johansson & Stefan Oscarson
(2004) Synthesis of the Branched Trisaccharide L‐Glycero‐α‐D‐manno‐heptopyranosyl‐(1 → 3)‐
[β‐D‐glucopyranosyl‐(1 → 4)]‐L‐glycero‐α‐D‐manno‐heptopyranose, Protected to Allow Flexible Access
to Neisseria and Haemophilus LPS Inner Core Structures, Journal of Carbohydrate Chemistry, 23:8-9,
443-452, DOI: 10.1081/CAR-200044580
To link to this article: http://dx.doi.org/10.1081/CAR-200044580
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JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 23, No. 8–9, pp. 443–452, 2004
Synthesis of the Branched Trisaccharide
L-Glycero-a-D-manno-heptopyranosyl-(1 ! 3)-
[b-D-glucopyranosyl-(1 ! 4)]-L-glycero-a-D-
manno-heptopyranose, Protected to Allow Flexible
Access to Neisseria and Haemophilus LPS Inner
Core Structures
Eva Segerstedt, Karin Mannerstedt, Mikael Johansson,
*
and Stefan Oscarson
Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University,
Stockholm, Sweden
CONTENTS
ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444
I. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444
II. RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . 445
III. EXPERIMENTAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448
ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . 451
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
*
Correspondence: Stefan Oscarson, Department of Organic Chemistry, Arrhenius Laboratory,
Stockholm University, Floor 6, S-106 91 Stockholm, Sweden; Fax: þ46 8 15 49 08; E-mail:
s.oscarson@organ.su.se.
443
DOI: 10.1081/CAR-200044580
0732-8303 (Print); 1532-2327 (Online)
www.dekker.com
Copyright # 2004 by Marcel Dekker, Inc.

444
Segerstedt et al.
ABSTRACT
An efficient synthesis of the protected branched trisaccharide (20S,30S)-(7-O-benzyl-6-
O-chloroacetyl-3,4-O-(20,30-dimethoxybutane-20,30-diyl)-2-O-p-methoxybenzyl-L-
glycero-a-D-manno-heptopyranosyl)-(1 ! 3)-[(2,3,4,6-tetra-O-benzoyl-b-D-gluco-
pyranosyl)-(1 ! 4)]-7-O-acetyl-1,6-anhydro-2-O-benzyl-L-glycero-b-D-manno-
heptopyranose, which is a key intermediate in the synthesis of inner core
structures of Haemophilus and Neisseria LPSs, is described. The heptoses were
formed by Grignard reactions using a benzyloxymethyl chloride or a commercial
vinyl reagent. The anhydro bridge was formed by treatment of a 6-OH methyl
a-heptoside precursor with FeCl3. The protecting group pattern allows modifi-
cations at the 2-, 3-, 4-, and 6-positions of the second heptose moiety and also,
after acetolysis of the anhydro bridge, elongation at the reducing end, all known
alterations found in the bacterial LPSs.
Key Words: Oligosaccharide synthesis; 1,6-Anhydro acceptors; Glycoconjugate
vaccines.
INTRODUCTION
The lipopolysaccharide (LPS) of the gram-negative bacteria Haemophilus influenzae
is extremely heterogeneous, which has made the analysis of its structure most complex.
The LPS is truncated and lacks the polymeric O-antigen. Through the phase-variable
expression of several transferases and kinases, the bacteria can build up a large number
of structures, possibly to be able to evade the human immune system. With the elucidation
of the bacterial genome, subsequent use of bacterial mutants, and new analysis
techniques, it has become possible during the last decade to determine many of these struc-
tures and the genetics behind them.[1,2] These results strongly indicated that the bacterium
makes a conservative core pentasaccharide (Fig. 1), which is linked to Lipid A and is
found in all bacteria so far investigated. This core is then modified in numerous ways
by glycans, various phosphate groups, amino acids, and acetates.
Glycoconjugate vaccines based on capsular polysaccharide structures have been most
successful.[3,4] Due to the heterogeneity of the H. influenzae LPS it is not possible to use
these carbohydrate structures for the construction of a conjugate vaccine. One conceivable
solution instead would be to use well-defined synthetic structures corresponding to
the conservative part of the LPS. Another advantage of this approach would be that detoxi-
fication of the Lipid A part is not needed. As part of a program directed toward LPS-based
vaccines against H. influenzae, we are trying to synthesize part structures of the LPS inner
core and evaluate these as vaccine candidates after conjugation to a carrier protein.
Figure 1. Structure of the inner core of dephosphorylated H. influenzae LPS.

Allowing Access to Neisseria and Haemophilus LPS
445
A key intermediate in these syntheses is the branched trisaccharide L-glycero-a-D-
manno-heptopyranosyl-(1 ! 3)-[b-D-glucopyranosyl-(1 ! 4)]-L-glycero-b-D-manno-
heptopyranose.
In an earlier publication we showed that the use of a 1,6-anhydro-heptose acceptor
facilitated the formation of the 3,4-branched trisaccharide.[5] Obviously, the confor-
mational change, from 4C1 to 1C4, in the acceptor released the steric strain in the transition
state to the target trisaccharide and allows for an efficient a-glycosylation of the free
3-position in derivative 8. Herein, a more efficient route to the 1,6-anhydro-heptose accep-
tor is presented, starting from commercial methyl a-D-mannopyranoside and using a com-
mercial vinyl Grignard reagent in the carbon elongation step. Furthermore, the synthesis
and introduction of a highly differentially protected heptose donor is reported, which
permits subsequent selective glycosylations or other modifications, for example phos-
porylation, in positions 2, 3, 4, and 6 in the second heptose residue, variations frequently
found in the native LPS of Neisseria and Haemophilus strains.
RESULTS AND DISCUSSION
Our earlier published synthesis of the 1,6-anhydro heptose acceptor 7, although effi-
cient, involved features that make a scale-up difficult. Ethyl 1-thio-a-D-mannopyranoside
(prepared in four steps from commercial D-mannose) was used as precursor and the labile
benzyloxymethyl chloride Grignard reagent was utilized in the carbon elongation, which
is not completely stereoselective (L/D 9 : 1). Thus, modifications were investigated that
would allow an efficacious large scale synthesis of donor 7 (Sch. 1).
˚
Aberg and Ernst have described the formation of 1,6-anhydrohexoses in good yields
from the corresponding methyl glycosides by simple treatment with FeCl3 in aceto-
nitrile,[6] which should make it possible to use commercially available methyl
a-D-mannopyranoside as heptose precursor. Regarding the carbon elongation reaction,
Scheme 1. i) a. (COCl)2, DMSO, THF, 2608C; b. DIPEA, rt; ii) CH2CHMgBr, THF, 2608C;
iii) FeCl3, CH3CN, reflux; iv) OsO4, NaIO4, acetone-H2O 5 : 1; v) NaBH4, EtOH; vi) Ac2O, pyridine;
vii) H2 Pd(OH)2/C, EtOH; viii) Me2C(OMe)2 CSA, DMF.

446
Segerstedt et al.
Dasser et al. reported already in 1990 the use of a vinyl Grignard reagent for this
purpose.[7] Although this approach involves additional steps (i.a. dihydroxylation and per-
iodate cleavage, excluding thioglycoside precursors), the advantages are several: a stable
(commercial) reagent and very high stereoselectivity. Recently, a large scale synthesis of
methyl L-glycero-D-manno-heptopyranoside employing this methodology on a BDA-
protected methyl mannoside was published.[8]
Methyl mannoside derivative 1 was prepared by known procedures.[9] One-pot Swern
oxidation and consecutive Grignard vinylation afforded the vinyl derivative 2 in 82% yield
and with complete stereoselectivity for the L-glycero form (Sch. 1). Treatment of 2 with
FeCl3 in acetonitrile at reflux then smoothly afforded the 1,6-anhydro derivative 3 (71%).
The alkene was dihydroxylated, the obtained diol was cleaved through periodate oxi-
dation, and the resulting aldehyde was reduced with NaBH4 to give 4 in an 88% overall
yield. Straightforward acetylation (!5, 92%), debenzylation (!6, 91%), and isopropy-
lidenation then afforded the desired heptose acceptor 7 (86%). As compared to the
earlier synthesis of 7, the yield is only slightly better (35% vs. 30% calculated from
the 6-OH mannose precursors), but the real advantage is the possibility to start from the
methyl mannoside in combination with the reproducibility of the Grignard reaction.
Compound 7 was then transformed as previously described into the 3-OH disacchar-
ide donor 8 (Sch. 2).[5]
As mentioned in the introduction, the second heptose residue is frequently heavily
substituted both in Neisseria and in Haemophilus LPS. In Neisseria meningitidis there
are glycosylations found in the 2- and 3-positions and phosphorylations at the 3-, 6-,
and 7-hydroxyl groups.[10] In H. influenzae there are glycan substituents found in the
2- and 3-positions, whereas phosphate groups are found mainly at the 6-hydroxyl
group.[1,2] To make later introduction of these substituents feasible, a heptosyl thioglyco-
side donor with orthogonal temporary protecting groups in the 2-, 3-, 4-, and 6-positions
was designed and synthesised (Sch. 3). Starting from ethyl 1-thio-a-D-mannopyranoside,
the known 3,4-BDA-acetal 9[11] was prepared. Attempted regioselective p-methoxybenzy-
lation at the 2-position, in accordance with benzylation results obtained by Yamasaki
et al.,[8] afforded instead mainly the 6-O-benzylated derivative. Interestingly, the same
selectivity was observed also for the corresponding O-methyl glycoside, which is the sub-
strate used by Yamasaki et al. Thus, regioselective benzylation of the 2,6-diol produced
2-O-selectivity, whereas p-methoxybenzylation gave 6-O-selectivity. Hence, the 2,6-
diol 9 was first silylated selectively at the 6-position (!10) and then p-methoxybenzy-
lated and desilylated to yield 11 in 48% overall yield. Swern oxidation to the aldehyde
Scheme 2. i) AgOTf, CH2Cl2, 08C, 77%; ii) HOAc (80% aq.), 808C, 85%; iii) a. Bu2SnO, benzene,
reflux, b. BnBr, Bu4NBr, reflux, 80%.

Allowing Access to Neisseria and Haemophilus LPS
447
Scheme 3. i) TBDMSCl, DMF, imidazole; ii) a. p-MBnBr, NaH, DMF; b. TBAF, THF; iii) a.
(COCl)2, DMSO, THF, 2608C; b. DIPEA, rt; iv) BnOCH2Cl, Mg, THF; v) ClAcCl, pyridine/
CH2Cl2.
followed by a Barbier reaction using benzyloxymethyl chloride afforded in 50% yield the
heptoside derivative 12, chloroacetylation of which afforded the desired thioglycoside
donor 13.
Dimethyl(methylthio)sulfonium triflate (DMTST)-promoted coupling of donor 13
and acceptor 8 yielded the trisaccharide 14 in 79% yield with complete a-selectivity
(JC,H 169 Hz), proving once more 8 to be an excellent acceptor for the construction of
these 3,4-branched trisaccharide structures (Sch. 4).
Trisaccharide 14 comprises the possibilities for the syntheses of a plethora of
Neisseria and Haemophilus LPS inner core structures. Removal of the p-methoxybenzyl
Scheme 4. i) DMST, Et2O; ii) Sc(OTf)3, Ac2O.

448
Segerstedt et al.
group allows for the introduction in the 20-position of a 2-acetamido-2-deoxy-a-D-
glucopyranosyl (N. meningitidis) or a third heptosyl (H. influenzae) moiety, whereas
removal of the chloroacetyl-protecting group permits formation of a 60-ethanolamine-
phosphate group (N. meningitidis and H. influenzae). Removal of the BDA-acetal make
30-substitution possible,[12] and acetolysis of the 1,6-anhydro bridge [!15 (78%),
Sch. 4] and subsequent transformation into a donor would enable elongation at the
reducing end as shown previously.[5]
EXPERIMENTAL
General methods. TLC was carried out on Merck precoated 60 F254 plates using
UV-light and/or 8% sulfuric acid for visualization. Column chromatography was per-
formed on silica gel (0.040–0.063 mm, Amicon). NMR spectra were recorded in CDCl3
(internal Me4Si, d ¼ 0.00) at 258C on a Varian 300 MHz or 400 MHz instrument. Organic
phases were dried over MgSO4 before evaporation, which was performed under reduced
pressure. Benzyloxymethyl chloride was synthesized according to the literature,[13] dried
over CaCl2, and stored without drying agent in a sealed container at 2188C.
Methyl 2,3,4-tri-O-benzyl-7,8-dideoxy-a-D-manno-oct-7-enopyranoside (2).
Oxalyl chloride (2.52 mL, 28.9 mmol) and dry THF (75 mL) were added to a 1000 mL
flask fitted with a dropping funnel and a stirrer. The solution was cooled to 2708C,
DMSO (4.1 mL, 57.8 mmol) in dry THF (10 mL) was added, and the mixture was stirred
for 15 min. Methyl 2,3,4-tri-O-benzyl-a-D-mannopyranoside[9] (1, 12.2 g, 26.3 mmol) in
dry THF (50 mL) was added drop-wise. The reaction mixture was stirred for 1 hr at
2608C and then for 1 hr at 2408C. N-Ethyldiisopropylamine (22.9 mL, 131 mmol) was
added, and the solution was slowly (overnight) brought to rt. The mixture was recooled
down to 2608C. Vinyl magnesium bromide (1 M in THF, 131 mmol) was added drop-
wise, and the reaction mixture was stirred for 2 hr at 2608C. The reaction was quenched
by the addition of ethanol (10 mL), and after adding saturated aqueous NH4Cl (25 mL),
the whole reaction mixture was warmed to rt. The mixture was extracted with EtOAc
(50 mL ꢀ 3), and the combined extracts were sequentially washed with 5% sodium hypo-
chlorite in H2O (50 mL ꢀ 4) and brine (50 mL ꢀ 2). After drying (MgSO4), the organic
solution was concentrated to a syrup, which was purified by silica gel chromatography
(toluene/EtOAc 9 : 1) to give compound 2 (10.6 g, 21.6 mmol, 82%); [a]D þ9
(c 1.0, CHCl3); 1H NMR d: 7.37–7.32 (m, 15H, Ar-H), 6.02 (m, 1H, H-7), 5.39 (dt, 1H,
J7,8a ¼ 17.3 Hz, J8a,8b ¼ 1.4 Hz, H-8a), 5.22 (dt, 1H, J7,8b ¼ 10.4 Hz, H-8b), 5.00 (d, 1H,
J ¼ 11 Hz, CHPh), 4.81–4.65 (m, 6H, 5 ꢀ CHPh, H-1), 4.44 (m, 1H, J6,7 ¼ 4.9 Hz, H-6),
4.17 (t, 1H, J4,5 ¼ 9.6 Hz, H-4), 3.92 (dd, 1H, J3,4 ¼ 9.3 Hz, H-3), 3.80 (dd, 1H,
J2,3 ¼ 3Hz, J1,2 ¼ 1.9, H-2), 3.59 (dd, J5,6 ¼ 1.7 Hz, H-5), 3.28 (s, 3H, OCH3); 13C
NMR: d 54.7, 70.7, 73.2, 72.9, 73.8, 74.7, 74.9, 75.3, 80.3, 99.4, 115.4, 127.6–128.5,
138.4, 138.5, 138.6, 138.7.
Anal. Calcd for C30H34O6: C, 73.45; H, 6.99; Found: C, 73.20; H, 7.06.
7-O-Acetyl-1,6-anhydro-2,3,4-tri-O-benzyl-L-glycero-b-D-manno-heptopyranose
(5). Compound 2 (450 mg, 0.91 mmol) was dissolved in dry CH3CN (10 mL), and FeCl3
(104 mg, 0.64 mmol) was added. The reaction mixture was refluxed for 1.5 hr and then
cooled to room temperature. Concentration followed by silica gel chromatography

Allowing Access to Neisseria and Haemophilus LPS
449
(toluene/EtOAc 6:1) yielded 1,6-anhydro-2,3,4-tri-O-benzyl-7,8-dideoxy-b-D-manno-
oct-7-enopyranose (3, 300 mg, 0.65 mmol, 71%); [a]D 237 (c 1.0, CHCl3); 13C NMR:
d 71.4, 71.5, 73.5, 74.4, 74.4, 76.3, 76.5, 78.7, 100.9, 116.8, 127.8–128.6, 137.3, 137.7,
138.0. Compound 3 (403 mg, 0.88 mmol) in acetone/H2O (5 : 1, 6 mL) was cooled to
08C and treated with NaIO4 (958 mg, 4.48 mmol) and 1% aqueous OsO4 (1.6 mL). After
stirring the reaction mixture for 24 hr at room temperature, H2O (10 mL) was added, and
the mixture was extracted with CH2Cl2 (10 mL ꢀ 2). The combined extracts were
washed with H2O (10 mL), dried (MgSO4), and concentrated to a syrup, which was then
treated with NaBH4 in EtOH (7 mL) for 15 hr at room temperature. The solution was con-
centrated, and the residue was dissolved in CH2Cl2 (10 mL) and washed with H2O
(5 mL ꢀ 2). The organic phase was separated, dried (MgSO4), and concentrated in
vacuo. The residue was purified by silica gel chromatography (toluene/EtOAc 4 : 1) to
yield 1,6-anhydro-2,3,4-tri-O-benzyl-L-glycero-b-D-manno-heptopyranose (4, 359 mg,
0.78 mmol, 88%); 13C NMR: d 63.8, 71.2, 71.3, 73.1, 73.9, 74.0, 75.4, 75.5, 76.0,
100.6, 127.6–128.4, 137.5, 137,6, 137.7. Compound 4 (235 mg, 0.51 mmol) was dis-
solved in Ac2O/pyridine (2 : 1, 3 mL), and the reaction mixture was stirred at room temp-
erature for 1.5 hr. MeOH was added and the mixture was concentrated. Silica gel
chromatography (toluene/EtOAc 6 : 1) of the residue afforded 5 (238 mg, 0.47 mmol,
92%); [a]D –32 (c 1.0, CHCl3); 1H NMR d: 7.37–7.23 (m, 15H, Ar-H), 5.50
(s, 1H, H-1), 4.65–4.53 (m, 7H, 6 ꢀ CHPh, H-6), 4.26 (s, 1H, H-5), 4.02 (m, 2H,
J6,7a ¼ 6.3 Hz, J6,7b ¼ 6.6 Hz, J7a,7b ¼ 11.0 Hz, H-7a, H-7b), 3.83 (m, 1H, J3,4 ¼ 1.6 Hz,
H-3), 3.56 (dd, 1H, J2,3 ¼ 5.2 Hz, J1,2 ¼ 1.6 Hz, H-2), 3.50 (s, 1H, H-4), 2.06
(s, 3H, CH3CO); 13C NMR: d 21.1, 65.2, 71.6, 71.7, 73.3, 73.6, 74.3, 74.3, 76.2, 76.4,
101.1, 128.0–128.128.8, 137.8, 138.0, 138.1, 170.9.
Anal. Calcd for C30H34O7: C, 71.41; H, 6.39; Found: C, 71.17; H, 6.37.
7-O-Acetyl-1,6-anhydro-2,3-O-isopropylidene-L-glycero-b-D-manno-heptopyra-
nose (7). Compound 5 (178 mg, 0.35 mmol) was dissolved in EtOH (4 mL). Pd(OH)2
(20%, 50 mg), 5 drops of H2O and 5 drops of EtOAc were added to the solution. The
mixture was hydrogenolyzed at 110 psi for 72 hr. The mixture was filtered through
Celite, concentrated, and purified on a silica gel column (CH2Cl2/MeOH 7 : 1) to give
7-O-acetyl-1,6-anhydro-L-glycero-b-D-manno-heptopyranose (6, 75 mg, 0.32 mmol,
91%); [a]D 297 (c 1.0, CHCl3); 13C NMR: d 20.8, 65.1, 65.7, 70.6, 71.5, 72.8, 78.0,
102.6, 171.3. Compound 6 (33 mg, 0.14 mmol) was dissolved in DMF (2 mL). 2,2-
Dimethoxypropane (69 mL, 0.56 mmol) was added and the solution was adjusted to pH
2 by addition of ( þ/2)-10-camphorsulfonic acid. The mixture was stirred for 3 hr at rt
and then concentrated. Toluene was added and the solution was neutralized by addition
of triethylamine. The solution was concentrated and co-evaporated three times with
toluene. Silica gel chromatography (toluene/EtOAc 3 : 1!1 : 1) yielded 7 (33 mg,
0.12 mmol, 86%), which NMR data were identical to the earlier prepared sample.[5]
(20S,30S)-Ethyl 3,4-O-(20,30-Dimethoxybutane-20,30-diyl)-2-O-p-methoxybenzyl-1-
thio-a-D-mannopyranoside (11). Compound 9[11] (4.3 g, 12.7 mmol) was treated with
tert-butyldiphenylsilyl chloride (2.3 g, 15.2 mmol) in dry DMF (50 mL) in the presence
of imidazole (2.16 g, 31.8 mmol) for 20 hr at rt. After quenching the reaction by adding
H2O (30 mL), the product was extracted with EtOAc (100 mL ꢀ 3). The combined
organic extracts were dried (MgSO4), concentrated, and purified on a silica gel column
(toluene/EtOAc 6 : 1 ! 1 : 1) to yield ethyl 6-O-tert-butyldimethylsilyl-3,4-O-(20,30-
dimethoxybutane-20,30-diyl)-1-thio-a-D-mannopyranoside (10, 4.68 g, 10.3 mmol, 81%);

450
Segerstedt et al.
13C NMR: d 25.1, 25.4, 14.8, 17.6, 17.8, 18.2, 24.7, 25.9, 47.8, 48.0, 61.7, 63.3, 69.1,
71.1, 71.8, 83.8, 99.8, 100.3. Compound 10 (3.96 g, 8.76 mmol) was dissolved in dry
DMF (17 mL) and added drop-wise to a chilled (08C) mixture of p-methoxybenzyl
bromide (2.9 mL, 17.5 mmol) and sodium hydride (660 mg, 95% in oil) in dry DMF
(5 mL). The ice bath was removed, and the reaction was allowed to continue until TLC
showed complete reaction. MeOH (2 mL) was added slowly and the mixture was stirred
overnight. Toluene was added, and the organic phase was washed with H2O, dried
(MgSO4), and concentrated. Silica gel chromatography (toluene/EtOAc) gave ethyl
6-O-tert-butyldimethylsilyl-3,4-O-(20,30-dimethoxybutane-20,30-diyl)-2-O-p-methoxyben-
zyl-1-thio-a-D-mannopyranoside (4.92 g, 8.59 mmol, 98%). This compound (5.58 g,
9.74 mmol) was treated with tetrabutylammonium fluoride (1 M in THF, 26 mL) in dry
THF (26 mL) for 24 hr at rt. The reaction mixture was diluted with H2O (100 mL) and
then extracted with EtOAc (100 mL ꢀ 2). The combined extracts were washed with
brine, dried (MgSO4), and concentrated. Purification by silica gel chromatography
yielded 11 (2.68 g, 5.84 mmol, 60%); [a]D þ226 (c 1.0, CHCl3); 13C NMR: d 15.0,
17.9, 17.9, 25.5, 48.0, 48.0, 55.4, 61.8, 64.2, 69.7, 71.3, 72.9, 77.6, 84.2, 99.7, 100.0,
113.8, 129.7–130.8, 159.3.
HRMS: Calcd for C22H33O8S: 457.1896; Found: 457.1895.
(20S,30S)-Ethyl 7-O-Benzyl-3,4-O-(20,30-dimethoxybutane-20,30-diyl)-2-O-p-meth-
oxybenzyl-1-thio-L-glycero-a-D-manno-heptopyranoside (12). Compound 11 (2.4 g,
5.23 mmol) was submitted to Swern oxidation according to the procedure described
above for compound 2 to give the corresponding aldehyde. Freshly activated magnesium
turnings (725 mg, 29.8 mmol), 50 mg of sublimed HgCl2 and dry THF (5 mL) were added
(under argon atmosphere) to a flame-dried flask equipped with an internal thermometer, a
stirrer, and a dropping funnel. The solution was stirred for 10 min. Benzyloxymethyl
chloride (2.17 mL, 15.7 mmol) was added to the dropping funnel, and a few drops were
added to the magnesium at rt. Once the exothermic reaction had started (about þ308C,
as monitored by the thermometer, which must extend into the reaction slurry), the flask
was partially immersed into an ice bath (08C). The aldehyde (approx. 5.23 mmol) dis-
solved in freshly distilled THF (10 mL) was added into the dropping funnel, and this alde-
hyde/alkyl halide mixture was added drop-wise while the inner flask temperature was kept
between 20–308C. The mixture was stirred overnight and then diluted by diethyl ether
(200 mL), whereafter freshly prepared saturated aqueous NH4Cl (400 mL) was added.
The organic phase was separated and washed with saturated aqueous NH4Cl, dried
(MgSO4), filtered, and concentrated to give ethyl 7-O-benzyl-3,4-O-(20,30-dimethoxy-
butane-20,30-diyl)-2-O-p-methoxybenzyl-1-thio-L-glycero-a-D-manno-heptopyranoside (12,
1.6 g, 2.61mmol, 50%) after silica gel column chromatography (toluene/EtOAc
12: 1 ! 8 : 1); [a]D þ211 (c 1.0, CHCl3); 13C NMR: d 14.8, 17.9, 25.2, 48.0, 48.0, 55.4,
63.1, 67.6, 69.9, 70.4, 71.8, 72.7, 73.4, 76.7, 84.0, 99.7, 100.0, 113.7, 127.7, 2130.8,
138.2, 159.2.
HRMS: Calcd for C30H41O9S: 577.2471; Found, 577.2462.
[(20S,30S)-7-O-Benzyl-6-O-chloroacetyl-3,4-O-(20,30-dimethoxybutane-20,30-diyl)-
2-O-p-methoxybenzyl-L-glycero-a-D-manno-heptopyranosyl]-(1 ! 3)-[(2,3,4,6-tetra-
O-benzoyl-b-D-glucopyranosyl)-(1 ! 4)]-7-O-acetyl-1,6-anhydro-2-O-benzyl-L-glycero-
b-D-manno-heptopyranose (14). Chloroacetyl chloride (448 mL, 5.62 mmol) was added
to a chilled (08C) solution of compound 12 (1.3 g, 2.25 mmol) in CH2Cl2/pyridine (15 : 1,
16 mL). The reaction was stirred at rt for 45 min. H2O (10 mL) was added, and the organic

Allowing Access to Neisseria and Haemophilus LPS
451
phase was separated, dried (MgSO4), and concentrated. Purification by silica gel column
chromatography gave ethyl 7-O-benzyl-6-O-chloroacetyl-3,4-O-(20,30-dimethoxybutane-
20,30-diyl)-2-O-p-methoxybenzyl-1-thio-L-glycero-a-D-manno-heptopyranoside (13, 1.18 g,
1.80mmol, 80%); 1H NMR d: 7.37–7.30 (m, 7H, Ar-H), 6.86 (d, 2H, Ar-H), 5.63, (m, 1H,
J6,7a ¼ 7.5 Hz, J6,7b ¼ 6.0 Hz, H-6), 5.30 (s, 1H, H-1), 4.82 (d, 1H, CHPh), 4.62–4.45
(m, 3H, J ¼ 11.5Hz, 3 ꢀ CHPh), 4.29 (dd, 1H, J5.6 ¼ 1.6 Hz, H-5), 4.19 (t, 1H,
J4,5 ¼ 10.1Hz, H-4), 4.05 (s, 2H, ClCH2CO) 4.00 (1H, J3,4 ¼ 10.1Hz, H-3), 3.80 (s, 3H,
PhOCH3), 3.78 (m, J2,3 ¼ 2.6 Hz, J1,2 ¼ 0.92Hz, H-2), 3.65 (m, 2H, J7a,7b ¼ 10.1Hz, H-7a,
H-7b), 3.25 (s, 3H, OCH3), 3.19 (s, 3H, OCH3), 2.52 (m, 2H, SCH2), 1.33 (s, 3H, CH3), 1.29
(s, 3H, CH3), 1.19 (t, 3H, SCH2CH3); 13C NMR: d 14.7, 17.8, 17.9, 25.2, 40.9, 47.9, 48.2,
55.3, 63.0, 67.9, 69.1, 69.8, 70.2, 73.4, 73.0, 76.7, 83.9, 99.9, 100.2, 113.6, 113.7, 127.6–
130.7, 137.8 159.2, 167.0. Compound 8 (297mg, 0.33mmol) and 13 (347mg, 0.53mmol)
˚
were dissolved in dry diethyl ether and powdered molecular sieves (4 A) were added. After
stirring at rt for 1 hr, DMTST (408mg, 1.58mmol) was added and the reaction was stirred
overnight. Triethylamine (300 mL) was added and the mixture was filtered through Celite
and concentrated to give 14 after silica gel column chromatography (toluene/EtOAc 5 : 1);
Yield: 393 mg (0.26 mmol, 79%); [a]D þ24 (c 1.0, CHCl3); 13C NMR: d 18.0, 18.0, 20.9,
41.0, 47.7, 48.2, 55.5, 62.6, 62.9, 63.9, 65.0, 68.4, 68.9, 69.4, 69.8, 70.2, 71.5, 71.8, 71.9,
72.7, 72.8, 73.1, 73.2, 73.6, 75.5, 75.6, 76.0, 97.4 (JC,H 169 Hz), 99.8, 99.9, 100.4 (JC,H
177 Hz), 100.6 (JC,H 161 Hz), 113.7, 127.2–133.6, 137.9, 138.0, 159.3, 165.1, 165.2, 165.9,
166.2, 167.0, 170.6.
Anal. Calcd for C80H83ClO26: C, 64.23; H, 5.59; Found: C, 64.07; H, 5.71.
[(20S,30S)-7-O-Benzyl-6-O-chloroacetyl-3,4-O-(20,30-dimethoxybutane-20,30-diyl)-2-
O-p-methoxybenzyl-L-glycero-a-D-manno-heptopyranosyl]-(1 ! 3)-[(2,3,4,6-tetra-
O-benzoyl-b-D-glucopyranosyl)-(1 ! 4)]-1,6,7-tri-O-acetyl-2-O-benzyl-L-glycero-a-D-
manno-heptopyranose (15). Compound 14 (72 mg, 0.048 mmol) was dissolved in Ac2O
(2 mL) and cooled to 2408C. Sc(OTf)3 (0.5mol%) was added and the mixture was stirred
at 2408C for 2 hr, whereafter CH2Cl2 and sat. aq. NaHCO3 were added. The aqueous layer
was extracted twice with CH2Cl2, and the combined organic layers were washed with ice
water, dried (MgSO4), filtrated, and concentrated. Purification by silica gel chromatography
(toluene : EtOAc 5 : 1) yielded 15 (60 mg, 0.038 mmol, 78%); [a]D þ45 (c 1.0, CHCl3); 13
C
NMR: d 18.0, 20.4, 20.9, 21.0, 21.6, 40.9, 47.7, 48.0, 55.3, 61.7, 62.7, 63.9, 66.8, 67.8, 68.8,
69.1, 69.3, 70.4, 72.1, 72.1, 72.2, 72.4, 72.8, 73.0, 73.4, 74.3, 74.6, 76.0, 91.0 (JC,H 175 Hz),
99.9, 100.1, 101.2, 101.5, 113.8, 125.4, 127.8–130.1, 131.5, 133.3, 133.3, 133.57, 133.6,
137.5, 137.6, 159.1, 164.9, 165.4, 165.6, 166.0, 197.1, 168.5, 169.9, 170.4.
ACKNOWLEDGEMENTS
Financial support from the Swedish Science Research Council is gratefully
acknowledged.
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