Synthesis of a common tetrasaccharide motif of Haemophilus influenzae LPS inner core structures

Article abstract of DOI:10.1039/b717564g

A conserved tetrasaccharide structure, l-glycero-α-d-manno- heptopyranosyl-(1→2)-(6-O-aminoethylphosphono-l-glycero-α-d-manno- heptopyranosyl)-(1→3)-[β-d-glucopyranosyl-(1→4)] -l-glycero-α-d-manno-heptopyranose, from the LPS inner core of Haemophilus influenzae has been synthesised as its ethylamino glycosides to allow later conjugations. Starting from a previously synthesised suitably protected trisaccharide intermediate, the third heptose and subsequently the spacer were introduced using thioglycoside donor chemistry. The phosphoethanolamine was formed employing a Boc-protected phosphoamidite. Final deprotection and conjugation to biotin gave conjugates that will be used to study the specificity of MAbs raised against native LPS structures.

Full text of DOI:10.1039/b717564g

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of a common tetrasaccharide motif of Haemophilus influenzae LPS
inner core structures†
Karin Mannerstedt, Eva Segerstedt, Johan Olsson and Stefan Oscarson*
Received 13th November 2007, Accepted 23rd January 2008
First published as an Advance Article on the web 14th February 2008
DOI: 10.1039/b717564g
A conserved tetrasaccharide structure, L-glycero-a-D-manno-heptopyranosyl-(1→2)-(6-O-
aminoethylphosphono-L-glycero-a-D-manno-heptopyranosyl)-(1→3)-[b-D-glucopyranosyl-(1→4)]-L-
glycero-a-D-manno-heptopyranose, from the LPS inner core of Haemophilus influenzae has been
synthesised as its ethylamino glycosides to allow later conjugations. Starting from a previously
synthesised suitably protected trisaccharide intermediate, the third heptose and subsequently the spacer
were introduced using thioglycoside donor chemistry. The phosphoethanolamine was formed
employing a Boc-protected phosphoamidite. Final deprotection and conjugation to biotin gave
conjugates that will be used to study the specificity of MAbs raised against native LPS structures.
LPS structures to a carrier protein. Another possibility would be to
Introduction
use synthetic oligosaccharide structures. Apart from being well-
The lipopolysaccharide (LPS) produced by the Gram-negative
defined vaccine candidates, these synthetic derivatives can also
bacteria Haemophilus influenzae is extremely heterogeneous, which
be of assistance in establishing immunodominant motifs, inter
makes its structural analysis most difficult. However, with the
alia, by aiding in the determination of the specificity of MAbs.
sequencing of the bacterial genome, subsequent use of bacterial
As part of a programme directed towards LPS-based vaccines
mutants and new analytical techniques, many structures, as well
as the genetics behind them, have recently been elucidated.1–3 The
against H. influenzae, we are involved in synthesising partial
structures of the LPS inner core and evaluating these as vaccine
results strongly indicates that the bacterium makes a conservative
candidates. Obvious primary target structures are motifs from the
core pentasaccharide (Fig. 1) linked to Lipid A via the Kdo moiety.
This conserved core is then modified in numerous ways by glycans,
various phosphate groups, amino acids and acetates.
conserved inner core. Herein, we describe the synthesis of the
outer tetrasaccharide part of the conserved inner core including
the phosphoethanolamino group on the middle heptose, an inner
core feature, which in molecular models appears to be exposed
and accessible, and thus, might be immunologically important.7–9
Results and discussion
The synthesis starts from a published trisaccharide precursor 110
containing features allowing all the transformation necessary to
reach the target structures, namely orthogonal protecting groups
in the 2ꢀ- and 6ꢀ-positions to permit the introduction of a heptose
Fig. 1 Structure of the conserved inner core of H. influenzae LPS.
Several glycoconjugate vaccines based on capsular polysaccha-
residue and a phosphoethanolamine group, respectively, and a
ride structures (CPS) are now commercial and have been most
1,6-anhydro bridge, both protecting the reducing end and making
successful, inter alia, against H. influenzae type b.4–6 However,
H. influenzae bacteria are frequently found as non-capsulated
(also referred to as non-typeable) H.influenzae (NTHi). Due to
the heterogeneity of the native LPS it is not possible to use
these carbohydrate structures for the construction of a conjugate
vaccine. One conceivable solution is to use LPS from bacterial
mutants expressing less variation.3 Still, there are several problems
connected with this approach, such as identification of protective
motifs, detoxification of the lipid A part and conjugation of the
elongation possible after acetolysis and transformation into a
thioglycoside donor.
Removal of the p-methoxybenzyl group from 1 by DDQ-
oxidation afforded the 2ꢀ-OH acceptor
2 in 77% yield
(Scheme 1). Coupling with the perbenzoylated thioglycoside 3
using NIS/AgOTf as promoter gave a high yield of the exclusively
a-linked tetrasaccharide 4, as proven by JC,H-couplings (>170 Hz)
of the anomeric carbon.11 Hydrolysis using TFA (90% aqueous)
removed the BDA-acetal without touching the anhydro linkage to
produce the 3ꢀ,4ꢀ-diol 5 (89%), leaving open the possibility for
substitution in these positions.12 Since this time we only were
interested in 6ꢀ-phosphorylation, a scandium triflate-mediated
acetolysis was performed to accomplish cleavage of the anhydro
linkage and (concurrently) acetylation of all free hydroxyl groups.
Department of Organic Chemistry, Arrhenius Laboratory, Stockholm Uni-
versity, S-106 91, Stockholm, Sweden. E-mail: s.oscarson@organ.su.se;
Fax: +46 8162 480; Tel: +46 8154 908
† Electronic supplementary information (ESI) available: Copies of 1H
NMR spectra of compounds 9 and 13. See DOI: 10.1039/b717564g
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 1087–1091 | 1087
©

View Article Online
Scheme 1 Synthesis of the protected tetrasaccahride spacer glycoside.
Reagents and conditions: (i) DDQ; (ii) NIS, AgOTf; (iii) 90% TFA;
(iv) Sc(OTf)3, Ac2O; (v) TMSSMe, TMSOTf; (vi) N-(benzyloxycarbonyl)-
aminoethanol, NIS, TfOH.
Scheme 2 Synthesis of target structures 9 and 13. Reagents and conditions:
(vii) NaOMe; (viii) Pd/C, HCl; (ix) hydrazine dithiocarbonate; (x) a. 11,
tetrazole. b. mCPBA.
Concomitant acetolysis of the primary benzyl group was also
observed to different extent. To get a single product the acetolysis
was allowed to go to completion to produce derivative 6 in almost
quantitative yield
smoothly without any indication of acetyl migration to yield
the 6ꢀ-OH derivative 10 (78%). When introducing the phospho-
ethanolamine substituent, a Boc-protection of the amino group
was chosen, so as to be able to differentiate this from the
spacer amino group in later conjugations. Preliminary attempts
using H-phosphonate chemistry were not successful, and so a
phosphoamidite, 11, was synthesised and tested. This time the
phosphotriester 12 was produced in 89% yield as a diastereomeric
mixture. Again, deprotection in two steps was effective to afford
the Boc-protected aminoethanolphosphorylated target structure
13 (87%).
Biotin conjugates were of interest for ELISA-screening of MAbs
produced against NTHi bacteria with a known specificity for
inner core structures. Treatment of compounds 9 and 13 with the
commercial NHS-ester of biotin, afforded derivatives 14 and 15,
respectively, in almost quantitative yield according to MALDI-
TOF MS (Scheme 3). Mild TFA-hydrolysis of the Boc-group in
15 then yielded the last target structure 16.
Efforts to convert this compound into the corresponding ethyl
thioglycoside were difficult to monitor both by TLC (no difference
in Rf value) and also by MALDI-TOF MS (almost the same
MW). Hence, the methyl thioglycoside, the formation of which
could easily be followed by MALDI-TOF MS, was synthesised by
treatment with TMSSMe/TMSOTf, affording 7 (78%). As proven
before with a similar trisaccharide donor, coupling with a spacer
acceptor yielded exclusively the a-linked spacer glycoside 8 (72%),
although a non-participating benzyl group was present in the 2-
position.13 These results are different from the ones obtained with
monosaccharide donors, where often a substantial amount of b-
linked product is produced.14–16
Compound 8 was deprotected in two effective steps, Zemple´n
deacylation followed by catalytic hydrogenolysis, to give the
non-phosphorylated target structure 9 in high yield (Scheme 2).
Selective removal of the chloroacetyl group from 8 was first tried
with a reagent, DABCO, reported in the literature to prevent acetyl
migration.17 However, in this case, this reagent caused substantial
acetyl migration and removal, whereas the reagent we normally
use, hydrazine dithiocarbonate,18 removed the chloroacetyl group
In conclusion, an effective synthesis of Haemophilus inner core
structures from the versatile precursor 1 has been performed. In
four steps and 45% overall yield a tetrasaccharide thioglycoside
intermediate 7 was prepared. This was then successfully converted
to the two target structures 9 and 13, in 30 and 20% overall
yield (from 1) respectively. The target structures are designed
1088 | Org. Biomol. Chem., 2008, 6, 1087–1091
This journal is
The Royal Society of Chemistry 2008
©

View Article Online
69.1, 69.4, 70.0, 70.1, 71.5, 71.8, 72.1, 72.7, 72.8, 72.8, 73.4, 75.6,
75.7 (C-2-7, C-2ꢀ-7ꢀ, C-2ꢀꢀ-6ꢀꢀ, PhCH2O), 98.1, 100.0, 100.3, 100.3,
100.4 (C-1, C-1ꢀ, C-1ꢀꢀ, C BDA), 127.4-137.9 (aromatic C), 165.1,
165.2, 165.9, 166.2, 167.0, 170.6 (CH3CO, PhCO, ClCH2CO).
(2ꢀS,3ꢀS)-(2,3,4,6,7-Penta-O-benzoyl-L-glycero-a-D-manno-hep-
topyransyl)-(1→2)-(7-O-benzyl-6-O-chloroacetyl-3,4-O-(2ꢀ,3ꢀ -di-
methoxybutane-2ꢀ,3ꢀ -diyl)-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-heptopyr-
anose (4). A solution of 2 (43 mg, 31.3 lmol) and 313 (48 mg,
˚
62.5 lmol) in CH2Cl2 containing powdered molecular sieves (4 A)
was stirred at room temperature under an argon atmosphere for
1 h. The mixture was cooled to −30 ◦C, NIS (14 mg, 62.5 lmol)
and AgOTf (cat.) were added, and the reaction was slowly brought
◦
to 5 C. After neutralisation with Et3N, the mixture was diluted
with CH2Cl2 and filtered through Celite. The filtrate was washed
with Na2S2O3 (10% aq) and water, dried and concentrated.
Purification by silica gel chromatography (toluene–EtOAc 10:1)
gave 4 (56 mg, 26.8 lmol, 86%): [a]D −16 (c 1.0, CHCl3); 13C
NMR d 17.7, 17.8 (CH3 BDA), 20.8 (CH3CO), 41.5 (ClCH2CO),
47.8, 48.2 (CH3O BDA), 61.7, 62.3, 65.2, 65.5, 68.7, 68.8, 69.4,
69.8, 70.2, 70.3, 70.7, 71.5, 71.6, 72.2, 72.8, 73.4, 73.7, 74.1, 74.9,
76.3 (C-2-6, C-2ꢀ-6ꢀ, C-2ꢀꢀ-6ꢀꢀ, PhCH2O), 97.1 (JC,H 171 Hz), 98.8
(JC,H 176 Hz), 99.0 (JC,H 163 Hz), 99.9, 100.2, 100.4 (JC,H 176 Hz)
(C-1, C-1ꢀ, C-1ꢀꢀ, C BDA), 127.5–138.2 (aromatic C), 164.7,
165.0, 165.2, 165.5, 165.5, 165.7, 165.8, 166.1, 166.9, 167.6, 170.8
(CH3CO, PhCO, ClCH2CO). HRMS calcd for C114H107ClO36 [M
+ Na]+ 2091.6129, found 2091.6204.
Scheme 3 Synthesis of biotin conjugates. Reagents and conditions:
(xi) (+)-biotin N-hydroxysuccinimide ester, Et3N, 0.1 M phosphate buffer
pH 7.0; xii: TFA(aq)
.
to allow selective activation and conjugation through the spacer
amino group. Biotin conjugates have been constructed and protein
conjugates are under way. The former will be used to investigate
the epitope specificity of MAbs, whereas the latter will be used in
immunological experiments and evaluated as vaccine candidates.
(2,3,4,6,7-Penta-O-benzoyl-L-glycero-a-D-manno-heptopyransyl)-
(1→2)-(7-O-benzyl-6-O-chloroacetyl-L-glycero-a-D-manno-hepto-
pyranosyl)-(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 (5). Compound 4 (148 mg, 70.9 lmol) was
dissolved in 90% TFA (aq, 5 mL) and the reaction mixture
Experimental
General methods
Organic solvents were dried over MgSO4 before concentration,
which was performed under reduced pressure at <40 ◦C (bath
temperature). TLC was carried out on Merck precoated 60 F254
plates with detection by UV-light and/or 8% sulfuric acid or
ammonium molybdate (100 g) : Ce(IV) sulfate (2 g) : sulfuric acid
(10%, 2 L). Column chromatography was performed on silica gel
(35–70 lm, Millipore), and reverse-phase chromatography was
performed on silica gel (C18 60A 40–60 lm). NMR spectra
were recorded in CDCl3 at 25 ◦C on a Varian 300 MHz or
400 MHz instrument, unless otherwise stated. MALDI-TOF
spectra were recorded on a Bruker Biflex III instrument using
2ꢀ,4ꢀ,6ꢀ-trihydroxyacetophenone trihydrate (THAP) as matrix.
was stirred at room temperature for
1 h, concentrated
and co-concentrated with toluene. Purification by silica gel
chromatography (toluene–EtOAc 1 : 1) gave 5 (124 mg, 62.8 lmol,
89%): [a]D −45◦ (c 1.0, CHCl3); 13C NMR d 20.9 (CH3CO), 41.1
(ClCH2CO), 60.7, 65.2, 65.4, 65.8, 67.2, 68.6, 68.8, 69.0, 70.2,
70.6, 70.7, 70.9, 71.3, 71.4, 71.8, 72.3, 72.7, 72.7, 72.9, 73.2, 73.5,
74.1, 74.7, 76.7 (C-2-7, C-2ꢀ-7ꢀ, C-2ꢀꢀ-6ꢀꢀ, C-2ꢀꢀꢀ-7ꢀꢀꢀ, PhCH2O), 96.2,
98.2, 99.3, 100.2 (C-1, C-1ꢀ, C-1ꢀꢀ, C-1ꢀꢀꢀ), 127.8–138.0 (aromatic
C), 164.9, 165.1, 165.2, 165.6, 165.7, 165.7, 165.7, 166.1, 166.9,
169.3, 170.9 (CH3CO, PhCO, ClCH2CO).
(2ꢀS,3ꢀS)-(7-O-Benzyl-6-O-chloroacetyl-3,4-O-(2ꢀ,3ꢀ-dimethoxy-
butane-2ꢀ,3ꢀ -diyl)-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 (2).
2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 45 mg,
0.197 mmol) and H2O (0.5 mL) were added to a solution of 110
(197 mg, 0.131 mmol) in CH2Cl2 (10 mL). The mixture was stirred
at room temperature for 4 h, then diluted with CH2Cl2, washed
with Na2S2O3 (5% aq), dried (Na2SO4) and concentrated. The
residue was purified by silica gel chromatography (toluene–EtOAc
1 : 1) to give 2 (138 mg, 0.100 mmol, 76%): [a]D +33 (c 1.0,
CHCl3); 13C NMR d 17.9 (CH3 BDA), 20.8 (CH3CO), 41.0
(ClCH2CO), 47.9, 48.2 (CH3O BDA), 62.1, 62.8, 64.9, 68.1, 68.2,
(2,3,4,6,7-Penta-O-benzoyl-L-glycero-a-D-manno-heptopyransyl)-
(1→2)-(3,4,7-tri-O-acetyl-6-O-chloroacetyl-L-glycero-a-D-manno-
heptopyranosyl)-(1→3)-[(2,3,4,6-tetra-O-benzoyl-b-D-glucopyran-
osyl)-(1→4)]-1,6,7-tri-O-acetyl-2-O-benzyl-L-glycero-a-D-manno-
heptopyranose (6). Sc(OTf)3 (0.5 mol%) was added to a solution
of 5 (51 mg, 25.8 lmol) in Ac2O and the mixture was stirred at
room temperature over night., The reaction was quenched by
the addition of MeOH whereafter the mixture was concentrated.
Purification by silica gel chromatography (toluene–EtOAc 2 : 1)
gave 6 (53 mg, 25.1 lmol, 97%): [a]D −15 (c 1.0, CHCl3); 13C
NMR d 20.4, 20.6, 20.7, 20.9, 21.0 (CH3CO), 41.1 (ClCH2CO),
61.7, 62.0, 63.8, 64.7, 65.5, 66.0, 66.3, 67.7, 68.9, 69.1, 69.2, 69.5,
69.8, 70.8, 71.2, 71.7, 71.9, 72.0, 73.1, 73.4, 73.8, 74.8, 75.4, 78.7
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 1087–1091 | 1089
©

View Article Online
(C-2-7, C-2ꢀ-7ꢀ, C-2ꢀꢀ-6ꢀꢀ, C-2ꢀꢀꢀ-7ꢀꢀꢀ, PhCH2O), 90.4 (JC,H 174 Hz),
99.8 (JC,H 173 Hz, 2C), 101.9 (JC,H 163 Hz) (C-1, C-1ꢀ, C-1ꢀꢀ,
C-1ꢀꢀꢀ), 128.2–137.1 (aromatic C), 164.2, 165.1, 165.2, 165.5, 165.6,
165.7, 165.9, 166.0, 166.3, 167.4, 169.0, 169.5, 169.9, 170.1, 170.5,
170.9 (CH3CO, PhCO, ClCH2CO). MALDI-TOF MS calcd for
C111H103ClO40 [M + Na]+ 2134,56, found 2134.52.
dissolved in EtOH, HCl (0.1 M, 98 lL) palladium on activated
carbon powder was added, and the mixture was hydrogenolyzed
at 110 psi over night, followed by filtration through Celite and
concentration. Purification by Biogel P2-column (1% BuOH in
1
H2O) gave 9 (7 mg, 8.75 lmol, 89%): [a]D +174 (c 0.5, H2O); H
NMR (D2O, 30 ◦ C) d 3.20–3.41 (7H), 3.57–4.04 (22 H), 4.11 (bs,
1H), 4.21 (t, J 10 Hz, 1H), 4.56 (d, J1,2 7.5 Hz, 1H), 4.88 (s, 1H),
5.13 (s, 1H), 5.68 (s, 1H). HRMS calcd for C36H59NO24 [M + H]+
800.2958, found 800.2991.
Methyl (2,3,4,6,7-penta-O-benzoyl-L-glycero-a-D-manno-hepto-
pyransyl)-(1→2)-(3,4,7-tri-O-acetyl-6-O-chloroacetyl-L-glycero-
a-D-manno-heptopyranosyl)-(1→3)-[(2,3,4,6-tetra-O-benzoyl-b-
D-glucopyranosyl)-(1→4)]-6,7-di-O-acetyl-2-O-benzyl-1-thio-L-
glycero-a-D-manno-heptopyranoside (7). TMSSMe (158 lL,
1.11 mmol) and TMSOTf (536 lL, 2.96 mmol) were added to a
stirred solution of 6 (277 mg, 0.131 mmol) in CH2Cl2, containing
molecular sieves (AW-300). The reaction mixture was stirred at
room temperature for 72 h (monitored by MALDI-TOF MS),
filtered through Celite and concentrated. Purification by silica
gel chromatography (toluene–EtOAc 3 : 1) gave 7 (214 mg,
0.102 mmol, 78%): [a]D −20 (c 1.0, CHCl3); 13C NMR d 13.9,
20.4, 20.7, 20.9, 20.9 (CH3CO), 41.3 (ClCH2CO), 61.3, 62.4, 64.1,
64.1, 65.6, 66.1, 67.9, 69.4, 69.7, 69.8, 69.9, 70.0, 70.1, 70.4, 70.9,
71.9, 72.0, 73.3, 73.3, 75.6, 76.0, 78.0 (C-2-7, C-2ꢀ-7ꢀ, C-2ꢀꢀ-6ꢀꢀ, C-
2ꢀꢀꢀ-7ꢀꢀꢀ, PhCH2O), 83.5, 99.6, 99.6, 102.0 (C-1, C-1ꢀ, C-1ꢀꢀ, C-1ꢀꢀꢀ),
127.9–137.6 (aromatic C), 164.5, 165.2, 165.2, 165.5, 165.6, 165.9,
166.0, 166.1, 167.7, 169.5, 170.0, 170.4, 170.5, 170.7 (CH3CO,
PhCO, ClCH2CO). HRMS calcd for C110H103ClO38S [M + Na]+
2121.5435, found 2121.5435.
2-(N -Benzyloxycarbonyl)aminoethyl (2,3,4,6,7-penta-O-ben-
zoyl-L-glycero-a-D-manno-heptopyransyl)-(1→2)-(3,4,7-tri-O-
acetyl-L-glycero-a-D-manno-heptopyranosyl)-(1→3)-[(2,3,4,6-
tetra-O-benzoyl-b-D-glucopyranosyl)-(1→4)]-6,7-di-O-acetyl-2-O-
benzyl-L-glycero-a-D-manno-heptopyranoside (10). Hydrazine
dithiocarbonate18 (71 lL, 26.7 lmol) was added to a stirred
solution of 8 (20 mg, 8.90 lmol) in DMF. After 1 h, the solution
was diluted with CH2Cl2, washed with 1M H2SO4, NaHCO3
(sat., aq), water, dried and concentrated. Purification by silica
gel chromatography (toluene–EtOAc 2 : 1) gave 10 (15 mg, 6.91
lmol, 78%): [a]D −18 (c 1.0, CHCl3); 13C NMR d 20.4, 21.0, 21.1,
21.2 (CH3CO), 40.8 (OCH2CH2N), 61.4, 64.0, 64.7, 65.8, 66.5,
66.7, 67.0, 67.5, 67.9, 68.3, 69.3, 69.9, 70.3, 70.5, 70.7, 71.0, 71.9,
72.1, 73.3, 73.5, 74.8, 75.1, 76.0, 78.4 (C-2-7, C-2ꢀ-7ꢀ, C-2ꢀꢀ-6ꢀꢀ,
C-2ꢀꢀꢀ-7ꢀꢀꢀ, PhCH2O, OCH2CH2N), 98.1, 99.1, 99.1, 102.1 (C-1,
C-1ꢀ, C-1ꢀꢀ, C-1ꢀꢀꢀ), 127.9–138.0 (aromatic C), 156.7, 164.3, 165.2,
165.4, 165.5, 165.6, 165.8, 166.0, 166.4, 166.4, 170.0, 170.6, 170.8,
171.1, 171.3 (CH3CO, PhCO, ClCH2CO).
2-(N-Benzyloxycarbonyl)aminoethyl
(2,3,4,6,7-penta-O-ben-
zoyl-L-glycero-a-D-manno-heptopyransyl)-(1→2)-(3,4,7-tri-O-
acetyl-6-O-chloroacetyl-L-glycero-a-D-manno-heptopyranosyl)-
(1→3)-[(2,3,4,6-tetra-O-benzoyl-b-D-glucopyranosyl)-(1→4)]-
6,7-di-O-acetyl-2-O-benzyl-L-glycero-a-D-manno-heptopyranoside
(8). A solution of 7 (35 mg, 16.7 lmol) and N-(benzyloxycar-
bonyl)aminoethanol (13 mg, 66.7 lmol) in Et2O containing
Benzyloxy-[2-(tert-butyloxycarbonylamino)ethoxy]-(N,N-diiso-
propylamino)phosphine (11). 2-(tert-Butyloxycarbonylamino)-
ethanol (0.343 g, 2.13 mmol) in CH2Cl2 was added to a stirred
solution of benzyloxybis(N,N-diisopropylamino)phosphine19
(1.08 g, 3.19 mmol) and tetrazole (75 mg, 1.07 mmol) in
CH2Cl2. The mixture was stirred at room temperature for 3 h
and concentrated. Purification by silica gel chromatography
(pentane–EtOAc–Et3N 90 : 10 : 5) gave 11 (0.611 g, 1.53 mmol,
72%): 1H NMR d 1.19 (dd, 12H), 1.42 (s, 9H), 3.31 (d, 2H), 3.67
(m, 4H), 4.71 (m, 2H), 4.95 (bs, 1H), 7.30 (m, 5H); 13C NMR d
24.7 (t, J 6.9 Hz), 28.5, 41.9 (d, J 5.94 Hz), 43.0 (d, J 12.2 Hz),
62.8 (d, J 16.8 Hz), 65.5 (d, J 18.3 Hz), 79.1, 127.1, 127.4, 128.4,
139.3 (d, J 7.63 Hz), 156.0; 31P NMR d 147.9.
˚
powered molecular sieves (4 A) was stirred at room temperature
◦
for 1 h. The reaction mixture was cooled to 10 C, NIS (7.5 mg,
33.4 lmol) and TfOH (cat.) were added, and the reaction mixture
was stirred for 3 h. After dilution with Et2O and filtration through
Celite, the organic phase was washed with Na2S2O3 (10% aq),
dried and concentrated. Purification by silica gel chromatography
(toluene–EtOAc 2 : 1) gave 8 (26 mg, 12.0 lmol, 72%): [a]D
−22 (c 1.0, CHCl3); 13C NMR d 20.4, 20.7, 21.0, 21.1 (CH3CO),
40.8, 41.2 (OCH2CH2N, ClCH2CO), 61.5, 62.2, 63.9, 64.8, 65.6,
66.0, 66.5, 67.0, 67.9, 68.3, 68.9, 69.4, 69.6, 69.9, 70.1, 70.8,
71.3, 71.9, 73.2, 73.5, 74.9, 75.9, 78.7 (C-2-7, C-2ꢀ-7ꢀ, C-2ꢀꢀ-6ꢀꢀ,
C-2ꢀꢀꢀ-7ꢀꢀꢀ, PhCH2O, OCH2CH2N), 97.9 (JC,H 176 Hz), 99.5 (JC,H
179 Hz), 99.8 (JC,H 171 Hz), 102.1 (JC,H 164 Hz) (C-1, C-1ꢀ, C-1ꢀꢀ,
C-1ꢀꢀꢀ), 128.0–137.7 (aromatic C), 156.7, 164.3, 165.2, 165.3, 165.5,
165.7, 165.8, 165.9, 166.4, 166.5, 167.4, 169.5, 169.9, 170.5, 170.8
(CH3CO, PhCO, ClCH2CO). HRMS calcd for C119H112ClNO41
[M + 2Na]2+ 1145.8097, found 1145.8025.
2-(N-Benzyloxycarbonyl)aminoethyl (2,3,4,6,7-penta-O-benzoyl-
L-glycero-a-D-manno-heptopyransyl)-(1→2)-(3,4,7-tri-O-acetyl-6-
O - [benzyl - 2 - (tert - butyloxycarbonylaminoethyl)phosphono] - L -
glycero-a-D-manno-heptopyranosyl)-(1→3)-[(2,3,4,6-tetra-O-ben-
zoyl-b-D-glucopyranosyl)-(1→4)]-6,7-di-O-acetyl-2-O-benzyl-L-
glycero-a-D-manno-heptopyranoside (12). Tetrazole (8 mg,
111 lmol) was added to a solution of 10 (48 mg, 22.1 lmol) and
11 (44 mg, 111 lmol) in dry CH2Cl2. The reaction mixture was
stirred at room temperature for 4 h, and after cooling to 0 ◦C,
m-chloroperbenzoic acid (17 mg, 66.3 lmol) was added. The
mixture was stirred for another hour at room temperature and the
solution was diluted with CH2Cl2, washed with NaHCO3 (sat.,
aq), dried (MgSO4), filtered and concentrated. Purification by
silica gel chromatography (toluene–EtOAc 2 : 1) gave 12 (49 mg,
19.7 lmol, 89%): 31P NMR d 8.08; MALDI-TOF MS calcd for
C131H131N2O45P [M + Na]+ 2506.78, found 2507.33.
2-Aminoethyl (L-glycero-a-D-manno-heptopyransyl)-(1→2)-(L-
glycero-a-D-manno-heptopyranosyl)-(1→3)-[(b-D-glucopyranosyl)-
(1→4)]-L-glycero-a-D-manno-heptopyranoside (9). Compound
8 (22 mg, 9.79 lmol) was dissolved in MeOH and the pH was
adjusted to 11 by treatment with 1 M NaOMe (in MeOH). The
mixture was stirred for 2 h, neutralised with Dowex 50 (H+)
ion exchange resin, filtered and concentrated. The residue was
1090 | Org. Biomol. Chem., 2008, 6, 1087–1091
This journal is
The Royal Society of Chemistry 2008
©

View Article Online
2-Aminoethyl (L-glycero-a-D-manno-heptopyransyl)-(1→2)-(6-
O-(benzyl-2-((tert-butyloxycarbonyl)aminoethyl)phosphono)-L -
glycero-a-D-manno-heptopyranosyl)-(1→3)-[(b-D-glucopyranosyl)-
(1→4)]-L-glycero-a-D-manno-heptopyranoside (13). Compound
12 (10 mg, 4.03 lmol) was dissolved in THF–EtOH 3 : 1, HCl
(0.1 M, 40 lL), palladium on activated carbon powder was
added and the mixture was hydrogenolyzed at 110 psi over night,
followed by filtration through Celite and concentration. The
residue was dissolved in MeOH and the pH was adjusted to 11
by treatment with 1 M NaOMe (in MeOH). The mixture was
stirred for 2 h, neutralised with Dowex 50 (H+) ion exchange
resin, filtered and concentrated. Purification by reverse-phase
chromatography (H2O–MeOH 1 : 0 → 1 : 1) gave 13 (3.6 mg, 3.52
lmol, 87%): [a]D +110 (c 0.1, H2O); 1H NMR (D2O, 30 ◦C) d 1.38
(s, 9H, (CH3)3), 3.20–3.41 (9H), 3.59–4.05 (23 H), 4.10 (bs, 1H),
4.23 (t, J 10 Hz, 1H), 4.42 (dd, 1H, H-6ꢀ), 4.56 (d, J1,2 7.5 Hz, 1H,
H-1ꢀꢀ), 4.88 (s, 1H), 5.12 (s, 1H), 5.77 (s, 1H) (H-1,1ꢀ,1ꢀꢀꢀ); 31P (D2O)
d 0.56. HRMS calcd for C131H131N2O45P [M + H]+ 1023.3567,
found 1023.3627.
Acknowledgements
We are thankful to the Swedish Research Council for financial
support. Dr Petter Tollba¨ck, Department of Analytical Chemistry,
SU, is acknowledged for recording the HRMS spectra.
References
1 J. C. Richards, A. D. Cox, E. K. H. Schweda, A. Martin, D. W. Hood
and E. R. Moxon, Adv. Exp. Med. Biol., 2001, 491, 515, and references
cited therein.
2 H. H. Yildirim, D. W. Hood, E. R. Moxon and E. K. H. Schweda,
Eur. J. Biochem., 2003, 270, 3153, and references cited therein.
3 E. R. Moxon, D. W. Hood, N. J. Saunders, E. K. H. Schweda and J. C.
Richards, Philos. Trans. R. Soc. London, Ser. B, 2002, 357, 109.
4 N. Ravenscroft and C. Jones, Curr. Opin. Drug Discovery Dev., 2000, 3,
222.
5 A. A. Lindberg, Vaccine, 1999, 17, S28.
6 V. Veres-Bencomo, V. Ferna´ndez-Santana, E. Hardy, M. E. Toledo,
M. C. Rodr´ıguez, L. Heynngnezz, A. Rodriguez, A. Baly, L. Herrera,
M. Izquierdo, A. Villar, Y. Valde´s, K. Cosme, M. L. Deler, M. Montane,
E. Garcia, A. Ramos, A. Aguilar, E. Medina, G. Toran˜o, I. Sosa,
I. Hernandez, R. Mart´ınez, A. Muzachio, A. Carmenates, L. Costa,
F. Cardoso, C. Campa, M. Diaz and R. Roy, Science, 2004, 305,
522.
2-[(+)-Biotinylamino]ethyl (L-glycero-a-D-manno-heptopyranosyl)-
(1→2)-(L-glycero-a-D-manno-heptopyranosyl)-(1→3)-[(b-D-glucopy-
7 D. W. Hood, M. E. Deadman, T. Allen, H. Masoud, A. Martin, J. R.
Brisson, R. Fleischman, J. C. Venter, J. C. Richards and E. R. Moxon,
Mol. Microbiol., 1996, 22, 951.
8 J. S. Plested, K. Makepeace, M. P. Jennings, M. A. J. Gidney, S. Lacalle,
J.-R. Brisson, A. C. Cox, A. Martin, A. G. Bird, C. M. Tang, F. M.
Mackinnin, J. C. Richards and E. R. Moxon, Infect. Immunol., 1999,
67, 5417.
9 M. A. J. Gidney, J. S. Plested, S. Lacalle, P. A. Coull, J. C. Wright, K.
Makepeace, J.-R. Brisson, A. C. Cox, E. R. Moxon and J. C. Richards,
Infect. Immunol., 2004, 72, 559.
10 E. Segerstedt, K. Mannerstedt, M. Johansson and S. Oscarson,
J. Carbohydr. Chem., 2004, 23, 443.
11 K. Bock and C. Pedersen, J. Chem. Soc., Perkin Trans. 2, 1973, 293.
12 H. Kubo, K. Ishii, H. Koshino, K. Toubetto, K. Naruchi and R.
Yamasaki, Eur. J. Org. Chem., 2004, 1202.
13 C. Bernlind and S. Oscarson, J. Org. Chem., 1998, 63, 7780.
14 S. Oscarson, Top. Curr. Chem., 1997, 187, 171.
15 P. J. Garegg, S. Oscarson, H. Ritze´n and M. Szo¨nyi, Carbohydr. Res.,
1992, 228, 121.
ranosyl)-(1→4)]-L-glycero-a-D-manno-heptopyranoside
(14).
(+)-Biotin N-hydroxysuccinimide ester (0.8 mg, 2.50 lmol) and
Et3N (50 lL) were added to 9 (1 mg, 1.25 lmol) in 0.1 M
phosphate buffer pH 7.0 (0.5 mL). After 30 min the reaction was
completed (monitored by MALDI-TOF MS) and the residue
was purified on a Biogel P2 column eluted with H2O (1%
n-BuOH), followed by lyophilisation to give 14: HRMS calcd for
C39H67N3O26S [M + Na]+ 1048.3626, found 1048.3613.
2-[(+)-Biotinylamino]ethyl (L-glycero-a-D-manno-heptopyransyl)-
(1→2)-(6-O-aminoetylphosphono-L-glycero-a-D-manno-heptopyra-
nosyl)-(1→3)-[(b-D-glucopyranosyl)-(1→4)]-L-glycero-a-D-manno-
heptopyranoside (16). Compound 13 (1 mg, 0.777 lmol) was
treated with biotin N-hydroxysuccinimide (0.7 mg, 1.96 lmol)
and Et3N (50 lL) in 0.1 M phosphate buffer pH 7.0 (0.5 mL)
as described for compound 14 to give compound 15. The
Boc-protecting group was subsequently removed by treatment of
15 with TFA (100 lL) in H2O (500 lL) for 2 h. Purification by
size-exclusion chromatography using a Biogel P2 column eluted
with H2O (1% n-BuOH) and lyophilisation gave 16: MALDI-TOF
MS calcd for C41H73N4O29PS [M + Na]+ 1171.28, found 1172.28.
16 S. Oscarson and H. Ritze´n, Carbohydr. Res., 1994, 254, 81.
17 D. J. Lefeber, J. P. Kamerling and F. G. Vliegenhart, Org. Lett., 2000,
2, 701.
18 C. A. A. van Boeckel and T. Beetz, Tetrahedron Lett., 1983, 24,
3775.
19 C. E. Dreef, C. J. J. Elie, P. Hoogerhout, G. A. von der Marel and J. H.
van Boom, Tetrahedron Lett., 1988, 29, 6513–6516.
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 1087–1091 | 1091
©