Synthesis of phosphorylated Neisseria meningitidis inner core lipopolysaccharide structures

Article abstract of DOI:10.1016/j.tetasy.2009.02.017

A phosphoethanolamine-substituted tetrasaccharide structure, 2-aminoethyl 2-acetamido-2-deoxy-α-d-glucopyranosyl-(1→2)-6-O-[2-(tert-butyloxycarbonylaminoethyl)-phosphono]-l-glycero-α-d-manno-heptopyranosyl-(1→3)-[β-d-glucopyranosyl-(1→4)]-l-glycero-α-d-ma

Full text of DOI:10.1016/j.tetasy.2009.02.017

Tetrahedron: Asymmetry 20 (2009) 879–886
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Synthesis of phosphorylated Neisseria meningitidis inner core
lipopolysaccharide structures
Johan D. M. Olsson a, Stefan Oscarson b,
*
a Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden
b Centre for Synthesis and Chemical Biology, UCD School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
A phosphoethanolamine-substituted tetrasaccharide structure, 2-aminoethyl 2-acetamido-2-deoxy-a-D-
Received 4 February 2009
Accepted 10 February 2009
Available online 9 March 2009
glucopyranosyl-(1?2)-6-O-[2-(tert-butyloxycarbonylaminoethyl)-phosphono]-L-glycero-a-D-manno-hepto-
pyranosyl-(1?3)-[b- -glucopyranosyl-(1?4)]- -glycero- -manno-heptopyranoside, corresponding to
D
L
a-D
the non-reducing part of the conserved part of Neisseria meningitidis lipopolysaccharides has been syn-
thesized. Orthogonal protection of the phosphoethanolamino group in combination with the presence
of a free amino-containing anomeric spacer allows conjugation to proteins to construct conjugate vaccine
candidates. The tetrasaccharide is built up using a linear strategy, where the introduction of the terminal
This paper is dedicated to Professor George
Fleet, on the occasion of his 65th birthday
a-GlcNAc moiety is performed using a 2-azido-thioglucoside as a donor and NIS/AgOTf as a promoter. The
synthetic pathway includes tetrasaccharide intermediates appropriately designed to permit other phos-
phorylation patterns as well as elongation at the reducing end.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
at the 6-position of the outer heptose residue, a determinant be-
lieved to be of immunological importance.4 The synthetic strategy
Efficient glycoconjugate vaccines against Neisseria meningitidis
based on the surface capsular polysaccharide (CPS) are already
commercial or under development for four, A, C, W-135 and Y, of
the five serotypes that are the main causes of meningococcal men-
ingitis.1 Type B, however, constitutes a problem, since the structure
of the CPS mimics a human structure.2 An alternative, which has
been pursued for some time, is to try to base a vaccine on lipopoly-
saccharide (LPS) structures instead.3 These structures conjugated
to a carrier protein should be good vaccine candidates since they
are genetically stable and have proven to be accessible to host im-
mune mechanisms and to induce protective antibodies.4 An advan-
tage with the LPS-structures is that they are conserved in all
clinically relevant strains across the species; LPS-based vaccine
protection should not be dependent on the capsular serotype of
the infecting bacteria. However, there are other issues with LPS-
based vaccines against Neisseria meningitidis, for example, the tox-
icity of the Lipid A part and a heterogeneity in the outer parts of the
LPS-molecule. Furthermore, the activation and subsequent protein
conjugation are not trivial. To try to identify protecting epitopes,
well-defined synthetic part structures would be an asset. Herein,
a synthesis of a tetrasaccharide 20, corresponding to the non-
reducing part of the conserved inner core structure of N. meningit-
idis LPS (Fig. 1), is presented. In addition phosphorylated structures
18 and 19 are reported, containing a phosphoethanolamine moiety
also allows for the introduction of a phosphoethanolamine in the
3-position at the same heptose residue, which is the other impor-
tant phosphorylation position found. The synthesis proceeds via a
tetrasaccharide thioglycoside donor 14. Glycosylation of this with
an aminospacer alcohol was performed to facilitate efficient conju-
gation of the target structures, but this intermediate is also de-
signed to be a vital building donor block in the formation of
larger structures containing the Kdo and LipidA parts.
2. Results and discussion
Starting from intermediate 7,5 Haemophilus influenzae structures
similar to the target N. meningitidis structures 18–20, differing only
in the non-reducing end moiety, being an
a-GlcNAc residue in Neis-
seria (Fig. 1) and an -LD-Hep residue in Haemophilus, have earlier
a
been efficiently synthesized in our group.6 However, initial experi-
ments showed that the coupling of glucosamine donors to the inter-
mediate 7 was not trivial (see below). Hence, a different pathway, a
2+2-block approach, was attempted to reach the desired tetrasac-
charide 9 (Scheme 1). Using the well-known trichloroacetimidate
donor 37 and TMSOTf-activation in a coupling to the easily accessi-
ble acceptor 2, a good yield and an excellent
were obtained to give disaccharide 4 (63%, only
a
-stereoselectivity
a
). But when glyco-
sylation reactions between disaccharide donor 4 and acceptor 65
were attempted, no tetrasaccharide product was formed at all in
spite of the use of a variety of promoters and coupling conditions.
It has been observed earlier that heptose donors are generally less
* Corresponding author. Tel.: +353 17162318; fax: +353 17162501.
E-mail address: stefan.oscarson@ucd.ie (S. Oscarson).
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.02.017

880
J. D. M. Olsson, S. Oscarson / Tetrahedron: Asymmetry 20 (2009) 879–886
β-D-Glc
α-D-Kdop
PEtN
1
↓
4
2
↓
4
|
3 or 6
α-D-GlcNAcp-(1→2)-L-α-D-Hepp-(1→3)- L-α-D-Hepp-(1→5)-α-D-Kdop-(2→6)-LipidA
Figure 1. Conserved inner core structure of N. meningitidis LPS.
iii
BnO
CAO
O
O
MeO
OR
O
OAc
OAc
N3
BnO
CAO
O
O
OAc
N3
BnO
CAO
O
O
OAc
O
O
O
AcO
OBz
OAc
OAc
OH
MeO
SEt
MeO
O
MeO
O
O
O
OBn
ii
1 R = -pMBn
2 R = H
O
O
BzO
BzO
i
O
OBz
6
MeO
4
5
SEt
MeO
SEt
OAc
O
O
AcO
O
CCl3
AcO
N3
X
NH
3
X
OAc
N3
BnO
CAO
O
O
OAc
O
O
OAc
MeO
O
O
CA = ClCH2CO
MeO
O
AcO
O
OBz
OBn
O
BzO
BzO
O
9
OBz
Scheme 1. Attempted block approach towards tetrasaccharide intermediate 9. Reagents and conditions: (i) DDQ, CH2Cl2/H2O (15:1), 94%; (ii) TMSOTf, 4 A MS, Et2O, 0 °C 63%;
(iii) m-CPBA, CH2Cl2, ꢀ60 °C, 61%.
reactive than hexose donors,8 but this disaccharide donor was
unusually difficult to be activated even by thiophilic promoters that
are considered to be very effective (Me2S2/Tf2O9 and Ph2SO/Tf2O10).
To try to increase the reactivity, the corresponding sulfoxide donor
was constructed, donors that have been advocated by Kahne et al.
to be most reactive.11 Using this donor, small amounts of tetrasac-
charide were obtained, but the results were difficult to be repro-
duced and even the best yields were quite disappointing (<15%).
We were therefore forced back to the earlier used linear (1+3)-
approach (Scheme 2 and Table 1). Glycosylations of acceptor 7
with the above mentioned donor 3 gave, with TMSOTf as promoter,
again no tetrasaccharide product at all, and with BF3-etherate low
yields and low stereoselectivity were obtained (22%,
tries vii–ix). With the corresponding bromide donor activated by
AgOTf/DTBMP, only slightly better yield and -selectivity (34%,
/b 1:1, entries x–xi) were observed. The thioglycoside donor 812
activated by DMTST or Me2S2/Tf2O also gave very poor results (en-
tries i–ii), however, surprisingly, activation by NIS/AgOTf in CH2Cl2
gave excellent yields of the tetrasaccharide 9/10, though once
a/b 1:1.6, en-
a
a
more with low stereoselectivity (entries iv–vi). The
a-selectivity
was found to increase with increase in temperature, but, again sur-
prisingly, a change of solvent from CH2Cl2 to Et2O (generally con-
sidered
a
-directing13) afforded complete b-selectivity (entry iii).
We have earlier obtained interesting results in glycosylation with
BnO
CAO
O
O
MeO
OH
O
OAc
OAc
OAc
MeO
O
AcO
N3
BnO
CAO
HO
HO
OAc
O
N3
O
O
O
AcO
AcO
OBz
O
O
O
OBz
OAc
OBn
BnO
CAO
O
O
OBn
O
OAc
BzO
BzO
O
MeO
O
O
BzO
BzO
O
O
O
O
OBz
OBz
OAc
AcO
AcO
CAO
AcO
7
i
ii
iii
MeO
O
O
O
AcO
OBz
AcO
O
O
O
O
OBz
OAc
OAc
OBn
OBn
O
OAc
O
O
O
N3
BzO
BzO
BzO
BzO
O
O
OAc
AcO
AcO
9 (
10 (
β)
12
α);
11
OBz
OBz
SEt
N3
8
Scheme 2. Synthesis of tetrasaccharide intermediate 12 using a linear approach. Reagents and conditions: (i) NIS, AgOTf, 4 A MS, CH2Cl2, 10 °C, 95% (a/b 1.5:1); (ii) 90% TFA
(aq), 86%; (iii) Sc(OTf)3, Ac2O, 96%.

J. D. M. Olsson, S. Oscarson / Tetrahedron: Asymmetry 20 (2009) 879–886
881
Table 1
Glycosylation investigation to optimize formation of tetrasaccharide 9
Entry
Donor
Acceptor
7
Promoter
DMTST
Solvent
Yield
OAc
O
AcO
AcO
i
Et2O (0 °C)
Traces
SEt
N3
8
OAc
O
AcO
AcO
ii
7
7
Me2S2/Tf2O
NIS/AgOTf
Et2O (0 °C)
Decomposition
SEt
SEt
N3
8
OAc
O
AcO
AcO
iii
Et2O (0 °C)
82% (a/b 0/1)
N3
8
OAc
O
AcO
AcO
iv
7
7
7
NIS/AgOTf
NIS/AgOTf
NIS/AgOTf
CH2Cl2 (ꢀ20 °C)
CH2Cl2 (0 °C)
CH2Cl2 (rt)
95% (
93% (
95% (
a
a
a
/b 1/1.4)
SEt
SEt
SEt
N3
8
OAc
O
AcO
AcO
v
/b 1/1)
N3
8
OAc
O
AcO
AcO
vi
/b 1.5/1)
N3
8
OAc
O
AcO
AcO
vii
viii
ix
7
7
7
BF3–OEt2
TMSOTf
TMSOTf
CH2Cl2 (ꢀ30 °C)
Et2O/CH2Cl2 (0 °C)
Et2O (0 °C)
22% (
a
/b 1/1.6)
O
O
O
CCl3
CCl3
CCl3
N3
3
NH
NH
NH
OAc
O
AcO
AcO
Traces
N3
3
OAc
O
AcO
AcO
Decomposition
Decomposition
N3
3
OAc
O
AcO
AcO
x
7
7
AgOTf
CH2Cl2 (ꢀ60 °C)
N3
Br
OAc
O
AcO
AcO
xi
AgOTf/DTBMP
CH2Cl2 (ꢀ35 °C)
34% (a/b 1/1)
N3
Br

882
J. D. M. Olsson, S. Oscarson / Tetrahedron: Asymmetry 20 (2009) 879–886
2-N-acetyl-2N,3O-oxazolidinone-protected glucosamine donors
activated by NIS/AgOTf, where the stereoselective outcome is
determined by the amount of AgOTf added, catalytic amounts
AcO
AcO
OBz
O
OBn
O
BzO
BzO
O
O
OBz
afforded b-linked products whereas 0.5 equiv gave
a-linked prod-
OAc
AcO
ucts.14 This methodology was applied to the present glycosylation
AcO
CAO
AcO
O
O
reaction and excellent yield (94%) and complete a-selectivity were
OAc
OAc
O
obtained.15 Unfortunately, the carbamate-protecting group is not
orthogonal to the chloroacetyl temporary-protecting group in the
molecule. To proceed, the best conditions found were applied to
the coupling reaction between donor 3 and acceptor 7 on a pre-
N3
OAc
12
i
parative scale to yield an
a/b-mixture, which was separated on a
silica gel column affording the desired tetrasaccharide 9 (57%)
and also 10 (38%).
AcO
OBz
OBn
AcO
O
BzO
BzO
Tetrasaccharide 9 was then converted to the target structures
18–20, partly using methodologies worked out for the synthesis
of the Haemophilus structures.6 Hydrolysis of the BDA-acetal
(?11, 86%) followed by acetolysis of the 1,6-anhydro-linkage using
Sc(OTf)3 as a catalyst very efficiently afforded the anomeric acetate
12 (96%). At this stage, chemoselective reduction of the azido
group was performed using Staudinger conditions. Acetylation of
the obtained amine yielded 13 (83%), which was converted into
the corresponding thioglycoside donor 14 (68%) by EtSH/BF3-
etherate treatment (Scheme 3). This donor will be a key intermedi-
ate in the synthesis of larger Neisseria LPS structures including the
Kdo- and lipid A parts of the native molecule. To test its donor
properties glycosylation with a spacer, N-Cbz-protected ethanol-
amine, using again NIS/AgOTf as promoter was performed which
afforded an 85% yield of spacer tetrasaccharide 15.
O
O
O
OBz
R
AcO
AcO
O
O
CAO
AcO
OAc
OAc
O
AcHN
OAc
13 R = OAc
14 R = SEt
ii
iii
AcO
AcO
OBz
OBn
O
O
BzO
BzO
O
O
OBz
From this intermediate, the non-phosphorylated deprotected
target structure 20 as well as the phosphorylated structures 18
and 19 was produced (Scheme 4). A two-step deprotection scheme
comprising Zemplen deacylation followed by catalytic hydrogenol-
ysis yielded tetrasaccharide 20 in 92% overall yield. To introduce
the phosphoethanolamine regioselectively in the O-600 position,
the chloroacetyl group was removed by treatment with ammonia
in MeOH/CH2Cl2 (?16, 75%). The phosphoethanolamine was intro-
duced by phosphoramidite chemistry using a Boc-protected amino
O
AcO
AcO
CAO
AcO
AcHN
NHCbz
O
O
OAc
OAc
O
OAc
15
Scheme 3. Synthesisof tetrasaccharide intermediate 15. Reagents and conditions: (i)
(a) PPh3, THF, H2O, reflux; (b) Ac2O/pyridine (1:1), 83%; (ii) EtSH, BF3–OEt2, CH2Cl2,
68%; (iii) N-Cbz-2-aminoethanol, NIS, AgOTf, CH2Cl2/Et2O (2:1), 4 Å MS, 0 °C, 85%.
AcO
AcO
AcO
OBz
OBz
OBn
O
OBn
O
AcO
O
O
BzO
BzO
BzO
O
O
BzO
O
O
OBz
OBz
i
O
O
AcO
AcO
CAO
AcO
AcHN
AcO
AcO
HO
AcO
AcHN
NHCbz
NHCbz
HO
HO
OH
O
O
O
OH
O
O
O
HO
O
O
OAc
OAc
OAc
OAc
HO
O
O
O
OH
O
HO
HO
O
OAc
OAc
NH2
15
16
O
O
P
v
OH
OH
ii
BocHN
O
HO
HO AcHN
O
OH
19
HO
AcO
AcO
OH
OBz
O
iv
OH
HO
OBn
O
HO
HO
BzO
O
O
O
O
O
O
O
BzO
OH
OBz
O
HO
HO
HO
HO
AcHN
O
AcO
AcO
O
AcO
AcHN
NH2
BocHN
NHCbz
O
HO
HO
O
O
O
OH
O
O
O
OH
O
P
HO
HO
OH
OH
OAc
OAc
O
O
O
BnO
iii
OH
OH
OAc
O
HO
20
17
NH2
HO
O
O
O
O
P
OH
OH
NH2
O
HO
AcHN
O
HO
OH
18
Scheme 4. Synthesis of target structures 18–20. Reagents and conditions: (i) NH3 (satd) MeOH/CH2Cl2 (2:1), 75%; (ii) a) (2-tert-butyloxycarbonyl aminoethyl)benzyl N,N-
diisopropyl phosphoramidite, tetrazole, CH2Cl2; (b) m-CPBA, CH2Cl2, 0 °C, 76%; (iii) (a) Pd/C (10%), H2, 0.1 M HCl, EtOAc/MeOH (1:1); (b) NaOMe, MeOH (c) Dowex-H+, MeOH,
40 °C, 68% over three steps; (iv) a) Pd/C (10%), H2, 0.1 M HCl, EtOAc/MeOH (1:1); (b) NaOMe, MeOH, 68% over two steps; (v) (a) NaOMe, MeOH; (b) Pd/C (10%), H2, 0.1 M HCl,
MeOH, 92% over two steps.

J. D. M. Olsson, S. Oscarson / Tetrahedron: Asymmetry 20 (2009) 879–886
883
group to attain orthogonality with the protection of the spacer ami-
4.3. (20S,30S)-Ethyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-
a-D-
no group. Treatment of alcohol 16 with (N-Boc-aminoethyl)benzyl
N,N-diisopropyl phosphoramidite6 and tetrazole followed by m-
CPBA oxidation gave phosphotriester 17 in 76% yield. Deprotection
of 17 was performed using the two-step procedure discussed above
but this time in reversed order to give target structure 19 (68%)
with a free spacer amino group which can be selectively activated
and conjugated in the presence of the Boc-protected phosphoetha-
nolamine group. Acidic workup after the deacylation step afforded
target structure 18 with two free amino groups.
glucopyranosyl-(1?2)-7-O-benzyl-6-O-chloroacetyl-3,4-O-
(20,30-dimethoxybutane-20,30-diyl)-1-thio-
heptopyranoside 4
L-glycero-a-D-manno-
2-Azido-3,4,6-tri-O-acetyl-2-deoxy-a-D-glucopyranosyl trichlo-
roacetimidate7 3 (128 mg, 0.269 mmol) and 2 (80 mg, 0.149 mmol)
were dissolved in dry Et2O (3 mL) and 4 A MS was added. After stir-
ring for 30 min under an Ar-atmosphere, the mixture was cooled to
0 °C and TMSOTf (0.015 mmol, from Et2O stock solution) was
added. After 2.5 h, Et3N (150 lL) was added and the mixture was
diluted with CH2Cl2 (6 mL), filtered through Celite and subjected
to normal workup. FC (toluene/EtOAc 9:1 ? 6:1) gave 4 (80 mg,
3. Conclusion
0.094 mmol, 63%); Rf 0.68 (toluene/EtOAc 2:1); [a]D = +16 (c 1.0,
In conclusion, an effective synthesis of a tetrasaccharide thio-
glycoside donor, corresponding to the outer part of the conserved
inner core LPS structure of N. meningitidis and allowing elongation
through glycosylation at the reducing end and regioselective phos-
phorylation at the 600-position, is reported. Spacer-equipped target
structures were produced, with or without phosphoethanolamine
substituents, ready for conjugation and following immunological
evaluation.
CHCl3); 13C NMR (100 MHz, CDCl3): d 14.7, 17.7, 17.9, 20.7, 20.8,
20.8, 25.2, 41.2, 48.1, 48.3, 61.2, 62.3, 62.7, 67.8, 68.1, 69.0, 69.1,
69.5 (2C), 69.6, 73.2, 75.2, 83.9, 98.4 (JC,H = 177 Hz), 100.1, 100.4,
127.7 (2C), 127.8, 128.5 (2C), 137.8, 167.3, 169.3, 170.1, 170.7;
1H NMR (400 MHz, CDCl3): d 1.22 (t, 3H, J = 7.6 Hz), 1.26 (s, 6H),
2.05 (s, 3H), 2.09 (s, 3H), 2.09 (s, 3H), 2.57 (m, 2H), 3.09 (s, 3H),
3.23 (s, 3H), 3.22 (dd, 1H, J = 3.6, 10.8 Hz), 3.60 (dd, 1H, J = 6.8,
9.6 Hz), 3.67 (dd, 1H, J = 6.8, 9.6 Hz), 4.04 (dd, 1H, J = 3.2, 9.6 Hz),
4.04–4.35 (m, 10H), 4.48 (benzylic d, 1H, Jgem = 11.6 Hz), 4.55 (ben-
zylic d, 1H, Jgem = 11.6 Hz), 5.01 (dd, 1H, J = 9.6, 9.6 Hz), 5.32 (s, 1H),
5.47 (d, 1H, J = 3.6 Hz), 5.51 (dd, 1H, J = 9.2, 10.8 Hz), 5.61 (dd, 1H,
J = 6.8, 6.8 Hz), 7.22–7.35 (m, 5H); HRMS: Calcd for C36H50ClN3
O16S: [M+Na]+ 870.2493; Found 870.2467.
4. Experimental
4.1. General methods
Normal workup means drying the organic phase with MgSO4 (s)
or Na2SO4 (s), filtering and evaporation of the solvent in vacuo at
ꢁ35 °C. CH2Cl2 was distilled over calcium hydride and collected
onto 4 Å predried MS. Thin Layer Chromatography (TLC) was car-
ried out on 0.25 mm precoated silica-gel plates (Merck silica-gel
60 F254); detected with UV-abs (254 nm) and/or by charring with
8% sulfuric acid or AMC (ammonium molybdate (10 g) and cerium
sulfate (2 g) dissolved in 10% H2SO4 (200 mL)) followed by heating
to ꢁ250 °C. FC means Flash Column chromatography using silica
gel (Amicon, (0.040–0.063 mm)). 1H NMR, 31P NMR and 13C NMR
spectra were performed on a Varian or Bruker instrument (300,
400 or 500 MHz) at 25 °C unless otherwise stated. Chemical shifts
are given in ppm relative to solvent peaks in CDCl3 (d = 77.17 for
13C and d = 7.26 for 1H) and in D2O (d = 4.79 for 1H) or TMS
(d = 0.00) for 13C NMR and 1H NMR. For 31P NMR, 85% H3PO4
(d = 0.00) was used as an external reference.
4.4. (20S,30S)-Ethyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-
a-D-
glucopyranosyl-(1?2)-7-O-benzyl-6-O-chloroacetyl-3,4-O-
(20,30-dimethoxybutane-2’,30-diyl)-1-thio-
heptopyranoside S-oxide 5
L-glycero-a-D-manno-
Compound 4 (67 mg, 0.079 mmol) was dissolved in dry CH2Cl2
(2 mL) and the solution was cooled to ꢀ60 °C followed by addition
of m-CPBA (14 mg, 0.083 mmol). After 30 min (ꢀ60 °C ? ꢀ20 °C),
the reaction was diluted with CH2Cl2 (5 mL), washed with NaHCO3
(satd, 10 mL) and subjected to normal workup followed by FC (tolu-
ene/EtOAc 1:1) to give 5 (42 mg, 0.048 mmol, 61%); Rf 0.26 (toluene/
EtOAc 1:1); [a]
D = +22 (c 1.0, CHCl3); 13C NMR (100 MHz, CDCl3): d
5.8, 17.7, 17.9, 20.7, 20.8, 20.9, 41.0, 43.6, 48.4, 48.5, 61.2, 61.5,
62.0, 66.4, 68.4, 68.9 (2C), 69.3, 69.5, 73.6, 74.1, 93.1, 98.7, 100.3,
100.5, 127.8 (2C), 128.1, 128.7 (2C), 137.4, 167.2, 169.7, 170.3, 171.1.
1H NMR (400 MHz, CDCl3): d 1.26 (t, 3H, J = 7.2 Hz), 1.28 (s, 3H),
1.30 (s, 3H), 2.05 (s, 3H), 2.10 (s, 3H), 2.11 (s, 3H), 2.65 (m, 1H), 2.88
(m, 1H), 3.11 (s, 3H), 3.27 (s, 3H), 3.27 (dd, 1H, J = 4.0, 10.4 Hz),
3.57–3.66 (m, 2H), 3.98–4.10 (m, 5H), 4.23–4.28 (m, 2H), 4.39
(dd, 1H, J = 10.0, 10.0 Hz), 4.49 (benzylic d, 1H, Jgem = 11.6 Hz),
4.53 (benzylic d, 1H, Jgem = 11.6 Hz), 5.55 (s, 1H), 4.70 (dd, 1H,
J = 1.2, 2.8 Hz), 5.02 (dd, 1H, J = 10.0, 10.0 Hz), 5.45 (dd, 1H,
J = 7.6, 7.6 Hz), 5.50 (dd, 1H, J = 9.2, 10.8 Hz), 5.64 (d, 1H,
J = 3.6 Hz), 7.25–7.38 (m, 5H); HRMS: Calcd for C36H50ClN3O17S:
[M+Na]+ 886.2442; Found 886.2384.
4.2. (20S,30S)-Ethyl 7-O-benzyl-6-O-chloroacetyl-3,4-O-(20,30-
dimethoxybutane-20,30-diyl)-1-thio-
heptopyranoside 2
L-glycero-a-D-manno-
(20S,30S)-Ethyl 7-O-benzyl-6-O-chloroacetyl-3,4-O-(20,30-dim-
ethoxybutane-20,30-diyl)-2-O-p-methoxybenzyl-1-thio-
-glycero-
-manno-heptopyranoside5
(381 mg, 0.58 mmol) was
L
a
-
D
1
dissolved in CH2Cl2/H2O (15:1, 8 mL) and DDQ (152 mg,
0.67 mmol) was added. After 3 h, the mixture was diluted with
CH2Cl2 (10 mL), washed with Na2S2O3 (5% aq, 10 mL) and subjected
to normal workup. FC (toluene/EtOAc 6:1 ? 3:1) gave 2 (260 mg,
4.5. (20S,30S)-3,4,6-tri-O-Acetyl-2-azido-2-deoxy-
nosyl-(1?2)-7-O-benzyl-6-O-chloroacetyl-3,4-O-(20,3’-dimethoxy-
butane-20,30-diyl)-
-glycero- -manno-heptopyranosyl-(1?3)-
[2,3,4,6-tetra-O-benzoyl-b- -glucopyranosyl-(1?4)]-7-O-acetyl-
1,6-anhydro-2-O-benzyl- -glycero- -manno-heptopyranose 9
and (20S,30S) 3,4,6-tri-O-acetyl-2-azido-2-deoxy-b-
-glucopyran-
osyl-(1?2)-7-O-benzyl-6-O-chloroacetyl-3,4-O-(20,30-dimethoxy-
butane-20,30-diyl)-
-glycero- -manno-heptopyranosyl-(1?3)-
[2,3,4,6-tetra-O-benzoyl-b- -glucopyranosyl-(1?4)]-7-O-acetyl-
1,6-anhydro-2-O-benzyl- -glycero- -manno-heptopyranose 10
a-D-glucopyra-
0.55 mmol, 94%); Rf 0.46 (toluene/EtOAc 2:1); [a]D = +17 (c 1.0,
L
a-D
CHCl3); 13C NMR (100 MHz, CDCl3): d 14.6, 17.7, 17.8, 24.9, 41.0,
48.1, 48.2, 62.6, 67.8, 68.7, 69.2, 70.1, 71.0, 73.1, 84.6, 100.2,
100.6, 127.7 (2C), 127.8, 128.4 (2C), 137.7, 167.0; 1H NMR
(400 MHz, CDCl3): d 1.22 (t, 3H, J = 7.6 Hz), 1.27 (s, 3H), 1.29 (s,
3H), 2.55 (m, 2H), 3.13 (s, 3H), 3.24 (s, 3H), 3.62 (dd, 1H, J = 6.4,
10.0 Hz), 3.68 (dd, 1H, J = 7.2, 10.0 Hz), 3.95 (dd, 1H, J = 2.8,
10.0 Hz), 4.00–4.16 (m, 4H), 4.35 (dd, 1H, J = 0.8, 10.0 Hz), 4.47
(benzylic d, 1H, Jgem = 11.6 Hz), 4.55 (benzylic d, 1H, Jgem = 11.6 Hz),
5.34 (s, 1H), 5.60 (ddd, J = 0.8, 1.6, 3.6 Hz), 7.22–7.34 (m, 5H);
HRMS: Calcd for C24H35ClO9S: [M+Na]+ 557.1583; Found 557.1611.
D
L
a-D
D
L
a-D
D
L
a-D
(20S,30S) 7-O-Benzyl-6-O-chloroacetyl-3,4-O-(20,30-dimethoxybu-
tane-20,30-diyl)-
-glycero- -manno-heptopyranosyl-(1?3)-[2,3,4,
L
a-D

884
J. D. M. Olsson, S. Oscarson / Tetrahedron: Asymmetry 20 (2009) 879–886
6-tetra-O-benzoyl-b-
D
-glucopyranosyl-(1?4)]-7-O-acetyl-1,6-anhy-
-manno-heptopyranose5
(61 mg,
0.044 mmol) and ethyl 2-azido-2-deoxy-3,4,6-tri-O-acetyl-1-thio-
-glucopyranoside12 8 (65 mg, 0.173 mmol) were dissolved in
130.0 (Ar-C), 133.5, 133.6, 133.7, 137.5, 138.0, 165.2, 165.3,
dro-2-O-benzyl-L-glycero-
a-D
7
166.1, 168.4, 169.8, 170.1, 170.7, 170.8; 1H NMR (400 MHz, CDCl3):
d = 1.97 (s, 3H), 2.04 (s, 3H), 2.08 (s, 3H), 2.08 (s, 3H), 3.36 (d, 1H,
J = 4.4 Hz), 3.37 (dd, 1H, J = 4.8, 10.4 Hz), 3.54 (dd, 1H, J = 3.6,
10.4 Hz), 3.55–3.61 (m, 2H), 3.84–3.88 (m, 2H), 3.92–3.96 (m,
2H), 4.00–4.04 (m, 2H), 4.10–4.34 (m, 13 H), 4.46 (dd, 1H, J = 3.6,
12.0 Hz), 4.72 (dd, 1H, J = 3.6, 12.0 Hz), 5.04 (dd, 1H, J = 9.6 Hz),
5.13–5.15 (m, 3H), 5.21–5.24 (m, 2H), 5.38 (dd, 1H, J = 9.6,
9.6 Hz), 5.57 (dd, 1H, J = 4.0, 10.0 Hz), 5.77 (dd, 1H, J = 10.0,
10.0 Hz), 5.95 (dd, 1H, J = 10.0, 10.0 Hz), 7.15–7.57 (m, 22H), 7.84
(dd, 2H, J = 1.2, 8.4 Hz), 7.89 (dd, 2H, J = 0.8, 8.0 Hz), 7.95 (dd, 2H,
J = 1.2, 8.4 Hz), 8.03 (dd, 2H, J = 0.8, 8.0 Hz); HRMS: Calcd for
C78H80ClN3O30: [M+Na]+ 1596.4413; Found 1596.4430.
a-D
dry CH2Cl2 (5 mL) and 4 A MS was added. After stirring for
30 min under an Ar-atmosphere, the mixture was cooled to
10 °C, followed by addition of NIS (41 mg, 0.180 mmol) and AgOTf
(cat.). After 30 min, Et3N (150 lL) was added and the mixture was
diluted with CH2Cl2 (10 mL), filtered through Celite, washed with
Na2S2O3 (10% aq, 15 mL) and subjected to normal workup. FC (pen-
tane/EtOAc 2:1 ? 3:2 ? 1:1) eluted the
0.025 mmol, 57%) followed by the b-anomer 10 (28 mg,
0.017 mmol, 38%). For 9 ( -anomer): Rf 0.46 (toluene/EtOAc 2:1);
D = +58 (c 1.0, CHCl3); 13C NMR (100.0 MHz, CDCl3): d 17.8,
a-anomer 9 (42 mg,
a
[a]
17.9, 20.6, 20.8 (2C), 21.0, 41.3, 47.7, 48.1, 60.5, 61.4, 62.1, 62.4,
65.1, 67.2, 68.1, 68.2, 69.3, 69.6, 69.7, 71.7 (2C), 72.1, 72.6, 72.8,
72.9, 73.3, 73.7, 74.8, 74.9, 75.3, 96.9 (JCH = 171 Hz), 98.3
4.7. 3,4,6-Tri-O-acetyl-2-azido-2-deoxy-
(1?2)-3,4,7-tri-O-acetyl-6-O-chloroacetyl-
heptopyranosyl-(1?3)-[2,3,4,6-tetra-O-benzoyl-b-
glucopyranosyl-(1?4)]-1,6,7-tri-O-acetyl-2-O-benzyl-
-manno-heptopyranose 12
a
-
D
-glucopyranosyl-
L-glycero- -manno-
a-D
-
D
(JCH = 178 Hz),
99.9,
100.0
(JCH = 168 Hz),
100.2,
100.5
L-glycero-
(JCH = 173 Hz), 127.5–129.9 (Ar-C), 133.5, 133.6, 137.8, 138.0,
165.1, 165.2, 165.9, 166.1, 167.3, 169.8, 170.1, 170.6, 170.8; 1H
NMR (400 MHz, CDCl3): d 1.26 (s, 6H), 1.98 (s, 3H), 2.04 (s, 3H),
2.08 (s, 3H), 2.10 (s, 3H), 3.09 (s, 3H), 3.16 (s, 3H), 3.31 (dd, 1H,
J = 3.6, 10.8 Hz), 3.38–3.42 (m, 2H), 3.50 (dd, 1H, J = 8.0, 10.0 Hz),
3.78 (dd, 1H, J = 6.4, 11.2 Hz), 3.91 (dd, 1H, J = 6.0, 11.2 Hz), 3.94
(m, 1H), 4.08–4.38 (m, 16H), 4.47 (dd, 1H, J = 3.6, 12.4 Hz), 4.79
(dd, 1H, J = 3.2, 12.0 Hz), 5.07 (dd, 1H, J = 9.6, 9.6 Hz), 5.12 (s,
1H), 5.15 (d, 1H, J = 8.0 Hz), 5.22 (s, 1H), 5.35 (dd, 1H, J = 6.4,
6.8 Hz), 5.45 (d, 1H, J = 3.6 Hz), 5.50 (dd, 1H, J = 9.6, 9.6 Hz), 5.58
(dd, 1H, J = 9.6, 10. Hz), 5.82 (dd, 1H, J = 9.6, 9.6 Hz), 5.96 (dd, 1H,
J = 9.6, 9.6 Hz), 7.17–7.57 (m, 22 H), 7.84 (d, 2H, J = 8.0 Hz), 7.89
(d, 2H, J = 8.0 Hz), 7.94 (d, 2H, J = 8.0 Hz), 8.03 (d, 2H, J = 8.0 Hz);
a-D
Compound 11 (59 mg, 0.037 mmol) was dissolved in Ac2O
(2 mL) and Sc(OTf)3 (1.0 mg, 0.002 mmol) was added. After 2 h,
the mixture was cooled to 0 °C and MeOH (3 mL) was added. The
solvent was evaporated, and the residue was co-evaporated with
toluene (2 ꢂ 5 mL). FC (toluene/EtOAc 3:1 ? 2:1) gave 12 (61 mg,
0.35 mmol, 96%); Rf 0.57 (toluene/EtOAc 1:1); [a]D = +40 (c 1.0,
CHCl3); 13C NMR (125 MHz, CDCl3): d 20.1, 20.2, 20.5, 20.6, 20.7
(4C), 20.8, 40.9, 61.1, 61.2, 61.3, 62.2, 63.4, 65.1, 67.5, 68.6, 68.7,
69.0 (2C), 69.1, 70.2, 70.3, 71.6, 71.9, 72.1, 72.8, 73.1, 73.3, 74.2,
74.5, 78.5, 90.4 (JCH = 175 Hz), 99.5 (JCH = 178 Hz), 100.0
(JCH = 171 Hz), 101.3 (JCH = 165 Hz), 128.3–130.1 (Ar-C), 133.4,
133.4, 133.7, 133.8, 137.1, 164.5, 165.4, 165.6, 165.7, 167.2,
168.6, 169.1, 169.6, 169.7, 169.7, 170.0, 170.3, 170.7, 170.8; 1H
NMR (500 MHz, CDCl3): d 1.62 (s, 3H), 1.75 (s, 3H), 1.94 (s, 3H),
2.01 (s, 3H), 2.08 (s, 3H), 2.09 (s, 3H), 2.11 (s, 3H), 2.13 (s, 3H),
2.18 (s, 3H), 3.51 (m, 1H), 3.56–3.59 (m, 2H), 3.67 (dd, 1H, J = 3.5,
11.0 Hz), 3.76 (dd, 1H, J = 7.0, 11.0 Hz), 3.92–3.98 (m, 2H), 4.05
(dd, 1H, J = 6.5, 11.0 Hz), 4.11–4.19 (m, 3H), 4.24 (benzylic d, 1H,
Jgem = 15.0 Hz), 4.33 (m, 1H), 4.34 (benzylic d, 1H, Jgem = 15.0 Hz),
4.43–4.55 (m, 5H), 4.64–4.72 (m, 2H), 4.92–4.97 (m, 2H), 5.25
(dd, 1H, J = 10.0, 10.0 Hz), 5.36–5.39 (m, 2H), 5.46–5.56 (m, 5H),
5.69 (s, 1H), 5.86 (dd, 1H, J = 10.0, 1.0 Hz), 6.14 (d, 1H, J = 1.5 Hz),
7.23–7.52 (m, 17H), 7.80 (d, 2H, J = 7.0 Hz), 7.91 (d, 2H, J = 7.5
Hz), 7.96 (d, 2H, J = 7.5 Hz), 8.05 (d, 2H, J = 7.0 Hz). HRMS: Calcd
for C81H86ClN3O36: [M+Na]+ 1734.4572; Found 1734.4499.
HRMS: Calcd for C84H90ClN3O32
:
[M+Na]+ 1710.5094; Found
1710.5138. For 10 (b-anomer): Rf 0.64 (toluene/EtOAc 1:1);
[a
]
D = ꢀ25 (c 1.0, CHCl3); 13C NMR (75.4 MHz, CDCl3): d 17.9,
18.0, 20.7, 20.8 (2C), 20.8, 41.0, 47.7, 48.0, 61.3, 62.2, 62.9, 63.9,
65.0, 68.0 (2C), 68.5, 69.4, 70.1, 70.1, 71.5, 71.6, 72.1, 72.4, 72.9,
72.8 (2C), 73.1, 73.3, 73.5, 75.4, 75.6, 77.0, 96.9 (JCH = 168 Hz),
99.9, 100.1, 100.3 (JCH = 178 Hz), 100.5 (JCH = 161 Hz), 101.2
(JCH = 166 Hz), 127.4–129.9 (Ar-C), 133.3, 133.4 (2C), 133.6, 137.8,
138.0, 165.2, 165.2, 165.9, 166.2, 167.0, 169.6, 170.2, 170.7,
170.8; 1H NMR (300 MHz, CDCl3): d = 1.23 (s, 3H), 1.25 (s, 3H),
1.99 (s, 3H), 2.00 (s, 3H), 2.08 (s, 3H), 2.09 (s, 3H), 3.07 (s, 3H),
3.20 (s, 3H), 3.37–3.60 (m, 4H), 3.76 (dd, 1H, J = 6.6, 11.1 Hz),
3.89–4.38 (m, 19H), 4.44 (dd, 1H, J = 4.2, 12.0 Hz), 4.69 (dd, 1H,
J = 2.7, 12.0 Hz), 4.78 (d, 1H, J = 8.1 Hz), 4.92–5.10 (m, 3H), 5.18
(s, 1H), 5.27 (s, 1H), 5.36 (m, 1H), 5.56 (dd, 1H, J = 7.5, 9.3 Hz),
5.72 (dd, 1H, J = 9.9, 9.9 Hz), 5.90 (dd, 1H, J = 9.9, 9.9 Hz), 7.19–
7.55 (m, 22H), 7.82 (dd, 2H, J = 1.5, 8.4 Hz), 7.88 (d, 2H, J = 1.5,
8.4 Hz), 7.93 (d, 2H, J = 1.2, 8.4 Hz), 8.03 (d, 2H, J = 1.5, 8.4 Hz);
4.8. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-
glucopyranosyl-(1?2)-3,4,7-tri-O-acetyl-6-O-chloroacetyl-
glycero- -manno-heptopyranosyl-(1?3)-[2,3,4,6-tetra-O-
benzoyl-b- -glucopyranosyl-(1?4)]-1,6,7-tri-O-acetyl-2-O-
benzyl- -glycero- -manno-heptopyranose 13
a-D-
L-
a
-D
D
HRMS: Calcd for C84H90ClN3O32
:
[M+Na]+ 1710.5094; Found
1710.5161.
L
a-D
4.6. 3,4,6-Tri-O-acetyl-2-azido-2-deoxy-
(1?2)-7-O-benzyl-6-O-chloroacetyl- -glycero-
heptopyranosyl-(1?3)-[2,3,4,6-tetra-O-benzoyl-b-
glucopyranosyl-(1?4)]-7-O-acetyl-1,6-anhydro-2-O-benzyl-
glycero- -manno-heptopyranose 11
a
-
D
-glucopyranosyl-
Derivative 12 (101 mg, 0.059 mmol) was dissolved in THF
(5 mL), and PPh3 (31 mg, 0.118 mmol) was added. The mixture
was refluxed for 6 h before H2O (1.5 mL) was added. The mixture
was refluxed for an additional 90 min and then evaporated. The
residue was dissolved in pyridine (2 mL) and cooled to 0 °C fol-
lowed by dropwise addition of Ac2O (2 mL). After 12 h, the mixture
was concentrated and the residue was co-evaporated with toluene
(2 ꢂ 4 mL). FC (toluene/EtOAc 2:1 ? 1:1 ? 1:2) gave 13 (85 mg,
L
a-D
-manno-
D-
L
-
a-D
Compound 9 (91 mg, 0.054 mmol) was dissolved in 90% TFA (aq,
4 mL). After stirring for 20 min, the mixture was evaporated fol-
lowed by co-evaporation with toluene (2 ꢂ 10 mL). FC (toluene/
EtOAc 2:1 ? 1:1) gave 11 (73 mg, 0.046 mmol, 86%); Rf 0.33 (tolu-
0.49 mmol, 83%); Rf 0.55 (toluene/EtOAc 1:2); [a]D = +20 (c 1.0,
CHCl3); 13C NMR (100 MHz, CDCl3): d 20.3, 20.5, 20.7, 20.8 (2C),
20.8 (3C), 21.1, 23.3, 40.7, 52.3, 61.4, 61.8, 61.9, 63.7, 65.4, 67.6,
68.1, 68.9 (2C), 69.2, 70.1, 70.5, 71.4, 71.5, 72.0 (2C), 73.1, 73.2,
73.5, 74.6, 74.9, 77.9, 90.3, 100.2 (2C), 101.7, 128.3–130.1 (Ar-C),
133.2, 133.3, 133.5, 133.7, 136.9, 164.8, 165.2, 165.7, 165.9,
ene/EtOAc 1:1); [a]
D = +12 (c 1.0, CHCl3); 13C NMR (100 MHz,
CDCl3): d 20.6, 20.8, 21.0, 21.0, 41.1, 61.8, 62.1, 62.6, 65.1, 67.6,
68.0, 68.5, 68.8, 69.4, 70.8, 71.0, 71.1, 71.5, 72.1, 72.6, 72.7, 72.8,
73.1, 73.5, 73.8, 75.0, 75.3, 79.9, 96.5, 99.2, 99.6, 100.2, 127.9–

J. D. M. Olsson, S. Oscarson / Tetrahedron: Asymmetry 20 (2009) 879–886
885
167.3, 168.8, 169.3, 169.8, 169.9, 170.1, 170.2 (2C), 170.8, 171.0,
171.3; 1H NMR (400 MHz, CDCl3): d 1.64 (s, 3H), 1.81 (s, 3H),
1.99 (s, 3H), 2.00 (s, 3H), 2.02 (s, 3H), 2.04 (s, 3H), 2.05 (s, 3H),
2.06 (s, 3H), 2.10 (s, 3H), 2.23 (s, 3H), 3.48 (m, 2H), 3.55 (d, 1H,
J = 9.6 Hz), 3.67 (dd, 1H, J = 6.8, 11.2 Hz), 3.79 (dd, 1H, J = 5.2,
11.2 Hz), 3.94 (dd, 1H, J = 2.0, 8.8 Hz), 4.02 (dd, 1H, J = 6.8,
11.2 Hz), 4.05–4.17 (m, 4H), 4.21–4.36 (m, 5H), 4.39 (dd, 1H,
J = 3.6, 12.0 Hz), 4.45 (benzylic d, 1H, Jgem = 12.8 Hz), 4.50 (dd, 1H,
J = 3.6, 9.6 Hz), 4.56 (dd, 1H, J = 0.8, 12.4 Hz), 4.65 (benzylic d,
1H, Jgem = 12.8 Hz), 4.69 (dd, 1H, J = 7.2, 12.4 Hz), 4.92 (d, 1H,
J = 8.0 Hz), 5.04 (m, 1H), 5.16 (d, 1H, J = 3.2 Hz), 5.26–5.54 (m,
7H), 5.89 (dd, 1H, J = 9.6, 9.6 Hz), 6.14 (d, 1H, J = 9.6 Hz), 6.16 (d,
1H, J = 2.0 Hz), 7.22–7.56 (m, 17H), 7.81 (d, 2H, J = 7.2 Hz), 7.89
(d, 2H, J = 8.0 Hz), 7.91 (d, 2H, J = 8.0 Hz), 8.05 (d, 2H, J = 7.6 Hz);
40.8 (2C), 52.4, 60.8, 62.1, 63.8, 65.6, 67.1, 67.2, 67.7, 68.3, 69.1
(2C), 69.3, 69.8, 70.2, 70.6, 71.5, 71.8, 72.1, 73.2, 73.3, 74.7, 75.0,
75.4, 77.5, 78.1, 97.6 (JCH = 170 Hz), 99.9 (JCH = 178 Hz), 100.2
(JCH = 174 Hz), 101.8 (JCH = 165 Hz), 128.2–130.2 (Ar-C), 133.4,
133.5, 133.6, 133.7, 136.7, 137.5, 156.8, 164.7, 165.3, 165.8,
166.0, 167.4, 169.4, 169.9, 169.9 (2C), 170.3 (2C), 170.8, 171.1,
171.4; 1H NMR (500 MHz, CDCl3): d = 1.56 (s, 3H), 1.71 (s, 3H),
2.00 (s, 3H), 2.05 (s, 6H), 2.05 (s, 3H), 2.06 (s, 3H), 2.10 (s, 3H),
2.24 (s, 3H), 3.09 (m, 1H), 3.35–3.39 (m, 2H), 3.49–3.51 (m, 3H),
3.60 (m, 1H), 3.73 (m, 1H), 3.93–3.99 (m, 2H), 4.03–4.14 (m, 5H),
4. 19 (benzylic d, 1H, Jgem = 15.0 Hz), 4.26–4.42 (m, 6H), 4.47–
4.56 (m, 2H), 4.62 (benzylic d, 1H, Jgem = 15.0 Hz), 4.71 (m, 1H),
4.85–4.88 (m, 2H), 5.02 (d, 1H, J = 3.5 Hz), 5.06–5.11 (m, 2H),
5.17 (d, 1H, J = 3.5 Hz), 5.29–5.31 (m, 2H), 5.35 (dd, 1H, J = 10.5,
10.5 Hz), 5.42–5.47 (m, 3H), 5.51 (dd, 1H, J = 8.0, 9.5 Hz), 5.87
(dd, 1H, J = 9.5, 9.5 Hz), 6.18 (d, 1H, J = 9.5 Hz), 7.24–7.50 (m,
22H), 7.80 (dd, 2H, J = 1.5, 8.0 Hz), 7.89–7.92 (m, 4H), 8.05 (dd,
2H, J = 1.5, 8.5 Hz); HRMS: Calcd for C91H99ClN2O38: [M+Na]+
1885.5457; Found 1885.5474.
HRMS: Calcd for C83H90ClNO37
1750.4688.
:
[M+Na]+ 1750.4772; Found
4.9. Ethyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
glucopyranosyl-(1?2)-3,4,7-tri-O-acetyl-6-O-chloroacetyl-
glycero- -manno-heptopyranosyl-(1?3)-[2,3,4,6-tetra-O-
benzoyl-b- -glucopyranosyl-(1?4)]-6,7-di-O-acetyl-2-O-
benzyl-1-thio- -glycero- -manno-heptopyranoside 14
a-D-
L
-
a
-D
D
4.11. 2-(N-Benzyloxycarbonyl)aminoethyl 2-acetamido-3,4,6-
L
a
-D
tri-O-acetyl-2-deoxy-
acetyl- -glycero- -manno-heptopyranosyl-(1?3)-[2,3,4,6-
tetra-O-benzoyl-b- -glucopyranosyl-(1?4)]-6,7-di-O-acetyl-2-
O-benzyl- -glycero- -manno-heptopyranoside-(1?3)-
[2,3,4,6-tetra-O-benzoyl-b- -glucopyranosyl-(1?4)]-6,7-di-O-
acetyl-2-O-benzyl- -glycero- -manno-heptopyranoside 16
a-D-glucopyranosyl-(1?2)-3,4,7-tri-O-
L
a-D
Compound 13 (83 mg, 0.048 mmol) was dissolved in dry CH2Cl2
(4 mL), EtSH (35 L, 1.25 mmol)
D
lL, 0.48 mmol) and BF3ꢃOEt2 (157
l
L
a-D
were added. After 30 h (reaction monitored by HRMS) under an Ar-
atmosphere, the mixture was diluted with CH2Cl2 (5 mL), washed
with NaHCO3 (satd, 10 mL) and subjected to normal workup fol-
lowed by FC (toluene/EtOAc 2:1 ? 1:1 ? 1:2) to give 14 (57 mg,
D
L
a-D
Compound 15 (14 mg, 7.5 lmol) was dissolved in CH2Cl2
0.033 mmol, 68%); Rf 0.28 (toluene/EtOAc 1:1); [
a
]
D = +28 (c 1.0,
(1 mL), the solution was cooled to ꢀ10 °C and MeOH saturated
with NH3 (2 mL) was added dropwise. After stirring for 30 min
(ꢀ10 °C ? 0 °C), the solvents were evaporated followed by FC (tol-
uene/EtOAc 1:3 ? EtOAc) of the residue to give 16 (10 mg,
CHCl3); 13C NMR (100 MHz, CDCl3): d 14.8, 20.4, 20.8, 20.9, 20.9
(2C), 21.0 (2C), 21.2, 23.4, 25.4, 40.9, 52.4, 61.0, 62.1, 63.9, 65.5,
67.8, 68.2, 69.2, 69.3, 69.4, 69.9, 70.2, 70.6, 71.4, 71.5, 72.1, 73.2,
73.2, 75.6, 75.8, 76.8, 77.4, 77.9, 81.7, 100.2 (2C), 101.9, 128.3–
130.3 (Ar-C), 133.4, 133.5, 133.6, 133.6, 137.4, 164.7, 165.3,
165.8, 166.0, 167.5, 169.4, 169.9, 170.0, 170.2 (2C), 170.4, 170.8,
171.0, 171.3; 1H NMR (400 MHz, CDCl3): d 1.14 (t, 3H, J = 7.2 Hz),
1.59 (s, 3H), 1.88 (s, 3H), 2.00 (s, 3H), 2.00 (s, 3H), 2.04 (s, 3H),
2.05 (s, 3H), 2.07 (s, 3H), 2.08 (s, 3H), 2.24 (s, 3H), 2.35–2.50 (m,
2H), 3.57 (m, 1H), 3.65 (m, 1H), 3.70 (dd, 1H, J = 7.2, 10.8 Hz),
3.85–3.88 (m, 2H), 3.98–4.50 (m, 13H), 4.53 (dd, 1H, J = 4.0,
12.4 Hz), 4.65–4.70 (m, 2H), 4.89 (d, 1H, J = 8.0 Hz), 5.07 (m, 1H),
5.14 (d, 1H, J = 2.8 Hz), 5.27–5.40 (m, 4H), 5.46 (dd, 1H, J = 2.8,
10.4 Hz), 5.50–5.55 (m, 4H), 5.88 (dd, 1H, J = 9.6, 9.6 Hz), 6.09 (d,
1H, J = 9.6 Hz), 7.24–7.51 (m, 17H), 7.81 (dd, 2H, J = 1.6, 8.4 Hz),
7.88–7.92 (m, 4H), 8.08 (dd, 2H, J = 1.2, 8.8 Hz); HRMS: Calcd for
C83H92ClNO35S: [M+Na]+ 1752.4751; Found 1752.4808.
5.5 lmol, 75%); Rf 0.44 (EtOAc); [a]
D = +17 (c 1.0, CHCl3); 13C
NMR (100 MHz, CDCl3): d 20.4, 20.9, 20.9, 21.0 (2C), 21.0 (2C),
21.2, 23.4, 40.7, 52.5, 60.9, 62.4, 63.8, 65.2, 66.8, 67.0 (2C), 67.3,
67.8, 68.6, 69.8, 70.4, 70.5, 70.6 (2C), 71.3, 72.1 (2C), 73.3 (2C),
75.1 (2C), 76.6, 77.5, 98.0, 99.7 (2C), 101.9, 128.0–130.2 (Ar-C),
133.4, 133.4, 133.6, 133.7, 136.8, 137.9, 156.8, 164.8, 165.3,
165.8, 166.1, 169.7, 169.9, 170.1, 170.5, 170.6, 170.8, 171.3,
171.4, 171.5; 1H NMR (400 MHz, CDCl3): d 1.57 (s, 3H), 1.99 (s,
3H), 2.03 (s, 6H), 2.04 (s, 6H), 2.08 (s, 3H), 2.12 (s, 3H), 2.22 (s,
3H), 3.10 (m, 1H), 3.35 (m, 3H), 3.49–3.65 (m, 6H), 3.75–3.83 (m,
2H), 4.02–4.14 (m, 6H), 4.27–4.52 (m, 6H), 4.63 (benzylic d, 1H,
Jgem = 13.2 Hz), 4.71 (m, 1H), 4.81 (s, 1H), 4.93 (d, 1H, J = 8.0 Hz),
5.01 (d, 1H, J = 3.2 Hz), 5.23 (d, 1H, J = 3.6 Hz), 5.33 (dd, 1H,
J = 10.0, 10.0 Hz), 5.40–5.57 (m, 7H), 5.87 (dd, 1H, J = 9.6, 9.6 Hz),
6.36 (1H, bs), 7.21–7.50 (m, 22H), 7.81 (dd, 2H, J = 1.2, 8.4 Hz),
7.89–7.92 (m, 4H), 8.06 7.81 (dd, 2H, J = 1.2, 8.4 Hz); HRMS: Calcd
for C89H98N2O37: [M+Na]+ 1809.5741; Found 1809.5720.
4.10. 2-(N-Benzyloxycarbonyl)aminoethyl 2-acetamido-3,4,6-
tri-O-acetyl-2-deoxy-
acetyl-6-O-chloroacetyl-
(1?3)-[2,3,4,6-tetra-O-benzoyl-b-
a
-
D
-glucopyranosyl-(1?2)-3,4,7-tri-O-
-glycero- -manno-heptopyranosyl-
-glucopyranosyl-(1?4)]-6,7-
-manno-heptopyranoside
L
a-D
D
4.12. 2-(N-Benzyloxycarbonyl)aminoethyl 2-acetamido-3,4,6-
di-O-acetyl-2-O-benzyl-
L
-glycero-
a
-
D
tri-O-acetyl-2-deoxy-
acetyl-6-O-[benzyl-2-(tert-butyloxycarbonylaminoethyl)-
phosphono]- -glycero- -manno-heptopyranosyl-(1?3)-
[2,3,4,6-tetra-O-benzoyl-b- -glucopyranosyl-(1?4)]-6,7-di-O-
acetyl-2-O-benzyl- -glycero- -manno-heptopyranoside 17
a-D-glucopyranosyl-(1?2)-3,4,7-tri-O-
15
L
a-D
Derivative 14 (33 mg, 0.019 mmol) and N-Cbz-2-aminoethanol
(11 mg, 0.057 mmol) were dissolved in dry CH2Cl2/Et2O (2:1,
3 mL) followed by addition of 4 A MS. After stirring for 30 min un-
der an Ar-atmosphere, the mixture was cooled to 0 °C followed by
addition of NIS (9 mg, 0.038 mmol) and AgOTf (cat.). After stirring
D
L
a-D
(2-tert-Butyloxycarbonyl aminoethyl)benzyl N,N-diisopropyl
phosphoramidite5 (11 mg, 28
mol) and 16 (10 mg, 5.6 mol)
were dissolved in dry CH2Cl2 (1 mL), followed by addition of tetra-
zole (2 mg, 28 mol). After stirring for 2 h, the solution was cooled
to 0 °C and m-CPBA (4 mg, 17 mol) was added. After an additional
l
l
for 45 min (0 °C ? rt), Et3N (70 lL) was added and the mixture was
diluted with CH2Cl2 (5 mL), filtered through Celite, washed with
Na2S2O3 (10% aq) and subjected to normal workup. FC (toluene/
EtOAc 2:1 ? 1:1 ? 1:2) gave 15 (30 mg, 0.016 mmol, 85%); Rf
l
l
15 min (0 °C ? rt), the solution was diluted with CH2Cl2 (4 mL),
washed with NaHCO3 (satd, 5 mL) and subjected to normal work-
0.50 (toluene/EtOAc 1:3); [
a]
D = +18 (c 1.0, CHCl3); 13C NMR
(100 MHz, CDCl3): d 20.4, 20.5, 20.9 (3C), 21.0 (2C), 21.2, 23.4,
up. FC (toluene/EtOAc 1:4) gave 17 (9 mg, 4.3 lmol, 76%) as an

886
J. D. M. Olsson, S. Oscarson / Tetrahedron: Asymmetry 20 (2009) 879–886
inseparable diastereomeric mixture; Rf 0.60 (EtOAc); 13C NMR
(100 MHz, CDCl3, diastereomeric mixture): d 20.2–21.1 (several
carbons), 23.1, 28.4 (3C), 28.5 (3C), 29.7, 40.5 (2C), 40.7, 40.9,
52.1, 55.2, 57.3, 60.4, 61.7, 63.5 (2C), 66.5 (2C), 67.0, 67.1, 67.4
(2C), 68.1, 68.3, 69.3 (3C), 69.9, 70.3, 71.3, 71.6, 71.8, 73.0 (2C),
73.1, 74.4, 74.6, 75.1, 77.4, 80.0, 97.6, 99.7, 100.0, 101.5, 127.9–
130.3, 133.2, 133.4, 133.5, 133.6, 135.9, 136.5, 137.3, 156.6,
164.5, 165.0, 165.6, 165.8, 169.7–171.2 (several carbons); 31P
NMR (decoupled, 162 Hz, CDCl3): d ꢀ1.15, ꢀ0.62; HRMS: Calcd
for C103H118N3O42P: [M+Na]+ 2122.6820; Found 2122.6731.
J = 5 Hz), 73.5, 74.1, 75.8, 76.5, 79.7, 99.1, 99.6, 99.9, 102.4, 158.2,
174.3. (Note: Cq from—NBoc not detected); 1H NMR (500 MHz,
D2O): d 1.46 (s, 9H), 2.07 (s, 3H), 3.11 (m, 2H), 3.27–3.39 (m, 4H),
3.46–3.52 (m, 3H), 3.62–4.13 (m, 23 H), 4.26 (dd, 1H, J = 10.0,
10.0 Hz), 4.53 (m, 1H), 4.54 (d, 1H, J = 8.0 Hz), 4.86 (s, 1H), 5.03
(d, 1H, J = 3.0 Hz), 5.64 (s, 1H); 31P NMR (decoupled, 162 Hz,
D2O): d = -0.57; HRMS: Calcd for C37H68N3O28P: [M+Na]+
1056.3619; Found 1056.3596.
4.15. 2-Aminoethyl 2-acetamido-2-deoxy-a-D-glucopyranosyl-
(1?2)- -glycero- -manno-heptopyranosyl-(1?3)-[b-D-
L
a-
D
4.13. 2-Aminoethyl 2-acetamido-2-deoxy-
(1?2)-6-O-(2-aminoethyl-phosphono)- -glycero-
heptopyranosyl-(1?3)-[b- -glucopyranosyl-(1?4)]-
-manno-heptopyranoside 18
a
-
D
-glucopyranosyl-
-manno-
-glycero-a-
glucopyranosyl-(1?4)]-L-glycero-a-D-manno-heptopyranoside
20
L
a-
D
D
L
D
Compound 15 (10 mg, 5.4 lmol) was dissolved in MeOH (1 mL)
and NaOMe (50 ll, 1 M solution) was added. After stirring for 3 h,
Derivative 17 (11 mg, 4.7
(1:1, 2 mL) followed by addition of 0.1 M HCl (104
l
mol) was dissolved in MeOH/EtOAc
the solution was neutralized with Dowex-H+ ion exchange resins,
filtered and evaporated. The resulting compound was dissolved
l
L, 10.0 mol)
l
and Pd/C (10%, cat.). The mixture was stirred under H2 pressure
in MeOH (2 mL) and 0.1 M HCl (107 ll, 11 lmol) and Pd/C (10%
(110 psi) for 14 h, then filtered through Celite and concentrated.
The residue was dissolved in MeOH (1 mL) and NaOMe (50 ll,
cat.) were added. The mixture was stirred under H2 pressure
(110 psi) for 18 h, then filtered through Celite and concentrated.
Reversed phase chromatography (H2O)of the residue and freeze-
1 M solution) was added. After stirring for 7 h, the temperature
was increased to 40 °C and the stirring was continued for an addi-
tional 45 min. Dowex-H+ ion-exchange resins were added (excess)
and the stirring was continued for additional 15 min at 40 °C. The
mixture was then filtered and concentrated. Reversed phase chro-
matography (H2O) of the residue and freeze-drying of the product
drying of the product gave 20 (4 mg, 4.9 l
mol, 92%); 13C NMR
(100 MHz, D2O): d = 22.0, 39.3, 54.4, 60.7, 61.5, 62.6, 62.8, 63.7,
66.5, 68.0, 69.0, 69.9, 70.1, 70.4, 70.5, 70.9, 71.0, 71.7, 72.4, 73.1,
73.7, 74.6, 76.0, 76.6, 80.1, 99.7, 100.0, 100.2, 102.6, 174.5; 1H
NMR (400 MHz, D2O): d = 2.07 (s, 3H), 3.25–3.31 (m, 2H), 3.39
(m, 1H), 3.46–3.56 (m, 3H), 3.62–3.85 (m, 10H), 3,89–4.00 (m,
6H), 4.06–4.14 (m, 5H), 4.26 (dd, 1H, J = 9.6, 9.6 Hz), 4.55 (d, 1H,
J = 8.0 Hz), 4.88 (s, 1H), 5.04 (d, 1H, J = 3.2 Hz), 5.53 (s, 1H); HRMS:
Calcd for C30H54N2O23: [M+Na]+ 833.3010; Found 833.2994.
gave 18 (3 mg, 3.2 l
mol, 68%); 13C NMR (125 MHz, D2O): d 21.9,
39.0, 40.0 (d, J = 6 Hz), 54.3, 60.3, 60.6, 60.9, 62.5 (d, J = 5 Hz),
62.4, 63.3, 67.8, 69.7, 69.8 (2C), 70.2, 70.7, 70.8 (d, J = 5 Hz), 72.2,
73.0, 73.5, 73.6 (d, J = 5 Hz) 74.2, 75.8, 76.5 (2C), 80.0, 99.2, 99.7,
99.8, 102.4, 174.3; 1H NMR (500 MHz, D2O): d 2.05 (s, 3H), 3.14
(m, 1H), 3.26–3.30 (m, 5H), 3.35–3.38 (m, 2H), 3.45–3.53 (m,
4H), 3.61–4.01 (m, 15H), 4.07–4.17 (m, 6H), 4.26 (dd, 1H, J = 10.0,
10.0 Hz), 4.54 (d, 1H, J = 8.0 Hz), 4.57 (m, 1H), 4.87 (s, 1H), 5.03
(d, 1H, J = 3.5 Hz), 5.62 (s, 1H); 31P NMR (decoupled, 162 Hz,
Acknowledgements
We thank the Swedish Research Council and Science Founda-
tion Ireland, for financial support.
D2O):
d
ꢀ0.98; HRMS: Calcd for C32H60N3O26P: [M
+
H]+
934.3286; Found 934.3285.
References
4.14. 2-Aminoethyl 2-acetamido-2-deoxy-a-D-glucopyranosyl-
(1?2)-6-O-[2-(tert-butyloxycarbonylaminoethyl)-phosphono]-
1. See for example, Canada Communicable Disease Report, Vol. 33. ACS-3 (http://
www.phac-aspc.gc.ca/publicat/ccdr-rmtc/07vol33/acs-03/index-eng.php) and
references cited therein.
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3. Jennings, H. J.; Lugowski, C.; Ashton, F. E. Infect. Immun. 1984, 43, 407–412.
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6. Mannerstedt, K.; Segerstedt, E.; Olsson, J. D. M.; Oscarson, S. Org. Biomol. Chem.
2008, 6, 1087–1091.
7. Grundler, G.; Schmidt, R. R. Liebigs Ann. Chem. 1984, 1826–1847.
8. Paulsen, H.; Heitmann, A. C. Liebigs Ann. Chem. 1989, 655–663.
9. Tatai, J.; Fugedi, P. Org. Lett. 2007, 9, 4647–4650.
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H.; van der Marel, G. A. Org. Lett. 2003, 5, 1519–1522.
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3044–3046.
L
-glycero- -manno-heptopyranosyl-(1?3)-[b-
a
-
D
D-
glucopyranosyl-(1?4)]-L-glycero-a-D-manno-heptopyranoside
19
Compound 17 (6 mg, 2.8
lmol) was dissolved in MeOH/EtOAc
(1:1, 2 mL) followed by addition of 0.1 M HCl (57
l
L, 5.7 mol)
l
and Pd/C (10%, cat.). The mixture was stirred under H2 pressure
(110 psi) for 14 h, filtered through Celite, concentrated and purified
by FC (CH2Cl2/MeOH 4:1). The resulting amine was dissolved in
MeOH (1 mL) and NaOMe (20 ll, 1 M solution) was added. After
stirring for 10 h, the solution was cooled to 0 °C and neutralized
with Dowex-H+ ion exchange resins, filtered and concentrated. Re-
versed phase chromatography (H2O ? H2O/MeOH 1:1) of the resi-
due and freeze-drying of the product gave 19 (2 mg, 1.9 lmol,
68%); 13C NMR (125 MHz, D2O): d 21.9, 27.7 (3C), 39.4, 40.7 (d,
J = 10 Hz), 54.3, 60.2, 60.7, 61.3, 62.5, 64.9, 65.3, 66.2, 67.8, 69.8,
69.8, 69.9, 70.2, 70.7, 70.8, 71.3 (d, J = 5 Hz), 72.0, 73.0, 73.4 (d,
15. Olsson, J. D. M.; Lahmann, M.; Oscarson, S. J. Org. Chem. 2008, 73, 7181–7188.