Synthesis of the repeating unit of the lipoteichoic acid of Streptococcus pneumoniae

Article abstract of DOI:10.1016/j.tet.2011.11.088

The lipoteichoic acid repeating unit of Streptococcus pneumoniae is a complex pseudopentasaccharide (3). It consists of one ribitol-phosphate, one 2-acetamino-4-amino-2,4,6-trideoxy-galactose, one glucose and two galactosamine residues each differently li

Full text of DOI:10.1016/j.tet.2011.11.088

Tetrahedron 68 (2012) 1052e1061
Contents lists available at SciVerse ScienceDirect
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Synthesis of the repeating unit of the lipoteichoic acid of
Streptococcus pneumoniae
,
d
,
a
b
b
Christian Marcus Pedersen a , Ignacio Figueroa-Perez , Artur J. Ulmer , Ulrich Zahringer ,
*
€
,c,
Richard R. Schmidt a
*
a
€
Universitat Konstanz, Fachbereich Chemie, Fach 725, D-78457 Konstanz, Germany
b
€
Forschungszentrum Borstel, Leibniz-Zentrum fur Medizin und Biowissenschaften, Parkallee 1-40, D-23845 Borstel, Germany
c Chemistry DepartmentdFaculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia
d Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 August 2011
Received in revised form 7 November 2011
Accepted 28 November 2011
Available online 3 December 2011
The lipoteichoic acid repeating unit of Streptococcus pneumoniae is a complex pseudopentasaccharide (3).
It consists of one ribitol-phosphate, one 2-acetamino-4-amino-2,4,6-trideoxy-galactose, one glucose and
two galactosamine residues each differently linked, but both carrying one phosphocholine substituent, at
position 6. Suitable building blocks (6e10) for efficient and diastereocontrolled ligations were designed,
thus providing, after complete deprotection, the target molecule in high purity. Biological tests revealed
that repeating unit 3, lacking the lipid moiety, did not stimulate a pro-inflammatory response in human
monocytes (hMNCs).
Keywords:
Gram-positive bacteria
Glycosylation
Cytokines
Ó 2011 Elsevier Ltd. All rights reserved.
Lipoteichoic acid
Carbohydrates
1. Introduction
the so called ‘repeating unit’, which is generally a negatively
charged, hydrophilic glycerophosphate or ribitolphosphate residue,
During the onset of bacterial infections, recognition of microbial
cell wall constituents occurs via pattern recognition receptors
(PRRs) of the innate immune system. The recognition of these
molecules of microbial origin, the so called pathogen-associated
molecular patterns (PAMPs), triggers signalling pathways that ac-
tivate transcription of pro-inflammatory cytokines, which partici-
pate in the generation of a rapid but nevertheless specific immune
response. The most important conserved PAMPs in Gram-negative
bacteria are the lipopolysaccharides (LPS, endotoxin). They are
found in the outer leaflet of the outer membrane in the Gram-
negative bacterial cell wall. Their potency to activate pro-
inflammatory reactions in cells of the myeloid lineage has been
known for a long time as it is extremely high.1,2
respectively.3,4
Streptococcus pneumoniae, one of the most common Gram-
positive pathogens also causes severe infections like otidis me-
dia, sinusitis and others.5e7 When reaching the lower respiratory
tract or bloodstream, S. pneumoniae infections may result even
in more life-threatening diseases like pneumonia, bacteraemia
and meningitis.5 The cell wall of S. pneumoniae consists of sev-
eral layers of peptidoglycan covalently linked to teichoic acid,
and of lipoteichoic acid, that is anchored in the cell mem-
brane.3,4,8 Structural analysis of pneumococcal LTA of the R6
strain (Scheme 1, 1) revealed that it contains phosphodiester
interlinked pseudopentasaccharide repeating units each carrying
two phosphocholine residues (3) and a glycolipid core structure
2 comprising a trisaccharide linked to diacylglycerol.9,10 This
structural analysis was confirmed by our recent total synthesis
of 1 with R¼H, X¼NHþ3 and n¼1;11 also details of the synthesis
of the core structure 2 were reported.12 Biological studies with 1
and 2 showed that both compounds stimulate interleukin-8 (IL-
8) release in human monocytes (hMNCs). Since this activity was
not mediated via toll-like receptor 2 (TLR2), the investigation of
the biological properties of the repeating unit 3 became of in-
terest. Hence, the overall strategy and execution of the synthesis
The corresponding immunostimulatory component of Gram-
positive bacteria was not clear for a long time. Yet, a structural
counterpart to LPS called lipoteichoic acid (LTA) was found in the
cell wall of Gram-positive bacteria. As LPS, LTA shares its amphi-
philic nature consisting of a lipid anchor, a core oligosaccharide and
* Corresponding authors. Tel.þ45 35320190 (C.M.P.); fax: þ49 7531 883135
(R.R.S.); e-mail addresses: cmp@chem.ku.dk (C.M. Pedersen), richard.schmidt@
uni-konstanz.de (R.R. Schmidt).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.11.088

C.M. Pedersen et al. / Tetrahedron 68 (2012) 1052e1061
1053
Scheme 1. Structure of the LTA of S. pneumoniae (1), the derived core structure 2 and the repeating unit 3. A retrosynthetic scheme for the synthesis of 3.
of 3 based on monosaccharide intermediates having different
anomeric configurations as well as some biological results are
reported in the present paper. Evidence is provided that, for
biological activity of LTA in hMNCs, the lipid anchor is in-
dispensable, whereas the pseudopentasaccharide 3 expresses no
such pro-inflammatory activity.
thus also cleaving all other O-benzyl protecting groups. Hence,
pseudodisaccharide 4 should be available from known ribitol de-
rivative 613 and 2-azidogalactosyl donor 7 and trisaccharide 5 from
previously prepared glycosyl donors 912 and 1014 and 4-O-un-
protected 2-azido-galactosyl thioglycoside 8 as acceptor, that is,
readily available from galactosamine (vide infra). After the assembly
of building blocks 8e10, transformation of the resulting trisaccharide
into the corresponding trichloroacetimidate based glycosyl donor 5
will be performed.14
2. Results and discussion
The retrosynthesis of pseudopentasaccharide 3 is displayed in
Scheme 1. For a convergent synthesis strategy, disconnection be-
tween sugar residues b and c was chosen leading to pseudodi-
saccharide 4 and trisaccharide donor 5. Hence, tert-butyldiphenylsilyl
(TBDPS) groups at 6-O of sugar residues b and c were introduced for
the regioselective attachment of the choline phosphate residues and
the 5-O-allyl group at ribitol residue a was chosen for an eventual
regioselective attachment of a phosphate residue as required for the
total synthesis of 1.11 The introduction of the 2-acetylamino groups in
sugar residues b, c and d is based on concomitant reduction of three
azido groups and their subsequent N-acetylation. Thereafter the
amino group in sugar residue d can be liberated by hydrogenolysis,
For the synthesis of galactose derived intermediates 7 and 8,
galactosamine was transformed into tetra-O-acetyl-2-azido de-
rivative 11 following a reported procedure (Scheme 2).15 Treat-
ment with thiophenol in the presence of boron trifluouride ether
complex afforded known phenyl thioglycoside 1216 as a 9:7
a/b-
mixture. Removal of the O-acetyl groups with sodium methoxide
in methanol and then treatment with benzaldehyde dimethyl
acetal in the presence of p-toluenesulfonic acid (p-TsOH) fur-
nished 4,6-O-benzylidene protected derivatives 13
be readily separated. Subjecting the 13 mixture to different
reaction sequences transformed it into the required donor 7 and
into the 8 mixture, which was subsequently used to prepare
a,b that could
a,b
a,b

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C.M. Pedersen et al. / Tetrahedron 68 (2012) 1052e1061
trisaccharide donor 5. 3-O-Benzylation of 13
a and 13b with
benzyl bromide and sodium hydride as base in DMF as solvent
(/14 , 14 ), followed by camphorsulfonic acid (CSA) catalyzed
cleavage of the 4,6-O-benzylidene group (/15 , 15 ), and then
regioselective 6-O-silylation with TBDPSeCl in the presence of
imidazole as base led to the desired building blocks 8 and 8 . 3-
O-Acetylation of 13 and 13 (/16 , 16 ), then reductive open-
ing of the 4,6-O-benzylidene group with excess borane$THF
complex in the presence of equiv of dibutylboron tri-
fluoromethanesulfonate (Bu2BOTf)17 furnished 4-O-benzyl pro-
tected derivatives 17 and 17 . 6-O-Silylation with TBDPSeCl and
imidazole (/18 , 18 ) and then treatment with N-bromo-succi-
a
b
a
b
a
b
a
b
a
b
1
a
b
a
b
nimide (NBS) and thereafter aqueous sodium bicarbonate led to
the 1-O-unprotected 2-azido-galactopyranose derivative 19 that
gave with trichloroacetonitrile in the presence of DBU as base the
desired glycosyl donor 7.
Scheme 3. Reagents and conditions: (a) TMSOTf (0.1 equiv), CH2Cl2 (86%), (b) NaOMe,
MeOH, then H2NeCH2eCH2eNH2, NaOMe, BuOH, then ZeCl, NaHCO3 (92%); (c)
TMSOTf (0.15 equiv), MeCN, ꢀ40 ꢁC (94%), (d) NBS, Me2CO, H2O, ꢀ15 ꢁC (91%); (e)
CCl3CN, DBU, CH2Cl2 (71%, two steps).
The pseudodisaccharide 4 was readily obtained from glycosyl
donor 7 and 1-O-unprotected ribitol derivative 6 (Scheme 4).13
Activation of 7 with catalytic TMSOTf as catalyst and employing
the nitrile effect19 afforded the desired
b(1-1)-linkage furnishing
glycoside 24 that on treatment with sodium methanolate in
methanol led to the desired acceptor 4.
Scheme 2. Reagents and conditions: (a) PhSH, BF3$OEt2, CH2Cl2 (84%); (b) NaOMe,
MeOH; PhCH(OMe)2, p-TsOH, DMF, 40 ꢁC (93%); (c) BnBr, NaH, DMF (88%); (d) CSA,
MeOH, CH2Cl2 (77%); (e) TBDPSeCl, imidazole, DMF (94%); (f) Ac2O, Pyr (97%); (g)
H3B$THF, Bu2BOTf, CH2Cl2 (74%); (h) TBDPSeCl, imidazole, DMF (92%); (i) NBS, Me2CO,
then aq NaHCO3 (86%); (j) CCl3CN, DBU, CH2Cl2 (91%).
For the construction of the trisaccharide donor 5, acceptors 8
a
and 8
b were glycosylated with donor 9 that was obtained from
glucosamine,12 with trimethylsilyl trifluoromethanosulfonate
(TMSOTf) as catalyst in dichloromethane as solvent at room
Scheme 4. Reagents and conditions: (a) TMSOTf (0.1 equiv), MeCN, ꢀ40 ꢁC (88%); (b)
temperature, thus affording due to the anomeric effect,
a(1-4)-
NaOMe, MeOH (quantitative).
linked disaccharides 20 and 20 , respectively (Scheme 3).
a
b
Cleavage of the 3d-O-benzoyl group with sodium methoxide in
methanol, then removal of the N-phthaloyl (Phth) group with
ethylenediamine18 and protection of the amino group with the
benzyloxycarbonyl (Z) group by treatment with ZeCl in aqueous
Glycosylation of acceptor 4 with trisaccharide donor 5 under
standard conditions for
a-anomeric stereocontrol afforded the de-
THF in the presence of sodium bicarbonate furnished 21
a
and 21
b
.
sired (1-3)-linkage between sugar residues b and c furnishing
a
Following glycosylation with glucosyl donor 1014 and TMSOTf as
catalyst in acetonitrile as solvent at ꢀ40 ꢁC, thus employing for the
anomeric stereocontrol the nitrile effect,19 led to trisaccharides
pseudopentasaccharide 25 in high yield (Scheme 5). Trans-
formation of the three azido groups into amino groups required
some experimentation;20 finally the use of hydrogen sulfide in
aqueous pyridine afforded very good results and, after N-acetyla-
tion with acetic anhydride in pyridine, compound 26 was obtained
in good overall yield. Selective 6b,6c-O-desilylation was readily
performed with HFepyridine complex furnishing compound 28.
Ligation with choline phosphate residues using phosphite building
block 2921 was performed with tetrazole as catalyst followed by
oxidation of the phosphite intermediate with tert-butylhydroper-
oxide to the phosphate stage. Treatment with dimethylamine led to
22
a and 22b. Their treatment with NBS and then with aqueous
sodium bicarbonate solution gave 1-O-unprotected intermediate
23 that was transformed with trichloroacetonitrile and DBU as
base into the desired trichloroacetimidate trisaccharide donor 5. It
was found that under the conditions employed for the synthesis of
7, 8a, 8b and 5 from precursors 13a and 13b, respectively, the
anomeric configuration of the intermediates had practically no
influence on the transformation yields.

C.M. Pedersen et al. / Tetrahedron 68 (2012) 1052e1061
1055
Table 1
1H, 13C NMR chemical shift assignments for 3a,b
Sugar
e (
b-Glc)
d (
a
-AAT)
c
b
a
(
a
-GalNAc)
(b-GalNAc) (Ribitol)
1-H
1-H0
2-H
3-H
4-H
5-H
5-H0
6-H
6-H0
4.61 (J1,2 7.9) 4.98 (J1,2 4.1) 5.20 (J1,2 3.9) 4.67 (J1,2 8.5) 3.98
d
d
d
d
4.03
4.02
3.80
3.72
3.68
3.83
3.34 (J2,3 9.0) 4.30
4.37
4.15
3.89
4.19
3.80
d
3.50 (J3,4 9.0) 4.42 (J3,4 9.2) 3.95
3.46 (J4,5 9.3) 3.91 4.14
4.81 (J4,5 1.8) 4.00
3.48
d
d
d
3.78
3.92
1.27 (J5,6 6.5) 4.03
4.10
4.10
4.06
C-1
C-2
C-3
C-4
C-5
C-6
104.7
73.3
76.3
69.7
76.0
60.9
98.9
48.8
75.2
55.5
63.8
15.9
94.0
49.9
67.3
77.2
101.8
51.1
74.8
63.7
70.0
71.1
72.5
72.4
71.0 (JC,P 6.1) 74.0 (JC,P 8.5) 62.9
64.0 (JC,P 4.3) 65.0 (JC,P 5.5) d
Choline residues
1b-H/H0
4.35 (J1,1 11.5,
C-1b
C-1c
60.0 (JC,P 4.9)
0
J1,2 4.0)c
1c-H/H0
4.29 (J1,1 11.2,
60.0
0
J1,2 3.8)c
3.69
3.69
2b-H/H0
2c-H/H0
C-2b
C-2c
66.3 (JC,P 4.9)
66.3 (JC,P 4.9)
54.5
þN(CH3)3 3.25
þN(CH3)3
þN(CH3)3 3.25
þN(CH3)3
54.5
N-Acetyl groups
NAc (C]O)
13C: 175.3,
175.2, 175.0
13C: 22.9,
NAc (CH3) 1H: 2.15,
2.12, 2.08
22.6, 22.5
a
d
(ppm); J (Hz).
b
Homo-(1H) and 1H, 13C-heteronuclear NMR spectra (HMQC) were recorded with
a Bruker Advance DPX-360 spectrometer at 360.1 MHz (1H) and 90.6 MHz (13C),
respectively, at 300 K in D2O; chemical shifts were referenced to internal sodium 3-
Scheme 5. Reagents and conditions: (a) TMSOTf, CH2Cl2 (89%); (b) H2S, Pyr, H2O, then
Ac2O, Pyr (84%); (c) HF, Pyr (91%); (d) tetrazole, MeCN, then t-BuO2H (72%); (e)
(Ph3P)3RuCl2 (0.05 equiv), DBU, EtOH, then HCl, Me2CO (26e27 77%; 30e31 87%); (f)
H2, Pd(OH)2, MeOH (44%).
trimethylsilyl-propionate-d4 (TSP, dH, dC 0.0).
c
Assignments may have to be reversed.
release indicating that the lipophilic part of 1 and 2 is necessary for
contributing to the biological activity identified (Fig. 1). It was also
found that, like 1 and 2, 3 did not sense toll-like receptor 2 (TLR2) as
well as TLR4/MD2/CD14 (data not shown), further indicating that
the preparations were free of contaminating bacterial LPS and lip-
opeptide, respectively.
In previous work we also showed that the glycolipid core
structure 2 consisting of a trisaccharide bound to the lipid anchor
exhibits qualitatively and quantitatively the same biological pro-
file as 1 and activates the release of IL-8 in hMNCs.12 From those
data we already concluded that the lipid anchor and part of the
attached oligosaccharide mediate the biological activities of LTA.
For 1 as well as for 2 and 3, it was found that this activity neither
correlated to TLR2 nor to TLR4 indicating that other receptors of
the innate immune system, such as the lectin pathway of the
complement system might be the most likely PRR for 1 and 2.
Based on the results obtained with the pseudopentasaccharide of
the repeating unit (3), this hypothesis has now gained further
credence.
removal of the cyanoethyl groups furnishing pseudopenta-
saccharide 30 carrying the desired phosphocholine residues at 6b-
O and 6c-O. The 5a-O-allyl group was cleaved by isomerization with
catalytic tris(triphenylphosphine)ruthenium dichloride22 and DBU
as base in ethanol, followed by acid treatment (p-TsOH in methanol
or HCl in acetone) furnishing compound 31. Similar treatment of
fully protected pseudopentasaccharide 26 afforded 6-O-deallylated
compound 27, that is useful for other modifications. Compound 27
was investigated for the introduction of the choline phosphate
residue at a late stage of the attempted total synthesis of S. pneu-
moniae LTA 1.20 However, the required TBDPS cleavage in the
presence of a phosphate was not selective. Hence, an alternative
route was chosen for the reported total synthesis of 1.11 The final
deprotection of 31 relied on hydrogenolytic O-debenzylation with
Pearlman’s catalyst. The purification of the crude material was
performed on reversed-phase silica gel (SEP-Pak C18) and gel phase
chromatography (GPC) in order to remove salt contaminants, thus
affording target molecule 3 whose structure was fully confirmed by
1H, 13C NMR spectroscopy and mass data (Table 1).
3. Conclusions
2.1. Biological studies
The pseudopentasaccharide repeating unit of S. pneumoniae R6
strain could be efficiently prepared from ribose, galactosamine,
glucosamine and glucose precursors based on O-glycosyl tri-
chloroacetimidates as glycosyl donors. For the anomeric stereo-
control the anomeric effect and the nitrile effect, respectively, were
employed. Final deprotection led to target molecule 3 in high pu-
rity. This molecule, lacking a lipid moiety did not induce an immune
The induction of interleukin-8 (IL-8) by 3 was tested in human
peripheral blood cells using stimulation of isolated human mono-
nuclear cells (hMNCs) as well as whole blood assay (data not given).
Both tests revealed that, by contrast to what was found for 1 and
2,11,12 the pentasaccharide representing the de-phosphorylated
repeating unit in S. pneumoniae LTA 3 did not stimulate IL-8

1056
C.M. Pedersen et al. / Tetrahedron 68 (2012) 1052e1061
ether/EtOAc, 2:1) to give 12 (12.3 g, 84%). The physical data were in
accordance with those reported in the literature.16
4.1.2. Phenyl 2-azido-4,6-O-benzylidene-1-thio-a- and -b-D-gal-
actopyranoside (13 and 13 ). Compound 12 (12.00 g, 28.34 mmol)
a
b
was dissolved in MeOH (250 mL) and 4 mL of a 0.2 M NaOMe
(MeOH) solution was added. The reaction mixture was stirred for 1 h
and after this time neutralized with ionic exchange resin (IR120Hþ
form) and the solvent evaporated in vacuo. The intermediate product
was dried for 1 h in high vacuo and then redissolved in DMF
(100 mL). Benzaldehyde dimethyl acetal (6.4 mL, 42.52 mmol) was
added to the solution followed by addition of p-TsOH (0. 488 g,
2.83 mmol). The reaction mixture was stirred overnight at 40 ꢁC;
after this time the mixture was poured into water and extracted with
EtOAc twice. The organic extracts were combined, washed with
water and brine, dried with sodium sulfate and the solvent was
evaporated in vacuo. The product was purified by flash chromatog-
raphy (petroleum ether/EtOAc, 3:1/1:1) and the anomeric mixture
separated to give 13a and 13b (9:7) (10.12 g, 93%). TLC (petroleum
ether/EtOAc, 2:1) Rf¼0.64
a-isomer, 0.15 b-isomer. The physical data
were in agreement with those reported in the literature.23
4.1.3. Phenyl 2-azido-3-O-benzyl-4,6-O-benzylidene-1-thio-a-D-gal-
actopyranoside (14a). To a solution of 13a (4.62 g,12.0 mmol) in DMF
Fig. 1. Induction of IL-8 release in human MNC by synthetic compound 3. As a control,
the cells were stimulated also with a native preparation of LTA of a lgt-mutant of
Staphylococcus aureus, LPS derived from Salmonella friedenau, and synthetic Pam3C-SK4.
After incubation for 16 h the release of IL-8 into the culture supernatant was determined
by ELISA. Each result represents the meanꢃSD of duplicate cultures.
(60mL), NaH60% (0.71 g,1.5equiv) wasadded at0 ꢁC and the reaction
mixture stirred for 30 min at that temperature. Benzyl bromide
(1.72 mL, 1.2 equiv) was then added and the reaction mixture stirred
for another 3 h. Methanol was added to quench the reaction and the
clear solution was poured in water and extracted twice with EtOAc.
The organic phase was washed with water, dried over MgSO4 and the
solvent evaporated in vacuo. Flash chromatography (petroleum
response via the innate immune system. Hence, further studies are
required to elucidate the influence of the LTA repeating unit on the
host’s immune defence system.
ether/EtOAc, 13:1) yielded 14a (5.0 g, 88%) as a colourless syrup. TLC
(petroleum ether/EtOAc, 8:1) Rf¼0.45. nmax(ATR) 2107 cmꢀ1; 1H NMR
4. Experimental section
4.1. General
(250 MHz, CDCl3)
d
7.60e7.20 (m, 15H, Ph), 5.80 (d, 1H, J1,2¼5.3 Hz),
5.50 (s, 1H, CHPh), 4.80 (d, 2H, CH2Ph), 4.50 (dd, 1H, J2,1¼5.3 Hz,
J2,3¼10.6 Hz), 4.20 (m, 4H, 4-H, 5-H, 6-H), 3.90 (dd, 1H, J3,2¼10.6 Hz,
J3,4¼3.4 Hz, 3-H).13C NMR (125MHz, CDCl3)
d 137.8, 137.7, 134.0, 131.3,
Solvents were dried according to standard procedures. NMR
spectroscopic measurements were performed at 22 ꢁC with Bruker
DRX600, Bruker Avance 600 cryo, Bruker 400 Avance, Varian Mer-
cury 300 and Bruker AC250 instruments. TMS of the resonances of
129.3, 129.3, 128.7, 128.4, 128.2, 128.0, 127.5, 126.4, 101.2, 87.8, 76.6,
73.1, 71.6, 69.6, 64.0, 59.5. MALDI-MS (positive mode, matrix DHB,
THF): [MþNa]þ m/z 498.2, found 498.1. C26H25N3O4S (475.56), calcd:
C 65.67, H 5.30, N 8.84, found: C 65.81, H 5.18, N 8.72.
the deuterated solvents were used as an internal standard. CDCl3 (
d
7.24 ppm) was used as an external standard; 85% of phosphoric acid
was used as an external standard for 31P spectra. IR was recorded on
a Bruker ALPHA instrument equipped with an ATR single reflection
diamond. MALDI mass spectrawere recorded with a Kratos Kompact
Maldi II spectrometer, 2,5-dihydroxybenzoic acid (DHB) or p-
nitroaniline and NaI were used as matrices for positive measure-
ments, and trihydroxyacetophenone (THAP) was used as matrix for
negative mode measurements. Optical rotation was recorded with
a PerkineElmer polarimeter 241/MS in a 1-dm cell at 22 ꢁC. Thin
layer chromatography (TLC) was performed on E. Merck Silica Gel 60
F254 plastic plates. The compounds were visualized by a treatment
with a solution of (NH4)6Mo7O24$4H2O (20 g) and Ce(SO4)2 (0.4 g) in
10% H2SO4 (400 mL). Flash chromatography was performed on J. T.
Baker Silica Gel 60 (0.040e0.063 mm) at a pressure of 0.3 bar.
4.1.4. Phenyl
anoside (15
2-azido-3-O-benzyl-2-deoxy-1-thio-
a-D-galactopyr-
a
). To a solution of 14 (4.4 g, 9.25 mmol) in CH2Cl2/
a
MeOH (1:1, 80 mL), CSA (0.43 g, 0.2 equiv) was added and the re-
action mixture stirred for 24 h. The mixture was diluted with
CH2Cl2 and washed with NaHCO3 saturated solution, the organic
phase was dried over sodium sulfate and the solvent evaporated in
vacuo. Flash chromatography (petroleum ether/EtOAc, 1:1) yielded
15
a (2.73 g, 77%) as a colourless syrup. TLC (petroleum ether/EtOAc,
22
1:1) Rf¼0.38. [
a
]
þ15.8 (c 1, CHCl3). 1H NMR (250 MHz, CDCl3)
D
d
7.20 (m, 10H, Ph), 5.65 (d, 1H, J1,2¼5.4 Hz, 1-H), 4.70 (2ꢂ d, 2H,
CH2Ph), 4.30 (m, 2H, 2-H, 5-H), 4.15 (m, 1H, 60-H), 3.90 (m, 1H, 6-H),
3.80 (m, 2H, 3-H, 4-H). MALDI-MS (positive mode, matrix DHB,
THF): [MþNa]þ m/z 410.1, found 410.2. C19H21N3O4S (387.45), calcd:
C 58.90, H 5.46, N 10.85, found: C 59.12, H 5.55, N 10.73.
4.1.1. Phenyl
3,4,6-tri-O-acetyl-2-azido-2-deoxy-1-thio-
a,
b-
D-gal-
4.1.5. Phenyl 2-azido-3-O-benzyl-6-O-tert-butyl-diphenylsilyl-2-
actopyranoside (12). To a solution of compound 1115 (12.91 g,
34.6 mmol) in CH2Cl2 (100 mL), thiophenol (4.3 mL; 41.5 mmol,
1.2 equiv) was added and the mixture cooled with ice bath, then
BF3$OEt2 (1.5 equiv) was added dropwise and the reaction mixture
stirred for 3 days at room temperature. The reaction mixture was
neutralized with saturated sodium bicarbonate solution and washed
withwater, dried withsodiumsulfateand thesolvent wasremovedin
vacuo. The product was purified by flash chromatography (petroleum
deoxy-1-thio-
a- and -b-D-galactopyranoside (8a and 8b). To a solu-
tion of 15 (2.23 g, 5.76 mmol) in DMF (20 mL), imidazole (0.705 g,
a
1.8 equiv) and TBDPSeCl (1.8 mL,1.2 equiv) were added. The reaction
mixturewas stirredfor 2 h and thenpouredin towaterand extracted
twice with EtOAc. The organic phase was washed with water, dried
over sodium sulfate and the solvent was removed in vacuo. Flash
chromatography (petroleum ether/EtOAc, 6:1) yielded 8
94%). TLC (petroleum ether/EtOAc, 5:1) Rf¼0.45. [
a
(3.4 g,
22
a]
þ12.3 (c 1,
D

C.M. Pedersen et al. / Tetrahedron 68 (2012) 1052e1061
1057
CHCl3). nmax(ATR) 3474(br), 2109 cmꢀ1
;
1H NMR (250 MHz, CDCl3)
1.2 equiv) were added. The reaction mixture was stirred for 1 h at
room temperature and then quenched with MeOH. The solvent was
evaporated in vacuo. Flash chromatography (petroleum ether/
d
7.40 (m, 20H, Ph), 5.60 (d,1H, J1,2¼5.1 Hz,1-H), 4.75 (d, 2H, CH2Ph),
4.33 (m, 2H, 2-H, 5-H), 4.20 (m,1H, 60-H), 3.90 (m, 2H, 4-H, 6-H), 3.70
(dd, 1H, J3,4¼3.0 Hz, J3,2¼10.0 Hz, 3-H), 1.05 (s, 9H, C(CH3)3).
EtOAc, 8:1) yielded 18
(petroleum ether/EtOAc, 5:1) Rf¼0.61. [
nmax(ATR) 2110 cmꢀ1 1H NMR (250 MHz, CDCl3)
d
a (4.6 g, 92%) as a colourless syrup. TLC
22
Via the same route was obtained 8
b
from 13
b
:
nmax(ATR) 3450
a
]
þ19.6 (c 1, CHCl3).
D
(br), 2109 cmꢀ1
;
1H NMR (250 MHz, CDCl3)
d
7.40 (m, 20H, Ph),
;
7.20 (m, 20H),
4.70 (s, 2H, CH2Ph), 4.35 (d, 1H, J1,2¼10.2 Hz, 1-H), 4.15 (m, 1H, 60-
H), 3.90 (m, 2H, 2-H, 5-H), 3.65 (t, 1H, 3J¼9.8 Hz, 6-H), 3.40 (m,
2H, 3-H, 4-H), 1.05 (s, 9H, C(CH3)3). MALDI-MS (positive mode,
matrix DHB, THF): [MþNa]þ m/z 632.3, found 632.2.
C35H39N3O4SSi (635.85), calcd: C 68.93, H 6.45, N 6.89, found: C
69.15, H 6.58, N 6.81.
5.45 (d, 1H, J1,2¼5.6 Hz, 1-H), 5.15 (dd, 1H, J3,2¼10.1 Hz, J3,4¼3.0 Hz,
3-H), 4.70 (2ꢂ d, 2H, CH2Ph), 4.45 (m, 2H, 4-H, 5-H), 4.25 (m, 1H, 60-
H), 3.80 (dd, 1H, J6,5¼8.1 Hz, Jgem¼10.1 Hz, 6-H), 3.60 (dd, 1H,
J2,1¼5.8 Hz, J2,3¼10.0 Hz, 2-H), 2.15 (s, 3H, CH3CO), 1.05 (s, 9H, t-Bu).
The same procedure was employed for the transformation of
17b into 18b d 7.20 (m, 20H), 4.85 (dd,
: 1H NMR (250 MHz, CDCl3)
1H, J3,2¼10.2 Hz, J3,4¼2.9 Hz, 3-H), 4.65 (2ꢂ d, 2H, CH2Ph), 4.45 (d,
1H, J1,2¼9.8 Hz), 4.10 (m, 1H, 60-H), 3.85 (m, 3H, 4-H, 5-H, 6-H), 3.60
(t, 1H, J2,1¼J2,3¼9.6 Hz, 2-H), 2.15 (s, 3H, CH3CO), 1.05 (s, 9H, t-Bu).
MALDI-MS (positive mode, matrix DHB, THF): [MþNa]þ m/z 690.2,
found 690.1. C37H41N3O5SSi (575.73), calcd: C 66.54, H 6.19, N 6.29,
found: C 66.38, H 6.27, N 6.42.
4.1.6. Phenyl 3-O-acetyl-2-azido-4,6-O-benzylidene-2-deoxy-1-thio-
a- and -b-D-galactopyranoside (16a and 16b). Compound 13a
(5.5 g, 14.3 mmol) was dissolved in pyridine/Ac2O (2:1, 150 mL). The
reaction mixture was stirred for 2 h and the solvent removed in
vacuo. The residue was dissolved in EtOAc and washed with a 1 M
HCl solution and water, dried with MgSO4 and the solvent was
removed in vacuo. Flash chromatography (petroleum ether/EtOAc,
4.1.9. 3-O-Acetyl-2-azido-4-O-benzyl-6-O-tert-butyl-diphenylsilyl-
3:1) yielded 16
ether/EtOAc, 3:1) Rf¼0.38. [
(250 MHz, CDCl3)
5.55 (s, 1H, PhCH), 5.15 (dd, 1H, J3,4¼3.4 Hz, J3,2¼11.1 Hz, 3-H), 4.55
(m, 2H, 4-H, 60-H), 4.15 (m, 3H, 2-H, 5-H, 6-H), 2.15 (s, 3H, CH3OC).
MALDI-MS (positive mode, matrix DHB, THF): [MþNa]þ m/z 450.1,
found 450.2. C21H21N3O3S (427.47), calcd: C 59.00, H 4.95, N 9.83,
found: C 58.84, H 5.02, N 9.93.
a
(5.90 g, 97%) as a colourless syrup. TLC (petroleum
2-deoxy-1-thio-
a,b-D-galactopyranose (19). A solution of 18 (a- or
22
a
]
þ22.3 (c 1, CHCl3). 1H NMR
b-anomer) (3.50 g, 5.24 mmol) in acetone (80 mL) was cooled to
D
d
7.20 (m, 10H, Ph), 5.80 (d, 1H, J1,2¼5.3 Hz, 1-H),
15 ꢁC and NBS (1.31 g, 1.4 equiv) was added and the reaction stirred
for 15 min in the dark. TLC (petroleum ether/EtOAc, 5:1) showed
that the reaction was finished. The reaction mixture was quenched
with sodium bicarbonate, diluted and extracted with ethyl acetate.
The organic phase was washed with brine and dried over MgSO4.
Flash chromatography (petroleum ether/EtOAc, 6:1) yielded 19
The same procedure was employed for the transformation of
(2.60 g, 86%) as a colourless syrup. TLC (petroleum ether/EtOAc,
22
22
13
d
b
into 16
b
: [a
]
ꢀ16.0 (c 1, CHCl3). 1H NMR (500 MHz, CDCl3)
5:1) Rf¼0.28. [
a
]
þ25.2 (c 1, CHCl3). 1H NMR (250 MHz, CDCl3)
D
D
7.66 (m, 2H, Ar), 7.36e7.16 (m, 8H, Ar), 5.42 (s, 1H, PhCH), 4.74 (dd,
d
7.70e7.20 (m, 30H,
a
/b
-Ph), 5.40 (dd, 1H, J3,2¼11.0 Hz, J3,4¼3.0 Hz,
1H, J¼3.3, 10.3 Hz, 3-H), 4.43 (d, 1H, J¼9.9 Hz, 1-H), 4.32 (dd, 1H,
J¼1.6, 12.4 Hz, 6-H), 4.27 (dd, 1H, J¼0.7, 3.3 Hz, 4-H), 3.95 (dd, 1H,
J¼1.6, 12.4 Hz, 60-H), 3.79 (dd, 1H, J¼9.9, 10.3 Hz, 2-H), 3.49 (m, 1H,
3a
-H), 5.30 (t, 1H, J1,2¼J1,OH¼3.0 Hz,
-H), 4.65 (m, 4H,
1H, J4,3¼6.0 Hz, J4,5¼7.9 Hz, 4 -H), 4.25e4.00 (m, 4H, 1
60 -H, 60 -H, 60 -H, 60
-H), 3.95e3.65 (m, 5H, 2 -H, 4 -H, 5
3.55 (dd, 1H, J2,1¼8.0 Hz, J2,3¼9.0 Hz, 2 -H), 3.30 (d, 1H, J¼6.0 Hz,
-OH), 2.05 (2s, 6H, -CH3CO), 1.05 (s,
1a
-H), 4.75 (dd, 1H,
-CH2Ph), 4.50 (dd,
-H, 5 -H,
-H),
J3,2¼10.8 Hz, J3,4¼3.1 Hz, 3
b
a/b
a
b
a
5-H), 2.03 (s, 3H, Ac). 13C NMR (125 MHz, CDCl3)
d
170.4 (C]O),
a
b
a
b
b
a
b
137.5 (Ar), 134.3 (2C, Ar), 130.1 (Ar), 129.3 (Ar), 129.1 (2C, Ar), 128.5
(Ar), 128.2 (Ar), 126.4 (2C, Ar), 101.0 (PhCH), 85.4 (C-1), 74.0 (C-3),
72.7 (C-4), 69.7 (C-5), 69.2 (C-6), 58.3 (C-2), 21.0 (Ar). HRMS:
[MþNa]þ m/z 450.1100; found 450.1024.
b
b-
OH), 2.85 (d, 1H, J¼3.1 Hz,
a
a/b
18H, t-Bu). MALDI-MS (positive mode, matrix DHB, THF): [MþNa]þ
m/z 598.2, found 598.1. C31H37N3O6Si (575.73), calcd: C 64.67, H
6.48, N 7.30, found: C 64.72, H 6.49, N 7.42.
4.1.7. Phenyl 3-O-acetyl-2-azido-4-O-benzyl-2-deoxy-1-thio-
a- and
-b
-D
-galactopyranoside (17 and 17 ). Compound 16 was dis-
a
b
a
4.1.10. O-(3-O-acetyl-2-azido-4-O-benzyl-6-O-tert-butyl-diphe-
solved in dry CH2Cl2 (60 mL), cooled to 0 ꢁC and 1 M borane tet-
rahydrofuran complex solution in THF (138 mL, 10 equiv) was
added. After 5 min dibutylboryl triflate solution (13.8 mL, 1 equiv,
1 M in THF) was added dropwise. The reaction was stirred for 1 h at
0 ꢁC and neutralized with Et3N: excess borane was quenched with
MeOH. The solvent was removed in vacuo and the residue coeva-
porated several times with MeOH. The product was purified by
nylsilyl-2-deoxy-a,b-D-galactopyranosyl) trichloroacetimidate (7). To
a solution of 19 (2.5 g, 4.34 mmol) in dry CH2Cl2 (50 mL), tri-
chloroacetonitrile (8.7 mL, 20 equiv) and DBU (0.065 mL, 0.1 equiv)
were added. The reaction mixture was stirred for 2 h and the sol-
vent evaporated in vacuo; the product was purified by fast flash
chromatography (petroleum ether/EtOAc, 5:1) yielding 7 (2.85 g,
91%) as colourless syrup. TLC (petroleum ether/EtOAc, 5:1) Rf¼0.52.
flash chromatography (petroleum ether/EtOAc, 3:1) to give 17
a
1H NMR (250 MHz, CDCl3)
b-isomer: d 8.70 (s,1H, NH), 7.40 (m, 15H,
(4.41 g, 74%) as a colourless syrup. TLC (petroleum ether/EtOAc, 3:1)
Ph), 5.65 (d, 1H, J1,2¼8.4 Hz, 1-H), 4.85 (dd, 1H, J3,2¼10.7 Hz,
J3,4¼3.0 Hz, 3-H), 4.60 (d, 2H, CH2Ph), 4.10 (m, 2H, 5-H, 60-H), 3.80
(m, 3H, 2-H, 4-H, 6-H), 2.15 (s, 3H, CH3CO),1.05 (s, 9H, t-Bu). MALDI-
MS (positive mode, matrix DHB, THF): [MþNa]þ m/z 741.2, found
741.3 and 598.0 (imidate loss). C33H37Cl3N4O6Si (720.11), calcd: C
55.04, H 4.18, N 7.78, found: C 54.89, H 4.46, N 7.83.
22
Rf¼0.22. [
a
]
þ17.3 (c 1, CHCl3). 1H NMR (250 MHz, CDCl3)
d 7.20
D
(m, 10H, Ph), 5.80 (d, 1H, J1,2¼5.3 Hz, 1-H), 4.80 (dd, 1H, J3,4¼3.0 Hz,
J3,2¼10.3 Hz, 3-H), 4.70 (d, 1H, CH2Ph), 4.50 (d, 1H, CH2Ph), 3.85 (m,
3H, 4-H, 5-H, 60-H), 3.55 (m, 2H, 2-H, 6-H), 2.15 (s, 3H, CH3OC).
MALDI-MS (positive mode, matrix DHB, THF): [MþNa]þ m/z 452.1,
found 452.2. C21H23N3O5S (429.49), calcd: C 58.73, H 5.40, N 7.98,
found: C 58.84, H 5.27, N 7.77.
4.1.11. Phenyl (2-azido-3-O-benzoyl-4-phthalimido-2,4,6-trideoxy-
-galactopyranosyl)-(1e4)-2-azido-3-O-benzyl-6-O-tert-butyl-di-
phenylsilyl-2-deoxy-1-thio- -galactopyranoside (20 ). Donor
(1.764 g, 3.11 mmol) and acceptor 8 (2.081 g, 3.33 mmol) were
a-
The same procedure was employed for the transformation of
D
16b
into 17
b
that was immediately transformed into 18
b
(see
a
-D
a
9
below).
a
coevaporated three times with toluene and dried overnight to-
ꢀ
4.1.8. Phenyl 3-O-acetyl-2-azido-4-O-benzyl-6-O-tert-butyl-diphe-
nylsilyl-2-deoxy-1-thio- and -galactopyranoside (18 and
18 ). Compound 17 (3.2 g, 7.4 mmol) was dissolved in dry CH2Cl2
(30 mL), and imidazole (0.921 g, 1.8 equiv) and TBDPSeCl (2.32 mL,
gether with 4 A MS. CH2Cl2 (30 mL) was added and the mixture
a-
-
b-
D
a
stirred for 1 h at room temperature before it was cooled to 0 ꢁC
and TMSOTf (56
reaction was quenched by the addition of Et3N, concentrated in
b
a
mL, 0.1 equiv) added by syringe. After 30 min the

1058
C.M. Pedersen et al. / Tetrahedron 68 (2012) 1052e1061
vacuo and purified by flash chromatography (petroleum ether/
EtOAc 5:1 to 2:1) to give disaccharide 20 as a white foam
galactopyranosyl)-(1e4)-2-azido-3-O-benzyl-6-O-tert-butyl-diphe-
a
nylsilyl-2-deoxy-1-thio-
a solution of acceptor 21
and donor 10 (1.25 g, 2.00 equiv) in acetonitrile (80 mL) TMSOTf
a- and -b-D-galactopyranoside (22b). To
b
containing inseparable impurities of amide and hydrolyzed do-
nor, which was successfully removed in the following step.
(0.850 g, 0.91 mmol) cooled to ꢀ40 ꢁC
nmax(ATR) 2107, 1719 cmꢀ1
;
1H NMR (400 MHz, CDCl3)
(16.5 mL, 0.1 mmol) was added dropwise. The reaction mixture was
d
7.80e7.00 (m, 29H, Ar), 5.75 (dd, 1H, J¼6.6, 11.2 Hz, 3-H), 5.50
stirred for 1 h at ꢀ40 ꢁC and Et3N was added. The solvent was
(d, 1H, J¼5.5 Hz, 1c-H), 5.39 (d, 1H, J¼4.0 Hz, 1d-H), 5.01 (dd, 1H,
J¼3.8, 6.7 Hz, 4d-H), 4.84e4.75 (m, 3H, 5d-H, Bn), 4.71 (dd, 1H,
J¼4.0, 11.3 Hz, 2d-H), 4.37 (d, 1H, J¼2.6 Hz, 4c-H), 4.33 (dd, 1H,
J¼5.5, 10.7 Hz, 2c-H), 4.14 (m, 2H, 6c-H, 5c-H), 3.62 (dd, 1H, J¼2.6,
10.7 Hz, 3c-H), 3.58 (m, 1H, 6c-H), 1.03 (s, 9H, t-Bu), 0.74 (d, 3H,
evaporated in vacuo. Flash chromatography (petroleum ether/
EtOAc 5:1) yielded 22
(petroleum ether/EtOAc 5:1) Rf¼0.24. [
NMR (600 MHz, CDCl3)
b (1.07 g, 81%) as a pale yellow syrup. TLC
22
a]
þ15.8 (c 1, CHCl3); 1H
D
d
7.70e7.10 (m, 45H, Ph), 5.12 (m, 2H, 1d-H,
CH2(Z)), 5.03 (m, 2H, CH2Ph, CH2(Z)), 4.92 (m, 2H, CH2Ph, NH), 4.82
(m, 3H, CH2Ph), 4.72 (m, 4H,1e-H, CH2Ph), 4.60 (m, 2H, CH2Ph), 4.38
(m, 1H, 5d-H), 4.25 (m, 3H, 1c-H, 4c-H, 4e-H), 4.10 (m, 2H, 6c-H, 3d-
H), 3.89 (m, 1H, 6e-H), 3.80 (m, 2H), 3.70 (m, 3H, 2c-H, 3d-H, 4d-H),
3.55 (m, 2H, 2e-H, 5e-H), 3.20 (m, 3H, 3c-H, 5c-H, 2d-H), 1.1 (m, 9H,
J¼6.4 Hz, 6d-H). 13C NMR (101 MHz, CDCl3)
d 168.8, 165.1,
137.5e123.5 (24C, Ar), 99.5, 87.8, 77.4, 73.3, 72.5, 71.5, 69.3, 64.2,
60.8, 60.6, 59.6, 52.4, 27.1, 19.4, 16.4. HRMS [MþNa]þ m/z
1052.3443, found 1052.3405.
C(CH3)3), 1.00 (d, 3H, 6d-H). 13C NMR (150.9 MHz, CDCl3)
d 157.00
4.1.12. Phenyl (2-azido-4-benzyloxycarbonylamino-2,4,6-trideoxy-
-galactopyranosyl)-(1e4)-2-azido-3-O-benzyl-6-O-tert-butyl-di-
phenylsilyl-2-deoxy-1-thio- - and - -galactopyranoside (21 and
21 ). Disaccharide 20 was dissolved in t-BuOH (25 mL) and 5
a
-
(CH2CO), 139.66e128.44 (Ph), 103.7 (C-1e), 98.5 (C-1d), 86.8 (C-1c),
84.7 (C-3e), 82.3 (C-5e), 79.6 (C-5c), 78.3 (C-3c), 77.8 (C-4d), 75.0
(2C, CH2Ph, C-2e), 74.7 (2C, CH2Ph), 74.3 (C-3d), 73.0 (CH2Ph), 72.5
(CH2Ph), 71.4 (C-4c), 68.7 (C-6e), 66.5 (CH2(Z)), 65.7 (C-5d), 61.6 (C-
2c), 60.9 (C-6c), 60.6 (C-2d), 55.4 (C-4d), 27.95 (2C, C-6d, CH3CO),
20.20 (C(CH3)3), 17.63 (C(CH3)3). MALDI-MS (positive mode, matrix
DHB, THF): [MþNa]þ m/z 1474.6, found m/z 1474.6. C83H89N7O13SSi
(1452.78): C 68.62, H 6.17, N 6.75, found C 68.49, H 5.99, N 7.01.
The same procedure was employed for the transformation of 10
D
a
b-
D
a
b
a
drops of MeONa solution was added to remove the benzoyl pro-
tecting group. After 2 h at room temperature ethylenediamine
(8.5 mL) was added and the reaction was refluxed for 1 h followed
by concentration on silica gel, filtration through silica gel and
concentration of the fractions containing the intermediate amine.
The amine was dissolved in a THF/water mixture (32 mL, 4:1)
containing NaHCO3 (3 equiv). ZeCl (0.27 mL, 1.5 equiv) was added
and the reaction followed by TLC (EtOAc); when all starting ma-
terial was consumed the reaction was diluted by EtOAc, washed
with HCl (1 M), brine, dried (MgSO4) and concentrated in vacuo.
Flash chromatography (petroleum ether/EtOAc 1:7 to 1:4) gave
and 21
a
into 22
a
: TLC (petroleum ether/EtOAc, 5:1) Rf¼0.45.
nmax(ATR) 2108, 1729 cmꢀ1; 1H NMR (600 MHz, CDCl3)
d
7.70e7.10
(m, 45H, Ph), 5.50 (br d, 1H, J¼5.2 Hz, 1c-H), 5.25 (d, 1H, J¼4.0 Hz,
1d-H), 5.13 (br d, 1H, J¼12.3 Hz, Z), 5.01 (br d, 1H, J¼12.3 Hz, Z),
4.96e4.45 (m, 15H, Bn, 1e-H, 5d-H, NH), 4.42 (br s, 1H, 4c-H), 4.32
(m, 2H, 3d-H, 4d-H), 4.28 (dd, 1H, J¼5.2, 10.6 Hz, 2c-H), 4.14 (m, 1H,
5c-H), 4.07 (m, 1H, 6c-H), 3.83e3.47 (m, 8H, 3c-H, 60c-H, 2e-H, 3e-
H, 4e-H, 5e-H, 6e-H, 60e-H), 3.11 (br d, 1H, J¼9.0 Hz, 2d-H), 1.09 (s,
9H), 0.99 (d, 3H, J¼6.4 Hz, 6d-H). 13C NMR (150.9 MHz, CDCl3)
21
21
a
(two steps, 92% overall yield) as a colourless syrup. Compound
22
a
: [
a]
þ30.8 (c 1, CHCl3); nmax(ATR) 2108, 1698 cmꢀ1
;
1H NMR
D
(600 MHz, CDCl3)
d
7.67e7.10 (m, 25H, Ar), 5.52 (d, 1H, J¼5.3 Hz,
1c-H), 5.17 (d, 1H, J¼3.8 Hz, 1d-H), 5.14 (d, 1H, J¼11.8 Hz, CH2, Z),
5.12 (d, 1H, J¼11.8 Hz, CH2, Z), 5.05 (d, 1H, J¼8 Hz, NH Z), 4.82 (s,
2H, Bn), 4.59 (m, 1H, 5d-H), 4.27 (d, 1H, J¼2.3 Hz, 4c-H), 4.28 (m,
2H, 2c-H, 3d-H), 4.14 (dd, 1H, J¼5.7, 9.7 Hz, 5c-H), 4.08 (dd, 1H,
J¼9.5, 9.9 Hz, 6c-H), 3.99 (br d, 1H, J¼8 Hz, 4d-H), 3.64 (dd, 1H,
J¼2.3, 10.6 Hz, 3c-H), 3.61 (dd, 1H, J¼5.5, 9.9 Hz, 6c-H), 3.14 (dd,
1H, J¼3.8, 10.5 Hz, 2d-H), 3.08 (s, 1H, OH), 1.07 (s, 9H, t-Bu), 0.90
d 156.5 (C]O, Z), 138.8e127.3 (Ph), 103.5 (C-1e), 98.7 (C-1d), 87.6
(C-1c), 86.0e67.0 (5ꢂ Bn, Z, C-3e, C-4e, C-6e), 82.6 (C-2c), 71.9 (C-
4c), 76.7 (C-3c), 73.2 (C-3d), 70.7 (C-5c), 65.9 (C-5d), 60.6 (C-6c),
60.5 (C-2e), 60.2 (C-2d), 55.4 (C-4d), 26.9 (C-6d),19.2 (C(CH3)3),16.6
(C(CH3)3). MALDI-MS (positive mode, matrix DHB, THF): [MþNa]þ
m/z 1474.6, found 1474.9.
(d, 3H, J¼6.4 Hz, 6d-H); 13C NMR (150.9 MHz, CDCl3)
d
¼158.5 (Z),
4.1.14. (2,3,4,6-Tetra-O-benzyl-
4-benzyloxycarbonylamino-2,4,6-trideoxy-
(1e4)-2-azido-3-O-benzyl-6-O-tert-butyl-diphenylsilyl-2-deoxy-
-galactopyranose (23). To a solution of 22 or 22 (1.07 g,
b
-
D
-glucopyranosyl)-(1e3)-(2-azido-
137.4e127.6 (30C, Ar), 98.6 (C-1d), 87.6 (C-1c), 77.2 (C-3c), 72.7
(Bn), 72.2 (C-4c), 70.9 (C-5c), 69.2 (C-3d), 67.7 (Z), 65.0 (C-5d), 61.1
(C-2d), 60.9 (C-2c), 60.6 (C-6c), 56.2 (C-4d), 27.1 (C(CH3)3), 19.4
(C(CH3)3), 16.6 (C-6d). HRMS [MþNa]þ m/z 952.3500, found
952.3522.
a-D
-galactopyranosyl)-
a,b-
D
a
b
0.72 mmol) cooled to ꢀ15 ꢁC in acetone (35 mL) NBS (0.180 g,
1.4 equiv) was added and the reaction mixture stirred for 3 h at that
temperature. The reaction was quenched with saturated NaHCO3
solution and extracted three times with CH2Cl2. The organic phase
was dried over sodium sulfate and the solvent removed in vacuo.
Flash chromatography (petroleum ether/EtOAc 3:1) yielded 23
The same procedure was employed for the transformation of
22
20
d
b
into 21
b: [
a]
ꢀ11.3 (c 1, CHCl3); 1H NMR (600 MHz, CDCl3)
D
7.70e7.30 (m, 25H, Ph), 5.16 (2 d, 2H, CH2(Z)), 5.06 (d, 1H, NH),
5.00 (d,1H, J1,2¼3.6 Hz,1d-H), 4.74 (2 d, 2H, CH2Ph), 4.43 (m,1H, 5d-
H), 4.25 (d, 1H, J1,2¼9.6 Hz, 1c-H), 4.17 (m, 2H, 4c-H, 6d-H), 4.05 (m,
1H, 3d-H), 3.89 (m, 2H, 4d-H, 6c-H), 3.65 (t, 1H, 3J¼10.2 Hz, 2c-H),
3.30 (dd, 1H, J2,1¼3.6 Hz, J2,3¼10.2 Hz, 2d-H), 2.70 (m, 1H, 5c-H),
3.23 (dd, 1H, J3,4¼2.4 Hz, J3,2¼10.2 Hz, 3c-H), 3.12 (d, 1H, OH), 1.09
(m, 9H, C(CH3)3), 0.92 (d, 3H, J¼6.4 Hz, 6d-H). 13C NMR (150.9 MHz,
(0.985 g, 91%) as a colourless wax. TLC (petroleum ether/EtOAc 3:1)
22
Rf¼0.28.
[
a
]
þ5.5 (c 1, CHCl3); nmax(ATR) 3356(br), 2109,
1720 cmꢀ1; 1DH NMR (250 MHz, CDCl3)
d 7.7e7.1 (m, 40H, Ph), 5.12
(m, 2H, CH2(Z)), 5.03 (m, 2H, CH2Ph, CH2(Z)), 4.92 (m, 2H, CH2Ph,
NH), 4.82 (m, 3H, CH2Ph), 4.72 (m, 4H, 1e-H, CH2Ph), 4.64 (m, 2H,
CH2Ph), 4.38 (m, 1H, 5d-H), 4.25 (m, 3H, 1c-H, 4c-H, 4d-H), 4.12 (m,
2H, 6c-H, 3d-H), 3.89 (m, 1H, 6f0-H), 3.81 (m, 2H, 6e-H), 3.73 (m, 3H,
2c-H, 3e-H, 4e-H), 3.55 (m, 2H, 2e-H, 5e-H), 3.22 (m, 3H, 3c-H, 5c-H,
2d-H), 1.15 (m, 9H, C(CH3)3), 1.0 (d, 3H, 6d-H). MALDI-MS (positive
mode, matrix DHB, THF): [MþNa]þ m/z 1382.6, found m/z 1382.6.
C77H85N7O14SSi (1360.62): C 67.97, H 6.30, N 7.21, found C 68.16, H
6.42, N 7.01.
CDCl3) d 159.5 (C]O X), 138.26e128.7 (Ph), 98.0 (C-1d), 85.6 (C-1c),
79.8 (C-3c), 78.2 (C-5c), 72.3 (CH2Ph), 71.6 (C-4c), 70.1 (C-3d), 67.6
(CH2(Z)), 64.7 (C-5d), 61.6 (C-2d), 61.1 (C-2c), 60.6 (C-6c) 55.9 (C-
4d), 27.9 (C-6d), 20.21 (C(CH3)3),17.5 (C(CH3)3). MALDI-MS (positive
mode, matrix DHB, THF): [MþNa]þ m/z 952.4, found 952.3.
C48H55N7O8SSi (930.15): C 63.27, H 5.96, N 10.54, found C 63.05, H
5.78, N 10.38.
4.1.13. Phenyl (2,3,4,6-tetra-O-benzyl-
b-
D-glucopyranosyl)-(1e3)-(2-
4.1.15. O-[(2,3,4,6-Tetra-O-benzyl-
b-D-glucopyranosyl)-(1e3)-(2-
azido-4-benzyloxycarbonylamino-2, 4, 6-trideoxy-
a
-
D
-
azido-4-benzyloxycarbonylamino-2, 4, 6-trideoxy-a-D -

C.M. Pedersen et al. / Tetrahedron 68 (2012) 1052e1061
1059
galactopyranosyl)-(1e4)-2-azido-3-O-benzyl-6-O-tert-butyl-diphe-
nylsilyl-2-deoxy- -galactopyranosyl] trichloroacetimidate (5). To
a solution of23(0.97 g, 0.79mmol) inCH2Cl2 (10 mL)CCl3CN(1.57 mL,
20 equiv) and DBU (11.7 L, 0.1 equiv) were added, the reaction
azido-4-O-benzyl-6-O-tert-butyl-diphenylsilyl-2-deoxy-
b
-
D
-gal-
a-D
actopyranosyl)- -ribitol (25). To a solution of acceptor 4 (0.315 g,
D
ꢀ
0.5 mmol) and donor 5 (0.485 g, 1 equiv) in CH2Cl2, 4 A molecular
sieves were added. After 60 min the reaction mixture was cooled to
0 ꢁC and TMSOTf (ca. 10 mg in 0.5 mL CH2Cl2) was added dropwise.
The reaction mixture was stirred for 1.5 h at 0 ꢁC and allowed to
reach 10 ꢁC before Et3N was added to neutralize. The solvent was
removed in vacuo. Flash chromatography (toluene/EtOAc 30:1)
m
mixture stirred for 1.5 h and the solvent evaporated in vacuo. Flash
chromatography (petroleum ether/EtOAc 3:1) yielded 5 (0.99 g, 91%)
as a pale yellow syrup. TLC (petroleum ether/EtOAc 5:1) Rf¼0.31. 1H
NMR (600 MHz, CDCl3) d 8.48 (s, 1H, NHeimidate), 7.7e7.1 (m, 40H,
Ph), 6.73 (s, 1H, 1c-H), 5.12 (m, 2H, 1d-H, CH2(Z)), 5.03 (m, 2H, CH2Ph,
CH2(Z)), 4.92(m, 2H, CH2Ph, NH), 4.82(m, 3H, CH2Ph), 4.72 (m, 4H,1e-
H, CH2Ph), 4.6 (m, 2H, CH2Ph), 4.38 (m, 1H, 5d-H), 4.25 (m, 2H, 4c-H,
4d-H), 4.10 (m, 2H, 6c-H, 3d-H), 3.89 (m, 1H, 6a0-H), 3.80 (m, 2H, 6e-
H), 3.70 (m, 3H, 2c-H, 3e-H, 4e-H), 3.55 (m, 2H, 2e-H, 5e-H), 3.25 (m,
3H, 3c-H, 5c-H, 2d-H), 1.10 (m, 9H, C(CH3)3), 1.00 (d, 3H, 6d-H).
MALDI-MS (positive mode, matrix DHB, THF): [MþNa]þ m/z 1525.5,
found 1474.6 (OH free). C79H85Cl13N8O14Si (1505.01): C 63.05, H 5.69,
N 7.45, found C 63.21, H 5.88, N 7.55.
yielded 25 (0.664 g, 89%) as a colourless amorphous solid. TLC
22
(toluene/EtOAc 20:1) Rf¼0.42. [
a]
e19.2 (c 1, CHCl3); nmax(ATR)
D
2109, 1725 cmꢀ1; 1H NMR (600 MHz, CDCl3)
d 7.7e7.1 (m, 70H, Ph),
5.75 (m, 1H, CHeAll), 5.3e5.1 (m, 4H, CH2eAll, 1d-H, 1c-H), 5.1e4.6
(m, 21H, 9ꢂ CH2Ph, CH2(Z),1e-H), 4.5 (m,1H, 5d-H), 4.45 (m,1H, 4c-
H), 4.3 (m, 2H, 4d-H, 3d-H), 4.17 (m,1H,1a-H), 4.1 (d,1H, J1,2¼7.6 Hz,
1b-H), 4.05 (m, 4H, 4b-H, 1a-H, 4e-H), 4.0e3.6 (m, 18H, 3e-H, 6e-H,
2a-H, 3a-H, 4a-H, 5a-H, CH2eAll, 6b-H, 6c-H, 2b-H, 2c-H, 5c-H, 5e-
H), 3.5 (m, 3H, 2e-H, 3b-H, 3c-H), 3.3 (m, 1H, 5b-H), 3.07 (m, 1H, 2d-
H), 1.05 (m, 18H, 2ꢂ C(CH3)3), 0.94 (d, 3H, 6g). 13C NMR (150.9 MHz,
4.1.16. 5-O-Allyl-2,3,4-tri-O-benzyl-1-O-(3-O-acetyl-2-azido-4-O-
CDCl3) d 158.0 (CO(Z)), 139.5 (CHeAll), 138e128 (CePh), 117.6
benzyl-6-O-tert-butyl-diphenylsilyl-2-deoxy-
b
-
D
-galactopyranosyl)-
(CH2eAll), 103.4 (C-1e), 102.1 (C-1b), 98.5 (C-1d), 94.5 (C-1c), 84.5
(C-4e), 82.5 (C-2e), 78.6e69.0 (C-1a, C-2a, C-3a, C-4a, C-5a, C-3b, C-
4b, C-5b, C-3c, C-4c, C-5c, C-3d, C-3e, C-5e, C-6e, CH2eAll), 66.8
(CH2(Z)), 65.8 (C-5d), 62.7 (C-2b), 61.7, 60.9 (C-6b, C-6c), 60.0 (C-
2d), 59.5 (C-2c), 55.4 (C-4d), 27.9 (C(CH3)3), 20.3 (C-6d). MALDI-MS
(positive mode, matrix DHB, THF): [MþNa]þ m/z 2342.0, found
2342.0. C135H150N10O22Si (2320.86): C 69.86, H 6.51, N 6.04, found C
70.09, H 6.73, N 6.21.
D-ribitol (24). Donor 7 (0.78 g, 1.2 equiv) and acceptor 6 (0.420 g,
0.91 mmol) were dried 1 h under vacuum and dissolved in aceto-
nitrile (15 mL), the solution was cooled to ꢀ40 ꢁC and TMSOTf
(24.6 mL, 0.15 equiv) was added dropwise. The reaction mixture was
stirred at ꢀ40 ꢁC for 2 h and Et3N was added to quench the reaction.
The solvent was evaporated in vacuo. Flash chromatography (pe-
troleum ether/EtOAc 3:1) yielded 24 (0.748 g, 81%) as a pale yellow
22
syrup. TLC (petroleum ether/EtOAc 8:1) Rf¼0.2. [
a]
þ8.1 (c 1,
D
CHCl3); 1H NMR (250 MHz, CDCl3)
d
7.60e7.05 (m, 30H, Ph), 5.80
4.1.19. 5-O-Allyl-2,3,4-tri-O-benzyl-1-O-[(2,3,4,6-tetra-O-benzyl-
- g l u c o p y r a n o s y l ) - ( 1 e3 ) - ( 2 - a c e t y l a m i n o - 4 -
benzyloxycarbonylamino-2,4,6-trideoxy- -galactopyranosyl)-
(1e4)-(2-acetylamino-3-O-benzyl-6-O-tert-butyl-diphenylsilyl-2-
deoxy- -galactopyranosyl)-(1e3)-(2-acetylamino-4-O-benzyl-6-
O-tert-butyl-diphenylsilyl-2-deoxy- -galactopyranosyl)]- -ribitol
b-
(m, 1H, CHeAll), 5.15 (m, 2H, CH2eAll), 4.80e4.50 (m, 9H, 4ꢂ
CH2Ph, 3b-H), 4.25 (d, 1H, J1,2¼8.1 Hz, 1b-H), 4.10e3.45 (m, 14H, 1a-
H, 2a-H, 3a-H, 4a-H, 5a-H, 2b-H, 4b-H, 5b-H, 6b-H, CH2eAll), 2.05
(s, 3H, CH3CO), 1.05 (s, 9H, C(CH3)). 13C NMR (101 MHz, CDCl3)
D
a-D
a-D
d
138.8, 138.7, 138.6, 138.6, 135.6, 135.0, 133.2, 133.2, 129.9, 129.8,
b
-
D
D
128.2, 128.2, 128.2, 128.1, 127.9, 127.9, 127.8, 127.8, 127.8, 127.5, 127.4,
116.6, 102.1, 81.6, 78.6, 78.5, 78.3, 74.9, 74.7, 73.8, 72.4, 72.3, 72.2,
72.0, 70.3, 68.7, 66.0, 63.2, 62.2, 26.9, 19.2, 15.5. MALDI-MS (positive
mode, matrix DHB, THF): [MþNa]þ m/z 1042.5, found 1042.6.
[MþK]þ m/z 1058.5, found 1058.5. C60H69N3O10Si (1020.29): C
70.63, H 6.82, N 4.12, found C 70.88, H 7.03, N 4.29.
(26). A solution of 25 (0.205 g, 0.088 mmol) in pyridine/water (3:1,
v/v, 4 mL) was saturated with H2S and the reaction mixture stirred
for 3 days until TLC showed complete conversion of the starting
material. The solvent was evaporated in vacuo and the residue
coevaporated twice with toluene. The intermediate product was
dissolved in pyridine/Ac2O (1.5:1, v/v, 3 mL) and the reaction mix-
ture stirred overnight. The solvent was evaporated in vacuo and
4.1.17. 5-O-Allyl-2,3,4-tri-O-benzyl-1-O-(2-azido-4-O-benzyl-6-O-
flash chromatography (toluene/EtOAc, 3:1) yielded 26 (0.176 g,
22
tert-butyl-diphenylsilyl-2-deoxy-
b
-
D
-galactopyranosyl)-
D
-ribitol
84%) as a pale yellow syrup. TLC (toluene/EtOAc 3:1) Rf¼0.27. [
a]
(4). Compound 24 (0.72 g, 0.71 mmol) was dissolved in MeOH and
a freshly prepared NaOMe solution (0.2 M) was added dropwise
until pH¼9. The reaction mixture was stirred for 2 h and amberlite
IR-120 acid resin was added until neutralization. The mixture was
filtrated and the solvent removed in vacuo, whichyielded 23 (0.69 g,
100%) as a pale yellow oil. TLC (petroleum ether/EtOAc 5:1) Rf¼0.32.
e13.1 (c 1, CHCl3); nmax(ATR) 3450(br), 3275(br), 1723, 1659 cmꢀD1
;
1H NMR (600 MHz, CDCl3)
d 7.70e7.10 (m, 70H, Ph), 6.26 (m, 1H, 2d-
NH), 6.11 (m, 1H, 2c-NH), 5.90 (m, 1H, CHeAll), 5.30e4.40 (m, 29H,
CH2eAll, 1c-H, 2c-H, 1d-H, 5d-H, 1e-H, 9ꢂ CH2Ph, CH2(Z), 2b-NH,
4d-NH), 4.40e3.35 (m, 29H, 1a-H, 2a-H, 3a-H, 4a-H, 5a-H, 1b-H, 2b-
H, 3b-H, 4b-H, 6b-H, 3c-H, 4c-H, 5c-H, 6c-H, 2d-H, 3d-H, 4d-H, 2e-
H, 3e-H, 4e-H, 5e-H, 6e-H, CH2eAll), 3.30 (m, 1H, 5b-H), 1.60e1.40
(3s, 9H, 3ꢂ CH3C(O)N),1.05 (m,18H, 2ꢂ C(CH3)3), 0.94 (d, 3H, 6d-H).
[
a
]
22 þ12.1 (c 1, CHCl3); nmax(ATR) 2112(s) cmꢀ1; 1H NMR (250 MHz,
D
CDCl3)
d 7.6e7.05 (m, 30H, Ph), 5.8 (m, 1H, CHeAll), 5.15 (m, 2H,
CH2eAll), 4.8e4.5 (m, 8H, 4ꢂ CH2Ph), 4.25 (d, 1H, J1,2¼8.1 Hz,1b-H),
4.1e3.55 (m, 14H, 1a-H, 2a-H, 3a-H, 4a-H, 5a-H, 2b-H, 4b-H, 5b-H,
6b-H, CH2eAll), 3.45 (dd, 1H, J4,3¼7.8 Hz, J4,5¼5.9 Hz, 3b-H), 1.05 (s,
13C NMR (150.9 MHz, CDCl3)
d 172e170 (COeAceN2b, COeAceN2c,
COeAceN2d), 158.0 (CO(Z)), 139.5 (CHeAll), 138e128 (C-Ph), 117.6
(CH2eAll), 103.4 (C-1e), 102.1 (C-1b), 98.5 (C-1d), 94.5 (C-1c), 84.5
(C-4e), 81.9 (C-2e), 78.6e69.0 (C-1a, C-2a, C-3a, C-4a, C-5a, C-3b, C-
4b, C-5b, C-3c, C-4c, C-5c, C-3d, C-3e, C-5e, C-6e), 67.7 (CH2eAll),
66.8 (CH2(Z)), 66.6 (C-5d), 62.7, 62.6 (C-6b, C-6c), 55.2 (C-4d), 52.9
(C-2b), 50.1 (C-2c, C-2d), 27.9 (C(CH3)3), 20.3 (C-6d). MALDI-MS
(positive mode, matrix DHB, THF): [MþNa]þ m/z 2390.1, found
2390.1. C141H162N4O25Si (2368.98): C 71.49, H 6.89, N 2.37, found C
71.18, H 6.73, N 2.69.
9H, C(CH3)). 13C NMR (101 MHz, CDCl3)
d 138.8e127.4 (Ar, CHeAll),
116.7 (All), 102.3 (C-1b), 78.7, 78.5, 78.5 (C-2a, C-3a, C-4a), 77.4, 75.5
(Bn), 75.4, 74.8 (C-4b, C-5b), 73.8 (Bn), 72.4 (C-3b), 72.2 (CH2eAll,
Bn), 70.3, 69.1 (C-1a, C-5a), 65.3 (C-2b), 61.8 (C-6b), 27.0 (C(CH3)3),
19.3 (C(CH3)3). MALDI-MS (positive mode, matrix DHB, THF):
[MþNa]þ m/z 1000.5, found m/z 1000.6. C60H67N3O9Si (978.25): C
71.21, H 6.90, N 4.30, found C 70.98, H 7.03, N 4.41.
4.1.18. 5-O-Allyl-2,3,4-tri-O-benzyl-1-O-[(2,3,4,6-tetra-O-benzyl-
-glucopyranosyl)-(1e3)-(2-azido-4-benzyloxycarbonylamino-2,4,6-
trideoxy- -galactopyranosyl)-(1e4)-(2-azido-3-O-benzyl-6-O-
tert-butyl-diphenylsilyl-2-deoxy- -galactopyranosyl)-(1e3)-(2-
b
-
4.1.20. 2,3,4-Tri-O-benzyl-1-O-[(2,3,4,6-tetra-O-benzyl-
pyranosyl)-(1e3)-(2-acetylamino-4-benzyloxycarbonylamino-2,4,6-
trideoxy- -galactopyranosyl)-(1e4)-(2-acetylamino-3-O-benzyl-6-
O-tert-butyl-diphenylsilyl-2-deoxy- -galactopyranosyl)-(1e3)-(2-
b-D-gluco-
D
a
-
D
a-D
a
-
D
a-D

1060
C.M. Pedersen et al. / Tetrahedron 68 (2012) 1052e1061
acetylamino-4-O-benzyl-6-O-tert-butyl-diphenylsilyl-2-deoxy-
galactopyranosyl)]- -ribitol (27). To solution of 26 (0.29 g,
0.122 mmol) in EtOH (9 mL) DBU (2.8 L, 0.15 equiv) and (Ph3P)3RuCl2
b
-
D
-
the reaction was stirred overnight at room temperature followed by
8 h at 45 ꢁC where TLC (CHCl3/MeOH/H2O 65:35:8, Rf¼0.3) showed
full conversion of starting material. The reaction mixture was cooled
D
a
m
(0.059 g, 0.5 equiv) were added and the reaction mixture was stirred
for 20 min at 90 ꢁC. The solvent was evaporated in vacuo and the
residue dissolved in a mixture of acetone/HCl 1 N (10 mL, 9:1 v/v). The
reaction was stirred for another 15 min and Et3N was added. The
solvent was removed in vacuo. Flash chromatography (toluene/ace-
in an ice bath and t-BuOOH (128 mL, 6 equiv) was added. After 30 min
the reaction was quenched with NaHSO3 (aq, 1 M) and the reaction
filtered, diluted with water and extracted with EtOAc (three times).
The combined organic phases were washed with brine, dried
(MgSO4) and concentrated in vacuo followed by deprotection of the
cyanoethyl group in Me2NH (33% in EtOH). After 4 h the reaction was
finished (determined from MS) and concentrated in vacuo on silica
gel. Flash chromatography (CHCl3/MeOH 80:20 to CHCl3/MeOH/H2O
65:35:8) yielded 30 (186 mg, 72%) as a colourless wax and the mono-
tone, 5:1) yielded 27 (0.22 g, 81%) as a colourless syrup. TLC (toluene/
22
acetone, 1:1) Rf¼0.38. [
a
]
ꢀ19.2 (c 1, CHCl3). nmax(ATR) 3294(br),
D
1721,1660 cmꢀ1; 1H NMR (600 MHz, CDCl3)
d 7.70e7.10 (m, 70H, Ph),
6.26 (br s, 1H, 2d-NH), 6.11 (br s, 1H, 2c-NH), 5.20 (br s, 2H, 1c-H, 4a-
NH), 5.10 (d, 1H, Jgem¼12.6 Hz, CH(Z)), 5.05e4.40 (m, 24H, 9ꢂ CH2Ph,
2b-NH, 2c-H,1e-H, 5d-H,1d-H, CH(Z)), 4.40e4.20 (m, 3H, 4c-H, 2a-H,
4a-H), 4.15 (m, 2H, 1b-H, 3d-H), 4.05 (m, 1H, 2b-H), 4.00e3.35 (m,
20H, 1a-H, 2a-H, 3a-H, 4a-H, 5a-H, 3b-H, 4b-H, 6b-H, 3c-H, 5c-H, 6c-
H, 3e-H, 4e-H, 5e-H, 6e-H), 3.30 (m, 1H, 5b-H), 1.60e1.40 (3s, 9H, 3ꢂ
CH3C(O)N), 1.05 (m, 18H, 2ꢂ C(CH3)3), 0.94 (d, 3H, 6a-H). 13C NMR
phosphocholine side product (60 mg, 25%). [
a]
D
22 þ95.6 (c 1, CHCl3);
nmax(ATR) 3420(br), 3273 (br), 1703, 1659 cmꢀ1; 1H NMR (600 MHz,
CDCl3) d 8.71 (br s, 1H, NH); 7.73 (br s, 1H, NH), 7.51e7.11 (m, 51H, Ar,
NH), 5.89 (m, 1H, All), 5.29 (br s, 1H, 1c-H), 5.25 (dd, 1H, J¼1.5,
17.2 Hz, All), 5.22 (br s,1H, NH), 5.17 (d,1H, J¼10.5 Hz, All), 5.11 (d,1H,
J¼12.3 Hz, CH2(Z)), 5.02 (d, 1H, J¼12.3 Hz, CH2(Z)), 4.92e4.40 (m, 4H,
1b-H, 1d-H), 4.81e4.44 (m, 24H, 1e-H, 2c-H, 2d-H, 5d-H, 10Bn), 4.34
(m, 1H, 2b-H), 4.30e3.38 (m, 35H, 2e-H, 3d-H, 4d-H, 3-6c-H, 1-5a-H,
CH2eAll, 2ꢂ OCH2CH2N(CH3)3), 2.98 (s, 9H, OCH2CH2N(CH3)3), 2.91
(s, 9H, OCH2CH2N(CH3)3), 1.90, 1.83, 1.61 (3ꢂ s, 9H, 3ꢂ NAc), 0.96 (br
d, 3H, Jz6 Hz, 6d-H). 13C NMR (150.9 MHz, CDCl3) (from HSQC)
(150.9 MHz, CDCl3)
d 172.00e170.00 (COeAcN-2b, COeAcN-2c,
COeAcN-2d), 158.00 (CO(Z)), 138.00e128.00 (CePh), 103.4 (C-1e),
102.1 (C-1b), 98.5 (C-1d), 94.5 (C-1c), 84.5 (C-4e), 81.9 (C-2e),
78.6e69.0 (C-1a, C-2a, C-3a, C-4a, C-3b, C-4b, C-5b, C-3c, C-4c, C-5c,
C-3d, C-3e, C-5e, C-6e), 66.8 (CH2(Z)), 66.6 (C-5d), 62.7, 62.6 (C-5a, C-
6b, C-6c), 55.2 (C-4d), 52.9 (C-2b), 50.1 (C-2c, C-2d), 27.9 (C(CH3)3),
20.3 (C-6d). MALDI-MS (positive mode, matrix DHB, THF): [MþNa]þ
m/z 2350.1, found 2350.1. C138H158N4O25Si2 (2328.91), calcd:C 71.17, H
6.84, N 2.41, found: C 71.13, H 6.93, N 2.65.
d
134.5 (CHeAll), 138e128 (Ar), 116.8 (CH2eAll), 103.6 (C-1e), 100.6
(C-1b), 97.4 (C-1d), 92.1 (C-1c), 84.3 (C-3e), 81.9 (C-2e), 78.3, 78.1,
77.9, 75.8, 75.1e71.9 (Bn), 74.5, 73.4, 73.1, 72.1, 72.9, 72.0, 70.1, 70.1,
69.8, 69.0, 66.4 (CH2(Z)), 65.9, 65.8, 65.2, 64.7, 59.3, 54.1 (3C,
OCH2CH2N(CH3)3), 53.9 (3C, OCH2CH2N(CH3)3), 49.9, 48.8, 22.9
(NAc), 22.9 (NAc), 22.5 (NAc), 16.5 (C-6g). 31P NMR (242 MHz, CDCl3)
4.1.21. 5-O-Allyl-2,3,4-tri-O-benzyl-1-O-[(2,3,4,6-tetra-O-benzyl-
glucopyranosyl)-(1e3)-(2-acetylamino-4-benzyloxycarbonylamino-
2,4,6-trideoxy- -galactopyranosyl)-(1e4)-(2-acetylamino-3-O-ben-
zyl-2-deoxy- -galactopyranosyl)-(1e3)-(2-acetylamino-4-O-benzyl-
2-deoxy- -galactopyranosyl)]- -ribitol (28). Pseudopentasacchari
de 26 (177 mg, 0.75 mmol) was dissolved in pyridine (1 mL) followed
by addition of HF$pyridine (3 mmol, 100 L, 60% solution in pyridine).
b-D-
d
ꢀ1.53, ꢀ1.72. HRMS (C119H150N6O31P2) [Mþ2H]2þ m/z 1112.0000,
a-D
found 1111.9996.
a-D
b-D
D
4.1.23. 2,3,4-Tri-O-Benzyl-1-O-[(2,3,4,6-tetra-O-benzyl-b-D-gluco-
pyranosyl)-(1e3)-(2-acetylamino-4-benzyloxycarbonylamino-2,4,6-
trideoxy- -galactopyranosyl)-(1e4)-(2-acetylamino-3-O-benzyl-2-
deoxy-6-O-phosphocholine- -galactopyranosyl)-(1e3)-(2-
acetylamino-4-O-benzyl-2-deoxy-6-O-phosphocholine- -gal-
actopyranosyl)]- -ribitol (31). Fully protected pseudopenta-
saccharide 30 (100 mg, 45 mol) was dissolved in EtOH (5 mL) and
(Ph3P)3RuCl2 (22 mg, 2.25 mol, 0.5 equiv) was added together with
m
a-D
The reaction mixture was stirred overnight where TLC (petroleum
ether/EtOAc 1:1þ5% MeOH) showed full conversion. The crude re-
action mixture was concentrated in vacuo on silica gel and purified by
flash chromatography (petroleum ether/EtOAc 1:1þMeOH gradient
a-D
b-D
D
m
m
0e5) to give diol 28 (129 mg, 91%) as a colourless syrup. [
a
]
22 þ71.1 (c
D
1, CHCl3); nmax(ATR) 3275(br), 1654 cmꢀ1; 1H NMR (600 MHz, CDCl3)
DBU (1 drop) followed by heating to reflux for 30 min. The reaction
mixture was diluted with MeOH, cooled on ice bath and TsOH$H2O
(10 mg) added. After 3.5 h at room temperature no further reaction
took place and the reaction was quenched with Et3N, concentrated
on silica gel and purified by flash chromatography (CHCl3/MeOH
80:20 to CHCl3/MeOH/H2O 65:35:8) to yield 31 (58 mg, 68%) as
a colourless wax and unreacted starting material (22 mg). Yield
d
7.45e7.15 (m, 50H, Ar), 6.50e5.80 (m, 1H, AcNH), 5.93 (m, 1H, All),
5.30 (d, 1H, J¼17.3 Hz, All), 5.22 (br s, 0.5H, 1d-H), 5.20 (d, 1H,
J¼10.5 Hz, All), 5.15 (br d, 1H, J¼3.4 Hz, 1c-H), 5.10e4.30 (m, 25H, 1b-
H, 1d-H (0.5H), 2c-H, 2d-H, 5d-H, 10Bn, 2AcNH), 4.60 (m, 2c-H), 4.18
(m, 1H, 1e-H), 3.55 (m, 3c-H), 3.45 (m, 1H, 2b-H), 4.25e3.15 (m, 22H,
1-5a-H, 3-6b-H, 4-6c-H, 2-4d-H, 3-6e-H), 3.97 (m, All), 2.03,1.84,1.18,
1.69, 1.61, 1.60 (6s, 9H, NAc), 1.10 (br d, 3H, J¼6 Hz, 6d-H). 13C NMR
based on recovered starting material 87%. [
a
]
D
22 þ95.3 (c 1, CHCl3);
(150.9 MHz, CDCl3)
d
171.5 (1C, 2NAc), 157.0 (Z), 138.7e126.8 (40C,
nmax(ATR) 3450(br), 3275(br),1658 cmꢀ1; 1H NMR (600 MHz, CDCl3)
Ar), 117.1 (All), 103.8 (C-1e), 102.5 (low intensity C-1d), 101.3 (C-1b),
96.6 (broad, C-1d), 94.3 (C-1c), 84.7, 82.2, 78.5 (2C), 77.9, 75.6, 75.1,
75.0, 74.9, 74.4, 74.0, 73.8, 73.3, 72.5, 72.3 (2C), 72.0, 71.5, 71.1, 70.7,
70.1, 69.0, 68.1, 66.4 (2C), 61.6, 60.5, 52.0, 49.9, 49.0, 23.4, 22.9, 22.8,
d 8.86 (br s, 0.6H, NH), 7.77 (br s, 0.55H, NH), 7.42e7.02 (m, 51H, Ph,
NH), 5.21 (br s, 1.5H, 1c-H, NH (Z)), 5.06 (d, 1H, J¼12.4 Hz, CH2(Z)),
4.95 (d,1H, J¼12.4 Hz, CH2(Z)), 4.80 (m,1H,1b-H), 4.76 (m,1H,1d-H),
4.66 (m, 1H, 1e-H), 4.60 (m, 1H, 2c-H); 4.52 (2b-H), 4.41 (5d-H),
4.85e4.40 (m, 18H, Bn), 4.15 (2d-H), 4.35e3.20 (m, 32H, 3-6e-H, 3-
4d-H, 3-6c-H, 3-6e H,1-5a-H, 2ꢂ OCH2CH2N(CH3)3), 3.35 (m,1H, 2e-
16.6. HRMS (C109H126N4O25
)
[Mþ2Na]2þ m/z 968.9264, found
968.9259.
H), 2.89 (br
s
9H, OCH2CH2N(CH3)3), 2.86 (br s, 9H,
4.1.22. 5-O-Allyl-2,3,4-tri-O-benzyl-1-O-[(2,3,4,6-tetra-O-benzyl-
glucopyranosyl)-(1e3)-(2-acetylamino-4-benzyloxycarbonylamino-
2,4,6-trideoxy- -galactopyranosyl)-(1e4)-(2-acetylamino-3-O-ben-
zyl-2-deoxy-6-O-phosphocholine- -galactopyranosyl)-(1e3)-(2-
acetylamino-4-O-benzyl-2-deoxy-6-O-phosphocholine- -galactopyr-
anosyl)]- -ribitol (30). Diol 28 (222 mg, 117 mol) was coevaporated
with toluene and dissolved in MeCN (5 mL), containing 4 A molec-
ular sieves (300 mg powder). 2-Cyanoethoxy-diisopropylamino-2-
trimethylammonium-ethoxy-phosphine (29, 223 mg, 4 equiv) was
b
-
D
-
OCH2CH2N(CH3)3), 1.81 (s, 3H, NAc), 176 (s, 3H, NAc), 1.52 (s, 3H,
NAc), 0.88 (m, 3H, 6d-H). 13C NMR (150.9 MHz, CDCl3)
d
171.6 (C]O
a-D
NAc),171.5 (C]O NAc),171.3 (C]O NAc),156.8 (C]O Z),138.9,138.8
(3C), 138.7, 138.5, 138.3, 138.0, 136.9, 128.7e127.6 (Ar), 104.0 (C-1e),
101.0 (C-1b), 97.3 (C-1d), 92.3 (C-1c), 84.6 (C-3e), 82.1 (C-2e), 79.9,
79.2, 78.7, 78.1, 77.9, 75.9, 75.4, 75.1, 74.9, 74.4, 74.0, 73.7 (2C), 73.6,
73.4, 73.1, 72.6, 72.4, 72.1, 71.4, 71.0, 70.5, 69.2, 67.8, 66.6, 66.2, 66.2,
66.1, 64.9, 61.5, 59.4, 59.3, 55.4 (C-3-6e, C-3-5d, C-3-6c, C-3-6b, C-1-
5a, 2ꢂ CH2CH2N(CH3)3, CH2 Z, 9ꢂ Bn), 54.2 (CH2CH2N(CH3)3), 53.6
(CH2CH2N(CH3)3), 50.0 (C-2d), 49.6 (C-2c), 49.1 (C-2b), 29.8 (NAc),
a-D
b-D
D
m
ꢀ
added together with tetrazole (0.45 M in MeCN, 520 mL, 2 equiv) and

C.M. Pedersen et al. / Tetrahedron 68 (2012) 1052e1061
1061
23.2 (NAc), 22.8 (NAc), 16.8 (C-6d). HRMS (C116H146N6O31P2)
References and notes
[Mþ2Na]2þ m/z 1113.9663, found 1113.9647.
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This work was supported by the University of Konstanz, the
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tet.2011.11.088.