Synthesis of α-d-glucosyl substituted methyl glycosides of 3-deoxy-α-d-manno- and d-glycero-α-d-talo-oct-2-ulosonic acid (Kdo/Ko) corresponding to inner core fragments of Acinetobacter lipopolysaccharide

Article abstract of DOI:10.1016/j.carres.2014.03.004

The α-d-glucopyranosyl-(1→5)-substituted methyl glycosides of 3-deoxy-α-d-manno-oct-2-ulosonic acid (Kdo), 3-deoxy-α-d-lyxo-hept- 2-ulosonic acid (Kdh), and d-glycero-α-d-talo-oct-2-ulosonic acid (Ko) were prepared using orthogonally protected glycosyl acceptor derivatives via glycosylation with a torsionally disarmed 4,6-O-benzylidene protected trifluoroacetimidate glucosyl donor followed by global deprotection. The related 6-O-phosphoryl-α-d-glucopyranosyl-(1→5)-substituted Kdo and Kdh derivatives were derived from a benzylidene-protected glucosyl intermediate using phosphoramidite and phosphoryl chloride-based phosphorylation steps, respectively. The deprotected disaccharides serve as ligands to study lectin binding of Acinetobacter lipopolysaccharide core oligosaccharides.

Full text of DOI:10.1016/j.carres.2014.03.004

Accepted Manuscript
Synthesis of α-D-glucosyl substituted methyl glycosides of 3-deoxy-α-D-man-
no- and D-glycero-α-D-talo-oct-2-ulosonic acid (Kdo/Ko) corresponding to
inner core fragments of Acinetobacter lipopolysaccharide
Barbara Pokorny, Sven Müller-Loennies, Paul Kosma
PII:
S0008-6215(14)00092-5
DOI:
http://dx.doi.org/10.1016/j.carres.2014.03.004
CAR 6699
Reference:
To appear in:
Carbohydrate Research
Received Date:
Revised Date:
Accepted Date:
20 January 2014
26 February 2014
5 March 2014
Please cite this article as: Pokorny, B., Müller-Loennies, S., Kosma, P., Synthesis of α-D-glucosyl substituted methyl
glycosides of 3-deoxy-α-D-manno- and D-glycero-α-D-talo-oct-2-ulosonic acid (Kdo/Ko) corresponding to inner
core fragments of Acinetobacter lipopolysaccharide, Carbohydrate Research (2014), doi: http://dx.doi.org/10.1016/
j.carres.2014.03.004
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Synthesis of α-D-glucosyl substituted methyl glycosides of 3-deoxy-α-D-manno- and D-
glycero-α-D-talo-oct-2-ulosonic acid (Kdo/Ko) corresponding to inner core fragments of
Acinetobacter lipopolysaccharide
1
2
1
Barbara Pokorny , Sven Müller-Loennies , Paul Kosma *
1
Department of Chemistry, University of Natural Resources and Life Sciences,
Muthgasse 18, A-1190 Vienna, Austria
2
Research Center Borstel, Parkallee 22, D-23845, Germany
Abstract− The α-D-glucopyranosyl-(1→5)-substituted methyl glycosides of 3-deoxy-α-
D-manno-oct-2-ulosonic acid (Kdo), 3-deoxy-α-D-lyxo-hept-2-ulosonic acid (Kdh) and
D-glycero-α-D-talo-oct-2-ulosonic acid (Ko) were prepared using orthogonally
protected glycosyl acceptor derivatives via glycosylation with a torsionally disarmed
4
,6-O-benzylidene protected trifluoroacetimidate glucosyl donor followed by global
deprotection. The related 6-O-phosphoryl-α-D-glucopyranosyl-(1→5)-substituted Kdo
and Kdh derivatives were derived from a benzylidene-protected glucosyl intermediate
using phosphoramidite-based and phosphoryl chloride promoted phosphorylation steps,
respectively. The deprotected disaccharides serve as ligands to study lectin binding of
Acinetobacter lipopolysaccharide core oligosaccharides.
Keywords: lipopolysaccharide, Kdo, Ko, oligosaccharide synthesis, Acinetobacter
*
Corresponding author. Phone: *43-1-47654-6055. Fax: *43-1-47654-6059. E-mail address:
paul.kosma@boku.ac.at
1

1
.
Introduction
Bacteria of the genus Acinetobacter have increasingly been implicated in nosocomial
1
infections which are difficult to eradicate due to resistance against major antimicrobial drugs.
Lipopolysaccharide (LPS) - located in the outer leaflet of the bacterial cell membrane - is a
major virulence factor contributing to bacterial evasion of adaptive and innate immune
2
responses. A general but not exclusive architecture of LPS comprises a bisphosphorylated
acylated diglucosamine backbone, termed lipid A, a core region and an antigenic
polysaccharide which is the main target of specific antibodies allowing the distinction of O-
3
serotypes and which is therefore called O-polysaccharide or O-antigen. The first sugar
connecting the core region and the lipid A is - in general - 3-deoxy-D-manno-oct-2-ulosonic
acid (Kdo) but it may be partially replaced by the isosteric, acid-stable 3-hydroxy-derivative
4
-7
D-glycero-D-talo-oct-2-ulosonic acid (Ko). In LPS from Acinetobacter haemolyticus NCTC
0305 in particular, a large fraction (~80%) contains Ko instead of Kdo (~20%), providing the
1
linkage to the lipid A. In addition, the core oligosaccharide contains several α- and β-
configured glucosyl as well as two 3-deoxy-D-lyxo-hept-2-ulosaric acid (Dha) residues (Fig
1
).
Previously, a 28 kDa murine serum protein has been described which binds to the inner core
region of LPS from this Acinetobacter strain but also to oligosaccharides containing L-
8
-10
glycero-D-manno-heptosyl-Kdo units. This « serum factor » has only recently been
recognized as mannose binding lectin-A (MBL-A). Most notably this MBL binds to isolated
LPS oligosaccharides with an unusual high affinity for the individual binding site (with
1
1
ELISA IC50 values in the mid to low nanomolar range). Extending our previous studies
1
2-15
detailing the interaction of the core region with antibodies and lectins
, we have set out to
prepare a first series of fragments of the Acinetobacter haemolyticus NCTC 10305 inner core
region in order to define the binding epitope in molecular detail. Since Smith degradation of
2

the isolated LPS core resulted in ligands which were still bound by the lectin with similar
5
affinity , non-natural disaccharides containing 3-deoxy-D-lyxo-hept-2-ulosonic acid (Kdh)
have been prepared in addition. The synthetic oligosaccharides serve as ligands in
forthcoming binding and STD NMR studies with C-type lectins such as human lung
surfactant protein D and mannose-binding lectins.
Fig. 1. Structure of the deacylated inner core LPS fraction from Acinetobacter haemolyticus
6
NCTC 10305
2
. Results and discussion
2
.1. Preparation of the disaccharides α-Glc-(1→5)-α-Kdo/Kdh
1
The glycosides were designed as methyl (Me) glycosides since the single H NMR signal of
the Me group serves as a suitable reference for integration of STD effects in binding studies.
16
Thus the previously reported Me α-Kdo glycoside 2 – easily available via the scalable
3

+
reaction of the peracetate 1 with MeOH – catalyzed by Dowex (H ) ion-exchange resin – was
employed for the preparation of the Kdo glycosyl acceptor derivative. The exocyclic 7,8-O
positions of 2 were protected by a 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl (TIPDS) group in
order to utilize its arming effect and to provide orthogonality of fluoride-induced silyl
1
7
2
deprotection. Thus, by using imidazole and TIPDSCl in DMF at -40 °C the bis-silyl ether
derivative 3 was obtained in 68% yield (Scheme 1). In order to generate an option for
eventual attachment of the lateral Kdo unit (Fig. 1) and to also activate 5-OH for subsequent
glycosylation, a p-methoxybenzyl group (PMB) was installed at position 4. The diol
derivative 3 was first reacted with dibutyltin oxide to give the intermediate stannylene
derivative followed by treatment with PMB-chloride / tetrabutyl ammonium iodide / DMF in
toluene. As previously reported, the activated stannylene acetal also induced formation of the
1
8
corresponding PMB ester 5 and the 1.5-lactone 6 as by-products. Treatment of the mixture
with sodium methoxide, however, eventually afforded the methyl ester derivative 4 in 70%
yield.
Scheme 1. Reagents and conditions: (a) TIPDSCl
Bu SnO, toluene, reflux, then DMF, PMBCl, Bu NI, toluene, 60 °C; mixture of 4, 5 and 6;
then (c) 0.1 M NaOMe, MeOH, rt, 70% for 4.
Previously, an α-(1→5)-linked glucosyl residue had been coupled to Kdo using an acetylated
2
, 1H-imidazole, DMF, -40 °C, 68%; (b)
2
4
1
9
2
-O-benzyl thioglycoside donor under promotion with DMTST in 85% yield. The use of the
4

2
0
known perbenzylated N-phenyl trifluoroacetimidate donor (NPTFA) 7α/β for the TMSOTf-
catalyzed glycosylation of 4, however, resulted in decreased anomeric selectivity and a
2
1,22
temperature dependant outcome.
Glycosylation of acceptor 4 promoted by 10% TMSOTf
at -5 °C in CH Cl
2
2
afforded the α-(1→5)-linked disaccharide 8α as the major anomer (α/β
ratio 2.4:1) accompanied by formation of the alcohols 9α and 9β (resulting from cleavage of
the acid-labile PMB group). By lowering the temperature to -20 °C and reducing the amount
of promoter (5% TMSOTf) the PMB cleavage could be largely suppressed. However, under
these conditions, the undesired 8β was formed preferentially (α/β ratio 1:1.4). The change of
the solvent to CH
ratio (1.7:1), albeit in poor yield. Presumably, the formation of an intermediate α-anomeric
triflate at lower temperature leads to an increased contribution of an S 2-type glycosylation
pathway. Alternatively, the torsionally disarmed 4,6-O-benzylidene trifluoroacetimidate
2 2 2
Cl :Et O (9:1) resulted in a sluggish reaction with slightly enhanced α/β
N
2
3
2
4
25
donor 10 was prepared from its hemiacetal precursor as a separable anomeric mixture. The
glycosylation of acceptor 4 with donor 10 in CH Cl at -5 °C provided the disaccharide 11 in
0% yield as the α-anomer only, irrespective of the anomeric configuration of the donor
Scheme 2). Due to milder reaction conditions (lower temperature and promoter
2
2
8
(
concentration) PMB cleavage could be completely suppressed without affecting the
stereochemical outcome. The PMB group of the α-linked disaccharide derivative 8α was
selectively removed by treatment with trifluoracetic acid (TFA) affording 9α, followed by
cleavage of the silyl ether group in order to generate the triol derivative 12. Treatment of 9α
with tetrabutylammonium fluoride (TBAF) produced compound 12 in near quantitative yield,
which was then fully deprotected by catalytic hydrogenation followed by alkaline hydrolysis
of the methyl ester group to furnish disaccharide 13 as sodium salt in 90% yield. Global
deprotection of the benzylidene protected disaccharide 11 provided disaccharide 13 in an
excellent overall yield of 65% (based on acceptor 4). TFA-treatment of 11 simultaneously
5

Scheme 2. Reagents and conditions: (a) TMSOTf, CH
for 8α, 14% for 8β, 15% for 9α, 7% for 9β, 80% for 11; (b) 99% TFA, CH
α, 88% for 14; (c) TBAF, THF, rt, 98% for 12, 97% for 15; (d) H (1 atm), 10% Pd-C,
MeOH, then 0.01 M aq NaOH, rt, 90% for 12→13, 95% for 15→13, 79% for 16→17; (e)
NaIO -SiO , CH Cl , -10 °C, then NaBH , MeOH, 0 °C, 34%.
2
Cl
2
, molecular sieves 4Å, -5 °C, 34%
2
Cl , 0 °C, 83% for
2
9
2
4
2
2
2
4
cleaved the benzylidene and the PMB group, respectively, to give triol 14 in 88% yield,
followed by TIPDS removal and ensuing full deprotection of the resulting derivative 15 to
give disaccharide 13.
6

In addition, the side-chain shortened analog was prepared by oxidative cleavage of the
exocyclic diol of 12 with sodium metaperiodate generating the heptulosonic glycoside 16. The
intermediate aldehyde formed upon oxidation could be analysed by NMR, but proved to be
rather labile upon attempted purification on silica gel. The ensuing borohydride reduction was
accompanied by concomitant ester reduction. The reaction was therefore not allowed to run
until completion, but was stopped when ester reduction was first detected on TLC. Global
deprotection of 16 eventually afforded disaccharide 17 in 79% yield.
2
.2. Preparation of the disaccharides α-Glc6P-(1→5)-α-Kdo/Kdh
Selective cleavage of the PMB-group of the benzylidene-protected disaccharide derivative 11
via DDQ-promoted oxidation afforded the glycosyl acceptor 18 (suitable for attachment of the
lateral Kdo unit) in 83% yield (Scheme 3). The orthogonal protecting group pattern also
allows for the selective introduction of the 6-phosphoester group as well as for additional
glucosyl extension at position 4 of the glucosyl unit upon reductive opening of the
benzylidene group (cf. Fig. 1). Reductive opening of the benzylidene towards the 6-OH
derivative 19, however, met with difficulties when applying various combinations of Lewis
2 3 3 3
acids and hydride sources (CoCl /BH .THF, TMSCl/NaCNBH , TMSOTf/BH ,
Bu BOTf/BH .THF). Lack of reactivity, loss of the PMB group or cleavage of the
2
3
benzylidene group with formation of additional by-products was observed under these
conditions. A modest conversion of compound 11 into the 4-O-benzyl ether 19 could
eventually be accomplished in the presence of PhBCl
2
/Et SiH in 43% yield. Thus, it was
3
envisaged to introduce the 6-O-phosphate group at the 4,6-diol intermediate, with the
additional option to utilize the remaining 4-hydroxyl group as acceptor site for future
extension by glucosyl residues. Hence, the benzylidene group of 11 was selectively removed -
without cleavage of the PMB group - using p-toluenesulfonic acid in dry MeOH to afford the
7

Scheme 3. Reagents and conditions: (a) DDQ, CH
Et SiH, CH Cl , -70 °C, 43%; (c) pTosOH, MeOH, 40 °C, 91%; (d) (BnO)
tetrazole, CH Cl , -5 °C/0 °C, molecular sieves 4Å, then mCPBA, 67% for 21, 10% for 22,
3% for 25; (e) (PhO) P(=O)Cl, DMAP, CH Cl , molecular sieves 4Å, 0 °C, 96%; (f) Ac O,
pyr., DMAP, 0 °C, 96%; (g) TFA, CH Cl , 0 °C, 88%; (h) 1 M TBAF, THF, rt, 92%; (i) H (1
2
Cl
2
/MeOH (3:1), rt, 83%; (b) PhBCl
2
,
3
2
2
2
PN(iPr) , 1H-
2
2
2
6
2
2
2
2
2
2
2
atm), 10% Pd-C, MeOH, then 0.01 M aq NaOH, rt, 98%.
diol 20 in 91% yield. Short reaction times at 40 °C gave better and reproducible yields
compared to prolonged reaction times at ambient temperature. Phosphorylation of the diol 20
was first approached using the phosphoramidite chemistry. Thus, treatment of 20 with
dibenzyl N,N-diisopropylphosphoramidite / 1H-tetrazole in the presence of ground molecular
2 2
sieves (4Å) in CH Cl followed by oxidation with mCPBA gave the 6-O-phosphotriester
derivative 21 in 67% yield and the corresponding 4-O-regioisomer 22 (10%). The
phosphoramidite protocol also proved to be appropriate for the phosphorylation of the triol 14
and furnished the 6-O-phosphotriester derivative 25 in 63% yield. The assignment of the
1
phosphate substitution at O-6 was based on the downfield shift of the H NMR signals of the
8

1
3
31
13
H-6 protons and the C- P coupling interaction leading to splitting of C NMR signals of C-
6
and C-5, respectively. To increase the selectivity in the phosphorylation step, a more
reactive phosphoryl halide was used at low temperature. Indeed, reaction of the diol 20 with
diphenyl phosphoryl chloride at 0 °C in the presence of 4-N,N-dimethylaminopyridine
afforded the corresponding 6-O-phosphotriester derivative 23 in 96% yield.
TFA-treatment of the dibenzylphosphate derivative 21 proved to be selective for the removal
of the PMB group and gave the diol derivative 25 in 88% yield. Subsequent reaction of 25
with TBAF afforded the tetraol derivative 26 (92%), which was subjected to hydrogenation
on 10% Pd-C followed by alkaline hydrolysis to furnish the target disaccharide phosphate 27
as sodium salt in 98% yield. Deprotection of the silyl group of diphenylphosphate 23,
however, required a modified protocol, since treatment with TBAF resulted in fluoride-
2
6
induced nucleophilic displacement of the phenoxy groups on the phosphoester. The 4-OH
group of 23 was thus acetylated to give compound 24 in order to prevent intramolecular
phosphate migration or cyclization, respectively. The silyl ether groups of 24 were then
removed by the action of triethylamine trihydrogen fluoride (TREAT), which afforded 28 in
8
2% yield with only minor (~10%) phosphate cleavage (Scheme 4). Compound 28 was used
for the preparation of the side-chain-shortened analogue 29 by periodate oxidation (36%)
followed by successive hydrogenolysis on Pd/C and PtO , and saponification to afford the
2
heptulosonic glycoside 30 in 52% yield. The poor yield was due to formation of an
unidentified side product, which had to be separated on a HILIC column. Alternatively,
dibenzylphosphate 26 was subjected to the oxidative degradation protocol affording 31
(
47%), which reacted cleanly to target compound 30 upon hydrogenolysis with Pd-C and
subsequent saponification.
9

Scheme 4. Reagents and conditions: (a) TREAT, CH
C → rt, then NaBH , MeOH, 0°C, 36% for 29, 47% for 31; (c) H
MeOH, then H (1 atm), PtO , MeOH, then 0.01 M aq NaOH, rt, 52%; (d) H
Pd-C, MeOH, then 0.01 M aq NaOH, rt, 87%.
2
Cl
2
, rt, 82%; (b) NaIO
(1 atm), 10% Pd-C,
(1 atm), 10%
4 2 2 2
-SiO , CH Cl , 0
°
4
2
2
2
2
2
.3. Preparation of the disaccharide α-Glc-(1→5)-α-Ko
The previously reported intermediate 32 was employed for the preparation of a suitably
2
7
protected methyl glycoside of D-glycero-α-D-talo-oct-2-ulopyranosylonic acid (Ko). The
epimeric 3-O-acetate 32 was subjected to base-induced anomeric methylation followed by de-
O-acetylation to produce 33. Ensuing Dess-Martin periodinane oxidation and reduction with
ammonia-borane complex gave the alcohol 34 in 65% overall yield (Scheme 5). Reaction of
3
4 with benzyl bromide/NaH in DMF afforded the 3-O-benzyl derivative 35 in 94% yield.
The reaction had to be performed in high dilution at 0 °C in order to prevent formation of the
corresponding benzyl ester derivative. Next, the 4,5-O-isopropylidene ketal was selectively
cleaved by the action of p-toluenesulfonic acid in wet acetone under equilibrating conditions.
10

Scheme 5. Reagents and conditions: (a) NaH, MeI, DMF, 0°C, then NaOMe, rt, 86%; (b)
Dess-Martin reagent, CH Cl , rt, then NH .BH , MeOH, 0°C, 75%; (c) NaH, BnBr, DMF,
°C, 94%; (d) pTosOH, wet acetone, rt, 71%; (e) Bu SnO, toluene, reflux, then DMF,
NI, toluene, 60 °C, 81%; (f) TMSOTf, CH Cl , molecular sieves 4Å, -30 °C,
(1 atm), 10% Pd-C, MeOH, then 0.01 M
2
2
3
3
0
2
PMBCl, Bu
4
2
2
4
2%; (g) pTosOH, MeOH, 0 °C → rt, 70%; (h) H
2
aq NaOH, rt, 92%.
The use of a defined amount of water was critical in order to prevent additional loss of the
,8-O-acetonide.This way diol 36 was obtained in 71% yield ready for further processing into
7
suitable glycosyl acceptor derivatives. Similar to the corresponding Kdo acceptor 4, the 4-O-
PMB group was installed via the respective stannylidene acetal intermediate and ensuing
treatment with PMBCl/Bu
7 in 81% yield. Coupling of 37 with the benzylidene-protected NPTFA donor 10 promoted
by TMSOTf in CH Cl at -5 °C afforded the α-linked disaccharide 38 in 42% yield. The
4
NI/DMF in toluene affording the orthogonally protected acceptor
3
2
2
reduced yield of the coupling step was due to the concomitant cleavage of the acetonide
during the glycosylation reaction. Deprotection of 38 was achieved by treatment with p-
toluenesulfonic acid hydrate in methanol for 24 h which furnished the tetraol 39 in 70% yield.
Hydrogenation of 39 with 10% Pd-C in methanol and final purification on Bio-Gel PD10
11

1
3
afforded the glucosyl-Ko disaccharide 40 as sodium salt in 92% yield. C NMR data of the
deprotected target disaccharides 13, 17, 27, 30 and 40 were fully assigned and confirmed the
respective structures (Table 1).
2
.4. Conclusions and outlook
A benzylidene-protected glucosyl NPTFA donor proved as efficient and α-selective glycosyl
donor for the glycosylation of OH-5 of orthogonally protected Kdo and Ko glycosides and
allowed for the regioselective introduction of the 6-O-phosphoryl group. Oxidative cleavage
of the exocyclic side chain of Kdo provided the Kdh containing fragments. Global
deprotection gave the disaccharide ligands related to the inner core region of Acinetobacter
LPS which are suited to perform various binding assays to elucidate structural details of the
interaction with MBL which are serum components and important in innate first-line immune
reactions such as complement activation. The synthesis of larger fragments is currently in
progress.
3
3
. Experimental
.1. General
All purchased chemicals were used without further purification unless stated otherwise.
Solvents were dried over activated 3 Å (acetone) or 4 Å (CH Cl , DMF, pyridine, toluene)
2
2
molecular sieves. THF was distilled on 4 Å molecular sieves shortly before use. Dry MeOH
+
(
secco solv) was purchased from Merck. Cation exchange resin DOWEX 50 H was
regenerated by consecutive washing with HCl (3 M), water and dry MeOH. Aqueous
solutions of salts were saturated unless stated otherwise. Concentration of organic solutions
was performed under reduced pressure < 40 °C. Optical rotations were measured with a
2
D
0
-1
2 -1
Perkin-Elmer 243 B polarimeter. [α]
values are given in units of 10 deg cm g . Thin layer
chromatography was performed on Merck precoated plates: generally on 5 x 10 cm, layer
12

thickness 0.25 mm, silica gel 60F254; alternatively on HPTLC plates with 2.5 cm
concentration zone (Merck). Spots were detected by dipping reagent (anisaldehyde-H
For column chromatography silica gel (0.040 – 0.063 mm) was used. HP-column
2
SO ).
4
chromatography was performed on pre-packed columns (YMC-Pack SIL-06, 0.005 mm, 250
x 10 mm and 250 x 20 mm). Size exclusion chromatography was performed on Bio-Gel® P-2
Gel extra fine < 45 µm (wet) (Bio-Rad, 45 – 90 µm) or on pre-packed PD-10 columns (GE
TM
Healthcare, Sephadex G-25 M). NMR spectra were recorded with a Bruker Avance III 600
1
13
31
instrument (600.22 MHz for H, 150.93 MHz for C and 242.97 MHz for P) using standard
1
Bruker NMR software. H spectra were referenced to 7.26 (CDCl
3
), 5.32 (CD
2
Cl
2
), 3.31
(
MeOD) and 0.00 (D
2
O, external calibration to 2,2-dimethyl-2-silapentane-5-sulfonic acid)
1
3
ppm unless stated otherwise. C spectra were referenced to 77.00 (CDCl
3
), 53.84 (CD
2
Cl
2
2
),
3
1
4
9.00 (MeOD) and 67.40 (D
2
O, external calibration to 1,4-dioxane) ppm. P spectra in D
O
were referenced to external ortho-phosphoric acid (0.00 ppm). ESI-MS data were obtained on
a Waters Micromass Q-TOF Ultima Global instrument.
3
.2. Methyl [methyl 3-deoxy-7,8-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-α-D-
manno-oct-2-ulopyranosid]onate (3).
2
A cooled mixture of 1H-imidazole (0.22 g, 3.228 mmol) and TIPDSCl (0.46 mL, 1.412
mmol) in dry DMF (7.4 mL) was added dropwise to a solution of 2 (0.36 g, 1.345 mmol) in
dry DMF (8.6 mL) at -40 °C. After 2 h at -40 °C another portion of 1H-imidazole (22 mg,
0
.323 mmol) and TIPDSCl
and after 30 min excessive reagent was scavenged by addition of dry MeOH (2 mL). The
mixture was allowed to warm up to ambient temperature, solid NaHCO (0.4 g) was added
and the suspension was concentrated. The residue was partitioned between EtOAc and aq
NaHCO , the aqueous phase was extracted with EtOAc (2 x 10 mL) and the combined
2
(46 µL, 0.141 mmol) in dry DMF (0.74 mL) was slowly added
3
3
13

organic layers were dried over MgSO
4
, filtered and concentrated. The residual oil was
purified by column chromatography (toluene/EtOAc 4:1 → 1:1) affording 3 (0.46 g, 68%) as
2
0
1
a colorless amorphous solid: [α]
NMR (CDCl ): δ 4.30 – 4.26 (m, 1H, H-7), 4.18 (dd, 1H, J8a,8b 12.1, J8a,7 1.7 Hz, H-8a), 4.06
4.04 (m, 1H, H-5), 4.03 – 3.98 (m, 1H, H-4), 3.84 (dd, 1H, J8b,7 7.0 Hz, H-8b), 3.78 (s, 3H,
CO CH ), 3.50 (dd, J6,7 8.0, J6,5 1.0 Hz, H-6), 3.22 (s, 3 H, OCH ), 2.15 (dd, 1H, J3eq,3ax 13.0,
3eq,4 5.1 Hz, H-3eq), 1.86 (dd, 1H, J3ax,4 11.5 Hz, H-3ax), 1.12 – 0.93 (m, 28 H, TIPDS); C
NMR (CDCl ): δ 168.53 (s, C-1), 99.36 (s, C-2), 74.08 (d, C-7), 71.35 (d, C-6), 66.81 (t, C-
), 66.67 (d, C-5), 66.17 (d, C-4), 52.52 (q, CO CH ), 51.17 (q, OCH ), 35.10 (t, C-3), 17.40,
7.35, 17.33, 17.23, 17.21 [q, 8C, Si-CH-(CH ], 13.31, 12.77, 12.48 [d, 4C, Si-CH-(CH ];
D
+ 36.4 (c 0.85, CHCl
3
); R
f
0.38 (toluene/EtOAc 1:1); H
3
–
2
3
3
13
J
3
8
1
2
3
3
3
)
2
)
3 2
+
ESI-TOF HRMS: m/z = 531.2409; calcd for C22
H
44
O
9
Si Na : 531.2416.
2
3
.3. Methyl [methyl 3-deoxy-4-O-(4-methoxybenzyl)-7,8-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)-α-D-manno-oct-2-ulopyranosid]onate (4)
A mixture of 3 (91 mg, 0.179 mmol) and dibutyltin oxide (49 mg, 0.197 mmol) in dry toluene
4.0 mL) was heated to reflux on a Dean-Stark apparatus for 2 h. To the cooled solution dry
(
DMF (166 µL, 2.146 mmol), 4-methoxybenzyl chloride (73 µL, 0.537 mmol) and
tetrabutylammonium iodide (73 mg, 0.197 mmol) were added successively. After 16 h at 60
°
C the solution was allowed to cool to ambient temperature. The solution was diluted with
EtOAc and consecutively washed with HCl (1 M), aq NaHCO , aq Na (50 g/L) and
brine. The organic phase was dried (MgSO ), filtered and concentrated. The residual oil was
taken up in dry MeOH (4.0 mL) and treated with 0.1 M NaOMe in dry MeOH (0.179 mmol,
3
2
S
2
O
3
4
1
.8 mL) at 0 °C. After 1 h at ambient temperature the solution was made neutral by adding
+
DOWEX 50 H resin, the suspension was filtered and the filtrate was concentrated. Column
chromatography of the residue (toluene/EtOAc 19:1 → 9:1) provided 4 (79 mg, 70%) as a
14

2
0
1
colorless oil: [α]
D
+ 24.5 (c 1.07, CHCl
3
); R
f
0.66 (toluene/EtOAc 3:1); H NMR (CDCl
3
):
δ 7.27 – 7.24 (m, 2H, Ar), 6.89 – 6.86 (m, 2H, Ar), 4.56 (d, 1H, J 11.5 Hz, CHHPh), 4.50 (d,
1
8
H, J 11.4 Hz, CHHPh), 4.36 – 4.32 (m, 1H, H-7), 4.25 (dd, 1H, J8a,8b 11.9, J8a,7 1.6 Hz, H-
a), 4.14 – 4.12 (m, 1H, H-5), 3.84 (ddd, 1H, J4,3ax 11.5, J4,3eq 5.2, J4,5 2.9, H-4), 3.80 – 3.75
(
m, 7H, H-8b, PhOCH3, CO
2
CH
3
), 3.38 (dd, 1H, J6,7 8.6, J6,5 1.0 Hz, H-6), 3.20 (s, 3H,
OCH
3
), 2.21 (bs, 1H, OH), 2.18 (dd, 1H, J3eq,3ax 12.8, H-3eq), 2.01 (dd, 1H, H-3ax), 1.18 –
1
3
0
.91 (m, 28H, TIPDS); C NMR (CDCl
29.28, 113.96 (d, 4C, Ar), 99.36 (s, C-2), 72.97 (d, 2C, C-4, C-7), 71.88 (d, C-6), 69.99 (t,
Ph), 67.16 (t, C-8), 63.77 (d, C-5), 55.26 (q, PhOCH ), 52.50 (q, CO CH ), 50.99 (q,
OCH ), 32.09 (t, C-3), 17.47, 17.41, 17.37, 17.27 [q, 8C, Si-CH-(CH
2.49 [d, 4C, Si-CH-(CH
51.2991.
3
): δ 168.60 (s, C=O), 159.38, 130.03 (s, 2C, Ar),
1
CH
2
3
2
3
3
3
)
2
], 13.29, 12.82, 12.54,
+
1
6
3
)
2
]; ESI-TOF HRMS: m/z = 651.2995; calcd for C30
H
52
O
10Si
2
Na :
2
0
1
Data for 5: colorless oil; [α]
CDCl ): δ 7.32 – 7.29 (m, 2H, Ar), 7.26 – 7.22 (m, 2H, Ar), 6.89 - 6.85 (m, 4H, Ar), 5.15 (s,
H, CH Ph), 4.53 (d, 1H, J 11.6 Hz, CHHPh), 4.49 (d, 1H, J 11.4 Hz, CHHPh), 4.33 (ddd,
H, J7,6 8.6, J7,8b 7.2, J7,8a 1.5 Hz, H-7), 4.24 (dd, 1H, J8a,8b 11.8 Hz, H-8a), 4.13 - 4.11 (m,
H, H-5), 3.84 - 3.79 (m, 7H, H-4, 2 x PhOCH ), 3.76 (dd, 1H, H-8b), 3.37 (d, 1H, H-6), 3.13
), 2.21 (d, 1H, JOH,5 2.6 Hz, OH), 2.15 (dd, 1H, J3eq,3ax 12.9, J3eq,4 4.9 Hz, H-
D
3 f
+ 18.2 (c 0.72, CHCl ), R 0.76 (toluene/EtOAc 3:1); H NMR
(
3
2
1
1
2
3
(
s, 3H, OCH
3
1
3
3
1
eq), 1.97 (dd, J3ax,4 11.7 Hz, H-3ax), 1.10 - 0.90 (m, 28H, TIPDS); C NMR (CDCl
3
): δ
67.77 (s, C=O), 159.74, 159.37 (s, 2C, Ar), 130.26 (d, 2C, Ar), 130.03 (s, Ar), 129.29 (d, 2C,
Ar), 127.57 (s, Ar), 113.94 (d, 4C, Ar), 99.18 (s, C-2), 73.06 (d, 2C, C-4, C-7), 71.84 (d, C-6),
6
5
1
C
9.98 (t, CH
2
Ph), 67.10 (t, C-8), 66.98 (t, CH
), 31.99 (t, C-3), 17.47, 17.40, 17.38, 17.27 [q, 8C, Si-CH-(CH
]; ESI-TOF HRMS: m/z = 757.3414; calcd for
2
Ph), 63.75 (d, C-5), 55.26 (q, 2C, 2 x PhOCH
3
),
0.94 (q, OCH
3
3 2
)
], 13.29,
3 2
2.81, 12.54, 12.51 [d, 4C, Si-CH-(CH )
+
37
58 2
H O11Si Na : 757.3410.
15

2
0
Data for 6: colorless amorphous solid; [α]
D
- 13.8 (c 0.82, CHCl
3
); R 0.59 (toluene/EtOAc
f
1
9
1
:1); H NMR (CDCl
3
): δ 7.26 - 7.23 (m, 2H, Ar), 6.89 – 6.86 (m, 2H, Ar), 5.00 – 4.99 (m,
H, H-5), 4.54 (d, 1H, J 11.9 Hz, CHHPh), 4.49 (d, 1H, J 11.8 Hz, CHHPh), 4.16 (dd, 1H,
8a,8b 12.1, J8a,7 1.7 Hz, H-8a), 3.93 (ddd, 1H, J7,6 9.5, J7,8b 7.6 Hz, H-7), 3.89 (app. td, 1H,
4,5 2.2 Hz, H-4), 3.80 (s, 3H, PhOCH ), 3.69 (dd, 1H, H-8b), 3.58 (s, 3H,
), 3.57 (d, 1H, H-6), 2.50 (dd, 1H, J3ax,3eq 14.7 Hz, H-3ax), 2.00 - 1.97 (m, 1H, H-3eq),
J
J
4,3ax 8.8, J4,3eq
J
3
OCH
.12 – 0.91 (m, 28H, TIPDS); C NMR (CDCl
d, 2C, Ar), 128.92 (s, Ar), 114.03 (d, 2C, Ar), 94.91 (s, C-2), 73.37 (d, C-6), 72.71, 72.68 (d,
C, C-5, C-7), 71.93 (d, C-4), 70.05 (t, CH Ph), 66.64 (t, C-8), 55.25 (q, PhOCH ), 52.71 (q,
OCH ), 38.72 (t, C-3), 17.40, 17.35, 17.32, 17.27, 17.20, 17.18 [q, 8C, Si-CH-(CH ], 13.20,
2.64, 12.45, 12.39 [d, 4C, Si-CH-(CH ].
3
13
1
3
): δ 168.24 (s, C=O), 159.54 (s, Ar), 129.36
(
2
2
3
3
)
3 2
1
)
3 2
3
.4. 2,3,4,6-Tetra-O-benzyl-α-D-glucopyranosyl-(1→5)-methyl [methyl 3-deoxy-4-O-(4-
methoxybenzyl)-7,8-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-α-D-manno-oct-2-
ulopyranosid]onate (8α), 2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl-(1→5)-methyl
[
methyl 3-deoxy-4-O-(4-methoxybenzyl)-7,8-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl)-α-D-manno-oct-2-ulopyranosid]onate (8β), 2,3,4,6-tetra-O-benzyl-α-D-
glucopyranosyl-(1→5)-methyl [methyl 3-deoxy-7,8-O-(1,1,3,3-tetraisopropyldisiloxane-
1
,3-diyl)-α-D-manno-oct-2-ulopyranosid]onate (9α), and 2,3,4,6-tetra-O-benzyl-β-D-
glucopyranosyl-(1→5)-methyl [methyl 3-deoxy-7,8-O-(1,1,3,3-tetraisopropyldisiloxane-
,3-diyl)-α-D-manno-oct-2-ulopyranosid]onate (9β)
1
A solution of glycosyl acceptor 4 (50 mg, 0.080 mmol) in dry CH
Å molecular sieves (75 mg) was stirred for 20 min followed by addition of glycosyl donor 7
85 mg, 0.120 mmol) in dry CH Cl (0.6 mL) under Ar. TMSOTf (1.4 µL, 0.008 mmol) in dry
CH Cl (50 µL) was added dropwise to the cold (-5 °C) mixture and after 5 min the reaction
2 2
Cl (0.6 mL) containing 4
2
2
(
2
2
2
2
16

3 2 2
was quenched by adding a solution of Et N (23 µL, 0.160 mmol) in dry CH Cl (0.3 mL).
Filtration of the suspension over Celite®, concentration of the filtrate and purification of the
residue by HP-column chromatography (n-hexane/EtOAc 9:1 → 2:1) provided target
compound 8α (31 mg, 34%) together with 8β (13 mg, 14%) and disaccharide alcohols 9α (14
mg, 15%) and 9β (6 mg, 7%) as colorless syrups.
2
0
1
Data for 8α: [α]
δ 7.36 – 7.20 (m, 20H, Ar), 7.10 – 7.07 (m, 2H, Ar), 6.85 – 6.81 (m, 2H, Ar), 5.28 (d, 1H,
1′,2′ 3.7 Hz, H-1′), 4.94 (d, 1H, J 12.0 Hz, CHHPh), 4.86 (d, 1H, J 11.7 Hz, CHHPh), 4.83 (d,
D
+ 68.4 (c 0.53, CHCl
3
); R
f
0.62 (n-hexane/EtOAc 3:1); H NMR (CDCl
3
):
J
1
4
H, J 11.1 Hz, CHHPh), 4.75 (d, 1H, J 10.7 Hz, CHHPh), 4.68 (d, 1H, J 12.0 Hz, CHHPh),
.54 (d, 1H, J 11.5 Hz, CHHPh), 4.51 (d, 1H, J 12.3 Hz, CHHPh), 4.50 – 4.45 (m, 2H, H-7,
CHHPh), 4.41 (d, 1H, J 11.0 Hz, CHHPh), 4.27 (d, 1H, J5,4 2.2 Hz, H-5), 4.24 – 4.19 (m, 2H,
H-5′, CHHPh), 4.06 (app. t, 1H, J3′,2′ = J3′,4′ 9.5 Hz, H-3′), 4.01 (dd, 1H, J8a,8b 13.1, J8a,7 1.9
Hz, H-8a), 3.86 – 3.80 (m, 4H, H-4, CO
2
CH
.4 Hz, H-4′), 3.64 – 3.60 (m, 3H, H-2′, H-6, H-8b), 3.40 (dd, 1H, J6′a,6′b 10.7, J6′a,5′ 2.1 Hz, H-
′a), 3.29 (s, 3H, OCH ), 3.12 (dd, 1H, J6′b,5′ 1.9 Hz, H-6′b), 2.21 (dd, 1H, J3eq,3ax 12.5, J3eq,4
.4 Hz, H-3eq), 2.16 (app. t, 1H, J3ax,4 12.2 Hz, H-3ax), 1.06 – 0.87 (m, 28H, TIPDS); C
): δ 168.80 (s, C=O), 159.07, 138.86, 138.78, 138.57, 138.11, 130.52 (s, 6C,
Ar), 129.01, 128.25, 128.23, 128.21, 128.09, 127.98, 127.85, 127.82, 127.50, 127.48, 127.36,
27.00, 126.77, 113.70 (d, 24C, Ar), 99.13 (s, C-2), 98.66 (d, C-1′), 81.82 (d, C-3′), 80.19 (d,
C-2′), 77.95 (d, C-4′), 75.10 (t, CH Ph), 74.78 (t, CH Ph), 73.62 (d, C-4), 73.29 (t, CH Ph),
2.53 (t, CH Ph), 71.82 (d, C-5), 71.75 (d, C-7), 71.39 (d, C-6), 70.23 (d, C-5′), 69.94 (t,
CH Ph), 67.79 (t, C-6′), 63.00 (t, C-8), 55.22 (q, PhOCH ), 52.34 (q, CO CH ), 51.12 (q,
OCH ), 33.21 (t, C-3), 17.60, 17.54, 17.50, 17.49, 17.29, 17.17, 17.09 [q, 8C, Si-CH-(CH
4.18, 13.55, 12.99, 12.75 [d, 4C, Si-CH-(CH ]; ESI-TOF HRMS: m/z = 1168.5848; calcd
3
), 3.79 (s, 3H, PhOCH
3
), 3.72 (app. t, 1H, J4′,5′
9
6
4
3
13
NMR (CDCl
3
1
2
2
2
7
2
2
3
2
3
3
3 2
) ],
1
3 2
)
+
for C64
H
86
O
15Si
2
NH : 1168.5844.
4
17

2
0
1
Data for 8β: [α]
δ 7.37 - 7.34 (m, 2H, Ar), 7.29 – 7.22 (m, 16H, Ar), 7.18 – 7.13 (m, 4H, Ar), 6.83 – 6.80 (m,
H, Ar), 5.31 (d, 1H, J1′,2′ 7.8 Hz, H-1′), 5.08 (d, 1H, J 11.6 Hz, CHHPh), 4.89 (d, 1H, J 10.9
Hz, CHHPh), 4.81 (d, 1H, J 11.0 Hz, CHHPh), 4.78 – 4.76 (m, 1H, H-5), 4.74 – 4.71 (m, 2H,
D
+ 14.4 (c 0.94, CHCl
3
); R
f
0.60 (n-hexane/EtOAc 3:1); H NMR (CDCl
3
):
2
2
4
1
–
x CHHPh), 4.62 (d, 1H, J 11.8 Hz, CHHPh), 4.52 – 4.45 (m, 4H, 1 x CHHPh, 3 x CHHPh),
.35 – 4.31 (m, 1H, H-7), 4.27 (dd, 1H, J8a,8b 12.0, J8a,7 2.0 Hz, H-8a), 3.94 (ddd, 1H, J4,3ax
1.7, J4,3eq 4.6, J4,5 1.8 Hz, H-4), 3.81 (dd, 1H, J8b,7 6.4 Hz, H-8b), 3.78 (s, 3H, PhOCH
3.69 (m, 1H, H-6′a), 3.65 – 3.55 (m, 2H, H-3′, H-6′b), 3.49 (s, 3H, CO CH ), 3.46 – 3.39
m, 3H, H-4′, H-5′, H-6), 3.34 (dd, 1H, J2′,3′ 9.1 Hz, H-2′), 3.20 (s, 3H, OCH ), 2.26 (app. t,
H, J3ax,3eq 12.7 Hz, H-3ax), 2.20 (dd, 1H, H-3eq), 1.11 – 0.87 (m, 28H, TIPDS); C NMR
CDCl ): δ 168.52 (s, C=O), 159.00, 138.95, 138.67, 138.22, 138.19, 130.32 (s, 6C, Ar),
3
), 3.73
2
3
(
3
1
3
1
(
3
1
1
28.66, 128.35, 128.29, 128.25, 128.15, 128.01, 127.96, 127.89, 127.68, 127.56, 127.51,
27.45, 127.19, 113.79 (d, 24C, Ar), 100.49 (d, C-1′), 99.36 (s, C-2), 84.73 (d, C-3′), 82.31 (d,
C-2′), 78.36 (d, C-4′), 75.81 (d, C-4), 75.75 (t, CH
4.09 (t, CH Ph), 73.28 (d, C-5′), 73.24 (t, CH Ph), 72.69 (d, C-7), 69.86, 69.83 (t, 2 C,
CH Ph, C6′), 66.57 (t, C-8), 64.98 (d, C-5), 55.24 (q, PhOCH ), 52.19 (q, CO CH ), 50.94 (q,
OCH ), 33.30 (t, C-3), 17.81, 17.79, 17.57, 17.48, 17.42, 17.34, 17.28 [q, 8C, Si-CH-(CH ] ,
2 2
Ph), 74.94 (t, CH Ph), 74.57 (d, C-6),
7
2
2
2
3
2
3
3
3 2
)
1
3.38, 12.79, 12.74 [d, 4C, Si-CH-(CH
3
)
2
]; ESI-TOF HRMS: m/z = 1168.5854; calcd for
+
C
64 86 2 4
H O15Si NH : 1168.5844.
2
0
1
Data for 9α: [α]
D
+ 34.3 (c 0.64, CHCl
3
); R
f
0.39 (n-hexane/EtOAc 3:1); H NMR (CDCl
3
):
δ 7.33 – 7.24 (m, 18H, Ar), 7.13 – 7.10 (m, 2H, Ar), 5.04 (d, 1H, J1′,2′ 3.4 Hz, H-1′), 4.93 (d,
1
4
H, J 11.0 Hz, CHHPh), 4.82 (d, 1H, J 11.0 Hz, CHHPh), 4.79 (d, 1H, J 10.7 Hz, CHHPh),
.75 (s, 2H, CH Ph), 4.54 (d, 1H, J 12.2 Hz, CHHPh), 4.52 (m, 1H, H-7), 4.45 (d, 1H, J 12.2
2
Hz, CHHPh), 4.44 (d, 1H, J 10.5 Hz, CHHPh), 4.16 (dd, 1H, J8a,8b 12.9, J8a,7 2.9 Hz, H-8a),
4
3
.09 – 3.99 (m, 5H, H-3′, H-5′
,
H-4
,
H-5, H-8b), 3.79 – 3.77 (m, 4H, H-6, CO
2
CH
3
), 3.61 –
.56 (m, 2H, H-6′a, H-6′b), 3.54 (dd, 1H, J2′,3′ 9.6 Hz, H-2′), 3.48 (dd, 1H, J 9.9, J 8.9 Hz, H-
18

4
′), 3.44 (d, 1H, JOH,4 9.6 Hz, OH), 3.31 (s, 3H, OCH
Hz, H-3eq), 1.86 (app. t, 1H, J3ax,4 12.5 Hz, H-3ax), 1.06 – 0.82 (m, 28H, TIPDS); C NMR
CDCl ): δ 168.57 (s, C=O), 138.67, 138.30, 137.96, 137.69 (s, 4C, Ar), 128.39, 128.38,
28.35, 138.33, 127.99, 127.89, 127.87, 127.80, 127.73, 127.58, 127.53, 127.45 (d, 20C, Ar),
9.46 (d, C-1′), 99.03 (s, C-2), 81.42 (d, C-3′), 78.74 (d, C-2′), 78.60 (d, C-5), 77.87 (d, C-4′),
5.37 (t, CH Ph), 75.08 (t, CH Ph), 73.50 (t, CH Ph), 73.08 (d, C-7), 72.56 (t, CH Ph), 72.05
CH ),
), 36.13 (t, C-3), 17.63, 17.54, 17.53, 17.50, 17.35, 17.22, 17.14 [q, 8C, Si-
3
), 2.13 (dd, 1H, J3eq,3ax 12.8, J3eq,4 4.4
13
(
3
1
9
7
2
2
2
2
(
d, C-6), 71.39 (d, C-5′), 68.67 (t, C-6′), 66.79 (d, C-4), 63.87 (t, C-8), 52.37 (q, CO
1.19 (q, OCH
CH-(CH ], 13.98, 13.60, 12.96, 12.77 [d, 4C, Si-CH-(CH
048.5273; calcd for C56
2
3
5
3
3
)
2
3
)
2
]; ESI-TOF HRMS: m/z =
+
1
H
78
O
14Si
2
NH
4
: 1048.5268.
); R
2
D
0
1
Data for 9β: [α]
+ 19.7 (c 0.85, CHCl
3
f
0.27 (n-hexane/EtOAc 3:1); H NMR (CDCl
3
):
δ 7.33 – 7.23 (m, 18H, Ar), 7.17 – 7.13 (m, 2H, Ar), 5.00 (d, 1H, J 11.0 Hz, CHHPh), 4.90
d, 1H, J 10.9 Hz, CHHPh), 4.83 (d, 1H, J 10.7 Hz, CHHPh), 4.78 (2d, 2H, J 10.7 Hz,
CHHPh, CHHPh), 4.70 (d, 1H, J1′,2′ 7.9 Hz, H-1′), 4.62 (d, 1H, J 11.9 Hz, CHHPh), 4.57 (d,
H, J 10.7 Hz, CHHPh), 4.41 (d, 1H, J 11.9 Hz, CHHPh), 4.38 (app. dt, 1H, J7,6 = J7,8a 8.7,
7,8b 2.5 Hz, H-7), 4.17 – 4.09 (m, 3H, H-5, H-8a, H-8b), 3.90 (app. ddt, 1H, J4,3ax = J4,OH
1.6, J4,3eq 4.8, J4,5 2.6 Hz, H-4), 3.81 (dd, 1H, J6′a,6′b 10.9, J6′a,5′ 3.2 Hz, H-6′a), 3.80 (app. t,
H, J4′,3′ = J4′,5′ 9.4 Hz, H-4′), 3.78 (s, 3H, CO CH ), 3.72 – 3.68 (m, 3H, H-3′, H-6′b, OH),
.63 (d, 1H, H-6), 3.55 (dd, 1H, J2′,3′ 9.1 Hz, H-2′), 3.41 – 3.37 (m, 1H, H-5′), 3.26 (s, 3H,
), 1.82 (dd, 1H, J3eq,3ax 12.7 Hz, H-3eq), 1.64 (app. t, 1H, H-3ax), 1.08 – 0.85 (m, 28H,
(
1
J
1
1
3
2
3
OCH
TIPDS); C NMR (CDCl
28.39, 128.34, 127.96, 127.94, 127.8, 127.71, 127.69, 127.67, 127.60 (d, 20C, Ar), 103.86
d, C-1′), 99.21 (s, C-2), 85.57 (d, C-3′), 82.12 (d, C-2′), 78.06 (d, C-4′), 77.23 (d, C-5), 75.53
t, CH Ph), 75.50 (t, CH Ph), 75.13 (d, C-5′), 74.90 (t, CH Ph), 73.43 (t, CH Ph), 72.73 (d, C-
), 71.12 (d, C-6), 68.75 (t, C-6′), 67.43 (d, C-4), 63.88 (t, C-8), 52.33 (q, CO CH ), 51.20 (q,
OCH ), 35.88 (t, C-3), 17.83, 17.74, 17.54, 17.47, 17.37, 17.34, 17.21, 17.15 [q, 8C, Si-CH-
3
13
3
): δ 168.73 (s, C=O), 138.15, 137.16 (s, 4C, Ar), 128.47, 128.44,
1
(
(
2
2
2
2
7
2
3
3
19

(
CH
053.4818; calcd for C56
Alternatively compound 9α was obtained by acid treatment of 8α:
A solution of 8α (17.6 mg, 0.015 mmol) in CH Cl (14.2 mL) was treated with 99% TFA (3.6
3
)
2
], 13.77, 13.67, 12.95, 12.84 [d, 4C, Si-CH-(CH
3
)
2
]; ESI-TOF HRMS: m/z =
+
1
78
H O
14Si Na : 1053.4822.
2
2
2
mL) at 0 °C for 10 min. The solution was concentrated and coevaporated with toluene.
Subsequent column chromatography of the residue (n-hexane/EtOAc 5:1) yielded 9α (13.0
mg, 83%) as a colorless oil.
3
.5. 2,3-Di-O-benzyl-4,6-O-benzylidene-α,β-D-glucopyranosyl (N-
phenyl)trifluoroacetimidate (10α/β)
2
8
2
,3-Di-O-benzyl-4,6-O-benzylidene-α,β-D-glucopyranose (128 mg, 0.285 mmol) was
dissolved in a mixture of dry CH Cl (2.0 mL) and dry acetone (0.9 mL) followed by
successive addition of K CO (79 mg, 0.570 mmol) and 2,2,2-trifluoro-N-phenylacetimidoyl
chloride (92 µL, 0.570 mmol). The heterogenous mixture was stirred at ambient temperature
for 16 h, filtered through a pad of Celite® and rinsed with CH Cl . After addition of one drop
of Et N the filtrate was concentrated and the crude residue was purified by chromatography
n-hexane/EtOAc 10:1) affording an anomeric mixture (α/ β ~ 1:8) of 10 (169 mg, 96%).
2
2
2
3
2
2
3
(
2
D
0
Data for 10β: colorless amorphous solid; [α]
3 f
+ 49.1 (c 2.57, CHCl ); R 0.75 (n-
1
hexane/EtOAc 4:1); H NMR (CD
2
Cl ): δ 7.54 – 7.46 (m, 2H, Ar), 7.42 – 7.27 (m, 15H, Ar),
2
7
.17 – 7.13 (m, 1H, Ar), 6.88 – 6.75 (m, 2H, Ar), 5.83 (bs, 1H, H-1), 5.60 (s, 1H, CHPh), 4.92
d, 1H, J 11.4 Hz, CHHPh), 4.84 (d, 1H, J 11.0 Hz, CHHPh), 4.81 (2d, 2H, J 11.1 Hz, 2 x
CHHPh), 4.38 – 4.29 (m, 1H, H-6a), 3.88 – 3.65 (m, 4H, H-2, H-3, H-4, H-6b), 3.45 (bs, 1H,
(
1
3
H-5); C NMR (CD
2
Cl
2
): δ 143.78, 138.94, 138.41, 137.84 (s, 4C, Ar), 129.37, 129.20,
1
28.75, 128.67, 128.59, 128.49, 128.41, 128.27, 128.06, 126.49, 124.88, 119.63 (d, 20C, Ar),
20

1
01.69 (d, CHPh), 81.50, 81.13, 81.11 (d, 3C, C-2, C-3, C-4), 75.71, 75.21 (t, 2C, 2 x
CH Ph), 68.91 (t, C-6), 67.06 (d, C-5).
Data for 10α: colorless oil; [α]
NMR (CD Cl ): δ 7.53 – 7.49 (m, 2H, Ar), 4.42 – 7.27 (m, 15H, Ar), 7.15 – 7.11 (m, 1H, Ar),
.80 – 6.74 (m, 2H, Ar), 6.45 (bs, 1H, H-1), 5.61 (s, 1H, CHPh), 4.94 (d, J 11.4 Hz, CHHPh),
.85 (d, J 11.3 Hz, CHHPh), 4.84 (d, J 11.8 Hz, CHHPh), 4.76 (d, J 11.9 Hz, CHHPh), 4.36
2
2
0
1
D
+ 43.0 (c 1.92, CHCl
3
); R 0.61 (n-hexane/EtOAc, 4:1); H
f
2
2
6
4
(
(
dd, 1H, J6a,6b 10.2, J6a,5 4.8 Hz, H-6a), 4.09 (app. t, 1H, J3,2 = J3,4 9.5 Hz, H-3), 4.03 – 3.96
1
3
m, 1H, H-5), 3.81 – 3.70 (m, 3H, H-2, H-4, H-6b); C NMR (CD
2
Cl
38.46, 137.89 (s, 4C, Ar), 129.37, 129.20, 128.83, 128.63, 128.60, 128.30, 128.27, 128.12,
27.96, 126.52, 124.69, 119.75 (d, 20C, Ar), 101.82 (d, CHPh), 81.77 (d, C-4), 79.14 (d, C-
), 78.62 (d, C-3), 75.50, 74.29 (t, 2C, 2 x CH Ph), 69.07 (t, C-6), 65.52 (d, C-5).
2
): δ 144.06, 139.23,
1
1
2
2
3
.6. 2,3-Di-O-benzyl-4,6-O-benzylidene-α-D-glucopyranosyl-(1→5)-methyl [methyl 3-
deoxy-4-O-(4-methoxybenzyl)-7,8-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-α-D-
manno-oct-2-ulopyranosid]onate (11)
2 2
A solution of donor 10α/β (74 mg, 0.119 mmol) in dry CH Cl (1.0 mL) was added to dry
acceptor 4 (50 mg, 0.080 mmol) under Ar followed by addition of 4 Å molecular sieves. After
stirring for 2.5 h at ambient temperature a solution of TMSOTf (0.4 µL, 0.002 mmol) in dry
CH
2
Cl
2
(0.1 mL) was added dropwise at -5 °C. The reaction was quenched after 15 min by
N (23 µL, 0.159 mmol) in dry CH Cl (0.7 mL). The mixture was allowed to
addition of Et
3
2
2
warm to ambient temperature and was filtered through a pad of Celite®. The filtrate was
concentrated and the residue was purified by chromatography (n-hexane/EtOAc 8:1→3:1,
2
D
0
with 0.1 % TEA) to give 11 (67 mg, 80%) as a coloress oil: [α]
+ 59.7 (c 0.53, CHCl
3
); R
f
1
0
1
.63 (n-hexane/EtOAc 3:1), H NMR (CDCl
3
): δ 7.51 - 7.47 (m, 2H, Ar), 7.42 - 7.21 (m,
5H, Ar), 6.88 - 6.84 (m, 2H, Ar), 5.51 (s, 1H, CHPh), 5.28 (d, 1H, J1′,2′ 3.8 Hz, H-1′), 5.08
21

(
d, 1H, J 11.8 Hz, CHHPh), 4.96 (d, 1H, J 11.3 Hz, CHHPh), 4.75 (d, 1H, J 11.0 Hz,
CHHPh), 4.66 (d, 1H, J 11.5 Hz, CHHPh), 4.57 (s, 2H, CH Ph), 4.42 - 4.39 (m, 1H, H-7),
.35 (app. dt, 1H, J5′,4′ = J5′,6′b 9.9, J5′,6′a 5.1 Hz, H-5′), 4.25 (d, 1H, J5,4 2.3 Hz, H-5), 4.19
app. t, 1H, J3′,2′ = J3′,4′ 9.5 Hz, H-3′), 3.96 (dd, 1H, J8a,8b 13.2, J8a,7 1.6 Hz, H-8a), 3.89 (dd,
H, J6′a,6′b 10.1 Hz, H-6′a), 3.85 (ddd, 1H, J4,3ax 11.6, J4,3eq 4.7, H-4), 3.81 (s, 3H, CO CH ),
.77 (s, 3H, PhOCH ), 3.67 (dd, 1H, H-2′), 3.64 - 3.59 (m, 3H, H-4′, H-6, H-8b), 3.55 (app. t,
H, J6′b,5′ 10.2 Hz, H-6′b), 3.28 (s, 3H, OCH ), 2.23 (dd, 1H, J3eq,3ax 12.5, H-3eq), 2.17 (app. t,
2
4
(
1
3
1
1
1
1
2
2
3
3
3
1
3
3
H, H-3ax), 1.14 - 0.77 (m, 28H, TIPDS); C NMR (CDCl ): δ 168.76 (s, C=O), 159.08,
38.93, 138.78, 137.79, 130.34 (s, 5C, Ar), 129.01, 128.69, 128.23, 128.10, 128.05, 127.91,
27.43, 126.96, 126.08, 113.85 (d, 19C, Ar), 100.99 (d, CHPh), 99.24 (d, C-1′), 99.16 (s, C-
), 82.96 (d, C-4′), 79.28 (d, C-2′), 78.04 (d, C-3′), 74.70 (t, CH
Ph), 71.65 (d, C-5), 71.54 (d, C-7), 71.23 (d, C-6), 70.11 (t, CH
t, C-8), 62.37 (d, C-5′), 55.19 (q, PhOCH ), 52.34 (q, CO CH ), 51.06 (q, OCH
C-3), 17.55, 17.49, 17.47, 17.44, 17.27, 17.16, 17.07 [q, 8C, Si-CH-(CH ], 14.22, 13.44,
]; ESI-TOF HRMS: m/z = 1076.5210; calcd for
2
Ph), 73.09 (d, C-4), 71.99 (t,
Ph), 69.08 (t, C-6′), 62.79
), 33.20 (t,
CH
2
2
(
3
2
3
3
3 2
)
1
3 2
2.99, 12.79 [d, 4C, Si-CH-(CH )
+
C
57
H
78
O
15Si
2
NH : 1076.5218.
4
3
.7. 2,3,4,6-Tetra-O-benzyl-α-D-glucopyranosyl-(1→5)-methyl (methyl 3-deoxy-α-D-
manno-oct-2-ulopyranosid)onate (12)
Compound 9α (16.0 mg, 0.016 mmol) was dissolved in dry THF (3.3 mL) and was treated
with TBAF (1 M in THF, 23 µL, 0.023 mmol) at ambient temperature for 20 min. After
addition of dry MeOH (1.7 mL) the solvent was removed in vacuo. Purification of the residue
2
D
0
by chromatography (toluene/EtOAc 1:1) afforded 12 (12.0 mg, 98%) as a colorless oil; [α]
1
+
36.0 (c 0.53, CHCl
3
); R
f
0.18 (toluene/EtOAc 1:1); H NMR (CDCl
3
): δ 7.39 - 7.24 (m,
1
8H, Ar), 7.18 – 7.15 (m, 2H, Ar), 4.96 (d, 1H, J 11.1 Hz, CHHPh), 4.93 (d, 1H, J 11.1 Hz,
22

CHHPh), 4.89 (d, 1H, J 11.6 Hz, CHHPh), 4.83 (d, 1H, J 10.7 Hz, CHHPh), 4.73 (d, 1H, J1′,2′
.7 Hz, H-1′), 4.64 (d, 1H, J 11.8 Hz, CHHPh), 4.52 (d, 1H, J 12.3 Hz, CHHPh), 4.49 (d, 1H,
J 10.7 Hz, CHHPh), 4.45 (d, 1H, J 12.1 Hz, CHHPh), 4.28 (d, 1H, J 5.6 Hz, OH), 4.07 (app. t,
H, J3′,2′ = J3′,4′ 9.5 Hz, H-3′), 4.03 – 3.95 (m, 4H, H-5′, H-4, H-5, H-7), 3.86 – 3.81 (m, 1H,
H-8a), 3.79 (s, 3H, CO CH ), 3.68 (dd, 1H, J6,7 8.9, J6,5 1.6 Hz, H-6), 3.66 – 3.61 (m, 1H, H-
b), 3.60 (dd, 1H, J6′a,6′b 10.4, J6′a,5′ 2.2 Hz, H-6′a), 3.58 (dd, 1H, H-2′), 3.55 (dd, 1H, J6′b,5′ 5.6
Hz, H-6′b), 3.54 (d, 1H, J 12.5 Hz, OH), 3.51 (app. t, 1H, J4′,5′ 9.6 Hz, H-4′), 3.18 (s, 3H,
OCH ), 2.16 – 2.11 (m, 1H, H-3eq), 2.08 – 2.00 (m, 1H, OH), 1.68 (app. t, 1H, J3ax,3eq = J3ax,4
3
1
2
3
8
3
1
3
1
4
1
3
2.3 Hz, H-3ax); C NMR (CDCl ): δ 168.20 (s, C=O), 138.15, 137.55, 137.48, 136.60 (s,
C, Ar), 128.85, 128.72, 128.61, 128.54, 128.53, 128.40, 128.03, 128.01, 127.89, 127.86,
27.83 (d, 20C, Ar), 100.65 (d, C-1′), 98.99 (s, C-2), 81.83 (d, C-3′), 80.06 (d, C-5), 78.83 (d,
C-2′), 78.31 (d, C-4′), 75.77 (t, CH
2
Ph), 75.37 (t, CH
1.98 (d, C-6), 71.75 (d, C-5′), 69.06 (d, C-7), 68.44 (t, C-6′), 65.85 (d, C-4), 64.32 (t, C-8),
2.46 (q, CO CH ), 51.02 (q, OCH ), 36.12 (t, C-3); ESI-TOF HRMS: m/z = 811.3299; calcd
13Na : 811.3300.
2 2 2
Ph), 74.96 (t, CH Ph), 73.51 (t, CH Ph),
7
5
2
3
3
+
52
for C44H O
3
.8. α-D-Glucopyranosyl-(1→5)-sodium (methyl 3-deoxy-α-D-manno-2-oct-2-
ulopyranosid)onate (13)
Compound 12 (11.2 mg, 0.014 mmol) was dissolved in dry MeOH (0.5 mL). The atmosphere
was exchanged to argon by alternating evacuation and flushing with argon. Then, palladium
on active charcoal (10%, 1 mg) was added to the flask followed by successive exchange of the
atmosphere to argon and hydrogen using the same method described before. The mixture was
stirred intensively for 3 h, diluted with MeOH and passed through a 0.45 µm syringe filter.
Concentration of the filtrate afforded debenzylated methyl ester which was saponified with aq
NaOH (0.01 M, 2.0 mL) at ambient temperature for 1.5 h. The solution was neutralized by
23

+
addition of freshly regenerated DOWEX 50 H , the ion-exchange resin was filtered off and
the filtrate was lyophilized. Purification by SEC (Bio-Gel P2 gel, 5% aq. EtOH) and freeze-
2
0
drying of pooled fractions provided 13 (5.6 mg, 90%) as a colorless amorphous solid; [α]
D
+
1
9
9.9 (c 0.56, D
2
O); H NMR (D
2
O): 5.11 (d, 1H, J1′,2′ 4.0 Hz, H-1′), 4.12 - 4.03 (m, 4H, H-4,
H-5, H-7, H-5′), 3.89 (dd, 1H, J8a,8b 11.8, J8a,7 2.8 Hz, H-8a), 3.79 - 3.68 (m, 3H, H-3′, H-6′a,
H-6′b), 3.62 (dd, 1H, J8b,7 6.1 Hz, H-8b), 3.56 – 3.54 (m, 1H, H-6), 3.49 (dd, 1H, J2′,3′ 10.0
Hz, H-2′), 3.41 (app. t, 1H, J 10.0, J 9.0 Hz, H-4′), 3.11 (s, 3H, OCH ), 1.99 – 1.95 (m, 1H,
3
1
3
H-3eq), 1.82 (app. t, 1H, J3ax,3eq = J3ax,4 12.5 Hz, H-3ax); C NMR data: see Table 1; ESI-
-
TOF HRMS: m/z = 413.1296; calcd for C15
H
25
O
13 : 413.1301.
3
.9. 2,3-Di-O-benzyl-α-D-glucopyranosyl-(1→5)-methyl [methyl 3-deoxy-7,8-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)-α-D-manno-oct-2-ulopyranosid]onate (14)
Compound 11 (50.0 mg, 0.047 mmol) was treated with trifluoroacetic acid (99%, 1.0 mL) in
2 2
CH Cl (8.0 mL) at 0 °C for 10 min. The solution was concentrated, coevaporated with
toluene and the crude product was purified by chromatography (n-hexane/EtOAc 3:1, then
2
0
1
:2) yielding 14 (40.2 mg, 88%) as a colorless oil; [α]
D
3 f
+ 79.6 (c 0.52, CHCl ); R 0.24 (n-
1
hexane-EtOAc 1:2); H NMR (CDCl
3
): δ 7.36 - 7.26 (m, 10H, Ar), 5.09 (d, 1H, J1′,2′ 3.4 Hz,
H-1′), 4.91 (d, 1H, J 11.6 Hz, CHHPh), 4.79 (d, 1H, J 12.4 Hz, CHHPh), 4.75 (d, 1H, J 12.5
Hz, CHHPh), 4.71 (d, 1H, J 11.5 Hz, CHHPh), 4.44 (app. td, 1H, J7,6 8.3, J7,8a = J7,8b 2.3 Hz,
H-7), 4.14 (dd, 1H, J8a,8b 12.8 Hz, H-8a), 4.10 - 4.05 (m, 2H, H-4, H-5), 4.03 - 3.97 (m, 2H,
H-5′, H-8b), 3.85 (app. t, 1H, J3′,2′ = J3′,4′ 8.6 Hz, H-3′), 3.81 - 3.76 (m, 5H, CO
2
CH
′a), 3.73 (dd, 1H, J6′b,6′a 11.7, J6′b,5′ 5.6 Hz, H-6′b), 3.53 (dd, 1H, H-2′), 3.49 (app. t, 1H, J4′,5′
.7 Hz, H-4′), 3.31 (s, 3H, OCH ), 2.17 (m, 1H, H-3eq), 1.90 (app. t, 1H, J3ax,3eq = J3ax,4 12.2
3
, H-6, H-
6
8
3
1
3
Hz, H-3ax), 1.07 - 0.81 (m, 28H, TIPDS); C NMR (CDCl
3
): δ 168.62 (s, C=O), 138.38,
1
38.02 (s, 2C, Ar), 128.58, 128.41, 127.96, 127.91, 127.67, 127.42 (d, 10C, Ar), 99.07 (s, C-
24

2
), 98.90 (d, C-1′), 80.20 (d, C-3′), 78.39 (d, C-2′), 77.62 (d, C-5), 74.73 (t, CH
C-5′), 72.91 (d, C-7), 72.55 (t, CH Ph), 71.79 (d, C-6), 69.91 (d, C-4′), 66.60 (d, C-4), 63.70
t, C-8), 62.19 (t, C-6′), 52.42 (q, CO CH ), 51.20 (q, OCH ), 36.15 (t, C-3), 17.56, 17.52,
7.50, 17.44, 17.31, 17.20, 17.12 [q, 8C, Si-CH-(CH
2
Ph), 73.12 (d,
2
(
2
3
3
1
3
)
2
], 14.00, 13.57, 12.93, 12.77 [d, 4C,
+
Si-CH-(CH
3
)
2
]; ESI-TOF HRMS: m/z = 873.3880; calcd for C42
H
66
O
14Si
2
Na : 873.3883.
3
.10. 2,3-Di-O-benzyl-α-D-glucopyranosyl-(1→5)-methyl (methyl 3-deoxy-α-D-manno-
oct-2-ulopyranosid)onate (15)
TBAF (1 M in THF, 28 µL, 0.028 mmol) was added to a solution of 14 (15.9 mg, 0.019
mmol) in dry THF (1.0 mL) at ambient temperature and stirred for 5 min. Dry MeOH (2.0
mL) was added followed by concentration. The residue was purified by chromatography
2
0
(
EtOAc, then EtOAc/methanol 19:1) to provide 15 (11.0 mg, 97%) as a colorless oil; [α]
D
+
1
5
1
4
4
6
3
0.6 (c 0.52, CHCl
3
); R
f
0.22 (EtOAc); H NMR (CDCl
3
): δ 7.41 - 7.30 (m, 10H, Ar), 4.98 (d,
H, J 11.4 Hz, CHHPh), 4.91 (d, 1H, J 11.6 Hz, CHHPh), 4.85 (d, 1H, J 11.4 Hz, CHHPh),
.77 (d, 1H, J1′,2′ 3.6 Hz, H-1′), 4.66 (d, 1H, J 11.5 Hz, CHHPh), 4.20 (d, 1H, J 5.8 Hz, OH),
.06 - 3.97 (m, 3H, H-4, H-5, H-7), 3.92 (app. t, 1H, J3′,2′ = J3′,4′ 9.3 Hz, H-3′), 3.86 - 3.78 (m,
H, CO
.57 (app. dt, 1H, J4′,5′ 9.3, J4′,OH 3.2 Hz, H-4′), 3.54 (dd, 1H, H-2′), 3.44 (d, 1H, J 11.7 Hz,
), 2.63 (d, 1H, JOH,4′ 3.7 Hz, OH), 2.20 (dd, 1H, J3eq,3ax 12.6, J3eq,4 4.3
2
CH , H-5′, H-6′a, H-8a), 3.75 - 3.68 (m, 2H, H-6′b, H-6), 3.66 - 3.61 (m, 1 H, H-8b),
3
OH), 3.19 (s, 3H, OCH
Hz, H-3eq), 2.14 - 2.07 (m, 2H, 2 x OH), 1.73 - 1.65 (m, 1H, H-3ax); C NMR (CDCl
68.40 (s, C=O), 138.17, 136.48 (s, 2C, Ar), 128.89, 128.80, 128.73, 128.63, 128.11, 127.90
d, 10C, Ar), 100.72 (d, C-1′), 99.04 (s, C-2), 81.33 (d, C-3′), 79.78 (d, C-5), 78.97 (d, C-2′),
5.60 (t, CH Ph), 74.86 (t, CH Ph), 73.01 (d, C-5′), 71.90 (d, C-6), 70.69 (d, C-4′), 69.06 (d,
C-7), 65.87 (d, C-4), 64.19 (t, C-8), 62.01 (t, C-6′), 52.62 (q, CO CH ), 51.10 (q, OCH ),
6.20 (t, C-3); ESI-TOF HRMS: m/z = 626.2804; calcd for C30
3
13
3
): δ
1
(
7
2
2
2
3
3
+
3
H
40
O
13NH : 626.2807.
4
25

Deprotection of 15
A solution of 15 (5.6 mg, 0.009 mmol) in dry MeOH (1.0 mL) was hydrogenated for 4 h with
1
0% Pd-C (1 mg) as described for 13. The suspension was diluted with MeOH and passed
through a 0.45 µm syringe filter. Concentration of the filtrate afforded the debenzylated
methyl ester which was treated with 0.01 M aq NaOH (1.0 mL) at ambient temperature for 12
+
h. The solution was made neutral by addition of DOWEX 50 H resin. The ion-exchange resin
was filtered off and the filtrate was lyophilized. Purification of the residue on Bio-Gel PD10
2
column (H O) and freeze-drying of pooled fractions provided 13 (3.8 mg, 95%) as a white
amorphous solid.
3
.11. 2,3,4,6-Tetra-O-benzyl-α-D-glucopyranosyl-(1→5)-methyl (methyl 3-deoxy-α-D-
lyxo-2-hept-2-ulopyranosid)onate (16)
2 2
A solution of 12 (32.0 mg, 0.041 mmol) in dry CH Cl (3.5 mL) was cooled to -10 °C.
Sodium metaperiodate on silica (15 w%, 116 mg, 0.082 mmol) was added and the suspension
was stirred for 1 h at -10 °C with exclusion of light. The excess of reagent was destroyed by
addition of ethylene glycol (3 w% in water, 84 µL, 0.041 mmol). The mixture was diluted
with CHCl
3
and extracted with distilled water. The aqueous phase was further extracted with
SO ), filtered and
CH Cl (3 x 5 mL) and the combined organic extracts were dried (Na
2
2
2
4
concentrated. The crude material was dissolved in dry MeOH (1.0 mL), cooled to 0 °C and
treated with sodium borohydride (2.5 mg, 0.065 mmol) for 1 h. Another portion of sodium
borohyride (1.0 mg, 0.026 mmol) was added at 0 °C and after 5 min the solution was diluted
with EtOAc and aq NH
the combined organic layers were dried (Na
product which was purified by HP-column chromatography (n-hexane/EtOAc 4:1 → 3:2) to
4
Cl. The aqueous solution was extracted with EtOAc (3 x 5 mL) and
2
SO ). Concentration of the solution gave a crude
4
26

2
0
afford 16 (10.4 mg, 34 %) as a colorless oil; [α]
D
+ 40.4 (c 0.96, CHCl
3
); R 0.54
f
1
(
toluene/EtOAc 1:2); H NMR (CDCl
3
): δ 7.39 - 7.26 (m, 18H, Ar), 7.18 – 7.15 (m, 2H, Ar),
Ph), 4.85 (d, J 11.8 Hz, CHHPh), 4.84 (d, J 10.6 Hz, CHHPh), 4.74
d, 1H, J1′,2′ 3.5 Hz, H-1′), 4.64 (d, 1H, J 11.9 Hz, CHHPh), 4.52 (d, 1H, J 11.8 Hz, CHHPh),
.49 (d, 1H, J 10.7 Hz, CHHPh), 4.45 (d, 1H, J 12.4 Hz, CHHPh), 4.06 (app. t, 1H, J3′,2′
3′,4′ 9.4 Hz, H-3′), 4.04 - 3.98 (m, 2H, H-5′, H-4), 3.92 – 3.86 (m, 2H, H-5 H-7a), 3.86 – 3.83
m, 1H, H-6), 3.82 - 3.78 (m, 4H, H-7b, CO CH ), 3.60 (dd, 1H, J6′a,6′b 10.3, J6′a,5′ 2.1 Hz, H-
′a), 3.58 - 3.54 (m, 2H, H-2′, H-6′b), 3.54 - 3.48 (m, 2H, H-4′, OH), 3.21 (s, 3H, OCH
4
.95 - 4.91 (m, 2H, CH
2
(
4
=
J
,
(
2
3
6
3
), 2.15
1
3
(
dd, 1H, J3eq,3ax 12.2, J3eq,4 4.5 Hz, H-3eq), 1.71 (app. t, 1H, J3ax,4 12.5 Hz, H-3ax); C NMR
CDCl ): δ 168.28 (s, C=O), 138.25, 137.63, 137.53, 136.83 (s, 4C, Ar), 128.82, 128.62,
28.51, 128.40, 128.02, 127.99, 127.88, 127.83, 127.80 (d, 20C, Ar), 100.47 (d, C-1′), 99.03
s, C-2), 81.74 (d, C-3′), 79.80 (d, C-5), 79.22 (d, C-2′), 78.11 (d, C-4′), 75.80 (t, CH Ph),
5.36 (t, CH Ph), 74.77 (t, CH Ph), 73.49 (t, CH Ph), 71.77 (d, C-5′), 71.59 (d, C-6), 68.49 (t,
C-6′), 65.95 (d, C-4), 60.64 (t, C-7), 52.50 (q, CO CH ), 51.01 (q, OCH ), 36.27 (t, C-3); ESI-
TOF HRMS: m/z = 781.3198; calcd for C43 12Na : 781.3194.
(
3
1
(
2
7
2
2
2
2
3
3
+
50
H O
3
.12. α-D-Glucopyranosyl-(1→5)-sodium (methyl 3-deoxy-α-D-lyxo-hept-2-
ulopyranosid)onate (17)
A solution of 16 (8.0 mg, 0.011 mmol) in dry MeOH (0.5 mL) was hydrogenated for 4 h with
1
0% Pd-C (1 mg) as described for 13. Another portion of catalyst (1 mg) was added and
stirring under H was continued for 19 h. The suspension was diluted with MeOH and passed
2
through a 0.45 µm syringe filter. Concentration of the filtrate afforded the debenzylated
methyl ester which was treated with 0.01 M aq NaOH (1.5 mL) at ambient temperature for 1
+
h. The solution was made neutral by addition of DOWEX 50 H resin. The ion-exchange resin
was filtered off and the filtrate was lyophilized. Purification of the residue on Bio-Gel P2 (5%
27

aq. EtOH) and freeze-drying of pooled fractions provided 17 (3.4 mg, 79%) as a colorless
2
0
1
amorphous solid; [α]
D
+ 170.7 (c 0.28, D
2
O); H NMR (D
2
O): 4.89 (d, 1H, J1′,2′ 3.8 Hz, H-
′), 4.10 (ddd, 1H, J3ax,4 12.3, J3eq,4 4.8, J5,4 3.0 Hz, H-4), 4.06 (ddd, 1H, J6′a,5′ 3.5, J6′b,5′ 2.6,
4′,5′ 10.3 Hz, H-5′), 3.96 (dd, 1H, J7a,7b 11.4, J7a,6 8.2 Hz, H-7a), 3.88 (br d, 1H, H-5), 3.81
dd, 1H, J7b,6 4.7 Hz, H-7b), 3.76-3.68 (m, 4H, H-6, H-3′, H-6′a, H-6′b), 3.48 (dd, 1H, J2′,3′
0.0 Hz, H-2′), 3.41 (app. t, 1H, J4′,3′ 9.1 Hz, H-4′), 3.14 (s, 3H, OCH ), 2.01 – 1.99 (m, 1H,
1
J
(
1
3
13
H-3eq), 1.82 (app. t, 1H, J3ax,3eq = J3ax,4 12.6 Hz, H-3ax); C NMR data: see Table 1; ESI-
-
TOF HRMS: m/z = 383.1193; calcd for C14
H
23
O
12 : 383.1195.
3
.13. 2,3-Di-O-benzyl-4,6-O-benzylidene-α-D-glucopyranosyl-(1→5)-methyl [methyl 3-
deoxy-7,8-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-α-D-manno-oct-2-
ulopyranosid]onate (18)
2
,3-Dichloro-4,5-dicyano-p-benzoquinone (26.2 mg, 0.116 mmol) was added in four portions
during 1 h to a solution of 11 (15.3 mg, 0.014 mmol) in dry CH Cl (0.9 mL) and dry MeOH
0.3 mL). After complete addition the mixture was stirred at ambient temperature for 1.5 h,
diluted with CHCl and washed with aq NaHCO . The aqueous phase was extracted with
CH Cl (2 x 5 mL) and the combined organic phases were washed with aq NaHCO , dried
MgSO ), filtered and concentrated. To remove aromatic impurities the crude product was
dissolved in toluene and subjected to chromatography (toluene→toluene/EtOAc 9:1) to afford
2
2
(
3
3
2
2
3
(
4
2
D
0
1
8 (11.2 mg, 83%) as a colorless oil; [α]
3 f
+ 52.2 (c 0.54, CHCl ); R 0.30 (n-hexane/EtOAc
1
3
:1); H NMR (CDCl
3
): δ 7.49 - 7.47 (m, 2H, Ar), 7.39 - 7.24 (m, 13H, Ar), 5.54 (s, 1H,
CHPh), 5.11 (d, 1H, J1′,2′ 3.9 Hz, H-1′), 4.93 (d, 1H, J 11.2 Hz, CHHPh), 4.90 (d, 1H, J 12.2
Hz, CHHPh), 4.79 (d, 1H, J 11.2 Hz, CHHPh), 4.75 (d, 1H, J 12.5 Hz, CHHPh), 4.47 (app.
dt, 1H, J7,6 = J7,8a 8.7, J7,8b 2.1 Hz, H-7), 4.24 (dd, 1H, J6′a,6′b 10.4, J6′a,5′ 4.9 Hz, H-6′a), 4.15 -
4
.07 (m, 5H, H-3′, H-5′, H-4, H-5, H-8a), 3.90 (dd, 1H, J8b,8a 12.9 Hz, H-8b), 3.80 (s, 3H,
28

CO
2
CH
4′,5′ 9.5 Hz, H-4′), 3.62 (dd, 1H, J2′,3′ 9.0 Hz, H-2′), 3.32 (s, 3H, OCH
.20 - 2.16 (m, 1H, H-3eq), 1.92 (app. t, 1H, J3ax,3eq = J3ax,4 12.2 Hz, H-3ax), 1.05 - 0.81 (m,
3
), 3.75 (d, 1H, H-6), 3.69 (app. t, 1H, J6′b,5′ 10.4 Hz, H-6′b), 3.65 (app. t, 1H, J4′,3′
=
J
2
2
1
3
), 2.85 (bs, 1H, OH),
13
8H, TIPDS); C NMR (CDCl
3
): δ 168.53 (s, C=O), 138.59, 138.39, 137.28 (s, 3C, Ar),
28.90, 128.27, 128.22, 127.96, 127.56, 127.46, 127.41, 125.97 (d, 15C, Ar), 101.13 (d,
CHPh), 100.05 (d, C-1′), 99.07 (s, C-2), 82.09 (d, C-4′), 78.26 (d, C-2′), 78.18 (d, C-3′), 77.25
d, C-5), 74.84 (t, CH Ph), 72.57 (t, CH Ph), 72.32 (d, C-7), 71.49 (d, C-6), 68.75 (t, C-6′),
6.50 (d, C-4), 63.37 (d, C-5′), 63.33 (t, C-8), 52.46 (q, CO CH ), 51.22 (q, OCH ), 36.28 (t,
C-3), 17.62, 17.53, 17.52, 17.49, 17.32, 17.18, 17.11 [q, 8C, Si-CH-(CH ], 14.12, 13.58,
]; ESI-TOF HRMS: m/z = 961.4190; calcd for
(
2
2
6
2
3
3
3 2
)
1
3 2
2.96, 12.81 [d, 4C, Si-CH-(CH )
+
C
49 70 2
H O14Si Na : 961.4196.
3
.14. 2,3,4-Tri-O-benzyl-α-D-glucopyranosyl-(1→5)-methyl [methyl 3-deoxy-4-O-(4-
methoxybenzyl)-7,8-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-α-D-manno-oct-2-
ulopyranosid]onate (19)
A suspension of compound 11 (20.0 mg, 0.019 mmol) and 4 Å molecular sieves (70 mg) in
dry CH
9.8 µL, 0.061 mmol) and dichlorophenylborane (7.2 µL, 0.055 mmol). After 1 h Et
µL) and dry MeOH (44 µL) were added to the cold solution. The mixture was diluted with
CHCl , allowed to warm up to ambient temperature and washed with aq NaHCO . The
aqueous layer was extracted with CH Cl (3 x 5 mL), the combined organic layers were dried
Na SO ) and concentrated. The residue was purified by column chromatography (n-
2
Cl
2
(0.8 mL) was cooled to -70 °C followed by consecutive addition of triethylsilane
(
3
N (44
3
3
2
2
(
2
4
2
0
hexane/EtOAc 9:1 → 3:1) to give 19 (8.6 mg, 43 %) as a colorless oil; [α]
D
+ 87.6 (c 0.78,
1
CHCl
3
); R
f
0.31 (n-hexane/EtOAc 3:1); H NMR (CDCl
3
): δ 7.36 - 7.21 (m, 17H, Ar), 6.84 -
6
.81 (m, 2H, Ar), 5.25 (d, 1H, J1′,2′ 3.7 Hz, H-1′), 4.93 (d, 1H, J 12.0 Hz, CHHPh), 4.88 – 4.82
29