Synthesis of Staphylococcus aureus type 5 capsular polysaccharide repeating unit using novel l-FucNAc and d-FucNAc synthons and immunochemical evaluation

Article abstract of DOI:10.1016/j.bmc.2012.08.048

Staphylococcus aureus is a major cause of nosocomial infections. Glycoconjugates of type 5 and 8 capsular polysaccharides have been investigated for vaccine application. The proposed structure of type 5 polysaccharide is: →4-β-d-ManNAcA-(1→4)-α-l-FucNAc(3OAc)-(1→3) -β-d-FucNAc-(1→. The stereocontrolled insertion of these three glycosydic bonds is a real synthetic challenge. In the present paper we report the preparation of two novel versatile l- and d-fucosamine synthons from commercially available starting materials. In addition we applied the two building blocks to the synthesis of type 5 trisaccharide repeating unit. The immunochemical properties of the synthesized trisaccharide were assessed by competitive ELISA and by immunodot blot analysis using sera of mice immunized with type 5 polysaccharide conjugated to CRM₁₉₇. The results suggest that although the type 5 S. aureus trisaccharide is recognized by specific anti polysaccharide antibodies in dot blot, structures longer than the trisaccharide may be needed in order to significantly compete with the native type 5 polymer in the binding with sera from mice immunized with S. aureus type 5 polysaccharide-CRM₁₉₇ conjugate.

Full text of DOI:10.1016/j.bmc.2012.08.048

Bioorganic & Medicinal Chemistry 20 (2012) 6403–6415
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of Staphylococcus aureus type 5 capsular polysaccharide
repeating unit using novel L-FucNAc and D-FucNAc synthons
and immunochemical evaluation
Elisa Danieli a, Daniela Proietti a, Giulia Brogioni a, Maria R. Romano a, Emilia Cappelletti a, Marta Tontini a,
Francesco Berti a, Luigi Lay b, Paolo Costantino a, Roberto Adamo a,
⇑
a Research Center, Novartis Vaccine and Diagnostics, Via Fiorentina 1, 53100 Siena, Italy
b Department of Organic and Industrial Chemistry, University of Milan, Via G. Venezian 21, 20133 Milan, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Staphylococcus aureus is a major cause of nosocomial infections. Glycoconjugates of type 5 and 8 capsular
Received 11 May 2012
Revised 21 August 2012
Accepted 24 August 2012
Available online 31 August 2012
polysaccharides have been investigated for vaccine application. The proposed structure of type 5 polysac-
charide is: ?4-b-
insertion of these three glycosydic bonds is a real synthetic challenge. In the present paper we report the
preparation of two novel versatile - and -fucosamine synthons from commercially available starting
D-ManNAcA-(1?4)-a-L-FucNAc(3OAc)-(1?3)-b-D-FucNAc-(1?. The stereocontrolled
L
D
materials. In addition we applied the two building blocks to the synthesis of type 5 trisaccharide repeating
unit. The immunochemical properties of the synthesized trisaccharide were assessed by competitive ELISA
and by immunodot blot analysis using sera of mice immunized with type 5 polysaccharide conjugated to
CRM197. The results suggest that although the type 5 S. aureus trisaccharide is recognized by specific anti
polysaccharide antibodies in dot blot, structures longer than the trisaccharide may be needed in order to
significantly compete with the native type 5 polymer in the binding with sera from mice immunized with
S. aureus type 5 polysaccharide–CRM197 conjugate.
Keywords:
Total synthesis
Staphylococcus aureus
D,L-Fucosamine
N-Acetyl mannuronic acid
Antigenicity
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
The structure of type 5 repeating unit was first determined by
Moreau et al.,6 then revised by Vann et al.7 and ultimately by Jones,8
Staphylococcus aureus is a major cause of nosocomial infections,
the most frequent and serious of which is bacteremia and its
complications such as endocarditis, arthritis, and osteomyelitis in
hospitalized patients.1 S. aureus accounts approximately for one
fourth of the isolates in patients affected by bacteremia, which is
often associated with high mortality due to the spreading antibi-
otic resistance.2
Based on the structure of the capsular polysaccharide, 12 types
have been isolated. However type 5 and 8 comprise the majority of
clinical isolates, and thereby represent an important target for the
development of a conjugate vaccine.3 An unadjuvanted bivalent
vaccine composed of S. aureus capsular polysaccharides types 5
and 8 chemically conjugated to the recombinant non-toxic mutant
of Pseudomonas aeruginosa exotoxin A (rEPA), showed to confer
60% protection for 10 months,4 however in further efficacy trials
it failed to induce long lasting protection in end stage renal disease
patients who are unusually susceptible to S. aureus bacteremia and
represent an important target population.5
who proposed the trisaccharide structure ?4-b-
(1?4)- -FucNAc(3OAc)-(1?3)-b- -FucNAc-(1?.
To our best knowledge, the synthesis of this structure has never
been described and represent a challenge for carbohydrate chem-
istry: not only it is composed of three very unusual sugars, but it
also presents a b-linkage to the ManNAcA (N-acetyl-mannuronic
D-ManNAcA-
a-
L
D
acid) unit, an
N-acetyl- -fucosamine) unit, and finally a b-linkage between the
-FucNAc (N-acetyl- -fucosamine) and the following sugar residue.
a glycosidic bond to the 3-OAc L-FucNAc (3-O-acetyl
L
D
D
D
-FucNAc has been also found as constituent of the O-chain of
Pseudomonas aeruginosa lipopolysaccharide antigens.9 The synthe-
sis of related benzyl protected glycosides has been reported by
Horton et al,10 who used as starting material 2-acetamido-2-
deoxy-
xyl group in position 4 and C-6 deoxygenation.
-FucNAc, besides being a component of the O-chain of the anti-
D-glucose in a strategy based on the inversion of the hydro-
L
genic lipopolysaccharide of Pseudomonas aeruginosa, has been ob-
tained from the polysaccharides of certain enteric bacteria11 and
by acidic hydrolysis of Pneumococcus type 512 and type 1213 capsular
polysaccharides. Its preparation by azidonitration of 3,4-
⇑
Corresponding author. Tel.: +39 0577 539393; fax: +39 0577 245307.
E-mail address: roberto.adamo@novartis.com (R. Adamo).
di-O-acetyl-L
-fucal was first reported by Anisuzzaman et al.14
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.048

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E. Danieli et al. / Bioorg. Med. Chem. 20 (2012) 6403–6415
HO
R1O
BnO
BnO
O
RO
O
O
O
c
a
BnO
BnO
BnO
OAll
OAll
O
O
NHAc
BnO
BnO
NHAc
OR2
O
O
OAc
R=Bn
8 R=Ac
O
O
HO
HO
NHAc
NHAc
9 R1=R2=OH
10 R1=TBS R2=OH
11 R1=TBS R2=Lev
7
6
d
e
b
O
OAc
O
OMe
HO
1
O
O
BnO
BnO
RO
BnO
O
O
f
h
OAll
OLev
12 R=H
BnO
BnO
BnO
O
OR
LevO
14 R=H
R=CNHCCl3
O
NHCbz
O
i
g
3
13
R=Bn
N3
O
O
BnO
BnO
N3
N3
O
Scheme 2. Synthesis of NAcManA building block 3. Reagents and conditions: (a)
AllOH, TMSOTf; then Ac2O, py, 88%; (b) ZnCl2, AcOH, 76%; (c) NaOMe/MeOH, 85%;
(d) TBSCl, Im, DMF, 94%; (e) LevOH, DCC, DMAP, CH2Cl2, 80%; (f) CrO3, H2SO4,
(CH3)2CO/H2O; g. BnBr, NaHCO3, CH2Cl2, 65% (2 steps); (h) PdCl2, NaOAc, AcOH; (i)
CCl3CN, DBU, CH2Cl2, 66% (2 steps).
OAc
O
BnO
2
O
BnO
BnO
BnO
HO
OAll
N3
O
O
O
OAc
4
The glucuronic donor 3 was prepared by glycosylation of allyl
alcohol with the known orthoester 6.20 The reaction afforded a
mixture of the allyl 2-O-acetyl and allyl 2-O-hydroxy derivatives,
as reported in literature,20 so acetylation of the reaction crude
was needed to provide 7 in 88% yield (Scheme 2). Following acetol-
ysis of the 6-O-benzyl ether at C-6 with ZnCl2 in acetic acid (76%
yield) gave the 6-O-acetylated derivative 8.
Deacetylation by Zemplen transesterification and subsequent
selective silylation of the primary alcohol 9 using t-butyldimethyl-
silyl chloride (TBSCl) and imidazole provided compound 10 (94%
yield). At this stage orthogonal protection at C-2 of 10 was inserted
by reaction with levulinic acid, N,N0-dicyclohexylcarbodiimide and
4-dimethylaminopyridine (80% yield).21
O
NHCbz
BnO
OCNHCCl3
N3
LevO
3
HO
5
Scheme 1. Retrosynthetic analysis of trisaccharide 1
In the present paper we disclose the synthesis of the two novel
versatile -FucNAc and -FucNAc synthons 4 and 5 (Scheme 1),
L
D
respectively, and their application for assembling S. aureus type 5
trisaccharide repeating unit. The immunochemistry of the trisac-
charide was evaluated by competitive ELISA and immunodot blot
analysis using sera of mice vaccinated with S. aureus type 5 poly-
saccharide–CRM197 conjugate.15
The 6-O-desilylation and C-6 oxidation of 11 were achieved
one-pot under Jones conditions,22 followed by mild benzylation
with BnBr and NaHCO3 to provide the glucuronic derivative 13 in
65% yield over two steps. Deallylation of the anomeric position by
reaction with PdCl2 and subsequent treatment of the foregoing
1-hydroxyl derivative with trichloroacetonitrile and 1,8-diazabicy-
clo[5.4.0]undec-7-ene (DBU) led to donor 3, almost exclusively as
2. Results and discussion
2.1. Synthesis of monosaccharide building blocks and
assembling of S. aureus type 5 trisaccharide repeating unit
Our retrosynthetic approach to target molecule 1 is outlined in
Scheme 1. Since very few preparations of L- and D-FucNAc building
a- anomer.
The preparation of acceptor 4 required a new reaction sequence
blocks have been previously reported, we designed and synthesized
the new precursors 4 and 5 as key intermediates. On the other hand,
the choice of a suitable synthon for the ManNAcA unit was not
trivial. The b-linkageis notoriously difficultto beinstalled in manno-
sides, moreover the problem is increased by the presence of a
carboxylic group, which further deactivates the glycosyl donor.
Though some methods have been reported for the insertion of
b-ManNAcA building blocks, the outcome of the reaction is often
unsatisfactory and strictly dependent on the substituents in the
donor.16 Very recently, an improved mannopyranosyl uronic acid
donor for b- glycosylation has been reported by van der Marel and
coworkers.17 Another option to introduce a b- mannosyl linkage is
the glycosylation with a glucopyranosyl donor, followed by epimer-
ization at C-2 to get the manno configuration.18 In our synthetic
strategy we pursued the latter approach, and we employed the
2-O-levulinoyl glucuronate donor 3 as a precursor of the b-ManN-
AcA unit.
(Scheme 3), and we envisaged
material, which could be converted into
sequential epimerization at C-4 and C-2. Accordingly, compound
15,23 easily obtained from commercial
-rhamnose, was epimer-
ized at C-4 by Swern oxidation and subsequent reduction with
NaBH4 to afford the talo derivative 17 in excellent yield (94%) with
complete stereoselectivity.24
L
-rhamnose as suitable starting
L
-FucNAc synthon by
L
OAll
OAll
OAll
a
O
O
c, d
O
e
R1
HO
O
O
O
O
O
R2
O
OH
16
15
R1=R2=(=O)
18
b
17 R1=H, R2=OH
OAll
N3
OAll
OAll
R1
O
O
O
Azides were chosen to mask N-acetyl groups of the three carbo-
hydrate units with the purpose of favouring the formation of the
g
h
N3
OAc
O
OH
O
HO
R2
HO
a- glycosidic bond between L- and D-FucNAc units, which repre-
sented an additional challenge. Moreover, azides would deliver the
amine functions during the hydrogenolytic debenzylation of trisac-
charide 2.
4
19
20
R1=H R2=OTf
R1=N3 R2=H
21
f
Scheme 3. Synthesis of L-FucNAc building block 4. Reagents and conditions: (a)
The D-FucNAc unit carries an anomerically-linked aminopropyl
(COCl)2; DMSO, CH2Cl2; (b) NaBH4, EtOH, 94% (2 steps); (c) 90% AcOH; (d)
CH3C(OCH3)2CH3, p-TsOH, 82% (2 steps); (e) Tf2O, py, CH2Cl2; (f) TBAN3, Tol, 64% (2
steps); (g) 80% AcOH, 86%; (h) AcCl, py, CH2Cl2, 99%.
tether N-protected as a benzyl carbamate (Cbz)19 for possible con-
jugation to a carrier protein.

E. Danieli et al. / Bioorg. Med. Chem. 20 (2012) 6403–6415
6405
OAll
N3
OAll
N3
OAll
N3
O
BnO
BnO
O
O
O
O
O
O
BnO
BnO
BnO
BnO
a
b
c
O
O
BnO
OAc
OAc
OAc
O
O
HO
BnO
BnO
LevO
3
OCNHCCl3
LevO
HO
4
22
23
OAll
N3
OCNHCCl3
N3
OH
O
O
BnO
BnO
O
BnO
O
O
N3
BnO
BnO
O
N3
N3
e
f
R2
O
O
OAc
O
O
OAc
BnO
BnO
O
OAc
O
BnO
BnO
R1
26
27a,b
24
25
R1=OTf R2=H
R1=H R2=N3
d
Scheme 4. Synthesis of disaccharide donor 27a,b. Reagents and conditions: (a) TMSOTf, CH2Cl2, ꢀ10 °C, 61%; (b) H2NNH2.AcOH, CH2Cl2/MeOH, 86%; (c) Tf2O, py, CH2Cl2; (d)
TBAN3, Tol, 70%; (e) PdCl2, MeOH, 78%; (f) CCl3CN, DBU, CH2Cl2, 98%.
A triplet at 3.49 ppm in the 1H NMR spectrum of 17 with
J = 5.6 Hz assigned to H-4 (to be compared with J3,4 = 7.1 Hz in
precursor 15) confirmed the axial orientation of C-4 hydroxyl and
the inversion of configuration. The shift of the isopropylidene
protection from position 2,3 to 3,4 was achieved by hydrolysis in
aqueous acetic acid, followed by isopropylidene re-insertion, mak-
ing the C-2 hydroxyl group of the resulting 18 available for the
inversion of configuration (82% yield over two steps). The treatment
of 18 with trifluoromethanesulfonic anhydride (Tf2O) in CH2Cl2 in
the presence of pyridine, followed by nucleophilic displacement
with freshly prepared tetrabutylammonium azide furnished the
obtained in reasonable 61% yield. The b-manno configuration was
then introduced by selective removal of the levulinoyl group and
inversion at C-2 through O-triflylation and nucleophilic displace-
ment of the O-triflate with tetrabutylammonium azide to obtain
25 in 70% yield. The b-configuration of the mannuronic unit was
evidenced by a singlet at 4.44 ppm in the 1H NMR spectrum and
a
signal at 101.03 ppm (JC-1,H-1 = 157 Hz) in the 13C NMR
spectrum.25
After cleavage of the anomeric allyl protection by PdCl2, com-
pound 26 was converted into the disaccharide trichloroacedimi-
date 27a,b by reaction with trichloroacetonitrile and DBU. The
2-azido
L
-fucoside 20 in 64% yield. A doublet of doublet at
a/b mixture of trichloroacedimidates 27a,b could be resolved by
3.37 ppm with J2,3 = 10.7 Hz and J1,2 = 3.7 Hz assigned to H-2
confirmed the fuco configuration.
chromatography and the reactivity of the two epimers was studied
separately.
Finally, isopropilydene removal by acidic hydrolysis with 80%
aqueous acetic acid to give 21 (86% yield), and regioselective acet-
ylation of C-3 hydroxyl group by dropwise addition of acetyl chlo-
ride in dichloromethane-pyridine at 0 °C provided the acceptor 4 in
nearly quantitative yield.
The synthesis of compound 1 was based on a (2+1) strategy,
where disaccharide donor 27 would be reacted with the D-Fuc (D-
Fucose) acceptor 5. To this end disaccharide 22 was assembled
by glycosylation of 4 with 3, using TMSOTf a as promoter at
ꢀ10 °C (Scheme 4).
The synthesis of D-FucNAc acceptor 5 commenced with glyco-
sylation of benzyl N-(3-hydroxypropyl)carbamate with
acetate to give 36 in 72% yield (Scheme 5).
D-Fuc per-
A doublet at 5.21 ppm and J1,2 = 7.8 Hz in the 1H NMR spectrum
was attributed to H-1 and confirmed the b- configuration of the
newly formed glycosidic bond. Deacetylation of 28 and following
introduction of the isopropylidene protection afforded compound
30, which was very efficiently inverted at C-2 by Swern oxidation
and subsequent reduction with NaBH4, leading stereoselectively
to the talo configuration in 32 (87% yield, over two steps).24
O-triflylation at C-2 followed by displacement with sodium
The optimization of the reaction conditions was a very challeng-
ing task, since the 3-O-acetyl group of 4 was prone to partial
migration from C-3 to C-4 under the acidic conditions of the glyco-
sylation. While low temperature showed to slow down this side
reaction, the glycosylation proceeded in sluggish way leading to
decomposition of the donor 3. Final conditions were the best
compromise to drive the reaction toward the desired product,
azide provided in high yield (90%) the 2-azido D-fucoside 34, which
possessed a b-anomeric configuration, as proved in the 1H NMR
spectrum by a doublet at 4.17 ppm with J1,2 = 8.6 Hz assigned to
H-1.
After removal of the isopropylidene protective group, the unfa-
voured introduction of a benzyl protective group at the axial C-4
AcO
AcO
AcO
AcO
O
O
O
a
O
b, c
O
NHCbz
O
NHCbz
O
OAc
OAc
OAc
29
OH
28
30
O
R2O
R2
O
O
d
h
O
NHCbz
O
O
NHCbz
R1O
R1
N3
31
32
33
34
R1=R2=(=O)
e
f
g
35 R1=R2=H
i
j
k
R1=H R2=OH
R1=H R2=OTf
R1=N3 R2=H
36
37
R1=PMB R2=H
R1=PMB R2=Bn
5 R1=H
R2=Bn
Scheme 5. Synthesis of
D
-FucNAc acceptor 5. Reagents and conditions: (a) HO(CH2)3NHCbz, BF3ꢁEt2O, 72%; (b) NaOMe/MeOH; (c) CH3C(OCH3)2CH3, p-TsOH, 87% (2 steps); (d)
(COCl)2, DMSO; CH2Cl2; (e) NaBH4, EtOH, 87%; (f) Tf2O, py, CH2Cl2; (g) NaN3, DMF, 90%; (h) 90% AcOH, 98%; (i) BuSn2O, PMBBr, TBAI, Tol, 76%; (j) BnBr, NaOH, 18-crown-6,
THF/H2O, 99%; (k) DDQ, CH2Cl2/H2O, 94%.

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E. Danieli et al. / Bioorg. Med. Chem. 20 (2012) 6403–6415
R2
O
R1
N3
BnO
O
BnO
HO
N3
O
O
OAc
BnO
O
O
NHCbz
BnO
27a
N3
R1 = H, R2 = OCCNHCl3
27b R1 = OCCNHCl3, R2 = H
5
a
R2O
O
BnO
O
O
R3
O
O
BnO
BnO
O
NHCbz
O
O
N3
R1
N3
O
R2O
R2O
O
N3
O
R1
R1
OAc
O
O
BnO
OAc
O
R2O
2 R1=N3 R2=Bn R3=NHCbz
1 R1=R3=NHAc R2=H
b, c
Scheme 6. Synthesis of trisaccharide 1. Reagents and conditions: (a) TMSOTf, CH2Cl2, ꢀ10 °C, 65%; (b) 10% Pd-C, MeOH/H2OAcOH; (c) Ac2O, MeOH/H2O, 40%.
hydroxyl required the temporary protection of the C-3 hydroxyl
group as p-methoxybenzyl ether (PMB) to give 35.25
analysis. For this purpose, S. aureus type 5 capsular polysaccharide
(SA5) was purified adapting a procedure described in litera-
ture.4a,27 After depolymerization and sizing of the capsular poly-
saccharide, the obtained fragments were oxidized in order to
introduce an aldehyde group and coupled to CRM197 via direct
reductive amination.28 The glycoconjugate, formulated with alu-
minium hydroxide as adjuvant, was then used for immunization
of CD1 mice. Sera containing anti-SA5 capsular polysaccharide
IgG antibodies, as determined by ELISA assay, were used for com-
petitive ELISA and dot blot.
As shown in Figure 1, in competitive ELISA on plates coated
with SA 5 polysaccharide, the native polysaccharide at a starting
concentration of 4.5 mg/ml fully inhibited the binding of sera from
mice immunized with SA5-CRM197 conjugate, whereas the syn-
thetic repeating unit did not exhibit significant inhibition (ꢃ15%)
at the same concentration. The synthetic trisaccharide 1 was then
tested in dot blot (Fig. 2), and while no staining was observed for
the negative control (the non S. aureus correlated b-glucan polysac-
charide laminarin29), antibodies directed against SA5 recognized
the native polysaccharide used as positive control (SA5) as well
as compound 1, although apparently with lower intensity.
Benzylation at C-4 was achieved in excellent yield using BnBr and
NaOH as base in the presence of crown ether in THF.25 Other meth-
ods, such as BnBr–NaHor BnBr–NaOH in DMF, producedbenzylation
also of the amide function. After selective oxidative cleavage of PMB
by use of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), com-
pound 37 furnished the key compound 5 in very good overall yield.
Glycosylation of D-Fuc acceptor 5 with the a disaccharide 27a,
in the presence of TMSOTf as promoter, at -20 °C (Scheme 6) led
to a mixture of the desired trisaccharide 2 and the product of b-
linkage formation 38 in a ratio ꢂ2.3:1 (Table 1, entry 1). The ste-
reochemical orientation of the newly formed anomeric center
was determined by 1H NMR: whereas the
a- product 2 presented
a doublet at 5.29 ppm with a J1,2 = 3.8 Hz, the b-anomer 38 showed
a doublet at 4.40 ppm with J1,2 = 8.3 Hz. When the temperature
was raised up to ꢀ10 °C, only very little improvement was
achieved (entry 2), while using diethyl ether as solvent instead of
dichloromethane gave very surprisingly the predominant forma-
tion of the b trisaccharide (2:38 = 1:2, entry 3). The use of b donor
27b and TMSOTf as promoter led to a mixture 2:38 ꢃ 2.3:1 at
ꢀ20 °C and 2.8:1 at ꢀ10 °C (entry 4 and 6, respectively). BF3ꢁEt2O
was then added as promoter at 0 °C, to favour the displacement
of b-trichloroacetidimoyl group of 27b by the acceptor 5 via SN2
process.26 Nevertheless, the reaction stereoselectivity was not im-
proved, and once more a mixture 2:38 = 2.4:1 was obtained (entry
5). Eventually, since our primary purpose was a preliminary anti-
genic evaluation of trisaccharide 1, we deemed the yield in entries
2 and 6 acceptable and no further investigation of this challenging
glycosylation was performed at this stage.
3. Conclusions
We report here the synthesis and the antigenic evaluation of the
repeating unit of the type 5 S. aureus polysaccharide, which to date
has never been described. Our synthetic goal was achieved by
exploiting the versatility of two novel synthons for
L-FucNAc and
D-FucNAc residues (4 and 5, respectively), synthesized from cheap
commercially available starting materials.
A (2+1) strategy based on glycosylation of the
Conversion of the azide groups to acetamide and simultaneous
deprotection of trisaccharide 2 was carried out by hydrogenation
in flow chemistry using a 10% Pd-C cartridge, followed by treatment
with acetic anhydride in MeOH. The anomeric region of the depro-
tected trisaccharide 1 in the 1H NMR was in a good agreement with
D
-FucNAc accep-
tor 5 with the protected b-ManNAcA-(1?4)-a-L-FucNAc(3OAc)
disaccharide donor 27a,b was applied.
The b-mannuronate of disaccharide 27a,b was in turn obtained
by coupling the -FucNAc synthon 4 and the b- glucopyranosyl do-
nor 3, followed by epimerization at C-2 of the b-gluco unit.
Glycosylation of 5 with 27a,b proved a moderate stereoselec-
reported NMR data,8 except for the
D-FucNAc unit which carries a N-
L
acetamidopropyl chain as aglycon in the synthesized repeating unit.
When we tried to selectively convert the azides of 2 into amines
without concomitant removal of the Cbz protection in the linker,
the lactamization of the mannuronic unit was the prevalent reaction
either under Staudinger conditions (trimethylphosphine and aque-
ous NaOH in THF) or by treatment with H2S in pyridine–water.
a
tivity allowing the synthesis of the desired trisaccharide 2, which
after deprotection provided the target molecule 1. The spectro-
scopic characterization of trisaccharide 1 confirmed previous
structure assignment.8
The immunochemical properties of trisaccharide 1 were evalu-
ated by competitive ELISA and immunodot blot analysis. While dot
blot indicated that a fragment as small as a single repeating unit is
recognized by anti S. aureus type 5 polysaccharide sera, competi-
tive ELISA result suggested that longer structures are necessary
2.2. Immunochemical analyses
The immunochemical properties of the synthesized trisaccha-
ride 1 were assessed by competitive ELISA and immunodot blot

E. Danieli et al. / Bioorg. Med. Chem. 20 (2012) 6403–6415
6407
Table 1
Tested conditions for glycosylation of acceptor 5a
Entry
Donor
Solvent
Promoter
Temperature (°C)
a/b = 2:38
1
2
3
4
5
6
27a
27a
27a
27b
27b
27b
CH2Cl2
CH2Cl2
Et2O
CH2Cl2
CH2Cl2
CH2Cl2
TMSOTf
TMSOTf
TMSOTf
TMSOTf
BF3ꢁEt2O
TMSOTf
ꢀ20
ꢀ10
ꢀ10
ꢀ20
0
2.3:1
2.8:1
1:2
2.3:1
2.4:1
2.8:1
ꢀ10
a
Ratios of trisaccharides 2 and 38 were determined by NMR analysis of isolated products. Yields were comprised in the range 63–70%.
to efficiently inhibit the binding between the native polymer and S.
aureus type 5 polysaccharide–CRM197 conjugate.
by TLC and NMR spectroscopy. Palladium chloride catalyst was
purchased from Sigma–Aldrich. Hydrogenation reactions were
performed in a continuous flow reactor H-Cube (Thalesnano)
instrument, using packed catalyst cartridges CatCart. Laminarin
polysaccharide was purchased from Sigma–Aldrich.
These findings highlight that synthetic glycans from S. aureus
type 5 capsular polysaccharide could be useful structures to be
studied as potential candidates for an anti S. aureus vaccine, how-
ever fragments longer than the single trisaccharide repeating unit
would be needed.
4.1.1. Allyl 2-O-acetyl-3,4,6-tri-O-benzyl-b-
D-glucopyranoside 7
Our conclusions are in line with results reported for other bac-
terial polysaccharides, as it has been observed that while in certain
cases small synthetic fragments are sufficient to elicit antibodies
that efficiently bind to the native polysaccharide,30 other examples
show that longer structures are required.31
3,4,6-Tri-O-benzyl-[1,2-O-(1-methoxyethylidene)]-a-D
-gluco-
pyranoside 6 (22.6 g, 0.045 mol) was dissolved under nitrogen
atmosphere in anhydrous allyl alcohol (47.5 ml). The solution was
cooled to 0 °C and TMSOTf (1.8 ml, 0.01 mmol) was slowly added.
The mixture was reacted at room temperature for 3 h, when TLC
(7:3 cyclohexane/EtOAc) showed the formation of a ꢂ1:1 (v/v) mix-
ture of the allyl 2-O-acetyl and allyl 2-O-hydroxy derivatives, as re-
ported in literature.20 The residue was dissolved in 4:1 (v/v) acetic
anhydride/pyridine (300 ml), and the mixture was stirred 3 h at
room temperature (TLC, 7:3 cyclohexane/EtOAc), when it was con-
centrated and purified on silica gel (9:1 cyclohexane/EtOAc), to give
4. Experimental section
4.1. General methods
All chemicals were of reagent grade, and were used without fur-
ther purification. Reactions were monitored by thin-layer chroma-
tography (TLC) on Silica Gel 60 F254 (Sigma–Aldrich); after
examination under UV light, compounds were visualized by heating
with 10% (v/v) ethanolic H2SO4. In the work up procedures, organic
solutions were washed with the amounts of the indicated aqueous
solutions, then dried with anhydrous Na2SO4, and concentrated un-
der reduced pressure at 30–50 °C on a water bath. Column chroma-
tography was performed on Silica Gel 60 (Sigma Aldrich, 0.040–
0.063 nm) or using pre-packed silica cartridges RediSep (Tele-
dyne-Isco, 0.040–0.063 nm) SiliaSep HP (Silicycle, 0.015–
0.040 nm) or Supelco (Sigma Aldrich, spherical silica 0.040–
0.075 nm). Unless otherwise specified, a gradient 0 ? 100% of the
elution mixture was applied in a Combiflash Rf (Teledyne-Isco)
instrument. 1H NMR spectra were measured at 400 MHz with a
Bruker Avance III 400 spectrometer; dH values are reported in
ppm, relative to internal Me4Si (dH = 0.00, CDCl3 and CD3OD); sol-
vent peak for D2O was calibrated at 4.79 ppm. 13C NMR spectra
were measured at 100 MHz with a Bruker AvanceIII 400 spectrom-
eter; dC values are reported in ppm relative to the signal of CDCl3
(dC = 77.0, CDCl3), CD3OD (dC = 49.0, CD3OD) or internal acetone
(dC = 30.9, D2O). Assignments of NMR signals were made by homo-
nuclear and heteronuclear 2-dimensional correlation spectroscopy,
run with the software supplied with the spectrometer. Assignment
of 13C NMR spectra of some compounds was aided by comparison
with spectra of related substances reported previously from this
laboratory or elsewhere.
compound 7 (21.2 g, 88% yield) as a pale yellow oil. ½a D23
ꢄ
= +4.8 (c
0.25, CHCl3). 1H NMR (CDCl3, 400 MHz): d = 7.35–7.16 (m, 15H,
Ph), 5.90–5.80 (m, 1H, CH@), 5.26–5.14 (m, 2H, CH2@), 5.03 (dd,
1H, J1,2 = 7.8 Hz, J2,3 = 9.03 Hz, H-2), 4.79, 4.67 (2 d, 2H,
2J = 11.4 Hz, CH2Ph), 4.78, 4.55 (d, 2H, 2J = 11.9 Hz, CH2Ph), 4.62,
4.55 (2 d, 2H, 2J = 11.9 Hz, CH2Ph), 4.41 (d, 1H, J1,2 = 7.8 Hz, H-1),
4.36–4.31 (m, 1H, CH2a), 4.10–4.04 (m, 1H, CH2b), 3.76–3.64 (m,
4H, H-3,4,6), 3.50–3.46 (m, 1H, H-5), 1.96 (s, 3H, CH3CO). 13C
NMR (CDCl3, 100 MHz): d = 169.40 (CO), 138.05, 137.99, 137.76
(Ar), 133.71 (CH@), 128.31, 128.25, 127.90, 127.71, 127.61,
127.50, 127.40 (Ar), 116.84 (CH2@), 99.75 (C-1), 82.86, 77.88 (C-
3,4), 75.04 (C-5), 74.91, 74.91 (CH2Ph), 73.36 (CH2Ph), 72.98 (C-2),
69.43 (CH2), 68.60 (C-6), 20.81 (CH3CO). ESI HR-MS (C32H36O7):
m/z = ([M+Na]+ found 555.2353; calcd 555.2359).
4.1.2. Allyl 2,6-di-O-acetyl-3,4-di-O-benzyl-b-D-glucopyranoside 8
Zinc chloride (5 g, 0.037 mol), freshly melted under a nitrogen
atmosphere, was dissolved in acetic acid (88 ml). A solution of com-
pound 7 (5.85 g, 0.011 mol) in acetic anhydride (175 ml) was then
added, and the mixture was stirred at room temperature for 12 h
when TLC (2:1 cyclohexane/EtOAc) showed complete reaction.
The mixture was then diluted with H2O (100 ml) and partitioned
with EtOAc (150 ml). The organic layer was separated and washed
with aq NaHCO3 (ꢅ4). Combined organic layers were concentrated,
and the crude residue was purified on silica gel (17:3 cyclohexane/
EtOAc) to give compound 8 (4.0 g, 76% yield) as pale yellow oil.
½
a 2D3
ꢄ
= ꢀ20.7 (c 0.20, CHCl3). 1H NMR (CDCl3, 400 MHz): d = 7.35–
When reporting assignments of NMR signals, sugar residues in
oligosaccharides are serially numbered, beginning with the one
bearing the aglycon, and are identified by a Roman numeral super-
script in listings of signal assignments. Nuclei associated with the
linker are denoted with a prime. Exact masses were measured by
electron spray ionization cut-off spectroscopy, using a Q-Tof micro
Macromass (Waters) instrument. Structures of these compounds
follow unequivocally from the mode of synthesis, NMR data and
m/z values found in their mass spectra, and their purity was verified
7.26 (m, 10H, Ph), 5.85–5.78 (m, 1H, CH@), 5.27–5.16 (m, 2H,
CH2@), 5.03 (dd, 1H, J1,2 = 8.3, J2,3 = 8.6 Hz, H-2), 4.83, 4.69 (2 d,
2H, 2J = 10.9 Hz, CH2Ph), 4.80, 4.57 (2 d, 2H, 2J = 11.2 Hz, CH2Ph),
4.42 (d, 1H, H-1), 4.37 (dd, 1H, J6a,6b = 11.9, J5,6a = 2.0 Hz, H-6a),
4.33–4.28 (m, 1H, CH2a), 4.21 (dd, 1H, J5,6b = 4.5 Hz, H-6b), 4.07–
4.00 (m, 1H, CH2b), 3.72–3.62 (m, 2H, H-3,4), 3.54–3.51 (m, 1H, H-
5), 2.04 (s, 3H, CH3CO), 1.98 (s, 3H, CH3CO). 13C NMR (CDCl3,
100 MHz): d = 170.76, 169.52 (CO), 137.93, 137.46 (Ar), 133.61

6408
E. Danieli et al. / Bioorg. Med. Chem. 20 (2012) 6403–6415
(C-2/3/4), 70.51 (CH2), 61.88 (C-6). ESI HR-MS (C23H28O6): m/
z = ([M+Na]+ found 423.1769 calcd 423.1784).
To a solution of 9 (4.75 g, 11.86 mmol) in dry DMF (95 ml), imid-
azole (1.21 g, 13.05 mmol) was added, under a nitrogen atmosphere.
The mixture was cooled down to 0 °C and t-butyldimethylsilyl chlo-
ride (1.97 g, 17.79 mmol) was added. The mixture was slowly
warmed up to room temperature and stirred for 2 days, when TLC
(3:1 cyclohexane/EtOAc) showed that the reaction was complete.
The mixture was diluted with H2O (50 ml), and partitioned with
EtOAc (100 ml ꢅ 2). Combined organic layers were washed with
aq NaHCO3, and concentrated. The crude residue was purified on sil-
ica gel (4:1 cyclohexane/EtOAc), to give compound 10 (5.77 g, 94%
yield) as pale yellow oil. ½a D23
ꢄ
= ꢀ15.5 (c 0.99, CHCl3). 1H NMR (CDCl3,
400 MHz): d = 7.39–7.26 (m, 10H, Ph), 5.99–5.89 (m, 1H, CH@),
5.33–5.20 (m, 2H, CH2@), 4.89, 4.86 (2 d, 2H, 2J = 11.2 Hz, CH2Ph),
4.85, 4.68 (2 d, 2H, 2J = 11.2 Hz, CH2Ph), 4.37–4.32 (m, 1H, CH2a),
4.30 (d, 1H, J1,2 = 7.5 Hz, H-1), 4.15–4.09 (m, 1H, CH2b), 3.87 (dd,
1H, J6a,6b = 11.5, J5,6a = 1.8 Hz, H-6a), 3.83 (dd, 1H, H-6b), 3.63–3.56
(m, 2H, H-3,4), 3.52 (dd, 1H, J2,3 = 7.9 Hz, H-2), 3.32–3.29 (m, 1H,
H-5), 2.30 (br s, 1H, OH-2), 1.26 (s, 9H, (CH3)3CSi), 0.07 (s, 3H, CH3Si),
0.06 (s, 3H, CH3Si). 13C NMR (CDCl3, 100 MHz): d = 138.58, 138.27
(Ar), 133.88 (CH@), 128.43 128.40, 127.96, 127.74, 127.71 (Ar),
117.79 (CH2@), 101.37 (C-1), 84.40, 77.28 (C-3,4), 76.05 (C-5),
75.18, 74.96 (CH2Ph), 74.60 (C-2), 69.85 (CH2), 62.17 (C-6), 25.84
((CH3)3CSi), 5.41 (CH3Si), 5.10 (CH3Si). ESI HR-MS (C29H42O6Si): m/
z = ([M+Na]+ found 537.2654; calcd 514.2648).
Figure 1. Competitive ELISA analysis of S. aureus type
5
polysaccharide
(IC50 = 3.4 ꢅ 10ꢀ5 mg/ml) and synthetic trisaccharide
1 at a starting inhibitor
concentrantion of 4.5 mg/ml and dilutions thereof. Laminarin was used as negative
control.
4.1.5. Allyl 3,4-di-O-benzyl-6-O-t-butyldimethylsilyl-2-O-
levulinoyl-b-D-glucopyranoside 11
Compound 10 (5.77 g, 11.21 mmol) was dissolved in dry CH2Cl2
(250 ml), then dicyclohexyl carbodiimide (3.0 g, 14.57 mmol), lev-
ulinic acid (1.69 g, 14.57 mmol), and dimethyl aminopyridine
(1.78 g, 15.57 mmol) were added. The mixture was stirred over-
night at room temperature (TLC, 2:1 cyclohexane/EtOAc). After fil-
tration, the solvent was evaporated and the crude was purified by
column chromatography (cyclohexane/EtOAc), to obtain com-
Figure 2. Immunodot blot assay with anti SA5 sera. A1: SA5 (100
l
g); B1: SA5
pound 11 (5.88 g, 85%) as a pale yellow oil. ½a D23
= ꢀ39.5 (c 1.08,
ꢄ
(10
(50
l
l
g); C1: laminarin (100
g each).
lg, negative control); A2, B2 and C2: trisaccharide 1
CHCl3). 1H NMR (CDCl3, 400 MHz): d = 7.35–7.25 (m, 10H, Ph),
5.88–5.81 (m, 1H, CH@), 5.25–5.14 (m, 2H, CH2@), 4.96 (dd, 1H,
J1,2 = 8.4, J2,3 = 8.6 Hz, H-2), 4.81, 4.67 (2 d, 1H, 2J = 10.2 Hz, CH2Ph),
4.80, 4.70 (2 d, 2H, 2J = 11.3 Hz, CH2Ph), 4.39 (d, 1H, J1,2 = 8.4 Hz, H-
1), 4.29–4.24 (m, 1H, CH2a), 4.11–4.03 (m, 1H, CH2b), 3.88–3.81 (m,
2H, H-6), 3.71–3.63 (m, 2H, H-3,4), 3.30–3.28 (m, 1H, H-5), 2.70–
2.67 (m, 2H, COCH2), 2.57–2.51 (m, 2H, CH2COO), 2.15 (s, 3H,
CH3CO), 1.60 (s, 9H, (CH3)3CSi), 0.06 (s, 3H, CH3Si) 0.05 (s, 3H,
CH3Si). 13C NMR (CDCl3, 100 MHz): d = 206.31 (CO), 171.48
(COO), 138.30, 138.17 (Ar), 133.98 (CH@), 128.77, 128.45, 128.35,
128.02, 127.94, 127.83, 127.65 (Ar), 117.03 (CH2@), 99.54 (C-1),
82.77, 77.66 (C-3,4), 79.94 (C-5), 75.05, 75.02 (CH2Ph), 73.55 (C-
2), 69.26 (CH2), 62.01 (C-6), 37.90 (CH2CO), 29.88 (CH3CO), 28.01
(COOCH2)), 25.88 ((CH3)3CSi), 5.40 (CH3Si), 5.06 (CH3Si). ESI HR-
MS (C34H48O8Si): m/z = ([M+Na]+ found 635.3047; calcd 635.3016).
(CH@), 129.32, 128.88, 128.52, 128.46, 128.15, 128.07 (Ar), 117.21
(CH2@), 99.72 (C-1), 82.98, 77.41 (C-3,4), 75.12, 75.04 (CH2Ph),
72.97 (C-2,5), 69.71 (CH2), 62.78 (C-6), 20.89, 20.84 (CH3CO). ESI
HR-MS (C27H32O8): m/z ([M+Na]+ found 507.1983; calcd 507.1995).
4.1.3. Allyl 3,4-di-O-benzyl-6-O-t-butyldimethylsilyl-b-D-
glucopyranoside 10
To a solution of compound 8 (4.0 g, 8.25 mol) in MeOH (150 ml),
a solution of NaOMe (1.8 g) in MeOH (20 ml) was added. The mix-
ture was stirred at 50 °C for 12 h, when TLC (2:1 cyclohexane/
EtOAc) showed the reaction was complete. The mixture was neu-
tralized with Dowex H+, then filtered and the solvent evaporated.
The crude mixture was chromatographed (cyclohexane/EtOAc) to
afford compound 9 (2.8 g, 85% yield).
4.1.6. (Allyl 3,4-di-O-benzyl-2-O-levulinoyl-b-D-glucopyranosid)
uronate 12
4.1.4. Allyl 3,4-di-O-benzyl-b-D-glucopyranoside 9
To a solution of 11 (4.68 g, 7.64 mmol) in acetone (230 ml) was
added Jones’ reagent at 0 °C [prepared by slow addition of H2SO4
(5.5 ml) to a solution of CrO3 (3.05 g, 30.56 mmol) in H2O (40 ml)
at 0 °C] and the mixture was stirred for 12 h at room temperature.
2-Propanol was then added until the colour of the solution turned
deep green/blue, then the mixture was filtered through Celite and
evaporated. The crude was chromatographated on silica gel (1%
AcOH/EtOAc) to give compound 12 (3.69 g, 94%) as a pale yellow
1H NMR (CDCl3, 400 MHz): d = 7.38–7.23 (m, 10H, Ph), 5.98–
5.88 (m, 1H, CH@), 5.34–5.20 (m, 2H, CH2@), 4.93, 4.85 (2 d, 2H,
2J = 11.2 Hz, CH2Ph), 4.87, 4.68 (2 d, 2H, 2J = 11.5 Hz, CH2Ph),
4.38–4.34 (m, 1H, CH2a), 4.35 (d, 1H, J1,2 = 7.7 Hz, H-1), 4.15–4.10
(m, 1H, CH2b), 3.86 (dd, 1H, J6a,6b = 12.0, J5,6a = 2.4 Hz, H-6a), 4.24
(dd, 1H, J5,6b = 4.4 Hz, H-6b), 3.63–3.51 (m, 3H, H-2,3,4), 3.39–
3.36 (m, 1H, H-5), 2.50 (br s, 1H, OH), 2.04 (br s, 1H, OH). 13C
NMR (CDCl3, 100 MHz): d = 138.48, 137.88 (Ar), 133.64 (CH@),
128.45 128.02, 127.89, 127.73 (Ar), 118.04 (CH2@), 101.80 (C-1),
84.23, 77.20 (C-2/3/4), 75.31 (C-5), 75.14, 75.03 (CH2Ph), 74.58
oil. ½a 2D3
ꢄ
= ꢀ45.5 (c 1.90, CHCl3). 1H NMR (CDCl3, 400 MHz):
d = 7.30–7.24 (m, 10H, Ph), 5.86–5.82 (m, 1H, CH@), 5.27–5.16
(m, 2H, CH2@), 5.04 (dd, 1H, J1,2 = 7.3, J2,3 = 8.2 Hz, H-2), 4.77–

E. Danieli et al. / Bioorg. Med. Chem. 20 (2012) 6403–6415
6409
4.62 (m, 4H, CH2Ph), 4.56 (d, 1H, J1,2 = 7.3 Hz, H-1), 4.34–4.28 (m,
1H, CH2a), 4.09–4.04 (m, 1H, CH2b), 4.04 (d, 1H, J4,5 = 8.4 Hz, H-5),
3.94 (dd, 1H, J3,4 = 8.8, J4,5 = 8.4 Hz, H-4), 3.72 (dd, 1H,
J2,3 = 8.3 Hz, H-3), 2.71–2.68 (m, 2H, CH2CO), 2.54–2.45 (m, 2H,
CH2COO), 2.17 (s, 3H, CH3CO). 13C NMR (CDCl3, 100 MHz):
d = 206.41 (CO), 172.66 (C-6), 171.33 (COO), 137.72, 137.23 (Ar),
133.21 (CH@), 128.38, 128.28, 128.02, 127.87, 127.83, 127.67,
127.61 (Ar), 117.54 (CH2@), 99.67 (C-1), 81.13 (C-3), 78.39 (C-4),
74.78, 74.56 (CH2Ph), 74.13 (C-5), 73.13 (C-2), 69.98 (CH2), 37.70
(CH2CO), 29.70 (CH3CO), 27.76 (CH2COO). ESI HR-MS (C28H32O9):
([M+Na]+ found 535.1921; calcd 535.1944).
To a solution of compound 14 (1.9 g, 3.4 mmol) in dry CH2Cl2,
trichloroacetonitrile (1.02 ml, 10 mmol) and DBU (51 l,
l
0.34 mmol) were added, under a nitrogen atmosphere. The mix-
ture was stirred at room temperature for 4 h, when TLC (1:1 cyclo-
hexane/EtOAc) showed complete reaction. The solvent was then
evaporated, and the crude residue was chromatographed (cyclo-
hexane/EtOAc, containing 0.5% Et3N), to give compound 3 (1.58 g,
66%) as a colourless oil. ½a D23
= ꢀ7.6 (c 1.10, CHCl3).
ꢄ
1H NMR (CDCl3, 400 MHz): d = 7.34–7.10 (m, 15H, Ph), 6.53 (d,
1H, J1,2 = 3.5 Hz, H-1), 5.16 (s, 2H, CH2Ph), 5.10 (dd, 1H,
J2,3 = 9.9 Hz, H-2), 4.81, 4.76 (2 d, 2H, 2J = 11.4 Hz, CH2Ph), 4.71,
4.45 (2 d, 2H, 2J = 10.6 Hz, CH2Ph), 4.46 (d, 1H, J4,5 = 10.0 Hz, H-5),
4.10 (dd, 1H, J3,4 = 9.4 Hz, H-3), 3.90 (dd, 1H, J4,5 = 9.7 Hz, H-4),
2.68–2.40 (m, 4H, CH2CH2) 2.14 (s, 3H, CH3CO). 13C NMR (CDCl3,
100 MHz): d = 205.90 (CO), 171.73 (COO), 168.08 (C-6), 160.60
(C@N), 137.83, 137.24, 134.78, 128.96, 128.53, 128.48, 128.34,
128.15, 127.94, 127.90, 127.84, 127.78 (Ar, CCl3), 93.43 (C-1),
78.90, 78.47 (C-4, C-3), 75.40, 75.35 (CH2Ph), 72.83 (C-5), 71.90
(C-2), 67.48 (CH2Ph), 37.57 (CH2CO), 29.73 (CH3CO), 27.46
(COOCH2). ESI HR-MS (C34H33Cl3NO9): m/z = ([M+K]+ 704.0936;
calcd 744.0965); ([M+Na]+ found 728.1135; calcd 728.1197).
4.1.7. Benzyl (allyl 3,4-di-O-benzyl-2-O-levulinoyl-b-D-glucopyr
anosid)uronate 13
To a solution of compound 12 (3.64 g, 7.10 mmol) in dry DMF
(37 ml), NaHCO3 (1.79 g, 21.3 mmol) and benzyl bromide (4.2 mL,
35.5 mmol) were added. The mixture was stirred at room temper-
ature for 4 h, monitoring by TLC (1:1 cyclohexane/EtOAc). The sol-
vent was then evaporated, and the obtained residue was dissolved
in EtOAc (50 mL) and washed with aq NaHCO3. Combined organic
layers were again concentrated, and the crude mixture was puri-
fied on silica gel (6:1 cyclohexane/EtOAc) to give compound 13
(2.8 g, 65% yield). White crystals from EtOAc: mp 44–45 °C.
4.1.10. Allyl 2,3-O-isopropylidene-
Compound 15 was prepared modifying a procedure described in
literature.23
suspension of rhamnose monohydrate (20 g,
a-L-rhamnopyranoside 15
½
a 2D3
ꢄ
= ꢀ47.7 (c 0.67, CHCl3). 1H NMR (CDCl3, 400 MHz): d = 7.32–
7.10 (m, 15H, Ph), 5.89–5.78 (m, 1H, CH@), 5.26–5.15 (m, 2H,
CH2@), 5.18 (s, 2H, CH2Ph), 5.05 (dd, 1H, J1,2 = 7.6, J2,3 = 9.1 Hz, H-
2), 4.84, 4.52 (2 d, 2H, 2J = 11.4 Hz, CH2Ph), 4.75, 4.73 (2 d, 2H,
2J = 10.7 Hz, CH2Ph), 4.53 (d, 1H, H-1), 4.32–4.27 (m, 1H, CH2a),
4.07–4.02 (m, 1H, CH2b), 3.98–3.85 (m, 2H, H-4,5), 3.68 (m, 1H,
H-3), 2.70–2.67 (m, 2H, COCH2), 2.55–2.40 (m, 2H, CH2COO), 2.15
(s, 3H, CH3CO). 13C NMR (CDCl3, 100 MHz): d = 206.16 (CO),
171.31 (COO), 168.19 (C-6), 137.93, 137.59, 136.00 (Ar), 133.41
(CH@), 128.57, 128.51, 128.34, 128.31, 128.15, 127.90, 127.83,
127.77, 127.72 (Ar), 117.46 (CH2@), 100.01 (C-1), 81.82 (C-3),
79.12 (C-4/5), 74.96, 74.84 (CH2Ph), 74.61 (C-4/5), 73.18 (C-2),
69.93 (CH2), 67.35 (CH2Ph), 37.79 (CH2CO), 29.81 (CH3CO), 27.85
(CH2COO). ESI HR-MS (C35H38O9): m/z = ([M]+ found 602.2532;
calcd 602.2516).
A
108 mmol) in AllOH (150 ml) was refluxed for 2 h in presence of
Dowex H+ (6 g), monitoring by TLC (1:1 CH2Cl2/acetone). The mix-
ture was filtered, and the filtrate was concentrated to be used for
the next step.
To a solution of the foregoing allyl rhamnoside (108 mmol) in
1:1 (v/v) 2,2-dimethoxypropane-acetone (160 ml), BF3ꢁEt2O
(0.5 ml) was added, and the mixture was stirred for 1 h, at which
time TLC (2:1 cyclohexane/EtOAc) showed that the reaction was
complete. The mixture was neutralized with triethylamine
(0.5 ml), filtered and the filtrate was concentrated. Chromatogra-
phy of the residue (4:1 cyclohexane/EtOAc) gave the product 15
(19.3 g, 73% over two steps), whose structure was confirmed by
NMR (spectra not reported in literature).22 1H NMR (CDCl3,
400 MHz): d = 5.89–5.86 (m, 1H, CH@), 5.29–5.17 (m, 2H, CH2@),
4.96 (s, 1H, H-1), 4.30–4.17 (m, 2H, CH2a, incl. d, 4.15, J2,3 = 5.5 Hz,
H-2), 4.06 (dd, 1H, J3,4 = 7.1 Hz, H-3), 4.02–3.97 (m, 1H, CH2b),
3.74–3.65 (m, 1H, H-5), 3.38–3.43 (ddd, 1H, J4,5 = 9.3, J4,OH = 5.4 Hz,
H-4), 2.64 (d, 1H, OH-4), 1.53 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.29 (d,
3H, J5,6, = 6.6 Hz, H-6). 13C NMR (CDCl3, 100 MHz): d = 133.53
(CH@), 117.80 (CH2@), 109.44 (C(CH3)2), 96.18 (C-1), 86.03 (C-3),
75.77 (C-2), 74.42 (C-4), 67.89 (CH2), 65.89 (C-5), 27.92, 26.08 (2
CH3), 17.41 (C-6).
4.1.8. Benzyl (3,4-di-O-benzyl-2-O-levulinoyl-a-D-glucopyrano
syl trichloroacetimidate) uronate 3
To a solution of compound 13 (2.4 g, 4.4 mmol) in 20:1 (v/v)
AcOH/H2O (134 ml), AcONa (22 g) and PdCl2 (1.8 g, 12.0 mmol)
were added. The mixture was stirred at room temperature for
24 h, when TLC (1:1 cyclohexane/EtOAc) showed that the reaction
was complete. The mixture was diluted with EtOAc and filtered
over celite. The recovered solution was washed with aq NaHCO3,
and combined organic layers were concentrated. The crude prod-
uct was chromatographed (cyclohexane/EtOAc), to give compound
4.1.11. Allyl 4-deoxy-2,3-O-isopropylidene-a-L-talopyranoside 16
14 (1.9 g, 77% yield) as a colourless oil mainly in
a
-configuration.
To a mixture of oxalyl chloride (10.2 ml, 122 mmol) in CH2Cl2
(50 ml), DMSO (17 ml, 244 mmol) in CH2Cl2 (50 ml) was added
drop-wise at ꢀ78 °C. After stirring at the same temperature for
30 min, the sugar (15 g, 61 mmol) dissolved in CH2Cl2 (150 ml)
was added drop-wise and stirring was continued for 30 min at
ꢀ78 °C. Di-i-propylethylamine (43 ml, 244 mmol) was added
drop-wise and stirring was continued for 12 h at room temperature.
After TLC (2:1 cyclohexane/EtOAc) showed complete reaction, the
crude mixture was washed with 10% Na2S2O3, and the organic lay-
ers concentrated to be used for the next step. A small portion
(50 mg) of the crude material was purified on silica gel (cyclohex-
ane/EtOAc) to be used for characterization. 1H NMR (CDCl3,
400 MHz): d = 5.94–5.86 (m, 1H, CH@), 5.34–5.24 (m, 2H, CH2@),
5.02 (s, 1H, H-1), 4.50–4.46 (m, 2H, H-2,3), 4.33–4.20 (m, 2H, H-5,
CH2a), 4.11–4.06 (m, 1H, CH2b), 1.53 (s, 3H, CH3), 1.40 (d, 3H,
J5,6, = 6.5 Hz, H-6), 1.37 (s, 3H, CH3). 13C NMR (CDCl3, 100 MHz):
d = 204.67 (CO), 132.90 (CH@), 118.26 (CH2@), 111.36 (C(CH3)2),
4.1.9. Benzyl 3,4-di-O-benzyl-2-O-levulinoyl-a-D-glucopyranosyl
uronate 14
1H NMR (CDCl3, 400 MHz): d = 7.33–7.12 (m, 15H, Ph), 5.43 (d,
1H, J1,2 = 3.1 Hz, H-1), 5.14 (s, 2H, CH2Ph), 4.87 (dd, 1H, J1,2 = 3.3,
J2,3 = 9.3 Hz, H-2), 4.77, 4.74 (2 d, 2H, 2J = 11.0 Hz, CH2Ph), 4.75,
4.48 (2 d, 2H, 2J = 11.0 Hz, CH2Ph), 4.51 (d, 1H, J4,5 = 9.1 Hz, H-5),
4.06 (dd, 1H, J2,3 = 9.3, J3,4 = 9.0 Hz, H-3), 3.84 (dd, 1H, H-4), 3.63
(br s, 1H, OH), 2.72–2.39 (m, 4H, CH2CH2), 2.14 (s, 3H, CH3CO).
13C NMR (CDCl3, 100 MHz): d = 206.88 (CO), 171.96 (COO),
169.13 (C-6), 138.14, 137.58, 134.89, 128.46, 128.36, 128.27,
128.21, 127.74, 127.64, 127.58 (Ar), 90.38 (C-1), 79.10 (C-4),
78.57 (C-3), 75.22, 74.81 (CH2Ph), 73.17 (C-2), 70.52 (C-5), 67.23
(CH2Ph), 37.81 (CH2CO), 29.66 (CH3CO), 27.78 (CH2COO). ESI HR-
MS (C32H34O9): m/z = ([M+K]+ found 601.1792; calcd 601.1840).

6410
E. Danieli et al. / Bioorg. Med. Chem. 20 (2012) 6403–6415
95.91 (C-1), 78.74 (C-2), 75.89 (C-3), 69.98 (C-5), 68.14 (CH2), 26.69,
25.44 (2 CH3), 15.80 (C-6). ESI MS (C12H18O5): m/z = found ([M+H]+
243.1260; calcd 243.1232).
The foregoing triflate 19 was re-dissolved in toluene (25 ml),
and tetrabutylammonium azide (8.5 g, 30 mmol) was added. The
resulting suspension was stirred for 12 h at 60 °C, when TLC (6:1
cyclohexane/EtOAc) showed the reaction was complete. The mix-
ture was concentrated and purified on silicagel (99:1 ? 9:1 cyclo-
hexane/EtOAc) to yield 1.75 g of product 20 as a sirup (64%).
The foregoing cheto compound 16 (62 mmol) was dissolved in
EtOH (250 ml), and NaBH4 (122 g, 62 mmol) was added portion
wise at 0 °C. The mixture was stirred for 1 h when TLC (4:1 cyclo-
hexane/EtOAc) showed the reaction was complete. The mixture
was filtered and the filtrate concentrated and purified on silica
gel (99:1 ? 7:3 cyclohexane/EtOAc) to give the talo sugar 17
½
a 2D3
ꢄ
= ꢀ48.1 (c 0.25, CHCl3).
1H NMR (CDCl3, 400 MHz): d = 5.94–5.85 (m, 1H, CH@), 5.33–
5.19 (m, 2H, CH2@), 4.86 (d, 1H, J1,2 = 3.4 Hz, H-1), 4.36 (dd, 1H,
J2,3 = 8.9, J3,4 = 5.2 Hz, H-3), 4.30–4.10 (m, 2H, CH2a, H-5), 4.06
(dd, 1H, J4,5 = 2.5 Hz, H-4), 4.03–3.98 (m, 1H, CH2b), 3.32 (dd, 1H,
H-2), 1.52 (s, 3H, CH3), 1.35–1.33 (m, 6H, CH3, H-6). 13C NMR
(CDCl3, 100 MHz): d = 133.24 (CH@), 117.74 (CH2@), 109.37
(C(CH3)2), 96.80 (C-1), 75.56 (C-4), 73.57 (C-3), 68.57 (CH2), 63.49
(C-5), 61.16 (C-2), 28.37, 26.26 (2 CH3), 16.24 (C-6). ESI HR-MS
(C12H19N3O4): m/z = found ([M-N2+H]+ 242.1332; calcd 242.1392).
FT-IR: 2108.78 cmꢀ1 (N3).
(14.1 g, 94%), as colourless sirup. ½a D23
= ꢀ53.0 (c 0.25, CHCl3).
ꢄ
1H NMR (CDCl3, 400 MHz): d = 5.90–5.80 (m, 1H, CH@), 5.27–
5.14 (m, 2H, CH2@), 5.02 (s, 1H, H-1), 4.18–4.11 (m, 2H, CH2a, incl.
t, 4.15, J = 6.5 Hz, H-3), 4.01–3.95 (m, 2H, CH2b, incl. d, 4.00,
J2,3 = 6.0 Hz, H-2), 3.83–3.78 (m, 1H, H-5), 3.49 (t, 1H, J = 5.6 Hz,
H-4), 2.23 (d, 1H, J4,OH = 6.0 Hz, OH-4), 1.52 (s, 3H, CH3), 1.32 (s,
3H, CH3). 1.26 (d, 3H, J5,6 = 6.6 Hz, H-6). 13C NMR (CDCl3,
100 MHz): d = 133.53 (CH@), 117.56 (CH2@), 109.05 (C(CH3)2),
96.43 (C-1), 73.09 (C-2), 72.78 (C-3), 68.01 (CH2), 66.64 (C-5),
64.25 (C-4), 25.64, 25.08 (2 CH3), 16.48 (C-6). ESI HR-MS
(C12H20O5): m/z = found ([M+Na]+ 267.1166; calcd 267.1208).
4.1.14. Allyl 2-azido-2-deoxy-a-L-fucopyranoside 21
A solution of fucopyranoside 20 (3.5 g, 13 mmol) in 9:1 (v/v)
AcOH–H2O (50 ml) was stirred for 6 h at 50 °C, when TLC (1:1 cyclo-
hexane/EtOAc) showed the reaction was complete. The mixture was
concentrated co-evaporating with toluene, and the residue was
purified on silicagel (cyclohexane/EtOAc) to afford 2.55 g of product
4.1.12. Allyl 6-deoxy-3,4-O-isopropylidene-a-L-talopyranoside 18
A solution of talopyranoside 17 (1 g, 0.61 mmol) in 10% AcOH
(10 ml) was stirred at 50 °C for 5 h, monitoring by TLC (1:1 cyclo-
hexane/EtOAc). The mixture was concentrated co-evaporating
with toluene, then the residue was re-dissolved in 1:1 (v/v) 2,2-
dimethoxypropane-acetone (20 ml), and p-toluensulfonic acid
(100 mg) was added. After stirring for 30 min, TLC (2:1 cyclohex-
ane/EtOAc) showed that the reaction was complete, and the mix-
ture was neutralized with triethylamine and concentrated.
Chromatography of the residue (cyclohexane/EtOAc) yielded
21 (86%). White crystals from EtOAc, mp 87–88 °C. ½a D23
= ꢀ37.0 (c
ꢄ
0.25, CHCl3). 1H NMR (CDCl3, 400 MHz): d = 5.99–5.87 (m, 1H,
CH@), 5.39–5.20 (m, 2H, CH2@), 4.96 (d, 1H, J1,2 = 3.7 Hz, H-1),
4.23–4.17 (m, 1H, CH2a), 4.11–3.95 (m, 3H, CH2b, H-5, incl. dd,
4.08, J2,3 = 10.7 Hz, J3,4 = 3.1 Hz, H-3), 3.82 (dd, 1H, H-4), 3.47 (dd,
1H, H-2), 2.93 (br s, 2H, OH-3,4), 1.29 (d, 3H, J5,6 = 6.6 Hz, H-6). 13
C
NMR (CDCl3, 100 MHz): d = 133.35 (CH@), 117.69 (CH2@), 97.03
(C-1), 71.82 (C-4), 68.62, 68.58 (C-3, CH2), 65.84 (C-5), 60.09 (C-2),
16.05 (C-6). ESI HR-MS (C9H15N3O4): m/z = found ([M-N2+H]+
202.1029; calcd 202.1079).
800 mg of syrupy product 18 (80%). ½a D23
= ꢀ16.5 (c 2.00, CHCl3).
ꢄ
1H NMR (CDCl3, 400 MHz): d = 5.99–5.89 (m, 1H, CH@), 5.34–5.18
(m, 2H, CH2@), 4.81 (d, 1H, J1,2 = 5.4 Hz, H-1), 4.52 (dd, 1H,
J2,3 = 3.5, J3,4 = 7.5 Hz, H-3), 4.29–4.24 (m, 1H, CH2a), 4.12 (dd, 1H,
J4,5, = 2.0 Hz, H-4), 4.08–4.03 (m, 1H, CH2b), 3.88–3.83 (m, 1H, H-
5), 3.71–3.74 (m, 1H, H-2), 2.37 (br s, 1H, OH-2), 1.53 (s, 3H,
CH3), 1.37 (s, 3H, CH3). 1.27 (d, 3H, J5,6, = 6.5 Hz, H-6). 13C NMR
(CDCl3, 100 MHz): d = 134.28 (CH@), 117.19 (CH2@), 109.89
(C(CH3)2), 99.91 (C-1), 76.10 (C-4), 73.51 (C-3), 68.72 (C-2), 68.49
(CH2), 65.09 (C-5), 26.01, 25.16 (2 CH3), 15.80 (C-6). ESI HR-MS
(C12H20O5): m/z = found ([M+Na]+ 267.1158; calcd 267.1208).
4.1.15. Allyl 3-O-acetyl-2-azido-2-deoxy-
To a solution of diol 21 (500 mg, 2.18 mmol) in 4:1 (v/v) dichlo-
romethane-pyridine (25 ml), a solution of AcCl (185 l, 2.62 mmol)
a-L-fucopyranoside 4
l
in dichloromethane (5 ml) was added drop-wise at 0 °C. After stir-
ring for 3 h at the same temperature, the reaction was complete
(TLC, 1:1 cyclohexane/EtOAc). The mixture was partitioned with
aq NaHCO3, and combined organic layers were filtered and concen-
trated. The residue was chromatographed (99:1 ? 4:1 cyclohex-
ane/EtOAc) to give 585 mg of product
4 as a sirup (99%).
4.1.13. Allyl 2-azido-2-deoxy-3,4-O-isopropylidene-a-L-
fucopyranoside 20
½
a 2D3
ꢄ
= ꢀ65.6 (c 2.00, CHCl3). 1H NMR (CDCl3, 400 MHz): d = 5.98–
5.88 (m, 1H, CH@), 5.38–5.21 (m, 3H, CH2=, incl. dd, 5.32,
J2,3 = 10.9, J3,4 = 2.9 Hz, H-3), 4.99 (d, 1H, J1,2 = 3.6 Hz, H-1), 4.24–
4.19 (m, 1H, CH2a), 4.12–4.09 (m, 2H, CH2b, H-5), 3.68 (dd, 1H, H-
2), 3.94 (d, 1H, H-4), 2.22–2.14 (m, 4H, CH3CO, OH-4), 1.28 (d,
3H, J5,6 = 6.5 Hz, H-6). 13C NMR (CDCl3, 100 MHz): d = 169.95
(CO), 133.34 (CH@), 117.91 (CH2@), 97.13 (C-1), 71.23 (C-3),
70.92 (C-4), 70.14 (CH2), 65.73 (C-5), 57.14 (C-2), 20.98 (CH3CO),
15.93 (C-6). ESI HR-MS (C11H17N3O5): m/z = found 244.2132 ([M-
N2+H]+; calcd 244.1185).
To a solution of compound 18 (2.5 g, 12.2 mmol) in 6:1 (v/v)
CH2Cl2–pyridine (28 ml), Tf2O (2.5 ml, 15 mmol) was added at
0 °C. The mixture was stirred for 30 min at room temperature,
when TLC (5:1 cyclohexane/EtOAc) showed the reaction was com-
plete. The mixture was washed with iced aq NaHCO3, and the com-
bined organic layers were concentrated at 30 °C, then at the high
vacuum pump to be used for the next step.
4.1.13.1.
Allyl
6-deoxy-3,4-O-isopropylidene-2-O-trif-
1H
-L-talopyranoside 19. NMR
luoromethansulfonyl-
a
4.1.16. 3-(Benzyloxycarbonyl)aminopropyl 2,3,4-tri-O-acetyl-b-
(CDCl3, 400 MHz): d = 5.93–5.83 (m, 1H, CH@), 5.39–5.22 (m, 2H,
CH2@), 5.00 (d, 1H, J1,2 = 5.7 Hz, H-1), 4.82 (dd, 1H, J2,3 = 3.1, H-2),
4.64 (dd, 1H, J3,4 = 7.4 Hz, H-3), 4.28–4.23 (m, 1H, CH2a), 4.19 (dd,
1H, J4,5 = 2.0 Hz, H-4), 4.08–4.02 (m, 1H, CH2b), 3.88–3.85 (m, 1H,
H-5), 1.53 (s, 3H, CH3), 1.37 (s, 3H, CH3), 1.26 (d, 3H, J5,6, = 6.7 Hz,
H-6). 13C NMR (CDCl3, 100 MHz): d = 133.41 (CH@), 118.36 (q,
CF3), 116.79 (CH2@), 111.28 (C(CH3)2), 95.81 (C-1), 83.41 (C-2),
75.54 (C-4), 71.56 (C-3), 68.75 (CH2), 65.62 (C-5), 25.86, 25.26 (2
CH3), 15.45 (C-6). ESI HR-MS (C13H19F3O7S): m/z = found
([M+H2O+H]+ 395.1354; calcd 395.0987).
D
-fucopyranoside 29
To a solution of fucose peracetate 28 (24 g, 72 mmol) and ben-
zyl N-(3-hydroxypropyl)carbamate (22.5 g, 108 mmol) in CH2Cl2
(100 ml), BF3ꢁEt2O (13.3 ml, 108 mmol) was added at 0 °C. The mix-
ture was stirred at room temperature for 2 h, when TLC (3:2 cyclo-
hexane/EtOAc) showed that the reaction was complete. After
neutralization with triethylamine (13.3 ml), the crude mixture
was concentrated and purified on silica gel (6:1 ? 1:1 cyclohex-
ane/EtOAc) to give 26.1 g of 29 (72%). ½a D23
= ꢀ31.9 (c 3.90, CHCl3).
ꢄ
1H NMR (CD3OD, 400 MHz): d = 7.40–7.29 (m, 5H, Ph), 5.22 (d, 1H,

E. Danieli et al. / Bioorg. Med. Chem. 20 (2012) 6403–6415
6411
J3,4 = J4,5 = 3.5 Hz, H-4), 5.18 (dd, 1H, J1,2 = 7.9, J2,3 = 10.5 Hz, H-2),
5.13–5.08 (m, 3H, NH, CH2Ph), 5.00, (dd, 1H, H-3), 4.42 (d, 1H,
J1,2 = 7.9 Hz, H-1), 3.98–3.92 (m, 1H, H-10a), 3.82–3.73 (m, 1H, H-
5), 3.59–3.54 (m, 1H, H-10b), 3.38–3.20 (m, 2H, H-30), 2.16, 2.02,
1.98 (3 s, 3H each, 3 CH3), 1.86–1.74 (m, 2H, H-20), 1.22 (d, 3H,
J5,6 = 6.7 Hz, H-6). 13C NMR (CD3OD, 100 MHz): d = 170.69,
170.22, 169.69, 156.58 (4 CO), 136.66, 128.47, 128.03 (Ph),
100.97 (C-1), 71.27 (C-3), 70.18 (C-4), 69.20 (C-2), 68.87 (C-5),
67.46 (C-10), 66.52 (CH2), 38.29 (C-30), 29.44 (C-20), 20.67, 20.62
(3 CH3), 16.00 (C-6). ESI HR-MS (C23H31NO10): m/z = found
([M+Na]+ 504.1874; calcd 504.1846).
TLC (3:1 CH2Cl2/acetone) showed the reaction was complete and
one product was formed. The mixture was diluted with CH2Cl2
(100 ml) and washed with brine. Combined organic layers were
concentrated and purified on silica gel (1:1 cyclohexane/EtOAc)
to give 11.3 g of product 32 (81%). ½a D23
= ꢀ20.3 (c 1.20, CHCl3).
ꢄ
1H NMR (CDCl3, 400 MHz): d = 7.36–7.26 (m, 5H, Ph), 5.35 (br t,
1H, J = 5.0 Hz, NH), 5.09 (s, 2H, CH2Ph), 4.44 (s, 1H, H-1), 4.21 (t, 1H,
J = 5.2 Hz, H-3), 4.04 (dd, 1H, J3,4 = 6.0, J4,5 = 2.1 Hz, H-4), 3.96–3.90
(m, 1H, H-10a), 3.83–3.72 (m, 2H, H-2,5), 3.64–3.59 (m, 1H, H-10b),
3.41–3.27 (m, 2H, H-30), 2.47 (d, H, J2,OH = 8.4 Hz, OH-2), 1.86–1.75
(m, 2H, H-20), 1.59 (s, 3H, CH3), 1.39 (d, 3H, J5,6 = 6.5 Hz, H-6), 1.36
(s, 3H, CH3). 13C NMR (CDCl3, 100 MHz): d = 156.53 (CO), 136.74,
128.51, 128.43, 128.09, 127.94 (Ph), 109.79 (C(CH3)2), 99.78 (C-
1), 74.16 (C-4), 73.82 (C-3), 68.45 (C-5), 67.39 (C-10), 66.41 (C-2),
66.34 (CH2), 38.45 (C-30), 29.48 (C-20), 25.57, 25.26 (CH3), 16.53
(C-6). ESI HR-MS (C20H29NO7): m/z = found ([M+Na]+ 396.2025;
calcd 396.2022).
4.1.17. 3-(Benzyloxycarbonyl)aminopropyl 3,4-O-
isopropylidene–D-fucopyranoside 30
To a solution of 3-(benzyloxycarbonyl)aminopropyl
D
-fucopyr-
anoside 29 (20.9 g, 43 mmol) in MeOH (100 ml), 1 M NaOMe in
MeOH was added until pH was 10. The mixture was stirred for
2 h, then TLC (1:1 cyclohexane/EtOAc) showed the reaction was
complete. The mixture was neutralized with Dowex H+, filtered
and the filtrate was concentrated.
4.1.19. 3-(Benzyloxycarbonyl)aminopropyl 2-azido-2-deoxy-
3,4-O-isopropylidene–D-fucopyranoside 34
The residue was re-dissolved in 2,2-dimethoxypropane
(100 ml), to which p-TsOH (1.2 g) was added. After 1 h (TLC, 1:1 tol-
uene/EtOAc or 4:1 CH2Cl2/acetone) the reaction went to comple-
tion. The mixture was concentrated and chromatography of the
residue (3:1 CH2Cl2/acetone) gave 14.8 g of product 30 (87%).
To a solution of compound 32 (4.4 g, 11.1 mmol) in 6:1 (v/v)
CH2Cl2/pyridine (60 ml), Tf2O (2.8 ml, 16.7 mmol) was added at
0 °C. The mixture was stirred for 30 min at room temperature, at
which time TLC (1:1 cyclohexane/EtOAc) showed that the reaction
was complete. The mixture was washed with iced aq NaHCO3, and
the combined organic layers were concentrated at 30 °C.
½
a 2D3
ꢄ
= ꢀ13.5 (c 0.40, CHCl3). 1H NMR (CDCl3, 400 MHz): d = 7.37–
7.15 (m, 5H, Ph), 5.14 (br t, 1H, J = 4.9 Hz, NH), 5.09 (s, 2H, CH2Ph),
4.11 (d, 1H, J1,2 = 8.4 Hz, H-1), 4.04 (dd, 1H, J2,3 = 10.5, J3,4 = 5.4 Hz,
H-3), 3.98 (dd, 1H, J4,5 = 2.1 Hz, H-4), 3.97–3.94 (m, 1H, H-10a),
3.87–3.81 (m, 1H, H-5), 3.63–3.57 (m, 1H, H-10b), 3.52 (d, 1H,
J = 7.0 Hz, H-2), 3.49–3.42 (m, 1H, H-3a0), 3.29–3.21 (m, 1H, H-
3b0), 3.00 (s, 1H, OH-2), 1.89–1.67 (m, 2H, H-20), 1.49 (s, 3H, CH3),
1.40 (d, 3H, J5,6 = 6.4 Hz, H-6), 1.31 (s, 3H, CH3). 13C NMR (CDCl3,
100 MHz): d = 156.66 (CO), 136.59, 129.04, 128.48, 128.21, 128.10
(Ph), 109.83 (C(CH3)2), 102.27 (C-1), 78.93 (C-3), 76.68 (C-4),
73.48 (C-2), 69.14 (C-5), 66.87 (C-10), 66.65 (CH2), 38.87 (C-30),
29.58 (C-20), 28.20, 26.33 (CH3), 16.54 (C-6). ESI HR-MS
(C20H29NO7): m/z = found ([M+H]+ 396.2017; calcd 396.2022).
The structure of the compound 30 was confirmed by acetyla-
tion. 1H NMR showed a shift of H-2 to 4.83 ppm (t, J = 8.0 Hz).
4.1.19.1. 3-(Benzyloxycarbonyl)aminopropyl 6-deoxy-3,4-O-iso-
propylidene-2-O-trifluoromethansulfonyl–
33.
D-talopyranoside
1H NMR1H NMR (CDCl3, 400 MHz): d = 7.28–7.13 (m, 5H,
Ph), 5.18 (br t, J = 5.0 Hz, 1H, NH), 5.06 (s, 2H, CH2Ph), 4.83 (t, 1H,
J = 5.7 Hz, H-3), 4.36 (s, 1H, H-1), 4.32 (d, 1H, J3,4 = 5.7 Hz, H-2),
3.99 (dd, 1H, J3,4 = 6.0, J4,5 = 2.7 Hz, H-4), 3.97–3.93 (m, 1H, H-
10a), 3.84–3.79 (m, 1H, H-5), 3.60–3.54 (m, 1H, H-10b), 3.42–3.35
(m, 1H, H-30a), 3.27–3.19 (m, 1H, H-30b), 1.77–1.68 (m, 2H, H-20),
1.58 (s, 3H, CH3), 1.44 (d, 3H, J5,6 = 6.7 Hz, H-6), 1.36 (s, 3H, CH3).
13C NMR (CDCl3, 100 MHz): d = 156.58 (CO), 136.75, 129.04,
128.48, 128.22, 127.95 (Ph), 118.48 (q, CF3), 110.82 (C(CH3)2),
97.58 (C-1), 78.79 (C-3), 72.74 (C-4), 71.06 (C-2), 69.80 (C-5),
66.97 (C-10), 66.47 (CH2), 37.63 (C-30), 29.38 (C-20), 25.65, 25.02
(CH3), 16.23 (C-6). ESI HR-MS (C21H28NF3O9S): m/z = found
([M+Na]+ 550.1346; calcd 550.1335).
4.1.18. 3-(Benzyloxycarbonyl)aminopropyl 6-deoxy-3,4-O-
isopropylidene–
D
-talopyranoside 32
The foregoing residue was dissolved in DMF (15 ml), to which
NaN3 (4.3 g, 66.6 mmol) was added. The mixture was stirred for
6 h at 60 °C, when TLC (1:1 cyclohexane/EtOAc) showed a new spot
had been formed. The mixture was concentrated and purified on
silica gel (7:3 cyclohexane/EtOAc) to give 4.2 g of product 34 as a
To a mixture of oxalyl chloride (6 ml, 70.8 mmol) in CH2Cl2
(40 ml), DMSO (10 ml, 141 mmol) in CH2Cl2 (40 ml) was added
drop-wise at ꢀ78 °C. After stirring at the same temperature for
30 min, compound 30 (14 g, 35.4 mmol) dissolved in CH2Cl2
(20 ml) was added drop-wise and stirring was continued for
30 min at ꢀ78 °C. DIPEA (24 ml) was gently dropped, and the mix-
ture was warmed up to room temperature. After stirring for 3 h TLC
(4:1 CH2Cl2/acetone) showed the reaction was complete. The crude
mixture was washed with 10% NaS2O3, and the organic layers were
sirup (90%).
½
a 2D3
ꢄ
= ꢀ10.1 (c 1.60, CHCl3). 1H NMR (CDCl3,
400 MHz): d = 7.38–7.30 (m, 5H, Ph), 5.18 (br t, 1H, J = 5.6 Hz,
NH), 5.09 (s, 2H, CH2Ph), 4.17 (d, 1H, J1,2 = 8.6 Hz, H-1), 4.00–3.95
(m, 1H, H-10a), 3.95 (d, 1H, J3,4 = 5.1, J4,5 = 2.1 Hz, H-4), 3.90 (dd,
1H, J2,3 = 8.2 Hz, H-3), 3.85–3.79 (m, 1H, H-5), 3.63–3.58 (m, 1H,
H-10b), 3.43–3.27 (m, 3H, H-30, incl. t, 3.37, J = 8.1 Hz, H-2), 1.88–
1.83 (m, 2H, H-20), 1.55 (s, 3H, CH3), 1.40 (d, 3H, J5,6 = 6.8 Hz, H-
6), 1.36 (s, 3H, CH3). 13C NMR (CDCl3, 100 MHz): d = 156.48 (CO),
136.66, 128.49, 128.13 (Ph), 110.22 (C(CH3)2), 101.41 (C-1), 77.30
(C-3), 75.47 (C-4), 69.05 (C-5), 67.38 (C-10), 66.53 (CH2), 65.12 (C-
2), 38.25 (C-30), 29.38 (C-20), 28.26, 26.20 (CH3), 16.51 (C-6). ESI
HR-MS (C20H28NO4): m/z = found ([M+H]+ 421.2131; calcd
421.2187); found ([M+Na]+ 443.1950; calcd 450.1907); found
([M+K]+ 459.1695; calcd 459.1646).
concentrated to afford compound 31. ½a D23
= ꢀ10.3 (c 1.2, CHCl3).
ꢄ
1H NMR (CDCl3, 400 MHz): d = 7.33–7.15 (m, 5H, Ph), 5.04–5.01
(m, 3H, CH2Ph, NH), 4.73 (s, 1H, H-1), 4.37 (d, 1H, J3,4 = 5.7 Hz, H-
3), 4.33 (dd, 1H, J4,5 = 1.8 Hz, H-4), 4.09–4.04 (m, 1H, H-5), 3.90–
3.84 (m, 1H, H-10a), 3.65–3.60 (m, 1H, H-10b), 3.32–3.21 (m, 2H,
H-30), 1.66–1.60 (m, 2H, H-20), 1.38 (d, 3H, J5,6 = 6.5 Hz, H-6), 1.37
(s, 3H, CH3), 1.32 (s, 3H, CH3). 13C NMR (CDCl3, 100 MHz):
d = 198.96 (CO), 156.53 (CONH), 136.42, 128.53, 128.44, 128.16,
127.94 (Ph), 111.07 (C(CH3)2), 99.15 (C-1), 80.38 (C-4), 77.62 (C-
3), 69.20 (C-5), 67.26 (C-10), 66.80 (CH2), 59.52 (C-2), 37.68 (C-
30), 29.33 (C-20), 27.14, 25.98 (CH3), 16.40 (C-6). ESI HR-MS
(C20H27NO7): m/z = found ([M+Na]+ 394.1887; calcd 394.1866).
The foregoing compound 31 was re-dissolved in dry EtOH
(80 ml), to which NaBH4 (10.5 g) was added at 0 °C. After 30 min
4.1.20. 3-(Benzyloxycarbonyl)aminopropyl 2-azido-2-deoxy–
D-
fucopyranoside 35
A solution of compound 34 (4.2 g, 10 mmol) in 9:1 (v/v) AcOH/
H2O (50 ml) was stirred at 60 °C for 5 h, when TLC (1:1 cyclohex-

6412
E. Danieli et al. / Bioorg. Med. Chem. 20 (2012) 6403–6415
ane/EtOAc) showed complete reaction. The mixture was concen-
trated and purified on silica gel (cyclohexane/EtOAc) to afford
3.7 g of deprotected product 35 (98%), as colourless syrup.
ESI HR-MS (C32H38N4O7): m/z = found ([M+Na]+ 629.2373; calcd
629.2378).
½
a 2D3
ꢄ
= ꢀ39.3 (c 0.72, CHCl3). 1H NMR (CDCl3, 400 MHz): d = 7.36–
4.1.23. 3-(Benzyloxycarbonyl)aminopropyl 2-azido-4-O-benzyl-
2-deoxy–D-fucopyranoside 5
7.29 (m, 5H, Ph), 5.20 (br t, 1H, J = 5.7 Hz, NH), 5.09 (s, 2H, CH2Ph),
4.22 (d, 1H, J1,2 = 7.0 Hz, H-1), 4.00–3.95 (m, 1H, H-10a), 3.67 (d, 1H,
J3,4 = J4,5 = 1.6 Hz, H-4), 3.70–3.66 (m, 1H, H-10b), 3.58–3.54 (m, 1H,
H-5), 3.46 (t, 1H, J = 8.1 Hz, H-2), 3.39–3.29 (m, 3H, H-3,30), 2.89 (br
s 1H, OH-4), 2.58 (br s, 1H, OH-3), 1.87–1.81 (m, 2H, H-20), 1.31 (d,
3H, J5,6 = 6.4 Hz, H-6). 13C NMR (CDCl3, 100 MHz): d = 156.52 (CO),
136.62, 128.48, 128.04, 127.33 (Ph), 102.04 (C-1), 73.35 (C-3),
70.84 (C-4), 70.54 (C-5), 67.52 (C-10), 66.58 (CH2), 64.03 (C-2),
38.24 (C-30), 29.47 (C-20), 16.02 (C-6). ESI HR-MS (C17H24N4O6):
m/z = found ([M+Na]+ 403.1593; calcd 403.1594); found ([M+K]+
419.1280; calcd 419.1333).
To a solution of the 3-O-PMB protected sugar 37 (1.5 g,
2.5 mmol) in CH2Cl2 (20 ml) moistened with water (2 ml), DDQ
(0.74 mg, 3.3 mmol) was added and the mixture was stirred for
2 h (TLC, 1:1 cyclohexane/EtOAc). The mixture was partitioned
with 10% sodium thiosulfate. Combined organic layers were con-
centrated and purified on silica gel (cyclohexane/EtOAc) to afford
1.13 g of product 5 (94%). White crystals from EtOAc: mp 86–
87 °C. ½a 2D3
ꢄ
= ꢀ3.5 (c 1.70, CHCl3). 1H NMR (CDCl3, 400 MHz):
d = 7.38–7.26 (m, 10H, Ph), 5.19 (br t, 1H, J = 4.8 Hz, NH), 5.09 (s,
2H, CH2PhCbz), 4.81, 4.70 (2 d, 2H, 2J = 11.3 Hz, CH2PhBn), 4.18 (d,
1H, J1,2 = 7.3 Hz, H-1), 3.99–3.93 (m, 1H, H-10a), 3.63–3.57 (m, 1H,
H-10b), 3.57–3.44 (m, 4H, H-2,3,4,5), 3.41–3.25 (m, 2H, H-30), 2.25
(br s, 1H, OH-3), 1.86–1.80 (m, 2H, H-20), 1.27 (d, 3H, J5,6 = 6.5 Hz,
H-6). 13C NMR (CDCl3, 100 MHz): d = 156.49 (CO), 131.87, 130.00,
129.63, 129.59, 128.46, 128.25, 128.13, 128.04, 127.99 (Ar),
102.02 (C-1), 74.38 (C-4), 75.94 (CH2Bn), 72.97 (C-3), 70.31 (C-5),
4.1.21. 3-(Benzyloxycarbonyl)aminopropyl 2-azido-2-deoxy-3-
O-p-methoxybenzyl–D-fucopyranoside 36
A solution of diol 35 (3.5 g, 9.2 mmol) and Bu2SnO (3.46 g,
13.8 mmol) in toluene (50 ml) containing pre activated 4 Å MS
was stirred under reflux for 1 h. Then temperature was decreased
to 60 °C and PMBBr (2 ml, 13.8 mmol) was added, followed by TBAI
(5.09 g, 13.8 mmol). After stirring for 3 h the reaction was com-
plete (TLC 1:1 cyclohexane/EtOAc). The mixture was filtered and
concentrated. The residue was chromatographed (cyclohexane/
Cbz
67.51 (C-10), 66.50 (CH2 ), 64.55 (C-2), 38.29 (C-30), 29.40 (C-20),
16.83 (C-6). ESI HR-MS (C24H30N4O6): m/z = found ([M+H]+
471.2230; calcd 471.2244); found ([M+Na]+ 493.2032; calcd
493.2063); = found ([M+K]+ 509.1833; calcd 509.1802).
EtOAc) to give 3.45 g of product 36 (76%). ½a D23
= ꢀ65.1 (c 2.05,
ꢄ
The structure of 5 was further confirmed by acetylation of 3-OH
which shifted H-3 to 4.61 ppm (dd, J2,3 = 10.3, J3,4 = 2.4 Hz).
CHCl3). 1H NMR (CDCl3, 400 MHz): d = 7.37–6.90 (m, 9H, Ar), 5.27
(br t, 1H, J = 5.0 Hz, NH), 5.09 (s, 2H, CH2PhCbz), 4.62 (s, 2H,
CH2PhPMB), 4.15 (d, 1H, J1,2 = 7.8 Hz, H-1), 3.99–3.94 (m, 1H, H-
10a), 3.81 (s, 3H, OCH3), 3.69 (br t, 1H, J = 2.5 Hz, H-4), 3.62–3.54
(m, 2H, H-10b, incl. dd, J2,3 = 10.0 Hz, H-2), 3.49–3.44 (m, 1H, H-
5), 3.41–3.26 (m, 3H, H-3,30), 2.40 (br s, 1H, OH-4), 1.86–1.80 (m,
2H, H-20), 1.33 (d, 3H, J5,6 = 6.5 Hz, H-6). 13C NMR (CDCl3,
100 MHz): d = 159.61 (Cq), 156.49 (CO), 136.75, 129.68, 129.15,
128.44, 128.02, 127.96, 114.02 (Ar), 101.72 (C-1), 79.23 (C-3),
71.68 (CH2PMB), 70.25 (C-5), 68.08 (C-4), 67.53 (C-10), 66.46
4.1.24. Allyl 3-O-acetyl-2-azido-4-O-(benzyl 3,4-di-O-benzyl-2-O
-levulinoyl-b-
fucopyranoside 22
TMSOTf (27 l, 0.15 mmol) dissolved in CH2Cl2 (100 ll) was
D-glucopyranosyl uronate)-2-deoxy-a-L-
l
slowly added at -10 °C to a mixture of acceptor 4 (400 mg,
1.47 mmol) and donor 3 (1.8 g, 2.54 mmol) in CH2Cl2 (7 ml). The
mixture was stirred at the same temperature for 2 h, then temper-
ature was slowly allowed to go 0 °C, monitoring by TLC (4:1 tolu-
ene/EtOAc). After 4 h the reaction was quenched with TEA
Cbz
(CH2 ), 62.31 (C-2), 55.27(OCH3), 38.39 (C-30), 29.32 (C-20),
16.30 (C-6). ESI HR-MS (C25H32N4O7): m/z = found ([M+H]+
501.2357; calcd 501.2349); found ([M+Na]+ 523.2188; calcd
523.2169); found ([M+K]+ 539.1921; calcd 539.1908).
(27 ll), and the mixture was concentrated and purified on silica
gel (99:1 ? 93:7 toluene/EtOAc) to afford 735 mg of disaccharide
22 as a sirup (61%). ½a D23
= ꢀ31.3 (c 1.2, CHCl3).
ꢄ
1H NMR (CDCl3, 400 MHz): d = 7.34–7.09 (m, 15H, Ph), 5.94–
5.89 (m, 1H, CH@), 5.33–5.19 (m, 2H, CH2@), 5.15 (s, 2H, CH2Ph),
5.13 (m, 1H, H-2II), 5.05 (dd, 1H, J2,3 = 11.3, J3,4 = 2.7 Hz, H-3I),
4.87 (d, 1H, J1,2 = 3.5 Hz, H-1I), 4.75, 4.70 (2 d, 2H, 2J = 11.0 Hz,
CH2Ph), 4.71, 4.45 (2 d, 2H, 2J = 11.2 Hz, CH2Ph), 4.37 (d, 1H,
J1,2 = 8.0 Hz, H-1II), 4.15 (m, 1H, CH2a), 4.05 (d, 1H, J3,4 = 2,7 Hz, H-
4I), 4.03–3.91 (m, 3H, CH2b, H-5I,4II), 3.86 (d, 1H, J4,5= 9.7 Hz, H-
5II), 3.75 (dd, 1H, H-2I), 3.66 (dd, 1H, J2,3 = 9.1, J3,4 = 9,1 Hz, H-3II),
2.72–2.48 (m, 4H, CH2CH2), 2.15 (s, 3H, CH3CO), 2,00 (s, 3H,
CH3CO), 1.19 (d, 3H, J5,6 = 6.6 Hz, H-6I). 13C NMR (CDCl3,
100 MHz): d = 206.12 (CO), 171.34 (CH2COO), 170.76 (CH3CO),
167.83 (C-6II), 137.92, 137.47, 134.67 (Ar), 133.38 (CH@), 128.67,
128.62, 128.33, 127.82, 127.71 (Ar), 117.62 (CH2@), 101.64 (C-
1II), 96.91 (C-1I), 81.73 (C-3II), 79.35 (C-4II), 75.77 (C-4I), 75.06
(CH2Ph), 74.91 (CH2Ph), 74.64 (C-5II), 73.21 (C-2II), 70.14 (C-3I),
68.52 (CH2), 67.55 (CH2Ph), 65.46 (C-5I), 56.95 (C-2I), 37.92
(CH2CO), 29.77 (CH3CO), 27.88 (CH2COO), 20.80 (CH3CO), 16.04
(C-6I). ESI HR-MS (C43H49N3O13): m/z = ([M+H]+ found 816.3300,
calcd 816.3344); ([M+Na]+ found 838.3083 calcd 838.3163).
4.1.22. 3-(Benzyloxycarbonyl)aminopropyl 2-azido-4-O-benzyl-
3-O-p-methoxybenzyl-2-deoxy–D-fucopyranoside 37
The substrate 36 (1.5 g, 3 mmol) was dissolved in THF (25 mol)
containing 0.5% of water. Then finely powdered NaOH (0.6 g,
15 mmol) was added, followed by BnBr (0.5 ml, 4.5 mmol) and a
catalytic amount of 18-crown-6. The mixture was stirred at room
temperature for 3 d when TLC (3:2 cyclohexane/EtOAc) showed a
new spot had been formed. The mixture was neutralized with 5%
HCl, then partitioned with water. Combined organic layers were
dried over NaSO4, filtered and the filtrated was concentrated. Chro-
matography of the residue (cyclohexane/EtOAc) yielded 1.76 g of
syrupy product 36 (99%). ½a D23
ꢄ
= ꢀ77.2 (c 0.85, CHCl3). 1H NMR
(CDCl3, 400 MHz): d = 7.36–6.90 (m, 14H, Ar), 5.29 (br t, 1H,
J = 4.8 Hz, NH), 5.08 (s, 2H, CH2PhCbz), 4.91, 4.64 (2 d, 2H,
2J = 11.5 Hz, CH2PhBn), 4.62 (s, 2H, CH2PhPMB), 4.13 (d, 1H,
J1,2 = 8.0 Hz, H-1), 3.98–3.93 (m, 1H, H-10a), 3.81 (s, 3H, OCH3),
3.77 (dd, 1H, J2,3 = 10.1 Hz, H-2), 3.59–3.53 (m, 1H, H-10b), 3.40 (d,
1H, J3,4 = J4,5 = 2.5 Hz, H-4), 3.41–3.34 (m, 1H, H-5), 3.33–3.25 (m,
3H, H-3,30), 1.84–1.78 (m, 2H, H-20), 1.17 (d, 3H, J5,6 = 6.3 Hz, H-6).
13C NMR (CDCl3, 100 MHz): d = 159.42 (Cq), 156.52 (CO), 136.15,
136.78, 130.26, 129.63, 129.51, 128.46, 128.22, 128.02, 127.94,
127.73, 114.73 (Ar), 101.97 (C-1), 80.72 (C-3), 74.73 (C-4), 74.63
(CH2Bn), 72.45 (CH2PMB), 70.07 (C-5), 67.60 (C-10), 66.45 (CH2Cbz),
62.88 (C-2), 55.28 (OCH3), 38.48 (C-30), 29.70 (C-20), 16.80 (C-6).
4.1.25. Allyl 3-O-acetyl-2-azido-4-O-(benzyl 3,4-di-O-benzyl-b-
glucopyranosyl uronate)-2-deoxy- -fucopyranoside 23
D-
a
-L
To a solution of disaccharide 28 (1.4 g, 1.71 mmol) in 20:1 (v/v)
CH2Cl2/MeOH (20 ml), hydrazine acetate (185 mg, 2.06 mmol) was
added and the mixture was stirred for 2 h (TLC, 4:1 toluene/EtOAc,
4:1 cyclohexane/acetone). The mixture was concentrated and puri-

E. Danieli et al. / Bioorg. Med. Chem. 20 (2012) 6403–6415
6413
fied on silica gel (99:1 ? 1:1 toluene/EtOAc) to give 1.10 g of
4.1.27. 3-O-Acetyl-2-azido-4-O-(benzyl 2-azido-2-deoxy-3,4-di-
O-benzyl-b- -mannopyranosyl uronate)-2-deoxy- -fucopyr
anosyl trichloroacetimidate 27a
To a solution of disaccharide 25 (380 mg, 0.5 mmol) in dry
MeOH (20 ml), PdCl2 (120 mg, 0.8 mmol) was added under nitro-
gen atmosphere. The mixture was stirred for 1 d, when TLC (7:3
cyclohexane/EtOAc) showed two new spots formed. The mixture
was filtered, and the filtrate purified on silicagel (4:1 cyclohex-
desired product 23 (86%). ½a D23
ꢄ
= ꢀ48.7 (c 1.35, CHCl3).
D
a-L
1H NMR (CDCl3, 400 MHz): d = 7.33–7.08 (m, 15H, Ph), 5.87 (m,
1H, CH@), 5.35–5.21 (m, 2H, CH2@), 5.15 (s, 2H, CH2Ph), 5.11 (dd,
1H, J2,3 = 11.1, J3,4 = 3.2 Hz, H-3I), 4.94 (d, 1H, J1,2 = 3.5 Hz, H-1I),
4.90, 4.82 (2 d, 2H, 2J = 11.4 Hz, CH2Ph), 4.74, 4.46 (2 d, 2H,
2J = 10.8 Hz, CH2Ph), 4.28 (d, 1H, J1,2= 7.9 Hz, H-1II), 4.18 (m, 1H,
CH2a), 4.11 (d, 1H, H-4I), 4.07–4.01 (m, 2H, CH2b, H-5I), 3.85 (m,
2H, H-4II,5II), 3.78 (dd, 1H, H-2I), 3.69 (dd, 1H, J2,3 = 8.2 Hz, H-2II),
3.58–3.44 (m, 1H, H-3II), 2.01 (s, 3H, CH3CO), 1.28 (d, 3H,
J5,6 = 6.7 Hz, H-6I). 13C NMR (CDCl3, 100 MHz): d = 170.63 (CO),
168.20 (C-6II), 138.37, 137.62, 134.65 (Ar), 133.15 (CH@), 128.56,
128.32, 128.24, 128.13, 127.67, 127.62 (Ar), 117.83 (CH2@),
104.07 (C-1II), 96.79 (C-1I), 83.63 (C-3II), 78.99 (C-4II/5II), 76.23
(C-4I), 75.18 (CH2Ph), 74.95 (CH2Ph), 74.59 (C-4II/5II), 74.36 (C-
2II), 70.01 (C-3I), 68.58 (CH2), 67.38 (CH2Ph), 65.81 (C-5I), 57.10
(C-2I), 20.73 (CH3CO), 15.91 (C-6I). ESI HR-MS (C38H43N3O11): m/
z = ([M+Na]+ found 740.2664, calcd 740.2795); ([M+K]+ found
756.2496, calcd 756.2536).
ane/EtOAc) to give 290 mg (78%) of product 26, as
a,b mixture.
4.1.27.1. 3-O-Acetyl-2-azido-4-O-(benzyl 2-azido-2-deoxy-3,4-
di-O-benzyl-b-D-mannopyranosyl uronate)-2-deoxy-L-fucopyra-
nose 26.
1H NMR (CDCl3, 400 MHz): d = 7.34–7.09 (m, Ph),
5.30 (s, J1,2 = 3.5 Hz, H-1I ), 5.21, 5.15 (2 d, 2J 12.0 Hz, CH2Ph),
a
5.12–5.08 (m, H-3Ib, incl. dd, 5.10, J2,3 = 11.2 Hz, J3,4 = 3.3 Hz, H-
3I ), 4.79, 4.46 (2 d, 2J = 10.7 Hz, CH2Ph), 4.70 (s, CH2Ph), 4.54 (d,
a
J1,2 = 8.4 Hz, H-1Ib), 4.49 (s, H-1II), 4.24 (m, H-5I ), 4.19 (d, H-4I ),
a
a
4.14 (d, J3,4 = 1.9 Hz, H-4Ib), 4.09–4.03 (m, H-2II,4II), 3.95 (dd,
J2,3 = 11.1 Hz, H-2I ), 3.83–3.71 (m, H-5II,2Ib), 3.66 (m, H-5Ib), 3.56
a
(dd, J2,3 = 9.3, J3,4 = 3.6 Hz, H-3II), 2.06 (s, CH3CO), 1.24 (d,
4.1.26. Allyl 3-O-acetyl-2-azido-4-O-(benzyl 2-azido-2-deoxy-
J5,6 = 6.4 Hz, H-6Ib), 1.17 (d, J5,6 = 6.6 Hz, H-6I ). 13C NMR (CDCl3,
a
3,4-di-O-benzyl-b-
fucopyranoside 25
To a solution of disaccharide 23 (900 mg, 1.26 mmol) in 6:1 (v/
v) CH2Cl2/pyridine (20 ml), Tf2O (320 l, 1.89 mmol) was added at
D
-mannopyranosyl uronate)-2-deoxy-
a-
L-
100 MHz): d = 170.86 (CO), 167.52 (C-6II), 137.73, 137.18, 134.77,
129.46, 128.75, 128.63, 128.32, 128.19, 127.86 (Ar), 101.03 (C-
1II), 96.06 (C-1Ib), 92.11 (C-1I ), 79.44 (C-3II), 75.73 (C-4I ), 75.43
a
a
l
(C-4II/2II), 75.42 (CH2Ph), 75.22 (C-5II), 72.64 (C-4Ib), 72.19 (CH2Ph),
0 °C. The mixture was stirred at room temperature for 30 min, at
which time the reaction was complete (TLC, 1:1 cyclohexane/
EtOAc). The mixture was washed with iced aq NaHCO3, and the
combined organic layers were concentrated at 30 °C and used for
the next step.
70.04 (C-5Ib), 69.88 (C-3I ), 69.642 (C-3Ib), 67.58 (CH2Ph), 65.38 (C-
a
5I ), 61.78 (C-2Ib), 60.97 (C-4II/2II), 57.60 (C-2I ), 20.85 (CH3CO),
a
a
16.59, 16.46 (C-6I ,b). ESI HR-MS (C35H38N6O10): m/z = ([M+Na]+
a
found 725.2487, calcd 725.2547); ([M+K]+ found 741.2251. calcd
741.2286).
To a solution of the disaccaride 26 (280 mg, 0.4 mmol) in CH2Cl2
4.1.26.1. Allyl 3-O-acetyl-2-azido-4-O-(benzyl 3,4-di-O-benzyl-2-
(10 ml), CCl3CN (400
ll, 4 mmol) was added, followed by DBU
O-trifluomethansulfonyl-b-
D
-glucopyranosyl uronate)-2-deoxy-
(60 l, 0.04 mmol). The mixture was stirred for 4 h, when TLC
l
a
-
L
-fucopyranoside 24.
1H NMR (CDCl3, 400 MHz): d = 7.33–
(1:1 cyclohexane/EtOAc) showed that the reaction was complete.
The mixture was concentrated and purified on silica gel (4:1 cyclo-
hexane/EtOAc) to give 330 mg of product (98%).
7.02 (m, 15H, Ph), 5.90 (m, 1H, CH@), 5.35–5.20 (m, 2H, CH2@),
5.16 (s, 2H, CH2Ph), 5.12 (dd, 1H, J2,3 = 11.1, J3,4 = 3.2 Hz, H-3I),
4.94 (d, 1H, J1,2 = 3,5 Hz, H-1I), 4.85, 4.79 (2 d, 2H, 2J = 10.6 Hz,
CH2Ph), 4.73 (dd, 1H, J1,2= 8.2, J2,3 = 9.1 Hz, H-2II), 4.64, 4.46 (2 d,
2H, 2J = 10.7 Hz, CH2Ph), 4.57 (d, 1H, H-1II), 4.19 (d, 1H,
J3,4 = 3.2 Hz, H-4I), 4.19–4.15 (m, 1H, CH2a), 4.11–4.01 (m, 2H,
CH2b, H-5I), 3.98 (dd, 1H, J3,4 = 9.4, J4,5 = 9.4 Hz, H-4II), 3.90 (d, 1H,
H-5II), 3.76 (dd, 1H, H-3II), 3.70 (dd, 1H, H-2I), 2.03 (s, 3H, CH3CO),
1.25 (d, 3H, J5,6 = 6.6 Hz, H-6I).
4.1.27.2. First eluted: 3-O-Acetyl-2-azido-4-O-(benzyl 2-azido-2-
deoxy-3,4-di-O-benzyl-b-D-mannopyranosyl uronate)-2-deoxy-
a
½
-
-fucopyranosyl
trichloroacetimidate
27a
(33%).
aꢄL2D3 = ꢀ31.4 (c 0.65, CHCl3). 1H NMR (CDCl3, 400 MHz): d = 8.69
(s, 1H, NH), 7.37–7.01 (m, 15H, 3 Ph), 6.38 (d, 1H, J1,2 = 3.6 Hz, H-
1I), 5.21, 5.15 (2 d, 2H, 2J = 11.9 Hz, CH2Ph), 5.10 (dd, 1H,
J2,3 = 11.1, J3,4 = 3.3 Hz, H-3I), 4.79, 4.47 (2 d, 2H, 2J = 10.5 Hz,
CH2Ph), 4.73, 4.69 (2 d, 2H, 2J = 12.3 Hz, CH2Ph), 4.50 (s, 1H, H-
1II), 4.29 (d, 1H, J4,5 = 3.0 Hz, H-4I), 4.23–4.16 (m, 2H, H-5I, incl.
dd, 4.17, H-2I), 4.06 (t, 1H, J = 9.5 Hz, H-4II), 4.01 (d, 1H,
J2,3 = 3.4 Hz, H-2II), 3.74 (d, 1H, J4,5 = 9.0 Hz, H-5II), 3.56 (dd, 1H,
J3,4 = 9.4 Hz, H-3II), 2.06 (s, 3H, CH3CO), 1.20 (d, 3H, J5,6 = 6.5 Hz,
H-6I). 13C NMR (CDCl3, 100 MHz): d = 170.68 (CO), 167.38 (C-6II),
160.72 (C@NH), 137.62, 137.06, 134.66, 128.66, 128.54, 128.23,
128.13, 127.79, 127.76 (Ar), 100.93 (C-1II), 94.73 (C-1I), 80.78 (C-
3II), 75.32 (CH2Ph), 75.15 (C-4II,5II), 75.00 (C-4I), 72.19 (CH2Ph),
69.94 (C-3I), 67.93 (C-5I), 67.52 (CH2Ph), 60.93 (C-2II), 56.27 (C-
2I), 20.78 (CH3CO), 16.38 (C-6II). ESI HR-MS (C37H38Cl3N7O10): m/
z = found ([M+Na]+ 868.1572; calcd 868.1643).
The foregoing triflate was re-dissolved in toluene (20 ml), and
tetrabutilammonium azide (1.07 g, 3.78 mmol) was added. The
mixture was stirred at 60 °C for 3 h, when TLC (1:1 cyclohexane/
EtOAc) showed a new spot had been formed.
The crude mixture was concentrated and purified on silica gel
(99:1 ? 4:1 toluene/EtOAc) to yield 625 mg of product 25 (70%).
½
a 2D3
ꢄ
= ꢀ17.4 (c 1.65, CHCl3). 1H NMR (CDCl3, 400 MHz): d = 7.28–
7.02 (m, 15H, Ph), 5.83 (m, 1H, CH@), 5.28–5.14 (m, 2H, CH2@),
5.15, 5.09 (2 d, 2H, 2J = 11.7 Hz, CH2Ph), 5.03 (dd, 1H, J2,3 = 11.2,
J3,4 = 3.2 Hz, H-3I), 4.85 (d, 1H, J1,2 = 3.5 Hz, H-1I), 4.71, 4.43 (2 d,
2H, 2J = 10.8 Hz, CH2Ph), 4.63 (s, 2H, CH2Ph), 4.44 (s, 1H, H-1II),
4.11 (d, 1H, J3,4 = 3.1 Hz, H-4I), 4.12–4.07 (m, 1H, CH2a), 4,01–3.94
(m, 4H, CH2b,
H-2II,4II,5I), 3.80 (dd, 1H, H-2I), 3.66 (d, 1H,
J4,5 = 9.7 Hz, H-5II), 3.48 (dd, 1H, J2,3 = 3.5, J3,4 = 9.4 Hz, H-3II), 1.97
(s, 3H, CH3CO), 1.10 (d, 3H, J5,6 = 6.5 Hz, H-6I). 13C NMR (CDCl3,
100 MHz): d = 170.81 (CO), 167.48 (C-6II), 137.72, 137.18, 134.76
(Ar), 133.24 (CH@), 128.72, 128.60, 128.29, 128.15, 127.83,
127.75 (Ar), 117.85 (CH2@), 101.03 (C-1II), 96.85 (C-1I), 79.44 (C-
3II), 75.81 (C-4I), 75.42 (C-4II/5I/2II), 75.38 (CH2Ph), 75.20 (C-5II),
72.19 (CH2Ph), 69.75 (C-3I), 68.67 (CH2), 67.54 (CH2Ph), 65.27 (C-
4II/5I/2II), 61.00 (C-4II/5I/2II), 56.89 (C-2I), 20.81 (CH3CO), 16.33
(C-6I). ESI HR-MS (C38H42N6O10): m/z = ([M+Na]+ found 765.2771,
calcd 765.2860); ([M+K]+ found 781.2612, calcd 781.2599).
4.1.27.3. Second eluted: 3-O-Acetyl-2-azido-4-O-(benzyl 2-
azido-2-deoxy-3,4-di-O-benzyl-b-D-mannopyranosyl uronate)-
2-deoxy-b-
(65%).
L
-fucopyranosyl
trichloroacetimidate
27b
½
a 2D3
ꢄ
= ꢀ57.1 (c 1.00, CHCl3). 1H NMR (CDCl3,
400 MHz): d = 8.63 (s, 1H, NH), 7.29–7.02 (m, 15H, 3 Ph), 5.50 (d,
1H, J1,2 = 8.3 Hz, H-1I), 5.14, 5.08 (2 d, 2H, 2J = 12.1 Hz, CH2Ph),
4.73, 4.40 (2 d, 2H, 2J = 10.9 Hz, CH2Ph), 4.66, 4.60 (2 d, 2H,
2J = 11.9 Hz, CH2Ph), 4.54 (dd, 1H, J2,3 = 10.4, J3,4 = 3.5 Hz, H-3I),
4.44 (s, 1H, H-1II), 4.08 (d, 1H, J3,4 = J4,5= 3.2 Hz, H-4I), 4.06 (d, 1H,

6414
E. Danieli et al. / Bioorg. Med. Chem. 20 (2012) 6403–6415
J2,3 = 10.4 Hz, H-2II), 4.00 (t, 1H, J = 9.5 Hz, H-4II), 3.96 (dd, 1H, H-
2I), 3.74–3.71 (m, 1H, H-5I), 3.74 (d, 1H, J4,5 = 9.0 Hz, H-5II), 3.49
(dd, 1H, J3,4 = 4.6 Hz, H-3II), 1.99 (s, 3H, CH3CO), 1.20 (d, 3H,
2I), 3.73 (d, 1H, J4,5 = 9.8 Hz, H-5III), 3.67 (dd, 1H, J2,3 = 3.3,
J3,4 = 9.3 Hz, H-3III), 3.66–3.48 (m, 4H, H-10b,4I,5II incl. dd, 3.51,
J2,3 = 9.5, J3,4 = 3.7 Hz, H-3I), 3.44–3.27 (m, 2H, H-30,5I), 1.84–1.81
(m, 2H, H-20), 1.60 (s, 3H, CH3CO), 1.27 (d, 3H, J5,6 = 6.3 Hz, H-6II),
1.15 (d, 3H, J5,6 = 6.3 Hz, H-6I). 13C NMR (CDCl3, 100 MHz):
d = 170.89 (CO), 167.53 (C-6III), 156.49 (CONH), 138.16, 137.74,
137.15, 136.77, 134.75, 128.75, 128.63, 128.44, 128.32, 128.18,
127.95, 127.84, 127.67 (Ar), 101.99 (C-1I), 101.12 (C-1III), 100.44
(C-1II), 80.00 (C-3III), 79.45 (C-3I), 75.52 (C-4I), 75.40 (CH2Bn, C-
4III), 75.23 (C-5III), 74.74 (CH2Bn), 74.37 (C-4II), 73.41 (C-3II), 72.08
J
5,6 = 6.3 Hz, H-6I). 13C NMR (CDCl3, 100 MHz): d = 170.66 (CO),
167.39 (C-6II), 160.09 (C@NH), 137.77, 137.64, 137.03, 134.68,
129.42, 128.93, 128.67, 128.55, 128.23, 128.13, 127.76, 127.19
(Ar), 100.85 (C-1II), 96.64 (C-1I), 79.30 (C-3II), 75.34 (CH2Ph, C-
4II), 75.16 (C-5II), 74.83 (C-4I), 72.49 (C-3I), 71.95 (CH2Ph), 70.81
(C-5I), 67.52 (CH2Ph), 60.58 (C-2II), 59.78 (C-2I), 20.69 (CH3CO),
16.38 (C-6II). ESI HR-MS (C37H38Cl3N7O10): m/z = found ([M+Na]+
868.1622; calcd 868.1643).
(CH2Bn), 70.46 (C-5I), 70.09 (C-5II), 67.58 (CH2 , C-10), 66.45
Bn
(CH2 ), 62.66 (C-2III), 60.89 (C-2I), 60.49 (C-2II), 38.37 (C-30),
Cbz
4.1.28. 3-(Benzyloxycarbonyl)aminopropyl 3-O-(3-O-acetyl-2-
29.68 (C-20), 20.81 (CH3CO), 16.69 (C-6II), 16.58 (C-6I). ESI HR-MS
(C59H66N10O15): m/z = found ([M+H]+ 1155.4757; calcd 1155.4787).
azido-4-O-[benzyl 2-azido-3,4-di-O-benzyl-2-deoxy-b-
nopyranosyl uronate]-2-deoxy- -fucopyranosyl)-2-azido-4-O-
benzyl-2-deoxy– -fucopyranoside 2
To a mixture of acceptor 5 (158 mg, 0.34 mmol) and disaccharide
donor 27a/b (140 mg, 0.165 mmol) in CH2Cl2 (4 ml), TMSOTf (3 l,
D-man
a-L
D
4.1.29. 3-Acetamidopropyl 2-acetamido-3-O-(3-O-acetyl-2-
acetamido-4-O-[2-acetamido-2-deoxy-b-
D
-mannopyranosyl
l
uronate]-2-deoxy-
a-
L
-fucopyranosyl)-2-deoxy–
D-
0.017 mmol) was added at -10 °C. The mixture was stirred for
fucopyranoside 1
30 min, when the donor was consumed (TLC, 3:2 cyclohexane/
Trisaccharide 2 (5 mg, 0.004 mmol) was dissolved in 9:1 (v/v)
MeOH/H2O (5 ml) and flown for 18 h in a H-Cube Thales-Nano
hydrogenator, over a Pd/C 10% cartridge at room temperature and
atmospheric pressure. The solvent was then evaporated, and the
crude material was dissolved in 4:1 (v/v) Ac2O–MeOH (1 ml). After
stirring overnight, the mixture was concentrated, and purification
of the residue on a G10 PD MiniTrapTM GE Healthcare cartridge affor-
EtOAc). The mixture was neutralized with triethylamine (3
ll) and
concentrated. Chromatography of the residue (99:1 ? 9:1 ?1:1
cyclohexane/EtOAc) afforded
trisaccharides.
a
mixture 2.4:1 of a- and -
4.1.28.1.
50%).
First
ꢄ
eluted
was
compound
2
(94 mg,
½
a 2D3
= ꢀ22.3 (c 1.20, CHCl3). 1H NMR1H NMR (CDCl3,
ded 1.35 mg of the final trisaccharide 1 (40%). ½a D23
ꢄ
= +24.0 (c 0.10,
400 MHz): d = 7.36–7.10 (m, 25H, Ph), 5.29 (d, 1H, J1,2 = 3.8 Hz, H-
1II), 5.20, 5.15 (2 d, 2H, 2J = 11.9 Hz, CH2PhBn), 5.12–5.08 (m, 3H,
NH, CH2PhCbz), 5.05 (dd, 1H, J2,3= 11.2, J3,4 = 3.0 Hz, H-3II), 4.88,
4.68 (2 d, 2H, 2J = 12.0 Hz, CH2PhBn), 4.78, 4.46 (2 d, 2H,
2J = 10.7 Hz, CH2PhBn), 4.72, 4.68 (2 d, 2H, 2J = 11.9 Hz, CH2PhBn),
4.46 (s, 1H, H-1III), 4.20 (d, 1H, J1,2 = 8.3 Hz, H-1I), 4.12 (d, 1H,
J3,4 = 3.3 Hz, H-4II), 4.06 (t, 1H, J = 9.7 Hz, H-4III), 4.01 (d, 1H,
J2,3 = 3.6 Hz, H-2III), 4.00–3.95 (m, 1H, H-10a), 3.89–3.80 (m, 3H, H-
2I,II,5II), 3.72 (d, 1H, J4,5 = 9.9 Hz, H-5III), 3.64–3.53 (m, 1H, H-10b),
3.54 (dd, 1H, J2,3 = 3.6, J3,4 = 9.3 Hz, H-3III), 3.52–3.46 (m, 2H, H-5I,
incl. dd, 3.47, J2,3 = 9.3, J3,4 = 3.6 Hz, H-3I), 3.42 (d, 1H, J3,4 = J4,5
=2.3, 1H, H-4I), 3.39–3.29 (m, 2H, H-30), 1.89–1.82 (m, 2H, H-20),
1.58 (s, 3H, CH3CO), 1.29 (d, 3H, J5,6 = 6.5 Hz, H-6I), 1.10 (d, 3H,
J5,6 = 6.5 Hz, H-6II). 13C NMR (CDCl3, 100 MHz): d = 170.74 (CO),
167.43, (C-6III), 156.48 (CONH), 137.84, 137.62, 137.19, 136.68,
134.75, 128.77, 128.63, 128.44, 128.32, 128.17, 128.03, 127.95,
127.84, 127.26 (Ar), 102.51 (C-1I), 101.01 (C-1III), 99.26 (C-1II),
79.46 (C-3III), 78.84 (C-3I), 77.32 (C-4I), 75.65 (C-4II), 75.41 (C-4III),
75.25 (C-5III), 75.00 (CH2Bn), 72.19, 70.87 (CH2Bn), 69.11 (C-5I),
H2O). 1H NMR (D2O, 323 K, 400 MHz): d = 5.00–4.98 (m, 2H, H-
1II,3II), 4.70 (br s, 1H, H-1III), 4.65 (br s, 1H, H-2III), 4.43 (d, 1H,
J1,2 = 8.5 Hz, H-1I), 4.08–4.4 (m, 3H), 3.88–3.66 (m, 6H), 3.62–3.55
(m, 3H), 3.82–3.55 (m, 2H), 2.19, 2.04, 2.02, 1.96, 1.86 (5 s, 15H,
CH3CO), 1.75–1.68 (m, 2H, H-20), 1.28 (d, 3H, J5,6 = 6.9 Hz, H-6I),
1.25 (d, 3H, J5,6 = 6.5 Hz, H-6II). ESI HR-MS (C31H50N4O17): m/
z = found ([M+H]+ 751.3278; calcd 751.3249).
4.2. Immunochemical analysis
4.2.1. Preparation of S. aureus type 5 (SA5)-CRM197
glycoconjugate
Purified S. aureus type 5 capsular polysaccharide4a,27 was dis-
solved in distilled water at 2 mg/ml. Acetic acid was added to a fi-
nal concentration of 2% (v/v) and the reaction kept at 90 °C for 3 h.
The solution was then neutralized with 1 M NaOH, and the hydro-
lyzed saccharide was purified on a gel-filtration column (per-
formed on an AktaTM system (G&E Healthcare) using a S300
Sephacryl resin (G&E Healthcare) eluting with 10 mM NaPi/
10 mM NaCl pH 7.2 buffer). The saccharide was detected at
215 nm. Pooled fractions were dialyzed against distilled water
using a 1 kDa membrane (SpectraPor). The depolymerized saccha-
ride was dissolved in distilled water at 2 mg/ml. NaIO4 was added
at a saccharide:NaIO4 ratio of 1:1 (w/w) and the reaction kept at
room temperature for 1-2 h in the dark. The solution was then dia-
lyzed against distilled water using a 1 kDa membrane (SpectraPor).
The oxidized saccharide was dissolved in a 200 mM NaPi/1 M
NaCl pH 7.2 buffer at a concentration of 10 mg/ml. CRM197 was
added to the solution at a saccharide:protein ratio of 4:1 (w/w)
and NaBH3CN (Aldrich) added at a saccharide:NaCNBH3 ratio of
2:1 (w/w). The solution was kept at 37 °C for 48 h.
67.96 (C-3II), 67.61 (CH2 ), 67.43 (C-10), 66.50 (CH2Cbz), 63.61 (C-
Bn
5II), 61.00 (C-2III), 56.64 (C-2I), 56.66 (C-2II), 38.13 (C-30), 29.49 (C-
20), 20.82 (CH3CO), 16.97 (C-6I), 16.48 (C-6II). ESI HR-MS
(C59H66N10O15): m/z = found ([M+H]+ 1155.4786; calcd 1155.4787).
4.1.28.2. Second eluted was compound: 3-(Benzyloxycar-
bonyl)aminopropyl 3-O-(3-O-acetyl-2-azido-4-O-[benzyl 2-
azido-3,4-di-O-benzyl-2-deoxy-b-
D-mannopyranosyl uronate]-
2-deoxy-b- -fucopyranosyl)-2-azido-4-O-benzyl-2-deoxy-b-D-
L
fucopyranoside 38 (28 mg, 15%).
½
a 2D3
ꢄ
= ꢀ32.6 (c 0.25,
CHCl3).
1H NMR (CDCl3, 400 MHz): d = 7.40–7.10 (m, 25H, Ph), 5.23 (br
t, 1H, J = 4.5 Hz, NH), 5.20, 5.14 (2 d, 2H, 2J = 11.9 Hz, CH2PhBn), 5.08
(s, 2H, CH2PhCbz), 5.00, 4.70 (2 d, 2H, 2J = 11.1 Hz, CH2PhBn), 4.78,
4.45 (2 d, 2H, 2J = 10.3 Hz, CH2PhBn), 4.70, 4.67 (2 d, 2H,
2J = 11.2 Hz, CH2PhBn), 4.60 (dd, 1H, J2,3= 10.3, J3,4 = 2.9 Hz, H-3II),
4.51 (s, 1H, H-1III), 4.40 (d, 1H, J1,2 = 8.3 Hz, H-1II), 4.12 (d, 1H,
J1,2 = 8.1 Hz, H-1I), 4.11 (d, 1H, J3,4 = 2.0 Hz, H-4II), 4.10–4.03 (m,
2H, H-2III incl. t, 4.05, J = 9.5 Hz, H-4III), 3.99–3.93 (m, 1H, H-10a),
3.92 (dd, 1H, J2,3 = 10.8 Hz, H-2II), 3.77 (dd, 1H, J2,3 = 10.5 Hz, H-
The conjugate was purified by gel-filtration chromatography
(performed on an AktaTM system G&E Healthcare) using a S300
Sephacryl resin (G&E Healthcare) with a 10 mM NaPi/10 mM NaCl
pH 7.2 buffer).
The glycoconjugate was characterized by SDS–PAGE using 3–8%
Tris–Acetate gels (NuPAGE, Invitrogen). Total saccharide in the
conjugate was determined by HPAEC-PAD analysis and protein
content by MicroBCA potein assay kit (Thermo Scientific).

E. Danieli et al. / Bioorg. Med. Chem. 20 (2012) 6403–6415
6415
4.2.2. Immunizations
Supplementary data
Animal experimental guidelines set forth by the Novartis
Animal Care Department were followed in the conduct of all
animal studies.
Supplementary data (copies of all described 1H NMR and 13C
NMR spectra) associated with this article can be found, in the on-
line version, at http://dx.doi.org/10.1016/j.bmc.2012.08.048.
A group of eight female CD1 mice (5–6 week old) were immu-
nized on days 1 and 14 with 2
gen formulated with Aluminium hydroxyde as adjuvant. All
immunizations were performed by administering a 200 l of vac-
cine via intraperitoneal route. Adjuvant alone was used for nega-
tive control groups. Sera were collected on days 0 (before the
first immunization), 15 (two weeks after the first immunization)
and 22 (eight days after the second immunization).
Pool serum obtained after the second immunization was
analyzed by ELISA assay. Anti-capsular IgG antibody titers were
calculated by interpolating an optical density (OD) of 1 as cutoff
and expressed in ELISA units (EU). Sera showing anti-SA5 IgG titers
(6400 EU) were used for competitive ELISA and dot blot.
lg of conjugated carbohydrate anti-
References and notes
l
1. (a) Diekema, D. J.; Pfaller, M. A.; Jones, R. N. Int. J. Antimicrob. Agents 2002, 20,
412; (b) Diekema, D. J.; Pfaller, M. A.; Schmitz, F. J.; Smayevsky, J.; Bell, J.; Jones,
R. N.; Beach, M. Clin. Infect. Dis. 2001, 32, S114.
2. Pfaller, M. A.; Jones, R. N.; Doern, G. V.; Kugler, K. Antimicrob. Agents Chemother.
1998, 42, 1762.
3. (a) Robbins, J. B.; Schneerson, R.; Horwith, G.; Naso, R.; Fattom, A. Am. Heart J.
2004, 147, 594; (b) Poutrel, B.; Boutennier, A.; Sutra, L.; Fournier, J. M. J. Clin.
Microbiol. 1998, 26, 38.
4. (a) Fattom, A. I.; Schneerson, R.; Szu, S. C.; Vann, W. F.; Shiloach, J.; Karakawa,
W. W.; Robbins, J. B. Infect. Immun. 1990, 58, 2367; (b) Fattom, A. I.; Schiloach,
J.; Bryla, D.; Fitzgerald, D.; Pastan, I.; Karakawa, W. W.; Robbins, J. B.;
Schneerson, R. Infect. Immun. 1992, 60, 584; (c) Fattom, A. I.; Schneerson, R.;
Watson, D. C.; Karakawa, W. W.; Fitzgerald, D.; Pastan, I.; Li, X.; Shiloach, J.;
Bryla, D. A.; Robbbins, J. B. Infect. Immun. 1993, 61, 1023; (d) Fattom, A. I.; Lit,
X.; Chol, Y. H.; Burns, A.; Hawwari, A.; Shepherd, S. E.; Coughlinl, R.; Winston,
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Infect. Immun. 1996, 64, 1659.
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880; (b) Fattom, A.; Fuller, S.; Propst, M.; Winston, S.; Muenz, L.; He, D.; Naso,
R.; Horwith, G. Vaccine 2004, 23, 656.
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W. F. Carbohydr. Res. 1990, 201, 285.
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Horowitz, M.A. Ed.; Liss, New York, 1988; Vol. 64, pp 187.
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9. Horton, D.; Rodemeyer, G. Carbohydr. Res. 1977, 55, 35.
10. Horton, D.; Safki, H. Carbohydr. Res. 1978, 63, 270.
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15. Giannini, G.; Rappuoli, R. Nucleic Acids Res. 1984, 25(12), 4063.
4.2.3. Competitive ELISA
The protocol described above was followed to prepare SA5-
coated plates. The plate was designed to contain (a) a blank column
with TPBS alone, without serum and inhibitors, and (b) a column
with serum alone, without inhibitors (b0); the other columns con-
tained both, the serum and the inhibitors, including SA5 and the
not correlated polysaccharide laminarin as positive and negative
controls, respectively.
The different competitors (SA5 and compound 1) were predilut-
ed to obtain the starting concentration of 4.5 mg/ml and eight 10-
fold dilutions were performed on the plate.
The competitors at different concentrations were mixed with an
equal volume of a fixed dilution of anti-SA5 immune serum, fol-
lowed by 2 h incubation at 37 °C. After addition of 100 ll/well of
1:10,000 in TPBS antimouse IgG alkaline phosphatase conjugated
(Sigma–Aldrich), the plates were incubated for 1 h at 37 °C. Plates
were then developed for 30 min at rt with 100 ll per well of 1 mg/
ˇ
16. (a) Vesely´, J.; Rolenová, A.; Dzoganová, M.; Trnka, T.; Tišlerová, I.; Šaman, D.;
ml p-nitrophenyl phosphate disodium (Sigma–Aldrich) in 1 M
diethanolamine (pH 9.8) and read at 405 nm with a microplate
spectrophotometer (Biorad). All OD lectures were subtracted from
the mean value of the blank column (b). The inhibition percentage
was expressed as follows:
Ledvina, M. Synthesis 2006, 4, 699–704; (b) Litjens, R. E. J. N.; Leewenburgh, M.
A.; van der Marel, G. A.; van Boom, J. H. Tetrahedron Lett. 2001, 42, 8693; (c) van
den Bos, L. J.; Duivenvoorden, B. A.; de Koning, M. C.; Filippov, D. V.; Overkleeft,
H. S.; van der Marel, G. A. Eur. J. Org. Chem. 2007, 116.
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%inhibition ¼ ½ðB0 ꢀ ODxÞ=B0ꢄ ꢅ 100;
where B0 is the mean values of the b0 column (serum without inhib-
itor) and ODx is the optical density corresponding to each inhibitor
concentration. IC50 was defined as the inhibitor concentration result-
ing in 50% inhibition of the main reaction. Fitting of inhibition curves
and calculation of IC50 values were performed on the Graphpad
Prism software using the variable slope model (Graphpad Prism Inc.).
19. Schneider, F.; Bernauer, K.; Guggisberg, A.; van den Broek, P.; Hesse, M.;
Schmid, H. Helv. Chim. Acta 1974, 57, 434.
20. Draghetti, V.; Poletti, L.; Prosperi, D.; Lay, L. J. Carbohydr. Chem. 2001, 20, 813.
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Kamerling, J. P.; Vliegenthart, J. F. G. Carbohydr. Res. 1998, 309, 175.
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24. Bundle, D. R.; Gerken, M.; Peters, T. Carbohydr. Res. 1988, 174, 239.
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4.2.4. Immunodot blot analysis
Trisaccharide 1 (50
l
g), S. aureus type 5 polysaccharide (100 and
g, negative control)
10
lg, positive control) and laminarin (100 l
were deposed onto the cellulose membrane using the vacuum sys-
tem for 30 min. Subsequently, the membrane were washed three
times with 20 ml of PBS with 0.05% Tween 20 (TPBS) at pH 7.2
and then, blocked with 5% bovine serum albumin (Fraction V, Sig-
ma–Aldrich) in TPBS for 1 h at room temperature. After three wash-
ing with 20 ml of TPBS, 20 ml of anti SA5-CRM197 polyclonal
immune mouse sera pre-diluted in TPBS were placed on the mem-
brane at the dilution of 1:100 and incubated for 1 h at room temper-
ature. The membrane was washed again with TPBS and 20 ml of
1:1000 of anti-mouse IgG alkaline phosphatase conjugated
(Sigma–Aldrich) were added and incubated for 30 min at room
temperature. Finally, the membrane was developed at room
temperature with 20 ml of colorimetric AP substrate reagent kit
(Biorad).