Synthesis and biological evaluation of a trisaccharide repeating unit derivative of Streptococcus pneumoniae 19A capsular polysaccharide

Article abstract of DOI:10.1016/j.bmc.2018.10.016

Streptococcus pneumoniae (SP) is a common human pathogen associated with a broad spectrum of diseases and it is still a leading cause of mortality and morbidity worldwide, especially in children. Moreover, SP is increasingly associated with drug resistance. Vaccination against the pathogen may thus represent an important strategy to overcome its threats to human health. In this context, revealing the molecular determinants of SP immunoreactivity may be relevant for the development of novel molecules with therapeutic perspectives as vaccine components. Serogroup 19 comprises the immune-cross reactive types 19F, 19A, 19B and 19C and it accounts for a high percentage of invasive pneumococcal diseases, mainly caused by serotypes 19F and 19A. Herein, we report the synthesis and biological evaluation of an aminopropyl derivative of the trisaccharide repeating unit of SP 19A. We compare two different synthetic strategies, based on different disconnections between the three monosaccharides which make up the final trisaccharide, to define the best approach for the preparation of the trisaccharide. Synthetic accessibility to the trisaccharide repeating unit lays the basis for the development of more complex biopolymer as well as saccharide conjugates. We also evaluate the binding affinity of the trisaccharide for anti-19A and anti-19F sera and discuss the relationship between the chemical properties of the trisaccharide unit and biological activity.

Full text of DOI:10.1016/j.bmc.2018.10.016

Bioorganic&MedicinalChemistry26(2018)5682–5690
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of a trisaccharide repeating unit
derivative of Streptococcus pneumoniae 19A capsular polysaccharide
Laura Morellia, Silvia Fallarinib, Grazia Lombardib, Cinzia Colomboc, Luigi Layc,
⁎
Federica Compostellaa,
b
c
a
Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Via Saldini 50, 20133 Milano, Italy
Dipartimento di Scienze del Farmaco, Università degli Studi del Piemonte Orientale, Largo Donegani 2, 28100 Novara, Italy
Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, 20133 Milano, Italy
A B S T R A C T
Streptococcus pneumoniae (SP) is a common human pathogen associated with a broad spectrum of diseases and it is still a leading cause of mortality and morbidity
worldwide, especially in children. Moreover, SP is increasingly associated with drug resistance. Vaccination against the pathogen may thus represent an important
strategy to overcome its threats to human health. In this context, revealing the molecular determinants of SP immunoreactivity may be relevant for the development
of novel molecules with therapeutic perspectives as vaccine components. Serogroup 19 comprises the immune-cross reactive types 19F, 19A, 19B and 19C and it
accounts for a high percentage of invasive pneumococcal diseases, mainly caused by serotypes 19F and 19A. Herein, we report the synthesis and biological evaluation
of an aminopropyl derivative of the trisaccharide repeating unit of SP 19A. We compare two different synthetic strategies, based on different disconnections between
the three monosaccharides which make up the final trisaccharide, to define the best approach for the preparation of the trisaccharide. Synthetic accessibility to the
trisaccharide repeating unit lays the basis for the development of more complex biopolymer as well as saccharide conjugates. We also evaluate the binding affinity of
the trisaccharide for anti-19A and anti-19F sera and discuss the relationship between the chemical properties of the trisaccharide unit and biological activity.
1. Introduction
required for killing less immunogenic serotypes. Serogroup 19, which
comprises the immune-cross reactive types 19F, 19A, 19B and 19C,
belongs to this group, and deserves particular attention since it globally
accounts for a high percentage of IPD. Serogroup 19 IPD are mainly
caused by serotypes 19F and 19A, and, in particular, type 19F is one of
the most common causes of IPD in children.5 The low immunogenicity
of this serotype can be explained by the thickness of the 19F capsule
and increased resistance to complement deposition, which is the event
required to opsonize pneumococci, facilitate phagocytosis and pa-
thogen clearance. Serogroup 19 has also attracted the interest of the
research community because it represents one of the most significant
cases to investigate cross-protective immunity. Capsules of serotypes
19F and 19A are isopolymers, differing only in one glycosidic linkage
(glucose to rhamnose, Fig. 1). The high similarity of the two capsular
structures suggested the inclusion of only SP 19F in the formulation of
the first glycoconjugate vaccine PCV7, since antibodies to some CPS
may cross-react with related types providing protection against addi-
tional types. Indeed, this is what happened for the vaccine-type 6B,
included in PCV7, since 6B-induced antibodies resulted able to cross
protect against the structurally similar 6A CPS, with high effectiveness
against 6A disease.6 Unfortunately, antibodies elicited by 19F antigen
present in PCV7 provided limited cross-reactive protection against 19A
Streptococcus pneumoniae (SP) represents a relevant cause of infec-
tions associated with high mortality and morbidity: invasive pneumo-
coccal disease (IPD) indeed still shows a high incidence especially in
children and in the elderly. Capsular polysaccharides (CPSs) are the
primary determinants of the pathogenicity of the bacterium, and ac-
count for the classification of SP in more than 90 serotypes.1 A limited
subset of serotypes is responsible for the majority of pneumococcal
infections, and representatives of such subsets are contained in com-
mercial licensed vaccines (for example PCV7, Prevnar 7 – Wyeth
Pharmaceuticals, contains serotypes 4, 6B, 9V, 14, 18C, 19F and 23F).
Indeed, capsular polysaccharides (CPSs) are immunogenic, and the
generation of type-specific antibodies to CPS is protective.2 The pattern
of predominant IPD associated serotypes, subjected to a natural fluc-
tuation over time, contains also serotypes of low immunogenicity, such
as 6, 14, 19 and 23, where low immunogenicity unfortunately does not
equate to low virulence, especially in immune-naive hosts.3 Conse-
quently, a lower vaccination efficacy has been observed for these ser-
otypes.4 This is probably not associated to the absolute antibody con-
centration generated by the vaccine towards each single different
serotype, but, more likely, to the increased amount of antibodies
⁎
Corresponding author.
E-mail address: federica.compostella@unimi.it (F. Compostella).
https://doi.org/10.1016/j.bmc.2018.10.016
Received 17 July 2018; Received in revised form 10 October 2018; Accepted 18 October 2018
Availableonline19October2018
0968-0896/©2018ElsevierLtd.Allrightsreserved.

L. Morelli et al.
Bioorganic&MedicinalChemistry26(2018)5682–5690
O
P
O
P
*
O-
O
*
O-
O
Me
HO
O
Me
HO
O
2
O 3
1'
HO
O
NHAc
O
OH
NHAc
O
HO
HO
*
HO
HO
*
1'
HO
O
HO
O
O
O
HO
O
HO
O
HO
HO
n
n
SP 19F CPS
SP 19A CPS
Fig. 1. Structures of serotypes 19F and 19A capsular polysaccharides.
disease, with the consequence of increasing non-vaccine 19A serotype
carriage and virulence among population in a process defined “serotype
replacement”.7 Indeed, most of the PCV7 recipients achieved a sig-
nificant concentration of antibodies for the vaccine-associated serotype,
but the absence of 19A opsonophagocytic activity indicates that such
antibodies are not-functional against 19A.8 The immunogenicity of
the19F vaccine serotype, and the level of cross-opsonophagocytic an-
tibodies can be influenced by the conjugation method used to connect
the antigenic saccharide fragment to the T-helper peptide, like re-
ductive amination vs cyanylation.9 The lack of antibody-related cross-
protection between serotypes 19F and 19A may be alternatively related
to conformational differences between the two CPS structures.10 Of
note, the problem to induce protection against 19A disease was over-
come after the replacement of PCV7 with PCV13, that contains anti-
genic CPSs of both serotypes 19A and 19F. Remarkably, a higher level
of serotype 19F IgG was found in the sera of patients immunized with
PCV13 with respect to PCV7 recipients, suggesting a contribution of
cross-reactive 19A antibodies to the higher 19F opsonophagocytic ac-
tivity titers induced by PCV13.8
Trisaccharide 1 showed a similar and moderate activity towards both
sera, indicating that a limited cross recognition exists at the level of the
single repeating unit.
2. Results and discussion
2.1. Chemistry
A key point in our synthetic strategy towards compound 1 has been
the preparation of protected trisaccharide 2 as the direct precursor of
the target derivative. Compound 2 is a very versatile molecule, which
allows access to both the trisaccharide repeating unit of SP 19A (the
goal of this work), and, in principle, to oligomeric and/or shifted
fragments of SP 19A CPS. Elongation at the upstream residue of the
trisaccharide can be performed after selective reductive opening of the
benzylidene group. The functionalization at the reducing end with a 3-
aminopropyl linker has been designed to allow conjugation to carrier
proteins11 or the preparation of multivalent systems12,13 appropriate for
the in vivo evaluation of the immunogenic activity of 19A CPS-related
saccharide antigens. In this frame of thoughts, we have planned the
synthesis of rhamnosyl acceptor 3, with the aminopropyl linker already
installed,14 in order to avoid the glycosylation of the aglycon acceptor
at a later stage of the synthetic route.
Molecular approaches investigating the structural and chemical de-
terminants of the cross reactivity between 19F and 19A serotypes have
never been reported. Nonetheless, this knowledge may be useful to elu-
cidate the mechanism responsible for immunoreactivity. 19F and 19A
CPSs are linear biopolymers made up of trisaccharide repeating units
linked through phosphodiester bridges. Each trisaccharide is composed
by a β-D-ManpNAc-(1 → 4)-α-D-Glcp disaccharide linked to C2 or C3 of an
α-L-Rha unit respectively (Fig. 1). In this framework, we report the
synthesis of compound 1, the trisaccharide repeating unit of SP 19A,
functionalized at the reducing end with an aminopropyl linker, in turn
obtained from protected trisaccharide 2 (Fig. 2). Our strategy is based on
the development of a new route for the synthesis of an aminopropyl
functionalized rhamnosyl acceptor, compound 3 (Scheme 1). Further-
more, in search of the most straightforward approach towards 19A tri-
saccharide, we explored two alternative synthetic strategies, based on
different disconnections between the three monosaccharides which make
up the final trisaccharide. In particular, trisaccharide 1 was assembled
with higher yields when the α-Glc-(1 → 3)-Rha disaccharide was glyco-
sylated with a glucose moiety, followed by epimerization at C2.
To this aim, tetraacetyl rhamnopyranoside 415 was glycosylated
with N-Z-3-aminopropanol in the presence of boron trifluoride etherate
to give the rhamnose aminopropyl glycoside 5 in 75% yield (Scheme 1).
Zemplen deacetylation afforded deprotected rhamnoside 6 (93%),
which was regioselectively tritylated at position 3 by treatment with
trityl chloride at high temperature, and then benzylated in 64% yield
over two steps. Finally, the trityl group was removed by treatment with
trifluoroacetic acid to give rhamnoside acceptor 3 in 90% yield.
Two different disconnection strategies are possible for the con-
struction of the 19A trisaccharide repeating unit (Fig. 2), and all the
syntheses previously reported are based on a A-B+C approach, where a
preformed β-ManNAc-(1 → 4)-Glc (A-B) disaccharide is coupled with a
rhamnosyl acceptor (C).16–19 Based on our previous experience on the
synthesis of the trisaccharide related to SP 19F CPS,20,21 we first fol-
lowed the alternative B-C+A pathway in which an α-Glc-(1 → 3)-Rha
(B-C) disaccharide is initially formed in high selectivity, then β-glyco-
sylated with a glucose moiety (A) which is finally epimerized to N-
acetyl-mannosamine.
Finally, we evaluated the binding affinity of trisaccharide 1 towards
anti-19A and anti-19F sera, to investigate the role of the carbohydrate
portion of the repeating unit in the antibody binding affinity.
Bn
O
NH3Cl
O
NCbz
Me
HO
Me
BnO
O
O
C
O
O
NHAc
O
NHAc
O
OH
OBn
HO
BnO
HO
HO
HO
O
Ph
HO
O
BnO
O
O
BnO
O
O
HO
BnO
A
B
1
2
Fig. 2. Structures of the target compound 1 and its precursor 2.
5683

L. Morelli et al.
Bioorganic&MedicinalChemistry26(2018)5682–5690
OAc
O
NHCbz
O
NHCbz
Me
AcO
O
Me
AcO
Me
HO
O
O
AcO
AcO
HO
OAc
OAc
OH
4
5
6
Bn
Bn
O
NCbz
O
NCbz
c
d
Me
BnO
O
Me
BnO
O
TrO
HO
OBn
7
OBn
3
Scheme 1. Reagents and conditions: a. N-Z-3-aminopropanol, BF3⋅Et2O, DCM, 0 °C to rt, 75%; b. MeONa, MeOH, 93%; c. TrCl, Py, 60 °C; BnBr, NaH, 64%; CF3COOH,
DCM/MeOH, 90%.
Rhamnosyl acceptor 3 was thus glycosylated at position 3 with 2,3-
O-benzyl-4,6-O-benzylidene glucosyl trichloroacetimidate donor 822
under the catalysis of triethylsilyl triflate (Scheme 2). The aminopropyl
disaccharide 9 was recovered in excellent yield (93%) and complete α-
selectivity. Reductive opening of the benzylidene acetal to the corre-
sponding 6-O-benzyl ether was next accomplished by treatment of 9
with triethylsilane in the presence of boron trifluoride-diethyl ether
complex to give disaccharide acceptor 10 in good yield.
glycosylation between the 2-O-acetyl-3-O-benzyl-4,6-O-benzylidene
glucosyl trichloroacetimidate donor 1123 and disaccharide acceptor 10
to give compound 12. The β-selectivity was guaranteed by the anchi-
meric assistance offered by the acetyl group at position 2 of glucose 11.
Trisaccharide 12 was finally subjected to the synthetic sequence that
allows gluco to manno epimerization. The acetyl group was initially
removed to give unprotected 13 through Zemplen de-acetylation.
Compound 13 was then reacted with sulfonyldiimidazole in the pre-
sence of sodium hydride to yield saccharide 14, which was subjected to
nucleophilic displacement with sodium azide to give mannoside 15.
The newly established manno configuration was confirmed by the broad
1H NMR singlet for the anomeric proton of mannose. Finally, the azido
group was reduced with Zinc in the presence of acetic acid/acetic an-
hydride to give the fully protected trisaccharide 2, which upon hy-
drogenolysis gave the target trisaccharide 1 in quantitative yield.
Overall, the desired trisaccharide 1 was obtained starting from the
properly protected monosaccharide donors 8 and 11 and the rhamnosyl
acceptor 3 in 18% overall yield over 8 steps.
The desired trisaccharide scaffold was obtained through a high yield
Bn
NCbz
O
Ph
O
O
O
BnO
Me
BnO
O
O
CCl3
NH
BnO
8
+
HO
3
OBn
a
Bn
O
NCbz
Me
BnO
O
With the goal of developing a solid protocol to trisaccharide 1, we
next planned to test the feasibility of the alternative A-B+C dis-
connection strategy, which offers the advantage to reduce the number
of steps on the already formed trisaccharide scaffold. To this aim, we
decided to exploit a new synthetic strategy to obtain thio-disaccharide
16 for the glycosylation of the aminopropyl rhamnosyl acceptor 3. This
approach is based on our consolidated protocols for the construction of
the β-mannoside linkage (Scheme 3). In this framework, disaccharide
18 was initially formed in a stereoselective fashion through a high yield
glycosylation (86%) between phenylthio glucoside 1724 and tri-
chloroacetimidate donor 11. Next, epimerization at the C2′ of dis-
accharide 18, and the introduction of the acetamido group gave com-
pound 16 in 35% overall yield over 4 steps. In detail, compound 18 was
initially de-acetylated to 19, then the hydroxy group was activated in
high yield as imidazylate and subjected to azide displacement with
sodium azide to give mannoside 21, followed by azide reduction and N-
acetylation. Glycosylation with rhamnosyl acceptor 3 was promoted
using silver triflate-N-iodosuccinimide system as previously de-
scribed,19 and gave protected trisaccharide 2 in satisfactory yields but
low stereoselectivity (α/β = 1:2). Compound 2 was finally quantita-
tively deprotected to the target compound 1. The overall yield of the
second synthetic strategy to compound 1, starting from the suitable
building blocks 11, 17 and 3, is 6% over 7 steps. In general, this A-B+C
strategy shows an efficient and easy linear synthesis of the β-manno-
sylated thioglycosyl-donor 16, but suffers from moderate yields and low
stereoselectivity in the final glycosylation of rhamnose 3.
O
OBn
BnO
BnO
RO
O
R1O
9. R, R1 = CHPh
10. R = H, R1 = Bn
b
O
Ph
O
O
BnO
c
O
CCl3
AcO
Bn
11
O
NCbz
NH
Me
O
BnO
O
OBn
BnO
O
O
Ph
O
BnO
O
O
BnO
BnO
OR
12. R = Ac
13. R = H
14. R = SO2Im
d
e
Bn
NCbz
f
O
Me
BnO
O
O
R
O
OBn
BnO
O
O
O
Ph
BnO
O
BnO
BnO
15. R = N3
2. R = NHAc
h
g
1
Scheme 2. Reagents and conditions: a. TESOTf, DCM, −20 °C, 93%; b. Et3SiH,
BF3⋅Et2O, DCM, 0 °C, ms, 60%; c. TMSOTf, DCM, −20 °C, ms, 88%; d. MeONa,
MeOH, DCM, 89%; e. Im2SO2, NaH, DMF, −40 °C, 85%; f. NaN3, DMF, 80 °C,
80%; g. Zn, AcOH/Ac2O, THF, 62%; h. H2, Pd(OH)2, HCl, AcOEt, MeOH, quant.
2.2. Biology
The ability of increasing concentrations (from 10−7 to 100 mg/mL)
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L. Morelli et al.
Bioorganic&MedicinalChemistry26(2018)5682–5690
OBn
O
be important for their biological activity. The low effectiveness of the
newly synthetized compound could be related to its relative weak
avidity, since short chain lengths saccharide antigens, like a tri-
saccharide, have decreased strength of antibody-antigen binding. The
single repeating unit of 19A polysaccharide displayed inhibitory prop-
erties also in 19F system. The trisaccharide was slightly both more ef-
fective and potent in 19A than in 19F system (41% and 32% of in-
hibition for 19A and 19F respectively; EC50 1.3 × 10−3 and 2.7 × 10−2
for 19A and 19F respectively). These data suggest that differences in
structures of the 19A and 19F trisaccharides are almost negligible at the
repeating unit level, and a level of cross reactivity exists. It is reasonable
to speculate that saccharide fragments with chain length longer than
compound 1, resulting in more complex structures, would contain
multiple epitopes leading to an increase in specificity for 19A serum
and a reduction in cross-reactivity versus 19F.
HO
SPh
BnO
BnO
17
a
OBn
O
Ph
Ph
O
O
O
O
SPh
SPh
BnO
BnO
OR
BnO
18. R = Ac
b
c
19. R = H
20. R = SO2Im
d
R
OBn
O
O
O
O
O
BnO
BnO
BnO
21. R = N3
16. R = NHAc
f
e
3. Conclusions
g
2
1
In conclusion, the synthesis of compound 1, an aminopropyl deri-
vative of the trisaccharide repeating unit of SP 19A, has been developed
exploiting rhamnosyl acceptor 3, already functionalized with an ami-
nopropyl linker. We developed a new and more efficient synthetic route
to the rhamnosyl acceptor, which allows to obtain compound 3 in 40%
overall yield over four steps. Two different synthetic strategies were
used to build trisaccharide 1, allowing a direct comparison among the
two protocols. Based on our results, we suggest that the protocol based
on the B-C+A strategy is more effective than the A-B+C one. The
overall yield of assembly was around 20% for the first protocol, in
contrast to the more modest 6% of the second approach, which is
limited by the low selectivity in the glycosylation between disaccharide
A-B and rhamnoside 3 (C). The results confirmed that the stereo-
selectivity of the reaction of α-glucosylation is a function of the pro-
tecting groups on glucose, and the use of 4,6-O-benzylidene glucosyl
donors, protected with no participating groups at the 2-position, are
usually α-selective.25 Indeed, the use of 4,6-O-benzylidene glucosyl
donor 8 allowed the formation of the α-product in excellent yield.
Overall, the first approach to trisaccharide 1 is solid and highly re-
producible. Furthermore, the protected trisaccharide 2 is a valuable
intermediate for the synthesis of shifted fragments of the CPS of SP 19A:
Scheme 3. Reagents and conditions: a. 11, TESOTf, DCM, −20 °C, ms, 86%; b.
MeONa, MeOH, DCM, 75%; c. Im2SO2, NaH, DMF, −40 °C, 86%; d. NaN3, DMF,
80 °C, 83%; e. Zn, AcOH/Ac2O, THF, 66%; f. 3, AgOTf, NIS, DCM, −35 °C to
−10 °C, α/β: 60%, α: 20%) g. H2, Pd(OH)2, HCl, AcOEt, MeOH, quant.
of the newly synthetized trisaccharide to inhibit the binding between
19A polysaccharide coated onto plates (positive control) and the anti
19A rabbit polyclonal antibody was evaluated by competitive ELISA. To
evaluate the cross-reactivity against 19F serotype, competitive ELISA
was done using native 19F polysaccharide and 19F reference serum.
Fig. 3 shows the inhibition curves obtained with compound 1, under
evaluation in both systems. The relative efficacy of compound 1 was
calculated by measuring the maximum effect elicited in each system,
while the concentration that produces 50% of the maximum effect
(EC50) was taken as indirect index of its relative potency (Fig. 3). As
expected the natural polysaccharide exhibited higher efficacy (100%
inhibition at 10−1 mg/mL) and affinity (EC50 = 9.1 × 10−5 mg/mL)
than synthetized compound (41% inhibition at 100 mg/mL and
EC50 = 1.3 × 10−3) confirming that saccharide chain length seems to
Compound
19A PS
1
EC50 ± SEM (mg/mL)
(9.1 ± 1.2) × 10-5
Max inhibitiona (%)
Compound
19F PS
1
EC50 ± SEM (mg/mL)
(1.7 ± 0.006) × 10-4
(2.7 ± 0.3) × 10-2
Max inhibitiona (%)
100
41
100
32
(1.3 ± 0.02) × 10-3
a The maximum inhibition elicited by each compound at 1 mg/ml.
a The maximum inhibition elicited by each compound at 1 mg/ml.
Fig. 3. Results of the Elisa experiments with compound 1. Concentration/response curves of compound 1 on the inhibition of the binding between the 19A (on the
left) or 19F (on the right) native polysaccharides, coated onto the plates, and the anti-19A or anti-19F antibodies, respectively, were evaluated by a competitive ELISA
method.
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Bioorganic&MedicinalChemistry26(2018)5682–5690
the elongation of the trisaccharide at the upstream residue is functional
for the synthesis of oligomers functionalized at the downstream residue
with the aminopropyl linker, useful for conjugation to proteins or
multivalent scaffolds. We have also showed that compound 1, which
possesses moderate inhibitory activity towards anti-19A antibodies,
displays a comparable activity also towards anti-19F antibodies. This
data suggests that the two sera are not capable of discriminating small
differences in the structure of 19F and 19A trisaccharides. Since dif-
ferences in conformational preferences have been described for the
repeating units of SP 19A and 19F,10 it is reasonable to assume that
longer and structured fragments are needed to significantly affect the
binding specificity of the antibodies to the saccharide antigens.
3.66 (dd, 1H, J2,3 = 3.3 Hz, J3,4 = 9.5 Hz, H-3), 3.61–3.55 (m, 1H, H-5),
3.47–3.41 (m, 1H, H-a), 3.38 (t, 1H, J3,4 = J4,5 = 9.5 Hz, H-4), 3.28–3.18
(m, 2H, 2H-c), 1.83–1.75 (m, 2H, 2H-b), 1.27 (d, 3H, J5,6 = 6.4 Hz, 3H-
6); 13C NMR (MeOD): δ = 157.5 (C]O), 137.0 (arom), 128.1–127.4 (5C
arom), 100.3 (C-1), 72.6 (C-4), 71.0 (C-3), 70.9 (C-2), 68.4 (C-5), 66.0
(CH2Ph), 64.5 (C-a), 37.6 (C-c), 29.4 (C-b), 16.6 (C-6). MS (ESI) m/z (%):
378.1 (100) [M+Na]+, 732.8 (12) [2M+Na]+. HRMS (ESI): m/z calcd
for C17H25NO7Na 378.1529 [M+Na]+, found 378.1526.
4.1.3. Synthesis of N-benzyl-N-benzyloxycarbonyl-3-aminopropyl 2,4-di-O-
benzyl-3-O-trityl-α-L-rhamnopyranoside (7)
A
mixture of 6 (1.60 g, 4.50 mmol), trityl chloride (2.51 g,
9.00 mmol) and dry pyridine (15 mL) was stirred at 60 °C for 20 h. After
the addition of Et3N (2 mL), the reaction was diluted with EtOAc
(50 mL) and washed with HCl 1 N (2 × 50 mL). The combined aqueous
phases were extracted with AcOEt (3 × 40 mL), and then the combined
organics were washed with satd. NaHCO3 soln. (1 × 60 mL), dried over
Na2SO4 and evaporated under reduced pressure. To a solution of the
crude and benzyl bromide (3.2 mL, 27 mmol) in dry DMF (50 mL), NaH
(60% in oil, 1.21 g, 31.5 mmol) was added portionwise at 0 °C. The
reaction was warmed to room temperature. After 5 h, an additional
amount of NaH (60% in oil, 0.34 g, 9.00 mmol) was added and the
reaction stirred for 12 h. The mixture was quenched by carefully ad-
dition of MeOH (5 mL), then diluted with HCl 1 N (100 mL), and ex-
tracted with AcOEt (3 × 100 mL). The combined organics were washed
with brine (2 × 150 mL), dried over Na2SO4 and evaporated. The crude
was purified through flash chromatography (hexane/AcOEt, 82:25) to
give product 7 (2.5 g, 64%) as a light yellow viscous oil. [α]D20 = −8.7
(c = 0.5 in chloroform). 1H NMR (CDCl3): δ = 7.65–7.10 (m, 35H,
arom.), 5.25–5.00 (m, 3H, CH2Ph), 4.80–4.65 (m, 1H, CH2Ph),
4.55–4.35 (m, 3H, H-1 and CH2Ph), 4.30–4.15 (m, 2H, CH2Ph), 4.10
(dd, 1H, J2,3 = 2.6 Hz, J3,4 = 9.2 Hz, H-3), 3.90–3.70 (m, 1H, H-4),
3.65–3.35 (m, 2H, H-5 and H-a), 3.35–3.05 (m, 3H, H-a and 2H-c),
2.45–2.25 (m, 1H, H-2), 1.75–1.55 (m, 2H, 2H-b), 1.33(d, 3H,
4. Experimental section
4.1. Synthetic procedures
Standard laboratory procedures were followed to carry out the re-
actions and to prepare dry solvents.26 Optical rotations were measured
with a Perkin-Elmer 241 polarimeter at 20 °C. 1H and 13C NMR spectra
were recorded with a Bruker AVANCE-500 spectrometer at a sample
temperature of 298 K.27 Mass spectrometric analyses were performed
on a Thermo Quest Finnigan LCQ™DECA ion trap mass spectrometer;
equipped with a Finnigan ESI interface. High-resolution mass spectra
were collected by electrospray ionization (ESI) spectroscopy on a QTof
SYNAPT G2Si Mass Spectrometer. NaH was washed with hexane three
times prior to use.
4.1.1. Synthesis of N-(benzyloxycarbonyl)aminopropyl 2,3,4-tri-O-acetyl-
α-L-rhamnopyranoside (5)
BF3⋅Et2O (5.0 mL, 39.45 mmol) was slowly added through a drop-
ping funnel to a solution at 0 °C under argon of compound 4 (2.28 g,
6.86 mmol) and N-CBz-aminopropanol (3.59 g, 17.15 mmol) in dry
CH2Cl2 (70 mL). The reaction was stirred at room temperature, mon-
itored by TLC (hexane/ethyl acetate, 1:1) and appeared to be complete
after 12 h. The reaction was washed with saturated NaHCO3 solution
(2 × 100 mL), and the combined aqueous phases extracted with AcOEt
(2 × 100 mL). The combined organic layers were dried over Na2SO4
and concentrated. Purification by flash chromatography (hexane/
J
5,6 = 6.2 Hz, 3H-6); 13C NMR (CDCl3): δ = 156.8 (C]O), 145.1–127.0
(42C, arom), 97.2 (C-1), 87.4 (C trityl), 80.5 (C-4), 77.9 (C-2), 75.3
(CH2Ph), 73.8 (C-3), 71.9 (CH2Ph), 69.1 (C-5), 67.2 (CH2Ph), 65.0 (C-
a), 51.0 (CH2Ph), 45.1–44.1 (m, C-c), 28.6–28.0 (m, C-b), 18.4 (C-6);
MS (ESI) m/z (%): 890.5 (100) [M+Na]+. HRMS (ESI): m/z calcd for
AcOEt, 6:4) gave pure
5
(2.48 g, 75%) as
a
colorless oil.
C
57H57NO7Na 890.4033 [M+Na]+, found 890.4029.
[α]D20 = −43.6 (c = 0.5 in chloroform). 1H NMR (CDCl3):
δ = 7.40–7.30 (m, 5H, arom.), 5.29 (dd, 1H,
J
2,3 = 3.5 Hz,
4.1.4. Synthesis of N-benzyl-N-benzyloxycarbonyl-3-aminopropyl 2,4-di-O-
benzyl-α-L-rhamnopyranoside (3)
J
3,4 = 10.0 Hz, H-3), 5.25 (dd, 1H, J1,2 = 1.7 Hz, J2,3 = 3.5 Hz, H-2),
5.13 (s, 2H, CH2Ph), 5.08 (t, 1H, J3,4 = J4,5 = 10.0 Hz, H-4), 4.95–4.88
(m, 1H, NH), 4.73 (br s, 1H, H-1), 3.91–3.83 (m, 1H, H-5), 3.81–3.74
(m, 1H, H-a), 3.54–3.47 (m, 1H, H-a′), 3.37–3.29 (m, 2H, 2H-c), 2.17 (s
,3H, CH3CO), 2.06 (s, 3H, CH3CO), 2.01(s, 3H, CH3CO), 1.92–1.80 (m,
2H, 2H-b), 1.24 (d, 3H, J5,6 = 6.3 Hz, 3H-6); 13C NMR (CDCl3):
δ = 170.2 (C]O), 170.0 (C]O), 169.9 (C]O), 156.4 (C]O, Cbz),
136.6 (arom), 128.5–128.1 (5C arom), 97.5 (C-1), 71.1 (C-4), 69.8 (C-
2), 69.1 (C-3), 66.7 (CH2Ph), 66.5 (C-5), 65.8 (C-a), 38.4 (C-c), 29.6 (C-
b), 20.9 (CH3), 20.8 (CH3), 20.7 (CH3), 17.4 (C-6). MS (ESI) m/z (%):
504.1 (100) [M+Na]+. HRMS (ESI): m/z calcd for C23H31NO10Na
504.1846 [M+Na]+, found 504.1836.
To a solution of compound 7 (0.90 g, 1.04 mmol) in 21 mL of DCM/
MeOH (6:1, v/v), trifluoroacetic acid (0.60 mL, 7.88 mmol) was added
dropwise. The reaction was stirred at room temperature for 5 h, then
quenched to neutrality through addition of TEA. The solvent was eva-
porated under reduced pressure, and the crude purified by flash chro-
matography (hexane/AcOEt, 82:25) to give rhamnoside 3 (0.58 g, 90%)
as a colorless oil. [α]D20 = −13.8 (c = 0.1 in chloroform). 1H NMR
(CDCl3): δ = 7.43–7.13 (m, 20H, arom), 5.23–5.14 (m, 2H, CH2Ph),
4.92 (d, 1H, J = 11.1 Hz, CH2Ph), 4.81–4.70 (m, 2H, H-1 and CH2Ph),
4.67 (d, 1H, J = 11.1 Hz, CH2Ph), 4.63–4.43 (m, 3H, CH2Ph),
3.97–3.87 (m, 1H, H-3), 3.74–3.17 (m, 3H, H-2,5 and H-a), 3.48–3.25
(m, 4H, H-4, H-a and 2H-c), 1.87–1.70 (m, 2H, 2H-b), 1.33 (d, 3H,
4.1.2. Synthesis of N-(benzyloxycarbonyl)aminopropyl α-L-
J
5,6 = 6.2 Hz, 3H-6); 13C NMR (CDCl3): δ = 156.2 (C]O), 138.6–127.3
rhamnopyranoside (6)
(24C, arom), 97.0 (C-1), 82.3 (C-4), 78.6 (C-2), 75.1 (CH2Ph), 73.0
(CH2Ph), 71.7 (C-3), 67.2 (2C, C-5 and CH2Ph), 65.0 (C-a), 50.5 and
50.7 (d, NCH2Ph), 44.5 and 43.7 (d, C-c), 28.3 and 27.8 (C-b), 18.0 (C-
6); MS (ESI) m/z (%): 684.4 (100) [M+Na]+. HRMS (ESI): m/z calcd
for C38H43NO7Na [M+Na]+ 648.2937, found 648.2936.
Compound
5 (2.40 g, 4.98 mmol) was dissolved in dry di-
chloromethane (50 mL) and sodium methoxide in dry methanol (0.2 M
solution, 12 mL) was added. The reaction was stirred for 3 h at room
temperature, then it was neutralized with an ion exchange resin (Dowex
50 × 8, H+ form), filtered and concentrated. The crude was subjected to
flash chromatography (CH2Cl2/MeOH, 9:1) to give compound 6 (1.64 g,
93%) as a colorless oil. [α]D20 = −38.5 (c = 0.5 in chloroform). 1H
NMR (MeOD): δ = 7.40–7.28 (m, 5H, arom.), 5.09 (br s, 2H, CH2Ph),
4.67 (br s, 1H, H-1), 3.83–3.80 (m, 1H, H-2), 3.77–3.70 (m, 1H, H-a),
4.1.5. Synthesis of N-benzyl-N-benzyloxycarbonyl-3-aminopropyl 2,3-di-O-
benzyl-4,6-O-benzylidene-α-D-glucopyranosyl-(1 → 3)-2,4-di-O-benzyl-α-L-
rhamnopyranoside (9)
A solution of glucosyl trichloroacetimidate 8 (0.70 g, 1.20 mmol)
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L. Morelli et al.
Bioorganic&MedicinalChemistry26(2018)5682–5690
and rhamnoside 3 (0.30 g, 0.48 mmol) in DCM (16 mL) was cooled at
−20 °C, then triethylsilyl trifluoromethanesulfonate (0.1 M solution in
DCM, 0.95 mL) was added dropwise. After 1.5 h the reaction was
quenched by the addition of TEA, and allowed to warm to room tem-
perature. The reaction was concentrated, then purified by flash chro-
matography (hexane/AcOEt, 8:2) to give disaccharide 9 (0.47 g, 93%)
as an oil. [α]D20 = +3.8 (c = 1 in chloroform). 1H NMR (CDCl3):
δ = 7.50–7.14 (m, 35H, arom.), 5.56 (s, 1H, CHPh), 5.18 (br s, 2H,
CH2Ph), 5.14 (d, 1H, J1′,2′ = 3.4 Hz, H-1′), 4.98–4.93 (m, 2H, CH2Ph),
4.85–4.41 (m, 9H, H-1 and CH2Ph), 4.21–4.04 (m, 4H, H-3, 6′ and 2Hs),
3.90–3.81 (m, 1H, H-2), 3.69–3.55 (m, 6H, H-a, 4, 5, 2′, 6′ and 1H),
3.44–3.22 (m, 3H, 1H-a and 2H-c), 1.84–1.69 (m, 2H, 2H-b), 1.31 (br d,
3H, 3H-6). 13C NMR (CDCl3): δ = 163.3 (C]O), 138.6–126.2 (42C,
arom.), 101.3 (PhCH), 98.1 (C-1), 96.3 (C-1′), 82.6, 80.2, 79.2, 78.5,
76.7 (C-3), 75.5 (2C, C-2 and CH2Ph), 75.1 (CH2Ph), 73.7 (CH2Ph),
73.2 (CH2Ph), 69.0 (C-6′), 68.4, 67.2 (CH2Ph), 65.1 (C-a), 63.0, 50.52
and 50.75 (NCH2Ph),43.73 and 44.50 (C-c), 27.81 and 28.30 (C-b),
18.0 (C-6). MS (ESI) m/z (%): 1079.1 (100) [M+1+Na]+. HRMS (ESI):
m/z calcd for C65H69NO12Na 1078.4717 [M+Na]+, found 1078.4712.
6b′, 3″, 6b″), 3.35–3.21 (m, 3H, 1H-a and 2H-c), 3.18–3.10 (m, 1H, H-
5″), 1.82 (s, 3H, COCH3), 1.81–1.67 (m, 2H, 2H-b), 1.22 (br d, 3H, 3H-
6). 13C NMR (CDCl3): δ = 168.9 (C]O), 139.3–126.0 (54C, arom.),
101.1 (CHPh), 100.8 (C-1″), 98.2 (C-1), 96.5 and 96.3 (C-1′), 81.6 (C-
4″), 80.1, 79.9, 79.2, 78.7, 77.4 (C-3), 76.7, 76.1 (C-2), 75.0 (CH2Ph),
74.9 (CH2Ph), 74.0 (CH2Ph), 73.6 (CH2Ph), 73.3 (2C, C-2″ and CH2Ph),
73.2 (CH2Ph), 70.7, 68.6 (C-6″), 68.2, 67.6 (C-6′), 67.2 (CH2Ph), 65.9
(C-5″), 65.1 (C-a), 50.8 and 50.5 (NCH2Ph), 44.6 and 43.7 (C-c), 28.3
and 27.9 (C-b), 20.8 (CH3CO), 18.0 (C-6). MS (ESI) m/z (%): 1463.5
(100) [M+1+Na]+
. HRMS (ESI): m/z calcd for C87H93NO18Na
1462.6290 [M+Na]+, found 1462.6276.
4.1.8. Synthesis of N-benzyl-N-benzyloxycarbonyl-3-aminopropyl 3-O-
benzyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1 → 4)-2,3,6-tri-O-benzyl-α-
D-glucopyranosyl-(1 → 3)-2,4-di-O-benzyl-α-L-rhamnopyranoside (13)
To a stirred solution of 12 (0.27 g, 0.19 mmol) in DCM/MeOH 1:1
(6 mL) sodium methoxide in methanol (1 M solution, 0.19 mL) was
added. The reaction was stirred for 48 h at room temperature, then it
was neutralized with an ion exchange resin (Dowex 50 × 8, H+ form),
filtered and concentrated. The crude product was subjected to flash
chromatography (hexane/AcOEt, 7:3) to give pure 13 (0.24 g, 89%) as
an amorphous solid. [α]D20 = +15.3 (c = 1 in chloroform). 1H NMR
(CDCl3): δ = 7.52–7.16 (m, 45H, arom.), 5.48 (s, 1H, PhCH), 5.24–5.11
(m, 2H, H-1′ and CH2Ph), 5.00–4.61 (m, 10H, H-1 and CH2Ph),
4.61–4.41 (m, 4H, CH2Ph), 4.37 (d, 1H, J1″,2″ = 7.5 Hz, H-1″),
4.33–4.28 (m, 1H, CH2Ph), 4.12–3.95 (m, 5H, H-3, 3′, 4′, 5′, 6a″),
3.89–3.80 (m, 1H, H-2), 3.80–3.74 (m, 1H, H-6a′), 3.74–3.58 (m, 5H,
H-a, 4, 5, 2′), 3.58–3.51 (t, 1H, J3″,4″ = J4″,5″ = 9.3 Hz, H-4″),3.51–3.37
(m, 3H, H-6b′, 3″, 6b″),3.37–3.21 (m, 4H, H-a, 2″ and 2H-c), 3.11–3.04
(m, 1H, H-5″), 1.85–1.60 (m, 3H, 2H-b and OH), 1.35 (d, 3H,
J = 5.7 Hz, 3H-6). 13C NMR (CDCl3): δ = 157.7 (C]O), 128.4–126.0
(54C, arom), 103.4 (C-1″), 101.2 (CHPh), 98.3 (C-1), 94.5 (br s, C-1′),
81.2 (C-4″), 80.6, 80.3 (C-3″), 80.0, 79.1 (C-2′), 77.4, 76.1 (C-3), 75.2
(2C, C-2, 2″), 75.1–73.3 (6C, CH2Ph), 70.0 (C-5′), 68.7 (C-6″), 68.4,
68.2 (C-6′), 67.2 (CH2Ph), 66.1 (C-5″), 65.2 (C-a), 50.6 (br s, NCH2Ph),
44.5 and 44.4 (C-c), 27.9 and 27.6 (C-b), 18.0 (C-6). MS (ESI) m/z (%):
4.1.6. Synthesis of N-benzyl-N-benzyloxycarbonyl-3-aminopropyl 2,3,6-tri-O-
benzyl-α-D-glucopyranosyl-(1 → 3)-2,4-di-O-benzyl-α-L-rhamnopyranoside
(10)
Compound 9 (0.45 g, 0.43 mmol) and 4 Å m.s. (0.45 g) were dis-
solved in DCM (10 mL), stirred at room temperature for 15 min, then
the suspension was cooled at 0 °C. Triethylsilane (0.63 mL, 4.30 mmol)
was added, followed by the slow dropwise addition of BF3·Et2O
(0.27 mL, 2.15 mmol). The reaction was stirred at 0 °C for 2 h, then
quenched with triethylamine, diluted with DCM, filtered over celite,
and concentrated in vacuo. The residue was purified by flash chroma-
tography (Hexane/AcOEt, 8:2) to afford compound 10 (0.27 g, 60%) as
an oil. [α]D20 = +10.8 (c = 1 in chloroform). 1H NMR (CDCl3):
δ = 7.40–7.20 (m, 35H, arom), 5.21–5.13 (m, 3H, H-1′ and CH2Ph),
5.00–4.80 (m, 2H, CH2Ph), 4.84–4.83 (m, 11H, H-1 and CH2Ph),
4.14–4.06 (m, 1H, H-3), 4.04–3.97 (m, 1H, H-5′), 3.96–3.80 (m, 2H, H-
2, 3′), 3.74–3.45 (m, 7H, H-a, 4, 5, 2′, 4′, 6′a, 6′b), 3.42–3.19 (m, 3H,
2H-c and 1H-a), 2.25–2.05 (br s, 1H, OH), 1.81–1.65 (m, 2H, 2H-b),
1.33 (br s, 3H, 3H-6). 13C NMR (CDCl3): δ = 156.4 (C]O),
138.8–127.3 (42C, arom.), 98.2 (C-1), 95.0 (C-1′), 81.3 (C-3′), 80.1,
79.4, 76.0 (C-3), 75.5 (C-2), 75.2 (2C, CH2Ph), 73.4 (CH2Ph), 73.2
(CH2Ph), 73.0 (CH2Ph), 71.2, 70.2 (C-5′), 69.5 (C-6′), 68.4, 67.2
(CH2Ph of Cbz), 65.1 (C-a), 50.5 and 50.8 (NCH2Ph), 43.7 and 44.5 (C-
c), 27.8 and 28.3 (C-b), 18.1 (C-6). MS (ESI) m/z (%): 1080.1 (100) [M
+Na]+. HRMS (ESI): m/z calcd for C65H71NO12Na 1080.4874 [M
+Na]+, found 1080.4883.
1420.7 (100) [M+NaM+Na]+
.
HRMS (ESI): m/z calcd for
C
85H91NO17Na 1420.6185 [M+Na]+, found 1420.6194.
4.1.9. Synthesis of N-benzyl-N-benzyloxycarbonyl-3-aminopropyl 3-O-benzyl-
4,6-O-benzylidene-2-O-(N-imidazole-1-sulfonyl)-β-D-glucopyranosyl-(1 → 4)-
2,3,6-tri-O-benzyl-α-D-glucopyranosyl-(1 → 3)-2,4-di-O-benzyl-α-L-
rhamnopyranoside (14)
NaH (60% in oil, 0.070 g, 1.76 mmol) was added to a stirred solu-
tion of compound 13 (0.23 g, 0.16 mmol) in dry DMF (3.5 mL) at room
temperature. After 1 h, the suspension was cooled at −40 °C and 1,1′-
sulfonyl-diimidazole (0.22 g, 1.12 mmol) in dry DMF (1.5 mL) was
added. After 24 h the reaction mixture was quenched with MeOH and
allowed to warm to room temperature, then diluted with water (40 mL).
The mixture was extracted with AcOEt (3 × 50 mL). The combined
organic layers were washed with brine, dried over Na2SO4, filtered and
evaporated. Flash chromatography (hexane/AcOEt, 7:3) of the crude
product gave trisaccharide 14 (0.21 g, 85%) as an amorphous solid.
[α]D20 = +1.8 (c = 1 in chloroform). 1H NMR (CDCl3): δ = 7.94 (br s,
1H, Im), 7.53–7.10 (m, 45H, arom), 7.00 (m, 2H, Im), 5.46 (s, 1H,
PhCH), 5.25–5.12 (m, 3H, H-1′ and CH2Ph), 4.97 (d, 1H, J = 11.1 Hz,
CH2Ph), 4.85–4.58 (m, 10H, H-1 and CH2Ph), 4.58–4.44 (m, 4H, H-2″
and CH2Ph), 4.32 (d, 1H, J1″,2″ = 7.9 Hz, H-1″), 4.23 (dd, 1H,
J5″,6″ = 4.9 Hz, J6a″,6b″ = 10.6 Hz, H-6a″), 4.16–4.09 (m, 1H, CH2Ph),
4.09–3.80 (m, 5H, H-2, 3, 3′, 4′, 5′), 3.75–3.52 (m, 5H, H-a, 4, 5, 2′,4″),
3.52–3.20 (m, 7H, H-a′, c, c′, 6a′, 6b′, 3″, 6b″), 3.10–2.98 (m, 1H, H-5″),
1.91–1.69 (m, 2H, 2H-b), 1.29–1.21 (br d, 3H, 3H-6). 13C NMR (CDCl3):
δ = 155.6 (C]O), 139.1–136.5 (9C, arom), 136.8 (C Im),129.2–126.0
(46C, arom.), 118.6 (C Im), 101.4 (CHPh), 98.6 (C-1″), 98.2 (C-1), 97.1
(br s, C-1′), 85.7 (C-2″), 81.9 (C-4″), 80.3 (br s, C-4), 79.5 (C-3′), 79.2
(C-2′), 78.4 (br s, C-3), 76.7 (C-3″), 76.5 (br s, C-2), 76.2 (C-4′), 75.2
4.1.7. Synthesis of N-benzyl-N-benzyloxycarbonyl-3-aminopropyl 2-O-acetyl-
3-O-benzyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1 → 4)-2,3,6-tri-O-benzyl-
α-D-glucopyranosyl-(1 → 3)-2,4-di-O-benzyl-α-L-rhamnopyranoside (12)
A suspension of 2-O-acetyl-glucosyl trichloroacetimidate 11 (0.42 g,
0.78 mmol), disaccharide 10 (0.23 g, 0.22 mmol) and 4 Å m.s. (0.23 g)
in DCM (7 mL) was stirred for 0.15 min at room temperature, then
cooled at −20 °C. Triethylsilyl trifluoromethanesulfonate (0.1 M solu-
tion in DCM, 0.44 mL) was added dropwise and the disappearance of
the starting material was followed by TLC (Toluene/Acetone, 7:3;
hexane/AcOEt, 7:3). After 1.5 h, the reaction was quenched with trie-
thylamine, diluted with DCM, filtered over Celite, and the solvent
evaporated. The crude product was purified by flash chromatography
(hexane/AcOEt, 8:2) to give 12 (0.28 g, 88%) as an amorphous solid.
[α]D20 = +5.9 (c = 1 in chloroform). 1H NMR (CDCl3): δ = 7.53–7.10
(m, 45H, arom.), 5.48 (s, 1H, PhCH), 5.23–5.13 (m, 3H, H-1′ and
CH2Ph), 4.97–4.82 (m, 4H, H-2″ and CH2Ph), 4.76 (d, 2H, CH2Ph),
4.72–4.53 (m, 7H, H-1 and CH2Ph), 4.52–4.44 (m, 3H, H-1″ and
NCH2Ph), 4.29–4.22 (m, 1H, CH2Ph), 4.17–4.11 (m, 1H, H-6a″),
4.08–3.99 (m, 1H, H-3), 3.99–3.91 (m, 3H, H-3′, 4′, 5′), 3.91–3.82 (m,
1H, H-2), 3.74–3.53 (m, 6H, H-a, 4, 5, 2′, 6a′, 4″), 3.51–3.36 (m, 3H, H-
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L. Morelli et al.
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(2C, CH2Ph), 74.6 (br s, CH2Ph), 74.3 (CH2Ph), 73.6 (CH2Ph), 73.1 (br
s, CH2Ph), 70.2 (C-5′), 68.4 (C-6″), 68.3 (C-5), 67.3 (C-6′), 67.2
(CH2Ph), 65.7 (C-5″), 65.2 (C-a), 50.8 and 50.5 (NCH2Ph), 44.6 and
43.7 (C-c), 28.4 and 27.9 (C-b), 18.0 (C-6). MS (ESI) m/z (%): 1550.3
(100) [M+NaM+Na]+. HRMS (ESI): m/z calcd for C88H93N3O19NaS
1550.6022 [M+Na]+, found 1550.6055.
4.1.12. Synthesis
of
phenyl
3-O-benzyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1 → 4)-2,3,6-tri-O-benzyl-1-thio-β-D-glucopyranoside (19)
To a stirred solution of 18 (0.28 g, 0.30 mmol) in DCM (6 mL) so-
dium methoxide in methanol (0.1 M solution, 0.60 mL) was added. The
reaction was stirred for 17 h at room temperature, then it was neu-
tralized with an ion exchange resin (Dowex 50 × 8, H+ form), filtered
and concentrated. The crude product was subjected to flash chroma-
tography (hexane/AcOEt, 8:2) to give pure 19 (0.20 g, 75%) as an
amorphous white solid. [α]D20 = +0.20 (c = 1 in chloroform). 1H
NMR (CDCl3): δ = 7.60–7.27 (m, 30H, arom.), 5.49 (s, 1H, CHPh), 5.33
(s, 1H, OH), 4.98–4.94 (m, 2H, CH2Ph), 4.87–4.78 (m, 3H, CH2Ph),
4.74–4.60 (m, 5H, H-1, 1′ and CH2Ph), 4.10–4.01 (m, 2H, H-3, 6a), 3.97
(dd, 1H, J5′,6′a = 5 Hz, J6′a,6′b = 10.4 Hz, H-6′a), 3.87–3.84 (m, 1H, H-
6b), 3.68 (dd, 1H, J3,4 = J4,5 = 8.8 Hz, H-4), 3.63–3.48 (m, 6H, H-2, 4,
5, 2′, 3′, 6′), 3.18–3.12 (m, 1H, H-5′). 13C NMR (CDCl3):
δ = 138.8–126.0 (36C, arom.), 103.6 (C-1′), 101.2 (CHPh), 87.5 (C-1),
85.5 (C-4), 81.3, 80.5, 80.4, 78.8, 77.0 (C-3), 75.5, 75.3 (2C, CH2Ph),
74.6 (CH2Ph), 73.6 (CH2Ph), 68.6 (C-6′), 68.5 (C-6), 66.4 (C-5′). MS
(ESI) 905.3 (100) [M+Na]+, 1787.9 (40) [2M+Na]+. HRMS (ESI): m/
z calcd for C53H54O10NaS 905.3335 [M+Na]+, found 905.3331.
4.1.10. Synthesis of N-benzyl-N-benzyloxycarbonyl-3-aminopropyl 2-
azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-β-D-mannopyranosyl-(1 →
4)-2,3,6-tri-O-benzyl-α-D-glucopyranosyl-(1 → 3)-2,4-di-O-benzyl-α-L-
rhamnopyranoside (15)
To a stirred solution of 14 (0.20 g, 0.13 mmol) in dry DMF (4 mL),
sodium azide (0.085 g, 1.30 mmol) was added and the resulting solu-
tion was heated at 80 °C. After 5 h, the reaction was cooled to room
temperature, diluted with H2O and extracted with AcOEt (3 × 40 mL).
The combined organic layers were dried over Na2SO4, filtered and
evaporated. The crude product was purified by flash chromatography
(hexane/AcOEt, 75:25) to give compound 15 (0.15 g, 80%) as colour-
less oil. [α]D20 = −4.1 (c = 1 in chloroform). 1H NMR (CDCl3):
δ = 7.63–7.08 (m, 45H, arom.), 5.52 (s, 1H, PhCH), 5.25–5.16 (m, 2H,
CH2Ph), 5.13 (d, 1H, J1′,2′ = 3.4 Hz, H-1′), 5.06 (d, 1H, J = 10.5 Hz,
CH2Ph), 4.92 (d, 1H, J = 11.5 Hz, CH2Ph), 4.88–4.82 (m, 2H, CH2Ph),
4.82–4.75 (m, 2H, CH2Ph), 4.74–4.45 (m, 8H, H-1 and CH2Ph), 4.34 (br
s, 1H, H-1″), 4.19–3.95 (m, 6H, H-3, 3′, 4′, 5′, 6a″ and 1H × CH2Ph),
3.88 (t, 1H, J3″,4″ = J4″,5″ = 9.4 Hz, H-4″), 3.85–3.76 (m, 1H, H-2),
3.75–3.58 (m, 4H, H-a, 4, 5, 2′), 3.56–3.48 (m, 2H, H-2″, 6b″),
3.48–3.20 (m, 6H, H-a′, 6a′, 6b′, 3″ and 2H-c), 3.00–2.92 (m, 1H, H-5″),
1.88–1.72 (m, 2H, 2H-b), 1.33 (d, 3H, J5,6 = 5.8 Hz, 3H-6). 13C NMR
(CDCl3): δ = 156.6 and 156.1 (C]O), 137.9–126.0 (54C, arom.), 101.5
(CHPh), 99.7 (C-1″), 98.4 (C-1), 95.5 (br s, C-1′), 80.5 (C-3′), 79.8 (C-4),
79.0 (C-2′), 78.5 (C-4″), 76.9 (C-3), 76.7 (C-4′), 76.4 (C-3″), 75.9 (C-2),
75.1 (CH2Ph), 74.7 (CH2Ph), 73.7 (CH2Ph), 73.6 (CH2Ph), 73.5
(CH2Ph), 72.5 (CH2Ph), 69.7 (C-5′), 68.6 (C-5), 68.4 (C-6″), 68.2 (C-6′),
67.2 (CH2Ph), 67.1 (C-5″), 65.2 (C-a), 63.2 (C-2″), 50.8 and 50.6
(NCH2Ph), 44.6 and 43.7 (C-c), 28.3 and 27.9 (C-b), 18.0 (C-6). MS
(ESI) m/z (%): 1446.4 (100) [M+1+Na]+. HRMS (ESI): m/z calcd for
4.1.13. Synthesis of phenyl 3-O-benzyl-4,6-O-benzylidene-2-O-(N-
imidazole-1-sulfonyl)-β-D-glucopyranosyl-(1 → 4)-2,3,6-tri-O-benzyl-1-
thio-β-D-glucopyranoside (20)
NaH (60% in oil, 0.13 g, 3.30 mmol) was added to a stirred solution of
compound 19 (0.19 g, 0.22 mmol) in dry DMF (6 mL) at room tempera-
ture. After 1 h, the suspension was cooled at −40 °C and 1,1′-sulfonyl-
diimidazole (0.44 g, 2.20 mmol) in dry DMF (3 mL) was added. After 2 h
the reaction mixture was quenched with MeOH and allowed to warm to
room temperature, then diluted with AcOEt (40 mL) and washed with
brine (2 × 30 mL). The organic layers were dried over Na2SO4, filtered
and evaporated. Flash chromatography (hexane/AcOEt, 8:2) of the crude
product gave compound 20 (0.19 g, 86%) as
a foamy solid.
[α]D20 = −10.6 (c = 1 in chloroform). 1H NMR (CDCl3): δ = 7.89 (s, 1H,
H imidazole), 7.59–7.22 (m, 31H, arom.), 7.03 (s, 1H, H imidazole), 5.49
(s,1H, CHPh), 4.89–4.75 (m, 6H, CH2Ph), 4.64–4.58 (m, 3H, H-1, 1′ and
1H of CH2Ph), 4.52 (t, 1H, J1′,2′ = J2′,3′ = 8.7 Hz, H-2′), 4.42 (d, 1H,
J = 11.8 Hz,1H of CH2Ph), 4.26 (dd, 1H, J5,6′a = 5.0 Hz, J6′a,6′b = 10.5 Hz,
H-6′a), 4.06 (t, 1H, J3,4 = J4,5 = 9.5 Hz, H-4), 3.70–3.44 (m, 7H, H-2, 3,
6a, 6b, 3′, 4′, 6′b), 3.18–3.07 (m, 2H, H-5, 5′). 13C NMR (CDCl3):
δ = 138.7–126.0 (38C, 2C imidazole and 36C arom), 118.5 (C imidazole),
101.5 (CHPh), 98.4 (C-1′), 87.5 (C-1), 85.5 (C-2′), 84.1, 81.9, 80.3, 78.1
(C-5), 77.0, 75.6, 75.5 (CH2Ph), 75.4 (CH2Ph), 74.5 (CH2Ph), 73.6
(CH2Ph), 68.4 (C-6′), 67.6 (C-6), 65.9 (C-5′). MS (ESI) m/z (%): 1035.2
(100) [M+Na]+. HRMS (ESI): m/z calcd for C56H56N2O12NaS2 1035.3172
[M+Na]+, found 1035.3165.
C
85H90N4O16Na 1445.6250 [M+Na]+, found 1445.6246.
4.1.11. Synthesis of phenyl 2-O-acetyl-3-O-benzyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1 → 4)-2,3,6-tri-O-benzyl-1-thio-β-D-glucopyranoside (18)
Glucosyl trichloroacetimidate 11 (0.31 g, 0.57 mmol), phenylthio
glucoside 17 (0.20 g, 0.37 mmol) and 4 Å molecular sieves (0.20 g)
were diluted in DCM (4 mL). The suspension was cooled at −20 °C,
then triethylsilyl trifluoromethanesulfonate (0.1 M solution in DCM,
0.74 mL) was added dropwise. The reaction was monitored by TLC
(toluene/acetone, 9:1). After 1 h the reaction was quenched by the
addition of TEA, diluted with AcOEt, and filtered over a Celite pad.
After evaporation of the solvent, then crude was purified by flash
chromatography (hexane/AcOEt, from 8:2 to 7:3) to give disaccharide
18 (0.29 g, 86%) as an amorphous white solid. [α]D20 = +20.6 (c = 1
in chloroform). 1H NMR (CDCl3): δ = 7.65–7.20 (m, 30H, arom), 5.50
(s, 1H, PhCH), 5.02–4.95 (m, 2H, H-2′ and CH2Ph), 4.92–4.70 (m, 5H,
CH2Ph), 4.68–4.62 (m, 3H, H-1, 1′ and CH2Ph), 4.52 (d, 1H,
J = 12.0 Hz, CH2Ph), 4.15 (dd, 1H, J5′,6′a = 5.0 Hz, J6′a,6′b = 10.5 Hz,
H-6′a), 3.98 (t, 1H, J3,4 = J4,5 = 9.4 Hz, H-4), 3.79–3.77 (m, 2H, 2H-6),
3.68 (t, 1H, J3′,4″ = J4′,5′ = 9.3 Hz, H-4′), 3.65–3.56 (m, 2H, H-3, 3′),
3.52–3.43 (m, 2H, H-2, 6′b), 3.38 (dt, 1H, J4,5 = 9.4 Hz, J5,6 = 2.6 Hz,
H-5), 3.21 (dt, 1H, J4′,5′ = 9.3 Hz, J5′,6′a = 5.0 Hz, J5′,6′b = 9.8 Hz, H-
5′), 1.97 (s, 3H, CH3). 13C NMR (CDCl3): δ = 169.1 (C]O),
138.2–126.0 (36C, arom.), 101.2 (CHPh), 100.8 (C-1′), 87.4 (C-1), 84.7
(C-3), 81.7 (C-4′), 80.2 (C-2), 79.0 (C-5), 78.6 (C-3′), 76.7 (C-4), 75.5
(CH2Ph), 75.4 (CH2Ph), 74.1 (CH2Ph), 73.6 (CH2Ph), 73.3 (C-2′), 68.6
(C-6′), 67.9 (C-6), 66.1 (C-5′), 20.9(CH3). MS (ESI) m/z (%): 947.6
(100) [M+Na]+. HRMS (ESI): m/z calcd for C55H56O11NaS 947.3441
[M+Na]+, found 947.3439.
4.1.14. Synthesis of phenyl 2-azido-3-O-benzyl-4,6-O-benzylidene-2-
deoxy-β-D-glucopyranosyl-(1 → 4)-2,3,6-tri-O-benzyl-1-thio-β-D-
glucopyranoside (21)
To a stirred solution of 20 (0.18 g, 0.18 mmol) in dry DMF (3.5 mL),
sodium azide (0.12 g, 1.80 mmol) was added and the resulting solution
was heated at 85 °C. After 4 h, the reaction was cooled to room tem-
perature, diluted with brine (30 mL) and extracted with AcOEt
(3 × 20 mL). The combined organic layers were dried over Na2SO4,
filtered and evaporated. The crude product was purified by flash
chromatography (hexane/AcOEt, 75:25) to give compound 21 (0.13 g,
83%) as a foamy white solid. [α]D20 = −23.9 (c = 1 in chloroform). 1H
NMR (CDCl3): δ = 7.60–7.28 (m, 30H, arom.), 5.53 (s, 1H, CHPh), 5.01
(d, 1H, J = 10.5 Hz, 1H of CH2Ph), 4.88–4.76 (m, 4H, CH2Ph),
4.73–4.70 (m, 2H, H-1′ and 1H of CH2Ph), 4.68–4.64 (m, 2H, H-1 and
1H of CH2Ph), 4.50 (d, 1H, J = 10.5 Hz, 1H of CH2Ph), 4.05–3.99 (m,
2H, H-3, 6′a), 3.94 (t, 1H, J3′,4′ = J4′,5′ = 9.5 Hz, H-4′), 3.86 (dd, 1H,
J
1′,2′ = 1.1 Hz, J2′,3′ = 3.6 Hz, H-2′), 3.81–3.78 (m, 2H, 2H-6), 3.72 (t,
1H, J3,4 = J4,5 = 8.9 Hz, H-3), 3.58–3.49 (m, 4H, H-2, 5, 3′, 6′b),
3.09–3.04 (m, 1H, H-5′). 13C NMR (CDCl3): δ = 138.8–126.0 (36C,
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L. Morelli et al.
Bioorganic&MedicinalChemistry26(2018)5682–5690
arom.), 101.5 (CHPh), 100.3 (C-1′), 87.5 (C-1), 85.0 (C-4), 80.3, 78.5,
76.5 (C-4′), 77.4 (C-3), 76.7, 75.5 (2C, CH2Ph), 73.7 (CH2Ph), 72.8
(CH2Ph), 68.8 (C-6), 68.3 (C-6′), 67.3 (C-5′), 63.6 (C-2′). MS (ESI) m/z
(%): 930.3 (100) [M+Na]+, 1837.5 (20) [2M+Na]+. HRMS (ESI): m/z
calcd for C53H53N3O9NaS 930.3400 [M+Na]+, found 930.3403.
3″ and 2H-c), 3.13–3.03 (m, 1H, H-5″), 1.87–1.73 (m ,5H, 2H-b and
CH3CO), 1.32 (d, 3H, J5,6 = 6.0 H, 3H-6). 13C NMR (CDCl3): δ = 170.25
(C]O), 156.6 and 156.1 (C]O), 139.4–126.1 (54C, arom.), 101.6
(CHPh), 99.4 (C-1″), 98.3 (C-1), 96.3 (C-1′), 80.8 (C-4′), 79.9 (C-4),
79.4 (C-2′), 78.7 (C-4″). 77.6 (br s, C-3′), 76.3 (C-2), 75.8 (C-3″), 75.7
(C-3), 74.9 (CH2Ph),74.8 (CH2Ph), 73.6 (CH2Ph), 73.4 (CH2Ph), 73.3
(CH2Ph), 71.3 (CH2Ph), 70.2 (C-5′), 68.7 (C-6″), 68.5 (C-5), 68.0 (C-6′),
67.2 (CH2Ph), 67.0 (C-5″), 65.1 (C-a), 50.8 and 50.6 (2C, C-2″ and
NCH2Ph), 44.6 and 43.7 (C-c), 28.3 and 27.9 (C-b), 23.1 (CH3CO), 18.1
(C-6). MS (ESI) m/z (%): 1461.9 (100) [M+Na]+. HRMS (ESI): m/z
calcd for C87H94N2O17Na 1461.6450 [M+Na]+, found 1461.6449.
4.1.15. Synthesis of phenyl 2-acetamido-3-O-benzyl-4,6-O-benzylidene-2-
deoxy-β-D-glucopyranosyl-(1 → 4)-2,3,6-tri-O-benzyl-1-thio-β-D-
glucopyranoside (16)
A mixture of 21 (0.12 g, 0.13 mmol) and Zinc (0.43 g, activated with
aq. 2% CuSO4) in THF/Ac2O/AcOH 3:2:1 (5 mL) was stirred for 1 h at
room temperature. The reaction was diluted with AcOEt and filtered
over a Celite pad. Satd. aq. NaHCO3 was added (30 mL) and, after se-
paration, the aqueous phases were extracted with AcOEt (2 × 20 mL).
The combined organics were dried over NaSO4, filtered and con-
centrated. Flash chromatography (Hexane/AcOEt, 6:4) of the crude
product gave pure 16 (0.080 g, 66%) as a foam. [α]D20 = −34.0 (c = 1
in chloroform). 1H NMR (CDCl3): δ = 7.60–7.24 (m, 30H, arom.), 5.58
(br d, 1H, J = 9.1 Hz, NH), 5.50 (s, 1H, CHPh), 4.89–4.86 (m, 3H,
CH2Ph), 4.77–4.67 (m, 5H, H-1′, 2′ and CH2Ph), 4.65 (d, 1H,
J1,2 = 9.7 Hz, H-1), 4.57–4.53 (m, 2H, CH2Ph), 4.18–4.06 (m, 2H, H-4,
6′a), 3.84–3.77 (m, 2H, 2H-6), 3.66–3.58 (m, 3H, H-3, 4′, 6′b),
3.54–3.51 (m, 2H, H-2, 3′), 3.46–3.44 (m, 1H, H-5), 3.22–3.15 (m, 1H,
H-5′), 1.87 (s, 3H, CH3). 13C NMR (CDCl3): δ = 170.4 (C]O),
139.0–126.1 (36C, arom.), 101.6 (CHPh), 100.1 (C-1′), 87.5 (C-1), 85.3,
80.6, 78.7 (C-5), 78.6, 76.5 (C-4), 75.8, 75.4 (CH2Ph), 75.2 (CH2Ph),
73.5 (CH2Ph), 71.5 (CH2Ph), 68.7 (C-6), 68.6 (C-6′), 67.1 (C-5′), 50.4
(C-2′), 23.2 (CH3).
4.1.17. Synthesis of 3-aminopropyl 2-acetamido-2-deoxy-β-D-
mannopyranosyl-(1 → 4)-α-D-glucopyranosyl-(1 → 3)-α-L-rhamnopyranoside
hydrochloride salt (1)
Compound 2 (0.080 g, 0.056 mmol) in AcOEt/MeOH/0.02 M HCl,
1:1:1 (9 mL) was hydrogenolyzed over Pd(OH)2 (0.070 g) for 4 days.
The mixture was filtered over pleated filter paper, the filtrate was
concentrated to 1 mL, and then lyophilized to give trisaccharide 1
(0.034 g, 97%) as an amorphous white solid. [α]D20 = −19.6 (c = 0.5
in water). 1H NMR (D2O): δ = 4.98 (d, 1H, J1′,2″ = 3.7 Hz, H-1′), 4.81
(br d, 1H, J1″,2″ = 1.3 Hz, H-1″), 4.77 (br d, 1H, J1,2 = 1.7 Hz, H-1),
4.47 (dd, 1H, J″,2″ = 1.4 Hz, J2″,3″ = 4.4 Hz, H-2″), 4.09–4.05 (m, 1H,
H-2), 3.98–3.92 (m, 1H, H-5′), 3.89–3.34 (m, 15H), 3.10–2.95 (m, 2H,
2H-c), 1.99 (s, 3H, CH3CO), 1.95–1.87 (m, 2H, H-b), 1.23 (d, 3H, 3H-6).
13C NMR (D2O): δ = 175.4 (C]O), 99.4 (2C, C-1, 1″), 95.5 (C-1′), 78.6,
76.5 (C-5″), 75.9 (C-3), 71.9 (C-3″), 71.5 (C-3′), 71.2 (C-2′), 70.2 (2C),
68.8, 66.8 (C-2), 66.7, 64.9 (C-a), 60.4 (C-6″), 59.7 (C-6′), 53.3 (C-2″),
37.4 (C-c), 26.7 (C-b), 22.0 (CH3CO), 16.7 (C-6). MS (ESI) m/z (%):
609.3 (100) [M+Na]+. HRMS (ESI): m/z calcd for C23H43N2O15
587.2663 [M+H]+, found 587.2664.
MS (ESI) m/z (%): 946.4 (100) [M+Na]+, 1869.7 (75) [2M+Na]+
.
HRMS (ESI): m/z calcd for C55H57NO10NaS 946.3601 [M+Na]+, found
946.3605.
4.2. Biological test
4.1.16. Synthesis of N-benzyl-N-benzyloxycarbonyl-3-aminopropyl 2-
acetamido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-β-D-mannopyranosyl-
(1 → 4)-2,3,6-tri-O-benzyl-α-D-glucopyranosyl-(1 → 3)-2,4-di-O-benzyl-α-
L-rhamnopyranoside (2)
Competitive ELISA assay: 96-well flat-bottomed plates were in-
cubated overnight at 4–8 °C with a mixture of S. pneumoniae CPS 19A
(1 mg/mL, Statents serum Institute, Artillerivej, Denmark) or 19F
(1 mg/mL, Sanofì-Aventis, France) and methylated human serum al-
bumin (1 mg/mL). A solution of foetal calf serum (5%) in phosphate-
buffered saline supplemented with Brij-35 (0.1%) and sodium azide
(0.05%) was applied to the plates for blocking of nonspecific binding
sites. The plates were incubated overnight at 4–8 °C with a solution
(1:200) of rabbit anti-19A or 19F, used as reference serum (Statents
serum Institute, Artillerivej, Denmark). When trisaccharide was tested,
it was added to each well immediately before the addition of the re-
ference serum. The plates were then incubated with alkaline phospha-
tase conjugate goat anti-rabbit IgG (Sigma-Aldrich, Milan, Italy),
stained with p-nitrophenylphosphate, and the absorbance was mea-
sured at 405 nm with an Ultramark microplate reader (Bio-Rad
Laboratories S.r.l., Milan, Italy).
From compound 15: A mixture of 15 (0.14 g, 0.10 mmol) and Zinc
(0.44 g, activated with aq. 2% CuSO4) in THF/Ac2O/AcOH 3:2:1 (5 mL)
was stirred for 3 h at room temperature. The reaction was diluted with
AcOEt and filtered over a Celite pad. Satd. aq. NaHCO3 was added
(30 mL) and, after separation, the aqueous phases were extracted with
AcOEt (2 × 20 mL). The combined organics were washed with brine,
dried over NaSO4, filtered and concentrated. Flash chromatography
(Hexane/AcOEt, 7:3) of the crude product gave pure 16 (0.091 g, 62%)
as an amorphous glassy solid.
From compound 16: A solution of 16 (0.06 g, 0.065 mmol) and 3
(0.08 g, 0.13 mmol) in dry DCM (2 mL) containing 4 Å molecular sieves
(0.15 g) was stirred at room temperature for 0.5 h. The suspension was
cooled to -35 °C, and then NIS (0.022 g, 0.097 mmol) followed by
AgOTf (8 mg, 0.033 mmol) were added. After the addition, the reaction
was allowed to warm to −10 °C and was stirred at that temperature.
After 0.45 h, TLC (Hexane/AcOEt, 6:4) showed the disappearances of
the donor. The reaction was diluted with DCM (30 mL) and filtered over
a Celite pad. The organic solution was then washed with 10% aq.
Na2S2O3 (30 mL) and satd aq. NaHCO3 (30 mL). The organics were then
dried over NaSO4, filtered and concentrated. The residue was purified
by flash chromatography (Hexane/AcOEt, 7:3) to give first α-2
(0.018 g), followed by β-2 (0.037 g) with an overall glycosylation yield
of 60%. [α]D20 = +0.77 (c = 0.5 in chloroform). 1H NMR (CDCl3):
δ = 7.59–7.01 (m, 45H, arom.), 5.53–5.43 (m, 2H, 1H × CH2Ph and
NH), 5.23–5.17 (m, 2H, CH2Ph), 5.15 (d, 1H, J1′,2′ = 2.9 Hz, H-1), 4.95
(d, 1H, J = 11.7 Hz, 1 × CH2Ph), 4.89–4.74 (m, 4H, CH2Ph), 4.74–4.46
(m, 10H, H-1, 2″ and CH2Ph), 4.43 (br s, 1H, H-1″), 4.25–4.11 (m, 2H,
H-6a″ and 1 × CH2Ph), 4.07–4.01 (m, 2H, H-3, 3′), 3.99–3.92 (m, 2H,
H-4′, 5′), 3.84 (br d, 1H, H-2), 3.75–3.58 (m, 5H, H-a, 4, 5, 2′, 6b″), 3.55
(t, 1H, J3″,4″ = J4″,5″ = 9.6 Hz, H-4″), 3.47–3.20 (m, 6H, H-a′, 6a′, 6b′,
Acknowledgements
This work was supported by the Italian Ministry of University and
Research (PRIN 2015 grant, prot. 2015RNWJAM, Nanoplatforms for
enhanced immune response).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2018.10.016.
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