Selectively charged and zwitterionic analogues of the smallest immunogenic structure of Streptococcus pneumoniae type 14

Article abstract of DOI:10.3390/molecules24183414

Zwitterionic polysaccharides (ZPs) have been shown in recent years to display peculiar immunological properties, thus attracting the interest of the carbohydrate research community. To fully elucidate the mechanisms underlying these properties and exploit

Full text of DOI:10.3390/molecules24183414

molecules
Article
Selectively Charged and Zwitterionic Analogues of
the Smallest Immunogenic Structure of Streptococcus
Pneumoniae Type 14
Tiziana Gragnani 1, Doretta Cuffaro 1, Silvia Fallarini 2, Grazia Lombardi 2, Felicia D’Andrea 1,
*
and Lorenzo Guazzelli 1,
*
1
Dipartimento di Farmacia, University of Pisa, Via Bonanno 6/33, 56126 Pisa, Italy;
mastgragnani@inwind.it (T.G.); doretta.cuffaro@for.unipi.it (D.C.)
Dipartimento di Scienze del Farmaco, University of Piemonte Orientale Amedeo Avogadro, Largo
Donegani 2, 28100 Novara, Italy; silvia.fallarini@uniupo.it (S.F.); grazia.lombardi@uniupo.it (G.L.)
Correspondence: felicia.dandrea@unipi.it (F.D.); lorenzo.guazzelli@unipi.it (L.G.)
2
*
Academic Editor: Ramón J. Estévez Cabanas
Received: 5 September 2019; Accepted: 17 September 2019; Published: 19 September 2019
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Zwitterionic polysaccharides (ZPs) have been shown in recent years to display peculiar
immunological properties, thus attracting the interest of the carbohydrate research community. To
fully elucidate the mechanisms underlying these properties and exploit the potential of this kind
of structures, in depth studies are still required. In this context, the preparation of two cationic, an
anionic, as well as two zwitterionic tetrasaccharide analogues of the smallest immunogenic structure
of Streptococcus pneumoniae type 14 (SP14) capsular polysaccharide are presented. By exploiting a
block strategy, the negative charge has been installed on the non-reducing end of the lactose unit of
the tetrasaccharide and the positive charge either on the non-reducing end of the lactosamine moiety
or on an external linker. These structures have then been tested by competitive ELISA, showing that
the structural variations we made do not modify the affinity of the neutral compound to binding
to a specific antibody. However, lower efficacies than the natural SP14 compound were observed.
The results obtained, although promising, point to the need to further elongate the polysaccharide
structure, which is likely too short to cover the entire epitopes.
Keywords: Streptococcus pneumoniae type 14; zwitterionic analogues; competitive ELISA
1. Introduction
Vaccination represents one of public health’s most cost-effective interventions, deeply
contributing to global health security and striving against antimicrobial resistance. In this context,
carbohydrate-based vaccines have been studied and developed for many years [1–4]. The cells of several
bacteria, virus, and fungi are surrounded by a complex, often specific, pattern of non-mammalian
glycan structures, which represent their primary virulence factor and can protect them from the hosts’
immune defenses. These pathogen-specific glycan structures act as epitopes, able to elicit specific
antibodies when in contact with the host immune cells, representing promising target structures for
the development of vaccines.
In general, polysaccharides are characterized by low immunogenic activities, they are able to trigger
B-cell-mediated immune responses without IgG class switching and memory development [5]. For this
reason, the two polysaccharide vaccines currently on the market are both conjugated to an immunogenic
carrier protein. The 10-valent (PCV10) vaccine is composed of capsular polysaccharides purified from
10 serotypes (1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F). Each capsular polysaccharide is conjugated to a
carrier protein, either protein D (an outer membrane protein from non-typeable Haemophilus influenzae),
Molecules 2019, 24, 3414; doi:10.3390/molecules24183414
www.mdpi.com/journal/molecules

Molecules 2019, 24, 3414
2 of 19
tetanus toxoid or diphtheria toxoid. PCV13 contains the capsular polysaccharides of serotypes 1, 3, 4, 5,
6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F, individually conjugated to a nontoxic diphtheria cross-reactive
material (CRM197) carrier protein. Both PCV10 and PCV13 have been shown to be safe, effective
and to have both direct (in vaccinated individuals) and indirect (in unvaccinated individuals living
in communities with vaccinated children) effects against pneumococcal diseases [6]. Literature data
have shown that particular polysaccharides, bearing on their structure both negatively and positively
charged functionalities, and thus called zwitterionic polysaccharides (ZPs), are endowed with peculiar
immunological properties [7]. ZPs, in fact, are able to be processed by the antigen presenting cells
(APC) and loaded into the class-II major histocompatibility complex (MCH II), for presentation to
T-cells and activation of immune responses [8].
Carbohydrate synthetic chemistry has become, during the years, a valuable tool for the preparation
of complex structures both in the search of protective epitopes [
analogues and mimics [19 21]. Due to the interest raised by ZPs in the carbohydrate-based vaccine
research area, several recent synthetic efforts have been devoted to the preparation of both natural
zwitterionic structures [21 23] and even to the transformation of otherwise neutral carbohydrate
9–18] and in the preparation of epitope
–
–
capsular fragments into their zwitterionic analogues [21]. From the synthetic perspective, the insertion
of charged species in the target structures represents an extra-challenging aspect.
Streptococcus pneumoniae (S. pneumoniae) is a Gram-positive bacterium responsible for invasive
and non-invasive infections in adults and children [24]. As mentioned previously, different
carbohydrate-based vaccines against S. pneumoniae (Prevenar®, Synflorix
™ (PCV10) [25] Prevenar
13® (PCV 1)) have been licensed and commercialized. S. pneumoniae type 14 (SP14) is one of the
serotypes with major worldwide clinical relevance. In 2008, Safari et al. [26] established that the
synthetic branched tetrasaccharide
of the SP14 capsular polysaccharide (CPS) (Figure 1), previously identified by Mawas et al. [27], elicits
a protective antibody response when conjugated with the immunogenic protein CRM197.
β
-d-Galp-(1
→
4)-
β
-d-Glcp-(1
→
6)-[
β
-d-Galp-(1
→
4)]-β-d-GlcpNAc (1)
Figure 1.
Identified smallest immunogenic fragment of S. pneumoniae type 14 capsular
polysaccharide (CPS).
This fragment of SP14 CPS thus represents the smallest structure for the development of a
synthetic vaccine and was further studied after conjugation with the bovine serum albumin (BSA)
carrier protein [28] or as part of future potential synthetic glycoconjugate vaccines in the case of gold
glyconanoparticles [29,30].
As part of an ongoing project aimed at further elucidating the molecular basis of the ZPS
immunological properties, we selected this well-known model tetrasaccharide with the intention
to study how its gradual zwitterionization affects the biological activity. Therefore, along with the
synthesis of the methyl glycoside of 1 [26,27], needed as a benchmark for biological evaluation, here
we report the preparation of an anionic, two cationic, and two zwitterionic tetrasaccharide analogues.
The prepared structures were evaluated by competitive ELISA to understand whether the introduction
of charges or the zwitterionization influence the ability to bind to specific antibodies.

Molecules 2019, 24, 3414
3 of 19
2. Results and Discussion
Several different synthetic approaches have been explored thus far for preparing tetrasaccharide
which is formally constituted by a lactose unit linked to an N-acetyl-lactosamine unit through a
beta 1 6 glycosidic linkage. Thanks to a long-lasting experience in modifying the structure of
lactose [31 34], we decided to use this natural disaccharide as the starting material for the preparation
1,
→
–
of lactose/lacturonic building block donors, while we built up lactosamine acceptors from suitably
protected monosaccharide derivatives. The general design of the planned synthetic strategy is reported
in Scheme 1, with the negative charge located on the galactose frame of the lactose unit (X) and the
positive charge inserted either on the galactose part of the lactosamine unit (Y) or on the external
linker (R).
Scheme 1. General approach for the synthesis of neutral tetrasaccharide
charged and zwitterionic target structures 3–7.
2 and of negatively/positively
2.1. Chemistry
2.1.1. Preparation of Required N-acetyl-glucosamine Acceptors 20–23 and Lacturonic Donor 28
-d-GlcNAc acceptors 20 23 (Scheme 2), characterized by the presence of an orthogonal protecting
group on the 6-OH in view of their further use in the preparation of lactosamine acceptors, were
synthesized starting from known 35] and 36]. The fully protected derivatives 10 15 (78–97%) were
β
–
8
[
9
[
–
obtained by the introduction of a 4,6-O-isopropylidene acetal (2-methoxypropene/CSA) and either
benzylation or benzoylation of OH-3.
Scheme 2. Synthesis of the target acceptors 20
2–4 h (10: 91%; 13: 88%); b) BzCl, Py, rt, 2–5 h (11: 88%) or BnBr, KOH, THF-H2O, 18-crown-6, rt, 4 h
12: 78%); (ii) 70% aq AcOH, 40 ◦C, 3–5 h (16: 96%; 17: 96%; 18: 89% from 13
19: 91% from 13); (iii)
TBDMSCl, Py, rt, 2–4 h (20: 81%; 21: 93%; 22: 91%; 23: 96%).
–23. Reagents and conditions: (i) a) 2-MP, CSA, DMF, rt,
(
;

Molecules 2019, 24, 3414
4 of 19
The acid hydrolysis of the acetal group of 10
–
15 lead to the corresponding diols 16
–19 (89–96%)
which were submitted to a regioselective 6-OH silylation affording target acceptors 20–23 (91–96%, see
Supporting Information File for full experimental details).
Lacturonic donor 28 (Scheme 3) was synthesized starting from known lactose derivative 24
[37],
prepared by benzylation at the C-20 of known 2,3:5,6:30,40-tri-O-isopropylidene-60-O-(1-methoxy-
1-methylethyl)lactose dimethyl acetal
[38] followed by mild acidic hydrolysis of the
6’-O-methoxyisopropyl acetal. Oxidation at the C-60 position of 24 (TEMPO, NaOCl-NaHCO3-Me2CO)
followed by protection of the carboxylic acid as benzyl ester (BnBr, KF, DMF) afforded 25 in an excellent
yield (93%). This was converted into 26
protections (80% aq AcOH, 80 C) and consecutive acetylation with acetic anhydride in pyridine.
(
α
/
β
ratio = 2:3, NMR) through acidic hydrolysis of acetal
◦
Selective removal of the acetyl group at the anomeric position of 26 under mild basic condition
(NH2NH2
DBU gave pure
·
AcOH, DMF) followed by treatment of 27 with trichloroacetonitrile in the presence of
α
-d-trichloroacetimidate 28 (70% from 25) (see Supporting Information File for full
experimental details).
Scheme 3. Synthesis of the lacturonic donor 28. Reagents and conditions: (i) TEMPO, 5% aq NaHCO3,
KBr, 13% aq NaOCl, Me2CO, rt, 15 min and then BnBr, KF, DMF, rt, overnight (93%); (ii) 80% aq ACOH,
80 ◦C, 4 h, and then 1:2 Ac2O-Py, rt, 12 h (98%); (iii): NH2NH2
(iv) CCl3CN, DBU, dry CH2Cl2, rt, 30 min (91%).
·
AcOH, dry DMF, 60 ◦C, 30 min (79%);
2.1.2. Synthesis of the Target Tetrasaccharides 2–7
Tetrasaccharide target structures were then built up from the prepared blocks. First, lactosamine
acceptors 36 40 were synthetized (Scheme 4). Glycosylation of the OH-4 of N-acetyl-glucosamines is
known to represent a challenging reaction [39 40]. Instead of changing the protecting group pattern on
–
,
the acceptors, and in particular the protecting group on the amino function which would ultimately
require the deprotection/acetylation sequence on larger structures, we decided to screen several reaction
conditions on this kind of structure (supporting information file).
The optimal conditions found were the following: trimethylsilyltriflate (TMSOTf, 0.5 eq) [41] as
the catalyst of the glycosylation reaction which was added at
acceptors 20–23 (1.0 eq) and known donors 29 [42] and 30 [43] (1.5 eq) in dry DCM. After 24–48 h the
desired -(1 4)-lactosamine derivatives 31 35 were obtained as the only products with satisfying
yields (40%–72%) for all substrates (Scheme 4). NMR data confirmed the disaccharide structures
−
30 ◦C to a strictly anhydrous solution of
β
→
–
0
0
and the high values (about 7.5 Hz) of the J1 ,2 coupling constants, in agreement with an axial-axial
disposition of H-10 and H-20, ascertained the desired beta configuration of the formed glycosidic bonds.
Disaccharide acceptors 36–40 were then prepared in good to excellent yields (83%–96%) through an
easy acid cleavage of the silyl protecting group by treating 31–35 with a 70% aq AcOH solution at
70 ◦C.

Molecules 2019, 24, 3414
5 of 19
Scheme 4. Synthesis of the
CH2Cl2, 300-AW MS,
30 ◦C to rt, 17–24 h (31: 62%, 32: 40%, 33: 47%, 34: 72%, 35: 57%); (ii) 70% aq
AcOH, 70 ◦C, 1–2 h (36: 83%, 37: 83%, 38: 96%, 39: 81%, 40: 92%).
β-(1→
4)-lactosamine acceptors 36–40. Reagents and conditions: (i) TMSOTf,
−
Lactose donors 28 and 41
lactosamine acceptors 36 40 (Scheme 5). As expected, this glycosylation step was less problematic
when compared to the previous -(1 4)-galactosylation reaction due to the higher accessibility and
reactivity of the primary 6-OH than the 4-OH in 20–23.
[43,44] were employed for the glycosylation reaction with the prepared
–
β
→
Scheme 5. Synthesis of tetrasaccharides 42
–
47. Reagents and conditions: (i): BF3 Et2O, AW-300 MS,
·
CH2Cl2, −15 ◦C to rt, 17–24 h (42: 70%; 43: 81%; 44: 89%; 45: 88%; 46: 75%; 47: 77%).
Thus, lactosamine acceptors 36
41 (1.5 eq) in CH2Cl2 using boron trifluoride etherate (BF3
of acid-washed molecular sieves. Tetrasaccharides 42 47 (Scheme 5) were isolated in good yields
–
40 (1.0 eq) were coupled with trichloroacetimidate donors 28 and
·
Et2O, 1.3 eq) as the catalyst in the presence
–
(70%–89%) after purification by flash chromatography on silica gel of crude products. The presence of
the acetate participating group on donors 28 and 41 allowed again for obtaining only the beta anomer.
It is worth noticing that no differences in reactivity were observed between the peracetylated lactose
trichloroacetimidate donor 41 and its C-6’ oxidized analogue 28.
The target point-charged tetrasaccharide analogues (anionic
structure 44], as well as zwitterionic analogues ( and ), were then prepared by the following
removal of the protecting groups (Scheme 6). Deprotection of compound 42 required a simple Zemplen
3, and cationic 4 and 6) of the neutral
2
[
5
7

Molecules 2019, 24, 3414
6 of 19
reaction (0.33M MeONa/MeOH) to afford the neutral tetrasaccharide
2
(72%) which, as mentioned
before, was needed as a benchmark for the biological evaluation of the charged structures. The desired
ammonium derivatives (71%) and (96%) were obtained by deprotection of compounds 43 and 44
4
6
respectively (Scheme 6), through a two-step procedure. After the basic hydrolysis of the ester groups
either by using Zemplen conditions (0.33M MeONa/MeOH) or by treating with a methanolic ammonia
solution (3.5N NH3-MeOH) [45], the partially deprotected disaccharides 48 and 49 were submitted to
catalytic hydrogenolysis (H2, 10% Pd/C) in MeOH in the presence of 1% HCl-MeOH.
Scheme 6. Synthesis of target tetrasaccharides 2–7. Reagents and conditions: (i) for
MeONa-MeOH, rt, 12-48 h ( : 72%; 48: 69%); for 49: 3.5N NH3-MeOH, rt (65%); for 50: H2, 10% Pd/C,
2.5:1 MeOH-EtOAc, 48 h (98%); for 51 and 52: H2, 10% Pd/C, 3:1:0.5 MeOH-CH2Cl2-H2O, 48 h (51: 70%;
52: 76%); (ii) for : 3.5N NH3-MeOH, 48 h (89%); for and : H2, 10% Pd/C, 3:1 MeOH-H2O, 1%
2 and 48: 0.33M
2
3
4
6
HCl-MeOH, 20 h (4: 71%; 6: 96%); for 5 and 7: 3.5N NH3-MeOH, rt, 48 h (5: 98%; 7: 86%).
Unfortunately, unsatisfactory results were obtained when the same deprotection protocol was
applied to tetrasaccharides 45 and 46. In fact, during the basic hydrolysis of the acetyl groups a side
trans-esterification reaction involving the 6”’ position occurred, and the corresponding methyl esters
of benzylic esters 45 and 46 were isolated. Therefore, a slightly different deprotection pathway was
followed (Scheme 6) by reversing the order of the two deprotection steps. The catalytic hydrogenolysis
(H2, 10% Pd/C) of 45 and 46 was performed in 2.5:1 MeOH-EtOAc or in a ternary solvent system
(3:1:0.5 MeOH-CH2Cl2-H2O) and in all cases, the purification by chromatographic mean, afforded
pure uronic acids 50 and 51 in good yields (70% and 98%, respectively). The following deacetylation
reaction with 3.5N NH3-MeOH gave the desired deprotected target tetrasaccharides
3 and 5 (89% and

Molecules 2019, 24, 3414
7 of 19
98%, respectively). As no drawbacks were encountered, this second protocol was also applied to 47
(Scheme 6), thus obtaining zwitterionic tetrasaccharide 7 (86%).
All compounds were characterized and their mono- and two-dimensional NMR analyses (1H,
13C, DEPT-135, COSY, HETCOR, HSQC) were consistent with their structures (please refer to the
experimental section).
2.2. Biological Tests
First of the all, we determined the biocompatibility of the newly synthesized compounds by
calcein-AM viability assay on the RAW 264.7 cell line [46]. No compounds resulted toxic at all
concentrations tested (1
×
10−5 –1
×
10−1 mg/mL), suggesting that the structural changes we have
introduced into the CPS fragments did not modify cell viability (data not shown). Competitive ELISA
were then performed to investigate the importance of the chain length, as well as of the structural
charges, on the antigenic properties of the newly synthesized compounds [20]. Experiments performed
with a specific rabbit anti-SP14 polyclonal antibody showed that the natural SP14 CPS (positive control)
and all synthesized oligosaccharides are recognized by the antibody in a concentration-dependent
manner. Colominic acid was always included as a negative control (Table 1 and Figure 2).
110
100
90
80
70
60
50
40
30
20
10
0
SP-14
2
3
6
110
100
90
80
70
60
50
40
30
20
10
0
SP-14
2
3
4
7
5
colominic acid
colominic acid
10-7 10-6 10-5 10-4 10-3 10-2 10-1 100
Log[compound] (mg/ml)
10-7 10-6 10-5 10-4 10-3 10-2 10-1 100
Log[compound] (mg/ml)
(b)
(a)
Figure 2. (a,b). Concentration-response curves of SP14 CPS fragments on the inhibition of SP-14 CPS
binding to an anti-SP-14 rabbit polyclonal antibody (competitive ELISA).
Table 1. Results of the competitive ELISA assay.
Compound
IC50 (mg/mL)
Maximum Inhibitiona (%)
28 × 10−5 ± 1.2
1.2 × 10−5 ± 0.5
1.4 × 10−5 ± 0.4
4.1 × 10−5 ± 0.3
5.1 × 10−5 ± 0.4
2.8 × 10−5 ± 0.2
3.8 × 10−5 ± 0.3
SP-14
100 ± 3
30 ± 5
31 ± 3
31 ± 2
31 ± 3
33 ± 4
34 ± 5
2
3
4
5
6
7
a
The maximum inhibition elicited by each compound at 1 mg/mL.
Data show that the relative affinities, expressed as IC50 values (mg/mL) resulted similar among
the different charged fragments
3
–
7. The introduction of either a positive
4
and
6 or a negative 3 charge
into these molecules did not modify the potency of neutral tetrasaccharide
2
(IC50 calculated values at

Molecules 2019, 24, 3414
8 of 19
10−5 order of magnitude for all compounds), suggesting that the presence of charged functionalities
within the repeating unit do not improve the ability of compounds to bind to a specific antibody.
The introduction of both positive and negative charges (ZPS compounds,
results, independently of the charge positions within the tetrasaccharide structure. All analogues (
exhibited similar efficacies (31 3), which are lower ( 70%) than the natural compound (100 3).
5 and 7) gave the same
2–7)
±
−
±
These results confirm that to obtain a high inhibition of the antibody binding to natural SP14 CPS, a
higher number of different CPS epitopes is required [47].
3. Materials and Methods
3.1. Chemistry
3.1.1. General
Optical rotations were measured on a Perkin-Elmer 241 polarimeter at 20
±
2 C. Melting points
were determined with a Kofler hot-stage apparatus and are uncorrected. 1H NMR spectra were
recorded in appropriate solvents with a Bruker Avance II operating at 250.13 MHz or a Bruker DRX
600 (biodrx600) spectrometer operating at 600 MHz. 13C NMR spectra were recorded with the above
spectrometers operating at 62.9 or 150 MHz. The assignments were made, when possible, with the
aid of DEPT, HETCOR, HSQC and COSY experiments. The first order proton chemical shifts
δ are
referenced to either residual CD3CN (δH 1.94, δC 1.28) or residual CD3OD (δH 3.31, δC 49.0) and
J-values are given in Hz. All reactions were followed by TLC on Kieselgel 60 F254 or Silica gel 60 RP-18
F254s or with detection by UV light and/or with ethanolic 10% phosphomolybdic or sulfuric acid, and
heating. Kieselgel 60 (E. Merck, 70–230 and 230–400 mesh, respectively) or Biotage reverse phase C18
silica columns was used for column and flash chromatography. Some of the flash chromatography
were conducted by the automated system Isolera Four (Biotage®, Uppsala, Sweden), equipped with a
UV detector with variable wavelength (200–400 nm). Unless otherwise noted, solvents and reagents
were obtained from commercial suppliers and were used without further purification. All reactions
involving air- or moisture-sensitive reagents were performed under an argon atmosphere by using
anhydrous solvents. Anhydrous dimethylformamide (DMF), dichloromethane (CH2Cl2) and methanol
(CH3OH) were purchased from Sigma–Aldrich. Other dried solvents were obtained by distillation
according to standard procedure [48] and stored over 4Å molecular sieves activated for at least 12 h at
200 ◦C. MgSO4 was used as the drying agent for solutions. Elemental analysis were obtained using an
Elementar Vario MICRO cube equipment.
Compound
8
[
35],
9
[
36] and donors 29
[
42], 30
[
–
43], 28
15 18
[
–
43
23
,
,
44] were prepared according to the
25 28 and 31 40 is reported in the
reported procedure. The synthesis of compounds 10
,
–
–
Electronic Supplementary Information.
3.1.2. General Procedure for the 6-O-glycosylation: Synthesis of the Tetrasaccharides 42–47
A mixture of the appropriate acceptors 36 40 (1.0 eq), excess of opportune donors 28 or 41 (1.5 eq)
and activated AW 300 MS (800 mg) in dry CH2Cl2 (20 mL), was stirred for 15 min at room temperature,
cooled to Et2O (1.3 eq) was added. The reaction mixture was allowed to slowly attain
15 ◦C and BF3
–
−
·
room temperature with stirring until the appropriate acceptor was disappeared (17–24 h, TLC, EtOAc
or 1:9 toluene-EtOAc) and the formation of a major UV visible spot had occurred. Et3N (1.0 mL) was
added and after 30 min the mixture was filtered through a short pad of Celite, diluted with CH2Cl2,
and concentrated under diminished pressure. Purification of crude product by flash chromatography
on silica gel afforded pure tetrasaccharides 42–47.
Methyl 2-acetamido-3-O-benzoyl-4-O-(2,3,4,6-tetra-O-acetyl-
β
-d-galactopyranosyl)-6-O-[4-O-(2,3,4,6-tetra-O-
-d-glucopyranoside (42). The
acetyl- -d-galactopyranosyl)-2,3,6-tri-O-acetyl- -d-glucopyranosyl]-2-deoxy-β
β
β
glycosylation of lactosamine acceptor 36 (155 mg, 0.23 mmol, 1 eq) with lactose donor 41 (269 mg, 0.345
.
mmol, 1.5 eq) was performed in dry CH2Cl2 (5.5 mL) with AW-300 MS (300 mg) and BF3 Et2O (36
µL,

Molecules 2019, 24, 3414
9 of 19
0.30 mmol, 1.3 eq) in dry CH2Cl2 (0.5 mL) in accordance with the general procedure. Purification of
crude product by flash chromatography on silica gel (5:95 hexane-EtOAc + 0.1% Et3N) afforded pure
tetrasaccharide 42 (208 mg, 70%) as a white foam, Rf 0.19 (5:95 hexane-EtOAc); (
CHCl3); 1H NMR (250.13 MHz, CD3CN):
8.00–7.96 (m, 2H, Ar-H), 7.58–7.55 (m, 1H, Ar-H), 7.49–7.42
(m, 2H, Ar-H), 6.54 (d,1H, J2,NH 9.5 Hz, NH), 5.30 (dd, 1H, J3
α)D -16.1 (c 0.93 in
δ
3.4 Hz, J4 ”,5 ” 1.0 Hz, H-4000), 5.25
0
0
0004000
0
0
(dd, 1H, J2,3 10.2 Hz, J3,4 8.5 Hz, H-3), 5.17 (dd, 1H, J2”,3” 9.7 Hz, J3”,4” 8.9 Hz, H-3”), 5.11 (dd, 1H, J4 ,5
1.1 Hz, J3 ,4 3.4 Hz, H-40), 5.03 (dd, 1H, J2 ”,3 ” 10.4 Hz, H-30”), 4.96 (dd, 1H, J2 ,3 10.4 Hz, H-30), 4.94
0
0
0
0
0
0
0
(dd, 1H, J1 ” 0,2 ” 7.8 Hz, H-20”), 4.84 (dd, 1H, J1 ,2 7.6 Hz, H-20), 4.83 (dd, 1H, J1”,2” 7.9 Hz, H-2”),
0
0
0
4.70 (d, 1H, H-100), 4.58 (d, 1H, H-1000),4.53 (d, 1H, H-10),4.46 (d, 1H, J1,2 8.5 Hz, H-1), 4.39 (dd, 1H,
J
6”a,6”b 12.2 Hz, J5”,6”b 2.2 Hz, H-6”b), 4.18-3.98 (m, 5H, H-4, H-5000, H-600a, H-6000a, H-6000b), 3.97-3.81
(m, 2 H, H-2, H-400), 3.80-3.63 (m, 5H, H-5, H-50, H-500, H-60a, H-60b), 3.42 (s, 3H, OMe), 3.39 (dd, 1H,
6a,6b 11.1 Hz, J5,6b 7.4 Hz, H-6b), 3.22 (dd, 1H, J5,6a 6.0 Hz, H-6a), 2.08, 2.07, 2.03, 2.02, 2.01, 2.00, 1.99,
1.94, 1.89, 1.87, 1.86 (11s, each 3H, 11
MeCOO), 1.68 (s, 3H, MeCON); 13C NMR (62.9 MHz, CD3CN):
171.5-170.4 (12
J
×
δ
×
MeCO), 166.5 (PhCO), 134.1, 130.3, 129.4 (Ar-CH), 131.1 (Ar-C), 102.3 (C-1), 101.4
(C-10, C-1”, C-1000), 77.6 (C-500), 77.0 (C-4), 74.5 (C-3), 75.0 (C-5), 73.4 (C-5000), 73.1 (C-300), 72.3 (C-200),
71.6 (C-3000), 71.5 (C-30), 71.4 (C-400), 71.1 (C-50), 69.9 (C-20, C-2000), 68.8 (C-6), 68.0 (C-4000), 67.7 (C-40),
62.9 (C-600), 61.9, 61.1 (C-60, C-6000), 57.2 (MeO), 54.5 (C-2), 23.0 (MeCON), 21.1-20.6 (11
×
MeCOO).
Elemental Analysis Found: C, 52.25; H, 5.75; N, 1.13. Calculated for C56H73NO33 (1288.17): C, 52.21; H,
5.71; N, 1.09.
3-Azidopropyl 2-acetamido-3-O-benzoyl-4-O-(2,3,4,6-tetra-O-acetyl-
β
-d-galactopyranosyl)-6-O-[4-O-(2,3,4,6-
-d-glucopyranoside (43).
tetra-O-acetyl- -d-galactopyranosyl)-2,3,6-tri-O-acetyl- -d-glucopyranosyl]-2-deoxy-β
β
β
The glycosylation of lactosamine acceptor 38 (338 mg, 0.457 mmol, 1 eq) with lactose donor 41 (535 mg,
.
0.685 mmol, 1.5 eq) was performed in dry CH2Cl2 (10 mL) with AW-300 MS (550 mg) and BF3 Et2O
(69
of crude product by flash chromatography on silica gel (15:85 hexane-EtOAc + 0.1% Et3N) afforded
pure tetrasaccharide 43 (505 mg, 81%) as a white foam, Rf 0.34 (1:9 toluene-EtOAc); ( -16.5 (c 0.98 in
CHCl3); 1H NMR (250.13 MHz, CD3CN):
8.02–7.96 (m, 2H, Ar-H), 7.62–7.55 (m, 1H, Ar-H), 7.50–7.43
(m, 2H, Ar-H), 6.46 (d,1H, J2,NH 9.6 Hz, NH), 5.30 (dd, 1H, J4 ”,5 ” 1.0 Hz, J3 ”,4 ” 3.5 Hz, H-40”), 5.26 (dd,
µL, 0.594 mmol, 1.3 eq) in dry CH2Cl2 (1.0 mL), as described in the general procedure. Purification
α
)
D
δ
0
0
0
0
0
0
1H, J2”,3” 10.4 Hz, J3”,4” 8.8 Hz, H-3”), 5.18 (dd, 1H, J2,3 9.6 Hz, J3,4 9.1 Hz, H-3), 5.11 (dd, 1H, J3 ,4 3.4
Hz, J4 ,5 1.1 Hz, H-40), 5.02 (dd, 1H, J2 ”,3 ” 9.3 Hz, H-30”), 5.00 (dd, 1H, J2 ,3 9.7 Hz, H-30), 4.89 (dd,
0
0
0
0
0
0
1H, J1”,2” 7.7 Hz, H-2”), 4.84 (dd, 1H, J1 ”,2 ” 7.8 Hz, H-20”), 4.83 (dd, 1H, J1 ,2 7.9 Hz, H-20), 4.70 (d,
1H, H-10), 4.56 (d, 1H, H-10”), 4.55 (d, 1H, J1,2 8.4, H-1), 4.53 (d, 1H, H-1”), 4.39 (dd, 1H, J6”a,6”b 12.4
Hz, J5”,6”b 2.1 Hz, H-6b”), 4.18-3.95 (m, 5H, H-60a, H-60 b, H-6”a, H-60”a, H-60”b), 3.94- 3.81 (m, 4 H,
H-2, H-4, H-4”, H-50”), 3.80-3.50 (m, 5H, H-5, H-50, H-5”, CH2O), 3.39 (dd, 1H, J6a,6b 11.1 Hz, J5,6b 7.4
Hz, H-6b), 3.36 (t, 2H, Jvic 6.8 Hz, CH2N3), 3.24 (m, 1H, J5,6a 5.9 Hz, H-6a), 2.08, 2.02, 2.01, 2.00, 1.99,
0
0
0
0
1.97, 1.94, 1.93, 1.89, 1.87, 1.86 (11s, each 3H, 11
×
MeCOO), 1.81 (m, 2H, CH2), 1.70 (s, 3H, MeCON);
MeCO), 166.5 (PhCO), 134.1, 130.4, 129.4 (Ar-CH),
13C NMR (62.9 MHz, CD3CN):
δ
171.5-170.4 (12
×
131.1 (Ar-C), 101.5 (C-1, C-1”), 101.4 (C-10, C-10”), 74.7 (C-3), 77.5 (C-5), 77.0 (C-4), 75.1 (C-50), 75.0
(C-50”), 73.5 (C-4”), 73.2 (C-3”), 71.6 (C-30”), 71.4 (C-30, C-2”), 69.9 (C-20, C-20”), 68.8 (C-6), 68.0 (C-40”),
67.7 (C-40), 54.7 (C-2), 67.1 (CH2O), 62.9, 61.9, 61.1 (C-60, C-6”, C-60”), 48.8 (CH2N3), 29.4 (CH2), 23.0
(MeCON), 21.1-20.7 (11
for C58H76N4O33 (1357.24): C, 51.33; H, 5.64; N, 4.13.
×
MeCOO). Elemental Analysis Found: C, 51.37; H, 5.67; N, 4.16. Calculated
Methyl 2-acetamido-3-O-benzyl-4-O-(2,3,4-tri-O-acetyl-6-azido-6-deoxy-β-d-galactopyranosyl)-6-O-[4-O-(2,3,4,6-
tetra-O-acetyl-β-d-galactopyranosyl)-2,3,6-tri-O-acetyl-β-d-glucopyranosyl]-2-deoxy-β-d-glucopyranoside (44).
The glycosylation of lactosamine acceptor 40 (97 mg, 0.152 mmol, 1 eq) with lactose donor 41 (178 mg,
·
0.228 mmol, 1.5 eq) was performed in dry CH2Cl2 (5.0 mL) with AW-300 MS (270 mg) and BF3 Et2O
(27
of crude product by flash chromatography on silica gel (1:9 hexane-EtOAc + 0.1% Et3N) afforded
pure tetrasaccharide 44 (170 mg, 89%) as a white foam, Rf 0.32 (1:9 hexane-EtOAc); ( -27.3 (c 1.2 in
µL, 0.196 mmol, 1.3 eq) in dry CH2Cl2 (0.5 mL), as described in the general procedure. Purification
α
)
D

Molecules 2019, 24, 3414
10 of 19
CHCl3); 1H NMR (250.13 MHz, CD3CN):
δ
7.35–7.29 (m, 5H, Ar-H), 6.52 (d,1H, J2,NH 9.3 Hz, NH), 5.28
(m, 2H, H-40, H-40”), 5.16 (dd, 1H, J2”,3” 8.8 Hz, J3”,4” 9.7 Hz, H-3”), 5.01 (m, 3H, H-30, H-20”, H-30”),
4.93 (dd, 1H, J1 ,2 7.8 Hz, J2 ,3 10.4 Hz, H-20), 4.86, 4.54 (AB system, 2H, JA,B 11.1 Hz, CH2Ph), 4.81 (dd,
0
0
0
0
1H, J1”,2” 7.9 Hz, H-2”), 4.68 (d, 1H, J1 ”,2 ” 7.9 Hz, H-10”),4.65 (d, 1H, H-10), 4.56 (d, 1H, H-1”), 4.26 (d,
1H, J1,2 7.8 Hz, H-1), 4.39 (dd, 1H, J6”a,6”b 12.1 Hz, J5”,6”b 1.9 Hz, H-6”b), 4.15-3.97 (m, 5H, H-6b, H-6”a,
H-50”, H-60”a, H-60”b), 3.86 (m, 2H, H-50, H-4”), 3.77–3.65 (m, 4H, H-2, H-4, H-6a, H-5”), 3.51 (m, 2H,
0
0
H-3, H-5), 3.37 (s, 3H, OMe), 3.26 (dd, 1H, J6 a,6 b 12.9 Hz, J5 ,6 b 7.4 Hz, H-60b), 3.13 (dd, 1H, J5 ,6 5.4
0
0
0
0
0
0
a
Hz, H-60a), 2.10, 2.08, 2.06, 2.03, 2.02, 2.01, 2.00, 1.99, 1.92, 1.89 (10s, each 3H, 10
×
MeCOO), 1.83 (1s, 3H,
C=O), 139.9 (Ar-C), 129.0–128.2 (Ar-CH),
MeCON); 13C NMR (62.9 MHz, CD3CN):
δ
171.4-170.4 (11
×
102.8 (C-1), 101.4 (C-1”, C-10”), 100.7 (C-10), 80.5 (C-3), 77.7 (C-4), 76.6 (C-4”), 76.9 (C-5), 74.2 (CH2Ph),
73.3 (C-5”), 73.1 (C-3”), 72.6 (C-50), 72.3 (C-2”), 71.3 (C-50”), 71.6 (C-30, C-30”), 70.1 (C-20”), 69.2 (C-6),
68.6 (C-40”), 68.0 (C-40), 68.8 (C-20), 62.9 (C-6”), 61.9 (C-60”), 56.9 (MeO), 54.6 (C-2), 50.7 (C-60), 23.2
(MeCON), 21.1-20.7 (10
C54H72N4O30 (1257.17): C, 51.59; H, 5.77; N, 4.46.
×
MeCOO). Elemental Analysis Found C, 51.63; H, 5.80; N, 4.49. Calculated for
Methyl 2-acetamido-3-O-benzyl-4-O-(2,3,4,6-tetra-O-acetyl-
β
-d-galactopyranosyl)-6-O-[4-O-(benzyl-(2-O-benzyl-
-d-glucopyranosyl]-2-deoxy- -d-glucopyranoside
45). The glycosylation of lactosamine acceptor 37 (159 mg, 0.24 mmol, 1 eq) with lacturonic donor 38
3,4-di-O-acetyl-β-d-galactopyranosyl)uronate)-2,3,6-tri-O-acetyl-
β
β
(
(321 mg, 0.36 mmol, 1.5 eq) was performed in dry CH2Cl2 (5.5 mL) with AW-300 MS (310 mg) and
.
BF3 Et2O (36.4
µL, 0.315 mmol, 1.3 eq) in dry CH2Cl2 (0.5 mL), as described in the general procedure.
Purification of crude product by flash chromatography on silica gel (2:8 hexane-EtOAc + 0.1% Et3N)
afforded pure tetrasaccharide 45 (292 mg, 88%) as a white foam, Rf 0.24 (2:8 hexane-EtOAc); ( -15.1
α
)
D
(c 1.1 in CHCl3); 1H NMR (250.13 MHz, CD3CN):
δ 7.67–7.23 (m, 15H, Ar-H), 6.48 (d, 1H, J2,NH 9.4
Hz, NH), 5.47 (dd, 1H, J3 ”,4 ” 3.7 Hz, J4 ”,5 ” 1.4 Hz, H-40”), 5.30 (dd, 1H, J2”,3” 10.3 Hz, J3”,4” 8.9 Hz,
H-30”), 5.32 (m, 1H, H-40), 5.15, 5.08 (AB system, 2H, JA,B 12.1 Hz, COOCH2Ph), 5.08 (m, 2H, H-30,
0
0
0
0
H-2 ), 5.00 (dd, 1H, J ”3 ” 10.0 Hz, H-30”), 4.84 (dd, 1H, J1”,2” 7.9 Hz, H-2”), 4.73 (d, 1H, H-1”), 4.77, 4.54
(AB system, 2H, JA,B 11.0 Hz, CH2Ph), 4.73, 4.57 (AB system, 2H, JA,B 11.3 Hz, CH2Ph), 4.68 (d, 1H,
0
0
J1 ,2 7.8 Hz, H-10), 4.48 (d, 1H, H-50”), 4.46 (d, 1H, J1 ”,2 ” 7.9 Hz, H-10”), 4.35 (dd, 1H, J6 a,6 b 12.1 Hz,
0
0
0
0
0
0
J5 ,6 b 2.0 Hz, H-60b), 4.28 (d, 1H, J1,2 7.9 Hz, H-1), 4.20 (dd, 1H, J5 ,6 4.9 Hz, H-60a), 4.08-3.79 (m, 6H,
0
0
0
0
a
H-6b, H-50, H-5”, H-4”, H-6”a, H-6”b), 3.77-3.65 (m, 3H, H-2, H-4, H-6a), 3.53 (dd, 1H, J2,3 9.5 Hz,
J
3,4 8.2 Hz, H-3), 3.50 (m, 1H, H-5), 3.49 (dd, 1H, H-20”), 3.41 (s, 3H, MeO), 2.10, 2.07, 2.03, 2.02, 1.98,
1.92, 1.88, 1.87, 1.85 (9s, each 3H, 9
171.3–170.5 (10
102.8 (C-10), 101.4 (C-1”), 101.1 (C-1), 80.6 (C-3), 78.2 (C-4), 77.3 (C-5), 77.0 (C-4”), 75.8, 74.0 (CH2Ph),
75.4 (C-5), 73.7 (C-5”), 72.9, 72.9, 72.8, 72.5, 72.4 (C-50, C-2”, C-3”, C-50”, C-30”), 71.6, 71.4 (C-30, C-20”),
(70.2 (C-20), 69.5 (C-40”), 69.2 (C-6), 68.1 (C-40), 67.8 (COOCH2Ph), 62.9 (C-60), 61.9 (C-6”), 57.0 (MeO),
×
MeCOO), 1.83 (s, 3H, MeCON); 13C NMR (62.9 MHz, CD3CN):
Ar-C), 129.5-129.3 (Ar-CH), 103.3 (C-10”),
δ
×
C=O), 167.0 (C-60”), 139.8, 138.9, 136.4 (3
×
54.6 (C-2), 23.3 (MeCON), 21.0–20.5 (9
Calculated for C66H81NO31 (1384.35) C, 57.26; H, 5.90; N, 1.01.
×
MeCOO). Elemental Analysis Found: C, 57.29; H, 5.94; N, 1.05.
3-Azidopropyl 2-acetamido-3-O-benzyl-4-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-6-O-[4-O-(benzyl-
(3,4-di-O-acetyl-2-O-benzyl-
β
-d-galactopyranosyl) uronate)-2,3,6-tri-O-acetyl-
β
-d-glucopyranosyl]-2-deoxy- -d-
β
glucopyranoside (46). The glycosylation of lactosamine acceptor 39 (71 mg, 0.098 mmol, 1 eq) with
lacturonic donor 28 (131 mg, 0.147 mmol, 1.5 eq) was performed in dry CH2Cl2 (3.0 mL) with AW-300
.
MS (175 mg) and BF3 Et2O (14.6
µL, 0.26 mmol, 1.3 eq) in dry CH2Cl2 (0.5 mL), as described in
the general procedure. Purification of crude product by flash chromatography on silica gel (3:7
hexane-EtOAc + 0.1% Et3N) afforded pure tetrasaccharide 46 (107 mg, 75%) as a clear syrup, Rf
0.23 (3:7 hexane-EtOAc); (α δ 7.39-7.24 (m,
)D -14.9 (c 1.2 in CHCl3); 1H NMR (250.13 MHz, CD3CN):
15H, Ar-H), 6.60 (d, 1H, J2,NH 9.3 Hz, NH), 5.48 (dd, 1H, J3 ”,4 ” 3.7 Hz, J4 ”,5 ” 1.4 Hz, H-40”), 5.33
(m, 1H, H-40), 5.29 (dd, 1H, J2”,3” 9.4 Hz, J3”,4” 8.0 Hz, H-3”), 5.16, 5.07 (AB system, 2H, JA,B 12.1 Hz,
0
0
0
0
COOCH2Ph), 5.10- 5.05 (m, 2H, H-30, H-20), 5.00 (dd, 1H, J2 ”,3 ” 10.2 Hz, H-30”), 4.82 (dd, 1H, J1”,2”
7.4 Hz, H-2”), 4.78, 4.57 (AB system, 2H, JA,B 11.7 Hz, CH2Ph), 4.73, 4.55 (AB system, 2H, JA,B 11.3 Hz,
0
0

Molecules 2019, 24, 3414
11 of 19
CH2Ph), 4.69 (d, 1H, H-1”), 4.67 (d, 1H, J1 ,2 7.9 Hz, H-10), 4.5 (d, 1H, H-50”), 4.46 (d, 1H, J1 ”,2 ” 7.9 Hz,
H-10”), 4.37 (d, 1H, J1,2 8.2 Hz, H-1), 4.36 (dd, 1H, J6”a,6”b 12.1 Hz, J5”,6”b 1.7 Hz, H-6”b), 4.20 (dd, 1H,
J5”,6”a 5.1 Hz, H-6”a), 4.08–3.86 (m, 9H, H-5, H-50, H-5”, H-4, H-4”, H-6a, H-60a, H-6b, H-60b), 3.85
(m, 2H, CH2O), 3.78–3.66 (m, 3H, H-2, H-4, H-4”), 3.55 (dd, 1H, J2,3 9.8 Hz, J3,4 8.4 Hz, H-3), 3.51 (m,
1H, H-5), 3.43 (dd, 1H, H-20”), 3.36 (t, 1H, Jvic 6.7 Hz, CH2N3), 2.10, 2.07, 2.04, 2.03, 1.98, 1.92, 1.89,
0
0
0
0
1.87, 1.86 (9s, each 3H, 9
×
MeCOO), 1.84 (s, 3H, MeCON), 1.77 (m, 2H, CH2); 13C NMR (62.9 MHz,
C=O), 167.0 (C-60”), 139.9, 139.0, 136.4 (3
Ar-C), 129.5-128.3 (Ar-CH),
CD3CN,): 171.4-170.3 (10
δ
×
×
103.3 (C-10”), 101.9 (C-1), 101.4 (C-1”), 101.2 (C-10), 80.6 (C-3), 78.2 (C-4”), 78.1 (C-4), 77.3 (C-20”), 75.6
(C-50”), 75.8, 74.1 (CH2Ph), 73.7 (C-50), 72.9 (C-3”), 72.9 (C-5”), 72.6 (C-30”), 72.4 (C-2”), 71.6 (C-30), 71.4
(C-5), 70.3 (C-20), 69.5 (C-40”), 69.0 (C-6), 68.1 (C-40), 67.9 (COOCH2Ph), 66.9 (CH2O), 62.9 (C-6”), 61.9
(C-60), 54.9 (C-2), 48.8 (CH2N3), 29.5 (CH2), 23.3 (MeCON), 21.1–20.5 (9
×
MeCOO). Elemental Analysis
Found: C, 56.23; H, 5.84; N, 3.88. Calculated for C68H84N4O31 (1453.42): C, 56.20; H, 5.83; N, 3.85.
Methyl 2-acetamido-3-O-benzyl-4-O-(2,3,4-tri-O-acetyl-6-azido-6-deoxy- -d-galactopyranosyl)-6-O-[4-O-
(benzyl-(3,4-di-O-acetyl-2-benzyl- -d-galactopyranosyl) uronate)-2,3,6-tri-O-acetyl- -d-glucopyranosyl]-2-deoxy-
-d-glucopyranoside (47). The glycosylation of lactosamine acceptor 40 (145 mg, 0.227 mmol, 1 eq)
β
β
β
β
with lacturonic donor 28 (303 mg, 0.34 mmol, 1.5 eq) was performed in dry CH2Cl2 (7.5 mL) with
.
AW-300 MS (400 mg) and BF3 Et2O (33.8
µL, 0.292 mmol, 1.3 eq) in dry CH2Cl2 (0.5 mL), as described
in the general procedure. Purification of crude product by flash chromatography on silica gel (2:8
hexane-EtOAc + 0.1% Et3N) afforded pure tetrasaccharide 47 (238 mg, 77%) as a white foam, Rf 0.35
1
(2:8 hexane-EtOAc); (
α
)
-18.64 (c 1.2 in CHCl3); H NMR (250.13 MHz, CD3CN):
δ
7.38–7.23 (m,
D
15H, Ar-H), 6.68 (d, 1H, J2,NH 9.4 Hz, NH), 5.47 (dd, 1H, J3 ”,4 ” 3.7 Hz, J4 ”, 5 ” 1,5 Hz, H-40”), 5.31
(m, 1H, H-40), 5.27 (dd, 1H, J2”,3” 9.6 Hz, J3”,4” 8.8 Hz, H-3”), 5.16–5.06 (AB system, 2H, JA,B 12.1 Hz,
COOCH2Ph), 5.09 (m, 2H, H-30, H-20), 5.00 (dd, 1H, H-30”), 4.84 (dd, 1H, J1”,2” 7.9 Hz, H-2”), 4.84, 4.55
(AB system, 2H, JA,B 11.0 Hz, CH2Ph), 4.75, 4.56 (AB system, 2H, JA,B 11.3 Hz, CH2Ph), 4.73 (d, 1H,
0
0
0
0
H-1”), 4.67 (d, 1H, J2 ,3 7.9 Hz, H-10), 4.49 (d, 1H, H-50”), 4.46 (d, 1H, J1 ”,2 ” 7.9 Hz, H-10”), 4.35 (dd,
1H, J6”a,6”b 12.1 Hz, J5”,6”b 1.5 Hz, H-6”b), 4.20 (dd, 1H, J5”,6”a 4.9 Hz, H-6”a), 4.27 (d, 1H, J1,2 7.8 Hz,
H-1), 4.01 (dd, 1H, J6a,6b 10.9 Hz, J5,6b 2.2 Hz, H-6b), 3.93 (dd, 1H, J5,6a 6.8 Hz, H-6a), 3.90 (m, 1H, H-5”),
3.90–3.65 (m, 3H, H-2, H-4, H-5), 3.87 (m, 1H, H-50), 3.84 (m, 1H, H-4”), 3.53 (dd, 1H, J2,3 9.4 Hz, J3,4
0
0
0
0
8.1 Hz, H-3), 3.43 (dd, 1-H, J2 ”,3 ” 10.1 Hz, H-20”), 3.41 (s, 3H, OMe), 3.27 (dd, 1H, J6 a,6 b 12.8 Hz,
0
0
0
0
J
5 ,6 b 7.3 Hz, H-60b), 3.12 (dd, 1H, J5 ,6 a 5.6 Hz, H-60a), 2.10, 2.06, 2.04, 2.03, 1.98, 1.91, 1.87, 1.85 (8s,
0
0
0
0
each 3H, 8 171.6-170.5 (9 C=O),
×
MeCOO), 1.83 (1s, 3H, MeCON); 13C NMR (62.9 MHz, CD3CN):
δ
×
167.1 (C-60”), 139.9, 138,9, 136.4 (Ar-C), 129.5-128.3 (Ar-CH), 103.3 (C-10”), 102.9 (C-1), 101.5 (C-1”),
100.9 (C-10), 80.6 (C-3), 77.9 (C-4”), 77.4 (C-20”), 77.0 (C-4), 75.9, 74.4 (CH2Ph), 75.4 (C-5”), 73.7 (C-50),
73.0 (C-50”), 72.9 (C-3”), 72.6 (C-5, C-30”), 72.4 (C-2”), 71.6 (C-30), 70.2 (C-20), 69.6 (C-40”), 69.3 (C-6),
68.6 (C-40), 67.9 (COOCH2Ph), 63.0 (C-6”), 57.0 (MeO), 54.7 (C-2), 50.8 (C-60), 23.3 (MeCON), 21.1–20.5
(8
×
MeCOO). Elemental Analysis Found: C, 56.26; H, 5.78; N, 4.14. Calculated for C64H78N4O29
(1367.33): C, 56.22; H, 5,75; N, 4.10.
3.1.3. Synthesis of 3-Azidopropyl 2-acetamido-4-O-β-d-galactopyranosyl-6-O-
[4-O-(β-d-galactopyranosyl)- β-d-glucopyranosyl]-2-deoxy-β-d-glucopyranoside (48)
A solution of tetrasaccharide 43 (200 mg, 0.147 mmol) in MeOH (1 mL) was cooled at 0 ◦C, treated
with a methanolic solution of MeONa (0.33M, 3 mL), and the reaction mixture was stirred at 0 ◦C until
TLC analysis (1:1 CHCl3-MeOH) showed the complete disappearance of the starting material (48 h).
The solution was neutralized with resin acid (Amberlist-15), filtered on a cotton pad, washed with
MeOH and concentrated under diminished pressure. Purification of crude product by reversed-phase
flash chromatography on C-18 silica gel (1:1 MeOH-H2O) followed by trituration with Et2O afforded
pure tetrasaccharide 48 (80 mg, 69%) as a white solid, Rf 0.20 (4:6 CHCl3-MeOH); mp 159–161 ◦C (from
MeOH-Isopropanol); (
α) δ 4.53 (d, 1H,
-6.05 (c 1.19 in MeOH); 1H NMR (250.13 MHz, CD3OD-D2O):
D
J1”,2” 8.0 Hz, H-1”), 4.47 (d, 1H, J1 ,2 7.8 Hz, H-10), 4.43 (d, 1H, J1,2 8.3 Hz, H-1), 4.35 (d, 1H, J1
0
0
0”,20”
7.7 Hz, H-10”), 3.95-3.70 (m, 13H, H-2, H-3, H-4, H-40, H-40”, H-5a, H-6b, H-60a, H-60b, H-6”a, H-6”b,

Molecules 2019, 24, 3414
12 of 19
H-60”a, H-60”b), 3.67-3.54 (m, 11H, CH2O, H-20, H-20”, H-4”, H-5, H-50, H-5”, H-50”, H-2”, H-3”),
3.51–3.3.34 (m, 2H, H-30, H-30”), 3.37 (t, 2H, Jvic 6.7 Hz, CH2N3), 2.00 (s, 3H, MeCON), 1.81 (m, 2H,
CH2); 13C NMR (62.9 MHz, CD3OD-D2O):
δ
170.7(C=O), 104.7 (C-10”), 104.4 (C-10), 104.1 (C-1”), 102.8
(C-1), 80.1 (C-4”), 79.6 (C-4), 76.8 (C-50), 76.6 (C-50”), 76.2 (C-5”), 75.9 (C-5), 74.9 (C-2”), 74.3–73.7 (C-30,
C-30”, C-3, C-3”), 72.4 (C-20, C-20”), 70.1 (C-40, C-40”), 68.6 (C-6), 67.7 (CH2O), 62.4 (C-60, C-60”), 61.5
(C-6”), 56.4 (C-2), 48.6 (CH2N3), 29.8 (CH2), 23.1 (MeCON). Elemental Analysis Found: C, 44.09; H,
6.41; N, 7.13. Calculated for C29H50N4O21 (790.73): C, 44.05; H, 6.37; N, 7.09.
3.1.4. Synthesis of Methyl 2-acetamido-3-O-benzyl-4-O-(6-azido-6-deoxy-β-d-galactopyranosyl)-6-O-
[4-O-(β-d-galactopyranosyl)-β-d-glucopyranosyl]-2-deoxy-β-d-glucopyranoside (49)
A solution of tetrasaccharide 44 (170 mg, 0.135 mmol) in MeOH (1.35 mL) was treated with
7N NH3-MeOH (1.35 mL) and the solution was stirred at room temperature until TLC analysis (3:7
MeOH-H2O, reversed-phase) revealed the complete disappearance of the starting material (20 h).
The solution was concentrated under diminished pressure and the purification of crude product by
reversed-phase flash chromatography on C-18 silica gel (3:7 MeOH-H2O) afforded pure tetrasaccharide
49 (73 mg, 65%) as a white foam, Rf 0.47 (3:7 MeOH-H2O); (
(250.13 MHz, CD3OD-D2O):
α)
-10.86 (c 1.04 in MeOH); 1H NMR
D
δ
7.36–7.19 (m, 5H, Ar-H), 4.95 (m, 2H, CH2Ph), 4.60 (d, 1H, J1”,2” 7.9 Hz,
H-1”), 4.42 (d, 1H, J1 ,2 7.8 Hz, H-10), 4.36 (d, 1H, J1,2 8.1 Hz, H-1), 4.34 (d, 1H, J1 ”,2 ” 7.9 Hz, H-10”),
4.25 (m, 1H, H-6b), 4.08-3.42 (m, 21H, H-2, H-3, H-4, H-5, H-6a, H-20, H-30, H-40, H-50, H-2”, H-3”, H-4”,
H-5”, H-6”a, H-6”b, H-20”, H-30”, H-40”, H-50”, H-60”b, H-60”a), 3.38-3.27 (m, 2H, H-60a, H-60b), 3.42
0
0
0
0
(s, 3H, MeO), 1.85 (s, 3H, MeCON); 13C NMR (62.9 MHz, CD3OD-D2O):
δ 173.5 (C=O), 140.2 (Ar-C),
129.1–128.4 (Ar-CH), 104.8 (C-1), 104.4, 104.2 (C-10, C-1”), 103.6 (C-10”), 81.9 (C-3), 80.3 (C-4”), 78.0
(C-4), 76.9 (C-5), 76.3, 76.2, 75.5, 74.5, 74.4, 74.3, 74.0, 72.6, 72.4, 69.9, 69.8, (C-20, C-30, C-40, C-50, C-2”,
C-3”, C-5”, C-20”,C-30”, C-40”, C-50”), 75.1 (CH2Ph), 68.1 (C-6), 62.4 (C-6”), 61.6 (C-60”), 57.4 (OMe),
55.8 (C-2), 51.6 (C-60), 23.3 (MeCON). Elemental Analysis Found: C, 48.84; H, 6.27; N, 6.74. Calculated
for C34H52N4O20 (836.80): C, 48.80; H, 6.23; N, 6.70.
3.1.5. Synthesis of Methyl 2-acetamido-4-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-6-O-
[4-O-(3,4-di-O-acetyl-β-d-galactopyranoyl uronic acid)-2,3,6-tri-O-acetyl-β-d-glucopyranosyl]-2-
deoxy-β-d-glucopyranoside (50)
Compound 45 (70.6 mg, 0.051 mmol) in 2.5:1 MeOH-EtOAc (3.5 mL) was treated with 10% Pd
on activated charcoal (8 mg). The suspension was stirred at room temperature under H2 atmosphere
until TLC analysis (8:2 CHCl3-MeOH) revealed the complete disappearance of the starting material
(48 h) and the formation of a product at Rf 0.16 (no UV visible spot). The suspension was filtered
on a cotton pad, washed with MeOH and concentrated under diminished pressure. Purification of
crude product by flash chromatography on silica gel (8:2 CHCl3-MeOH) gave the uronic acid pure 50
(55.7 mg, 98%) as a white foam, Rf 0.16 (8:2 CHCl3-MeOH); (
(250.13 MHz, CDCl3-CD3OD):
α)
+4.74 (c 1.16 in CHCl3); 1H NMR
D
δ
5.61 (bd, 1H, H-40”), 5.38 (m, 1H, H-40), 5.20-4.90 5.11-4.90 (m, 4H,
H-3”, H-30”, H-20, H-30), 4.80 (m, 3H, H-10, H-1”, H-2”), 4.40–4.20 (m, 3H, H-10”, H-1, H-60b,), 4.10–4.02
(m, 2H, H-60a, H-6”b H-50”, H-6”a), 4.00-3.88 (m, 2H, H-50, H-6a), 3.77–3.15 (m, 8H, H-6b, H-5”, H-4,
H-4”, H-2, H-3, H-5, H-20”), 3.43 (s, 3H, MeO), 2.13, 2.07, 2.06, 1.98 (4s, each 3H, 4
×
MeCOO), 2.02 (s,
9H, 3
×
MeCOO), 1.95 (s, 6H, 2
×
MeCOO), 1.64 (s, 3H, MeCON); 13C NMR (62.9 MHz, CDCl3-CD3OD):
δ
172.4-170.2 (10
×
C=O, C-60”), 104.3 (C-10”), 101.8 (C-1), 101.5 (C-10), 100.9 (C-1”), 81.9 (C-3), 77.0
(C-4”), 75.2 (C-5), 74.2 (C-3”), 74.1 (C-5”), 74.0 (C-30), 73.6 (C-30”), 73.1 (C-4), 72.6 (C-50), 72.2 (C-2”),
71.3 (C-50”), 71.2 (C-20), 69.2 (C-20”), 69.8 (C-40”), 69.0 (C-6), 67.4 (C-40), 63.0 (C-60), 61.7 (C-6”), 56.7
(MeO), 55.2 (C-2), 22.9 (MeCON), 20.9-20.6 (9
N, 1.29. Calculated for C45H63NO31 (1113.98): C, 48.52; H, 5.70; N, 1.26.
×
MeCOO). Elemental Analysis Found: C, 48.55; H, 5.74;

Molecules 2019, 24, 3414
13 of 19
3.1.6. Synthesis of 3-Ammoniumpropyl 2-acetamido-4-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-
6-O-[4-O-(3,4-di-O-acetyl-2-benzyl-β-d-galactopyranosyl uronate)-2,3,6-tri-O-acetyl-β-d-
glucopyranosyl]-2-deoxy-β-d-glucopyranoside (51)
A solution of 46 (142.4 mg, 0.098 mmol) in 3:1:0.5 MeOH-CH2Cl2-H2O (14 mL) was treated with
10% Pd on activated charcoal (74.4 mg). The suspension was stirred at room temperature under H2
atmosphere until TLC analysis (7:3 CHCl3-MeOH) revealed the complete disappearance of the starting
material (48 h). The suspension was filtered on a cotton pad, washed with MeOH and concentrated
under diminished pressure. Purification of crude product by flash chromatography on silica gel (7:3
CHCl3-MeOH) gave pure zwitterionic tetrasaccharide 51 (80 mg, 70%) as a white solid, Rf 0.23 (7:3
CHCl3-MeOH); mp 193–195 ◦C (after trituration with Et2O); (
α
)D +2.57 (c 1.13 in MeOH); 1H NMR
4.72 (m, 2H, H-10, H-1”), 4.46 (d, 1H,
J1 ”,2 ” 7.9 Hz, H-10”), 4.36 (d, 1H, J1,2 8.1 Hz, H-1), 4.01 and 3.63 (2m, each 1H, CH2O), 3.15 (bt, 1H,
vic 6.8 Hz, CH2NH3+), 2.15, 2.11, 2.10, 2.07, 2.05, 2.03, 1.98, 1.95, 1.94 (9s, each 3H, 9
MeCOO), 1.91
(s, 3H, MeCON), 1.88 (m, 2H, CH2); 13C NMR (62.9 MHz, CD3OD): 174.1 (C-60”), 173.0-171.2 (10
(250.13 MHz, CD3OD) led to identify only some signals as:
δ
0
0
J
×
δ
×
C=O), 104.9 (C-10”), 102.4 (C-1), 101.9 (C-10), 101.8 (C-1”), 82.1 (C-4”), 78.0 (C-4), 75.0 (C-5”), 74.7 (C-5,
C-2”, C-3”, C-50”), 74.2 (C-50), 73.3 (C-3), 72.2, 72.1, 70.9, 70.4 (C-20”, C-30”, C-30, C-20), 69.9 (C-40”), 69.7
(C-6), 68.6 (C-40), 68.5 (CH2O), 63.9 (C-6”), 62.7 (C-60), 56.4 (C-2), 39.3 (CH2NH3+), 28.1 (CH2), 23.0
(MeCON), 21.3-20.5 (9
C47H68N2O31 (1157.04): C, 48.79; H, 5.92; N, 2.42.
×
MeCOO). Elemental Analysis Found: C, 48.82; H, 5.95; N, 2.44. Calculated for
3.1.7. Synthesis of Methyl 2-acetamido-4-O-(6-ammonium-2,3,4-tri-O-acetyl-6-deoxy-β-d-
galactopyranosyl)-6-O-[4-O-(3,4-di-O-acetyl-β-d-galactopyranosyl uronate)-2,3,6-tri-O-acetyl-
β-d-glucopyranosyl]-2-deoxy-β-d-glucopyranoside (52).
A solution of 47 (238 mg, 0.174 mmol) in 3:1:0.5 MeOH-CH2Cl2-H2O (13.5 mL) was treated with
10% Pd on activated charcoal (131 mg). The suspension was stirred at room temperature under H2
atmosphere until TLC analysis (7:3 CHCl3-MeOH) revealed the complete disappearance of the starting
material (48 h). The suspension was filtered on a cotton pad, washed with MeOH and concentrated
under diminished pressure. Purification of crude product by reversed-phase flash chromatography on
C-18 silica gel (4:6 MeOH-H2O) gave pure zwitterionic tetrasaccharide 52 (141 mg, 76%) as a white
solid, Rf 0.40 (4:6 MeOH-H2O, reversed-phase); mp 195–197 ◦C (after trituration with Et2O); (
α
)
D –1.75
(c 1.03 in MeOH); 1H NMR (250.13 MHz, CD3OD-D2O):
δ
5.49 (m, 1H, H-40”), 5.31 (m, 1H, H-40), 5.23
(m, 1H, H-3”), 5.12-4.91 (m, 2H, H-30, H-20), 4.90–4.70 (m, 2H, H-30”, H-2”), 4.75 (bd, 2H, J 7.5 Hz,
H-1”, H-10), 4.54-4.20 (m, 5H, H-10”, H-50”, H-1, H-6”b, H-6”a), 4.13- 3.88 (m, 4H, H-6b, H-6a, H-5”,
H-50), 3.82- 3.40 (m, 6H, H-2, H-4, H-5, H-4”, H-3, H-20”), 3.47 (s, 3H, OMe), 3.10-2.95 (m, 2H, H-60a,
H-60b), 2.10-1.88 (m, 27H, 9
×
MeCO); 13C NMR (62.9 MHz, CD3OD-D2O):
δ
174.6-172.3 (9
×
C=O,
C-60”), 104.3 (C-10”), 103.0 (C-1), 101.9 (C-1”), 101.3 (C-10), 79.9 (C-3), 77.0 (C-4”), 75.4 (C-2”), 74.6 (C-4,
C-50, C-5”), 73.8, 73.5, 73.2, 72.0 (C-50”, C-3”, C-30”, C-5), 71.0 (C-30, C-20”), 70.5 (C-20), 69.9 (C-6), 69.7
(C-40”), 69.5 (C-40), 64.0 (C-6”), 57.5 (OMe), 56.5 (C-2), 40.8 (C-60), 23.1 (MeCON), 21.5-20.7 (8
MeCOO).
Elemental Analysis Found: C, 48.26; H, 5.87; N, 2.65. Calculated for C43H62N2O29 (1070.95): C, 48.23;
H, 5.84; N, 2.62.
×
3.1.8. Synthesis of Methyl 2-acetamido-4-O-β-d-galactopyranosyl-6-O-[4-O-(β-d-galactopyranosyl)-
β-d-glucopyranosyl]-2-deoxy-β-d-glucopyranoside (2)
A solution of 42 (99.7 mg, 0.0774 mmol) in MeOH (1 mL) was treated with a methanolic solution
of MeONa (0.33 M, 3 mL) and the reaction mixture was stirred at 0◦C until TLC analysis (9:1
CHCl3-MeOH) showed the complete disappearance of the starting material (12 h). The solution
was neutralized with resin acid (Amberlist-15), filtered on a cotton pad, washed with MeOH and
concentrated under diminished pressure. Crystallization of the crude product with EtOH-H2O afforded
pure tetrasaccharide
2
(40 mg, 72%) as a white solid, Rf 0.10 (9:1 CHCl3-MeOH); mp 158-160 ◦C (from
+1 (c 1 in H2O); Lit [49] ( –4.40 (c 2.75 in H2O);
EtOH-H2O); ( –14.42 (c 1.2 in H2O); Lit [44] (
α
)
D
α
)
D
α)
D

Molecules 2019, 24, 3414
14 of 19
1H NMR (600 MHz, D2O): 4.45 (d, 1H, J1”,2” 8.02 Hz, H-1”), 4.43 (d, 1H, J1 7.8 Hz, H-10), 4.36 (d,
δ
0,20
1H, J1,2 8.3 Hz, H-1), 4.34 (d, 1H, J1 ”,2 ” 8.2 Hz, H-10”), 4.19 (dd, 1H, J6a,6b 11.1 Hz, J5,6b 1.2 Hz, H-6b),
3.85 (dd, 1H, J5,6a 3.7 Hz, H-6a), 3.87 (m, 1H, H-6”a), 3.82 (m, 1H, H-40”), 3.81 (m, 1H, H-40), 3.71 (m,
1H, H-6”b), 3.65 (m, 4H, H-60a, H-60b, H-60”a, H-60”b), 3.74 (m, 1H, H-4), 3.63 (m, 1H, H-2), 3.61 (m,
1H, H-5), 3.59 (m, 1H, H-50), 3.57 (m, 1H, H-3”), 3.567 (m, 1H, H-50”), 3.56 (m, 4H, H-3, H-40”, H-30”,
H-30), 3.49 (m, 1H, H-5”), 3.43 (m, 2H, H-20, H-20”), 3.40 (s, 3H, OMe), 3.27 (m, 1H, H-2”), 1.93 (s, 3H,
0
0
MeCON); 13C NMR (D2O):
δ
174.3 (C=O), 102.8 (C-10”), 102.6 (C-10), 102.2 (C-1”), 102.0 (C-1), 78.3
(C-4”), 77.7 (C-4), 75.2 (C-50), 74.6 (C-5”), 74.4 (C-50”), 74.2 (C-30, C-30”), 73.4 (C-5), 72.6 (C-2”), 72.4
(C-3), 72.3 (C-3”), 70.9 (C-20, C-20”), 68.5 (C-40, C-40”), 67.1 (C-6), 61.0 (C-60, C-60”), 59.9 (C-6”), 57.7
(MeO), 54.9 (C-2), 23.1 (MeCON). Elemental Analysis Found: C, 44.98; H, 6.59; N, 1.97. Calculated for
C27H47NO21 (721.67): C, 44.94; H, 6.56; N, 1.94.
3.1.9. Synthesis of Methyl 2-acetamido-4-O-β-d-galactopyranosyl-6-O-[4-O-(β-d-galactopyranosyl
uronate)-β-d-glucopyranosyl]-2-deoxy-β-d-glucopyranoside Ammonium Salt (3)
A solution of 50 (40.7 mg, 0.0365 mmol) in MeOH (0.5 mL) was treated with 7N NH3-MeOH
(0.5 mL) and the reaction mixture was stirred at room temperature until TLC analysis (6:4 CHCl3-MeOH)
showed the complete disappearance of the starting material (48 h). The solution was concentrated
under diminished pressure and purification of crude product by reversed-phase flash chromatography
on C-18 silica gel (1:3 MeOH-H2O) gave pure tetrasaccharide
(8:2 CHCl3-MeOH); mp 241–243 ◦C (from isopropanol-MeOH); (
(600 MHz, D2O):
4.46 (d, 1H, J1”,2” 7.99 Hz, H-1”), 4.43 (d, 1H, J1 ,2 7.87 Hz, H-10), 4.36 (d, 1H, J1,2
3
(24.4 mg, 89%) as a white solid, Rf 0.0
α
)
D –50.6 (c 0.9 in MeOH); 1H NMR
0
0
δ
8.37 Hz, H-1), 4.34 (d, 1H, J1 ”,2 ” 7.76 Hz, H-10”), 4.20 (dd, 1H, J6a,6b 10.9 Hz, J5,6b 1.1 Hz, H-6b), 4.11
(m, 1H, H-40”), 3.99 (m, 1H, H-50”), 3.85 (dd, 1H, J5,6a 3.1 Hz, H-6a), 3.81 (m, 1H, H-40), 3.74 (m, 1H,
H-4), 3.72 (m, 1H, H-6”b), 3.66 (m, 1H, H-6”a), 3.64 (m, 1H, H-2), 3.63 (m, 2H, 60a, H-60b), 3.62 (m,
1H, H-5), 3.60 (m, 1H, H-30”), 3.60 (m, 1H, H-50), 3.59 (m, 1H, H-3”), 3.58 (m, 2H, H-3, H-30), 3.52 (m,
1H, H-4”), 3.507 (m, 1H, H-5”), 3.48 (m, 1H, H-20”), 3.45 (m, 1H, H-20), 3.44 (s, 3H, MeO), 3.29 (m, 1H,
0
0
H-2”), 1.93 (s, 3H, MeCON); 13C NMR (150 MHz, D2O): 178.2, 175.4 (MeCO, C-60”), 102.5 (C-10), 102.5
δ
(C-10”), 102.3 (C-1”), 102.0 (C-1), 78.9 (C-4”), 77.6 (C-4), 75.5 (C-40”), 75.2 (C-50), 75.1 (C-50”), 74.6 (C-5”),
74.4 (C-30), 73.5 (C-20”), 73.4 (C-5), 73.4 (C-20), 72.6 (C-2”), 72.4 (C-3, C-3”), 70.0 (C-30”), 68.6 (C-40), 67.2
(C-6), 61.0 (C-60), 59.9 (C-6”), 54.9 (C-2), 58.2 (MeO), 22.2 (MeCON). Elemental Analysis Found: C,
43.12; H, 6.46; N, 3.75. Calculated for C27H48N2O22 (752.67): C, 43.09; H, 6.43; N, 3.72.
3.1.10. Synthesis of 3-Aminopropyl 2-acetamido-4-O-β-d-galactopyranosyl-6-O-[4-O-(β-d-
galactopyranosyl)-β-d-glucopyranosyl]-2-deoxy-β-d-glucopyranoside Hydrochloride (4)
A solution of 48 (59.3 mg, 0.075 mmol) in 3:1 MeOH-H2O (4 mL) was turned to pH 3 adding few
drops of 1% methanolic HCl (0.46 mL) and treated with 10% Pd on activated charcoal (42.2 mg). The
suspension was stirred at room temperature under H2 atmosphere until TLC analysis (2:8 MeOH-H2O,
reversed-phase) revealed the complete disappearance of the starting material (20 h). The suspension
was filtered on a cotton pad, washed with MeOH and concentrated under diminished pressure.
Crystallization of the crude product with EtOH afforded pure tetrasaccharide
4 (43 mg, 71%) as a
white solid, Rf 0.98 (2:8 MeOH-H2O, reversed-phase); mp 183–185 ◦C (from EtOH); (
α
)
–4.3 (c 0.72 in
D
MeOH); 1H NMR (600 MHz, D2O):
δ
4.45 (d, 1H, J1”,2” 8.1 Hz, H-1”), 4.43 (d, 1H, J1 ,2 8.03 Hz, H-10),
0
0
4.42 (d, 1H, J1,2 8.3 Hz, H-1), 4.34 (d, 1H, J1 ”,2 ” 7.85 Hz, H-10”), 4.19 (dd, 1H, J6a,6b 10.9 Hz, J5,6b 1.1 Hz,
H-6b), 3.87 (m, 1H, H-6”b), 3.85 (dd, 1H, J5,6b 3.1 Hz, H-6a), 3.82 (m, 1H, H-40”), 3.81 (m, 1H, H-40),
3.75 (m, 1H, H-4), 3.71 (m, 1H, H-6”a), 3.68 (m, 2H, CH2O), 3.65 (m, 2H, H-60a, H-60b), 3.64 (m, 3H,
H-2, H-60”a, H-60”b,), 3.61 (m, 2H, H-5, H-50”), 3.60 (m, 1H, H-50), 3.59 (m, 1H, H-3), 3.57 (m, 2H, H-4”,
H-3”), 3.56 (m, 1H, H-30), 3.55 (m, 1H, H-30”), 3.50 (m, 1H, H-5”), 3.44 (m, 2H, H-20, H-20”), 3.27 (m, 1H,
H-2”), 3.01 (bt, 2H, Jvic 6.8 Hz, CH2N3), 2.01 (s, 3H, MeCON), 1.91 (m, 2H, CH2); 13C NMR (150 MHz,
0
0
D2O):
δ
175.7 (C=O), 102.9 (C-10”), 102.6 (C-10), 102.3 (C-1”), 101.3 (C-1), 78.3 (C-4”), 77.5 (C-4), 75.3
(C-50, C-50”), 74.6 (C-5”), 74.2 (C-30, C-30”), 73.3 (C-5), 72.6 (C-2”), 72.5 (C-3”), 72.2 (C-3), 70.9 (C-20”,

Molecules 2019, 24, 3414
15 of 19
C-20), 68.5 (C-40”, C-40), 68.3 (CH2O), 67.1 (C-6), 61.0 (C-60”), 60.9 (C-60), 59.8 (C-6”), 55.1 (C-2), 38.6
(CH2N3), 27.6 (CH2), 23.2 (MeCON). Elemental Analysis Found: C, 43.51; H, 6.69; N, 3.54. Calculated
for C29H52ClN2O21 (801.19): C, 43.48; H, 6.67; N, 3.50.
3.1.11. Synthesis of 3-Ammoniumpropyl 2-acetamido-4-O-β-d-galactopyranosyl-6-O-
[4-O-(β-d-galactopyranosyl uronate)-β-d-glucopyranosyl]-2-deoxy-β-d-glucopyranoside (5)
A solution of 51 (60.1 mg, 0.052 mmol) in MeOH (1.0 mL) was treated with 7N NH3-MeOH
(1.0 mL) and the solution was stirred at room temperature until TLC analysis (4:6 MeOH-H2O,
reversed-phase) revealed the complete disappearance of the starting material (48 h). The solution was
concentrated under diminished pressure and the purification of crude product by reversed-phase flash
chromatography on C-18 silica gel (H2O) afforded pure tetrasaccharide
5
(39.8 mg, 98%) as a white
solid, Rf 0.95 (H2O); mp 194–195 ◦C (after trituration with MeOH); (
α
)
D –38.8 (c 0.99 in H2O); 1H NMR
0
0
(600 MHz, D2O):
δ
4.47 (d, 1H, J1”,2” 7.97 Hz, H-1”), 4.43 (d, 1H, J1 ,2 7.76 Hz, H-10), 4.42 (d, 1H, J1,2
8.3 Hz, H-1), 4.33 (d, 1H, J1 ”,2 ” 7.86 Hz, H-10”), 4.19 (dd, 1H, J6a,6b 10.9 Hz, J5,6b 1.2 Hz, H-6b), 4.12
(m, 1H, H-40”), 3.98 (m, 1H, H-50”), 3.85 (dd, 1H, J5,6a 3.7 Hz, H-6a), 3.86 (m, 1H, H-6”b), 3.81–3.72 (m,
3H, H-40, H-4, H-6”a), 3.67–3.51 (m, 13H, H-30”, H-2, H-60a, H-60b, CH2O, H-5, H-3, H-50, H-3”, H-30,
H-4”, H-5”), 3.47 (m, 1H, H-20”), 3.43 (m, 1H, H-20), 2.98 (bt, 1H, Jvic 6.8 Hz, CH2NH3+), 3.27 (m, 1H,
0
0
H-2”), 1.91 (s, 3H, MeCON), 1.82 (m, 2H, CH2); 13C NMR (150 MHz, D2O):
δ
175.7 (C=O), 175.6 (C-60”),
102.6 (C-10), 102.4 (C-10”), 102.2 (C-1”), 101.4 (C-1), 78.7 (C-4”), 77.5 (C-4), 75.6 (C-50”), 75.0 (C-50), 74.6
(C-5”), 73.3 (C-5), 72.7 (C-2”), 72.4 (C-3”, C-30”), 72.3 (C-3, C-30), 70.9 (C-20), 70.4 (C-20”), 69.9 (C-40”),
68.4 (C-40), 68.3 (CH2O), 67.1 (C-6), 60.9 (C-60), 59.9 (C-6”), 55.0 (C-2), 38.6 (CH2NH3+), 27.6 (CH2), 23.1
(MeCON). Elemental Analysis Found: C, 44.76; H, 6.49; N, 3.64. Calculated for C29H50N2O22 (778.71):
C, 44.73; H, 6.47; N, 3.60.
3.1.12. Synthesis of Methyl 2-acetamido-4-O-(6-amino-6-deoxy-β-d-galactopyranosyl)-6-O-
[4-O-(β-d-galactopyranosyl)-β-d-glucopyranosyl]-2-deoxy-β-d-glucopyranoside Hydrochloride (6)
A solution of 49 (52 mg, 0.062 mmol) in 3:1 MeOH-H2O (2.5 mL) was turned to pH 3 adding
few drops of 1% methanolic HCl (0.51 mL) and treated with 10% Pd on activated charcoal (50 mg).
The suspension was stirred at room temperature under H2 atmosphere until TLC analysis (1:9
MeOH-H2O, reversed-phase) revealed the complete disappearance of the starting material (20 h).
The suspension was filtered on a cotton pad, washed with MeOH and concentrated under diminished
pressure. Purification of crude product by reversed-phase flash chromatography on C-18 silica gel (1:9
MeOH-H2O) afforded pure tet◦rasaccharide
6
(45 mg, 96%) as a white solid, Rf 0.47 (7:3 MeOH-H2O,
1
reversed-phase); mp 183–186 C (from MeOH-isopropanol); (
α
)
–19.9 (c 1.0 in MeOH); H NMR
D
(250.13 MHz, CD3OD-D2O):
4.67 (d, 1H, J1”,2” 7.6 Hz, H-1”), 4.42 (d, 1H, J1 ,2 7.8 Hz, H-10), 4.39
0 0
δ
(d, 1H, J1 ”,2 ” 7.9 Hz, H-10”), 4.34 (d, 1H, J1,2 8.2 Hz, H-1), 4.28 (m, 1H, H-6b), 4.10-3.51 (m, 21H, H-2,
0
0
H-3, H-4, H-5, H-6a, H-20, H-30, H-40, H-50, H-2”, H-3”, H-4”, H-5”, H-6”a, H-6”b, H-20”, H-30”, H-40”,
H-50”, H-60”b, H-60”a), 3.45 (s, 3H, MeO), 3.38–3.21 (m, 2H, H-60a, H-60b), 1.99 (s, 3H, MeCON); 13
C
NMR (62.9 MHz, CD3OD-D2O):
δ
173.3 (MeCO), 104.8 (C-1), 103.8, 103.5, 103.4 (C-10, C-1”, C-10”), 80.2
(C-4”), 77.8 (C-4), 76.7 (C-3, C-5), 76.1, 75.0, 74.4, 74.4, 74.0, 73.9, 72.2, 71.9, 71.6, 70.9, 69.9 (C-20, C-30,
C-40, C-50, C-2”, C-3”, C-5”, C-20”, C-30”, C-40”, C-50”), 67.7 (C-6), 62.2 (C-6”), 61.4 (C-60”), 57.1 (OMe),
56.2 (C-2), 41.7 (C-60), 22.6 (MeCON). Elemental Analysis Found: C, 42.86; H, 6.55; N, 3.72. Calculated
for C27H49ClN2O20 (757.13): C, 42.83; H, 6.52; N, 3.70.
3.1.13. Synthesis of Methyl 2-acetamido-4-O-(6-ammonium-6-deoxy-β-d-galactopyranosyl)-6-O-[4-O-
(β-d-galactopyranosyl uronate)-β-d-glucopyranosyl]-2-deoxy-β-d-glucopyranoside (7)
A solution of 52 (39.1 mg, 0.0365 mmol) in MeOH (1.0 mL) was treated with 7N NH3-MeOH (1.0 mL)
and the solution was stirred at room temperature until TLC analysis (H2O, reversed-phase) revealed
the complete disappearance of the starting material (48 h). The solution was concentrated under
diminished pressure and the purification of crude product by reversed-phase flash chromatography

Molecules 2019, 24, 3414
16 of 19
on C-18 silica gel (H2O) afforded pure tetrasaccharide
7
(23 mg, 86%) as a white solid, Rf 0.95 (H2O,
reversed-phase); mp 226–228 ◦C (from MeOH-EtOH); ( )D –16.67 (c 1.05 in H2O), 1H NMR (250.13
α
4.46 (d, 1H, J1”,2” 7.9 Hz, H-1”), 4.45 (d, 1H, J1 ,2 7.3 Hz, H-10), 4.40 (d, 1H, J1,2 8.4 Hz,
0 0
MHz, D2O): δ
H-1), 4.37 (d, 1H, J1 ”,2 ” 8.1 Hz, H-10”), 4.12 (m, 1H, H-6b), 4.03 (m, 1H, H-40”), 3.91 (m, 1H, H-50”),
3.85-3.63 (m, 4H, H-40, H-4, H-5, H-6b), 3.60-3.41 (m, 12H, H-20”, H-30, H-4”, H-30”, H-3”, H-6”a, H-6”b,
H-50, H-5”, H-2, H-3, H-6a,), 3.40-3.10 (m, 5H, H-20, H-2”, H-2, H-60a, H-60b), 3.33 (s, 3H, OMe), 1.85
0
0
(s, 3H, MeCON); 13C NMR (62.9 MHz, D2O):
δ
175.7, 175.6 (C=O, C-60”), 103.9 (C-10), 103.7(C-10”),
103.4 (C-1”), 103.0 (C-1), 80.1 (C-4”), 79.3 (C-4), 75.7 (C-50”), 75.5 (C-50), 74.5 (C-5”, C-20”), 73.9 (C-2”),
73.8-73.7 (C-5, C-3”, C-3), 73.4 (C-30”), 73.3 (C-30), 71.8 (C-20), 71.4 (C-40”), 69.9 (C-40), 68.4 (C-6), 61.1
(C-6”), 58.3 (OMe), 56.0 (C-2), 40.8 (C-60), 23.2 (MeCON). Elemental Analysis Found: C, 44.17; H, 6.34;
N, 3.84. Calculated for C27H46N2O21 (734.66): C, 44.14; H, 6.31; N, 3.81.
3.2. Biological Methods
3.2.1. Test ELISA
◦
96-Well flat-bottomed plates were incubated overnight at 4–8 C with a mixture of SP14 CPS
(1 mg/mL) (American Type Culture Collection, ATCC, Manassas, VA, USA) and methylated human
serum albumin (1 mg/mL). A solution of fetal 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-SP14
(Thermo Fisher Scientific, Rockford, IL, USA), used as reference serum. When SP14 analogous were
tested, they were added to each well immediately before the addition of the reference serum. The
plates were then incubated with alkaline phosphatase conjugate goat anti-rabbit IgG stained with
p-nitrophenylphosphate (Sigma Aldrich, Milan Italy), and the absorbance was measured at 405 nm
with an Ultramark microplate reader.
3.2.2. Statistical Analysis
Results are expressed as the mean + SEM of at least five different experiments run in triplicate.
Statistical significance was evaluated by Student0s t-test for unpaired varieties. Differences were
considered statistically significant when p ≤ 0.05.
4. Conclusions
A synthetic approach for the synthesis of charged analogues of the smallest immunogenic structure
of Streptococcus pneumoniae type 14 (SP14) capsular polysaccharide was explored. Suitable lacturonic
and lactose donors were coupled with modified lactosamine acceptors which allowed, after the
final deprotection, to obtain two cationic, an anionic as well as two zwitterionic tetrasaccharide
target structures. From the synthetic perspective, the proposed block strategy proved to be feasible,
permitting to build up several point-charged structures. The biological data we reported for the
prepared tetrasaccharides clearly demonstrate that the introduction of positive and/or negative charges
into the basic unit of SP14 CPS do not modify the affinity of a neutral compound to binding to a specific
antibody. In our opinion, these results would represent an encouraging starting point for future studies
on the ability of these charged compounds to stimulate immune cell responses. All analogues exhibited
similar efficacies, which are lower than the natural SP-14 compound. This might be related to the
length of synthetic fragments, too short to cover the entire SP14 CPS epitopes.
Supplementary Materials: The following are available online, experimental procedures and characterization data
of compound 10–23, 25–28 and 31–40. NMR spectra of new compounds.
Author Contributions: F.D.A. and L.G. designed the study and analyzed the data, T.G. and D.C. performed the
chemical experimental, S.F and G.L. performed the biological experiments, F.D.A., L.G., D.C., S.F and G.L. wrote
the main text. All authors reviewed and accepted the final version of the manuscript.

Molecules 2019, 24, 3414
17 of 19
Funding: This work was supported by MIUR-Italy (PRIN 2010, prot. 2010JMAZML_008 and PRIN 2015, prot.
2015RNWJAM_003).
Acknowledgments: The authors would like to thank Professor Giorgio Catelani who inspired the whole project,
Professor Alba Silipo (University of Napoli) for registering high-field NMR spectra of compounds 2–5, and
undergraduates Chiara Esposto, Lara Zampini and Beatrice Tamassia (University of Pisa) for the contribution to
the synthesis of compounds 42, 43, 3 and 6.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Verez-Bencomo, V.; Fernández-Santana, V.; Hardy, E.; Toledo, M.E.; Rodríguez, M.C.; Heynngnezz, L.;
Rodriguez, A.; Baly, A.; Herrera, L.; Izquierdo, M.; et al. A Synthetic Conjugate Polysaccharide Vaccine
Against Haemophilus influenzae Type b. Science 2004, 305, 522–525. [CrossRef]
2.
Schumann, B.; Anish, C.; Pereira, C.L.; Seeberger, P.H. Carbohydrate Vaccines. In Biotherapeutics: Recent
Developments using Chemical and Molecular Biology; Jones, L., McKnight, A.J., Eds.; RSC Drug Discovery Series
No. 36. ed. RSC: Cambridge, UK, 2013; pp. 68–104.
3.
4.
5.
6.
Micoli, F.; Costantino, P.; Adamo, R. Potential targets for next generation antimicrobial glycoconjugate
vaccines. FEMS Microbiol. Rev. 2018, 42, 388–423. [CrossRef]
Colombo, C.; Pitirollo, O.; Lay, L. Recent Advances in the Synthesis of Glycoconjugates for Vaccine
Development. Molecules 2018, 23, 1712. [CrossRef]
Nishat, S.; Andreana, P.R. Entirely Carbohydrate-Based Vaccines: An Emerging Field for Specific and
Selective Immune Responses. Vaccines 2016, 4, 19. [CrossRef]
Mondiale de la Sant
under 5 years of age: WHO position paper–February 2019–Vaccins antipneumococciques conjugu
nourrissons et les enfants de moins de 5 ans: note de synth se de l’OMS–f vrier 2019. Weekly Epidemiological
Record=Relevé épidémiologique hebdomadaire 2019, 94, 85–103.
é
O, World Health Organization. Pneumococcal conjugate vaccines in infants and children
és chez les
è
é
7.
8.
9.
Tzianabos, A.O.; Onderdonk, A.; Rosner, B.; Cisneros, R.; Kasper, D.L. Structural features of polysaccharides
that induce intra-abdominal abscesses. Science 1993, 262, 416–419. [CrossRef]
Cobb, B.A.; Wang, Q.; Tzianabos, A.O.; Kasper, D.L. Polysaccharide processing and presentation by the
MHCII pathway. Cell 2004, 117, 677–687. [CrossRef]
Van der Put, R.M.F.; Kim, T.H.; Guerreiro, C.; Thouron, F.; Hoogerhout, P.; Sansonetti, P.J.; Westdijk, J.;
Stork, M.; Phalipon, A.; Mulard, L.A. A Synthetic Carbohydrate Conjugate Vaccine Candidate against
Shigellosis: Improved Bioconjugation and Impact of Alum on Immunogenicity. Bioconjug. Chem. 2016, 27,
883–892. [CrossRef]
10. Hu, Z.; Bongat White, A.F.; Mulard, L.A. Efficient Iterative Synthesis of O-Acetylated Tri- to
Pentadecasaccharides Related to the Lipopolysaccharide of Shigella flexneri Type 3 a through Di- and
Trisaccharide Glycosyl Donors. Chem. Asian J. 2017, 12, 419–439. [CrossRef]
11. Carboni, F.; Adamo, R.; Fabbrini, M.; De Ricco, R.; Cattaneo, V.; Brogioni, B.; Veggi, D.; Pinto, V.; Passalacqua, I.;
Oldrini, D.; et al. Structure of a protective epitope of group B Streptococcus type III capsular polysaccharide.
Proc. Natl. Acad. Sci. USA 2017, 114, 5017–5022. [CrossRef]
12. Guazzelli, L.; Ulc, R.; Rydner, L.; Oscarson, S. A synthetic strategy to xylose-containing thioglycoside
tri-and tetrasaccharide building blocks corresponding to Cryptococcus neoformans capsular polysaccharide
structures. Org. Biomol. Chem. 2015, 13, 6598–6610. [CrossRef]
13. Guazzelli, L.; McCabe, O.; Oscarson, S. Synthesis of part structures of Cryptococcus neoformans serotype C
capsular polysaccharide. Carbohydr. Res. 2016, 433, 5–13. [CrossRef]
14. Morelli, L.; Fallarini, S.; Lombardi, G.; Colombo, C.; Lay, L.; Compostella, F. Synthesis and biological evaluation
of a trisaccharide repeating unit derivative of Streptococcus pneumoniae 19A capsular polysaccharide.
Bioorg. Med. Chem. 2018, 26, 5682–5690. [CrossRef]
15. Menova, P.; Sella, M.; Sellrie, K.; Pereira, C.L.; Seeberger, P.H. Identification of the Minimal Glycotope of
Streptococcus pneumoniae 7F Capsular Polysaccharide using Synthetic Oligosaccharides. Chem.: A Eur. J. 2018
,
24, 4181–4187. [CrossRef]

Molecules 2019, 24, 3414
18 of 19
16. Seeberger, P.H.; Pereira, C.L.; Khan, N.; Xiao, G.; Diago-Navarro, E.; Reppe, K.; Opitz, B.; Fries, B.C.;
Witzenrath, M. A Semi-Synthetic Glycoconjugate Vaccine Candidate for Carbapenem-Resistant Klebsiella
pneumoniae. Angew. Chem. Int. Ed. 2017, 56, 13973–13978. [CrossRef]
17. Zhang, G.-L.; Yang, L.; Wei, M.; Yan, W.; Xiong, D.-C.; Ye, X.-S. Synthesis and Antigenic Evaluation of
Oligosaccharide Mimics of Vi Antigen from Salmonella typhi. Chem. Eur. J. 2017, 23, 10670–10677. [CrossRef]
18. Zhang, G.-L.; Wei, M.; Song, C.; Ma, Y.-F.; Zheng, X.-J.; Xiong, D.-C.; Ye, X.-S. Chemical synthesis and
biological evaluation of penta- to octa- saccharide fragments of Vi polysaccharide from Salmonella typhi.
Org. Chem. Front. 2018, 5, 2179–2188. [CrossRef]
19. Calloni, I.; Unione, L.; Jimenez-Oses, G.; Corzana, F.; Del Bino, L.; Corrado, A.; Pitirollo, O.; Colombo, C.;
Lay, L.; Adamo, R.; et al. The Conformation of the Mannopyranosyl Phosphate Repeating Unit of the
Capsular Polysaccharide of Neisseria meningitidis Serogroup A and Its Carba-Mimetic. Eur. J. Org. Chem.
2018, 33, 4548–4555. [CrossRef]
20. Fallarini, S.; Buzzi, B.; Giovarruscio, S.; Polito, L.; Brogioni, G.; Tontini, M.; Berti, F.; Adamo, R.; Lay, L.;
Lombardi, G. A Synthetic Disaccharide Analogue from Neisseria meningitidis A Capsular Polysaccharide
Stimulates Immune Cell Responses and Induces Immunoglobulin G (IgG) Production in Mice When
Protein-Conjugated. ACS Infect. Dis. 2015, 1, 487–496. [CrossRef]
21. Fusari, M.; Fallarini, S.; Lombardi, G.; Lay, L. Synthesis of di- and tri-saccharide fragments of Salmonella
typhi Vi capsular polysaccharide and their zwitterionic analogues. Bioorg. Med. Chem. 2015, 23, 7439–7447.
[CrossRef]
22. Zhang, Q.; Overkleeft, H.S.; van der Marel, G.A.; Codée, J.D.C. Synthetic zwitterionic polysaccharides.
Curr. Opin. Chem. Biol. 2017, 40, 95–101. [CrossRef]
23. Pfister, H.B.; Paoletti, J.; Poveda, A.; Jimenez-Barbero, J.; Mulard, L.A. Zwitterionic Polysaccharides of Shigella
sonnei: Synthetic Study toward a Ready-for-Oligomerization Building Block Made of Two Rare Amino
Sugars. Synthesis 2018, 50, 4270–4282. [CrossRef]
24. Bridy-Pappas, A.E.; Margolis, M.B.; Center, K.J.; Isaacman, D.J. Streptococcus pneumoniae: Description of
the Pathogen, Disease Epidemiology, Treatment, and Prevention. Pharmacotherapy 2005, 25, 1193–1212.
[CrossRef]
25. Marchetti, F.; Sottana, F.; La Torre, G. Caratteristiche biologiche e dati clinici disponibili per il nuovo vaccino
antipneumococcico Synflorix™ (PHiD-CV). Ital. J. Public Health 2009, 6, s59–s72.
26. Safari, D.; Dekker, H.A.T.; Joosten, J.A.F.; Michalik, D.; Carvalho de Souza, A.; Adamo, R.; Lahmann, M.;
Sundgren, A.; Oscarson, S.; Kamerling, J.P.; et al. Identification of the Smallest Structure Capable of Evoking
Opsonophagocytic Antibodies against Streptococcus pneumoniae Type 14. Infect. Immun. 2008, 76, 4615–4623.
[CrossRef]
27. Mawas, F.; Niggemann, J.; Jones, C.; Corbel, M.J.; Karmeling, J.P.; Vliegenthart, J.F.G. Immunogenicity in a
Mouse Model of a Conjugate Vaccine Made with a Synthetic Single Repeating Unit of Type 14 Pneumococcal
Polysaccharide Coupled to CRM197. Infect. Immun. 2002, 70, 5107–5114. [CrossRef]
28. Kurbatova, E.A.; Akhmatova, N.K.; Akhmatova, E.A.; Egorova, N.B.; Yastrebova, N.E.; Sukhova, E.V.;
Yashunsky, D.V.; Tsvetkov, Y.E.; Gening, M.L.; Nifantiev, N.E. Neoglycoconjugate of Tetrasaccharide
Representing One Repeating Unit of the Streptococcus pneumoniae Type 14 Capsular Polysaccharide Induces
the Production of Opsonizing IgG1 Antibodies and Possesses the Highest Protective Activity As Compared
to Hexa- and Octasaccharide Conjugates. Front. Immunol. 2017, 8, 659. [CrossRef]
29. Safari, D.; Marradi, M.; Chiodo, F.; Th Dekker, H.A.; Shan, Y.; Adamo, R.; Oscarson, S.; Rijkers, G.T.;
Lahmann, M.; Kamerling, J.P.; et al. Gold nanoparticles as carriers for a synthetic Streptococcus pneumoniae
type 14 conjugate vaccine. Nanomedicine 2012, 7, 651–662. [CrossRef]
30. Vetro, M.; Safari, D.; Fallarini, S.; Salsabila, K.; Lahmann, M.; Penadés, S.; Lay, L.; Marradi, M.;
Compostella, F. Preparation and immunogenicity of gold glyco-nanoparticles as antipneumococcal vaccine
model. Nanomedicine 2017, 12, 13–23. [CrossRef]
31. Guazzelli, L.; Catelani, G.; D0Andrea, F. Lactose as an inexpensive starting material for the preparation of
aldohexos-5-uloses: Synthesis of l-ribo and d-lyxo derivatives. Carbohydr. Res. 2010, 345, 369–376. [CrossRef]
32. Bianchini, R.; Rolla, M.; Isaad, J.; Catelani, G.; Guazzelli, L.; Corsi, M.; Bonanni, M. Efficient double
glycoconjugation to naturalize high molecular weight disperse dyes. Carbohydr. Res. 2012, 356, 104–109.
[CrossRef]

Molecules 2019, 24, 3414
19 of 19
33. Pistarà
, V.; Corsaro, A.; Rescifina, A.; Catelani, G.; D0Andrea, F.; Guazzelli, L. Prevalence of Oxetanose
Forms in the Tautomeric Equilibrium of
9444–9449. [CrossRef]
β-Hydroxy-1,5-dicarbonyl Monosaccharides. J. Org. Chem. 2013, 78,
34. Guazzelli, L.; Catelani, G.; D0Andrea, F.; Gragnani, T.; Griselli, A. Stereoselective Access to the
β
-d-N-Acetylhexosaminyl-(1 4)-1-deoxy-d-nojirimycin Disaccharide Series Avoiding the Glycosylation
→
Reaction. Eur. J. Org. Chem. 2014, 29, 6527–6537. [CrossRef]
35. Gudmundsdottir, A.V.; Nitz, M. Protecting Group Free Glycosidations Using p-Toluenesulfonohydrazide
Donors. Org. Lett. 2008, 10, 3461–3463. [CrossRef]
36. Willems, M.M.; Zom, G.G.; Meeuwenoord, N.; Ossendorp, F.A.; Overkleeft, H.S.; van der
Marel, G.A.; Codée, J.D.C.; Filippov, D.V. Design, automated synthesis and immunological evaluation
of NOD2-ligand–antigen conjugates. Beilstein, J. Org. Chem. 2014, 10, 1445–1453. [CrossRef]
37. Yoshino, T.; Reuter, G.; Kelm, S.; Schauer, R. Facile synthesis of 20-substituted lactoses. Glycoconj. J. 1986, 3,
7–14. [CrossRef]
38. Barili, P.L.; Catelani, G.; D0Andrea, F.; De Rensis, F.; Falcini, P. Improved preparation of
2,3:5,6:30,40-tri-O-isopropylidenelactose dimethyl acetal and its 60-O-(1-methoxy-1-methylethyl) derivative.
Carbohydr. Res. 1997, 298, 75–84. [CrossRef]
39. Wang, J.-W.; Asnani, A.; Auzanneau, F.-I. Synthesis of a BSA-Lex glycoconjugate and recognition of Lex
analogues by the anti-Lex monoclonal antibody SH1: The identification of a non-cross-reactive analogue.
Bioorg. Med. Chem. 2010, 18, 7174–7185. [CrossRef]
40. Wang, A.; Hendel, J.L.; Auzanneau, F.-I. Convergent syntheses of LeX analogues. Beilstein, J. Org. Chem.
2010, 6, 17. [CrossRef]
41. Debost, J.L.; Gelas, J.; Horton, D. Selective preparation of mono-and diacetals of d-mannitol. J. Org. Chem.
1983, 48, 1381–1382. [CrossRef]
42. Cheng, H.; Cao, X.; Xian, M.; Fang, L.; Cai, T.B.; Ji, J.J.; Tunac, J.B.; Sun, D.; Wang, P.G. Synthesis and
Enzyme-Specific Activation of Carbohydrate-Geldanamycin Conjugates with Potent Anticancer Activity.
J. Med. Chem. 2005, 48, 645–652. [CrossRef] [PubMed]
43. Fais, M.; Karamanska, R.; Allman, S.; Fairhurst, S.A.; Innocenti, P.; Fairbanks, A.J.; Donohoe, T.J.; Davis, B.G.;
Russell, D.A.; Field, R.A. Surface plasmon resonance imaging of glycoarrays identifies novel and unnatural
carbohydrate-based ligands for potential ricin sensor development. Chem. Sci. 2011, 2, 1952–1959. [CrossRef]
44. Koeman, F.A.W.; Meissner, J.W.; van Ritten, H.R.P.; Karmeling, J.P.; Vliegenthart, J.F.G. Synthesis of Structural
Elements of the Capsular Polysaccharide of Streptococcus Pneumoniae Type 14. J. Carbohydr. Chem. 1994, 13,
1–25. [CrossRef]
45. Neilson, T.; Werstiuk, E.S. Oligoribonucleotide Synthesis. II. Preparation of 20-O-tetrahydropyranyl
Derivatives of Adenosine and Cytidine Necessary for Insertion in Stepwise Synthesis. Can. J. Chem.
1971, 49, 493–499. [CrossRef]
46. Manea, F.; Bindoli, C.; Fallarini, S.; Lombardi, G.; Polito, L.; Lay, L.; Bonomi, R.; Mancin, F.; Scrimin, P.
Multivalent, Saccharide-Functionalized Gold Nanoparticles as Fully Synthetic Analogs of Type A Neisseria
meningitidis Antigens. Adv. Mat. 2008, 20, 4348–4352. [CrossRef]
47. Costantino, P.; Norelli, F.; Giannozzi, A.; D0Ascenzi, S.; Bartoloni, A.; Kaur, S.; Tang, D.; Seid, R.; Viti, S.;
Paffetti, R.; et al. Size fractionation of bacterial capsular polysaccharides for their use in conjugate vaccines.
Vaccine 1999, 17, 1251–1263. [CrossRef]
48. Perrin, D.D.; Armarengo, W.L.F.; Perrin, D.R. Purification of Laboratory Chemicals, 2nd ed.; Pergamon Press:
Headington Hill Hall, Oxford, UK, 1980.
49. Kochetkov, N.K.; Nifant0ev, N.E.; Backinowsky, L.V. Synthesis of the capsular polysaccharide of Streptococcus
Pneumoniae type 14. Tetrahedron 1987, 43, 3109–3121. [CrossRef]
Sample Availability: Samples of the compounds are not available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).