Regioselective Glycosylation Strategies for the Synthesis of Group Ia and Ib Streptococcus Related Glycans Enable Elucidating Unique Conformations of the Capsular Polysaccharides

Article abstract of DOI:10.1002/chem.201903527

Group B Streptococcus serotypes Ia and Ib capsular polysaccharides are key targets for vaccine development. In spite of their immunospecifity these polysaccharides share high structural similarity. Both are composed of the same monosaccharide residues and

Full text of DOI:10.1002/chem.201903527

A Journal of
Accepted Article
Title: Regioselective strategies for the synthesis of Group Ia and
Ib Streptococcus related glycans enable elucidating unique
conformations of the capsular polysaccharides
Authors: Linda Del Bino, Ilaria Calloni, Davide Oldrini, Maria Michelina
Raso, Rossella Cuffaro, Ana Arda, Jeroen Codée, Jesús
Jiménez-Barbero, and Roberto Adamo
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To be cited as: Chem. Eur. J. 10.1002/chem.201903527
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Regioselective glycosylation strategies for the synthesis of Group
Ia and Ib Streptococcus related glycans enable elucidating unique
conformations of the capsular polysaccharides
Linda Del Bino[a], Ilaria Calloni[b], Davide Oldrini[a], Maria Michelina Raso[a], Rossella Cuffaro[a],
Ana Ardá[b], Jeroen D. C. Codée[c], Jesús Jiménez-Barbero[b], and Roberto Adamo*[a]
Dedication ((optional))
in phase I/II clinical trials[4] with the ultimate goal of
Abstract: Group B Streptococcus serotypes Ia and Ib capsular
developing a maternal vaccination strategy.[1, 5] Multivalent
polysaccharides are key targets for vaccine development. In spite of
formulations with six different serotypes are currently under
their immunospecifity these polysaccharides share high
clinical testing[6].
structural similarity. Both are composed of the same
GBS serotypes Ia, Ib and III account for the majority of GBS
related diseases[7]. CPS Ia and Ib are structurally very
monosaccharide residues and differ only in the connection of the
Neu5Acα2-3Gal side chain to the GlcNAc unit, which is a β1-4
linkage in serotype Ia and a β1-3 linkage in serotype Ib. We
described development of efficient regioselective routes for
GlcNAcβ1-3[Glcβ1-4)]Gal synthons giving access to different
GBS Ia and Ib repeating unit frameshifts. These glycans were
used to probe the conformation and molecular dynamics of the
two polysaccharides, highlighting the different presentation of the
protruding Neu5Acα2-3Gal moieties on the polysaccharide
backbones and a higher flexibility of Ib polymer compared to Ia which
similar. Both composed of the same monosaccharide
residues and differ only in the linkage between the
Neu5Acα2-3Gal side chain and the GlcNAc unit: a β1-4
linkage in type Ia and a β1-3 linkage in type Ib[8]. This
difference is critical in determining the immunospecificity
(Figure 1).[3, 9]
The repeating units of CPS Ia and Ib can be described by the
branched
1
-
3
and linear
2
-
4
frameshifts depicted in Figure 1.
share identical
Intriguingly, the latter pentasaccharides
3-4
monosaccharide composition with milk oligosaccharides,
which have recently been proposed to inhibit GBS
can impact epitope exposure
.
colonization[10]
.
The availability of well-defined GBS CPS glycans is key to
explore interactions with serotype specific monoclonal
antibodies in order to identify relevant glycoepitopes for
elucidating the mechanism of action of the polysaccharide
conjugates and for the development of synthetic
carbohydrate based vaccines.[11] The most studied of GBS
polysaccharides is type III. This CPS is known to form a
helical structure,[12] and this feature has an impact on epitope
exposure.[13] Our group has recently synthesized CPSIII
oligosaccharides that were used along with fragments
obtained from CPS depolymerization to map a sialylated
structural epitope spanning two repeating units.[14] Since
neither chemical nor enzymatic depolymerization reactions
are available for CPS Ia and Ib, chemical synthesis is the
only approach to obtain homogeneous oligosaccharidesfrom
the CPS.
Introduction
Group B Streptococcus (GBS) is a leading cause of
pneumonia, sepsis, meningitis and death in neonates1. It has
also been associated with high rates of invasive diseases in
the elderly.[1] On the basis of variation in polysaccharide
composition,
the
GBS
sialic
acid-rich
capsular
polysaccharides (CPSs) are divided into ten serotypes (Ia,
Ib, and II–IX).[2] GBS CPSs are key virulence factors and
considered the prime vaccine candidate to combat GBS
infections[3]. Monovalent conjugate vaccines prepared with
GBS type-specific polysaccharide representing the most
frequent disease-causing serotypes (Ia, Ib, II, III and V), as
well as a trivalent combination (Ia, Ib, III), have been tested
While synthesis of GBS CPS Ia repeating unit has been
reported[15], the preparation of the pentasaccharide repeating
unit of GBS CPS Ib has not been achieved. When
approaching the synthesis of CPS Ia and Ib fragments, we
envisaged the formation of the disaccharide GlcNAcβ1-3Gal
motif as a key step to enable convergent syntheses of a
variety of structures depicted in Figure 1. Typically,
installation of the GlcNAcβ1-3Gal disaccharide within more
complex glycans has been achieved with the 4-hydroxyl
group of the Gal acceptor either protected[16],[17] or already
engaged in a glycosidic linkage[18]. Particularly, in the
preparation of CPS Ia repeating units[19] a 4,6-O-benzylidene
protected Gal acceptor was used for glycosylation with a
glucosamine trichloroacetimidate donor, and subsequent
regioselective ring opening before further glycosylation of
position 4 for the construction of the trisaccharide GlcNAcβ1-
3[Glcβ1-4)]Gal could take place. There is need of
expeditious procedures for the construction of complex
glycans, and regio- and stereoselective reactions
[a]
Dr. Linda Del Bino, Dr. Davide Oldrini, Dr. Maria Michelina Raso,
Dr. Rossella Cuffaro, Dr. Roberto Adamo
GSK
Via Fiorentina 1
53100 Siena, Italy
E-mail: roberto.x.adamo@gsk.com
Dr. Ilaria Calloni, Dr. Ana Ardá, Prof. Jesus-Jimenez Barbero
Chemical Glycobiology Laboratory
CIC bioGUNE
Bizkaia Technology Park, Building 800, 48160 Derio, Spain
Institution
Prof. Jeroen D. C. Codée.
Department of Bioorganic Synthesis
Leiden University
[b]
[c]
2333 Leiden, The Netherlands
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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Figure 1. (A) Chemical structure of GBS CPS Ia and Ib. (B) Chemical structure of the target fragments from GBS CPS Ia (1-2) and CPS Ib (3-4). Synthesis of an unsialylated form of CPS
Ia repeating units 5 was also envisaged.
distinguishing among diverse deprotected hydroxyls are highly Neu5Acα2-3Gal donor. This approach envisages the challenging
desirable to simplify the oligosaccharide assembly.[20] We stereoselective α-sialylation of the upstream galactose at an early
reasoned that regioselective glycosylation of Gal 3-OH would stage of the synthesis.[22] Alternative use of a Gal donor would
be key for accelerating the synthesis of the GlcNAcβ1-3Gal enable synthesis of 5. In this design faster and efficient access to a
disaccharide and rendering the 4-OH available for further GlcNAcβ1-3Gal disaccharide building block plays a central role to
glycosylation
without
the
need
of
tedious obtain the trisaccharide acceptor without a temporary protection at
position 4 for further assembly of GBS CPS Ia fragments. To
protection/deprotection sequences.[21]
Herein we report tactics to achieve regioselective syntheses achieve its regioselective synthesis, we investigated the effect of
of protected GlcNAcβ1-3Gal building blocks and the use of arming benzyl and disarming benzoyl groups[23], [24] at position 2 and
these key synthons in convergent routes towards a series of 6 of the Gal acceptors in tuning the reactivity of the 3- and 4-OH,
fragments from CPS Ia and Ib repeating units with a built-in respectively, in combination with various protecting and leaving
aminopropyl linker amenable for future conjugation to carrier groups in the glucosamine donors. Despite the expected higher
proteins (Figure 1). Furthermore, combination of NMR data reactivity of the equatorial 3-OH versus the axial 4-OH,
from the synthetic GBS CPS Ia and Ib repeating units in their regioselective glycosylation of the position 3 has been shown not
branched form
1 and 3
, respectively, and molecular dynamics to be trivial[16]. Accordingly, we synthesized a series of glucosamine
simulation allowed to shed light on how the variation of a single thioglycoside and trichloroacetimidate donors with the amine
sugar connection dramatically affects the conformational protected by the participating phthalimido (Phth) or trichloroethyl
properties of CPS Ia and Ib polysaccharides, and hence carbamate (Troc) group (experimental procedures are provided as
exposition of potential epitopes for antibody recognition.
SI).
Levulinoyl (Lev) and Fluorenylmethyloxycarbonyl (Fmoc) were
selected for temporary protection of either position 3 or 4.
Alternatively, a 4,6-O-benzylidene was used to lock the 4 and 6
hydroxyls to be subjected to regioselective ring opening delivering
the 4-OH at a later stage of the synthesis (Scheme 1). The
prepared donors and acceptors were then coupled under several
Results and Discussion
Optimization of regioselective glycosylation of galactose.
According to our retrosynthetic design (Figure 1), the target glycans
1-4 can be obtained through a [2+3] convergent strategy based on
the glycosylation of a suitable trisaccharide acceptor with a
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Scheme 1. A) Preparation of disaccharide intermediates 12-15 for the synthesis of GBS CPS Ia repeating unit. Promoters and conditions are described in Table 1. B)
Preparation of disaccharide intermediates 21-25 for the synthesis of GBS PSIb repeating unit. Promoters and conditions are described in Table 2.
Table 1. Reaction of glucosamine donors 6-9 with Gal acceptors 10-11
acceptor 11 with both donor 6 or 7 under NIS/AgOTf mediated
activation (Entry 7-8, Table 1), which gave 14a and 15a in yield of
53 and 65%, respectively, or the imidate 9 and acceptor 11 (Entry
9, Table 1) which enabled the attainment of 14a in 77% yield.
Similarly, conditions for the preparation of a GlcNAcβ1-3Gal
synthon with a temporary group at its C3’-OH, to allow the ensuing
assembly of GBS CPSIb fragments, were explored (Table 2 and
Scheme 1). The glycosylation of di-O-benzyl acceptor 10 with
donor 16 using NIS with either TfOH or AgOTf as co-promoters
gave variable mixtures of the β1-3 21a and β1-4 21b disaccharides
(Entry 1-2, Table 2). Again, the di-O-benzoyl acceptor 11 in
presence of NIS/AgOTf activation at -30°C allowed achieving a
yield of 68% (Entry 4, Table 2), confirming the improved capacity of
the benzoyl substituents to govern the regioselectivity of the
Entry
Donor
Acceptor
Promoter,
Yield
temperature
NIS/TfOH, - 30 °C
NIS/Ag(OTf), 30°C
1
2
6
6
10
10
nda
12a (43%), 12b
(26%)
3
7
10
NIS/Ag(OTf), - 30°C
13a (40%), 13bb
(28%)
4
5
6
7
8
9
10
8
9
6
6
7
8
9
10
10
11
11
11
11
11
TMSOTf, - 10°C
TMSOTf, -10°C
NIS/TfOH, - 30°C
NIS/Ag(OTf), - 30°C
NIS/Ag(OTf), - 30°C
TMSOTf, -10°C
12a (31%)
13a (45%)
nda
14a (53%)
15a (65%)
14a (77%)
15a (33%)
TMSOTf, -10°C
[a]. DCM was the solvent in all tested conditions. b. nd = not determined, product
could not be detected; [b]. The formation of the β1-4 linkage was confirmed by reaction compared to benzyl substituents. These conditions were
acetylation of 13b. In the 1H NMR spectrum a shift from 3.32 to 4.69 ppm of the
H-3 signal of Gal, appearing as a doublet of doublets with J2,3 = 10.3 Hz and J3,4
yield (Entry 6, Table 2). When the trichloroacetimidate 18 was used,
= 2.5 Hz was observed, confirming occurrence of glycosylation at position 4.
also efficient for the GlcNTroc donor 17 which gave 23a in 65%
the yield was increased up to 70% (Entry 7, Table 2), corroborating
the potential of this type of donor for the regioselective control of
glycosylation conditions (Table 1 and Scheme 1) to optimize the
the reaction. Finally, trifluoroacetimidate glucosamine 20 bearing a
synthesis of the GlcNAcβ1-3Gal building block. The most efficient
4,6-O-silylidene protection in presence of TMSOTf as promoter
routes proved to be the combination of the 2,6- di-O-benzoyl
afforded the target disaccharide 25a in 62% yield.[25] The slighty
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higher flexibility or lower hindering effect of the silylidene compared reacted with disaccharide donors 12a and 14a. to furnish
to benzylidene group favored the reaction. Overall, these results trisaccharides 27a and 27b in 75% and 68% yield, respectively,
indicated that the regioselectivity of the glycosylation benefits from
the decreased nucleophilicity of the axial 4-hydroxyl, which is
intrinsically lower reactive than the 3-hydroxyl group, induced by
the electron withdrawing effect of the 2,6-O-benzoyl as compared
to 2,6-di-O-benzyl substituents in the Gal acceptor.
with the newly formed glycosidic bond was in -configuration, as
expected by the presence of a participating group.
Despite the deactivating effect of the 6-O-benzoyl ester compared
to the 6-O-benzyl ether, the reaction proceeded with almost
identical efficiency (Scheme 3), whereas
a peracetylated
In addition, mild activation conditions (NIS/AgOTf) for the
thioglycoside donor or the torsional disarming effect of the
benzylidene/silylidene group for the imidate donors appears to
favor the regioselectivity of glycosylation at position 3.
trichloroacetimidate glucose donor with TMSOTf activation was
ineffective for glycosylation of the 4-OH. Considering the higher
regioselectivity and yield achieved in making disaccharide 14a, the
resulting trisaccharide 27b was advanced in the GBS CPS Ia
repeating unit construction and subjected to regioselective opening
of the 4,6-O-benzylidene acetal with BF3·Et2O and Me3N·BH3 to
provide the acceptor 28 (70%).
Table 2. Reaction of glucosamine donors 16-20 with Gal acceptors 10-11
Entry
Donor
Acceptor
Promoter,
Yield
In order to complete the pentasaccharide construction, the sialo-
temperature
NIS/TfOH, - 30°C
27]
galactosyl trifluoroacetimidate donor 29[14a,
and thioglycoside
1
2
16
16
10
10
21a (30%), 21b
(<5%)
21a (38%), 21b
(26%)
22a (40%)
22a (68%)
nda
23a (65%)
23a (70%)
24a (50%)
30[28] were tested. Of these two disaccharides, 30 can be prepared
with a higher -stereoselectivity, whereas 29 is easily accessible
from a commercial disaccharide precursor.[14a]
NIS/AgOTf, - 30°C
3
4
5
6
7
8
16
16
17
17
18
19
11
11
11
11
11
11
NIS/TfOH, - 30°C
NIS/AgOTf, - 30°C
NIS/TfOH, - 30°C
NIS/AgOTf, - 30°C
TMSOTf, - 10°C
TMSOTf, - 10°C
Glycosylation of trisaccharide 28 with 29 under TMSOTf activation
gave the protected pentasaccharide 31 in 75% yield, while the use
of disaccharide 30 in presence of NIS/TfOH led to the protected
pentasaccharide 32 in a similar yield (73%). Compound 30 was
deprotected by a four-step procedure[18], including (i) saponification
of the methyl ester of Neu5Ac with lithium iodide in pyridine; (ii)
reaction with ethylenediamine in refluxing ethanol for concomitant
removal of the O-acetates and the NPhth protecting group; (iii)
reacetylation with acetic anhydride/pyridine to install the acetamide
group of the GlcNAc residue along with acetyl esters; (iv)
methanolysis and final catalytic hydrogenation over Pd/charcoal to
provide the target branched pentasaccharide 1.
9
20
11
TMSOTf, - 10°C
25a (62%)
[a]. DCM was the solvent in all tested conditions. [b]. nd = not determined, product
could not be detected.
Synthesis of GBS CPS Ia linear and branched repeating units.
Having identified the two glycosylation partners giving the
GlcNAcβ1-3Gal motif in a regioselective fashion, we elongated the
disaccharide building block to assemble the pentasaccharide
repeating unit of GBS CPS Ia. To this end, glucose donor 26[26] was
Scheme 3. Assembly of GBS CPS Ia repeating unit. Reagent and conditions: a) TMSOTf, DCM dry, -10°C, (-) 75% from 12a; 68% (-) from 14a; b) Me3N·BH3,
BF3·Et2O, ACN, 0°C, 70% ; c) TMSOTf, DCM dry, 0°C, (-) 75%; d) TfOH, NIS, DCM dry, -40°C, (-) 73%; e) LiI, Py, 120°C; H2NCH2CH2NH2, EtOH, 90°C; Ac2O, Py;
MeONa, MeOH; H2, Pd-C, 40% (over five steps) ; f) 3M NaOH, THF, reflux; Ac2O, MeOH, H2, Pd-C, 45% (over three steps).
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Pentasaccharide 32 was first subjected to saponification with (Scheme 4). NMR data of the synthesized fragments were in
NaOH in refluxing THF, followed by amine reacetylation with a 2:3 excellent agreement when compared to CPS Ia samples.[8]
acetic anhydride/methanol mixture.
From acceptor 35 a desialylated CPS Ia linear fragment for future
Hydrogenation over Pd/charcoal afforded the target branched mapping studies was also obtained by glycosylation (72% yield)
pentasaccharide 1 equipped with the aminopropyl linker suitable for with the trifluoroacetimidate 38, prepared from the known 1-OH
conjugation. After purification by size exclusion chromatography, compound 37[18]. After global deprotection tetrasaccharide 5 was
the final compound was obtained in 40% overall yield from 31 and obtained in 42% yield (Scheme 4).
45% from overall yield 32, respectively (Scheme 3).
Next, we extended the same regioselective approach to the
synthesis of the linear frameshift 2 of the serotype Ia repeating unit
(Scheme 4). In this case, the benzoylated lactose 33 and the
glucosamine donor 8 were chosen as glycosylation partners
affording the linear trisaccharide acceptor 34 in 68% yield with
complete regioselectivity. Following benzylidene opening, the
trisaccharide acceptor 35 was glycosylated with the two donors 29
and 30. The first glycosylation promoted by TMSOTf at 0°C
Synthesis of GBS CPS Ib linear and branched repeating unit.
Differently than the GBS CPS Ia pentasaccharides, the two Ib
frameshifts 3-4 required a glucosamine building block bearing a
temporary protecting group at its C3-OH and the creation of the
Galβ1-3GlcNAc linkage, which had a strong impact on our synthetic
design. Initial attempts to prepare the branched pentasaccharide 3
using a NPhth protected trisaccharide acceptor, similarly as done
for the CPS Ia branched unit, were unsuccessful (SI, Scheme S9).
afforded the target linear pentasaccharide 36 in 65% yield, with - The C3-OH of the glucosamine appeared significantly less reactive
stereo- and regioselectivity at C-4 of GlcNAc over the C-4 of Gal. than the C4-OH, likely due to the presence of the bulky NPhth
The presence of the free galactose 4-OH throughout all stages of group that could hinder the glycosylation reaction at the C3-OH. We
the synthesis, from trisaccharide 34 to pentasaccharide 36, was anticipated that its replacement with a Troc protection would result
monitored by following the signal of the Gal H-4, which appeared at in a higher nucleophilicity of the vicinal hydroxyl. Disaccharides 23a
3.97 ppm (d, J = 2.7 Hz) in the 1H NMR and HSQC of all synthetic and 25a, which differ only in the cyclic protecting group blocking the
intermediates. This confirmed the regioselectivity of the two glucosamine C4,6-OH’s, were selected to be elongated to the
glycosylations performed. Unexpectedly, reaction of 35 with the branched pentasaccharide 3 (Scheme 5). Glycosylation of the two
tolyl thioglycoside 30 under NIS/TfOH activation at -40°C yielded acceptors with the armed Glc donor 26 under TMSOTf activation at
only traces of the corresponding pentasaccharide, while mainly 0°C afforded the trisaccharides 40 and 41 in 63% and 70% yield,
decomposition of the glycosyl donor was observed, as revealed by
LC-MS analysis. The linear pentasaccharide 36 was subjected to
the five-step deprotection protocol previously described for
compound 31. The target oligosaccharide 2 was purified by size
exclusion chromatography and obtained in 33% overall yield
respectively, as -anomers. After Fmoc removal by treatment with
10% piperidine in DCM (92%), glycosylation with the
sialogalactoside donor 29 of the two
Scheme 4. Assembly of linear GBS PS Ia fragments 2. Reagent and conditions: a) TMSOTf, DCM dry, -10°C, (-) 68%; b) Me3N·BH3, BF3·Et2O, ACN, 0°C, 65%; c)
TMSOTf, DCM dry, 0°C, (-) 65%; d) LiI, Py, 120°C; H2NCH2CH2NH2, EtOH, 90°C; Ac2O, Py; MeONa, MeOH; H2, Pd-C, 33% (over five steps); e) TFACl, Cs2CO3,
DCM, 61%; f) TMSOTf, -20°C, DCM, (-) 72%; g) PdCl2, MeOH; H2NCH2CH2NH2, EtOH, 90°C; Ac2O, Py; MeONa, MeOH; H2, Pd-C, 42% (over five steps).
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Scheme 5. Assembly of GBS PSIb pentasaccharide branched unit 3. Reagent and conditions: a) TMSOTf, DCM dry, 0°C; (-) 63% for 41, (-) 70% or 42; b) Piperidine,
DCM dry, 92%; c) TMSOTf, DCM dry, 0°C, (-) 80%; d) TfOH, NIS, DCM dry, -40°C, (-) 65%; e) HF/Pyridine, 0°C; 3 M NaOH, THF, reflux; Ac2O/MeOH; H2/Pd-C,
40%.
Scheme 6. Synthetic route to type Ib linear repeating unit. Reagent and conditions: a) TMSOTf, DCM dry, 0°C, (-) 55%; b) Piperidine, DCM, 90%; c) TMSOTf, DCM
dry, 0°C, (-) 66%; d) TfOH, NIS, DCM dry, -40°C, (-) 40%; d) HF/pyridine; 3 M NaOH, THF, reflux; Ac2O/MeOH; H2/Pd-C, 40%.
acceptors 42 and 43 was tested.
leading to complete recovery of the unreacted acceptor. In contrast,
Reaction of the 4,6-O-benzylidene trisaccharide 42 and 29 with reaction of acceptor 43, bearing the more flexible 4,6-O-silylidene
TMSOTf as promoter failed to afford the target pentasaccharide, ketal, with 29 in the presence of TMSOTf gave the target
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pentasaccharide 44 in 80% yield (Scheme 5). This result suggests
that the glycosylation of 42 was prevented by the steric and
torsional constrain of the 4,6-O-benzylidene ring. Trisaccharide 43
was also efficiently -glycosylated with disaccharide donor 30 by
NIS/TfOH activation, affording 45 in 65% yield (Scheme 5). Despite
a slightly lower yield in this step, the overall efficiency of the
synthesis of GBS serotype Ib branched repeating unit was superior
using the thioglycoside 30 with respect to the imidate 29 due to the
better α-stereoselectivity of the glycosylation leading to 30.[27-28]
Pentasaccharides 44 and 45 were then deprotected by a four-step
protocol (Scheme 5): (i) desilylation by treatment with HF.pyridine,
(ii) saponification with NaOH in refluxing THF, for concomitant
hydrolysis of the acyl esters, the Troc group and the 5-N,4-O-
oxazolidinone protecting group and Neu5Ac methyl ester; (iii)
reacetylation of the amines by a 2:3 acetic anhydride/methanol
mixture; (iv) hydrogenation over Pd/charcoal. The target branched
pentasaccharide 3 was obtained in 40% yield.
Figure 2. Superimposition of the major conformations for pentasaccharides 1
(lime) and 3 (grey), with the exo-anti- geometry around the Neu5Ac2-3Gal
linkage.
Finally, we extended our regioselective approach to the synthesis
of the linear frameshift 4 of the GBS serotype Ib repeating unit
(Scheme 6). For this purpose, benzoylated lactose acceptor 33 was
glycosylated with the 4,6-O-silylidene glucosamine imidate 20
under TMSOTf activation to give the target trisaccharide 46 with full
For the Gal1-4GlcNAc linkage, there is a perfect agreement for
the H1Gal-H4GlcNAc distance with slight discrepancies for the
H1Gal-H6,H6’GlcNAc ones, probably due to the MD bias around
β1-3 stereo- and regioselectivity (55%). Following Fmoc the GlcNAc  torsion angle (SI, Figure S2). These data support the
deprotection with piperidine in DCM, the obtained acceptor 47 was
-glycosylated with imidate 29 to attain the linear protected conformation, as predicted by the MD simulation.
existence of a single population around the exo-syn-/syn
pentasaccharide 48 (66%). Reaction with thioglycoside 30 in TfOH
and NIS reaction conditions also provided the analogous
pentasaccharide 49 (40%). The obtained pentasaccharides were
deprotected and purified as described above. NMR data of the
synthesized CPS Ib fragments were in excellent agreement with
NMR data from samples of the bacterial polysaccharide.[8a]
For the Neu5Ac2-3Gal linkage, MD simulations predict three
different populations, 180⁰/-30⁰, -60⁰/-20⁰ and -60⁰/-50⁰. The
interglycosidic interproton distances for each population are
gathered in Table 3. There is a remarkable difference for the
H3Gal-H3axNeuNAc distance, being shorter according to NMR,
and indicating that the MD simulation has a bias for the
conformational ensemble towards exo-syn- populations. Indeed,
according the NOE-derived distance, the exo-anti- population
should be the major one, representing around 75% of the total
ensemble. Two representative conformations for the CPS Ia
pentasaccharide 1 are shown in the SI, differing in the Neu5Ac2-
3Gal linkage (SI, Figure S3). The analysis for the CPS Ib
pentasaccharide 3 yielded similar results (SI, Table S3), although
the GlcNAc1-3Gal linkage could not be fully characterised due to
the overlapping between the H1GlcNAc and H3Gal protons. The
linkage between Gal and GlcNAc, now 1-3 instead of 1-4,
populates a minimum around the exo-syn-/syn(-)- conformation
(SI, Figure S4). Two representative conformations for the CPS Ib
pentasaccharide 3 are shown in the SI, which also differ in the
orientation around the Neu5Ac2-3Gal  torsion (SI, Figure S5). A
superimposition of representative 3D structures for the CPS Ia and
Ib pentasaccharides 1 and 3, with the major conformation exo-anti-
 around the Neu5Ac2-3Gal linkage is shown in Figure 2. The
conformational behavior of the polysaccharides was then analyzed
following a similar protocol. A model for the polysaccharide was
built with 10 repeating units (50 monosaccharides) and MD
simulations were run for 2.5 s.
Conformational analysis. The conformational properties of the
CPS Ia and Ib branched repeating unit pentasaccharides 1 and
3 were studied by a combination of NMR and modelling tools,[29]
and compared to those of the corresponding polysaccharides.
Interglycosidic interproton distances for 1 and 3 were estimated
from ROESY spectra. The obtained experimental distances were
compared with those derived from a 200 ns MD simulation. Table
3 gathers the results for the CPS Ia pentasaccharide 1. The
comparison reflects a good agreement between the NMR- and the
MD-derived distances for the glycosidic linkages GlcNAc1-3Gal
and Glc1-4Gal (defined by the interproton distances H1GlcNAc-
H3Gal and H1Glc-H4Gal, respectively). The / population
analysis from the MD simulation showed a single population for 
fulfilling the exo-anomeric effect (exo-syn-)[30-31]
populations around  for both linkages (SI, Figure S1).
, and two
Table 3. Interglycosidic interproton (Å) distances for the CPS Ia pentasaccharide
(1)
NMR
MD
180/-30
Total
average
4.2
-60/20
4.4
-60/-50
4.6
The analysis of the glycosidic linkages was carried out for the 49
glycosidic bonds, revealing that the behavior for every glycosidic
bond type is reproducible along the polysaccharide (Figure 3).
These populations are comparable to those of the corresponding
pentasaccharide for every glycosidic linkage, and thus, the
resulting interglycosidic interproton distances are very similar (SI,
table). Remarkably, for both GBS serotype Ia and Ib, the HSQC
spectra of the polysaccharide and the pentasaccharide were very
similar, with the obvious exception for the Glc1-4 linked moiety
(E), which is not glycosylated at O4 in the pentasaccharides (Figure
4). The analysis of the interglycosidic NOE (from NOESY spectra
H3Gal-
none
2.7
3.4
2.1
4.8
H3eqNeuNAc
H3Gal-
3.6
3.9
4.3
4.0
H3axNeuNAc
H3Gal-
Very
weak
2.4
2.7
3.1
3.7
3.4
H8NeuNAc
H1Gal-H4GlcNAc
H1Gal-H6GlcNAc
H1Gal-
2.4
3.0
3.9
H6´GlcNAc
H1Glc-H4Gal
H1GlcNAc-H3Gal
2.6
2.5
2.5
2.4
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major exo-syn- form (ca. 55%). The model structures for the
polysaccharides with all Neu5Ac2-3Gal linkages in exo-anti- (Ia)
and exo-syn- (Ib) are displayed in Figure 6, showing different
preferential shapes for the two polysaccharides. The Neu5Ac2-
3Gal branches of GBS PSIII have been shown to be strongly
engaged in antibody recognition.[14b] The favored presentation of
the different epitopes for the major conformation is rather different.
However, given their intrinsic flexibility, especially for Ib, both
molecules could be accommodated to interact with the monoclonal
binding pockets without a major entropy penalty.[32]
Figure 3. Glycosidic linkage analysis for GBS Ia (A) and Ib (B) polysaccharides:
/ plots for representative glycosidic bonds of a 10 repeating unit model along
the 2.5 μs MD simulation.
Conclusions
at 20 ms mixing time) was consistent with the MD derived
populations. The only discrepancies arose again for the Neu5Ac2-
3Gal linkages. Interestingly, for the Ia polysaccharide the NOE-
derived distance for H3axNeu5Ac-H3Gal is 2.4Å, shorter than that
in the pentasaccharide. At the same time, there is a clear NOE
To have fast access to homogeneous oligosaccharide antigens
from GBS serotypes Ia and Ib and gain insights on the
conformational difference among these structurally similar
polymers, we developed a highly convergent synthetic strategy
based on the regioselective glycosylation of a galactose C3,4-diol
to obtain GlcNAcβ1-3Gal disaccharide building blocks.
Investigation of the different reactivities of the C3- and C4-
hydroxyls allowed us to reduce the number of protective groups
manipulations and synthetic steps to the final fragments, therefore
simplifying the overall synthetic design.
between
H3eqNeu5Ac-H3Gal,
not
observed
for
the
pentasaccharide. On the contrary, for the Ib polysaccharide the
distance H3axNeu5Ac-H3Gal is longer, 3.3 Å, while the NOE
between H3eqNeu5Ac-H3Gal does not exist (Figure 5 A and B). At
the same time, the distance H8Neu5Ac-H3Gal is slightly shorter for
the Ib than for the Ia polysaccharide (Figure 5 C and D). These data
suggest that for the Ia polysaccharide, the major conformation
around the Neu5Ac2-3Gal fragment is the exo-anti- (ca. 85%),
while for the Ib polysaccharide, there is a larger flexibility, with a
Figure 4. 1H-13C-HSQC spectrum recorded for the pentasaccharide repeating unit of GBS Ia at 600 MHz, 298 K, D2O (A and B) ad for GBS CPS Ib at 800 MHz, 318
K, D2O, showing the assignment of the 1H and 13C NMR signals. As expected, the matching is excellent except for some signals of residue E.
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Figure 5. The key regions of the NOESY spectra recorded for the GBS CPS Ia (A and C) and GBS CPS Ib (B and D) polysaccharides showing the essential inter
residue cross peaks.
Particularly, the use of a 2,6-O-benzoyl galactose diol resulted
in improved regioselectively as compared to the 2,6-di-O-benzyl
counterpart. In addition, mild activation conditions (NIS/AgOTf)
for the glucosamine thiol donors or the torsional disarming
effect of the benzylidene group for the trichloroacetimidate
donors appear to favor the glycosylation reaction. The
Experimental Section
Experimental procedures for the synthesis of oligosaccharides
1-5 and their characterization, Molecular Dynamics simulations
and NMR spectra are provided as Supplemental Information.
regioselective glucosamine incorporation was successfully
applied to the synthesis of GBS CPS Ia and Ib branched
Conflicts of interest
L.D.B. D.O., M.M.R., R.C. and R.A. are employees of GSK
groups companies. L.D.B. and R.A. are inventors of a patent
related to the topic. R.A. is owner of GSK stocks.
repeating units (1-2). Their linear frameshifts (3-4) and a non-
sialylated CPS Ia form (5) were also synthesized achieving an
additional regioselective glycosylation of the Gal C3-OH over
the C4-OH residue.
These results support the general applicability of the method to
a variety of medically relevant glycans. Importantly, the
structures synthesized through regioselective glycosylation
appear extendible at the 4-OH position of the Gal residue, thus
potentially enabling synthesis of longer and more complex GBS
oligosaccharide structures.
Acknowledgements
We thank the Horizon 2020 research and innovation
programme of the European Union for support through the
Marie Skłodowska-Curie grant agreement Glycovax No 675671.
Conformation analysis studies of the prepared oligosaccharides
by NMR and MD simulations showed the impact of the
GlcNAcβ1-3Gal vs GlcNAcβ1-4Gal connectivity in the
orientation of the Neu5Ac2-3Gal branching. The model,
established from the single synthetic pentasaccharide repeating
units, was used to study the conformational behavior of the GBS
Ia and Ib polysaccharides, showing different preferential shape
for each polysaccharide with the Neu5Ac2-3Gal linkages in
exo-anti- for Ia and exo-syn- for Ib. These unique structural
features are expected to influence antibody recognition and
immunospecificity. Studies are ongoing to map the relevant
glycoepitopes. Moreover, all glycans were designed with a
chemical handle for conjugation to carrier proteins for
immunological evaluation. Results on structural and
immunogenic studies will be reported in due course.
Author Contributions
Conceptualization, R.A., J.J.B., J.D.C.C., L.D.B., A.A., I.C.;
Methodology, L.D.B., I.C., D.O., M.M.R. R.C.; Writing-Original
Draft Preparation, R.A., J.J.B., J.D.C.C., L.D.B, A.A., I.C.;
Writing-Review & Editing, all authors.
Keywords: Carbohydrates • Regioselectivity • Conformation •
Therapeutics
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Linda Del Bino, Ilaria Calloni, Davide
Oldrini, Maria Michelina Raso, Rossella
Cuffaro, Ana Ardá, Jeroen D. C. Codée,
Jesús Jiménez-Barbero, and Roberto
Adamo*
Regioselectively made Group B Streptococcus complex glycans: A panel of
GBS serotype Ia and Ib fragments was prepared via combined regioselective-
stereoselective approaches and used to unravel polysaccharide conformations
responsible for immune specificity.
Page No. – Page No.
Title
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