Synthesis of glycoimmunogen Tn-Thr-PS A1 via hydrazone bond and stability optimization of PS A1 monosaccharide mimics under vaccine development conditions

Article abstract of DOI:10.1080/07328303.2019.1709975

Previously, our group constructed several immunogens utilizing oxime linkage to conjugate a T-cell stimulatory zwitterionic polysaccharide PS A1 and tumor associated carbohydrate antigens (TACAs) in acetate buffer. Here, a semi-synthetic immunogen was synthesized using hydrazone conjugation between PS A1 and a glycopeptide hydrazide (α-D-GalNAc-L-Thr-NH-NH₂) with an excellent loading in PBS buffer. To get robust immune response, the retention of zwitterionic character of PS A1 under vaccine construction conditions is essential. In this regard, the stability of embedded pyruvate acetal moiety in tetrasaccharide repeating unit of PS A1 can validate the retention of the dual charges. Therefore, rather than utilizing this highly immunogenic PS A1 fully, stability studies were performed with synthetic 1-thiophenyl-4,6-O-pyruvate acetal-D-galactopyranose in varying acetate buffer pHs and time intervals. Furthermore, 1-propyl-D-galactofuranose was synthesized to mimick the D-Galf of PS A1 to examine regioselective hydrazone and oxime formation with α-D-GalNAc-L-Thr-NH-NH₂ and α-D-GalNAc-ONH₂ moieties respectively.

Full text of DOI:10.1080/07328303.2019.1709975

Journal of Carbohydrate Chemistry
ISSN: 0732-8303 (Print) 1532-2327 (Online) Journal homepage: https://www.tandfonline.com/loi/lcar20
Synthesis of glycoimmunogen Tn-Thr-PS A1 via
hydrazone bond and stability optimization of
PS A1 monosaccharide mimics under vaccine
development conditions
F. Hossain, S. Nishat, S. Ghosh, S. Boga, G. T. Hymel & P. R. Andreana
To cite this article: F. Hossain, S. Nishat, S. Ghosh, S. Boga, G. T. Hymel & P. R. Andreana
(2020): Synthesis of glycoimmunogen Tn-Thr-PS A1 via hydrazone bond and stability optimization
of PS A1 monosaccharide mimics under vaccine development conditions, Journal of Carbohydrate
Chemistry, DOI: 10.1080/07328303.2019.1709975
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JOURNAL OF CARBOHYDRATE CHEMISTRY
https://doi.org/10.1080/07328303.2019.1709975
Synthesis of glycoimmunogen Tn-Thr-PS A1 via
hydrazone bond and stability optimization of PS A1
monosaccharide mimics under vaccine
development conditions
F. Hossain, S. Nishat, S. Ghosh, S. Boga, G. T. Hymel, and P. R. Andreana
Department of Chemistry and Biochemistry, University of Toledo, Toledo, OH, USA
ABSTRACT
ARTICLE HISTORY
Received 22 December 2019
Accepted 25 December 2019
Previously, our group constructed several immunogens utiliz-
ing oxime linkage to conjugate a T-cell stimulatory zwitter-
ionic polysaccharide PS A1 and tumor associated carbohydrate
antigens (TACAs) in acetate buffer. Here, a semi-synthetic
immunogen was synthesized using hydrazone conjugation
between PS A1 and a glycopeptide hydrazide (a-D-GalNAc-L-
Thr-NH-NH2) with an excellent loading in PBS buffer. To get
robust immune response, the retention of zwitterionic charac-
ter of PS A1 under vaccine construction conditions is essential.
In this regard, the stability of embedded pyruvate acetal moi-
ety in tetrasaccharide repeating unit of PS A1 can validate the
retention of the dual charges. Therefore, rather than utilizing
this highly immunogenic PS A1 fully, stability studies were
performed with synthetic 1-thiophenyl-4,6-O-pyruvate acetal-
D-galactopyranose in varying acetate buffer pHs and time
intervals. Furthermore, 1-propyl-D-galactofuranose was synthe-
sized to mimick the D-Galf of PS A1 to examine regioselective
hydrazone and oxime formation with a-D-GalNAc-L-Thr-NH-
NH2 and a-D-GalNAc-ONH2 moieties respectively.
KEYWORDS
Carbohydrate-based immu-
nogen; hydrazone
conjugation; zwitterionic
polysaccharide; stability
study; regioselective
conjugation
GRAPHICAL ABSTRACT
CONTACT P. R. Andreana
peter.andreana@utoledo.edu
Department of Chemistry and Biochemistry,
University of Toledo, 2801 W. Bancroft Street, Toledo, OH 43606, USA.
Supplemental data for this article can be accessed on the publisher’s website.
ß 2020 Taylor & Francis Group, LLC

2
F. HOSSAIN ET AL.
Introduction
Tumor associated carbohydrate antigens (TACAs),[1,2] as well as carbohy-
drate antigens of pathogens (bacterium, virus and fungus),[3] are significant
bio-markers for the development of therapeutic and preventive vaccines
because they are prominent on cell surfaces.[4] However, these B cell
dependent, poorly immunogenic carbohydrate antigens are not able to
induce cellular T (cluster of differentiation, CD 4þ and/or CD8þ) cell
dependent immunity on their own, critical for the presentation on major
histocompatibility class (MHC) molecules of antigen presenting cells.[5]
Over the past few years, a number of strategies have been devised to over-
come the T-cell independent barrier for development of semi[6] or fully
synthetic, multi-component carbohydrate-based cancer vaccines.[7] For
example, conjugating TACAs (Tn, TF, STn, GM2, GD2, GD3, fucosyl-
GM1, Globo-H and Lewisy) with i) protein carriers such as keyhole limpet
hemocyanin (KLH), tetanus toxoid (TT), bovine serum albumin (BSA),
diphtheria toxin (CRM197),[4] ii) polysaccharides like zwitterionic polysac-
charides (ZPSs), Hemophilus influenzae type b (Hib), Streptococcus pneumo-
niae (Spn),[5,8] iii) Toll like receptor 2 (TLR2) ligand, Pam3CysSerK4,[9] iv)
immunological epitopes (T-epitope)[10] have been achieved. During opti-
mization of vaccines, major challenges include antigen stability, high anti-
gen loading levels, and construct/immunogen shelf-life stability. Herein, we
have carried out stability tests by studying simple hydrolytic effects with
synthesized monosaccharide units of the valuable immunogenic PS A1
polysaccharide T-cell stimulant.
In order to compensate for the T-cell independent nature of carbohydrate
antigens, our group has been pursuing zwitterionic polysaccharide (ZPS), PS
A1, as a carrier. This ZPS was isolated as a capsular polysaccharide from the
commensal anaerobe Bacteroides fragilis (ATCC 25285/NCTC 9343) and con-
sists of a tetrasaccharide core repeating unit (ꢀ120 repeat units, overall MW
ꢀ110 kD).[11–13] PS A1 was discovered to invoke MHC II mediated T-cell
dependent immune responses.[14] The monosaccharide units of PS A1 are 4,6-
O-pyruvate acetal D-galactopyranose, 2-acetamido-4-amino-2,4,6-trideoxy-D-
galactose, 2-acetamido-D-galactopyranose and D-galactofuranose.[15]
Among a known number of TACAs, the Thomsen-nouveau (Tn) antigen
(GalNAc-a-1-O-Ser/Thr) is the simplest and most widely studied.[16]
Previously, our group prepared a Tn-PS A1 immunogen by conjugating a
aminooxy a-D-GalNAc to an oxidized PS A1 via an oxime bond.[17] An
immunogenicity profile of the vaccine was examined using C57BL/6 mice
rendering a robust immune response against the Tn antigen.[17]

JOURNAL OF CARBOHYDRATE CHEMISTRY
3
Although, oxime/hydrazone bond formation is reversible, the linkages
are comparatively stable and faster than their corresponding imines due to
the a-effect.[18,19] Because of the relative high stability, oxime conjugation
strategies have been utilized in many biorthogonal conjugation reactions.
Oximes, with greater inherent stability than hydrazones, are typically uti-
lized for more robust linkage strategies, whereas acid labile hydrazones,
which succumb to hydrolysis under more acidic conditions, are preferen-
tially utilized for the release of biologically active molecules such as in anti-
body-drug conjugates.[20] In both cases, however, the commonly observed
resistance to hydrolysis can be explained by the inductive effect of the
more electronegative oxygen (vN ¼ 3.5) compared to the nitrogen atom
(vN ¼ 3.0) (Fig. 1).[21]
Figure 1. Hydrolysis of oxime/hydrazone/amine bonds.
Figure 2. (a) Previously synthesized Tn-hydrazides,[22] (b) the structure of[23] glycan-bishydrazide
conjugate,[24] (c) biotin reactive derivative,[25] (d) antibody immobilization with GlycoLinkTM
resin,[26] (e) conjugating protein with oxide-passivated silicon photonic microring resonators,[27]
(f) the structure of H40-P (LA-DOX)-b-PEG-OH/FA copolymer.[20]

4
F. HOSSAIN ET AL.
Scheme 1. Synthesis of Tn-Thr-PS A1 via hydrazone linkage. Reagents and conditions: (a) 0.1
M acetate buffer (pH 5.1), 2 mM NaIO4 solution, dark room, rt, 90 min; (b) a-D-GalNAc-L-Thr-NH-
NH2, 1X PBS buffer (pH 7.2), rt, overnight.
Previously, our group synthesized a complete stereochemical set of a-
and b-linked D-GalNAc-O-Ser/Thr antigens, with the aim of understanding
structural aspects of the Tn antigen for immune recognition (Fig. 2a).[22]
Currently, we are endeavoring to develop a vaccine through conjugation
with one Tn antigen (a-D-GalNAc-L-Thr-NH-NH2) and PS A1 (Sch. 1)
employing a hydrazone link rather than an oxime. The aldehyde-reactive
hydrazine group is commonly used for labeling and bioconjugation with a
variety of biomolecules.[23] For example, with the aim of lectin binding and
pathogen detection, glycoconjugates of bishydrazides were synthesized by
conjugating a,x-bishydrazides of carboxymethylated hexa (ethylene glycol)
with unprotected saccharides at one end and a carrier protein (BSA) at the
other (Fig. 2b).[24] For a variety of assay-based reasons, bacterial polysac-
charides and glycoproteins have been labeled with biotin using hydrazide-
biotin reactive derivatives (Fig. 2c).[25] Similarly, functionally modified anti-
bodies or other proteins have been attached to affinity resins (e.g.
GlycoLinkTM coupling resin) (Fig. 2d)[26] or other solid support via the
hydrazide moiety (Fig. 2e).[27] Furthermore, the utility of these acid-sensi-
tive hydrazone linkages can be recognized by examining their application
in various drug delivery vehicles such as nanoparticles, polymers, den-
drimers, micelles, liposomes, and utilized in pH-responsive drug delivery
(Fig. 2f).[20]
Results and discussions
PS A1 isolation
Bacteroides fragilis (ATCC 25285/NCTC 9343) was cultured in sterile and
anaerobic conditions to isolate crude PS A1 and the crude was purified uti-
1
lizing a previous protocol.[28] Recorded H NMR at 22 ꢁC and 60 ꢁC and

JOURNAL OF CARBOHYDRATE CHEMISTRY
5
COSY spectra of the purified PS A1 was comparable exactly with reported
literature values.[28] It was then used for conjugation with a-D-GalNAc-L-
Thr-NH-NH2 to get our desire immunogen.
Synthesis of Tn-Thr-PS A1
The vaccine construct was assembled with a slight modification of our pre-
viously reported protocol.[29] We observed less antigen loading as com-
pared to our previous oxime-based Tn-PS A1. To improve the loading, we
conducted conjugation reactions in 1X PBS buffer (pH 7.2), which resulted
in an increased loading of the Tn-Thr antigen to PS A1 (Sch. 1). The con-
jugation was validated by the presence of hydrazone doublets correspond-
ing to the E and Z isomers at d 7.47 and 7.12 ppm, respectively.
Considering the molar fraction of the hydrazone doublets with respect to
the molar fraction of PS A1 methyl group present on the pyruvate acetal of
1
PS A1 and then analyzing the integration of H NMR spectrum, we found
a 9:1 E/Z ratio and an antigen loading of 8% (see Supplementary
Information).
Factors effecting hydrolysis of pyruvate acetal on the D-galactopyranose
The tetrasaccharide repeating unit of PS A1 includes a pyruvate acetal ring,
which contains an electron withdrawing carboxylate group adjacent to the
acetal carbon center. A few factors, such as buffer,[30] solvent[30] and neigh-
boring group participation,[31,32] can influence cleavage of this pyruvate
acetal via a hydrolysis reaction.[33,34] However, it is known that the zwitter-
ionic charge character is essential for immunogenicity of PS A1.[35] Since
eliminating the carboxylate of the pyruvate acetal group via hydrolysis ren-
ders PS A1 non-zwitterionic, i.e. non-immunogenic,[35] so our focus turned
to the stability of the pyruvate acetal group under biological conditions,
including acetate buffer condition (pH 5.5). Hydrolysis of acetals involves
Figure 3. Hydrolysis of pyruvate acetal on the D-galactopyranose sugar.

6
F. HOSSAIN ET AL.
protonation followed by decomposition to alcohol and carbonium ion or
oxocarbenium ion (Fig. 3). Generally, acetal bond cleavage is favored in the
presence of a good leaving group or the stability of intermediate carbonium
and oxocarbenium ion.[36,37] Furthermore, intramolecular participation of
the neighboring acetamido group[31] or carboxyl group[32] is known to
facilitate hydrolysis of glycosides and phenolic acetals. Therefore, as pyru-
vate acetal has a carboxyl functionality that might undergo neighboring
group participation by intramolecular H-bonding with the oxygen, as noted
in Figure 3a, hydrolysis of the acetal can be promoted even in the absence
of hydronium ions.[38] Fortunately, carboxylic is a poor leaving group, and
the presence of this electron withdrawing group adjacent to the intermedi-
ate carbonium or oxocarbenium center usually destabilize the ions,[39]
which can potentially lead to the cleavage of this particular acetal difficult
(Fig. 3b).
Since promising immunological responses have been obtained from
entirely carbohydrate based Tn-PS A1 vaccine,[17] we always assumed that
Tn-PS A1 was stable under various conditions. However, up until this
point, our assumption was never experimentally proven. In comparison,
many comparative hydrolytic stability studies were carried out by several
groups on a series of hydrazone and oxime conjugates.[40,41] Kalia et al.
reported that acceleration of oxime and hydrazone bond formation is acid-
catalyzed, but slows at low pH due to protonation of either heteroatoms.[41]
However, in order to address our concern regarding acetal stability, we
designed and synthesized 1-thiophenyl-4,6-O-pyruvate acetal-D-galctopyra-
nose (11) to perform analogous stability studies.
Synthesis of phenyl 4,6-O-pyruvate acetal-1-thio-D-galactopyranosidee (11)
The synthesis of compound 11 commenced from monosaccharide D-galac-
topyranose 4, which was peracetylated by treatment with excessive acetic
anhydride in pyridine in the presence of a catalytic amount of DMAP
(Sch. 2). Peracetylated saccharide 5 was subsequently treated with thiophe-
nol and boron trifluoride dietherate in anhydrous DCM at 0 ꢁC overnight
to afford compound 6.[42] Following purification of compound 6, Zemplen
ꢀ
conditions were employed using freshly prepared NaOMe/MeOH from
sodium metal and methanol to deprotect the acetate groups. After comple-
R
tion of reaction, NaOMe was neutralized using DOWEXV 50X8-100 and
the resulting reaction mixture was evaporated to dryness. Subsequently, a
4,6-benzylidine group was installed by treatment of 7 with benzaldehyde
dimethylacetal in the presence of camphorsulphonic acid to yield com-
pound 8 in a 77% yield. Reaction of benzoyl chloride with compound 8 in
the presence of catalytic DMAP in pyridine, followed by deprotection of

JOURNAL OF CARBOHYDRATE CHEMISTRY
7
Scheme 2. Synthesis of 1-thiophenyl-4,6-O-pyruvate acetal-D-galactopyranose. Reagents and
.
conditions: (a) Ac2O (10 eq), pyr, DMAP (cat.), rt, overnight, 75%; (b) BF3 OEt2, thiophenol,
anhyd. DCM, 0 ꢁC, 80%; c) NaOMe, MeOH, rt, 1 h, 90%; (d) benzaldehyde dimethylacetal (1.2
eq), camphorsulphonic acid (pH ꢀ 4–5), DMF, rt, 5 h, 77%; (e) (i) BzCl (7 eq), pyr, DMAP (cat.),
.
overnight, rt, 82%; (ii) pTSA (cat.), MeOH, rt, 81%; (f) methyl pyruvate (2 eq), BF3 OEt2 (2 eq),
CH3CN, rt, 2.5h, 50%; (g) saturated NaOH solution, reflux, 2 h, 50%.
benzylidine acetal with p-toluene sulfonic acid in methanol, produced com-
pound 9 in a very good overall yield for two steps. The 4,6-pyruvate acetal
.
was installed employing the reaction with methyl pyruvate and BF3 OEt2 in
acetonitrile to generate (R)-pyruvate acetal 10 in a 50% yield.[13,43]
Saponification of compound 10 at room temperature resulted in the target
compound 11, which, after purification, was used in stability studies.
NMR based stability study
We utilized compound 11 to examine the stability of the pyruvate acetal, as
it is similar to the saccharide moiety present in the repeating unit of PS A1
(see Sch. 1). We understood that if cleavage of the acetal group took place,
the chemical shift value of H-4, H-6, C-4 and C-6 of compound 11 would
move upfield due to the loss of the electron withdrawing carboxylic group.
1H and 13C NMR spectra of compound 11 were recorded in deuterated
acetate buffer (pD 5.51) at different time intervals for a total of 21 h. After
the allotted time, no shift of the signals was noticed, which ensures no ace-
tal cleavage (see Supplementary Information). The quaternary carbon peak
of pyruvate acetal in the 13C NMR spectra confirmed no hydrolysis of the
ring either. Furthermore, HMBC spectrum revealed the correlation of H-6
(at d 3.90–3.88) with quaternary carbon (at d 100.84) present in the

8
F. HOSSAIN ET AL.
Figure 4. (a) HMQC spectrum indicated the presence of a quaternary carbon 21 h after heating
compound 11 in acetate buffer (pD 5.5), (b) HPLC results at different time intervals of 11 in
acetate buffer (pH ¼ 5.5).
Table 1. NMR based stability studies of 11.
Acetate buffer (pD)
Temperature (ꢁC)
Time (h)
Significant signal shift
5.51
1.3
22
22
37
50
70
85
21
48
24
6
1
4
No
No
No
No
No
Yes
pyruvate acetal ring (Fig. 4a). To determine the stability under more acidic
conditions, NMR spectra of compound 11 were recorded at pD 1.3 using
deuterated chloroacetic acid buffer under a normal probe temperature
(22 ꢁC) in varying time intervals for 48 h (see Supplementary Information).
No peak shifts were observed, indicating that bond cleavage did not take
place. As pH decreased with an increase in temperature, the NMR probe
temperature was raised to 37 ꢁC and spectra were recorded multiple times
within 24 h. The temperature was further increased to 50 ꢁC for 6 h, fol-
lowed by 70 ꢁC for 1 h. The compound was stable under all the temperature
conditions noted. Finally, the temperature was elevated to 85 ꢁC and spectra
were recorded at varying time intervals for 12 h. At time zero, no cleavage
was observed; however, at around the 4 h time-frame, approximately 50%
of cleavage was noted. This was confirmed by comparing the integration of
1
new peaks to the actual peaks in recorded H NMR spectrum of the com-
pound (see Table 1).
We observed upfield shifts of relevant proton and carbon signals. After
4 h of heating, we analyzed 1 D (1H, 13C, DEPT135) and 2 D (H-H COSY,
HMQC, HMBC) spectra of the compound to identify the peaks properly.
The H-4 signal was observed to move from d 4.20 to 3.97 and H-6 shifted
from d 4.00 to 3.73–3.70. The H-3 and H-5 peaks were also shifted upfield,
respectfully. Similarly, a 13C-4 signal shifted from d 71.53 to 67.95 and the

JOURNAL OF CARBOHYDRATE CHEMISTRY
9
C-6 peak shifted from d 65.21 to 60.87. In addition, a new quaternary car-
bon signal appeared at d 124.61, which represents a signal related to a
hydrated acetal of the pyruvic acid. Considering all the chemical shifts, it is
clear that pyruvate acetal is very stable at pD 5.51 and the stability is nega-
tively correlated with temperature and positively correlated with pH.
HPLC based stability study
High performance liquid chromatography (HPLC) was also used to trace
the stability of compound 11 in 0.1 M acetate buffer (pH ¼ 5.5) at different
time intervals by closely followung a previously reported hydrolytic
study.[44] A 600 mM solution of compound 11 was prepared in acetate buf-
fer then approximately 10 mL of the sample was injected in HPLC after 1 h,
14 h, 21 h, and 96 h. We observed a corresponding single major peak with
minor decrease in area and shift in retention time after 21 h compared to
the initial injection. However, after 96 h, we observed multiple peaks.
One of these peaks had a similar retention time (RT) with compound 7
(Fig. 4b), which might indicate hydrolysis.
Regioselective conjugation
To achieve regioselective conjugation of Tn-hydrazide antigen with PS A1,
we conducted oxidation of PS A1 with 2 mM sodium metaperiodate solu-
tion. We anticipated that under this condition, only the vicinal cis-diol
would be oxidized to leave the vicinal trans diol intact. In order to reveal
the regioselective cleavage of cis-diol over trans- diol of galactofuranose, we
synthesized propyl D-galactofuranoside 13 and subjected it to low to harsher
oxidation conditions and analyzed the outcome. Thus, galactofuranose was
converted into allyl galactofuranoside 12 by stirring with allyl bromide and
sodium hydride in DMPU for 72 h (Sch. 3), which was followed by 10% Pd/
C-catalyzed hydrogenolysis in methanol under 40 psi hydrogen pressure to
produce 13, which was purified by a silica gel column.
Thereafter, 13 was treated with a 4.5 mM NaIO4 solution in 100 mM
acetate buffer. The reaction was allowed to continue with stirring in the
dark (NaIO4 is light sensitive)[45] for 3 h in order to form the aldehyde.
Only a 30% yield of aldehyde 14 was obtained as a result of the cis-diol
cleavage. No trans-diol cleavage was observed (see Supplementary
Information, compound 14 with a single aldehyde peak at 9.6 ppm), which
is indicative of the selective oxidation under this reaction condition. Tn-
hydrazide 15[22] and aminoxy Tn 16[17] were synthesized following previ-
ous literatures. Finally, the conjugation was examined by dissolving alde-
hyde 14 in acetate buffer (pH 5.0), followed by treatment with Tn-

10
F. HOSSAIN ET AL.
Scheme 3. Synthesis of 1-propyl-D-galactofuranose and compound 17 and 18. Reagents and
conditions: (a) Allyl bromide (4 eq), NaH (3 eq), DMPU, rt, 72 h, 60%; (b) 10% Pd–C/H2, MeOH,
rt, 3 h, 70%; (c) NaIO4 (1 eq, 4.5mM), 100 mM NaOAc buffer (pH 5.0), rt, 6 h, dark, 30%; (d) 15,
1X PBS buffer (pH 7.2), rt, overnight; (e) 16, 0.1 M acetate buffer (pH ¼ 5.5), rt, overnight.
hydrazide (15)[22] and aminoxy Tn (16) separately. Hydrazone and oxime
formation was first detected by ESI-mass spectroscopy. Presence of peaks at
m/z 531.2 and 432.9 corresponding to the (M þ Na) peak of compound 17
and 18 were detected. Compound 17 and 18 were purified with a P2-
1
Biogel column using water as eluent. H NMR spectra of 17 and 18
revealed the presence hydrazone and oxime doublets at d 7.62 and 7.38,
respectively. Therefore, the synthesis of 1-allyl-galactofuranose and subse-
quent oxidation and conjugation validated the reaction outcome that was
used for Tn-PS A1 conjugation. Furthermore, 1 D TOSCY of 18 was taken
by radiating the peak at 7.34 ppm to confirm the oxime conjugation
between aminooxy Tn and C-5 in compound 18.
Conclusion
In summary, the Tn-Thr-PS A1 conjugate was synthesized by coupling a
hydrazine derivative of a-D-GalNAc-O-L-Thr with oxidized PS A1, giving rise
to the formation of a hydrazone conjugation. The synthesis of phenyl 4,6-O-
pyruvate acetal-1-thio-D-galctopyranoside was achieved to mimic PS A1 and
used to evaluate the stability of pyruvate acetal. Stability studies were per-
formed with NMR using varying pD, time, and temperature. The pyruvate ace-
tal was revealed to be very stable at pD 5.1 and 1.3 at room temperature. The
compound was stable for hours even after being heated to 70 ꢁC at pD 1.3, but

JOURNAL OF CARBOHYDRATE CHEMISTRY
11
cleavage was observed at pD 1.3 when the compound was heated at 85ꢁC.
Another stability study was carried out with HPLC using varying time and
showed that the pyruvate acetal was stable after our desired reaction time at
pH 5.5. Finally, to reveal the cleavage of cis-diol over trans-diol in galactofura-
nose, propyl D-galactofuranoside was synthesized and subjected to oxidation
conditions. Only the cis-diol was found to be cleaved with 4.5 mM NaIO4 in
100mM acetate buffer. These observations suggested that our strategy to oxi-
dize PS A1 and conjugate it with hydrazine- or aminooxy-based antigens will
not alter the structure of PS A1 and regioselective conjugation can be achieved
to synthesize vaccines following this strategy.
Experimental
Materials and methods
All reagents and solvents were purchased from Aldrich, AK Scientific,
Fisher Scientific, Chem Impex and EMD Millipore. All the solvents and
reagents were used without further purification, unless otherwise stated.
Thin layer chromatography (TLC) was performed on 0.25 mm Dynamic
Adsorbents, L.L.C. pre-coated silica gel (particle size 0.03–0.07 mm, catalog
no. 84111, lot # LA2006) to monitor the progress of reaction. TLC plates
were visualized using UV light or by staining with p-anisaldehyde or nin-
ꢀ
hydrin solution followed by charring. Whatman Purasil 60 Å (230–400
mesh ASTM) silica gels were used for normal phase column chromatog-
1
raphy. H and 13C NMR, DEPT135, HMQC, COSY spectra were acquired
using Bruker 600 MHz spectrometers. The residual CDCl3 singlet at d
7.27 ppm (1H NMR) and residual triplet at d 77.23 ppm (13C NMR) were
1
used as the standard for H NMR and 13C NMR spectra respectively. The
residual D2O was referenced to 4.79 when spectra were taken in D2O
Signal patterns are indicated as s: singlet; d: doublet; t: triplet; q: quartet;
m: multiplet; dd: doublet of doublets; br: broad and coupling constants are
reported in hertz (Hz). Low resolution mass spectra (LRMS) were recorded
on an Esquire-LC electrospray ionization (ESI) mass spectrometer. High
performance liquid chromatography (HPLC) chromatograms were analyzed
with Agilent Technologies 1100 Series. Hypersil GOLD, 5 lm, 150 ꢂ 3 mm
column from Thermo Fisher scientific, 254 nm wavelength and ACN/H2O
mobile phase were utilized elute the samples.
Isolation and purification of PS A1
B. fragilis ATCC 25285/NCTC 9343 was purchased from Presque Isle
Cultures, which was lined on Blood Agar and Bacteroids Bile Esculin
(BBE) plates.[15,46] An anaerobic glove bag was used to prepare the plate.

12
F. HOSSAIN ET AL.
Initial bacterial growth was started by transferring agar plates to anaerobic
jar filled with nitrogen (N2) in presence of oxygen (O2) indicator strips and
incubated at 37 ꢁC. The cultured bacteria were stored at ꢃ80 ꢁC to use
in future.
Culture of bacteria
1 L of nanopure water containing 20 g proteose-peptone, 5 g yeast extract,
5 g NaCl and 1 mg resazurin was autoclaved and 25% glucose, 25% potas-
sium phosphate, 5% cysteine, 0.5% Hemin in 1 N NaOH and 0.5% Vitamin
K1 in absolute ethanol solutions were prepared. Then 2 mL glucose, 2 mL
potassium phosphate, 1 mL cysteine, 100 mL Hemin and 50 mL vitamin K1
were added to the autoclaved broth. Resazurin in presence of oxygen made
the color of the entire solution red. To get the anaerobic condition the
entire solution was degassed with nitrogen until the red color of the solu-
tion changed. After achieving the anaerobic condition, 5 mL of liquid media
containing B. fragilis was transferred by cannulation to the degassed jar.
The bacteria inoculated jar was incubated at 37 ꢁC for 24 h.
Extraction of PS A1
After 48 h, the media was centrifuged at 8000 X g for 20 minutes. A 1X
PBS buffer was prepared with 137 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, and 1.8 mM KH2PO4. Then the pH was adjusted to 7.4. After
washing with PBS solution, the cells were suspended with water and equal
amount of 70% phenol was added to the suspension with continuous stir-
ring at 68 ꢁC. After 30 minutes of stirring the phenol layer was separated
from aqueous layer by centrifuging at 5000 X g for 30 minutes at 4 ꢁC. The
aqueous layer after repeated extraction with diethyl ether was evaporated to
dryness, dissolved in minimal amount of water and subjected to dialysis.
After 3 days of dialysis followed by lyophilization, the crude was treated
with DNase and RNase in 0.1 M acetate buffer. Then pH of the solution
was adjusted to 7.0 by 2 M NaOH solution and then the solution was
treated with pronase. Dialysis followed by lyophilization resulted crude
polysaccharide, which was purified to obtain PS A1.
Purification of PS A1
The crude was subjected to purification by size exclusion column. At first
sephacryl S-300 column was used and the crude was applied to the column
by dissolving it in 0.5% sodium deoxycholate, 50 mM glycine and 10 mM
EDTA (pH¼ 9.8).[47] Small fractions were pooled to measure UV absorb-
ance at 220, 260 and 280 nm. Fractions having more than 0.1 absorbance at
260 and 280 nm were treated further with DNase, RNase and Pronase and

JOURNAL OF CARBOHYDRATE CHEMISTRY
13
were subjected to extra purification. But the fractions having absorbance at
220 nm were pooled and dialyzed. Finally, it was purified by anion
exchange column. Before loading on to the column, the crude PS A1 was
treated with 3% acetic acid for 1 h at 70 ꢁC. 50 mM Tris-HCl (pH 7.3) and
increasing NaCl solution upto 2 M concentration was used as eluent. Pure
PS A1 was obtained at the earlier fractions, before eluting with NaCl. PS
A1 fractions were subjected to dialysis against distilled water for 3 days to
remove buffer. After lyophilization, pure PS A1 was obtained as white fluffy
1
solid. H-NMR spectra of lyophilized PS A1 was recorded at 22 ꢁC and
60 ꢁC temperature and compared with the literature to determine and
ascertain the presence and purity of the product.
Synthesis of designed conjugates and model compounds
Tn-Thr-PS A1 conjugate
PS A1 (0.001 g, 9.1ꢂ10ꢃ9 mol) was dissolved in 0.4 mL of 100 mM NaOAc
buffer (pH 5.0) in an amber vial. To this solution was added 0.1 mL of
10 mM NaIO4 to obtain overall 2 mM NaIO4 concentration in the reaction
mixture and the reaction was stirred at room temperature in dark for
60 minutes to afford PS A1 aldehyde. The reaction mixture was purified
using a spin column with 3k molecular weight cut off. Nano-pure water
was used to drain any residual NaIO4 through the column by centrifuging
it at 12000 rpm for 20 minutes and the process was repeated 4 times. Pure
PS A1 aldehyde was dissolved in 2 mL of 1X PBS buffer (pH 7.2) and
0.5 mg Tn hydrazide (0.0014 mmol, 3 equiv. Considering PS A1 has an
average MW of 110 K g/mol and 120 repeating units per mole) was added
to the solution. The reaction was continued at room temperature for over-
night in the dark and after that the desired product was purified using a
10k molecular weight cut off spin column. Nano pure water was used for
spin column and the washing was repeated 5 times. The compound was
1
lyophilized and H NMR spectra was recorded. Formation of conjugate was
confirmed by observing hydrazone doublets corresponding to the E and Z
isomers at d 7.47 and d 7.12 ppm respectively.
The % loading was calculated based on integration value of hydrazone
1
protons on H-NMR spectrum and compared it with methyl proton pre-
sent on pyruvate ring acetal of PS A1. The loading of antigen was obtained
as about 8% by using the following formula:
½MW of Tn=ðMW of PSA1 þ MW of TnÞꢄ
ꢂ ðMole fraction of oxime HÞ ꢂ 100,
where mole fraction of hydrazone H ¼ Hydrazone H integration/(methyl
proton integration/3).

14
F. HOSSAIN ET AL.
1,2,3,4,6-penta-O-Acetyl-b-D-galactopyranose (5). Galactopyranose 4 (4 g,
22.22 mmol) was treated with Ac2O (10 eq) and catalytic amount of DMAP
in pyridine at room temperature and stirred overnight.[48] After completion
of the reaction as noted by TLC, the solvent was evaporated and co-evapo-
rated with toluene and partitioned with ethyl acetate (100 mL) and water
(200 mL). The ethyl acetate layer was concentrated, and compound was
purified over silica gel column using 40% ethyl acetate in hexane as eluent
and peracetylated galactopyranose 5 was obtained as a white solid (6.5 g,
1
16.67 mmol, 75%). H NMR (600 MHz, CDCl3): d 5.64 (d, 1H, J ¼ 5 Hz, H-
1), 5.42 (d, 1H, J ¼ 5 Hz, H-4), 5.34–5.30 (m, 1H, H-2), 5.07 (dd, J ¼ 6 Hz,
H-3), 4.15–4.11 (m, 1H, H-6), 4.05–4.04 (m, 1H, H-5), 2.15 (s, 3H, OCH3),
2.11 (s, 3H, OCH3), 2.03 (s, 3H, OCH3), 1.98 (s, 3H, OCH3) ppm; 13C
NMR (150 MHz, CDCl3): d 170., 170.1, 169.9, 169.4,168.9, 92.1, 71.7, 70.8,
67.8, 66.8, 61.0, 20.8, 20.6, 20.6, 20.5 ppm; ESIMS [(M þ Na)þ] calcd for
C20H24NaO9S is 463.1, found 462.8; ESIMS [(M þ Na)þ] calcd for
C16H22NaO11 is 390.34, found 390.1.
Phenyl 2,3,4,6-tetra-O-acetyl-1-thio-b-D-galactopyranoside (6). Compound
5 was again dissolved in anhydrous DCM (65 mL), thiophenol (1.5 eq) was
added to it and stirred at 0 ꢁC temperature.[48] Boron trifluoride dietherate
(5 eq) was added to the cooled reaction mixture and the reaction was
stirred for 24 h, as reaction competition was noted by TLC. Then the reac-
tion mixture was quenched by saturated NaHCO3 solution (until pH shows
neutral solution) and partitioned with DCM (150 mL). The DCM layer was
collected, dried over anhydrous Na2SO4, filtered and the filtrate was con-
centrated under reduced pressure. Silica gel column using 30% ethyl acetate
in hexane as eluent resulted major beta product, 1-thiophenyl peracetylated
1
galactopyranose 6, as a pale-yellow solid (5.87 g, 13.34 mmol, 80%). H
NMR (600 MHz, CDCl3): d 7.51–7.49 (m, 1H, Ar-H), 7.32–7.26 (m, 2H,
Ar-H), 5.41 (dd, J1 ¼ 6 Hz, J2 ¼ 1.2 Hz, 1H, H-4), 5.23 (t, J ¼ 12 Hz, 1H, H-
2), 5.05 (dd, J1 ¼ 3.6 Hz, J2 ¼ 6 Hz, 1H, H-3), 4.71 (d, J ¼ 12 Hz, 1H, H-1),
4.19–4.16 (m, 1H, H-6), 4.12–4.09 (m, 1H, H-60), 3.95–3.92 (m, 1H, H-5),
2.51 (s, 3H, OCH3), 2.08 (s, 3H, OCH3), 2.03 (s, 3H, OCH3), 1.96 (s, 3H,
OCH3) ppm; 13C NMR (150 MHz, CDCl3): d 170.4, 170.2, 170.1,169.4,
132.5, 132.5, 128.9, 128.2. 86.–128.15, 86.58, 71.98, 67.22–67.20, 61.63,
20.86–20.59 ppm; ESIMS [(M þ Na)þ] calcd for C20H24NaO9S is 463.1,
found 462.8.
Phenyl 1-thio-b-D-galactopyranoside (7). NaOMe was prepared in situ by
dissolving 60 mg Na in anhydrous methanol at 0 ꢁC. Compound 6 (4.5 g,
10.2 mmol) was dissolved in minimum volume of anhydrous methanol
(30 mL) and NaOMe solution (until pH shows strong basic around 9–10)
was slowly added to it. After completion of the reaction, the reaction mix-
ture was quenched with amberlite 15 resin. Filtration followed by solvent

JOURNAL OF CARBOHYDRATE CHEMISTRY
15
evaporation and co-evaporation with anhydrous toluene resulted fully
22
deacetylated sugar 7 in 90% yield (2.5 g, 9.2 mmol). [a]D ¼ ꢃ52.1 (c ¼ 1.0,
MeOH);[49] 1H NMR (600 MHz, CDCl3): d 7.42–7.41 (d, J ¼ 6 Hz, 1H, Ar-
H), 7.26–7.21 (m, 3H, Ar-H), 4.62 (d, J ¼ 6Hz, 1H), 3.84 (d, J ¼ 6 Hz, 1H),
3.61–3.57 (m, 4H), 3.55–3.46 (m, 1H) ppm; HRMS: TOF [(M þ Na)þ] calcd
for C12H16NaO5S is 295.0616, found 295.0615.
Phenyl 4,6-O-benzylidene-1-thio-b-D-galactopyranoside (8). Compound 7
was subsequently dissolved in DMF (20 mL) and further treated with ben-
zaldehyde dimethylacetal (1.7 mL, 11.0 mmol) and camphor sulfonic acid
(CSA) until solution got acidic pH. The reaction mixture was stirred for
5 h at 50 ꢁC. After completion of the reaction, triethylamine (Et3N) was
added to quench CSA. The reaction mixture was evaporated to dryness and
was purified over silica gel column using 45% EtOAc in hexane as the elu-
1
ent to furnish pure compound 8 (2.5 g, 6.9 mmol, 77%) as a white solid. H
NMR (600 MHz, DMSO): d 7.55–7.54 (m, 1H, Ar-H), 7.44–7.37 (m, 2H,
Ar-H), 7.28–7.24 (m, 1H, Ar-H), 5.57–5.52 (m, 1H), 4.70–4.67 (m, 1H),
4.15 (d, J ¼ 6 Hz, 1H), 4.07–4.02 (m, H), 3.65 (d, J ¼ 12 Hz, 1H), 3.55–3.50
(m, 1H), 2.50 (d, J ¼ 6 Hz, 1H) ppm; 13C NMR (150 MHz, DMSO): d
138.6, 133.9, 130.5, 128.7, 127.9, 126.5, 126.4, 99.8, 86.6, 76.1, 73.1, 69.3,
68.61, 67.88 ppm; HRMS: TOF [(M þ Na)þ] calcd for C19H20NaO5S is
383.0929, found 383.0925.
Phenyl 2,3-di-O-benzoyl-1-thio-b-D-galactopyranoside (9). Compound 8
(0.9 g, 2.5 mmol) was dissolved in pyridine (12 mL). Benzoyl chloride
(2.5 mL, 15 mmol) and catalytic amount of DMAP were added to the solu-
tion and stirred overnight at room temperature. After completion of the
reaction, solvent was evaporated in reduced pressure and the crude solid
was partitioned with DCM (30 mL) and saturated NaHCO3 solution
(50 mL). Organic layer was collected, dried over anhydrous Na2SO4 and
concentrated under reduced pressure to give the crude product, which was
purified over silica gel column using 25% EtOAc in hexane to afford Bz
protected compound in 82% yield. To a solution of Bz protected compound
(1.4 g, 2.5 mmol) in MeOH catalytic amount of TsOH was added and
stirred at room temperature until full consumption of starting material was
noticed by TLC. Then reaction mixture was quenched with Et3N and
evaporated to dryness. Crude mixture was subjected to silica gel column
using 40% EtOAc in hexane as eluent to afford compound 9 (1.2 g,
22
2.5 mmol, 81%) as a white solid. [a]D ¼ þ105 (c ¼ 1.0, CHCl3);[50] 1
H
NMR (600 MHz, CDCl3): d 7.54–7.51 (m, 4H, Ar-H), 7.43–7.38 (m, 4H,
Ar-H), 7.36–7.34 (m, 4H, Ar-H), 7.33–7.28 (m, 3H, Ar-H), 5.83–5.80 (m,
1H, H-2), 5.35 (dd, J ¼ 3.42, 6.78 Hz, 1H, H-3), 5.00 (d, J ¼ 10.02 Hz, 1H,
H-1), 4.46–4.44 (m, 1H, H-4), 4.08–4.05 (m, 1H, H-6), 3.96 (dd, J ¼ 4.2,
7.86 Hz, 1H, H-60), 3.86–3.84 (m, 1H, H-5) ppm; 13C NMR (150 MHz,

16
F. HOSSAIN ET AL.
CDCl3): d 165.8, 165.3, 133.5, 133.3, 132.7, 132.3, 129.9, 129.8, 129.4, 129.0,
128.5, 128.4, 128.2, 86.6, 78.1, 75.4, 68.7, 67.8, 63.0 ppm; HRMS:TOF
[(M þ Na)þ] calcd for C26H24NaO7S is 503.1140, found 503.1136.
Phenyl
4,6-O-[1-(R)-(methoxycarbonyl)-ethylidene]-2,3-di-O-benzoyl-1-
thio-b-D-galactopyranoside (10). Compound 9 (0.25 g, 0.52 mmol) was dis-
solved in minimum volume of acetonitrile and the solution was cooled to
.
0 ꢁC. Methyl pyruvate (0.094 mL, 1.04 mmol) and BF3 OEt2 (0.13 mL,
1.04 mmol) was added to the cooled reaction mixture and stirred at room
temperature for 2.5 h. After completion of the reaction, the reaction mix-
ture was diluted with DCM (15 mL) and quenched with saturated NaHCO3
(25 mL) solution. The compound was extracted with DCM from the aque-
ous layer and washed with water and brine. The organic layer was dried
over anhydrous Na2SO4 and concentrated under reduced pressure. The
crude mass was purified over silica gel column chromatography using 25%
EtOAc in hexane as eluent to afford compound 10 as a white solid (0.15 g,
22
1
0.27 mmol, 50%). [a]D þ145; H NMR (600 MHz, CDCl3): d 8.03–7.99
(m, 4H, Ar-H), 7.62–7.60 (m, 2H, Ar-H), 7.57–7.51 (m, 2H, Ar-H),
7.44–7.36 (m, 4H, Ar-H), 7.35–7.33 (m, 3H, Ar-H), 5.80–5.77 (m, 1H, H-
2), 5.23 (dd, J ¼ 3.42, 9.96 Hz, 1H, H-3), 4.93 (d, J ¼ 9.90 Hz, 1H, H-1), 4.58
(d, J ¼ 3.3 Hz, 1H, H-4), 4.23–4.21 (m, 1H, H-6), 4.06–4.04 (m, 1H, H-6),
3.67–3.66 (m, 1H, H-5) ppm; 13C NMR (150 MHz, CDCl3): d 170.1, 166.1,
165.0, 133.6, 133.3, 133.2, 131.5, 129.9, 129.8, 129.6, 129.3, 128.8, 128.4,
128.4, 128.3, 98.6, 85.7, 74.0, 69.2, 69.0, 67.0, 65.3, 52.5, 25.6 ppm; HRMS:
TOF [(M þ Na)þ] calcd for C30H28NaO9S is 587.1364, found 587.1352.
Phenyl 4,6-O-[1-(R)-(methoxycarbonyl)-ethylidene]-1-thio-b-D-galactopyra-
noside (11). Compound 10 (0.15 g, 0.27 mmol) was stirred with saturated
NaOH solution at room temperature for 2 h. After that, reaction mixture
was quenched with DOWEX 50WX8-100 ion exchange (Hþ) resin.
Following neutralization, the reaction mixture was filtered, and the filtrate
was evaporated to dryness. Silica gel column purification using 10% MeOH
in DCM as eluent resulted compound 11 (0.06 g, 0.18 mmol, 50%) as a
white solid. 1H NMR (600 MHz, D2O): d 7.60–7.59 (m, 2H, Ar-H),
7.40–7.36 (m, 3H, Ar-H), 4.74 (d, J ¼ 9.02Hz, 1H, H-1), 4.17 (d,
J ¼ 3.00 Hz, 1H, H-4), 3.97–3.95 (m, 1H, H-6), 3.90–3.88 (m, 1H, H-60),
3.70–3.64 (m, 2H, H-2, H-3), 3.60 (br s, 1H, H-5), 1.42 (s, 3H, CH3) ppm;
13C NMR (150 MHz, D2O): d 176.1, 132.1, 132.0, 129.4, 128.3, 100.8, 87.2,
73.0, 71.3, 69.5, 68.4, 65.2, 25.4 ppm; HRMS: TOF [(M þ NH)þ] calcd for
C15H19O7S is 343.0852, found 343.0851.
Allyl a-D-galactofuranoside (12). D-galactopyranose (500 mg, 2.78 mmol)
was partially dissolved in 7 mL DMPU in an inert atmosphere. Sodium
hydride (NaH) (200 mg, 8.33 mmol) was added to the suspension and the
reaction mixture was cooled to 0 ꢁC. To the cooled reaction, allyl bromide

JOURNAL OF CARBOHYDRATE CHEMISTRY
17
(1 mL, 11.12 mmol) was added slowly and the reaction mixture was stirred
at room temperature for 72 h. After completion of the reaction, the reaction
mixture was cooled to 0 ꢁC and NaH was quenched with MeOH by adding
dropwise (1 mL). Subsequently DMPU was extracted with hexane from the
solution. A viscous sirup was obtained which was subjected to silica gel col-
umn chromatography using 10% MeOH in DCM as eluent to afford com-
22
pound 12 (367 mg, 1.67 mmol, 60%) as a light-yellow solid. [a]D þ95.6 (c
1, H2O);[51] 1H NMR (600 MHz, MeOD): d 5.98–5.95 (m, 1H, –CH¼CH2),
0
5.37–5.33 (m, 1H, –CH ¼ CH2), 5.20–5.17 (m, 1H, –CH ¼ CH2 ), 4.91 (d,
J ¼ 4.56 Hz, 1H, H-1), 4.33–4.29 (m, 1H, –CH2–CH ¼ CH2), 4.14–4.08 (m,
2H, H-3, –CH02–CH ¼ CH2), 3.98 (dd, J ¼ 4.56, 7.92 Hz, 1H, H-2),
3.75–3.73 (m, 1H, H-6), 3.66–3.62 (m, 2H, H-4, H-60), 3.58–3.55 (m, 1H,
H-5) ppm; 13C NMR (150 MHz, MeOD): d 134.3, 116.1, 100.5, 82.2, 77.5,
74.9, 73.1, 68.4, 62.7 ppm; HRMS: TOF [(M þ Na)þ] calcd for C9H16NaO6
is 243.0845 found 243.0813.
Propyl a-D-galactofuranoside (13). To a solution of compound 12 (100 mg,
0.45 mmol) in anhydrous methanol (10 mL) was added 10% Pd–C (50 mg) and
the mixture was stirred for 3 h at room temperature in 40 psi hydrogen pres-
R
V
sure. The reaction mixture was filtered through a Celite -545 bed, then washed
with plenty of MeOH and the combined filtrate was evaporated under reduced
pressure to obtain compound 13 (70 mg, 0.32mmol, 70%) as a white solid.
22
1
[a]D ¼ ꢃ12 (c 1, MeOH) H NMR (600 MHz, D2O): d 4.97 (d, J ¼ 4.19 Hz,
1H, H-1), 4.12–4.08 (m, 2H, H-2, H-6), 3.76–3.67 (m, 4H, H-3, H-5, H-60,
CH3–CH2–CH2), 3.58–3.55 (m, 1H, H-4), 3.48–3.44 (m, 1H, CH3–CH2–CH02),
1.61–1.55 (m, 2H, CH3–CH2–CH2), 0.88 (t, J ¼ 7.29 Hz, CH3–CH2–CH2) ppm.
13C NMR (150MHz, D2O): d 100.79, 81.09, 76.29, 74.55, 72.99, 70.25, 62.08,
22.06, 9.75 ppm; EIMS [(M þ Na)þ] calcd for C9H18NaO6 is 245.1 found 244.9.
Propyl a-D-xylo-pentodialdo-1,4-furanoside (14). To a solution of com-
pound 13 (20 mg, 0.09 mmol) in 20 mL 100 mM NaOAc buffer (pH 5.0),
NaIO4 (19.3 mg, 0.09 mmol) was added slowly to obtain overall 4.5 mM
NaIO4 concentration in the reaction mixture and the reaction was stirred
at room temperature in dark for 3 h. Thin layer chromatography revealed
partial consumption of starting material. The product was extracted with
EtOAc from the buffer solution, evaporated to dryness and finally purified
over silica gel column using 10% MeOH in DCM as eluent to afford com-
1
pound 14 (5.1 mg, 0.03 mmol, 30%) as colorless gum. H NMR (600 MHz,
CDCl3): d 9.71 (s, 1H, CHO), 5.17 (d, J ¼ 4.34 Hz, 1H, H-1), 4.36–4.35
(m, 1H, H-3), 4.22–4.21 (m, 1H, H-4), 4.12–4.11 (m, 1H, H-2), 3.89–3.86
(m, 1H, CH3–CH2–CH2), 3.58–3.54 (m, 1H, CH3–CH2–CH02), 1.67–1.62
(m, 2H, CH3–CH2–CH2), 0.94 (t, J ¼ 7.43 Hz, 1H, CH3–CH2–CH2) ppm.
13C NMR (150 MHz, CDCl3): d 201.53, 102.82, 87.14, 78.46, 78.15, 71.22,

18
F. HOSSAIN ET AL.
23.09, 10.84 ppm; ESIMS [(M þ Na)þ] calcd for C8H14NaO5 is 213.1,
found 213.3.
(Propyl a-D-xylofuranoside)-5-aldehyde {O-[2-(acetylamino)-2-deoxy-a-D-
galactopyranosyl]-L-threonyl}hydrazone (17). Compound 14 (3 mg,
0.015 mmol) was dissolved in 100 mM acetate buffer (pH 5.0) and Tn
hydrazide 15 (5.3 mg, 0.015 mmol) was added to it. The reaction mixture
was stirred overnight and formation of the hydrazone was monitored by
mass spectroscopy. After obtaining the desired hydrazide, the reaction mix-
ture was lyophilized and subjected to P2-Biogel column using water as elu-
1
ent for the purification of product 17. H NMR (600 MHz, CD3OD): d
7.55, (d, J ¼ 6.0 Hz, 1H, N ¼ CH), 4.85 (d, J ¼ 6.0 Hz, 1H), 4.25–4.24 (d,
J ¼ 6.0 Hz, 1H), 4.03–4.00 (m, 1H), 3.95–3.93 (m, 1H), 3.87 (d, J ¼ 6 Hz,
1H), 3.76 (dd, J ¼ 6.0 Hz, 1H), 3.67–3.63 (m, 2H), 3.54–3.44 (m, 2H), 1.95
(s, 3H), 1.28–1.27 (s, 3H); ESIMS [(M þ Na)þ] calcd for C20H36N4NaO11 is
531.2, found 531.5.
(Propyl a-D-xylofuranoside)-5-aldehyde O-[2-(acetylamino)-2-deoxy-a-D-
galactopyranosyl]oxime (18). Compound 14 (3.6 mg, 0.016 mmol) was dis-
solved in 100 mM acetate buffer (pH 5.2) and aminooxy Tn (4.1 mg,
0.016 mmol) was added to it. The reaction mixture was stirred overnight,
and formation of the oxime was monitored by mass spectroscopy. After
obtaining the desired oxime compound 18 the reaction mixture was sub-
jected to P2-Biogel column using water as eluent for the purification of
1
product and finally lyophilized. H NMR (600 MHz, CD3OD): d 7.11–7.10
(d, J ¼ 6.80 Hz, 1H, N ¼ CH), 4.94–4.93 (d, J ¼ 6 Hz, 1H), 4.11–4.10 (d,
J ¼ 6 Hz, 1H), 4.04–4.01 (m, 1H), 3.60–3.57 (m, 1H), 3.41–3.40 (m, 1H),
2.65 (s, 3H), 2.50–2.49 (m, 1H), 2.40–2.30 (m, 4H), 1.50–1.48 (q, 2H),
0.80–0.78 (m, 2H) ppm. 13C NMR (150 MHz, CDCl3): d 181.46, 149.15,
174.84, 172.58, 146.52, 101.15, 80.41, 70.49, 28.92, 26.15, 23.18, 9.78 ppm.
ESI-MS [(M þ Na þ H)þ] calcd for C20H37N4NaO11 is 432.4, found 432.9.
Stability study
Preparation of buffer solutions
To prepare 100 mM acetate buffer of pD 5.50, 20 mg NaOH was dissolved
in D2O and pH was adjusted by adding deuterated acetic acid (CD3COOD)
dropwise and pH was measured using a pH meter. When the pH meter
reading showed pH 5.10, the volume of the entire the following solution
was made 5 mL. Then pD of the solution was calculated as 5.51 using
formula:
pD ¼ pH meter reading þ 0:41

JOURNAL OF CARBOHYDRATE CHEMISTRY
19
653.5 mg trichloroacetic acid was dissolved in 5 mL D2O and the pH was
adjusted to 0.89 by slowly adding 1 M NaOH solution (prepared in D2O).
After obtaining the desired pH, the volume of the entire solution was made
10 mL (overall trichloroacetic acid concentration was 0.40 M). Then pD of
the solution was calculated as 1.30 using the above formula.
NMR analysis at different temperatures
NMR spectra were recorded at 22 ꢁC only when stability of compound 11
was evaluated at acetate buffer of pD 5.51. However, variable temperatures
were used to examine the stability of compound 11 at pD 1.30. At first,
spectra were recorded at 22 ꢁC in different time interval for 48 h. Then
temperature was increased to 37 ꢁC and the solution was heated in an incu-
bator for 24 h at the same temperature. Subsequently temperature was
increased to 50 ꢁC and allowed to continue the same temperature for 6 h in
a water bath. As no change was noticed in the NMR peak, temperature was
again increased to 70 ꢁC followed by 85 ꢁC in water bath. NMR spectra
were recorded after 4 h of heating at 85 ꢁC temperature. A small fraction of
pyruvate acetal cleavage was observed; therefore, the solution was again
heated at 85 ꢁC for 12 h and NMR spectra were recorded.
HPLC analysis at different temperatures
0.1 M of acetate buffer at pH 5.2 was prepared following Henderson-
Hasselbalch equation. Then, around 0.86 mg of compound 11 was dissolved
in 5 mL of this acetate buffer to get 500 mM concentration. From the same
vial 10 mL of solution was injected to the HPLC system after 1 h, 14 h, 21 h
respectively and recorded the chromatogram. Since, most of Tn-Ps A1 con-
jugation were carried out for overnight, we also decided to monitor the
chromatogram till 21 h. However, after 96 h, 50 mL was injected to get bet-
ter observations of the multiple peaks. Most of the samples were eluted
around 40/60 water and acetonitrile mobile phase system.
Disclosure statement
No potential conflict of interest was reported by the authors.
References
[1] Slovin, S.F.; Keding, K.S.; Ragupathi, R. Carbohydrate vaccines as immunotherapy
for cancer. Immunol. Cell Biol. 2005, 83, 418–428. doi: 10.1111/j.1440-1711.2005.
01350.x.
[2] Hossain, F.; Andreana, P.R. Developments in carbohydrate-based cancer therapeu-
tics. Pharmacetules 2019, 12, 84. doi: 10.3390/ph12020084.

20
F. HOSSAIN ET AL.
[3] Silverman, G.J. Human antibody responses to bacterial antigens: studies of a model
conventional antigen and a proposed model B-cell superantigen. Int. Rev. Immunol.
1992, 9(1), 57–78. doi: 10.3109/08830189209061783.
[4] Feng, D.; Shaikh, A.S.; Wang, F. recent advance in tumor-associated carbohydrate
antigens (TACAs)-based antitumor vaccines. ACS Chem. Biol. 2016, 11(4), 850–863.
doi: 10.1021/acschembio.6b00084.
[5] Guo, Z.; Wang, Q. Recent development in carbohydrate-based cancer vaccines.
Curr. Opin. Chem. Biol. 2009, 13(5–6), 608–617. doi: 10.1016/j.cbpa.2009.08.010.
[6] Dey, S.; Bajaj, S.O.; Tsai, T.-I.; Lo, H.-J.; Wu, K.; Wong, C.-H. Synthesis of modular
building blocks using glycosyl phosphate donors for the construction of asymmetric
N-glycans. Tetrahedron. 2018, 74(41), 6003–6011. doi: 10.1016/j.tet.2018.08.039.
[7] Danishefsky, S.J.; Shue, Y.-K.; Chang, M.N.; Wong, C.-H. Development of globo-H
cancer vaccine. Acc. Chem. Res. 2015, 48(3), 643–652. doi: 10.1021/ar5004187.
[8] Nishat, S.; Andreana, P. Entirely carbohydrate-based vaccines: an emerging field for
specific and selective immune responses. Vaccines. 2016, 4(2), 19. doi: 10.3390/
vaccines4020019.
[9] Wilkinson, B.L.; Day, S.; Malins, L.R.; Apostolopoulos, V.; Payne, R.J. Self-adjuvant-
ing multicomponent cancer vaccine candidates combining per-glycosylated MUC1
glycopeptides and the toll-like receptor 2 agonist Pam3CysSer. Angew. Chem. Int.
Ed. 2011, 50(7), 1635–1639. doi: 10.1002/anie.201006115.
[10] Lakshminarayanan, V.; Thompson, P.; Wolfert, M.A.; Buskas, T.; Bradley, J.M.;
Pathangey, L.B.; Madsen, C.S.; Cohen, P.A.; Gendler, S.J.; Boons, G.-J. Immune rec-
ognition of tumor-associated mucin MUC1 is achieved by a fully synthetic aber-
rantly glycosylated MUC1 tripartite vaccine. Proc. Natl. Acad. Sci. USA 2012,
109(1), 261–266. doi: 10.1073/pnas.1115166109.
[11] Tzianabos, A.O.; Onderdonk, A.B.; Rosner, B.; Cisneros, R.L.; Kasper, D.L.
Structural features of polysaccharides that induce intra-abdominal abscesses. Science
1993, 262(5132), 416–419. doi: 10.1126/science.8211161.
[12] Cobb, B.A.; Wang, Q.; Tzianabos, A.O.; Kasper, D.L. Polysaccharide processing and
presentation by the MHCII pathway. Cell 2004, 117(5), 677–687. doi: 10.016/j.cell.
2004.05.001.
[13] Eradi, P.; Ghosh, S.; Andreana, P.R. Total synthesis of zwitterionic tetrasaccharide
repeating unit from Bacteroides fragilis ATCC 25285/NCTC 9343 capsular polysac-
charide PS A1 with alternating charges on adjacent monosaccharides. Org. Lett.
2018, 20(15), 4526–4530. doi: 10.1021/acs.orglett.8b01829.
[14] Tzianabos, A.; Wang, J.Y.; Kasper, D.L. Biological chemistry of immunomodulation
by zwitterionic polysaccharides. Carbohydr. Res. 2003, 338(23), 2531–2538. doi: 10.
1016/j.carres.2003.06.005.
[15] Baumann, H.; Tzianabos, A.O.; Brisson, J.R.; Kasper, D.L.; Jennings, H.J. Structural
elucidation of two capsular polysaccharides from one strain of Bacteroides fragilis
using high-resolution NMR spectroscopy. Biochemistry. 1992, 31(16), 4081–4089.
doi: 10.1021/bi00131a026.
[16] Ju, T.; Otto, V.I.; Cummings, R.D. The Tn antigen-structural simplicity and bio-
logical complexity. Angew. Chem. Int. Ed. 2011, 50(8), 1770–1791. doi: 10.1002/anie.
201002313.
[17] De Silva, R.A.; Wang, Q.; Chidley, T.; Appulage, D.K.; Andreana, P.R.
Immunological response from an entirely carbohydrate antigen: design of synthetic
vaccines based on Tn ꢃ PS A1 conjugates. J. Am. Chem. Soc. 2009, 131(28),
9622–9623. doi: 10.1021/ja902607a.

JOURNAL OF CARBOHYDRATE CHEMISTRY
21
~
ꢀ
[18] Morales, S.; Acena, J.L.; Garcıa Ruano, J.L.; Cid, M.B. Sustainable synthesis of
oximes, hydrazones, and thiosemicarbazones under mild organocatalyzed reaction
conditions. J. Org. Chem. 2016, 81(20), 10016–10022. doi: 10.1021/acs.joc.6b01912.
[19] Zhang, Z.; He, C.; Chen, X. Hydrogels based on pH-responsive reversible carbon–ni-
trogen double-bond linkages for biomedical applications. Mater. Chem. Front. 2018,
2(10), 1765–1778. doi: 10.1039/C8QM00317C.
[20] Fang, Y.; Xue, J.; Gao, S.; Lu, A.; Yang, D.; Jiang, H.; He, Y.; Shi, K. Cleavable
PEGylation: a strategy for overcoming the “PEG Dilemma” in efficient drug deliv-
ery. Drug Delivery 2017, 24(2), 22–32. doi: 10.1080/10717544.2017.1388451.
€
[21] Kolmel, D.K.; Kool, E.T. Oximes and hydrazones in bioconjugation: mechanism and
catalysis. Chem. Rev. 2017, 117(15), 10358–10376. doi: 10.1021/acs.chemrev.7b00090.
[22] Shaik, A.A.; Nishat, S.; Andreana, P.R. Stereoselective synthesis of natural and non-
natural thomsen-nouveau antigens and hydrazide derivatives. Org. Lett. 2015,
17(11), 2582–2585. doi: 10.1021/acs.orglett.5b00512.
[23] King, T.P.; Zhao, S.W.; Lam, T. Preparation of protein conjugates via intermolecular
hydrazone linkage. Biochemistry 1986, 25(19), 5774–5779. doi: 10.1021/bi00367a064.
[24] Adak, A.K.; Leonov, A.P.; Ding, N.; Thundimadathil, J.; Kularatne, S.; Low, P.S.;
Wei, A. Bishydrazide glycoconjugates for lectin recognition and capture of bacterial
pathogens. Bioconjugate Chem. 2010, 21(11), 2065–2075. doi: 10.1021/bc100288c.
[25] Wilchek, M.; Bayer, E.A. The avidin-biotin complex in bioanalytical applications.
Anal. Biochem. 1988, 171(1), 1–32. doi: 10.1016/0003-2697(88)90120-0.
[26] O’Shannessy, D.J.; Dobersen, M.J.; Quarles, R.H. A novel procedure for labeling
immunoglobulins by conjugation to oligosaccharide moieties. Immunol. Lett. 1984,
8, 273–277. doi: 10.1016/0165-2478(84)90008-7.
[27] Byeon, J.-Y.; Limpoco, F.T.; Bailey, R.C. Efficient bioconjugation of protein capture
agents to biosensor surfaces using aniline-catalyzed hydrazone ligation. Langmuir
2010, 26(19), 15430–15435. doi: 10.1021/la1021824.
[28] Shi, M.; Kleski, K.A.; Trabbic, K.R.; Bourgault, J.-P.; Andreana, P.R. Sialyl-Tn poly-
saccharide A1 as an entirely carbohydrate immunogen: synthesis and immunological
evaluation. J. Am. Chem. Soc. 2016, 138(43), 14264–14272. doi: 10.1021/jacs.
6b05675.
[29] Ghosh, S.; Nishat, S.; Andreana, P.R. Synthesis of an aminooxy derivative of the tet-
rasaccharide repeating unit of Streptococcus dysgalactiae 2023 polysaccharide for a
PS A1 conjugate vaccine. J. Org. Chem. 2016, 81(11), 4475–4484. doi: 10.1021/acs.
joc.6b00195.
[30] Fife, T.H. Participation of solvent and general acids in acetal hydrolysis. Hydrolysis
of 2-(para-substituted phenyl)-4,4,5,5-tetramethyl-1,3-dioxolanes. J. Am. Chem. Soc.
1967, 89(13), 3228–3231. doi: 10.1021/ja00989a024.
[31] Piszkiewicz, D.; Bruice, T.C. Glycoside hydrolysis. I. Intramolecular acetamido and
hydroxyl group catalysis in glycoside hydrolysis. J. Am. Chem. Soc. 1967, 89(24),
6237–6243. doi: 10.1021/ja01000a044.
[32] Capon, B.; Smith, M.C. Intramolecular catalysis in acetal hydrolysis. Chem.
Commun. (London) 1965 (21), 523–524. doi: 10.1039/c19650000523.
[33] Dey, S.; Jayaraman, N. Glycosidic bond hydrolysis in septanosides: a comparison of
mono-, di-, and 2-chloro-2-deoxy-septanosides. Carbohydr. Res. 2014, 399, 49–56.
doi: 10.1016/j.carres.2014.05.013.
[34] Kancharla, P.K.; Crich, D. Influence of side chain conformation and configuration
on glycosyl donor reactivity and selectivity as illustrated by sialic acid donors

22
F. HOSSAIN ET AL.
epimeric at the 7-position. J. Am. Chem. Soc. 2013, 135(50), 18999–19007. doi: 10.
1021/ja410683y.
[35] Surana, N.K.; Kasper, D.L. The yin yang of bacterial polysaccharides: lessons learned
from B. fragilis PSA. Immunol. Rev. 2012, 245(1), 13–26. doi: 10.1111/j.1600-065X.
2011.01075.x.
[36] Anderson, E.; Fife, T.H. General acid catalysis of ketal hydrolysis. The hydrolysis of
tropone diethyl ketal. J. Am. Chem. Soc. 1969, 91(25), 7163–7166. doi: 10.1021/
ja01053a046.
[37] Mayer, S.C.; Gallaway, W.; Kulishoff, J.; Yin, M.; Gadamasetti, V.; Mitchell, R.
Stability studies of C-40,60 acetal benzylmaltosides synthesized as inhibitors of
smooth muscle cell proliferation. Bioorg. Med. Chem. Lett. 2004, 14(11), 2829–2833.
doi: 10.1016/j.bmcl.2004.03.049.
[38] Fife, T.H.; Anderson, E. Intramolecular carboxyl group participation in acetal
hydrolysis. J. Am. Chem. Soc. 1971, 93(24), 6610–6614. doi: 10.1021/ja00753a047.
[39] Bohe, L.; Crich, D. A propos of glycosyl cations and the mechanism of chemical gly-
cosylation; the current state of the art. Carbohydr. Res. 2015, 403, 48–59. doi: 10.
1016/j.carres.2014.06.020.
[40] Zatsepin, T.S.; Stetsenko, D.A.; Arzumanov, A.A.; Romanova, E.A.; Gait, M.J.;
Oretskaya, T.S. Synthesis of peptide ꢃ oligonucleotide conjugates with single and
multiple peptides attached to 20-aldehydes through thiazolidine, oxime, and hydra-
zine linkages. Bioconjugate Chem. 2002, 13(4), 822–830.
[41] Kalia, J.; Raines, R.T. Hydrolytic stability of hydrazones and oximes. Angew. Chem.
Int. Ed. 2008, 47(39), 7523–7526. doi: 10.1002/anie.200802651.
[42] Ngoje, G.; Addae, J.; Kaur, H.; Li, Z. Development of highly stereoselective GalN3
donors and their application in the chemical synthesis of precursors of Tn antigen.
Org. Biomol. Chem. 2011, 9(19), 6825–6831. doi: 10.1039/c1ob05893b.
[43] Ziegler, T. Rhizobial saccharides 2. Selective synthesis of both diastereomers of 4,6-
O-pyruvylated d-glycopyranosides. Tetrahedron Lett. 1994, 35(37), 6857–6860. doi:
10.1016/0040-4039(94)85023-2.
[44] Salmani, J.; Asghar, S.; Lv, H.; Zhou, J. Aqueous solubility and degradation kinetics
of the phytochemical anticancer thymoquinone; probing the effects of solvents, pH
and light. Molecules 2014, 19(5), 5925–5939. doi: 10.3390/molecules19055925.
€
[45] Liimatainen, H.; Sirvio, J.; Pajari, H.; Hormi, O.; Niinimaki, J. Regeneration and
recycling of aqueous periodate solution in dialdehyde cellulose production. J. Wood
Chem. Tech. 2013, 4, 258–266. doi: 10.1080/02773813.2013.783076.
[46] Pantosti, A.; Tzianabos, A.O.; Onderdonk, A.B.; Kasper, D.L. Immunochemical char-
acterization of two surface polysaccharides of Bacteroides fragilis. Infec. Immun.
1991, 59, 2075–2082.
[47] Kalka-Moll, W.M.; Wang, Y.; Comstock, L.E.; Gonzalez, S.E.; Tzianabos, A.O.;
Kasper, D.L. Immunochemical and biological characterization of three capsular pol-
ysaccharides from a single ltem Bacteroides fragilis strain. Infect. Immun. 2001,
69(4), 2339–2344. doi: 10.1128/IAI.69.4.2339-2344.2001.
[48] Ohlsson, J.; Magnusson, G. Galabiosyl donors; efficient synthesis from 1,2,3,4,6-
penta-O-acetyl-b-D-galactopyranose. Carbohydr. Res. 2000, 329(1), 49–55. doi: 10.
1016/S0008-6215(00)00154-3.
[49] Oßwald, M.; Lang, U.; Friedrich-Bochnitschek, S.; Pfrengle, W.; Kunz, H.
Regioselective glycosylation of glucosamine and galactosamine derivates using O-
pivaloyl galactosyl donors. J Charbohydr. Sci. 2003, 58, 764. doi: 10.1515/znb-2003-
0808.

JOURNAL OF CARBOHYDRATE CHEMISTRY
23
ꢀ
[50] Gold, H.; Boot, R.G.; Aerts, J.M.F.G.; Overkleeft, H.S.; Codee, J.D.C.; van der Marel,
G.A. A concise synthesis of globotriaosylsphingosine. Eur. J. Org. Chem. 2011,
2011(9), 1652–1663. doi: 10.1002/ejoc.201001690.
ꢀ
[51] Gola, G.; Libenson, P.; Donadıo, L.; Rodriguez, C. Synthesis of 2,3,5,6-tetra-O-ben-
zyl-D-galactofuranose for a-glycosidation. ARKIVOC 2005, 12, 234–242.