Synthesis of an aminooxy derivative of the GM3 antigen and its application in oxime ligation

Article abstract of DOI:10.1021/acs.joc.0c00320

The anomeric aminooxy GM3 trisaccharide cancer antigen (Neu5Acα2,3Galβ1,4Glcβ-ONH₂) has been chemically synthesized using a linear glycosylation approach. The key step involves a highly α(2,3)-stereoselective sialylation to a galactose acceptor

Full text of DOI:10.1021/acs.joc.0c00320

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Note
Synthesis of an aminooxy derivative of the GM3
antigen and its application in oxime ligation
Kristopher A. Kleski, Mengchao Shi, Matthew Lohman,
Gabrielle T. Hymel, Vinod K. Gattoji, and Peter R. Andreana
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.0c00320 • Publication Date (Web): 22 Apr 2020
Downloaded from pubs.acs.org on April 25, 2020
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Page 1 of 12
The Journal of Organic Chemistry
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Synthesis of an aminooxy derivative of the GM3 antigen and its appli-
cation in oxime ligation.
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Kristopher A. Kleski, Mengchao Shi, Matthew Lohman, Gabrielle T. Hymel, Vinod K. Gattoji, Peter R. Andreana*
Department of Chemistry and Biochemistry and School of Green Chemistry and Engineering, University of Toledo, 2801
West Bancroft Street, Toledo, Ohio 43606, United States.
*
peter.andreana@utoledo.edu
Supporting Information Placeholder
2
ABSTRACT: The anomeric aminooxy GM3 trisaccharide cancer antigen (Neu5Acα2,3Galβ1,4Glcβ-ONH ) has been chemically
synthesized using a linear glycosylation approach. The key step involves a highly α(2,3)-stereoselective sialylation to a galactose
acceptor. The Neu5Acα2,3Gal intermediate was functionalized as a donor for a [2 + 1] glycosylation including a glucose acceptor
that featured an O-succinimidyl group on the reducing end as an aminooxy precursor. The fully deprotected anomeric aminooxy
GM3 trisaccharide was then conjugated to the immunologically relevant zwitterionic polysaccharide PS A1 via an oxime link.
Oxime ligation has emerged as an attractive conjugation
method in vaccine formulation for a number of reasons: 1) ox-
ime bonds are hydrolytically stable in physiological condi-
Figure 1. General structure of TACA-PS A1 glycoconju-
gates and aminooxy derivatives of TACAs.
1
-3
tions, 2) the oxime bond is immunologically “silent” as com-
4-5
pared to other conjugation linkages, 3) the condensation of
aminooxy functions are fast, quantitative, and only produce wa-
6-7
ter as a byproduct.
Our group has used the oxime ligation
strategy to create entirely carbohydrate immunogens capable of
generating protective immune responses against tumor associ-
ated carbohydrate antigens (TACAs).8-11 These glycoconju-
gates feature the unique zwitterionic polysaccharide PS A1
which is isolated from the commensal organism Bacteroides
fragilis (ATCC 25285/NCTC 9343). The alternating charge
character on the repeating unit of PS A1 provides distinct prop-
erties that permit binding to the MHC II complex on antigen
presenting cells and downstream activation of CD4+ T cells
12-13
through the MHC II:αβTCR complex pathway.
Formation
of these immunogens is materialized by the oxime condensation
of a chemically derived aldehyde on PS A1 and with an ami-
nooxy derivative of a chosen TACA (Figure 1). This vaccine
also positively correlated with tumor malignancy leading to
1
6
poor prognosis. Due to these characteristics, GM3 has been
prioritized within the top 50 cancer antigens for immune inter-
8
, 14
platform has been successfully demonstrated with Tn-PS A1
1
7
1
0
vention.
and STn-PS A1 where we observed robust IgG antibody pro-
duction, cellular immunity, and tumor cell lysis.
The biological relevance of the GM3 antigen implicates ne-
cessity for preparative ease and availability for immunological
experiments. Indeed, both chemo-enzymatic and chemical
methods exist for the production of the GM3 antigen, but they
Progress of these initial developments led us to expand our
TACA toolbox to incorporate the ganglioside GM3
(Neu5Acα2,3Galβ1,4Glcβ-Cer). GM3 is overexpressed in car-
1
8-25
have limitations.
Chemo-enzymatic methods offer excel-
cinomas of the breast and skin and has implications in tumor
cell proliferation and metastasis.1
5-16
GM3 overexpression is
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lent regio- and stereo-selective control of glycan production but

The Journal of Organic Chemistry
Page 2 of 12
are typically demonstrated on smaller scales.19, 26 On the other
Scheme 1. Retrosynthetic Analysis of the Aminooxy
GM3 Glycan
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hand, synthetic reports of the GM3 glycan relay poor stereose-
lectivity forming the challenging α(2→3) glycosidic bond. Up
to date, only one synthesis of the aminooxy GM3 glycan has
been reported through a sialyltransferase catalyzed glycosyla-
1
9
tion. Herein, we describe our chemical synthetic platform to
produce the aminooxy GM3 glycan by using a linear approach
that features a high α-selective sialylation in good yield. The
final step involves a microwave assisted global deprotection of
-OAc/-OBz protecting groups and an O-succinimidyl (OSu)
protecting group to achieve the corresponding alcohol and ami-
nooxy functions, respectively. The fully deprotected aminooxy
GM3 glycan was then conjugated to PS A1 by oxime ligation
to form the GM3-PS A1 glycoconjugate.
0
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The synthetic route to the aminooxy GM3 glycan (1) must
feature high α-selectivity in the Neu5Ac2,3Gal bond forming
step. To achieve this, we elected to use the α-directing trans-
fused 4O,5N-acetyl oxazolidinone ring system on the sialic acid
1
0, 27-28
donor.
This moiety exhibits strain on the cyclic system
which destabilizes the formation of the glycosyl oxocarbenium
ion intermediate, and instead a β-triflate intermediate is more
likely formed. This pushes the mechanism of glycosylation to-
wards more of an S
to an unselective S
a highly reactive anomeric leaving group, such as a phosphate
N
2-like associative mechanism as opposed
29
N
1 dissociative mechanism. Additionally,
30-31
10
ester,
was desired based on our previous experiences.
forming step, but feature a greater number of overall synthetic
These two features led us to adopt the sialyl phosphate donor 3
(Scheme 1, Scheme S1). The synthetic strategy can be further
broken down through pathway A or pathway B (Scheme 1).
Reports that describe pathway A in the synthesis of the GM3
glycan discuss high yields and α-selectivity in the α(2→3) bond
24
steps than pathway B. Contemporary reports of GM3 synthe-
sis describe pathway B with the use of lactosyl acceptors, but
these reports describe low yields in the α(2→3) bond forming
25
step. We must also account for the introduction of -OSu on
the reducing end of the protected trisaccharide as the precursor
Table 1. Optimization of α(2,3)-Glycosylationa
b
c
entry
1
acceptor
product
yield α:β ratio
68%
81%
52%
89%
87%
1:5
1:4
2
3
4
5
1:1
8:1
16:1
a
2 2
Typical conditions: 1.5 equivalents of donor, 1.0 equivalent of acceptor, 1.5 equivalents of TMSOTf, in dry CH Cl at -78 °C
b
c
1
for 1 hour. Isolated yield. Determined by H NMR spectroscopic analysis of the unpurified reaction mixture.
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Page 3 of 12
The Journal of Organic Chemistry
for an aminooxy group.9-10 This requires limited use of benzyl
ether protecting groups, as long exposure times of hydrogena-
tion can cause undesired cleavage of the -OSu N-O bond. We
proceeded to screen various glycosyl acceptors for the α(2→3)
bond forming step using sialyl phosphate 3 as the donor (Table
Scheme 3. Attempted 2 + 1 Glycosylation Through In-
termediate 13
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2
1
).
We commenced with screening lactose acceptors 4 (Table 1,
entry 1) and 5 (Table 1, entry 2, Scheme S2). Both acceptors
feature a 3’,4’-diol and differ on their reducing ends with 4 con-
taining thiophenol and 5 containing an O-succinimidyl. Both
acceptors 4 and 5 provided the β-anomer as the major product
with α:β ratios of 1:5 and 1:4 respectively. Poor selectivity is
0
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in line with previous reports and is a consequence of the dis-
25, 33
arming effect of acetyl group protections.
We then looked
toward the 3, 2’, 3’, 4’-unprotected lactoside 6 (Table 1, entry
3) which has been described to provide improved α-formation
as compared to 4 and 5 due to the relatively weaker disarming
effect of pivaloyl groups.34 Acceptor 6 resulted in an improved
α:β ratio at 1:1, although this ratio remained unsatisfactory.
Based on these observed limitations of lactose acceptors, we in-
vestigated the use of galactose acceptor 735 which featured a
3
,4-diol with a 2-O-benzoyl-6-O-benzyl protection strategy
(Table 1, entry 4). The resulting α:β ratio was drastically im-
proved to provide the α-anomer in an 8:1 ratio in 89% yield.
The greatly improved α:β ratio led us to explore galactose ac-
NIS/TMSOTf activation conditions. TLC analysis of the reac-
tion mixture revealed partial consumption of the donor (Rf =
0.4) accompanied by the presence of a new spot (Rf = 0.2). In-
vestigation of the reaction revealed the new spot to be the gly-
cosyl succinimide product (S6). Indeed, this activation system
produces the weakly nucleophilic succinimide as a by-product,
which can outcompete weakly nucleophilic or sterically hin-
dered alcohols on glycosyl acceptors. This phenomenon has
been described for donor/acceptor pairs with mismatched reac-
31
ceptor 8 which contains the conformation restricting 4,6-O-
benzylidene acetal (Table 1, entry 5). Galactose acceptor 8 pro-
vided excellent α-selectivity with an α:β ratio of 16:1 in 87%
yield. It was logical at this point to proceed through pathway A
using galactosyl acceptor 8.
With disaccharide 13 in hand, we turned our attention to the
D-glucose acceptor 16 as shown in Scheme 2. Here, thio-donor
1
4 was activated using N-iodosuccinimide (NIS) and trime-
36
tivities. Notably, we also discovered partial proton exchange
thylsilyl trifluoromethanesulfonate (TMSOTf) in the presence
of N-hydroxysuccinimide (NHS) to afford compound 15 in 84%
yield as a pure β-anomer. Selective ring opening of the 4,6-
benzylidene acetal, on compound 15, was performed with tri-
with trimethyl silane on acceptor 16. We attempted the [2 + 1]
coupling using different activation systems such as
NIS/trifluoromethanesulfonic acid (TfOH), dimethyl disulfide-
triflic anhydride (Me
TCA) donor (18). The results led to a glycosyl succinimide
by-product, a complex mixture, and the Chapman rearrange-
2 2 2
S -Tf O), and a trichloroacetamidate
ethylsilane (Et
3
SiH) as the hydride source and boron trifluoride
(
Scheme 2. Synthesis of Glc-OSu Acceptor 16
3
7
ment respectively (Scheme 3). These experiments offer
Scheme 4. Synthesis of the Aminooxy GM3 Glycan
3 2
diethyl etherate (BF ·Et O) as the catalyst. Successful regiose-
lective ring opening to afford 16 was confirmed using HMBC
as the C-6 position nicely correlated with the benzylic protons
on the benzyl ether.
Disaccharide 13 (Table 1, entry 5) was subjected to Zemplén
sodium methoxide conditions to remove the oxazolidinone, and
the resulting free hydroxyls were acetylated to provide com-
pound 17 as shown in Scheme 3. Interestingly, the C-2 O-ben-
zoylated protection on compound 13 was not removed during
the Zemplén method. This is likely due to the steric hinderance
2
1
of this position, especially within disaccharide systems. With
disaccharide donor 17 and glucose acceptor 16 in hand, we
sought to perform this [2 + 1] glycosylation under typical
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The Journal of Organic Chemistry
Page 4 of 12
further evidence of the torsional disarming effect from 4,6-O-
deprotection of 21. Repetitive cycling (flow-chemistry tech-
acetal bicyclic donors.38
niques) of the aforementioned conditions led to MS analysis
clearly depicting fully deprotected 21. The reaction mixture
was then concentrated, resuspended in water, and passed
through a P2 BioGel® column using water as the eluent. Frac-
tions identified to contain compound 1 by TLC staining were
collected, frozen, and lyophilized. The resulting white solid
was characterized and compound 1 was definitively confirmed
by NMR and HRMS (See supporting information).
1
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We sought to deviate the synthetic route through the more
armed disaccharide donor compound 12 (Table 1, entry 4). As
illustrated in Scheme 4, preparation of the disaccharide donor
20 mirrored the preparation of donor 17, with the C-2-O-ben-
zoyl protection remaining intact. Donor 20 was then activated
with the NIS/TfOH system in the presence of acceptor 16 and
provided the desired, fully protected trisaccharide 2 in 87%
yield as the β-anomer. No evidence of the glycosyl succinimide
was present.
With the aminooxy GM3 glycan complete, we pursued oxime
ligation with PS A1 (Scheme 5, 22). The repeating unit of PS
0
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o
o
A1 contains the side chain D-galactofuranose with a 1 and 2
vicinal diol that can be chemo- and regio-selectively oxidized
Deprotection of key intermediate 2 began with the cleavage
of the benzyl ethers using hydrogen gas at 1 atm and a suspen-
sion of Pearlman’s catalyst. TLC indicated full consumption of
starting material after 1 hour of mixing and the reaction was
purged with argon gas to remove dissolved hydrogen gas. The
reaction was filtered, concentrated, and passed through a short
silica column to afford the debenzylated product S7 in 89%
yield with no evidence of -OSu N-O cleavage. Deprotection of
the Neu5Ac C-1 methyl ester was accomplished using the Krap-
8-11
by sodium periodate to afford an aldehyde.
After PS A1 ox-
idation, the aldehyde moiety was then subjected to four equiva-
lents of 1 and subsequently stirred for 18 hours in a 0.1 M so-
dium acetate buffer at a pH of 5. The reaction mixture was then
filtered and rinsed through a 10 kDa molecular weight cutoff
filter. The filtered material was then resuspended in water, fro-
zen, and lyophilized to produce 23 as a white (cotton-like) sub-
stance. NMR analysis of this material at 60 °C provided the
evidence we needed to note successful conjugation with PS A1.
The defining peaks included an oxime proton peak (δ = 7.99-
1
0
cho-like demethylation by lithium iodide in pyridine. This
mixture was refluxed at 110 °C for 3 hours in the absence of
light. Pyridine was then removed in vacuo and the crude mate-
rial was purified by silica flash chromatography to afford com-
pound 21 in 68% yield.
8
.00), an additional anomeric proton peak (δ = 5.37), an addi-
tional N-acetyl peak (δ = 2.31), and the presence of the Neu5Ac
H-3eq (δ = 3.03-3.06) and H-3ax peaks (δ = 2.04-2.08). Based
on integration analysis of the Neu5Ac H-3eq and PS A1 AAT
methyl peak, we determined the relative ratio of GM3:PS A1 to
be 1:2.33, which translates into approximately 51.5 GM3 con-
jugation sites per PS A1.
In the final deprotection step, we utilized excess hydrazine-
hydrate to remove the remaining -OAc/-OBz protecting groups
and to remove -OSu to expose the aminooxy functionality.
However, based on the results of Zemplén sodium methoxide
conditions as described for 12 and 13 we did not expect depro-
tection of the C-2-O-benzoylatd positions using hydrazine hy-
In conclusion, we have discussed the first fully chemical syn-
thetic method to provide the aminooxy GM3 glycan. This syn-
thesis features highly α-selective chemistries in the formation
of the Neu5Ac(2→3)Gal bond. In these experiments we
showed the benefit of using the armed galactose acceptors as
opposed to the disarmed lactose acceptors, which gave undesir-
able α:β ratios. This synthesis also features a 3-step deprotec-
tion strategy of the fully protected trisaccharide. Notably, the
final step utilized microwave assisted chemistry to deprotect the
sterically hindered C-2 O-benzoylations in excellent yield, after
reiterations. Lastly, we highlighted the utility of the aminooxy
functionality by conjugating the GM3 glycan to the immuno-
logically relevant PS A1 to generate the GM3-PS A1 glycocon-
jugate. The GM3-PS A1 vaccine candidate will be investigated
for its immunological characteristics in due time.
drate at room temperature. Additionally, literature precedent
suggested long reaction times24 and elevated temperatures25 in
hydroxide or methoxide conditions for C-2-O-benzoyl removal,
which would then cause an undesirable ring opening of the in-
stalled -OSu group and failure of aminooxy deprotection. To
overcome this limitation, we turned to microwave assistance in
the final deprotection step. Compound 22 was dissolved in eth-
anol followed by the addition of excess hydrazine hydrate. The
mixture was then stirred for 1 hour at room temperature and MS
analysis did not detect any formation of product. The reaction
was then subjected to microwave irradiation at 200 Watts with
constant temperature of 40 °C for 20 minutes. Once again, MS
analysis indicated little-to-no deprotection of compound 21.
We then increased the temperature to a constant 90 °C for 30
minutes with no change in maximum Watts. Following this
procedure, MS analysis of the reaction mixture revealed partial
EXPERIMENTAL SECTION
General Information. Relevant reagents and solvents were
purchased from commercial sources and used without further
purification. Molecular sieves (4 Å) were dried overnight in a
vacuum oven set to 140 °C, and further dried under high vac-
uum using a heat gun for 5 minutes and cooled to room temper-
ature under high vacuum. Synthesized compounds were puri-
fied using flash chromatography with SiliCycle Inc. 60 Å 230-
Scheme 5. Synthesis of GM3-PS A1
4
00 mesh silica gel or size exclusion chromatography with Bio-
Gel P-2 (Bio-Rad Laboratories, Inc). Thin layer chromatog-
raphy (TLC) was performed with SiliCycle Inc. silica gel TLC
2
50 µm w/h F-254. Proton and carbon NMR spectra were rec-
orded using a Bruker Avance III 600 Ultrafield Cryoprobe spec-
trometer. CDCl , CD OD, and D O were used as solvents, and
3
3
2
the chemical shifts were referenced relative to residual solvent.
Low resolution mass spectrometry data were taken on an LCQ
Deca ESI-MS machine. High resolution mass spectrometry
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The Journal of Organic Chemistry
then dissolved in 2 0ꢀ mL of dry CH
data were collected using an Orbitrap Fusion Tribrid Mass
2
2
Cl followed by the addition
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Spectrometer.
of 4 Å molecular sieves (4 g) and dibutyl phosphate (1.75 mL,
8.80 mmol, 5.0 equiv). Gas in the reaction vessel was ex-
changed with Ar and this solution was then stirred for 30
minutes at room temperature, followed by the addition of NIS
Experimental Procedures
Methyl
(phenyl-5-acetamido-7,8,9-tri-O-acetyl-5-N,4-O-
carbonyl-3,5-dideoxy-2-thio-D-glycero-α,β-D-galacto-non-2-
ulopyranoside)onate (S2). Compound S1 (5.20 g, 8.91 mmol,
(
1.19 g, 5.28 mmol, 3.0 equiv). Gas was then exchanged with
Ar again and the reaction was stirred at room temperature for
2 hours. After 12 hours, the reaction was diluted with CH Cl
30 mL) and quenched with a saturated solution of Na and
(30 mL). The quenched reaction was then filtered,
28
1
.0 equiv) was suspended in 75 mL of MeOH with vigorous
1
(
2
2
stirring. To the suspension was added MeSO H (2.90 mL, 44.6
3
2 2 3
S O
mmol, 5.0 equiv) at room temperature and the suspension was
refluxed for 24 hours. The mixture was then cooled to room
temperature and the reaction quenched with excess TEA. The
solution was then concentrated under reduced pressure and
dried under vacuum. The resulting crude concentrate was then
dissolved in 50 mL MeCN and 100 mL H
addition of NaHCO (3.74 g, 44.6 mmol, 5.0 equiv). The mix-
ture was stirred vigorously to a uniform suspension and cooled
to 0 °C. A separate solution of triphosgene (7.93 g, 26.7 mmol,
NaHCO
3
and the collected organic layer was washed with saturated
Na and NaHCO (30 mL, 2X). The resulting organic layer
was washed with brine, dried over Na SO , filtered, and then
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
S
2
O
3
3
2
4
concentrated under reduced pressure. The crude compound was
then purified via flash chromatography using hexanes:EtOAc
(1:1) to give sialyl donor 3 as an anomeric mixture and as a
ꢀ
ꢀ
2
O followed by the
3
1
yellow oil (1.05 g, 1.57 mmol, 89% yield). (3α) H NMR
(
1
CDCl
.68 (4H, m), 2.06 (3H, s), 2.13 (3H, s), 2.17 (3H, s), 2.52 (3H,
s), 2.71 (1H, t, J = 12.7 Hz), 3.03 (1H, dd, J = 4.0, 12.2 Hz),
.86 (3H, s), 3.89 (1H, t, J = 10.1 Hz), 4.14 (6H, m), 4.43 (1H,
dd, J = 2.8, 12.3 Hz), 4.78 (1H, dd, J = 1.4, 9.5 Hz), 5.34 (1H,
3
, 600 MHz): δ = 0.96 (6H, t, J = 7.4 Hz), 1.43 (4H, m),
ꢀ
3
.0 equiv) dissolved in 50 mL MeCN was then added to the
vigorously stirred suspension via dropping funnel at a slow rate
of ~1 drop/5 seconds. The reaction was maintained at 0 °C for
5 hours. EtOAc (100 mL) was then added to the mixture and
3
ꢀ
vigorously stirred for 10 minutes. The organic and aqueous lay-
ers were separated, and the aqueous layer was extracted with
13
1
3
m), 5.69 (1H, dd, J = 1.4, 7.5 Hz). C{ H} NMR (CDCl , 150
MHz): δ = 13.6, 18.6, 18.7, 20.8, 20.8, 21.0, 24.7, 32.1, 32.1,
32.1, 35.9, 35.9, 53.5, 58.3, 62.5, 68.0, 68.1, 68.1, 68.2, 69.8,
ꢀ
EtOAc (100 mL, 2X). The combined organic layers were
2 4
washed with brine, dried over Na SO , and concentrated under
71.5, 74.2, 98.2, 98.2, 153.6, 167.3, 167.3, 169.9, 170.0, 170.7,
reduced pressure. The resulting residue was passed through a
short, thick bed of silica eluting with EtOAc:MeOH (9:1). Frac-
tions containing the intermediate were pooled and concentrated
under reduced pressure and then set under high vacuum over-
night to give the crude intermediate (3.02 g, 7.56 mmol, 85%
yield of crude). The crude material was then dissolved in pyri-
dine (35 mL) followed by the addition of DMAP (0.18 g, 1.5
mmol, 0.2 equiv). Gas was exchanged with Ar and the reaction
+
171.9. LRMS (ESI) m/z: [M + Na] calcd for C27
H
42NO16PNa
1
6
90.2; Found 690.4. (3β) H NMR (CDCl
6H, dt, J = 4.2, 7.4 Hz), 1.42 (4H, m), 1.68 (4H, m), 2.05 (3H,
s), 2.12 (3H, s), 2.15 (3H, s), 2.32 (1H, dt, J = 2.8, 12.7 Hz),
3
, 600 MHz): δ = 0.96
(
2
9
1
.52 (3H, s), 2.91 (1H, dd, J = 3.7, 12.8 Hz), 3.79 (1H, dd, J =
.5, 11.3 Hz), 3.88 (3H, s), 4.13 (5H, m), 4.56 (1H, dd, J = 2.6,
2.2 Hz), 4.60 (1H, td, J = 3.7, 11.4, 12.6 Hz), 4.75 (1H, dd, J
=
2.0, 9.5 Hz), 5.28 (1H, m), 5.64 (1H, dd, J = 2.0, 4.0 Hz).
C{ H} NMR (CDCl , 150 MHz): δ = 13.6, 18.6, 18.6, 20.8,
3
mixture was cooled to 0 °C with vigorous stirring. Ac
2
O (14.3
13
1
mL, 151 mmol, 20 equiv) was then injected into the reaction
mixture and the reaction was stirred for 12 hours while the re-
action was allowed to slowly warm to room temperature. The
reaction was then quenched with MeOH at 0 °C and then con-
centrated under reduced pressure followed by co-distillation
with toluene (3X). The crude material was then dissolved in
21.0, 24.7, 32.1, 32.1, 32.1, 32.2, 36.1, 36.1, 53.5, 58.9, 62.9,
68.4, 68.4, 68.5, 68.6, 71.8, 72.6, 74.0, 76.7, 98.8, 98.9, 153.5,
165.6, 169.8, 170.6, 170.7, 172.2. LRMS (ESI) m/z: [M + Na]+
calcd for C27
H
42NO16PNa 690.2; Found 690.4.
Phenyl
(2,6-di-O-acetyl-β-D-galactopyranosyl)-(1→4)-
2,3,6-tri-O-acetyl-1-thio-β-D-glucopyranoside (4). Acceptor 4
CH
X). The resulting organic layer was washed with brine, dried
with Na SO , filtered, and concentrated under reduced pressure.
2 2 3
Cl (50 mL) and washed with saturated NaHCO (50 mL,
3
9
was prepared as previously described. Acceptor 4 was puri-
fied by silica flash chromatography using 2% MeOH in CH Cl
as the eluent to provide 4 as a white solid (0.50 g, 0.78 mmol).
3
2
2
2
4
The crude material was then purified by silica flash chromatog-
raphy eluting with hexanes:EtOAc (1:1) to give compound S2
1
H NMR (CDCl
3H, s), 2.10 (3H, s), 2.11 (3H, s), 3.59 (1H, dd, J = 3.5, 9.8 Hz),
3.62 (1H, t, J = 6.5 Hz), 3.69 (2H, m), 3.86 (1H, d, J = 3.0 Hz),
3
, 600 MHz): δ = 2.03 (3H, s), 2.08 (3H, s), 2.09
(
as a white solid (4.00 g, 7.05 mmol, 79% yield over 3 steps).
1
H NMR (CDCl
3H, s), 2.34 (1H, t, J = 13.0 Hz), 2.53 (3H, s), 2.91 (1H, dd, J
3.7, 13.1 Hz), 3.63 (3H, s), 3.76 (1H, dd, J = 9.2, 11.3 Hz),
3
, 600 MHz): δ = 1.96 (3H, s), 2.09 (3H, s), 2.15
4
4
.16 (1H, dd, J = 5.5, 11.8 Hz), 4.22 (1H, dd, J = 6.6, 11.4 Hz),
.32 (2H, m), 4.51 (1H, dd, J = 1.9, 12.0 Hz), 4.69 (1H, d, J =
(
=
10.1 Hz), 4.89 (1H, dd, J = 8.0, 9.8 Hz), 4.93 (1H, t, J = 9.7 Hz),
3
4
5
.90 (1H, dd, J = 8.2, 12.1 Hz), 4.37 (1H, dd, J = 2.5, 12.0 Hz),
.78 (1H, td, J = 4.3, 15.0 Hz), 4.89 (1H, dd, J = 11.7, 6.6 Hz),
.00 (1H, td, J = 2.2, 8.2 Hz), 5.56 (1H, t, J = 2.3 Hz), 7.36 (2H,
1
3
1
5.19 (1H, t, J = 8.9 Hz), 7.31 (3H, m), 7.47 (2H, m). C{ H}
NMR (CDCl , 150 MHz): δ = 21.1, 21.1, 21.2, 21.2, 62.7, 68.8,
70.4, 72.6, 73.0, 73.7, 74.1, 76.5, 77.1, 85.9, 101.2, 128.5,
29.2, 133.0, 169.9, 170.8, 170.8, 171.3, 171.7. HRMS (ESI)
3
13
1
3
m), 7.43 (1H, m), 7.50 (2H, m). C{ H} NMR (CDCl , 150
MHz): δ = 20.7, 20.8, 21.1, 24.7, 36.0, 52.8, 59.6, 62.9, 72.7,
73.9, 75.1, 75.7, 88.4, 128.2, 129.2, 130.2, 136.8, 153.6, 167.8,
169.7, 170.4, 171.2, 172.5. LRMS (ESI) m/z: [M + Na] calcd
for C25H29NO12SNa 590.1; Found 590.2.
1
+
m/z: [M + Na] calcd for C28
667.1769.
36
H O15SNa 667.1667; Found
+
Trichloroacetimidatyl
(2,6-di-O-acetyl-3,4-O-isopropy-
lidene-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-β-D-
Methyl (5-acetamido-7,8,9-tri-O-acetyl-5-N,4-O-carbonyl-
2-(dibutylphosphoryl)-3,5-dideoxy-D-glycero-α,β-D-galacto-
non-2-ulopyranoside)onate (3). Compound S2 (1.00 g, 1.76
mmol, 1.0 equiv) was dried by co-distillation with toluene (3X)
followed by high vacuum for 3 hours. The dry compound was
3
9
glucopyranoside (S4). Compound S3 (0.67 g, 0.98 mmol, 1.0
ꢀ
equiv) was dissolved in an acetone:H
2
O (4:1, 10 mL) mixture
and then cooled to 0 °C. While stirring the mixture, NBS (0.52
g, 2.9 mmol, 3.0 equiv) was added to the reaction gradually over
the duration of 30 minutes. The reaction was then stirred for an
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 6 of 12
additional 30 minutes at room temperature. The solution was
then concentrated under reduced pressure until the solution be-
S5 (69 mg, 0.10 mmol, 1.0 equiv) was dissolved in 5 mL of an
AcOH:H O (4:1) mixture and was heated at 80 °C for 1 hour
with a condenser attached to the reaction flask. The reaction
was then concentrated under reduced pressure followed by co-
distillation with toluene under reduced pressure (3X). The
crude was then purified by silica flash chromatography using
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
came turbid. The turbid mixture was diluted with CH
mL) and then quenched and washed with saturated Na
NaHCO
brine, dried with Na
2
Cl
S O
2 2 3
2
(20
and
3
(15 mL, 3X). The organic layer was then washed with
SO , filtered, and then concentrated under
2
4
reduced pressure to give the crude hemiacetal intermediate as a
white solid (0.51 g, 0.86 mmol, 88% yield of crude). The crude
hemiacetal was then co-distilled with toluene under reduced
pressure (3X) and set under high vacuum for 3 hours. The crude
2% MeOH in CH
5 as a white solid (55 mg, 0.084 mmol, 84% yield). H NMR
(CDCl , 600 MHz): δ = 2.08 (3H, s), 2.11 (3H, s), 2.11 (3H, s),
2 2
Cl as the eluent to provide lactosyl acceptor
1
3
2.12 (3H, s), 2.13 (3H, s), 2.73 (4H, s), 3.00 (1H, br), 3.28 (1H,
br), 3.61 (1H, dd, J = 2.7, 9.4 Hz), 3.64 (1H, t, J = 6.5 Hz), 3.75
(1H, m), 3.84 (1H, br), 4.10 (1H, t, J = 9.4 Hz), 4.21 (2H, m),
4.36 (1H, dd, J = 6.4, 11.5 Hz), 4.40 (1H, d, J = 7.9 Hz), 4.47
(1H, dd, J = 2.3, 12.0 Hz), 4.86 (1H, dd, J = 8.0, 9.6 Hz), 5.13
ꢀ
hemiacetal was then dissolved in dry CH Cl (10 mL) followed
2 2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
by the addition of 4 Å molecular sieves (2 g) and trichloroace-
tonitrile (345 μL, 3.44 mmol, 4.0 equiv), and then gas was ex-
changed with Ar. The solution was stirred at room temperature
for 30 minutes and was then cooled to 0 °C followed by the
addition of DBU (26 μL, 0.17 mmol, 0.2 equiv). The reaction
was then stirred for 1 hour at 0 °C. The reaction was filtered
and then concentrated under reduced pressure to provide the
crude lactosyl donor. The crude material was then purified by
silica flash chromatography using toluene:acetone (3:2) as the
1
3
1
3
(1H, d, J = 6.4 Hz), 5.19 (2H, m). C{ H} NMR (CDCl , 150
MHz): δ = 20.8, 21.0, 21.0, 21.0, 21.0, 25.5, 62.1, 62.4, 68.4,
70.1, 72.3, 72.7, 72.9, 73.1, 73.9, 75.7, 101.0, 102.7, 169.6,
170.2, 170.4, 170.7, 171.2, 171.8. HRMS (ESI) m/z: [M + Na]+
calcd for C26H35NO18Na 672.1746; Found 672.1750.
Phenyl (6-O-pivaloyl-β-D-galactopyranosyl)-(1→4)-2,6-di-
eluent to provide lactosyl donor S4 as a white solid (0.44 g, 0.60
O-pivaloyl-1-thio-β-D-glucopyranoside (6). Acceptor 6 was
1
mmol, 62% yield over 2 steps). H NMR (CDCl
3
, 600 MHz): δ
34
prepared as previously described. Acceptor 6 was purified by
=
1.31 (3H, s), 1.54 (3H, s), 2.01 (3H, s), 2.07 (3H, s), 2.08 (3H,
silica flash chromatography using toluene:acetone (1:1) as the
s), 2.09 (3H, s), 2.12 (3H, s), 3.83 (1H, t, J = 9.8 Hz), 3.93 (1H,
m), 4.14 (3H, m), 4.19 (1H, dd, J = 4.5, 12.2 Hz), 4.31 (2H, m),
4.41 (1H, d, J = 7.3 Hz), 4.43 (1H, dd, J = 2.0, 12.2 Hz), 4.85
(1H, dd, J = 6.1, 7.3 Hz), 5.06 (1H, dd, J = 3.8, 10.1 Hz), 5.55
1
eluent to provide 6 as a white solid (0.31 g, 0.45 mmol).
NMR (CDCl
9H, s), 3.46 (1H, t, J = 9.2 Hz), 3.60 (1H, m), 3.66 (1H, m),
.77 (4H, m), 3.91 (1H, t, J = 3.3 Hz), 4.16 (2H, m), 4.27 (3H,
H
3
, 600 MHz): δ = 1.19 (9H, s), 1.21 (9H, s), 1.26
(
3
(
1H, t, J = 9.8 Hz), 6.48 (1H, d, J = 3.8 Hz), 8.64 (1H, s).
C{ H} NMR (CDCl , 150 MHz): δ = 20.7, 21.0, 21.0, 21.0,
3
m), 4.36 (1H, dd, J = 4.1, 11.9 Hz), 4.50 (1H, d, J = 3.8 Hz),
4.69 (1H, d, J = 10.9 Hz), 4.72 (1H, d, J = 10.2 Hz), 4.90 (1H,
1
3
1
2
7
1
1.1, 26.2, 27.4, 61.9, 63.3, 69.3, 70.1, 71.0, 71.2, 72.9, 73.1,
5.8, 77.0, 90.8, 93.1, 100.8, 111.0, 161.1, 169.4, 169.8, 170.3,
70.5, 170.9. LRMS (ESI) m/z: [M + Na]+ calcd for
13
1
t, J = 9.7 Hz), 7.28 (3H, m), 7.50 (2H, m). C{ H} NMR
CDCl , 150 MHz): δ = 27.1, 27.1, 27.2, 38.7, 38.8, 38.9, 63.4,
3.7, 68.6, 70.7, 70.9, 73.2, 73.3, 74.4, 76.8, 81.5, 86.2, 104.2,
27.8, 128.9, 132.2, 133.3, 176.8, 178.8, 179.2. LRMS (ESI)
(
3
6
1
C
27
H
36Cl
3
NO16Na 758.1; Found 758.3.
+
Succinimidyl (2,6-di-O-acetyl-3,4-O-isopropylidene-β-D-ga-
m/z: [M + Na] calcd for C33
50
H O13SNa 709.3; Found 709.2.
lactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-β-D-glucopyra-
Phenyl (6-O-benzyl-2-O-benzoyl-1-thio-β-D-galactopyra-
ꢀ
35
noside (S5). Lactosyl donor S4 (80 mg, 0.11 mmol, 1.0 equiv)
noside (7). Acceptor 7 was prepared as previously described.
Acceptor 7 was purified by silica flash chromatography using
and NHS (63 mg, 0.55 mmol, 5.0 equiv) were co-distilled with
toluene under reduced pressure (3X) followed by high vacuum
overnight. The mixture was then dissolved in 5.0 mL of dry
CH Cl and then 4 Å molecular sieves (0.2 g) were added to the
2 2
solution. Gas was then exchanged with Ar and the solution was
stirred at room temperature for 30 minutes. TMSOTf (4.0 μL,
hexanes:EtOAc (1:1) as the eluent to provide 7 as a white solid
1
(
0.80 g, 1.7 mmol). H NMR (CDCl
3
, 600 MHz): δ = 2.95 (1H,
d, J = 3.6 Hz), 3.21 (1H, d, J = 7.6 Hz), 3.78 (1H, t, J = 4.8 Hz),
.83 (1H, m), 3.88 (2H, d, J=5.10 Hz), 4.14 (1H, t, J = 3.5 Hz),
3
4.64 (2H, br), 4.84 (1H, d, J = 10.0 Hz), 5.23 (1H, t, J = 9.6 Hz),
0
.022 mmol, 0.2 equiv) was then introduced into the mixture at
7.28 (3H, m), 7.37 (5H, m), 7.50 (4H, m), 7.63 (1H, m), 8.11
1
3
1
room temperature and the reaction was stirred for 30 minutes.
The reaction was then quenched with TEA, filtered, and then
concentrated under reduced pressure. The crude material was
then purified by silica flash chromatography using 1% MeOH
(
7
1
2H, m). C{ H} NMR (CDCl
3
, 150 MHz): δ = 69.7, 69.8,
2.2, 73.8, 74.1, 77.2, 86.0, 127.8, 127.9, 128.0, 128.5, 128.5,
28.9, 129.5, 130.1, 132.6, 132.6, 133.5, 137.7, 167.0. LRMS
+
(
26 6
ESI) m/z: [M + Na] calcd for C26H O SNa 489.1; Found
in CH
2 2
Cl to provide compound S5 as a white solid (69 mg, 0.10
4
89.0.
Phenyl (4,6-O-benzylidene-2-O-benzoyl-1-thio-β-D-galacto-
pyranoside) (8). Acceptor 8 was prepared as previously de-
1
mmol, 90% yield). H NMR (CDCl
3
, 600 MHz): δ = 1.31 (3H,
s), 1.53 (3H, s), 2.08 (3H, s), 2.08 (3H, s), 2.10 (3H, s), 2.12
(3H, s), 2.12 (3H, s), 2.73 (4H, s), 3.74 (1H, m), 3.94 (1H, m),
4.10 (1H, dd, J = 8.5, 10.0 Hz), 4.14 (2H, m), 4.19 (1H, dd, J =
3
1
scribed. Acceptor 8 was purified by silica flash chromatog-
raphy using hexanes:EtOAc (1:1) as the eluent to provide 8 as
5
4
1
.8, 12.0 Hz), 4.27 (1H, dd, J = 7.5, 11.7 Hz), 4.33 (1H, dd, J =
.7, 11.7 Hz), 4.39 (1H, d, J = 7.7 Hz), 4.41 (1H, dd, J = 2.3,
2.0 Hz), 4.86 (1H, dd, J = 6.4, 7.6 Hz), 5.13 (1H, d, J = 6.3
1
a white solid (1.70 g, 3.66 mmol). H NMR (CDCl
3
, 600 MHz):
δ = 2.65 (1H, d, J = 10.8 Hz), 3.61 (1H, s), 3.91 (1H, td, J =
10.1, 3.6 Hz), 4.06 (1H, d, J = 12.3 Hz), 4.26 (1H, d, J = 3.5
Hz), 4.43 (1H, d, J = 12.3 Hz), 4.84 (1H, d, J = 9.8 Hz), 5.28
(1H, t, J = 9.7 Hz), 5.55 (1H, s), 7.28 (2H, m), 7.34 (1H, m),
1
3
1
Hz), 5.17 (1H, t, J = 6.8 Hz), 5.20 (1H, t, J = 7.9 Hz). C{ H}
NMR (CDCl , 150 MHz): δ = 20.8, 20.9, 21.0, 21.0, 25.5, 26.3,
7.5, 62.1, 63.3, 70.2, 71.1, 72.5, 72.8, 73.1, 73.2, 75.5, 77.0,
00.7, 102.7, 111.0, 169.4, 169.6, 169.9, 170.3, 170.6, 171.0.
3
2
1
1
3
1
7
.45 (7H, m), 7.60 (3H, m), 8.09 (2H, m).
C{ H} NMR
(
CDCl , 150 MHz): δ = 69.2, 70.1, 70.7, 73.0, 75.7, 84.9, 101.5,
3
+
LRMS (ESI) m/z: [M + Na] calcd for C29
Found 712.1.
H39NO18Na 712.2;
1
1
26.6, 128.3, 128.3, 128.5, 128.9, 129.5, 129.9, 130.0, 131.2,
33.3, 134.0, 137.5, 166.0. LRMS (ESI) m/z: [M + Na] calcd
+
Succinimidyl
(2,6-di-O-acetyl-β-D-galactopyranosyl)-
24 6
for C26H O SNa 487.1; Found 487.1.
(1→4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside (5). Compound
ACS Paragon Plus Environment

Page 7 of 12
The Journal of Organic Chemistry
General procedure for sialylations in Table 1. Acceptors
24.9, 25.5, 36.8, 53.1, 59.3, 62.1, 63.2, 68.9, 69.8, 69.8, 71.0,
72.2, 72.5, 72.9, 73.7, 74.5, 74.9, 75.9, 76.4, 79.3, 99.8, 101.6,
103.3, 154.5, 166.7, 168.9, 169.5, 170.0, 170.2, 170.7, 170.7,
170.8, 171.0, 171.3, 173.0. HRMS (ESI) m/z: [M + Na] calcd
58 2
for C45H N O30Na 1129.2967; Found 1129.2948.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(1.0 equiv) were co-distilled with toluene under reduced pres-
sure (3X) and then were combined with freshly prepared sialyl
phosphate donor 3 (1.5 equiv) followed by high vacuum for 3
hours. The donor acceptor pair were then dissolved in dry
+
2 2
CH Cl (volume calculated to make 20 mM solution of accep-
Phenyl (methyl 5-acetamido-7,8,9-tri-O-acetyl-5-N,4-O-car-
bonyl-3,5-dideoxy-D-glycero-α-D-galacto-non-2-ulopyra-
nosylonate)-(2→3)-(6-O-pivaloyl-β-D-galactopyranosyl)-
tor), 4 Å molecular sieves were added (mass = 4 x acceptor
mass), gas was exchanged with Ar, and the resulting solution
was stirred at room temperature for 1 hour. The reaction mix-
ture was cooled to -78 °C and TMSOTf (1.5 equiv) was added
to initiate the reaction. The reaction was stirred at -78 °C for 1
(1→4)-2,6-di-O-pivaloyl-1-thio-β-D-glucopyranoside
(11).
Compound 11 was purified by silica flash chromatography us-
ing toluene:acetone (1:1) as the eluent to provide 11 as a white
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
hour and the reaction was quenched with saturated NaHCO
3
1
solid (0.16 g, 0.14 mmol, 52% yield). H NMR (CDCl
3
, 600
with vigorous stirring as the reaction slowly warmed to room
temperature. The quenched reaction mixture was then diluted
MHz): δ = 1.20 (9H, s), 1.21 (9H, s), 1.26 (9H, s), 2.05 (3H, s),
.13 (3H, s), 2.16 (3H, s), 2.21 (1H, t, J = 13.0 Hz), 2.49 (1H,
2
with CH
2 2
Cl (10 mL) and the organic layer was dried over
d, J = 2.64Hz), 2.52 (3H, s), 3.00 (1H, dd, J = 3. 5, 12.3 Hz),
3.20 (1H, d, J = 1.7 Hz), 3.52 (1H, t, J =9.2 Hz), 3.70 (2H, m),
3.78 (4H, m), 3.84 (3H, s), 4.04 (2H, m), 4.09 (1H, dd, J = 7.9,
12.1 Hz), 4.15 (1H, dd, J = 3.3, 9.6 Hz), 4.23 (1H, dd, J = 8.1,
Na SO , filtered, and concentrated under reduced pressure. The
2
4
1
crude product was then analyzed by H NMR to determine α:β
ratios based on the integrals of the Neu5Ac C3 equatorial pro-
tons. Crudes were then purified by silica flash chromatography
using appropriate solvent mixtures to isolate α:β mixtures of the
products to obtain the isolated yield. Isolated and pure α
anomers were then characterized.
1
1.9 Hz), 4.31 (1H, d, J = 1.0 Hz), 4.40 (1H, dd, J = 4.2, 11.9
Hz), 4.47 (1H, dd, J = 2.6, 12.3 Hz), 4.51 (1H, d, J = 7.9 Hz),
.65 (1H, dd, J = 1.1, 9.5 Hz), 4.71 (1H, d, J = 10.3 Hz), 4.88
1H, dd, J = 1.5, 12.1 Hz), 4.94 (1H, dd, J = 9.4, 10.1 Hz), 5.55
(1H, td, J = 4.1, 10.7 Hz), 5.70 (1H, dd, J = 1.1, 8.5 Hz), 7.28
4
(
Phenyl (methyl 5-acetamido-7,8,9-tri-O-acetyl-5-N,4-O-car-
bonyl-3,5-dideoxy-D-glycero-α-D-galacto-non-2-ulopyra-
nosylonate)-(2→3)-(2,6-di-O-acetyl-β-D-galactopyranosyl)-
13
1
3
(3H, m), 7.51 (2H, m). C{ H} NMR (CDCl , 150 MHz): δ =
20.7, 20.9, 21.2, 24.7, 27.1, 27.1, 27.2, 36.6, 38.7, 38.8, 53.5,
58.8, 63.2, 63.3, 63.6, 67.9, 68.8, 68.9, 70.7, 71.2, 72.6, 74.4,
74.7, 76.7, 76.9, 77.2, 82.2, 86.6, 97.9, 104.2, 127.6, 128.9,
131.8, 134.0, 153.3, 168.3, 169.8, 170.4, 170.7, 172.0, 176.7,
177.9, 178.6. HRMS (ESI) m/z: [M + Na]+ calcd for
(
1→4)-2,3,6-tri-O-acetyl-1-thio-β-D-glucopyranoside
(9).
Compound 9 was purified by silica flash chromatography using
toluene:acetone (1:1) as the eluent to provide 9 as a white solid
1
(0.29 g, 0.26 mmol, 68% yield). H NMR (CDCl
3
, 600 MHz):
δ = 2.01 (3H, s), 2.02 (3H, s), 2.03 (3H, s), 2.07 (6H, s), 2.08
3H, s), 2.10 (3H, s), 2.12 (1H, m), 2.16 (3H, s), 2.49 (3H, s),
.70 (1H, d, J = 5.0 Hz), 2.83 (1H, dd, J = 3.5, 12.1 Hz), 3.67
3H, m), 3.77 (2H, m), 3.81 (4H, m), 4.01 (2H, m), 4.09 (1H, t,
J = 3.8 Hz), 4.14 (1H, dd, J = 6.3, 11.91 Hz), 4.40 (1H, dd, J =
.8, 12.2 Hz), 4.51 (2H, m), 4.59 (1H, dd, J = 1.5, 9.4 Hz), 4.68
1H, d, J = 10.1 Hz), 4.87 (2H, m), 5.16 (1H, dd, J = 7.9, 10.2
Hz), 5.20 (1H, t, J = 9.2 Hz), 5.36 (1H, dt, J = 2.8, 7.6 Hz), 5.56
C
52
H
73NO25SNa 1166.4085; Found 1166.4072.
(
2
(
Phenyl (methyl 5-acetamido-7,8,9-tri-O-acetyl-5-N,4-O-car-
bonyl-3,5-dideoxy-D-glycero-α-D-galacto-non-2-ulopyra-
nosylonate)-(2→3)-2-O-benzoyl-6-O-benzyl-1-thio-β-D-galac-
topyranoside (12). Compound 12 was purified by silica flash
chromatography using hexanes:EtOAc (1:1) as the eluent to
2
(
1
provide 12 as a white solid (1.30 g, 1.41 mmol, 89% yield). H
NMR (CDCl
3
, 600 MHz): δ = 1.48 (3H, s), 2.02 (3H, s), 2.07
1
3
1
(1H, dd, J = 1.6, 7.7 Hz), 7.29 (3H, m), 7.46 (2H, m). C{ H}
NMR (CDCl , 150 MHz): δ = 20.8, 20.9, 20.9, 21.0, 21.0, 21.0,
21.2, 21.6, 24.8, 35.8, 53.5, 53.6, 59.0, 62.5, 63.0, 63.5, 66.4,
(
(
(
1H, t, J = 6.3 Hz), 2.13 (3H, s), 2.44 (3H, s), 2.74 (1H, br), 2.86
1H, dd, J = 3.4, 12.0 Hz), 3.55 (1H, dd, J = 9.4, 11.2 Hz), 3.73
3H, s), 3.85 (5H, m), 3.95 (1H, dd, J = 7.4, 12.2 Hz), 4.43 (1H,
3
6
9
1
9.3, 69.7, 70.4, 71.9, 72.8, 73.5, 73.9, 74.8, 76.0, 76.1, 85.4,
9.3, 100.9, 128.3, 129.0, 132.0, 133.0, 153.6, 168.4, 169.4,
dd, J = 2.5, 12.3 Hz), 4.49 (1H, dd, J = 1.8, 9.4 Hz), 4.52 (1H,
dd, J = 3.1, 9.5 Hz), 4.61 (2H, m), 4.96 (1H, d, J = 10.0 Hz),
5.43 (1H, t, J = 9.7 Hz), 5.49 (1H, dd, J = 1.9, 8.7 Hz), 5.55
69.8, 169.9, 170.2, 170.3, 170.4, 170.5, 171.2, 172.3. HRMS
+
(
ESI) m/z: [M + Na] calcd for C47
H
59NO27SNa 1124.2887;
(1H, td, J = 2.4, 8.7 Hz), 7.25 (3H, m), 7.35 (5H, m), 7.49 (4H,
Found 1124.2898.
m), 7.61 (1H, t, J = 7.4 Hz), 8.18 (2H, dd, J = 1.0, 8.1 Hz).
1
3
1
Succinimidyl (methyl 5-acetamido-7,8,9-tri-O-acetyl-5-N,4-
O-carbonyl-3,5-dideoxy-D-glycero-α-D-galacto-non-2-ulopy-
ranosylonate)-(2→3)-(2,6-di-O-acetyl-β-D-galactopyranosyl)-
3
C{ H} NMR (CDCl , 150 MHz): δ = 20.2, 20.8, 21.3, 24.6,
35.9, 53.3, 58.8, 63.4, 67.5, 68.5, 68.9, 69.1, 71.5, 73.6, 74.9,
75.2, 75.5, 76.5, 86.7, 97.5, 127.7, 127.7, 127.8, 128.4, 128.5,
(
1→4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside (10).
Com-
128.8, 130.1, 130.2, 132.6, 132.9, 133.3, 138.1, 153.4, 165.3,
168.5, 170.0, 170.5, 170.8, 171.8. LRMS (ESI) m/z: [M + Na]+
pound 10 was purified by silica flash chromatography using tol-
uene:acetone (1:1) as the eluent to provide 10 as a white solid
calcd for C45H49NO18SNa 946.3; Found 946.2.
1
(0.13 g, 0.12 mmol, 81% yield). H NMR (CDCl
3
, 600 MHz):
Phenyl (methyl 5-acetamido-7,8,9-tri-O-acetyl-5-N,4-O-car-
bonyl-3,5-dideoxy-D-glycero-α-D-galacto-non-2-ulopyra-
nosylonate)-(2→3)-2-O-benzoyl-4,6-O-benzylidene-1-thio-β-
D-galactopyranoside (13). Compound 13 was purified by silica
flash chromatography using hexanes:EtOAc (1:1) as the eluent
δ = 2.00 (1H, t, J = 12.8 Hz), 2.07 (3H, s), 2.09 (3H, s), 2.11
(3H, s), 2.11 (3H, s), 2.14 (9H, s), 2.23 (3H, s), 2.50 (3H, s),
2
(
.74 (4H, s), 2.97 (1H, dd, J = 3.2, 12.0 Hz), 3.47 (1H, br), 3.73
3H, m), 3.81 (3H, s), 3.89 (2H, m), 3.98 (1H, dd, J = 7.1, 12.3
Hz), 4.18 (1H, t, J = 9.3 Hz), 4.23 (1H, dd, J = 6.3, 11.9 Hz),
.27 (2H, d, J = 6.2 Hz), 4.36 (1H, dd, J = 3.0, 10.0 Hz), 4.42
1H, dd, J = 2.0, 11.8 Hz), 4.48 (1H, dd, J = 2.4, 12.2 Hz), 4.53
1H, dd, J = 1.7, 9.4 Hz), 4.64 (1H, d, J = 7.9 Hz), 5.05 (1H, dd,
to provide 13 as a white solid (0.96 g, 1.0 mmol, 87% yield).
4
(
(
1
3
H NMR (CDCl , 600 MHz): δ = 1.76 (1H, dd, J = 12.3, 13.5
Hz), 1.82 (3H, s), 2.05 (3H, s), 2.21 (3H, s), 2.46 (3H, s), 2.92
1H, dd, J = 3.2, 12.1 Hz), 3.48 (3H, s), 3.53 (1H, dd, J = 9.5,
1.2 Hz), 3.73 (1H, br), 3.75 (1H, m), 4.01 (1H, m), 4.12 (1H,
d, J = 3.2 Hz), 4.17 (1H, dd, J = 1.4, 12.0 Hz), 4.39 (1H, dd, J
(
1
J = 8.1, 9.9 Hz), 5.12 (1H, t, J = 6.6 Hz), 5.17 (1H, t, J = 7.2
13
1
Hz), 5.24 (1H, t, J = 8.1 Hz), 5.60 (2H, m). C{ H} NMR
(CDCl , 150 MHz): δ = 20.8, 20.8, 20.9, 20.9, 20.9, 21.0, 21.2,
3
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=
1.4, 12.1 Hz), 4.47 (2H, m), 4.62 (1H, dd, J = 3.5, 9.7 Hz), m), 3.95 (1H, dd, J = 3.9, 10.6 Hz), 4.06 (1H, t, J = 9.2 Hz),
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5.00 (1H, d, J = 9.8 Hz), 5.38 (1H, s), 5.44 (1H, t, J = 9.7 Hz),
5.57 (2H, m), 7.25 (2H, m), 7.31 (1H, m), 7.38 (3H, m), 7.43
(2H, m), 7.50 (2H, t, J = 7.8 Hz), 7.61 (3H, m), 8.16 (2H, d, J =
4.65 (2H, q, J = 11.5 Hz), 5.33 (1H, d, J = 7.8 Hz), 5.5 (1H, t, J
= 9.1 Hz), 5.70 (1H, dd, J = 7.9, 9.1 Hz), 7.33 (1H, m), 7.40
(8H, m), 7.55 (2H, m), 8.02 (2H, d, J = 7.1 Hz), 8.08 (2H, d, J
1
3
1
13
1
7
2
7
1
1
.1 Hz). C{ H} NMR (CDCl
3
, 150 MHz): δ = 20.7, 21.0,
3
= 7.1 Hz). C{ H} NMR (CDCl , 150 MHz): δ = 25.4, 69.7,
1.6, 24.8, 37.2, 53.0, 58.9, 63.9, 68.1, 68.3, 69.3, 69.6, 71.6,
3.0, 73.9, 75.0, 75.1, 85.2, 96.9, 101.0, 126.7, 128.1, 128.3,
28.6, 128.8, 129.2, 130.1, 130.5, 131.4, 133.3, 134.1, 137.9,
69.9, 70.6, 73.8, 75.5, 76.6, 103.5, 127.7, 127.9, 128.4, 128.5,
128.5, 128.8, 129.1, 130.0, 130.1, 133.4, 133.7, 137.8, 165.4,
167.2, 170.1. HRMS (ESI) m/z: [M + Na]+ calcd for
53.5, 165.0, 168.8, 170.3, 170.6, 171.1, 172.1. LRMS (ESI)
C
31
H
29NO10Na 598.1684; Found 598.1680.
+
m/z: [M + Na] calcd for C45
H
47NO18SNa 944.2; Found 944.1.
Phenyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dide-
Succinimidyl (4,6-O-benzylidene-2,3-di-O-benzoyl-β-D-glu-
copyranoside (15). Compound 1440 (1.58 g, 2.78 mmol, 1.0
equiv) and NHS (1.60 g, 13.9 mmol, 5.0 equiv) were co-dis-
tilled with toluene under reduced pressure (3X) followed by
high vacuum for 3 hours. The mixture was then dissolved in 10
mL of dry CH Cl and 4 Å molecular sieves (5 g) were added
to the solution. Gas was then exchanged with Ar and the solu-
tion was stirred at room temperature for 30 minutes. The mix-
ture was then cooled to -10 °C using a salted ice bath and then
NIS (1.88 g, 8.34 mmol, 3.0 equiv) was quickly added to the
reaction vessel. Gas was exchanged with Ar again, and
TMSOTf (0.10 mL, 0.56 mmol, 0.2 equiv) was then introduced
into the mixture and the reaction was stirred at -10 °C for 30
minutes and then at 0 °C for 1 hour. The reaction was then
diluted with CH
lution of Na
tion was then filtered, and the resulting organic layer was
washed with saturated Na and NaHCO (20 mL, 2X). The
collected organic layer was then washed with brine, dried over
Na SO , filtered, and then concentrated under reduced pressure.
oxy-D-glycero-α-D-galacto-non-2-ulopyranosylonate)-(2→3)-
2-O-benzoyl-4,6-O-benzylidene-1-thio-β-D-galactopyranoside
(17). Compound 13 (0.66 g, 0.72 mmol, 1.0 equiv) was dis-
solved in MeOH (9 mL) and cooled to 0 °C. Freshly prepared
1.0 M NaOMe in MeOH (1 mL) was then introduced into the
reaction mixture to generate a 0.1 M NaOMe solution with the
substrate, and was stirred for 15 minutes at 0 °C. The reaction
was stirred at room temperature for 1 hour and then quenched
with Amberlite® IR120 acidic resin, filtered, and concentrated
under reduced pressure to reveal a white solid in quantitative
yield. The solid was dried by co-distillation with toluene under
reduced pressure (3X) followed by high vacuum for 3 hours.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀ
2
2
ꢀ
Dry intermediate was then dissolved in pyridine (10 mL) fol-
lowed by the addition of DMAP (18 mg, 0.14 mmol, 0.2 equiv).
Gas was exchanged with Ar and then the mixture was cooled to
2
Cl
2
(10 mL) and quenched with a saturated so-
2
S
2
O
3
and NaHCO
3
(20 mL). The quenched reac-
2
0 °C. Once cool, Ac O (1.4 mL, 14 mmol, 20 equiv) was intro-
duced and the reaction was stirred for 12 hours while gradually
warming to room temperature. The reaction was then cooled to
0 °C again and quenched with excess MeOH and stirred for 10
minutes. The crude was concentrated under reduced pressure,
co-distilled with toluene under reduced pressure (3X) and was
2
S
2
O
3
3
2
4
The crude material was then purified by silica flash chromatog-
raphy using hexanes:EtOAc (4:1) as the eluent with 10%
CH
pound 15 was isolated as a white solid (1.34 g, 2.34 mmol, 84%
yield). H NMR (CDCl
reconstituted in CH (20 mL). The crude was then washed
2
Cl
2
2
Cl
2
to ensure compound solubility on the column. Com-
with saturated NaHCO (20 mL, 3X) and the resulting organic
3
2 4
layer was washed with brine, dried over Na SO , filtered, and
concentrated under reduced pressure. The crude was then puri-
fied by silica flash chromatography using toluene:acetone (3:1)
1
3
, 600 MHz): δ = 2.72 (4H, s), 3.91 (1H,
m), 4.02 (1H, t, J = 10.3 Hz), 4.41 (1H, dd, J = 4.9, 10.7 Hz),
4
5
.53 (1H, t, J = 9.3 Hz), 5.49 (1H, d, J = 5.9 Hz), 5.65 (1H, s),
.76 (2H, m), 7.35 (3H, m), 7.44 (6H, m), 7.56 (2H, m), 8.04
as the eluent to reveal 17 as a white solid (0.51 g, 0.55 mmol,
1
76% yield over 2 steps). H NMR (CDCl
3
, 600 MHz): δ = 1.69
1
3
1
(2H, d, J = 7.1 Hz), 8.10 (2H, d, J = 7.1 Hz). C{ H} NMR
(CDCl , 150 MHz): δ = 25.4, 66.6, 68.3, 70.9, 72.3, 77.3, 101.5,
103.1, 126.2, 128.3, 128.4, 128.5, 128.9, 129.2, 129.2, 130.0,
30.1, 133.4, 133.6, 136.7, 165.0, 165.5, 170.4. HRMS (ESI)
(1H, t, J = 12.7 Hz), 1.82 (3H, s), 1.83 (3H, s), 1.95 (3H, s), 2.07
(3H, s), 2.23 (3H, s), 2.56 (1H, dd, J = 4.5, 12.9 Hz), 3.56 (3H,
s), 3.67 (1H, br), 3.90 (2H, m), 4.00 (1H, dd, J = 6.2, 12.2 Hz),
4.05 (1H, d, J = 3.4 Hz), 4.13 (1H, d, J = 11.2 Hz), 4.35 (1H,
dd, J = 2.5, 12.4 Hz), 4.39 (1H, d, J = 12.0 Hz), 4.61 (1H, dd, J
= 3.4, 9.7 Hz), 4.71 (1H, m), 4.99 (1H, d, J = 9.8 Hz), 5.06 (1H,
br), 5.25 (1H, d, J = 9.5 Hz), 5.38 (1H, s), 5.43 (1H, t, J = 9.8
Hz), 5.52 (1H, m), 7.25 (3H, m), 7.38 (3H, m), 7.44 (2H, m),
3
1
+
m/z: [M + Na] calcd for C31
96.1529.
Succinimidyl (2,3-di-O-benzoyl-6-O-benzyl-β-D-glucopyra-
H27NO10Na 596.1527; Found
5
noside (16). Compound 15 (0.80 g, 1.4 mmol, 1.0 equiv) was
co-distilled with toluene under reduced pressure (3X), set under
high vacuum for 3 hours, and was then dissolved in 10 mL of
7
.51 (2H, t, J = 7.7 Hz), 7.60 (3H, m), 8.16 (2H, d, J = 7.1 Hz).
13
1
C{ H} NMR (CDCl
3
, 150 MHz): δ = 20.6, 20.8, 20.9, 21.5,
ꢀ
2
3.2, 38.2, 49.1, 52.8, 62.6, 66.9, 67.6, 68.1, 68.8, 69.3, 69.6,
dry CH
was cooled to 0 °C followed by the addition of Et
mL, 4.2 mmol, 3.0 equiv) into the reaction vessel. This mixture
was then stirred for 10 minutes at 0 °C and then BF Et O (0.69
mL, 5.6 mmol, 4.0 equiv) was introduced into the reaction drop-
wise. The reaction was then stirred for 1.5 hours at 0 °C. The
reaction was then quenched with saturated NaHCO
The aqueous layer was extracted with CH Cl (20 mL, 3X) and
the combined organic layers were washed with brine, dried with
Na SO , filtered, and concentrated under reduced pressure. The
crude was then purified by silica flash chromatography using
toluene:EtOAc (4:1) as the eluent with 10% CH Cl to ensure
compound solubility. Compound 16 was isolated as a white
2
Cl
2
. Gas was then exchanged with Ar and the solution
72.3, 73.3, 73.6, 85.3, 96.7, 101.0, 126.6, 127.9, 128.1, 128.4,
128.7, 129.1, 130.0, 130.4, 131.6, 133.1, 133.7, 137.8, 164.9,
3
SiH (0.67
168.8, 170.1, 170.2, 170.3, 170.8, 170.9. HRMS (ESI) m/z: [M
3
2
+
+
Na] calcd for C46
H
51NO18SNa 960.2719; Found 960.2715.
Succinimidyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-
3
(20 mL).
3,5-dideoxy-D-glycero-α-D-galacto-non-2-ulopyranosylonate)-
(2→3)-2-O-benzoyl-4,6-O-benzylidene-1-amino-β-D-galacto-
pyranoside (S6). Donor 17 (50 mg, 0.053 mmol, 1.5 equiv) and
2
2
ꢀ
2
4
acceptor 16 (21 mg, 0.036 mmol, 1.0 equiv) were combined and
co-distilled with toluene under reduced pressure (3X) and set
under high vacuum for 3 hours. The donor and acceptor pair
2
2
2 2
were dissolved in dry CH Cl (5 mL) followed by the addition
of 4 Å molecular sieves (0.2 g) and gas was exchanged with Ar.
The mixture was stirred at room temperature for 1 hour before
1
solid (0.65 g, 1.1 mmol, 81% yield). H NMR (CDCl
MHz): δ = 2.67 (4H, m), 3.41 (1H, br), 3.80 (1H, m), 3.85 (1H,
3
, 600
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Page 9 of 12
The Journal of Organic Chemistry
cooling the mixture to 0 °C. NIS (36 mg, 0.16 mmol, 4.5 equiv)
was then introduced into the reaction mixture and gas was ex-
changed with Ar again followed by the addition of TMSOTf
(2.0 μL, 0.011 mmol, 0.3 equiv). The reaction was then stirred
1H NMR (CDCl
, 600 MHz): δ = 1.79 (1H, t, J = 12.7 Hz), 1.90
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(3H, s), 1.96 (3H, s), 2.06 (3H, s), 2.16 (3H, s), 2.21 (3H, s),
2.59 (1H, dd, J = 4.4, 12.9 Hz), 3.52 (3H, s), 4.08 (3H, m), 4.14
(1H, br), 4.21 (1H, d, J = 12.5 Hz), 4.26 (1H, dd, J = 2.7, 12.5
Hz), 4.32 (1H, d, J = 12.6 Hz), 4.42 (1H, d, J = 3.1 Hz), 4.78
(1H, m), 5.11 (1H, d, J = 9.4 Hz), 5.18 (1H, dd, J = 3.3, 10.7
Hz), 5.37 (1H, dd, J = 1.5, 9.8 Hz), 5.47 (1H, s), 5.49 (1H, m),
5.70 (1H, dd, J = 3.3, 10.7 Hz), 6.79 (1H, d, J = 3.3 Hz), 7.37
(3H, m), 7.47 (2H, t, J = 7.8 Hz), 7.54 (2H, d, J = 6.7 Hz), 7.59
(1H, t, J = 7.4 Hz), 8.07 (2H, d, J = 7.1 Hz), 8.49 (1H, s).
at 0 °C for 1.5 hours. The reaction was then diluted with CH
5 mL) and quenched with a saturated solution of Na
NaHCO (10 mL). The quenched reaction was then filtered,
and the resulting organic layer was washed with saturated
Na and NaHCO (10 mL, 2X). The resulting organic layer
was washed with brine, dried with Na SO , filtered, and con-
2
Cl
2
(
2 2 3
S O
and
3
2
S
2
O
3
3
2
4
13
1
centrated under reduced pressure. The resulting crude solid was
then purified by silica flash chromatography using toluene:ace-
tone (3:1) as the eluent to reveal compound S6, and not desired
compound. This procedure was repeated using TfOH as the
3
C{ H} NMR (CDCl , 150 MHz): δ = 20.8, 20.9, 21.4, 23.2,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
38.7, 49.5, 52.9, 62.5, 65.2, 67.0, 67.4, 68.0, 68.6, 69.0, 72.7,
73.5, 91.2, 95.1, 96.6, 100.6, 126.3, 128.2, 128.3, 128.5, 129.0,
129.1, 129.6, 129.8, 133.4, 137.7, 160.4, 165.5, 169.0, 169.8,
1
+
acid catalyst and resulted in the same product. H NMR
170.0, 170.3, 170.7, 171.0. HRMS (ESI) m/z: [M + Na] calcd
(
1
CDCl
.93 (3H, s), 1.97 (3H, s), 2.09 (3H, s), 2.24 (3H, s), 2.53 (1H,
br), 2.56 (1H, dd, J = 4.4, 12.9 Hz), 2.65 (3H, m), 3.49 (3H, s),
.72 (1H, s), 3.97 (2H, m), 4.04 (1H, dd, J = 6.2, 12.4 Hz), 4.15
2H, m), 4.24 (1H, dd, J = 1.4, 12.3 Hz), 4.34 (1H, dd, J = 2.6,
2.4 Hz), 4.65 (1H, dd, J = 3.3, 9.9 Hz), 4.70 (1H, m), 5.06 (1H,
br), 5.28 (1H, d, J = 10.2 Hz), 5.37 (1H, s), 5.50 (1H, td, J =
.0, 12.2 Hz), 5.53 (1H, d, J = 9.2 Hz), 6.36 (1H, t, J = 9.6 Hz),
3
, 600 MHz): δ = 1.68 (1H, t, J = 12.8 Hz), 1.84 (3H, s),
3 2
for C42H47Cl N O19Na 1011.2; Found 1011.1.
Phenyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dide-
oxy-D-glycero-α-D-galacto-non-2-ulopyranosylonate)-(2→3)-
3
(
1
4
-O-acetyl-2-O-benzoyl-6-O-benzyl-1-thio-β-D-galactopyra-
noside (20). Compound 12 (1.3 g, 1.4 mmol, 1.0 equiv) was
dissolved in MeOH (18 mL) and cooled to 0 °C. Freshly pre-
pared 1.0 M NaOMe in MeOH (2.0 mL) was then introduced to
the reaction mixture to generate a 0.1 M NaOMe solution with
the substrate, and was stirred for 15 minutes at 0 °C. The reac-
tion was then stirred at room temperature for 1 hour and
quenched with Amberlite® IR120 acidic resin, filtered, and
concentrated under reduced pressure to reveal a white solid in
quantitative yield. The solid was dried by co-distillation with
toluene under reduced pressure (3X) followed by high vacuum
for 3 hours. Dry intermediate was then dissolved in pyridine
ꢀ
3
7.34 (1H, m), 7.39 (2H, t, J = 7.3 Hz), 7.47 (2H, t, J = 7.8 Hz),
7.58 (1H, t, J = 7.4 Hz), 7.66 (2H, d, J = 7.0 Hz), 8.02 (2H, d, J
1
3
1
= 7.1 Hz). C{ H} NMR (CDCl
3
, 150 MHz): δ = 20.7, 20.8,
2
6
1
1
0.9, 21.5, 23.2, 27.9, 38.6, 49.3, 52.8, 62.6, 66.8, 67.0, 67.6,
8.2, 68.7, 68.7, 72.5, 72.7, 72.9, 78.7, 96.7, 101.7, 127.1,
28.3, 128.6, 129.2, 129.6, 129.9, 133.4, 137.9, 165.4, 168.8,
70.1, 170.3, 170.8, 171.0, 174.8, 176.2. HRMS (ESI) m/z: [M
+
+
Na] calcd for C44
H
50
N
2
O
20Na 949.2849; Found 949.2838.
(20 mL) followed by the addition of DMAP (35 mg, 0.28 mmol,
Trichloroacetimidatyl (methyl 5-acetamido-4,7,8,9-tetra-O-
0.2 equiv). Gas was exchanged with Ar and the mixture was
cooled to 0 °C. Once cool, Ac O (3.3 mL, 35 mmol, 25 equiv)
acetyl-3,5-dideoxy-D-glycero-α-D-galacto-non-2-ulopyra-
nosylonate)-(2→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-ga-
lactopyranoside (18). Compound 17 (0.41 g, 0.44 mmol, 1.0
ꢀ
2
equiv) was dissolved in an acetone:H O (4:1) mixture (10 mL)
and then cooled to 0 °C. While stirring, NBS (94 mg, 0.53
mmol, 1.2 equiv) was added gradually to the reaction and the
mixture was stirred for 30 minutes. After 30 minutes of mixing,
NBS (78 mg, 0.44 mmol, 1.0 equiv) was added and the mixture
was stirred at 0 °C for another 1 hour. The reaction was then
concentrated under reduced pressure until the solution became
2
was introduced and the reaction was stirred for 12 hours while
gradually warming to room temperature. The reaction was then
cooled to 0 °C again and quenched with excess MeOH and
stirred for 10 minutes. The crude was concentrated under re-
duced pressure, co-distilled with toluene under reduced pres-
sure (3X) and was reconstituted in CH
was then washed with saturated NaHCO
resulting organic layer was washed with brine, dried over
Na SO , filtered, and concentrated under reduced pressure. The
2
Cl
2
(20 mL). The crude
3
(20 mL, X 3) and the
2
4
turbid. The turbid mixture was diluted with CH
and then quenched and washed with saturated Na
HCO
brine, dried with Na
2
Cl
2
(20 mL)
and Na-
crude was then purified by silica flash chromatography using
2
S
2
O
3
toluene:acetone (3:1) as the eluent to reveal 20 as a white solid
1
3
(15 mL, 3X). The organic layer was then washed with
SO , filtered, and then concentrated under
(1.02 g, 1.04 mmol, 74% yield over 2 steps). H NMR (CDCl
3
,
2
4
600 MHz): δ = 1.43 (3H, s), 1.74 (1H, t, J = 12.4 Hz), 1.80 (3H,
s), 1.99 (3H, s), 2.02 (3H, s), 2.08 (3H, s), 2.17 (3H, s), 2.56
(1H, dd, J = 4.6, 12.6 Hz), 3.51 (1H, dd, J = 6.1, 10.0 Hz), 3.61
(2H, m), 3.82 (1H, m), 3.86 (3H, s), 3.90 (1H, dd, J = 5.7, 12.5
Hz), 3.98 (1H, t, J = 6.2 Hz), 4.27 (1H, dd, J = 2.4, 12.5 Hz),
4.48 (1H, d, J = 11.6 Hz), 4.56 (1H, d, J = 11.6 Hz), 4.76 (1H,
dd, J = 3.2, 9.6 Hz), 4.85 (1H, m), 4.96 (1H, d, J = 10.3 Hz),
5.10 (2H, m), 5.19 (1H, dd, J = 2.8, 9.5 Hz), 5.32 (1H, t, J = 9.8
Hz), 5.57 (1H, m), 7.25 (3H, m), 7.35 (5H, m), 7.50 (4H, m),
reduced pressure. The crude hemiacetal was passed through a
short bed of silica using toluene:acetone (2:1) as the eluent to
give the crude hemiacetal intermediate as a white solid (0.31 g,
0.36 mmol, 82% crude). The crude hemiacetal was then co-
distilled with toluene under reduced pressure (3X) and set under
high vacuum for 3 hours. The crude hemiacetal was then dis-
2 2
solved in dry CH Cl (5 mL) followed by the addition of 4 Å
molecular sieves (1 g), trichloroacetonitrile (144 μL, 1.44
mmol, 4.0 equiv), and the gas was exchanged with Ar. The so-
lution was stirred at room temperature for 30 minutes. The mix-
ture was then cooled to 0 °C followed by the addition of DBU
1
3
1
7.59 (1H, t, J = 7.4 Hz), 8.19 (2H, d, J = 7.1 Hz). C{ H} NMR
(CDCl , 150 MHz): δ = 20.2, 20.8, 20.8, 21.5, 23.2, 37.4, 48.8,
3
53.2, 62.3, 66.5, 67.4, 68.4, 68.5, 69.3, 69.4, 71.8, 72.4, 73.5,
75.8, 86.5, 96.8, 127.7, 127.7, 127.7, 128.3, 128.4, 128.8, 130.2,
130.4, 132.3, 133.0, 133.2, 138.0, 165.3, 168.0, 170.1, 170.3,
ꢀ
(
10 μL, 0.072 mmol, 0.2 equiv) and the reaction was then stirred
for 1.5 hours at 0 °C. The reaction was filtered and then con-
centrated under reduced pressure to provide the crude donor.
The crude material was then purified by silica flash chromatog-
raphy using toluene:acetone (3:1) as the eluent to provide donor
18 as a white solid (0.32 g, 0.32 mmol, 73% yield over 2 steps).
+
170.3, 170.5, 170.7, 170.9. HRMS (ESI) m/z: [M + Na] calcd
for C48H55NO19SNa 1004.2981; Found 1004.2967.
Succinimidyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-
,5-dideoxy-D-glycero-α-D-galacto-non-2-ulopyranosylonate)-
3
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 10 of 12
(
2→3)-(4-O-acetyl-2-O-benzoyl-6-O-benzyl-β-D-galactopyra- 12.0 Hz), 2.97 (1H, dd, J = 6.4, 12.0 Hz), 3.50 (1H, t, J = 6.7
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
nosyl)-(1→4)-2,3-di-O-benzoyl-6-O-benzyl-β-D-glucopyra-
noside (2). Donor 20 (0.36 g, 0.37 mmol, 1.5 equiv) and accep-
tor 16 (0.14 g, 0.25 mmol, 1.0 equiv) were combined and co-
distilled with toluene under reduced pressure (3X) and set under
high vacuum for 3 hours. The donor acceptor pair were dis-
Hz), 3.56 (1H, td, J = 3.1, 9.3 Hz), 3.69 (1H, dd, J = 2.7, 10.7
Hz), 3.76 (5H, m), 3.83 (1H, q, J = 10.4 Hz), 3.90 (1H, dd, J =
7.9, 12.2 Hz), 4.37 (1H, t, J = 8.8 Hz), 4.46 (1H, dd, J = 2.5,
12.2 Hz), 4.55 (1H, dd, J = 3.4, 10.1 Hz), 4.69 (1H, d, J = 3.2
Hz), 4.73 (1H, td, J = 6.1, 14.9 Hz), 4.91 (1H, d, J = 7.9 Hz),
4.96 (1H, d, J = 10.3 Hz), 5.13 (1H, dd, J = 2.6, 9.6 Hz), 5.22
(2H, m), 5.62 (2H, m), 5.70 (1H, td, J = 3.9, 12.1 Hz), 7.41 (4H,
q, J = 8.2 Hz), 7.53 (4H, m), 7.60 (1H, t, J = 7.3 Hz), 8.02 (4H,
2 2
solved in dry CH Cl (5 mL) followed by the addition of 4 Å
molecular sieves (1 g) and gas was exchanged with Ar. The
mixture was stirred at room temperature for 1 hour before cool-
ing the mixture to 0 °C. NIS (0.25 g, 1.1 mmol, 4.5 equiv) was
then introduced into the reaction mixture and gas was ex-
changed with Ar again followed by the addition of TfOH (6 μL,
0.07 mmol, 0.3 equiv). The reaction was then stirred at 0 °C for
1.5 hours and the reaction was diluted with CH
then quenched with saturated Na and NaHCO
The organic layer was separated, filtered, and washed with sat-
urated Na and NaHCO (20 mL, 2X). The resulting or-
ganic layer was washed with brine, dried with Na SO , filtered,
and concentrated under reduced pressure. The resulting crude
solid was then purified by silica flash chromatography using
toluene:acetone (3:1) as the eluent to afford compound 2 as a
white solid (0.32 g, 0.22 mmol, 87% yield). Only β anomer was
observed due to anchimeric assistance. H NMR (CDCl
13
1
3
d, J = 8.3 Hz), 8.26 (2H, d, J = 7.1 Hz). C{ H} NMR (CDCl ,
150 MHz): δ = 20.3, 20.6, 20.8, 20.8, 21.4, 23.1, 25.3, 37.4,
48.7, 53.3, 59.9, 60.5, 63.3, 67.0, 67.1, 68.1, 69.2, 70.1, 71.1,
71.1, 71.9, 73.0, 74.5, 76.3, 96.8, 100.9, 104.1, 128.2, 128.3,
128.4, 128.5, 129.1, 129.1, 129.8, 129.9, 130.0, 130.4, 133.2,
133.2, 133.3, 164.9, 165.2, 165.2, 168.1, 170.2, 170.3, 170.4,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
Cl
2
(15 mL) and
2
S
2
O
3
3
(20 mL).
+
170.6, 170.8, 171.7, 171.9. HRMS (ESI) m/z: [M + Na] calcd
2
S
2
O
3
3
for C59
H
66
N
2
O
29Na 1289.3643; Found 1289.3627.
2
4
Succinimidyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dide-
oxy-D-glycero-α-D-galacto-non-2-ulopyranosylonic
acid)-
(2→3)-(4-O-acetyl-2-O-benzoyl-β-D-galactopyranosyl)-
(
1→4)-2,3-di-O-benzoyl-β-D-glucopyranoside (21).
Com-
ꢀ
pound S7 (0.25 g, 0.20 mmol, 1.0 equiv) was dissolved in 10
1
3
, 600
mL pyridine followed by the addition of LiI (0.54 g, 4.0 mmol,
MHz): δ = 1.41 (3H, s), 1.71 (1H, t, J = 12.4 Hz), 1.78 (3H, s),
1.94 (3H, s), 1.97 (3H, s), 2.09 (3H, s), 2.13 (3H, s), 2.56 (5H,
m), 2.80 (1H, t, J = 9.2 Hz), 2.91 (1H, dd, J = 4.8, 9.5 Hz), 3.46
2
0 equiv) in the dark. This solution was then refluxed in the
dark for 3 hours, after which pyridine was removed under re-
duced pressure followed by co-distillation with toluene under
reduced pressure (3X). This crude was then purified by silica
(2H, m), 3.55 (1H, dd, J = 2.8, 10.7 Hz), 3.65 (1H, d, J = 9.2
Hz), 3.71 (1H, t, J = 7.6 Hz), 3.78 (3H, s), 3.81 (1H, q, J = 10.5
Hz), 3.96 (1H, dd, J = 6.1, 12.5 Hz), 4.02 (1H, d, J = 11.7 Hz),
4
4
2 2
flash chromatography using 5% MeOH in CH Cl as the eluent
to afford compound 21 as a white solid (0.18 g, 0.14 mmol, 68%
yield). Note: The white solid pertains to the protonated form of
the compound. A yellow oily material may result as the lithi-
ated species. This species has identical NMR characterization
as the protonated form but exhibits the lithiated form in MS
.10 (1H, d, J = 11.8 Hz), 4.24 (2H, br), 4.34 (1H, t, J = 9.3 Hz),
.38 (1H, dd, J = 2.28, 12.42 Hz), 4.58 (1H, dd, J = 3.2, 10.1
Hz), 4.83 (1H, td, J = 4.6, 11.9 Hz), 4.94 (1H, d, J = 3.0 Hz),
.98 (2H, t, J = 8.9 Hz), 5.19 (2H, dd, J = 7.6, 10.0 Hz), 5.24
1H, d, J = 7.4 Hz), 5.56 (1H, t, J = 7.6 Hz), 5.67 (2H, t, J = 8.6
4
(
+
1
analysis i.e. [MLi + Na] . H NMR (CD
.58 (1H, br), 1.70 (3H, br), 1.78 (3H, s), 1.92 (3H, s), 2.04 (3H,
s), 2.08 (3H, s), 2.10 (3H, s), 2.51 (1H, br), 2.66 (4H, s), 3.52
1H, br, J = 9.6 Hz), 3.62 (2H, m), 3.69 (4H, m), 3.90 (1H, br),
.94 (1H, dd, J = 5.6, 11.0 Hz), 4.04 (1H, dd, J = 6.2, 12.3 Hz),
3
OD, 600 MHz): δ =
Hz), 7.18 (2H, t, J = 7.3 Hz), 7.27 (3H, m), 7.32 (7H, m), 7.39
(2H, t, J = 7.8 Hz), 7.50 (4H, m), 7.57 (1H, t, J = 7.3 Hz), 8.02
1
13
1
(4H, t, J = 6.7 Hz), 8.26 (2H, d, J = 7.2 Hz). C{ H} NMR
CDCl , 150 MHz): δ = 20.3, 20.7, 20.8, 20.9, 21.5, 23.1, 25.3,
7.2, 48.7, 53.2, 62.6, 66.4, 66.6, 67.2, 67.3, 68.7, 69.5, 70.4,
(
3
(
3
3
7
1
1
1
1
4.21 (1H, br), 4.32 (1H, t, J = 9.4 Hz), 4.39 (1H, dd, J = 2.3,
12.4 Hz), 4.46 (1H, br, J = 9.1 Hz), 4.94 (1H, d, J = 8.00 Hz),
5.23 (2H, br), 5.33 (1H, d, J = 8.00 Hz), 5.44 (1H, t, J = 8.6 Hz),
1.0, 71.7, 71.7, 72.8, 72.9, 74.0, 75.0, 75.7, 96.9, 101.3, 102.9,
27.4, 127.4, 127.4, 127.5, 128.2, 128.3, 128.3, 128.3, 128.6,
29.1, 129.2, 129.8, 129.9, 130.0, 130.1, 130.5, 133.0, 133.2,
38.0, 138.4, 164.9, 165.0, 165.2, 167.9, 170.0, 170.0, 170.2,
5
.54 (1H, br), 5.66 (1H, t, J = 9.2 Hz), 7.43 (4H, m), 7.56 (4H,
m), 7.65 (1H, t, J = 7.4 Hz), 7.99 (4H, m), 8.17 (2H, d, J = 7.5
+
70.3, 170.4, 170.8, 170.8. HRMS (ESI) m/z: [M + Na] calcd
13
1
Hz). C{ H} NMR (CD
3
OD, 150 MHz): δ = 19.4, 19.5, 20.1,
78 2
for C73H N O29Na 1469.4582; Found 1469.4562.
2
1.2, 25.0, 48.2, 49.1, 59.7, 62.4, 62.8, 67.5, 68.1, 70.7, 71.0,
Succinimidyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-
,5-dideoxy-D-glycero-α-D-galacto-non-2-ulopyranosylonate)-
71.2, 72.3, 73.0, 73.5, 74.5, 76.1, 101.0, 103.3, 128.0, 128.1,
128.4, 129.3, 129.5, 129.5, 129.5, 129.7, 129.8, 130.0, 133.0,
3
(2→3)-(4-O-acetyl-2-O-benzoyl-β-D-galactopyranosyl)-
133.0, 165.5, 165.8, 170.3, 170.4, 170.5, 170.9, 170.9, 171.3,
171.9, 172.0. HRMS (ESI) m/z: [M + Na]+ calcd for
(1→4)-2,3-di-O-benzoyl-β-D-glucopyranoside (S7).
Com-
pound 2 (0.32 g, 0.22 mmol, 1.0 equiv) was dissolved in a mix-
ture of MeOH:EtOAc (1:3, 10 mL) followed by the addition of
palladium hydroxide on carbon (55 mg, 0.25 g/mmol substrate)
at room temperature. The reaction was then stirred for 1 hour
2
under an atmosphere of H gas (balloon, 1 atm). After 1 hour
the reaction was diluted with 10 mL of the same MeOH:EtOAc
mixture followed by bubbling Ar gas into the reaction solution.
The reaction was then filtered over a bed a Celite® followed by
a short bed of silica using 1% MeOH in CH
Compound S7 was then concentrated under reduced pressure to
C
58
H
63
N
2
O
29LiNa 1281.3569; Found 1281.3561. LRMS (ESI):
-
ꢀ
[M] calcd for C58 29 1252.4; Found 1252.1.
64 2
H N O
Aminooxy (5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-
non-2-ulopyranosylonic acid)-(2→3)-(β-D-galactopyranosyl)-
ꢀ
(1→4)-β-D-glucopyranoside (1). Compound 21 (20 mg, 0.016
mmol, 1.0 equiv) was dissolved in EtOH (1 mL) in a microwave
ꢀ
reaction tube. Hydrazine hydrate (30 μL, 0.96 mmol, 60 equiv)
was then introduced into the vessel which was then capped and
stirred at room temperature for 1 hour. Compound 21 was then
subjected to microwave conditions (200 W, 90 °C) for 1 hour
total in two 30 minute repetitive cycles. MS analysis revealed
2 2
Cl as the eluent.
1
3
a white solid (0.25 g, 0.20 mmol, 89% yield). H NMR (CDCl ,
600 MHz): δ = 1.46 (3H, s), 1.74 (1H, t, J = 12.5 Hz), 1.80 (3H,
s), 1.98 (3H, s), 2.02 (3H, s), 2.13 (3H, s), 2.18 (3H, s), 2.52
(1H, dd, J = 4.5, 12.6 Hz), 2.68 (4H, s), 2.73 (1H, dd, J = 6.8,
-
a single peak at 687.6 [M] . This peak is the acetone adduct of
the aminooxy GM3 which spontaneously forms with residual
acetone in the injector of the MS instrument. The reaction
ACS Paragon Plus Environment

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The Journal of Organic Chemistry
mixture was then concentrated under reduced pressure and then
passed through a P2 biogel column using H O as the eluent.
Fractions were found to contain final compound by staining sil-
ica gel plates. These fractions were frozen and the lyophilized
Support for this work was provided by the National Institutes of
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Health (NIH NCI R01 CA156661) & (NIH NIGMS U01
GM125271). We acknowledge Dr. Yong-Wah Kim in the De-
partment of Chemistry and Biochemistry at the University of
Toledo for providing assistance with the NMR instrument. We
also acknowledge Dr. Dragan Isailovic and David Baliu-Rodri-
guez for their assistance with high resolution mass spectrome-
try.
2
to reveal compound 1 as a white solid (8.0 mg, 0.012 mmol,
1
7
7% yield). H NMR (D
2
O, 600 MHz): δ = 1.70 (1H, t, J = 12.1
Hz), 1.94 (3H, s), 2.66 (1H, dd, J =4.6, 12.4 Hz), 3.26 (1H, m),
.57 (12H, m), 3.77 (4H, m), 3.86 (1H, d, J = 3.1 Hz), 3.92 (1H,
dd, J = 2.1, 12.2 Hz), 4.02 (1H, dd, J = 3.2, 9.9 Hz), 4.43 (1H,
3
13
1
d, J = 7.9 Hz), 4.51 (1H, d, J = 8.3 Hz). C{ H} NMR (D
2
O,
50 MHz): δ = 22.0, 39.6, 51.6, 59.9, 61.0, 62.5, 67.4, 68.1,
8.3, 69.3, 71.3, 71.7, 72.8, 74.3, 74.7, 75.1, 75.4, 78.0, 99.8,
REFERENCES
1
6
(1) Johnson, R. W.; Stieglitz, J. The Velocity of Hydrolysis of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
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7
8
9
0
Stereoisomeric Hydrazones and Oximes. J. Am. Chem. Soc. 1934,
56, 1904-1908.
+
102.6, 104.8, 173.9, 175.0. HRMS (ESI) m/z: [M + Na] calcd
19Na 671.2117; Found 671.2106.
GM3-PS A1 (23). Polysaccharide PS A1 (22) was isolated
PS A1 (1.0 mg, 9.1 X
10 mol) was dissolved in NaOAc buffer (1.0 mL, 0.1 M, pH =
5). To this solution was added 55 µL of 10 mM NaIO solution
5.5 x 10 mol). The reaction was allowed to shake gently for
0 min in the dark at room temperature. Excess NaIO was
for C23
H
40
N
2
O
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8, 41
and purified as previously described.
-9
4
-7
(
9
4
quenched by adding ethylene glycol and the mixture was con-
tinually shaken for another 20 min in the dark. The oxidized PS
A1 was purified with a centrifugal filter (Vivaspin, MWCO =
3
0 kDa). PS A1 was redissolved in NaOAc buffer (1.0 mL, 0.1
(5) Yin, Z.; Chowdhury, S.; McKay, C.; Baniel, C.; Wright, W.
-6
M, pH = 5). Subsequently, 2.9 mg (4.4 x 10 mol) of β-ami-
nooxy GM3 (1) was added and the reaction was gently shaken
for 18 hours in the dark at room temperature. Unreacted β-ami-
nooxy GM3 (1) was removed using a 30 kDa centrifugal filter
and purified GM3-PS A1 (23) conjugate was obtained as a ly-
ophilized white foam (1.0-1.1 mg).
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cessed with Bruker TopSpin 4.0.3. GM3-PS A1 H spectra
1
958, 80, 4585-4588.
were obtained with a probe temperature of 60 °C using the zg30
pulse program (D1 = 3.0 sec, AQ = 2.7 sec, NS = 128). Inte-
grations of the key “diagnostic” peaks were obtained using an
automated baseline correction. These integrations suggested a
(8) De Silva, R. A.; Wang, Q.; Chidley, T.; Appulage, D. K.;
Andreana, P. R. Immunological Response from an Entirely
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1
:2.33 ratio between GM3:PSA1 repeating unit. On average,
(9) Bourgault, J. P.; Trabbic, K. R.; Shi, M.; Andreana, P. R.
12
PS A1 has at least 120 repeating units which translates to ap-
proximately 51.5 GM3 conjugation sites per PS A1 molecule.
Synthesis of the tumor associative alpha-aminooxy disaccharide of
the TF antigen and its conjugation to a polysaccharide immune
stimulant. Org. Biomol. Chem. 2014, 12, 1699-1702.
(10) Shi, M.; Kleski, K. A.; Trabbic, K. R.; Bourgault, J.-P.;
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Carbohydrate Immunogen: Synthesis and Immunological
Evaluation. J. Am. Chem. Soc. 2016, 138, 14264-14272.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
NMR spectra for new or appropriate compounds (.pdf)
(11) Kleski, K. A.; Trabbic, K. R.; Shi, M.; Bourgault, J.-P.;
Andreana, P. R. Enhanced Immune Response Against the
Thomsen-Friedenreich Tumor Antigen Using a Bivalent Entirely
Carbohydrate Conjugate. Molecules 2020, 25, 1319.
AUTHOR INFORMATION
(12) Kalka-Moll, W. M.; Tzianabos, A. O.; Bryant, P. W.;
Corresponding Author
Niemeyer, M.; Ploegh, H. L.; Kasper, D. L. Zwitterionic
Polysaccharides Stimulate T Cells by MHC Class II-Dependent
Interactions. J. Immunol. 2002, 169, 6149-6153.
(13) Cobb, B. A.; Wang, Q.; Tzianabos, A. O.; Kasper, D. L.
Polysaccharide Processing and Presentation by the MHCII
Pathway. Cell 2004, 117, 677-687.
(14) De Silva, R. A.; Appulage, D. K.; Pietraszkiewicz, H.;
Bobbitt, K. R.; Media, J.; Shaw, J.; Valeriote, F. A.; Andreana, P.
R. The entirely carbohydrate immunogen Tn-PS A1 induces a
cancer cell selective immune response and cytokine IL-17. Cancer
Immunol., Immunother. 2012, 61, 581-585.
*Email = peter.andreana@utoledo.edu
ORCID
Peter R. Andreana: 0000-0002-5300-6700
Kristopher A. Kleski: 0000-0003-2418-5321
Mengchao Shi: 0000-0002-8174-3302
Matthew Lohman: 0000-0003-2823-6819
Notes
The authors declare no competing financial interest.
(15) Xia, L.; Schrump, D. S.; Gildersleeve, J. C. Whole-Cell
Cancer Vaccines Induce Large Antibody Responses to
ACKNOWLEDGMENT
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