Design and synthesis of trivalent Tn glycoconjugate polymers by nitroxide-mediated polymerization

Article abstract of DOI:10.1016/j.tet.2019.130776

A new synthetic method for preparing Tn glycoconjugate polymers, containing tumor-associated carbohydrate antigens, by controlled living radical polymerization is reported. To mimic the authentic structures of Tn glycopeptide antigens and to explore the controlled living radical polymerization, three tumor-associated carbohydrate antigens (GalNAc, GalNAcα1-O-Ser, and GalNAcα1-O-Thr) were attached to a styrene-type monomer through a diethylene glycol spacer. Under nitroxide-mediated polymerization, controlled living radical polymerization proceeded to afford defined glycopeptide polymers with different Tn densities and compositions. The polydispersity index (PDI) and molecular weights were increased and conversions were decreased upon increasing the concentration of Tn glycoconjugate monomers. The resulting Tn glycoconjugate polymers were characterized by NMR and IR. The spectral data indicate that the Tn glycoconjugate moiety did attach to the polymer chain and Tn glycoconjugate density could be adjusted through the nitroxide-mediated polymerization conditions. The number of Tn units containing in the polymer chains could be estimated by NMR integration. This synthetic approach provides a new and efficient tool for constructing novel Tn glycoconjugate polymers.

Full text of DOI:10.1016/j.tet.2019.130776

Tetrahedron xxx (xxxx) xxx
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Design and synthesis of trivalent Tn glycoconjugate polymers by
nitroxide-mediated polymerization
Si-Xian Liu, Yun-Tzu Tsai, Yu-Tung Lin, Jia-Yue Li, Che-Chien Chang*
Department of Chemistry, Fu Jen Catholic University, 510, Zhongzheng Rd., Xinzhuang Dist., New Taipei City, 24205, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
A new synthetic method for preparing Tn glycoconjugate polymers, containing tumor-associated car-
bohydrate antigens, by controlled living radical polymerization is reported. To mimic the authentic
structures of Tn glycopeptide antigens and to explore the controlled living radical polymerization, three
Received 18 September 2019
Received in revised form
2 November 2019
Accepted 6 November 2019
Available online xxx
tumor-associated carbohydrate antigens (GalNAc, GalNAca1-O-Ser, and GalNAca1-O-Thr) were attached
to a styrene-type monomer through a diethylene glycol spacer. Under nitroxide-mediated polymeriza-
tion, controlled living radical polymerization proceeded to afford defined glycopeptide polymers with
different Tn densities and compositions. The polydispersity index (PDI) and molecular weights were
increased and conversions were decreased upon increasing the concentration of Tn glycoconjugate
monomers. The resulting Tn glycoconjugate polymers were characterized by NMR and IR. The spectral
data indicate that the Tn glycoconjugate moiety did attach to the polymer chain and Tn glycoconjugate
density could be adjusted through the nitroxide-mediated polymerization conditions. The number of Tn
units containing in the polymer chains could be estimated by NMR integration. This synthetic approach
provides a new and efficient tool for constructing novel Tn glycoconjugate polymers.
Keywords:
Glycopolymers
Carbohydrate antigens
Glycoconjugates
Nitroxide-mediated polymerization
Tumor-associated
Tn antigens
© 2019 Elsevier Ltd. All rights reserved.
1. Introduction
production of long-lasting antibodies.
Fully synthetic carbohydrate vaccines with defined composi-
The carbohydrate Tn antigen (GalNAc
a
1-O-Ser/Thr), an
tions of carbohydrate ligands have showed promising results in
cancer immunotherapy [4]. Pioneered by Danishefsky, the syn-
thesis of multivalent anticancer vaccines bearing five different
tumor-associated carbohydrate antigens (TACAs) through peptide
bond formation has been reported [5]. The installation of an
appropriate linker, the conjugation to a carrier protein, Keyhole
Limpet Hemocyanin (KLH, an immunoenhancer), and the ratio of
glycopeptide: KLH (228:1) were determined to be an appropriate
chemical structure to serve as an efficient vaccine for the treatment
of breast and ovarian cancer (Scheme 1) [6]. These anticancer
vaccines are currently in clinical trials by OBI Pharma [7].
Instead of fully synthetic carbohydrate vaccines, several poly-
meric methods for preparing Tn glycoconjugates were also inves-
tigated in attempts to mimic synthetic anticancer vaccines [8e10].
Li and Kunz reported on the synthesis of synthetic polymer
glycopeptide conjugates by reversible addition-fragmentation
chain-transfer polymerization (RAFT) and click chemistry [8] (see
Scheme 2). The polymer-based glycopeptide vaccines were found
to induce significant immune reactions and elicit the production of
antibodies that were able to recognize breast tumor cells.
abnormal mucin-type O-glycan, is exclusively found in tumor cells
and its expression is associated with various types of cancers and
several human disorders [1]. It has a simple core structure,
composed of N-acetyl-D-galactosamine with a glycosidic linkage to
serine/threonine residues in animal glycoproteins (Fig. 1). Because
of the association of the Tn antigen with cancers and diseases,
development of Tn-based vaccines and other therapeutic ap-
proaches based on Tn-related studies were gradually gained
attention [2]. Tn antigens, along with other tumor-associated car-
bohydrate antigens (TACAs), provide ideal targets for the develop-
ment of anticancer vaccines [3]. However, the isolation of these
carbohydrate antigens in sufficient quantities and high purity from
natural sources is extremely difficult. Anticancer vaccines with
synthetic tumor-associated carbohydrate antigens are emerging as
an alternative approach to the treatment of cancers by enhancing
the immunogenicity of these carbohydrate antigens upon the
* Corresponding author.
E-mail address: 080686@mail.fju.edu.tw (C.-C. Chang).
Davis and Cameron have also reported on a polymerizable
https://doi.org/10.1016/j.tet.2019.130776
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molecular weights, and defined PDIs continues to be a challenge for
biomedical chemists. Several methods have been developed for
preparing carbohydrate polymers, including atom transfer radical
polymerization (ATRP) [12], reversible addition-fragmentation
chain transfer (RAFT) [13], and nitroxide-mediated polymeriza-
tion (NMP) [14]. Among these polymerization methods, NMP
techniques have been attracted
a great deal of interest by
biomedical chemists, since the NMP method can be used to prepare
metal-free or sulfur-free carbohydrate polymers. Hence, carbohy-
drate polymers prepared by the NMP method are much more
compatible with biological and physiological conditions. Further-
more, to be eventually used for the administration to animals or in
human testing, an ideal polymerization method that is both metal-
free and sulfur-free will be needed.
Herein, we report on a practical and synthetic method for the
preparation of Tn glycoconjugate polymers containing tumor-
associated carbohydrate antigens. Our approach involved the use
of nitroxide-mediated polymerization for preparing well-
controlled glycoconjugate polymers with tunable Tn densities,
defined molecular weights, and PDIs. The living character of
controlled radical polymerization could be further explored to
prepare novel glycoconjugates polymers containing trivalent Tn
glycoconjugates ligands. With a suitable spacer between the
glycosyl amino acid and the polymer backbone, well-defined Tn
glycoconjugates polymers bearing Tn tumor-associated carbohy-
drate antigen were designed. (Scheme 3).
Fig. 1. The structures of Tn carbohydrate antigens.
version of a truncated Tn-antigen glycan (without an amino acid
moiety), prepared by reversible addition-fragmentation chain
transfer (RAFT) polymerization [9]. The truncated Tn glycopolymer
was then conjugated with gold nanoparticles, to produce
multicopy-multivalent nanoscale glycoconjugates. Immunological
studies indicated that these nanomaterials generated the strong
and long-lasting production of antibodies. These results demon-
strated a simple approach toward preparing synthetic anticancer
vaccines based on nanomaterials without the need for a typical
vaccine protein component. Huang developed several ligation
methods for enhancing the immunogenicity of these carbohydrate
antigens to produce an immune response with a significant amount
of antibodies [10], which could be recognized by Tn expressing
tumor cells. These methods included (1) azide-alkyne cycloaddition
reactions (click chemistry) to produce covalent bonding between
Tn moiety and the tobacco mosaic virus (TMV) capsid as a carrier
[10a], (2) polymers prepared by cyanoxyl-mediated free radical
polymerization, followed by conjugation (amide bond formation)
with a Tn antigen [10b], (3) polymers produced by atom transfer
radical polymerization (ATRP), followed by ligation with a Tn
moiety [10c].
Although the preparation of synthetic glycopeptide conjugates
polymer have been previously reported, the main problems with
the current synthetic methods are that the polymerization process
and conjugation or ligation methods [11] between the polymers
and Tn carbohydrate moieties cannot be easily controlled. To pre-
pare strong and longest-lasting antibodies by these tumor-
associated carbohydrate antigens, a general method for preparing
well-defined and tunable carbohydrate polymers containing Tn
carbohydrate antigens with different compositions, desired
2. Results and discussion
2.1. Synthesis of Tn glycoconjugate monomers
A practical method was designed for the synthesis of Tn glyco-
conjugate monomers. Tn glycoconjugate monomers are composed
of three parts, namely, a Tn carbohydrate moiety, a spacer, and a
monomer. Because styryl-TEMPO could only regulate styrene or n-
butyl acrylate in a controlled manner, we decided to simply
examine the styryl-TEMPO-mediated polymerization of Tn glyco-
conjugate monomers using polystyrene as the polymer backbone.
Diethylene glycol (1) and 2-(2-aminoethoxy)ethanol (4) were used
as the spacers between the styrene and the Tn carbohydrate moiety
to avoid possible steric hindrance during the polymerization re-
actions and also to mimic the glycopeptide bonds on the surface of
Scheme 1. A pentavalent carbohydrate-based anticancer vaccine made by peptide bond formation.
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Scheme 2. Polymer-based glycopeptide conjugate prepared by RAFT polymerization and Click Chemistry.
Scheme 3. Synthesis of Tn glycoconjugate polymers by nitroxide-mediated polymerization.
tumor cells. Diethylene glycol (1) was reacted with 4-
chloromethylstyrene (2) under basic conditions to afford the sty-
rene derivative 3 as the first spacer monomer with a hydroxyl
group as its terminal [15] (Scheme 4). For the other spacer, the
amino group of 2-(2-aminoethoxy)ethanol (4) was first protected
by a Boc group, followed by reaction with 4-chloromethylstyrene,
and then deprotected to produce the second spacer monomer 7
with an amino group as its terminal.
bromide 10 by reaction with HBr/HOAc. Reductive elimination by a
reaction with zinc power in an aqueous buffer solution afforded the
triacetyl galactal 11 [16]. Compound 11 was then converted to 12
via an azidochlorination reaction [17]. Meanwhile, two glycosyl
acceptors 14a and 14b were prepared by esterifying Fmoc-
Ser(OH)eOH (13a) and Fmoc-Thr(OH)eOH (13b) with the t-butyl
trichloroimidate reagent in a co-solvent system of EtOAc/cyclo-
hexane [18]. (Scheme 5).
The Tn carbohydrate moieties were composed of N-acetyl
Silver ion-mediated glycosylation reactions [19] between the
glycosyl donor 12 and the glycosyl acceptors 14a/14b gave the
galactosamine that was attached to serine/threonine, through a
a-
glycosidic bond linkage. Starting from -galactose (8), all of the
D
glycosylation products 15a/15b. However, the a- and b-isomers
hydroxyl groups were reacted with acetic anhydride in pyridine to
could not be successfully separated at this stage. To overcome this
give the pentaacetate 9, which was then converted to the glycosyl
problem, the reduction of the azido groups to N-acetyl groups with
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Scheme 4. Synthesis of spacers.
Scheme 5. Synthesis of glycosyl donor and acceptors.
Zn power in presence of acetic anhydride gave the key Tn carbo-
hydrate derivatives 16a/16b, which are versatile synthons for syn-
thesis of a variety of Tn related structures. For example: (1)
deprotection of the acetyl groups on 16 gave a structure that could
serve as another glycosyl acceptor for the second glycosylation
reactions to produce tumor-associated disaccharides STn or TF
carbohydrate antigens [20]; (2) deprotection of the Fmoc group on
16 would generated a free amino group, which could be used for
the synthesis of an N-terminal Tn glycopeptide; (3) deprotection of
the t-Bu group on 16 would generate a free carboxylic acid group
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that could be used for the solid-phase synthesis of C-terminal Tn
glycoproteins. In this study, deprotection of the t-butyl group using
TFA in anisole [16] produced Tn glycosyl amino acid 17a/17b, which
could then be used in coupling reactions with the spacer monomer
7 to produce Tn glycoconjugate monomers (Scheme 6).
to protect the amino group to afford the desired Tn glycoconjugate
monomers 22a/22b (Tn1/Tn2). Herein, we report on the first syn-
thesis of Tn glycoconjugate monomers, including GalNAc (trun-
cated Tn), GalNAc-Ser (Tn1), and GalNAc-Thr (Tn2). With these
advanced Tn glycoconjugate monomers, the preparation of Tn
glycoconjugate polymers containing tumor-associated carbohy-
drate antigens will now be easier than before. Meanwhile, with
these synthetic methods in hand, the preparation of other tumor-
associated glycoconjugate polymers, such as STn and TF will also
be feasible.
Three different Tn glycoconjugate monomers, truncated Tn
(GalNAc), Tn1 (GalNHAc-Ser), and Tn2 (GalNAc-Thr), were designed
to study their polymerization reactions, shown in Scheme 7. To
prepare the truncated Tn glycopolymer (GalNAc, without an amino
acid moiety), the silver ion-mediated glycosylation reaction [19]
between the glycosyl donor 12 with the spacer 3 generated the
glycomonomer 18. To convert 18 into an N-acetyl galactosamine
derivative, the azido group was reduced to an amino group with
Zinc power in presence of acetic anhydride/acetic acid [16] to
directly give the truncated Tn glycomonomer 19. With Tn glycosyl
amino acids (17a/17b) and the spacer monomer 7 in hand, we are
now ready to prepare two Tn glycoconjugate monomers using
standard peptide bond formation reactions. After testing a variety
of coupling reagents [21], PyBOP/HOBt/DIPEA was found to be the
best coupling reagent for this amide bond formation to produce the
Tn glycoconjugate monomers 20a/20b. Preliminary studies in the
radical polymerization using the Tn glycoconjugate monomers 20a/
20b were not successful. NMR and GPC spectra indicated that no
polymerization occurred. The unsuccessful results were attributed
to the Fmoc protecting group on compounds 20a/20b, which are
not a stable functional group under conditions of Styryl-TEMPO
polymerization reactions. The higher temperature (125 ꢀC) used
in these polymerization reactions caused the degradation of the
Fmoc carbamate group, which is a normal process in carbamate
synthesis [22]. To cope with this situation and to continue our
studies on the Tn glycoconjugate polymerization, alternative ap-
proaches were examined. The deprotection of the Fmoc group us-
ing 5% piperidine in DMF [23] and gave compounds 21a/21b with a
free amino group. The amino group on compounds 21a/21b could
be regarded as starting compounds for producing peptide-bonds in
Tn glycopeptide polymers. Finally, a simple acetyl group was used
2.2. Polymerization studies
The polymerization reactions were conducted in sealed tubes
using 1 mol% of alkoxyamine and 40 wt % of dimethylformamide
(DMF) as the co-solvent with different compositions of Tn glyco-
conjugate monomers and styrene and the results are presented in
Table 1. In some experiments (entries 1e6, and 9), styryl-TEMPO
was used as the regulator. In other experiments (entries 7e8),
polymeric alkoxyamines were used as regulators in the living
radical polymerizations. The Tn glycoconjugate polymers were
collected by centrifugation and dried at 60 ꢀC for 12 h in a vacuum-
drying cabinet. Conversions were evaluated gravimetrically. Mo-
lecular weights and polydispersity index (PDI) were determined by
size exclusion chromatography (SEC). Because the synthesis of the
Tn glycoconjugate monomer 22a and 22b are laborious [24], at this
stage, only a ratio (10/90; ratio to styrene) of monomer composi-
tions in the polymerizations were studied (entries 5e8). In one
experiment (entry 9), the co-polymerization of three different Tn
glycoconjugate monomers was also studied.
The copolymerization reactions of the truncated glycoconjugate
monomer 19 and styrene using different ratios were studied first
(entry 1e4) at 125 ꢀC and an optimized reaction time (24 h) was
used. A conversion of 70% and a narrow PDI of 1.13 were obtained
when a ratio of 10/90 (monomer 19/styrene) was used in the sty-
rene polymerization (entry 1). When the concentration of
Scheme 6. Synthesis of Tn and glycosyl amino acids.
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Scheme 7. Synthesis of Tn glycoconjugate monomers.
truncated glycoconjugate monomer 19 in the styrene polymeriza-
tion reaction was increased, fluctuations in conversions between 45
and 70% were observed and narrow PDIs between 1.09 and 1.16
were detected. When the concentration of glycoconjugate mono-
mer 19 was increased to 75%, a broader PDI of 1.40 was observed
(entry 4). In general, the results showed that increasing the con-
centration of the Tn glycoconjugate monomer in the reactions
resulted in a decrease in conversion, a decrease in molecular weight
(Mn), and an increase in PDIs.
We then continued our studies on the polymerization of the Tn
glycoconjugate monomers 22a and 22 b at 125 ꢀC. As expected that
when Tn glycoconjugate monomers 22a and 22b were attached on
the styrene, the reactivity was decreased. The results could be
referred to the steric effect. Bulky Tn molecule caused inefficient
polymerization, compared to the monomer 19. Longer reaction
time was needed. To obtain reasonable conversions, the reaction
time was increased from 24 h to 30 h. Because the synthesis of the
monomers 22a and 22b were laborious and highly dense Tn gly-
coconjugate polymers are not suitable for synthetic mimics of po-
tential anticancer vaccines [25]. Therefore, only a ratio of 10/90 in
the polymerization reactions was studied. Similar results for the
polymerization of the Tn glycoconjugate monomers 22a and 22b
were obtained (entry 5, conversion ¼ 89%; PDI ¼ 1.08) and (entry 6,
conversion ¼ 79%; PDI ¼ 1.27). At this stage, commercially available
styryl-TEMPO could be used to regulate controlled radical poly-
merizations and Tn glycoconjugate polymers could be prepared
with reasonable conversions (~80%) and narrow PDIs (~1.2).
We next turned our attention to achieve our synthetic goal, i.e.,
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Table 1
Copolymerization of Tn Glycoconjugate Monomers and Styrenea
.
Entry
Glycoconjugate monomer
Ratio of glycoconjugate monomer/styrene
Time (h)
Conversion (%)
Mn (g/mol)
PDI
1
2
3
4
5
6
19
19
19
19
22a
22b
22a
19
10/90
25/75
50/50
75/25
10/90
10/90
10/90
10/90
24
24
24
24
30
30
30
30
30
70
45
57
41
89
79
95
90
59
7651
8031
5868
3280
14163
13171
19947
35347
31806
1.13
1.09
1.16
1.40
1.08
1.28
1.46
1.61
1.09
7b
8c
9
19þ22aþ22b
10/9/11/70
a
The ratio of styryl-TEMPO to the total monomers is 1/100, ratios of different monomers in polymerization are indicated in the table, and DMF was used as co-solvent (40 wt
%).
b
The polymerization was initiated by the polymeric alkoxyamine a, obtained from entry 6 under radical living conditions.
The polymerization was initiated by the polymeric alkoxyamine b, obtained from entry 7 under radical living conditions.
c
to produce three different Tn glycoconjugate carbohydrate moieties
The result was successful, with a conversion of 59%, Mn ¼ 32K, and
a PDI of 1.09 being obtained. This is the first report of the applica-
tion of a TEMPO-mediated polymerization to produce novel Tn
glycoconjugate polymers bearing unimolecular trivalent tumor-
associated carbohydrate ligands with the desired molecular
weights and defined PDIs. This synthetic method provides an
alternative approach for the rapid synthesis of Tn glycoconjugate
polymers with multivalent tumor-associated carbohydrate ligands
in one single polymerization sequence. Furthermore, these Tn
glycoconjugate polymers, carrying a TEMPO unit as its terminal,
constitute a living radical site for further polymerization reactions.
Block copolymerization in the presence of three Tn glycoconjugate
monomers in a one-pot polymerization reaction could be used to
produce a variety of novel Tn glycoconjugate polymers with the
desired sugar compositions. Since multivalent tumor-associated
carbohydrate anticancer vaccines were recently used in clinical
trials for immunocancer therapy, these results provide an alterna-
tive and practical approach for the preparation of tumor-associated
Tn glycoconjugate polymers with the desired molecular weights
and controlled PDIs. This appears to be the first report of the use of
nitroxide-mediated polymerization reactions to produce a poly-
meric mimic of tumor-associated Tn glycoconjugate glycopolymers.
NMP provides a powerful method for preparing these novel tumor-
associated Tn glycoconjugate polymers.
attached on one single polymer chain to produce a mimic for
tumor-associated carbohydrate anticancer vaccines bearing unim-
olecular trivalent Tn ligands. Two polymerization methods using
these three Tn glycoconjugate monomers (19, 22a, and 22b) were
examined, as shown in Scheme 8. One is a radical living polymer-
ization using polymeric alkoxyamines (entry 6 / 7 / 8) and the
other is a one-pot random polymerization reaction using three Tn
glycoconjugate monomers (entry 9). In general, when % Tn glyco-
monomer was increased, both conversion and Mn decreased, it
indicated that polymerization reaction rates were slowed down
when Tn glycomonomer was added. It also indicated that poly-
merization rates of Tn glycoonomer is slow than simple styrene.
In subsequent radical living polymerization reactions, once the
Tn glycoconjugate polymer was prepared, this polymer chain (a
polymeric alkoxyamine) contained a TEMPO unit as its terminal,
which could be used as a living radical site. When another potion of
monomer was added, this polymer chain would be expected to
grow again, as shown in Scheme 8. This is the advantage of
nitroxide-mediated polymerization (NMP).
To achieve a living, controlled radical polymerization, the po-
lymerizations of the Tn glycoconjugate monomer 22a and styrene
were initiated and regulated by the polymeric alkoxyamine A
(obtained from entry 6) to delivery a second polymeric alkoxy-
amine B bearing an unimolecular divalent Tn ligand (entry 7). The
polymer molecular weight was indeed increased (Mn ¼ 13K /
20K) and the PDI became broader (1.28 / 1.46). This glyco-
conjugate polymeric alkoxyamine B could be further polymerized
in the presence of the glycoconjugate monomer 19 and styrene to
provide a novel polymeric alkoxyamine C bearing an unimolecular
trivalent Tn ligand with a molecular weight of 35K and a PDI of 1.61
(entry 8). In one-pot random reaction, the polymerization of three
Tn glycoconjugate monomers (19, 22a and 22b) and styrene in a
ratio of (10/9/11/70) were initiated and regulated by styryl-TEMPO.
2.3. Characterization of Tn glycopolymers by NMR
1H NMR measurements were also carried out to confirm that the
Tn carbohydrate moieties were, in fact, attached to the polystyrene
chain, as shown in Fig. 2. In spectrum 2a, the characteristic peaks of
the Tn glycoconjugate monomer 22b appear at around 5.3, 5.1, and
4.8 ppm, and are assigned to the H4, H3, and H1 protons of the
GalNAc carbohydrate moieties, respectively. The characteristic
singlet peak around 4.5 ppm is assigned to the benzyl protons
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Scheme 8. Preparation of Novel Glycoconjugate Polymers with Trivalent Ligands Bearing a Tumor-Associated Carbohydrate Antigen (Tn).
(OCH2Ar), para-to the styrene group. The multiplets around
3.7e3.4 ppm are assigned to the methylene protons of the spacer
on the 2(2-aminoethoxy)ethanol group. Peaks corresponding to
five acetyl groups (OAc) appear at around 2.0e2.2 ppm. The char-
acteristic peaks of the Tn glycoconjugate A in spectrum 2b appear at
around 5.3, 5.1, and 4.8 ppm, and are assigned to the H4, H3, and H1
protons of the GalNAc carbohydrate moieties. The peaks at 4.5 ppm
of benzyl protons (OCH2Ar), the peaks at 3.7e3.4 ppm of the 2-
aminoethoxy group, and the peaks at around 2.0 ppm of the
acetyl groups are all present in the spectrum. These peaks serve to
confirm polymers containing a Tn glycoconjugate were, in fact,
prepared. The Tn glycoconjugate polymer B containing a divalent
ligand and polymer C (trivalent ligands) are also shown in spectra
2c and 2d, respectively. As shown in Fig. 2e, random polymerization
in the presence of three Tn glycoconjugate monomers gave the Tn
glycoconjugate polymer D containing trivalent ligands. During the
living polymerization sequence (A / B / C), characteristic peaks
with a relatively low intensity corresponding to the Tn glyco-
conjugate, compared to the characteristic beaks around
7.3e6.3 ppm of polystyrene, were observed. This indicates that the
polymerization efficiency indeed decreased as the polymers grew
in size.
carbohydrate specific peak (~5.35 ppm, 1H, H4) and the aromatic
protons (6.3e7.3 ppm, 5H, Ph), we could determine the Tn units per
100 units of styrene. (Please see the Supporting Information for
NMR integration and detail calculation).
When the ratio of glycomonomer/styrene is 10/90 in polymer-
ization reactions (entry 6, 7, and 8), it supposed to have 10 Tn units
per 100 units of styrene. However, by NMR integration, there are
about 3.37 units in Tn glycopolymer A (entry 6); 3.14 Tn units in Tn
glycopolymer B (entry 7), 2.29 units in Tn glycopolymer C (entry 8).
In general, it only has around 2e3 Tn units per 100 units of styrene
in the real products (entry 6e8).
When the ratio of glycomonomer/styrene is 30/70 in polymer-
ization reactions (entry 9), it supposed to have 30 Tn units per 100
units of styrene. However, there are around 22e23 Tn units per 100
units of styrene in Tn glycopolymer D (entry 9). Another example is,
when the ratio of glycomonomer/styrene is 50/50 in polymeriza-
tion reactions (entry 3), it supposed to have 50 Tn units per 100
units of styrene. However, by NMR integration, there are about 45
units per 100 units of styrene in glycopolymers (entry 3). The more
concentrated Tn glycomonomers in polymerization reactions, the
more accurate is the NMR integration used to determine Tn
numbers in the real products.
Regarding the numbers of Tn containing in polymer chains, we
could also estimate by NMR integration. Integration of the
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Fig. 2. a NMR spectrum of the Tn glycoconjugate monomer 22b, b NMR spectrum of the Tn glycoconjugate polymer A, c NMR spectrum of the Tn glycoconjugate polymer B, d NMR
spectrum of the Tn glycoconjugate polymer C, e NMR spectrum of the Tn glycoconjugate polymer D.
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Fig. 2. (continued).
2.4. Characterization of Tn glycopolymers by IR
in the spectra of the Tn glycoconjugate polymer (entry 6/black line,
entry 7/blue line, entry 8/brown line, entry 9/peach red line).
However, in the case of the Tn glycoconjugate polymer, the char-
acteristic bands of the polystyrene backbone and aromatic carbon-
carbon double bonds appeared at 2928 cmꢁ1 (C(sp3)ꢁH bond) and
1600 cmꢁ1 (C]C double bond), respectively. These IR spectra
further confirm that these polymers indeed contain Tn glyco-
conjugate units.
IR experiments were also performed to confirm that the poly-
mers prepared from NMP indeed contain Tn glycoconjugate moi-
eties. (Fig. 3) The characteristic bands [26] for the carbonyl
absorption bands appear at 1744 cmꢁ1 (ester) and 1680 cmꢁ1
(amide), indicating that the Tn conjugate monomer 22b contained
OAc and NHAc functional groups (red line), respectively. Similarly,
characteristic bands for the carbonyl absorption were also observed
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11
Fig. 3. IR spectrum of the Tn glycoconjugate monomer 22b (red line), polymer A (entry 6/black line), polymer B (entry 7, blue line), polymer C (entry 8, brown line), and polymer D
(entry 9, peach red line).
3. Conclusions
approach presented here provides a demonstration to show that
nitroxide-mediated polymerization is a powerful method for pre-
paring novel Tn glycoconjugate polymers with specific tumor-
associated carbohydrate ligands. The preparation of carbohydrate
polymers containing other tumor-associated carbohydrate anti-
gens, such as TF and STn, could also be prepared using presented
methods. However, there is an issue that the current Tn glyco-
conjugate polymers could not be used for cell-based assays or hu-
man clinical trials. Because these Tn glycoconjugate polymers
contained polystyrene, which may become the major epitope to
induce a strong immune response. Hence, to produce Tn glyco-
conjugate polymers containing water soluble monomers or bio-
compatible monomers may be another choice for the next gener-
ation of developing synthetic anticancer vaccines. To produce water
soluble monomers or bio-compatible monomers should rely on
using low temperature nitroxide-mediated polymerization tech-
niques [27], which is currently underway. The whole synthetic
approaches may provide an alternative method that can be applied
to research, related to cancer immunotherapy.
To simply examine the synthesis of Tn glycoconjugate polymers
could be prepared by nitroxide-mediated polymerization, the first
synthetic methods for preparing Tn glycoconjugate monomers
containing different Tn glycopeptide antigens were successfully
developed. Three tumor-associated Tn antigens (GalNAc, Gal-
NAca1-O-Ser, and GalNAca1-O-Thr) were attached to a styrene-
type monomer through a diethylene glycol spacer unit. The
nitroxide-mediate polymerization of these three monomers
resulted in the production of some defined Tn glycoconjugates
polymers that could simply mimic the authentic structures of Tn
glycoconjugate carbohydrate antigens. Controlled living radical
polymerization afforded defined glycoconjugate polymers with
different Tn densities and different compositions. The PDIs and
molecular weights were increased and conversions were decreased
with increasing the concentration of the Tn glycoconjugate
monomers. The resulting Tn glycoconjugate polymers were char-
acterized by NMR and IR. The spectra indicate that the Tn glyco-
conjugate moiety was attached to the polymer chain and that the
density and composition of the Tn glycoconjugate could be
adjusted though a controlled-living radical polymerization. The
number of Tn units containing in the polymer chains could be
estimated by NMR integration. This is the first report of the pro-
duction of Tn glycoconjugate polymers by NMP, which provide an
alternative method to afford Tn glycoconjugate polymers. These
preliminary results provide an alternative synthetic approach for
the rapid synthesis of multiple tumor-associated carbohydrates
antigens in one single living polymerization sequence. To the best
of our knowledge, this is the first report of the preparation of
tumor-associated carbohydrate polymers bearing unimolecular
trivalent Tn ligands with defined molecular weights and PDIs. The
4. Experimental section
4.1. General
1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were recor-
ded on a Bruker AVIII -300 MHz. The NMR spectra were recorded in
CDCl3 or CD3OD. Chloroform
(
d
¼ 7.26 ppm in 1H NMR;
d
¼ 77.0 ppm in 13C NMR) and methanol (
d
¼ 3.31 ppm in 1H NMR;
d
¼ 49.00 ppm in 13C NMR) were used as internal standard,
respectively. Splitting patterns were reported as following: s:
singlet, d: doublet, t: triplet, q: quartet, m: multiplet. Coupling
constant (J) was reported in Hz. IR were recorded on a Perkin Elmer
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12
S.-X. Liu et al. / Tetrahedron xxx (xxxx) xxx
Spectrum 100 FT-IR spectrometer and reported in cmꢁ1. High res-
olution mass spectrometry (HRMS) were recorded on a Shimadzu
LCMS-IT-TOF spectrometer (ESI-MS). Optical rotations were
measured on a Horiba SEPA-300 Digital polarimeter. TLC (Merck
Art. 60 F254, 0.25 mm) precoated sheet was used. The reaction
products were isolated by flash chromatography performed on
Merck Art. Geduran Si 60 (0.040e0.063 mm) silica gel. Yields of
products refer to chromatographically purified products unless
otherwise stated. The benzene used for radical cyclizations was
deoxygenated by passing a gentle stream of argon through for
30 min before use. All reactions were performed under a blanket of
N2 or Ar. The carbohydrate polymers were collected by centrifugal
sedimentation on Eppendorf Centrifuge 5810 R as a rotation rate of
8000 rpm for 10 min and further dried in a vacuum-drying cabinet
at 60 ꢀC for 12 h. Size exclusion chromatography (SEC) was carried
out with THF as eluent at a flow rate of 1.0 mL/min at room tem-
perature on a system consisting of a PU-1580 isocratic pump
(Jasco), a KF-804L column (Shodex), and a RI-71 refractometer
detector (Shodex). Data were analyzed with Elite EC2000 software
based upon calibration curves built upon polystyrene standards
(Polymer Standards Service) with peak molecular weights ranging
from 1800 to 56000 g/mol.
J ¼ 11.1, 0.6 Hz, 1H, ArCH]CH2), 5.02 (br s, 1H, NHBoc), 4.54 (s, 2H,
ArCH2O), 3.65e3.56 (m, 4H, CH2 ꢂ 2), 3.53 (t, J ¼ 5.1 Hz, 2H, CH2),
3.31 (q, J ¼ 5.3 Hz, 2H, CH2), 1.43 (s, 9H); 13C NMR (75 MHz, CDCl3)
d
155.9 (C), 137.6 (C), 137.0 (C), 136.4 (CH), 127.9 (CH ꢂ 2), 126.2
(CH ꢂ 2), 113.7 (CH2), 79.1 (C), 72.9 (CH2), 70.3 (CH2), 70.2 (CH2),
69.2 (CH2), 40.3 (CH2), 28.3 (CH3 ꢂ 3); HRMS (ESIþ): m/z calcd for
C
18H28NO4 [MþH] þ : 322.2013; found: 322.2008.
4.4. 2-[2-(4-Vinylbenzyloxy)ethoxy] ethanamine (7)
To a stirred solution of compound 6 (0.33 g, 1.03 mmol) in
anhydrous CH2Cl2 (10.3 mL) was slowly added trifluoroacetic acid
(0.95 mL) at 0 ꢀC. The reaction mixture was stirred at room tem-
perature for 1.5 h, then quenched by addition of 10 mL of 15% NaOH
solution at 0 ꢀC. The resulting mixture was diluted with CH2Cl2
(120 mL). The organic layer was washed, first with water
(24 mL ꢂ 2) and brine (24 mL), dried over anhydrous sodium sul-
fate, and concentrated under reduced pressure to give the crude
product 7 (0.22 g, 94%) as a pale yellow oil.
IR (neat) 3017, 2971, 2947, 2866, 1739, 1630, 1571, 1512, 1443,
1407, 1366, 1353, 1265, 1229, 1217, 1134, 1095, 1041, 1016, 992, 946,
913, 844, 829, 731, 702; 1H NMR (300 MHz, CDCl3)
d 7.38 (d,
J ¼ 8.1 Hz, 2H, para-), 7.30 (d, J ¼ 7.8 Hz, 2H, para-), 6.70 (dd, J ¼ 17.7,
10.8 Hz, 1H, ArCH]CH2), 5.74 (d, J ¼ 17.7 Hz, 1H, ArCH]CH2), 5.23
(d, J ¼ 10.8 Hz, 1H, ArCH]CH2), 4.55 (s, 2H, ArCH2O), 3.63 (s, 4H,
CH2 ꢂ 2), 3.50 (t, J ¼ 5.3 Hz, 2H, CH2), 2.86 (t, J ¼ 4.8 Hz, 2H, CH2),
4.2. N-[2-(2-Hydroxyethoxy)ethyl]carbamic acid tert-butyl ester
(5)
To
a
stirred solution of 2-(2-aminoethoxy)-ethanol (2 g,
1.60 (br s, 2H, NH2); 13C NMR (75 MHz, CDCl3)
d 137.7 (C), 136.9 (C),
19.02 mmol) in anhydrous CH2Cl2 (32 mL) was added di-tert-butyl
dicarbonate (4.94 g mL, 22.63 mmol, 1.2 equiv) and trimethylamine
(2.47 g, 24.39 mmol) at 0 ꢀC. The reaction mixture was stirred for
3.5 h at room temperature. The solution was then diluted with
CH2Cl2 (100 mL) and washed with brine (20 mL). The organic layer
was dried over anhydrous sodium sulfate, and concentrated under
reduced pressure to give the crude product 5 (>99%) as a pale
yellow oil, which was directly used for the next step.
136.5 (CH), 127.9 (CH ꢂ 2), 126.2 (CH ꢂ 2), 113.8 (CH2), 73.4 (CH2),
72.9 (CH2), 70.3 (CH2), 69.3 (CH2), 41.7 (CH2); HRMS (ESIþ): m/z
calcd for C13H20NO2 [MþH] þ : 222.1489; found: 222.1486.
4.5. 1,2,3,4,6-Penta-O-acetyl-
D-galactopyranose (9)
To a solution of -galactose (5.00 g, 27.75 mmol) and DMAP
D
(0.34 g, 2.78 mmol) in dry pyridine (35 mL) was added dropwise
acetic anhydride (26.24 mL, 277.50 mmol) at 0 ꢀC. The reaction
mixture was stirred at the room temperature for 3.5 h and then
worked up by addition of cold water, then concentrated to dryness
in vacuum. The residue was diluted with EtOAc (500 mL), and
washed with 1 M HCl (100 mL ꢂ 2), saturated NaHCO3(aq)
(100 mL ꢂ 3), water (100 mL), and brine (100 mL). The organic layer
was dried over MgSO4 and concentrated to give a crude product,
which was purified by flash chromatography with the eluent of
EtOAc/hexanes ¼ 4/6 to give the desired product 9 (11 g, > 99%) as a
white solid.
IR (neat) 3443, 3056, 2981, 2938, 2872, 2306, 1706 (C]O), 1507,
1456, 1423, 1393, 1368, 1265, 1171, 1125, 1065, 896, 863, 732,
730 cmꢁ1 1H NMR (300 MHz, CDCl3)
; d 5.03 (br s, 1H, NHBoc),
3.76e3.69 (m, 2H), 3.59e3.51 (m, 4H), 3.32 (q, J ¼ 5.3 Hz, 2H), 2.46
(t, J ¼ 5.4 Hz, 1H, OH), 1.43 (s, 9H); 13C NMR (75 MHz, CDCl3)
d 156.1
(C), 79.4 (C), 72.2 (CH2) 70.3 (CH2), 61.7 (CH2), 40.3 (CH2), 28.4
(CH3 ꢂ 3).
4.3. tert-Butyl (2-(2-((4-vinylbenzyl)oxy)ethoxy)ethyl)carbamate
(6)
1H NMR (300 MHz, CDCl3)
d
6.35 (d, J ¼ 1.2 Hz, 1H), 5.48 (d,
To a solution of compound 5 (0.98 g, 4.76 mmol) in DMF
(47.6 mL) was slowly added sodium hydride (0.23 g, 5.71 mmol) at
0 ꢀC. The resulting white suspension was stirred magnetically at the
same temperature for 30 min, followed by addition of 4-
vinylbenzyl chloride (1.34 mL, 9.51 mmol). The reaction mixture
was stirred at room temperature for overnight. The reaction was
quenched by addition of 1 mL of MeOH, and then concentrated
under reduced pressure to give a crude residue. The residue was
diluted with CH2Cl2 (180 mL) and then washed with water
(36 mL ꢂ 2) and brine (36 mL), dried over anhydrous sodium sul-
fate, filtered, and concentrated to give a crude product, which was
purified by flash chromatography with the eluent of EtOAc/hex-
anes ¼ 3/7 to give the desired product 6 (0.77 g, 51% over two steps)
as a yellow oil.
J ¼ 0.9 Hz, 1H), 5.31 (d, J ¼ 1.2 Hz, 2H), 4.32 (t, J ¼ 6.6 Hz, 1H),
4.01e4.14 (m, 2H), 2.14 (s, 6H), 2.02 (s, 3H), 2.00 (s, 3H), 1.98 (s, 3H);
13C NMR (75 MHz, CDCl3)
(C), 89.7 (CH), 68.7 (CH), 67.4 (CH), 67.3 (CH), 66.4 (CH), 61.2 (CH),
d
170.3 (C), 170.1 (C ꢂ 2), 169.8 (C), 168.9
20.9 (CH3), 20.6 (CH3 ꢂ 3), 20.5 (CH3).
4.6. 2,3,4,6-Tetra-O-acetyl-a-D-galactopyranosyl bromide (10)
To a solution of compound 9 (8.14 g, 20.86 mmol) in dry CH2Cl2
(41.72 mL) was added HBr (33% in acetic acid, 24.4 mL). The reaction
mixture was stirred at the room temperature for 5 h. The resulting
mixture was diluted with CH2Cl2 (250 mL), washed with saturated
NaHCO3 (aq) (60 mL ꢂ 3), and brine (60 mL). The organic layer was
dried over MgSO4 and concentrated to give the crude product 3
(7.72 g, 90%) as a pale yellow oil, which was directly used for the
next step.
IR (neat) 3442, 2980, 2868, 1708 (C]O), 1630, 1506, 1456, 1392,
1366, 1352, 1276, 1248, 1171, 1135, 1095, 1039, 1016, 992, 911, 845,
829, 779, 734 cmꢁ1;1H NMR (300 MHz, CDCl3)
d
7.38 (d, J ¼ 8.1 Hz,
1H NMR (300 MHz, CDCl3)
d
6.67 (d, J ¼ 3.9 Hz, 1H, H1), 5.49 (dd,
2H, para), 7.27 (d, J ¼ 8.1 Hz, 2H, para), 6.70 (dd, J ¼ 17.6, 11.0 Hz, 1H,
J ¼ 3.0, 0.6 Hz, 1H), 5.37 (dd, J ¼ 10.5, 3.3 Hz, 1H), 5.02 (dd, J ¼ 10.8,
ArCH]CH2), 5.73 (dd, J ¼ 17.7, 0.6 Hz, 1H, ArCH]CH2), 5.23 (dd,
4.1 Hz, 1H), 4.46 (t, J ¼ 6.6 Hz, 1H, H3), 4.16 (dd, J ¼ 11.4, 6.0 Hz, 1H,
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13
0
H6), 4.08 (dd, J ¼ 11.4, 6.9 Hz, 1H, H6 ), 2.13 (s, 3H, Ac), 2.09 (s, 3H,
IR (neat) 3368 (OeH), 2980, 1721 (C]O), 1515, 1450, 1370, 1310,
1223 (CeO), 1154, 1057, 839, 731 cmꢁ1 1H NMR (300 MHz, CDCl3)
Ac), 2.03 (s, 3H, Ac), 1.99 (s, 3H, Ac); 13C NMR (75 MHz, CDCl3)
;
d
170.2 (C), 170.0 (C), 169.8 (C), 169.7 (C), 88.1 (CH, anomeric car-
d
7.76 (d, J ¼ 7.2 Hz, 2H, Fmoc), 7.62 (d, J ¼ 7.5 Hz, 2H, Fmoc), 7.40 (t,
bon), 71.0 (CH), 68.0 (CH), 67.7 (CH), 66.7 (CH), 60.7 (CH2), 2.7 (CH3),
20.5 (CH3 ꢂ 3).
J ¼ 7.5 Hz, 2H, Fmoc), 7.31 (t, J ¼ 7.5 Hz, 2H, Fmoc), 5.66 (br d,
J ¼ 9.0 Hz, 1H, NHFmoc), 4.41 (d, J ¼ 7.2 Hz, 2H, Fmoc-CH2),
4.36e4.19 (m, overlapped with one t at 4.23, J ¼ 6.8 Hz, included
Fmoc-CH, Ha, and Hb), 2.25 (br s, 1H, OH), 1.49 (s, 9H, t-Bu), 1.25 (d,
4.7. 3,4,6-Tri-O-acetyl-D-galactal (11)
J ¼ 6.3 Hz, 3H, Hg); 13C NMR (75 MHz, CDCl3)
d 170.3 (C), 156.8 (C),
To a solution of compound 10 (9.20 g, 22.37 mmol) in acetone
(44.4 mL) was added saturated NaH2PO4 solution (89.5 mL) and
zinc dust (18.29 g, 279.63 mmol). The reaction mixture was stirred
at the room temperature for overnight. The mixture was then
diluted with EtOAc and filtered through a pad of celite, then
concentrated to dryness in vacuum. The residue was diluted with
EtOAc (500 mL), washed with saturated NaHCO3(aq) (100 mL ꢂ 2),
water (100 mL), and brine (100 mL). The organic layer was dried
over MgSO4 and concentrated to give a crude product, which was
purified by flash chromatography with the eluent of EtOAc/hex-
anes ¼ 3/7 to give the desired product 11 (5.40 g, 89%) as a colorless
oil.
143.9 (C), 143.8 (C), 141.3 (C ꢂ 2), 127.7 (CH ꢂ 2), 127.1 (CH ꢂ 2),
125.2 (CH), 120.0 (CH ꢂ 2), 82.7 (C), 68.3 (CH), 67.2 (CH2), 59.9 (CH),
47.2 (CH ꢂ 2), 28.0 (CH3 ꢂ 3), 20.0(CH3); HRMS: m/z calcd for
[MþH]þ: 398.1962; found: 398.1959.
4.10. O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-
galactopyranosyl)-N-(9-fuorenylmethoxycarbonyl)-L-serine tert-
butyl ester (16a)
To a two-neck round-bottom flask containing iron trichloride
(3.30 g, 20.00 mml) and sodium azide (0.86 g, 13.35 mmol) was
added a solution of the galcal 11 (1.82 g, 6.67 mmol) in acetonitrile
(56 mL) at ꢁ30 ꢀC, followed by dropwise addition of 35% hydrogen
peroxide (2.63 mL, 30.03 mmol) at the same temperature. The re-
action mixture was stirred at the same temperature for 3.5 h. After
TLC analysis indicated that the starting material was consumed
completed, the reaction mixture was allowed to warm to room
temperature. The mixture was concentrated to give a residue, and
diluted with DCM (250 mL). The ether solution was washed with
water (50 mL ꢂ 4), saturated NaHCO3 (50 mL ꢂ 3), brine
(50 mL ꢂ 2), dried over MgSO4, filtered, and concentrated to give a
crude residue, which was purified by column chromatography to
give the glycosyl chloride 12 (1.792 g, 77%), as a yellow oil.
To a three-neck round-bottom flask containing silver perchlo-
rate (80.8 mg, 0.390 mmol) and 4 Å molecular sieves (1.71 g) was
dried by heating the flask to 100e110 ꢀC under reduced pressure for
overnight. This flask was then cooled down to the room tempera-
ture, and was added solid silver carbonate (0.726 g, 2.660 mmol).
The whole system was added a solution of compound 14a (0.487 g,
1.270 mmol) in CH2Cl2/toluene (3.9 mL/3.9 mL) at 0 ꢀC and stirred
for 30 min. Followed by addition of another solution of previous
glycosyl chloride 12 (1.772 g, 5.067 mmol) in CH2Cl2/toluene
(3.9 mL/3.9 mL) at 0 ꢀC. The reaction mixture was stirred at room
temperature and protected from light for 15.8 h. After TLC analysis
indicated that the reaction progress was completed. The reaction
mixture was then filtered through a pad of celite, and washed with
CH2Cl2. The filtrate was concentrated and then diluted again with
CH2Cl2 (200 mL). The solution was washed with water (50 mL ꢂ 2),
saturated NaHCO3(aq) (65 mL ꢂ 2), and brine (65 mL). The organic
layer was dried over MgSO4 and concentrated to give a crude
product, which was preliminarily purified by flash chromatography
with the eluent of EtOAc/hexanes ¼ 3/7 to 4/6 to give the cure
product (0.84 g, crude yield, 95%) as a colorless oil.
1H NMR (300 MHz, CDCl3)
d
6.46 (dd, J ¼ 6.3, 1.5 Hz, 1H),
5.58e5.53 (m, 1H), 5.42 (dt, J ¼ 4.8, 1.7 Hz, 1H), 4.73 (ddd, J ¼ 6.3,
2.9, 1.7 Hz, 1H), 4.35e4.17 (m, 3H), 2.13 (s, 3H), 2.08 (s, 3H), 2.03 (s,
3H); 13C NMR (75 MHz, CDCl3)
d 170.6 (C), 170.1 (C), 170.3 (C), 170.1
(C), 145.4 (CH), 98.8 (CH), 72.8 (CH), 63.8 (CH), 63.7 (CH), 61.9 (CH2),
20.8 (CH3), 20.7 (CH3), 20.6 (CH3).
4.8. (S)eN-(Fluoren-9-ylmethoxycarbonyl)-
(14a)
L-serine tert-butyl ester
To a solution of Fmoc-L-Ser(OH)eOH (1.5 g, 4.58 mmol) in dry
EtOAc (34.0 mL) was added another solution of tert-butyl 2,2,2-
trichloroacetimidate (1.475 mL, 6.412 mmol) in cyclohexane
(4.5 mL). The reaction mixture was stirred at the 60 ꢀC for 24.5 h.
The reaction mixture was then concentrated to give a crude prod-
uct, which was first purified by recrystallization with DCM and
hexane. The collected solid was purified again by flash chroma-
tography with the eluent EtOAc/hexanes ¼ 4/6 to give the product
14a (1.438 g, 82%) as a white solid.
IR (neat) 3408 (OeH), 1729 (C]O), 1264 (CeO), 1157, 1081, 833,
729, 703 cmꢁ1
;
1H NMR (300 MHz, CDCl3)
d
7.77 (d, J ¼ 7.5 Hz, 2H,
Fmoc), 7.61 (d, J ¼ 7.2 Hz, 2H, Fmoc), 7.40 (t, J ¼ 7.5 Hz, 2H, Fmoc),
7.32 (t, J ¼ 7.2 Hz, 2H), 5.72 (br d, J ¼ 5.7 Hz, 1H, NH), 4.42 (d,
J ¼ 6.9 Hz, 2H, CO2CH2), 4.33 (br s, 1H, -H), 4.23 (t, J ¼ 6.9 Hz, 1H),
a
3.93 (br d, J ¼ 1.2 Hz, 2H,
b
d
-H), 1.90 (br s, 1H, OH), 1.49 (s, 9H, t-Bu);
169.4 (C), 156.3 (C), 143.8 (C), 143.7 (C),
13C NMR (75 MHz, CDCl3)
141.3 (C), 141.3 (C), 127.7 (CH ꢂ 2), 127.1 (CH ꢂ 2), 127.0 (CH ꢂ 2),
125.0 (CH ꢂ 2),120.0 (CH), 83.0 (C), 67.1 (CH2), 63.7 (CH2), 56.6 (CH),
47.1 (CH3 ꢂ 3).
4.9. (S)eN-(Fluoren-9-ylmethoxycarbonyl)-
L-threonine tert-butyl
ester (14b)
To a solution of this cure product (0.840 g, 1.206 mmol) in THF
(24.1 mL) was added zinc dust (1.621 g, 24.119 mmol), acetic an-
hydride (1.14 mL, 12.06 mmol), and acetic acid (0.69 mL,
12.06 mmol). The reaction was stirred at room temperature for
7.1 h. After TLC analysis indicated that the starting material was
consumed completely. The reaction mixture was then filtrated
through a pad of celite, and washed with EtOAc. The filtrate was
concentrated and then diluted again with EtOAc (180 mL). The so-
lution was washed with saturated NaHCO3(aq) (45 mL ꢂ 2), and
brine (45 mL). The organic layer was dried over MgSO4 and
concentrated to give a crude product, which was purified by flash
chromatography with the eluent of EtOAc/hexanes ¼ 7/3 to 8/2 to
give the product 16a (0.860 g, 74%) as a colorless oil.
To a solution of Fmoc-L-Thr(OH)eOH (3.00 g, 8.63 mmol) in dry
EtOAc (64 mL) was added another solution of tert-butyl 2,2,2-
trichloroacetimidate (2.16 mL, 12.08 mmol) in cyclohexane
(9.0 mL). The reaction was stirred at the room temperature. The
reaction progress was monitored by TLC analysis. Another part of
tert-butyl 2,2,2-trichloroacetimidate (2.00 mL, 11.16 mmol) was
added every 24 h until the starting material was consumed
completed at 94 h. The reaction mixture was concentrated to give a
crude product, which was first purified by recrystallization with
DCM and hexane. The collected solid was purified again by flash
chromatography with the eluent EtOAc/hexanes ¼ 35/65 to give the
product compound 14b (2.820 g, 82%) as a white solid.
IR (neat) 3341, 2936, 1744 (C]O), 1673 (C]O), 1523, 1450, 1369,
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S.-X. Liu et al. / Tetrahedron xxx (xxxx) xxx
1223 (CeO), 1154, 1133, 1048, 834 cmꢁ1; 1H NMR (300 MHz, CDCl3)
63%), as a colorless oil.
d
7.77 (d, J ¼ 7.5 Hz, 2H), 7.62 (d, J ¼ 7.2 Hz, 2H), 7.41 (t, J ¼ 7.2 Hz,
IR (neat) 3413, 3038, 2973, 2936, 1745 (C]O), 1682 (C]O), 1514,
2H), 7.33 (t, J ¼ 7.5 Hz, 2H), 5.82 (br d, J ¼ 8.4 Hz, 2H, NHAc,
NHFmoc), 5.37 (d, J ¼ 3.0 Hz, 1H, H4), 5.12 (dd, J ¼ 11.1, 3.3 Hz, 1H,
H3), 4.87e4.80 (m, 1H, H1), 4.64e4.54 (td, J ¼ 10.5, 3.6 Hz, 1H, H2),
4.43 (d, J ¼ 7.2 Hz, 2H, Fmoc-CH2), 4.25 (t, J ¼ 6.9 Hz, 1H, Fmoc-CH),
4.15e4.01 (m, 3H), 4.01e3.81 (m, 2H, H6 ꢂ 2), 2.16 (s, 3H, NHAc),
2.00 (s, 3H, OAc), 1.99 (s, 3H, OAc), 1.93 (s, 3H, OAc), 1.49 (s, 9H, t-
1450, 1370, 1311, 1266, 1222 (CeO), 1154, 1046, 909, 729 cmꢁ1 1H
;
NMR (300 MHz, CDCl3)
d
7.78 (d, J ¼ 7.5 Hz, 2H), 7.64 (d, J ¼ 7.5 Hz,
2H), 7.41 (t, J ¼ 7.2 Hz, 2H), 7.33 (t, J ¼ 7.4 Hz, 2H), 6.04 (d, J ¼ 9.9 Hz,
1H, NHAc), 5.65 (d, J ¼ 9.6 Hz, 1H, NHFmoc), 5.38 (d, J ¼ 2.1 Hz, 1H,
H4), 5.09 (dd, J ¼ 11.4, 3.0 Hz, 1H, H3), 4.88 (d, J ¼ 3.3 Hz, 1H, H1),
4.61 (td, J ¼ 10.7, 3.1 Hz, 1H, H2), 4.54e4.37 (m, 2H, Fmoc-CH2),
4.32e4.13 (m, 4H, included H5, Ha, Hb, and Fmoc-CH), 4.13e4.00 (m,
2H, H6), 2.17 (s, 3H, NHAc), 2.04 (s, 3H, OAc), 2.00 (s, 3H, OAc), 1.99
(s, 3H, OAc), 1.45 (s, 9H, t-Bu), 1.32 (d, J ¼ 6.3 Hz, 3H, Hg ꢂ 3); 13C
Bu); 13C NMR (75 MHz, CDCl3)
d 170.9 (C), 170.4 (C), 170.3 (C), 170.1
(C), 169.0 (C), 155.8 (C), 143.7 (C ꢂ 2), 141.3 (C ꢂ 2), 127.8 (CH ꢂ 2),
127.1 (CH ꢂ 2), 125.0 (CH ꢂ 2), 120.0 (CH ꢂ 2), 98.8 (CH, anomeric
carbon), 83.1 (C), 69.2 (CH2), 68.4 (CH), 67.2 (CH ꢂ 2), 61.9 (CH2 ꢂ 2),
54.8 (CH), 47.5 (CH), 47.1 (CH), 28.0 (CH3 ꢂ 3, t-Bu), 23.2 (CH3), 20.7
(CH3), 20.7 (CH3), 20.6 (CH3).
NMR (75 MHz, CDCl3)
d
170.9 (C), 170.3 (C ꢂ 4), 170.0 (C), 156.4 (C),
143.7 (C), 141.3 (C ꢂ 2), 127.7 (CH ꢂ 2), 127.1 (CH ꢂ 2), 125.1 (CH),
125.0 (CH), 120.0 (CH ꢂ 2), 99.9 (CH, anomeric carbon), 83.2 (C),
77.0 (CH), 68.7 (CH), 67.3 (CH, CH2), 62.1 (CH2), 58.9 (CH), 47.3 (CH),
47.1 (CH ꢂ 2), 28.1 (CH3 ꢂ 3, t-Bu), 23.2 (CH3), 20.7 (CH3 ꢂ 2), 20.6
(CH3), 18.5 (CH3); HRMS (ESIþ): m/z calcd for C37H47O13N2 [MþH]:
727.3073; found: 727.3057.
4.11. O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-
galactopyranosyl)-N-(9-fuorenylmethoxycarbonyl)-L-threonine
tert-butyl ester (16b)
To a two-neck round-bottom flask containing iron trichloride
(5.38 g, 33.15 mml) and sodium azide (1.44 g, 22.16 mmol) was
added a solution of the galcal 11 (3.02 g, 11.05 mmol) in acetonitrile
(92 mL) at ꢁ30 ꢀC, followed by dropwise addition of 35% hydrogen
peroxide (3.88 mL, 44.21 mmol). The reaction mixture was stirred
at the same temperature for 4 h. After TLC analysis indicated that
the starting material was consumed completed, the reaction
mixture was allowed to warm to room temperature. The mixture
was concentrated to give a residue, and diluted with ether
(300 mL). The ether solution was washed with water (80 mL ꢂ 4),
saturated NaHCO3 (80 mL ꢂ 2), brine (80 mL ꢂ 2), dried over
MgSO4, filtered, and concentrated to give a crude residue, which
was purified by column chromatography to give the glycosyl
chloride 12 (1.749 g, 45%), as a yellow oil.
To a three-neck round-bottom flask containing silver perchlo-
rate (0.125 mg, 0.600 mmol) and 4 Å molecular sieves (0.80 g) was
dried by heating the flask to 100e110 ꢀC under reduced pressure for
overnight. The flask was then cooled down to the room tempera-
ture, and was added solid silver carbonate (1.059 g, 4.083 mmol).
The whole system was added a solution of compound 14b (0.955 g,
2.402 mmol) in CH2Cl2/toluene (3.0 mL/3.0 mL) at 0 ꢀC and stirred
for 30 min. Followed by addition of another solution of compound
12 (1.750 g, 3.003 mmol) in CH2Cl2/toluene (3.0 mL/3.0 mL) at 0 ꢀC.
The reaction mixture was stirred at room temperature and pro-
tected from light for 48 h. After TLC analysis indicated that the re-
action progress was completed. The reaction mixture was then
filtered through a pad of celite, and washed with CH2Cl2. The filtrate
was concentrated and then diluted again with CH2Cl2 (300 mL). The
solution was washed with water (60 mL ꢂ 1), saturated NaHCO3(aq)
(60 mL ꢂ 3), and brine (60 mL). The organic layer was dried over
MgSO4 and concentrated to give a crude product, which was pre-
liminarily purified by flash chromatography with the eluent of
EtOAc/hexanes ¼ 4/6 to give the crude product (1.237 g, crude yield,
74%), as a colorless oil.
4.12. 2-[2-(4-Vinylbenzyloxy)ethoxy]ethyl-O-2-azido-2-deoxy-
3,4,6-triacetate-a-D-galactopyranoside (18)
To a two-neck round-bottom flask containing iron trichloride
(1.50 g, 6.17 mml) and sodium azide (0.80 g,12.34 mmol) was added
a solution of the galcal 11 (1.68 g, 6.17 mmol) in acetonitrile (21 mL)
at ꢁ30 ꢀC, followed by dropwise addition of 35% hydrogen peroxide
(5.76 mL, 61.70 mmol). The reaction mixture was stirred at the
same temperature for 12 h. After TLC analysis indicated that the
starting material was consumed completed, the reaction mixture
was allowed to warm to room temperature. The mixture was
concentrated to give a residue, and diluted with ether (210 mL). The
ether solution was washed with water (40 mL ꢂ 3), saturated
NaHCO3 (40 mL), brine (40 mL), dried over MgSO4, filtered, and
concentrated to give a crude residue, which was purified by column
chromatography to give the glycosyl chloride 12 (1.51 g, 70%), as a
yellow oil.
To a three-neck round-bottom flask containing silver perchlo-
rate (0.20 g, 0.95 mmol) and 4 Å molecular sieves (2.00 g) was dried
by heating the flask to 100e110 ꢀC under reduced pressure for
overnight. The flask was then cooled down to the room tempera-
ture, and was added solid silver carbonate (1.04 g, 3.80 mmol). The
whole system was added a solution of compound 12 (0.42 g,
1.90 mmol) in toluene (7.0 mL) at 0 ꢀC and stirred for 30 min. Fol-
lowed by addition of another solution of compound 3 (1.26 g,
5.69 mmol) in CH2Cl2 (6.5 mL) at 0 ꢀC. The reaction mixture was
stirred at room temperature and protected from light for 21 h. After
TLC analysis indicated that the reaction progress was completed.
The reaction mixture was then filtered through a pad of celite, and
washed with CH2Cl2. The filtrate was concentrated and then diluted
again with CH2Cl2 (240 mL). The solution was washed with water
(45 mL), saturated NaHCO3(aq) (45 mL ꢂ 3), and brine (50 mL). The
organic layer was dried over MgSO4 and concentrated to give a
crude product, which was preliminarily purified by flash chroma-
tography with the eluent of EtOAc/hexanes ¼ 3/7 to 45/55 to give
the product 18 (0.64 g, 64%), as a colorless oil.
To a solution of this cure product (1.237 g, 1.740 mmol) in THF
(34.8 mL) was added zinc dust (2.29 g, 34.80 mmol), acetic anhy-
dride (1.64 mL, 17.40 mmol), and acetic acid (1.00 mL, 17.40 mmol).
The reaction was stirred at room temperature for 2.4 h. After TLC
analysis indicated that the starting material was consumed
completely. The reaction mixture was then filtrated through a pad
of celite, and washed with EtOAc. The filtrate was concentrated and
then diluted again with EtOAc (300 mL). The solution was washed
with saturated NaHCO3(aq) (80 mL ꢂ 2), and brine (80 mL). The
organic layer was dried with MgSO4 and concentrated to give a
crude product, which was purified by flash chromatography with
the eluent of EtOAc/hexanes ¼ 7/3 to give the product 16b (0.794 g,
IR (neat) 3409, 3056, 2983, 2924, 1747 (C]O), 1644, 1512, 1424,
1371, 1321, 1226 (CeO), 1130, 1067, 1043, 1017, 971, 943, 915, 846,
822, 732, 702, 624, 612, 603, 591, 575, 551 cmꢁ1; HRMS (ESIþ): m/z
calcd for C25H33O10N3 [MþNa]þ: 558.2058; found: 558.2055.
[17]
a
-form: [
a]
þ 90.45 (c ¼ 0.37, CHCl3); 1H NMR (300 MHz,
D
CDCl3)
d
7.39 (d, J ¼ 8.1 Hz, 2H), 7.30 (d, J ¼ 8.1 Hz, 2H), 6.71 (dd,
J ¼ 17.4, 10.8 Hz, 1H, ArCH]CH2), 5.74 (dd, J ¼ 17.7, 0.6 Hz, 1H,
ArCH]CH2, cis to Ph), 5.45e5.35 (m, 2H, H4, H3), 5.23 (dd, J ¼ 10.8,
0.6 Hz, 1H, ArCH]CH2, trans to Ph), 5.08 (d, J ¼ 3.3 Hz, 1H, H1), 4.55
(s, 2H, ArCH2O), 4.30 (t, J ¼ 6.9 Hz, 1H, H5), 4.09 (dd, J ¼ 11.4, 6.5 Hz,
Please cite this article as: S.-X. Liu et al., Design and synthesis of trivalent Tn glycoconjugate polymers by nitroxide-mediated polymerization,
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S.-X. Liu et al. / Tetrahedron xxx (xxxx) xxx
15
1H, H6a or H6b), 4.05 (dd, J ¼ 11.4, 6.9 Hz, 1H, H6a or H6b), 3.90e3.58
moisture, as well as the compound 7.
(m, 9H, (OCH2CH2)2O, and H2), 2.14 (s, 3H), 2.05 (s, 3H), 2.02 (s, 3H);
Afterward, to a solution of PyBOP (0.526 g, 1.011 mmol), HOBt
(0.137 g, 1.011 mmol) in CH2Cl2 (1.4 mL), was added a solution of
previous free acid 17a in CH2Cl2 (4.0 mL), another solution of
compound 7 (0.217 g, 0.981 mmol) in CH2Cl2 (4.0 mL), and then N,
N-diisopropylethylamine (0.233 mL, 1.348 mmol) at the room
temperature. The reaction mixture was stirred for 15 h, and diluted
the result with CH2Cl2 (150 mL). The solution was washed with
water (30 mL), saturated NaHCO3(aq) (40 mL ꢂ 2), and brine
(40 mL), dried with MgSO4, and concentrated to give a crude
product, which was purified by flash chromatography with the
eluent of MeOH/CH2Cl2 ¼ 1/16 to give the desired product com-
pound 20a (0.456 g, 79%) as a colorless oil.
13C NMR (75 MHz, CDCl3)
d 170.4 (C), 170.0 (C), 169.8 (C), 137.8 (C),
136.9 (C), 136.5 (CH), 127.9 (CH ꢂ 2), 126.2 (CH ꢂ 2), 113.7 (CH2),
98.2 (CH), 72.9 (CH2), 70.7 (CH2), 70.1 (CH2), 69.4 (CH2), 68.1 (CH),
67.7 (CH2), 67.6 (CH), 66.5 (CH), 61.6 (CH2), 57.4 (CH), 20.6 (CH3 ꢂ 3).
[26]
b
-form was reported: [
a]
D ꢁ 12.68 (c ¼ 0.32, CHCl3); 1H NMR
(300 MHz, CDCl3)
d
7.39 (d, J ¼ 8.1 Hz, 2H), 7.30 (d, J ¼ 8.1 Hz, 2H),
6.71 (dd, J ¼ 17.7, 10.8 Hz, 1H, ArCH]CH2), 5.74 (d, J ¼ 17.7 Hz, 1H,
ArCH]CH2, cis to Ph), 5.29 (dd, J ¼ 3.3, 0.9 Hz, 1H, H4), 5.23 (d,
J ¼ 11.1, 0.9 Hz, 1H, ArCH]CH2, trans to Ph), 4.74 (dd, J ¼ 10.8,
3.3 Hz, 1H, H3), 4.55 (s, 2H, ArCH2O), 4.48 (d, J ¼ 8.1 Hz, 1H, H1),
4.15e4.00 (m, 2H, H6a, H6b), 3.87e3.51 (m, 10H, (OCH2CH2)2O, H2,
and H5), 2.13 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H); 13C NMR (75 MHz,
IR (neat) 1746 (C]O),1723 (C]O),1681 (C]O),1514,1450,1370,
CDCl3) d 170.4 (C),170.0 (C), 169.8 (C), 137.8 (C), 137.0 (C), 136.5 (CH),
1266, 1245, 1225, 1154, 1048, 730 cmꢁ1; 1H NMR (300 MHz, CDCl3)
128.0 (CH ꢂ 2), 126.2 (CH ꢂ 2), 113.8 (CH2), 102.5 (CH), 73.0 (CH2),
71.0 (CH), 70.7 (CH2), 70.6 (CH), 70.4 (CH2), 69.4 (CH2 ꢂ 2), 66.4
(CH), 61.3 (CH2), 60.7 (CH), 20.7 (CH ꢂ 2), 20.6 (CH3).
d
7.76 (d, J ¼ 7.2 Hz, 2H), 7.59 (d, J ¼ 7.2 Hz, 2H), 7.45e7.21 (m, 8H,
included Hf), 6.76e6.61 (m, 2H, Hg and NHꢁspeacer), 6.07 (d,
J ¼ 9.6 Hz, 1H, NHAc), 5.80 (d, J ¼ 7.5 Hz, 1H, NHFmoc), 5.73 (d,
J ¼ 17.4 Hz, 1H, Hh), 5.36 (d, J ¼ 3.0 Hz, 1H, H4), 5.23 (d, J ¼ 11.3 Hz,
1H, Hh), 5.07 (dd, J ¼ 11.3, 2.9 Hz, 1H, H3), 4.81 (d, J ¼ 3.3, 1H, H1),
4.63e4.47 (m, overlapped with 1 s at 4.50, 3H, H2 and He), 4.44 (d,
J ¼ 6.6, 2H, Fmoc-CH2), 4.32 (br s, 1H, Ha), 4.22 (t, J ¼ 6.9 Hz, 1H,
Fmoc-CH), 4.15e3.97 (m, 3H, H6 and H5), 3.92e3.69 (m, 2H, Hb),
3.67e3.37 (m, 8H, Ha-d), 2.16 (s, 3H, NHAc), 1.99 (s, 3H, OAc), 1.98 (s,
4.13. 2-[2-(4-Vinylbenzyloxy)ethoxy]ethyl-O-2-acetamido-2-
deoxy-3,4,6-triacetate-a-D-galactopyranoside (19)
To a solution of compound 18 (0.24 g, 0.45 mmol) in THF
(9.00 mL) was added zinc dust (0.59 g, 9.00 mmol), acetic anhy-
dride (0.43 mL, 4.50 mmol), and acetic acid (0.26 mL, 4.50 mmol).
The reaction was stirred at room temperature for 3 h. After TLC
analysis indicated that the starting material was consumed
completely. The reaction mixture was then filtrated through a pad
of celite, and washed with EtOAc. The filtrate was concentrated and
then diluted again with EtOAc (100 mL). The solution was washed
with saturated NaHCO3(aq) (20 mL ꢂ 2), and brine (20 mL). The
organic layer was dried with MgSO4 and concentrated to give a
crude product, which was purified by flash chromatography with
the eluent of EtOAc to give the product 19 (0.17 g, 68%), as a
colorless oil.
3H, OAc), 1.95 (s, 3H, OAc); 13C NMR (75 MHz, CDCl3)
d 170.8 (C),
170.4 (C), 170.3 (C ꢂ 2), 169.1 (C), 155.9 (C), 143.6 (C ꢂ 2), 141.3
(C ꢂ 2), 137.2 (C), 136.3 (CH), 128.0 (CH ꢂ 2), 127.8 (CH ꢂ 2), 127.1
(CH ꢂ 2), 126.3 (CH ꢂ 2), 125.0 (CH), 120.0 (CH ꢂ 2), 114.1 (CH2), 99.1
(CH), 72.9 (CH2), 70.1 (CH2), 69.5 (CH2), 69.2 (CH2 ꢂ 2), 68.5 (CH),
67.2 (CH2), 67.2 (CH ꢂ 2), 61.8 (CH2), 54.5 (CH), 47.4 (CH), 47.1 (CH),
39.4 (CH2), 23.1 (CH3), 20.7 (CH3 ꢂ 2), 20.6 (CH3); HRMS (ESIþ): m/z
calcd for C45H54N3O14 [MþH] þ :860.3600; found: 860.3591.
4.15. N-(9H-Fluoren-9-yl)-O-(2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-
a-D-galactopyranosyl)-L-threonine 2-[2-(4-vinylbenzyloxy)
Due to difficulty of products separation, only major product (a-
ethoxy]ethyl ester (20b)
[26]
form) was reported: [
a
]
D þ 2.11 (c ¼ 2.97, CHCl3); IR (neat) 3762,
3594, 2983, 3436, 3302, 3056, 2871, 1745 (C]O), 1667 (C]O)
amide, 1513, 1429, 1408, 1371, 1221 (CeO), 1161, 1130, 1045, 1018,
993, 949, 918, 845, 828, 732, 702, 622, 590, 557 cmꢁ1; found:
To a stirred solution of compound 16b (0.120 g, 0.166 mmol) in
anisole (0.436 mL) was slowly added trifluoroacetic acid (4.36 mL)
at 0 ꢀC. The reaction mixture was stirred for 1.2 h at room temper-
ature. The reaction solution was then concentrated under reduce
pressure. To this flash containing free acid 17b was co-evaporated
with toluene/benzene for three times to efficiently remove mois-
ture, as well as the compound 7.
Afterward, to a solution of PyBOP (0.103 g, 0.199 mmol), HOBt
(0.027 g, 0.199 mmol) in CH2Cl2 (0.26 mL), was added a solution of
previous free acid 17b in CH2Cl2 (0.7 mL), another solution of
compound 7 (0.609 g, 0.275 mmol) in CH2Cl2 (0.7 mL), and then
N,N-Diisopropylethylamine (0.060 mL, 0.347 mmol) at the room
temperature. The reaction mixture was stirred for 14.8 h, and
diluted the result with CH2Cl2 (60 mL). The solution was washed
with water (30 mL), saturated NaHCO3(aq) (30 mL ꢂ 2), and brine
(30 mL), dried with MgSO4, and concentrated to give a crude
product, which was purified by flash chromatography with the
eluent of EtOAc to give the desired product compound 20b (0.107 g,
60%) as a colorless oil.
553.2436; 1H NMR (300 MHz, CDCl3)
d
7.38 (d, J ¼ 8.1 Hz, 2H), 7.28
(d, J ¼ 8.1 Hz, 2H), 6.70 (dd, J ¼ 17.4, 10.8 Hz, 1H, ArCH]CH2), 5,87
(br d, J ¼ 9.6 Hz,1H, NHAc), 5.74 (d, J ¼ 17.4 Hz,1H, ArCH]CH2, cis to
Ph), 5.35 (d, J ¼ 3.0 Hz 1H, H4), 5.23 (dd, J ¼ 10.7, 0.3 Hz, 1H, ArCH]
CH2, trans to Ph), 5.17 (dd, J ¼ 11.4, 3.3 Hz,1H, H3), 4.89 (d, J ¼ 3.6 Hz,
1H, H1), 4.63e4.54 (m, 1H, H2) 4.53 (s, 2H, ArCH2O), 4.23 (t,
J ¼ 6.3 Hz, 1H, H5), 4.14e4.01 (m, 2H, H6a, H6b), 3.89e3.58 (m, 8H,
(OCH2CH2)2O), 2.15 (s, 3H, NHCOCH3), 2.02 (s, 3H), 1.98 (s, 3H), 1.90
(s, 3H); 13C NMR (75 MHz, CDCl3)
d 170.9 (C), 170.4 (C), 170.3 (C),
170.1 (C), 137.5 (C), 137.1 (C), 136.4 (CH), 127.9 (CH ꢂ 2), 126.2
(CH ꢂ 2), 113.9 (CH2), 98.0 (CH), 72.9 (CH2), 70.75 (CH2), 69.8 (CH2),
69.2 (CH2), 68.5 (CH), 67.6 (CH2), 67.3 (CH), 66.7 (CH), 61.9 (CH2),
47.6 (CH), 23.2 (CH3), 20.7 (CH3 ꢂ 3); HRMS (ESIþ): m/z calcd for
C
27H38O11N [MþNa]þ: 552.2439.
4.14. N-(9H-Fluoren-9-yl)-O-(2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy- -galactopyranosyl)- -serine 2-[2-(4-vinylbenzyloxy)
ethoxy]ethyl ester (20a)
a
-D
L
IR (neat) 3340, 2980, 1740 (C]O), 1721 (C]O), 1683 (C]O),
1515. 1450, 1370, 1311, 1272, 1224 (CeO), 1154, 839, 731 cmꢁ1 1H
;
NMR (300 MHz, CDCl3)
d
7.77 (d, J ¼ 7.2 Hz, 2H), 7.63 (d, J ¼ 6.9 Hz,
To a stirred solution of compound 16a (0.480 g, 0.674 mmol) in
anhydrous CH2Cl2 (6.74 mL) was slowly added trifluoroacetic acid
(4.84 mL) at 0 ꢀC. The reaction mixture was stirred at room tem-
perature for 5.0 h. The reaction solution was then concentrated
under reduce pressure. To this flash containing free acid 17a was co-
evaporated with benzene for three times to efficiently remove
2H), 7.46e7.23 (m, 8H), 6.74e6.60 (m, 2H, Hg and NHꢁspeacer),
6.43 (d, J ¼ 9.0 Hz, 1H, NHAc), 5.73 (d, J ¼ 17.4 Hz, 1H, Hh), 5.64 (d,
J ¼ 8.7 Hz,1H, NHFmoc), 5.38 (s,1H, H4), 5.23 (d, J ¼ 10.8 Hz,1H, Hh),
5.05 (dd, J ¼ 11.3, 3.0 Hz, 1H, H3), 4.89 (s, 1H, H1), 4.64e4.36 (m, 5H,
included H2 and He), 4.31e4.15 (m, 3H, H5 and Нa), 4.15e3.97 (m,
3H, H6 and Hb), 3.68e3.37 (m, 8H, Ha-d), 2.16 (s, 3H, NHAc), 2.01 (s,
Please cite this article as: S.-X. Liu et al., Design and synthesis of trivalent Tn glycoconjugate polymers by nitroxide-mediated polymerization,
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16
S.-X. Liu et al. / Tetrahedron xxx (xxxx) xxx
6H, OAc ꢂ 2), 1.98 (s, 3H, OAc), 1.23 (d, J ¼ 6.3 Hz, Hg); 13C NMR
stirred at room temperature for 2.8 h, and concentrated to give a
crude product, which was purified by flash chromatography with
the eluent of MeOH/CH2Cl2 ¼ 1/15 to give the compound 22b
(0.10 g, 83%) as a pale yellow oil.
(75 MHz, CDCl3)
d
170.8 (C), 170.7 (C), 170.3 (C ꢂ 2), 169.9 (C), 156.6
(C), 143.7 (C ꢂ 2), 141.3 (C ꢂ 2), 137.3 (C), 137.2 (C), 136.3 (CH), 127.9
(CH ꢂ 2), 127.8 (CH ꢂ 2), 127.1 (CH ꢂ 2), 126.3 (CH ꢂ 2), 126.2
(CH ꢂ 2), 125.1 (CH), 120.0 (CH ꢂ 2), 114.0 (CH2), 100.0 (CH), 77.4
(CH), 72.9 (CH2), 70.3 (CH2), 69.3 (CH2), 69.2 (CH), 68.8 (CH), 67.4
(CH2), 67.3 (CH ꢂ 2), 62.0 (CH2), 58.5 (CH), 47.5 (CH), 47.1 (CH ꢂ 2),
39.4 (CH2), 23.0 (CH3), 20.7 (CH3 ꢂ 2), 20.6 (CH3), 17.7 (CH3); HRMS
(ESIþ): m/z calcd for C46H55N3O14 [MþH]: 874.3757; found:
874.3753.
IR (neat) 2986, 1745 (C]O), 1723(C]O), 1681 (C]O), 1515, 1450,
1370, 1310, 1266, 1245, 1154, 1135, 1046, 909, 842, 730 cm-1; 1H
NMR (300 MHz, CDCl3)
d
7.40 (d, J ¼ 8.1 Hz, 2H, Hf), 7.29 (d,
J ¼ 8.1 Hz, 2H, Hf), 6.96 (d, J ¼ 4.8 Hz, 1H), 6.70 (dd, J ¼ 4.8 Hz, 1H,
Hg), 6.61e6.47 (m, 2H, NHAc ꢂ 2), 5.75 (d, J ¼ 17.7 Hz, 1H, Hh), 5.35
(s, 1H, H4), 5.24 (d, J ¼ 11.1 Hz, 1H, Hh), 5.07 (dd, J ¼ 11.4, 3.0 Hz, 1H,
H3), 4.88 (d, J ¼ 3.3 Hz, 1H, H1), 4.66e4.45 (m, 4H, included H2 and
Ha), 4.22 (t, J ¼ 6.3 Hz, 1H, H5), 4.15e3.96 (m, 3H, H6 and Hb),
3.70e3.33 (m, 8H), 2.15 (s, 3H, NHAc), 2.09 (s, 3H, NHAc), 2.01 (s,
6H, OAc ꢂ 2), 1.98 (s, 3H, OAc), 1.22 (d, J ¼ 6.3 Hz, Hg); 13C NMR
4.16. N-(9-Acetyl)-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-
galactopyranosyl)-L-serine 2-[2-(4-vinylbenzyloxy)ethoxy]ethyl
ester (22a)
(75 MHz, CDCl3)
d
171.0 (C), 170.7 (C ꢂ 2), 170.4 (C ꢂ 2), 170.4 (C),
To a two neck round-bottom flask containing compound 20a
(0.125 g, 0.146 mmol) was treated with 5% piperidine/DMF (0.073/
1.5 mL) to remove the N-Fmoc protecting group. The reaction
mixture was stirred at room temperature for 2.3 h, and then
concentrated under reduce pressure to give a crude product as a
pale yellow oil. To this flash containing free amine 21a was co-
evaporated with benzene for three times to efficiently remove
moisture.
170.1 (C), 137.2 (C ꢂ 2), 136.3 (CH), 128.0 (CH ꢂ 2), 126.3 (CH ꢂ 2),
114.1 (CH2), 100.4 (CH), 78.1 (CH), 73.0(CH2), 70.3 (CH2), 69.3
(CH2 ꢂ 2), 69.0 (CH), 67.3 (CH2), 62.2 (CH2), 56.5 (CH), 47.4 (CH),
40.2 (CH2), 23.2 (CH3), 23.0 (CH3), 20.8 (CH3), 20.7 (CH3 ꢂ 2), 18.0
(CH3); HRMS (ESIþ): m/z calcd for C33H47N3O13 [MþH]: 694.3182;
found: 694.3177.
4.18. Typical procedure for polymerization of carbohydrate
polymers
To a solution of this free amine 21a in CH2Cl2 (1.66 mL) was
added triethylamine (0.030 mL, 0.219 mmol) and acetic anhydride
(0.021 mL, 0.219 mmol). The reaction mixture was stirred at room
temperature for 30 min, and concentrated to give a crude product,
which was purified by flash chromatography with the eluent of
MeOH/CH2Cl2 ¼ 1/20e1/15 to give the compound 22a (0.092 g,
A Schlenk-tube (thick ¼ 1.6 mm) was charged with styryl-
TEMPO (1%), carbohydrate monomer, styrene, and N,N-dime-
thylformamide (40 wt%). The tube was subjected to three freeze-
thaw cycles and sealed off under argon. The polymerization was
carried out under argon at 125 ꢀC for an indicated period (please
see Table 1). The resulting mixtures were cooled to room temper-
ature and precipitated in methanol, diethyl ether, or hexane. The
carbohydrate polymers were collected by centrifugal sedimenta-
tion and dried in a vacuum-drying cabinet at 60 ꢀC for 12 h. Con-
version was evaluated gravimetrically. Molecular weight and
polydispersity index (PDI) were determined by size exclusion
chromatography (SEC).
92%) as a pale yellow oil.
[23]
[a
]
þ81.88 (c ¼ 1.66, CHCl3); IR (neat) 3085, 2935, 2875,
D
1748 (C]O), 1661 (C]O), 1539, 1434, 1407, 1373, 1235, 1133, 1080,
1050, 949, 912, 848, 830, 720 cmꢁ1 1H NMR (300 MHz, CDCl3)
7.40 (d, J ¼ 8.1 Hz, 2H, para), 7.29 (d, J ¼ 8.1 Hz, 2H, para), 6.81 (br s,
;
d
1H, C(O)NHCH2), 6.70 (dd, J ¼ 17.7, 10.8 Hz, 1H, ArCH]CH2), 6.59 (d,
J ¼ 7.8 Hz, 1H, NHAc), 6.20 (d, J ¼ 9.9 Hz, 1H, NHAc), 5.75 (d,
J ¼ 17.3 Hz, 1H, ArCH]CH2), 5.35 (d, J ¼ 2.7 Hz, 1H, H4), 5.25 (d,
J ¼ 11.1 Hz, 1H, ArCH]CH2), 5.07 (dd, J ¼ 11.1, 3.3 Hz, 1H, H3), 4.80
(d, J ¼ 3.6 Hz, 1H, H1), 4.65e4.50 (m, 4H, overlapped 1 s at 4.56, H2,
Ha and ArCH2O), 4.15e3.98 (m, 3H, H5 and H6 ꢂ 2), 3.84 (dd,
Acknowledgment
0
J ¼ 10.2, 4.2 Hz, 1H, Hb), 3.71e3.52 (m, 7H, Hb and CH2 ꢂ 3),
We wish to thank the Ministry of Science and Technology,
Taiwan (grant number: MOST 107-2113-M-030-005), and Depart-
ment of Chemistry, Fu Jen Catholic University for financial supports.
SXL and YTT both held Department of Chemistry Master Student
Fellowships. JYL held a university scholarship for excellent main-
land Chinese students. Mr. Chih-Hao Luo is acknowledged for
processing NMR integration.
3.51e3.36 (m, 2H, NHCH2), 2.15 (s, 3H), 2.04 (s, 6H),1.98 (s, 3H), 1.96
(s, 3H); 13C NMR (75 MHz, CDCl3)
d 170.9 (C), 170.5 (C), 170.4 (C),
173.3 (C), 170.2 (C), 169.3 (C), 137.3 (C), 137.2 (C), 136.3 (CH), 128.1
(CH ꢂ 2), 126.3 (CH ꢂ 2), 114.1 (CH2), 99.1 (CH, anomeric carbon),
73.0 (CH2), 70.2 (CH2), 69.3 (CH2 ꢂ 3), 68.6 (CH), 67.1 (CH ꢂ 2), 61.8
(CH2), 52.6 (CH), 47.3 (CH), 39.4 (CH2), 23.2 (CH3), 23.1 (CH3), 20.8
(CH3), 20.7 (CH3 ꢂ 2); HRMS (ESIþ): m/z calcd for C32H46N3O13
[MþH]þ:680.3025; found: 680.3020.
Appendix A. Supplementary data
4.17. N-(9-Acetyl)-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-
galactopyranosyl)-L-threonine 2-[2-(4-vinylbenzyloxy)ethoxy]ethyl
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tet.2019.130776.
ester (22b)
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To a two neck round-bottom flask containing compound 20b
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concentrated under reduce pressure to give a crude product as a
pale yellow oil. To this flash containing free amine 21b was co-
evaporated with benzene for three times to efficiently remove
moisture.
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Tetrahedron, https://doi.org/10.1016/j.tet.2019.130776