Synthetic mimics of carbohydrate-based anticancer vaccines: Preparation of carbohydrate polymers bearing unimolecular trivalent carbohydrate ligands by controlled living radical polymerization

Article abstract of DOI:10.1039/c4ra04907a

Synthetic methods for preparation of three different styrene-type carbohydrate monomers, containing mannose, sialic acid and N-acetyllactosamine were successfully developed. Diethylene glycol was used as the spacer between the styrene and carbohydrate moi

Full text of DOI:10.1039/c4ra04907a

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Synthetic mimics of carbohydrate-based
anticancer vaccines: preparation of carbohydrate
polymers bearing unimolecular trivalent
carbohydrate ligands by controlled living radical
polymerization†
Cite this: RSC Adv., 2014, 4, 47066
*
Teng-Yuan Kuo, Li-An Chien, Ya-Chi Chang, Shuang-Yu Liou and Che-Chien Chang
Synthetic methods for preparation of three different styrene-type carbohydrate monomers, containing
mannose, sialic acid and N-acetyllactosamine were successfully developed. Diethylene glycol was used
as the spacer between the styrene and carbohydrate moieties. Under the conditions of nitroxide-
mediated polymerizations, controlled living radical polymerizations could be accomplished, affording
well defined carbohydrate polymers with different sugar compositions. The PDIs were increased and
conversions were decreased upon increasing the concentrations of carbohydrate monomers. The
resulting carbohydrate polymers were characterized by NMR. Novel carbohydrate polymers bearing
unimolecular trivalent carbohydrate ligands could also be achieved through the living radical process
used in this study.
Received 24th May 2014
Accepted 17th September 2014
DOI: 10.1039/c4ra04907a
www.rsc.org/advances
enhanced via the use of multivalent ligands. Carbohydrate
polymers with multiple carbohydrates ligands are capable of
exhibiting a cluster effect, thus increasing their binding ability.6
Due to the increasing interest in articial materials for a variety
of biomedical uses, scientic reports have been published on
their use as macromolecular drugs, drug delivery systems, a
stationary phase for the separation of carbohydrates binding
proteins, bioassays, surface modiers, articial tissues.7
Introduction
Cell surface carbohydrates are involved in a variety of biological
and medical functions, including cellular recognition, adhe-
sion, cell growth regulation, cancer cell metastasis, and
inammation.1 They also serve as recognition sites for infec-
tious bacteria, viruses, toxins, and hormones that result in the
development of a wide variety of diseases.2 Studies of their
biological and medical functions are limited because of the
unavailability of sufficient amounts from natural sources or
difficulties associated with preparing these complex oligosac-
charides. Although the great improvements have been achieved
in the chemical and enzymatic synthesis of oligosaccharides,
practical and efficient synthetic methods for the preparation of
these complex structures are still highly demanded.3
Carbohydrate polymers are synthetic polymers with a non-
carbohydrate main chain, containing carbohydrate moieties as
the terminals.4 Carbohydrate polymers can be considered as
alternative structures to oligosaccharides, which have been
shown to mimic oligosaccharides in interactions between
carbohydrates and lectins.5 The interaction between carbohy-
drates and lectins is usually weak and binding ability can be
Several methods have been developed for preparing carbo-
hydrate polymers, such as atom transfer radical polymerization
(ATRP),8 reversible addition–fragmentation chain transfer
(RAFT),9 and nitroxide-mediated polymerization (NMP).10 NMP
techniques have attracted a great deal of attention from
biomedical chemists, since NMP is a method that can be used
to prepare metal-free or sulfur-free carbohydrate polymers.
Carbohydrate polymers prepared by NMP are much more
compatible with biological and physiological conditions.11
Several synthetic methods have been reported for preparing
carbohydrate polymers containing cell surface carbohydrates,
along with their biomedical applications.12 However, the main
limitation and disadvantages of current synthetic methods are
that the polymerization processes or efficient ligation methods
between polymers and carbohydrates moieties cannot be easily
controlled. Therefore, the successful preparation of well-
dened carbohydrate polymers containing cell surface carbo-
hydrates with different compositions of carbohydrates moieties,
desired molecular weights and dened PDI is still a challenge
for biomedical chemists. Fully synthetic carbohydrate antigens
with dened compositions of carbohydrate ligands showed
Department of Chemistry, Fu Jen Catholic University, 510, Zhongzheng Rd., Xinzhuang
Dist., New Taipei City, 24205 Taiwan, Republic of China. E-mail: 080686@mail.u.
edu.tw; Fax: +886-2-29023209; Tel: +886-2-29053562
† Electronic supplementary information (ESI) available: Experimental procedures;
characterization data; 1H, 13C/DEPT/2D NMR spectra. See DOI:
10.1039/c4ra04907a
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promising results in cancer immunotherapy.13 Synthesis of a
pentavalent carbohydrate vaccine bearing ve different tumor-
associated carbohydrates antigens (TACAs) has been repor-
ted.14 Installation of an appropriate linker, conjugation to a
carrier protein, Keyhole Limpet Hemocyanin (KLH, an immu-
noenhancer), and a ratio of glycopeptides : KLH (228 : 1) were
determined to be an appropriate structure for an efficient
vaccine in the treatment of breast and ovarian cancer.14a
Experimental section
Materials
Styrene was distilled from CaH2 under reduced pressure to
remove the stabilizer and was stored at 4 ꢀC under an argon
atmosphere. THF were distilled by reuxing over traces of
sodium metal using benzophenone as indicator under N2.
Benzene, dichloromethane, pyridine, N,N-dimethylformamide
(DMF), and 1,8-diazavicyclo[5.4.0]undec-7-ene (DBU) were dried
over CaH2 and then distilled before use. Acetic anhydride, acetyl
chloride, and borotriuoride diethyletherate (BF3$OEt2)
were directly distilled before use. 2,2,6,6-Tetramethyl-1-
piperidinyloxy free radical (TEMPO), 4-chloromethylstyrene,
diethylene glycol, mannose, sialic acid, galatose, and N-acetyl-
glucosamine were used as received.
Herein, we report on a practical and synthetic method for the
preparation of carbohydrate polymers containing different cell
surface carbohydrates using nitroxide-mediated polymerization
(Scheme 1). Three different cell surface carbohydrates, i.e.,
mannose (Man), sialic acid (Sia), and N-acetyllactosamine
(LacNAc) were attached to the polymer backbone. Carbohydrate
polymers containing mannose have been used to investigate
interactions between human dendritic cells associated with
lectins and HIV envelop glycoproteins.15 Carbohydrate polymers
containing sialic acid have been shown to inhibit the spread of
inuenza virus infections.16 Carbohydrate polymers bearing
N-acetyllactosamine have been prepared to inhibit hemagglu-
tination by Erythrina corallodendron and the specic binding
ability of lectins.17 However, only a few of these carbohydrate
polymers could be prepared in a well-controlled manner in
polymerization reactions or with different sugar composi-
tions.18 Furthermore, to be eventually used for potential
administration in animals or in human testing, an ideal poly-
merization method that is both metal-free and sulfur-free will
be needed. Our approach involved the use of nitroxide-
mediated polymerization for preparing well-controlled carbo-
hydrate polymers with dened molecular weights and PDIs. The
living character of controlled radical polymerization could be
further explored to prepare novel carbohydrate polymers con-
taining multivalent carbohydrate ligands. With a suitable
spacer between the carbohydrate and the polymer backbone,
carbohydrate polymers bearing unimolecular trivalent carbo-
hydrate ligands that mimic unimolecular trivalent
carbohydrates-based anticancer vaccines were designed.14
General
1H NMR (800, 500, and 300 MHz) and 13C NMR (200, 125, and 75
MHz) spectra were recorded on a Bruker AVIII-800 MHz, a
Bruker AVIII-500 MHz, and a Bruker Advance-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 Spectrum 100 FT-IR spec-
trometer and reported in cmꢁ1. High resolution mass spec-
trometry (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 ash chromatography performed on Merck Art.
Geduran Si 60 (0.040–0.063 mm) silica gel. Yields of products
refer to chromatographically puried products unless otherwise
stated. The benzene used for radical cyclizations was deoxy-
genated 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
Scheme 1 Synthetic mimics of unimolecular trivalent carbohydrate-based anticancer vaccines.
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sedimentation on Eppendorf Centrifuge 5810 R as a rotation layers were dried over MgSO4, ltered, and concentrated in
rate of 8000 rpm for 10 min and further dried in a vacuum- vacuum. The crude product was puried by silica gel chroma-
drying cabinet at 60 ꢀC for 12 h. Size exclusion chromatog- tography (EtOAc : hexane ¼ 7 : 3) to give the product as a
raphy (SEC) was carried out with THF as eluent at a ow rate of colorless oil (6.37 g, 95%). IR (neat) 3420 (OH), 3087, 3006, 2868,
1.0 mL minꢁ1 at room temperature on a system consisting of a 1629, 1512, 1454, 1406, 1351, 1319, 1288, 1243, 1212, 1093,
PU-1580 isocratic pump (Jasco), a KF-804L column (Shodex), 1067, 1016, 991, 907, 845, 827, 764, 729, 719, 628 cmꢁ1; 1H NMR
and a RI-71 refractometer detector (Shodex). Data were analyzed (300 MHz, CDCl3) d 7.39 (d, J ¼ 8.1 Hz, 2H, para-), 7.39 (d, J ¼ 8.1
with Elite EC2000 soware based upon calibration curves built Hz, 2H, para-), 6.70 (dd, J ¼ 17.7, 10.8 Hz, 1H, ArCH]CH2), 5.74
upon polystyrene standards (Polymer Standards Service) with (d, J ¼ 17.7 Hz, 1H, ArCH]CH2), 5.23 (d, J ¼ 10.8 Hz, 1H,
peak molecular weights ranging from 1800 to 56 000 g molꢁ1
.
ArCH]CH2), 4.55 (s, 2H, ArCH2O), 3.78–3.58 (m, 8H, CH2 ꢂ 4),
2.79 (br s, 1H, OH); 13C NMR (75 MHz, CDCl3) d 137.5 (s), 137.0
(s), 136.4 (d), 127.9 (d ꢂ 2), 126.2 (d ꢂ 2), 72.9 (t), 72.4 (t), 70.3
(t), 69.3 (t), 61.7 (t); HRMS (ESI+): m/z calcd for C13H18O3 [M +
H]+: 223.1334; found: 223.1325.
Typical procedure for polymerization of carbohydrate
polymers
A Schlenk-tube (thick ¼ 1.6 mm) was charged with styryl-
TEMPO (1%), carbohydrate monomer, styrene, and N,N-di-
methylformamide (40 wt%). The tube was subjected to three
freeze–thaw cycles and sealed off under argon. The polymeri-
2-[2-(4-Vinylbenzyloxy)ethoxy]ethyl 2,3,4,6-tetra-O-acetyl-a-D-
glucopyranoside (3). To a solution of compound 2 (ref. 19) (6.16
˚
g, 11.82 mmol) and freshly activated 4 A molecular sieves (9 g) in
CH2Cl2 (136 mL) was added a solution of compound 1 (3.15 g,
ꢀ
ꢀ
zation was carried out under argon at 125 C for an indicated
14.17 mmol) in CH2Cl2 (100 mL) at ꢁ10 C. Aer being stirred
period (please see Table 1). The resulting mixtures were cooled
to room temperature and precipitated in methanol, diethyl
ether or hexane. The carbohydrate polymers were collected by
centrifugal sedimentation and dried in a vacuum-drying
for another 30 minutes, BF3$OEt2 (0.30 mL, 2.36 mmol) was
added dropwise at ꢁ10 ꢀC. The reaction mixture was stirred for
another 3.5 h at the same temperature, then the reaction was
quenched by slow addition of MeOH (100 mL). The resulting
mixture was ltrated and the residue was washed carefully with
CH2Cl2 and MeOH. The combined organic phases were washed
with concentrated NaHCO3 solution (150 mL), brine (150 mL),
dried over MgSO4, ltered, and concentrated in vacuum. The
crude product was puried by silica gel chromatography (hex-
ane : EtOAc ¼ 1 : 1) to give the product as a colorless oil (4.47 g,
69%). [a]2D6 +13.23 (c ¼ 1.45, CHCl3); IR (neat) 2893, 1746 (C]O),
1630, 1513, 1434, 1407, 1369, 1219, 1173, 1135, 1083, 1045,
1017, 979, 912, 847, 829, 793, 735, 689, 638 cmꢁ1; 1H NMR (300
ꢀ
cabinet at 60 C for 12 h. Conversion was evaluated gravimet-
rically. Molecular weight and polydispersity index (PDI) were
determined by size exclusion chromatography (SEC).
2-[2-(4-Vinylbenzyloxy)ethoxy]ethanol (1). To a solution of
diethylene glycol (76.0 mL, 795 mmol) was added a solution of
4-vinylbenzyl chloride (4.70 mL, 30.00 mmol), NaOH (1.20 g,
30.00 mmol), and water (0.54 mL, 30.00 mmol). The reaction
mixture was stirred at 60 ꢀC for 24 h. Aer cooling to room
temperature, H2O (100 mL) was added and the resulting
mixture was extracted with Et2O (3 ꢂ 250 mL). The organic
Table 1 Copolymerization of carbohydrate monomers and styrenea
Carbohydrate
monomer
Ratio of carbohydrate
monomer/styrene
Entry
Time (h)
Conversion (%)
Mn (g molꢁ1
)
PDI
1
2
3
4
5
6
7
8
3
3
3
3
3
3
3
3
5
5
5
5
20/80
20/80
20/80
20/80
40/60
60/40
80/20
100/0
20/80
40/60
60/40
80/20
100/0
25/75
75/25
20/80
20/80
24
30
36
42
42
42
42
42
48
48
48
48
48
48
48
42
42
6
36
70
72
74
59
80
58
31
19
16
22
9
6237
7267
1.11
1.10
1.14
1.12
1.21
1.19
1.37
1.30
1.13
1.55
1.68
1.74
1.22
1.15
1.38
1.24
1.52
11 961
14 301
25 738
20 302
39 818
20 831
11 115
8363
10 835
11 997
4497
27 348
28 692
15 082
24 045
9
10
11
12
13
14
15
16b
17c
5
10b
10b
5
65
19
33
65
10b
a
The ratio of styryl-TEMPO to total monomers is 1/100, ratios of different monomers in polymerization are indicated as table, and DMF was used as
co-solvent (40 wt%). b The polymerization was initiated by the polymeric alkoxyamine I, obtained from entry 4 under radical living conditions. c The
polymerization was initiated by the polymeric alkoxyamine II, obtained from entry 16 under radical living conditions.
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MHz, CDCl3) d 7.38 (d, J ¼ 8.1 Hz, 2H, para-), 7.29 (d, J ¼ 8.1 Hz, solution of 2-[2-(4-vinylbenzyloxy)ethoxy]ethyl 2-acetamido-3,4,6-
2H, para-), 6.70 (dd, J ¼ 17.4, 10.8 Hz, 1H, RCH]CH2), 5.73 (d, J tri-O-acetyl-2-deoxy-b-D-glucopyranoside (obtained from previous
¼ 17.7 Hz, 1H, RCH]CH2), 5.36 (dd, J ¼ 9.9, 3.3 Hz, 1H), 5.19– step) (0.12 g, 0.22 mmol) in MeOH (4.40 mL) was added sodium
5.32 (m, 3H, RCH]CH2), 4.87 (d, J ¼ 1.5 Hz, 1H, H1), 4.55 (s, 2H, methoxide (0.006 g, 0.110 mmol). The reaction mixture was stirred
ArCH2O), 4.28 (dd, J ¼ 12.9, 5.7 Hz, 1H, H6), 4.04–4.12 (m, 2H), at room temperature for 6 h. The solution was then neutralized
3.77–3.87 (m, 1H), 3.63–3.72 (m, 2H), 2.14 (s, 3H, Ac), 2.09 (s, with Amberlite IR 120 (H+) ion-exchange resin, the resin ltered
3H, Ac), 1.99 (s, 3H, Ac), 1.98 (s, 3H, Ac); 13C NMR (75 MHz, off and washed with MeOH. The resulting solution was concen-
CDCl3) d 170.6 (s), 170.0 (s), 169.8 (s), 169.7 (s), 137.8 (s), 136.9 trated under reduced pressure, and then puried by silica gel
(s), 136.5 (d), 127.9 (d ꢂ 2), 126.2 (d ꢂ 2), 113.7 (t), 97.7 (d), 72.9 chromatography (DCM : MeOH ¼ 7.5 : 1) to give the product as a
(t), 70.7 (t), 70.0 (t), 69.5 (d), 69.4 (t), 69.1 (d), 68.3 (d), 67.4 (t), colorless and viscous liquid (0.06 g, 85%), over two steps from
66.1 (d), 62.4 (t), 20.8 (q), 20.7 (q), 20.6 (q ꢂ 2); HRMS (ESI+): m/z compound 6. [a]D25 ꢁ69.78 (c ¼ 0.33, CH3OH); IR (neat) 3340, 2945,
calcd for C27H36O12 [M + H]+: 575.2105; found: 575.2112.
2835, 1650 (C]O), 1566, 1450, 1408, 1378, 1318, 1258, 1211, 1078,
Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-2,3,5-trideoxy-2-{2- 1022, 942, 829 cmꢁ1; 1H NMR (300 MHz, CD3OD) d 7.37 (d, J ¼ 8.3
[2-(4-vinylbenzyloxy)ethoxy]ethyl}-D-glycero-a-D-galacto-2-non- Hz, 2H, para-), 7.27 (d, J ¼ 8.3 Hz, 2H, para-), 6.68 (dd, J ¼ 17.7,
ulopyranosonate (5). To a solution of compound 4 (ref. 20) (0.95 11.0 Hz, 1H, ArCH]CH2), 5.73 (dd, J ¼ 17.7, 0.8 Hz, 1H,
˚
g, 1.86 mmol), compound 1 (4.15 g, 18.60 mmol) and 4 A ArCH]CH2), 5.17 (d, J ¼ 11.0 Hz, 1H, ArCH]CH2), 4.49 (s, 2H,
0
molecular sieve (2 g) in acetonitrile (1.9 mL) was added zinc ArCH2O), 4.42 (d, J ¼ 8.4 Hz, 1H, H1, b-form), 3.92 (m, 1H, H1 a),
bromide (0.84 g, 3.74 mmol) at room temperature. The reaction 3.82 (dd, J ¼ 11.9, 1.7 Hz, 1H, H6a), 3.70–3.52 (m, 9H), 3.40–3.32
mixture was stirred at same temperature for 19 h. The precipi- (m, 1H), 3.30–3.19 (m, 2H), 1.89 (s, 3H, NHAc); 13C NMR (75 MHz,
tation was ltered off on celite and washed with CH2Cl2. The CD3OD) d 174.0 (s), 139.2 (s), 138.7 (s), 138.0 (d), 129.4 (d ꢂ 2),
ltrate was washed with water, 50% aqueous NaHCO3, dried 127.4 (d ꢂ 2), 114.2 (t), 102.9 (d), 78.2 (d), 76.5 (d), 74.0 (t), 72.2 (d),
over MgSO4, and concentrated in vacuum. The crude product 71.8 (t ꢂ 2), 70.8 (t), 70.1 (t), 62.9 (t), 57.5 (d), 23.2 (q); HRMS (ESI+):
was puried by silica gel chromatography (hexane : EtOAc ¼ m/z calcd for C21H31NO8 [M + H]+: 426.2128; found: 426.2138.
1 : 9) to give the product as a colorless solid (0.96 g, 75%, a : b ¼
2-[2-(4-Vinylbenzyloxy)ethoxy]ethyl
2-acetamido-6-O-tert-
91 : 9). However, only a-anomer was successfully characterized. butyldiphenylsilyl-2-deoxy-b-D-glucopyranoside (8). To a solu-
IR (neat) 3264 (NH), 3010, 2957, 1743 (C]O), 1659 (C]O), 1546, tion of compound 7 (1.49 g, 3.50 mmol) and imidazole (0.72 g,
1445, 1407, 1369, 1303, 1220, 1199, 1177, 1130, 1095, 1074, 10.50 mmol) in dry DMF (17.5 mL) was added TBDPSCl (1.82
1039, 995, 942, 912 (CH2]CHR), 855, 828, 787, 754, 733, 666, mL, 7.00 mmol) dropwise. The reaction mixture was stirred at
648 cmꢁ1; 1H NMR (300 MHz, CDCl3) d 7.37 (d, J ¼ 8.1 Hz, 2H, room temperature for 5 h. The excess TBDPSCl was quenched
para-), 7.28 (d, J ¼ 8.1 Hz, 2H, para-), 6.69 (dd, J ¼ 17.7, 11.0 Hz, by addition of MeOH (1 mL). The resulting mixture was allowed
1H, ArCH]CH2), 5.72 (d, J ¼ 17.7 Hz, 1H, ArCH]CH2), 5.42– to stir for another 30 minutes, and then concentrated under
5.27 (m, overlapped with one dd at 5.30, J ¼ 8.3, 1.4 Hz, 3H, H8, reduced pressure to give a crude product, which was then
H7, NHAc), 5.21 (d, J ¼ 11.0 Hz, 1H, ArCH]CH2), 4.91–4.77 (m, puried by silica gel chromatography (DCM : MeOH ¼ 15 : 1) to
1H, H4), 4.53 (s, 2H, ArCH2O), 4.29 (dd, J ¼ 12.5, 2.6 Hz, 1H, afford the product as a pale yellow oil (1.75 g, 76%). [a]2D5 ꢁ46.51
00
H9a), 4.12–4.01 (m, 3H, H5, H6, H9b), 3.94–3.83 (m, 1H, H1 a), (c ¼ 2.66, CHCl3); IR (neat) 3311 (OH), 3072, 3010, 2930, 2858,
3.75 (s, 3H, CO2CH3), 3.69–3.56 (m, 6H, CH2 ꢂ 3), 3.51–3.40 (m, 1907, 1826, 1652 (C]O), 1513, 1472, 1462, 1428, 1406, 1374,
00
1H, H1 b), 2.60 (dd, J ¼ 12.8, 4.7 Hz, 1H, H3-eq.), 2.15–1.89 (m, 1361, 1312, 1238, 1216, 1110, 1061, 998 (RCH]CH2), 938, 908
1
13H, overlapped with four s at 2.12, 2.11, 2.01, 2.00, Ac ꢂ 4 and (RCH]CH2), 850, 824, 750, 702, 666, 622, 604 cmꢁ1; H NMR
H
3-ax), 1.86 (s, 3H, Ac); 13C NMR (75 MHz, CDCl3) d 170.9 (s), (300 MHz, CDCl3) d 7.76–7.66 (m, 4H, Ph), 7.45–7.33 (m, 8H, Ph,
170.6 (s), 170.2 (s), 170.1 (s ꢂ 2), 168.2 (s), 137.8 (s), 136.9 (s), para-), 7.29 (d, J ¼ 8.1 Hz, 2H, para-), 6.94 (d, J ¼ 5.7 Hz, 1H,
136.5 (d), 127.9 (d ꢂ 2), 126.2 (d ꢂ 2), 113.7 (t), 98.8 (s), 72.9 (t), NH), 6.70 (dd, J ¼ 17.7, 11.1 Hz, 1H, ArCH]CH2), 5.75 (dd, J ¼
72.4 (d), 70.5 (t), 70.1 (t), 69.3 (t), 69.1 (d), 68.6 (d), 67.3 (d), 64.4 17.7, 0.5 Hz, 1H, ArCH]CH2), 5.25 (dd, J ¼ 11.1, 0.5 Hz, 1H,
(t), 62.3 (t), 52.7 (q), 49.3 (d), 37.9 (t), 23.1 (q), 21.0 (q), 20.8 (q ꢂ ArCH]CH2), 4.57 (d, J ¼ 12.0 Hz, 1H, ArCH2O), 4.53–4.48 (m,
2), 20.7 (q); HRMS (ESI+): m/z calcd for C33H45NO15 [M + H]+: overlapping with one d at 4.50, J ¼ 8.4 Hz, 2H, H1 and ArCH2O),
696.2867; found: 696.2885, [M
718.2681.
+
Na]+: 718.2687; found: 3.99 (dd, J ¼ 11.0, 3.2 Hz, 1H), 3.95–3.83 (m, 2H), 3.77 (dd, J ¼
8.9, 2.3 Hz, 1H), 3.73–3.49 (m, 10H), 3.46–3.33 (m, 3H), 1.94 (s,
2-[2-(4-Vinylbenzyloxy)ethoxy]ethyl-2-acetamido-2-deoxy-b-D- 3H, NHAc), 1.05 (s, 9H, t-Bu); 13C NMR (75 MHz, CDCl3) d 173.1
glucopyranoside (7). To a solution of compound 1 (0.40 g, 1.81 (s), 137.4 (s), 136.8 (s), 136.2 (d), 135.6 (d ꢂ 4), 133.3 (s), 133.2
˚
mmol), compound 6 (ref. 21) (0.82 g, 2.26 mmol), and 4 A (s), 129.6 (d ꢂ 2), 128.3 (d ꢂ 2), 127.6 (d ꢂ 4), 126.3 (d ꢂ 2),
molecular sieve (0.5 g) in dry DCM (3.3 mL) was allowed to stirred 114.2 (t), 101.2 (d), 76.7 (d), 75.6 (d), 73.1 (t), 72.0 (d), 70.9 (t),
for 30 minutes at room temperature. The reaction mixture was 69.3 (t), 68.5 (t), 64.1 (t), 57.4 (d), 26.7 (q ꢂ 3), 22.9 (q), 19.2 (s);
then added mercuric cyanide (0.66 g, 4.75 mmol) and stirred at HRMS (ESI+): m/z calcd for C37H49NO8Si [M + H]+: 664.3305;
same temperature for 30 h. The precipitation was ltered off on found: 664.3328; [M + Na]+: 686.3125; found: 686.3102.
celite and diluted with CH2Cl2 (150 mL). The ltrate was washed
2-[2-(4-Vinylbenzyloxy)ethoxy]ethyl-O-(2,3,4,6-tetra-O-acetyl-
with aqueous 10% NaHCO3, brine, dried over MgSO4, and D-galactopyranosyl)-(1 / 4)-2-acetamido-6-O-tert-butyldiphe-
concentrated in vacuum. The crude product was pure enough and nylsilyl-2-de-oxy-b-D-glucopyranoside (10a and 10b). To a solu-
directly used for the next step without further purication. To a tion of compound 9 (ref. 22) (1.17 g, 2.25 mmol), compound 8
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˚
(0.99 g, 1.50 mmol), and 4 A molecular sieve (2.20 g) in anhy- CDCl3) d 170.6 (s), 170.3 (s), 170.0 (s), 170.0 (s), 169.0 (s), 137.3
drous DCM (25 mL) was cooled to ꢁ40 ꢀC. The reaction mixture (s), 137.0 (s), 136.3 (d), 135.9 (d ꢂ 2), 135.4 (d ꢂ 2), 133.5 (s),
was allowed to stir for 20 minutes. Borontriuoride dieth- 132.5 (s), 129.7 (d ꢂ 2), 128.2 (d ꢂ 2), 127.7 (d ꢂ 2), 127.5 (d ꢂ
yletherate (0.38 mL, 3.00 mmol) was added into the solution. 2), 126.3 (d ꢂ 2), 114.0 (t), 101.0 (d), 100.9 (d), 79.9 (d), 74.5 (d),
The resulting mixture was allowed to stir at the same temper- 73.1 (t), 72.7 (d), 71.1 (t), 71.1 (d), 70.8 (d), 70.5 (t), 69.5 (t), 68.8
ature for 5 h. The reaction mixture was added aqueous NaHCO3 (d), 68.0 (t), 66.8 (d), 61.8 (t), 61.0 (t), 55.8 (d), 26.7 (q ꢂ 3), 23.2
and stirred for 10 minutes. The organic layers were washed with (q), 20.5 (q ꢂ 2), 20.4 (q), 20.2 (q), 19.2 (s); HRMS (ESI+): m/z
brine, dried over MgSO4, ltered, and concentrated under calcd for C51H67NO17Si [M + H]+: 994.4256; found: 994.4280, [M
reduced pressure. The crude residue was puried by silica gel + Na]+: 1016.4076; found: 1016.4069.
chromatography (EtOAc : hexane ¼ 9 : 1) to afford the minor
isomer (a-form) 10a as a white solid and the major isomer (b-
Results and discussion
Synthesis of styrene derivative 1 and carbohydrate monomer
form) 10b as a colorless oil (1.75 g, 66%, a : b ¼ 26% : 40%):
a-isomer 10a: mp 158.2–161.1 ꢀC; [a]D25 +38.52 (c ¼ 1.01,
3, 5, and 10
CHCl3); IR (neat) 3519 (OH), 3273, 2860, 1743, (C]O), 1653
(C]O), 1568, 1428, 1372, 1259, 1226, 1111, 1086, 1030, 959, 909,
To fundamentally study the controlled living radical polymeri-
zation of carbohydrate polymers containing cell surface carbo-
hydrates, we used polystyrene as the backbone. To avoid steric
hindrance during the polymerization and gain more hydrophilic
interactions in aqueous solution as biomaterials, diethylene
glycol was used as a spacer between the styrene and carbohydrate
moieties.23 The reaction of 4-chloromethylstyrene with diethylene
glycol afforded the styrene derivative 1 in 71% yield (Scheme 2).24
To prepare the mannose-containing carbohydrate monomer, a
glycosylation reaction between the mannosyl donor 2 (ref. 19)
and the styrene derivative 1 was carried out using borontri-
uoride-diethyletherate25 to complete the synthesis of the
carbohydrate monomer 3 containing mannose as its terminal in
69% yield (only a-form). The synthesis of a carbohydrate mono-
mer containing sialic acid has been intensively studied.26 Even-
tually, zinc bromide (ZnBr2)27 was found to be an efficient catalyst
for the glycosylation between the sialyl donor 4 (ref. 20) and the
styrene derivative 1. Under optimized conditions, a partial sepa-
rable mixture of carbohydrate monomers 5 in a ratio of 91 : 9
(a : b) was produced in 78% yield. More synthetic steps were
needed for the synthesis of carbohydrate monomers 10 con-
taining the disaccharide N-acetyllactosamine. The glycosylation
reaction between the glycosyl donor 6 (ref. 21) and compound 1
was catalyzed by mercury cyanide [Hg(CN)2].28 Deacetylation
using sodium methoxide29 afforded the carbohydrate monomer
7, which contained an N-acetylglucosamine (GluNAc) unit. To
complete the synthesis of the LacNAc-derived carbohydrate
monomer, selective silylation at the 6-position of GluNAc
moiety30 on the compound 7 gave the glycosyl acceptor 8. A
second glycosylation reaction between the donor 9 (ref. 22) and
the acceptor 8 was successfully carried out to give the disaccha-
ride LacNAc carbohydrate monomers 10 in 66% yield (a-form:
26%; b-form: 40%).31 It is fortunate that the major product (the b-
isomer) functions as an efficient substrate for many cell surface
recognition processes. Three carbohydrate monomers containing
mannose, sialic acid, N-acetyllactosamine were successfully
prepared. The methods presented herein permit the efficient
preparation of carbohydrate monomers on a gram scale.
880, 853, 825, 810, 765, 743, 705 cmꢁ1 1H NMR (500 MHz,
;
CDCl3) d 7.67–7.63 (two overlapping d at 7.66, J ¼ 6.5 Hz, 7.64, J
¼ 6.6 Hz, 4H, Ph), 7.43–7.31 (m, 8H, Ph, para-), 7.28 (d, J ¼ 8.0
Hz, 2H, para-), 6.70–6.62 (dd at 6.68, J ¼ 17.6, 10.9 Hz, 1H,
ArCH]CH2, overlapped with one d at 6.62, J ¼ 8.15 Hz, 1H,
NHAc), 5.71 (d, J ¼ 17.6 Hz, 1H, ArCH]CH2), 5.33 (d, J ¼ 4.5 Hz,
0
1H H4 ), 5.22 (d, J ¼ 10.9 Hz, 1H, ArCH]CH2), 4.99–4.89 (m,
0
0
overlapped with one d at 4.91, J ¼ 3.9 Hz, 3H, H3 , H1 , a-form),
4.86 (d, J ¼ 8.3 Hz, 1H, H1, b-form), 4.57 (s, 2H, ArCH2O), 4.16
0
(dd, J ¼ 8.1, 5.7 Hz, 1H, H6 a), 4.09–3.93 (m, overlapped with one
0
0
dd at 3.97, J ¼ 10.9, 5.4 Hz, 3H, H5 , H6 b), 3.89 (dt, J ¼ 11.9, 3.4
Hz, 1H), 3.81 (td, J ¼ 11.0, 3.7 Hz, 1H), 3.77–3.71 (m, 1H), 3.71–
3.58 (m, overlapped with one dd at 3.68, J ¼ 11.6, 6.0 Hz, 8H),
0
3.52 (dd, J ¼ 17.7, 8.5 Hz, 1H, H2), 3.46–3.39 (m, 1H, H2 ), 2.06 (s,
3H, Ac), 2.01 (s, 3H, Ac), 1.98 (s, 3H, Ac), 1.97 (s, 3H, Ac), 1.87 (s,
3H, Ac), 1.02 (s, 9H, t-Bu); 13C NMR (125 MHz, CDCl3) d 171.1 (s),
170.6 (s), 170.5 (s), 170.4 (s), 170.0 (s), 137.4 (s), 137.2 (s), 136.4
(d), 135.6 (d ꢂ 2), 135.5 (d ꢂ 2), 133.3 (s), 133.2 (s), 129.6 (d),
129.6 (d), 127.9 (d ꢂ 2), 127.6 (d ꢂ 2), 127.6 (d ꢂ 2), 126.3 (d ꢂ
2), 114.0 (t), 100.8 (d), 100.4 (d), 80.3 (d), 74.4 (d), 72.9 (t), 71.3
(d), 70.9 (t), 70.3 (t), 70.1 (d), 69.5 (t), 68.4 (t), 67.7 (d), 66.9 (d),
66.6 (d), 63.1 (t), 61.0 (t), 56.6 (d), 26.7 (q ꢂ 3), 23.4 (q), 20.8 (q),
20.7 (q), 20.6 (q), 20.5 (q), 19.2 (s); HRMS (ESI+): m/z calcd for
C
51H67NO17Si [M + H]+: 994.4256; found: 994.4268.
b-isomer 10b: [a]3D1 ꢁ19.41 (c ¼ 1.88, CHCl3); IR (neat) 3395
(OH), 3007, 2933, 2859, 1755 (C]O), 1737 (C]O), 1677 (NHCO),
1540, 1429, 1367 (t-Bu), 1297, 1248, 1218, 1141, 1110, 1060,
1039, 954, 911, 853, 826, 794, 761, 745, 706, 665, 606 cmꢁ1; H
1
NMR (500 MHz, CDCl3) d 7.73–7.65 (m, 4H, Ph), 7.42–7.31 (m,
8H, Ph), 7.26 (d, J ¼ 8.1 Hz, 2H, para-), 6.69 (dd, J ¼ 17.6, 11.0
Hz, 1H, ArCH]CH2), 6.20 (d, J ¼ 8.4 Hz, 1H, NHAc), 5.73 (d, J ¼
0
17.6 Hz, 1H, ArCH]CH2), 5.33 (d, J ¼ 3.4 Hz, 1H, H4 ), 5.22 (d, J
0
¼ 11.0 Hz, 1H, ArCH]CH2), 5.17 (dd, J ¼ 10.5, 8.1 Hz, 1H, H2 ),
0
0
4.94 (dd, J ¼ 10.5, 3.4 Hz, 1H, H3 ), 4.68 (d, J ¼ 8.1 Hz, 1H, H1 , b-
form), 4.65 (d, J ¼ 8.3 Hz, 1H, H1, b-form), 4.54 (d, J ¼ 12.0 Hz,
1H, ArCH2O, AB), 4.50 (d, J ¼ 12.0 Hz, 1H, ArCH2O, AB), 4.15–
4.07 (two overlapping dd at 4.13, J ¼ 11.3, 6.3 Hz, and 4.10, J ¼
0
0
00
0
11.3, 7.0 Hz, 2H, H6 a,6 b), 3.92–3.82 (m, 3H, H1 a, H6a, H5 ), 3.81–
00
00
3.65 (m, 5H, H6b, H4, H2 a, H2), 3.64–3.57 (m, 6H, CH2 ꢂ 2, H2 b
,
Polymerization studies
00
H
1 b), 3.52 (t, J ¼ 9.2 Hz, 1H, H3), 3.30 (d, J ¼ 8.1 Hz, 1H, H5),
2.12 (s, 3H, Ac), 2.02 (s, 3H, Ac), 1.95 (s, 3H, Ac), 1.94 (s, 3H, The polymerizations were conducted in sealed tubes using 1
Ac),1.69 (s, 3H, Ac), 1.04 (s, 9H, t-Bu); 13C NMR (125 MHz, mol% of alkoxyamine and 40 wt% of dimethylforamide (DMF)
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Scheme 2 Preparation of carbohydrate monomers 3, 5, and 10.
as the co-solvent with different ratios of carbohydrate mono- regulator. In other experiments (entries 16 and 17), polymeric
mers and styrene. The results are presented in Table 1. In some alkoxyamines were used as regulators in the living radical
experiments (entries 1–15), styryl-TEMPO was used as the polymerizations. The carbohydrate polymers were collected by
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centrifugation and dried in a vacuum-drying cabinet at 60 ꢀC for
We then continued our studies on polymerization of carbo-
ꢀ
12 h. Conversions were evaluated gravimetrically. Molecular hydrate monomer 5 at 125 C for 48 h. Compared to the poly-
weights and polydispersity indices (PDI) were determined by merization of carbohydrate monomer 3, relatively low
size exclusion chromatography (SEC). Five different ratios (20/ conversions were obtained. Increasing the concentration of
80, 40/60, 60/40, 80/20, 100/0; ratio to styrene) of monomer carbohydrate monomer 5 in the styrene polymerization resulted
compositions in polymerizations were studied in the cases of a in a decrease in conversion, from 31% to 9% and an increase in
series of carbohydrate monomers 3 and 5, in which a mannose the PDI from 1.13 to 1.74 (entry 9–13). The reasons for the low
and a sialic acid are attached to its terminals, respectively. conversion can be attributed to the bulky effect of the carbo-
Because the synthesis of carbohydrate monomer 10b bearing an hydrate itself (sialic acid) in polymerizations or ineffective
N-acetyllactosamine was laborious, only two different ratios (25/ precipitation caused by the highly polar protecting groups (six
75 and 75/25) were examined.
acetyl groups and one ester group) on the sialic acid molecule. It
The copolymerization reactions of the carbohydrate monomer should be noted that a low conversion (9%) and a narrow PDI
3 and styrene using a ratio of 20/80 were studied rst (entry 1–4) (1.22) were observed in the polymerization, when 100% of the
at 125 ꢀC. As expected, the conversion was increased (from 6% to carbohydrate monomer 5 was used (entry 13).
72%) upon extending reaction time (from 24 h to 42 h) while the
The next series of experiments involved the use of the
PDIs remained narrow (1.10–1.14). The optimized reaction time carbohydrate monomer 10b (b-isomer) since it is considerably
(42 h), a conversion of 72% and a narrow PDI of 1.12 were more useful than compound 10a (a-isomer) in cell surface
obtained (entry 4). The polymerization behavior of different recognition interactions. Polymerizations at two different
concentrations in the reactions of carbohydrate monomer 3 with concentrations were studied (entry 14–15). A low concentration
styrene (entry 4–8) was studied next. When the concentration of (25/75, ratio to styrene) of carbohydrate monomer 10b led to a
carbohydrate monomer 3 in the styrene polymerization was good conversion of 65% and a low PDI of 1.15 (entry 14). This
increased, uctuations in conversions between 58 and 80% and good conversion can be attributed to the effect of the hydro-
in PDIs between 1.12 and 1.37 were observed. Surprisingly, a phobic tert-butyldiphenylsiyl group, which enhanced the yield
polymerization reaction using 100% of the carbohydrate mono- of precipitated product. A high concentration (75/25, ratio to
mer 3, resulted in the formation of mannosyl-glycopolymers with styrene) of carbohydrate monomer 10b in the polymerization
a conversion of 58% and a PDI of 1.30 (entry 8). At this stage, we resulted in decreased conversion (19%), and a larger PDI of 1.38
can conrm that, using commercially available styryl-TEMPO, (entry 15). In general, the results showed that increasing the
controlled radical polymerizations can be achieved to produce concentration of carbohydrate monomers in the reaction
carbohydrate polymers with different compositions, reasonable resulted in a decrease in conversion and an increase in PDIs.
conversions and dened PDIs.
Scheme 3 Preparation of novel carbohydrate polymers bearing unimolecular trivalent carbohydrate ligands.
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The nal polymerization reactions were intended to produce
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novel carbohydrate polymers bearing unimolecular multivalent
ligands (Scheme 3). To show the living character of controlled
radical polymerization, the polymerizations of the carbohydrate
monomer 5 and styrene were initiated and regulated by the
polymeric alkoxyamine I (obtained from entry 4) to delivery
polymeric alkoxyamine II bearing a unimolecular divalent
carbohydrate ligand (entry 16). The polymer weight was indeed
increased and the PDI remained low (1.12 / 1.24). This
carbohydrate polymeric alkoxyamine II could be further poly-
merized in the presence of the carbohydrate monomer 10b and
styrene to provide a novel polymeric alkoxyamine III bearing a
unimolecular trivalent carbohydrate ligand with a molecular
weight of 24 K and PDI of 1.52 (entry 17). This is the rst report
of an application of TEMPO-mediated polymerization to
produce novel carbohydrate polymers bearing unimolecular
trivalent cell surface carbohydrate ligands with desired molec-
ular weights and PDIs. This synthetic approach provided an
alternative method for the rapid synthesis of carbohydrate
polymers with multivalent carbohydrate ligands in one single
polymerization sequence. Furthermore, these novel carbo-
hydrate polymers carrying a TEMPO as its terminal, which
constitutes a living radical site for further polymerization
reactions. One might imagine that using this approach, block
copolymerization in the presence of three carbohydrate mono-
mers in a one-pot polymerization reaction could be used to
produce a variety of novel carbohydrate polymers with desired
sugar compositions. Since multivalent carbohydrate vaccines
were recently used in clinical trials for cancer therapy, these
results provided an alternative and practical approach for the
generation of carbohydrate polymers with desired molecular
weights and controlled PDIs. Different sugar compositions of
carbohydrate polymers could also be achieved to mimic tumor-
associated carbohydrate antigens.
Characterization of carbohydrate polymers by NMR
1H NMR measurements were also carried out to verify that the
carbohydrate moieties were, in fact, attached to the polystyrene
backbone, shown in Fig. 1. In spectrum 2a, the characteristic
peaks of the polymer obtained from polymerization with 20%
carbohydrate monomer 3 appear at around 4.8 ppm, and are
assigned to the anomeric proton (H1 of mannose), and four
acetyl groups (OAc) at around 2.0–2.2 ppm. Characteristic peaks
of carbohydrate polymers containing sialic acid (20% with
styrene) in spectrum 2b appear at 3.7 and 2.6 ppm, and are
assigned to the methyl ester (OMe) and the equatorial proton
(H3) of the sialic acid moiety, respectively. In spectrum 2c, the
peaks at 7.4 and 7.7 ppm of diphenyl group, 4.7 ppm of two
anomeric protons, and 1.0 ppm of tert-butylsilyl group clearly
are present, thus verifying that carbohydrate polymers con-
taining N-acetyllactosamine were successfully produced. The
polymeric alkoxyamines containing divalent and trivalent
ligands (II and III) are also shown in spectra 2d and e, respec-
tively. The relatively low intensity of the characteristic peaks
corresponding to sialic acid are shown in spectrum 2d,
consistent with the relatively low extent of polymerization in the
Fig. 1 1H NMR Spectra of carbohydrate polymers in CDCl3 (a) polymer
from entry 4. (b) Polymer from entry 9. (c) Polymer from entry 14. (d)
Polymer from entry 16. (e) Polymer from entry 17.
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´
York, 2008; (d) H.-J. Gabius, H.-C. Siebert, S. Andre,
series of carbohydrate polymer 5. Peaks for carbohydrate poly-
mers obtained from the polymerization in entry 17 are shown in
spectrum 2e, indicating that a polymer bearing a unimolecular
trivalent carbohydrate ligand was successfully produced.
´
¨
J. Jimebez-Barbero and H. Rudiger, ChemBioChem, 2004, 5,
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2 (a) H.-J. Gabius, S. Andre, H. Kaltner and H.-C. Siebert,
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Conclusions
¨
3 (a) B. Ernst, G. W. Hart and P. Sinay, Carbohydrates in
Three different styrene-type monomers containing cell surface
carbohydrates were successfully prepared for the studies of
nitroxide-mediated polymerization. Diethylene glycol was used
as the spacer. These new synthetic methods could be used to
synthesize carbohydrate monomers on a gram scale. Under the
conditions of TEMPO-mediated polymerization, controlled
living radical polymerization could be achieved to produce
carbohydrate polymers with dened molecular weights and
PDIs. Carbohydrate polymers with different sugar compositions
could also be produced through this approach. The PDIs were
increased and conversions were decreased when increasing
concentrations of carbohydrate monomers in polymerizations
were used. Meanwhile, a radical “living” character could be
exhibited to produce three different carbohydrates moieties
attached to the polymers chains. To the best of our knowledge,
this is the rst report of the preparation of carbohydrate poly-
mers bearing unimolecular trivalent carbohydrate ligands with
dened molecular weights and PDIs. The approach presented
here provides a platform to show that controlled living radical
polymerization is a powerful method for preparing novel
carbohydrate polymers with specic carbohydrate ligands.
Polymer and sugar compositions could be adjusted through
polymerization with different concentrations of initiators and
carbohydrate monomers. These preliminary results provide an
alternative synthetic approach for the rapid synthesis of
multiple tumor-associated carbohydrates antigens in one single
living polymerization sequence. Since multivalent carbohydrate
vaccines are currently used in clinical trials for cancer
therapy,13,14 the preparation of carbohydrate polymers contain-
ing tumor-associated carbohydrate antigens using well-
established nitroxides techniques32 is currently underway. Our
approaches should provide a potentially rewarding concept that
can be applied to research related to cancer immunotherapy.
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Acknowledgements
This work was generously supported by the Ministry of Science
and Technology, Taiwan and the Department of Chemistry, Fu
Jen Catholic University. CCC thanks to Professor Armido Studer
¨
(Organisch-Chemisches Institut, Westfalische Wilhelms-Uni-
¨
¨
versitat Munster) for preliminary discussions and valuable
suggestions.
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