Total synthesis of dansyl and biotin functionalized ganglioside GM3 by chemoenzymatic method

Article abstract of DOI:10.1007/s11426-013-4838-4

Ganglioside GM3, as well as other gangliosides, offers a variety of modifications in its sialic acid and ceramide moieties GM3 exhibits various types of important biological activities, due to the inability to effectively observe the trafficking of ganglioside GM3, developing sensitive research tools for specific monitoring of GM3 expression and activity is thus desirable. The total synthesis of a dansyl and biotin bifunctionalized fluorescent ganglioside GM3 were reported in this article. From lactose after 13 reaction steps, the compound of 2″-biotinoylaminoethyl-6-N-dansylamido-6-deoxy-β-D- galatopyranosyl-(1→4)-β-D-glucopy-ranoside was obtained in total yield of 16.2%. Sialylation of dansyl and biotin functionalized lactose by enzymatic method gave dansyl and biotin labeled ganglioside GM3. The fluorescent property of this compound was also investigated.

Full text of DOI:10.1007/s11426-013-4838-4

SCIENCE CHINA
Chemistry
July 2013 Vol.56 No.7: 933–938
doi: 10.1007/s11426-013-4838-4
• ARTICLES •
Total synthesis of dansyl and biotin functionalized ganglioside
GM3 by chemoenzymatic method
SUN Bin* & JIANG HeYan
Research Center of Medicinal Chemistry & Chemical Biology, Chongqing Key Laboratory of Catalysis and Functional Organic Molecules,
Chongqing Technology and Business University, Chongqing 400067, China
Received November 8, 2012; accepted January 7, 2013; published online March 18, 2013
Ganglioside GM3, as well as other gangliosides, offers a variety of modifications in its sialic acid and ceramide moieties GM3
exhibits various types of important biological activities, due to the inability to effectively observe the trafficking of ganglioside
GM3, developing sensitive research tools for specific monitoring of GM3 expression and activity is thus desirable. The total
synthesis of a dansyl and biotin bifunctionalized fluorescent ganglioside GM3 were reported in this article. From lactose after
13 reaction steps, the compound of 2′-biotinoylaminoethyl-6-N-dansylamido-6-deoxy-β-D-galatopyranosyl-(1→4)-β-D-glu-
copy-ranoside was obtained in total yield of 16.2%. Sialylation of dansyl and biotin functionalized lactose by enzymatic
method gave dansyl and biotin labeled ganglioside GM3. The fluorescent property of this compound was also investigated.
fluorescence, biotin, chemoenzymatic synthesis, ganglioside GM3
fluorescence-based research tools and probes can play a
1 Introduction
central role. Various fluorescence-based molecular biology
techniques have over the recent years evolved into powerful
and sensitive means of studying protein expression and ac-
tivity, but this technique was seldom used in the research of
gangliosides expression and activity. Herein, we design and
synthesize a dansyl and biotin functionalized ganglioside
GM3 by chemo-enzymatic method.
Ganglioside GM3 is a ubiquitous metabolite with a variety
of cellular activities that have been reviewed, including dif-
ferentiation [1], the modulation of several receptors and
other enzymes [2, 3], immunosupression [4]. In recent years,
ganglioside GM3 has attracted great attention as an im-
portant tumor-associated carbohydrate antigen, as it was
found to be over expressed in some tumor tissues, such as
small-cell lung cancer, human breast cancer, melanomas,
and so on, but it had a more restricted distribution in normal
tissues [57]. As a good target for cancer-active immuno-
therapy, the GM3-based vaccine has been efficiently de-
veloped for preclinical/clinical study by several research
groups [814]. However, due to the inability to effectively
observe the trafficking of ganglioside GM3, developing
sensitive research tools for specific monitoring of GM3 ex-
pression and activity is thus desirable. Within this context,
2 Results and discussion
Our journey commenced from the preparation of
per-acetylated lactose from the commercially available lac-
tose (Scheme 1). Acetylation of lactose (2) in the presence
of acetic anhydride and anhydrous sodium acetate and re-
fluxing the mixture gave per-acetylated lactose 3 (76.7%)
[1517], which underwent boron trifluoride diethyl etherate
promoted glycosylation with chloroethanol to provide the
desired β-2’-chloroethyl-lactoside 4 in 77.9% yield [16].
SN2 displacement of the chloride with sodium azide fol-
*Corresponding author (email: sunbin@ctbu.edu.cn)
© Science China Press and Springer-Verlag Berlin Heidelberg 2013
chem.scichina.com www.springerlink.com

934
Sun B, et al. Sci China Chem July (2013) Vol.56 No.7
lowed by Zemplén deacetylation, regioselective benzylidene
formation and reacetylation left hydroxyl groups produced
β-2’-azidoethyl-lactoside 5. Selective opening of benzyli-
dene acetal in the presence of NBS and anhydrous of cal-
cium acetate in dry tetrachloromethane refluxing 90 min
gave 6-bromo-6-deoxy-β-2-azidoethyl lactose 6 [18, 19].
Due to the bromide is very ease to remove under reductive
circumstance, reducing azido group to amine and keeping
bromide is a key step for the synthesis of the target product.
After several alternative reaction routes, we found that the
mixture of trimethylphosphine and compound 6 in aqueous
THF stirred overnight at 60 °C to successfully reduce the
azide group and obtain desired 6-bromo-6-deoxy-β-2-
aminoethyl-lactose 7 in 92% yield [20].
azide followed by Staudinger reduction and coupling with
dansyl chloride gave a protected dansyl and biotin function-
alized lactose 12 [22, 23]. Compound 12 was treat with so-
dium methoxide in dry methanol at 5 °C, when ESI-MS
spectrum showed that benzoyl and acetyl groups were com-
pletely removed, the reaction mixture was neutralized with
a IR (H+) resin to give depreotected dansyl and biotin la-
beled lactose 13.
Contrary to the previously reported lactoside and other
synthetic substrates for glycosyltransferases, compound 13
is poorly soluble in water: it forms gel, and as such is not
amenable for enzymatic modification. The solubility prop-
erties of compound 13 appeared to be different from those
described before. Thus, it dissolved readily in methanol
while clearly forming gel in water. To obtain a suitable me-
dium for the reactions with sialyltransferase, compounds 13
is first dissolved in 100% MeOH. After the addition of
aqueous solutions of the other components, we obtained a
The aminoethyl functionalized lactose 7 reacted with bi-
otin 8 in the presence of N-(3-dimethyl-aminopropyl)-
N’-ethylcarbodiimide (EDC) to produce biotin labeled lac-
tose 10 [21]. SN2 displacement of the bromide with sodium
Scheme 1 The synthetic route of 6-bromo-6-deoxy-β-2-aminoethyl-lactose 7. Reagents and condition: (a) NaOAc/OAc2, reflux 2 h, 76.7%; (b)
2-chloroethanol/BF3·Et2O/CH2Cl2, 0 °Crt, overnight, 77.9%; (c) NaN3/NaI/DMF, 80 °C, overnight, 100%; (d) NaOMe/MeOH; (e) PhCH(OCH3)2/CSA/
THF, reflux, 3 h (CSA: (±)-camphor 10-sulfonic acid); (f) Ac2O/Py, 81.6% (4 steps); (g) NBS/CCl4, reflux 90 min, 91.2%; (h) PMe3, THF/H2O, rt, overnight,
92%.
Scheme 2 The synthetic route of dansyl and biotin funcationalized ganglioside GM3. Reagents and conditon: (a) biotin 8 (1 eq), EDC (1 eq), DMAP (1 eq),
DMF, rt, N2, 4 h, 65.5%; (b) NaN3/DMF, 70 °C, 24 h, 97.8%; (c) PMe3, THF, 60 °C, overnight, 91%; (d) dansyl chloride 11, N,N-diisopropylethylamine/
CH2Cl2, rt, 5 h, 67.8%; (e) NaOMe/MeOH, 20 °C, IR(H+) resin, 100%; (f) CMP-NeuAc, 2,3-alpha sialyltransferase, 30% of methanol in aqueous buffer,
37 °C, 24 h, 92%.

Sun B, et al. Sci China Chem July (2013) Vol.56 No.7
935
mixture of methanol/water (30/70, v/v) that was compatible
with sialyltransferase, which used to add NeuAc. The mix-
ture of respective compound 13, sialyltransferase and
CMP-NeuAc was kept at 37 °C for 24 h, after which TLC
analysis unambiguously showed that the lactoside was con-
verted completely. In a separate experiment, the target dan-
syl and biotin labeled gangliside GM3 was subsequently
bound on a SepPak column, which was then washed with
H2O to elute hydrophilic compounds (such as the buffer and
the nucleotide) and finally the dansyl and biotin funcation-
alized ganglioside GM3 was eluted with MeOH in 92%
yield.
(Waters Corporation, Milford, MA). The fluorescence was
measured on a FLS900 fluorescence spectrometer. The op-
tical rotations were measured using the sodium D line.
3.2 Synthesis and characterization
2-Azido-ethyl-6-bromo-6-deoxy-2,3-di-O-acetyl-4-O-benzoyl-
β-D-gala-topyranosyl-(1 → 4)-2,3,6-tri-O-acetyl-β-D-glucop-
yranoside (6)
Anhydrous sodium acetate (12.5 g, 0.152 mol) was added
into acetic anhydride (200 mL). The mixture was heated to
the boiling point of the solution. Lactose 2 (50 g, 0.146 mol)
was added into the mixture in portions to maintain the re-
fluxing of reaction mixture. After completion of the addi-
tion, the reaction mixture was refluxed for another 10
minutes. The reaction mixture was cooled to room temper-
ature and poured into water (1500 mL). After stirring for 15
minutes, the water was removed and the organic layer was
washed 5 times with water (1000 mL) to give a white solid.
Recrystallization of the solid from 95% ethanol afforded the
white solid 3 (56 g, 62%). BF3·Et2O (5 mL) was added
dropwise into the mixture of lactose octa-acetate 3 (10 g,
14.7 mmol) and chloroethanol (1.5 mL) in dichloromethane
(200 mL) at 0 °C for 30 minutes. The reaction mixture was
stirred for 45 h from 0 °C to room temperature. Then the
mixture was diluted with dichloromethane (400 mL), which
was then poured into ice-water (500 mL) and stirred for 30
minutes. The aqueous layer was removed and the organic
layer was washed with a saturated solution of Na2CO3. The
organic layer was dried over anhydrous Na2SO4. The sol-
vent was removed and the residue was purified by column
chromatography (hexanes:ethyl acetate = 1:1 ) to give 4 (7.9
g, 75%). The mixture of compound 4 (7 g, 10 mmol), so-
dium azide (10 eq) in DMF (20 mL) was stirred overnight at
80 °C. After cooling to room temperature, the mixture was
diluted with ethyl acetate (200 mL) and washed with brine
for 3 times. The organic layer was dried over anhydrous
Na2SO4 and purified by flash chromatography (hex-
anes:ethyl acetate = 2:1) to give compound 5 (7 g, 99%).
Sodium methoxide (1 eq) was added to the mixture of 5 (7 g,
9.9 mmol) in methanol (200 mL) and was stirred for 5 h.
The progress of the reaction was monitored by ESI-MS.
When all acetate groups were removed, IR-120 (H+) resin
was added to neutralize the reaction. This was followed by
filtration and solvent removal to give deacetylation product.
The resulting residue was dissolved in toluene (50 mL),
followed by the addition of camphorsulphonic acid (CSA,
135 mg, 0.6 mmol) and benzaldehyde dimethyl acetal (3.05
g, 20 mmol). The reaction mixture was stirred at 80 °C for 3
h and then cooled to room temperature. The reaction mix-
ture was neutralized with triethylamine and then poured into
ethyl acetate (250 mL). The mixture was washed with brine
(2 times) and the organic layer was dried over anhydrous
sodium sulfate. The solvent was removed and the resulting
residue was purified by column chromatography (hex-
We also investigated the fluorescent property of this
compound in methanol solution and found that which fluo-
rescence was exc = 333 nm and em = 570 nm (Figure 1).
3 Experimental section
3.1 General experimental procedures
All reactions were carried out under nitrogen with anhy-
drous solvents in flame-dried glassware, unless otherwise
noted. 2,3-alpha sialyltransferase(from Pasteurella Mutocida,
recombinant, expressed in E. Coli; BL21) and CMP-
Neu5Ac (Cytidine-5′-monophospho-N-acetylneuraminic acid
sodium salt) were purchased from Sigma-Aldrich. Chemi-
cals used were reagent grade as supplied except where not-
ed. Analytical thin-layer chromatography was performed
using silica gel 60 F254 glass plates; Compound spots were
visualized by UV light (254 nm) and by staining with a yel-
low solution containing Ce(NH4)2(NO3)6 (0.5 g) and
(NH4)6Mo7O24·4H2O (24.0 g) in 6% H2SO4 (500 mL).
Flash column chromatography was performed on silica gel
60 (230–400 mesh). NMR spectra were referenced using
Me4Si (0 ppm), residual CHCl3 (1H NMR 7.26 ppm, 13C
NMR 77.0 ppm). Peak and coupling constant assignments
1
1
are based on H NMR and H–1H gCOSY. High-resolution
mass spectra were recorded on a Q-TOF Ultima API
LC-MS instrument with Waters 2795 Separation Module
Figure 1 Fluorescence spectrum of dansyl and biotin labeled lactose.
Concentration = 1.04 × 105 M.

936
Sun B, et al. Sci China Chem July (2013) Vol.56 No.7
anes:EtOAc = 1:1) to afford benzylidene lactose (3.72g,
75%). Acetic anhydride was added dropwise into the sol-
vent of the benzylidene lactose (3.72 g, 7.4 mmol) dissolved
in pyridine (6 mL) at 0 °C, the mixture was stirred overnight
from 0 °C to room temperature. The mixture was diluted
with ethyl acetate (200 mL) and washed with aqueous solu-
tion of copper sulfate (5%) 3 times. The organic layer was
dried over anhydrous sodium sulfate and purified by flash
column chromatography (hexanes:ethyl acetate = 3:1) to
give compound 5 (4.12 g, 78%). A solution of 5 (4.12 g, 5.8
mmol) in tetrachloromethane (100 mL) was treated with
fresh recrystallized N-bromosuccinimide (1.08 g, 6 mmol)
and barium carbonate (2.15 g, 11.1 mmol). The mixture was
heated to reflux and stirred for 3 h. After the mixture was
cooled to room temperature and filtered, it was dried over
anhydrous sodium sulfate, concentrated and purified by
flash column chromatography (hexanes:ethyl acetate = 3:1)
21.1, 20.9, 20.5. ESI-HRMS calcd. for C31H40Br NO16Na
([M + Na]+) 784.1428, found 784.1445.
2-Biotinoylaminoethyl-6-bromo-6-deoxy-2,3-di-O-acetyl-4-
O-benzoyl-β-D-gala-topyranosyl-(1 → 4)-2,3,6-tri-O-acetyl-
β-D-glucopyranoside (9)
To a solution of compound 7 (1.9 g, 2.5 mmol) in CH2Cl2
(20 mL), biotin (8) (732.9 mg, 3 mmol), N-ethyl-N,N-
dimethylaminopropyl carbodiimide hydrochloride (EDC)
(0.96 g, 5 mmol) and 4-N,N-dimethylamino pyridine
(DMAP) (88 mg, 0.72 mmol) were added and the reaction
was stirred overnight at room temperature. The reaction
mixture was diluted with CH2Cl2 (100 mL) and extracted
with saturated NaHCO3 solution. The organic layer was
evaporated to dryness and compound 9 (1.62 g) was ob-
tained in 65.5 % yield after flash column chromatographic
1
purification. H NMR δ (400 MHz, CDCl3, ppm) 8.20 (d, J
1
= 8.4 Hz, 2H), 7.96 (t, J = 7.5 Hz, 1H), 7.42 (t, J = 7.5 Hz,
2H), 6.92 (s, 1 H, NH), 6.36 (s, 1 H, NH), 5.69 (s, 1 H, NH),
5.65 (d, J = 8.4 Hz, 1H), 5.52 (d, J = 3.6 Hz, 1H), 5.25 (t, J
= 8.4 Hz, 1H), 5.205.00 (m, 2H), 4.61 (d, J = 8.4Hz, 1H),
4.544.51 (m, 1H), 4.484.44 (m, 2H), 4.154.04 (m, 3H),
3.903.83 (m, 2H), 3.773.70 (m, 3H), 3.373.32 (m, 2H),
3.102.90 (m, 3H), 2.83 (s, 1H), 2.742.65 (m, 1H),
2.302.19 (m, 2H), 2.13 (s, 3H), 2.12 (s, 3H), 2.08 (s, 3H),
2.03 (s, 3H), 1.97 (s, 3H), 1.701.35 (m, 6H). 13C NMR δ
(100 MHz, CDCl3, ppm) 173.6, 171.4, 170.4, 170.3, 169.3,
166.3, 164.1, 152.4, 131.5, 130.0, 129.6, 128.4, 127.1,
101.2, 100.3, 77.1, 75.2, 73.2, 73.1, 72.4, 71.5, 71.3, 69.9,
69.2, 67.2, 62.6, 62.4, 55.6, 45.8, 43.6, 40.7, 25.7, 21.4,
21.2, 21.1, 20.9, 20.5, 18.4. ESI-HRMS calcd. for
to give 6 (4.18 g, 91.2%). H NMR δ (400 MHz, CDCl3,
ppm) 8.19 (d, J = 8.6 Hz, 2H), 7.60 (t, J = 7.5 Hz, 1H), 7.42
(t, J = 7.5 Hz, 2H), 5.66 (d, J = 8.4 Hz, 1H), 5.34 ( d, J =
3.6 Hz, 1H), 5.24 (t, J = 8.7 Hz, 1H), 51.35.08 (m, 1H),
5.065.02 (m, 1H), 4.954.92 (m, 1H), 4.484.43 (m, 2H),
4.154.03 (m, 3H), 3.883.82 (m, 2H), 3.773.69 ( m, 3H),
3.373.32 (m, 2H), 2.15 (s, 3H), 2.12 (s, 3H), 2.09 (s, 3H),
2.01 (s, 3H), 1.96 ( s, 3H). 13C NMR δ (100 MHz, CDCl3,
ppm) 173.2, 171.3, 170.5, 170.2, 169.8, 164.5, 152.3, 131.4,
130.0, 129.5, 128.6, 127.2, 101.2, 100.4, 77.1, 75.3, 73.2,
73.1, 72.4, 71.6, 71.3, 69.9, 69.2, 67.3, 62.6, 62.3, 59.6,
42.6, 21.5, 21.2, 21.1, 20.9, 20.5. ESI-HRMS calcd. for
C31H38BrN3O16Na ([M + Na]+) 810.1333, found 810.1309.
2-Amino-ethyl-6-bromo-6-deoxy-2,3-di-O-acetyl-4-O-benz-
oyl-β-D-gala-topyrano-syl-(1 → 4)-2,3,6-tri-O-acetyl-β-D-gl-
ucopyranoside (7)
C41H54BrN3O18SNa ([M
1010.2224.
+
Na]+) 1010.2204, Found
A 1 M solution of trimethylphosphine in THF (21 mL, 7 eq)
was added to the solution of compound 6 (2.37 g, 3 mmol)
dissolved in aqueous THF (THF:H2O = 9:1) (10 mL) then
stirred at 60 °C overnight. The mixture was then concen-
trated and the resulting residue was dissolved in dichloro-
methane (100 mL) and extracted twice with H2O. The or-
ganic layer was dried over anhydrous Na2SO4 and concen-
trated to dryness. The crude product was purified by silica
gel column chromatography (ethyl acetate:hexane = 2:1) to
2-Biotinoylaminoethyl-6-amino-6-deoxy-2,3-di-O-acetyl-4-
O-benzoyl-β-D-gala-topyranosyl-(1 → 4)-2,3,6-tri-O-acetyl-β-
D-glucopyranoside (10)
The mixture of compound 9 (1.48 g, 1.5 mmol) and sodium
azide (10 eq) in DMF (10 mL) was stirred at 80 °C for 5 h.
After cooling to room temperature, the mixture was diluted
with ethyl acetate (100 mL) and washed with brine for 3
times. The organic layer was dried over anhydrous Na2SO4
and purified by flash chromatography (hexanes/ethyl acetate,
v/v = 1/2). A 1 M solution of trimethylphosphine in THF
(10.5 mL, 7 eq) was added to the solution of the result
compound was dissolved in aqueous THF (H2O:THF, v/v =
9/1) and stirred overnight at 60 °C, The mixture was then
concentrated and the resulting residue was dissolved in di-
chloromethane (60 mL) and extracted twice with water. The
organic layer was dried over anhydrous Na2SO4 and con-
centrated to dryness. The crude product was purified by
silica gel column chromatography (ethyl acetate/hexane, v/v
1
give 7 (2.1 g, 92%). H NMR δ (400 MHz, CDCl3, ppm)
8.18 (d, J = 8.6 Hz, 2H), 7.64 (t, J = 7.5 Hz, 1H), 7.45 (t, J
= 7.5 Hz, 2H), 5.70 (d, J = 8.4 Hz, 1H), 5.35 (d, J = 3.6 Hz,
1H), 5.21 (t, J = 9.3 Hz, 1H), 5.12 (dd, J = 8.4, 3.6 Hz, 1H),
4.96 (dd, J = 7.5, 3.0 Hz, 1H), 4.91 (d, J = 7.5 Hz, 1H), 4.56
(d, J = 7.5 Hz, 1H), 4.50 (d, J = 7.5 Hz, 1H), 4.154.03 (m,
2H), 3.903.83 (m, 3H), 3.803.72 (m, 3H), 3.363.32 (m,
2H), 2.13 (s, 3H), 2.12 (s, 3H), 2.08 (s, 3H), 2.03 (s, 3H),
1.97 (s, 3H). 13C NMR δ (100 MHz, CDCl3, ppm) 173.0,
171.1, 170.4, 170.2, 169.5, 166.0, 152.4, 131.2, 129.8,
128.5, 127.1, 101.3, 100.4, 77.2, 75.3, 73.14, 73.1, 72.2,
71.5, 71.2, 69.8, 69.2, 67.0, 62.5, 62.3, 60.5, 42.6, 21.3, 21.2,
1
= 2/1) to give 10 (1.25 g, 90% for two steps). H NMR δ
(400 MHz, CDCl3, ppm) 8.19 (d, J = 8.4 Hz, 2 H), 7.97 (t, J
= 7.5 Hz, 1H), 7.44 (t, J = 7.5 Hz, 2H), 6.93 (s, 1H, NH),

Sun B, et al. Sci China Chem July (2013) Vol.56 No.7
937
6.36 (s, 1H, NH), 5.68 (s, 1H, NH), 5.65 (d, J = 8.4 Hz, 1H),
5.54 (d, J = 3.6 Hz, 1H), 5.25 (t, J = 8.4 Hz, 1H), 5.195.00
(m, 2H), 4.61 (d, J = 8.4 Hz, 1H), 4.594.52 (m, 1H),
4.484.43 (m, 2H), 4.154.01 (m, 3H), 3.903.82 (m, 2H),
3.783.72 (m, 3H), 3.423.36 (m, 2H), 3.112.90 (m, 3H),
2.84 (s, 1H), 2.742.65 (m, 1H), 2.302.19 (m, 2H), 2.15 (s,
3H), 2.13 (s, 3H), 2.09 (s, 3H), 2.03 (s, 3H), 1.98 (s, 3H),
1.701.35 (m, 6H). 13C NMR δ (100 MHz, CDCl3, ppm)
173.5, 171.3, 170.4, 170.3, 169.5, 166.3, 164.2, 152.3,
131.5, 129.9, 129.7, 128.4, 127.3, 101.2, 100.3, 77.2, 75.2,
73.2, 73.1, 72.4, 71.5, 71.3, 70.0, 69.2, 67.2, 62.6, 62.4,
60.6, 55.6, 45.8, 40.7, 26.0, 21.5, 21.2, 21.1, 20.9, 20.5,
18.5. ESI-HRMS calcd. for C41H56N4O18SNa ([M + Na]+)
947.3208, Found 947.3225.
8.27 (d, J = 8.4 Hz, 1H), 8.18 (d, J = 7.2 Hz, 1H), 7.947.92
( m, 2H), 7.15 (d, J = 7.2 Hz, 1H), 4.60 (d, J = 7.6 Hz, 1H),
4.56 (d, J = 8.0 Hz, 1H), 4.36 (dd, J = 10.0 , 3.2 Hz, 1H),
4.304.29 (m, 1H), 4.094.06 (m, 1H), 4.02 (dd, J = 12.4,
2.2 Hz), 1H), 3.893.80 (m, 3H), 3.773.70 (m, 3H),
3.683.65 (m, 1H), 3.643.60 (m, 3H), 3.513.35 (m, 2H),
3.112.92 (m, 2H), 2.89 (s, 6 H), 2.85 (s, 1 H), 2.752.65
(m, 1H), 2.322.20 (m, 2H), 1.711.39 (m, 6H). 13C NMR
δ(100 MHz, CD3OD, ppm) 166.3, 164.1, 152.4, 139.0,
134.1, 128.4, 128.0, 126.9, 125.0, 123.7, 118.9, 116.7, 105.5,
105.2, 83.0, 81.5, 77.9, 77.8, 77.3, 75.6, 72.2, 71.6, 69.8, 64.0,
63.1, 62.8, 62.4, 53.9, 45.9, 43.8, 39.7, 36.2, 30.4, 28.6, 28,
25.7. ESI-HRMS calcd. for C36H53N5O14S2Na([M + Na]+)
866.2928, found 866.2942.
2-Biotinoylaminoethyl-6-N-dansylamido-6-deoxy-2,3-di-O-
acetyl-4-O-benzoyl-β-D-gala-topyranosyl-(1 → 4)-2,3,6-tri-
O-acetyl-β-D-glucopyranoside(12)
2-Biotinoylaminoethyl-(5-acetamido-3,5-dideoxy-D-glyceroa-
D-galacto-non-2-ulopyranosylonic acid)-(23)-6-N-
dansylamido-6-deoxy-β-D-galatopyranosyl-(14)-β-D-
glucopyranoside (1)
The mixture of compound 10 (0.99 g,
1 mmol),
N,N,-diisopropylethylamin (0.2 mL, 1.1 mmol) and dansyl
chloride (11) (297 mg, 1.1 mmol) in dichloromethane (15
mL) was stirred at room temperature for 5 h, then the mix-
ture was diluted with methylenchloride (100 mL) and
washed with water 3 times. The organic layer was dried
over anhydrous sodium sulfate and purified by flash chro-
matography (ethyl acetate/hexane, v/v = 1/1) to give 10(0.79
g, 67.8%). 1H NMR δ (400 MHz, CDCl3, ppm) 8.32 (d, J =
8.4 Hz, 1H), 8.31 (d, J = 8.8 Hz, 1H), 8.19 (d, J = 7.2 Hz,
1H), 7.987.95 (m, 2H), 7.627.44 (m, 5H), 7.17 (d, J = 7.6
Hz, 1H), 6.92 (s, 1H, NH), 6.36 (s, 1H, NH), 5.77 (s, 1H,
NH), 5.67 (s, 1H, NH), 5.50 (d, J = 3.2 Hz, 1H), 5.205.00
(m, 3 H), 4.61(d, J = 8 Hz, 1H), 4.594.35 (m, 3H),
4.354.01 (m, 3H), 3.963.90 (m, 1H), 3.853.60 (m, 3H),
3.523.35 (m, 2H), 3.102.91 (m, 2H), 2.87 (s, 6H), 2.84 (s,
1H), 2.742.65 (m, 1H), 2.302.19 (m, 2H), 2.13 (s, 3H),
2.06 (s, 3H), 2.04(s, 3H), 1.90 (s, 3H), 1.77 (s, 3H),
1.701.35 (m, 6H). 13C NMR δ(100 MHz, CDCl3, ppm)
173.5, 171.3, 170.4, 170.3, 169.5, 166.3, 164.1, 152.3,
134.9, 134.2, 131.0, 130.6, 130.3, 129.9, 129.8, 129.1,
123.7, 119.3, 115.7, 101.4, 100.3, 77.1, 75.1, 73.2, 73.1,
72.0, 71.5, 70.1, 69.7, 69.1, 62.9, 62.2, 60.6, 55.6, 45.8,
43.6, 40.7, 39.7, 36.2, 30.1, 28.4, 28.2, 25.7, 21.4, 21.3,
21.1, 21.06, 20.9. ESI-HRMS calcd. for C53H67N5O20S2Na
([M + Na]+) 1180.3719, found 1180.3735.
Compound 13 (100 mg, 0.118 mmol) was first dissolved in
10 mL of methanol, then 30 mL of CMP-Neu5Ac (150 mg,
0.224 mmol, dissolved in water), 2.5 mL of 1 M Hepes
buffer (pH 7.5), 0.5 mL of 1 M MgCl and 2.5 mL (14 U) of
2,3-alpha sialyltransferase were added. The reaction was
kept at 37 °C for 24 h, after which 100% of the starting ma-
terial was converted to product. The reaction mixture was
centrifuged and loaded on a Sep-Pak column equilibrated
with methanol. Hydrophilic material was washed off with
water and the product was eluted with methanol. Appropri-
ate ractions were collected and evaporated to give 123 mg
(92%) of compound 1 as white powder. []D20= +2 (c = 0.5,
H2O). 1H NMR δ(400 MHz, CD3OD, ppm) 8.30 (d, 1H, J =
8.4 Hz), 8.27 (d, 1H, J = 8.4 Hz), 8.20 (d, 1H, J = 7.2 Hz),
7.947.92 ( m, 2H), 7.15 (d, 1H, J = 7.2 Hz), 4.61 (d, 1H, J
= 7.6 Hz), 4.56 (d, 1H, J = 7.6 Hz), 4.36 (dd, 1H, J = 10.2,
3.2 Hz), 4.304.29 (m, 1H), 4.094.06 (m, 1H), 4.03 (dd,
1H, J = 10.2, 3.2 Hz), 3.903.81 (m, 3H), 3.773.35 (m,
16H), 3.112.92 (m, 2H), 2.89 (s, 6H), 2.85 (s, 1H),
2.752.65 (m, 1H), 2.56 (dd, 1H, J = 12.0, 4.0 Hz),
2.322.30 (m, 2H), 1.83 (s, 3H), 1.74 (t, 1H, J = 12.0 Hz),
1.711.39 (m, 6H). 13C NMR δ(100 MHz, CD3OD, ppm)
177.8, 176.4, 167.3, 164.1, 152.4, 139.0, 134.1, 128.4,
128.0, 127.9, 127.6, 126.9, 125.0, 123.7, 118.9, 116.7,
105.2, 103.9, 103.0, 83.0, 81.5, 77.9, 77.8, 77.3, 75.6, 73.7,
72.2, 71.6, 71.3, 71.1, 69.8, 66.3, 65.5, 64.0, 63.1, 62.8,
62.4, , 54.7, 53.9, 45.9, 43.8, 42.3, 39.7, 36.2, 30.4, 28.6,
28.0, 25.7, 24.9. ESI-HRMS calcd. for 1135.4063
([M + H]+), found 1135.4089.
2-Biotinoylaminoethyl-6-N-dansylamido-6-deoxy-β-D-gala-
atopyranosyl-(1→4)-β-D-glucopyranoside (13)
Sodium methoxide (1 eq) was added to the mixture of 12
(0.58 g, 0.5 mmol) in methanol (50 mL) and was stirred at
20 °C for 5 h. The progress of the reaction was monitored
by ESI-MS. When benzoyl group and all acetate groups
were removed, IR-120 (H+) resin was added to neutralize
the reaction. This was followed by filtration and solvent
4 Conclusion
1
removal to give deprotected product 13 (0.43 g, 100%). H
In conclusion, from lactose after 13 steps reaction, a dansyl
and biotin functionalized lactose was obtained in 16.2%
NMR δ(400MHz, CD3OD, ppm) 8.29 (d, J = 8.4 Hz, 1H),

938
Sun B, et al. Sci China Chem July (2013) Vol.56 No.7
based vaccines and anti-idiotype antibodies for active immunotherapy
against cancer, Expert Rev Vaccines, 2003, 2: 817–823
12 Gabri MR, Mazorra Z, Ripoll GV, Mesa C, Fernandez LE, Gomez
DE, Alonso DF. Complete antitumor protection by perioperative
immunization with GM3/VSSP vaccine in a preclinical mouse mela-
noma model. Clin Cancer Res, 2006, 12: 7092–7098
total yield. This result lactose was sialylated by enzymatic
method to achieve a dansyl and biotin labeled ganglioside
GM3. This will be very useful for preparing more complex
fluorescent gangliosides and for trafficking of the biological
process of ganglioside GM3.
13 Oliva JP, Valdes Z, Casaco A, Pimentel G, Gonzalez J, Alvarez I,
Osorio M, Velazco M, Figueroa M, Ortiz R, Escobar X, Orozco M,
Cruz J, Franco S, Diaz M, Roque L, Carr A, Vazquez AM, Mateos C,
Rubio MC, Perez Rfernandez LE. Clinical evidences of GM3 (NeuGc)
ganglioside expression in human breast cancer using the 14F7 mono-
clonal antibody labelled with Tc-99m. Breast Cancer Res Treat, 2006,
96:115–121
This work was supported by Chongqing Municipal Commission of Education
(KJ110704), Chongqing Technology & Business University (2010-56-01),
Natural Science Foundation Project of CQ CSTC (2010BB5083) and
Chongqing Innovative Research Team Development Program in University
(KJTD201020).
14 Bada AM, Casaco A, Mancebo A, Fuentes D, Gonzalez B,
Hernandez O, Subiros N, Artega M, Hernandez J, Carr A, Fernandez
LE. Acute and repeated dose intramuscular toxicity of GM3 cancer
vaccine in SD rats. Pak J Biol Sci, 2005, 8: 1045–1050
15 Sun B, Pukin A, Visser GM, Zuilhof H. An efficient glycosylation
reaction for the synthesis of asialo GM2 analogues. Tetrahedron Lett,
2006, 47: 7371–7374
16 Sun B, Jiang H, An efficient approach for total synthesis of ami-
nopropyl functionalized ganglioside GM1b. Tetrahedron Lett, 2012,
53: 5711–5715
1
Saito M. Bioactive gangliosides: Differentiation inducers for hema-
topoietic cells and their mechanism(s) of actions. Adv Lipid Res, 1993,
25: 303–327
Ledeen RW, Wu G. Ganglioside function in the neuron. Trends Gly-
cosci Glycotech, 1992, 4: 174–187
Hakomori SI, Igarashi Y. Gangliosides and glycosphingolipids as
modulators of cell growth, adhesion, and transmembrane signaling.
Adv Lipid Res, 1993, 25:147–162
2
3
4
Ladisch S, Hasegawa A, Li R, Kiso M. Immunosuppressive activity
of chemically synthesized gangliosides Biochemistry, 1995, 34:
1197–1202
Fuentes R, Allman R, Mason MD. Ganglioside expression in lung
cancer cell lines. Lung Cancer, 1997, 18: 21–33
Ritter G, Livingston PO. Ganglioside antigens expressed by human
cancer cells. Semin Cancer Biol, 1991, 2: 401–409
Hersey P. Ganglioside antigens in tissue sections of skin, naevi, and
melanoma-implications for treatment of melanoma. Cancer Treat Res,
1991, 54: 137–151
Azuma Y, Ishikawa Y, Kawai S, Tsunenari T, Tsunoda H, Igawa T,
Iida S, Nanami M, Suzuki M, Irie RF, Tsuchiya M, Yamada-Okabe H.
Recombinant human hexamer-dominant IgM monoclonal antibody to
ganglioside GM3 for treatment of melanoma. Clin Cancer Res, 2007,
13: 2745–2750
Bitton RJ, Guthmann MD, Gabri MR, Carnero AJ, Alonso DF, Fain-
boim L, Gomez DE. Cancer vaccines: An update with special focus
on ganglioside antigens. Oncol Rep, 2002, 9: 267–276
17 Sun B, Yang B, Huang X. Total synthesis of the aminopropyl func-
tionalized ganglioside GM1. Sci China Chem, 2012, 55: 31–35
18 Crich D, Yao Q, Bowers AA. On the regioselectivity of the Hanes-
sian-Hullar reaction in 4,6-O-benzylidene protected galactopyra-
nosides. Carbohydr Res, 2006, 341: 1748–175
19 Lindhorst TK. Essentials of Carbohydrate Chemistry and Biochemis-
try (2nd ed.), Wiley-VCH, 2003
20 Wang Z, Zhou L, El-boubbou K, Ye XS, Huang X. Multi-component
one-pot synthesis of the tumor-associated carbohydrate antigen Glo-
bo-H based on preactivation of thioglycosyl donors. J Org Chem,
2007, 72: 6409–6420
21 Wong LS, Khan F, Micklefield J. Selective covalent protein immobi-
lization: Strategies and applications. Chem Rev, 2009, 109:
4025–4053; and references there
22 Sigal GB, Mammen M, Dahmann G, Whitesides GM. Polyacryla-
mides bearing pendant alpha-sialoside groups strongly inhibit agglu-
tination of erythrocytes by influenza virus: The strong inhibition re-
flects enhanced binding through cooperative polyvalent interactions.
J Am Chem Soc, 1996, 118: 3789–3800
23 Braun M, Camps X, Vostrowsky O, Hirsch A, Endress E, Bayerl TM,
Birkert O, Gauglitz G. Synthesis of a biotinated lipofullerene as a
new type of transmembrane anchor. Eur J Org Chem, 2000, 7:
1173–1182
5
6
7
8
9
10 Diaz Y, Gonzalez A, Lopez A, Perez R, Vazquez AM, Montero E.
Anti-ganglioside anti-idiotypic monoclonal antibody-based cancer
vaccine induces apoptosis and antiangiogenic effect in a metastatic
lung carcinoma. Cancer Immunol Immunother, 2009, 58: 1117–1128
11 Fernandez LE, Alonso DF, Gomez DE, Vazquez AM. Ganglioside-