An efficient glycosylation reaction for the synthesis of asialo GM2 analogues

Article abstract of DOI:10.1016/j.tetlet.2006.08.008

We investigated the coupling reaction of glycosyl donors N-trichloroethoxycarbonyl-galactosamine-O-trichloroacetimidate (2a) and N-p-nitrobenzyloxycarbonyl-galactosamine-O-trichloroacetimidate (2b) with the 4′-OH of lactose derivatives (3a-d) to synthesiz

Full text of DOI:10.1016/j.tetlet.2006.08.008

Tetrahedron Letters 47 (2006) 7371–7374
An efficient glycosylation reaction for the synthesis
of asialo GM2 analogues
Bin Sun, Aliaksei V. Pukin, Gerben M. Visser* and Han Zuilhof
Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB Wageningen, The Netherlands
Received 16 May 2006; revised 27 July 2006; accepted 2 August 2006
Available online 24 August 2006
Abstract—We investigated the coupling reaction of glycosyl donors N-trichloroethoxycarbonyl-galactosamine-O-trichloroacetimi-
date (2a) and N-p-nitrobenzyloxycarbonyl-galactosamine-O-trichloroacetimidate (2b) with the 40-OH of lactose derivatives (3a–d)
to synthesize key intermediates of asialo GM2 analogues, and found that the glycosylation yield with 2a was 90% or more in all
investigated cases.
Ó 2006 Elsevier Ltd. All rights reserved.
Oligosaccharides containing b-D-glycosides of N-acetyl-
2-amino-2-deoxy units are widely distributed in living
organisms.1 In this respect the biologically active ganglio-
sides GM1 and GM2, which both contain an N-acetyl-2-
amino-2-deoxy-D-galactopyranosyl residue b-linked to
O-40 of lactose,2 are typical examples.
and PNZ6 proved to be stable under the glycosylation
conditions (ꢀ20 °C, in diethyl ether and TMSOTf
(0.10 equiv) as catalyst), and both could be easily and
selectively cleaved.7 On the other hand, since O-glycosyl
trichloroacetimidate was shown to be superior com-
pared to the glycosyl donors containing halides (Cl or
Br) at the anomeric centre,8 we selected N-trichloroeth-
oxycarbonyl-galactosamine-O-trichloroacetimidate (2a)
and N-p-nitrobenzyloxycarbonyl-galactosamine-O-tri-
chloroacetimidate (2b) as glycosyl donors to investigate
their coupling efficiency with 40-OH lactose derivatives.
Asialo GM2 (GA2) has been widely studied as a tumor-
associated marker and a monoclonal antibody,3 and
various synthetic efforts to produce asialo GM2 have
been reported with moderate yields.4 However, the gly-
cosylation step required for its synthesis has only been
reported with low yields (<50%). Given its interesting
biological activity, we therefore set out to develop a
more efficient synthesis of asialo GM2 analogues 1
(see Fig. 1).
Scheme 1 outlines the syntheses of these donor units.
Treatment of galactosamine hydrochloride 4 with
1 equiv of NaOMe in MeOH, followed by the addition
of trichloroethyl chloroformate or p-nitrobenzyl chloro-
formate (1 equiv)–TEA (1 equiv) and O-acetylation
(Ac2O–pyridine) afforded compounds 5a (92%) or 5b
(92%) in good yields. Finally, regioselective deacetyl-
ation at O-1 with hydrazine acetate in DMF followed
by treatment of the reducing sugar with trichloroaceto-
nitrile in the presence of DBU, produced the Teoc-tri-
chloroacetimidate 2a (79%; overall yield from 4 is
73%).9 The PNZ analogue 2b was obtained in 75% yield
(overall yield from 4 is 69%).
As indicated in Figure 1, the retrosynthesis of asialo
GM2 involves assembly of two key building blocks 2
and 3. Compound 2 is an efficient glycosyl donor, in
which the NH2 group needs to be protected. The phthal-
imido (Phth) and azido groups have been the most
widely used.5 Recently, p-nitrobenzyloxycarbonyl6
(PNZ) and trichloroethoxycarbonyl (Teoc) groups were
applied in the synthesis of glycoconjugates containing 2-
acetamido glycoside units.7 However, the linkage of N-
phthalimido or azido halide donors and OH-40 lactose
derivatives have been reported to yield asialo GM2 in
low to moderate yields (20–54%).4 In our hands, Teoc
As stated above, the 40-OH lactose derivatives showed a
very low reactivity as glycosyl acceptors. As benzyl
ethers are able to enhance the reactivity of neighbouring
hydroxyl groups in glycosylation reactions,10 we selected
the benzyl group as the protecting group for the lactose
units of 3a–d (Scheme 2).
*
Corresponding author. Tel.: +31 317 484810; fax: +31 317 484914;
e-mail: Gerben.Visser@wur.nl
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.08.008

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B. Sun et al. / Tetrahedron Letters 47 (2006) 7371–7374
OH
OH
Asialo GM2 Analogues
O
O
HO
AcHN
OH
OH
HO
O
OH
O
HO
R'
O
OAc
O
AcO
AcO
OH
OAc
O
2
AcO
AcO
HN
O
OC(NH)CCl3
R
RHN
OBn
O
OBn
BnO
+
OBn
BnO
O
R'
O
OBn
OH
BnO
OBn
OBn
1
BnO
O
O
R'
O
OBn
OBn
3
O
C
O
C
R = Teoc; PNZ
R' = OCH3; OCH2CH2N3; SPh; OCH2(CH2)8CH=CH2
O
CH2
NO2
O
CH2 CCl3
Teoc =
PNZ =
Figure 1. Retrosynthesis of the key intermediates of asialo GM2 analogues.
OAc
OAc
OAc
OH
OH
HO
OAc
i, ii (a or b), iii
iv, v
O
O
O
OH
OAc
AcO
AcO
NH
NH
NH2*HCl
OC(NH)CCl3
R
R
4
5a R = Teoc; 92%
5b R = PNZ; 92%
2a R = Teoc; 79%
2b R = PNZ; 75%
Scheme 1. Reagents and conditions: (i) NaOMe (1 equiv)/MeOH, room temperature, 30 min; (ii) a. trichloroethyl chloroformate (1 equiv), Et3N
(1 equiv), room temperature, 2 h; b. p-nitrobenzyl chloroformate (1 equiv), Et3N (1 equiv, room temperature, 2 h); (iii) Ac2O/Py, room temperature,
overnight; (iv) hydrazine acetate, DMF, 0 °C, 2 h; (v) CCl3CN (10 equiv)/DBU, CH2Cl2, ꢀ10 °C, 5 h.
OH
O
OAc OAc
O
OAc
O
OAc
AcO
OH
HO
OH
O
OAc
O
OAc
O
i
ii (a; b; c; d)
HO
AcO
AcO
OH
OAc
R'
AcO
O
O
O
OAc
OAc
OH
OH
OAc
OAc
6
8
7
Ph
O
OH
O
OBn
O
OBn
OBn
iii, iv, v
vi
BnO
O
OBn
BnO
8a, 9a, 3a: R' = OCH3
R'
R'
BnO
O
BnO
O
O
O
8b, 9b, 3b: R' = OCH2CH2N3
8c, 9c, 3c: R' = SPh
OBn
OBn
OBn
8d, 9d, 3d: R' = OCH2(CH2)8CH=CH2
9
3
Scheme 2. Reagents and conditions: (i) Ac2O/NaOAc, reflux, 2 h, 77%; (ii) a. 24% NH2NH2ÆH2O, CH3CN, 24 h, room temperature, 86%; CH3I/
Ag2O/CH3CN, room temperature, 24 h, 8a, 84%; b. 2-azidoethanol (2.5 equiv), BF3ÆC2H5OC2H5, CH2Cl2, 0 °C to room temperature, 24 h, 8b, 80%;
c. thiophenol (2.5 equiv), BF3ÆC2H5OC2H5, CH2Cl2, 0 °C to room temperature, 24 h, 8c, 75%; d. 33% HBr in AcOH, 0 °C–rt, 5 h, 72%; undec-10-en-
1-ol, AgOTf, dry toluene, ꢀ78 °C to room temperature, overnight, 8d, 84%; (iii) NaOMe/MeOH, IR resin (H+); (iv) PhCH(OCH3)2/CSA/THF,
reflux, 3 h; (v) NaH/BnBr/DMF, overnight, room temperature, 9a (50%); 9b (57%), 9c (54%), 9d (60%) (three steps); (vi) NaCNBH3/HCl in Et2O/dry
THF, 30 min, 3a (78%), 3b (78%), 3c (76%), 3d (77%).
In Scheme 2, the synthetic route to the lactose acceptors
is depicted. Heating a mixture of lactose 6, acetic anhy-
Table 1. Glycosylation yields of glycosyl donors 2a–b and acceptors
3a–d
dride and anhydrous sodium acetate afforded peracetyl-
lactose 7 (yield: 77%).11 After treatment with hydrazine,
Glycosyl
donor
Lactose
derivative
Molar ratio
of 2:3
Product
Isolated
yield, %
the anomeric acetyl group in 7 was selectively deprotec-
ted, and the product was treated with CH3I and silver
oxide to give 8a (72%).12 Alternatively, compound 7
was reacted with 2-azidoethanol or thiophenol in the
presence of BF3-diethyl etherate to afford 8b (80%)
and 8c (75%), respectively. Bromination of 7 with
hydrogen bromide 33% (w/w) in acetic acid at 0 °C
and subsequent coupling with undec-10-en-1-ol gave
2a
2b
2a
2b
2a
2a
3a
3a
3b
3b
3c
3d
1.5
1.5
1.5
1.5
1.5
1.5
1a
1b
1c
1d
1e
1f
91
69
90
71
90
90

B. Sun et al. / Tetrahedron Letters 47 (2006) 7371–7374
7373
OAc
O
OAc
OAc
OBn
OH
OAc
O
OBn
O
OBn
i
AcO
O
BnO
O
O
OBn
+
R'
HN
R
AcO
BnO
BnO
O
R'
O
BnO
HN
R
OBn
O
OC(NH)CCl3
OBn
OBn
OBn
2a R = Teoc
2b R = PNZ
3a R' = OCH3
1a R = Teoc, R' = OCH3
1b R = PNZ, R' = OCH3
3b R' = OCH2CH2N3
3c R' = SPh
1c R = Teoc, R' = OCH2CH2N3
1d R = PNZ, R' = OCH2CH2N3
1e R = Teoc, R' = SPh
3d R' = OCH2(CH2)8CH=CH2
1f R = Teoc, R' = OCH2(CH2)8CH=CH2
OAc
O
OH
OAc
OH
HO
O
OBn
OH
ii, iii, iv
AcO
TeocHN
O
O
OBn
OH
HN
BnO
O
O
O
HO
O
yield over 3 steps: 73%
OCH3
OCH3
BnO
HO
O
Ac
O
OBn
OH
OBn
OH
1a
10
Scheme 3. Reagents and conditions: (i) TMSOTf/dry Et2O, ꢀ20 °C, 5 h; (ii) active Zn powder/Ac2O, room temperature, 5 h; (iii) NaOMe/MeOH,
room temperature; (iv) H2 (50 psi), Pd (10%)/C, room temperature, 6 h.
8d (84%). Compounds 8a–d were characterized by NMR
and LC–MS. Compounds 8a–d were deacetylated using
NaOMe in MeOH, followed by selective protection of
OH-40 and OH-60 with a,a-dimethoxytoluene under
mild acidic conditions in anhydrous THF. The remain-
ing OH-groups were benzylated with NaH and benzyl
bromide in DMF to give 9a–d (50%; 57%; 54%; 60%).
Selective cleavage13 of the benzaldehyde acetal in 9a–d
with NaBH3CN–HCl in dry THF afforded acceptor
compounds 3a–d in good yields (76–78%).14
program coordinated by the Dutch Ministry of
Economic Affairs.
References and notes
1. (a) Barasch, J.; Kiss, B.; Prince, A.; Saiman, L.; Gruenert,
D.; Al-Awquati, Q. Nature 1991, 352, 70; (b) Imundo, L.;
Barasch, J.; Prince, A.; Al-Awqati, Q. Proc. Natl. Acad.
Sci. U.S.A. 1995, 92, 3019; (c) Coligan, J. E.; Kindt, T. J.;
Krause, R. M. Immunochemistry 1978, 15, 755; (d)
Siddiqui, B.; Hakomori, S. J. Biol. Chem. 1971, 246,
5766.
2. (a) Bhattacharya, S. K.; Danishefsky, S. J. J. Org. Chem.
2000, 65, 144; (b) Kwon, O.; Danishefsky, S. J. J. Am.
Chem. Soc. 1998, 120, 1588; (c) Fang, J.; Chen, X.; Zhang,
W.; Wang, J.; Andreana, P. R.; Wang, P. G. J. Org. Chem.
1999, 64, 4089; (d) Paulsen, H.; Paal, M. Carbohydr. Res.
1985, 137, 39.
3. Hakomori, S. Annu. Rev. Biochem. 1981, 50, 733.
4. (a) Wessel, H. P.; Iversen, T.; Bundle, D. R. Carbohydr.
Res. 1984, 130, 5; (b) Sugimoto, M.; Horisaki, T.; Ogawa,
T. Glycoconjugate J. 1985, 11–15; (c) Paulsen, H.; Paal, M.
Carbohydr. Res. 1985, 137, 39.
5. (a) Banoub, J.; Boullanger, P.; Lafont, D. Chem. Rev.
1992, 92, 1167; (b) Barresi, F.; Hindsgaul, O. Carbohydr.
Res. 1995, 14, 1043.
Finally, glycosyl donors 2a–b were reacted with accep-
tors 3a–d at ꢀ20 °C in dry diethyl ether in the presence
of TMSOTf (0.11 equiv) to give GM2 analogues 1a–f.15
The glycosylation yields are listed in Table 1.
The results in Table 1 clearly show that the glycosylation
efficiency of glycosyl donor N-trichloroethoxycarbonyl-
galactosamine-O-trichloroacetimidate (2a) was better
than that of N-p-nitrobenzyloxycarbonyl-galactos-
amine-O-trichloroacetimidate (2b). Replacement of the
N-Teoc group in 1a by an N-acetyl group with active
Zn powder in acetic anhydride7 followed by deacetyl-
ation with NaOMe in MeOH and debenzylation with
H2 (50 psi) and Pd/C (10%) in MeOH at room temper-
ature gave b-D-GalNAc-(1 ! 4)-b-D-Gal-(1 ! 4)-b-D-
Glc-OMe (methyl asialo GM2, 10)16 in 73% yield (three
steps), see Scheme 3.
6. (a) Qian, X.; Hindsgaul, O. Chem. Commun. 1997, 1059;
(b) Liao, W.; Piskorz, C. F.; Locke, R. D.; Matta, K. L.
Bioorg. Med. Chem. Lett. 2000, 10, 793.
7. (a) Dullenkopf, W.; Castro-Palomino, J. C.; Manzoni, L.;
Schmidt, R. R. Carbohydr. Res. 1996, 296, 135; (b)
Higashi, K.; Susaki, H. Chem. Pharm. Bull. 1992, 40, 2019.
8. Lindhorst, T. K. Essentials of Carbohydrate Chemistry and
Biochemistry, 2nd ed.; Wiley-VCH, 2003; p 86.
In conclusion, we have prepared and investigated
two glycosyl donors 2a and 2b, which could be linked
efficiently to lactose acceptors 3a–d, and showed unam-
biguously that N-trichloroethoxycarbonyl-galactos-
amine-O-trichloroacetimidate was an efficient donor
with glycosylation yields of 90% or more.
9. Compound 2a. 1H NMR (300 MHz, CDCl3), all couplings
in Hz. d (ppm): 8.74 (s, 1H, C@NH), 6.40 (d, 1H, J = 3.6,
H-1), 5.46 (d, 1H, J = 2.6, H-4), 5.22 (dd, 1H, J1 = 3.2,
J2 = 11.4, H-3), 5.01 (d, 1H, J = 9, NHTeoc), 4.70 and
4.64 (ABq, J = 9.5, 1H each, Cl3CCH2OCO), 4.46–4.47
(m, 1H, H-2), 4.01–4.32 (m, 3H, H-5, H-6), 2.11 (s, 3H,
COCH3), 1.96 (s, 6H, 2 COCH3). HRMS [C17H2035Cl5-
37Cl1N2O10+Na]+ 646.91582 (calcd 646.91173, D ppm =
6.33).
Acknowledgements
The authors thank Peter van Galen (Radboud Univer-
sity, Nijmegen) for HRMS measurements. This work
was supported by the Human Frontier Science Program
(HFSP) and by NanoNed, a national nanotechnology
10. Paulsen, H.; Bunsch, A. Carbohydr. Res. 1982, 101, 21.
¨
11. Hudson, C. S.; Johnson, J. M. J. Am. Chem. Soc. 1915, 37,
1270.

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B. Sun et al. / Tetrahedron Letters 47 (2006) 7371–7374
12. Gebbie, S. J.; Gosney, I.; Harrison, P. R.; Lacan, I. M. F.;
Sanderson, W. R.; Sankey, J. P. Carbohydr. Res. 1998,
308, 345.
disappeared, the reaction was quenched with triethylamine
and was filtered through Celite, washed with CH2Cl2 and
concentrated. Column chromatography (3:7, ethyl ace-
tate:petroleum ether) gave trisaccharide 1a (280 mg, 91%).
13C NMR d (C6D6, ppm): 170.3, 170.0, 154.6, 140.0, 139.6,
139.3, 129.5, 129.2, 129.1, 129.0, 128.9, 128.8, 128.7, 128.2,
127.9, 127.8, 105.3, 103.1, 102.3, 83.2, 83.1, 82.6, 81.3,
76.2, 76.0, 75.3, 74.7, 73.9, 73.6, 71.4, 68.8, 67.2, 61.6, 60.3,
56.3, 53.8, 20.8, 20.6, 20.4.
13. Mackie, I. D.; Ro¨hrling, J.; Gould, R. O.; Pauli, J.; Ja¨ger,
C.; Walkinshaw, W.; Potthast, A.; Rosenau, T.; Kosma,
P. Carbohydr. Res. 2002, 337, 161.
14. Compound 3a. HRMS [M+Na]+ 919.40567 (calcd
919.40333, D ppm = 2.54).
15. Typical glycosylation procedure: a mixture of compound
2a (238 mg, 0.33 mmol), 3a (200 mg, 0.22 mmol) and
active powdered 4 A molecular sieves (1.0 g) in dry diethyl
16. Compound 10. NMR (400 MHz, D2O), all couplings in
1
˚
Hz. H d (ppm): 4.54 (d, J = 8, 1H), 4.35 (d, J = 8, 1H),
ether (6 mL) was stirred for 1 h at room temperature
under N2. After cooling to ꢀ20 °C, 150 lL of TMSOTf
solution (50 lL TMSOTf dissolved in 2.0 mL dry diethyl
ether) (0.0237 mmol) was injected into the reaction, and
the mixture was stirred for about 4 h at ꢀ20 °C. The
reaction was monitored by TLC (3:7, ethyl acetate:petro-
leum ether (40–60)). When acceptor 3a had almost
4.33 (d, J = 8, 1H), 4.01 (d, J = 2.8, 1H), 3.90 (d, J = 12,
1H), 3.84–3.78 (m, 2H), 3.77–3.46 (m, 15 H), 3.26–3.29 (m,
1H), 3.24–3.18 (m, 1H), 1.97 (s, 3H). 13C d (ppm): 175.34,
103.43, 103.35, 103.05, 78.83, 76.51, 75.20, 75.15, 74.73,
74.70, 73.09, 72.78, 71.43, 71.37, 68.17, 61.42, 61.05, 60.37,
57.58, 53.03, 22.77. HRMS [M+Na]+ 582.20233 (calcd
582.20100, D ppm = 2.28).