Synthesis of (1→6)-β-D-glucosamine hexasaccharide, a potential antitumor and immunostimulating agent

Article abstract of DOI:10.1016/S0040-4039(02)01812-9

(1→6)-β-D-Glucosamine hexasaccharide was synthesized convergently using isopropyl thioglycosides as glycosyl donors in all coupling steps. The target compound showed good antitumor activity based on mice S₁₈₀ model studies.

Full text of DOI:10.1016/S0040-4039(02)01812-9

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 7561–7563
Synthesis of (1“6)-b- -glucosamine hexasaccharide, a potential
D
antitumor and immunostimulating agent
Feng Yang, Hongmei He and Yuguo Du*
Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 100085 Beijing, China
Received 19 April 2002; revised 9 August 2002; accepted 23 August 2002
Abstract—(1“6)-b-D-Glucosamine hexasaccharide was synthesized convergently using isopropyl thioglycosides as glycosyl donors
in all coupling steps. The target compound showed good antitumor activity based on mice S180 model studies. © 2002 Elsevier
Science Ltd. All rights reserved.
Linear oligosaccharides are widely distributed in natu-
ral products. For example, linear -mannan from Can-
octa-furanoside using an unprotected glycoside as the
acceptor. Thus, coupling of imidate 1 and partially
protected acceptor 28 under standard glycosylation con-
ditions furnished disaccharide 3 in 30% yield (Scheme
1). About 54% of 2 was recovered after column separa-
tion. Compound 3 was benzoylated and subsequently
deacetylated with 5% HCl (gas) in methanol to give
triol 5 (76%). The critical regioselective glycosylation of
1 and 5 was not straightforward and a 35% yield of
trisaccharide 6 was obtained. We realized that the rapid
consumption of donor 1 might be responsible for the
low yields. Adding a second portion of 1 to the reac-
tions did indeed improve the yields (ꢀ50%). Unfortu-
nately, the removal of 4-methoxyphenyl (PMP) from 6
using DDQ or CAN9 was also a low yielding step in
this strategy.
D
dida albicans and Pseudomonas Syringae pv. Ciccaronei,
interacts with mannan-binding proteins of higher ani-
mals and elicits a specific immune response with forma-
tion of antibodies, which recognize oligosaccharide
fragments of the mannose polymer.1 (1“3)-b-
D
-Glu-
cans are believed to have immunomodulatory activity.2
Sulfated linear -galactopyranose derivatives exhibit
-(1“4)-N-
D
anti-HIV activity in vitro.3 Furthermore, b-
D
acetyl glucosamine has been used as a bifidus factor to
promote animal growth.4 The synthesis of linear sugar
chains has been extensively explored,5 but to the best of
our knowledge, (1“6)-b-
been synthesized so far.6 Herein we report the first
-glucosamine hexasaccharide
D-hexaglucosamine has not
synthesis of (1“6)-b-
D
and its bioactivities as an antitumor and an immunos-
timulating agent.
We then turned our attention to the stepwise synthesis
as shown in Scheme 2. N-Phthaloyl glucosamine 7 was
regioselectively silylated on C-6 with TBSCl in pyridine
and acetylated in situ. BF3·Et2O-catalyzed desilyation
The initial strategy was designed based on our previous
work7 on the regioselective synthesis of a (1“5)-linked
Scheme 1. Reagents and conditions: (a) TMSOTf, CH2Cl2/CH3CN (4:1), −15 to 0°C, 30% (3); 35–50% (6); (b) BzCl, Py; (c) 5%
HCl (gas), MeOH, 76%.
Keywords: carbohydrate; glycosylation; glucosamine; antitumor agent; immunostimulant.
* Corresponding author. Fax: 86-10-62923563; e-mail: ygdu@mail.rcees.ac.cn
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)01812-9

7562
F. Yang et al. / Tetrahedron Letters 43 (2002) 7561–7563
Scheme 2. Reagents and conditions: (a) NaOMe, MeOH; TBSCl, Py; Ac2O, 70%; (b) BF3·Et2O, CH2Cl2, 95% (9); 89% (14); 84%
(16); 88% (20); 85% (22); (c) from 7: Ac2O, Py; 2-propanethiol, BF3·Et2O, CH2Cl2, 86%; (d) NaOMe, MeOH; (e) TBSCl, Py;
Ac2O, 78%; (f) NIS/TMSOTf, CH2Cl2, −20°C, 95% (13), 94% (15), 86% (17), 80% (19), 92% (21), 90% (23); (g) NH3, MeOH, 91%.
in 55% overall yield (from 14). N-Iodosuccinimide
(NIS) and TMSOTf-catalyzed condensation of 18 and
22, followed by deacylation in ammonia-saturated
methanol afforded hexasaccharide 2410 in excellent
yield (82%, two steps).
of 8 gave acceptor 9 smoothly. It is noteworthy that
TBAF promoted desilylation causes acyl migration
from C-4 to C-6. A modified Helfrich reaction of fully
acetylated 7 and 2-propanethiol in CH2Cl2 under reflux
gave isopropyl thioglycoside 10 (86%) and protection
group manipulation furnished the latent glycosyl donor
12 in 78% yield. Condensation of 12 and 9 was
achieved with great success and disaccharide 15 was
obtained in 94% yield. Cleavage of TBS from 15 using
2.0 equiv. of BF3·Et2O was carried out smoothly and
further glycosylation with 10 yielded 17 which was
converted to isopropyl thioglycoside 18 (67%) as
described in the preparation of 10. Convergently, 12
was reacted with octanol in the presence of NIS and
TMSOTf to give 13 (95%) which was treated with
BF3·Et2O to yield 14 (89%). Reiterative coupling and
desilylation of 12 and 14 gave trisaccharide acceptor 22
Kun Min mice weighing about 20 g were used for the
bioassay. Seven-day-old Sarcoma-180 ascites (0.05 mL,
about 6×106 cells) was transplanted into the right groins
of mice. The test samples, dissolved in distilled water,
were injected daily for 14 days starting 24 h after tumor
implantation. At the end of the 14th day, the mice were
killed, and the tumors were extirpated and weighted.
Haemoglobin (Hb), red blood cell (RBC), white blood
cell (WBC) and marrow cell counts were recorded on
SYSMEX. The results (Table 1), compared to lentinan
and CTX in the parallel test, suggest that compound 24
may be a potent antitumor agent.
Table 1. Preliminary studies on antitumor activity of hexaglucosamine 24
Sample
Dose (mg/kg
mouse)
Tumor growth
inhibition (%)
Body weight (g) Hb (g/L)
RBC (×1012
)
WBC (×108)
Marrow cell
(×108)
Control
CTXa
24
0
70
2
5
0
75
50
25
75
27.494.09
22.193.87
29.893.91
26.093.23
25.793.39
116.6913.0
108.2915.80
128.8918.80
92.8912.03
107.1912.38
7.291.01
6.990.78
8.691.13
6.390.70
7.490.89
12.792.86
5.892.12
12.993.12
6.692.26
12.796.28
5.091.45
1.990.94
5.991.63
5.691.25
3.791.35
Lentinan
CTX+24b
70+2
a 35 mg on the first and third days, respectively.
b 36 mg on the first and third days, respectively.

F. Yang et al. / Tetrahedron Letters 43 (2002) 7561–7563
7563
In conclusion, a highly efficient and practical method
was developed for the preparation of (1“6)-b- -glu-
Takeo, K.; Maki, K.; Wada, Y.; Kitamura, S. Carbohydr.
Res. 1993, 245, 81–93; (e) Zhu, Y.; Kong, F. Carbohydr.
Res. 2001, 332, 1–21; (f) Hinou, H.; Umino, A.; Mat-
suoka, K.; Terunuma, D.; Takahashi, S.; Esumi, Y.;
Kuzuhara, H. Tetrahedron Lett. 1997, 38, 8041–8044.
6. (a) Du, Y.; Yang, F. Chin. Pat. Appl. 01136484x, 2001;
(b) In our manuscript preparation, Dr. Baasov published
a tetra-glucosamine oligosaccharide synthesis: Fridman,
M.; Solomon, D.; Yogev, S.; Baasov, T. Org. Lett. 2002,
4, 281–283.
D
cosamine oligosaccharides. Isopropyl thioglycosides
were shown to be suitable donors in target synthesis
compared to the corresponding trichloroacetimidates.
Acceptors with anomeric acetyl groups, which could
simplify the protection strategy, are compatible to NIS/
TMSOTf promoted coupling reactions. Using this pro-
cedure to build up more complicated glucosamine
oligosaccharides is currently underway in our group.
7. Du, Y.; Pan, Q.; Kong, F. Synlett 1999, 5, 1648–1650.
8. Nakano, T.; Ito, Y.; Ogawa, T. Carbohydr. Res. 1993,
243, 43–69.
Acknowledgements
9. (a) Fukuyama, T.; Laud, A. A.; Hotchkiss, L. M. Tetra-
hedron Lett. 1985, 26, 6291–6294; (b) Tanaka, T.;
Oikawa, Y.; Hamada, T.; Yonemitsu, O. Tetrahedron
Lett. 1986, 27, 3651–3654.
This work was supported by NNSF of China (Projects
39970179, 29972053), RCEES of CAS and Beijing
YFAX Sci-tech Ltd. We thank Professor Zhikui Wu of
Guang-An-Meng Hospital for mice experiments.
10. (a) Selected physical data for 23: [h]2D5 +37 (c 1, CHCl3);
1H NMR (400 MHz, CDCl3): l 0.81 (t, 3 H, J 7.2 Hz,
CH2CH3), 0.85–1.28 (m, 12 H, 6 CH2), 1.770, 1.776,
1.778, 1.788, 1.794, 1.870, 1.936, 1.937, 1.942, 1.945,
1.950, 2.052, 2.164 (13 s, 39 H, COCH3), 3.20 (m, 1 H,
OCHaHb), 3.46 (dd, 1 H, J 10.8, 6.4 Hz), 3.16 (m, 1 H,
OCHaHb), 3.40–3.80 (m, 14 H), 3.90–3.96 (m, 2 H),
4.11–4.24 (m, 6 H), 4.36–4.41 (m, 2 H), 4.70–4.82 (m, 3
H), 4.93 (t, 1 H, J 9.2 Hz), 5.13 (d, 1 H, J 8.4 Hz, H-1I),
5.21 (t, 1 H, J 9.2 Hz), 5.25 (d, 1 H, J 8.4 Hz, H-1II), 5.27
(d, 2 H, J 8.4 Hz, H-1III, H-1IV), 5.33 (d, 1 H, J 8.4 Hz,
H-1V), 5.51 (d, 1 H, J 8.4 Hz, H-1VI), 5.57–5.67 (m, 5 H),
6.07 (dd, 1 H, J 10.8, 9.0 Hz), 7.71–7.92 (m, 24 H, Ph);
13C NMR (100 MHz, CDCl3): l 97.51, 97.56, 97.60 (2C),
97.66, 98.00 (6 C-1). MALDI TOF-MS calcd for
References
1. (a) Ogawa, T.; Yamamoto, H. Carbohydr. Res. 1982, 104,
271–283; (b) Weis, W. I.; Drickamer, K.; Hendrickson,
W. A. Nature 1992, 360, 127–134.
2. (a) Lowman, D.; Ensley, H.; Williams, D. Carbohydr.
Res. 1998, 306, 559–562; (b) Witczak, Z. J. Curr. Med.
Chem. 1995, 1, 392–405.
3. (a) Takahashi, T.; Adachi, M.; Matsuda, A.; Doi, T.
Tetrahedron Lett. 1999, 41, 2599–2603; (b) Amornrut, C.;
Toida, T.; Imanari, T.; Woo, E.; Park, H.; Linhardt, R.
J.; Wu, S. J.; Kim, Y. S. Carbohydr. Res. 1999, 321,
121–127.
C
118H122N6O50: 2422.72 [M]. Found: 2445.6 [M+Na]+,
2461.6 [M+K]+; (b) To the NH3 saturated MeOH (200
mL) was added 23 (1 g). The mixture was stirred at rt for
7 days, then concentrated. The residue was dissolved in
H2O (5 mL) and then purified by charcoal chromatogra-
phy (H2O) to give 24 as a white solid. Selected 13C NMR
(100 MHz, D2O) for 24: 103.05, 103.51, 103.59, 103.63,
103.67, 103.7 (6 C-1). MALDI TOF-MS calcd for
C44H84N6O25: 1096.55 [M]. Found: 1119.2 [M+Na]+.
4. Austin, P. R.; Brine, C. J.; Castle, J. E.; Zikakis, J. P.
Science 1981, 212, 749–753.
5. (a) Randolph, J. T.; McClure, K. F.; Danishefsky, S. J. J.
Am. Chem. Soc. 1995, 117, 5712–5719; (b) Kovac, P.;
Taylor, R. B.; Glaudemans, C. P. J. J. Org. Chem. 1985,
50, 5323–5333; (c) Verduyn, R.; Douwes, M.; van der
Klein, P. A. M.; Mo¨singer, E. M.; van der Marel, G. A.;
van Boom, J. H. Tetrahedron 1993, 49, 7301–7316; (d)