The first synthesis of tetraglucosyl glucitol having phytoalexin-elicitor activity in rice cells based on a sequential glycosylation strategy

Article abstract of DOI:10.1016/S0040-4039(01)01944-X

We describe a highly convergent approach for the synthesis of the tetraglucosyl glucitol 1 and its derivatives 4, 5 and 6 which exhibit phytoalexin-elicitor activity in rice cells based on sequential glycosylation.

Full text of DOI:10.1016/S0040-4039(01)01944-X

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 9191–9194
The first synthesis of tetraglucosyl glucitol having
phytoalexin-elicitor activity in rice cells based on a sequential
glycosylation strategy
Toru Amaya,a Hiroshi Tanaka,a Takeshi Yamaguchi,b Naoto Shibuyab and Takashi Takahashia,*
aDepartment of Applied Chemistry, Graduate School of Science and Engineering, Tokyo Institute of Technology, 2-12-1
Ookayama, Meguro, Tokyo 152-8552, Japan
bBiochemistry Department, Institute of Plant Sciences, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba,
Ibaraki 305-8602, Japan
Received 15 August 2001; revised 9 October 2001; accepted 12 October 2001
Abstract—We describe a highly convergent approach for the synthesis of the tetraglucosyl glucitol 1 and its derivatives 4, 5 and
6 which exhibit phytoalexin-elicitor activity in rice cells based on sequential glycosylation. © 2001 Elsevier Science Ltd. All rights
reserved.
The production of phytoalexin is one of various defense
responses in higher plants. b-Glucan and some of its
hydrosates composed of b-(1,6) and b-(1,3) glucose are
known to demonstrate the phytoalexin-elicitor activity.
Recently, Yamaguchi and co-workers have purified
and characterized the elicitor-active tetraglucosyl glu-
citols 1, 2, and 3 after partial acid/enzymatic hydroly-
sis of the cell walls of the rice blast disease fungus
Pyricularia oryzae (Magnaporthe grisea) (Scheme 1).
For example, hepta-b-D-glucopyranosyl-D-glucitol con-
taining a b-(1,6) pentasaccharide backbone is a well-
known elicitor in soybean.1 Because of the difficulty of
the purification and the structural determination of these
oligosaccharides, the chemical synthesis of the b-glucan
elicitor and related compounds has contributed to the
study of their structure–activity relationships.2
These oligosaccharides contain
a
b-(1,3) tetra-
saccharide backbone with a b-(1,6) branched glucose
at different glucose units. The position of a b-(1,6)
branched glucose might influence the elicitor activity
because the elicitor activity of 1 is much stronger than
that of 2 and 3.3
Scheme 1. The structures and synthetic strategy of phytoalexin elicitors in rice cells.
Keywords: b-glucan; phytoalexin elicitor; branched oligosaccharide; sequential glycosylation; thioglyciside.
* Corresponding author. Tel.: +81-3-5734-2120; fax: +81-3-5734-2884; e-mail: ttakashi@o.cc.titech.ac.jp
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01944-X

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T. Amaya et al. / Tetrahedron Letters 42 (2001) 9191–9194
The synthesis of intermediate 7 is shown in Scheme 2.
Reductive cleavage of acetal 109 (90%), followed by
glycosylation at the 6 position with glycosyl bromide
1110 (84%) and deprotection of the silyl ether provided
the key intermediate 7 in 79% yield. We first examined
Method A to synthesize 1 and 4. Selective activation of
glycosyl bromides 12a10 and 12b10 in the presence of
thioglycoside 7 was achieved using silver triflate to
afford branched saccharide 13a and 13b in 68 and 43%
yields. In the next glycosylation a triacetal disaccharide
1411 was chosen as an acceptor because using the cyclic
acetal group we could utilize a highly reactive hydroxyl
group at the C3 position. Coupling of thioglycoside 13a
and acceptor 14 was accomplished in the presence of
NIS/TfOH to provide pentasaccharide 15 in 83% yield
as a single product. In order to synthesize elicitor 1,
glycosylation of monosaccharide 16a12 (see Scheme 3)
with tetrasaccharide donor 13b was examined. How-
ever, the coupling reaction of the two units 13b and 16a
did not proceed in the presence of NIS/TfOH. Thin
layer chromatography (TLC) analysis showed the thio-
glycoside 13b decomposed under the conditions. This
result indicates that the tetrasaccharide donor 13b
would not be reactive enough to glycosylate the accep-
tor 16a. Deprotection of the silyl ether in 15 and
hydrolysis of the esters, followed by hydrogenolysis of
the benzyl ethers and benzylidene acetals afforded the
fully deprotected pentasaccharide 4 in 38% overall
yield. Analysis of the 13C NMR spectra of 4 indicated
that the four glycosidic linkages except for that in the
reduced end were formed with b configuration.13
Synthetic studies of the soybean elicitor having a b-(1,6)
backbone have been reported by many research
groups.2a,4,9 We have already developed one-pot
sequential glycosylation5 to synthesize the soybean elic-
itor.6 Elongation of the b-(1,6) backbone could be
achieved by glycosylation of a reactive primary alcohol
at 6 position. However, the synthesis of b-(1,3) linked
backbone should require repeat glycosylation of less
reactive secondary alcohol at 3 position.7 Herein we
wish to report the total synthesis of rice elicitor 1 and
the related oligosaccharide 4, 5 and 6 having b-(1,3)
backbone based on sequential glycosylation.
Our synthetic strategy for the phytoalexin elicitor 1 and
derivatives 4, 5 and 6 is illustrated in Scheme 1. 3-
Hydroxy thioglycoside 7 having a branched glucose was
designed as a key intermediate, which can be connected
with various oligosaccharide units at the C1 and C3
hydroxyl groups independently by sequential glycosyla-
tion. A phenylthio group was chosen as a leaving group
for 7 because it is one of the most powerful leaving
groups. Furthermore, it is stable to the conditions of
activation for several other leaving groups such as
glycosyl bromide.5a It is possible to elongate the b-(1,3)
glucan chain in two ways using the key intermediate 7.
Method A: chemoselective glycosylation of intermedi-
ate 7 with glycosyl bromide 8a, followed by activation
of the resulting thioglycoside to couple glycosyl accep-
tor 9. Method B: site-selective glycosylation of acceptor
9 with thioglycoside 7 followed by glycosylation of the
resultant alcohol in 7 with thioglycoside 8b.8 For the
success of the Method A, it is necessary that the
glycosyl donor prepared by glycosylation of 7 would
have the high reactivity enough to form the b-(1,3)
linkage. On the other hand, in Method B the oligosac-
charide resulting from the first glycosylation would be
used as the glycosyl acceptor for the second glycosyla-
tion. However, the acceptor for the first glycosylation
should be more reactive than 7.
We next applied Method B to the synthesis of rice
elicitors 1 and 6, as shown in Scheme 3. We envisaged
that the reactivity of a benzyl alcohol and the C3-OH in
a monoglucose unit is higher than that of the secondary
hydroxyl in 7, thus allowing for a site-selective glycosy-
lation. Coupling of thioglycoside 7 and acceptor 16a12
or benzyl alcohol 16b was achieved in the presence of
NIS/TfOH to afford the corresponding oligosaccha-
,
Scheme 2. Reagents and conditions: (a) BH3·NMe3, AlCl3, CH2Cl2, Et2O, 0°C, 1 h, 90%; (b) 11, AgOTf, toluene, CH2Cl2, 4 A MS,
,
−40°C, 84%; (c) 40% aq. HF, CH3CN, 12 h, 79%; (d) 12, AgOTf, toluene, CH2Cl2, 4 A MS, −40°C, 10 min, 68% (for 13a), 43%
,
(for 13b); (e) 7, NIS, TfOH, CH2Cl2, 4 A MS, −20°C, 35 min, 83%; (f) 40% aq. HF, CH3CN, 12 h; (g) NaOMe, MeOH, 4 h; (h)
Pd(OH)2, MeOH, H2O, H2 (1 atm), 6 h, 37% (three steps).

T. Amaya et al. / Tetrahedron Letters 42 (2001) 9191–9194
9193
Fonds for Promoting Science and Technology from the
Ministry of Education, Culture, Sports, Science, and
Technology. We are also grateful to Dr. Tadashi Ishii
and Dr. Hiroshi Ono for their help in recording the 800
MHz NMR spectra.
References
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Scheme 3. Reagents and conditions: (a) NIS, TfOH, CH2Cl2, 4
,
A MS, 0°C, 69% (for 17a), 87% (for 17b); (b) NIS, TfOH,
,
CH2Cl2, 4 A MS, 0°C, 10 min, 76% (for 19a), 87% (for 19b);
(c) 40% aq. HF, CH3CN, 12 h; (d) NaOMe, MeOH, 4 h; (e)
Pd(OH)2, MeOH, H2O, H2, 6 h, 38% (for 6), 48% (for 5)
(three steps); (f) NaBH4, MeOH–H2O, 84%.
rides 17a and 17b in 69 and 87% yields. It should be
noted that self-condensation of 7 was not observed in
the reaction. Sequential glycosylation with disaccharide
donor 1814 carried out in the presence of NIS/TfOH
provided penta- or tetrasaccharide 19a and 19b in 76
and 87% yields, respectively. The deprotection of 19a
and 19b was accomplished by removal of the ester
groups, followed by hydrogenolysis of the benzyl ether
to afford the fully deprotected penta- and tetra-
saccharides 6 and 5 in 38 and 48% yields. Reduction of
pentasaccharide 6 was achieved by treatment with
sodium borohydride in water to afford the reported
glucitol 1 in 84% yield. The analytical data (1H NMR,
MS, HPLC) of the synthetic glucitol 115 were identical
with those of the isolated material.
5. (a) Yamada, H.; Harada, T.; Takahashi, T. Tetrahedron
Lett. 1994, 35, 3979–3982; (b) Yamada, H.; Kato, T.;
Takahashi, T. Tetrahedron Lett. 1999, 40, 4581–4584; (c)
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Soc. 1994, 116, 7919–7920; (b) Yamada, H.; Takimoto,
H.; Ikeda, T.; Tsukamoto, H.; Harada, H.; Takahashi, T.
Synlett 2001, 1751.
In conclusion, we have described the first synthesis of
the tetraglucosyl glucitol 1 and derivatives 4, 5 and 6
having phytoalexin-elicitor activity for rice based on a
sequential glycosylation protocol. Our key intermediate
7 allows for easy access to b-(1,3) linked oligosaccha-
rides 4, 5 and 6 with a b-(1,6) branched glucose in two
ways. The phytoalexin-elicitor activity of these
oligosaccharides is currently being explored.
7. (a) Du, Y.; Zhang, M.; Kong, F. Org. Lett. 2000, 24,
3797–3800; (b) Yang, G.; Kong, F. Synlett 2000, 10,
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Acknowledgements
9. Nicolaou, K. C.; Winssinger, N.; Pastor, J.; DeRoose, F.
J. Am. Chem. Soc. 1997, 119, 449–450.
This work was supported by Special Coordination

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T. Amaya et al. / Tetrahedron Letters 42 (2001) 9191–9194
10. Glycosyl bromide 11, 12a, and 12b were prepared by the
bromination ((COBr)2, DMF, CHCl3, rt) of the corre-
sponding C1-hydroxyl substrate. For a related bromina-
tion procedure, see: van Boeckel, C. A. A.; Beetz, T.;
Vos, J. N.; de Jong, A. J. M.; van Alest, S. F.; van den
Bosch, R. H.; Mertens, J. M. R.; van der Vlugt, F. A. J.
Carbohydr. Chem. 1985, 4, 293–321.
14. The glycosyl donor 18 was prepared by the coupling of
glycosyl bromide 12a and phenylthio 2-O-benzoyl-4,6-di-
benzyl-b-D-glucoside, which was prepared from the 6-O-
benzylation (BnBr, TBAI, then NaH, 0°C, 1 h) followed
by the removal of the silyl ether (40% aq. HF, CH3CN)
as donor and acceptor, respectively (AgOTf, CH2Cl2,
,
PhMe, 4 A MS).
15. 1: [h]2D7=−17.5 (c=0.21 in H2O); 1H NMR (800 MHz,
D2O): l=3.27 (dd, J=9.22, 8.14 Hz, 1H), 3.31 (dd,
J=8.1, 9.3 Hz, 1H), 3.34 (dd, J=9.5, 9.5 Hz, 1H), 3.36
(dd, J=9.5, 9.5 Hz, 1H), 3.40–3.49 (m, 7H), 3.50 (dd,
J=8.2, 9.0 Hz, 1H), 3.51 (dd, J=9.5, 9.5 Hz, 1H), 3.55
(dd, J=8.3, 9.0 Hz, 1H), 3.60–3.70 (m, 7H), 3.73 (dd,
J=8.4, 8.9 Hz, 1H), 3.74 (dd, J=9.1, 9.6 Hz, 1H), 3.80
(dd, J=2.7, 12.0 Hz, 1H), 3.83–3.88 (m, 5H), 3.97 (dt,
J=3.5, 6.4 Hz, 1H), 4.00 (dd, J=1.5, 6.6 Hz, 1H), 4.18
(dd, J=2.1, 11.4 Hz, 1H), 4.44 (d, J=7.9 Hz, 1H), 4.64
(d, J=8.0 Hz, 1H), 4.70 (d, 7.9 Hz, 1H), 4.75 (d, 8.0 Hz,
1H); 13C NMR (99.6 MHz, D2O): l=61.0, 61.1, 62.2,
62.9, 68.4×2, 68.6, 69.7, 69.9×2, 70.3, 71.1, 73.0, 73.4×2,
73.5, 73.8, 74.6, 75.9×2, 76.2×2, 76.3, 79.1, 84.2, 84.6,
102.7×2, 103.1, 103.2; IR (solid): w=3364.9, 1616.2,
11. The glycosyl acceptor 14 was prepared by the hydrolysis
(NaOMe, MeOH) of benzyl 2,4,6,2%,3%,4%,6%-hepta-O-ace-
tyl-b-D-laminaribioside followed by the 2,2%:4,6:4%,6%-tri-
O-benzylidenation (PhCH(OMe)2, CSA, DMF), see:
Wang, L.-X.; Sakairi, N.; Kuzuhara, H. J. Carbohydr.
Chem. 1991, 10, 349–361. For a related benzylidanation
procedure, see: Sakairi, N.; Okazaki, Y.; Furukawa, J.;
Kuzuhara, H.; Nishi, N.; Tokura, S. Bull. Chem. Soc.
Jpn. 1998, 71, 679–683.
12. The glycosyl acceptor 16a was prepared by the glycosyla-
,
tion of BnOH (NIS, TfOH, CH2Cl2, 4 A MS) with
phenylthio glucoside 10, followed by the removal of the
silyl ether (HF·Py, Py).
13. Selected physical data 4: 13C NMR (67.8 MHz, D2O):
l=94.7 (a anomer of the reduced end), 98.4 (b anomer of
the reduced end), 105.2–105.5 (other b anomer). Agrawal,
P. K. Phytochemistry 1992, 31, 3307–3330.
1456.2, 1080.9 cm−1
[C30H34O26+Na+] 853.2796, found 853.2796.
; HRMS (ESI-TOF): calcd for