Synthesis, (1→3)-β-d-glucanase-binding ability and phytoalexin-elicitor activity of (R)-2,3-epoxypropyl (1→3)-β-d- pentaglucoside

Article abstract of DOI:10.1016/j.bmcl.2004.09.076

The (1→3)-β-d-pentaglucoside was synthesized as its (R)-2,3-epoxypropyl glycoside via 2 + 3 strategy. The disaccharide donor 8 was obtained by 3-selective coupling of 2 with 4, followed by deallylation, and trichloroacetimidation. Meanwhile, the trisaccha

Full text of DOI:10.1016/j.bmcl.2004.09.076

Bioorganic & Medicinal Chemistry Letters 14 (2004) 6027–6029
Synthesis, (1!3)-b-D-glucanase-binding ability and
phytoalexin-elicitor activity of (R)-2,3-epoxypropyl
(1!3)-b-D-pentaglucoside
Gang-Liang Huang,a,* Xin-Ya Mei,b Man-Xi Liua and Tian-Cai Liua
aSchool of Life Science and Technology, Huazhong University of Science and Technology (East Campus),
1037 Luoyu Lu, Wuhan 430074, China
bSchool of Chemistry, Central China Normal University, 100 Luoyu Lu, Wuhan 430079, China
Received 6 August 2004; revised 11 September 2004; accepted 27 September 2004
Available online 18 October 2004
Abstract—The (1!3)-b-D-pentaglucoside was synthesized as its (R)-2,3-epoxypropyl glycoside via 2 + 3 strategy. The disaccharide
donor 8 was obtained by 3-selective coupling of 2 with 4, followed by deallylation, and trichloroacetimidation. Meanwhile, the tri-
saccharide acceptor 12 was prepared by coupling of 10 with 4, followed by deacetylation. Condensation of 8 with 12, followed by
epoxidation, and deprotection, gave the target pentaoside. The results of these bioassays demonstrated that the (1!3)-b-D-gluca-
nase was obviously inactivated by 15 with kapp = 3.79 · 10ꢀ4 minꢀ1. At the same time, we found that the 15 was more active as com-
pared to the laminaripentaose in eliciting phytoalexin accumulation in tobacco cotyledon tissue, and it could be kept longer time
than laminaripentaose, which indicated it is much more stable than laminaripentaose.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Higher plants have the ability to initiate various defence
reactions such as the production of phytoalexins, anti-
microbial proteins, reactive oxygen species and rein-
forcement of the cell wall when they are infected by
pathogens such as fungi, bacteria and viruses. If these
reactions occur in a timely manner, the infection will
not proceed further. However, if the defence reactions
occur too late or are suppressed, the infection process
will proceed successfully.1 Thus, it is critically important
for plant to detect infecting pathogens effectively and de-
liver such information intracellularly/intercellularly to
activate their defence machinery.
been discussed.2–4 Oligosaccharides derived from fungal
and plant cell wall polysaccharides are one class of well
characterized elicitors that, in some cases, can induce de-
fence responses at a very low concentration, for exam-
ple, nanomoles. At the same time, the elicitor activity
of oligosaccharides is dependent on the structural prop-
erties of oligosaccharide molecules, such as the number
of saccharide units, the length of the aglycone chain
and the molecule configuration.5
However, the elicitor-active oligosaccharides can be
hydrolyzed by endo- and exohydrolases from higher
plants, and give elicitor-inactive oligosaccharide frag-
ments.6 Therefore, improving the stability of the elici-
tor-active oligosaccharides is the key to developing the
biological pesticide of oligosaccharides.
It is believed that the detection of pathogens is mediated
by chemical substances secreted/generated by the patho-
gens. Various types of such compounds (elicitor mole-
cules) including oligosaccharides, (glyco)proteins,
(glyco)peptides and lipids, have been shown to induce
defence responses in plant cells and their involvement
in the detection of (potential) pathogens in plant has
The use of epoxyalkyl glycosides as active-site-directed
inhibitors has been invaluable in delineating the mecha-
nism of action for a variety of hydrolases, for example,
b-D-glucan endo- and exohydrolases.7,8 The epoxyalkyl
glycoside moiety targets the inhibitor to the substrate-
binding site and if the length of the alkyl chain is correct,
the epoxide group is brought into the vicinity of the cat-
alytic amino acids. Protonation of the epoxide oxygen
opens the epoxide ring and results in the formation of
Keywords: (R)-2,3-Epoxypropyl (1!3)-b-D-pentaglucoside; Synthesis;
(1!3)-b-D-Glucanase-binding ability; Phytoalexin-elicitor activity.
*
Corresponding author. Tel.: +86 2787554296; fax: +86
2787464570; e-mail: hgl226@126.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.076

6028
G.-L. Huang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 6027–6029
a stable ester linkage between the inhibitor and the cat-
alytic nucleophile. It has been well demonstrated9 that
the chain length of aglycone in the mechanism-based
epoxide-bearing inhibitors have a significant effect on
their activity.
ity to (1!3)-b-D-glucanase, as well as phytoalexin-elici-
tor activity in tobacco cotyledon tissue, herein we
present a very facile and convergent synthesis of (R)-
2,3-epoxypropyl (1!3)-b-D-pentaglucoside, with benzyl-
idenated glucose derivatives as the key intermediates. It
is an analogue of laminaripentaose, where the (R)-2,3-
epoxypropyl has been introduced at the reducing ends
of the aglycones. There are also some reports of
synthetic studies on phytoalexin-elicitor oligosac-
charides.11,12
Structure activity studies with laminarin, laminarin oligo-
mers, high molecular weight b-1,3–1,6 glucans from
fungal cell walls and the b-1,6–1,3 heptaglucan showed
that the elicitor effects observed in tobacco with b-glu-
cans are specific to linear b-1,3 linkages, with laminari-
pentaose being the smallest elicitor-active structure.10
With the aim of improving the stability of elicitor-active
laminaripentaose and studying the protein-binding abil-
Retrosynthetic analysis revealed that the best way to
synthesize the target 15 was to first prepare the b-
(1!3)-linked disaccharide and trisaccharide fragments,
Ph
O
Ph
O
Ph
Ph
O
O
O
O
O
O
O
c
O
O
a
b
O
AcO
AcO
HO
BzO
HO
BzO
BzO
HO
OC(NH)CCl3
OAll
OAll
OAll
1
2
3
4
Ph
Ph
O
O
a
O
O
O
OAll
O
OAll
HO
HO
BzO
HO
5
6
Ph
Ph
O
O
Ph
Ph
O
O
O
O
O
O
O
O
c
O
O
O
O
d
BzO
2+4
BzO
AcO
AcO
BzO
BzO
OC(NH)CCl3
OAll
7
8
Ph
Ph
O
O
Ph
Ph
O
O
O
O
O
O
OAll
O
O
OAll
e
d
O
O
O
O
6+4
BzO
BzO
HO
AcO
BzO
BzO
9
Ph
10
Ph
O
Ph
O
O
Ph
O
O
O
O
OAll
O
O
Ph
OAll
O
O
Ph
O
O
O
O
O
O
O
O
BzO
e
O
d
O
BzO
O
BzO
4+10
HO
BzO
AcO
BzO
BzO
11
12
Ph
O
Ph
O
O
O
O
O
OAll
Ph
Ph
O
O
O
O
O
Ph
O
O
O
BzO
O
O
O
O
O
BzO
d
O
AcO
O
BzO
8+12
BzO
BzO
O
Ph
13
H
H
O
Ph
O
O
O
O
O
Ph
Ph
O
O
O
H
Ph
O
O
O
BzO
O
O
O
O
BzO
f
O
AcO
O
BzO
O
BzO
H
H
HO
HO
BzO
14
HO
O
O
HO
HO
HO
O
H
HO
O
O
HO
HO
HO
O
O
HO
HO
O
g
O
O
HO
HO
HO
HO
15
Scheme 1. Conditions and reagents: (a) PhCOCl, CH2Cl2, 0ꢁC; (b) Ac2O, pyridine, rt; (c) PdCl2, CH3OH, 40ꢁC/CCl3CN, CH2Cl2, K2CO3, rt; (d)
TMSOTf, CH2Cl2, 0ꢁC; (e) HBF4, THF, rt, 4h; (f) m-CPBA, CH2Cl2, rt; (g) 90% HOAc–H2O, reflux, 3h; CH3ONa–CH3OH, rt.

G.-L. Huang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 6027–6029
6029
Table 1. Binding and phytoalexin-elicitor activity of (R)-2,3-epoxy-
propyl (1!3)-b-D-pentaglucoside (15)
then connect them at the C-3 of the glucose residue of
the trisaccharide backbone. The previous studies13 indi-
cated that in (1!3)-glucosylation the glycosyl bond
originally present in either donor or acceptor controlled
the stereoselectivity of the forthcoming bond, that is, the
newly formed glycosidic linkage had the opposite ano-
meric configuration of that of either the donor or accep-
tor. In addition, some reports14,15 revealed that with 4,
6-O-benzylidenated glucose derivatives as either donor or
acceptor, b-linked oligosaccharides are readily obtained.
Thus, in the present research, the benzylidenated glucose
derivatives were applied as the key intermediates. As
outlined in Scheme 1, 1 and 5, obtained from 4,6-O-benz-
ylidenation of allyl a,b-D-glucopyranoside, were mono-
benzoylated to afford 2 and 6 in high yield (75%).
Acetylation of 2 with acetic anhydride in pyridine fur-
nished 3 in high yield (95%). Deallylation of 3 with
PdCl2 in methanol,16 followed by trichloroacetimidation
with Cl3CCN, gave 4. The 4 was coupled with the accep-
tor 2 or 6 in the presence of TMSOTf to afford a unique
disaccharide 7 or 9 in 53% yield. Deallylation of 7, fol-
lowed by trichloroacetimidation, again gave 8. Deacetyl-
ation of 9 with HBF4 gave the disaccharide acceptor 10
in satisfactory yield (80%). The 4 was again coupled with
the acceptor 10 in the presence of TMSOTf to give 11.
Compound 11 was deacetylated with HBF4 to gave 12.
Then coupling of 8 with the trisaccharide acceptor 12
gave 13 as the sole product in 82% yield. The reaction
of 13 with m-chloroperoxybenzoic acid (m-CPBA) in
dichloromethane at room temperature gave the corre-
sponding 14. Finally, deprotection of 14 in turn in
90% HOAc–H2O and sodium methoxide–methanol
solution gave the target 15. The analytical data (1H
NMR, 13C NMR, ESI-MS, anal. found) of the synthetic
1517 was identical with those of the isolated material.
Epoxidation of 13 introduced new chiral centres at C-2
of the aglycone. In the case, the major isomer was iso-
lated and purified by column chromatography on silica
Substance
kapp (minꢀ1 a
)
Biological activity
(EC50, lmol/L)b
22h
44h
66h
Laminaripentaose
15
90
50
500
80
900
160
3.79 · 10ꢀ4
a Value was means of at least three independent determinations.
b Determined by measuring the absorbance (A) at 285nm.
Acknowledgements
The project was financially supported by ÔThe Scaling
the Height ProgramÕ of the State Science and Technol-
ogy Committee of China.
References and notes
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384.
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1
gel, and the H NMR spectrum indicated that C-2 of
˚
12. Fugedi, P.; Birberg, W.; Garegg, P. J.; Pilotti, A. Carbo-
the aglycone was R configuration (R:S = 5:1). It indi-
cated that the anomeric proton of this major compound
15 (C-2R) resonates at higher field (d = 3.09) than the
minor diastereoisomer (C-2S, d = 3.24).
¨
hydr. Res. 1987, 164, 297–312.
13. (a) Zeng, Y.; Ning, J.; Kong, F. Tetrahedron Lett. 2002,
43, 3729–3733; (b) Zeng, Y.; Ning, J.; Kong, F. Carbo-
hydr. Res. 2003, 338, 307–311.
14. Yang, G.; Kong, F. Synlett 2000, 1423–1426.
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88.
Seeing from the above-mentioned synthetic route, the
method was simple and practical, and it should be pos-
sible to apply the process to large-scale synthesis of 15.
The results of these bioassays, shown in Table 1, demon-
strated that the (1!3)-b-D-glucanase was obviously
inactivated by 15 with kapp = 3.79 · 10ꢀ4 minꢀ1. At the
same time, we found that the 15 was more active as com-
pared to the laminaripentaose (purchased from Fluka
Chemical Company) in eliciting phytoalexin accumula-
tion in tobacco cotyledon tissue, and it could be kept
longer time than laminaripentaose, which indicated it
is much more stable than laminaripentaose. The biolog-
ical activity of 15 fell at late time points, which was pos-
sible because the 15–hydrolase complex was unstable, so
the 15 would been hydrolyzed gradually by hydrolases.
17. Analytical data for 15: syrup; [a]D +86.3 (c 1.1, CHCl3);
1H NMR (D2O, 400MHz) d 5.24 (1H, d, J1,2 3.5Hz, a-H-
1I), 4.62–4.54 (5H, m, H-1V, IV, III, II), 3.92–3.85 (5H, m),
3.75–3.33 (19H, m), 3.25–3.19 (6H, m), 3.09 (1H, m,
–CH(O)CH2), 2.76–2.68 (2H, m, –CH(O)CH2); 13C NMR
(D2O, 75MHz) d 103.8, 103.65 (C-11, 15), 103.4 (3C) (C-
12, 13, 14), 85.4 (C-31), 85.2 (C-32), 85.0 (2C) (C-33, 34), 6.8
(C-55), 76.45, 76.4 (5C) (C-35, 51, 52, 53, 54), 74.3 (C-25),
74.1(3C) (C-2 2, 23, 24), 73.6 (C-21), 70.4 (C-45), 69.0, 68.9
(4C) (C-41, 42, 43, 44), 61.55 (5C) (C-61, 62, 63, 64, 65), 50.4
(–CH(O)CH2), 44.2, 44.1(–CH(O) CH2); ESI-MS m/z (%)
907 [M+Na]+; Anal. Calcd for C33H56O27: C, 44.80; H,
6.33. Found: C, 44.67; H, 6.40.