Large-scale preparation of the phytoalexin elicitor glucohexatose and its application as a green pesticide

Article abstract of DOI:10.1021/jf020683x

Large-scale preparation of the phytoalexin elicitor was achieved through a highly regio- and sterereoselective synthesis using 2,3,4,6-tetra-O-benzoyl-D-glucopyranosyl trichloroacetimidate (1), 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (2), and 6-O-ac

Full text of DOI:10.1021/jf020683x

J. Agric. Food Chem. 2003, 51, 987−991 987
Large-Scale Preparation of the Phytoalexin Elicitor
Glucohexatose and Its Application as a Green Pesticide
‡
JUN NING,† FANZUO KONG,*,† BANGMAO LIN,‡ AND HUIDE LEI
Research Center for Eco-Environmental Sciences, Academia Sinica, P.O. Box 281,
Beijing 100085, China, and Citrus Research Institute, Chinese Academy of Agricultural Sciences,
Chongqing 400712, China
Large-scale preparation of the phytoalexin elicitor was achieved through a highly regio- and
sterereoselective synthesis using 2,3,4,6-tetra-O-benzoyl-D-glucopyranosyl trichloroacetimidate (1),
1,2:5,6-di-O-isopropylidene-R-D-glucofuranose (2), and 6-O-acetyl-2,3,4-tri-O-benzoyl-R-D-glucopy-
ranosyl trichloroacetimidate (3) as the synthons. Coupling of 1 with 2 gave the 1f3-linked disaccharide;
subsequent selective removal of 5,6-O-isopropylidene to give 5 followed by selective 6-O-glycosylation
with 1 afforded the trisaccharide 6. Hydrolysis to remove the 1,2-O-isopropylidene was accompanied
by ring expansion, giving 3,6-branched pyranosyl trisaccharide. Acetylation, selective 1-O-deacet-
ylation, and activation with trichloroacetonitrile gave the trisaccharide donor 7. The trisaccharide
acceptor 9 was prepared from condensation of the disaccharide 5 with 3 and subsequent
6-O-deacetylation. Coupling of the trisaccharide donor 7 with the trisaccharide acceptor 9 and
subsequent deprotection afforded the glucohexatose elicitor. The cost of the produced glucohexatose
should be low enough to allow its applications in agriculture as a green pesticide. At a concentration
of 5-10 mg/L, the resultant elicitor was used to treat growing orange trees and harvested oranges,
giving very encouraging results, comparable with those obtained using commercial pesticides at a
concentration of 1400 mg/L (Topsin-M) for growing trees and 900 mg/L (Tecto) for harvested oranges,
respectively. Treatment of tomato leaves against Botrytis cinerea with the synthetic elicitor at a
concentration of 10 mg/L gave 82% inhibition, comparable with the inhibition of 84% by Wanmeiling
at a concentration of 1000 mg/L. Treatment of tea leaves also showed promising results.
KEYWORDS: Phytoalexin elicitor; glucohexatose; green pesticide
INTRODUCTION
minimum structural element required for high elicitor activity
(3). It is to be noted that, although much of this work was done
with soybean cotyledons, it was established that the glucan
elicitor also elicited the synthesis of different phytoalexins in a
wide range of other plant species (4).
Over the past decades, a variety of methods have been applied
to protect growing crops from attack by different pathogens.
Generally, various synthetic and naturally derived fungicides,
bactericides, and antiviral agents have been used to treat crops,
and these agents protect the crops from infection by pathogens
without deleteriously affecting the growth and ultimate harvest-
ing of the crops. While many such compositions are effective,
there has nonetheless been a growing concern from consumers
recently regarding the potential harmful side effects of chemical
antipathogenic agents. This, in turn, has led people to pay more
attention to products of natural origin, which are far less likely
to cause adverse side effects.
It is known that partial acid hydrolysis of mycelial walls of
the fungus Phytophthora megasperma f. sp. Glycinea gives a
mixture of oligosaccharides that are capable of stimulating the
formation of phytoalexins in soybeans (1). The most active
heptasaccharide (A) is effective in very low doses, approxi-
mately 0.1 pmol per cotyledon (2). Biological assays of several
oligosaccharides revealed that D-glucohexatose (B) is the
While the use of pathogenic agents on growing crops to elicit
natural defenses is theoretically interesting, it has nonetheless
† Academia Sinica.
‡ Chinese Academy of Agricultural Sciences.
10.1021/jf020683x CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/15/2003

988 J. Agric. Food Chem., Vol. 51, No. 4, 2003
Ning et al.
Scheme 1
isopropylidene group of 6 was accompanied by ring expansion.
Subesequent acetylation, selective 1-O-deacetylation, and trichloroace-
timidation gave the trisaccharide donor 7. The trisaccharide acceptor 9
was obtained by condensation of 5 with 6-O-acetyl-2,3,4-tri-O-benzoyl-
R-D-glucopyranosyl trichloroacetimidate (3) and then selective 6-O-
deacetylation. It is noted that compound 3 was prepared from
benzoylation of 1,6-anhydro-â-D-glucopyranose (levoglucosan), an
inexpensive material obtained from pyrolysis of cellulose (6), followed
by acetolysis, 1-O-deacetylation, and trichloroacetimidation. Condensa-
tion of the trisaccharide donor 7 with the trisaccharide acceptor 9
followed by deprotection gave the target glucohexatose.
not yet been a practical reality. There is concern that, by the
time the cell material of a growing crop produces the needed
antipathogenic agent, the growth of the pathogen has often
advanced to the point at which such antipathogenic agents are
of little effect. Another obstacle for the practical use of the
elicitor is that the successful use of elicitors on plants in an
attempt to “trick” a plant into producing antipathogenic agents
has been limited to wounded plants (5).
We have been engaged in research and development of new,
nontoxic, and nonpollutant pesticides for years. In view of its
high efficiency and nontoxicity, we selected glucohexatose as
a good preventive pesticide against harmful fungi. Provided that
regular treatments of “healthy plants” are performed, this should
allow the plants to produce enough phytoalexins to inhibit
growth and infection of the harmful pathogens. Its relatively
low molecular weight also suggested that glucohexatose should
have a good ability to penetrate into plants, and therefore
wounding of plants before treatment with the elicitor could be
unnecessary. Thus, the glucohexatose elicitor could be applied
directly to plants without the use of auxiliary agents such as
penetration agent, surfactant, and buffer. Simple spraying of a
water solution of glucohexatose elicitor onto plants would
represent the best approach. A major challenge for practical use
of the elicitor is to develop a simple, low-cost method for the
large-scale preparation of glucohexatose. This article describes
this preparation and the practical application of the elicitor in
orange growth and storage.
Preparation and Characterization of Important Intermediates.
Compound 6. To a stirred solution of 2,3,4,6-tetra-O-benzoyl-â-D-
glucopyranosyl-(1f3)-1,2-O-isopropylidene-R-D-glucofuranose (5, 80
g, 0.1 mol) and 2,3,4,6-tetra-O-benzoyl-R-D-glucopyranosyl trichloro-
acetimidate (1, 80 g, 0.108 mol) in dichloromethane (250 mL) was
added trimethylsilyl trifluoromethanesulfonate (TMSOTf, 200 µL) at
room temperature. After 3 h, triethylamine was added to the solution
to quench the reaction. The solution was concentrated, and the residue
was subjected to column chromatography with 2:1 petroleum ether-
ethyl acetate as the eluent to give the trisaccharide 6 (114.2 g, 83%):
1
[R]D + 15.3° (c 1.0, CHCl3); H NMR (400 MHz, CDCl3) δ 8.06-
7.28 (m, 40 H), 5.88 (t, 1 H), 5.87 (t, 1 H), 5.69 (t, 1 H), 5.64 (t, 1 H),
5.53 (dd, 1 H), 5.43 (dd, 1 H), 5.41 (d, 1 H), 4.96 (d, 1 H), 4.93 (d, 1
H), 4.68 (dd, 1 H), 4.48 (dd, 1 H), 4.67 (dd, 1 H), 4.35 (dd, 1 H),
4.34-3.65 (m, 8 H), 1.26, 1.03 (2 s, 6 H).
Compound 7. Compound 6 (50 g, 0.036 mol) was added to 80%
aqueous acetic acid solution (500 mL), and the mixture was heated
under reflux for 5 h. The mixture was concentrated, and the residue
was acetylated with acetic anhydride (250 mL) in pyridine (280 mL)
overnight. The resultant trisaccharide was dissolved in 3:1 THF-CH3-
OH (500 mL) containing ammonia (1.2 mol), and the solution was
stirred at room temperature for 2 h. The solution was concentrated,
and the residue was dissolved in dichloromethane (200 mL). To the
solution were added K2CO3 (20 g) and CCl3CN (10 mL), and the
mixture was stirred at room temperature overnight. The mixture was
filtered, the filtrate and washings were concentrated, and the residue
MATERIALS AND METHODS
Glucohexatose Preparation. Condensation of benzoylated glucosyl
trichloroacetimidate 1 with 1,2:5,6-di-O-isopropylidene-R-D-glucofura-
nose 2 afforded the disaccharide 4; selective removal of the 5,6-O-
isopropylidene group to give 5 and selective 6-O-glycosylation of 5
with 1 gave the trisaccharide 6 (Scheme 1). Removal of the 1,2-O-

Phytoalexin Elicitor Glucohexatose as a Green Pesticide
J. Agric. Food Chem., Vol. 51, No. 4, 2003 989
was subjected to column chromatography, giving the trisaccharide
donor 7 as a solid (40.0 g, 71%): [R]D + 23.3° (c 1.0, CHCl3); H
Table 1. Prevention of Colletotrichum glocosporioides Penz. in
1
Growing Treesa
NMR (400 MHz, CDCl3) δ 8.33 (s, 1 H, CNHCCl3), 8.07-7.19 (m,
40 H, 8 PhH), 6.19 (d, 1 H,), 5.91 (t, 1 H,), 5.85 (t, 1 H), 5.62 (t, 1 H),
5.61 (t, 1 H), 5.46 (dd, 1 H), 5.42 (dd, 1 H), 4.97 (d, 1 H), 4.96 (d, 1
H), 4.85 (t, 1 H), 4.67-4.59 (m, 3 H), 4.50-4.37 (m, 2 H), 4.19-4.02
(m, 4 H), 3.91 (dd, 1 H), 3.69 (dd, 1 H), 1.94, 1.78 (2 s, 6 H).
disease index after treatment
small district
concn
(mg/L)
PE
(%)
treatment
1
2
3
4
av
SE
SE
SE
MET
control
5
3
1
3.06
3.61
4.72
3.06
2.78
3.33
4.17
2.50
3.33
3.06
4.17
2.22
2.78
3.33
3.89
2.50
2.99 75.53
3.33 72.75
4.24 65.30
2.57 78.97
Compound 9. Using the same procedure as described for the
preparation of 6 from 1 and 5, the trisaccharide 8 (105.1 g, 80%) was
prepared from 3 (73.2 g, 0.108 mol) and 5 (80 g, 0.1 mol). Compound
8 (100 g, 0.076 mol) was dissolved in 250 mL of CH3OH, 8 mL of
acetyl chloride was added, and the reaction was carried out at room
temperature for 8 h. After neutralization and concentration, the residue
was subjected to column chromatography, giving compound 9 as a solid
1400
13.06 11.67 12.22 11.94 12.22
a SE, synthetic elicitor; MET, Topsin-M (obtained from 70% Topsin by 500 times
dilution with water); control, blank water; PE, prevention efficiency.
1
(87.1 g, 90%): [R]D + 12.6° (c 1.0, CHCl3); H NMR (400 MHz,
CDCl3) δ 8.05-7.26 (m, 35 H), 5.91 (t, 1 H), 5.90 (t, 1 H), 5.73 (t, 1
H), 5.56 (t, 1 H), 5.54-5.42 (m, 3 H), 4.99 (d, 1 H), 4.95 (d, 1 H),
4.75-3.77 (m, 12 H), 1.33, 1.05 (2 s, 6 H).
Table 2. Prevention of Diseases in Oranges during Storagea
treatment
Compound 10. A solid hexasaccharide 10 (77.0 g, 90%) was prepared
from 7 (50 g, 0.032 mol) and 9 (40.7 g, 0.032 mol) under the same
conditions as described for the preparation of 6 from 1 and 5. For 10:
[R]D + 6.6° (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.04-7.18
(m, 75 H), 6.13, 5.88, 5.83, 5.74 (4 t, 4 H), 5.69, 5.65, 5.62, 5.57 (4 t,
4 H), 5.50, 5.48, 5.44, 5.34 (4 dd, 4 H), 5.45 (d, 1 H), 5.07, 4.94, 4.83,
4.80 (4 d, 4 H), 1.95, 1.87 (2 s, 6 H), 1.33, 1.08 (2 s, 6 H); 13C NMR
(100 MHz, CDCl3) δ 169.4, 168.2 (2 CH3CO), 166.1-164.7 (15 PhCO),
105.0, 101.5, 101.1, 101.0, 100.9, 100.2 (C-1), 82.9, 82.5 (C-3), 20.85,
20.51 (2 CH3CO), 14.2 (C(CH3)2).
SE
SE
SE
Tecto
(1 mg/L) (5 mg/L) (10 mg/L) (900 mg/L) control
applied amount of
effective composition
(g/ton orange)
0.19
0.96
1.92
173.1
after 30 days decay (%) 1.75
PE (%) 46.15
after 60 days decay (%) 5.75
PE (%) 41.03
after 90 days decay (%) 13.5
PE (%) 46.02
1.5
53.85
4.5
53.85
11.75
53.08
1.25
61.53
2.25
76.92
6.5
0.75
76.92
1.75
82.05
3.75
85.03
3.23
9.75
25
74.07
The Glucohexatose Elicitor (B). Compound 10 (50 g, 0.0187 mol)
was dissolved in 80% acetic acid solution (250 mL), and the mixture
was heated under reflux for 6 h. Concentration of the mixture followed
by deacylation in ammonia-saturated methanol at room temperature
for 24 h gave the target glucohexatose elicitor (B) as a powder (17.6
g, 95%): [R]D -15.1° (c 0.2, MeOH); 13C NMR (200 MHz, D2O) δ
102.6-102.3 (6 C-1), 84.0 (C-3 ), 69.2 (C-6 ), 60.4 (C-6); ESMS m/z
989.5 [M - 1]+; FW ) 990.86.
a SE, synthetic elicitor; Tecto, obtained from 45% Tecto by 500 times dilution
with water; control, blank water; PE, prevention efficiency.
Table 3. Prevention Efficiency of Synthetic Elicitor for Botrytis cinrea
on Tomato Leavesa
treatment
Antifungi Test for Orange Growth and Storage. The test was
conducted in an orange garden located in the suburb of Chongqing
City of southwest China, where the terrain was smooth, soil was lightly
sandy, and there was good sunshine. The tested trees were 16 years
old, and there were 66 trees in a 660 m2 area, with a distance of 4 m
between two trees and 2.5 m between two tree lines. The growing
posture of the trees was typical. Before the test was started, orange
trees were treated once only with mitecide in May. At the time the test
began, there was no disease on any of the fruit. Spraying was done
using a back-carried MATABI-16 sprayer. The working pressure of
the sprayer was 3 Pa, the diameter of the spray nozzle was 1 mm, the
rate of solution spraying was 0.5 L/min, and the sprayed fog drops
had a diameter of 80-100 µm. The spraying amount was 2 kg of
solution per tree, and 1980 kg per hectare. There were four sprayings:
on Oct 17, Oct 22, Oct 27, and Nov 2. There were five treatment
groups: 5, 3, and 1 mg/L of the glucohexatose, 1400 mg/L of Topsin-M
(thiophanate-methyl), and blank water. There were four small districts
per treatment, and each district had two trees randomly arranged and
isolated by a protection tree. Since no infection had occurred at the
time when the test began, the study on infection base was not needed.
On Nov 30, 2000, just before the harvesting, a classified investigation
of the fruits was carried out. Sampling was conducted on the east, west,
north, south, and center portion of each tree, respectively. Four oranges
were investigated in each portion, and there were 40 oranges for
investigation in each small district. The disease index and prevention
effect were calculated on the basis of the investigation using the DMRT
method for determination of markedness. There were nine classes: class
0, no disease spot; class 1, one or two disease spots per orange; class
3, three or four disease spots per orange; class 5, five or six disease
spots per orange; class 7, seven or eight disease spots per orange, and
the spots were partially coupled, occupying one-fifth of the area of the
orange surface; class 9, more than nine disease spots per orange, and
SE
SE
SE
SE
(0.5 mg/L) (1 mg/L) (5 mg/L) (10 mg/L) Wanm. control
concn of effective
composition (mg/L)
disease index
PE (%)
0.5
1
5
10
500
0
30.1
69.38
26.6
72.94 78.03
21.6
15.0
84.74 83.01
16.7
98.3
a SE, synthetic elicitor; Wanm., Wanmeiling (obtained from 50% Wanmeiling
by 1000 times dilution with water); control, blank water; PE, prevention efficiency.
the spots were coupled, occupying more than one-fourth of the area of
the orange surface. The pesticide efficiency was calculated as follows:
disease index )
Σ(no. of infected oranges × corresponding class order)
× 100
total no. of investigated oranges × 9
prevention efficiency )
disease index in control area - disease index in testing area
× 100
disease index in control area
A test of the use of the synthetic elicitor as an orange (Glorioius
orange) antidecay agent was carried out in a storehouse with natural
ventilation. In the storehouse there were four layers of iron racks with
wooden fruit boxes on each rack. Fifteen days before the test, the
storehouse and the boxes were sterlized. The oranges, of uniform size
and without mechanical damage, had not been treated with any
bactericides before the testing. On Dec 1, the freshly harvested oranges
were treated with the synthetic elicitor at concentrations of 1, 5, and
10 mg/L, with Tecto suspension (Monsanto) at a concentration of 173
mg/L, and with blank water. There were four runs for each treatment,
and 100 oranges were used for each run. The tested oranges in each

990 J. Agric. Food Chem., Vol. 51, No. 4, 2003
Ning et al.
Table 4. Effect of Synthetic Elicitor on Feeding Amount and Weight of Ectropic obliqua Prout
feed
decrement
(%)
weightb of
the larvae
(mg/single)
weight
change
(±%)
weightb of
the pupae
(mg/single)
weight
change
(±%)
feed amountb
(mg/single)
property of
difference
property of
difference
property of
difference
treatmenta
SE, 0.1 mg/kg
SE, 1 mg/kg
SE, 10 mg/kg
Bt, 500 times dilution
blank
76.79
65.26
71.85
75.09
77.78
1.27
16.10
7.60
Aa
Ab
Aab
Aa
Aa
96.37
81.33
83.98
56.21
89.17
8.07
−8.79
−5.82
Aa
Ab
Aab
Bc
Aab
86.52
73.72
74.17
70.44
80.85
7.01
−8.82
−8.26
−12.9
Aa
BCc
BCc
Cc
3.46
−36.92
Abb
2
a Test area for each treatment was 32.3 m . Spraying used back-carried sprayer at an amount of 750 kg/hectare. Starting from Oct 16, fresh tea leaves were taken for
feeding Ectropic obliqua Prout. The leaves were replaced every other day with newly collected ones. Each treatment is divided into five groups, and 15 larvae of two instar
for each group. b Average value for five groups.
run were dipped in a specified solution for 1 min and then taken out to
dry under natural air flow and put into a wooden fruit box. The boxes
were randomly arranged for different treatments when put onto the iron
rack in the storehouse at room temperature. One week later, the oranges
were packed in small plastic bags. During the test, the average
temperature was 9.8 °C; the highest temperature was 16.2 °C, and the
lowest temperature was 5.6 °C. The average relative humidity was
83.8%. Infections by Pencillium italicum Wehmer, Pencillium digitatum
Sacc., Colletotrichum glocosporioides Penz., Phoopsis citri Fawc.,
Phytophthora parasitica Dastur., and Oospora citri aurautii Sacc. were
detected. The first detection was conducted 30 days after treatment,
and the second and the third, 60 days and 90 days after treatment,
respectively. No changes in the appearance and taste of oranges were
observed after testing. Calculations were done according to the
following equations:
hexatose have be prepared within a 2-month period in our
laboratory, and the construction of a model factory to produce
200 kg/year is under consideration.
The Synthetic Elicitor Has a High Efficiency of Prevention
of Fungi Invention. Colletotrichum glocosporioides Penz. is
one of the major diseases in oranges, occurring usually near
harvest time. Table 1 shows that the synthetic glucohexatose
at a concentration of 5 mg/L gave inhibition of Colletotrichum
glocosporioides Penz. similar to that of Topsin-M at a concen-
tration of 1400 mg/L. Even at concentrations as low as 1 mg/
L, the synthetic elicitor was still effective for inhibition of
Colletotrichum glocosporioides Penz., indicating the high
potency of the synthetic elicitor as a pesticide.
Table 2 shows that at a concentration of 10 mg/L (corre-
sponding to 1.92 g/ton of oranges), the synthetic elicitor was
an effective antidecay agent. Better results may be possible at
higher concentrations.
no. of decayed oranges
no. of totally treated oranges
decay (%) )
× 10
decay in control - decay in treated
prevention efficiency )
× 10
decay in control
Table 3 shows that the synthetic elicitor was very effective
for prevention of B. cinerea on tomato. At a concentration of
10 mg/L it gave 82% inhibition, comparable with the inhibition
of 84% by Wanmeiling at a concentration of 1000 mg/L. Large
area tests for the prevention of disease in cucumber, eggplant,
and tomato are in progress.
The synthetic elicitor was also tested for prevention of Botrytis
cinerea on tomato leaves. The tested aqueous solutions were 0.5, 1, 5,
and 10 mg/L solutions of the synthetic elicitor, 500 mg/L Wanmeiling,
and blank water. Tomato leaves of similar size were taken from the
plant body, sprayed with the test solution, and then dried under natural
air flow (for about 1 day), inoculated with B. cinerea, and incubated
at room temperature with a certain moisture and alternative light and
dark. Three days after the inoculation, detection was conducted by
determination of disease spot diameter and number of infected leaves,
and calculations were done according to the following equations:
Table 4 shows the test of the synthetic glucohexatose against
Ectropis obliqua Prout in tea leaves using Bacillus thuringiensis
(Bt) and blank water as comparisons. Before feeding of E.
obliqua Prout, the tea trees were sprayed on Oct 9 and Oct 16
with 0.1, 1, and 10 mg/L solutions of the synthetic elicitor, 500
times diluted Bt, and blank water, respectively. It was found
that at a concentration of 1 mg/L, the synthetic elicitor showed
some feed-antagonizing effect for E. obliqua Prout (feed
decrement 16.1%). However, its influence on body weight of
E. obliqua Prout was worse than Bt’s effect (-8.8 vs -36.9),
indicating that the synthetic elicitor is not a good prevention
pesticide for E. obliqua Prout.
disease index )
Σ(no. of infected leaves × corresponding class order)
× 100
total no. of tested leaves × the highest class number
prevention efficiency )
disease index in control - disease index in treated
× 100
disease index in control
RESULTS AND DISCUSSION
Conclusion. Large-scale production of the glucohexatose
elicitor at low cost is possible. The synthetic elicitor can be
applied as a green pesticide to protect growing oranges and
tomatoes from attack by harmful fungi and can be used as an
antidecay agent to keep oranges fresh.
Large-Scale Production of the Glucohexatose Elicitor Is
Possible. The de novo chemical synthesis of the oligosaccharide
elicitor (7-9) is a rapidly developing field; however, this mainly
serves the investigation of structure-activity relationships and
has not been of practical use. We have reported a highly regio-
and stereoselective method to prepare the glucohexatose elicitor
through ortho ester formation-rearrangement (10, 11). Sub-
stantial improvements have been made to this method to develop
a new and efficient way to prepare the elicitor on a large scale
(see Scheme 1). This new route contains several multistep one-
pot reactions. In terms of simplicity and efficiency, the above-
described synthesis can be applied as a practical production
method (12) at very low cost. Hundreds of grams of gluco-
ACKNOWLEDGMENT
This work was supported by the Beijing Natural Science
Foundation (6021004), the Natural Science Foundation of China
(59973026 and 29905004), the Ministry of Science and Tech-
nology (2001AA246014), and the Chinese Academy of Sciences
(KZCX3-J-08).

Phytoalexin Elicitor Glucohexatose as a Green Pesticide
J. Agric. Food Chem., Vol. 51, No. 4, 2003 991
LITERATURE CITED
(7) Plante, O. J.; Palmacci, E. R.; Seeberger, P. H. Automated solid-
phase synthesis of oligosaccarides. Science 2001, 291, 1523-
1527.
(8) Sears, P.; Wong, C.-H. Toward automated synthesis of oligosac-
charides and glycoproteins. Science 2001, 291, 2344-2350.
(9) Cheong, J.-J.; Birberg, W.; Fugedi, P.; Pilloti, A.; Garegg, P. J.;
Hong, N.; Ogawa, T.; Hahn, M. G. Structure-activity relation-
ships of oligo-â-glucoside elicitors of phytoalexin accumulation
in soybean. Plant Cell 1991, 3, 127-136.
(10) Wang, W.; Kong, F. Regio- and stereoselective of biologically
important oligosaccharides. J. Org. Chem. 1999, 64, 5091-5095.
(11) Wang, W.; Kong, F. A highly convergent synthesis of the elicitor
hexasaccharide. Tetrahedron Lett. 1998, 39, 1937-1940.
(12) Ning, J.; Kong, F. Preparation of the glucohexatose elicitor. WO
00254.
(1) Sharp, J. K.; McNeil, M.; Albersheim, P. The primary structure
of one elicitor-active and seven elicitor-inactive hexa(â-D-
glucopyranosyl)-D-glucitols isolated from the mycelial walls of
Phytophthora megasperma f. sp. glycinea. J. Biol. Chem. 1984,
259, 11321-11326.
(2) Sharp, J. K.; Valent, B.; Albersheim, P. Purification and partial
characterization of a â-glucan fragment that elicits phytoalexin
accumulation in soybean. J. Biol. Chem. 1984, 259, 11312-
11320.
(3) Cheong, J.-J.; Hahn, M. G. A specific, high-affinity binding site
for the hepta-â-glucoside elicitor exists in soybean membranes.
Plant Cell 1991, 3, 137-147.
(4) Darvill, A. G.; Alberseim, P. Phytoalexins and their elicitors. A
defense against microbial infection in plants. Annu. ReV. Plant
Physiol. 1984, 35, 243-275.
(5) Kolattukudy, P. E.; O’Neill, R. A.; Poulose, A. J. Protection of
growing crops from pathogens by treatment with enzymes. WO
91/06313.
(6) Scott, D. S.; Piskorz, J.; Radlein, D. Yields of chemicals from
biomass-based fast pyrolysis oils. Energy, Biomass, Waters 1993,
16, 797-809.
Received for review June 19, 2002. Revised manuscript received
November 18, 2002. Accepted November 24, 2002.
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