Chemoselective Glycosylation Strategy for the Convergent Assembly of Phytoalexin-Elicitor Active Oligosaccharides and Their Photoreactive Derivatives

Article abstract of DOI:10.1021/jo990836o

A highly convergent route for the synthesis of branched glucosides having phytoalexin-elicitor activity has been developed. The readily available building blocks 2, 3, and 6 were used for the assembly of a core trisaccharide in two chemo- and regioselective glycosylations. Compound 7 was converted into the artificial spacer-containing glycosyl acceptor 9. Regioselective condensation of fragment 7 with 9 led to the formation of fully protected hexasaccharide 14. Deprotection of hexasaccharide 14 followed by the reaction with 4-azidosalicylate gave compound 1b. It was established that compound 1b is a photoreactive compound. Reaction time and conditions were established for photolysis of compound 1b and labeling with radioactive iodine. A soybean root binding site for an elicitor-active hepta-β-glucoside bound compound 1b with the same affinity as the underivatized hepta-β-glucoside.

Full text of DOI:10.1021/jo990836o

7828
J . Org. Chem. 1999, 64, 7828-7835
Ch em oselective Glycosyla tion Str a tegy for th e Con ver gen t
Assem bly of P h ytoa lexin -Elicitor Active Oligosa cch a r id es a n d
Th eir P h otor ea ctive Der iva tives
Richard Geurtsen, Franc¸ois Coˆte´, Michael G. Hahn, and Geert-J an Boons*
The University of Georgia, Complex Carbohydrate Research Center, 220 Riverbend Road,
Athens, Georgia 30602-4712
Received May 21, 1999
A highly convergent route for the synthesis of branched glucosides having phytoalexin-elicitor
activity has been developed. The readily available building blocks 2, 3, and 6 were used for the
assembly of a core trisaccharide in two chemo- and regioselective glycosylations. Compound 7 was
converted into the artificial spacer-containing glycosyl acceptor 9. Regioselective condensation of
fragment 7 with 9 led to the formation of fully protected hexasaccharide 14. Deprotection of
hexasaccharide 14 followed by the reaction with 4-azidosalicylate gave compound 1b. It was
established that compound 1b is a photoreactive compound. Reaction time and conditions were
established for photolysis of compound 1b and labeling with radioactive iodine. A soybean root
binding site for an elicitor-active hepta-â-glucoside bound compound 1b with the same affinity as
the underivatized hepta-â-glucoside.
In tr od u ction
is not yet clear which of the proteins present in the
affinity-purified fractions are responsible for elicitor-
binding activity. Photoaffinity labeling is one method that
has often been used to identify polypeptides carrying a
specific binding site for a ligand of interest. Previously
published photoaffinity labeling experiments carried out
on soybean membrane extracts were performed with a
photoreactive derivative prepared by a series of chemical
modifications of the hepta-â-glucoside.3a,b,4 The conversion
of the arylamine to the azido derivative carries a great
risk of affecting the integrity of the hepta-â-glucoside
because of the exposure of the oligosaccharide to sulfuric
acid. Therefore, we decided to synthesize a de novo
photosensitive oligoglucoside ligand where the synthetic
steps for introduction of the photosensitive moiety can
be performed under mild and controlled reaction condi-
tions.
We report here the successful synthesis of a hexa-â-
glucoside (1a ) mimicking the effect of the original hepta-
â-glucoside and its conversion into a photoreactive 4-azi-
dosalicylate derivative (1b). Additionally, we confirmed
that the time and conditions for the photolysis of this aryl
azido derivative were the same as for similar azido
compounds. Moreover, we established the conditions for
labeling of the 4-azidosalicylate moiety with radioactive
iodine. Finally, we determined that these de novo oligo-
glucosides have the ability to competitively inhibit bind-
ing of radiolabeled hepta-â-glucoside to soybean root
membrane proteins with a similar efficiency as the
unlabeled hepta-â-glucoside. These controls are essential
for the validation of the synthesis of these analogues and
establish the conditions for performing the photoaffinity
experiments that will identify the proteins carrying a
specific binding site for the hepta-â-glucoside ligand.
Oligosaccharides such as fungal â-glucans are regarded
as essential players in the cellular communication be-
tween host plants and fungal pathogens. This com-
munication is based on specific recognition of signaling
or eliciting molecules at the plant cell surface, which
requires the functional expression of a receptor capable
of transmitting the signal to the inside of the cell.
Subsequent transmission of the signal within the cell
leads to the induced biosynthesis of antimicrobial phyto-
alexins and possibly activation of other plant defense
responses. Specific binding proteins for a branched hepta-
â-glucoside elicitor have been the subject of extensive
studies in several laboratories, because this molecule was
one of the first oligosaccharide signals to be purified to
homogeneity and structurally characterized.1 The exist-
ence of a specific plasma membrane binding site for this
molecule has been established by binding studies that
utilized, as the labeled ligand, the hepta-â-glucoside
covalently coupled to iodinatable functional groups.2
Binding of the radiolabeled hepta-â-glucoside to the root
membranes from the soybean displays characteristics
typical of physiological receptors: saturability, high
affinity [apparent dissociation constant (Kd ≈ 1 nm)], low
abundance [binding site concentration (Bmax ≈ 1 pmol/
mg protein)], and high degree of specificity.2
Previous efforts to purify the hepta-â-glucoside binding
proteins from the mixture of proteins solubilized from
soybean root membranes have primarily relied on affinity
chromatography using a matrix carrying an immobilized
mixture of elicitor-active glucan fragments.3 However, it
(1) Sharp, J . K.; Valent, B.; Albersheim, P. J . Biol. Chem. 1984, 259,
11312-11320.
(2) (a) Cosio, E. G.; Frey, T.; Verduyn, R.; Van Boom, J .; Ebel, J .
FEBS Lett. 1990, 271, 223-226. (b) Cheong, J .-J ., Hahn, M. G. Plant
Cell 1991, 3, 137-147. (c) Cheong, J .-J .; Alba, R.; Coˆte´, F.; Enkerli, J .;
Hahn, M. G. Plant Physiol. 1993, 103, 1173-1182.
(3) (a) Cosio, E. G.; Frey, T.; Ebel, J . Eur. J . Biochem. 1992, 204,
1115-1123. (b) Mitho¨fer, A.; Lottspeich, F.; Ebel, J . FEBS Lett. 1996,
381, 203-207. (c) Umemoto, N.; Kakitani, M.; Iwamatsu, A.; Yamaoka,
N.; Ishida, I. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 1029-1034.
Resu lts a n d Discu ssion
The artificial spacer-containing derivative 1a is a
suitable derivative for the selective and controlled incor-
(4) Frey, T.; Cosio, E. G.; Ebel, J . Phytochemistry 1993, 32, 543-
550.
10.1021/jo990836o CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/25/1999

Phytoalexin-Elicitor Active Oligosaccharides
J . Org. Chem., Vol. 64, No. 21, 1999 7829
Sch em e 1
poration of the photoreactive 4-azidosalicylate moiety to
give the target compound 1b (Figure 1).5 We have
designed a synthetic plan in which all acceptors and
donors that are required for the preparation of target
compound 1a are obtained from key building blocks 2 and
3 (Scheme 1). Furthermore, the core trisaccharide 7 was
used as the starting material for the preparation of the
spacer-containing trisaccharide 9 but was also used for
the extension of the latter derivative to give a hexasac-
charide.
Thioglycosides were selected as glycosyl donors and
acceptors. These sugar derivatives are stable to a wide
(5) For other synthetic approaches leading to oligomeric â-glucosides
see: (a) Ossowski, B. P.; Pilotti, Å; Garegg, P. J .; Lindberg, B. Angew.
Chem., Int. Ed. Engl. 1983, 10, 793. (b) Ossowski, B. P.; Pilotti, Å;
Garegg, P. J .; Lindberg, B. J . Biol. Chem. 1984, 259, 11337. (c) Fu¨gedi,
P.; Birberg, W.; Garegg, P. J .; Pilotti, Å J . Carbohydr. Res. 1987, 164,
297. (d) Fu¨gedi, P.; Garegg, P. J .; Kvarnstro¨m, I.; Svansson, L.
Carbohydr. Chem. 1988, 7, 389. (e) Birberg, W.; Fu¨gedi, P.; Garegg,
P. J .; Pilotti, Å J . Carbohydr. Res. 1989, 8, 47. (f) Hong, N.; Ogawa, T.
Tetrahedron Lett. 1990, 31, 3179. (g) Lorentzen, J . P.; Helpap, B.;
Oswald, L. Angew. Chem., Int. Ed. Engl. 1991, 12, 1681. (h) Verduyn,
M.; Douwes, P. A. M.; van der Marel, G. A.; van Boom, J . H.
Tetrahedron 1993, 49, 7301. (i) Hong, N.; Nakahara, Y.; Ogawa, T.
Proc. J pn. Acad. 1993, 69B, 55. (j) Yamada, H.; Harada, T.; Takahashi,
T. J . Am. Chem. Soc. 1994, 116, 7919-7920. (k) Timmers, C. M.;
Turner, J . J .; Ward, C. M.; van der Marel, G. A.; Kouwijzer, M. L. C.
E.; Grootenhuis, P. D. J .; van Boom, J . H. Chem. Eur. J . 1997, 3, 920.
(l) Nicolaou, K. C.; Winssinger, N.; Pastor, J .; Deroose, F. J . Am. Chem.
Soc. 1997, 119, 449-550. (m) Nicolaou, K. C.; Watanabe, N.; Li, J .;
Pastor, J .; Winssinger, N. Angew. Chem., Int. Ed. 1998, 37, 1559-
1561.
F igu r e 1.
range of reaction conditions commonly used in carbohy-
drate chemistry. Therefore, the anomeric thio alkyl group
first can act as effective protecting group; however, at
an appropriate stage they can readily be activated with
a wide range of thiophilic reagents to give O-glycosides
in high yields.
Another attractive feature of thioglycosyl donors and
acceptors is that they can be assembled to oligosaccha-
rides by chemoselective glycosylation strategies. This
approach is based on the observation that protecting-

7830 J . Org. Chem., Vol. 64, No. 21, 1999
Geurtsen et al.
Sch em e 2a
group patterns can control anomeric reactivities of thiogly-
cosides. Thus, it is possible that a thioglycosyl donor can
be coupled with a thioglycosyl acceptor of lower anomeric
reactivity to give a well-defined product. In a subsequent
glycosylation, the resulting product can act as a glycosyl
donor and react with a thioglycosyl acceptor that has
even a lower reactivity. This process can be repeated
several times to give rather complex structures without
the need for intermediate protecting-group chemistry.
The attractiveness of this approach has been further
enhanced by the creation of a database that makes it
possible to predict the relative reactivities of a large
number of p-methylphenyl thioglycosides.6 In general, an
electron-donating ether substituent at C-2 activates and
an electron-withdrawing ester functionality deactivates
the reactivity of an anomeric leaving group. Furthermore,
cyclic acetals and ketals reduce anomeric reactivities, and
thioglycosides protected with this type of protecting group
have reactivities between that of acylated- and ben-
zylated derivatives.
a
(i) Benzaldehyde dimethylacetal, CSA, DMF; (ii) H2SO4 (cat.),
HOAc/Ac2O (1/1); (iii) EtSH, TMSOTf, CH2Cl2.
Sch em e 3a
Control of the reactivity of thioglycosides by a C-2
protecting group imposes limitations because this func-
tionality is a critical determinant of the anomeric selec-
tivity of a glycosylation. To address this problem, we have
introduced an approach whereby anomeric reactivities
are controlled by the size of an alkyl thio leaving group.7
Target compound 1a was assembled by a novel chemo-
selective glycosylation strategy whereby the reactivities
of thioglycosyl donors and acceptors are controlled by a
new combination of properly selected protecting groups
and use of either an anomeric thioethyl or bulky dicy-
clohexylmethanethio group. We envisaged that the elec-
tronically deactivated (C-2 benzoyl) thioglycoside 3 is of
higher reactivity than the sterically (bulky anomeric thio
group) and torsionally (benzylidene group) deactivated
thioglycoside 2. Furthermore, it was expected that the
bulky dicyclohexylmethane thio group would sterically
block the C-2 hydroxyl. Thus, a chemo- and regioselective
glycosylation of 2 with 3 should yield 4. Disaccharide 4
can easily be transformed into glycosyl acceptor 5, which
will be glycosylated with donor 6 to give core trisaccha-
ride 7. In the latter reaction, we will take advantage of
the lower reactivity of the sterically and electronically
deactivated thioglycoside 5 compared to that of the
electronically deactivated compound 6. The well-known
higher reactivity of a primary sugar hydroxyl over a
secondary one is also exploited in this reaction.
The synthesis of monosaccharide building blocks 2, 3,
and 6 was undertaken as detailed in Scheme 2. Thiogly-
cosyl acceptor 2 was obtained in 82% yield by the
treatment of dicyclohexylmethyl 1-thio-â-D-glucopyrano-
side (10)7 with benzaldehyde dimethyl acetal and a
catalytic amount of camphorsulfonic acid (CSA) in DMF.
Ethyl 2-O-benzoyl-3,4,6-tri-O-benzyl-1-thio-â-D-glucopy-
ranoside (3) was easily prepared by a known procedure.8
Compound 3 was also the precursor for the synthesis of
6. Thus, treatment of 3 with a catalytic amount of H2-
SO4 in HOAc/Ac2O (1/1, v/v)9 resulted in acetolysis of the
anomeric ethyl thio moiety and 6-O-benzyl group to give
a
(i) NIS, TMSOTf, CH2Cl2/Et2O (1/1), -70 °C; (ii) BzCl, DMAP,
py; (iii) TFA, H2O, CH3CN/CH2Cl2 (2/1).
12 in a yield of 54%. Compound 11 was isolated in high
yield (80%) when the reaction time was shortened. This
observation shows that the anomeric ethyl thio group is
more acid labile than the benzyl ether at C-6. Reaction
of 12 with ethanethiol in the presence of trimethylsilyl
triflate gave the required glycosyl donor 6 (61%).
With significant quantities of building blocks in hand,
the hexasaccharide was assembled. NIS/TMSOTf-medi-
ated coupling of 2 with 3 in dichloromethane (DCM)/
diethyl ether at -70 °C furnished disaccharide 4 in an
acceptable 40% yield along with a regioisomer in which
glycosylation had taken place at C-2 (15%) (Scheme 3).
The two regioisomers could be separated by silica gel
column chromatography. When the glycosylation was
performed at room temperature, a loss of regioselectivity
accompanied by the formation of a considerable amount
of trisaccharide was observed. However, no side products
resulting from activation of the bulky anomeric dicyclo-
hexylmethane thio group could be detected. Benzoylation
of 4 with benzoyl chloride and N,N-(dimethylamino)-
pyridine in pyridine (f 13), followed by removal of the
benzylidene group in DCM/acetonitrile with TFA/H2O,
yielded disaccharide acceptor 5 in an 87% overall yield.
1
The H NMR spectrum of 13 showed a strong downfield
shift of H-2, demonstrating that the glycosylation had
taken place at C-3.
The glycosyl donor 6 was used in a second NIS/
TMSOTf-mediated chemo- and regioselective glycosyla-
tion with the acceptor 5 at -70 °C in DCM to afford key
trisaccharide 7 in a 50% yield (Scheme 4). In this
reaction, we exploit the fact that the electronically
deactivated compound 6 has a significant higher ano-
meric reactivity than compound 5, which is deactivated
electronically by a C-2 benzoyl group and sterically by
the bulky dicyclohexylmethane thio moiety. No side
products resulting from glycosylation at C-4 of 5 could
(6) Zhiyuan, Z.; Ollmann, I. R.; Ye, X.-S.; Wischnat, R.; Baasov, T.;
Chi-Huey Wong J . Am. Chem. Soc. 1999, 121, 734-753.
(7) (a) Boons, G.-J .; Geurtsen, R.; Holmes, D. S. Tetrahedron Lett.
1995, 36, 6325. (b) Geurtsen, R.; Holmes, D. S.; Boons, G.-J . J . Org.
Chem. 1997, 62, 8145-8154.
(8) (a) Zhang, Y.-M.; Brodzky, A.; Sinay¨, P.; Saint-Marcoux, G.;
Perly, B. Tetrahedron: Asymmetry 1995, 6, 1195-1216. (b) Ekelo¨f, K.;
Oscarson, S. J . Org. Chem. 1996, 61, 7711-7718.
(9) Eby, R.; Sondheimer, S. J .; Schuerch, C. Carbohydr. Res. 1979,
73, 273-276.

Phytoalexin-Elicitor Active Oligosaccharides
J . Org. Chem., Vol. 64, No. 21, 1999 7831
Sch em e 4a
F igu r e 2. UV light-induced photodecomposition of hexaglu-
coside 1b. The sample was dissolved in a 90% (v/v) ethanol
solution and was irradiated for various periods of time (as
indicated) with a UV light source as described in the Experi-
mental Section.
a
(i) NIS, TMSOTf, CH2Cl2, -70 °C; (ii) NIS, TMSOTf, HO(CH2)3-
NHZ; (iii) KOtBu, CH2Cl2/MeOH (9/1).
Sch em e 5a
1
be detected. The H NMR spectrum of 5 showed that the
H-4′ coupled to a hydroxyl signal. The hydroxyl signal
disappeared on exposure to D2O. These experiments
prove the regioselectivty of the glycosylation.
The 4-hydroxyl function of glycosyl donor 7 is signifi-
cantly less reactive than primary hydroxyls as a result
of the steric hindrance by the neighboring glycosyl
residues and therefore was left unprotected. Condensa-
tion of 7 with 3-(benzyloxycarbonylamino)-1-propanol10
in the presence of NIS/TMSOTf in DCM afforded the
spacer-containing trisaccharide 8 in 74% yield. Next, we
focused on the selective deacetylation of the 6′-OAc
moiety of trisaccharide 8. In general, O-acetyl groups are
more labile than O-benzoyl groups under both acidic and
basic reaction conditions. Critical to our studies was the
observation that the acetyl group could be cleaved
selectively in the presence of three benzoyl groups.
Fortunately, selective deacetylation of 8 could easily be
achieved by treatment with a catalytic amount of KOtBu
in DCM\ MeOH to afford glycosyl acceptor 9 in a 69%
yield. It is useful to note that selectivity was maintained
even when a relatively large amount of KOtBu was used
(0.4 equiv, 5 min).
a
(i) NIS, TMSOTf, CH2Cl2; (ii) KOtBu, CH2Cl2/MeOH (9/1); (ii)
Pd(OAc)2, H2(1 atm), EtOH, H2O; (iv) triethylammonium bicarbon-
ate buffer (1.0 M), N-hydroxysuccinimido-4-azidosalicylate, DMF.
Regioselective NIS/TMSOTf-mediated condensation of
7 with 9 in DCM at room temperature resulted in the
formation of hexamer 14 in an excellent 75% yield
(Scheme 5). All glycosylations leading to target compound
1a proceeded with exclusive â-anomeric stereoselectivity
as a result of neighboring group participation of C-2
benzoyl groups.
Deprotection of oligomer 14 was accomplished by
treatment with KOtBu in DCM/MeOH (f 16), followed
by hydrogenation in 10% aqueous EtOH in the presence
of Pd(OAc)2 to give the deprotected substrate 1a in 77%
overall yield. The O-acetyl protecting group of 14 could
selectively be deprotected by employing KOtBu in
DCM\ MeOH to give 15. The structural identity of 1a was
proven by homo- and heteronuclear NMR spectroscopy
and high-resolution MS. Finally, a photoreactive group
was introduced by reaction of 1a with succinimido
4-azidosalicylate11 in triethylammonium bicarbonate
buffer/DMF to furnish 1b in a 52% yield.
Compound 1b shows an absorption maximum at 269
nm (Figure 2), which is consistent with data from other
compounds carrying a similar functionality.11 Ultraviolet
irradiation of compound 1b induces a marked decrease
in A269 after 30 s. The decrease in A269 reaches a
maximum after 5 min of irradiation. Further irradiation
for an additional 5 min only causes marginal diminution
in A269. These results were interpreted as evidence of the
light-induced decomposition of the aryl azido function in
hexaglucoside 1b to give a nitrene species, which then
undergoes further reactions.12
Further validation of the hexaglucosides 1a and 1b as
valuable mimics of the hepta-â-glucoside elicitor involved
testing the compounds as inducers of phytoalexin ac-
cumulation in a biological assay, as competitive inhibitors
of the binding of the radiolabeled hepta-â-glucoside
elicitor to soybean root membrane proteins, and as
ligands in direct binding assays with the same proteins.
(10) Berntson, P.; Bra¨ndstro¨m, A.; J unggren, H.; Palme, L., Sjo¨s-
trand, S. E.; Sundell, G. Acta Pharm. Suec. 1977, 14, 229.
(11) Dupuis, G. Can. J . Chem. 1987, 65, 2450.
(12) Ruoho, A. E.; Rashidbaigi, A.; Roeder, P. E. Membranes,
Detergents and Receptor Solubilization; Venter, J . C., Harrison, L. C.,
Eds.; Alan R. Liss, Inc.: New York, 1984; pp 119-160.

7832 J . Org. Chem., Vol. 64, No. 21, 1999
Geurtsen et al.
combination of regio- and chemoselective glycosylations
were shown to be highly effective in the preparation of
oligosaccharides. Optimal tuning of the substrates (by
torsional, electronic, and anomeric sterical deactivation)
combined with the differences in the reactivity of various
unprotected hydroxyl functions led to the construction
of branched hexasaccharide analogues in a highly con-
vergent fashion.
Exp er im en ta l Section
Gen er a l Meth od s. Gravity column chromatography was
performed on silica gel 60 (Merck, 70-230 mesh), and reac-
tions were monitored by TLC on Kieselgel 60 F254 (Merck).
Detection was effected by examination under UV light and by
charring with a solution of 20% (v/v) sulfuric acid in methanol
or with a molybdate solution (0.02 M solution of ammonium
cerium(IV) sulfate dihydrate and ammonium molybdate(VI)
tetrahydrate in aqueous 10% (v/v) H2SO4). Size exclusion
column chromatography was performed on Sephadex LH-20
or LH-60 (both Pharmacia Biotech AB, Uppsala, Sweden) with
dichloromethane/methanol (1/1, v/v) as the eluent or on
Sephadex G-15 (Pharmacia Biotech AB, Uppsala, Sweden) and
deionized water as the eluent. Extracts were evaporated under
reduced pressure at 40 °C (bath). All solvents were distilled
in the presence of appropriate drying agents; dichloromethane
and toluene were distilled over P2O5 and stored over molecular
sieves (4 Å). Diethyl ether was distilled over CaH2, redistilled
from LiAlH4, and stored over sodium wire. N,N-Dimethylform-
amide was stirred with CaH2 for 16 h, distilled under reduced
pressure, and stored over molecular sieves (4 Å). Methanol was
dried by refluxing with magnesium methoxide, distilled, and
stored over molecular sieves (3 Å), and pyridine was dried by
refluxing with CaH2, then distilled, and stored over molecular
sieves (4 Å).
F igu r e 3. Competitive inhibition, by unlabeled oligogluco-
sides, of binding of 125I-labeled tyraminylated hepta-â-glucoside
elicitor to soybean root membranes. Radiolabeled tyraminyl-
ated hepta-â-glucoside (1 nM) was incubated with soybean
membranes (0.1 mg of protein) in the presence of increasing
amounts of hepta-â-glucoside (O), hexaglucoside 1a (0), and
hexaglucoside 1b (b). The numbers in the legend refer to the
relative binding activity (IC50), which is defined as the
concentration of an oligoglucoside required to give 50% inhibi-
tion of the binding of radiolabeled hepta-â-glucoside to its
binding site on soybean root membranes. These values were
obtained by a linear regression analysis of the central portion
of the curves. Competitive binding assays were carried out as
described in the Experimental Section. The amount of radio-
labeled elicitor remaining bound to the membranes was
normalized to the amount bound in the absence of any
unlabeled oligoglucoside.
Hexaglucoside 1a was an equally effective inducer of
phytoalexin accumulation in soybean cotyledons as the
hepta-â-glucoside elicitor (data not shown). Furthermore,
both hexaglucosides 1a and 1b were as effective as the
unmodified hepta-â-glucoside in ligand competition assay
with soybean membranes (Figure 3). The IC50 values
(defined in the figure legend) are 10.7, 5.1, and 3.8 nM
for the hepta-â-glucoside elicitor, hexaglucosides 1a and
1b respectively. These data confirm the results from our
previous structure-activity relationship studies where
we showed that the hexaglucoside and heptaglucoside
were equally biologically active. We had concluded then
that the reducing end of the hepta-â-glucoside was not
important for maximum biological activity; however, the
three terminal nonreducing glucoses are essential for
both the recognition by plant binding proteins and
maximum biological activity.2b,13
Dicycloh exylm eth yl 4,6-O-Ben zylid en e-1-th io-â-D-glu -
cop yr a n osid e (2). To a solution of dicyclohexylmethyl 1-thio-
â-D-glucopyranoside 10 (3.0 g, 7.9 mmol) in DMF (25 mL) were
added benzaldehyde dimethyl acetal (1.8 mL, 11.9 mmol) and
camphorsulfonic acid (70 mg, 0.3 mmol). The solution was
stirred for 18 h at room temperature under reduced pressure
(20 mmHg). The reaction mixture was diluted with Et2O (100
mL) and washed with aqueous 1 M NaHCO3 (1 × 10 mL) and
H2O (3 × 15 mL). The organic phase was dried (MgSO4) and
filtered. The filtrate was concentrated in vacuo and co-
concentrated with toluene (3 × 15 mL). The residue was
purified by silica gel column chromatography (CH2Cl2/acetone,
95/5, v/v) to yield 2 as an amorphous white solid (3.0 g, 82%):
Rf 0.55 (CH2Cl2/MeOH, 6/1, v/v); [R]25 -40.6 (c 1); FAB-MS
D
m/z (%) 485 [M + Na]+ (100); 13C NMR (CDCl3, 75 MHz) δ
137.0, 129.3-126.4 (C6H5CH), 101.9 (C6H5CH), 89.2 (C-1), 80.4,
74.5 (2×), 70.4 (C-2,3,4,5), 68.6 (C-6), 61.2 (SCH), 41.1, 39.8,
1
31.8-26.5 (2 C6H11); H NMR (CDCl3, 300 MHz) δ 7.50-7.20
Finally, a radioiodinated derivative of hexaglucoside
1b was prepared and used in binding assays to demon-
strate that this radioligand binds to soybean root mem-
brane proteins as well as the radiolabeled hepta-â-
glucoside used previously. Binding of radiolabeled 1b
could be competed by 98-99% with a 1000-fold excess of
either unlabeled hepta-â-glucoside elicitor or unlabeled
hexaglucoside 1b. These data indicate that the soybean
elicitor binding site has an affinity for the de novo
hexaglucosides 1a and 1b very similar to the affinity for
the hepta-â-glucoside.
(m, 5H, C6H5CH), 5.54 (s, 1H, C6H5CH), 4.37 (d, 1H, H-1, J 1,2
4.4 Hz), 4.30 (dd, 1H, H-6a, J 5,6a 5.0, J 6a,6b 10.3 Hz), 3.84 (dt,
1H, H-3, J 2,3J 3,4 9.2 Hz), 3.78 (t, 1H, H-6b, J 5,6bJ 6a,6b 10.3 Hz),
3.60 (t, 1H, H-4, J 3,4J 4,5 9.2 Hz), 3.50-3.41 (m, 2H, H-2,5), 2.71
(d, 1H, OH, J 1.8 Hz), 2.60 (d, 1H, OH, J 1.8 Hz), 2.45 (t, 1H,
SCH, J 5.5 Hz), 2.00-0.90 (m, 22H, 2 C6H11); HR-FAB MS
calcd for C26H38NaO5S 485.2338, found 485.2346.
Eth yl 6-O-Acetyl-2-O-ben zoyl-3,4-d i-O-ben zyl-1-th io-â-
D-glu cop yr a n osid e (6). Ethyl 2-O-benzoyl-3,4,6-tri-O-benzyl-
1-thio-â-D-glucopyranoside 3 (2.0 g, 3.3 mmol) was dissolved
in HOAc/Ac2O (1/1, v/v, 15 mL). A 2% (v/v) solution of H2SO4
in HOAc/Ac2O (1/1, v/v, 0.2 mL) was added, and the solution
was stirred for 24 h at room temperature. Next, the solution
was diluted with CH2Cl2 (100 mL) and neutralized with
aqueous NaOAc (10%, w/v). The organic phase was washed
with H2O (3 × 15 mL), dried (MgSO4), and filtered. The filtrate
was concentrated in vacuo, and the residue was purified by
silica gel column chromatography to give 12 as an amorphous
white solid (1 g, 54%): Rf 0.41 (CH2Cl2/acetone, 98/2, v/v).
Compound 12 (950 mg, 1.73 mmol) was dissolved in CH2Cl2
Con clu sion s
The new methodology is a reliable approach for the
construction of complex carbohydrates. Furthermore, the
(13) Cheong, J .-J .; Birberg, W.; Fu¨gedi, P.; Pilotti, Å.; Garegg, P.
J .; Hong, N.; Ogawa, T.; Hahn, M. G. Plant Cell 1991, 3, 127-136.

Phytoalexin-Elicitor Active Oligosaccharides
J . Org. Chem., Vol. 64, No. 21, 1999 7833
(30 mL). EtSH (0.3 mL, 4.1 mmol) and TMSOTf (50 µL, 0.28
mmol) were added, and the solution was stirred for 18 h. Next,
the solution was neutralized with TEA (0.1 mL), diluted with
CH2Cl2 (100 mL), and washed with H2O (3 × 15 mL). The
organic phase was dried (MgSO4) and filtered. The filtrate was
concentrated in vacuo, and the residue was purified by silica
gel column chromatography (CH2Cl2/acetone, 99/1, v/v) to give
6 as a colorless syrup (586 mg, 61%): Rf 0.38 (CH2Cl2/acetone,
99/1, v/v); FAB-MS m/z (%) 573 [M + Na]+ (100); 13C NMR
(CDCl3, 75 MHz) δ 170.9 (CH3CO), 165.5 (C6H5CO), 137.8-
128.0 (2 C6H5CH2, C6H5CO), 84.6, 83.9, 77.7, 77.4, 72.6 (C-
1,2,3,4,5), 75.6, 75.4 (2 C6H5CH2), 63.3 (C-6), 24.4 (SCH2CH3),
21.1 (CH3CO), 15.2 (SCH2CH3); 1H NMR (CDCl3, 300 MHz) δ
8.07-7.10 (m, 15H, 2 C6H5CH2, C6H5CO), 5.31 (t, 1H, H-2,
4.26 (dd, 1H, H-6a, J 5,6a 5.0, J 6a,6b -10.5 Hz), 4.19 (t, 1H, H-3,
J 3,4 8.6 Hz), 3.83 (t, 1H, H-4, J 4,5 9.0 Hz), 3.62 (dd, 1H, H-6′a,
J 5′,6′a 2.0, J 6′a,6′b -11.0 Hz), 3.59 (t, 1H, H-3′), 3.53 (dd, 1H,
H-6′b, J 5′,6′b 11.0 Hz), 3.40 (m, 1H, H-5), 3.37 (m, 1H, H-5′),
2.20 (t, 1H, SCH, J 5.9 Hz), 1.90-0.60 (m, 22H, 2 C6H11); HR
FAB-MS calcd for C67H74NaO12S 1125.4799, found 1125.4763.
Dicycloh exylm eth yl 3-O-(2-O-Ben zoyl-3,4,6-tr i-O-ben -
zyl-â-D-glu cop yr a n osyl)-2-O-ben zoyl-1-th io-â-D-glu cop y-
r a n osid e (5). To a stirred solution of compound 13 (2.3 g, 2.1
mmol) in CH3CN/CH2Cl2 (50 mL, 2/1, v/v) were added H2O
(0.3 mL) and TFA (0.2 mL). After stirring for 3 h at room
temperature, the mixture was washed with aqueous 1 M
NaHCO3 (1 × 10 mL) and H2O (3 × 10 mL). The organic layer
was dried (MgSO4) and filtered. The filtrate was concentrated
in vacuo, and the residue was purified by silica gel column
chromatography (CH2Cl2/acetone, 98/2, v/v) to give 5 as an
amorphous white solid (1.9 g, 89%): Rf 0.41 (CH2Cl2/acetone,
J
1,2J 2,3 9.6 Hz), 4.88-4.58 (m, 4H, 2 C6H5CH2), 4.55 (d, 1H,
H-1), 4.39 (dd, 1H, H-6a, J 5,6a 1.8, J 6a,6b 11.8 Hz), 4.22 (dd, 1H,
H-6b, J 5,6b 4.6 Hz), 3.87, 3.68 (2 t, 2H, H-3,4), 3.61 (m, 1H,
H-5), 2.69 (m, 2H, SCH2CH3, J 7.4 Hz), 2.04 (s, 3H, CH3CO),
1.23 (t, 3H, SCH2CH3); HR FAB-MS calcd for C31H34NaO7S
573.1923, found 573.1903.
95/5, v/v); [R]25 +30.3 (c 1); FAB-MS m/z (%) 1037.4 [M +
D
Na]+ (90); 13C NMR (CDCl3, 100 MHz) δ 165.0, 164.8 (2
C6H5CO), 137.5-127.7 (3 C6H5CH2, 2 C6H5CO), 101.2 (C-1′),
88.1 (C-1), 86.3 (C-3), 82.9 (C-3′), 79.6 (C-5), 77.7, 74.7, 70.3
(C-4,4′,5′), 75.1, 75.0, 73.7 (3 C6H5CH2), 73.5 (C-2′), 72.0 (C-
2), 69.0 (C-6′), 63.6 (C-6), 62.6 (SCH), 40.7, 39.2, 31.8-26.0 (2
C6H11); 1H NMR (CDCl3, 400 MHz) δ 7.70-6.90 (m, 25H, 3
C6H5CH2, 2 C6H5CO), 5.21 (dd, 1H, H-2′, J 1′,2′ 7.9, J 2′,3′ 9.0 Hz),
5.08 (dd, 1H, H-2, J 1,2 10.0, J 2,3 9.0 Hz), 4.76-4.45 (m, 6H, 3
C6H5CH2), 4.64 (d, 1H, H-1′), 4.36 (s br, 1H, OH-4), 4.35 (d,
1H, H-1), 3.89 (m br, 1H, H-6a), 3.79 (t, 1H, H-3, J 3,4 9.0 Hz),
3.75-3.66 (m, 3H, H-6b,3′,6′a), 3.66-3.57 (m, 3H, H-4,4′,5′),
3.54 (dd, 1H, H-6′b, J 5′,6′a 6.0, J 6′a,6′b -10.0 Hz), 3.35 (m, 1H,
H-5), 2.12 (t br, 2H, SCH, OH-6), 1.90-0.45 (m, 22H, 2 C6H11);
Dicycloh exylm eth yl 3-O-(2-O-Ben zoyl-3,4,6-tr i-O-ben -
zyl-â-D-glu cop yr a n osyl)-4,6-O-ben zylid en e-1-th io-â-D-glu -
cop yr a n osid e (4). To a stirred mixture of compound 3 (244
mg, 0.41 mmol), compound 2 (236 mg, 0.51 mmol), and
molecular sieves (4 Å, 2 g) in CH2Cl2/Et2O (1/1, v/v, 6 mL) at
-80 °C were added NIS (107 mg, 0.48 mmol) and TMSOTf (4
µL, 0.02 mmol). After stirring for 30 min at -70 °C, the
reaction mixture was quenched with TEA (0.02 mL), diluted
with CH2Cl2 (40 mL) and washed with aqueous Na2S2O3 (2 ×
15 mL, 15%, w/v). The organic phase was dried (MgSO4) and
filtered. The filtrate was concentrated in vacuo, and the
residue was purified by silica gel column chromatography
(CH2Cl2/acetone, 99/1, v/v) to yield 4 as a white glass (161 mg,
HR FAB-MS calcd for
1037.4483.
C60H70NaO12S 1037.4486, found
40%): Rf 0.51 (CH2Cl2/acetone, 99/1, v/v); [R]25 +1.60 (c 1);
D
Dicycloh exylm eth yl 6-O-(6-O-Acetyl-2-O-ben zoyl-3,4-
d i-O-ben zyl-â-D-glu cop yr a n osyl)-3-O-[(2-O-ben zoyl-3,4,6-
t r i-O-b en zyl-â-D-glu cop yr a n osyl)]-2-O-b en zoyl-1-t h io-â-
D-glu cop yr a n osid e (7). To a stirred mixture of ethyl 6-
O-acetyl-2-O-benzoyl-3,4-di-O-benzyl-1-thio-â-D-glucopyrano-
side 6 (373 mg, 0.68 mmol), compound 5 (630 mg, 0.62 mmol),
and molecular sieves (4 Å, 3.5 g) in CH2Cl2 (20 mL) at -80 °C
were added NIS (182 mg, 0.81 mmol) and TMSOTf (15 µL,
0.08 mmol). After stirring for 60 min at -70 °C, the reaction
mixture was quenched with TEA (0.05 mL), diluted with CH2-
Cl2 (40 mL), and washed with aqueous Na2S2O3 (2 × 15 mL,
15%, w/v). The organic phase was dried (MgSO4) and filtered.
The filtrate was concentrated in vacuo, and the residue was
purified by silica gel column chromatography (CH2Cl2/acetone,
98/2, v/v) to give 7 as a white glass (467 mg, 50%): Rf 0.59
FAB-MS m/z (%) 1021.5 [M + Na]+ (100); 13C NMR (CDCl3,
75 MHz) δ 165.7 (C6H5CO), 138.3-126.2 (3 C6H5CH2, C6H5-
CO, C6H5CH), 101.5 (C6H5CH), 101.1 (C-1′), 89.0 (C-1), 82.8,
81.8, 79.4, 77.8, 75.2, 74.6, 74.2, 70.8 (C-2,3,4,5,2′,3′,4′,5′), 75.0,
74.9, 73.6 (3 C6H5CH2), 68.6(2x) (C-6,6′), 60.9 (SCH), 41.0, 39.9,
1
31.8-26.5 (2 C6H11); H NMR (CDCl3, 500 MHz) δ 8.20-7.30
(m, 25H, 3 C6H5CH2, C6H5CO, C6H5CH), 5.57 (s, 1H, C6H5CH),
5.43 (dd, 1H, H-2′, J 1′,2′ 7.7, J 2′,3′ 9.0 Hz), 5.05 (d, 1H, H-1′),
4.90-4.56 (m, 6H, 3 C6H5CH2), 4.365 (d, 1H, H-1, J 1,2 10.0 Hz),
4.357 (d, 1H, H-6′a, J 5′,6′a 5.0, J 6′a6′b -10.3 Hz), 3.96-3.90 (m,
3H, H-3,4,3′), 3.83 (t, 1H, H-6′b, J 5′6′b 10.0 Hz), 3.78 (t, 1H,
H-4′, J 3′4′J 4′5′ 9.2 Hz), 3.75 (dd, 1H, H-6a, J 5,6a 4.3, J 6a,6b -11.0
Hz), 3.70 (dd, 1H, H-6b, J 5,6b 2.0 Hz), 3.56 (m, 1H, H-5), 3.52
(m, 1H, H-2), 3.46 (dt, 1H, H-5′, J 4′,5′ 9.2 Hz), 2.70 (d, 1H, OH,
J 2,OH 2.3 Hz), 2.49 (t, 1H, SCH, J 5.5 Hz), 2.00-1.10 (m, 22H,
2 C6H11); HR FAB-MS calcd for C60H70NaO11S 1021.4537,
found 1021.4545.
(CH2Cl2/acetone, 95/5, v/v); [R]25 +24.1 (c 1); FAB-MS m/z
D
(%) 1526 [M + Na]+ (100);13C NMR (CDCl3, 125 MHz) δ 170.8
(CH3CO), 165.0, 164.7, 164.2 (3 C6H5CO), 137.8-127.6 (5 C6H5-
CH2, 3 C6H5CO), 101.2 (C-1′,1′′), 87.3 (C-1), 85.9, 82.9 (C-3,3′),
83.1 (C-3′′), 80.6 (C-5), 77.7(2x), 74.7, 73.0 (C-4′,5′,4′′,5′′), 75.11,
75.09, 75.0, 74.8, 73.7 (5 C6H5CH2), 74.1 (C-2′′), 73.4 (C-2′),
72.0 (C-2), 68.9 (C-6′′), 68.74 (C-4), 68.67 (C-6), 63.1 (C-6′), 61.0
(SCH), 40.9, 39.6, 31.7-26.0 (2 C6H11), 20.9 (CH3CO); 1H NMR
(CDCl3, 500 MHz) δ 8.10-7.00 (m, 40H, 5 C6H5CH2, 3 C6H5-
CO), 5.29 (dd, 1H, H-2′′, J 1′′,2′′ 7.9, J 2′′,3′′ 9.2 Hz), 5.19 (dd, 1H,
H-2′, J 1′,2′ 7.9, J 2′,3′ 9.0 Hz), 4.92 (dd, 1H, H-2, J 1,2 10.0, J 2,3 9.0
Hz), 4.87-4.47 (m, 10H, 5 C6H5CH2), 4.81 (d, 1H, H-1′′), 4.58
(d, 1H, H-1′), 4.38 (dd, 1H, H-6′a, J 5′,6′a 2.2, J 6′a,6′b -11.9 Hz),
4.25 (dd, 1H, H-6′b, J 5′,6′b 5.0 Hz), 4.23 (d, 1H, H-1), 4.13 (d,
1H, OH-4, J 4,OH 1.5 Hz), 4.10 (dd, 1H, H-6a, J 5,6a 1.4, J 6a,6b
-12.5 Hz), 3.86 (t, 1H, H-3′′, J 3′′,4′′ 9.0 Hz), 3.82 (dd, 1H, H-6b,
Dicycloh exylm eth yl 3-O-(2-O-Ben zoyl-3,4,6-tr i-O-ben -
zyl-â-D-glu cop yr a n osyl)-2-O-ben zoyl-4,6-O-ben zylid en e-
1-th io-â-D-glu cop yr a n osid e (13). To a stirred solution of
compound 4 (2.1 g, 2.1 mmol) in pyridine (60 mL) were added
DMAP (77 mg, 0.6 mmol) and BzCl (0.7 mL, 6.3 mmol). After
stirring for 18 h at room temperature, the mixture was diluted
with CH2Cl2 (60 mL) and washed with H2O (3 × 15 mL). The
organic layer was dried (MgSO4) and filtered. The filtrate was
concentrated in vacuo, and the residue was co-concentrated
with toluene, MeOH, and CH2Cl2, respectively (3 × 15 mL
each). The residue was purified by silica gel column chroma-
tography (CH2Cl2/acetone, 99/1, v/v) to yield 13 as a white glass
(2.3 g, 98%): Rf 0.51 (CH2Cl2/acetone, 99/1, v/v); [R]25 +19.3
D
(c 1); FAB-MS m/z (%) 1125 [M + Na]+ (100); 13C NMR
(CDCl3, 125 MHz) δ 164.9, 164.5 (2 C6H5CO), 138.5-127.8 (3
C6H5CH2, 2 C6H5CO, C6H5CH), 101.4 (C6H5CH), 101.0 (C-1′),
88.2 (C-1), 83.0 (C-3′), 79.5 (C-4), 78.4 (C-3), 77.9 (C-4′), 75.3
(C-5′), 74.9, 74.6, 73.5 (3 C6H5CH2), 74.0 (C-2′), 73.3 (C-2), 70.7
(C-5), 69.2 (C-6′), 68.6 (C-6), 62.0 (SCH), 40.8, 39.6, 31.7-26.1
(2 C6H11); 1H NMR (CDCl3, 500 MHz) δ 8.10-7.00 (m, 30H, 3
C6H5CH2, 2 C6H5CO, C6H5CH), 5.47 (s, 1H, C6H5CH), 5.23 (dd,
1H, H-2, J 1,2 10.0, J 2,3 8.6 Hz), 5.19 (dd, 1H, H-2′, J 1′,2′ 7.7,
J 2′,3′ 8.6 Hz), 4.69-4.42 (m, 6H, 3 C6H5CH2), 4.47 (d, 1H, H-1),
J
5,6b 6.6 Hz), 3.72-3.58 (m, 6H, H-3,3′,4′,5′,4′′,5′′), 3.53 (m, 1H,
SCH), 2.07 (s, 3H, CH3CO), 1.90-0.40 (m, 22H, 2 C6H11); HR
FAB-MS calcd for C89H98NaO19S 1525.6321, found 1525.6306.
3-(Ben zyloxyca r bon yla m in o)-1-p r op yl 6-O-(6-O-Acetyl-
2-O-ben zoyl-3,4-d i-O-ben zyl-â-D-glu cop yr a n osyl)-3-O-[(2-
O-b en zoyl-3,4,6-t r i-O-b en zyl-â-D-glu cop yr a n osyl)]-2-O-
ben zoyl-â-D-glu cop yr a n osid e (8). To a stirred mixture of
3-(benzyloxycarbonylamino)-1-propanol (21 mg, 0.10 mmol),
compound 7 (111 mg, 0.07 mmol), and molecular sieves (4 Å,
0.4 g) in CH2Cl2 (3 mL) were added NIS (34 mg, 0.15 mmol)

7834 J . Org. Chem., Vol. 64, No. 21, 1999
Geurtsen et al.
and TMSOTf (3 µL, 0.02 mmol). After stirring for 3 min at
room temperature, the reaction mixture was quenched with
TEA (0.02 mL), diluted with CH2Cl2 (30 mL), and washed with
aqueous Na2S2O3 (2 × 15 mL, 15%, w/v). The organic phase
was dried (MgSO4) and filtered. The filtrate was concentrated
in vacuo, and the residue was purified by silica gel column
chromatography (CH2Cl2/acetone, 95/5, v/v) to yield 8 as a
white glass (82 mg, 74%): Rf 0.37 (CH2Cl2/acetone, 95/5, v/v);
silica gel column chromatography (CH2Cl2/acetone, 98/2, v/v)
to yield 14 as a white glass (274 mg, 75%): Rf 0.36 (CH2Cl2/
acetone, 94/6, v/v); [R]25D +11.0 (c 1); FAB-MS m/z (%) 2771.8
[M + Na]+ (100); 13C NMR (CDCl3, 125 MHz) δ 170.7 (CH3CO),
165.1-164.0 (6 C6H5CO), 156.3 (C6H5CH2OCO), 138.1-127.3
(10 C6H5CH2, 6 C6H5CO, C6H5CH2OCO), 101.5 (C-1′), 101.41
(C-1′′′′), 101.36 (C-1′′′′′), 101.1 (C-1′′), 100.9 (C-1′′′), 100.6
(C-1), 85.3-82.8, 77.9-76.8, 75.4-73.0 (10 C6H5CH2, C-3,5,-
2′,3′,4′,5′,2′′,3′′,4′′,5′′,3′′′, 4′′′/5′′′′,5′′′,2′′′′,3′′′′,4′′′′,2′′′′′,3′′′′′,4′′′′′,5′′′′′),
72.5(2x) (C-2,2′′′), 69.5 (C-4), 69.3 (C-6′′′a), 69.2 (C-4′′′/5′′′′),
68.9(2x) (C-6,6′), 68.7 (C-6′′′′′), 67.3 (C-6′′), 66.3 (OCH2), 66.1
(C6H5CH2OCO), 63.0 (C-6′′′′), 37.9 (CH2NHZ), 29.2 (CH2CH2-
[R]25 +10.5 (c 1); FAB-MS m/z (%) 1522.8 [M + Na]+ (100);
D
13C NMR (CDCl3, 100 MHz) δ 170.7 (CH3CO), 165.1, 164.7,
164.3 (3 C6H5CO), 156.3 (C6H5CH2OCO), 137.7-127.6 (5 C6H5-
CH2, 3 C6H5CO, C6H5CH2OCO), 101.3 (2×) (C-1′,1′′), 100.6 (C-
1), 85.1, 82.8, 77.7, 77.5, 74.7, 73.1 (C-3,4′,5′,3′′,4′′,5′′), 82.8 (C-
3′), 75.4 (C-5), 75.1 (2×), 75.0 (2×), 73.7 (5 C6H5CH2), 73.8 (C-
2′), 73.4 (C-2′′), 72.3 (C-2), 69.3 (C-4), 69.1, 68.9 (C-6,6′), 66.2
(OCH2, C6H5CH2OCO), 63.0 (C-6′′), 37.8 (CH2NHZ), 29.2
(CH2CH2CH2), 20.8 (CH3CO); 1H NMR (CDCl3, 400 MHz) δ
8.00-6.90 (m, 45H, 5 C6H5CH2, 3 C6H5CO, C6H5CH2OCO),
5.29 (dd, 1H, H-2′, J 1′,2′ 7.9, J 2′,3′ 9.1 Hz), 5.19 (dd, 1H, H-2′′,
J 1′′,2′′ 7.9, J 2′′,3′′ 9.1 Hz), 5.03 (s, 2H, C6H5CH2OCO), 4.90 (m,
2H, CH2NHZ, H-2), 4.85-4.47 (m, 10H, 5 C6H5CH2), 4.62 (d
br, 1H, H-1′), 4.58 (d br, 1H, H-1′′), 4.37 (dd, 1H, H-6′′a, J 5′′,6′′a
2.0, J 6′′a,6′′b -12.0 Hz), 4.27-4.18 (m, 4H, H-1,6a,6′′b,OH-4),
3.81 (t, 1H, H-3′, J 3′,4′ 9.1 Hz), 3.73-3.51 (m, 9H, H-3,-
6b,4′,5′,6′a,6′b,3′′,4′′,5′′), 3.46-3.36 (m, 2H, H-4,5), 3.33 (m, 1H,
OCH2), 3.08 (m, 1H, OCH2), 2.85 (m, 2H, CH2NHZ), 2.01 (s,
3H, CH3CO), 1.28 (m, 2H, CH2CH2CH2); HR FAB-MS calcd
for C87H89NNaO22 1522.5774, found 1522.5746.
1
CH2), 20.9 (CH3CO); H NMR (CDCl3, 500 MHz) δ 8.00-6.90
(m, 85H, 10 C6H5CH2, 6 C6H5CO, C6H5CH2OCO), 5.30 (dd, 1H,
H-2′′′′, J 1′′′′′,2′′′′′ 7.9, J 2′′′′′,3′′′′′ 9.5 Hz), 5.26 (dd, 1H, H-2′, J 1′,2′ 8.0,
J 2′,3′ 9.2 Hz), 5.21 (dd, 1H, H-2′′′′′, J 1′′′′′,2′′′′′ 8.0, J 2′′′′′,3′′′′′ 9.2 Hz),
5.10 (dd, 1H, H-2′′, J 1′′,2′′ 8.0, J 2′′,3′′ 9.2 Hz), 5.02 (pt, 1H, H-2′′′,
J 1′′′,2′′′ 7.9, J 2′′′,3′′′ 8.6 Hz), 5.02, 4.99 (2 d, 2H, C6H5CH2OCO,
J 12.5 Hz), 4.91 (pt, 1H, H-2, J 1,2J 2,3 8.8 Hz), 4.87 (m, 1H,
CH2NHZ), 4.82-4.38 (m, 18H, 9 C6H5CH2), 4.64 (d, 1H, H-1′′′′),
4.62 (d, 1H, H-1′), 4.58 (d, 1H, H-1′′′′′), 4.40 (d, 1H, H-1′′), 4.33
(dd, 1H, H-6′′′′a, J 5′′′′,6′′′′a 2.2, J 6′′′′a,6′′′′b -11.9 Hz), 4.31 (d, 1H,
H-1′′′), 4.22 (dd, 1H, H-6′′′′b, J 5′′′′,6′′′′b 5.0 Hz), 4.20 (s br, 1H,
OH-4′′′), 4.18 (d br, 1H, H-1), 4.17-4.05 (m, 4H, C6H5CH2, OH-
4, H-6′′′a), 4.07 (d br, 1H, H-6a, J 6a,6b -10.5 Hz), 3.83 (t, 1H,
H-3′′′′, J 3′′′′,4′′′′ 9.0 Hz), 3.76 (t, 1H, H-3′, J 3′,4′ 9.0 Hz), 3.75-
3.26 (m, 16H, H-3,4′′,5′,6′a,6′b,3′′,6′′a,3′′′,5′′′,6′′′b,4′′′′,3′′′′′,4′′′′′,-
5′′′′′,6′′′′′a, OCH2(1H)), 3.16 (m, 1H, H-5′′), 3.03 (m, 1H,
OCH2), 2.83 (m, 2H, CH2NHZ), 2.01 (s, 3H, CH3CO), 1.27 (m,
2H, CH2CH2CH2); HR FAB-MS calcd for C161H161NNaO40
2771.0493, found 2771.0473.
3-(Ben zyloxyca r bon yla m in o)-1-p r op yl 6-O-(2-O-Ben -
zoyl-3,4-d i-O-ben zyl-â-D-glu cop yr a n osyl)-3-O-[(2-O-ben -
zoyl-3,4,6-tr i-O-ben zyl-â-D-glu cop yr a n osyl)]-2-O-ben zoyl-
â-D-glu cop yr a n osid e (9). To a stirred solution of compound
8 (290 mg, 0.20 mmol) in CH2Cl2/MeOH (3 mL, 9/1, v/v) was
added KOtBu (9 mg, 0.08 mmol). After stirring for 4 min at
room temperature, the reaction mixture was neutralized with
camphorsulfonic acid and concentrated in vacuo. The residue
was co-concentrated with CH2Cl2 (2 × 5 mL) and purified by
silica gel column chromatography (CH2Cl2/acetone, 92/8, v/v)
to yield 9 as a white glass (194 mg, 69%): Rf 0.35 (CH2Cl2/
acetone, 91/9, v/v); [R]25D +6.60 (c 1); FAB-MS m/z (%) 1480.7
[M + Na]+ (100); 13C NMR (CDCl3, 100 MHz) δ 165.1, 164.7,
164.3 (3 C6H5CO), 156.4 (C6H5CH2OCO), 137.9-127.6 (5 C6H5-
CH2, 3 C6H5CO, C6H5CH2OCO), 101.3 (2×) (C-1′,1′′), 100.7 (C-
1), 85.0, 82.8, 77.7 (2×), 75.6, 75.5, 74.7 (C-3,5,4′,5′,3′′,4′′,5′′),
82.5 (C-3′), 75.1, 75.0 (2×), 74.7, 73.6 (5 C6H5CH2), 73.8 (C-
2′), 73.4 (C-2′′), 72.3 (C-2), 69.1 (2×) (C-4,6), 68.9 (C-6′), 66.4
(OCH2), 66.2 (C6H5CH2OCO), 61.9 (C-6′′), 37.8 (CH2NHZ), 29.2
(CH2CH2CH2); 1H NMR (CDCl3, 400 MHz) δ 8.00-6.90 (m,
45H, 5 C6H5CH2, 3 C6H5CO, C6H5CH2OCO), 5.26 (dd, 1H, H-2′,
J 1′,2′ 7.9, J 2′,3′ 9.2 Hz), 5.21 (dd, 1H, H-2′′, J 1′′,2′′ 7.9, J 2′′,3′′ 9.1
Hz), 5.03 (m, 2H, C6H5CH2OCO), 4.94 (m, 2H, CH2NHZ, H-2),
4.87-4.48 (m, 10H, 5 C6H5CH2), 4.65 (d br, 1H, H-1′), 4.60 (d,
1H, H-1′′), 4.33 (s br, 1H, OH-4), 4.24 (d, 1H, H-1, J 1,2 8.0 Hz),
4.22 (d br, 1H, H-6a, J 6a,6b -11.0 Hz), 3.92-3.39 (m, 13H,
H-3,4,5,6b,4′,5′,6′a,6′b,3′′,4′′,5′′,6′′b,OCH2(1H)), 3.89 (d br, 1H,
H-6′′a, J 6′′a,6′′b -11.0 Hz), 3.82 (t, 1H, H-3′, J 3′,4′ 9.2 Hz), 3.12
(m, 1H, OCH2), 2.86 (m, 2H, CH2NHZ), 2.25 (m br, 1H, OH-
3-(Ben zyloxyca r bon yla m in o)-1-p r op yl 6-O-(6-O-(6-O-
(2-O-Ben zoyl-3,4-d i-O-b en zyl-â-D-glu cop yr a n osyl)-3-O-
[(2-O-ben zoyl-3,4,6-tr i-O-ben zyl-â-D-glu cop yr a n osyl)]-2-
O-b e n zoyl-â-D-glu cop yr a n osyl)-2-O-b e n zoyl-3,4-d i-O-
b en zyl-â-D-glu cop yr a n osyl)-3-O-[(2-O-b en zoyl-3,4,6-t r i-
O-ben zyl-â-D-glu cop yr a n osyl)]-2-O-ben zoyl-â-D-glu cop y-
r a n o- sid e (15). To a stirred solution of compound 14 (163
mg, 0.06 mmol) in CH2Cl2/MeOH (7 mL, 9/1, v/v) was added a
0.1 M solution of KOtBu in CH2Cl2/MeOH (0.1 mL, 1/1, v/v).
After stirring for 30 min at room temperature, the reaction
mixture was neutralized with camphorsulfonic acid and
concentrated in vacuo. The residue was co-concentrated with
CH2Cl2 (2 × 5 mL) and purified by silica gel column chroma-
tography (CH2Cl2/acetone, 94/6, v/v) to give 15 as a white glass
(85 mg, 53%): Rf 0.49 (CH2Cl2/acetone, 90/10, v/v); [R]25D +2.80
(c 1); FAB-MS m/z (%) 2729.9 [M + Na]+ (100); 13C NMR
(CDCl3, 125 MHz) δ 165.1-164.0 (6 C6H5CO), 156.4 (C6H5-
CH2OCO), 138.0-126.8 (10 C6H5CH2, 6 C6H5CO, C6H5CH2-
OCO), 101.9 (C-1′′′′), 101.4 (2×) (C-1′′,1′′′′′), 101.1 (2×)
(C-1′,1′′′), 100.6 (C-1), 85.3-73.5 (10 C6H5CH2, C-3,5,2′,3′,4′,-
5′,2′′,3′′,4′′,5′′,3′′′,4′′′/5′′′′,5′′′,2′′′′,3′′′′,4′′′′,2′′′′′,3′′′′′,4′′′′′,5′′′′′), 72.5
(2×) (C-2,2′′′), 69.6 (C-4), 69.5 (C-6′′′), 69.1 (C-4′′′/5′′′′′), 68.9
(2×) (C-6,6′′), 68.7 (C-6′′′′′), 67.7 (C-6′), 66.3 (OCH2), 66.1
(C6H5CH2OCO), 37.9 (CH2NHZ), 29.2 (CH2CH2CH2); 1H NMR
(CDCl3, 500 MHz) δ 8.00-6.90 (m, 85H, 10 C6H5CH2, 6 C6H5-
CO, C6H5CH2OCO), 5.30 (dd, 1H, H-2′′′′′, J 1′′′′′,2′′′′′ 8.0, J 2′′′′′,3′′′′′
9.2 Hz), 5.26 (dd, 1H, H-2′′′′′, J 1′′′′′,2′′′′′ 8.0, J 2′′′′′,3′′′′′ 9.2 Hz), 5.22
(dd, 1H, H-2′′, J 1′′,2′′ 8.0, J 2′′,3′′ 9.2 Hz), 5.13 (dd, 1H, H-2′, J 1′,2′
7.9, J 2′,3′ 9.2 Hz), 5.022 (dd, 1H, H-2′′′, J 1′′′,2′′′ 8.0, J 2′′′,3′′′ 9.2 Hz),
5.019, 4.98 (2 d, 2H, C6H5CH2OCO, J -12.0 Hz), 4.93 (m, 2H,
H-2, CH2NHZ), 4.85-4.38 (m, 18H, 9 C6H5CH2), 4.653 (d, 1H,
H-1′′′′′), 4.645 (d, 1H, H-1′′′′′), 4.60 (d, 1H, H-1′′), 4.43 (d br,
1H, H-1′), 4.35 (d, 1H, H-1′′′), 4.32 (s br, 1H, OH-4′′′), 4.25,
4.178 (2 d, 2H, C6H5CH2, J -11.0 Hz), 4.21 (s br, 1H, OH-4),
4.180 (d, 1H, H-1, J 1,2 8.0 Hz), 4.08 (d br, 1H, H-6′′′a, J 6′′′a,6′′′b
-11.0 Hz), 4.07 (d br, 1H, H-6a, J 6a,6b -10.0 Hz), 3.87 (dd, 1H,
H-6′′′′′a, J 5′′′′′,6′′′′′a 2.5, J 6′′′′′a,6′′′′′b -12.0 Hz), 3.85 (t, 1H, H-3′′′′′,
J 3′′′′′,4′′′′′ 9.2 Hz), 3.77 (t br, 1H, H-3′′′′′, J 3′′′′′,4′′′′′ 9.2 Hz), 3.75-
3.55 (m, 16H, H-3,3′,6′a,3′′,4′′,5′′,6′′a,3′′′,5′′′,6′′′b,4′′′′′,6′′′′′b,4′′′′′,-
5′′′′′,6′′′′′a,6′′′′′b), 3.53 (dd, 1H, H-6′′b, J 5′′,6′′b 6.1, J 6′′a,6′′b -10.1
Hz), 3.48-3.28 (m, 8H, H-4,5,6b,4′,6′b,4′′′,5′′′′′,OCH2(1H)), 3.23
(m, 1H, H-5′), 3.05 (m, 1H, OCH2), 2.84 (m, 2H, CH2NHZ), 2.58
(s br, 1H, OH-6′′′′′), 1.29 (m, 2H, CH2CH2CH2); HR FAB-MS
calcd for C159H159NNaO39 2729.0387, found 2729.0436.
6′), 1.32 (m, 2H, CH2CH2CH2); HR FAB-MS calcd for C85H87
NNaO21 1480.5668, found 1480.5673.
-
3-(Ben zyloxyca r bon yla m in o)-1-p r op yl 6-O-(6-O-(6-O-
(6-O-Acet yl-2-O-b en zoyl-3,4-d i-O-b en zyl-â-D-glu cop yr a -
n osyl)-3-O-[(2-O-ben zoyl-3,4,6-tr i-O-ben zyl-â-D-glu cop y-
r a n osyl)]-2-O-ben zoyl-â-D-glu cop yr a n osyl)-2-O-ben zoyl-
3,4-d i-O-b en zyl-â-D-glu cop yr a n osyl)-3-O-[(2-O-b en zoyl-
3,4,6-tr i-O-ben zyl-â-D-glu cop yr a n osyl)]-2-O-ben zoyl-â-D-
glu cop yr a n osid e (14). To a stirred mixture of compound 7
(240 mg, 0.16 mmol), compound 9 (194 mg, 0.13 mmol), and
molecular sieves (4 Å, 1 g) in CH2Cl2 (20 mL) were added NIS
(63 mg, 0.28 mmol) and TMSOTf (10 µL, 0.06 mmol). After
stirring for 3 min at room temperature, the reaction mixture
was quenched with TEA (0.03 mL), diluted with CH2Cl2 (40
mL) and washed with aqueous Na2S2O3 (2 × 15 mL, 15%, w/v).
The organic phase was dried (MgSO4) and filtered. The filtrate
was concentrated in vacuo, and the residue was purified by

Phytoalexin-Elicitor Active Oligosaccharides
J . Org. Chem., Vol. 64, No. 21, 1999 7835
3-(Ben zyloxyca r bon yla m in o)-1-p r op yl 6-O-(6-O-(6-O-
(3,4-Di-O-b en zyl-â-D-glu cop yr a n osyl)-3-O-[(3,4,6-t r i-O-
ben zyl-â-D-glu cop yr a n osyl)]-â-D-glu cop yr a n osyl)-3,4-d i-
O-ben zyl-â-D-glu cop yr a n osyl)-3-O-[(3,4,6-tr i-O-ben zyl-â-
D-glu copyr an osyl)]-â-D-glu copyr an oside (16). To a solution
of 14 (56 mg, 0.02 mmol) in CH2Cl2/MeOH (9/1, v/v, 2 mL) was
added KOtBu (3 mg, 0.03 mmol). The solution was stirred for
18 h at room temperature. After neutralization with camphor-
sulfonic acid the mixture was concentrated in vacuo. The
residue was purified by silica gel column chromatography
(CH2Cl2/acetone, 7/3, v/v) followed by size exclusion column
chromatography (LH-20, CH2Cl2/MeOH, 1/1, v/v) to give 16
as a white glass (36 mg, 85%): Rf 0.56 (CH2Cl2/acetone, 7/3,
v/v); [R]25D -8.90 (c 1); FAB-MS m/z (%) 2106 [M + Na]+ (100);
1H NMR (CDCl3, 500 MHz) δ 7.40-7.00 (m, 55H, 10 C6H5-
CH2, C6H5CH2OCO), 5.09, 5.03 (2 d, 2H, C6H5CH2OCO), 4.98-
4.31 (m, 20H, 10 C6H5CH2), 4.46-4.28 (m, 6H, 6 H-1), 4.16-
3.19 (m, 27 H, CH2NHZ(1H), OCH2, 6 H-3,4,5,6), 3.64-3.46
(m, 6H, 6 H-2), 3.24 (m, 1H, CH2NHZ(1H)), 1.75 (m, 2H, CH2-
CH2CH2); HR FAB-MS calcd for C117H135NNaO33 2104.8814,
found 2104.8771.
dure described below. Although a small absorbance shift was
observed upon iodination, photosensitivity was not affected.
Ra d ioiod in a tion of Tyr a m in yla ted Hep ta -â-glu cosid e
a n d Hexa glu cosid e 1b. The tyramine conjugate of the hepta-
â-glucoside elicitor was iodinated using a published protocol.14
The protocol was slightly modified to use a molar ratio of iodine
to tyramine conjugate of 5:2. Consequently, 400 ng of Iodogen
(Pierce Chemicals, Rockford, IL) were used to coat the bottom
of a glass test tube. This procedure typically yielded radioio-
dinated oligosaccharides with a specific radioactivity of ∼1000
Ci/mmol.
Hexaglucoside 1b was iodinated according to a protocol
modified from Chizzonite et al.15 Radioactive iodine (1 mCi
Na125[I] in 10 µL; Amersham, Arlington Heights, IL) was added
to 40 µL PBS (20 mM sodium phosphate, pH 7.2 containing
150 mM NaCl) in a prewetted 12 mm × 50 mm glass tube
precoated with 50 µg Iodo-gen. The iodine was allowed to react
for 6 min, while the tube was flicked every 30 s. The solution
was then immediately transferred to a tube containing 500
pmol of hexaglucoside 1b (in 1.5 µL). The solution was mixed
every 30 s for 9 min, then applied to a Sephadex G-10
(Pharmacia) gel filtration column (0.5 cm × 15 cm), and eluted
with bis-Tris buffer (25 mM, pH 7.0). The fractions corre-
sponding to the void volume of the column and containing the
radioactive oligosaccharide were pooled and used in binding
assays.
P r ep a r a tion of Soybea n Root Mem br a n es a n d Bio-
logica l a n d Bin d in g Assa ys. Foundation quality soybean
(Glycine max L. cv. Williams 82) was obtained from Illinois
Foundation Seeds, Inc. (Champaign, IL), and the seedlings
were grown as described.12 The biological assay used to
determine the ability of the synthesized compounds to elicit
the synthesis and accumulation of phytoalexins in soybean
tissue was performed as described.14 Briefly, cotyledons from
young soybean plants were excised and surface-sterilized. A
thin layer of tissue was cut from the bottom surface of each
cotyledon with a razor blade. Samples (90 µL) were applied to
the wound of each cotyledon, and the cotyledons were incu-
bated in the dark at 26 °C for 20 h. The absorbance at 285 nm
of wound-droplet solutions was measured, which is linearly
correlated with the phytoalexin content. Crude total cellular
membranes were prepared from the roots of 10-day-old soy-
bean seedlings as described,2b,2c except that 25 mM bis-Tris,
pH 7.0 (4 °C), containing 2 mM MgCl2, 2 mM DTT, 5 mM
ascorbic acid, 8% (w/w) sucrose, 10 mM KCl, 10 mM EDTA,
200 µM AEBSF, 10 µg/mL leupeptin, 1 µM pepstatin, and 1
µg/mL aprotinin was used as the homogenization buffer at a
ratio of 2 mL per g (fresh weight) of roots.
The assays to determine binding of radiolabeled hepta-
â-glucoside or hexaglucoside 1b to intact membranes were
carried out as described in 96-well microtiter plates,2c except
that the binding assay buffer was modified (25 mm bis-Tris,
pH 7.0 (4 °C), 10 mM MgCl2, 2 mM dithiothreitol (DTT), 100
mM NaCl, 7.5 mM â-D-thioglucoside, and 7.5 mM D-gluconic
acid lactone). The incubation time for the binding assays was
2 h. Competitive binding assay were carried out by mixing
increasing amounts of competitive ligands (0.1-3000 times the
amount of radioligand) with constant amount of radiolabeled
hepta-â-glucoside and membrane proteins, and incubating for
4 h.
3-Am in o-1-p r op yl 6-O-(6-O-(6-O-(â-D-Glu cop yr a n osyl)-
3-O-[(â-D-glu cop yr a n osyl)]-â-D-glu cop yr a n osyl)-â-D-glu -
cop yr a n osyl)-3-O-[(â-D-glu cop yr a n osyl)]-â-D-glu cop yr a -
n osid e (3-Am in o-1-p r op yl 32,34-Di-â-D-glu cop yr a n osyl-
gen tiotetr a osid e) (1a ). Compound 16 (36 mg, 0.02 mmol)
was dissolved in 10% (v/v) aqueous EtOH (2 mL) and treated
with hydrogen at 1 atm in the presence of Pd(OAc)2 (20 mg,
0.09 mmol). After stirring for 18 h at room temperature, the
solution was filtered through Celite. The filtrate was concen-
trated, and the residue was purified over Sephadex G-15. The
final solution was freeze-dried to give 1a as a colorless fluffy
glass (16 mg, 90%): Rf 0.70 (EtOH/t-BuOH/H2O, 2/5/4, v/v/v);
FAB-MS m/z (%) 1048.5 [M]+ (100); 13C NMR (D2O, 125 MHz)
δ 105.5, 105.4, 105.3, 104.6 (6 C-1), 86.9, 78.6, 78.5, 78.2, 78.1,
77.4, 77.2, 77.03, 76.96, 76.03, 75.98, 75.7, 75.6, 75.3, 75.24,
72.17, 72.1, 72.0, 70.6 (6 C-2,3,4,5), 71.5, 71.4, 71.2, 63.3 (3×)
1
(6 C-6), 70.5 (OCH2), 40.1 (CH2NH2), 29.2 (CH2CH2CH2); H
NMR (D2O, 500 MHz) δ 4.67-4.62 (2H) and 4.48-4.39 (4H)
(2 m, 6H, 6 H-1), 4.12 (m, 3H) and 3.83 (d, 3H) (6 H, 6 H-6a),
3.95 (m, 1H, OCH2), 3.82-3.72 (m, 4H) and 3.65-3.60 (m, 3H)
(2 m, 7H, OCH2(1H), 6 H-6b), 3.69-3.20 (m, 24H, 6 H-2,3,4,5),
3.07 (t, 2H, CH2NH2), 1.92 (m, 2H, CH2CH2CH2); HR FAB-
MS calcd for C39H69NO31 1048.3932, found 1048.3955.
N-(4-Azid osa licyloyl)-3-a m in o-1-p r op yl 6-O-(6-O-(6-O-
(â-D-Glu cop yr a n osyl)-3-O-[(â-D-glu cop yr a n osyl)]-â-D-glu -
cop yr a n osyl)-â-D-glu cop yr a n osyl)-3-O-[(â-D-glu cop yr a -
n osyl)]-â-D-glu copyr an oside (N-(4-Azidosalicyloyl)-3-am i-
n o-1-p r op yl 32,34-Di-â-D-glu cop yr a n osyl-gen t iot et r a o-
sid e) (1b). To a solution of 1a (5 mg, 5 µmol) in triethylam-
monium bicarbonate buffer (1 mL, 1.0 M, pH 8.5, Sigma) was
added a soltion of N-hydroxysuccinimido-4-azidosalicylate (3
mg, 11 µmol) in DMF (1 mL). The mixture was stirred for 18
h in the dark at room temperature. The solvent was removed
in vacuo, and the residue was purified over Sephadex G15 to
yield 1b as an amorphous white fluffy solid (3 mg, 52%): Rf
0.38 (n-BuOH/EtOH/H2O, 2/1/1, v/v/v); FAB-MS m/z (%)
1231.4 [M + Na]+ (100); 1247.4 [M + K]+ (15); 1H NMR (D2O,
500 MHz) δ 7.73 (d, 1H, HAr, J 8.3 Hz), 6.35 (s, 1H, HAr), 6.30
(d, 1H, HAr), 4.10-3.20(m), 2.60(s) and 1.25(d) (44H, OCH2, 6
H-1,2,3,4,5,6a,6b,), 2.45-2.25 (m, 2H, CH2NH), 1.88 (m, 2H,
CH2CH2CH2).
P h otor ea ctivity of Hexa glu cosid e 1b. The compound 1b
was dissolved in water to a final concentration of 0.33 mM
and diluted 10-fold with ethanol. Absorbance was measured
in the range 240-360 nm in a quartz cuvette. The quartz
cuvette was placed at a distance of 10 cm from an UV lamp
(UVGL-52, UVP products, CA) and irradiated with short-wave
UV (252 nm) for 30 s. The absorbance of the solution was then
scanned. The solution was then irradiated for an additional
time period. After each irradiation period increment, the
absorbance of the solution was rescanned. Control irradiation
experiments were performed with 4-[p-azidosalicylamido] bu-
tylamine (ASBA; Pierce Chemicals, Rockford, IL). ASBA was
also iodinated with nonradioactive iodine by using the proce-
Ack n ow led gm en t. This work was supported by the
University of Georgia Faculty start-up funds (to G.J .B.)
and by a grant (to M.G.H.) from the U.S. National
Science Foundation (MCB-9723685).
J O990836O
(14) Hahn, M. G.; Darvill, A.; Albersheim, P.; Bergmann, C.; Cheong,
J .-J .; Koller, A.; Lo`, V.-M. Molecular Plant Pathology (Volume II): A
Practical Approach; Gurr, S., McPherson, M., Bowles, D. J ., Eds.;
Oxford University Press: Oxford, 1992; pp 103-147.
(15) Chizzonite, R.; Truitt, T.; Podlaski, F. J .; Wolitzky, A. G.; Quinn,
P. M.; Nunes, P.; Stern, A. S.; Gately, M. K. J . Immunology 1991, 147,
1548-1556.