Note: Descriptions are shown in the official language in which they were submitted.
CA 02668905 2009-06-15
B&P File No. 13764-86/PF
TITLE: METHOD FOR INHIBITING BRASSININ OXIDASE
FIELD OF THE DISCLOSURE
The present disclosure relates to a method for inhibiting brassinin oxidase.
In
particular, the method relates to inhibiting brassinin oxidase in using
specific phytoalexin
detoxification inhibitors (paldoxins).
BACKGROUND OF THE DISCLOSURE
Phytoalexins are antimicrobial secondary metabolites produced by plants in
response to stress, including bacterial and fungal infection, heat, heavy
metal salts, and
UV radiation.' In general, cruciferous phytoalexins, produced by plants of the
family
Brassicaceae (common name crucifer), are biosynthesized from tryptophan and
are
produced as blends whose composition depends on the plant species and on the
particular elicitor (stress factor).2 Brassinin (1) is an essential
phytoalexin due to its role
as biosynthetic precursor of other cruciferous phytoalexins and its
antimicrobial activity.
The dithiocarbamate group of brassinin (1) is the toxophore responsible for
its fairly
broad antifungal activity.3 To the detriment of many agriculturally important
crops,
several pathogenic fungi of crucifers are able to overcome phytoalexins, such
as
brassinin' by detoxification.4 These detoxification reactions are induced only
in the
presence of the phytoalexin, but can rather quickly deprive the plant of its
defense
chemicals and facilitate an outcome favoring the fungal pathogen.
Cruciferous species include a wide variety of crops cultivated worldwide, for
example, the oil seeds canola (Brassica napus and B. rapa L.) and rapeseed (B.
napus
and B. rapa) and many vegetables, such as rutabaga (B. napus ssp. napobrassica
L.),
turnip (B. rapa ssp. raps L.) and cauliflower (B. oleraceae var. botrytis).
Economically
significant diseases of the oil seeds canola and rapeseed caused by fungi such
as the
"blackleg" fungi [Leptosphaeria maculans (asexual stage Phoma lingam) and L.
biglobosa] are a global issue. L. maculans is a pathogen with well-established
stratagems to invade crucifers, including production of specific enzymes in
response to
the phytoalexin, such as the phytoalexin brassinin (1), that detoxify
essential plant
defenses.4
The compounds 5-fluorocamalexin, 6-fluorocamalexin, 1 -naphtha lenyl isoth
iazole,
5
1-naphthalenylisothiazole and camalexin have been studied as antifungal
agents., 6
CA 02668905 2009-06-15
SUMMARY OF THE DISCLOSURE
The present disclosure relates to a method for inhibiting brassinin oxidase in
plant pathogens.
Accordingly, the method of the present disclosure includes a method for
inhibiting
brassinin oxidase (BO), comprising treating a pathogen that is producing BO,
with an
effective amount of:
(a) a compound of Formula I:
R1 R3
R2
wherein,
A is a 5- or 6-membered unsaturated ring containing carbon and, optionally,
nitrogen
atoms;
R1 and R2 are independently or simultaneously selected from H, F, Ci-C4alkyl,
Cl, NO2,
OH, OC,-C4alkyl, NH2, N(H)(C,-C4alkyl) and N(C1-C4alkyl)(C1-C4alkyl); and
R3 is a 5- or 6-membered unsaturated ring containing carbon and, optionally,
nitrogen
and/or sulfur atoms, that is bonded to a carbon atom in A, provided that when
R3 is
phenyl, R1 and R2 are not both H;
(b) a salt or solvate of the compound of Formula I;
(c) a compound selected from cyclobrassinin and isobrassinin;
(d) a salt or solvate of cyclobrassinin and isobrassinin; or
(e) a mixture of one or more of the compounds in (a), (b), (c) and (d).
In another embodiment of the disclosure, there is provided a use for
inhibiting
brassinin oxidase (BO) in a pathogen that is producing BO, of
(a) a compound of Formula I:
R1
Rs
/ A (I)
R2
wherein,
A is a 5- or 6-membered unsaturated ring containing carbon and, optionally,
nitrogen
atoms;
R1 and R2 are independently or simultaneously selected from H, F, C1-C4alkyl,
Cl, NO2,
OH, OCR-C4alkyl, NH2, N(H)(C,-C4alkyl) and N(C,-C4alkyl)(C1-C4alkyl); and
2
CA 02668905 2009-06-15
R3 is a 5- or 6-membered unsaturated ring containing carbon and, optionally,
nitrogen
and/or sulfur atoms, that is bonded to a carbon atom in A, provided that when
R3 is
phenyl, R1 and R2 are not both H;
(b) a salt or solvate of the compound of Formula I;
(c) a compound selected from cyclobrassinin and isobrassinin;
(d) a salt or solvate of cyclobrassinin and isobrassinin; or
(e) a mixture of one or more of the compounds in (a), (b), (c) and (d).
Also included in the present disclosure are agricultural kits comprising
(I) one or more compounds selected from:
(a) a compound of Formula I:
R1
R3
I A
R2
wherein,
A is a 5- or 6-membered unsaturated ring containing carbon and, optionally,
nitrogen
atoms;
R1 and R2 are independently or simultaneously selected from H, F, C,-C4alkyl,
Cl, NO2,
OH, OCR-C4alkyl, NH2, N(H)(C1-C4alkyl) and N(C1-C4alkyl)(C1-C4alkyl); and
R3 is a 5- or 6-membered unsaturated ring containing carbon and, optionally,
nitrogen
and/or sulfur atoms, that is bonded to a carbon atom in A, provided that when
R3 is
phenyl, R1 and R2 are not both H;
(b) a salt or solvate of the compound of Formula I;
(c) a compound selected from cyclobrassinin and isobrassinin; and
(d) a salt or solvate of cyclobrassinin and isobrassinin,
said compounds, salts and/or solvates being optionally combined with one or
more
agriculturally acceptable carriers or agriculturally acceptable excipients,
and
(II) instructions for use of the one or more compounds to inhibit brassinin
oxidase (BO)
in a pathogen that is producing BO.
The enzyme involved in the oxidative detoxification of brassinin (1) by L.
maculans, brassinin oxidase (BO), was purified and shown to catalyze the
transformation of brassinin (1) to indole-3-carboxaldehyde (3, Scheme 1), a
nonantifungal metabolite. This transformation appears to have no counterpart
in the
microbial metabolism of dithiocarbamates, despite the wide use of
dithiocarbamates as
3
CA 02668905 2009-06-15
fungicides for many decades.' BO was stable and appeared to exhibit substrate
specificity.
Scheme 1
S
CHO
H N )" SCH3
N C6N
H H
~1) (3)
Accordingly, in another embodiment of the disclosure, there is provided a use
of
a purified and isolated BO enzyme for the identification of inhibitors of the
BO.
In another embodiment of the disclosure, there is also provided a method for
assaying and identifying inhibitors of the BO comprising:
i) contacting a purified and isolated BO with a substrate in the presence of
one or
more test substances; and
ii) determining an amount of conversion of the substrate in the presence of
the
one or more test substances compared to a control,
wherein if conversion of the substrate is less in the presence of the one or
more test
substances compared to the control, then the one or more test substances are
inhibitors
of BO.
Other features and advantages of the present disclosure will become apparent
from the following detailed description. It should be understood, however,
that the
detailed description and the specific examples while indicating preferred
embodiments
of the disclosure are given by way of illustration only, since various changes
and
modifications within the spirit and scope of the disclosure will become
apparent to those
skilled in the art from this detailed description.
DETAILED DESCRIPTION OF THE DISCLOSURE
DEFINITIONS
4
CA 02668905 2009-06-15
The term "C1_nalkyl" as used herein means a straight and/or branched chain,
saturated alkyl group containing from one to "n" carbon atoms and includes
(depending
on the identity of n) methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl,
isobutyl and t-butyl,
where the variable n is an integer representing the largest number of carbon
atoms in
the alkyl radical.
The term "OCi_nalkyl" as used herein means a straight and/or branched chain,
saturated alkoxy group containing from one to "n" carbon atoms and includes
(depending on the identity of n) methoxy, ethoxy, propoxy, isopropoxy, n-
butoxy, s-
butoxy, isobutoxy and t-butoxy, where the variable n is an integer
representing the
largest number of carbon atoms in the alkyl radical.
The term "unsaturated" as used herein means that the referenced group contains
at least one unsaturated bond. An "unsaturated" bond is a double or triple
bond.
Suitably the unsaturated bond is a double bond. Unsaturated rings include non-
aromatic and aromatic rings.
Cyclobrassinin is a compound of the formula:
N
)-SCH3
S
N
H
Isobrassinin is a compound of the formula:
~SCH3
HN--'~\
S
Thiabendazole is a compound of the formula:
CN S
H N
5
CA 02668905 2009-06-15
Camalexin is a compound of the Formula:
N\ S
1011 a N
H
The term "salt" as used herein means any plant compatible organic or inorganic
acid or basic addition salt of any neutral compound. Illustrative inorganic
acids which
form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric
acids, as
well as metal salts such as sodium monohydrogen orthophosphate and potassium
hydrogen sulfate. Illustrative organic acids that form suitable salts include
mono-, di-,
and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic,
glutaric,
fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic,
cinnamic and
salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and
methanesulfonic
acids. Illustrative inorganic bases which form suitable salts include lithium,
sodium,
potassium, calcium, magnesium or barium hydroxide. Illustrative organic bases
which
form suitable salts include aliphatic, alicyclic or aromatic organic amines
such as
methylamine, trimethylamine and picoline, alkylammonias or ammonia. Either the
mono
or di-acid/base salts can be formed, and such salts may exist in either
solvated or
substantially anhydrous form. In general, salts are more soluble in water and
various
hydrophilic organic solvents, and generally demonstrate higher melting points
in
comparison to their free base forms. The selection of the appropriate salt
will be known
to one skilled in the art. The formation of a desired compound salt is
achieved using
standard techniques. For example, the neutral compound is treated with an acid
or
base in a suitable solvent and the formed salt is isolated by filtration,
extraction or any
other suitable method
The term "solvate" as used herein means a compound or its pharmaceutically
acceptable salt, wherein molecules of a suitable solvent are incorporated in
the crystal
lattice. A suitable solvent is plant tolerable at the dosage administered.
Examples of
suitable solvents are ethanol, water and the like. When water is the solvent,
the
molecule is referred to as a "hydrate". The formation of solvates of compounds
will vary
6
CA 02668905 2009-06-15
depending on the compound and the solvate. In general, solvates are formed by
dissolving the compound in the appropriate solvent and isolating the solvate
by cooling
or using an antisolvent. The solvate is typically dried or azeotroped under
ambient
conditions.
As used herein, the phrase "effective amount" means an amount effective of one
or more compounds, at dosages and for periods of time necessary to achieve the
desired result. For example in the context of inhibiting BO activity, an
effective amount is
an amount that, for example, inhibits BO activity compared to the response
obtained
without administration or use of the compound(s). Effective amounts may vary
according
to factors such as the identity of the pathogen, the identity of the plant,
the identity of the
one or more compounds and the environmental conditions, and the like, but can
nevertheless be routinely determined by one skilled in the art.
As used herein, to "inhibit" or "suppress" or "reduce" a function or activity,
such
BO activity, is to reduce the function or activity when compared to a control,
for
example, otherwise same conditions except for a condition or parameter of
interest, or
alternatively, as compared to another condition. The terms "inhibitor" and
"inhibition", in
the context of the present disclosure, are intended to have a broad meaning
and
encompass compounds which directly or indirectly (e.g., via reactive
intermediates,
metabolites and the like) act on BO.
In understanding the scope of the present disclosure, the term "comprising"
and
its derivatives, as used herein, are intended to be open ended terms that
specify the
presence of the stated features, elements, components, groups, integers,
and/or steps,
but do not exclude the presence of other unstated features, elements,
components,
groups, integers and/or steps. The foregoing also applies to words having
similar
meanings such as the terms, "including", "having" and their derivatives.
Finally, terms of
degree such as "substantially", "about" and "approximately" as used herein
mean a
reasonable amount of deviation of the modified term such that the end result
is not
significantly changed. These terms of degree should be construed as including
a
deviation of at least 5% of the modified term if this deviation would not
negate the
meaning of the word it modifies.
METHODS OF THE DISCLOSURE
Selective BO inhibitors have been developed to prevent fungal detoxification
of
brassinin (1) in infected plants. Paldoxin is a term coined to address this
new class of
7
CA 02668905 2009-06-15
fungal enzyme inhibitors (phyloalexin detoxification inhibitors), that
conceptualizes a
new generation of chemicals designed, for example, for sustainable treatments
of
agricultural crops. Paldoxins are envisioned to inhibit unique metabolic
reactions in
fungal phytopathogens and, therefore, are less likely to affect non-targeted
organisms
and, thus, are expected to have minimal impact on cultivated ecosystems.
Having, on
hand, BO that has been purified to homogeneity has allowed the identification,
for the
first time, compounds that will selectively inhibit this enzyme and thereby
inhibit the
detoxification of brassinin oxidase by plant pathogens. This allows the plant
to maintain
its defense mechanism against pathogens that produce the detoxifying enzyme,
BO, in
response to the plant's production of brassinin.
The present disclosure accordingly relates to a method for inhibiting
brassinin
oxidase (BO), comprising treating a pathogen that is producing BO, with an
effective
amount of
(a) a compound of Formula I:
R1
R3
R2
wherein,
A is a 5- or 6-membered unsaturated ring containing carbon and, optionally,
nitrogen
atoms;
R1 and R2 are independently or simultaneously selected from H, F, C1-C4alkyl,
Cl, NO2,
OH, OC1-C4alkyl, NH2, N(H)(Ci-C4alkyl) and N(Ci-C4alkyl)(C1-C4alkyl); and
R3 is a 5- or 6-membered unsaturated ring containing carbon and, optionally,
nitrogen
and/or sulfur atoms, that is bonded to a carbon atom in A, provided that when
R3 is
phenyl, R1 and R2 are not both H;
(b) a salt or solvate of the compound of Formula I;
(c) a compound selected from cyclobrassinin and isobrassinin;
(d) a salt or solvate of cyclobrassinin and isobrassinin; or
(e) a mixture of one or more of the compounds in (a), (b), (c) and (d).
In another embodiment of the disclosure, there is provided a use for
inhibiting
brassinin oxidase (BO) in a pathogen that is producing BO, of
(a) a compound of Formula I:
8
CA 02668905 2009-06-15
R1
R3
A (~)
R2
wherein,
A is a 5- or 6-membered unsaturated ring containing carbon and, optionally,
nitrogen
atoms;
R1 and R2 are independently or simultaneously selected from H, F, C1-C4alkyl,
Cl, NO2,
OH, OC1-C4alkyl, NH2, N(H)(C1-C4alkyl) and N(C1-C4alkyl)(Ci-C4alkyl); and
R3 is a 5- or 6-membered unsaturated ring containing carbon and, optionally,
nitrogen
and/or sulfur atoms, that is bonded to a carbon atom in A, provided that when
R3 is
phenyl, R1 and R2 are not both H;
(b) a salt or solvate of the compound of Formula I;
(c) a compound selected from cyclobrassinin and isobrassinin;
(d) a salt or solvate of cyclobrassinin and isobrassinin; or
(e) a mixture of one or more of the compounds in (a), (b), (c) and (d).
In an embodiment of the methods and uses of the disclosure, R1 and R2 in the
compounds of Formula I are independently or simultaneously selected from H, F,
C1-
C2alkyl, Cl, NO2, OH, OC1-C4alkyl, NH2, N(H)(Ci-C2alkyl) and N(Ci-C2alkyl)(C1-
C2alkyl).
In a further embodiment, R1 and R2 are independently or simultaneously
selected from
H, F and OC1-C2alkyl. In another embodiment, R1 and R2 are independently or
simultaneously selected from H, F and OCi-C2alkyl. In a further embodiment, R1
and R2
are independently or simultaneously selected from H, F and OCH3.
In an embodiment of the methods and uses of the present disclosure, the
pathogen that is producing BO is treated with an effective amount of one or
more
compounds selected from:
9
CA 02668905 2009-06-15
N~,
S S S S
F H3CO
H F H H H3CO
H
N
S N SCH3
SCH3 HN4
F H H H 0::H
N-S
N
S
(:::CN SD H N and
and salts and solvates thereof.
In the uses and methods of the present disclosure the compounds inhibit
brassinin oxidase (BO) in a pathogen that is producing BO. In an embodiment,
the
inhibition of the pathogen is performed by administration of the compound(s)
to a plant
that has been infected with a pathogen and the pathogen has been induced to
produce
BO. BO is induced only when the pathogen has been challenged or exposed to
brassinin. The pathogen may be any organism that produces BO in response to
exposure to brassinin. In an embodiment, the pathogen is a fungus. In a
further
embodiment, the pathogen is Leptosphaeria maculans.
Also included in the present disclosure are agricultural kits comprising
(I) one or more compounds selected from:
(a) a compound of Formula I:
R1
R3
A
R2
wherein,
A is a 5- or 6-membered unsaturated ring containing carbon and, optionally,
nitrogen
atoms;
CA 02668905 2009-06-15
R1 and R2 are independently or simultaneously selected from H, F, Cl-C4alkyl,
Cl, NO2,
OH, OC1-C4alkyl, NH2, N(H)(C1-C4alkyl) and N(Ci-C4alkyl)(C1-C4alkyl); and
R3 is a 5- or 6-membered unsaturated ring containing carbon and, optionally,
nitrogen
and/or sulfur atoms, that is bonded to a carbon atom in A, provided that when
R3 is
phenyl, R1 and R2 are not both H;
(b) a salt or solvate of the compound of Formula I;
(c) a compound selected from cyclobrassinin and isobrassinin; and
(d) a salt or solvate of cyclobrassinin and isobrassinin,
said compounds, salts and/or solvates being optionally combined with one or
more
agriculturally acceptable carriers or agriculturally acceptable excipients,
and
(II) instructions for use of the one or more compounds to inhibit brassinin
oxidase (BO)
in a pathogen that is producing BO.
In an embodiment of disclosure, the agricultural kits comprise a compound of
Formula I in which R1 and R2 are independently or simultaneously selected from
H, F,
Cl-C2alkyl, Cl, NO2, OH, OCR-C4alkyl, NH2, N(H)(Ci-C2alkyl) and N(C,-
C2alkyl)(C1-
C2alkyl). In a further embodiment, R1 and R2 are independently or
simultaneously
selected from H, F and OC1-C2alkyl. In another embodiment, the agricultural
kits
comprise a compound of Formula I in which R1 and R2 are independently or
simultaneously selected from H, F and OC1-C2alkyl. In a further embodiment,
the
agricultural kits comprise a compound of Formula I in which R1 and R2 are
independently or simultaneously selected from H, F and OCH3.
In another embodiment, the agricultural kits of the present disclosure,
comprise
one or more compounds selected from:
11
CA 02668905 2009-06-15
S S IS
F H3CO
H F / H H H3CO H
N
S N SCH3
\ \ \ )--SCH3 HN~
s
SN S
F N N
H H H H
N-S
N
S
and
and salts and solvates thereof.
An agriculturally acceptable carrier may be solid, liquid or both. Solid
carriers are
essentially: mineral earth such as silicas, silica gels, silicates, talc,
kaolin,
montmorillonite, attapulgite, pumice, sepiolite, bentonite, limestone, lime,
chalk, bole,
loes, clay, dolomite, diatomaceous earth, calcite, calcium sulfate, magnesium
sulfate,
magnesium sulfate, magnesium oxide, sand, ground plastics, ferilizers such as
ammonium sulfate, ammonium phosphate, ammonium nitrate, ureas, and crushed
products of vegetable origin such as cereal meal, tree bark meal, wood meal
and
nutshell meal, cellulose powders, or other solid carriers. Optional excipients
include
organic solvents, water, surface active agents, and granular or particulate
solid carriers.
Optionally included in the kits are other substances, such as a stabilizer, an
emetic agent, a disintegrating agent, an antifoaming agent, a wetting agent, a
dispersing
agent, a binding agent, dye(s), fillers, carriers, surface active compounds
(surfactants),
and optionally solid and/or liquid auxiliaries and/or adjuvants such as
wetters,
adhesives, dispersants or emulsifiers. In an embodiment of the disclosure,
such
substances are included in a composition with the one or more compounds and
carriers/excipients.
Accordingly, in another embodiment of the disclosure, there is provided a use
of
a purified and isolated BO enzyme for the identification of inhibitors of the
BO.
12
CA 02668905 2009-06-15
In another embodiment of the disclosure, there is also provided a method for
assaying and identifying inhibitors of the BO comprising:
i) contacting a purified and isolated BO with a substrate in the presence of
one or
more test substances; and
ii) determining an amount of conversion of the substrate in the presence of
the
one or more test substances compared to a control,
wherein if conversion of the substrate is less in the presence of the one or
more test
substances compared to the control, then the one or more test substances are
inhibitors
of BO.
In an embodiment, the control is an equivalent reactions set up, except for
the
absence of the one or more test substances. The one or more test substances
can be
any compound which one wishes to screen for inhibition of BO including, but
not limited
to, proteins (including antibodies), peptides, nucleic acids, fragments of
proteins,
peptides, carbohydrates, organic compounds, inorganic compounds and natural
products. The one or more test compounds may also be, for example, a reaction
mixture, library extracts, combinatorial libraries, bodily fluids and other
samples that one
wishes to test for the BO actitivity.
The following non-limiting examples are illustrative of the present
disclosure:
EXAMPLES
Materials and Methods
All solvents were HPLC grade and used as such. Organic extracts were dried
over anhydrous Na2SO4 and solvents removed under reduced pressure in a rotary
evaporator. HPLC analysis was carried out with a high performance liquid
chromatograph equipped with quaternary pump, automatic injector, and diode
array
detector (wavelength range 190-600 nm), degasser, and a Hypersil ODS column (5
pm
particle size silica, 4.6 W. x 200 mm), equipped with an in-line filter.
Mobile phase: 75%
H20-25% CH3CN to 100% CH3CN, for 35 min, linear gradient, and a flow rate 1.0
mUmin. Fourier transform IR spectra were obtained on a Bio-Rad FTS40
spectrometer
in KBr. NMR spectra were recorded on 500 MHz spectrometers; S values were
referenced as follows: for 1H (500 MHz), CDCI3, 7.27 ppm; for 13C (125 MHz),
CDCI3,
77.23 ppm. Mass spectra (MS) were obtained on a VG 70 SE mass spectrometer
using
a solids probe or on a Q Star XL, Applied Biosystems.
All synthetic compounds were purified using flash column chromatography (FCC)
13
CA 02668905 2009-06-15
on silica gel; satisfactory spectroscopic data identical to those previously
reported were
obtained for all previously reported compounds. Synthesis of brassinin and
cyclobrassinin (1) was carried out as reported in the literature.2
Thiabendazole was
purchased from Sigma Aldrich, Oakville, Ontario, Canada.
Example 1: Compound 1(a)
Compound I(a) was synthesized as reported in the literature.5
S
F ~
/ N I(a)
H
Example 2: Compound 1(b)
Compound I(b) was synthesized as reported in the literature.9
N\ S
F hNi I(b)
Example 3: Compound 1(c)
Compound I(c) was synthesized as reported in the literature.10
N s
H3CO
H I(c)
Example 4: Compound 1(d)
Compound I(d) was synthesized as reported in the literature.10
14
CA 02668905 2009-06-15
Id\ S
H3CO H I(d)
Example 5: Compound 1(e)
Compound I(e) was synthesized as reported in the literature."
F / H I(e)
Example 6: Compound 1(f)
Compound 1(f) was synthesized as reported in the literatures
N-S
i i 1(f).
Example 7: Compound 1(g)
Compound I(g) was synthesized as reported in the literature. 6
N
S
1(g).
Example 8: Compound 1(h)
Compound I(h) was synthesized as reported in the literature.10
CA 02668905 2009-06-15
N S
S
H H I(h).
Example 9: Isobrassinin
Isobrassinin was synthesized as reported in the literature.13
SCH3
HN4
H isobrassinin
Example 10: Fungal Cultures
Liquid cultures of L. maculans (virulent isolate BJ-125, IBCN collection,
AAFC)
were handled as described in the literature.13 Fungal spores were subcultured
on V8
agar under continuous light at 23 VC; after 15 days, fungal spores were
collected
aseptically and stored at -20 C.14 Liquid cultures were initiated by
inoculating minimal
media (100 mL)15 with fungal spores at 107/mL in Erlenmeyer flasks, followed
by
incubation on a shaker under constant light at 23 1 C.
For purification of brassinin oxidase, 600 mL of 48 h old liquid cultures
prepared
as described above, were incubated with 3-phenylindole (19, 0.05 mM final
concentration in cultures to induce BO) for an additional 24 h and then
gravity filtered to
separate mycelia from culture broth. The mycelia was stored at -20 C up to 72
h and
used to obtain protein extracts containing BO activity.
For analysis of BO induction, 72 h old liquid cultures (20 mL) were co-
incubated
with test compounds (final concentrations in culture 0.10, 0.20, and 0.50 mM),
and after
an additional incubation for 24 h, the mycelia were separated from the culture
broth by
filtration.
Example 11: Preparation of Protein Extracts for Analysis of 80
Frozen mycelia (0.3-1.4 g) from L. maculans were suspended in ice-cold
extraction buffer (1 mL) and ground (mortar) for 5 min. The extraction buffer
consisted
of diethanolamine (DEA, 25 mM, pH 8.3), 5% (v/v) glycerol, D,L-dithiothreitol
(DTT, 1
16
CA 02668905 2009-06-15
mM), and 1/200 (v/v) protease inhibitor cocktail (P-8215, Sigma-Aldrich
Canada). The
homogenate was centrifuged at 4 C for 30 min at 50000 g. The resulting
supernatant
was used for determination of specific activity of BO. Protein concentrations
were
determined as described by Bradford16 using the Coomassie Brilliant Blue
method with
BSA as a standard.
Example 12: BO Activity Assay
The reaction mixture contained DEA (20 mM, pH 8.3), DTT (1 mM), 0.1 % (v/v)
Triton X-100, brassinin (1, 1.0 mM),, phenazine (0.50 mM), and protein extract
(50-100
pL) in a total volume of 500 pL. The reaction was carried out at 24 C for 20
min. A
control reaction was stopped by the addition of EtOAc (2 mL) at t = 0. The
product was
extracted with EtOAc (2 mL) and concentrated to dryness. The extract was
dissolved in
CH3CN (200 pL) and analyzed by HPLC-DAD. The amounts of brassinin (1) and
indole-
3-carboxaldehyde (3) in the reaction assay were determined using calibration
curves
built with pure cyclobrassinin. One enzyme unit (U) is defined as the amount
of the
enzyme that catalyzes the conversion of one micromole of substrate per minute
(pmol=min-1 = U).
Example 13: Chromatographic Purification of BO and Inhibitory Effect of
Paldoxins
The purification of BO was performed in four steps as previously described.17
The purified enzyme was used for screening test compounds for inhibitory
activity. To
determine potential inhibitors of BO, inhibition experiments were carried out
using
brassinin (1, 0.10 mM final concentration) and test compounds (0.10 and 0.30
mM final
concentrations). Standard deviation values for assays were determined from
four
independent experiments.
Discussion
The inhibitory effects of test compounds on BO activity were tested at 0.10 mM
and 0.30 mM using brassinin (1) as substrate (0.10 mM) and purified BO.
Thiabendazole, a common fungicide, was used as the reference compound due to
its
commercial availability and BO inhibitory activity (ca. 25% at 0.30 mM).17 The
concentrations of inhibitors were based on the Km of BO for brassinin (1, 0.15
mM
under the enzyme assay conditions), the natural substrate. Results of these
enzymatic
assays are summarized in Table 1.
17
CA 02668905 2009-06-15
Relative to camalexin, 5-methoxycamalexin I(c) was the most potent inhibitor
of
BO activity (ca. 72% at 0.30 mM), followed by 5-fiuorocamalexin I(a) and 6-
methoxycamalexin I(b), a natural phytoalexin2 (ca. 63% at 0.30 mM). The
inhibitory
effect of the 6-fluoro derivative I(b) (46%, 0.30 mM) on BO activity was
similar to that of
camalexin (53%, 0.30 mM).
Example 14: Inducer Effect of Paldoxins
Mycelial cultures of L. maculans were incubated with the test compounds (0.10,
0.20, and 0.50 mM) for 24 h to evaluate potential induction of BO activity.
The cultures
were filtered, the mycelia were extracted with extraction buffer, and the
resulting cell-
free extracts were analyzed for BO activity using brassinin (1) as substrate.
The total
protein content of each cell-free extract was determined using a calibration
curve built
using BSA.
Discussion
The results of these analyses are summarized in Table 2. In general, relative
to
control cultures all tested compounds induced BO activity, although there were
substantial differences in the percentage of induction. For example, relative
to controls,
camalexin induced the highest amount of BO activity at 0.20 mM (6.9 0.3).
Compound
added to mycelial cultures
18
CA 02668905 2009-06-15
Table 1: Inhibitory Effect of Paldoxins on BO
Compound Name Structure Inhibition
0.10 mm 0.30 mM
Thiabendazole \-~ 16 3 25 7
N
S
Camalexin 30 t 4 53 t 4
NS
H SCH3
HN(
Isobrassinin I \ s 11 t 5 23 t 6
N
H
N
~-SCH3
Cyclobrassinin I \ s 23 t 6 37 8
N
H
S
5-Fluor i caamalexin F \ 47 t 5 63 2
N
H
\
N S
6-Fluorocamalexin 29 t 10 46 2
1(b)
F N
H
N\ S
5-Methoxycamalexin H3CO 51 # 4 72 1
(c) \
N
H
S
6-Methoxycamalexin 41 t 6 63 5
NSN
I(d) ( H3C0 H
19
CA 02668905 2009-06-15
6-Fluoro-3- 11 t 4 17 6
phenylindole I(e)
F N
H
N-S
1-naphthalenyl- 16 4 21 6
isothiazole 1(f)
(",,N
2-naphthalenyl- s 29 2 42 3
isothiazole I(g)
N\ S
Camalexin I(h) 30 t 4 53 7
N
H
CA 02668905 2009-06-15
Table 2: Inducer and Antifungal Activity of Inhibitors
Compound Added to Concentration Relative Relative
mycelial cultures (#) (mM) Specific Activity Protein
of BO
control culture 1.0 1.0
camalexin 0.10 4.5 0.5 0.28
0.20 6.9+0.3 0.24
0.50 2.5 0.1 0.10
5-fluorocamalexin I(a) 0.10 5.0 0.2 0.30
0.20 4.9 0.4 0.23
0.50 7.9 0.6 0.19
6-fluorocamalexin 1(b) 0.10 3.9 0.1 0.36
0.20 4.0 0.2 0.28
0.50 1.8 0.3 0.24
5-methoxycamaiexin I(c) 0.10 1.7 0.3 0.28
0.20 5.8 0.6 0.24
0.50 1.6 0.3 0.13
6-methoxycamalexin I(d) 0.10 2.3 0.1 0.27
0.20 3.7 0.2 0.16
0.50 4.0 0.1 0.11
thiabendazole 0.10 1.9 0.2 0.35
0.20 4.1 0.2 0.28
0.50 4.9 0.3 0.06
2-naphthalenyl-isothiazole 1(f) 0.1 2.6 0.2 0.26
0.2 8.5 0.1 0.17
0.5 16.3 3.3 0.16
21
CA 02668905 2009-06-15
FULL CITATIONS FOR DOCUMENTS REFERRED TO IN THE SPECIFICATION
(1) Bailey, J. A.; Mansfield, J. W. Phytoalexins; Bailey, Blackie and Son:
Glasgow, U.K.,
1982; P 334.
(2) Pedras, M. S. C.; Zheng, Q. A.; Sarma-Mamillapalle, V. K. The phytoalexins
from
Brassicaceae: Structure, biological activity, synthesis, and biosynthesis.
Nat. Prod.
Commun. 2007, 2, 319330.
(3) Pedras, M. S. C. The chemical ecology of crucifers and their fungal
pathogens:
Boosting plant defenses and inhibiting pathogen invasion. Chem. Rec. 2008, 8,
109-
115.
(4) Pedras, M. S. C.; Ahiahonu, P. W. K. Metabolism and detoxification of
phytoalexins
and analogs by phytopathogenic fungi. Phytochemistry 2005, 66, 391-411.
(5) Pedras, M. S. C.; Liu, J. Designer phytoalexins: probing camalexin
detoxification
pathways in the phytopathogen Rhizoctonia solani. Org. Biomol. Chem. 2004, 2,
1070-1076.
(6) Pedras, M. S. C.; Suchy, M. Design, synthesis and antifungal activity of
inhibitors of
brassinin detoxification in the plant pathogenic fungus Leptosphaeria
maculans.
Bioorg. Med. Chem. 2006, 14, 714-723.
(7) Pedras, M. S. C.; Gadagi, R.S.; Jha, M.; Sarma-Mamillapalle, V. K.
Detoxification of
the phytoalexin brassinin by isolates of Leptosphaeria maculans pathogenic on
brown mustard involves an inducible hydrolase. Phytochemistry 2007, 68, 1572-
1578.
(8) Russell, P. E. A century of fungicide evolution. J. Agric. Sci. 2005, 143,
11-25.
(9) Pedras, M. S. C.; Ahiahonu, P. W. K. Probing the phytopathogenic stem rot
fungus
with phytoalexins and analogues: unprecedented glucosylation of camalexin and
6-
methoxycamalexin. Bioorg. Med. Chem. 2002, 10, 3307-3312.
(10) Ayer, W. A.; Craw, P. A; Ma, Y. T.; Miao, S. Synthesis of camalexin and
related
phytoalexins. Tetrahedron 1992, 48, 2919-2924.
(11) Pedras, M. S. C.; Hossain, M. Design, synthesis, and evaluation of
potential
inhibitors of brassinin g I ucosyltra nsfe rase, a phytoalexin detoxifying
enzyme from
Sclerotinia sclerotiorum. Bioorg. Med. Chem. 2007, 15, 5981-5996.
(12) Pedras, M. S. C.; Suchy, M.; Ahiahonu, P. W. K. Unprecedented chemical
structure
and biomimetic synthesis of erucalexin, a phytoalexin from the wild crucifer
22
CA 02668905 2009-06-15
Erucastrum gallicum. Org. Biomol. Chem. 2006, 4, 691-701.
(13) Pedras, M. S. C.; Suchy, M. Detoxification pathways of the phytoalexins
brassilexin
and sinalexin in Leptosphaeria maculans: Isolation and synthesis of the
elusive
intermediate 3-formylindolyl2-sulfonic acid. argo Biomol. Chem. 2005,3,2002-
2007.
(14) Pedras, M. S. C.; Khan, A. Q. Biotransformation of the brassica
phytoalexin
brassicanal A by the blackleg fungus. J. Agric. Food Chem. 1996,44,3403-3407.
(15) Pedras, M. S. C.; Biesenthal, C. J. Production of the host-selective
toxin phomalide
by isolation of Leptosphaeria maculans and its correlation with sirodesmin PL
production. Can. J. Microbiol. 1998,44, 547-553.
(16) Bradford, M. M. A rapid and sensitive method for quantitation of
microgram
quantities of protein utilizing the principle of protein-dye-binding. Anal.
Biochem.
1976, 72, 248-254.
(17) Pedras M. S. C.; Minic, Z. Jha, M. Brassinin oxidase, a fungal
detoxifying enzyme
to overcome a plant defense - Purification, characterization and inhibition.
FEBS J.
2008, 275, 3691-3705.
23
