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1397
and the components eluted with CH3CN after scraping each band.
The components eluted from the plate were analyzed by LC-HRMS,
HPLC-DAD (see Fig. S1), and 1H NMR (CD2Cl2). S-Methyl dithiocar-
bamate (6) was stable in the solvents used for TLC separation and
extraction at least for 24 h.
Mycelial growth inhibition assays of L. maculans isolate BJ 125
were carried out as previously described23 using S-methyl dithio-
carbamate (6) at three concentrations (0.50, 0.25, 0.12 mM). The
mycelial growth inhibition was determined relative to control cul-
tures grown in the absence of compound 6, but otherwise identical
conditions.
indolylmethyl)carbamate (5a) as a white solid (27 mg, 73% yield).
1H NMR (500.3 MHz, CD3CN): d 9.17 (br s, NH), 7.62 (d, J = 8 Hz,
1H), 7.40 (d, J = 8 Hz, 1H), 7.18 (s, 1H), 7.14 (dd, J = 8, 8 Hz, 1H),
7.06 (J = 8, 8 Hz, 1H), 5.78 (br s, NH), 3.6 (s, 3H) (only dideuterated
5a was detected). 13C NMR (125.8 MHz, CD3CN): d 158.3 (s), 137.9
(s), 127.9 (s), 124.8 (d), 123.0 (d), 120.4 (d), 120.0 (s), 114.3 (s),
112.7 (d), 52.7 (quartet), 36.8 (quintet, JC–D = 21.5 Hz). HRMS-EI:
m/z measured 206.1028 (206.1024 calcd for C11H102H2N2O2).
4.3.4. S-Methyl dithiocarbamate (6)
S-Methyl dithiocarbamate was prepared after modification of a
previously published procedure.25 MeI (321 mg, 2.26 mmol) was
added dropwise to a cooled suspension of ammonium dithiocarba-
mate (250 mg, 1.97 mmol) in EtOH (3 mL) at 0 °C and the reaction
mixture was stirred under argon for 4 h. The reaction mixture was
concentrated (ca. 1/2 volume), diluted with water and the resulting
suspension was filtered. The solid was dissolved in Et2O (5 mL) and
precipitated by adding n-pentane. Filtration of the precipitate
afforded S-methyl dithiocarbamate (152 mg, 72% yield) with a
mp 42–44 °C (lit value: 41 °C,25). HRMS-EI: m/z measured
106.9865 (106.9863 calcd for C2H5NS2).
4.3. Synthesis
4.3.1. [10,10-2H2]Brassinin (1a)
A solution of 3-indolecarbonitrile (50 mg, 0.35 mmol) in THF
(0.5 mL) was added to a suspension of lithium aluminum deuteride
(30 mg, 0.70 mmol) in dry THF (0.5 mL) and stirred at 55–60 °C for
4 h. The reaction mixture was cooled to room temperature, diluted
with NaOH (1 M, 0.5 mL) and filtered. The filtrate was diluted with
water (10 mL) and extracted with EtOAc (3 ꢁ 20 mL). The com-
bined organic extract was dried over Na2SO4 and concentrated.
The residue was separated (SiO2, CH2Cl2/MeOH/NH4OH) to afford
[10,10-2H2]indolyl-3-methanamine as a white solid (27 mg, 55%
yield). Carbon disulfide (12 mg, 0.152 mmol) was added to a solu-
tion of [10,10-2H2]indolyl-3-methanamine (15 mg, 0.10 mmol) and
Et3N (21 mg, 0.202 mmol) in pyridine (1 mL) and stirred at rt for
15 min. Next, MeI (180 mg, 1.27 mmol) was added and stirring
was continued for another 20 min. The reaction mixture was di-
luted with toluene (2 mL) and concentrated under reduced pres-
sure. The crude product was separated (SiO2, EtOAC/hexanes) to
yield [10,10-2H2]brassinin (14 mg, 58%). 1H NMR (500.3 MHz,
CD3CN): d 9.26 (br s, NH), 8.24 (br s, NH), 7.63 (d, J = 8 Hz, 1H),
7.42 (d, J = 8 Hz, 1H), 7.31 (s, 1H), 7.16 (dd, J = 8, 8 Hz, 1H), 7.07
(dd, J = 8, 8 Hz, 1H), 2.55 (s, 3H). Minor peaks of a rotamer were ob-
served at d 2.67 and 8.55 (only dideuterated brassinin was de-
tected). 13C NMR (125.8 MHz, CD3CN): d 199.1 (s), 137.7 (s),
128.1 (s), 126.1 (s), 123.2 (d), 120.7 (d), 120.0 (d), 112.8 (d),
111.8 (s), 42.9 (quintet, JC–D = 21.5 Hz), 18.5 (q). HRMS: m/z mea-
sured 238.0560 (238.0567 calcd for C11H102H2N2S2).
4.3.5. Co-factor PMSH
PMS (ca. 1 mmol in H2O) was treated with an aqueous solution
of sodium dithionite (Na2S2O4, ca. 2 mmol) for 3 min, the reaction
mixture was extracted with ethyl acetate and the extract analyzed
by HPLC. PMSH oxidized spontaneously to PMS under exposure to
aerobic conditions.
4.4. Fungal cultures and chromatographic purification of BOLm
Cultures of L. maculans (isolate BJ-125, IBCN collection, AAFC)
were carried out as described previously.2 For isolation and puri-
fication of BOLm, liquid cultures (600 mL) were induced with 3-
phenylindole (0.05 mM), the cultures were incubated for an addi-
tional 24 h and then gravity filtered to separate mycelia from
culture broth. The purification of BOLm was performed in four
steps as previously described.26 Protein concentrations were
determined as described by Bradford26 using the Coomassie Bril-
liant Blue method with bovine serum albumin (BSA) as
standard.
a
4.3.2. [10-2H]Indole-3-carboxaldehyde
BOLm standard activity assay. The reaction mixture contained
DEA (diethanol amine, 20 mM, pH 8.2), DTT (D,L-dithiothreitol,
1.0 mM), 0.1% (v/v) Triton X-100, brassinin (1.0 mM), PMS
A solution of indole (200 mg, 1.71 mmol) in DMF-d7 (0.6 mL)
was added to a cooled solution of freshly distilled POCl3 (288 mg,
1.88 mmol) in DMF-d7 (0.4 mL) and stirred at 40 °C for 60 min.
The reaction mixture was poured into ice-cold water and added
dropwise to an ice-cold solution of NaOH (1%, w/v, 3 mL). The sus-
pension was extracted with EtOAc (3 ꢁ 20 mL), the combined or-
ganic extract was dried over Na2SO4 and concentrated to dryness
to yield [10-2H]indole-3-carboxaldehyde (221 mg, 89% yield).24 1H
NMR (CD3CN) d: 1H NMR (500.3 MHz, CD3CN): d 10.07 (br s, NH),
8.17 (d, J = 7.6 Hz, 1H), 8.00 (s, 1H), 7.54 (d, J = 8 Hz, 1H), 7.31–
7.24 (m, 2H). 13C NMR (125.8 MHz, CD3CN): d 186.3 (t, JC–
D = 25.6 Hz), 138.7, 138.4 (s), 125.7 (s), 125.2 (d), 123.8 (d), 122.5
(1.0 mM) and protein extract (50–100
lL) in a total volume of
1000 L. The reaction was carried out at 20 °C for 20 min. A control
l
reaction was stopped by addition of EtOAc (2 mL) at t = 0. The
product was extracted with EtOAc (2 mL) and concentrated to dry-
ness. The extract was dissolved in CH3CN (200 lL) and analyzed by
HPLC-DAD.2 The amounts of brassinin (1), indole-3-carboxalde-
hyde (2), and S-methyl dithiocarbamate (6) in the reaction assay
were determined using calibration curves built with pure com-
pounds. Brassinin (1), indole-3-carboxaldehyde (2), and S-methyl
dithiocarbamate (6) were stable in the buffer and solvents used
for extraction and HPLC analysis at least for 24 h.
2
(d), 120.1 (t, JC–D = 3.7 Hz), 113.5 (d) HREIMS: m/z measured 144
(144. calcd for C9H62HNO).
4.3.3. Methyl [10,10-2H2]-N0-(3-indolylmethyl)carbamate (5a)
4.5. Origin of aldehydic oxygen
To
a
solution of [10,10-2H2]indolyl-3-methanamine (27 mg,
For the enzymatic experiments in H218O, all the reaction com-
ponents were dissolved in 50% of H218O. The incubation was car-
ried out in a saturated concentration of brassinin or dideuterated
brassinin (1.0 mM). Incorporation of 18O from H218O into indole-
3-carboxaldehyde was determined by HRMS. A control sample
was prepared using same experimental conditions but replacing
H218O with natural abundance H2O.
0.18 mmol) in CH2Cl2 (1 mL) was added methyl chloroformate
(19 mg, 0.20 mmol) and triethylamine (39 mg, 0.39 mmol) and
the reaction mixture was stirred at rt for 20 min. The reaction mix-
ture was diluted with water (10 mL), extracted with EtOAc
(3 ꢁ 20 mL) and the combined organic extract was dried over
Na2SO4 and concentrated to dryness. Purification of the crude
product (SiO2, EtOAC/hexanes) afforded methyl [10,10-2H2]-N0-(3-