Journal of Natural Products
Note
pH 2. The organic phase was separated, and the aqueous layer was
with deionized water, and cultivated at 24 °C for 72 h in the dark. The
next phase of this procedure was carried out under a green safe light in
a darkroom. The roots from the seedlings were cut off, and the residual
parts, consisting of two cotyledons and a hypocotyl, were placed on a 5
cm Petri dish (20 explants per dish) with filter paper soaked with 1 mL
extracted with EtOAc (3 × 30 mL). The combined organic phases
were washed with saturated NaHCO (15 mL), water (15 mL), and
3
brine (15 mL), dried over Na SO , and evaporated under a vacuum.
2
4
The residue was purified by column chromatography (silica gel,
petroleum ether−EtOAc, 7:3) to afford 5 (1.2 g, 68%) as a white,
of incubation medium containing 10 mM Na HPO /KH PO , pH 6.8,
2 4 2 4
1
−8
−4
amorphous solid: H NMR (500 MHz, CDCl ) δ 1.28 (3H, t, J = 7.2
5 mM tyrosine, and the test compound (10 −10 M, dissolved in
DMSO). The dishes were cultivated at 24 °C for 48 h in the dark. The
resulting betacyanin was extracted by repeated freezing and thawing of
the plant material in 4 mL of 3.33 mM acetic acid, and its
concentration was determined from the difference between
3
Hz, −CH ), 1.66−1.71 (1H, m), 2.14−2.18 (1H, m), 2.52 (1H, bs,
3
−
OH), 3.58−3.63 (1H, m), 3.71 (1H, dt, J = 11.8, 4.5 Hz), 4.20−4.24
(
3H, m), 4.43 (2H, ddd, J = 30.3, 10.6, 7.0 Hz), 4.51−4.55 (1H, m),
.69 (1H, d, J = 7.6 Hz, −NH), 7.32 (2H, t, J = 7.5 Hz, H ), 7.40 (2H,
5
Ar
1
6
t, J = 7.6 Hz, H ), 7.59 (2H, dd, J = 7.3, 2.4 Hz, H ), 7.76 (2H, d, J =
absorbances at 537 and 625 nm.
Ar
Ar
1
3
7
5
1
3
2
.3 Hz, H ); C NMR (125 MHz, CDCl ) δ 14.2, 35.9, 47.3, 51.2,
8.4, 61.9, 67.3, 120.1, 120.1, 125.1, 125.2, 127.2, 127.9, 141.4, 143.7,
43.9, 156.9, 172.7; (+)-ESIMS m/z (rel int) 370.8 [M + H] (80),
92.7 [M + Na] (45), 408.7 [M + K] (15); HPLC purity, 97.1% (t
Wheat Leaf Senescence Bioassay. Wheat seeds, Triticum
aestivum L. cv. Aranka, were washed under running water for 24 h,
sown in verticulite soaked with a Hoagland solution, and grown in a
cultivation chamber (16 h day/8 h night, 7000 lx) at 22 °C for 1 week.
Tip cuttings of fully developed first leaves, ca. 3.5 cm long, were taken.
The leaf segments were trimmed and combined (four pieces, total
weight 0.1 g per well), immersed by basal part in a well containing test
compound (150 μL/well), and cultivated in a closed plastic box with
damp paper tissue to prevent drying out at 24 °C for 96 h in the dark.
The residual chlorophyll was extracted by heating the leaf segments in
5 mL of 80% (v/v) ethanol at 80 °C for 10 min. The volume of the
extracts was then restored to 5 mL, and the absorbance at 665 nm was
measured. The values were compared with values from extracts of
fresh leaves (stored at −80 °C after detachment) and extracts of leaves
cultivated in deionized water.
Ar
3
+
+
+
R
6.45 min).
Ethyl (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4-
bromobutanoate (6). To a solution of alcohol 4 (1 g, 2.7 mmol,
equiv) in dry CH Cl (27 mL) was added CBr (0.98 g, 3 mmol, 1.1
1
2
2
4
equiv), and the solution was cooled to 0 °C. PPh (0.79 g, 3 mmol, 1.1
3
equiv) was added in portions over 15 min, and the mixture was
allowed to reach room temperature and stirred for 3 h. Solvent was
evaporated under vacuum, and the residue was purified by column
chromatography (silica gel, petroleum ether/EtOAc, 7:3) to afford
1
compound 6 (0.72 g, 62%) as a white, amorphous solid: H NMR
(
300 MHz, CDCl ) δ 1.32 (3H, t, J = 7.1 Hz, −CH ), 2.26 (1H, dd, J
3
3
=
−
−
14.4, 7.6 Hz, −CHaHb−), 2.47 (1H, dd, J = 13.3, 5.9 Hz,
Live-Cell Cytokinin Binding Assay. The binding assay was
CHaHb−), 3.42 (2H, t, J = 6.9 Hz, −CH ), 4.22−4.28 (3H, m,
performed according to a slightly modified method published by
2
1
5
CH , −CH), 4.45−4.51 (3H, m, −CH , −CH), 5.39 (1H, d, J = 7.7
Romanov et al. E. coli strains KMI001 carrying a pIN-III/AHK4 or a
pSTV28/AHK3 plasmid were grown in liquid M9 medium
supplemented with 0.1% casamino acids and antibiotics (ampicillin,
2
2
Hz, NH), 7.34 (2H, t, J = 7.4 Hz, H ), 7.43 (2H, t, J = 7.3 Hz, H ),
Ar
Ar
7
.62 (2H, d, J = 7.3 Hz, H ), 7.79 (2H, d, J = 7.5 Hz, H );
A
r
A
r
+
+
−1
−1
(
+)-ESIMS m/z (rel int) 432.5 [M + H] (93), 434.5 [M + H]
100 μg·mL for CRE1/AHK4; chloramphenicol, 20 μg·mL for
AHK3), with shaking (150 rpm) at 25 °C overnight to an OD600 of ca.
0.5−0.7. For the assay, 1 mL bacterial culture aliquots were incubated
(
100); HPLC purity 99.9% (t 29.42 min).
(
R
+)-Discadenine (1). To a solution of iP (0.100 g, 0.5 mmol, 1
equiv) in dry DMA (3 mL) was added bromo compound 5 (0.250 g,
.58 mmol, 1.16 equiv) in one portion. This mixture was heated to 85
C and stirred at this temperature for 48 h. The mixture was cooled to
3
with 3 nM 2-[ H]tZ and competitors tested at 4 °C for 30 min.
0
°
Samples were then centrifuged (8000 rpm, 6 min, 4 °C), the
supernatant was carefully removed, and the pellet was resuspended in
1 mL of scintillation cocktail (Ultima-Flo M, PerkinElmer, Waltham,
MA, USA). Residual radioactivity was measured on a Hidex 300 SL
scintillation counter (Hidex, Turku, Finland). For distinction between
specific and nonspecific bindings, 10 μM nonlabeled tZ for the
room temperature, and DMA was evaporated under vacuum as much
as possible. NaOH (1 M, 3 mL) followed with same amount of water
was added, and mixture was stirred at rt for 2 h. EtOH was removed in
vacuo, and the aqueous solution was extracted with EtOAc (3 × 10
mL). The pH of the aqueous solution was then adjusted to 6 with 3 M
HCl. After cooling in an ice bath, a white crystal precipitate was
filtered off and recrystallized from EtOH to afford discadenine (1) (91
17
competition was used, and this value was deducted from all data.
Bacterial Receptor Assay. This assay was performed using
transgenic E. coli strains KMI001 harboring a pINIII/AHK4 or a
pSTV28/AHK3 plasmid and expressing the β-galactosidase gene
7
mg, 60%) as a white solid: mp 192.5−194.0 °C (EtOH) (lit. 193−195
1
8
2
5
7
26
(
ΔrcsC, cps::lacZ) under the control of cytokinin receptors. Bacterial
°
C); [α] +23.9 (c 0.38, 0.1 M HCl) (lit. [α] +28 (c 1, 0.1 N
D
D
1
precultures were grown in liquid M9 medium supplemented with 0.1%
HCl); H NMR (500 MHz, D O/DMSO-d ) δ 1.57 (6H, s, 2 ×
2
6
−1
casamino acids and antibiotics (ampicillin, 100 μg·mL for CRE1/
−
−
−
CH ), 2.40−2.49 (2H, m, −CH −), 4.00 (1H, dd, J = 7.5, 6.0 Hz,
3
2
−1
AHK4, and chloramphenicol, 20 μg·mL for AHK3), with shaking
CH−), 4.09 (2H, d, J = 7.3 Hz, −CH −), 4.56−4.50 (2H, m,
2
(
300 rpm) at 25 °C for 24 h. Expression of the β-galactosidase gene
CH −), 5.20 (1H, t, J = 7.0 Hz, −CH−), 8.22 (1H, s, −H ), 8.46
2
Ar
13
was induced by cultivation of 200 μL precultures diluted by M9
(
1H, s, −H ); C NMR (75 MHz, D O/DMSO-d ) δ 17.3 (CH ),
Ar
2
6
3
medium with antibiotics (1:600) and test compounds (50, 10, 1, and
24.8 (CH ), 29.2 (CH ), 39.5 (CH ), 46.5 (CH), 50.0 (CH ), 111.1
3 2 2 2
0
.1 μM) with shaking (450 rpm) at 25 °C for 17 h. At the end of the
(
1
C), 117.3 (CH), 139.5 (C), 144.0 (CH), 146.2 (C), 148.3 (CH),
51.3 (C), 170.5 (C); (+)-ESIMS m/z (rel int) 305.2 [M + H]+
incubation period, 50 μL of the bacterial cultures was transferred to a
new 96-well plate and the activity of β-galactosidase was determined
(
100); HPLC purity >98% (t 10.72 min).
R
by measuring the fluorescence (λex/ − 365/460 nm) after incubation
Bioassays. Standard cytokinin bioassays were performed according
em
15
with 2 μL of 10 mM (25 mM for AHK3) chromogenic substrate
to Holub et al., with several modifications.
(
MUG) at 37 °C for 10 min (AHK4) and 30 min (AHK3),
Tobacco Callus Bioassay. Cytokinin-dependent tobacco callus
cells (Nicotiana tabacum L. cv. Winsconsin 38) were cultivated on solid
MS medium (3 mL/well) containing different concentrations of the
respectively, and addition of 100 μL of Stop buffer (132 mM glycine,
8
3 mM Na CO ).
2 3
−9
−4
test compound (10 −10 M, dissolved in DMSO) in six-well plates
ASSOCIATED CONTENT
Supporting Information
(
0.1 g of callus divided into 3 pieces/well) at 24 °C for 4 weeks in the
■
dark. The biological activity of discadenine (1) was determined as an
increase in the callus fresh weight. N -Benzylaminopurine was used as
S
*
6
a positive control.
Amaranthus Bioassay. Seeds of Amaranthus caudatus var.
atropurpurea were surface sterilized (10% sodium hypochloride for
1
13
1
1
0 min; washed five times with water; 70% ethanol 10 min; washed
H NMR/ C NMR for new compound 5 and H NMR
for known compounds 1, 3, 4, and 6; 1 C NMR for
3
five times with water), placed on a Petri dish with paper tissue sodden
D
J. Nat. Prod. XXXX, XXX, XXX−XXX