A. de Saint Germain, et al.
Phytochemistry168(2019)112112
afford 370 mg of ( )-2′-epi-GR24 containing no P270. The aqueous
phase was acidified until pH 2 and extracted with CH2Cl2 (3 × 20 mL).
The combined organic layers were dried (Na2SO4), filtered and eva-
porated under reduced pressure to afford 79 mg of crude product con-
taining a small amount of ( )-2′-epi-GR24, ABC = CHOH and P270.
P270 was purified by semi-preparative HPLC which was performed
using an Interchim puriFlash® 4250 instrument, combined with a
fraction collector with integrated ELSD, a PDA and a Phenomenex Luna
C
18, 250 × 21.2 mm, 5 μm column (0.1% formic acid in CH3CN (solvent
B) and 0.1% formic acid in water (solvent A). A/B (7/3) isocratic 5 min
then linear gradient to A/B (2/8) in 20 min at a flow rate of 1 mL/min.
P270 was obtained as
0.018 mmol, 1.4%).
a white solid after lyophilisation (5 mg,
P270: 1H NMR (500 MHz, CDCl3) δ: 7.82 (dd, J = 12.0 Hz,
J = 10.2 Hz, 1H), 7.53 (d, J = 7.0 Hz, 1H), 7.37 (dd, J = 8.0 Hz,
J = 7.2 Hz, 1H), 7.30–7.27 (m, 2H), 6.71 (d, J = 12.0 Hz, 1H), 5.96 (d,
J = 7.5 Hz, 1H), 4.15–4.09 (m, 1H), 3.69 (dd, J = 17.0 Hz, J = 10.0 Hz,
1H), 2.97 (dd, J = 17.0 Hz, J = 2.5 Hz, 1H), 2.11 (s, 3H). 13C NMR
(125 MHz, CDCl3) δ: 172.2 (Cq), 169.3 (Cq), 149.1 (Cq), 147.9 (Cq),
144.4 (Cq), 140.2 (Cq), 133.0 (CH), 132.0 (CH), 131.1 (CH), 128.4
(CH), 127.3 (CH), 126.3 (CH), 86.9 (CH), 40.6 (CH), 40.3 (CH2), 21.9
(CH3). IR ν (film, cm−1): 3600-2400 (br, COOH), 2935, 1747 (C]O),
1638. HRMS (ESI): Calculated for C16H13O4 [M − H]: 269.0814.
Found: 269.0811.
Fig. 7. Effect of ( )-contalactone on symbiotic ability of the AM fungus
Rhizophagus irregularis. This fungus was inoculated on Medicago truncatula SL-
deficient mutants, in the absence (CTL0) or presence of SL analogs at 10−7 M.
The number of infection points was recorded three weeks post-inoculation. Bars
represent the mean
SE of 9–12 replicates per condition. ***P < 0.001,
**P < 0.01, Mann-Whitney test, compared to control values (CTL0).
4.3. Preparation and isolation of ( )-contalactone
To solid K2CO3 (1.851 g, 13.4 mmol) dried under reduced pressure
was added at room temperature under argon anhydrous acetone
(27 mL). After 10 min, a mixture of ABC = CHOH (1.354 g, 6.7 mmol)
and D-Br (1.778 g, 10.5 mmol) in anhydrous acetone (67 mL) was
added dropwise to the preceding solution. The resulting reaction mix-
ture was stirred for 20 h at room temperature under argon and acetone
evaporated under reduced pressure. The residue was diluted in EtOAc
and filtered. This reaction was performed at this scale several times to
4.5. Crystallographic data collection, structure determination and
refinement
X-ray structure determination for contalactone (FDB2980F1) was
carried out at low temperature (173 K) using a RIGAKU XtaLabPro
diffractometer equipped with a Mo microfocus sealed tube generator
coupled to a double-bounce confocal Max-Flux® multilayer optic and a
HPAD PILATUS3 R 200 K detector. Data collection and processing were
performed with the CrysalisPro software (Rigaku, 2015). The structure
was solved by intrinsic phasing methods (SHELXT program) (Sheldrick,
2015b) then refined by full-matrix least-squares methods (SHELXL-
by anisotropic refinement, whereas H atoms bonded to C atoms were
included in the structure at idealized positions, and refined using a
riding model, with Uiso(H) = 1.2Ueq(C) and C–H = 0.95–0.99–1.00 Å
for aromatic, methylene, and methine H atoms, respectively, whereas
for methyl groups, Uiso(H) = 1.5Ueq(C) and C–H = 0.98 Å. A second
polymorph coexists in the crystallization medium, more massive than
the elongated platelet, and was characterized at 173 K as a triclinic
crystal. The diastereomer that was determined subsequently differs
from the monoclinic one at the level of the lactone tail (torsion angles
C9’-O14’ -C2′-O1′ 110.8° (triclinic) vs −77.6° (monoclinic)) (see the
model overlay in Fig. 1C). Crystallographic data (including structure
factors) for the structures, FDB2980F1 dia1 and FDB2980F1 dia2, re-
ported in this paper have been deposited with the Cambridge Crystal-
lographic Data Centre as supplementary publication no. CCDC-
1870390-1870391 respectively. Copies of the data can be obtained free
of charge on application to CCDC, 12 Union Road, Cambridge CB21EZ,
obtain
a
crude product (38.39 g) which contains
a
mixture of
(
)-GR24,
(
)-2′-epi-GR24 and
(
)-contalactone (45.5:45.5:9)
(ratio determined by 1H NMR). The crude mixture was purified by
medium pressure chromatography on silica gel and HPLC (Interchim
Uptisphere Strategy SI, 250 × 21.2 mm, 5 μm column) (Heptane/
EtOAc: 1/1) to furnish pure
(
(
)-GR24,
(
)-2′-epi-GR24 and
)-con-
)-contalactone. However, the two diastereomers of
(
talactone can be separated by HPLC using a HypercarbTM porous gra-
phitic carbon column (100 × 4.6 mm, 5 μm) (MeOH/iPrOH 1/1,
Formic acid 0.1%, 2 mL/min) to furnish contalactoneF1 (1.5 mg)
(> 99%) and contalactoneF2 (0.7 mg) (> 95%) after 30 injections and
evaporation under reduced pressure.
(
)-Contalactone: mixture of two diastereomers (1:1): M.p.
185.0–209.4 °C. 1H NMR (300 MHz, CDCl3) δ: 7.92 (d, J = 12.0 Hz,
1H), 7.55 (d, J = 7.0 Hz, 1H), 7.40–7.24 (m, 3H), 7.02–6.95 (m, 2H),
6.72 (d, J = 12.0 Hz, 1H), 6.00 (d, J = 8.0 Hz, 1H), 4.11–4.02 (m, 1H),
3.69 (dd, J = 16.5 Hz, J = 10.0 Hz, 1H), 2.97 (dd, J = 16.5 Hz,
J = 3.0 Hz, 1H), 2.18 (s, 3H), 2.03 (s, 3H). 13C NMR (75.5 MHz, CDCl3)
δ: 171.1 (Cq), 170.1 (Cq), 164.6 (Cq), 143.1 (Cq), 142.4 (Cq), 142.0
(CH), 138.7 (Cq), 136.6 (Cq), 134.9 (Cq), 134.8 (CH), 134.7 (CH),
134.49 (Cq), 134.46 (Cq), 131.7 (CH), 130.5 (CH), 127.9 (CH), 126.7
(CH), 125.2 (CH), 97.1 (CH), 93.1 (CH), 85.9 (CH), 39.82 (CH2), 39.77
(CH2), 21.6 (CH3), 10.90 (CH3). IR ν (film, cm−1): 2952, 2924, 2853
(CH), 1780 and 1748 (C]O). HRMS (ESI): Calculated for C21H18O6Na
[M + Na+]: 389.1001. Found: 389.0996.
4.6. Plant material and growth conditions
4.4. Preparation and isolation of ( )-P270
Pea (Pisum sativum) branching mutant plants used in this study were
derived from various cultivars of pea after ethyl methanesulfonate
1997). The rms1-10 (M3T-884) and rms3-5 (M2T-32) mutants were
obtained from the dwarf cv Térèse. Plants were grown in a greenhouse
All A. thaliana plants used in this study originated from the
Columbia (Col-0) ecotype background and have been described pre-
To a sample of ( )-2′-epi-GR24 purified by flash chromatography
(10 mL) was added a phosphate buffered saline (PBS buffer, pH 6.8)
solution (10 mL) and dropwise a aqueous solution of KOH (1 M) until
pH 9.5. The resultant solution was stirred for 10 h at room temperature
and extracted with CH2Cl2 (3 × 20 mL). The combined organic layer
was dried (Na2SO4), filtered and evaporated under reduced pressure to
7