S. Ortiz, et al.
BioorganicChemistry104(2020)104243
4. Material and methods
with methyl iodide (MeI, 13.5 equiv.) and potassium carbonate (K2CO3,
6.6 equiv.) in dried acetone at room temperature for 1 h. The crude
reaction mixtures were precipitated and washed with ice-cold water.
The resulting residues were solubilized with EtOAc (10 mL) and treated
with 1 N HCl solution (3 mL). The final products were extracted with
EtOAc (3x10 mL) and the organic phases were washed with saturated
NaCl solution and dried over Na2SO4. After filtration, and solvent
evaporation, methylated compounds were purified by silica gel chro-
matography, using a mixture of CH2Cl2/MeOH (99.1/0.1 to 95/5 v/v)
as eluent. Identification of resulting pure compounds was confirmed by
comparison of 1H, 13C NMR and MS analysis with literature data.
Methylation of 50 mg (0.20 mmol) of 5,7-dihydroxyflavone
(chrysin, 1) afforded 2 (48.5 mg, yield 92%) [28].
4.1. Chemical and reagents
Mass spectrometry analyses of compounds were performed on a
Waters ZQ 2000 mass spectrometer (Saint-Quentin, France). Silica gel
60 25–40 µm (Macherey-Nagel GmbH & Co, Düren, Germany) was used
for column chromatography (CC). H and 13C NMR experiments were
1
performed in deuterated chloroform on a Bruker AC 300 MHz or a
Bruker 400 MHz spectrometer (Wissembourg, France) using Bruker
pulse programs. All the reagents and solvents were purchased from
Carlo Erba-SDS (Chaussée du Vexin, France). Cyclohexane and ethyl
acetate were distilled before use. Deionized water was used for all ex-
periments.
(apigenin, 3) gave 4 (31.6 mg, 60%) and 5 (8.3 mg, 15%) with a ratio of
4.2. Flavonoids library
Methylation of 100 mg (0.35 mmol) of 5,7,3′,4′-tetrahydroxy-
flavone (kaempferol, 6) gave 9 (71.1 mg, 62%) and 10 (14.3 mg, 12%)
with a ratio of 5/1 [30].
Flavonoids library is composed of either isolated, commercial or
semi-synthetic derivatives. Compounds 7, 11, 12, 23, 26, 27, 30 and 31
were isolated from bud exudates of Gardenia oudiepe (Rubiaceae); fla-
vones 20, 21, 24 and 25 were isolated from aerial parts of Pentzia
monodiana (Asteraceae) [11,12]. Coumpounds 1, 3, 6 and 15 were
Louis, MO, USA). Compounds 7–8, 13–14 and 32–33 were previously
semisynthetized [12]. Compound 2 was obtained by methylation of 1;
likewise, compound 4 and 5 were obtained from 3; 9 and 10 from 6;
16–19 from 15; 22 from 20 and 28–29 from 27 (see Section 4.5).
Methylation of 150 mg (0.5 mmol) of 3,5,7,3′,4′-pentahydroxy-
flavone (quercetin, 15) gave 16 (15.6 mg, 10%), 17 (15.6 mg, 9.5%),
18 (97.8 mg, 55%) and 19 (17.0, 9.2%) with a ratio of 1/1/6/1
Methylation of 12 mg (0.032 mmol) of 27 gave 28 (7.5 mg, 61%)
and 29 (3.9 mg, 28%) with a ratio of 2/1 [35].
4.3. Plant material
4.6. Biomimetic heme-binding assay by MS
G. oudiepe (Rubiaceae) buds were collected in October 2008 in Forêt
Plate, North Province of New Caledonia. Voucher specimens (POU-
0290 respectively) are kept at the Herbarium of the Botanical and
Tropical Ecology Department of the IRD Center, Noumea, New
Caledonia.
Hemin (iron-containing porphyrin with chlorine counterion) and
compounds were dissolved in DMSO at 5 mM. Citric buffer saturated
octanol (CBSO) [36] was prepared by mixing 5 mL of citric acid 50 mM
pH 5.2 and 15 mL of n-octanol (anhydrous, ≥99%) and letting it to
settle at 23 °C for 30 min. Upper phase of CBSO was used. An auto
sampler (G1367E 1260 Hip ALS, Agilent Technologies, Les Ulis, France)
was used to realize the automatic mixing in 384 well-plates of: test
compound 5 µL + heme 5 µL + tween 20 (10 µM in water)
5 µL + CBSO 85 µL. Five microliters of the sample were injected in
infusion mode (direct injection) in a 6530 Accurate-Mass QToF LC/MS
instrument (Agilent Technologies) with the following settings: positive
ESI mode, 2 GHz acquisition rate. Ionization source conditions were:
drying gas temperature 325 °C, drying gas flow rate 11 L/min, nebulizer
35 psig, fragmentor 175 V, skimmer 65 V. Range of m/z was 200–1700.
In the positive-ion mode, purine C5H4N4 [M + H]+ ion (m/z
121.050873) and the hexakis (1H,1H,3H-tetrafluoropropoxy)-phos-
phazene C18H18F24N3O6P3 [M + H]+ ion (m/z 922.009 798) were used
as internal lock masses. Full scans were acquired at a resolution of 11
000 (at m/z 922). On the same spectrum (spectral windows) appears
heme at m/z 616.16; adducts at m/z 616.16 + mass of ligand and
2heme + mass of ligand; heme + DMSO at m/z 694.19; heme dimer at
m/z 1231.32 [2heme – H]+. Chloroquine 5 mM in water (chloroquine
diphosphate, Sigma-Aldrich) was used as positive control [37].
Aerial parts of P. monodiana (Asteraceae) were collected in the
Algerian Southern Sahara, in Tassili Ahaggar National Park nearby the
Tahat mountain (23° 8″39.95″, 5°43″46.28″, alt.: 2019 m), Wilaya of
Tamanrasset, Algeria, in September 2013. The collection agreement
was issued by the director of the Ahaggar National Park Office (OPNA),
n°965/DPLBCVPC/MC/13 and 111/SDV/MC/13. Identification was
done under supervision of M. Belghoul, head of Department of Study of
Natural Resources from the Ahaggar National Park. A voucher specimen
is stored at the Herbarium of the Department of Pharmacognosy, Paris
Descartes University, France (voucher specimen MKOSB2013KDHPM).
4.4. Extraction and isolation
Fifty-five grams of G. oudiepe exudate were dissolved in 1 L of
CH2Cl2 and paper-filtered in order to remove vegetal pieces. The re-
sidual gum was subjected to several steps of silica gel chromatographic
separation using various mixtures of solvents of increasing polarity as
eluent (cyclohexane/CH2Cl2, EtOAc/CH2Cl2 and CH2Cl2/MeOH) to
provide compounds 7, 11, 12, 23, 26, 27, 30 and 31.
Identification of isolated compounds was confirmed by NMR and
MS analysis and by comparison with literature data [26,27].
4.7. Collision induced dissociation of non-covalent adducts
4.5. Semisynthesis of compounds 2, 4–5, 9–10, 16–19, 22, 28 and 29.
Dissociation voltage at 50% (DV50) was determined by applying
increasing collision energy from 0 to 32 eV. MSMS targeted mode, with
isolation width of ~1.3 m/z and flow injection analysis mode (FIA)
were used. MSMS source parameters were the same as described above.
Results were plotted and a sigmoidal regression curve was calculated
using GraphPad Prism software. DV50 is the collision energy dis-
sociating 50% of the adduct [37,38].
Semisynthesis of compound 8 from 7; 13 and 14 from 12; 32 and 33
from 31 were previously reported, using dimethyl sulfate (Me2SO4, 4
equiv.) and 1,8-diazobicyclo[5.4.0]undec-7-en (DBU, 2 equiv.) in dried
acetone at room temperature [12]. Methylation of isolated compounds
20 and 27 to provide 22, 28 and 29 and methylation of commercially
available compounds 1, 3, 6 and 15 to provide compounds 2, 4–5,
9–10, 16–19, respectively, were performed by stirring the substrate
5