Photodegradation of Myrigalone A, an Allelochemical from Myrica gale: Photoproducts and Effect of Terpenes

Article abstract of DOI:10.1021/acs.jafc.9b01722

This study investigated the environmental fate of myrigalone A, a light absorbing natural herbicide found on leaves and fruits of Myrica gale. Myrigalone A was irradiated in water and as a dry solid deposit to simulate reactions on leaves, alone and in the presence of the terpenes generated by Myrica gale. The phototransformation was fast (t_1/2 = 35 min in water). Analyses by liquid chromatography coupled to high resolution orbitrap electrospray mass spectrometry (MS) and gas chromatography-MS revealed the formation of 11 photoproducts in water and solid and 9 in gaseous phase. Some were detected in the leaf glands and oil covering the fruits of Myrica gale, which suggested that photodegradation occurred in the field. Moreover, myrigalone A photoinduced the oxidation of terpenes that in turn protected it against photolysis. This highlights the need for additional research on the effect of terpenes on the photodegradation of pesticides on vegetation.

Full text of DOI:10.1021/acs.jafc.9b01722

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Agricultural and Environmental Chemistry
Photodegradation of myrigalone A, an allelochemical
from Myrica gale: photoproducts and effect of terpenes
Amina Khaled, Mohamad Sleiman, Etienne Darras, Aurelien Trivella,
Cedric Bertrand, Nicolas Inguimbert, Pascale Goupil, and claire Richard
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01722 • Publication Date (Web): 12 Jun 2019
Downloaded from http://pubs.acs.org on June 13, 2019
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Photodegradation of myrigalone A, an allelochemical from Myrica gale: photoproducts
and effect of terpenes
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Amina Khaled1, Mohamad Sleiman1, Etienne Darras1, Aurélien Trivella2, Cédric Bertrand3,4,
Nicolas Inguimbert3, Pascale Goupil5, Claire Richard1*
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(1) Université Clermont Auvergne, CNRS, SIGMA Clermont, Institut de Chimie de
Clermont-Ferrand, F-63000 Clermont-Ferrand, France
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(2) UMR CNRS 5805 EPOC - OASU, Equipe LPTC, IUT de Périgueux, Rue du Doyen
Lajugie, 24000 Périgueux, France
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(3) USR 3278 CRIOBE, PSL Research University, EPHE-UPVD-CNRS, Université de
Perpignan Via Domitia, Laboratoire d’Excellence « CORAIL ». Bâtiment T, 58 avenue P.
Alduy, 66860 Perpignan, France.
(4) AkiNaO SAS, F-66860 Perpignan, France
(5) Université Clermont Auvergne, INRA, PIAF, F-63000 Clermont-Ferrand, France
* corresponding author: Claire Richard; Claire.richard@uca.fr
Abstract :
This study investigated the environmental fate of myrigalone A, a light absorbing natural
herbicide found on leaves and fruits of Myrica gale. Myrigalone A was irradiated in water and
as a dry solid deposit to simulate reactions on leaves, alone and in the presence of the terpenes
generated by Myrica gale. The phototransformation was fast (t1/2 = 35 min in water). Analyses
by liquid chromatography coupled to high resolution orbitrap electrospray mass spectrometry
(MS) and gas chromatography-MS revealed the formation of 11 photoproducts in water and
solid and 9 in gaseous phase. Some were detected in the leaf glands and oil covering the fruits
of Myrica gale suggesting that photodegradation occurs in the field. Moreover, myrigalone A
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photoinduced the oxidation of terpenes that in turn protected it against photolysis. This
highlights the need for additional research on the effect of terpenes on the photodegradation of
pesticides on vegetation.
Key-words: biopesticide, fate, photolysis, medium, H-donor additives
1. Introduction
Myrigalone A (2,2,4-trimethyl-6-(3-phenylpropanoyl)cyclohexane-1,3,5-trione) is a
secondary metabolite of Myrica gale (Myricaceae).1,2 Myrigalone A received great attention
due to its interesting biological activities. It was reported to be antioxidant, antifungal,
antimicrobial, and to inhibit germination and seedling growth of various species.2-6 It was also
proposed to be responsible for the allelopathic properties of Myrica gale, i.e. for the capacity
of this plant to eliminate concurrent species growing in its surrounding area.7
Myrigalone A is contained in droplets of resin located on leaves and fruits of Myrica
gale. Myrigalone A is the main resin constituent7, but a variety of terpenes (-pinene,
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eucalyptol, p-cymene, limonene), sesquiterpenes (germacrone, -elemenone) and other
flavonoids were also identified in the oil extracted from the leaf resin and fruits of this plant.1-
3,8
Leptospermone, isoleptospermone, flavesone and grandiflorone are natural herbicides
bearing a cyclohexane-1,3,5-trione as myrigalone A (Scheme 1).9,10 A recent study showed
that they undergo photodegradation when exposed to simulated solar light.11,12 In myrigalone
A structure, the cyclohexane-1,3,5-trione moiety is substituted by 3 methyl groups against 4
methyl groups in the case of leptospermone, isoleptospermone, flavesone and grandiflorone.
The presence of an H atom on C4 instead of a CH3 group is expected to increase the
enolisation possibilities compared to the other triketones, to red-shift the maximum of
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absorption by extending electron delocalization and to improve the overlapping of the
absorption spectrum of the molecule with the solar spectrum. Therefore, myrigalone A should
undergo photodegradation both in surface waters that it can reach by washing off and on the
leaves and the fruits where it is naturally present.
In this study, our objective was to determine the photodegradation reactions of
myrigalone A under relevant environmental conditions from the kinetic and analytical points
of view. Myrigalone A was irradiated in aqueous solution to determine its fate in surface
waters and as a pure solid to simulate reactions on leaves. To approach real conditions,
myrigalone A was also irradiated in the presence of terpenes present in the leaf glands,
germacrone and eucalyptol.
2. Materials and methods
2.1. Chemicals
Myrigalone synthesis was performed using two different approaches. They are
described in details in Scheme SI-1. The first synthesis consists in a Friedel-Craft
condensation of phloroglucinol 1 with dihydrocinnamoylchloride 2 leading to the 2,4,6-
trihydoxydihydochalcone 3 (23% yield) and indanone 4 (75%).13 After separation of the two
products, compound 3 was methylated using iodomethane in the presence of potassium
tertiobutylate to afford myrigalone A (75%). The second method involves the methylation of
2,4,6-trihydroxyacetophenone to deliver compounds 6 (74%) and 7 (20%).14 Compound 6 is
further protected with trimethylsilyldiazomethane to yield compound 8 (74%). Claisen-
Schmidt condensation with benzaldehyde leads to compound 10 (69%) which was reduced
using palladium on carbon and the enol ether was finally deprotected using concentrated
hydrochloric acid to deliver Myrigalone A (47%). The latter obtained by the two methods
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have identical NMR and MS spectra to the natural compound (Scheme SI-1 and Figure SI-1).
Method 1 and method 2 are equivalent with overall yield of 17% and 18%, respectively.
Water was purified using a reverse osmosis RIOS 5 and Synergy (Millipore) device
(resistivity 18 M cm, DOC < 0.1 mg L-1). All solvents or other reactants were of the highest
grade available.
2.2 Irradiation of myrigalone A in solution
The stock solutions of myrigalone A were prepared in acetonitrile (ACN) and kept in
the dark at 4°C before use. The solutions were renewed every week. Solutions of myrigalone
A were prepared in a mixture water-ACN (90-10%, v/v) by diluting the stock solutions with
purified water. Due to its poor solubility in pure water, myrigalone A was solubilized in
water-ACN. These solutions were irradiated using a polychromatic device equipped with six
fluorescent tubes TLAD 15W05 Philips (300-450 nm, maximum emission at 365 nm) (Figure
SI-2) in a 20 ml capacity Pyrex-glass cylindrical reactor (1.4 cm, i.d.) closed by a silicon
stopper. Aliquots of 1 ml were withdrawn after selected irradiation times, analyzed by HPLC
to monitor the loss of myrigalone A and by high resolution UPLC-HRMS-ESI and UPLC-
HRMS2-ESI to characterize the main photoproducts. The stability of myrigalone A in the dark
was controlled.
2.3 Irradiation of Myrigalone A as a solid
For the irradiation in solid form, 2.2 mg of myrigalone A were dissolved in 5 ml of
ACN. 3.5 ml of this solution were poured in 7 glass dishes (500 l in each). Then, ACN was
let to totally evaporate for 2 h under a ventilator. These dishes were inserted in a cylindrical
Pyrex reactor (600 ml) equipped with inlet/outlet enabling the sampling of the gaseous phase.
The reactor was irradiated using two fluorescent tubes TLAD 15W05 Philips located 20 cm
above it. An aluminium foil was placed below the reactor to increase the photon flux received
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by the samples. The dishes were irradiated for 4h and 19 h. After irradiation, the gaseous
phase was sampled for 4 h at a flow of 100 ml/min (25 l) and the volatile photoproducts were
trapped using a Carbotrap 300 sorbent (Sigma-Aldrich). The solid remaining in the dishes was
dissolved in 2 ml of ACN and analyzed by HPLC-UV and UPLC-HRMS-ESI.
2.4 Irradiation of Myrigalone A in the presence of additives
The irradiation of myrigalone A in the presence of the additives was conducted in the
device described in §2.2. Eucalyptol and germacrone were added to ACN solutions of
myrigalone A while 2,4,6-trimethylphenol to water-ACN and ACN myrigalone A solutions.
All were at a final concentration of 10-4 M. Irradiated solutions of eucalyptol-myrigalone A,
of 2,4,6-trimethylphenol and of 2,4,6-trimethylphenol-myrigalone A mixtures were analyzed
by HPLC-UV. Irradiated solutions of germacrone alone (10-4 M) and germacrone-myrigalone
A mixtures were analyzed by HPLC and high resolution UPLC-HRMS-ESI.
2.5. Analyses
UV-vis spectra were recorded using a Varian Cary 3 spectrophotometer in a 1-cm quartz cell.
Analysis of the irradiated solutions were performed using liquid chromatography (Shimadzu
NEXERA XR HPLC) coupled to a photodiode array detector. HPLC-UV separation was
conducted using a Phenomenex reversed phase column C18 grafted silica, (100 mm length, 2.1
mm i.d., 1.7 μm particle size) and 55:45 acetonitrile: water (0.1% orthophosphoric acid) mobile
phase, at a flow rate of 0.2 ml min-1. The identification of degradation products was performed
using High resolution mass spectrometry (HRMS) constituted of an Orbitrap Q-Exactive
(Thermoscientific) coupled to an ultra-high performance liquid chromatography (UPLC)
instrument Ultimate 3000 RSLC (Thermoscientific). Analyses were carried out in both negative
and positive electrospray modes (ESI+ and ESI-). UPLC separations were performed using the
same column and elution gradient as previously indicated. The binary solvent system used was
composed of acetonitrile (ACN) and water acidified with 0.5‰ v/v formic acid. The gradient
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program was 5% ACN for the 7 first min, followed by a linear gradient to 99% in 7.5min and
kept constant until 20 min. The flow rate was set at 0.45 mL/min and injection volume was 5
μL. Identification of photoproducts was based on structural elucidation of mass spectra and the
use of accurate mass determination obtained with the Orbitrap high resolution. MS-MS was
done by the HCD technique (35 eV).
After sampling, Carbotrap 300 tubes were thermally desorbed at 300°C for 10 min at
50 mL/min of Helium, using a Perkin Elmer Automated Thermal Desorber (ATD) coupled to
an Agilent 6890 gas chromatograph and Agilent 5973 mass-spectrometry detector. Separation
of desorbed volatiles was carried out using a HP-5 ms column (30 m×0.25 mm×0.25 μm)
operated initially at 50°C over 1 min, followed by a 10° C/min ramp to reach 250° C. Mass
spectra were scanned between m/z 50 and 350 with a source temperature set at 230° C.
2.6 Extracts of Myrica gale
Myrica gale plants were bought from “Le jardin du pic vert” France. Young and old leaves
were taken and rinsed with methanol for 30 s. The extracts were analyzed by UPLC-HRMS-
ESI as previously described. Fruits extract was obtained by ethanolic maceration (solid/liquid
ratio : 1/10). The oil was analyzed by UPLC-HRMS-ESI and by GC-MS (injection
temperature: 300°C).
3. Results and discussion
3.1. Configuration of Myrigalone A
Previous NMR investigations showed that myrigalone A undergoes keto-enol
tautomerization with coexistence of mono and dienol forms depending on the solvent.1,5 Since
this tautomerization should affect the absorption characteristics of the molecule, we recorded
the absorption spectrum in water-ACN (90-10%, v/v) and in ACN and in solid form (Figure
1). In water/ACN (90:10), the maximum of absorption of myrigalone A is located at 352 nm
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( = 11300 M-1 cm-1), in ACN two maxima of absorption at 272 ( = 6380 M-1 cm-1) and 327
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nm ( = 5700 M-1 cm-1) are observed and in solid form on a quartz plate, the maximum of
absorption is located at 340 nm. Whatever the medium, the absorption extends up to 400 nm
explaining the yellow color of myrigalone A. The absorption characteristics in water/ACN
(90:10) are similar to those formerly found in ethanol. NMR spectrum in deuterated methanol
revealed that myrigalone A had the structure shown in Scheme 2.1 The high conjugation of
this dienolic structure is consistent with the long wavelength absorption maximum observed
in water: ACN. The structure in the water: ACN solvent should be therefore the same as the
one in alcohols. On the other hand, the absorption spectra of myrigalone A in ACN and in
solid form show some similarities. The maxima of absorption are blue-shifted compared to
water/ACN (90:10) and the tautomers should be therefore less enolized than that in
water/ACN (90:10). Acetonitrile was chosen as a solvent to test the effect of terpenes on the
photolysis of myrigalone A (in the last part of this work).
3.2. Kinetics of photodegradation of Myrigalone A
The kinetics of myrigalone A (10-4 M) photodegradation in water:ACN (90:10) is shown
Figure 2. Myrigalone disappeared quite fast. The initial decay rate is proportional to the
myrigalone A concentration within the wavelength range (1- to 3×10-4 M) (Figure SI-3) and
the kinetics follows a first order kinetics. In solid form, myrigalone A underwent fast
phototransformation too. In the two-lamp device, myrigalone A was totally consumed after 4
h of irradiation.
3.3 Photoproducts identification
The irradiation of myrigalone A led to numerous photoproducts. A typical UPLC-
HRMS-ESI chromatogram obtained after one hour of irradiation in water is shown in Figure
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SI-4. Detailed data obtained by UPLC-HRMS2-ESI and UV absorption are given in Table 1
and Figure SI-5. The ions selected for MS2 are shown by an asterisk. Compounds 1 and 1’ are
both detected at m/z= 315.1240 in ES- and 317.1378 in ES+ and correspond to C18H20O5 and
indicating a gain of an oxygen atom. The isomer 1 eluted at 2 min is already present at t0
while the other is formed during the reaction. A compound showing the same formula was
previously described.5 Using NMR, it was shown that the oxygen atom was added on the
carbon bearing the methyl group (Scheme 3) and the authors considered that myrigalone A
oxidation took place during the isolation procedure. UPLC-HRMS2-ESI analysis of 1 is
consistent with this structure. Indeed, the presence of the fragment at m/z = 181.0502 in ES-
confirms that the addition of the O atom took place on the cyclohexane ring. Then, the
fragment at m/z= 297.1140 corresponds to the loss of water with formation of an ethylenic
structure. The one at m/z= 273.1139 results from the loss of C2H2O due to the release of the
oxidized C4 atom with the methyl group while the one at m/z= 255.1030 to the loss of another
water molecule. The absorption characteristics of 1 (256 and 280 nm) are very different from
those of myrigalone A. In accordance, the oxidation of the C4 carbon atom makes impossible
enolization of the adjacent carbonyls, C5=O and C3=O, and thus drastically reduces electrons
delocalization. The isomer 1’ eluted at 3.6 min shows very close characteristics. The
oxidation can take place on the C6 atom or on the methyl groups. In this latter case, the
absorption characteristics should be the same for 1’ as for myrigalone A because the oxidation
of CH3 into CH2OH should not change enolization possibilities and thus the molecule
conjugation. This is not observed, the structure of 1’ is therefore likely the one given in
Scheme 3.
Compound 2 detected at m/z = 331.1176 in ES- and 333.1324 in ES+ corresponds to
C18H20O6 and thus to the addition of two oxygen atoms on myrigalone A. The fragment at m/z
= 181.0502 in ES- show that the two oxygen atoms have been added on the cyclohexane ring
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and the chemical structure given in Scheme 3 looks highly probable. Another possibility is the
formation of an hydroperoxide on C4 as previously proposed.5 However, the stability of such
a species is questionable.
Compound 3 detected at m/z= 289.1429 in ES+ and 287.1292 in ES- corresponds to
C17H20O4 and to the loss of a C atom compared to myrigalone A. A possible pathway of
formation could be decarbonylation of 1 and/or 1’. The fragments at m/z=271.1324 and
253.1218 in ES+ indicate that the molecule can lose up to two molecules of H2O. The
structure given in Scheme 3 is consistent with these data. Interestingly, compound 4 shows
the same m/z= 271.1322 in ES+ as the fragment of 3. A compound in Myrica gale extracts
showing the same mass and the same UV-vis absorption as compound 4 was already
observed.1 Based on NMR data, the 5-carbon ring structure shown in Scheme 3 was proposed.
Compound 4 is more conjugated than compound 3 and should absorb at longer wavelength.
Yet, the maximum of absorption in the former case is at 245 nm against 278 nm in the latter
one. A detailed observation of the absorption of 4 shows a weak absorption extending up to
350 nm suggesting the presence of another maximum around 300 nm, i.e. at longer
wavelength than that of 3.
Compound 5 with m/z = 271.0980 in ES- and 273.1116 in ES+ has lost C2H4 compared
to myrigalone A. Again, this product could be formed from 1 the main hydroxylated
compound, after the release of HO-C4-CH3. This will yield the triketonic cyclopentane
depicted in Scheme 3. In accordance, the fragment at m/z=139.0392 in ES- (C7H7O3)
corresponds to this dimethylated 5-carbon ring.
Compound 6 has a m/z corresponding to C9H10O2. The molecule is only detected in
negative mode and does not give any fragment. The most likely structure is that of the phenyl
propanoic acid shown in Scheme 4 for which decarboxylation would lead phenyl propane,
undetectable in UPLC-HRMS-ESI.
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Compounds 7 and 9 with m/z = 203.1063 and 217.1221 in ES+, respectively, and 8 and 10
with m/z = 131.03378 and 117.0182 in ES-, respectively, differ each by the presence of a CH3
substituent at C-4 and C-2. However, at m/z value they differ by CH3-H, and by the
substitution of hydrogen by the methyl group. Moreover, the sums of 7 + 8 and 9 + 10 both
correspond to the addition of H2O2 on myrigalone A. There are therefore some analogies in
the formation of these photoproducts. As shown in Scheme 4, 7 could be the result of a double
scission of bonds C1-C6 and C3-C4 with subsequent formation of a 3-carbon ring. The CO-
C(CH3)-CO diradical leaving group could generate a dicarboxylic molecule (8) after oxidation
and addition of OH on the CO. Similarly, 9 could be also the result of a double scission of
bonds C2-C3 and C5-C6 with also formation of a 3-carbon ring. The same rearrangements as
those described for 7 and 8 would lead to 9 and 10. Compound 11 corresponds to C14H18O2,
i.e., to the addition of two H atoms on 9. This molecule is not firmly identified. It might be
derived from 9 through the 3-carbon ring opening.
The kinetics of formation of photoproducts 1’ to 5 in water:ACN are shown Figure SI-
6. Compound 1’ reaches a maximum of formation after 30 min of irradiation when
myrigalone A decay is of 54% and it is fully photodegraded after 120 min when myrigalone A
is also fully transformed. Compound 3 already present in the starting solution reaches a
maximum concentration after 60 min of irradiation and then slowly decays. Photoproducts 4
and 5 accumulate during the whole decay period of myrigalone A and then start to decay
when myrigalone is totally consumed.
Myrigalone A was also irradiated in solid form in a hermetically closed reactor with
the objective to detect volatile photoproducts in the gaseous phase. The analysis of the
gaseous phase was performed after 4h of irradiation and the full disappearance of myrigalone
A. Many compounds were detected (see Table 2). Most of them derived from the phenyl
moiety of myrigalone A and were not or poorly oxygenated. Among them, benzaldehyde was
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the major contributor and acetic acid was also detected in significant amounts. The formation
of volatile photoproducts upon irradiation of a pesticide on leaves was already observed in the
case of acibenzolar-S-methyl.15
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3.4 Irradiation of Myrigalone in the presence of selected additives
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In the leaf glands where myrigalone A is found, other chemicals such as terpenes and
flavonoides are also present.1-3,8 To determine whether these molecules could have an effect
on myrigalone A photodegradation, we conducted a set of experiments in which myrigalone
A was irradiated in the presence of selected additives: eucalyptol or 1,8-cineole and
germacrone, two terpenes detected in the Myrica gale leaf glands. In this case due to the poor
water solubility of the terpenes, experiments were conducted using solution made in ACN.
In the presence of eucalyptol, the photodegradation of myrigalone A is slowed down
with a t1/2 of 60 min against 25 min alone (Figure 3A). In the presence of germacrone, the t1/2
of myrigalone A is equal to 38 min against 25 min alone and the photodegradation of
germacrone is slightly accelerated (Figure 3A). When it is irradiated alone, germacrone yields
several photoproducts, in particular an isomer eluted after germacrone and several oxidation
photoproducts : the ones showing retentions of 3.31 and 4.29 min correspond to the addition
of two O atom on germacrone while the one with a retention time of 3.09 min to the addition
of one O atom and the loss of 2 H atoms (see Figure SI-7). The doubly oxidized products
might be hydroperoxides as such photoproducts have been described in the photooxidation of
terpenes.16 When germacrone is irradiated in the presence of myrigalone A, the same
photoproducts are detected but those corresponding to G+2O are 9-fold more concentrated
than for germacrone irradiated alone while those corresponding to G+O-2H show the same
area as for germacrone irradiated alone. The higher formation of G+2O in the presence of
myrigalone A confirms that myrigalone A photoinduces the photooxidation of germacrone.
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Then, we tested the capacity of myrigalone A to induce the photodegradation of 2,4,6-
trimethylphenol chosen as a H-donor probe. Hence, in surface waters in which it could be in
contact with a lot of other natural molecules or contaminants, myrigalone could act as a
sensitizer and promote the photodegradation of number of these chemicals. In water:ACN,
2,4,6-trimethylphenol that disappears negligibly when irradiated alone showed a t1/2 of 130
min in the presence of myrigalone A (Figure 3B). This shows the capacity of myrigalone to
induce the photodegradation of other molecules.
3.5 Mechanism of Myrigalone photolysis
In the previous study on natural -triketones, the dominant photoproducts were
derivatives hydroxylated at C6 position.11 Only for grandiflorone that differs from myrigalone
A by the additional methyl group on C4, a molecule with two O atoms, reminiscent to 2 for
myrigalone A was also observed at a conversion extent of 50%. The photolysis of myrigalone
A yields much more photoproducts than the other -triketones and the reaction mechanism is
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296
297
298
299
thus more complex. Two types of reaction are observed: oxidation as for the natural -
triketones and Norrish cleavage.
The oxidation of myrigalone A requires the abstraction of an H atom as the initial step,
and the hydrogen atoms of enolic functions could be good candidates (Scheme 5). The
oxidant species might be an excited state such as triplet excited carbonyl, although no
transient species could be detected by laser flash photolysis in the nanosecond timescale.
Addition of oxygen on the radicalar oxidized form of myrigalone A and further
transformation of the peroxyl radical into alcohol can then take place as shown in Scheme 5
for the formation of 2. The dark oxidation of myrigalone A confirms that this molecule bears
very labile hydrogen atoms. Typically, in the course of these oxido-reduction reactions, the
radical formed by reduction of the oxidant, is expected to give back the ground state oxidant
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301
302
303
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306
307
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310
311
312
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314
315
316
317
318
319
320
321
322
323
324
after having transfer the additional H atom to molecular oxygen. Such a reaction is likely to
take place here leading to the regeneration of myrigalone A.
The radicals generated during this reaction, in particular the hydroperoxyl and the
peroxyl radicals, can also abstract an H atom from myrigalone A and propagate the oxidation.
The inhibiting effects of additives such as 2,4,6-trimethylphenol, eucalyptol and germacrone
on myrigalone A photolysis can be therefore explained by their H-donor properties and their
ability to trap the excited state of myrigalone A or the different radicals shown in Scheme 5 in
place to myrigalone A itself through H-atom transfer (Scheme SI-2). They would be oxidized
instead of myrigalone A and thus act as sacrifical agents.
The second type of reaction is a Norrish type I cleavage, a typical rearrangement of
ketones.17 In this reaction, the cleavage of a CO-C bond yields two radicals. The formation of
6 by this pathway is shown in Scheme 6A. Volatile or semi-volatile detected in the gaseous
phase are also likely due to the Norrish cleavage of the CO-C bond of the aliphatic chain.
Several other CO-C bonds can cleave in myrigalone A generating various photoproducts.
Another possibility is the cleavage of two CO-C bonds, consequently or simultaneously. This
explains the formation of 7 and 8 for instance (Scheme 6B).
3.6 Detection of Myrigalone photoproducts in leaf glands and on fruits of Myrica gale
Photoproducts characterized in this work were searched in the yellow leaf glands and
in the oil covering fruits of Myrica gale plants. The old leaves contained 1, 1’, 2, 3 and 5, the
young leaves 1, 1’ and 5 and the oil 1, 1’, 2, and 5. In general, chromatographic area of these
photoproducts were higher in the old leaves and in the oil than in the young leaves which is
consistent with a photochemical formation during sunny days. Moreover, germacrone was
detected in young and old leaves and in oil, while oxidized germacrone essentially in old
leaves and oil. In the oil, we detected the same volatiles by GC-MS as in laboratory
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329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
experiments, in particular acetic acid, benzaldehyde and acetophenone. Even though these
compounds could be produced by the plant itself at late stages of the plant growth, their
detection in old leaves and in oil together with the fast photoproduction in laboratory
experiments strongly suggest that they are formed by photochemistry.
Conclusions
This study showed that myrigalone A undergoes a fast photodegradation yielding a
variety of volatile and non-volatile products. The reaction pattern is complex and involves
both oxidation reactions and/or Norrish cleavage, resulting in photoproducts that are both
oxidized and polar or apolar and semi-volatile. The photoproducts detected in laboratory
experiments were also found in natural samples showing that photolysis can take place in the
real conditions. Interestingly, terpenes also produced by Myrica gale were found to exhibit an
inhibiting effect on myrigalone A, suggesting that they play a significant role in extending the
lifetime of myrigalone A. Such an effect of terpenes on the photochemical reactions of
chemicals was not reported to our knowledge. Further research is needed to investigate the
phytotoxic activity of the volatile and non-volatile photoproducts of myrigalone A or of
terpenes and to determine whether they may have a role in the allelopathic activity of Myrica
gale.
Acknowlegments: This action is led by the Ministry for Agriculture and Food and the
Ministry for an Ecological and Solidary Transition, with the financial support of the French
Biodiversity Agency on « Call for reseach & innovation projects on the development of
alternative solutions to plant protection in gardens, green spaces and infrastructure » research
call, with the fees for diffuse pollution coming from the Ecophyto plan.
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351
352
Supporting Information Available: Detailed description of myrigalone A synthesis with
NMR spectra of intermediates; UPLC-HRMS-ESI chromatogram of an irradiated solution;
UV absorption spectra and UPLC-HRMS2-ESI analysis of the main photoproducts.
353
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357
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References
1- Anthonsen, T.; Falkenberg, I.; Laake, M.; Midelfart, A.; Mortensen, T. Some unusual
flavonoids from Myrica gale. Acta Chem. Scand. 1971, 25, 1929-1930.
2- Popovici, J.; Bertrand, C.; Bagnarol, E.; Fernandez, M.P.;Comte, G. Chemical composition
of essential oil and headspace-solid microextracts from fruits of Myrica gale L. and antifungal
activity. Nat. Prod. Res. 2008, 22, 1024-1032.
3- Mathiesen, L.; Malterud, K.E.; Sund, R.B. Antioxidant activity of fruit exudate and C-
methylated dihydrochalcones from Myrica gale. Planta Med. 1995, 61, 515-518.
366
367
368
369
4- Voegele, A. ; Graeber, K. ; Oracz, K. ; Tarkowská, D. ; Jacquemoud, D. ; Turečková, V. ;
Urbanová, T. ; Strnad, M. ; Leubner-Metzger, G. Embryo growth, test permeability, and
endosperm weakening are major targets for the environmentally regulated inhibition of
Lepidium sativum seed germination by myrigalone A. J. Exp. Botany 2012, 63, 5337-5350.
370
371
372
373
5- Killeen, D. P.; Larsen, L.; Dayan, F.E.; Gordon, K. C.; Perry, N. B.; van Klink, J. W.
Nortriketones: Antimicrobial trimethylated acylphloroglucinols from Manuka (Leptospermum
scoparium). J. Nat Prod. 2016, 79, 564-569.
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376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
6- Oracz, K. ; Voegele, A. ; Tarkowska´, D. ; Jacquemoud, D. ; Turecˇkova´, V. ;
Urbanova´, T. ; Strnad, M. ; Sliwinska, E. ; Leubner-Metzger, G. Myrigalone A Inhibits
Lepidium sativum Seed Germinationby Interference with Gibberellin Metabolism and
Apoplastic Superoxide Production Required for Embryo Extension Growth and Endosperm
Rupture. Plant Cell Physiol. 2012, 53, 81-95.
7- Popovici, J. ; Bertrand, C. ; Jacquemoud, D. ; Bellvert, F. ; Fernandez, M. P. ; Comte, G. ;
Piola F. An Allelochemical from Myrica gale with Strong Phytotoxic Activity against Highly
Invasive Fallopia x bohemica Taxa. Molecules 2011, 16, 2323-2333.
8- Carlton, R. R.; Waterman, P. G.; Gray, A. I. Variation of leaf gland volatile oil within a
population of sweet gale (Myrica gale) (Myricaceae). Chemecology 1992, 3, 45-54.
9- Dayan, F. E; Howell, J. L; Marais, J. P; Ferreira, D.; Koivunen, M. Manuka oil, a natural
herbicide with preemergence activity. Weed Science 2011, 59, 464-469.
10- Beaudegnies, R.; Edmunds, A. J. F.; Fraser, T. E. M.; Hall, R. G.; Hawkes, T. R.;
Mitchell, G.; Schaetzer, J.; Wendeborn, S.; Wibley, J. Herbicidal 4-hydroxyphenylpyruvate
dioxygenase inhibitors-A review of the triketone chemistry story from a Syngenta
perspective. Bioorg. Med.. Chem. 2009, 17, 4134-4152.
11- Trivella, A. ; Stawinoga, M. ; Dayan, F.E. ; Cantrell, C.L. ; Mazellier, P. ; Richard, C.
Photolysis of natural triketonic herbicides in water. Water Res. 2015, 78, 28-36.
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401
402
403
404
12- Dumas, E.; Giraudo, M.; Goujon, E.; Halma, M.; Knhili, E.; Stauffert, M.; Batisson, I.; Besse-
Hoggan; P., Bohatier, J.; Bouchard, P.; Celle-Jeanton, H.; Delbac, F.; Forano, C.; Goupil, P.; Guix,
N.; Husson, P.; Ledoigt, G.; Mallet, C.; Mousty, C.; Prévot, V.; Richard, C.; Sarraute, S. Fate and
ecotoxicological impact of new generation herbicides from the triketone family: an overview for the
assessment of environmental risks. J. Hazard. Mat. 2017, 325, 136-156.
405
406
407
13- Uyar, T.; Malterud, K.E; Anthonsen, T. Two new dihydrochalcones from Myrica gale.
Phytochemistry 1978, 17, 2011-2013.
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409
410
411
412
413
414
415
416
14- Misirlioglu, H.; Stevens, R.; Meikle, T. Synthesis of dihydrochalcones of Myrica gale.
Phytochemistry, 1978, 17, 2015-2019.
15- Sleiman, M.; de Sainte-Claire, P.; Richard, C. Heterogeneous photoreactivity of sprayed
agrochemicals: A case study of the plant activator Acibenzolar-S-methyl. J Agric. Food
Chem. 2017, 65, 7653-7660.
16- Chiron, F.; Chalchat, J. C.; Garry, R.P.; Pilichowski , J.F.; Lacoste, J. Photochemical
hydroperoxidation of terpenes I. Synthesis and characterization of -pinene, -pinene and
limonene hydroperoxides. J. Photochem. Photobiol. A: Chem. 1997, 111, 75-86.
417
418
419
420
421
17- Wang, Z. (2010). Norrish Type I Reaction. In Comprehensive Organic Name Reactions
and Reagents, Z. Wang (Ed.).
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Schemes
424
425
426
O
2
O
O
O
O
O
O
O
O
4
6
O
O
O
Leptospermone
Grandiflorone
Isoleptospermone
O
O
O
O
4
6
2
O
O
O
O
Flavesone
Myrigalone A
427
428
429
Scheme 1: Structure of natural -triketones
430
431
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433
O
O
O
OH
O
O
O
OH
434
435
436
437
438
Scheme 2: Main tautomer of myrigalone A proposed to coexist in alcohols1
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OH
O
O
OH
O
O
OH
O
O
O
HO
O
HO
O
oxidation
OH
O
OH
OH
+
O
OH
2
1
1'
- C2OH4
-CO
O
HO
HO
O
HO
O
OH
O
3
5
-H2O
O
O
O
4
440
441
Scheme 3: Photooxidation of myrigalone A
442
443
444
445
446
447
448
449
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OH
O
O
OH
O
O
O
O
HO2C
6
7
9
+
+
CO2H
CO2H
CO2H
CO2H
10
8
450
451
Scheme 4: Norrish type I reactions of myrigalone A
452
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454
O
+ HO2
O2
OH
O
O
O
*
O
OH
+
+
O
OH
O
OH
O
O
O
O
O
O
O2
HO
OO
O
OH
O
OH
O
OH
2
455
456
457
458
459
460
461
462
463
464
465
466
467
Scheme 5 : Formation of 2
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OH
OH
O
O
h
+
O
OH
O
OH
O2
O
HO
468
469
Scheme 6A: Formation of photoproduct 6 by Norrish type I photocleavage
470
471
OH
OH
O
O
h
+
O
OH
O
OH
O2
O
HO
472
473
Scheme 6B: Formation of photoproducts 7 and 8 by Norrish type I photocleavage
474
475
476
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478
2,0x104
1,5x104
1,0x104
5,0x103
0,0
200
250
300
350
400
Wavelength/nm
479
480
481
482
483
484
485
Figure 1: UV-Vis absorption spectra of myrigalone A dissolved in water-ACN (90-10) at pH
5.2 (solid line) and in ACN (dashed line) and in solid (dotted line)
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487
488
1.0
0.8
0.6
0.4
0.2
0.0
0
10
20
30
40
50
Irradiation time/min
489
490
491
492
493
494
495
496
497
498
Figure 2: Consumption profile of myrigalone A (10-4 M) in water:ACN (90:10, v/v) () upon
irradiation with polychromatic light
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500
A)
O
1.0
0.8
0.6
0.4
0.2
0.0
O
0
10
20
30
40
50
Irradiation time/min
501
502
B)
1.0
0.5
0.0
0
100
200
Irradiation time/min
503
504
505
506
507
Figure 3 : Effect of eucalyptol and germacrone on myrigalone A photolysis (A) and effect of
myrigalone A on 2,4,6-trimethylphenol photodegradation (B). [M]=10-4 M in ACN for A and
[M]=1.6×10-4 M in water:ACN for C; [additive]=10-4 M. -- : consumption of myrigalone A
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509
510
511
or of 2,4,6-trimethylphenol alone and -- : consumption of myrigalone A in the presence of
germacrone; --: consumption of myrigalone A in the presence of eucalyptol
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513
Table 1 : UPLC-HRMS-ESI and absorption data for myrigalone A (M) and photoproducts
Label
Retention time
ES-
ES+
Fragmentsa
Formula
of the neutral
compound
C18H20O4
/nm
M
1
6.2
238/277/335
2.0
256/280 sh
3.6
299.1291
315.1240*
315.1240
NA
317.1378
317.1378
ES-
C18H20O5
C18H20O5
297.1140
273.1139
255.1030
181.0502
ES-
1’
242/280
2
2.0
331.1176*
333.1324
C18H20O6
199.0607
181.0501
149.0597
ES+
271.1324
253.1218
ES+
253.1218
225.1270
ES-
3
4
5
3.5
232/278
287.1292
269.1190
271.0980*
289.1429*
271.1322*
273.1116
C17H20O4
C17H18O3
C16H16O4
5.5
245
1.9
255/326
173.0602
139.0392
6
7
149.0598
NA
NA
203.1063
C9H10O2
C13H14O2
3.98
3.3
8
9
131.03378
NA
NA
C5H8O4
C14H16O2
C4H6O4
217.1221
NA
10
117.0182
11
NA
219.1375
C14H18O2
514
515
a The ion with* was selected for MS2
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516
517
518
Table 2: Volatile photoproducts detected in the gaseous phase upon irradiated of myrigalone
A in solid form.
519
520
Retention m/z
time/min
Proposed
structure
3.66
5.02
5.50
6.70
10.3
12.9
3.9
6.5
7.45
8.54
92
106
104
106
122
100
60
112
98
120
Toluene
Ethylbenzene
Styrene
Benzaldehyde
Benzoic acid
C5H8O2
Acetic acid
C6H8O2
C5H6O2
Acetophenone
521
522
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