Solar irradiation of the seed germination stimulant karrikinolide produces two novel head-to-head cage dimers

Article abstract of DOI:10.1039/c2ob25090j

Karrikinolide is a naturally derived potent seed germination stimulant that is responsible for triggering the germination of numerous plant species from various habitats around the world. We now report that solar irradiation of karrikinolide yields two novel head-to-head cage photodimers with the formation, stability and bioactivity of both presented herein.

Full text of DOI:10.1039/c2ob25090j

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PAPER
Solar irradiation of the seed germination stimulant karrikinolide produces
two novel head-to-head cage dimers†
Adrian Scaffidi,a Mark T. Waters,a Brian W. Skelton,b Charles S. Bond,a Alexandre N. Sobolev,a
Rohan Bythell-Douglas,a Allan J. McKinley,a Kingsley W. Dixon,c,d Emilio L. Ghisalberti,a Steven M. Smitha
and Gavin R. Flematti*a
Received 12th January 2012, Accepted 26th March 2012
DOI: 10.1039/c2ob25090j
Karrikinolide is a naturally derived potent seed germination stimulant that is responsible for triggering the
germination of numerous plant species from various habitats around the world. We now report that solar
irradiation of karrikinolide yields two novel head-to-head cage photodimers with the formation, stability
and bioactivity of both presented herein.
Introduction
The discovery of the potent seed germination stimulant 3-
methyl-2H-furo[2,3-c]pyran-2-one 1 from smoke,1 more com-
monly known as karrikinolide (Fig. 1), has generated significant
research interest due to the diverse range of plant species present
in both fire and non-fire prone regions that respond to this intri-
guing small molecule.2–4 Several other compounds with similar
Fig. 1 Karrikinolide seed germination stimulant.
structure to 1 have also been identified in smoke leading to the
collective name ‘karrikins’.5 The broad species recognition and
highly potent characteristics of 1 at concentrations as low as one
part-per-billion,1,6 provide a unique chemical tool for studying
seed dormancy7,8 and will enable significant cost benefits for
plant restoration programs through enhanced seed germination
and seedling establishment.9–12 Therefore, understanding the
environmental stability and fate of 1 is critical to implementing a
successful transition from research to commercial applications.
In an attempt to shed light on these challenges, we set out to
Fig. 2 General photodimer structures of 4-pyrones.
head-to-tail or head-to-head orientation.15–18 Furthermore, each
photodimer can result in either cis–syn–cis or cis–anti–cis
isomers making outcome prediction difficult (Fig. 2).
In addition, a limited number of cage photodimers resulting
investigate the environmental fate, and more specifically, the UV
stability of the seed germination stimulant 1.
It has previously been observed that similar compounds con-
taining 4-pyrone rings can undergo various photochemical reac-
tions.13,14 One of the main processes is
a [2 + 2]
from a second cyclization of cis–syn–cis head-to-tail photodi-
mers have also been reported,17,19–21 however, to the best of our
knowledge, no head-to-head cage photodimers of 4-pyrones
have been unequivocally characterised.20 Interestingly, for-
mations of the head-to-tail cage photodimers via a double
[2 + 2] cycloaddition have been shown to occur not only in sol-
ution but also in the solid state if the molecular arrangement of
the monomer crystal permits.18,22 More specifically, the centre-
to-centre distances between the two reacting alkenyl groups must
be within the limits proposed by Schmidt of 4.2 Å to initiate the
[2 + 2] photocycloaddition.23,24
photocycloaddition to furnish cyclobutane photodimers of either
aSchool of Chemistry and Biochemistry, The University of Western
Australia 35 Stirling Highway, Crawley, 6009 WA, Australia. E-mail:
gavin.flematti@uwa.edu.au
bCentre for Microscopy, Characterisation and Analysis, The University
of Western Australia, 35 Stirling Highway, Crawley, 6009 WA, Australia
cSchool of Plant Biology, The University of Western Australia,
35 Stirling Highway, Crawley, 6009 WA, Australia
dKings Park Botanic Garden, Fraser Avenue, West Perth, 6005 WA,
Australia
†Electronic supplementary information (ESI) available: NMR spectra.
CCDC 837152 and 837151. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/c2ob25090j
The solar stability of 1 was initially evaluated by irradiating a
dilute solution with natural sunlight. The reaction was monitored
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Fig. 4 The concentrations of 1 (primary axis), 2 and 3 (secondary
axis) after irradiating a 100 μg mL−1 solution of 1.
Fig. 3 Karrikinolide cage photodimers 2 and 3. (a) Structure of mol-
ecule 1 of cage photodimer 2 (second molecule similar). (b) Structure of
cage photodimer 3. All atoms have been drawn with arbitrary radii.
by high performance liquid chromatography (HPLC) with the
complete consumption of 1 observed within hours; however, it
was not clear whether 1 simply degraded or if products resulted
from a photochemical reaction. Further investigations by gas
chromatography-mass spectrometry (GC-MS) indicated the pres-
ence of two dimeric species with nuclear magnetic resonance
(NMR) spectroscopy confirming two cage photodimers due to
the absence of any downfield signals.
Fig. 5 Concentration of cage photodimer 2 and 3 (primary axis) pro-
duced at various wavelengths and UV absorbance spectrum of 1, 2 and 3
(secondary axis).
Given the symmetry generated by the formation of cage
photodimers, NMR spectroscopy does not discriminate between
head-to-head and the corresponding head-to-tail cage structures.
For example, head-to-head cage dimers with a mirror plane of
symmetry cannot be distinguished from head-to-tail cage dimers
with a two-fold axis of rotation. Likewise, head-to-head cage
dimers with a two-fold axis of rotation cannot be distinguished
from head-to-tail cage dimers with an inversion centre of sym-
metry. As a result, single crystal X-ray structure elucidation was
required to unequivocally assign two novel karrikinolide head-
to-head cage photodimers 2 and 3 (Fig. 3).
The unique cage photodimer 2 is a result of a double photocy-
clisation of two molecules of 1 superimposed with one molecule
rotated 180° along the C(3)–O(6) line. In contrast, two superim-
posed molecules of 1 resulted in the cage photodimer 3. The
crystal structure confirms the geometry of cage photodimer 2 as
having a pseudo two-fold axis relating the two butenolide rings
in comparison with the pseudo-mirror symmetry in the structure
of cage photodimer 3. There are two independent molecules in
the asymmetric unit of 2. Due to the lack of precision of the
determination, meaningful comparisons of the geometries with
those of structure 3 were not possible. The angles between the
two butenolide rings are 85.0(8) and 87.9(9)° for molecules 1
and 2 of cage photodimer 2 respectively, compared to 82.05(5)°
for those of cage photodimer 3.
indicated the presence of two cage photodimers formed within
the first hour with the reaction requiring a full day of irradiation
to see complete consumption of the starting material (Fig. 4).
The formation of both cage photodimers reached a maximum
around nine hours and were also found to degrade upon further
exposure to solar radiation with no further products observed by
GC-MS.
We then turned our attention towards identifying the optimal
wavelength to afford the cage photodimers by irradiating a sol-
ution of 1 with a high pressure xenon–mercury lamp fitted with
a monochromator. The reaction was monitored by GC-MS and it
was found that the optimal wavelength for cage photodimer for-
mation was within the range of 300 nm to 320 nm, consistent
with the UVabsorbance of 1 (Fig. 5).
Having successfully isolated two head-to-head cage photo-
dimers from irradiation of a dilute solution of 1, we set about
investigating whether similar transformations could occur in the
solid state as previously reported for 4-pyrones.18,22 To this end,
a crystal of 1 was irradiated with the solar simulator for several
hours but no dimerisation was observed by X-ray crystallogra-
phy. This result was not surprising as although the centre-to-
centre distances between the two reacting alkenyls have been
determined to be within the limits proposed by Schmidt,23,24 the
orientations are such that one unit would need to rotate approxi-
mately 60° to orientate anti-parallel along the C(3)–O(6) line.25
During efforts to optimise GC-MS conditions for the detection
of karrikinolide 1 and the two cage photodimers 2 and 3, we
observed that the amount of each compound detected had an
optimal injection temperature (Fig. 6). Above this temperature, a
reduced quantity of each compound was detected indicating that
all three butenolides were sensitive to elevated temperatures.
To investigate further the degradation of 1 and the formation
of cage photodimers 2 and 3 by means of solar radiation, a
dilute solution of 1 was irradiated under controlled conditions
employing a solar lamp simulator. This allowed the reaction to
be monitored at constant power output and removed variations
associated with natural solar UV indices. Once again, GC-MS
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that dimerization of 1 is restricted in the solid state. In addition,
some of the 4800 compounds29 produced during a fire may
protect 1 from UV degradation, allowing it to remain in the
environment for prolonged periods of time.
Conclusion
In summary, our study demonstrates that upon irradiation with a
solar light source, the seed germination stimulant 1 affords two
novel head-to-head cage photodimers 2 and 3. In addition to
reporting the first example of the unique cage moiety, it has been
shown that the facile double photocycloaddition is reversible
under elevated temperatures enabling the photodimers to revert
back to the monomer unit. Moreover, we have highlighted the
reactivity of karrikinolide 1 to solar radiation which suggests that
suitable formulations containing UV stabilisers may be required
to enable 1 to be used effectively in plant regeneration and land
conservation programs.
Fig. 6 Total-ion-count for a 10 μg mL−1 solution of 1, 2 and 3 at
various temperatures (primary axis), and total-ion-count for 1 produced
from 2 and 3 at various temperatures (secondary axis).
Experimental
General
1H and 13C nuclear magnetic resonance (NMR) spectra were
obtained on a Bruker AV600 (600 MHz for δH and 150.9 MHz
for δC) spectrometer. Hexadeuteroacetone ((CD3)2CO) was used
as the solvent with residual (CH3)2CO (δH 2.05) or (CD3)2CO
(δC 29.84) being employed as internal standards. Melting points
(m.p.) were determined on a Reichert hot stage melting point
apparatus. High-resolution mass spectra (HR-MS) were recorded
on a Waters GCT spectrometer using chemical ionisation (45
eV) (CI) technique. Gas chromatography-mass spectrometry
(GC-MS) were recorded on an Agilent 5973 instrument using
electron ionisation (EI) technique. UV-Vis data was collected on
a Perkin-Elmer Lambda 25 spectrophotometer. Flash chromato-
graphy was performed on Merck silica gel 60 with the specified
solvents and thin-layer chromatography (TLC) was effected on
Merck silica gel 60 F254 aluminium-backed plates. Karrikino-
lide 1 was prepared as previously described.30
Fig. 7 Germination of Solanum orbiculatum seeds in response to cage
photodimers 2 and 3.
Moreover, a rise in injection temperature resulted in increased
amounts of 1 detected from each of the cage photodimers indi-
cating the thermal reversibility of the facile double photocyclisa-
tion reaction required to furnish 2 and 3.
The reduction of 1 observed for injection temperatures above
240 °C is of particular interest given 1 is produced from burning
plant material.1 Since wildfire flame temperatures can reach
several hundred degrees,26 we speculate that 1 is mainly pro-
duced in the cooler parts of the fire and must be removed from
the source of the fire in the smoke or condensate fraction allow-
ing it to ultimately stimulate germination.
The two cage photodimers were purified by semi-preparative
HPLC prior to screening for stimulatory activity against
Solanum orbiculatum11 seeds under a range of concentrations.
Somewhat surprisingly, given recent structure–activity investi-
gations suggest substitutions at C4 significantly reduce activity,27
both photodimers appeared to promote germination above
control levels at higher concentrations (Fig. 7). However, given
that 1 is active below nanomolar concentrations1 and given the
reversibility of the cage photodimers at elevated temperatures,
we cannot rule out the presence of trace amounts of 1 which
may account for this unusual stimulatory ability.
Our results suggest that exposure to solar radiation after a
wildfire may result in smoke-derived 1 being consumed within a
relatively short period of time. However, soil exposed to plant-
derived smoke has been shown to promote germination several
years after a fire event28 and we have detected the presence of 1
in soil collected several months after a wildfire. In contrast, we
have not detected 2 or 3 in the same samples, which confirms
Cage photodimer isolation
In a silica glass reaction vessel was dissolved karrikinolide 1
(50 mg, 0.3 mmol) in a mixture of methanol and water (30 mL,
2 : 1) and the resulting solution irradiated for 24 h with an Oriel
Instruments Research Arc Lamp (Newport: 66907) fitted with a
xenon lamp (18.2 V, 5.5 A) and AM1.5G filter. The solution was
concentrated under vacuum followed by flash chromatography
(30%–70% ethyl acetate–hexanes) to furnish the cage photodi-
mer 2 as colourless needles after recrystallisation (dichloro-
1
methane–hexanes) (6.0 mg, 12%), m.p. > 250 °C; H NMR δ
5.05 (dd, 3J(H5/5′,H7′/7) = 8.2 Hz and 3J(H4/4′,H5/5′) = 8.1 Hz,
1H; H5/5′), 5.01 (dd, 4J(H4′/4,H7/7′) = 2.46 Hz 1H; H7/7′), 4.52
(dd, 1H; H4/4′), 1.77 (s, 3H; H3b/3′b). 13C NMR δ 173.21 (C2/
2′), 154.18 (C3a/3′a), 121.59 (C3/3′), 85.51, 78.84, 68.24 (C5/5′,
C7/7′, C7a/7′a), 44.68 (C4/4′), 8.54 (C3b/3′b). UV/Vis (metha-
nol): λmax(ε) = 235 nm (12 180); CI-HRMS: m/z 301.0712, ([M
+ H]+ C16H13O6 requires 301.0717). EI-MS: m/z (%) 254(5),
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150(25), 121(28), 84(100). Next to elute was the cage photodi-
mer 3 yielding needles after recrystallisation (dichloromethane–
hexanes) (6.0 mg, 12%), m.p. > 250 °C; 1H NMR δ 5.10 (s, 1H;
H7/7′), 4.95 (m, 1H; H5/5′), 4.47 (m, 1H; H4/4′), 1.74 (s, 3H;
H3b/3′b). 13C NMR δ 173.27 (C2/2′), 155.07 (C3a/3′a), 120.98
(C3/3′), 86.83, 76.05, 72.49 (C5/5′, C7/7′, C7a/7′a), 38.73 (C4/
4′), 8.34 (C3b/3′b). UV/Vis (methanol): λmax(ε) = 235 nm
(11 840); CI-HRMS: m/z 301.0705, ([M + H]+ C16H13O6
requires 301.0717). EI-MS: m/z (%) 300(M+,7), 227(45), 216
(30), 150(41), 122(24), 121(90), 84(100), 65(24). Semi-prepara-
tive HPLC was conducted using a Hewlett Packard 1050 system
equipped with a multiple wavelength detector (MWD). Separ-
ation was achieved using a 250 × 10 mm i.d., 5 μm, Apollo C18
reversed phase column (Grace-Davison) with a 33 mm × 7 mm
guard column of the same material. The column was eluted at
4 mL min−1 with 30% (v/v) acetonitrile–water and 1 mL was
injected. UV absorbance was monitored at wavelengths of 210,
254 and 325 nm. Photodimer 2 Rt = 23.67 min, photodimer 3
Rt = 24.48 min.
twin with approximately 50/50 occupancy. The geometries of
the two independent molecules were restrained to be similar. All
atoms were refined with isotropic displacement parameters after
anisotropic refinement resulted in many atoms with unacceptable
ellipsoids even with the application of reasonable restraints.
Attempts to obtain a disordered model for either of the molecules
or a superior solution in a space group of lower symmetry were
not successful.
Crystal data for 3: Formula C16H13O6, Mr = 300.26, monocli-
nic, P21/c, a = 6.8238(2) Å, b = 11.0492(3) Å, c = 16.3885(4)
Å, β = 100.760(2)°, V = 1213.93(6) Å3, Z = 4, ρcalcd = 1.643 g
cm−3, μ = 0.127 mm−1, 2θmax = 63°, 22 968 reflections col-
lected, 4234 unique, Rint = 0.040, R1 = 0.039, wR2 = 0.106.
CCDC No. = 837151. Anisotropic displacement parameters were
employed for the non-hydrogen atoms.
Seed bioassay
Germination experiments were performed using Solanum orbicu-
latum seeds collected in the Shark Bay region (Western Austra-
lia) and stored at −80 °C until use. Assays were conducted using
Millipore water obtained by filtration through a Milli-Q ultrapure
water system (Millipore, Australia). Solutions were tested for
germination activity by adding 2.5 mL to two layers of
Whatman no. 1 filter paper (7.0 cm) in plastic Petri dishes
(9.0 cm) followed by approximately 20–30 seeds. The Petri
dishes were sealed with a layer of plastic wrap and stored in a
light-proof container for 7 days at 20 ( 1 °C) with all experi-
ments conducted in triplicate.
The authors thank the Australian Research Council
(LP0882775) for financial support and the Australian Synchro-
tron, Victoria, Australia for use of the PX2 beamline. We also
thank Jeremiah Toster for use of the solar simulator and E.D.
Goddard-Borger for helpful discussions.
Wavelength optimisation
A solution of 1 (1.0 mL, 0.1 mg mL−1) in methanol was ir-
radiated for 6 h at different wavelengths (240–400 nm) using a
high pressure mercury/xenon lamp (1000 W, Newport: 6293)
fitted with a monochromator. The radiation intensity was
measured at each wavelength with a laser power meter (Ophir
AN/2) with all measurements normalized to the intensity at
300 nm. The resulting solutions were analysed by GC-MS using
a Varian factor four column (VF-5 ms, 30 m × 0.25 mm i.d.,
0.25 μm) with the initial oven temperature set at 50 °C and held
for 1 min before increasing at 15 °C min−1 to 250 °C, then 5 °C
min−1 to 300 °C, and held for 10 min (inlet temperature 260 °C;
transfer line 280 °C). The ion source was set at 200 °C and the
spectrometer was set to record in selective ion monitoring (SIM)
mode using ions m/z 121 and 150 for 1 and m/z 84 and 121 for
the cage photodimers 2 and 3. Quantitation was achieved using
m/z 121 for 1 and m/z 84 for both cage photodimers. Rt (photo-
dimer 2) = 19.98 min, Rt (photodimer 3) = 22.37 min.
Notes and references
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100 K, the Beamline PX2 at the Australian Synchrotron31 (λ =
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least-squares using the program SHELXL-97.32 All H-atoms
were added at calculated positions and refined by use of a riding
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the parent atom.
Crystal data for 2: Formula C16H13O6, Mr = 300.26, monocli-
nic, P21/c, a = 17.0210(4) Å, b = 11.9940(10) Å, c = 13.4560
(5) Å, β = 113.283(4), V = 2523.3(2) Å3, Z = 8, ρcalcd = 1.581 g
cm−3, μ = 0.123 mm−1, 2θmax = 50°, 16 855 reflections col-
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