The synthesis and biological evaluation of labelled karrikinolides for the elucidation of the mode of action of the seed germination stimulant

Article abstract of DOI:10.1016/j.tet.2010.11.006

Karrikins are a novel class of naturally occurring plant growth regulators that promote seed germination among a diverse range of species. Currently little is known about the mechanism by which these compounds overcome seed dormancy and initiate germination. The preparation of various karrikinolide derivatives provides an opportunity to investigate the karrikinolide mode of action at the cellular and molecular level. The first synthesis and biological evaluation of analogues suitable for use in metabolic labelling, affinity chromatography, photoaffinity labelling and NanoSIMS experiments are reported.

Full text of DOI:10.1016/j.tet.2010.11.006

Tetrahedron 67 (2011) 152e157
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
The synthesis and biological evaluation of labelled karrikinolides for the
elucidation of the mode of action of the seed germination stimulant
,
a
a
,
,
,b
Adrian Scaffidi a , Gavin R. Flematti , David C. Nelson b, Kingsley W. Dixon c d, Steven M. Smith a
,
*
Emilio L. Ghisalberti a
a School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Crawley, Western Australia 6009, Australia
b Plant Energy Biology, The University of Western Australia, Crawley, Western Australia 6009, Australia
c Kings Park and Botanic Garden, West Perth, Western Australia 6005, Australia
d School of Plant Biology, The University of Western Australia, Crawley, Western Australia 6009, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 August 2010
Received in revised form 1 October 2010
Accepted 1 November 2010
Available online 5 November 2010
Karrikins are a novel class of naturally occurring plant growth regulators that promote seed germination
among a diverse range of species. Currently little is known about the mechanism by which these
compounds overcome seed dormancy and initiate germination. The preparation of various karrikinolide
derivatives provides an opportunity to investigate the karrikinolide mode of action at the cellular and
molecular level. The first synthesis and biological evaluation of analogues suitable for use in metabolic
labelling, affinity chromatography, photoaffinity labelling and NanoSIMS experiments are reported.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Karrikinolide
Karrikins
Germination stimulant
Smoke
Seed dormancy
1. Introduction
O
3
Plant-derived smoke has a remarkable ability to increase the
germination success of more than 1200 species from over 80 gen-
era1 located in various regions around the world including Aus-
tralia,2 South Africa,3 North America4 and Europe.5 Interestingly,
this response is not limited to species present in fire-prone regions
as smoke-stimulated germination has also been observed for spe-
cies from regions not frequented by fire.6
The key germination stimulant from smoke was first isolated
and identified by Flematti et al. as 3-methyl-2H-furo[2,3-c]pyran-2-
one 1, now termed karrikinolide 1 (Fig. 1).7 This naturally occurring
germination stimulant displays activity in a variety of species at
concentrations as low as one-part-per-billion.1,8 Recently it has
been revealed that at least five other analogues are present in
smoke leading to the collective name ‘karrikins’.9
O
1
4
5
7
O
1
Fig. 1. Karrikinolide.
seed dormancy and germination, thus facilitating broader environ-
mental and commercial applications. The potential for promoting
a more uniform release from seed dormancy, improving germina-
tion rates and widening the range of species that respond, will allow
for more effective plant regeneration and conservation in restora-
tion programs. In addition, novel approaches to weed control
through initiating germination followed by treatment with knock-
down herbicides offers great potential for reducing weed seed levels
in agricultural settings.1
The mode of action of karrikinolide remains unknown, however
interactions with factors influencing germination such as light,
gibberellic acid and abscisic acid have all been observed.10,11
Unlocking the secrets behind karrikinolide perception may result
in a better understanding of the processes involved in regulating
Since the discovery of 1, only four syntheses have been pub-
lished. These include the preparation of 1 from pyromeconic acid
2,12 ethyl-4-methyl-2-oxo-2,5-dihydro-furan-3-carboxylate 3,13 2-
* Corresponding author. E-mail address: adrian.scaffidi@uwa.edu.au (A. Scaffidi).
furfurylmethanol 414 and -xylose 515 (Scheme 1).
D
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.11.006

A. Scaffidi et al. / Tetrahedron 67 (2011) 152e157
153
and a detection tag, for example, biotin. The Staudinger ligation23,24
and Huisgen 1,3-dipolar cycloaddition25,26 are examples of bio-
orthoganol reactions that have been successfully used in a number
of systems. The Staudinger ligation involves the reaction between
an azide and triphenylphosphine modified with a methyl ester to
result in the formation of a stable amide linkage between the
chemical reporter and detection tag. In a similar fashion, the
Huisgen 1,3-dipolar cycloaddition also allows for the covalent at-
tachment between an azide/alkyne containing chemical probe and
an alkyne/azide modified detection tag through the formation of
a triazole functionality, and has recently been used to label a pa-
pain-like cystine protease in Arabidopsis thaliana.27
Affinity chromatography is another common technique used for
the separation of complex biological mixtures, and has previously
been shown to be effective in the isolation of soluble and mem-
brane bound abscisic acid binding proteins.28 The identification of
interactions between two molecules is made possible by the at-
tachment of a molecular probe to a solid support. This is commonly
achieved by tagging the molecular probe with biotin, usually via
a polyethylene glycol linker to avoid steric hindrance and increase
solubility, prior to the attachment to an avidin or streptavidin solid
support. This methodology exploits the high affinity of avidin and
streptavidin for biotin (Kd¼10ꢀ14e10ꢀ15 M).29
O
O
HO
EtO2C
O
O
2
3
O
O
O
1
O
HO
HO
HO
OH
O
OH
4
5
Scheme 1.
A variation on the synthesis of 1 from
hexose -glucuronic acid -lactone 6, results in the addition of
D
-xylose 5 utilising the
D
g
The use of photoaffinity labelling for the isolation of binding
proteins requires the synthesis of a bioactive molecular probe
containing a photoreactive group, which upon irradiation with
a light source, will result in a covalent cross-link between the probe
and a nearby protein.19 Arylazides,30 diazirines31 and free radical
generating ketones32 upon irradiation, have all been shown to ex-
hibit suitable photochemical properties for labelling experiments.
Besides the attachment to a photoreactive group, bioactive probes
must also contain a suitable label for downstream detection with
the most common method involving the use of radioisotopes.
However, recent reports have seen the development of radioiso-
tope-free photoaffinity labelling by utilising alkyl azides and al-
kynes for subsequent Staudinger ligation and Huisgen
cycloadditions with various detection tags.33
a hydroxymethyl moiety at the C5 position of 1 affording the al-
cohol 7 (Scheme 2).15 Compound 7 provides an excellent scaffold
for preparing analogues to investigate the karrikinolide mode of
action as structure-activity relationships reveal that C5 derivatives
retain a high level of bioactivity with test species Solanum orbicu-
latum compared with analogues substituted at other positions
of 1.16
O
HO
O
OH
OH
O
O
O
OH
O
6
7
In addition to gaining information about the binding in-
teractions of bioactive molecules at the molecular level, a number
of techniques allow for visualising the location of bioactive mole-
cules at the sub-cellular level. NanoSIMS is a modern technique that
detects the difference in the isotope ratio of secondary ions gen-
erated by bombarding the surface of a sample with a primary ion
source.20 The use of an isotope-enriched molecular probe results in
a different isotope ratio at the location of the probe. The isotope
enrichment removes the need for structural modification of the
bioactive probe, which may adversely affect the bioactivity, thus
providing an advantage over other scanning techniques.
Scheme 2.
The identification of the fundamental relationships between
small bioactive molecules and their binding proteins, for example,
a ligand and receptor, presents a challenging task that often relies
on the nature of the interaction. As a result, a number of methods
have been developed to recognise both covalent or non-covalent
interactions including metabolic labelling,17 affinity chromatogra-
phy,18 photoaffinity labelling19 and NanoSIMS,20 with each method
requiring a uniquely prepared bioactive molecule.
Metabolic labelling, as the name suggests, involves the labelling
of a cellular target with a modified bioactive molecule using the
natural reaction mechanisms of the target biomolecule. The success
of this method relies on the stable attachment of the bioactive
molecule to a cellular target through a covalent linkage.21 It has
recently been proposed that 1 may adopt a similar mechanism to
that proposed for strigolactone bioactivity based on their related
methyl substituted butenolide rings and Michael acceptor charac-
teristics.22 If this proves to be the case, the interaction of 1 with
a putative receptor would result in a covalent attachment detect-
able via metabolic labelling techniques.
One method of metabolic labelling that has attracted significant
attention in recent years involves the incorporation of a non-eva-
sive chemical reporter onto the bioactive molecule, for example, an
azide or alkyne moiety, which is stable under most biological
conditions. This allows for the identification of proteins that are
covalently attached to a bioactive molecule of interest through
highly specific bioorthoganol reactions between the reporter tag
2. Results and discussion
A karrikinolide analogue suitable for use in metabolic labelling
studies was investigated. Studies have shown that analogues
modified at C3, C4 or C7 (Fig. 1) resulted in reduced germination
activity, while derivatives substituted at C5 were better tolerated.16
Thus, it was proposed that the incorporation of an azide moiety at
C5 would yield an analogue that should retain good bioactivity in
addition to one, that is, amenable to further functionalisation with
detection tags. To this end, the alcohol 715 was converted to the
mesylate followed by displacement with azide to furnish the kar-
rikin 8 (Scheme 3).
The germination activity of 8 was evaluated using Brassica
tournefortii seeds that had low control germination levels (<10%)
and were stimulated to over 90% germination with 1. As expected,
compound 8 showed excellent activity at concentrations as low as
one hundred-parts-per-billion (Fig. 2).

154
A. Scaffidi et al. / Tetrahedron 67 (2011) 152e157
alcohol 715 was alkylated with 1-azido-9-bromononane 11 to fur-
nish the azide 12 (Scheme 5).
The germination response of 12 was screened using B. tourne-
fortii seeds and revealed low activity, which was deemed sufficient
at higher concentrations to proceed to labelling with biotin (Fig. 3).
O
O
O
O
a
(81%)
OH
N3
O
O
7
8
Scheme 3. (a) (i) MsCl, Et3N, CH2Cl2 (ii) NaN3, DMF.
100
80
60
40
20
0
Fig. 3. Germination activity of 12.
104
0
1
10
concentration (
102
g/L)
103
This was achieved via the treatment of azide 12 with biotin al-
kyne 1327 under Huisgen 1,3-dipolar cycloaddition conditions to
return the triazole 14 in modest yield (Scheme 6).
µ
Fig. 2. Germination activity of 8.
With the successful synthesis of an active karrikinolide de-
O
rivative for metabolic labelling, attention was then directed to-
wards the preparation of an analogue suitable for affinity
chromatography. The attachment of a detection tag to the karriki-
nolide scaffold through a suitable linker appeared the most at-
tractive method in achieving this goal. The alcohol 715 was again
chosen as the starting material, however, a conscious effort was
made to reduce the number of subsequent synthetic steps given the
relatively low overall yield in obtaining the alcohol 7.15 As a result,
a triethylene glycol linker containing a halide leaving group for
alkylation onto the karrikin backbone, along with an azido moiety
for further functionalisation with biotin, was prepared.34 With the
bromide 934 in hand, the alcohol 715 was alkylated under standard
conditions resulting in the azide 10 (Scheme 4). The bioactivity of
10 was assessed prior to the addition of biotin but no activity was
observed with B. tournefortii seeds.
O
HN
H
NH
H
O
a
+
HN
S
N3
9
O
(52%)
O
O
12
13
O
O
S
O
HN
H
NH
H
N
N
H
N
N
9
O
O
O
14
Scheme 6. (a) CuSO4.5H2O, sodium ascorbate, EtOH, H2O.
O
Unfortunately 14 did not stimulate the germination of B. tour-
nefortti seeds under the range of concentrations tested. Although
the lack of activity might be due to the additional steric elements of
14, the possible low permeability properties could also contribute
to this effect and as a result, the triazole 14 may still be suitable for
in vitro affinity studies.
The design of a suitable photoaffinity probe relied on the highly
active benzyl derivative 15 when tested against S. orbiculatum
(Fig. 4).16
O
O
O
a
OH
O
O
(9%)
O
N3
7
3
Br
N3
10
O
2
9
Scheme 4. (a) NaH, DMF.
O
O
Based on recent structure-activity relationships,16 it was pro-
posed that the loss of activity may be a result of the increased
polarity of the triethylene glycol linker and that substitution with
an alkyl chain may return some bioactivity. To test this theory, the
O
O
O
15
O
Fig. 4. Karrikinolide benzyl derivative.
O
O
The modification of the benzyl ring to include an aryl azide for
photoactivation, in addition to a bioorthoganol alkyl azide for fur-
ther functionalisation with a detection tag, would yield a com-
pound with all the necessary requirements for a radioisotope-free
photoaffinity probe. Hosoya et al. recently reported the modifica-
tion of cervastatin with the bromide 1635 for use in photolabelling
the catalytic domain of human 3-hydroxy-3-methylglutaryl co-
enzyme A (HMG-CoA).33 It was revealed that the alkyl azide moiety
a
OH
O
N3
9
O
(15%)
O
7
N3
9
Br
12
11
Scheme 5. (a) NaH, DMF.

A. Scaffidi et al. / Tetrahedron 67 (2011) 152e157
155
remained intact during photoactivation of the aryl azide thus
allowing for chemoselective ligation with a fluorescent tag for de-
tection. This methodology was adopted for the synthesis of 17
resulting from the alkylation of the alcohol 715 with the bromide
1635 (Scheme 7).
identification of a karrikin binding protein. Various labelling ex-
periments have now been made possible allowing the metabolic
fate of karrikinolide to be investigated.
4. Experimental
O
O
4.1. General experimental
1H and 13C nuclear magnetic resonance (NMR) spectra were
obtained on a Bruker ARX500 (500 MHz for dH and 125.7 MHz for
O
OH
dC) or a Bruker AV600 (600 MHz for dH and 150.9 MHz for dC
spectrometer. Unless otherwise stated, deuterochloroform (CDCl3)
was used as the solvent with residual CHCl3 dH 7.26) or CDCl3 dC
)
O
N3
O
a
7
(
(
77.0) being employed as internal standards. Spectrums run in tet-
radeuteromethanol (CD3OD) used residual CH3OH (dH 3.34) or
CD3OD (dC 49.0) as internal standards.
(24%)
O
N3
O
Br
N3
17
Melting points (mp) were determined on a Reichert hot stage
melting point apparatus. High-resolution mass spectra (HRMS)
were recorded with a VG-Autospec spectrometer using electron
impact (70 eV) ionisation (EI) technique.
N3
16
Scheme 7. (a) NaH, DMF.
All experiments were carried out under an inert atmosphere
and all solvents were dried prior to use. [13C]5-
-Xylose 18 was
D
Once again the germination activity of the azide 17 was evalu-
ated with B. tournefortti seeds with assay results indicating no
stimulatory response. It was thought that any modification of 17 to
render an active photoaffinity probe would result in limited success
and therefore endeavours to prepare a bioactive photoaffinity
probe were no longer pursued.
obtained from Omicron Biomedical Inc. and [3H]-enriched water
was acquired from Amersham. A ‘usual workup’ refers to dilution
with water, repeated extraction into an organic solvent, sequential
washing of the combined extracts with hydrochloric acid (1 M,
where appropriate), saturated aqueous sodium bicarbonate and
brine solutions, followed by drying over anhydrous magnesium
sulfate, filtration and evaporation of the solvent by means of a ro-
tary evaporator at reduced pressure.
The preparation of karrikinolide from [13C]5-
-xylose 18 fol-
D
lowing the procedure of Goddard-Borger et al.15 allowed for the
isolation of an isotope-enriched karrikinolide 19 suitable for the use
in NanoSIMS experiments (Scheme 8). As expected, the ability of
compound 19 to stimulate the germination of B. tournefortti seeds
was identical to that observed for 1.
Flash chromatography was performed on Merck silica gel 60
with the specified solvents. Thin-layer chromatography (TLC) was
effected on Merck silica gel 60 F254 aluminium-backed plates that
were stained by heating (>200 ꢁC) with 0.25 M ceric sulfate in 2 M
sulfuric acid.
O
Semi-preparative high pressure liquid chromatography (HPLC)
was performed on an Agilent 1050 system using a 250ꢂ10 mm i.d.,
O
O
HO
HO
OH
5
m
m, Apollo C18 reversed-phase column (Grace-Davison).
Percentage yields for chemical reactions as described are quoted
only for those compoundsthat were purified by recrystallisation or by
O
19
OH
=
13C
18
chromatographyandthepurityassessedby TLC orNMR spectroscopy.
Scheme 8.
In addition to preparing analogues for binding protein elucida-
tion and location, a compound suitable for identifying metabolism
products and stability of 1 within the seed and during applications
to soil was sought. Sun et al. recently demonstrated that the
treatment of 1 with LiHMDS followed by the addition of an elec-
trophile, results in the formation of C7 substituted analogues.13
Following this methodology, the synthesis of a radiolabeled karri-
kinolide 20 was achieved through the use of [3H]-enriched water as
the electrophile source (Scheme 9).
4.2. Seed bioassay
All germination experiments were performed using B. tourne-
fortii seeds collected in the Meckering region of Western Australia
and stored at ꢀ80 ꢁC until use. All assays were conducted using
Millipore (MP) water obtained by filtration through a Milli-Q ultra-
pure water system (Millipore, Australia). A 1% acetone solution was
used as a control.
Stock solutions of 100 ppm were prepared by dissolving 1.0 mg
of compound in 100 mL of acetone prior to adding 9.9 mL of MP
O
O
O
O
water. Subsequent dilutions gave concentrations of 10 ppm, 1 ppm,
100 ppb, 10 ppb and 1 ppb. The solutions were tested for germi-
nation activity by adding 3 mL to one piece of Filtech glass micro-
fiber filter paper (8.2 cm) in plastic Petri dishes followed by
approximately 20e30 seeds. The Petri dishes were sealed with
a layer of plastic wrap and stored in a light proof container for 3
days at 20ꢃ1 ꢁC. All experiments were conducted in triplicate.
a
(27%)
O
1
[3H]
O
20
Scheme 9. (a) (i) LiHMDS, THF (ii) [3H]eH2O.
3. Conclusion
4.3. Synthesis
In summary, the synthesis and biological evaluation of the first
generation of labelled karrikinolides prepared from 715 is reported.
The diversity of labelling techniques allows the use of a variety of
different methods for the potential localisation, isolation and
4.3.1. 5-Azidomethyl-3-methyl-2H-furo[2,3-c]pyran-2-one
(8). Triethylamine (55 mg, 0.5 mmol) in dichloromethane (0.5 mL)
was added to the alcohol 715 (28 mg, 0.2 mmol) in dichloromethane
(2.0 mL) at 0 ꢁC and the solution stirred (10 min). To this solution

156
A. Scaffidi et al. / Tetrahedron 67 (2011) 152e157
was added, methanesulfonyl chloride (32 mg, 0.3 mmol) in
dichloromethane (0.5 mL) and the solution left to stand (30 min).
The reaction was subjected to a usual workup (dichloromethane) to
leave a white powder that was dissolved in dimethylformamide
(2.0 mL) followed by the addition of sodium azide (70 mg,
1.1 mmol). The mixture was stirred at room temperature (30 min)
before being subjected to a usual workup (dichloromethane) and
flash chromatography (ethyl acetateehexane 3:7) to return the
azide 8 (26 mg, 81%) as a tan powder. Rf (ethyl acetateehexane 1:1)
0.8; mp 118e120 ꢁC; dH (600 MHz) 7.41 (1H, s, H7), 6.52 (1H, s, H4),
4.17 (2H, s, H8), 1.95 (3H, s, H3b). dC (150.9 MHz) 171.03 (C2), 154.66
(C5), 141.69, 139.76 (C3a, C7a), 126.10 (C7), 101.80 (C3), 101.35 (C4),
51.39 (C8), 7.71 (C3b); HRMS (EI): m/z found 205.0488. C9H7N3O3
requires 205.0487.
108e110 ꢁC; dH (500 MHz, CD3OD) 7.83 (1H, s, H70/H4), 7.78 (1H, s,
H4/H70), 6.88 (1H, s, H40), 4.48 (1H dd, J3a ,6a ¼7.9 Hz, J6 ,6a ¼5.0 Hz,
00
00
00
00
H6a00), 4.41 (2H, s, H80/H6), 4.32 (2H, s, H6/H80), 4.37 (2H, t,
0
00
0
0
00 00
J17 ,18 ¼7.1 Hz, H18 ), 4.28 (1H, dd, J3a ,4 ¼4.4 Hz,00H3a ), 3.55 (2H, t,
0
0
0
J10 ,11 ¼6.5 Hz, H10 ), 3.20e3.15 (1H, m, H4 ), 2.91 (1H, dd,
00
00
00 00
00
00
J6 ,6 ¼12.7 Hz, H6 ), 2.70 (1H, d, H6 ), 2.23 (1H, t, J9 ,10 ¼7.3 Hz,
H1000), 1.90 (3H, s, H3b0), 1.90e1.85, 1.75e1.53, 1.45e1.25 (20H, 3m,
H110, H120, H130, H140, H150, H160, H170, H700, H800, H900). dC
(150.9 MHz) 175.95, 173.65, 166.09 (C20, C1100, C1200), 160.10 (C50),
146.28 (C5), 143.16, 142.79 (C3a0, C7a0), 128.99 (C70), 124.14 (C4),
101.92 (C40), 100.52 (C30)72.39, 69.84 (C80, C100), 63.33 (C3a00), 61.63
(C6a00), 56.99 (C400), 51.34 (C180), 41.05 (C600), 36.55, 35.60, 31.27,
30.64, 30.46, 30.32, 29.99, 29.70, 29.44, 27.42, 27.09, 26.70 (C6, C110,
C120, C130, C140, C150, C160, C170, C700, C800, C900, C1000), 7.44 (C3b0).
HRMS (EI): m/z found 628.3049. C31H44N6O6S requires 628.3043.
4.3.2. 5-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)methyl-3-methyl-2H-
furo[2,3-c]pyran-2-one (10). Sodium hydride (60% dispersion in
mineral oil, 30 mg, 0.8 mmol) was added to a stirred solution of 1-
(2-azidoethoxy)-2-(2-bromoethoxy)ethane 934 (500 mg, 2.1 mmol)
and the alcohol 715 (75 mg, 0.4 mmol) in dimethylformamide
(2.0 mL) at 0 ꢁC and the solution stirred (10 min). The reaction was
allowed to warm to room temperature (3 h) before being quenched
with methanol (0.5 mL). A usual workup (dichloromethane) fol-
lowed by flash chromatography (ethyl acetateetoluene 3:7) and
semi-preparative HPLC (C18, acetonitrile-water 9:11) yielded the
azide 10 (10 mg, 7%) as a colourless glass. Rf (ethyl aceta-
teedichloromethane 1:9) 0.6; dH (500 MHz) 7.43 (1H, s, H7), 6.62
(1H, s, H4), 4.37 (2H, s, H8), 3.76e3.67 (10H, m, H10, H11, H13, H14,
H16), 3.40 (2H, t, J16,17¼5.2 Hz, H17), 1.62 (3H, s, H3b). dC
(125.7 MHz) 171.46 (C2), 157.40 (C5), 141.82, 140.61 (C3a, C7a),
126.22 (C7), 100.57 (C4), 100.42 (C3), 70.74, 70.71, 70.66, 70.08,
69.29 (C10, C11, C13, C14, C16, C17) 50.67 (C8), 7.71 (C3b); HRMS
(EI): m/z found 337.1274. C15H19N3O6 requires 337.1274.
4.3.5. 5-(3-Azido-5-(azidomethyl)benzyloxy)methyl-3-methyl-2H-
furo[2,3-c]pyran-2-one (17). Sodium hydride (60% dispersion in
mineral oil, 55 mg, 1.4 mmol) was added to a stirred solution of the
bromide 1635 (300 mg, 1.1 mmol) and alcohol 715 (190 mg,
1.1 mmol) in dimethylformamide (5.0 mL) at 0 ꢁC and the solution
stirred (2 h). The reaction was quenched with methanol (1.0 mL)
followed by a usual workup (ethyl acetate) and flash chromatog-
raphy (ethyl acetateehexane 1:3) yielded the azide 17 (92 mg, 24%)
as a light yellow powder. Rf (ethyl acetateehexane 1:2) 0.3; mp
60e63 ꢁC; dH (600 MHz) 7.42 (1H, s, H7), 7.08, 7.02, 6.94 (3H, 3s,
H12, H14, H16), 6.58 (1H, s, H4), 4.63 (2H, s, H10), 4.36, 4.33 (4H, 2s,
H8, H17), 1.98 (3H, s, H3b). dC (150.9 MHz) 171.26 (C2), 156.66 (C5),
141.74, 141.08, 140.24, 139.71, 137.80 (C3a, C7a, C11, C13, C15), 126.11
(C7), 123.35, 118.12, 117.70 (C12, C14, C16), 100.86 (C4), 100.79 (C3),
72.27, 68.36 (C8, C10), 54.05 (C17), 7.64 (C3b); HRMS (EI): m/z found
366.1084. C17H14N6O4 requires 366.1077.
4.3.6. 3a,4,5,7,7a-[13C]5-3-Methyl-2H-furo[2,3-c]pyran-2-one
(19). Compound 19 was prepared in a manner previously described
for the preparation of karrikinolide 1 with the exception that 13C5-
4.3.3. 5-(9-Azidononoxy)methyl-3-methyl-2H-furo[2,3-c]pyran-2-
one (12). Sodium hydride (60% dispersion in mineral oil, 90 mg,
2.2 mmol) was added to a stirred solution of 1-azido-9-bromono-
nane 11 (850 mg, 3.4 mmol) and the alcohol 715 (210 mg, 1.2 mmol)
in dimethylformamide (5.0 mL) at 0 ꢁC and the solution stirred
(10 min). The reaction was allowed to warm to room temperature
(3 h) before being quenched with methanol (1.0 mL). A usual
workup (dichloromethane) followed by flash chromatography
(ethyl acetateedichloromethane 3:97) afforded the azide 12
(60 mg, 15%) as a light yellow powder. Rf (ethyl acetateehexanes
1:1) 0.6; mp 28e30 ꢁC; dH (600 MHz) 7.41 (1H, s, H7), 6.55 (1H, s,
H4), 4.26 (2H, s, H8), 3.55 (2H, t, J10,11¼6.6 Hz, H10), 3.25 (2H, t,
J17,18¼7.0 Hz, H18), 1.92 (3H, s, H3b), 1.65e1.55, 1.40e1.30 (14H, 2m,
H11, H12, H13, H14, H15, H16, H17). dC (150.9 MHz) 171.37 (C2),
157.65 (C5), 141.72, 140.54 (C3a, C7a), 126.12 (C7), 100.25 (C4),
100.23 (C3), 71.76, 68.79 (C8, C10), 51.34 (C18), 29.44, 29.26, 29.17,
28.96, 28.70, 26.57, 25.89 (C11, C12, C13, C14, C15, C16, C17), 7.61
(C3b); HRMS (EI): m/z found 347.1853. C18H25N3O4 requires
347.1845.
D
-xylose 18 was used as the starting material.15 Rf (ethyl aceta-
teehexane 1:2) 0.3; mp 118e120 ꢁC; da (600 MHz) 7.61e7.59,
H
7.28e7.25 (1H, 2m, H7), 7.51e7.46, 7.18e7.13 (1H, 2m, H5),
6.68e6.63, 6.39e6.35 (1H, 2m, H4), 1.93 (3H, d, JH3b,C3a¼4.7, H3b).
db (150.9 MHz) 171.13 (m, C2), 147.86 (dddd, J4,5¼71.5 Hz, C5),
C
142.16 (dddd, J7,7a¼97.0 Hz, J3a,7a¼46.6 Hz, H7a), 139.60 (dddd,
J3a,4¼57.3 Hz, H3a), 126.68 (br d, H7), 103.31 (dddd, C4), 100.19 (m,
C3), 7.56 (C3b). HRMS (EI): m/z found 155.0489. 12C133C5H6O3 re-
a
quires 155.0485. Multiplets split by 13C coupling. bOnly one bond
coupling assigned.
4.3.7. 7-[3H]-3-Methyl-2H-furo[2,3-c]pyran-2-one (20). Lithium bis
(trimethylsilyl)amide (1 M solution in tetrahydrofuran, 2.0 mL,
2.0 mmol) was added to karrikinolide 1 (150 mg, 1.0 mmol) in
tetrahydrofuran (10 mL) at ꢀ78 ꢁC and the resulting brown solution
stirred (15 min). [3H]eWater (5.0 mCi/mL, 50
mL, 2.5 mmol) was
added to the solution and the resulting mixture allowed to warm to
room temperature (1 h). The reaction was subjected to flash chro-
matography (ethyl acetateehexane 1:3) to return an orange pow-
der. This powder was treated with activated charcoal, filtered and
concentrated. Recrystallisation from hexane returned the radio-
labelled karrikinolide 20 (40 mg, 27%) as light yellow cyrstals.
Specific activity 85 mCi/mol.
4.3.4. N-(1-(9-((3-Methyl-2-oxo-2H-furo[2,3-c]pyran-5-yl)methoxy)
nonyl-1H-1,2,3-triazol-4-yl)methyl)-5-[(3aS,4S,6aR)-2-oxohexahy-
dro-1H-thieno[3,4-d]imidazol-4-yl]pentanamide (14). Copper (II)
sulfate pentahydrate (0.1 M solution, 50
sodium ascorbate (0.1 M solution, 200
(18 mg, 52
m
L, 5.0
m
m
mol) was added to
mol), the azide 12
mol) and biotin alkyne 1327 (15 mg, 53
m mmol) in
mL, 20
a mixture of ethanol and water (1:1, 4.0 mL) and the reaction
allowed to stand overnight at room temperature. The reaction was
concentrated followed by flash chromatography (meth-
anoledichloromethane 1:19e1:9) and semi-preparative HPLC (C18,
acetonitrileewater 1:1) to furnish the triazole 14 (17 mg, 52%) as
a white powder. Rf (methanoledichloromethane 3:17) 0.4; mp
Acknowledgements
This work was supported by the Australian Research Council
(grant nos. LP0882775, FF0457721, DP0667197, DP0880484) and
the Centres of Excellence Program of the Government of Western
Australia.

A. Scaffidi et al. / Tetrahedron 67 (2011) 152e157
18. Bailon, P.; Weber, D. V. Nature 1988, 335, 839.
157
References and notes
19. Kotzyba-Hibert, K.; Kapfer, I.; Goeldner, M. Angew. Chem., Int. Ed. 2003, 34, 1296.
20. Guerquin-Kern, J. L.; Wu, T. D.; Quintana, C.; Croisy, A. Biochim. Biophys. Acta
2005, 228.
21. Raghavana, A. S.; Hang, H. C. Drug Discovery Today 2009, 14, 178.
22. Zwanenburg, B.; Mwakaboko, A. S.; Reizelman, A.; Anilkumar, G.; Sethu-
madhavan, D. Pest Manage. Sci. 2009, 65, 478.
1. Dixon, K. W.; Merritt, D. J.; Flematti, G. R.; Ghisalberti, E. L. Acta Hortic. 2009,
813, 155.
2. Roche, S.; Dixon, K. W.; Pate, J. S. Aust. J. Bot. 1997, 45, 783.
3. Brown, N. A. C. S. Afr. J. Bot. 2004, 70, 559.
4. Keeley, J. E.; Fotheringham, C. J. Ecology 1998, 79, 2320.
23. Prescher, J. A.; Dube, D. H.; Bertozzi, C. R. Nature 2004, 430, 873.
24. Chang, P. V.; Prescher, J. A.; Hangauer, M. J.; Bertozzi, C. R. J. Am. Chem. Soc. 2007,
129, 8400.
25. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed.
2002, 41, 2596.
ꢀ
ꢀ
ꢀ
ꢀ
5. Perez-Fernandez, M. A.; Rodrõguez-Echeverrõa, S. J. Chem. Ecol. 2003, 29, 237.
6. Pierce, S. M.; Esler, K.; Cowling, R. M. Oecologia 1995, 102, 520.
7. Flematti,G. R.;Ghisalberti, E. L.;Dixon, K. W.;Trengove, R. D. Science2004, 305, 977.
8. Stevens, J. C.; Merritt, D. J.; Flematti, G. R.; Ghisalberti, E. L.; Dixon, K. W. Plant
Soil 2007, 298, 113.
26. Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.; Sharpless, K. B.; Finn, M. G. J. Am.
9. Flematti, G. R.; Ghisalberti, E. L.; Dixon, K. W.; Trengove, R. D. J. Agric. Food
Chem. Soc. 2003, 125, 3192.
Chem. 2009, 57, 9475.
27. Kaschani, F.; Verhelst, S. H. L.; Van Swieten, P. F.; Verdoes, M.; Wong, C.-S.;
Wang, Z.; Kaiser, M.; Overkleeft, H. S.; Bogyo, M.; van der Hoorn, R. A. L. The
Plant J. 2009, 57, 373.
28. Nyangulu, J. M.; Galka, M. M.; Jadhav, A.; Gai, Y.; Graham, C. M.; Nelson, K. M.;
Cutler, A. J.; Taylor, D. C.; Banowetz, G. M.; Abrams, S. R. J. Am. Chem. Soc. 2005,
127, 1662.
10. Nelson, D. C.; Riseborough, J.-A.; Flematti, G. R.; Stevens, J.; Ghisalberti, E. L.;
Dixon, K. W.; Smith, S. M. Plant Physiol. 2009, 149, 863.
11. Merritt, D. J.; Kristiansen, M.; Flematti, G. R.; Turner, S. R.; Ghisalberti, E. L.;
Trengove, R. D.; Dixon, K. W. Seed Science Research 2006, 16, 29.
12. Flematti, G. R.; Ghisalberti, E. L.; Dixon, K. W.; Trengove, R. D. Tetrahedron Lett.
2005, 46, 5719.
29. Green, N. M.; Konieczny, L.; Toms, E. J.; Valentine, R. C. Biochem. J. 1971, 125, 781.
30. Nielson, P. E.; Buchardt, O. Photochem. Photobiol. 1982, 35, 317.
31. Hashimoto, M.; Hatanaka, Y. Eur. J. Org. Chem. 2008, 2513.
32. Galardy, R. E.; Craig, L. C.; Printz, M. P. Nat. New Biol. 1973, 242, 127.
33. Hosoya, T.; Hiramatsu, T.; Ikemoto, T.; Nakanishi, M.; Aoyama, H.; Hosoya, A.;
Iwata, T.; Maruyama, K.; Endo, M.; Suzuki, M. Org. Biomol. Chem. 2004, 2, 637.
34. Amavam-Zollo, P. H.; Sinay, P. Carbohydr. Res. 1986, 150, 199.
35. Hosoya, T.; Hiramatsu, T.; Ikemoto, T.; Aoyama, H.; Ohmae, T.; Endoc, M.; Su-
zuki, M. Bioorg. Med. Chem. Lett. 2005, 15, 1289.
13. Sun, K.; Chen, Y.; Wagerle, T.; Linnstaedt, D.; Currie, M.; Chmura, P.; Song, Y.; Xu,
M. Tetrahedron Lett. 2008, 49, 2922.
14. Nagase, R.; Katayama, M.; Mura, H.; Matsuo, N.; Tanabe, Y. Tetrahedron Lett.
2008, 49, 4509.
15. Goddard-Borger, E. D.; Ghisalberti, E. L.; Stick, R. V. Eur. J. Org. Chem. 2007, 3925.
16. Flematti, G. R.; Scaffidi, A.; Goddard-Borger, E. D.; Heath, C. H.; Nelson, D. C.;
Commander, L. E.; Stick, R. V.; Dixon, K. W.; Smith, S. M.; Ghisalberti, E. L. J.
Agric. Food Chem. 2010, 58, 8612.
17. Beynon, R. J.; Pratt, J. M. Mol. Cell. Proteomics 2005, 4, 857.