New and scalable access to karrikin (KAR1) and evaluation of its potential application on corn germination

Article abstract of DOI:10.1002/hlca.201800081

Karrikin (KAR₁), also named ‘karrikinolide’, was isolated in 2004 from smoke water and identified as a very potent germination stimulant as well as controlling early seedling development for a large variety of plant species. We reported herein an unprecedented and scalable synthesis of Karrikin (KAR₁) based on an elegant and efficient 6-endo-dig cyclization. Moreover, we evaluated the effect of KAR₁ on corn seeds germination, we carried out additional studies on the uptake of this butenolide into corn seeds, and the main soil chemiodynamic properties (i.e., persistence and mobility) were calculated or estimated. Finally, we provided a rationalization of the experimental results by building a homology model of corn KAI2, using as template the X-ray of the KAI2 protein from Arabidopsis thaliana.

Full text of DOI:10.1002/hlca.201800081

Article Type: Full Paper
New and scalable access to Karrikin (KAR1) and evaluation of its
potential application on corn germination
Mathilde Lachia,a Raymonde Fonné-Pfister,a Claudio Screpanti,a Stefano Rendinea, Peter Renold,
David Witmer, Alexandre Lumbroso,a Edouard Godineau,a Damien Hubert,a and Alain De
Mesmaeker*a
aSyngenta Crop Protection AG, Crop Protection Research, Schaffhauserstrasse, CH-4332 Stein,
Switzerland
alain.de_mesmaeker@syngenta.com
Karrikin (KAR1) also named “karrikinolide” was isolated in 2004 from smoke water and identified as a
very potent germination stimulant as well as controlling early seedling development for a large
variety of plant species. We reported herein an unprecedented and scalable synthesis of Karrikin
KAR1 based on an elegant and efficient 6-endo-dig cyclisation. Moreover we evaluated the effect of
KAR1 on corn seeds germination, we carried out additional studies on the uptake of this butenolide
into corn seeds and the main soil chemiodynamic properties (i.e. persistence and mobility) were
calculated or estimated. Finally we provided a rationalization of the experimental results by building
a homology model of corn KAI2, using as template the X-ray of the KAI2 protein from Arabidopsis
thaliana.
Keywords: Karrikin • KAR1 • corn germination • 6-endo-dig cyclisation • Soil Stability • Strigolactone
Introduction
Karrikin (KAR1) also named “karrikinolide” was isolated in 2004i from smoke water and identified as a
potent germination stimulant as well as controlling early seedling development for a large variety of
plant species (Figure 1)ii. As the well-known phytohormones strigolactones,iii Karrikins bear a
butenolide ring and share similar signaling pathway (Figure 1).iia Indeed, whereas D14 is the
strigolactone receptor, a very close paralog known as KARRIKIN INSENSITIVE 2 (KAI2) and also named
D14-like has been proven to be involved in the Karrikin signaling and both pathways (strigolactone
and Karrikin) interact with the same F-box protein named MAX2. Five other karrikin analogs have
been so far detected and identified in smoke water named KAR2-KAR6 (structure variation based on
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their methyl-substitution, Figure 1)iv and several synthetic analogs have been prepared and reported
in the literature.v KAR1 is by far the major component among the Karrikins contained in smoke water
and usuallly the most potent seed germination inducer.ii Consequently, we decided to concentrate
our study on KAR1.
Karrikins compound identified
in smokewater
Examples of natural and synthetic
strigolactones
O
O
O
O
O
O
GR-24
O
O
O
O
O
1
O
Synthetic analog
KAR1
KAR2
O
O
5-Deoxystrigol
O
O
O
O
O
O
O
O
O
KAR3
KAR4
MeO
Me
O
Me
O
zealactone3f
O
O
O
O
O
O
O
O
O
KAR6
KAR6
corn natural product
Me
Figure 1. KAR1-6 and some strigolactones
Considering the promising biological effects reported in the literature and some chemical structure
diversity within the family of Karrikins, we were interested to explore further the potential
applications of Karrikins in agriculture for the production of healthier and more vigorous crops
through mainly the promotion of germination and early seedling development. We used corn as the
most important commercial crop. In fact previous investigations specifically disclosed the promotion
effects of KAR1 on corn.iie,f
In pratical terms any potential use of Karrikins will be in soil either directly applied (in-furrow or
sprayed) or as seed treatment. Thus, KAR1 needs to be sufficiently bioavailable in soil to be effective
under a broad range of agronomical conditions. Soil mobility and soil persistence are two key
components to evaluate the bioavailable of chemicals in soil. Previous reports indicated that other
butenolides derivatives such as simplified strigolactones or strigolactams were highly instable in
soil.iiic
Alongside a favorable soil behaviour, Karrikins need to be taken up within the seed by passing across
the seed coat to be effective. Despite seed uptake of exogenous chemicals is critical, limited studies
attempted to understand what mechanisms are involved in the processes.vi Early reports disclosed
simple and sufficiently precise methods to study the seed uptake of herbicides in different species.vii
A similar methodology was used in this study to evaluate the seed uptake of KAR1.
A careful examination of the literature revealed that no efficient synthesis of such molecule has been
reported yet especially on large scale that would be needed for its biological evaluation on crop of
interest such as corn. As depicted in Scheme 1, the first approach developed by Flematti et al. starts
from kojic acid and use a thio-Wittig cyclisation as key steps.viii However, the synthesis suffers from
low yields making this method not suitable for scale-up and access to large quantities (Scheme 1).
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Others reported synthetic routes such as Goddard,ix Shindo,x Dupontxi and Tanabexii,xiii either required
multi-steps synthesis (approx. 10 steps)ix,x or also suffer from low yielding steps.xi,xii,xiii
Hence, the main aims of the present study were: i) to present a new efficient and scalable synthesis
of KAR1; ii) to evaluate its biological effect on different commercial hybrids of corn considering
different temperature conditions; iii) to provide new information about the soil bioavailablity of KAR1
by evaluating its mobility and persistence in soil; iv) to compare the corn seed uptake of KAR1 vs
other compounds at two temperatures. Finally we studied the molecular interactions of KAR1
occuring at the receptor level by using an homology model of KAI2 in corn.
P2S5
51%
92%
4 steps from kojic acid
PPh3, NaOAc
Ac2O
22%
Scheme 1. Flematti’s approach to KAR1
Results and Discussion
We started to investigate a new synthesis of KAR1. In order to have a more reliable and efficient
access, our retrosynthetic analysis of KAR1 (1) would involve a key 6-endo-dig cyclisation to form the
pyranone ring (Scheme 2). The enol precursor 2 used in the cyclisation would be then easily
synthesized from tetronic acid 3 (Scheme 2).
1
2
3
Karrikin / KAR1
Scheme 2: Retrosynthetic analysis
We started our synthesis from ethyl-2-methyl acetoacetate which was easily converted to tetronic
acid 3 in good yield (60%, Scheme 3) via an efficient bromination/acid-catalyzed cyclisation
developed by Stolz.xiv The acetylene moiety was then introduced by a Sonogashira coupling between
TMS or TBS acetylene and the corresponding triflate 4, affording 5 in excellent yield (89%, Scheme
3).xv,xvi
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1- Br2
Tf2O
2- HBr
Et3N
CH2Cl2
4, 80%
3, 60%
CuI (2 mol%)
PdPPh3Cl2 (1 mol%)
iPr2NH,
dioxane
5, 89%
Scheme 3: Preparation of the alkynyl-intermediate 5
Attempts to formylate butenolide 5 via deprotonation and reaction with ethyl formate failed
(attempt done with R = TMS, Scheme 4). These butenolides are indeed known to dimerize very
quickly under basic conditions.xvii However, to our delight, stirring the compound 5 with the more
robust TBS protecting group with Bredereck’s reagent furnished dimethyl enamine 6 (Scheme 4) and
7 was finally efficiently obtained in only 6 steps and in high overall yield, even on large scale (Scheme
4).
with R = TMS
TBAF, THF
7, 67%
5
6, 87%
Bredereck's reagent
with R = TBS
hydrolysis
see Table 1
cyclisation
see Table 1
1, KAR1
2
Scheme 4: Access to key intermediate 7, direct precursor of Karrikin 1.
We then tried to hydrolyze the enamine 7 to the corresponding enol. Although we had evidence that
enol 2 was formed upon treatment with aqueous HCl, we could not isolate it due to its high
instability. We decided then to try a one-pot hydrolysis/cyclisation cascade and first considered an
acid catalyzed addition of the enol into the viniloguous acetylene ester. However, neither HCl nor
TsOH were able to trigger the cyclisation (Entries 1 and 2, Table 1). We also considered a base
catalyzed process in which the enolate would add on the activated acetylene but addition of
potassium carbonate to the reaction mixture after the hydrolysis did not led to the formation of the
expected Karrikin (KAR1) 1.
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Acid and base mediated 6-endo-dig cyclisation of enol and in particular phenol to acetylene have
often been reported in the literature for the synthesis of chromones but the alkyne moiety from 7 is
weakly activated and the enol too sensitive for these conditions. Metal-catalyzed activation of the
acetylene was then considered and in particular gold (I) complexes, as already been intensively
studied and used.xviii Gold triphenylphosphine triflimidate (10 mol %) was added to the reaction
mixture after completion of the hydrolysis and to our delight, 17% of 1 were isolated. Introduction of
the catalyst from the start of the hydrolysis led to a strong increase of the final yield (67%) which was
further improved by replacing TsOH with MsOH (75%, entries 5, 6, Table 1). Control reaction showed
that gold triphenylphosphine triflimidate alone was not enough to trigger the cyclisation in the
absence of acid, suggesting that the latter is required at least for the hydrolysis of the enamine and
possibly assisting the cyclisation step (entry 7, Table 2).
With this first results in hand, we then look at optimizing the conditions of the final metal-catalyzed
cyclization step and in particular to find a cost-attractive catalyst. Different metals reported to
successfully activate alkynes were investigated and the use of Cu(I), Cu(II), Ag, Pd, Pt or Fe did not
allow to obtain KAR1 1 or only in traces (entries 1/2, 11-20, Table 2). Gold catalysts MeAu(PPh3)
appeared to be the only suitable one for the reaction and gave by far the best result even with only 5
mol% (entry 6,table 2).
Table 1: Optimization of the hydrolysis/cyclisation sequence – Access to Karrikin (KAR1) 1
hydrolysis
cyclisation
1, KAR1
7
2
Entry
Hydrolysis
Cyclisation
Yield (Isolated)
Decomposition
Decomposition
Decomposition
17%
1
2
3
4
5
6
7
Aq. HCl, THF
Aq. HCl
TsOH
TsOH, aq.toluene
Aq. HCl, THF
K2CO3
TsOH, aq.toluene
Then AuNTf2PPh3
(One-pot)
(One-pot)
(One-pot)
TsOH, AuNTf2PPh3, (10%), aq. toluene
MsOH, AuNTf2PPh3, (10%), aq. toluene
AuNTf2PPh3, (10%), aq. toluene
62%
75%
-
Table 2: Optimization of the metal-catalyzed cyclisation to Karrikin (KAR1) 1
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MsOH (1.1 equiv), cat (10 mol%)
toluene, reflux
1
7
entries
Catalyst
PtCl2
t(h)
3.5
4
Yield (%)a
entries
11
Catalyst
CuI
Yield (%)a
1
2
3
4
7a
11a
-
PtCl4
12
CuOTf
10a
11a
16a
[(η2-C2H4)PtCl2]2
AuClb
4
6a
13
CuCl2, 2H2Ob
CuOTf2
22
Traces
14
e
5
MeAu(PPh3)
2
86%
15
CuOTf2
17a
b
6
7
MeAu(PPh3)c
MeAu(PPh3)b,d
HAuCl4
2
3
86%
35%a
traces
-
16
17
18
19
20
AgNO3
5a
2a
4a
-
PdCl2
8
3.5
4.5
2
Pd(OAc)2
b
b
9
AuCl3
FeCl3
10
AuPPh3Cl
traces
FeCl2, 4H2Ob
-
aNMR yield; b1.1 equiv of MsOH was added again after 1h; c5 mol% catalyst were used; d2 mol% catalyst were used; e20 mol % catalyst were
used.
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In order to evaluate the bioavailability of KAR1 in soil we measured or calculated different
parameters related to the mobility and persistence (Table 4). We estimated the soil organic carbon-
water partitioning coefficient (Koc) starting from the Log P value and by using the Briggs’ equationiiid.
The Koc is useful in predicting the mobility of small organic molecules in soil and low Koc
values indicate high mobility. The estimated Koc value for KAR1 was very low less than 50
suggesting a very high mobility. The low Koc value was also consistent with the very high
water solubility of KAR1. Soil half-life is another important parameter to ensure that
compound persists sufficiently long to delivery the desired biological effect. The half-life of
KAR1 under lab conditions at 25°C was 37 days which can be considered sufficiently long.
These results together with the long hydrolytical half-life suggest that KAR1 is relativly stable
against the main biotic and abiotic mechanisms of degradation. It is worth to mention that
conversly, other biologically active butenolides belonging to the class of strigolactones such
as GR-28 or GR-24, displayed a very short soil persistence, in the range of a few hours.iiic
This was hystorically considered one of the main limitations to use this other class of
butenolides in agriculture. Thus, the combination of soil persistence and high mobility
suggests that KAR1 can be sufficiently bioavailable once is applied in soil directly or via seed
treatment.
Table 4. Chemiodynamic properties of KAR1.
a Sand soil (pH 7.6); b Estimated using Log P.
Hydrolytic
stability
(T1/2)
Water
Log P
Koc
Soil stabilitya
(DT50)
(estimated)b
Solubility
To test the biological effect of KAR1 we used corn as crop
model and we studied the dose-response of KAR1 on
different commercial corn hybrids encompassing different
maturity groups and tolerance to cold stresses. Since the
>100 h
37 days
1622 ppm
0.96
23
corn seeds used in this study did not have any dormant phenotype we wanted to check whether KAR1 had any benefit in
supporting the germination under normal or slightly stressful condition represented by low temperature. In more details, we
wanted to evaluate whether KAR1 can alleviate any quiencensce physiological status caused by low temperature and we carried
out dynamic analysis of corn seed populations by studying the kinetic of germination. The curve of germinated seeds over time
allowed us to calculated the speed (T50) and uniformity of germination (slope).
Under our conditions we did not observed any consistent improvement of the kinetic of germination
across hybrids both at 15 or 23 °C. No obvious dose-response were observed for KAR1. As expected
and already indicated from previous studies,iiie at highest concentrations we could observed an
inhibitoring effect particulary for the speed of germination.
Our results differ from previous reportsiief disclosing that KAR1 or smoke water applied as liquid or
aerosol indeed improved germination and vigor of corn seeds. This divergence in results could be
linked to the different genetics and seed batches used in their experiments compared to our ones.
Indeed in both studies they reported to have very low germinabiltiy of corn < 70% in the control
treatment,iief which is not relevant with potential agronomic applications. In our experiments the
germination was always higher than 95%.
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In order to further understand the results of the germination assays we studied the uptake of KAR1
alongside other two compounds chlorppropham (commercially use as anti germinating aggent for
potatoes) and GR-24, into corn seeds. FFor the experiment, corn seeds were incubated at different
temperatures (T = 7 °C and 23 °C) in a solution containing the chemical and the decrease in
concentration was monitored over a periiod of 96 h. By defining the uptake as
ꢇ
ꢊ
ꢒꢓꢓꢔ
ꢐꢑ
ꢀꢁꢂꢃꢄꢅ ꢆ 100% ∗ ꢎ1 ꢏ ꢇ
ꢛ (eq.1)
ꢊ
ꢐꢑ
ꢕꢖꢗꢘꢙꢖꢚ
ꢇ
ꢊ
ꢇ
ꢊ
is the concentration of an AI in a solution containing seed and ꢈꢉ
ꢜꢝꢞꢟꢠꢝꢡ
where ꢈꢉ
the
ꢋꢌꢌꢍ
concentration of an AI in a control soluttion where no seed is present, only the relative decrrease in
concentration is considered, allowing forr compensation of processes other than related to seed, such
as degradation processes and adsorptioon to glass. The uptake behaviour of the three chemicals
differs significantly (Figures 2 and 3). While chlorpropham was taken up readily and nearly
thoroughly, an observation in good accordance with the results reported by Rieder and
collaborators,viic the uptake of GR-24 and KAR1 was less complete.
After 96h, only around 30% of KAR1 waas taken up by the seed at both temperatures, a loww value
indicating that bioavailability of this substance might be a critical factor. The fact that uptake of all
compounds was not only observed aat 23 °C but also at 7°C, albeit slower as expected by
thermodynamics, suggesting that a passive uptake mechanism might be the dominating one for all
three compounds.
Figure 2. Uptake of GR-24, KAR1 and chloropropham into seed aat 23 °C. The bars represent the standard errors of the mean.

Figure 3. Uptake of GR-24, KAR1 and chloropropham into seed aat 7 °C. The bars represent the standard errors of the mean.
Figure 4. Active site of AtKAI2 (PDB 4JYM, orange carbons). The amino acids differing in ZmKAI2 are shown in green carbons.
We also tried to rationalize the experimmental results by building a homology model of corn KAI2,
using as template the X-ray of the KAI2 protein from Arabidopsis thaliana (PDB ID 4JYM xix
M, see
Supporting Material), reported to positively respond to Karrikins in germination.xx The active site in
corn differs by only three amino acids (Figure 4 and Table 2), only one of which (Ala161Cys) is located
in proximity to the catalytic triad (Ser955/His246/Asp217) and the putative binding region of KAR1,
increasing the polar character of the biding pocket in corn. On the other hand, Ile146MMet and
Gly190Ala are located at higher distances (ca. 13Å and 8Å, respectively) from KAR1 and likely hhave no
influence on the ligand binding.
Corn and Arabidopsis sequences were also compared to other KAI2 genes from parasitic weeds,
which were reported to have different germination responses to karrikins, according to Conn et
al.xxiTable 2).

Interestingly, the clade gene KAI2c in Phelipanche aegyptiaca (PaKAI2c), which is described to confer
germination response to KAR1, shows a 100% active site sequence identity compared to corn KAI2.
This seemingly indicates that the same Ala161Cys, Ile146Met and Gly190Ala differences in PaKAI2c
are not affecting KAR1 binding and would likewise allow karrikins to bind in the identical binding site
in the corn receptor. Our result clearly suggest that KAR1 could bind to its KAI2 receptor in corn.
However, the previously described induction of corn seeds germinationiief has been performed with
low germinating seeds which are not relevant for commercial applications. In contrast, we performed
our study with three commercial corn seeds varieties demonstrating that KAR1 does not improve
their germination. This has very important consequences for the hypothetized value of KAR1 and
commercial use.
Table 2: Active site sequence alignment of KAI2 in corn (Zea mais, ZmKAI2) with Arabidopsis thaliana (AtKAI2), Striga hermontica (ShKAI2) and Phelipanche
aegyptiaca (PaKAI2). Genes not conferring germination responses to KAR1 are shaded in grey. Amino acid differences between corn and Arabidopsis are
Active site amino acids
12
4
13
9
13
4
14
2
14
5
14
6
15
3
15
7
15
8
16
1
17
4
17
8
19
0
19
3
19
4
21
7
21
9
24
6
24
7
25
26
27
94
95
96
97
AtKAI2
G
G
G
G
G
G
F
F
F
F
F
F
G
G
G
G
G
G
H
H
H
H
H
H
S
S
S
S
S
S
V
V
L
S
S
S
S
S
S
Y
Y
L
L
V
L
L
L
F
F
F
F
F
F
L
L
L
L
L
L
A
A
A
A
A
A
I
W
W
W
W
L
F
F
A
A
A
A
A
A
A
C
F
F
F
F
F
F
L
L
L
L
L
L
G
A
F
I
I
I
I
I
I
F
F
D
D
D
D
D
D
A
A
A
A
A
V
H
H
H
H
H
H
L
L
L
L
I
ZmKAI2a
ShKAI2i
M
I
F
F
V
F
PaKAI2c
ShKAI2d1
PaKAI2d3
V
L
Y
M
M
M
F
C
A
V
V
F
M
S
F
M
A
H
H
L
L
M
L
highlighted in red. a Absence of response in the case of ZmKAI2 might be due to genetics and seed batches quality (see discussion).
Conclusion
We have developed an unprecedented and scalable synthesis of 1 based on an elegant and efficient
6-endo-dig cyclisation. This represents an important and essential requirement to ensure that some
promising chemicals can be synthetically produced for potential practical applications. We have also
shown that conversely to other butenolides derivatives, KAR1 is sufficiently stable and mobile in soil,
suggesting that the compound should be bioavailable. We can expect a certain degree of flexiblility in
the application and due the high water solubility and stabiltiy the compound can be used both for in-
furrow or seed treatment applications.
Despite other studiesii,iii indicated that KAR1 promotes the germination and/or subsequent
development of young seedlings we could not
distinctly observed these effects in our studies. In several studies it is reported that KAR1 is able to
break primary or secondary dormancy in diverse species; in most of the cases it is about a wide range
of weeds
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or A. thaliana iig. A few papers reported the effects on corn cultivars. In these studies, the corn
displayed a surprisingly low germination level which may indicate that low vigor seed batches were
used. In high vigor and commercial corn hybrids we could not observed any effect with KAR1 in a
wide range of rates and at two temperatures. The high vigor of these seed batches might have
masked any effect of KAR1.
Surprisingly seed uptake of KAR1 appeared suboptimal compared to other molecules. This might be
an additional factor to explain the lack of biological effect of KAR1 in corn.
Furtherer work to better understand the role of KAR1 in the development of corn or other field crops
will be needed. Evaluating the potential benefit effect of KAR1 in low vigour crop seed batches could
be technically very interesting, althought of dubious commercial applicability, untill when only the
high vigour seed batches can be released and commercialized.
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Table 3: Effect of 1 (Karrikin, KAR1) on the germination of three corn seeds varieties at various concentrations and T°C.
Effect on the germination slope
Effect on the germination T50
(% compared to control)b
Corn variety
T°C
[c]
(% compared to control)a
2
10
50
250
2
+3.9
-1.2
-0.7
+0.1
+0.1
-1.3
Falkone
23°C
+6.5
+4.1
-18.9
-7.3
+5.3c
-0.7
10
50
250
2
Falkone
Famoso
Famoso
Multitop
Multitop
15°C
15°C
23°C
15°C
23°C
-10
+1.5
+5.1c
+1.1
-0.1
-19.1
+8.9
-0.6
10
50
250
2
+10.2
+10.0
+28.2
-0.9
+0.9
+8.0c
-2.6
10
50
250
2
-1.3
+7.3
+10.6
+7.4
+5.2
+5.1
+8.1
+21.4
-12.3
+9.2
-3.2
+13.6c
-0.1
10
50
250
2
0
+0.8
+3.7c
-1.0
10
50
250
+6.3 c
+4.5 c
+7.8 c
-5.5
a
b
Positive values mean a more homogeneous germination and vice versa for negative values; Positive values mean a slower germination and vice versa for
negative values; c Data in bold are statistically validated.
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Experimental Section
General Aspects and Materials
All reactions were carried out under an argon atmosphere with dry solvents unless otherwise noted. Reagents were purchased at highest
available commercial quality and used without further purification unless otherwise stated. Preparative flash chromatography was
performed using silica gel 60 (40–63 lm, E. Merck). NMR spectra were recorded on Brucker ultrashield 400 PLUS instrument. Chemical
shifts are given in parts per million (δ) and calibrated using residual undeuterated solvent as an internal reference. The relative
stereochemistry was determined by ROESY correlations. Compound
3 was prepared according to literature known procedurexiv and analytics were consistent with those reported.
3-methylfuro[2,3-c]pyran-2-one, 1 (KAR1): To a solution of enamine 7 (0.5g, 3 mmol) in toluene, was added MsOH (1.1 eq) and
MeAu(PPh3) (5 mol%, 0.067 g) and the resulting reaction mixture was stirred at reflux for 2 hours. After cooling to room temperature, the
solution was filtered on a pad of Celite® and concentrated under reduce pressure. The resulting crude reaction product was purified by
flash chromatography on SiO2 (EtOAc/CyHexane) affording 1 (KAR1) as a pure yellowish solid in 86% yield (2.43 mmol, 0.42 g). Analytical
data were consistent with those previously reported in the literature.xxii
(5Z)-5-(dimethylaminomethylene)-4-ethynyl-3-methyl-furan-2
one, 7: To a solution of 6 (10 g) in THF (100 mL) was added dropwise
tertrabutylammonium fluoride (TBAF 1M in THF, 38 mL) at room temperature and the resulting
solution was stirred for 12 hours. Saturated NaClaq. solution was then added and the reaction
mixture
diluted with ethyl acetate (200 mL). The phases were separated, the aqueous layer extracted with
EtOAc (2 x 100 mL) and the combined organic fractions were dried over sodium sulfate and
concentrated under vacuum. The resulting crude solid was washed with cyclohexane, dried and used
1
without further purification in the gold-catalyzed cyclization step (67%, 4.1 g, 23 mmol). H NMR
(400MHz, CDCl3) δ (ppm) 6.07 (s, 1H), 3.6 (s, 1H), 3.15 (s, 6H), 1.97 (s, 3H).13C NMR (101MHz, CDCl3) δ
(ppm) 170.6, 131.8, 127.8, 124.4, 116.3, 88.6, 73.1, 42.4, 9.0; 13C (101MHz, CDCl3) δ (ppm) 170.6,
131.8, 127.8, 124.4, 116.3, 88.6, 73.1, 42.4, 9.0; HRMS (M+H+):178.0872 (calc. 178.0790 for M+H+).
(5Z)-4-[2-[tert-butyl(dimethyl)silyl]ethynyl]-5-(dimethylamino-methylene)-3-methyl-furan-2-one,
6: To a solution of 5 (150 g, 636 mmol) in toluene (1.5 L) was added 221.2 g of Bredereck’s reagent.
The reaction mixture was then stirred for 12 hours at room temperature and quenched with 1L of
water. The phases were then separated, the aqueous layer extracted with TBME (2 x 500 mL) and the
combined organic fractions were dried over sodium sulfate, concentrated under vacuum and 6 was
obtained as a viscous gum in 87% yield (161 g) which was used without further purification. 1H NMR
(400MHz, CDCl3) δ (ppm) 6.04 (s, 1H), 3.14 (s, 6H), 1.97 (s, 3H), 1.00 (s, 9H), 0.20 (s, 6H).13C (101MHz,
CDCl3) δ (ppm) 171.0, 133.2, 128.1, 124.5, 115.7, 106.2, 94.5, 42.6, 25.9, 16.4, 9.3, -4.9 ; HRMS
(M+H+): 292.1759 (calc. 292.1654 for M+H+).
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3-[2-[tert-butyl(dimethyl)silyl]ethynyl]-4-methyl-2H-furan-5-one, 5: Compound 4 (225 g, 914 mmol)
was dissolved in dioxane (2.25 L) and the resulting solution was degassed with nitrogen. Palladium
catalyst (PdPPh3Cl2, 5 mol%, 32.4 g, 45.7 mmol) followed by CuI (5 mol%, 8.7 g, 45.7 mmol) and
diisopropylamine (1.5 eq, 194 mL, 1371 mmol) were then added to the reaction mixture. To the
resulting black solution was added dropwised at room temperature tert-butyl-ethynyl-dimethyl-
silane (1.1 eq, 191 mL, 1005 mmol). The reaction mixture was stirred for 1 hour at room
temperature, filtered through a pad of Celite® (filter cake washed with ethyl acetate) and the
resulting solution was concentrated under vacuum. The resulting crude gum was then suspended in
1
TBME and 5 was isolated as a brown solid (192 g, 615 mmol) in 89% yield. H NMR (400MHz, CDCl3)
δ (ppm) 4.5 (m, 2H), 1.8 (m, 3H), 0.78 (s, 9H), 0.0 (s, 6H). 13C (101MHz, CDCl3) δ (ppm) 174.2, 139.4,
133.2, 111.3, 94.8, 71.1, 26.0, 16.5, 10.2, -4.9; HRMS (M+H+): 237.1303 (calc. 237.1233 for M+H+).
(4-methyl-5-oxo-2H-furan-3-yl) trifluoromethanesulfonate, 4: 3 (220 g, 1928 mmol) was solved in
dichloromethane (1.1 L) and the resulting solution was cooled to -78°C. Triethylamine (1.5 eq, 403
mL, 2892 mmol) was then added, followed by triflic anhydride (1.2 eq, 391 mL, 2314 mmol)
dropwise. After complete addition, the reaction mixture was slowly warmed to 0°C and stirred at this
temperature for 1 hour. Water was added (1 L), the phases separated and the aqeous layer was
extracted with dichlorormethane (3 x 500 mL). The combined organic extracts were washed with aq
HCl solution (1M), dried over sodium sulfate and concentrated under vacuum. Compound 4 was
isolated as a brown liquid in 80% yield (380 g, 1544 mmol). 1H NMR (400MHz, CDCl3) δ (ppm) 4.90 (m,
2H), 1.92 (t, 3H); 13C (101MHz, CDCl3) δ (ppm) 170.5, 160.6, 120.2, 117.4, 66.9, 7.8; HRMS (M+H+):
246.9876 (calc. 246.9810 for M+H+)
Supplementary Material
Supporting information for this article
http://dx.doi.org/10.1002/MS-number.
is available
on the WWW under
Entry for the Table of Contents
endo dig
Through 6-
-
cyclization
Karrikin / KAR1
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References
i G. R., Flematti, E. L. Ghisalberti, K.W. Dixon, R. D. Trengove Science, 2004, 305, 977.
ii (a) C. De Cuyper, S. Struk, Braem, L., K. Gevaert, G. De Jaeger, S.Goormachtig, Plant, Cell and Environment 2017, 40, 1691; (b) D. C. Nelson, J.-A. Riseborough,
G. R. Flematti, J. Stevens, E. L. Ghisalberti, K. W.Dixon, S. M. Smith, Plant Physiology 2009, 149, 863; (c) W. Li, K. H. Nguyen, Chuc, H. D., Ha, C. V., Watanabe, Y.,
Osakabe, Y., M. A. Levva-González, M. Sato, K. Toyooka, L. Voges, M. Tanaka, M. G. Mostofa, M. Seki, M. Seo, S. Yamaguchi, D. C. Nelson, C. Tian, L. Herrera-
Estrella, L.-S. Phan Tran PLoS Genet 2017, 13(11): e1007076; (d) S. D. S. Chiwocha, K. W. Dixon, G. R. Flematti, E. L. Ghisalberti, D. J. Merritt, J. C. Stevens, Plant
Science, 2009, 177, 252. (e) J. van Staden, S. Sparg, M.G. Kulkarni, & M.E. Light, Field Crops Res 2006, 98, 98. (f) S. G. Sparg, M. G. Kulkarni & J. van Standen.
Crop Sci. 2006, 46, 1336. (g) E. Villedieu-Percheron, M. Lachia, P. M. J. Jung, C. Screpanti, R. Fonné-Pfister, S. Wendeborn, D, Zurwerra, & A. De Mesmaeker.
CHIMIA, 2014, 68, 654.
iii (a) C. Screpanti, R. Fonné-Pfister, A. Lumbroso, S. Rendine, M. Lachia, A. De Mesmaeker, Bioorg. Med. Chem. Lett. 2016, 26, 2392; (b) S. Al-Babili, H. J.
Bouwmeester, Annu. Rev. Plant Biol. 2015, 66, 161; (c) A. Lumbroso, E. Villedieu‐Percheron, D. Zurwerra, C. Screpanti, M. Lachia, P.Y. Dakas, & A. Beck, Pest
Manag. Sci. 2016, 72, 2054; (d) G. G. Briggs, J. Agric. Food Chem. 1981, 29, 1050; (e) T. Kretzschmar, W. Kohlen, J. Sasse, L. Borghi, M. Schlegel, J. Bachelier, D.
Reinhardt, R. Bours, H. Bouwmeester & E. Martinoia, Nature 2012, 483, 341; (f) T. V. Charnikova, K. Gaus, A. Lumbroso, M. Sanders, J.-P. Vincken, A. De
Mesmaeker, C. P. Ruyter-Spira, C. Screpanti, H.J. Bouwmeester, Phytochemistry 2017, 137, 123.
iv G. R. Flematti, E. L. Ghisalberti, K. W. Dixon, R. D. Trengove, J. Agric. Food Chem. 2009, 57, 9475.
v For example of synthetic analogs, see G. R. Flematti, A. Scaffidi, E. D. Goddard-Borger, C. H. Heath, D. C. Nelson, L. E. Commander, R. V. Stick, K. W. Dixon, S.
M. Smith, E. L. Ghisalberti, J. Agric. Food Chem. 2010, 58, 8612.
vi S. Niemann, M. Burghardt, C. Popp, M. Riederer, Plant, cell & environment 2013, 36, 1027.
vii (a) G. Rieder, K.P. Buchholtz, C.A. Kust, Weed Science 1970, 18, 101: (b) H. D. Scott, R. E. Phillips, Weed Science 1971, 128.
viii G. R. Flematti, E. D. Goddard-Borger, D. J. Merritt, E. L. Ghisalberti, K. W. Dixon, R. D. Trengove, J. Agric. Food Chem. 2007, 55, 2189
ix E. D. Goddard-Borger, E. L. Ghisalberti, R. V. Stick, Eur. J. Org. Chem. 2007, 3925.
x K. Matsuo, M. Shindo, Tetrahedron 2011, 67, 971.
xi K. Sun, Y. Chen, T. Wagerle, D. Linnstaedt, M. Currie, P. Chmura, Y. Song, M. Xu, Tetrahedron Lett. 2008, 49, 2922.
xii R. Nagase, M. Katayama,H. Mura,N. Matsuo, Y. Tanabe, Tetrahedron Lett. 2008, 49, 4509.
xiii Low yield was obtained when scaling-up Tanabe’s approach in our lab.
xiv U. K. tambar, T. Kano, J. F. Zepernick, P. M. Stolz, J. Org. Chem. 2006, 8357.
xv R. Grigg, P. Kennewell, V. Savic, Tetrahedron 1994, 50, 5489.
xvi R. H. Pouwer, H. Schill, C. M. Williams, P. V. Berhardt, Eur. J. Org. Chem. 2007, 4699.
xvii S. I. Clarke, B. Kasum, R. H. Prager, A. D. Ward, Aust. J. Chem. 1993, 36, 3483.
xviii (a) R. Dorel, A. M. Echavarren Chem. Rev. 2015, 115, 9028; (b) C. Jiang, Z. Xiong, S. Jin, P. Gao, Y. Tang, Y. Wang, C. Du, X. Wang, Y. Liu, B. Lin, Y. Liu, M.
Cheng, Chem. Commun. 2016, 52, 11516.
xix Y. Guo, Z. Zheng, J. J. La Clair, J. Chory, J. P. Noel, Proceedings of the National Academy of Sciences 2013, 110, 8284.
xx M.T. Waters, D.C. Nelson, A. Scaffidi, G.R Flematti, Y.K. Sun, K.W. Dixon, & S.M. Smith, Development 2012, 139, 1285.
xxi C. E. Conn, R.Bythell-Douglas, D. Neumann, S. Yoshida, B. Whittington, J. H. Westwood, K. Shirasu, C. S. Bond, K. A. Dyer, D. C. Nelson, Science 2015, 349,
540.
xxii G. R. Flematti, E. L. Ghisalberti, K. W. Dixon, R. D. Trengove, Tetrahedron Lett. 2005, 46, 5719.
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