Total Synthesis and Biological Evaluation of Zealactone 1a/b

Article abstract of DOI:10.1002/hlca.202000017

Strigolactones are plant hormones, which play pivotal roles in plant growth and development with potential application in sustainable agriculture. Recently, zealactone 1a/b has been identified as the major strigolactone from the root exudates of corn. Although zealactone is a promising molecule affecting signaling in the rhizosphere as well as in planta, evaluating its biological activities has been hampered by its low natural abundance and its relative chemical instability. Herein, we present the total synthesis of zealactone 1a/b based on our studies employing a [2+2]-cycloaddition strategy and a chemoselective Baeyer-Villiger oxidation to forge the γ-butyrolactone fragment. Furthermore, we disclose the biological activities of zealactone 1a/b on corn and in soil in comparison with related synthetic analogues.

Full text of DOI:10.1002/hlca.202000017

HELVETICA
chimica acta
Accepted Article
Title: Total Synthesis and Biological Evaluation of Zealactone 1a/b
Authors: Alain De Mesmaeker, Michael Dieckmann, Pierre-Yves
Dakas, Raymonde Fonné-Pfister, Claudio Screpati, Katrin
Hermann, Stefano Rendine, Pierre Quinodoz, Masahiko
Yoshimura, Saron Catak, and Beyza Horoz
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To be cited as: Helv. Chim. Acta 10.1002/hlca.202000017
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10.1002/hlca.202000017
Helvetica Chimica Acta
HELVETICA
Total Synthesis and Biological Evaluation of Zealactone 1a/b
Masahiko Yoshimura,a Michael Dieckmann,b Pierre-Yves Dakas,b Raymonde Fonné-Pfister,b Claudio
Screpanti,b Katrin Hermann,b Stefano Rendine,b Pierre Quinodoz,b Beyza Horoz,c Saron Catak,c and
Alain De Mesmaeker*,b
a Laboratorium für Organische Chemie, Department of Chemistry and Applied Biosciences, ETH Zürich, 8093 Zürich, Switzerland
b Syngenta Crop Protection AG, Chemical Research, Schaffhauserstrasse 101, CH-4332 Stein, Switzerland
c Bogazici University, Department of Chemistry, Bebek, 34342 Istanbul, Turkey
e-mail: alain.de_mesmaeker@syngenta.com
Dedicated to Professor Jacques Lebreton (University of Nantes, France) for his 60th birthday
ABSTRACT: Strigolactones are plant hormones, which play pivotal roles in plant growth and development with potential application in
sustainable agriculture. Recently, zealactone 1a/b has been identified as the major strigolactone from the root exudates of corn. Although
zealactone is considered to be a promising molecule affecting signaling in the rhizosphere as well as in planta, evaluating its biological
activities has been hampered by its low natural abundance and its relative chemical instability. Herein, we present the total synthesis of
zealactone 1a/b based on our studies employing a [2+2]-cycloaddition strategy and a chemoselective Baeyer-Villiger oxidation to forge the γ-
butyrolactone fragment. Furthermore, we disclose the biological activities of zealactone 1a/b on corn and in soil in comparison with related
synthetic analogues.
Keywords: strigolactone • total synthesis • ketene addition • soil stability • crop enhancement • sustainable agriculture
Phelipanche ramosa, and root development in corn.[10] However, low
Introduction
natural abundance, relative chemical instability and lack of synthetic
Strigolactones are terpenoid derived natural products acting as
accessibility of zealactone have hampered in-depth biochemical
plant signaling molecules, which control several physiological and
investigation and a thorough evaluation of potential agronomical
developmental processes in crops.[1-3] Many of these processes are
applications of this molecule. Herein, we describe the first total
related to productivity, nutrient assimilation efficiency and resilience
synthesis of zealactone 1a/b (1) and some related analogues, and
to a range of abiotic stresses, like drought or cold. All these positive
disclose data confirming that the mixture of C6 epimers is formed
effects play
a
pivotal role in the promotion of sustainable
during its biosynthesis and does not result from an epimerization
during its isolation/purification from corn roots. Furthermore, we
unveil their bioavailability in soil and biological activity on leaf
senescence and seed germination of corn, as well as their affinity for
corn strigolactone receptor ZmD14 through in vitro assays.
agriculture.[4-7] Therefore strigolactones represent
a
promising
starting point to develop innovative technologies with potential
applications for a more sustainable crop production. In nature,
several plant species synthesize specific strigolactones. In many
cases, they are structurally similar and belong to a class of canonical
strigolactones that are composed of tricyclic lactone (ABC-rings) with
O
O
OMe
O
biologically essential butenolide ring (D-ring) (Figure 1).[8]
C
a
6
A
R
B
O
O
2’
O
O
D
Intriguingly corn seems to produce a completely different type of
strigolactones, which are called non-canonical. This suggests that
perception and signaling of strigolactones in corn may differ in great
extent from other species. Previously, we have identified zealactone
1a/b (1) as the most abundant species of corn-derived strigolactones
(Figure 1).[9] Zealactone 1a/b (1) consists of a γ-butyrolactone
containing two adjacent quaternary sp3-carbons, connected to the D-
ring via a diene-chain. Zealactone has been isolated as 1:1-mixture
of diastereomers (1a/b) at C6.
O
O
R’
O
O
Canonical strigolactones
Zealactone 1a/b
(1)
Figure 1. Chemical structure of canonical strigolactones and zealactone 1a/b
(1).
Our retrosynthetic plan consisted of C3-C4-disconnection of
zealactone 1a/b (1) leading to vinyl stannane 9 and vinyliodide 6 by a
Stille coupling, following our previous work on the synthesis of
carlactonic acid 2, methylcarlactonoate
3 and heliolactone 7
Recently, Ravazzolo and coworkers reported the potential
activities of zealactone on seed germination of parasitic weed,
(Scheme 1).[11,
Vinyl stannane 9 was envisaged to arise from
12]
1
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10.1002/hlca.202000017
Helvetica Chimica Acta
HELVETICA
Scheme 2. Synthesis of ketene precursor 16.
hydrostannylation and chemoselective Baeyer-Villiger oxidation of
cyclobutanone 10 that in turn was planned to be accessible by [2+2]
cycloaddition of novel ketene 11 with isobutene as coupling partner.
Indeed, 11 would be the first example of vinyl-acetylenyl ketene and
its reactivity towards olefins had to be assessed.
TBS
Pd(PPh3)2Cl2
CuI, NEt3
1,4-dioxane
O
OR
O
OMe
X
O
OMe
Br2, DBU
CH2Cl2
23 °C, 2 h
[53%]
50 °C, 90 min
TBS
13a (X = Br) [< 5%]
13b (X = I)
12
13a/b
[91%]
(R = Me)
(R = OH)
15
16
LiOH (aq), tBuOH
23 °C, [70 %] (2 cycles)
Me2CuLi
THF, –45 °C
90 min;
O
OMe
Scheme 1. Retrosynthesis of zealactone 1a/b (1).
then I2,
–45 to 0 °C, 3 h
[95%]
Previous report 1: total synthesis of CLA and MeCLA (2018)[11]
O
R
O
O
OR
O
14
I
SnBu3
O
O
With ketene precursor 16 in hand, we investigated the key [2+2]-
cycloaddition, to synthesize the highly functionalized strained
cyclobutanone. Inspired by results from Dreiding et al.[16] using α-
vinyl ketene species and olefins in [2+2]-cycloadditions, we
attempted the cycloaddition of unprecedented α,α’-vinyl-acetylenyl
ketene 19 with isobutene, a moderately reactive olefinic substrate.3
Unfortunately, the preliminary results obtained on this reaction led to
very low yield of the desired cyclobutanone 25 (Scheme 3). The
dimers 22 and 23 were isolated as major products and the
reproducibility of the reaction was not optimal.
O
O
4
R= OH
R = OMe MeCLA 3
CLA
2
R= OH
R = OMe
5
6
Previous report 2: total synthesis of heliolactone (2019)[12]
O
OMe
O
OMe
I
SnBu3
O
O
O
O
O
O
O
O
Heliolactone 7
8
6
This work
O
OMe
O
4
OMe
3
3
6
SnBu3
I
O
O
O
2’
O
4
O
O
O
O
O
O
Scheme 3. Plausible intermediates and side products in the [2+2]
Zealactone 1a/b
9
6
(1)
cycloaddition.
O
C
R
R
OH
X
Base
NR3
Halogenating
reagent
O
6
C
R
R
O
PG
O
O
6
O
PG
X = Cl 17
X = Br 18
11
10
HX
16
19
17/18
R, R = Cyclopentyl 24
R = Me 25
NR3
NR3
NR3
NR3
NR3
Results and Discussion
–O
O
NR3
Synthesis of ketene precursor, carboxylic acid 16, started with α-
bromination of methylester 12 (Scheme 2).[13] Subsequent
Sonogashira coupling of vinylbromide 13a with mono-TBS-acetylene
resulted in poor yield of the desired enyne 15 employing a variety of
conditions.[14, 15] 1 Fortunately, the analogous vinyliodide 13b could be
synthesized by carbocupration-iodination of 14 in excellent yield and
provided high reactivity in the subsequent Sonogashira coupling.
Saponification of methylester 15 was achieved by using aqueous
lithium hydroxide in tert-butanol. Concomitant TBS-cleavage could be
mostly suppressed if the saponification was stopped after 50-60%
conversion and the recovered starting material was submitted to a
second cycle.2
X–
HX
21
20
TBS
O
O
O
O
O
TBS
22
23
To optimize the reaction conditions, a liquid and more reactive
substrate, methylenecyclopentane, was used as a model coupling
partner (Table 1).[16] Carboxylic acid 16 was converted to the
corresponding acid chloride in situ using oxalyl chloride and catalytic
amount of N,N-dimethylformamide (DMF), then cycloaddition with
methylenecyclopentane was performed by slow addition of the base.
1
Catalytic systems: PdCl2(PPh3)2/ CuI/ piperidine, PdCl2(NCPh)2/
CuI/ piperidine and PdCl2(PPh3)2/ CuI/ nBuNH2 were tested.
2
3
Other tested systems of solvent/water/MOH (solvent: MeOH, THF;
Initial experiments employing highly reactive ketene-iminium
base: Li/Na/KOH, Ba(OH)2) provided significant amounts of TBS-
cleavage and/ or 1,4-addition of hydroxide and subsequent retro-
aldol reaction giving TBS-CC-CH2CO2H.
intermediates with isobutene in [2+2]-cycloadditions revealed
incompatibility of keteniminum species with α-alkenyl-substituent as
well as α-alkynyl-substituent.
2
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10.1002/hlca.202000017
Helvetica Chimica Acta
HELVETICA
Table 1. Effect of the nature of the acyl halide and of the base.
The base could trigger the formation of the reactive ketene 19 as well
as ammonium enolate 21 depending on the base used (Scheme 3).
Since ammonium enolate 21 might react with ketene species 19 to
form the undesired dimer products 22 and 23, we investigated the
effect of the base on the [2+2]-cycloaddition.[16] At first, in order to
avoid the formation of ammonium enolate, we attempted less
nucleophilic bases such as proton sponge, collidine derivative and
Hünig’s base. Whereas proton sponge and collidine gave the
undesired dimer product and no conversion of starting material,
1) Halogenating
reagent
(1.1 eq.)
X
OH
2)
O
O
(20 eq.)
base (2.2 eq.)
CH2Cl2
23 °C, 30 min
slow addition
(4 mL/1h)
CH2Cl2
23 °C, 16 h
16
17/18
TBS
O
O
O
O
O
O
respectively, Hünig’s base provided the desired product in
a
TBS
moderate yield (Table 1, entries 1-3). Recent flow analysis reported
that ketene would be formed from the corresponding acyl halide
through the corresponding ammonium enolate species.[17] The rate of
ketene formation and its potential reaction with the nucleophilic
ammonium enolate could profoundly affect the outcome of the
desired [2+2] ketene addition on an olefin (Scheme 3). Thus, by fine-
tuning the nucleophilicity and basicity of trialkylamine, we tried to
control the equilibrium between ketene 19 and ammonium enolate
21. Amongst trialkylamines we tested, triethylamine showed
excellent conversion of carboxylic acid 16 into the desired
cyclobutanone 24 in 95% overall yield (entry 4). We also assessed
the effect of acyl halide reagents on our [2+2]-cycloadditions.
Although the combination of Ghosez’s reagent and triethylamine
produced the desired cyclobutanone 24 in 78% overall yield, the
reproducibility of these conditions depended on the quality of
Ghosez’s reagent (entry 10). Use of oxalyl bromide also provided the
corresponding cyclobutanone 24 through the formation of the
corresponding acylbromide intermediate, but several by-products
were observed, which decreased the overall yield (entry 11).
24
22
23
Halogenating
Yield:
24/22/23
Entry
Base
reagent[1]
1
(COCl)2
proton sponge
trace/major/–
2
3
4
5
6
7
8
9
(COCl)2
(COCl)2
(COCl)2
(COCl)2
(COCl)2
(COCl)2
(COCl)2
(COCl)2
2,6-di-tert-butyl-4-methylpyridine
N,N-diisopropylethylamine
triethylamine
–/–/–
67%/–/–
95%/–/–
56%/–/26%
79%/–/–
46%/10%/–
–/–/–
N-methylmorpholine
N-methylpiperidine
N-methylpyrrolidine
quinuclidine
N-ethylpiperidine
88%/–/–
Ghosez’s
reagent
10
triethylamine
78%/–/–
11
(COBr)2
triethylamine
67%/–/–
[1] Catalytic amount of DMF was used except for entry 10. Proton sponge: 1,8-
bis(dimethylamino)naphthalene.
Scheme 4. Optimal conditions of [2+2]-cycloaddition with isobutene.
O
1)
2)
Cl
(55 eq.)
Cl
O
triethylamine (2.2 eq.)
slow addition
(4 mL/1h)
OH
Cl
(1.1 eq.)
cat. DMF
Optimal conditions were then applied to [2+2]-cycloaddition with
isobutene, which resulted in 23% yield of the desired product due to
the lower reactivity of isobutene (Scheme 4, Condition: Table 1 entry
4). Thus, we had to optimize again the conditions and ultimately we
achieved 78% overall yield under diluted conditions at higher
temperature (Scheme 4). The dilution enabled dissolution of up to 55
equivalents of isobutene into the reaction solution which hampered
the undesired dimerization reaction between ketene 19 and
ammonium enolate 21. We reasoned that in addition to the rate
acceleration of [2+2]-cycloaddition, higher temperature might speed
up the decomposition of ammonium enolate species 21 into ketene
19. To our delight, these optimal conditions are applicable to the
gram scale synthesis of the desired cyclobutanone 25. Details of the
reaction conditions optimization and its application to the large scale
synthesis are described in the Supporting Information (Table S1 and
Figure S5).
O
O
O
1,2-dichloroethane
(c = 0.2 M), 23 °C, 30 min
1,2-dichloroethane
(c = 0.02 M) 60 °C, 5 h
16
17
25
[79%]
A computational study based on density functional theory (DFT)
was conducted to elucidate the mechanism in Scheme 3 by which
cyclobutanone 25 is obtained leading to the synthesis of zealactones
1a/b. Moreover, the [2+2] cycloaddition reaction was also modelled
to gain insight into the reactivity difference between olefins isobutene
(IB) and methylenecyclopentane (MC) with ketene 19. All reactions
were modelled with the Gaussian 16 program package (G16,
Revision A.03).[18] ω-B97X-D long-range corrected density functional,
known to give accurate results in kinetics and thermochemistry, was
employed.[19] The solvent environment was modeled employing the
self-consistent reaction field (SCRF) theory[20] with IEF-PCM[21, 22] in
dichloromethane (ε=8.93).
3
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10.1002/hlca.202000017
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Scheme 5. Plausible intermediates and side products in the [2+2]
cycloaddition. Solvent-optimized (IEF-PCM, CH2Cl2) transition state structures
and relative free energies of activation (ΔG‡) at the ω-B97X-D/6-31+G(d,p)
level of theory; free energies in kcal mol-1; critical distances in Å.
ketene 19’ were investigated, the activation free energy for the
preferred (less crowded side) attack face, was found to be ~5 kcal
mol-1 less than that of the unfavorable attack face (Figure 2). There
are two consecutive steps in the [2+2] cycloaddition of ketenes, the
initial attack of C3 to C1 results in the formation of a highly activated
intermediate. This step was previously shown to be the rate-
determining step for [2+2] cycloadditions,[23] as is the case for the
systems studied herein. Subsequently, the C2-C4 bond formation
generates the desired cyclobutanones 24’ and 25’ (Figure 2).
OH
Cl
O
O
NMe3
Oxalyl chloride
C
C
O
O
HCl
HCl
s-cis 19’
16’
17’
s-trans 19’
TS3
NMe3
NMe3
TS1
NMe3
R
R
R
R
NMe3
NMe3
O
–O
Preferred attack
C1
C2
Unfavorable attack
O
O
NMe3
TS2
Cl–
TMS
HCl
21’
20’
R, R = Cyclopentyl 24’
R = Me 25’
ketene 19’
TMS
C4 C3
C4 C3
IB
MC
O
O
O
O
O
TMS
O
22’
O
23’
TMS
25’
TMS
24’
TS1
= 8.3 kcal mol-1 ΔG‡
TS2
TS3
ΔG‡
= 14.7 kcal mol-1 ΔG‡ = 6.2 kcal mol-1
forward
forward
forward
ΔG‡
ΔG‡
= 6.9 kcal mol-1
ΔG‡
= 5.2 kcal mol-1
= 8.8 kcal mol-1
reverse
reverse
reverse
TS1IB
TS1MC
ΔG‡1 = 21.5 kcal mol-1
ΔGrxn = -9.5 kcal mol-1
ΔG‡ = 18.7 kcal mol-1
1
In favor of mimicking the experimental conditions, trimethylamine
ΔGrxn= -10.0 kcal mol-1
and trimethylsilyl were used as base and protecting group,
respectively. Relative free energies for possible s-cis and s-trans
conformers of carboxylic acid 16’, intermediate 17’, 20’, 21’ and
ketene 19’ were calculated and persistent intermediates were
reported. Relative free energies for ketene 19’ showed the s-trans
conformer to be 1.6 kcal mol-1 lower in energy than the s-cis, which
lacks a co-planar structure with respect to its single bond. Free
energy of activations for the forward and reverse reactions were
reported with regard to separate reactants for TS2 and TS3. Due to
high polarizability of the chloride ion, free energy of activation for TS1
Figure 2. Relative activation (ΔG‡) and reaction free energies (ΔGrxn) for
transition state structures of the first step, TS1IB, TS1MC in the [2+2]
,
cycloaddition of isobutene (IB) and methylenecyclopentane (MC) with ketene
19’. (ω-B97X-D/6-31+G(d,p) with IEF-PCM in CH2Cl2; critical distances in Å;
Free energies with respect to separate reactants; all energies relative to s-
trans ketene 19’).
Figure 2 shows free energies of activation and reaction for the [2+2]
cycloaddition of IB and MC with ketene 19’. Computed results show
a notable difference in activation free energies between IB and MC,
was calculated with respect to the pre-reactive (ΔG‡
= 8.3 kcal
forward
mol-1) and product complexes (ΔG‡reverse = 6.9 kcal mol-1) to ensure
more accurate energy barriers (Scheme 5). Computed free energy of
activations show the forward barrier leading to ketene 19’ from
ammonium enolate 21’ to be 2.6 kcal mol-1 lower than the reverse
barrier, hence by controlling this step, the yield of cyclobutanones 24’
and 25’ may be enhanced, hindering the formation of dimers 22’ and
23’. Note that, a concerted mechanism, between ketene 19’ and
intermediate 20’, could not been located.
ΔΔG‡ = 2.8 kcal mol-1. These results are in agreement with the
1
experimental yields of cyclobutanone 25 (23%) and cyclobutanone
24 (95%, Table 1). The main difference between the reactivity of the
two olefins lies in their HOMO energies (HOMOIB = -8.907 eV,
HOMOMC = -8.704 eV, ΔEHOMO = 0.203 eV), this is also reflected in
the slightly larger critical bond length in TS1MC, indicating the earlier
nature of the transition state, which leads to a lower barrier (Figure
2).
The [2+2] cycloaddition reactions of ketene 19’ with olefins
isobutene (IB) and methylenecyclopentane (MC) were modeled at
the same level of theory in CH2Cl2. Two possible attack faces to
We next investigated the anticipated Baeyer-Villiger oxidation
using cyclobutanone 24 as model substrate (Table 2). Several
conditions reported to selectively oxidize ketones to esters in
4
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10.1002/hlca.202000017
Helvetica Chimica Acta
HELVETICA
presence of olefins either gave no conversion or full decomposition
(Table 2, entries 1-3).[24, 25] H2O2 in acidic conditions reacted slowly
giving desired γ-butyrolactone 26 in low yield after 36 hours (entry 4).
Basic conditions using NaOH/H2O2 in ethanol gave 26 in 29% within
30 minutes at room temperature (entry 5). mCPBA was found to give
different chemoselectivity depending on the base employed (entries
6-9). Whereas without base or in the presence of NaHCO3, the
epoxides 27, 28 were formed in significant amounts besides the
desired lactone 26, NBu4OH provided high selectivity for lactone
formation in 76% isolated yield and only traces of 27 and 28 were
formed (entry 9).
The optimized oxidation protocol for cyclobutanone 24 could be
applied successfully to cyclobutanone 25 giving γ-butyrolactone 29 in
61% yield, again with high selectivity for lactone formation (Scheme
6). TBS-deprotection using n-tetrabutyl ammonium fluoride provided
terminal alkyne 30 which was hydrostannylated with HSnBu3 and
catalytic amount of AIBN to give the corresponding stannane 9 in
high isolated yield and as a single trans isomer. Robust Stille
coupling conditions with vinyliodide
6 have been developed
previously[11, 12] and could also successfully be applied to synthesize
zealactone 1a/b (1) and its related derivatives. Catalytic system
Pd2dba3/P(furyl)3 in dioxane at 120 °C provided the racemic mixture
of 1 in 80% yield (Scheme 6). 1H,13C-NMR and HRMS data of our
synthetic molecule were in full accordance with natural zealactone
1a/b (Supporting Information; Figures S3 and S4).[9] The present
synthetic route starting from 14 provided racemic zealactone 1 in 8
steps (longest linear sequence) with an overall yield of 21%. Since
vinyliodide (R)-6 is readily accessible as enantiomeric pure
compound, zealactone 1a/b (1) could be synthesized as the naturally
Table 2. Chemoselective Baeyer-Villiger oxidation of 24.
B -V oxidation
condition
O
O
O
O
O
O
O
O
24
26
27
28
Entry
Conditions
Yield: 26/27/28
1
2
3
4
5
6
7
8
Mg(monoperoxyphthalate)2, MeOH/H2O, 50°C, 24 h
Oxone, DMF, 23°C or 50°C, 36 h
H2O2 (aq), cat. Li+B(C6F5)4–, DCE, 23°C, 24 h
H2O2 (aq), AcOH, 23°C, 36 h
decomp.
no conversion
decomp.
occurring C6-diasteriomeric material (Scheme 7, Supporting
[11]
Information; Figure S7).
Furthermore, vinyliodide (S)-6 having
20%/–/–
opposite absolute configuration provided the corresponding C2’
epimer of zealactone (Scheme 7, Supporting Information; Figure
S10).
H2O2 (aq), NaOH, EtOH, 23°C, 30 min
mCPBA, CH2Cl2, 23°C, 24 h
29%/–/–
–/15%/45%
15%/13%/16%
mCPBA, NaHCO3, CH2Cl2, 23°C, 24 h
mCPBA (1.5 eq), NBu4OH (2.0 eq),
CH2Cl2/MeOH, 23°C, 1 h
65%/– / –
76%/ –/–
Having this route established, the related analogue 35 could be
synthesized adapting the procedure developed for the total synthesis
of zealactone 1a/b (1). The cyclopentyl lactone 26 could be
transformed to the corresponding stannane 34 in excellent yield
(Scheme 7). Furthermore, Stille coupling with vinyliodide 31 provided
the analogues 32 and 36, the latter having an additional methyl group
on the D-ring improving its soil stability.
9
mCPBA (4.0 eq), NBu4OH (5.0 eq),
CH2Cl2/H2O, 23°C, 1 h
Scheme 6. Synthesis of a mixture of all four stereoisomers of zealactone 1a/b
(1).
HSnBu3 (3.0 eq.)
AIBN (0.15 eq.)
mCPBA (6.0 eq.)
Bu4NOH (7.0 eq.)
SnBu3
O
O
O
O
O
CH2Cl2
23 °C, 1 h
[61%]
toluene
80 °C, 18 h
[97%]
Scheme 8. Synthesis of zealactone analogues.
9
25
R = TBS 29
TBAF (2.0 eq.)
THF
23 °C, 30 min
[96%]
HSnBu3 (3.0 eq.)
AIBN (0.15 eq.)
TBAF (2.0 eq.)
R = H
OMe
30
SnBu3
O
O
TBS
H
THF
23 °C, 30 min
[91%]
O
toluene
80 °C, 18 h
[97%]
O
O
O
O
OMe
O
Pd2dba3 (7.5 mol%)
P(furyl)3 (30 mol%)
SnBu3
I
34
26
33
O
O
O
R
O
O
O
O
1,4-dioxane,
120 °C 30 min
O
R
O
O
O
OMe
O
I
OMe
Pd2dba3 (10 mol%)
P(furyl)3 (40 mol%)
9
R = H [80%]
R = Me [90%]
R = H
R = Me 31
6
R = H rac-1
R = Me 32
SnBu3
O
O
1,4-dioxane,
120 °C 30 min
O
R
O
R
O
O
O
O
O
O
Scheme 7. Synthesis of natural zealactone 1a/b (1) and C2’ epimer of
zealactone.
R = H [63%]
R = Me [94%]
R = H 35
R = Me 36
34
R = H
R = Me 31
6
q.)
(1
.5 e
O
OMe
O
O
I
OMe
O
Pd2dba3 (7.5 mol%)
P(furyl)3 (30 mol%)
SnBu3
O
O
O
1,4-dioxane,
120 °C 30 min
[61%]
O
O
O
O
Fortunately, zealactone 1a/b (1) could be separated by HPLC on a
O
9
(R)-6
chiral stationary phase (chiral HPLC) (Supporting Information;
Figures S8 and S9). 1H,13C-NMR analysis prove that the fast moving
isomer should be zealactone 1a and the other is zealactone 1b
(Supporting Information; Figures S3 and S4). Stereochemistry of
optically pure building blocks 9 and of the corresponding vinyliodides
47 (Supporting Information; Scheme S1) could be assigned by the
Zealactone 1a/b
(1)
(natural)
O
OMe
O
O
I
OMe
O
Pd2dba3 (7.5 mol%)
P(furyl)3 (30 mol%)
SnBu3
O
O
O
1,4-dioxane,
120 °C 30 min
[66%]
O
O
O
O
O
9
(S)-6
C2’ epimer of zealactone 1a/b
5
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10.1002/hlca.202000017
Helvetica Chimica Acta
HELVETICA
R
measurement of circular dichroism (CD) spectra coupled with
computational simulation, which allowed the determination of the C6-
stereochemistry of zealactone 1a and 1b (Supporting Information;
Figures S17-27).
COOMe
Oxidation
β-Carotene
O
O
O
O
O
HO
O
38
40
Carlactone 39: R = CH3
Carlactonoic acid 2: R = COOH
Methylcarlactonoate 3: R = COOMe
Oxidation
Epoxidation
+H2O
COOMe
O
Table 3. Potential epimerization of zealactone 1a.
COOMe
O
OMe
O
O
OMe
O
O
OMe
O
O
HO
HO
O
O
O
O
O
O
6
6
O
O
O
O
O
O
O
42
41
O
O
O
O
O
Asymmetric
epoxidation
+H2O
Fragmentation
COOMe
zealactone 1a
zealactone 1b
37
COOMe
Entry
Conditions
CH2Cl2
pH
–
t1/2
Epimerization
OH
O
HO
O
O
HO
HO
O
O
1
2
3
4
No decomp.
No decomp.
5 days
No
No
No
O
O
O
CH3CN
–
43
42*
H2O/CH3CN (9/1)
7.0
5.8
Lactonization
Fragmentation
½ strength Hoaland solution with
10% CH3CN (extraction cond.)
H2O/CH3CN (9/1) with 1% formic
acid (purification cond.)
> 1 week
Epimerization
No
O
OMe
COOMe
5
2.3
22 h
No
OH
O
O
O
HO
O
O
O
O
O
O
43*
zealactone 1a/b
The C6 stereogenic center of zealactone 1a/b (1) could undergo
(1)
Epimerization
Lactonization
epimerization through heterolytic cleavage of the lactone bond
leading to the formation of the corresponding carboxylate and the
carbocation intermediate 37 highly stabilized by delocalization on the
two adjacent allylic moieties. To clarify whether the natural mixture of
C6-isomers is created in the biosynthetic pathway or during the
isolation/purification process, we analyzed the racemization reaction
of zealactone 1a that was purified by chiral HPLC in the aqueous
conditions that are used for isolation/purification of natural
zealactone. Zealactone 1a did not epimerize in organic solvents
within 7 days at room temperature and can be stored without
alteration for biological testing (Table 3, entries 1 and 2). Some
degradation of zealactone 1a was observed under aqueous
conditions. However, no racemization was detected for at least 7
days under the buffered conditions used for the isolation of
zealactone from corn roots (entries 3-5, Supporting Information;
Figures S13-S16).[9] In addition, no epimerization at C6 was detected
in the presence of 1% formic acid in H2O/MeCN, the conditions used
for zealactone HPLC purification after root extraction.[9] These
findings suggest that both C6-isomers are synthesized in planta. A
plausible biosynthetic pathway depicting how the mixture of C6-
isomers could be generated is described in Figure 3.[9] Unselective
epoxidation of compound 41 followed by fragmentation or
epimerization of compound 43* generated after asymmetric
epoxidation could provide the epimeric mixture 43, leading to
zealactone 1a and 1b.
O
OMe
O
O
O
O
O
zealactone 1a or 1b
Figure 3. Plausible biosynthetic pathway of zealactone 1a/b (1).
With the series of zealactone analogues in hand, we evaluated
their biological activity against corn and their stability in soil.
In Arabidopsis thaliana, the mechanism of action of strigolactone
has been well studied, and two essential genes DWARF14 (D14) and
MORE AXILLARY GROWTH2 (MAX2) play an important role in the
strigolactone-dependent plant responses.[26, 27] These genes encode
a family of α/β-hydrolase fold proteins and a F-box protein of
ubiquitin E3 ligase, respectively. The plausible molecular mechanism
of strigolactone signaling is depicted in Figure 4A. Binding of
strigolactone to the receptor protein D14 initiates the strigolactone
signaling.[28] Conformational change of D14 after strigolactone
binding facilitates formation of the complex with MAX2 protein, which
triggers the ubiquitination of specific repressors followed by their
proteasomal degradation.[29,
This promotes the strigolactone-
30]
responsive genes affecting plant growth and development. [31-33]
Since the strigolactone perception by D14 protein triggers the
activation of strigolactone signaling in planta, we assessed the
binding affinity of synthetic compounds to ZmD14 (D14 orthologue in
corn). Several technologies for assessing the binding activity of small
molecules to D14 have recently been developed. Yoshimulactone
Green (YLG) is a useful molecular tool that can generate a strong
fluorescent product upon the hydrolysis by ZmD14.[34] The
competitive inhibition of YLG-hydrolysis allows the evaluation of the
binding affinity of our synthetic molecules to ZmD14 (Figure 4B).[12, 35]
6
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10.1002/hlca.202000017
Helvetica Chimica Acta
HELVETICA
Figure 4. Molecular mechanism of strigolactone signaling and our in vitro assays. A) Molecular mechanism of strigolactone signaling in Arabidopsis thaliana. B)
YLG displacement assay. The binding affinity of small molecule can be evaluated by the decrease of the YLG-dependent fluorescence. C) DSF assay. The
strigolactone-induced conformational change of ZmD14 can be measured by the thermal shift of ZmD14 denaturation temperature. D) Alpha screen assay. The
strigolactone-induced association of ZmD14 and ZmMAX2 can be detected by the emission at 520-620 nm. At and Zm mean Arabidopsis thaliana and Zea maize,
respectively.
Table 4. The half inhibition concentration (IC50) of compounds against the ZmD14 dependent YLG hydrolysis, the compound-induced thermal shift of ZmD14
denaturation temperature in DSF assay and the relative value of compound-induced association of ZmD14 and ZmMAX2 in alpha screen assay.
O
O
OMe
O
OMe
O
OMe
O
OMe
O
6
6
6
11
O
2’
O
O
R
O
O
2’
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
R
R
R
spiro-zealactone
GR24
MeCLA
R = H
R = Me 45
heliolactone
R = H
R = Me 46
zealactone
R = H
R = Me 32
R = H 35
R = Me 36
7
1
44
3
DSF analysis
YLG displacement
Structures
R
Configuration
Compound[a]
Alpha screen[d]
assay [IC50/µM][b]
[ΔTm/°C][c]
GR24[e]
–
H
mixture of all four stereoisomers
44
3
1.76
1.10
4.81
-4.4
–
4.54
4.01
2.26
racemic
MeCLA
Me
racemic
45
–
H
H
mixture of all four stereoisomers
6S, 11R
7
7
1.35
0.30
0.29
3.35
2.53
–
5.60
5.14
4.91
0.90
3.52
-8.0
-8.0
-0.5
–
heliolactone
H
6R, 11R
7
H
mixture of (S)-C11 diastereomers
mixture of all four stereoisomers
7
Me
46
H
H
mixture of all four stereoisomers
mixture of (R)-C2’ diastereomers
mixture of (S)-C2’ diastereomers
mixture of all four stereoisomers
1
1
1.30
0.59
-3.7
-3.9
-0.2
-3.9
4.88
6.13
0.93
5.30
zealactone
H
1
32.42
2.14
Me
32
H
H
mixture of all four stereoisomers
mixture of (R)-C2’ diastereomers
mixture of all four stereoisomers
35
35
36
0.72
0.39
1.99
-1.4
-4.6
-3.7
5.69
5.13
5.45
spiro-zealactone
Me
[
a] The analytic data of 44, 3, 45, 7 and 46 were referenced from our previous report[12] [b] 1 µM of YLG was used for the YLG displacement assay. [c] 50 µM of each
.
compound were used for the DSF analysis. [d] The values mean relative activity score (higher value means stronger interaction). [e] GR24 is a most commonly used
synthetic strigolactone. Natural isomers of heliolactone and zealactone were highlighted by bold fonts.
7
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The racemic mixture of zealactone 1a/b (1) has comparable
activity to heliolactone 7 and Methylcarlactonoate (MeCLA) 3 (Table
4). Interestingly, spiro-cyclopentyl substituted analogue of zealactone
35 shows the better binding affinity to ZmD14 in comparison to
zealactone. Introduction of additional methyl group on the butenolide
moiety reduces the binding activity to the receptor protein as
previously observed.[12] Although the stereochemistry at C2’ in
zealactone 1a/b (1) was still ambiguous, it was assumed to be (R)-
configured in analogy with typical C2’-configuration of natural
strigolactones.[9] The YLG displacement assay showed that C2’-(R)
diastereoisomers of zealactone 1 is much more active than the
others, which confirms that C2’-(R) isomers are the natural ones of
zealactone 1a/b (1). These findings are supported by another
analysis, differential scanning fluorometry (DSF)(Figure 4C).[36] We
also evaluated small molecule induced ZmD14 to ZmMAX2
interaction by alpha screen analysis (Figure 4D). The obtained
results are well consistent with the binding affinity to the receptor
protein supported by the YLG displacement assay and the DSF.
Altogether, our synthetic zealactone and its analogues can induce
the conformational change of ZmD14 upon their binding then
promote the association of ZmD14 with ZmMAX2, which might
effectively activate the signal transduction in planta.
butyrolactone, whereas the gem-dimethyl faces Trp153, leaving
additional room for the spiro-cyclopentyl derivative to bind.
The putative binding mode of zealactone 1a and 35 to ZmD14 was
assessed through molecular modelling studies. A homology model
for ZmD14 (Uniprot accession number A0A317YGV1) was built with
MODELLER,[37] using the crystal structure of rice D14 (PDB 3W04)
as template. Zealactone 1a was docked into the active site using the
software GOLD.[38] The butenolide ring of zealactone 1a establishes
a hydrogen bond with the backbone of Phe26, located next to the
catalytic Ser95 at the bottom of the active site (Figure 5A). The C2’-
(R) diastereoisomer orients the rest of the molecule in a way to
establish a hydrogen bond network with surrounding amino acids. In
this binding conformation, the carbonyl ester interacts with Cys189,
whereas the oxygens of the vinyl ester and the γ-butyrolactone (A-
ring) interact with the sidechains of Tyr157 and Ser218, respectively.
This specific hydrogen bond pattern is only found for the C2’-(R)
diastereoisomers, possibly explaining the affinity difference between
C2’-(R) and C2’-(S) diastereomers observed in our in vitro analysis.
The gem-dimethyl group of zealactone 1a extends at the edge of the
active site in a hydrophobic environment, leaving additional room for
the spiro-cyclopentyl analogue. Such structural modification allows
the spiro-cyclopentyl moiety of zealactone 35 to establish further van
der Waals contacts with Phe134, leading to a slight improvement in
binding affinity, consistently with YLG displacement assay. The
docking of zealactone 1b (Figure 5B) shows a binding mode in line
with that of zealactone 1a, with a similar hydrogen bond network with
Phe26, Cys189 and Tyr157. The change in stereochemistry at the A
ring leads to a different orientation of the γ-butyrolactone at the wide
entrance of the binding cavity, but doesn’t affect its overall binding. In
this case, Ser218 interacts with the carbonyl oxygen of the γ-
Figure 5. Docking pose of zealactone 1a/b in the active site of corn ZmD14
receptor. Hydrogen bonds are represented in dotted yellow lines. A) Docking
pose of zealactone 1a. B) Docking pose of zealactone 1b.
We next assessed our synthetic zealactone derivatives for impact
on promotion of corn leaf senescence and stimulation of corn seed
germination.
Leaf senescence is self-attrition system to relocate nutrients from
aging leaves that are no longer required to newly developing plant
tissues. Since leaf senescence is associated with crop productivity,
chemical controlling leaf senescence enables accelerating nutrients
relocation, which leads to optimization of grain quality and quantity.
[39]
Leaf senescence is regulated by several plant hormones and it
has recently been reported that strigolactones can also accelerate
leaf senescence through the D14-dependent pathway.[40-42] Herein,
we assessed synthetic non-canonical strigolactones induced leaf
senescence of corn by following the previously developed protocol.[12]
As shown in Table 5, corn leaf senescence was activated by the
treatment of our synthetic zealactone derivatives except for C2’-(S)
diastereoisomers of zealactone 1. Their activities are well correlated
8
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10.1002/hlca.202000017
Helvetica Chimica Acta
HELVETICA
with the binding affinity to ZmD14. The A-ring structure of non-
canonical strigolactones affects the promotion of leaf senescence in
corn. Zealactone scaffolds have a better activity on leaf senescence
than heliolactone and MeCLA, and the spiro-cyclopentyl substituted
zealactone showed the highest activity among non-canonical
strigolactones. Since precisely chemical control of nutrients
remobilization enables the optimization of grain productivity,
senescence control with our developed molecules would be an
innovative technology towards sustainable agriculture.
Table 5. The half maximal senescence acceleration concentration (SC50 in µM)
Figure 6. Kinetic curves of seed germination in the presence (red line) or
absence (black) of small molecule. T50 value means the time point at the 50%
seed population germination and was calculated from the logistic germination
curve. The slope value was determined at the point of T50 in the logistic
germination curve. All the values in Table 6 are expressed as percentages
compared to an untreated control.
measured for corn leaf discs treated with our synthetic molecules.
Leaf
senescence
Structures
R
Configuration
Compound[a]
[SC50/ µM]
mixture of all four
stereoisomers
racemic
GR24
–
44
0.1
H
3
1.8
MeCLA
As shown in Table 6, all strigolactones we tested accelerated the
seed germination of corn in comparison with untreated control.
Regarding the speed and uniformity in the corn seed germination,
non-canonical type strigolactones are much more active than GR24
44. Interestingly, the series of zealactone analogues effectively
shortened the time required for the seed population germination
compared to MeCLA and heliolactone analogues. In addition, the
zealactone analogues have a comparable activity to MeCLA on the
parameter of germination homogeneity. Amongst non-canonical
strigolactones tested, analogues that have an additional methyl
group on D-ring showed the better accelerating activity on both
factors of corn germination compared with the corresponding mono
methyl D-ring molecules, which might be due to their improved
stability. As we mentioned above, the binding affinity of (S) C2’
diastereomers of zealactone 1 to ZmD14 is quite low. Nevertheless
(S) C2’ diastereomers of zealactone 1 can also stimulate the seed
germination of corn at the same level of (R) C2’ diastereomers of
zealactone 1, which suggests that (S) C2’ diastereomers of
zealactone 1 activates the corn seed germination through non-
ZmD14 signaling pathway and might indicate that strigolactone-
dependent seed germination of corn is not only regulated by the
ZmD14-dependent pathway.
Me
racemic
45
7
mixture of all four
stereoisomers
H
7
0.8
heliolactone
mixture of all four
stereoisomers
Me
46
0.09
mixture of all four
H
H
1
1
0.14
0.17
7.1
stereoisomers
mixture of (R)-C2’
diastereomers
zealactone
mixture of (S)-C2’
H
1
diastereomers
mixture of all four
Me
H
32
35
35
36
0.15
0.04
0.058
0.43
stereoisomers
mixture of all four
stereoisomers
spiro-
mixture of (R)-C2’
H
diastereomers
zealactone
mixture of all four
Me
stereoisomers
[
a] The data of 3, 45, 7 and 46 were referenced from the previous reported
paper[12]. Natural isomer of zealactone was highlighted by bold fonts.
Controlling seed germination and plant growth in early stage
largely contributes to the improvement of crop production. Breaking
the seed dormancy is triggered by several phytohormones and
Whereas natural isomers of strigolactones, which have C2’(R)
configuration at the D-ring, do not regulate the primary root growth
after emergence, the opposite epimer showed significant effect on
the primary root growth. (S) C2’ epimer of zealactone 1 and (S) C11
epimer of heliolactone 7 have the strongest primary corn root growth
enhancing and suppressing activity, respectively. Apparently, the
structure activity relationship of our synthetic molecules on the
primary root growth is not correlated with the in vitro binding affinity
to ZmD14, the leaf senescence activity and the seed germination
activity. These findings might suggest that the primary root growth
environmental factors.[43] Strigolactones are well known as
a
germination stimulant of parasitic weeds,[44] and can also activate the
seed germination of various species and even of crops.[43, 45-47] Next
we investigated our synthetic zealactones on the germination of corn
at 15 °C which is a mimic temperature of an agronomically relevant
soil. Two different kinetic parameters, the speed (T50) and uniformity
(slope), were extracted from the kinetic curve of seed population
germination. In addition to rates of seed germination, the speed of
primary root growth after emergence was studied (Figure 6).
9
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10.1002/hlca.202000017
Helvetica Chimica Acta
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after emergence is regulated on a different way from the signaling of
seed germination and ZmD14-dependent signaling. Interestingly, our
synthetic molecules (S) C2’ diastereomers of zealactone 1 and (S)
primary root growth. In addition, (S) C2’ diastereomers of zealactone
1 showed the effective stimulation on the seed germination and the
acceleration of the plant growth in early stage, which can be a
promising lead molecule enhancing crop productivity.
C11 diastereomers of heliolactone
7 work as an agonist and
antagonist of this signaling pathway, respectively, which could be
useful molecular tools for elucidating the signaling pathway of the
Table 6. Effect of non-canonical strigolactones on the germination and on the root growth kinetic parameters of corn at various concentrations and 15 °C.
O
O
O
OMe
O
OMe
O
OMe
O
OMe
O
6
6
6
11
O
2’
O
O
R
O
O
2’
O
O
O
O
O
O
O
O
O
O
O
O
O
O
R
R
R
spiro-zealactone
GR24
MeCLA
R = H
R = Me 45
heliolactone
R = H
R = Me 46
zealactone
R = H
R = Me 32
R = H 35
R = Me 36
7
1
44
3
Germination T50
(% compared to control)[c]
Germination slope
Primary root growth slope
(% compared to control)
(% compared to control)[b]
Structures
R
Chirality
Compound[a]
50
250
50
250
50
250
44
3
–
H
mixture of all four stereoisomers
racemic
–
-3.4
-7.6
–
50.4
158.4
135.3
101.5
96.1
–
–
GR24
-1.0
-2.8
-2.7
-2.9
-2.7
-4.7
-4.2
-3.0
-4.3
-5.3
-6.6
-8.0
-7.2
10.9
36.5
23.8
25.5
34.6
23.9
38.0
11.9
39.8
9.5
3.8
7.5
-0.2
1.3
-5.3
-5.7
5.7
4.4
-0.2
12.2
5.8
0.1
1.8
3.9
MeCLA
45
7
Me
H
racemic
-7.1
6.6
mixture of all four stereoisomers
6S, 11R
-8.6
-2.2
-5.0
-3.7
-13.4
-5.1
8.6
7
H
-8.7
7
H
6R, 11R
-9.0
11.2
heliolactone
7
H
mixture of (S)-C11 diastereomers
mixture of all four stereoisomers
mixture of all four stereoisomers
mixture of (R)-C2’ diastereomers
mixture of (S)-C2’ diastereomers
mixture of all four stereoisomers
mixture of all four stereoisomers
mixture of (R)-C2’ diastereomers
-9.1
118.3
132.9
116.8
119.0
120.8
149.3
63.4
46
1
Me
H
-7.9
-12.0
-12.0
-13.0
-13.5
-10.3
-11.6
1
H
7.3
zealactone
1
H
15.2
-1.9
1.4
32
35
35
36
Me
H
31.7
17.4
29.9
spiro-
zealactone
H
101.4
-2.8
Me
mixture of all four stereoisomers
-8.4
-9.8
27.2
114.3
4.8
-9.1
[a]
The data of 44, 3, 45, 7 and 46 were referenced from the previous reported paper.[12, 48] Positive values mean a more homogeneous germination and vice
[b]
versa for negative values. [c] Positive values mean a slower germination and vice versa for negative values. Data in bold are statistically validated.
In our previous work, we demonstrated that natural and close
The Table 7 reports the soil half-life of a series of non-canonical
strigolactones. An estimated value of Log P is reported as well. Log
P is an intrinsic property of the molecules which provides some
indirect indications regarding how strongly the molecule can be
bound to soil particles, especially to the organic matter fraction. For
comparison purposes, alongside the zealactone 1 and its derivatives
we also reported the soil half-life of heliolactone 7, MeCLA 3 and
others close analogs disclosed in a previous study.[12] A soil stability
ratio has been defined in order to compare the soil stability of all
these compounds with GR24 44. These results confirm the very short
soil persistence of strigolactones. All tested compounds showed a
soil half-life shorter than 10 hours. In this study we observed a very
low recovery of zealactone 1 already at the timing zero where we
were able to extract less than one fourth of the initial treated amount.
For this reason, we were not able to calculate reliably the soil half-life
of zealactone. Thus, this experimental data suggests that zealactone
synthetic analogs of strigolactones can have a very short half-life in
49]
soil, as short as a few hours.[12,
Soil half-life is an important
chemodynamic property of the molecule and provides indication
whether compounds are stable enough and sufficiently long
bioavailable in soil to deliver a given desirable biological activity. This
represents an important technical attribute for practical application. In
previous investigations we showed also that specific structural
changes/substitutions in different parts of the core scaffold of a
natural strigolactone like heliolactone can improve their stability.[12]
These findings suggested that we could optimize by structural
modification the soil persistence of strigolactones. Due to the
interesting in vivo and in vitro activities of zealactone, we were
strongly interested to evaluate whether this specific corn
strigolactone and others very close derivatives were sufficiently
stable in soil under laboratory conditions.
10
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10.1002/hlca.202000017
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1 degrades very fast. Interestingly, the zealactone derivatives 32, 35,
36 displayed slightly higher stability in soil. As already observed for
the heliolactone 7 and its derivative 46, an additional methyl group on
the D-ring led to a substantial increase of the soil half-life by a factor
6 or more. Similarly, the spiro-zealactone derivative 35, 36 with a
cyclopentyl substitution increased the soil half-life. It is noteworthy
that the combined substitutions of both cyclopentyl group and
additional methyl on the D-ring did not result in further improvement
of the soil stability. Most likely other parts of the core scaffold can be
still prone to metabolism, like for instance the methyl ester which
might undergo hydrolysis.
All together, these results confirm the labile nature of
strigolactones in soil. This can have implications for the practicability
of using strigolactones in agriculture. Despite the highly intrinsic
biological activity of strigolactones, for agronomical applications the
improvement of soil stability remains critical. More stable compounds
would allow the use of very low doses in the field and keep the
synthetic costs per hectare sufficiently low to make this technology
affordable for growers.
Table 7. Log P and soil half-life of non-canonical strigolactones in a non-sterile soil.
O
O
OMe
O
OMe
O
OMe
O
OMe
O
6
6
6
11
O
2’
O
O
R
O
O
2’
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
R
R
R
spiro-zealactone
GR24
MeCLA
R = H
R = Me 45
heliolactone
R = H
R = Me 46
zealactone
R = H
R = Me 32
R = H 35
R = Me 36
7
1
44
3
Structures
R
Compound[a]
Log P[b]
t1/2 [h]
r2
Soil Stability Ratio[c]
Remarks
H
Me
H
3
45
7
4.48
4.57
3.23
3.38
10.5
27.0
2.5
0.88
0.62
0.99
0.99
3.1
7.9
0.7
1.0
Data from a previous study[12]
Data from a previous study[12]
Data from a previous study[12]
Data from a previous study[12]
MeCLA
heliolactone
zealactone
Me
46
9.7
Very low recovery at T0 (i.e. 23%) did not allow to estimate
H
1
2.8
–
–
–
the t1/2, suggesting the compounds is highly unstable
Me
H
32
35
36
3.0
3.3
3.5
7.56
6.47
7.81
0.85
0.97
0.85
5.9
5.1
6.1
–
–
–
spiro-zealactone
Me
[a] All tested compounds are racemic mixture. [b] The log P values were calculated. [c] Soil stability ratio is the quotient of the measured soil half-life of the non-
canonical strigolactone divided by the t1/2 of the GR24 44 measured in the same study, 3.4 hours in previous study[12] and 1.3 hours in present study.
zealactone could be a lead candidate for optimization. For instance,
Conclusions
we have demonstrated that small changes/substitutions in the core
chemotype led to an increase of in vitro and in vivo activity in
In conclusion, a total synthesis of zealactone 1a/b (1) has been
relevant assays and a general improvement of soil stability. In the
developed providing this most abundant strigolactone from the root
optic of a more sustainable crop production, synthetic derivatives
exudates of corn in eight steps (longest linear sequence) and an
closely inspired by natural corn strigolactones like zealactone could
overall yield of 21%. As key-transformations a [2+2]-cycloaddition
play a critical role.
strategy using an unprecedented type of ketene could be employed
followed by a chemoselective Baeyer-Villiger oxidation to synthesize
Experimental Section
the challenging γ-butyrolactone fragment. Our novel procedure
allowed also the synthesis of closely related analogues.
General Experimental Procedures. All chemicals and solvents
were purchased from common suppliers and used as received.
Chromatographic purifications were done on an Rf-machine
CombiFlash Rf 200i Teledyne ISCO equipped with standard silica
columns (silica gel 60, 0.040−0.063 mm: CHROMABOND® Flash
from MACHEREY-NAGEL or silica gel 60, 0.015−0.040 mm:
RediSep®Rf GOLD from Teledyne ISCO). Thin-layer chromatography
was performed on precoated TLC glass plates SIL G-254 or
precoated TLC sheets ALUGRAM SIL G-UV254 with fluorescence
Taking all these results together, we could confirm that
strigolactones represent
a promising area with many potential
applications in agriculture. As we expected, the unique structure of
zealactone 1a/b (1) as non-canonical strigolactones gave some
specificity to the perception and signaling in corn, compared with
other non-canonical type strigolactones studied in the past. The in
vitro results showed that zealactone and its close derivatives were as
active as or even more active than GR24 44. Consequently,
11
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10.1002/hlca.202000017
Helvetica Chimica Acta
HELVETICA
indicator UV254. Staining agents were either KMnO4 (40 g of K2CO3 +
6 g of KMnO4 in 600 mL of water, then 5 mL of 10% NaOH added) or
CAM (2 g Ce(SO4)2 + 60 g (NH4)6Mo7O24·4H2O in 360 mL of water,
then 40 mL of H2SO4 conc added) and subsequent heating of TLC
Synthesis of Methyl 2-iodo-3-methyl-but-2-enoate 13b. To a
suspension of copper iodide (388 mg, 2.04 mmol, 2.00 equiv.) in THF
(5.00 mL) under argon atmosphere at 0 °C was added methyl lithium
(1.6 M in Et2O, 2.50 mL, 4.08 mmol, 4.00 equiv.). The obtained clear
colorless solution was cooled to -45 °C and a solution of methyl but-
2-ynoate (100 mg, 1.02 mmol, 1.00 equiv.) in THF (5.00 mL) was
slowly added. The opaque white solution was stirred for 90 min at -
1
plates treated with these staining solutions. H and 13C NMR spectra
were recorded on a Bruker spectrometer operating at 600 or 400 and
150 or 100 MHz, respectively, unless otherwise stated. Chemical
shifts are given in ppm. The following abbreviations describe the
multiplets: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; q,
quadruplet; quin, quintet; sext. sextet; m, multiplet. Mass spectra
were recorded on a High Resolution Mass Spectrometer (Q-Exactive,
Orbitrap analyzer) from Thermofischer equipped with an electrospray
source, (Polarity: positive mode, Capillary: 3.8 kV, Capillary
temperature: 320°C, S-lens RF level: 55, Sheath gas flow rate: 40,
Mass range: 100 to 1000 m/z) coupled to a 1200 Infinity series UPLC
from Agilent: Solvent degasser, binary pump, heated column
compartment and diode-array detector (DAD). DAD Wavelength
range (nm): 190 to 400. Column: Waters Acquity UPLC BEH C18,
1.7 µm, 50 x 2.1 mm, Temp: 60 °C. Solvent Gradient: A = water +
5% MeOH + 0.05 % HCOOH, B= Acetonitrile + 0.05 % HCOOH.
Infrared spectra were recorded on a Perkin Elmer UATR two FI-TR
spectrometer. Melting points were measured on a Büchi Melting
Point B-540 apparatus. The CD measurements were performed on a
JASCO J-1500 CD Spectrophotometer in MeCN at 20 °C in a 1115F-
QS Hellma cuvettes (10 mm light path) with a scan rate of 50
nm/min. All solutions were prepared and measured under air
45 °C, molecular iodine (776 mg, 3.06 mmol, 3.00 equiv.) as
a
solution in THF (3.00 mL) was slowly added and the clear yellowish
solution was stirred for 1 h at -45 °C, afterwards 2 h at 0 °C. An
aqueous saturated solutions of NH4Cl (10.0 mL) and Na2S2O3 (10.0
mL) were added and diethyl ether (20.0 mL) were added. The
mixture was extracted with diethyl ether (3 x 25 mL), combined
organic layers were dried over MgSO4, filtered and concentrated in
vacuo. Purification by flash column chromatography (SiO2,
cyclohexane/ ethyl acetate = 19/1) gave the title compound as
colorless oil (233 mg, 969 mmol, 95%). Rf = 0.57 (cyclohexane/ ethyl
acetate = 19/1); 1H NMR (400 MHz, CDCl3) δ ppm 3.78 (s, 3 H), 2.13
(s, 3 H), 2.09 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ ppm 165.9,
152.3, 83.8, 52.7, 31.8, 22.0; HRMS: calc. for C6H10IO2 [M+H]+:
240.9725, found: 240.9719; IR (cm-1): 2997, 2950, 2916, 1710, 1600,
1433, 1367, 1236, 1212, 1090, 1010, 856, 769.
Synthesis of Methyl 2-[2-[tert-butyl(dimethyl)silyl]ethynyl]-3-
methyl-but-2-enoate 15. To
a suspension of copper iodide
(60.3 mg, 317 mmol, 0.20 equiv.) in dioxane (16.0 mL) and triethyl
amine (8.00 mL) under argon atmosphere at room temperature was
added vinyl iodide (13b) (380 mg, 1.58 mmol, 1.00 equiv.) obtained
from above and tert-butyl-etynyl-dimethyl-silane (888 mg, 6.33 mmol,
4.00 equiv.). The clear colorless mixture was degassed by bubbling
argon through the solution for 15 min. PdCl2(PPh3)2 (111 mg, 158
mmol, 0.10 equiv.) was added and the mixture was stirred and
heated to 50 °C for 90 min. The mixture was allowed to cool to room
temperature and was filtered over a plug of Celite (wash with
cyclohexane/ ethyl acetate = 19/1, 40 mL). The solvent was
removed in vacuo and the crude was purification by flash column
chromatography over silica gel (cyclohexane/ ethyl acetate = 80/1 to
19/1) gave the title compound as yellowish oil (360 mg, 1.43 mmol,
91%). Rf = 0.15 (cyclohexane/ ethyl acetate = 80/1); 1H NMR (400
MHz, CDCl3) δ ppm 3.76 (s, 3 H), 2.23 (s, 3 H), 2.16 (s, 3 H), 0.97 (s,
9 H), 0.15 (s, 6 H); 13C NMR (100 MHz, CDCl3) δ ppm 165.8, 161.2,
112.6, 101.4, 97.7, 51.8, 26.1, 26.0, 22.4, 16.7, -4.6; HRMS: calc. for
C14H25O2Si [M+H]+: 253.1624, found: 253.1620; IR (cm-1): 2953,
2930, 2857, 2149, 1725, 1605, 1435, 1284, 1229, 1156, 1086, 827,
775.
saturated
conditions
if
not
otherwise
stated.
ZmD14
(Zm00001d048146),
a
maize orthologue of the Arabidopsis
strigolactone receptor D14, was expressed as a N-terminal 6x His-
SUMO fusion protein by cloning into the pET-SUMO vector (Thermo
Scientific) which was used to transform E.coli One Shot BL21(DE3)
cells (Thermo Fisher Scientific). For expression of ZmMAX2
(Zm00001d045313), the full-length sequence was codon-optimised
for E.coli and gene synthesized as an N-terminal maltose binding
protein (MBP)-TEV fusion which was inserted into a pET-DUET
expression vector. Expression was carried out in E.coli BL21 Star
(DE3) using auto-induction media. For purification, cells were lysed
using a cell disruptor and the clarified lysate was applied to a 1 mL
MBPTrap column (GE Healthcare) equilibrated in 5 mL PBS, 150 mM
NaCl, 0.5 mM TCEP, pH 7.4. The column was washed with 20ml of
the same buffer and bound protein was eluted using PBS pH 7.4,
150 mM NaCl, 0.5 mM TCEP, 10 mM D-Maltose monohydrate.
Elution fractions were analyzed by SDS-PAGE and those containing
the protein of interest were pooled and loaded onto a Superdex 200
16/60 column equilibrated in PBS, 150 mM NaCl, 0.5 mM TCEP, pH
7.4. Fractions containing the target protein were re-applied to the
MBPTrap column equilibrated in PBS, 150 mM NaCl, 0.5 mM TCEP,
pH 7.4 and eluted with a linear gradient over 10mL of the same
buffer containing 10 mM D-Maltose monohydrate. Samples were
stored at -80°C until use.
Synthesis of 2-[2-[tert-butyl(dimethyl)silyl]ethynyl]-3-methyl-but-
2-enoic acid 16. To a solution of methyl ester (15) (200 mg, 792
mmol, 1.00 equiv.) obtained from above dissolved in tert-butanol
(20.0 mL) was added an aqueous solution of lithium hydroxide (1.0
M, 5.00 mL, 6.30 equiv.) at room temperature. The mixture was
stirred for 22 h at room temperature. An aqueous saturated solution
12
This article is protected by copyright. All rights reserved.

10.1002/hlca.202000017
Helvetica Chimica Acta
HELVETICA
of NH4Cl (30 mL), acetic acid (2.0 mL and dichloromethane (50 mL)
were added, the aqueous layer was extracted with dichloromethane
(4x 50 mL), combined organic layers were dried over MgSO4, filtered
and concentrated in vacuo. Purification by flash column
(16 mL) under argon at room temperature. After premix solutions
descried above were additionally added to the reaction solution, the
resulting mixture was stirred for 1 h at room temperature. After
quenched with 10% Na2S2O3 solution, the aqueous layer was
extracted with dichloromethane three times. The combined organic
layers were dried over Na2SO4, filtered and concentrated in vacuo.
Purification by flash column chromatography over silica gel
(cyclohexane/ ethyl acetate = 19/1) gave the title compound 26
(160 mg, 0.50 mmol, 76%) as colorless oil. Rf = 0.13 (cyclohexane/
ethyl acetate = 40/1); 1H NMR (400 MHz, CDCl3) δ ppm: 5.32 (s,
1 H), 5.09 (s, 1 H), 2.72 (d, J = 16.9 Hz, 1 H), 2.48 (d, J = 16.9 Hz, 1
H), 2.10-2.03 (m, 1 H), 1.89 (s, 3 H), 1.80-1.74 (m, 2 H), 1.70-1.63
(m, 3 H), 1.57-1.51 (m, 1 H), 1.39-1.34 (m, 1 H), 0.94 (s, 9 H), 0.12
(s, 6 H); 13C NMR (100 MHz, CDCl3) δ ppm: 175.0, 140.3, 114.3,
102.9, 91.4, 78.9, 55.6, 43.2, 35.2, 33.0, 26.0, 24.4, 23.4, 20.2, 16.6,
-4.8; HRMS: calc. for C19H31O2Si [M+H]+: 319.2093, found: 319.2084;
IR (cm-1): 2953, 2929, 2857, 2150, 1793, 1718, 1471, 1250, 1218,
1198, 948, 912, 839, 826, 813, 777.
chromatography over silica gel (cyclohexane/ ethyl acetate
6/1+1%AcOH) gave the title compound (95.0 mg, Rf 0.20,
cyclohexane/ ethyl acetate 6/1+1%AcOH) besides recovered
=
=
=
starting material (59.0 mg, Rf = 0.59, cyclohexane/ ethyl acetate =
6/1+1%AcOH) that was resubmitted to the same reaction conditions
as described. Two cycles gave desired acid 16 (133 mg, 558 mmol,
70%) in total as colorless crystalline solid. Rf
=
0.20
(cyclohexane/ethyl acetate = 6/1+1%AcOH); mp = 131-132 °C; 1H
NMR (400 MHz, CDCl3) δ ppm: 2.28 (s, 3 H), 2.20 (s, 3 H), 0.98 (s, 9
H), 0.17 (s, 6 H); 13C NMR (100 MHz, CDCl3) δ ppm: 169.3, 164.3,
162.2, 112.1, 101.0, 98.5, 26.7, 26.1, 22.8, 16.9, -4.6; HRMS: calc.
for C13H23O2Si [M+H]+: 239.1467, found: 239.1462; IR (cm-1): 3077,
2951, 2928, 2893, 2857, 2150, 1695, 1584, 1408, 1285, 1249, 910,
839, 828, 774, 734, 666.
Synthesis
of
(rac)-3-[2-[tert-butyl(dimethyl)silyl]ethynyl]-3-
Synthesis
of
(rac)-1-ethynyl-1-isopropenyl-2-
isopropenyl-spiro[3.4]octan-2-one 24. To a solution of acid (16)
(2.50 g, 10.5 mmol, 1.00 equiv.) dissolved in dichloromethane (200
mL) was added a solution of oxalyl chloride (1.0 mL, 11.5 mmol, 1.10
equiv.) and N,N-dimethylformamide (40 µL, 0.52 mmol, 0.05 equiv.)
under argon at room temperature. The mixture was stirred for 30 min
at room temperature. The reaction was monitored by TLC after
oxaspiro[4.4]nonan-3-one 33. To a solution of TBS-protected
alkyne 26 (1.20 g, 3.80 mmol, 1.00 equiv.) in THF (40 mL) was
added a solution of TBAF (1.0 M in THF; 7.5 mL, 7.5 mmol, 2.00
equiv.) under argon at room temperature. The mixture was stirred for
30 min at room temperature. An aqueous saturated solution of NH4Cl
and dichloromethane were added, the aqueous layer was extracted
with dichloromethane three times, combined organic layers were
dried over Na2SO4, filtered and concentrated in vacuo. Purification by
flash column chromatography over silica gel (cyclohexane/ ethyl
acetate = 19/1) gave the title compound 33 (697 mg, 3.41 mmol,
91%) as colorless oil. Rf = 0.20 (cyclohexane/ ethyl acetate = 19/1);
1H NMR (400 MHz, CDCl3) δ ppm: 5.34 (s, 1 H), 5.11 (s, 1 H), 2.74
(d, J = 16.9 Hz, 1 H), 2.68 (s, 1 H), 2.49 (d, J = 16.9 Hz, 1 H), 2.08-
2.03 (m, 1 H), 1.91 (s, 3 H), 1.84-1.77 (m, 2 H), 1.73-1.60 (m, 3 H),
1.59-1.54 (m, 1 H), 1.42-1.36 (m, 1 H); 13C NMR (100 MHz, CDCl3) δ
ppm: 174.7, 140.2, 114.6, 87.2, 81.3, 75.8, 55.5, 42.8, 35.0, 33.0,
24.2, 23.2, 20.1; HRMS: calc. for C13H17O2 [M+H]+: 205.1228, found:
205.1225; IR (cm-1): 3266, 2962, 2877, 1789, 1213, 948, 913, 676.
Synthesis of (rac)-1-isopropenyl-1-[(E)-2-tributylstannylvinyl]-2-
oxaspiro [4.4]nonan-3-one 34. To a solution of alkyne 33 (650 mg,
3.18 mmol, 1.00 equiv.) in toluene (30 mL), tributylstannane (2.5 mL,
9.5 mmol, 3.0 equiv.) and AIBN (80 mg, 0.47 mmol, 0.15 equiv.)
under argon at room temperature. The mixture was stirred and
heated to 80 °C for 18 h under argon. Purification by flash column
chromatography over silica gel (cyclohexane/ ethyl acetate = 80/1 to
19/1) gave the title compound 34 (1.53 g, 0.97 mmol, 97%) as
quenched
with
methanol
to
form
methyl
ester.
Methylenecyclopentane (22 mL, 210 mmol, 20.0 equiv.) was added,
and then triethylamine (3.2 mL, 23.1 mmol, 2.20 equiv.) was slowly
added by a syringe pump (rate of addition; 4 mL/h). The resulting
solution was stirred for 16 h at room temperature. An aqueous
saturated solution of NH4Cl and dichloromethane were added, the
aqueous layer was extracted with dichloromethane three times,
combined organic layers were dried over Na2SO4, filtered and
concentrated in vacuo. Purification by flash column chromatography
over silica gel (cyclohexane/ ethyl acetate = 40/1) gave the title
compound 24 (3.01 g, 9.95 mmol, 95%) as yellowish oil. Rf = 0.30
(cyclohexane/ ethyl acetate = 40/1); 1H NMR (400 MHz, CDCl3) δ
ppm: 5.03 (s, 1 H), 4.99 (s, 1 H), 3.12 (d, J = 17.6 Hz, 1 H), 2.89 (d, J
= 17.6 Hz, 1 H), 2.25-2.18 (m, 1 H), 1.82-1.68 (m, 6 H), 1.81 (s, 3 H),
1.55-1.51 (m, 1 H), 0.94 (s, 9 H), 0.10 (s, 6 H); 13C NMR (100 MHz,
CDCl3) δ ppm: 203.2, 140.2, 113.7, 102.1, 90.6, 71.2, 57.7, 47.3,
37.2, 34.6, 26.1, 24.4, 24.0, 20.9, 16.7, -4.6; HRMS: calc. for
C19H31OSi [M+H]+: 303.2144, found: 303.2144; IR (cm-1): 2954, 2930,
2857, 2150, 1789, 1723, 1250, 1224, 1007, 838, 826, 812, 775.
Synthesis
of
(rac)-1-[2-[tert-butyl(dimethyl)silyl]ethynyl]-1-
1
colorless oil. Rf = 0.25 (cyclohexane/ ethyl acetate = 40/1); H NMR
isopropenyl-2-oxaspiro[4.4]nonan-3-one 26. Premix solution of
mCPBA (<77%; 246 mg, 1.32 mmol, 2.00 equiv.) and tetrabutyl
ammonium hydroxide (56% w/w solution in H2O, 760 mg, 1.65 mmol,
2.50 equiv.) was added to a clear yellowish solution of cyclobutanone
24 (200 mg, 0.66 mmol, 1.00 equiv.) dissolved in dichloromethane
(400 MHz, CD2Cl2) δ ppm: 6.38-6.20 (m, 2 H), 5.05 (s, 1 H), 4.95 (t, J
= 1.5 Hz, 1 H), 2.35-2.28 (m, 2 H), 1.98-1.91 (m, 1 H), 1.84 (s, 3 H),
1.74-1.70 (m, 2 H), 1.69-1.59 (m, 3 H), 1.54-1.46 (m, 6 H), 1.44-1.40
(m, 2 H), 1.30 (sext., J = 7.0 Hz, 6 H), 0.95-0.91 (m, 6 H), 0.88 (t, J =
13
This article is protected by copyright. All rights reserved.

10.1002/hlca.202000017
Helvetica Chimica Acta
HELVETICA
7.3 Hz, 9 H); 13C NMR (100 MHz, CD2Cl2) δ ppm: 175.9, 145.4,
144.1, 128.3, 112.3, 93.5, 55.0, 42.5, 34.4, 34.0, 29.7, 27.8, 24.0,
23.6, 21.3, 14.1, 10.1; HRMS: calc. for C25H45O2Sn [M+H]+:
497.2442, found: 497.2447; IR (cm-1): 2956, 2925, 2872, 2853, 1787,
1457, 1216, 1185, 997, 958.
diastereomers give very similar signals in 1H- and 13C-NMR are
reported as one compound: 1H NMR (400 MHz, CD2Cl2) δ ppm: 7.56
(s, 1H), 6.85 (dd, J = 1.1, 16.1 Hz, 1H), 6.35 (dd, J = 4.4, 16.1 Hz,
1H), 5.97 (s, 1H), 5.38-5.26 (m, 1H), 5.16-5.05 (m, 1H), 4.94 (d, J =
1.5 Hz, 1H), 3.73 (s, 3H), 2.43 (d, J = 16.9 Hz, 1H), 2.21 (dd, J = 2.0,
16.7 Hz, 1H), 2.00 (d, J = 0.7 Hz, 3H), 1.89-1.86 (m, 1H), 1.87-1.79
(m, 5H), 1.23 (s, 3H), 1.06 (s, 3H); 13C NMR (100 MHz, CD2Cl2) δ
ppm: 175.62, 175.59, 171.32, 171.30, 166.91, 166.89, 153.73,
153.69, 153.35, 153.27, 144.12, 144.09, 133.07, 133.02, 128.61,
128.55, 117.96, 117.91, 102.78, 102.64, 92.5, 55.28, 55.25, 52.1,
42.4, 34.49, 34.44, 34.0, 23.8, 23.6, 11.75, 11.73, 8.9; HRMS: calc.
for C23H29O7 [M+H]+: 417.1913, found: 417.1905; IR (cm-1): 2955,
2926, 2876, 1779, 1716, 1640, 1610, 1437, 1357, 1315, 1254, 1193,
1121, 1085, 983, 749.
Synthesis of (rac)-/ C2’-(R) diastereomers of methyl (E,2E)-4-(1-
isopropenyl-3-oxo-2-oxaspiro[4.4]
oxo-2H-furan-2-yl)oxymethylene]but-3-enoate 35. To a solution of
vinyl iodide (25 mg, 0.077 mmol, 1.00 equiv.), tris(2-
nonan-1-yl)-2-[(4-methyl-5-
6
furyl)phosphine ( 7.1 mg, 0.031 mmol, 0.40 equiv.) in 1,4-dioxane
(0.8 mL) was added vinylstannane 34 (57 mg, 0.12 mmol, 1.50
equiv.) under argon atmosphere. The solution was degassed by
bubbling argon through the solution for 10 min. Pd2dba3 (7.0 mg, 7.7
µmol, 0.10 equiv.) was added, the reaction vial was sealed and
heated to 120 °C for 30 min. The clear yellow mixture was allowed to
cool to rt and was filtered over Celite (wash with cyclohexane/ ethyl
acetate = 2/1, 15 mL). The solvent was removed in vacuo and the
crude was purified by flash column chromatography (SiO2,
cyclohexane/ ethyl acetate = 2/1). The corresponding coupling
product was obtained as colorless oil (63 mg, 0.049 mmol, 63 %).
The coupling with (R)-6 produced C2’-(R) disastereomers of
compound 35. HPLC analyses of them are described in Supporting
Information. Rf = 0.17 (cyclohexane/ ethyl acetate = 2/1); Both
diastereomers give very similar signals in 1H- and 13C-NMR are
reported as one compound: 1H NMR (400 MHz, CD2Cl2) δ ppm: 7.56
(s, 1 H), 6.99-6.97 (m, 1 H), 6.89 (d, J = 16.1 Hz, 1 H), 6.34 (m, 1 H),
6.17-6.16 (m, 1 H), 5.06 (s, 1 H), 4.95 (s, 1 H), 3.73 (s, 3 H), 2.35 s,
2 H), 1.99-1.98 (m, 3 H), 1.92-1.88 (m, 1 H), 1.82 (s, 3 H), 1.76-1.69
(m, 2 H), 1.68-1.58 (m, 4 H), 1.42-1.40 (m, 1 H); 13C NMR (100 MHz,
CD2Cl2) δ ppm: 175.7, 170.9, 166.9, 153.7, 144.2, 141.8, 136.2,
133.2, 117.8, 112.7, 112.0, 101.1, 92.5, 55.3, 52.1, 42.5, 34.5, 34.0,
23.8, 23.7, 21.3, 17.1, 11.0; HRMS: calc. for C22H27O7 [M+H]+:
403.1756, found: 403.1752; IR (cm-1): 2954, 1782, 1716, 1650, 1437,
1190, 1012, 953. Chiral HPLC analyses are described in Supporting
Information.
Synthesis
of
(rac)-2-[2-[tert-butyl(dimethyl)silyl]ethynyl]-2-
isopropenyl-3,3-dimethyl-cyclobutanone 25. To a solution of acid
(16) (4.00 g, 16.8 mmol, 1.00 equiv.) dissolved in dichloromethane
(40 mL) was added a solution of oxalyl chloride (1.6 mL, 18.5 mmol,
1.10 equiv.) and N,N-dimethylformamide (70 µL, 0.84 mmol, 0.05
equiv.) under argon at room temperature. The mixture was stirred for
30 min at room temperature. The reaction was monitored by TLC
after quenched with methanol to form methyl ester. The reaction
solution was diluted with 760 mL of dichloromethane. After cooling to
-78 °C, isobutene gas (bp: -6.9 °C; 83 mL, 922 mmol, 55.0 equiv.)
through a dry column with MgSO4 and CaCl2 as drying reagents was
dissolved into the reaction mixture. In order not to get isobutene
away, a cooling condenser (-78 °C) was installed and then the
mixture solution was heated to 60 °C. Triethylamine (5.2 mL,
36.9 mmol, 2.20 equiv.) was slowly added by a syringe pump (rate of
addition; 4 mL/h), followed by the stirring for 5 h at 60 °C. The clear
yellow solution was allowed to cool to rt and the solvents was
removed in vacuo. An aqueous saturated solution of NH4Cl and
dichloromethane were added, the aqueous layer was extracted with
dichloromethane three times, combined organic layers were dried
over Na2SO4, filtered and concentrated in vacuo. Purification by flash
column chromatography over silica gel (cyclohexane/ ethyl acetate =
40/1) gave the title compound 25 (3.65 g, 13.2 mmol, 79%) as
Synthesis of (rac)-methyl (E,2E)-2-[(3,4-dimethyl-5-oxo-2H-furan-
2-yl)oxymethylene]-4-(1-isopropenyl-3-oxo-2-
1
yellowish oil. Rf = 0.27 (cyclohexane/ ethyl acetate = 40/1); H NMR
oxaspiro[4.4]nonan-1-yl)but-3-enoate 36. To a solution of vinyl
iodide 31 (25 mg, 0.074 mmol, 1.00 equiv.), tris(2-furyl)phosphine
(6.8 mg, 0.029 mmol, 0.40 equiv.) in 1,4-dioxane (0.8 mL) was added
vinylstannane 34 (55 mg, 0.11 mmol, 1.50 equiv.) under argon
atmosphere. The solution was degassed by bubbling argon through
the solution for 10 min. Pd2dba3 (6.7 mg, 7.4 µmol, 0.10 equiv.) was
added, the reaction vial was sealed and heated to 120 °C for 30 min.
The clear yellow mixture was allowed to cool to rt and was filtered
over Celite (wash with cyclohexane/ ethyl acetate = 2/1, 15 mL). The
solvent was removed in vacuo and the crude was purified by flash
column chromatography (SiO2, cyclohexane/ ethyl acetate = 2/1).
The title compound 36 was obtained as colorless oil (29 mg, 0.069
mmol, 94 %). Rf = 0.17 (cyclohexane/ ethyl acetate = 2/1); Both
(400 MHz, CD2Cl2) δ ppm: 4.98 (s, 1 H), 4.96-4.95 (m, 1 H), 3.03 (d,
J = 17.1 Hz, 1 H), 2.71 (d, J = 17.1 Hz, 1 H), 1.78 (s, 3 H), 1.49 (s, 3
H), 1.19 (s, 3 H), 0.94 (s, 9 H), 0.11 (s, 6 H); 13C NMR (100 MHz,
CD2Cl2) δ ppm: 203.3, 141.1, 113.6, 103.0, 91.4, 72.4, 58.0, 36.1,
26.41, 26.39, 24.7, 20.7, 17.1, -4.4; HRMS: calc. for C17H29OSi
[M+H]+: 277.1987, found: 277.1986; IR (cm-1): 2954, 2929, 2857,
2149, 1787, 1717, 1471, 1463, 1368, 1287, 1249, 1171, 1086, 1007,
826, 812, 775.
Synthesis
of
(rac)-5-[2-[tert-butyl(dimethyl)silyl]ethynyl]-5-
29. Premix
isopropenyl-4,4-dimethyl-tetrahydrofuran-2-one
solution of mCPBA (<70%; 89 mg, 0.36 mmol, 2.00 equiv.) and
tetrabutyl ammonium hydroxide (40% w/w solution in H2O, 275 mg,
14
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10.1002/hlca.202000017
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HELVETICA
0.42 mmol, 2.33 equiv.) was added to a clear yellowish solution of
cyclobutanone 25 (50 mg, 0.18 mmol, 1.00 equiv.) dissolved in
dichloromethane (2.0 mL) under argon at room temperature. After
premix solutions descried above were additionally added to the
reaction solution twice, the resulting mixture was stirred for 1 h at
room temperature. After quenched with 10% Na2S2O3 aqueous
solution, the aqueous layer was extracted with dichloromethane three
times. The combined organic layers were dried over Na2SO4, filtered
and concentrated in vacuo. Purification by flash column
chromatography over silica gel (cyclohexane/ ethyl acetate = 19/1)
gave the title compound 29 (32 mg, 0.11 mmol, 61%) as colorless oil.
Rf = 0.29 (cyclohexane/ ethyl acetate = 19/1); 1H NMR (400 MHz,
CD2Cl2) δ ppm: 5.23 (s, 1 H), 5.05 (s, 1 H), 2.72 (d, J = 16.7 Hz, 1 H),
2.28 (d, J = 16.7 Hz, 1 H), 1.90 (s, 3 H), 1.40 (s, 3 H), 0.98 (s, 3 H),
0.95 (s, 9 H), 0.14 (s, 6 H); 13C NMR (100 MHz, CD2Cl2) δ ppm:
174.9, 140.5, 113.6, 102.7, 93.3, 89.1, 44.8, 44.2, 26.4, 24.7, 23.4,
20.6, 17.0, -4.6; HRMS: calc. for C17H29O2Si [M+H]+: 293.1936,
found: 293.1928; IR (cm-1): 2955, 2930, 2858, 1795, 1471, 1464,
1251, 1232, 1198, 1119, 1099, 1042, 979, 915, 827, 778.
0.041 mmol, 2.5 equiv) and Pd(PPh3)4 (1.0 mg, 0.81 µmol, 0.05
equiv) were added under argon. The mixture was stirred and heated
to 50 °C for 1 h. The solution was allowed to cool to room
temperature and was filtered over Celite, the filter cake was washed
with dichloromethane. The resulting solvent was washed with a 10%
LiCl aqueous solution three times. The organic phase was dried over
anhydrous Na2SO4, filtrated and concentrated. The crude was
purified by flash column chromatography (SiO2, cyclohexane) to give
the corresponding stannane compound 9 (4.7 mg, 0.010 mmol, 61%)
1
as a colorless oil.; H NMR (400 MHz, CD2Cl2) δ ppm: 6.35-6.15 (m,
2 H), 5.08 (s, 1 H), 4.94 (virt. t, J = 1.5 Hz, 1 H), 2.41 (d, J = 16.5 Hz,
1 H), 2.16 (d, J = 16.5 Hz, 1 H), 1.86-1.85 (m, 3 H), 1.54-1.46 (m, 6
H), 1.30 (sext., J = 7.5 Hz, 6 H), 1.25 (s, 3 H), 1.05 (s, 3 H), 0.95-
0.91 (m, 6 H), 0.88 (t, J = 7.3 Hz, 9 H); 13C NMR (100 MHz, CD2Cl2)
δ ppm: 175.4, 144.0, 142.9, 128.0, 111.4, 93.6, 43.5, 42.6, 29.1,
27.2, 24.3, 23.4, 20.6, 13.5, 9.5; HRMS: calc. for C23H43O2Sn [M+H]+:
471.2285, found: 471.2290; IR (cm-1): 2958, 2927, 2872, 2853, 1790,
1465, 1235, 1200, 1090, 1000,989, 905, 667.
Synthesis of (rac)-zealactone 1, C2’-(S) zealactone
1 and
Synthesis
of
(rac)-5-ethynyl-5-isopropenyl-4,4-dimethyl-
zealactone 1a/b.
tetrahydrofuran-2-one 30. To a solution of TBS-protected alkyne 29
(220 mg, 0.75 mmol, 1.00 equiv.) in THF (7.5 mL) was added a
solution of TBAF (1.0 M in THF; 1.5 mL, 1.5 mmol, 2.00 eq) under
argon at room temperature. The mixture was stirred for 30 min at
room temperature. An aqueous saturated solution of NH4Cl and
dichloromethane were added, the aqueous layer was extracted with
dichloromethane three times, combined organic layers were dried
over Na2SO4, filtered and concentrated in vacuo. Purification by flash
column chromatography over silica gel (cyclohexane/ ethyl acetate =
19/1) gave the title compound 30 (125 mg, 0.70 mmol, 93%) as
To a solution of vinyl iodide 6 (28 mg, 0.086 mmol, 1.00 equiv.),
tris(2-furyl)phosphine (8.0 mg, 0.034 mmol, 0.40 equiv.) in 1,4-
dioxane (0.9 mL) was added vinylstannane 9 (101 mg, 0.21 mmol,
2.50 equiv.) under argon atmosphere. The solution was degassed by
bubbling argon through the solution for 10 min. Pd2dba3 (7.9 mg, 8.6
µmol, 0.10 equiv.) was added, the reaction vial was sealed and
heated to 120 °C for 30 min. The clear yellow mixture was allowed to
cool to rt and was filtered over Celite (wash with cyclohexane/ ethyl
acetate = 2/1, 15 mL). The solvent was removed in vacuo and the
crude was purified by flash column chromatography (SiO2,
cyclohexane/ ethyl acetate = 2/1). The corresponding coupling
product was obtained as colorless oil (25.9 mg, 0.069 mmol, 80%).
The coupling with (R)-6 or (S)-6 produced C2’-(R) or C2’-(S)
disastereomers of zealactone 1, respectively. HPLC analyses of
them are described in Supporting Information. Chiral separation of
(6S, 2’R) and (6R, 2’R) of zealactone 1 and CD spectrum of them are
also in Supporting Information. Rf = 0.14 (cyclohexane/ ethyl acetate
= 2/1); Both diastereomers give very similar signals in 1H- and 13C-
1
colorless oil. Rf = 0.15 (cyclohexane/ ethyl acetate = 19/1); H NMR
(400 MHz, CD2Cl2) δ ppm: 5.25 (s, 1 H), 5.08-5.07 (m, 1 H), 2.80 (s,
1 H), 2.74 (d, J = 16.7 Hz, 1 H), 2.30 (d, J = 16.7 Hz, 1 H), 1.90-1.89
(m, 3 H), 1.41 (s, 3 H), 0.99 (m, 3 H); 13C NMR (100 MHz, CD2Cl2) δ
ppm: 174.7, 140.3, 113.9, 88.6, 80.9, 77.7, 44.7, 44.1, 24.5, 23.4,
20.4; HRMS: calc. for C11H15O2 [M+H]+: 179.1072, found: 179.1070;
IR (cm-1): 3263, 2977, 2933, 1788, 1232, 1199, 1120, 1085, 1042,
978, 959, 916, 665.
1
NMR.: H NMR (600 MHz, CD2Cl2) δ ppm: 7.56 (s, 1 H), 6.98 (virt. t,
Synthesis
of
(rac)-5-isopropenyl-4,4-dimethyl-5-[(E)-2-
tributylstannyl vinyl]tetrahydrofuran-2-one 9.
J = 1.6 Hz, 1 H), 6.97 (virt. t, J = 1.6 Hz, 1 H), 6.87 (d, J = 16.1 Hz, 1
H), 6.34 (d, J = 16.1 Hz, 1 H), 6.33 (d, J = 16.1 Hz, 1 H), 6.17-6.16
(m, 1 H), 5.08 (s, 1 H), 5.07 (s, 1 H), 4.94 (s, 1 H), 3.73 (s, 3 H), 2.44
(d, J = 16.8 Hz, 1 H), 2.43 (d, J = 16.8 Hz, 1 H), 2.22 (d, J = 16.8 Hz,
1 H), 2.21 (d, J = 16.8 Hz, 1 H), 1.99-1.98 (m, 3 H), 1.85-1.83 (s, 3
H), 1.24 (s, 3 H), 1.06 (s, 3 H); 13C NMR (150 MHz, CD2Cl2) δ ppm:
175.46, 170.92, 166.86, 153.71, 143.47, 143.43, 141.78,
141.75,136.25,136.21, 132.32, 117.72, 117.64, 112.29, 111.91,
101.10, 101.07, 93.19, 93.17, 52.06, 44.22, 44.21, 43.44, 43.41,
24.73, 24.14, 24.12, 21.19, 11.03, 11.02; The NMR comparison of
(6S, 2’R) and (6R, 2’R) of zealactone 1 are described in Supporting
Protocol A: To a solution of alkyne 30 (120 mg, 0.67 mmol, 1.00
equiv.) in toluene (7 mL), tributylstannane (0.27 mL, 1.00 mmol, 3.0
equiv.) and AIBN (17 bmg, 0.10 mmol, 0.15 equiv.) under argon at
room temperature. The mixture was stirred and heated to 80 °C for
18 h under argon. Purification by flash column chromatography over
silica gel (cyclohexane/ ethyl acetate = 80/1 to 19/1) gave the title
compound 9 (302 mg, 0.64 mmol, 96%) as colorless oil. Rf = 0.24
(cyclohexane/ ethyl acetate = 40/1).
Protocol B: To a solution of vinyl iodide 47 (5.0 mg, 0.016 mmol, 1.0
equiv) in N-methylpyrrolidone (0.15 mL), Bis(tributyltin) (23.8 mg,
15
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10.1002/hlca.202000017
Helvetica Chimica Acta
HELVETICA
Information. HRMS: calc. for C20H25O7 [M+H]+: 377.1600, found:
377.1593; IR (cm-1): 2960, 2929, 1783, 1716, 1611, 1354, 1255,
1237, 1198, 1125, 1093, 1045, 1015, 990, 951.
1772, 1643, 1605, 1466, 1418, 1376, 1291, 1234, 1200, 1123, 1094,
1044, 989, 954, 917, 799, 619, 543.
Soil stability analysis
Synthesis of (rac)-methyl (E,2E)-2-[(3,4-dimethyl-5-oxo-2H-furan-
2-yl)oxymethylene]-4-(2-isopropenyl-3,3-dimethyl-5-oxo-
tetrahydrofuran-2-yl)but-3-enoate 32. To a solution of vinyl iodide
31 (25 mg, 0.074 mmol, 1.00 equiv.), tris(2-furyl)phosphine (6.8 mg,
0.029 mmol, 0.40 equiv.) in 1,4-dioxane (0.75 mL) was added
Soil stability assay was carried out following the procedures as
described in Lumbroso et al., 2016.[49] Briefly, a loam soil (pH in H2O
8, % OM 4.5) was collected from a field near Stein Research Center
in Switzerland and was sieved 0-3 mm. Prior the experiment soil was
mixed with sand in a ratio 1:2 and humidity was adjusted to the 13%
w/w. Soil was pre-incubated for 1 week at 20°C in the dark prior the
chemical treatments. 10 g of dried soil-sand mix was transferred in
50 mL polypropylene tubes and treated with a solution of the test
compounds to reach a final concentration of 3.5 µg ai g-1 dry soil.
Treated samples were incubated in the dark at 20 ± 0.5 ° C 80% RH.
The following incubation timings were considered to study the kinetic
of disappearance of parent compounds: 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8
and 25 hours after treatment. Compound extraction was carried out
vinylstannane
9 (52 mg, 0.11 mmol, 1.50 equiv.) under argon
atmosphere. The solution was degassed by bubbling argon through
the solution for 10 min. Pd2dba3 (6.7 mg, 7.3 µmol, 0.10 equiv.) was
added, the reaction vial was sealed and heated to 120 °C for 30 min.
The clear yellow mixture was allowed to cool to rt and was filtered
over Celite (wash with cyclohexane/ ethyl acetate = 2/1, 15 mL). The
solvent was removed in vacuo and the crude was purified by flash
column chromatography (SiO2, cyclohexane/ ethyl acetate = 2/1).
The title compound 32 was obtained as colorless oil (26 mg, 0.66
mmol, 90 %). Rf = 0.14 (cyclohexane/ ethyl acetate = 2/1); Both
diastereomers give very similar signals in 1H- and 13C-NMR and are
reported as one compound: 1H NMR (400 MHz, CD2Cl2) δ ppm: 7.55
(s, 1 H), 6.86 (dd, J = 2.0, 16.0 Hz, 1H), 6.36 (dd, J = 5.0, 16.0 Hz,
1H), 5.97 (br s, 1H), 5.08-5.03 (m, 1H), 4.95 (br s, 1H), 3.72 (s, 3H),
2.35 (s, 2H), 2.00 (d, J = 0.7 Hz, 3H), 1.92-1.87 (m, 1H), 1.86 (s, 3H),
1.82 (s, 3H), 1.77-1.49 (m, 7H); 13C NMR (100 MHz, CD2Cl2) δ ppm:
175.45, 175.43, 171.3, 166.92, 166.89, 153.74, 153.69, 153.33,
153.26, 143.39, 143.35, 132.24, 132.21, 128.63, 128.58, 117.88,
117.84, 112.34, 112.29, 112.0, 102.8, 102.6, 93.1, 92.5, 52.1, 44.2,
24.7, 24.1, 24.0, 21.19, 21.16, 17.74, 11.71, 8.9; HRMS: calc. for
C21H27O7 [M+H]+: 391.1757, found: 391.1752; IR (cm-1): 2961, 2927,
1779, 1716, 1641, 1610, 1437, 1358, 1254, 1237, 1196, 1122, 1085,
1044, 982, 748.
by using Acetonitrile 30 mL and shake for
1 hour at room
temperature using a vertical mechanical shaker. Each timing point for
each compounds were tested in triplicate. Analyses were carried out
by using an UPLC-MS/MS (Waters Acquity UPLC-MS/MS I-Class
System). A C18 UPLC column (ACQUITY UPLC® column HSS T3
2.1ꢀ×ꢀ30ꢀmm, 1.8ꢀµm particle size; Waters) was used.
The mobile phase was as follows: H2O–MeOH (95/5)ꢀ+ꢀ0.1% HCOOH
(solvent A) and MeCNꢀ+ꢀ0.1% HCOOH (solvent B); 20–100% of
solvent B for 1.2ꢀmin, then 100% of solvent B for 0.20ꢀmin and then
down to 20% solvent
B up to 1.5ꢀmin; average flow rate
0.75ꢀmLꢀmin−1. The injection volume was 2 µl. A tested selective ion
recording method (SIR) depending on molecular mass of each
compound was set up by MS detector. The level of the compound
was determined by comparing chromatogram area counts of sample
extracts versus area counts of the standard solution of the
corresponding compound. The half-lives were calculated by an
exponential regression analysis (single first-order kinetic model)
plotting the percentage of recovered compound in soil against time.
Yoshimulactone green (YLG) displacement assay
(E)-5-(2-iodovinyl)-4,4-dimethyl-5-(prop-1-en-2-yl)dihydrofuran-
2(3H)-one 47. To a solution of stannane 9 (250 g, 0.53 mmol, 1.0
equiv) in dichloromethane (10 mL), iodine (150 mg, 0.59 mmol, 1.1
equiv) was added. The mixture was stirred at room temperature for 5
min. The reaction mixture was diluted with dichloromethane and
water, and the layers were separated. The aqueous layer was
extracted with dichloromethane three times, and the combined
organic layers were dried over anhydrous Na2SO4, filtrated and
concentrated. The crude was purified by flash column
chromatography (SiO2, cyclohexane/ethyl acetate = 3/1) to give the
title compound (120 mg, 0.39 mmol, 74%) as a clear oil; Rf = 0.60
(cyclohexane/ ethyl acetate = 3/1): 1H NMR (400MHz, CDCl3) δ ppm
6.86 (d, J = 14.3 Hz, 1H), 6.92-6.80 (m, 1H), 6.48 (d, J = 14.7 Hz,
1H), 5.12 (s, 1H), 5.00 (s, 1H), 2.46 (d, J = 16.0 Hz, 1H), 2.29 (d, J =
16.0 Hz, 1H, 1.81 - 1.74 (m, 1H), 1.70 (s, 3H), 1.85 (s, 3H), 1.27 (s,
3H), 1.07 (s, 3H); 13C NMR (100MHz, CDCl3) δ ppm 174.7, 143.0,
141.2, 113.1, 94.6, 77.5, 43.5, 43.0, 24.4, 23.84, 20.8; HRMS calcd
C11H15O2I [M+H]+ 307.01950, found 307.01911. IR (cm-1): 2971, 1789,
Yoshimulactone Green (YLG) is a fluorogenic agonist for the
strigolactone receptor D14 and was used in competition assays to
assess the binding affinity of test compounds as previously described
with minor modifications. ZmD14 (1 µg) was incubated in the
presence and absence of test compounds in buffer (PBS, 0.1% BSA)
for 5 min at room temperature followed by the addition of YLG to 1
µM. The final reaction volume was 150 µL and all wells contained 1%
DMSO. The extent of YLG hydrolysis by D14 was assessed by
measuring the fluorescence intensity (excitation: 480 nm, emission:
520 nm) in a Mithras LB 940 plate reader (Berthold Technologies) at
a single time-point during the linear phase of the reaction. The half-
maximal inhibitory concentration (IC50) values were determined from
the normalized fluorescence intensities relative to DMSO controls for
test compounds over an 8-point dilution curve (30 µM top
concentration, 3-fold dilution).
16
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10.1002/hlca.202000017
Helvetica Chimica Acta
HELVETICA
ALPHA screen luminescence proximity assay
The effect of compounds was evaluated on the germination of
untreated corn seeds under cold stress as follows. Corn NK Falkone
seeds were supplied by Syngenta Seeds SAS (Saint-sauveur
France) with Batch 12345408. Seeds were placed in 24 well plates
(each plate was considered as one experimental unit or replicate).
Germination was initiated by the addition of 250 µL for corn seeds, of
DMSO/water (0.5% (v/v)) for the check and solution at 2, 10, 50 and
250 µM of each molecules. 8 replicates (ie, 8 plates) were used for
each treatment characterization. Plates were sealed using seal foil
(Polyolefin Art. Nr. 900360) from HJ-BIOANALYTIK. All plates were
placed horizontally on trolleys in a climatic chamber at 15 °C. The
experiment was laid out in a completely randomized design with 75%
relative humidity under complete darkness. Foils were pierced, one
hole per well using syringes after 72 hours. Germination was
followed over time by taking photographs at different time points
using an EOS Canon 70D camera. Image analysis was performed
automatically with a macro which was developed using the Image J
software. A dynamic analysis of germination was carried out by fitting
a logistic curve. Three parameters were calculated from the logistic
curve: T50; the slope and the plateau. All the values are expressed as
percentages compared to an untreated control. Data in bold indicate
statistically significant differences between treated seeds and
untreated control (p < 0.05).
AlphaScreen technology (Amplified Luminescent Proximity
Homogenous Assay) was used to assess the ability of compounds to
promote interaction between ZmD14 and ZmMAX2. ZmD14 (75 nM)
was incubated in assay buffer (PBS, 0.1% BSA) in the presence and
absence of test compound (5 µM) for 5 min at room temperature
followed by the addition of ZmMAX2 (75 nM) and Triton X100 to
0.02% (w/v). After 15 min incubation at room temperature, 416
µg/mL Anti-Maltose-Binding Protein acceptor beads in assay buffer
were then added followed 5 min later by 416 µg/mL Nickel Chelate
Donor beads. Plates were incubated for 1 h at room temperature
protected from light and then measured in a Mithras LB 940 plate
reader (Berthold Technologies) equipped with an AlphaScreen
module for laser excitation at 680nm.
Differential Scanning Fluorimetry (DSF)
DSF was performed using a Bio-Rad CFX96 qPCR machine with
SyproOrange as the reporter dye. 2 µg of purified ZmD14 was
combined with 1 µL of test compound in DMSO and SyproOrange
dye at 5x final concentration) in PBS to final volume of 25 µL. After 1
min incubation at 20 °C samples were heat denatured using a linear
20 – 96 °C gradient, at a rate of 1 °C per min. Compounds were
tested in duplicate at a concentration of 50 µM (5% DMSO final
concentration). Melting temperatures were determined from the peak
of the first derivatives of the melting curves.
Dark induced corn leaf senescence assay
Supplementary Material
The effect of compounds was evaluated for their ability to promote
senescence of untreated corn leaves as follows. Corn Multitop seeds
were supplied by Syngenta Seeds SAS (Saint-sauveur France) with
Batch 11876872. Plants were raised up to V6 stage following,[50] in
greenhouse at 25/22 °C (day/night), humidity 80%, light intensity
between 100-150 µmol.m-2.s-1, photoperiod 14 h day/ 10 h dark in
pots. Compounds were solubilized in DMSO/water (0.5% (v/v)).
Compounds were tested at 100, 10, 1, 0.1, 0.01, 0.001 µM and 1 mL
of each solution was distributed into 24-well microtiterplates. 12
replicates were used for each treatment characterization. 10 mm leaf
discs were cut from the 6th leaf and placed axial side on the surface
of each well. Plates were sealed using seal foil (Polyolefin Art. Nr.
900360) from HJ-BIOANALYTIK. Foils were immediately pierced,
one hole per well using syringe needles. All plates were placed
horizontally on trolleys in a climatic chamber and placed in the dark
at 22°C with 80% relative humidity. Senescence was followed over
time by taking photographs at different time points using an EOS
Canon 70D camera Plates. Image analysis was performed
automatically with a macro which was developed using Image J
software. Changes in the grey scale pixel intensity were analyzed
and transformed to data points corresponding the senescence
development in leaf tissue. A dynamic analysis of senescence was
carried out by fitting a logistic curve. S50 parameter was calculated
from the logistic curve.
Spectral data and NMR spectra for all new compounds; HPLC
analysis for rac-zealactone, zealactone 1a/b, (S) C2’ diastereomers
of zealactone 1, rac-35, (R) C2’ diastereomers of 35, 47; CD studies
for zealactone 1a, zealactone 1b, (R)-/(S)-9, (R)-/(S)-47 (PDF).
Acknowledgements
We thank Prof. Jeffrey W. Bode for fruitful discussions and for
hosting Dr. Masahiko Yoshimura in his laboratory. We acknowledge
Prof. Christof Sparr, Mr. Frederic Bourgeois (Basel University) for CD
measurements and Dr. Daniel Emery for simulation of CD spectra.
We would like to thank Ms. Kristin Hoefer, Ms. Miesja. Yogo and Mr.
Gabriel Schmied for their support for the soil stability studies. Dr.
David Brocklehurst and Pauline Heinimann are acknowledged for
assistance with enzymatic assays and Maud Simonin for corn
germination assays. We further thank Dr. Peter Renold, Mr.
Alexander Schuschkowski and Ms. Juliane Vogt for HPLC
separations and measurement of HRMS spectra. Mr. Roman Staiger,
Dr. Dylan Dagoneau, Dr. Amandine Kolleth Krieger, Dr. Mathilde
Lachia and Dr. Alexandre Lumbroso are acknowledged for the fruitful
discussions. We thank also Prof. Harro Bouwmeester (Amsterdam
University) for the initial development of the leaf senescence assay
and for highly valuable collaboration.
Corn seed germination and root growth assays
17
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10.1002/hlca.202000017
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HELVETICA
[16]
[17]
D. A. Jackson, M. Rey, A. Dreiding, ‘2-Vinylcyclobutanones by
Cycloaddition of Vinylketenes to Simple Olefins’, Helv. Chim. Acta
1983, 66, 2330-2341.
Author Contribution Statement
A. J. Oosthoek-De Vries, P. J. Nieuwland, J. Bart, K. Koch, J. W. G.
Janssen, P. J. M.Van Bentum, F. P. J. T. Rutjes, H. J. G. E.Gardeniers,
A. P. M. Kentgens, ‘Inline Reaction Monitoring of Amine-Catalyzed
Acetylation of Benzyl Alcohol Using a Microfluidic Stripline Nuclear
Magnetic Resonance Setup’, J. Am. Chem. Soc. 2019, 141, 5369–
5380.
M. Y. and M. D. contributed equally to the synthesis of zealactone
1a/b and its derivatives. The project and experiments were designed
by M. Y., M. D., P.-Y. D., R. F.-P., C. S., P. Q., S. C. and A. De M.
The experimental work was performed by M. Y., M. D., P.-Y. D., C.
S., K. H, S. R., P. Q., B. H. All authors contributed to the manuscript
writing and review process.
[18]
Gaussian 16, Revision A.03, M. J. Frisch, G. W. Trucks, H. B. Schlegel,
G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone,
G. A. Petersson, H. Nakatsuji, X. Li, M. Caricato, A. V. Marenich, J.
Bloino, B. G. Janesko, R. Gomperts, B. Mennucci, H. P. Hratchian, J.
V. Ortiz, A. F. Izmaylov, J. L. Sonnenberg, D. Williams-Young, F. Ding,
F. Lipparini, F. Egidi, J. Goings, B. Peng, A. Petrone, T. Henderson, D.
Ranasinghe, V. G. Zakrzewski, J. Gao, N. Rega, G. Zheng, W. Liang,
M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T.
Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, K. Throssell, J. A.
Montgomery Jr., J. E. Peralta, F. Ogliaro, M. J. Bearpark, J. J. Heyd, E.
N. Brothers, K. N. Kudin, V. N. Staroverov, T. A. Keith, R. Kobayashi,
J. Normand, K. Raghavachari, A. P. Rendell, J. C. Burant, S. S.
Iyengar, J. Tomasi, M. Cossi, J. M. Millam, M. Klene, C. Adamo, R.
Cammi, J. W. Ochterski, R. L. Martin, K. Morokuma, O. Farkas, J. B.
Foresman, D. J. Fox, Gaussian, Inc., Wallingford CT, 2016.
J-D. Chai, M. Head-Gordon, ‘Long-range corrected hybrid density
functionals with damped atom-atom dispersion corrections’, Phys. Chem.
Chem. Phys. 2008, 10, 6615–6620.
References
[1]
[2]
[3]
S. Al-Babili, H. J. Bouwmeester, ‘Strigolactones, a novel carotenoid-
derived plant hormone’, Annu. Rev. Plant Biol. 2015, 66, 161-186.
B. Zwanenburg, D. Blanco-Ania ‘Strigolactones: new plant hormones in
the spotlight’, J. Exp. Bot. 2018, 69, 2205-2218.
C. De Cuyper, S. Struk, L. Bream, K. Gevaert, G. De Jaeger, S.
Goormachtig, ‘Strigolactones, karrikins and beyond’, Plant Cell Environ.
2017, 40, 1691-1703.
[4]
[5]
H. J. Bouwmeester, R. Fonne-Pfister, C. Screpanti, A. De Mesmaeker,
‘Strigolactones. A new plant hormone with promising features’, Angew.
Chem. Int. Ed. 2019, 55, 12778-12786.
C. Screpanti, R. Fonné-Pfister, A. Lumbroso, S. Rendine, M. Lachia, A.
De Mesmaeker, ‘Strigolactone derivatives for potential crop
enhancement applications’, Bioorg. Med. Chem. Let., 2016, 26, 2392-
2400.
[19]
[20]
[21]
J. Tomasi, B. Mennucci, R. Cammi, ‘Quantum mechanical continuum
solvation models’, Chem. Rev. 2005, 105, 2999–3093.
[6]
A. De Mesmaeker, C. Screpanti, R. Fonné-Pfister, M. Lachia, A.
Lumbroso, H. J. Bouwmeester, ‘Design, synthesis and biological
evaluation of strigolactone and strigolactam derivatives for potential
crop enhancement applications in modern agriculture’, Chimia 2019,
73, 549-560.
B. Mennucci, E. Cancès, J. Tomasi, ‘Evaluation of Solvent Effects in
Isotropic and Anisotropic Dielectrics and in Ionic Solutions with a
Unified Integral Equation Method: Theo- retical Bases, Computational
Implementation, and Numerical Applications’, J. Phys. Chem. B 1997,
101, 10506– 10517.
[7]
M. G. Mostofa, W. Li, K. H. Nguyen, M. Fujita, L. S. P. Tran,
‘Strigolactones in plant adaptation to abiotic stresses: An emerging
avenue of plant research’, Plant Cell Environ. 2018, 41, 2227-2243.
Y. Wang, H. J. Bouwmeester, ‘Structural diversity in the strigolactones’,
J. Exp. Bot. 2018, 69, 2219–2230.
[22]
[23]
[24]
[25]
[26]
[27]
J. Tomasi, B. Mennucci, E. Cancès, ‘The IEF version of the PCM
solvation method: an overview of a new method addressed to study
molecular solutes at the QM ab initio level’, J. Mol. Struct. 1999, 464,
211–226.
[8]
[9]
T. V. Charnikhova, K. Gaus, A. Lumbroso, M. Sanders, J-P. Vincken, A.
De Mesmaeker, C. P. Ruyter-Spira, C. Screpanti, H. J. Bouwmeester,
‘Zealactones. Novel natural strigolactones from maize’, Phytochemistry
2017,137, 123–131.
A. Kolleth, A. Lumbroso, G. Tanriver, S. Catak, S. Sulzer-Mossé, A. De
Mesmaeker,
‘Synthesis
of
Amino-Cyclobutanes
via
[2+2]
Cycloadditions Involving Keteniminium Intermediates’, Tetrahedron
Lett. 2016, 57 , 2697–2702.
[10]
L. Ravazzolo, S. Trevisan, A. Manoli, S. Boutet-Mercey, F. Perreau, S.
Quaggiotti ‘The control of zealactone biosynthesis and exudation is
involved in the response to nitrogen in maize root’, Plant Cell Physiol.,
2019, 60, 2100-2112.
S. Zarrabi, N. O. Mahmoodi, K. Tabatabaeian, M. A. Zanjanchi,
‘Baeyer–Villiger oxidation of cyclic ketones utilizing potassium
peroxydisulfate (K2S2O8) or sodium perborate (NaBO3) in acidic media’,
Chin. Chem. Lett. 2009, 20, 1400-1404.
[11]
[12]
M. C. Dieckmann, P. Y. Dakas, A. De Mesmaeker, ‘Synthetic Access to
Noncanonical Strigolactones: Syntheses of Carlactonic Acid and Methyl
Carlactonate’, J. Org. Chem. 2018, 83, 125–135.
M. Uyanik, D. Nakashima, K. Ishihara, ‘Baeyer-Villiger oxidation and
oxidative cascade reactions with aqueous hydrogen peroxide catalyzed
by lipophilic Li[B(C6F5)4] and Ca[B(C6F5)4]2’, Angew, Chem. Int. Ed.
2012, 51, 9093-9096.
M. Yoshimura, R. Fonné-Pfister, C. Screpanti, K. Hermann, S. Rendine,
M. Dieckmann, P. Quinodoz, A. De Mesmaeker, ‘Total synthesis and
biological evaluation of heliolactone’, Helv. Chim. Acta 2019, 102,
e1900211.
T. Arite, M. Umehara, S. Ishikawa, A. Hanada, M. Maekawa, S.
Yamaguchi, J. Kyozuka, ‘d14, a strigolactone-insensitive mutant of rice,
shows an accelerated outgrowth of tillers’, Plant Cell Physiol. 2009, 50,
1416−1424.
[13]
M. Lloveras, I. Ramos, E. Molins, A. Messequer, Tetrahedron, 2000, 56,
3391-3397.
D. C. Nelson, A. Scaffidi, E. A. Dun, M. T. Waters, G. R. Flematti, K. W.
Dixon, C. A. Beveridge, E. L. Ghisalberti, S. M. Smith, ‘F-box protein
MAX2 has dual roles in karrikin and strigolactone signaling in
[14]
[15]
D. Guillerm, G. Linstrumelle, Tetrahedron. Lett. 1985, 26, 3811-3812.
M. Alami, G. Linstrumelle, Tetrahedron. Lett. 1991, 43, 6109-6112.
18
This article is protected by copyright. All rights reserved.

10.1002/hlca.202000017
Helvetica Chimica Acta
HELVETICA
Arabidopsis thaliana’, Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 8897−
[39]
K. S. V. Jagadish, P. B. Kavi Kishor, R. N. Bahuguna, N. von Wirén, N.
Sreenivasulu, ‘Staying alive or going to die during terminal senescence
— An enigma surrounding yield stability.’ Front. Plant Sci. 2015, 6, 1–
14.
8902.
[28]
C. Hamiaux, R. S. M. Drummond, B. J. Janssen, S. E. Ledger, J. M.
Cooney, R. D. Newcomb, K. C. Snowden, ‘DAD2 is an α/β hydrolase
likely to be involved in the perception of the plant branching hormone,
strigolactone’, Curr. Biol. 2012, 22, 2032−2036.
[40]
[41]
Y. Yamada, M. Umehara, ‘Possible roles of strigolactones during leaf
senescence’, Plants 2015 4, 664–677.
Y. Yamada, S. Furusawa, S. Nagasaka, K. Shimomura, S. Yamaguchi,
M. Umehara, ‘Strigolactone signaling regulates rice leaf senescence in
response to a phosphate deficiency’, Planta 2014 240, 399-408.
H. Ueda, M. Kusaba ‘Strigolactone regulate leaf senescence in concert
with ethylene in arabidopsis’, Plant Physiol. 2015 169, 138-147.
J. M. Bradow, W. J. Connick, A. B. Pepperman, ‘Comparison of the
seed germination effects of synthetic analogs of strigol, gibberellic acid,
cytokinins, and other plant growth regulators’, J Plant Growth Regul
1988 7, 227-239.
[29]
[30]
R. Yao, Z. Ming, L. Yan, S. Li, F. Wang, S. Ma, C. Yu, M. Yang, L. Chen, L.
Chen, et al. ‘DWARF14 is
a non-canonical hormone receptor for
strigolactone’, Nature 2016, 536, 469−473.
[42]
[43]
A. de Saint Germain, G. Clavé, M.-A. Badet-Denisot, J.-P. Pillot, D.
Cornu, J.-P. Le Caer, M. Burger, F. Pelissier, P. Retailleau, C. Turnbull,
et al. ‘An histidine covalent receptor and butenolide complex mediates
strigolactone perception’, Nat. Chem. Biol. 2016, 12, 787−794.
[31]
[32]
[33]
F. Zhou, Q. Lin, L. Zhu, Y. Ren, K. Zhou, N. Shabek, F. Wu, H. Mao, W.
Dong, L. Gan, et al. ‘D14-SCFD3-dependent degradation of D53 regulates
strigolactone signalling’, Nature 2013, 504, 406−410.
[44]
[45]
C. E. Cook, L. P. Whichard, B. Turner, M. E. Wall, G. H. E.
‘Germination of witchweed (Striga lutea Lour.): Isolation and properties
of a potent stimulant’, Science 1966,154, 1189–1190.
L. Jiang, X. Liu, G. Xiong, H. Liu, F. Chen, L. Wang, X. Meng, G. Liu, H.
Yu, Y. Yuan, et al. ‘DWARF 53 acts as a repressor of strigolactone
signalling in rice’, Nature 2013, 504, 401−405.
J. M. Bradow, W. J. Connick, A. B. Pepperman, L. H. Wartelle,
‘Germination stimulation in wild oats (Avena fayua L.) by synthetic
strigol analogs and gibberellic acid’, J Plant Growth Regul 1990, 9, 35-
41.
L. Wang, B. Wang, L. Jiang, X. Liu, X. Li, Z. Lu, X. Meng, Y. Wang, S.
M. Smith, J. Li, ‘Strigolactone signaling in Arabidopsis regulates shoot
development by targeting D53-Like SMXL repressor proteins for
ubiquitination and degradation’, Plant Cell 2015, 27, 3128−3142.
Y. Tsuchiya, M. Yoshimura, Y. Sato, K. Kuwata, S. Toh, D. Holbrook-
Smith, H. Zhang, P. McCourt, K. Itami, T. Kinoshita, et al. ‘Probing
strigolactone receptors in Striga hermonthica with fluorescence’,
Science 2015, 349, 864−868.
[46]
[47]
E. Villedieu-Percheron, M. Lachia, P. M. J. Jung, C. Screpanti, R.
Fonné-Pfister, S. Wendeborn, D. Zurwerra, A. De Mesmaeker,
‘Chemicals inducing seed germination and early seedling development’,
Chimia 2014, 68, 654-663.
[34]
[35]
[36]
M. Lachia, R. Fonné-Pfister, C. Screpanti, S. Rendine, P. Renold, D.
Witmer, A. Lumbroso, E. Godineau, D. Hueber, A. De Mesmaeker,
‘New and scalable access to karrikin (KAR1) and evaluation of its
potential application on corn germination’, Helv. Chem. Acta. 2018, 101,
e201800081.
M. Yoshimura, A. Sato, K. Kuwata, Y. Inukai, T. Kinoshita, K. Itami, Y.
Tsuchiya, S. Hagihara, ‘Discovery of shoot branching regulator
targeting strigolactone receptor DWARF14’, ACS Cent. Sci. 2018, 4,
230−234.
[48]
A. De Mesmaeker, A. Lumbroso, C. Screpanti, K. Hermann, M. Lachia,
R. Fonné-Pfister, ‘Design, synthesis and biological evaluation of
strigolactones derivatives for Crop Enhancement’ in Recent Highlights
in the Discovery and Optimization of Crop Protection Products, Elsevier
book, 2020, in press.
L. H. Zhao, X. E. Zhou, W. Yi, Z. Wu, Y. Liu, Y. Kang, L. Hou, P. W. de
Waal, S. Li, Y. Jiang, et al. ‘Destabilization of strigolactone receptor
DWARF14 by binding of ligand and E3-ligase signaling effector
DWARF3’, Cell Research 2015, 25, 1219-1236.
[49]
A. Lumbroso, E. Villedieu-Percheron, D. Zurwerra, C. Screpanti, M.
Lachia, P. Y. Dakas, L. Castelli, V. Paul, H. C. Wolf, D. Sayer, A. Beck,
S. Rendine, R. Fonné-Pfister, A. De Mesmaeker, ‘Simplified
strigolactams as potent analogues of strigolactones for the seed
germination induction of Orobanche cumana Wallr’, Pest. Manag.
Sci. 2016, 72, 2054-2068.
[37]
[38]
A. Sali, T. L. Blundell, ‘Comparative protein modelling by satisfaction of
spatial restraints’, J. Mol. Biol. 1993, 234, 779-815.
CCDC, version 5.2, Development and Validation of a Genetic Algorithm
for Flexible Docking; G. Jones, P. Willett, R. C. Glen, A. R. Leach, R.
Taylor, ‘Development and validation of a genetic algorithm for flexible
docking’, J. Mol. Biol.1997, 267, 727-748.
[50]
‘Growth stages of mono- and dicotyledonous plants: BBCH Monograph'
Ed. U. Meier, Julius Kühn-Institute (JKI), Quedlinburg 2018.
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