Carlactone-type strigolactones and their synthetic analogues as inducers of hyphal branching in arbuscular mycorrhizal fungi

Article abstract of DOI:10.1016/j.phytochem.2016.05.012

Hyphal branching in the vicinity of host roots is a host recognition response of arbuscular mycorrhizal fungi. This morphological event is elicited by strigolactones. Strigolactones are carotenoid-derived terpenoids that are synthesized from carlactone and its oxidized derivatives. To test the possibility that carlactone and its oxidized derivatives might act as host-derived precolonization signals in arbuscular mycorrhizal symbiosis, carlactone, carlactonoic acid, and methyl carlactonoate as well as monohydroxycarlactones, 4-, 18-, and 19-hydroxycarlactones, were synthesized chemically and evaluated for hyphal branching-inducing activity in germinating spores of the arbuscular mycorrhizal fungus Gigaspora margarita. Hyphal branching activity was found to correlate with the degree of oxidation at C-19 methyl. Carlactone was only weakly active (100?ng/disc), whereas carlactonoic acid showed comparable activity to the natural canonical strigolactones such as strigol and sorgomol (100?pg/disc). Hydroxylation at either C-4 or C-18 did not significantly affect the activity. A series of carlactone analogues, named AD ester and AA'D diester, was synthesized by reacting formyl Meldrum's acid with benzyl, cyclohexylmethyl, and cyclogeranyl alcohols (the A-ring part), followed by coupling of the potassium enolates of the resulting formylacetic esters with the D-ring butenolide. AD ester analogues exhibited moderate activity (1?ng–100?pg/disc), while AA'D diester analogues having cyclohexylmethyl and cyclogeranyl groups were highly active on the AM fungus (10?pg/disc). These results indicate that the oxidation of methyl to carboxyl at C-19 in carlactone is a prerequisite but BC-ring formation is not essential to show hyphal branching activity comparable to that of canonical strigolactones.

Full text of DOI:10.1016/j.phytochem.2016.05.012

Phytochemistry xxx (2016) 1e9
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Carlactone-type strigolactones and their synthetic analogues as
inducers of hyphal branching in arbuscular mycorrhizal fungi
Narumi Mori, Kenta Nishiuma, Takuya Sugiyama, Hideo Hayashi, Kohki Akiyama*
Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Hyphal branching in the vicinity of host roots is a host recognition response of arbuscular mycorrhizal
fungi. This morphological event is elicited by strigolactones. Strigolactones are carotenoid-derived ter-
penoids that are synthesized from carlactone and its oxidized derivatives. To test the possibility that
carlactone and its oxidized derivatives might act as host-derived precolonization signals in arbuscular
mycorrhizal symbiosis, carlactone, carlactonoic acid, and methyl carlactonoate as well as mono-
hydroxycarlactones, 4-, 18-, and 19-hydroxycarlactones, were synthesized chemically and evaluated for
hyphal branching-inducing activity in germinating spores of the arbuscular mycorrhizal fungus Gigaspora
margarita. Hyphal branching activity was found to correlate with the degree of oxidation at C-19 methyl.
Carlactone was only weakly active (100 ng/disc), whereas carlactonoic acid showed comparable activity
to the natural canonical strigolactones such as strigol and sorgomol (100 pg/disc). Hydroxylation at either
C-4 or C-18 did not significantly affect the activity. A series of carlactone analogues, named AD ester and
AA’D diester, was synthesized by reacting formyl Meldrum’s acid with benzyl, cyclohexylmethyl, and
cyclogeranyl alcohols (the A-ring part), followed by coupling of the potassium enolates of the resulting
formylacetic esters with the D-ring butenolide. AD ester analogues exhibited moderate activity (1 ng
e100 pg/disc), while AA’D diester analogues having cyclohexylmethyl and cyclogeranyl groups were
highly active on the AM fungus (10 pg/disc). These results indicate that the oxidation of methyl to
carboxyl at C-19 in carlactone is a prerequisite but BC-ring formation is not essential to show hyphal
branching activity comparable to that of canonical strigolactones.
Received 28 March 2016
Received in revised form
23 May 2016
Accepted 27 May 2016
Available online xxx
Keywords:
Arbuscular mycorrhizal symbiosis
Glomeromycota
Gigaspora margarita
Hyphal branching
Strigolactone
Carlactone
Carlactonoic acid
Synthetic analogue
© 2016 Elsevier Ltd. All rights reserved.
1. Introduction
and morphological responses in AM fungi before physical interac-
tion (Akiyama et al., 2005; Besserer et al., 2006). Originally iden-
Arbuscular mycorrhizal (AM) fungi belonging to the phylum
Glomeromycota form symbiotic association with the roots of more
than 80% of land plants (Smith and Read, 2008). AM fungi are
ancient, obligate symbionts that rely on carbon provided by their
hosts to complete their life cycle and, in return, supply inorganic
nutrients, especially phosphate, to the host plants. Root coloniza-
tion by AM fungi requires mutual recognition of diffusible signals
released from the two partners during preinfection stages (Gutjahr
and Parniske, 2013). Strigolactones (SLs) exuded from plant roots
are host-derived precolonization signals that elicit physiological
tified as seed germination stimulants of root parasitic weeds (Cook
et al., 1966), SLs have now been shown to be a class of plant hor-
mones regulating several developmental processes that adapt plant
architecture to nutrient availability (Gomez-Roldan et al., 2008;
Umehara et al., 2008; Xie et al., 2010). Canonical SLs consists of a
methylbutenolide ring (D ring) connected to a tricyclic lactone (ABC
ring) via an enol ether bond (Fig. 1) (Al-Babili and Bouwmeester,
2015). To date, at least 22 naturally occurring canonical SLs have
been characterized from root exudates of various plant species, and
a number of structural analogues have been synthesized (Tokunaga
et al., 2015; Lopez-Obando et al., 2015). An extensive structure-
activity relationship study of SLs for induction of hyphal branch-
ing in AM fungi has demonstrated that the C-D part and the intact
ABC ring are essential for strong hyphal branching activity
(Akiyama et al., 2010).
* Corresponding author.
E-mail addresses: sv201042@edu.osakafu-u.ac.jp (N. Mori), mt201042@edu.
osakafu-u.ac.jp (K. Nishiuma), ss201019@edu.osakafu-u.ac.jp (T. Sugiyama),
hayashi@biochem.osakafu-u.ac.jp (H. Hayashi), akiyama@biochem.osakafu-u.ac.jp
(K. Akiyama).
Canonical SLs are biosynthesized from carotenoids via
http://dx.doi.org/10.1016/j.phytochem.2016.05.012
0031-9422/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Mori, N., et al., Carlactone-type strigolactones and their synthetic analogues as inducers of hyphal branching in
arbuscular mycorrhizal fungi, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.05.012

2
N. Mori et al. / Phytochemistry xxx (2016) 1e9
R3
O
1
O
C
R19
16
17
6
O
O
2
8
1
A
4
7
8b
8a
8
3
2
10
7
6
9
A
A
B
3a
O 11
12
O
D
3
O 14
4a
5
18
4
2'
O
5
O
D
D
O
R1 R2
O
13
15
4: R1=OH, R2=H, R3=H
5: R1=H, R2=OH, R3=H
6: R1=H, R2=H, R3=OH
1: R=CH3
2: R=COOH
3: R=COOMe
Canonical strigolactone
O
O
O
O
O
O
R2
O
O
O
O
O
O
R
R1
O
O
O
12
7: R=H
9: R1=H, R2=H
8: R=OH
10: R1=OH, R2=H
11: R1=H, R2=OH
COOMe
H
O
O
O
HO
O
O
O
O
O
O
14
O
13
Fig. 1. Structures of canonical and carlactone-type strigolactones. Carlactone (1); carlactonoic acid (2); methyl carlactonoate (3); 4-hydroxycarlactone (4); 18-hydroxycarlactone (5);
19-hydroxycarlactone (6); (ꢀ)-4-deoxyorobanchol (7); (ꢀ)-orobanchol (8); (þ)-5-deoxystrigol (9); (þ)-strigol (10); (þ)-sorgomol (11); (þ)-GR24 (12); avenaol (13); heliolactone
(14).
carlactone (CL, 1) (Fig. 1) (Al-Babili and Bouwmeester, 2015). CL (1),
a SL that contains the A and D rings and the enol ether bridge but
lacks the B and C rings was first identified as a product of three SL
None of the known canonical SLs was detected in the root exudates
of the both plants, suggesting that avenaol and heliolactone are
involved in rhizosphere communication with AM fungi. So far,
however, CL-type SLs have not been examined for hyphal
branching-inducing activity in AM fungi.
To test the possibility that CL-type SLs might act as host-derived
precolonization signals in AM symbiosis, natural CL (1), CLA (2), and
MeCLA (3), and monohydroxy-CLs, 4-, 18-, and 19-hydroxy-CLs (4,
5, 6) as well as synthetic CL analogues (32e37) were chemically
synthesized, and tested for hyphal branching-inducing activity in
germinating spores of the AM fungus Gigaspora margarita in this
study. It was found that the oxidation of methyl to carboxyl at C-19
in CL is a prerequisite, but BC-ring formation is not essential for
strong hyphal branching activity on the AM fungus.
biosynthetic enzymes, a b-carotene isomerase DWARF27, CAROT-
ENOID CLEAVAGE DIOXYGENASE 7 (CCD7), and CCD8, in vitro
(Alder et al., 2012), and was later shown to be an endogenous
biosynthetic precursor for canonical SLs (Seto et al., 2014). In Ara-
bidopsis, CL undergoes three step oxidation at C-19 by the cytosolic
cytochrome P450 MAX1 to carlactonoic acid (CLA, 2), which is
further converted to methyl carlactonoate (MeCLA, 3) which had
been tentatively identified as SL-LIKE 1 (Seto et al., 2014; Abe et al.,
2014). CL (1) and CLA (2) have also been identified in the roots of
rice, where the both compounds are converted to the canonical SLs,
4-deoxyorobanchol (7) and orobanchol (8) (Seto et al., 2014; Abe
et al., 2014).
According to the proposal by Al-Babili and Bouwmeester (2015),
CL-type SLs can be defined as SLs with a CL-type structure lacking
the canonical tricyclic ABC ring system. Three CL-type SLs, CL (1),
CLA (2), and MeCLA (3), although less active than a synthetic SL
analogue, GR24 (12), induce seed germination of the root parasitic
weeds Striga hermonthica and Orobanche minor in a concentration-
dependent manner (Alder et al., 2012; Abe et al., 2014). Two other
CL-type SLs, avenaol (13) and heliolactone (14), have been isolated
as a germination stimulant from root exudates of black oat (Avena
strigosa Schreb.) and sunflower (Helianthus annuus L.), respectively,
which are hosts of AM fungi (Kim et al., 2014; Ueno et al., 2014).
2. Results and discussion
2.1. Synthesis of carlactone and its oxidized derivatives
Naturally occurring CL (1), CLA (2), and MeCLA (3) were syn-
thesized as a racemate from
b-ionone according to the method
reported previously (Seto et al., 2014; Abe et al., 2014). A mono-
hydroxy derivative of CL, 19-hydroxy-CL (6), was also prepared as
described (Abe et al., 2014). To examine the effect of a hydroxyl
group at the corresponding position of strigol (C-5, 10) and oro-
banchol (C-4, 8) on hyphal branching activity, 4- and 18-hydroxy-
Please cite this article in press as: Mori, N., et al., Carlactone-type strigolactones and their synthetic analogues as inducers of hyphal branching in
arbuscular mycorrhizal fungi, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.05.012

N. Mori et al. / Phytochemistry xxx (2016) 1e9
3
CL (4 and 5) were synthesized for the first time in this study.
4-Hydroxy-CL (4) was synthesized as shown in Scheme 1 by
to give formylacetic esters 42 (Sato et al., 1986). O-Alkylation of the
potassium enolates of formylacetates 42 with bromobutenolide 20
furnished AD esters 32, 34, and 36 in very low yields probably due
to notable instability of formylacetates (Sato et al., 1986) and low O-
alkylation efficiency. The (E)-geometry of the enol ether double
bond in AD esters was determined on the basis of the coupling
constant (J ¼ 12.4 Hz for 32, 34, and 36). Although both (E)- and (Z)-
isomers of methyl 3-(4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)
acrylate were obtained previously in a ratio of about 2:3 (Umehara
et al., 2015), the (Z)-isomer was not found in the respective reaction
mixtures.
Other members of new CL analogues 33, 35, and 37 named AA’D
diesters were obtained serendipitously in the reaction mixture
during the synthesis of AD esters. AA’D diester analogues consist of
two A-ring esters and an enol ether bridged D-ring. These ana-
logues can be formed through self aldol condensation of for-
mylacetate 42 to (E)-4-formylpent-2-enedioic acid diester followed
by O-alkylation with bromobutenolide 20.
starting from 4-hydroxy-b-ionone (15) that was obtained in two
steps from -ionone as described previously (Ye et al., 2009). After
a
protection of the hydroxyl group by an isopropyldimethylsilyl
(IPDMS) group, the resultant silyl-protected ionone 16 was con-
verted to IPDMSO-C14-aldehyde 18 via the CoreyeChaykovsky
epoxidation with dimethylsulfonium methylide, followed by
methylaluminium
bis(4-bromo-2,6-di-tert-butylphenoxide)
(MABR)-promoted rearrangement of epoxide to aldehyde. The
IPDMS group of 18 was partially removed during the purification
process with C18 reversed-phase column chromatography, yielding
the desired hydroxy-aldehyde 19. Finally, the aldehyde 19 was
coupled with bromobutenolide 20 in the presence of 18-crown-6-
ether to yield a mixture of 4-hydroxy-CL (4) (together with its
(9E) geometric isomer in a ratio 1:2). Purification of semi-
preparative C18 reversed-phase HPLC afforded 4-hydroxy-CL (4)
as a mixture of two epimers at C-11 that was not separable under
the conditions used.
To synthesize 18-hydroxy-CL (5) (Scheme 2), cyclo-
hexanecarboxylic acid methyl ester 21 (Wada et al., 1994) was
converted to the triflate 22, which was subjected to the Heck re-
2.3. Hyphal branching-inducing activity
Naturally occurring CL-type SLs 1e3 and three monohydroxy-
CLs 4e6 were tested for hyphal branching-inducing activity in
germinating spores of the arbuscular mycorrhizal fungus Gigaspora
margarita by paper disc assay (Table 1). The activity was evaluated
by determining the minimum effective concentration using serial
dilutions of the test compounds. CL (1) showed weak activity
(100 ng per disc). CLA (2) was the most potent among the three
naturally occurring CL-type SLs, inducing hyphal branching at
100 pg per disc. The activity of CLA (2) was comparable to that of
the natural SLs such as strigol (10) and sorgomol (11) (Akiyama
et al., 2010). MeCLA (3) was 10-fold less active (1 ng per disc)
than CLA (2) presumably due to its relative instability. 19-Hydroxy-
CL (6) showed intermediate activity between CL (1) and CLA (2)
(10 ng per disc), whereas the activities of 4- and 18-hydroxy-CL (4
action with methyl vinyl ketone to afford the b-ionone derivative 23
having a carbomethoxy group at C-13. After protection of the keto
group of methyl ester 23 by ketalization with ethylene glycol, the
resultant ketal ester 24 was reduced to the alcohol 25 with lithium
aluminum hydride followed by TBDMS protection to give the sily-
lated ketal 26. Deprotection of ketal catalyzed by molecular iodine
in acetone gave 13-TBDMSO-b-ionone 27. The silylated ketone 27
was converted to TBDMSO-C14-aldehyde 29 via the Cor-
eyeChaykovsky epoxidation, followed by MABR-promoted rear-
rangement as above. O-Alkylation of the potassium enolate of
aldehyde 29 with bromobutenolide 20 in the presence of 18-
crown-6-ether furnished the silylated 18-hydroxy-CL 30 and its
(9E) geometric isomer 31 in a ratio 1:7. After purification by pre-
parative HPLC, 19-TBDMSO-CL (30) was deprotected with aqueous
acetic acid to afford 18-hydroxy-CL (5).
and 5) were less than or comparable to that of CL (1) (1 mg and
100 ng per disc). The 19-oxidized derivatives of CL (1), CLA (2),
MeCLA (3) and 19-hydroxy-CL (6) induced the formation of low
order branches, mainly consisting of long tertiary hyphae, as
observed for the natural SLs such as 5-deoxystrigol (9) and strigol
(10), whereas CL, 4- and 18-hydroxy-CLs only induced short tertiary
from the treated secondary hyphae as observed for GR24 (12)
(Akiyama et al., 2010).
To gain the insight into the structural requirements of CL-type
SLs for hyphal branching activity, CL analogues, AD esters (32, 34,
and 36) and AA’D diesters (33, 35, and 37), were tested for hyphal
branching activity in G. margarita (Table 1). AD ester 32 showed
moderate activity, inducing hyphal branching at 1 ng per disc.
2.2. Synthesis of carlactone analogues
New CL analogues 32, 34, and 36 named AD esters were
designed by truncating the BC-rings from GR24 (12) and 4-
deoxyorobanchol (7) as a lead structure (Fig. 1). The AD esters
consist of a hydroxymethyl-substituted A-ring ester with formyl-
acetic acid connecting to a methylbutenolide (the D-ring) via an
enol ether bridge. To synthesize AD esters (Scheme 3), formyl
Meldrum’s acid (38) was reacted with benzyl (39), cyclo-
hexylmethyl (40), and cyclogeranyl (41) alcohols (the A-ring part)
O
O
O
O
IPDMSCl
imidazole
trimethylsulfonium iodide
dimsyl sodium
DMF
78%
THF, DMSO
quant.
OH
OIPDMS
16
OIPDMS
15
17
O
Br
O
20
t-BuOK
MABR
Chromatorex ODS
O
O
O
O
CH2Cl2
18-crown-6-ether
phenotiazine
THF
MeCN-H2O
2.6% in 2 steps
O
OH
OIPDMS
OH
1.6%
18
4
19
Scheme 1. Synthesis of 4-hydroxycarlactone (4).
Please cite this article in press as: Mori, N., et al., Carlactone-type strigolactones and their synthetic analogues as inducers of hyphal branching in
arbuscular mycorrhizal fungi, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.05.012

4
N. Mori et al. / Phytochemistry xxx (2016) 1e9
O
O
O
O
NaH
Tf2O
HOCH2CH2OH
TsOH
OTf
O
LAH
ether
79%
ether
65%
Pd(PPh3)2Cl2
Pd(OAc)2
Et3N, DMF
46%
(MeO)3C
100%
COOMe
COOMe
COOMe
COOMe
21
22
23
24
O
O
O
O
I2
O
O
trimethylsulfonium iodide
dimsyl sodium
TBDMSCl
PPTS
THF
DMSO
quant.
imidazole
DMF
89%
acetone
24%
OTBDMS
OH
OTBDMS
28
OTBDMS
26
25
27
O
O
Br
20
MABR
AcOH
t-BuOK
O
O
O
O
CH2Cl2
35%
O
18-crown-6-ether
phenotiazine
THF
O
THF
H2O
69%
O
OTBDMS
29
OH
OTBDMS
30
1.9%
5
O
O
O
OTBDMS
31
13%
Scheme 2. Synthesis of 18-hydroxycarlactone (5).
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
32
33
O
O
Br
O
O
O
O
O
O
O
O
H
O
O
O
20
ROH, Toluene
39: R=Benzyl
40: R=Cyclohexanemethyl
41: R=β-Cyclogeranyl
R
O
K CO
THF or Toluene
O
O
O
O
H
42
38
34
35
O
O
O
O
O
O
O
O
36
37
Scheme 3. Synthesis of carlactone analogues (32e37).
Replacement of the benzene ring in 32 with cyclohexane as in 34
increased activity by 10-fold (100 pg per disc). Analogue 36 having
comparable activity to CLA (2) (100 pg per disc). AA’D diester
analogue 33 showed activity at 1 ng per disc. Analogues 35 and 37
the
b-ionone ring (cyclogeranyl) as in CL (1) also showed
having cyclohexane and b-ionone ring, respectively, were highly
Please cite this article in press as: Mori, N., et al., Carlactone-type strigolactones and their synthetic analogues as inducers of hyphal branching in
arbuscular mycorrhizal fungi, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.05.012

N. Mori et al. / Phytochemistry xxx (2016) 1e9
5
Table 1
and are expected to act as a host-derived precolonization signal in
AM symbiosis. Further studies are needed to show the existence of
CLA (2) and MeCLA (3) in the root exudates of AM host plants and
also to investigate the occurrence and distribution of known and
novel CL-type SLs in the plant kingdom. These studies could pro-
vide new insight into the molecular evolution of CL-type SLs and
canonical SLs as signals for AM fungi and root parasitic plants.
Minimum effective concentrations (MECs) of carlactone-type strigo-
lactones (1e6), carlactone analogues (32e37), and canonical strigolactones
(7e12) for hyphal branching-inducing activity in Gigaspora margarita.
Tested compound
MEC (pg/disc)a
Carlactone (1)
Carlactonoic acid (2)
Methyl carlactonoate (3)
4-Hydroxycarlactone (4)
18-Hydorxycarlactone (5)
19-Hydorxycarlactone (6)
32
100,000
100
1000
1,000,000
100,000
10,000
1000
1000
100
4. Experimental
4.1. General procedures
33
34
Mass spectra were recorded on a JMS-700 instrument (JEOL),
NanoFrontier L (Hitachi High-Technologies), or a GCMS-QP2010
Plus instrument (Shimadzu) in the direct injection mode. 1H and
13C-NMR spectra were obtained with a JNM-AL400 NMR spec-
trometer (JEOL). Chemical shifts were referenced to tetrame-
thylsilane as an internal standard. Column chromatography (CC)
was performed with Wakogel C-200 (Wako Pure Chemical Ind.),
Kieselgel 60 (Merck), Chromatorex ODS (Fuji Silysia Chemical),
35
36
37
10
100
10
(ꢀ)-4-Deoxyorobanchol (7)
(ꢀ)-Orobanchol (8)
(þ)-5-Deoxystrigol (9)
(þ)-Strigol (10)
(þ)-Sorgomol (11)
(þ)-GR24 (12)
3b
1b
3b
100b
100b
100b
a
Inertsil ODS-3 (
C18 (
10 ꢁ 250 mm, 5
10 ꢁ 250 mm, 5 m; GL Sciences).
4
10 ꢁ 250 mm, 5
mm; GL Sciences), InertSustain
Determined by serial 10-fold dilutions.
Tested previously (Akiyama et al., 2010).
b
4
m
m; GL Sciences) and Inertsil SIL-100A (4
m
active on the AM fungus, showing activity at 10 pg per disc. All
analogues tested induced long tertiary hyphae from the treated
secondary hyphae as observed for 19-oxidized CLs.
4.2. Synthesis of 4-hydroxy-CL (4)
4.2.1. (3E)-4-(3-isopropyldimethylsilyloxy-2,6,6-trimethylcyclohex-
The results obtained for CL-type SLs indicate that hyphal
branching activity correlates with the degree of oxidation at C-19
and that the BC ring closure to complete canonical SL structure is
not necessary for the activity. Hydroxylation at either C-4 or C-18 in
CL did not significantly affect the activity. By contrast, (þ)-strigol
(10) and (ꢀ)-orobanchol (8), the canonical SLs hydroxylated at the
corresponding position, exhibited weaker and stronger activity
than their respective parent SLs, (þ)-5-deoxystrigol (9) and (ꢀ)-4-
deoxyorobanchol (7) (Akiyama et al., 2010). All the CL analogues
having the A ring and the enol ether bridged D ring linked to a
carboxyl group, but lacked the B and C-rings induced hyphal
1-en-1-yl)but-3-en-2-one (16)
To a stirred solution of hydroxy-b-ionone 15 (14.7 g, 70.5 mmol)
in N,N-dimethylformamide (DMF, 179 ml) at 0 ꢂC were added
imidazole (19.2 g, 282 mmol) and isopropyldimethylchlorosilane
(19.3 g, 141 mmol), and the mixture was stirred for 2.5 h at room
temperature under Ar. The reaction mixture was quenched by
adding 5% (wt/vol) NaHCO3 solution (200 ml), and extracted with
Et2O and n-hexane. The organic phase was washed with H2O, dried
over anhydrous Na2SO4, and concentrated in vauo. Purification by
silica gel CC eluted stepwise with n-hexane and EtOAc [6% (vol/vol)
increments] gave isopropyldimethylsilyloxy-
b
-ionone 16 (16.9 g,
54.9 mmol, 78%). 1H-NMR (CDCl3, 400 MHz)
d: 0.10 (6H, s, Si-
branching in G. margarita. The analogues containing the b-ionone
ring were more active than those with benzene ring. Even though
an additional bulky A-ring ester is linked to the enol ether double
bond, AA’D diester analogues showed activity comparable to or
higher than that of their respective AD ester analogues. These facts
suggest that the putative SL receptor in the AM fungus has suffi-
(CH3)2), 0.81e0.90 (1H, m, SieCH), 0.97 (3H, d, J ¼ 7.0 Hz,
SieCHeCH3), 0.98 (3H, d, J ¼ 7.1 Hz, SieCHeCH3), 1.02 (3H, s, 60-
CH3), 1.08 (3H, s, 60-CH3), 1.38e1.87 (4H, m, H-40 and H-50), 1.76 (3H,
s, 20-CH3), 2.30 (3H, s, H-1), 4.03 (1H, t, J ¼ 5.6 Hz, H-30), 6.12 (1H, d,
J ¼ 16.5 Hz, H-3), 7.19 (1H, d, J ¼ 16.5 Hz, H-4); 13C-NMR (CDCl3,
cient flexibility to accommodate such a large ligand and that the b-
100 MHz)
d
: ꢀ3.8, ꢀ3.5, 14.9, 17.0, 18.3, 27.3, 28.3, 29.0, 34.6, 35.3,
70.8, 132.9, 135.4, 138.2, 143.2, 198.6; EI-MS m/z: 308 [M]þ, 293,
265; HREIMS m/z: 308.2164 [M]þ (calcd. for C18H32O2Si, m/z
308.2172).
ionone ring rather than benzene ring in the A-ring part is preferred
for binding to the receptor. Taken together, the oxidation of methyl
to carboxyl at C-19 in CL is a prerequisite but BC-ring formation is
not essential to show hyphal branching activity comparable to that
of canonical SLs.
4.2.2. (E)-isopropyldimethyl((2,4,4-trimethyl-3-(2-(2-
methyloxiran-2-yl)vinyl)cyclohex-2-en-1-yl)oxy)silane (17)
3. Concluding remarks
To a solution of trimethylsulfonium iodide (11.1 g, 54.3 mmol) in
DMSO (47 mL) was added tetrahydrofuran THF (47 mL) under Ar to
yield a finely divided suspension of sulfonium salt. This mixture
was then cooled to ꢀ5 ꢂC and treated with a solution of dimsyl
sodium (4.4 M, 13 mL, 57.2 mmol). The resulting gray colored sus-
pension was treated with a solution of isopropyldimethylsilyloxy-
In this study, it was shown that not only canonical SLs, but also
CL-type SLs can induce hyphal branching in AM fungi. The naturally
occurring 19-oxidized CLs, CLA (2) and MeCLA (3) are moderate
inducers for hyphal branching in G. margarita. So far, CLA (2) and
MeCLA (3) have been detected in root extracts, but their exudation
from roots has not yet been examined. CLA (2) and MeCLA (3) could
act as a symbiotic signal for AM fungi if they are exuded from AM
host roots at sufficient levels to trigger the fungal response. Avenaol
(13) and heliolactone (14), other members of CL-type SLs, discov-
ered from the root exudates of AM host plants fullfil the structural
requirements for induction of hyphal branching in AM fungi as
revealed by the previous work (Akiyama et al., 2010) and this study,
b
-ionone 16 (11.2 g, 36.2 mmol) in THF (11.2 mL). After stirring at
ꢀ5 ꢂC for 45 min, the mixture was warmed to room temperature,
quenched by successively adding H2O (200 mL) and n-hexane
(200 mL). The organic phase was washed with H2O, dried over
anhydrous Na2SO4, and concentrated in vacuo to give the epoxide
17 (11.1 g, 36.1 mmol). 1H-NMR (CDCl3, 400 MHz)
d
: 0.09 (6H, s, Si-
(CH3)2), 0.80e0.90 (1H, m, SieCH), 0.95 (3H, d, J ¼ 11.0 Hz,
SieCHeCH3), 0.96 (3H, s, 4-CH3), 0.97 (3H, d, 7.1 Hz,
J
¼
Please cite this article in press as: Mori, N., et al., Carlactone-type strigolactones and their synthetic analogues as inducers of hyphal branching in
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6
N. Mori et al. / Phytochemistry xxx (2016) 1e9
SieCHeCH3), 1.02 (3H, s, 4-CH3), 1.34e1.82 (4H, m, H-5 and H-6),
1.49 (3H, s, 200-CH3),1.69 (3H, s, 2-CH3), 2.73 (1H, d, J ¼ 5.4 Hz, H-300),
2.83 (1H, d, J ¼ 5.4 Hz, H-300), 4.00 (1H, t, J ¼ 5.4 Hz, H-1), 5.28 (1H, d,
J ¼ 16.4 Hz, H-20), 6.15 (1H, d, J ¼ 16.4 Hz, H-10); 13C-NMR (CDCl3,
(34), 91 (100); HREIMS m/z: 318.1827 [M]þ (calcd. for C19H26O4, m/z
318.1831).
4.3. Synthesis of 18-hydroxy-CL (5)
100 MHz)
d: ꢀ3.7, ꢀ3.5, 15.0, 17.0 and 17.1, 18.0 and 18.1, 19.69 and
19.74, 28.2, 29.2, 34.4 and 34.5, 35.08 and 35.13, 55.7 and 55.9, 71.0,
129.7, 130.9, 134.9, 135.0, 139.4 and 139.5; MS data was not
obtainable due to instability of the compound.
4.3.1. Methyl 3,3-dimethyl-2-(((trifluoromethyl)sulfonyl)oxy)
cyclohex-1-ene-1-carboxylate (22)
A solution of ester 21 (10.3 g, 55.9 mmol) in Et2O (90 ml) was
added to a suspension of NaH (1.75 g, 72.8 mmol) in Et2O (120 ml)
at 0 ꢂC under Ar. After stirring at 0 ꢂC for 30 min, triflic anhydride
(32.5 g, 115 mmol) was added, and the mixture was stirred at the
same temperature for 2 h. The reaction mixture was quenched by
adding H2O (150 ml), and extracted with CH2Cl2. The organic phase
was washed with brine, dried over anhydrous MgSO4, and
concentrated in vacuo. Purification by silica gel CC eluted with n-
hexane and EtOAc: n-hexane gave the triflate 22 (14.0 g, 44.3 mmol,
4.2.3. (E)-4-(3-hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl)-2-
methylbut-3-enal (19)
To
a solution of 2,6-di-tert-butyl-4-bromophenol (20.1 g,
72.2 mmol) in CH2Cl2 (318 mL) was added at room temperature a
1.4 M hexane solution of trimethylaluminium (Me3Al, 26 mL,
36.1 mmol), and the solution was stirred at room temperature for
1 h under Ar. To a solution of the MABR (36 mmol) in CH2Cl2 was
added a solution of epoxide 17 (11.1 g, 36.1 mmol) in CH2Cl2 (24 mL)
at ꢀ78 ꢂC, and the resulting mixture was stirred at ꢀ78 ꢂC for
30 min under Ar. The reaction mixture was poured into H2O, and
extracted with n-hexane. The organic phase was washed with
saturated aqueous NaHCO3, dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue containing IPDMSO-C14-alde-
hyde 18 was subjected to ODS CC eluted stepwise with 70e100%
(vol/vol) CH3CN in H2O [5% (vol/vol) increments]; 75e80% CH3CN
eluates were combined, evaporated to H2O in vacuo, extracted with
n-hexane, and evaporated to give the hydroxy aldehyde 19 (208 mg,
79%). 1H-NMR (CDCl3, 400 MHz)
(4H, m, H-4 and H-5), 2.49 (2H, t, J ¼ 6.2 Hz, H-6), 3.77 (3H, s,
d: 1.22 (6H, s, 3-CH3 x 2), 1.60e1.78
OCH3); 13C-NMR (CDCl3, 100 MHz)
d: 17.9, 26.2, 28.1, 39.6, 52.3,
118.4, 123.0, 155.1, 166.2; EI-MS m/z: 316 [M]þ, 285, 167; HREIMS m/
z: 316.0592 [M]þ (calcd. for C11H15F3O5S, m/z 316.0592).
4.3.2. Methyl (E)-3,3-dimethyl-2-(3-oxobut-1-en-1-yl)cyclohex-1-
ene-1-carboxylate (23)
A mixture of triflate 22 (14.0 g, 44.3 mmol), Et3N (17.9 g,
177 mmol), methyl vinyl ketone (6.21 g, 88.6 mmol), bis(-
triphenylphosphine) palladium(II) dichloride (240 mg, 0.34 mmol),
and palladium(II) acetate (480 mg, 2.2 mmol) in DMF (100 mL) was
stirred at 75 ꢂC for 24 h under Ar. The reaction mixture was poured
into ice-cooled 1 N HCl (100 ml), and extracted with n-hexane. The
organic phase was washed with brine and H2O, dried over anhy-
drous MgSO4, and concentrated in vacuo. Purification by silica gel
CC eluted stepwise with n-hexane and EtOAc [5% (vol/vol) in-
crements] gave the ester 23 (4.8 g, 20.3 mmol, 46%). 1H-NMR
2.6%). 1H-NMR (CDCl3, 400 MHz) : 0.98 (3H, s, 60-CH3), 1.01 (3H, s,
d
60-CH3), 1.36e1.72 (4H, m, H-40 and H-50), 1.80 (3H, br.s, 20-CH3),
3.17 (1H, m, H-2), 3.99 (1H, t, J ¼ 4.6 Hz H-30), 5.39 and 5.40 (1H, dd,
J ¼ 7.8, 16.2 Hz, H-3), 6.02 and 6.03 (1H, d, J ¼ 16.2 Hz, H-4), 9.616
and 9.620 (1H, d, J ¼ 1.5 Hz, H-1); 13C-NMR (CDCl3,100 MHz)
d: 13.6
and 14.2, 18.36 and 18.40, 21.1, 27.19 and 27.21, 28.4 and 28.8, 34.3
and 34.4, 50.6 and 50.7, 60.4, 69.93 and 69.94,130.0 and 131.1,130.5,
141.3, 171.2, 201.6; EI-MS m/z: 222 [M]þ, 207, 166; HREIMS m/z:
222.1593 [M]þ (calcd. for C14H22O2, m/z 222.1620).
(CDCl3, 400 MHz) d: 1.10 (6H, s, 3-CH3 x 2), 1.52e1.55 (2H, m, H-4),
1.65e1.73 (2H, m, H-5), 2.29 (3H, s, H-40), 2.32 (2H, dt, J ¼ 2.1, 6.3 Hz,
4.2.4. 5-(((1Z,3E)-4-(3-hydroxy-2,6,6-trimethylcyclohex-1-en-1-
yl)-2-methylbuta-1,3-dien-1-yl)oxy)-3-methylfuran-2(5H)-one (4-
hydroxy-CL (4))
To a mixture of 4-HO-C14-aldehyde 19 (104 mg, 0.468 mmol),
phenotiazine (1.6 mg), ( )-4-bromo-2-methyl-2-buten-4-olide 20
H-6), 3.65 (3H, s, OCH3), 6.02 (1H, d, J ¼ 16.2 Hz, H-20), 7.32 (1H, d,
J ¼ 16.2 Hz, H-10); 13C-NMR (CDCl3,100 MHz)
d: 18.2, 27.2, 27.5, 28.1,
34.7, 38.4, 51.7,129.8,131.0,143.4,146.6,170.1,198.0; EI-MS m/z: 236
[M]þ, 193, 179, 177, 163; HREIMS m/z: 236.1430 [M]þ (calcd. for
C14H20O3, m/z 236.1412).
(52.9 ml, 0.59 mmol), and 18-crown-6-ether (88.4 mg, 0.336 mmol)
in THF (3.9 mL), potassium tert-butoxide (75.5 mg, 0.620 mmol)
was added slowly under Ar, and the reaction mixture was stirred at
room temperature under Ar. After stirring for 45 min, the mixture
was poured into H2O (20 mL) and extracted with Et2O. The organic
phase was washed with brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was subjected to silica gel CC
eluted stepwise with n-hexane and Et2O [5% (vol/vol) increments].
The 30e40% Et2O in hexane eluates containing 4-HO-CL was pu-
4.3.3. Methyl (E)-3,3-dimethyl-2-(2-(2-methyl-1,3-dioxolan-2-yl)
vinyl)cyclohex-1-ene-1-carboxylate (24)
A mixture of ester 23 (4.8 g, 20.3 mmol), ethylene glycol (12.6 g,
203 mmol), trimethyl orthoformate (4.3 g, 40.6 mmol), p-toluene-
sulfonic acid monohydrate (58 mg, 0.3 mmol) was stirred overnight
at room temperature under Ar. The reaction mixture was quenched
by adding saturated aqueous NaHCO3 (50 ml), and extracted with
Et2O. The organic phase was dried over anhydrous MgSO4, and
concentrated in vacuo to give the ketal 24 (5.7 g, 20.3 mmol, 100%).
rified by a semi-preparative Inertsil ODS-3 HPLC (
m; GL Sciences), using isocratic elution with CH3CN: H2O (60:40
v/v) at a flow rate of 4.0 mL/min and monitored at 285 nm to give 4-
HO-CL 4 [2.43 mg, 7.64 mol, 1.6%, retention time 16.6 min]. 4-HO-
CL (as a mixture with 11-epimer): 1H-NMR (C6D6, 400 MHz)
: 0.96,
4
10 ꢁ 250 mm,
5
m
1H-NMR (CDCl3, 400 MHz) : 1.04 (6H, s, 3-CH3 x 2), 1.46 (3H, s, 200-
d
CH3), 1.47e1.53 (2H, m, H-4), 1.64e1.70 (2H, m, H-5), 2.26 (2H, dt,
J ¼ 2.1, 6.3 Hz, H-6), 3.65 (3H, s, OCH3), 3.87e4.00 (4H, m, H-400 and
H-500), 5.41 (1H, d, J ¼ 15.7 Hz, H-20), 6.36 (1H, dt, J ¼ 16.5, 2.1 Hz, H-
m
d
0.99, 1.01 and 1.04 (total 6H, s, CH3-16 or 17), 1.22e1.30 (1H, m, H-
2), 1.35 and 1.35 (3H, t, J ¼ 1.5 Hz, CH3-15), 1.54e1.58 (2H, m, H-3),
1.66e1.73 (1H, m, H-2), 1.587 and 1.591 (3H, s, CH3-18), 1.90 and
1.91 (3H, br.s, CH3-19), 3.75 (1H, br.d, H-4), 5.18 (1H, br.s, H-11),
5.77e5.78 (1H, m, H-12), 5.98 (1H, s, H-10), 6.08 (1H, d, J ¼ 16.3 Hz,
10); 13C-NMR (CDCl3, 100 MHz)
d: 18.5, 24.9, 27.5, 28.1, 34.1, 38.2,
51.3, 64.4, 107.0, 115.8, 127.5, 133.3, 146.6, 171.4; EI-MS m/z: 280
[M]þ, 223, 193, 179, 87; HREIMS m/z: 280.1659 [M]þ (calcd. for
C16H24O4, m/z 280.1674).
H-7), 6.90 (1H, d, J ¼ 16.3 Hz, H-8); 13C-NMR (C6D6, 100 MHz)
d
:
4.3.4. (E)-(3,3-Dimethyl-2-(2-(2-methyl-1,3-dioxolan-2-yl)vinyl)
cyclohex-1-en-1-yl)methanol (25)
A solution of ketal 24 (5.7 g, 20.3 mmol) in Et2O (25 ml) was
added to a suspension of LiAlH4 (1.9 g, 47 mmol) in Et2O (40 ml) at
0 ꢂC under Ar, and the mixture was stirred at the same temperature
10.2, 14.3, 18.8, 27.62 and 27.67, 28.95 and 29.00, 29.05 and 29.11,
30.4, 34.8 and 34.9, 69.91 and 69.94, 100.0, 116.3 and 116.4, 126.3,
128.6, 130.8, 134.6, 139.41 and 139.42, 141.29 and 141.31, 141.6,
170.5; EI-MS m/z (rel. int): 318 [M]þ (11), 300 (2.0), 221 (62), 97
Please cite this article in press as: Mori, N., et al., Carlactone-type strigolactones and their synthetic analogues as inducers of hyphal branching in
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N. Mori et al. / Phytochemistry xxx (2016) 1e9
7
for 2 h. The reaction mixture was cooled to 0 ꢂC, and quenched by
successively adding H2O (2 ml), 1 N NaOH (2 ml) and H2O (6 ml).
The mixture was extracted with Et2O. The organic phase was
washed with H2O, dried over anhydrous MgSO4, and concentrated
in vacuo. Purification by silica gel CC eluted stepwise with n-hexane
and EtOAc [10% (vol/vol) increments] gave the alcohol 25 (3.85 g,
15.3 mmol, 65%). This compound was readily transformed by
intramolecular cyclization into 7,7-dimethyl-1-((2-methyl-1,3-
stirring at 0 ꢂC for 1 h, the mixture was warmed to room temper-
ature, quenched by successively adding H2O (10 mL) and n-hexane
(30 mL). The organic phase was washed with H2O, dried over
anhydrous MgSO4, and concentrated in vacuo to give the epoxide 28
(522 mg, 1.55 mmol, 100%). 1H-NMR (CDCl3, 400 MHz)
d: 0.00 (6H,
s, Si-(CH3)2), 0.86 (9H, s, SieC(CH3)3), 0.959 (3H, s, 3-CH3), 0.961
(3H, s, 3-CH3), 1.40e1.45 (2H, m, H-4), 1.46 (3H, s, 200-CH3),
1.55e1.60 (2H, m, H-5), 2.07e2.13 (2H, m, H-6), 2.69 (1H, dd, J ¼ 0.5,
5.4 Hz, H-300), 2.80 (1H, d, J ¼ 5.4 Hz, H-300), 4.08 (1H, d, J ¼ 11.5 Hz,
OCH2), 4.12 (1H, d, J ¼ 11.5 Hz, OCH2), 5.24 (1H, d, J ¼ 16.1 Hz, H-20),
dioxolan-2-yl)methyl)-1,3,4,5,6,7-hexahydroisobenzofuran
(see
supplement) when dissolved in CDCl3. 1H-NMR (CDCl3, 400 MHz)
d:
1.05 (3H, s, 7-CH3), 1.09 (3H, s, 7-CH3), 1.33e1.50 (2H, m, H-6), 1.45
(3H, s, 20-CH3), 1.67e1.75 (2H, m, H-5), 1.79 (1H, dd, J ¼ 9.9, 14.5 Hz,
20-CH2), 1.88e1.97 (2H, m, H-4), 2.07 (1H, dd, J ¼ 2.2, 14.5 Hz, 20-
CH2), 3.96e4.00 (4H, m, H-400 and H-500), 4.36 (1H, m, H-3), 4.52
6.13 (1H, dt, J ¼ 16.5, 1.9 Hz, H-10); 13C-NMR (CDCl3, 100 MHz)
d:
ꢀ5.2, 18.4, 19.0, 19.9, 26.0, 27.5, 28.6, 34.0, 39.0, 55.6, 55.9, 64.5,
128.8, 132.7, 134.9, 139.1; EI-MS m/z: 336 [M]þ, 321, 305, 279;
HREIMS m/z: 336.2504 [M]þ (calcd. for C20H36O2Si, m/z 336.2484).
(1H, m, H-3), 4.92e5.00 (1H, m, H-1); 13C-NMR (CDCl3, 100 MHz)
d:
19.0, 22.2, 24.2, 27.8, 28.2, 31.1, 40.4, 44.5, 64.37, 64.44, 76.1, 82.5,
109.5, 131.4, 139.9; EI-MS m/z: 252 [M]þ, 237, 207, 151, 87; HREIMS
m/z: 252.1741 [M]þ (calcd. for C15H24O3, m/z 252.1726).
4.3.8. (E)-4-(2-(((tert-Butyldimethylsilyl)oxy)methyl)-6,6-
dimethylcyclohex-1-en-1-yl)-2-methylbut-3-enal (29)
To a solution of 2,6-di-tert-butyl-4-bromophenol (183 mg,
0.64 mmol) in CH2Cl2 (12 ml) was added at room temperature a
1.4 M hexane solution of trimethylaluminium (Me3Al, 0.23 ml,
0.32 mmol), and the solution was stirred at room temperature for
1 h under argon. To a solution of the MABR (0.32 mmol) in CH2Cl2
was added a solution of epoxide 28 (522 mg, 1.55 mmol) in CH2Cl2
(1 ml) at ꢀ78 ꢂC, and the resulting mixture was stirred at ꢀ78 ꢂC for
30 min under Ar. The reaction mixture was poured into H2O, and
extracted with n-hexane. The organic phase was washed with
saturated aqueous NaHCO3, dried over anhydrous MgSO4, and
concentrated in vacuo. The residue was subjected to ODS CC eluted
stepwise with 70e100% (vol/vol) CH3CN in H2O [5% (vol/vol) in-
crements]; 85e90% CH3CN eluates were combined, evaporated to
H2O in vacuo, extracted with n-hexane and Et2O, and evaporated to
give the aldehyde 29 (188 mg, 0.56 mmol, 35%). 1H-NMR (CDCl3,
4.3.5. (E)-tert-Butyl((3,3-dimethyl-2-(2-(2-methyl-1,3-dioxolan-2-
yl)vinyl)cyclohex-1-en-1-yl)methoxy)dimethylsilane (26)
To a stirred solution of alcohol 25 (3.85 g, 15.3 mmol) in DMF
(40 mL) was added imidazole (4.17 g, 61.2 mmol) and tert-butyl-
dimethylchlorosilane (4.64 g, 30.8 mmol), and the mixture was
stirred for 3 h at room temperature under Ar. The reaction mixture
was taken up in 2% (vol/vol) Et2O in n-hexane, washed with H2O,
dried over anhydrous MgSO4, and concentrated in vauo. Purification
by silica gel CC eluted with n-hexane and 10% Et2O in n-hexane gave
the silyl protected alcohol 26 (4.99 g, 13.6 mmol, 89%). 1H-NMR
(CDCl3, 400 MHz) d: 0.03 (6H, s, Si-(CH3)2), 0.89 (9H, s, SieC(CH3)3),
0.98 (6H, s, 3-CH3 x 2), 1.46e1.49 (1H, m, H-4 or H-5), 1.51 (3H, s, 200-
CH3), 1.54e1.58 (3H, m, H-40 and H-50), 2.12 (2H, dt, J ¼ 1.7, 6.3 Hz,
H-6), 3.93e4.02 (4H, m, H-400 and H-500), 4.12 (2H, s), 5.35 (1H, d,
J ¼ 15.9 Hz, H-20), 6.19 (1H, br.d, J ¼ 15.9 Hz, H-10); 13C-NMR (CDCl3,
400 MHz) d: e0.002 (6H, s, Si-(CH3)2), 0.86 (9H, s, SieC(CH3)3), 0.96
(6H, s, 60-CH3 x 2), 1.22 (3H, d, J ¼ 7.1 Hz, 2-CH3), 1.45e1.48 (2H, m,
H-50), 1.53e1.57 (2H, m, H-40), 2.11 (2H, dt, J ¼ 1.8, 6.2 Hz, H-30), 2.45
(1H, m, H-2), 5.32 (1H, dd, J ¼ 7.8, 15.4 Hz, H-3), 6.00 (1H, br.d,
J ¼ 15.4 Hz, H-4), 9.59 (1H, d, J ¼ 1.7 Hz, H-1); 13C-NMR (CDCl3,
100 MHz)
d
: ꢀ5.2, 19.0, 22.6, 25.9, 27.3, 28.6, 31.6, 33.9, 39.0, 64.47,
107.4, 127.1, 132.4, 134.4, 138.7; EI-MS m/z: 366 [M]þ, 351, 309, 265,
251, 87; HREIMS m/z: 366.2613 [M]þ (calcd. for C21H38O3Si, m/z
366.2590).
100 MHz)
d
: ꢀ5.2, 13.6, 18.4, 18.9, 26.0, 27.5, 28.5, 33.9, 39.0, 50.6,
64.6, 130.1, 130.8, 132.9, 139.4, 201.5; EI-MS m/z: 336 [M]þ, 321, 305,
279; HREIMS m/z: 336.2466 [M]þ (calcd. for C20H36O2Si, m/z
336.2484).
4.3.6. (E)-4-(2-(((tert-Butyldimethylsilyl)oxy)methyl)-6,6-
dimethylcyclohex-1-en-1-yl)but-3-en-2-one (27)
A mixture of silyl protected alcohol 26 (2.50 g, 6.8 mmol), pyr-
idinium p-toluenesulfonate (170 mg, 0.68 mmol), and iodine
(173 mg, 0.68 mmol) in acetone (20 ml) was stirred at room tem-
perature for 1 h under Ar. The reaction mixture was diluted with
Et2O (50 ml), washed with 5% aqueous Na2S2O3 and brine, dried
over MgSO4, and concentrated in vacuo. Purification by silica gel CC
eluted stepwise with n-hexane and Et2O [2% (vol/vol) increments]
gave the ketone 27 (532 mg 1.65 mmol, 24%). 1H-NMR (CDCl3,
4.3.9. 5-(((1Z,3E)-4-(2-(((Tert-Butyldimethylsilyl)oxy)methyl)-6,6-
dimethylcyclohex-1-en-1-yl)-2-methylbuta-1,3-dien-1-yl)oxy)-3-
methylfuran-2(5H)-one (18-TBDMSO-CL 30 and 18-TBDMSO-(9E)-
CL 31)
To a mixture of TBDMSO-C14-aldehyde 29 (178 mg, 0.53 mmol)),
phenotiazine (2.7 mg), ( )-4-bromo-2-methyl-2-buten-4-olide 20
(94 mL, 0.96 mmol), and 18-crown-6-ether (155 mg, 0.59 mmol) in
400 MHz)
d
: 0.04 (6H, s, Si-(CH3)2), 0.89 (9H, s, SieC(CH3)3), 1.05
THF (6.8 ml) was added slowly potassium tert-butoxide (120 mg,
1.07 mmol) under Ar, and the reaction mixture was stirred at room
temperature under Ar. After stirring for 1 h, the mixture was poured
into H2O (20 mL) and extracted with Et2O. The organic phase was
washed with H2O, dried over anhydrous MgSO4, and concentrated
in vacuo. The residue was subjected to silica gel CC eluted stepwise
with n-hexane and Et2O [3% (vol/vol) increments]. The 9%, 12% and
15% Et2O eluates containing TBDMSO-CL was purified by a semi-
(6H, s, 60-CH3 x 2), 1.47e1.51 (2H, m, H-50), 1.55e1.59 (2H, m, H-40),
2.19 (2H, br.t, J ¼ 6.1 Hz, H-30), 4.11 (2H, s, OCH2), 6.10 (1H, d,
J ¼ 16.1 Hz, H-3), 7.20 (1H, d, J ¼ 16.1 Hz, H-4); 13C-NMR (CDCl3,
100 MHz)
d: ꢀ5.2, 18.3, 18.7, 25.9, 27.6, 28.2, 28.7, 34.2, 39.1, 64.2,
132.5, 137.1, 138.2, 142.2, 198.2; EI-MS m/z: [M]þ (m/z 322) was not
observed, 307, 279, 265.
4.3.7. (E)-tert-Butyl((3,3-dimethyl-2-(2-(2-methyloxiran-2-yl)
vinyl)cyclohex-1-en-1-yl)methoxy)dimethylsilane (28)
preparative InertSustain C18 HPLC (
4
10 ꢁ 250 mm, 5
mm; GL
Sciences), using isocratic elution with 90% CH3CN in H2O at a flow
rate of 4.0 mL/min and monitored at 300 nm to give 18-TBDMSO-CL
30 (4.4 mg, 0.010 mmol, 1.9%, retention time 23.4 min) and 18-
TBDMSO-(9E)-CL 31 (29.7 mg, 0.069 mmol, 13.0%, retention time
To a solution of trimethylsulfonium iodide (458 mg, 2.3 mmol)
in DMSO (2 ml) was added THF (2 ml) under argon to yield a finely
divided suspension of sulfonium salt. This mixture was then cooled
to 0 ꢂC and treated with a solution of dimsyl sodium (4.4 M, 0.63 ml,
2.7 mmol). The resulting gray colored suspension was treated with
a solution of ketone 27 (500 mg, 1.55 mmol) in THF (0.26 ml). After
24.3 min).18-TBDMSO-CL 30; 1H-NMR (C6D6, 400 MHz)
d: 0.07 (6H,
s, Si-(CH3)2), 0.98 (9H, s, SieC(CH3)3), 1.03 (3H, s, CH3-16 or 17), 1.04
(3H, s, CH3-16 or 17), 1.41 (3H, t, J ¼ 1.6 Hz, CH3-15), 1.40e1.45 (2H,
Please cite this article in press as: Mori, N., et al., Carlactone-type strigolactones and their synthetic analogues as inducers of hyphal branching in
arbuscular mycorrhizal fungi, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.05.012

8
N. Mori et al. / Phytochemistry xxx (2016) 1e9
m, H-2), 1.55e1.64 (2H, m, H-3), 1.60 (3H, d, J ¼ 1.2 Hz, CH3-19),
2.22e2.38 (2H, m, H-4), 4.31 (1H, d, J ¼ 11.1 Hz, H-18 ), 4.37 (1H, d,
J ¼ 11.1 Hz, H-18 ), 5.19 (1H, m, H-12 or 10), 5.85 (1H, m, H-12 or
monitored at 236 nm to give benzyl AD analogue 32 (3.3 mg,
0.012 mmol, 0.7% in 2 steps, retention time 14.9 min) and dibenzyl
AA’D analogue 33 (5.0 mg, 0.012 mmol, 0.7% in 2 steps, retention
time 22.4 min). Benzyl (E)-3-((4-methyl-5-oxo-2,5-dihydrofuran-
a
b
10), 5.95 (1H, br.d, J ¼ 1.0 Hz, H-11), 6.12 (1H, br d, J ¼ 16.2 Hz, H-7),
6.85 (1H, d, J ¼ 16.6 Hz, H-8); 13C-NMR (CDCl3, 100 MHz)
d
: ꢀ5.1,:
2-yl)oxy)acrylate 32; 1H-NMR (CDCl3, 400 MHz):
d 2.00 (3H, t,
ꢀ5.0, 10.2, 14.4, 18.6, 19.4, 26.2, 28.2, 28.85, 28.89, 34.5, 39.5, 65.1,
100.0, 115.9, 125.8, 128.7, 133.0, 134.5, 139.5, 140.8, 141.6, 170.5;
EIMS m/z: 432 [M]þ, 375, 335, 203, 97; HREIMS m/z: 432.2669 [M]þ
(calcd. for C25H40O4Si, m/z 432.2696). 18-TBDMSO-(9E)-CL 31; 1H-
J ¼ 1.5 Hz, 40-CH3), 5.18 (2H, s, OCH2), 5.62 (1H, d, J ¼ 12.4 Hz, H-2),
6.10 (1H, t, J ¼ 1.5 Hz, H-20), 6.91 (1H, t, J ¼ 1.5 Hz, H-30), 7.31e7.41
(5H, m, C6H5), 7.60 (1H, d, J ¼ 12.4 Hz, H-3); 13C-NMR (CDCl3,
100 MHz):
d 10.7, 66.1, 99.2, 102.3, 128.22, 128.24, 128.6, 135.4,
NMR (C6D6, 400 MHz)
d: 0.08 (6H, s, Si-(CH3)2), 0.99 (9H, s,
135.9, 141.0, 158.1, 166.4, 170.4; EIMS m/z (rel. int.): 274 [M]þ(0.1),
256 (4.0), 97 (100), 91 (47); HRESIMS m/z 297.0741 [MþNa]þ (calcd
for C15H14O5Na, m/z 297.0739). Dibenzyl (2E,4E)-4-(((4-methyl-5-
oxo-2,5-dihydrofuran-2-yl)oxy)methylene)pent-2-enedioate 33;
SieC(CH3)3), 1.07 (3H, s, CH3-16 or 17), 1.08 (3H, s, CH3-16 or 17),
1.40 (3H, t, J ¼ 1.5 Hz, CH3-15), 1.44e1.48 (2H, m, H-2), 1.60e1.68
(2H, m, H-3), 1.84 (3H, d, J ¼ 1.2 Hz, CH3-19), 2.28e2.36 (2H, m, H-
4), 4.27 (1H, d, J ¼ 10.9 Hz, H-18
a
), 4.33 (1H, d, J ¼ 10.9 Hz, H-18
b
),
1H-NMR (CDCl3, 400 MHz):
d
2.03 (3H, t, J ¼ 1.5 Hz, 40-CH3), 5.20
5.22 (1H, m, H-12 or 10), 5.81 (1H, m, H-12 or 10), 6.09 (1H, br d,
(2H, s, OCH2), 5.21 (1H, d, J ¼ 12.4 Hz, OCH2), 5.24 (1H, d, J ¼ 12.4 Hz,
OCH2), 6.18 (1H, t, J ¼ 1.5 Hz, H-20), 6.72 (1H, d, J ¼ 16.2 Hz, H-2),
6.97 (1H, t, J ¼ 1.5 Hz, H-30), 7.30e7.41 (10H, m, C6H5 x 2), 7.59 (1H,
d, J ¼ 16.2 Hz, H-3), 7.80 (1H, s, 4-CH); 13C-NMR (CDCl3, 100 MHz):
J ¼ 15.9 Hz, H-7), 6.14 (1H, d, J ¼ 15.9 Hz, H-8), 6.33 (1H, br.d,
J ¼ 1.0 Hz, H-11); 13C-NMR (C6D6, 100 MHz)
: ꢀ5.0, 9.8, 10.3, 14.2,
d
18.6, 19.6, 26.2, 28.5, 28.9, 29.0, 34.5, 39.5, 60.0, 65.2, 100.0, 118.5,
123.6, 132.5, 133.1, 134.6, 141.2, 141.6, 142.7, 170.4; EIMS m/z: 432
[M]þ, 375, 335, 203, 97; HREIMS m/z: 432.2674 [M]þ (calcd. for
d
10.8, 66.3, 66.7, 100.6, 110.5, 121.7, 128.16, 128.20, 128.38, 128.44,
128.5,128.7,133.5,135.6,136.0,136.1,140.8,157.6,165.1,167.3, 170.1;
EIMS m/z (rel. int.): [M]þ was not observed, 373 (4.6), 343 (2.2), 283
(2.8), 231 (5.2), 229 (3.8), 181 (7.2), 97 (43), 91 (100); HRESIMS m/z
457.1249 [MþNa]þ (calcd for C25H22O7Na, m/z 457.1263).
C
25H40O4Si, m/z 432.2696).
4.3.10. 5-(((1Z,3E)-4-(2-(Hydroxymethyl)-6,6-dimethylcyclohex-1-
en-1-yl)-2-methylbuta-1,3-dien-1-yl)oxy)-3-methylfuran-2(5H)-
one (18-hydroxy-CL (5))
4.4.2. Cyclohexylmethyl (E)-3-((4-methyl-5-oxo-2,5-dihydrofuran-
2-yl)oxy)acrylate (34) and bis(cyclohexylmethyl) (2E,4E)-4-(((4-
methyl-5-oxo-2,5-dihydrofuran-2-yl)oxy)methylene)pent-2-
enedioate (35)
To a solution of formyl Meldrum’s acid 38 (215 mg, 1.25 mmol)
in toluene (2.5 ml) was added cyclohexanemethanol 40 (184 ml,
1.50 mmol) under Ar. After refluxing for 2 h under Ar, the reaction
mixture was cooled and concentrated in vacuo. The residue was
dissolved in THF (2.0 ml). Then, K2CO3 (138 mg, 0.75 mmol) and 5-
To a solution of 18-TBDMSO-CL 30 (4.4 mg, 0.010 mmol) in THF
(0.6 mL) was added at room temperature acetic acid (1.8 mL) and
H2O (0.6 mL), and the mixture was stirred for 9 h. The reaction
mixture was poured into H2O and extracted with Et2O. The organic
phase was washed with H2O, dried over anhydrous Na2SO4, and
concentrated in vacuo. Purification by silica gel CC eluted stepwise
with n-hexane and Et2O [10% (vol/vol) increments] gave 18-
hydroxy-CL
400 MHz)
5
(2.2 mg, 0.069
m
mol, 69%). 1H-NMR (C6D6,
d
: 1.01 (3H, s, CH3-16 or 17), 1.04 (3H, s, CH3-16 or 17),
bromo-3-methylfuran-2(5H)-one 20 (150 ml, 1.53 mmol) was added
1.36 (3H, t, J ¼ 1.6 Hz, CH3-15), 1.37e1.42 (2H, m, H-2), 1.50e1.57
(2H, m, H-3), 1.57 (3H, d, J ¼ 1.5 Hz, CH3-19), 2.14 (2H, td, J ¼ 6.3,
1.5 Hz, H-4), 4.09 (2H, s, H-18), 5.18 (1H, dd, J ¼ 1.2, 2.4 Hz, H-12 or
10), 5.81 (1H, m, H-12 or 10), 5.96 (1H, br.d, J ¼ 0.7 Hz, H-11), 6.09
(1H, br d, J ¼ 16.0 Hz, H-7), 6.86 (1H, d, J ¼ 16.0 Hz, H-8); 13C-NMR
at 0 ꢂC under Ar, and stirred at the same temperature for 1 h. After
stirring overnight at room temperature, the mixture was poured
into 0.1 N HCl, and extracted with EtOAc. The organic phase was
dried over anhydrous Na2SO4, and concentrated in vacuo. The res-
idue was subjected to silica gel CC eluted stepwise with n-hexane
and acetone (5% increments). The 15% acetone eluate was purified
(C6D6, 100 MHz) d: 10.2, 14.3, 19.4, 28.4, 28.86, 28.91, 34.4, 39.4,
64.6, 100.0, 116.8, 125.8, 128.5, 133.2, 134.5, 139.4, 141.7, 170.6 (one
carbon peak was missing due to overlapping with solvent peaks);
EIMS m/z (rel. int): 318 [M]þ (22), 221 (46), 203 (73), 97 (100);
HREIMS m/z: 318.1836 [M]þ (calcd. for C19H26O4, m/z 318.1831).
by
a
semi-preparative Inertsil SIL-100A HPLC column
m, GL Scieneces), using isocratic elution with 2%
(4
10 ꢁ 250 mm, 5
m
EtOH in n-hexane at a flow rate of 2.8 ml minꢀ1 and monitored at
236 nm to give cyclohexylmethyl AD analogue 34 (1.9 mg,
0.0068 mmol, 0.5% in 2 steps, retention time 23.8 min) and bis(-
cyclohexylmethyl) AA’D analogue 35 (7.3 mg, 0.016 mmol, 1.3% in 2
steps, retention time 31.9 min). Cyclohexylmethyl (E)-3-((4-
methyl-5-oxo-2,5-dihydrofuran-2-yl)oxy)acrylate 34; 1H-NMR
4.4. Synthesis of CL analogues
4.4.1. Benzyl (E)-3-((4-methyl-5-oxo-2,5-dihydrofuran-2-yl)oxy)
acrylate (32) and dibenzyl (2E,4E)-4-(((4-methyl-5-oxo-2,5-
dihydrofuran-2-yl)oxy)methylene)pent-2-enedioate (33)
To a solution of formyl Meldrum’s acid 38 (300 mg, 1.74 mmol)
(CDCl3, 400 MHz): d 0.91e1.05 (2H, m),1.10e1.33 (4H, m), 1.58e1.80
(5H, m), 2.01 (3H, t, J ¼ 1.5 Hz, 40-CH3), 3.94 (2H, d, J ¼ 6.6 Hz, OCH2),
5.58 (1H, d, J ¼ 12.4 Hz, H-2), 6.11 (1H, t, J ¼ 1.5 Hz, H-20), 6.92 (1H, t,
J ¼ 1.5 Hz, H-30), 7.56 (1H, d, J ¼ 12.4 Hz, H-3); 13C-NMR (CDCl3,
in toluene (3.5 ml) was added benzyl alcohol 39 (208 ml, 2.00 mmol)
under Ar. After refluxing for 2 h under argon, the reaction mixture
was cooled and concentrated in vacuo. The residue was dissolved in
THF (2.0 ml). Then, K2CO3 (103 mg, 0.75 mmol) and 5-bromo-3-
100 MHz): d 10.7, 25.6, 26.3, 29.7, 37.1, 69.5, 99.1, 102.6, 135.4, 141.1,
157.6, 166.7, 170.5; EIMS m/z (rel. int.): [M]þ was not observed, 236
(0.4), 185 (5.5), 184 (2.8), 97 (100); HRESIMS m/z 303.1219 [MþNa]þ
(calcd for C15H20O5Na, m/z 303.1208). Bis(cyclohexylmethyl)
(2E,4E)-4-(((4-methyl-5-oxo-2,5-dihydrofuran-2-yl)oxy)methy-
methylfuran-2(5H)-one 20 (113 m
l, 1.15 mmol) was added at 0 ꢂC
under Ar, and the reaction mixture was stirred at the same tem-
perature for 1 h. After stirring overnight at room temperature, the
mixture was poured into 0.1 N HCl, and extracted with EtOAc. The
organic phase was dried over anhydrous Na2SO4, and concentrated
in vacuo. The residue was subjected to silica gel CC eluted stepwise
with n-hexane and acetone (5% increments). The 15% acetone
eluate was purified by a semi-preparative Inertsil SIL-100A HPLC
lene)pent-2-enedioate 35; 1H NMR (CDCl3, 400 MHz):
d 0.93e1.07
(4H, m), 1.10e1.34 (6H, m), 1.58e1.80 (12H, m), 2.05 (3H, t,
J ¼ 1.5 Hz, 40-CH3), 3.97 (2H, d, J ¼ 6.6 Hz,OCH2), 4.01 (2H, d,
J ¼ 6.4 Hz, OCH2), 6.22 (1H, t, J ¼ 1.5 Hz, H-20), 6.65 (1H, dd, J ¼ 0.6,
16.2 Hz, H-2), 7.00 (1H, t, J ¼ 1.5 Hz, H-30), 7.53 (1H, d, J ¼ 16.2 Hz, H-
3), 7.76 (1H, d, J ¼ 0.6 Hz, 4-CH); 13C-NMR (CDCl3, 100 MHz):
d 10.7,
column (
4
10 ꢁ 250 mm, 5
m
m, GL Scieneces), using isocratic elution
25.6, 25.7, 26.3, 26.4, 29.65, 29.73, 37.1, 37.2, 69.6, 70.1, 100.5, 110.8,
with 2% EtOH in n-hexane at a flow rate of 3.8 ml minꢀ1 and
122.0, 133.0, 135.9, 140.9, 156.9, 165.4, 167.6, 170.1; EIMS m/z (rel.
Please cite this article in press as: Mori, N., et al., Carlactone-type strigolactones and their synthetic analogues as inducers of hyphal branching in
arbuscular mycorrhizal fungi, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.05.012

N. Mori et al. / Phytochemistry xxx (2016) 1e9
9
int.): 446 [M]þ (0.7), 349 (0.5), 253 (3.8), 237 (18), 141 (12), 97
and Applied Researches for Innovations in Bio-oriented Industry
and the Science and Technology Research Promotion Program for
Agriculture, Forestry, Fisheries and Food Industry.
(100); HRESIMS m/z 469.2212 (calcd for
C25H34O7Na, m/z
469.2202).
4.4.3. (2,6,6-Trimethylcyclohex-1-en-1-yl)methyl (E)-3-((4-methyl-
5-oxo-2,5-dihydrofuran-2-yl)oxy)acrylate (36) and bis((2,6,6-
trimethylcyclohex-1-en-1-yl)methyl) (2E,4E)-4-(((4-methyl-5-oxo-
2,5-dihydrofuran-2-yl)oxy)methylene)pent-2-enedioate (37)
To a solution of formyl Meldrum’s acid 38 (288 mg, 1.67 mmol)
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.phytochem.2016.05.012.
in toluene (10 ml) was added
b-cyclogeraniol 41 (346 ml,
2.00 mmol) under Ar. After heating under refluxconditions for 2 h
under Ar, the reaction mixture was cooled to 0 ꢂC. Then, K2CO3
(276 mg, 2.00 mmol) and a solution of 5-bromo-3-methylfuran-
References
2(5H)-one 20 (295
ml, 3.00 mmol) in toluene (2 ml) was added
Abe, S., Sado, A., Tanaka, K., Kisugi, T., Asami, K., Ota, S., Kim, H.I., Yoneyama, K.,
Xie, X., Ohnishi, T., Seto, Y., Yamaguchi, S., Akiyama, K., Yoneyama, K.,
Nomura, T., 2014. Carlactone is converted to carlactonoic acid by MAX1 in
Arabidopsis and its methyl ester can directly interact with AtD14 in vitro. Proc.
Natl. Acad. Sci. U. S. A. 111, 18084e18089.
Akiyama, K., Matsuzaki, K., Hayashi, H., 2005. Plant sesquiterpenes induce hyphal
branching in arbuscular mycorrhizal fungi. Nature 435, 824e827.
Akiyama, K., Ogasawara, S., Ito, S., Hayashi, H., 2010. Structural requirements of
strigolactones for hyphal branching in AM fungi. Plant Cell Physiol. 51,
1104e1117.
under Ar, and stirred at 0 ꢂC for 1 h. After stirring at room tem-
perature for 1 h, the mixture was poured into 0.1 N HCl, and
extracted with Et2O. The organic phase was dried over anhydrous
Na2SO4, and concentrated in vacuo. The residue was subjected to
silica gel CC eluted stepwise with n-hexane and EtOAc (5% in-
crements). The 25% acetone eluate was purified by a semi-
preparative Inertsil SIL-100A HPLC column (
GL Scieneces), using isocratic elution with 2% EtOH in n-hexane at a
flow rate of 2.8 ml minꢀ1 and monitored at 236 nm to give
cyclogeranyl AD analogue 36 (1.1 mg, 0.0031 mmol, 0.2% in 2 steps,
retention time 23.5 min) and bis( -cyclogeranyl) AA’D analogue 37
(13.0 mg, 0.025 mmol, 1.5% in 2 steps, retention time 31.1 min).
(2,6,6-Trimethylcyclohex-1-en-1-yl)methyl (E)-3-((4-methyl-5-
oxo-2,5-dihydrofuran-2-yl)oxy)acrylate 36; 1H-NMR (CDCl3,
400 MHz):
1.00 (6H, s, 600-CH3 x 2), 1.44e1.49 (2H, m, H-500),
4
10 ꢁ 250 mm, 5
mm,
Al-Babili, S., Bouwmeester, H.J., 2015. Strigolactones, a novel carotenoid-derived
plant hormone. Annu. Rev. Plant Biol. 66, 161e186.
Alder, A., Jamil, M., Marzorati, M., Bruno, M., Vermathen, M., Bigler, P., Ghisla, S.,
b
-
Bouwmeester, H., Beyer, P., Al-Babili, S., 2012. The path from
b-carotene to
carlactone, a strigolactone-like plant hormone. Science 335, 1348e1351.
b
ꢀ
Besserer, A., Puech-Pages, V., Kiefer, P., Gomez-Roldan, V., Jauneau, A., Roy, S.,
ꢁ
ꢁ
Portais, J.-C., Roux, C., Becard, G., Sejalon-Delmas, N., 2006. Strigolactones
stimulate arbuscular mycorrhizal fungi by activating mitochondria. PLoS Biol. 4,
1239e1247.
Cook, C.E., Whichard, L.P., Turner, B., Wall, M.E., Egley, G.H., 1966. Germination of
witchweed (Striga lutea Lour.): isolation and properties of a potent stimulant.
Science 154, 1189e1190.
d
1.59e1.64 (2H, m, H-400), 1.67 (3H, s, 200-CH3), 1.97e2.04 (2H, m, H-
300), 2.01 (3H, t, J ¼ 1.5 Hz, 40-CH3), 4.65 (2H, s, OCH2), 5.58 (1H, d,
J ¼ 12.3 Hz, H-2), 6.11 (1H, t, J ¼ 1.2 Hz, H-20), 6.91 (1H, t, J ¼ 1.6 Hz,
H-30), 7.54 (1H, d, J ¼ 12.3 Hz, H-3); 13C-NMR could not be measured
due to the scarcity of the compound; EIMS m/z (rel. int.): 320
[M]þ(0.8), 302 (0.9), 250 (0.6), 223 (0.5), 206 (11), 153 (12), 136
(100), 121 (80), 97 (68); HRESIMS m/z 343.1536 [MþNa]þ (calcd for
ꢀ
Gomez-Roldan, V., Fermas, S., Brewer, P.B., Puech-Pages, V., Dun, E.A., Pillot, J.-P.,
ꢁ
Letisse, F., Matusova, R., Danoun, S., Portais, J.-C., Bouwmeester, H., Becard, G.,
Beveridge, C.A., Rameau, C., Rochange, S.F., 2008. Strigolactone inhibition of
shoot branching. Nature 455, 189e194.
Gutjahr, C., Parniske, M., 2013. Cell and developmental biology of arbuscular
mycorrhizal symbiosis. Annu. Rev. Cell Dev. Biol. 29, 593e617.
Kim, H.I., Kisugi, T., Khetkam, P., Xie, X., Yoneyama, K., Uchida, K., Yokota, T.,
Nomura, T., McErlean, C.S., Yoneyama, K., 2014. Avenaol, a germination stimu-
lant for root parasitic plants from Avena strigosa. Phytochemistry 103, 85e88.
Lopez-Obando, M., Ligerot, Y., Bonhomme, S., Boyer, F.D., Rameau, C., 2015. Strigo-
lactone biosynthesis and signaling in plant development. Development 142,
3615e3619.
Sato, M., Yoneda, N., Katagiri, N., Watanabe, H., Kaneko, C., 1986. General and ver-
satile synthetic method of formylacetic esters. Synthesis 8, 672e674.
Seto, Y., Sado, A., Asami, K., Hanada, A., Umehara, M., Akiyama, K., Yamaguchi, S.,
2014. Carlactone is an endogenous biosynthesis precursor for strigolactones.
Proc. Natl. Acad. Sci. U. S. A. 111, 1640e1645.
C
18H24O5Na, m/z 343.1521). Bis((2,6,6-trimethylcyclohex-1-en-1-
yl)methyl) (2E,4E)-4-(((4-methyl-5-oxo-2,5-dihydrofuran-2-yl)
oxy)methylene)pent-2-enedioate 37; 1H NMR (CDCl3, 400 MHz):
1.00 (12H, s, 600-CH3 x 2, 6000-CH3 x 2), 1.42e1.49 (4H, m, H-500 and
d
H-5000), 1.56e1.64 (4H, m, H-400 and H-4000), 1.96e2.04 (4H, m, H-300
and H-3000), 2.04 (3H, br.t, 40-CH3), 4.68 (2H, s, OCH2), 4.71 (2H, s,
OCH2), 6.20 (1H, br.t, H-20), 6.66 (1H, d, J ¼ 16.3 Hz, H-2), 6.98 (1H, t,
J ¼ 1.2 Hz, H-30), 7.51 (1H, d, J ¼ 16.3 Hz, H-3), 7.69 (1H, s, 4-CH); 13C-
Smith, S.E., Read, D.J., 2008. Mycorrhizal Symbiosis, third ed. Academic Press, San
Diego.
Tokunaga, T., Hayashi, H., Akiyama, K., 2015. Medicaol, a strigolactone identified as a
putative didehydro-orobanchol isomer, from Medicago truncatula. Phytochem-
istry 111, 91e97.
NMR (CDCl3, 100 MHz):
d 10.7, 19.2, 19.80, 19.84, 28.3, 32.88, 32.90,
33.99, 34.01, 39.2, 60.9, 61.4, 100.4, 111.0, 122.1, 131.8, 132.2, 132.9,
135.9, 136.1, 136.68,136.74, 141.0, 156.7, 165.6,167.9, 170.2; EIMS m/z
(rel. int.): [M]þ was not observed, 389 (0.9), 229 (9.2), 136 (100), 121
(40), 97 (47); HRESIMS m/z 549.2811 (calcd for C31H42O7Na, m/z
549.2828).
Ueno, K., Furumoto, T., Umeda, S., Mizutani, M., Takikawa, H., Batchvarova, R.,
Sugimoto, Y., 2014. Heliolactone,
a non-sesquiterpene lactone germination
stimulant for root parasitic weeds from sunflower. Phytochemistry 108,
122e128.
Umehara, M., Hanada, A., Yoshida, S., Akiyama, K., Arite, T., Takeda-Kamiya, N.,
Magome, H., Kamiya, Y., Shirasu, K., Yoneyama, K., Kyozuka, J., Yamaguchi, S.,
2008. Inhibition of shoot branching by new terpenoid plant hormones. Nature
455, 195e200.
4.5. Hyphal branching assay
Umehara, M., Cao, M., Akiyama, K., Akatsu, T., Seto, Y., Hanada, A., Li, W., Takeda-
Kamiya, N., Morimoto, Y., Yamaguchi, S., 2015. Structural requirements of stri-
golactones for shoot branching inhibition in rice and Arabidopsis. Plant Cell
Physiol. 56, 1059e1072.
Wada, A., Sakai, M., Kinumi, T., Tsujimoto, K., Yamauchi, M., Ito, M., 1994. Retinoids
and related compounds. 17. Conformational study of retinochrome chromo-
phore: synthesis of 8, 18-ethanoretinal and a new retinochrome analog. J. Org.
Chem. 59, 6922e6927.
Hyphal branching activity on a germinating spores of Gigaspora
marigarita Becker & Hall (MAFF 520054) was conducted as re-
ported previously (Akiyama et al., 2010). The purity of each tested
compound was checked either by analytical silica gel TLC (n-hex-
ane-EtOAc/acetone)
or
normal
(n-hexane-EtOH)-/reversed
(CH3CNeH2O)-phase HPLC before assay.
Xie, X., Yoneyama, K., Yoneyama, K., 2010. The strigolactone story. Annu. Rev.
Phytopathol. 48, 93e117.
Ye, H., Deng, G., Liu, J., Qiu, F.G., 2009. Expedient construction of the Ziegler inter-
mediate useful for the synthesis of forskolin via consecutive rearrangements.
Org. Lett. 11, 5442e5444.
Acknowledgements
This work was supported by the Program for Promotion of Basic
Please cite this article in press as: Mori, N., et al., Carlactone-type strigolactones and their synthetic analogues as inducers of hyphal branching in
arbuscular mycorrhizal fungi, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.05.012