Synthesis of fluorescent molecular probes based on cis-cinnamic acid and molecular imaging of lettuce roots

Article abstract of DOI:10.1016/j.tet.2016.08.060

We synthesized azo dye- and fluorescence-labeled cis-cinnamic acid analogues possessing inhibitory activity against lettuce root growth and a trans-isomer without bioactivity as a control probe. The radicles incubated with the azo dye-labeled analogue were stained red, with their tips especially deeply dyed. The fluorescent images of the radicles incubated with each of these molecular probes depicted that the root cap was fluorescence-stained. However, images of the control radicles prepared by staining with the trans-isomer fluorescent probe did not show emission at the root cap. These contrasts suggest specific localization of the cis-cinnamate analogue at the columella cells.

Full text of DOI:10.1016/j.tet.2016.08.060

Tetrahedron 72 (2016) 6492e6498
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of fluorescent molecular probes based on cis-cinnamic
acid and molecular imaging of lettuce roots
Hiroshi Fukuda b, Keisuke Nishikawa a, Yukihiro Fukunaga a, Katsuhiro Okuda a,
,
Kozue Kodama a, Kenji Matsumoto a, Arihiro Kano a, Mitsuru Shindo a
*
a Institute for Materials Chemistry and Engineering, Kyushu University, 6-1, Kasuga-koen, Kasuga 816-8580, Japan
b Interdisciplinary Graduate School of Engineering Sciences, Kyushu University, 6-1, Kasuga-koen, Kasuga 816-8580, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 July 2016
Received in revised form 20 August 2016
Accepted 20 August 2016
Available online 22 August 2016
We synthesized azo dye- and fluorescence-labeled cis-cinnamic acid analogues possessing inhibitory
activity against lettuce root growth and a trans-isomer without bioactivity as a control probe. The rad-
icles incubated with the azo dye-labeled analogue were stained red, with their tips especially deeply
dyed. The fluorescent images of the radicles incubated with each of these molecular probes depicted that
the root cap was fluorescence-stained. However, images of the control radicles prepared by staining with
the trans-isomer fluorescent probe did not show emission at the root cap. These contrasts suggest
specific localization of the cis-cinnamate analogue at the columella cells.
Keywords:
Allelopathy
Azo dye
Ó 2016 Elsevier Ltd. All rights reserved.
cis-cinnamic acid
Fluorescent probe
Molecular imaging
Oxime ether
1. Introduction
germinated seedlings of lettuce (Lactuca sativa L.).3 A crucial
structure responsible for the bioactivity of 1 is cis-cinnamic acid
Allelopathy is an important self-defense system in higher plants,
which is based on the production of secondary metabolites that
show inhibitory or stimulatory interactions with other plants, in-
cluding microorganisms.1 Allelochemicals would provide insights
into the molecular mechanisms of bioactivity and enable the design
of useful bioactive compounds, especially agrochemicals.2 While
numerous plant ecological and plant physiological studies on al-
lelopathy have been reported, studies on the underlying molecular
mechanisms still remain unexplored.
(cis-CA, cis-2), which also works as an effective inhibitor of lettuce
root growth similar to 1, whereas trans-cinnamic acid (trans-CA,
trans-2), which would be a cis-2 precursor as well as a common
plant metabolite, is not effective for such inhibition.4 While trans-2
is considered as a weak antagonist of auxin,5e7 cis-2 has an auxin-
like activity.8 While mechanistic studies based on molecular bi-
ology have been stated,9e12 the molecular mechanisms of the in-
hibitory activity as well as target molecules of cis-2 have not yet
been explored. Recently, we published the structureeactivity re-
lationship (SAR) studies on cis-CA growth inhibition,13 including
the substituent effect of cis-2 as well as more potent synthetic
analogues.14 We also found cis-2 selective suppressors, the bio-
activities of which were distinct from those of both auxin and anti-
auxin, suggesting mechanistic insights of auxin-independent sig-
naling pathways.10b,15 The subsequent issues for mechanistic in-
vestigations are to clarify the plant target sites for cis-2. Fluorescent
molecular probes are a powerful tool for visualization of target or
localized sites of bioactive compounds.16 We report herein the
synthesis of fluorescence-labeled cis-2 as a molecular probe as well
as its molecular imaging, which indicates the localization of cis-2 in
a lettuce radicle.
1-O-cis-Cinnamoyl-b-D-glucopyranose (1) (Fig. 1), isolated by
Hiradate and Fujii from Spiraea thunbergii as a potent alleloche-
mical, shows growth-inhibitory activity on root elongation of
Fig. 1. Cinnamates.
* Corresponding author. E-mail address: shindo@cm.kyushu-u.ac.jp (M. Shindo).
http://dx.doi.org/10.1016/j.tet.2016.08.060
0040-4020/Ó 2016 Elsevier Ltd. All rights reserved.

H. Fukuda et al. / Tetrahedron 72 (2016) 6492e6498
6493
M)a
Table 1
2. Results and discussion
Growth inhibitory activities of meta-substituted cis-2 analogues
2.1. Design of molecular probes for bioimaging
Entry
Compounds
EC50 (m
1
2
3
4
5
6
cis-3a
cis-3b
cis-3c
cis-3d
cis-4
1.1
9.3
150
110
20
Our previous SAR studies revealed that the essential structural
components for bioactivity were the cis-configuration of the al-
kene, a carboxylate, and a planar ring. Furthermore, the substituent
effect on the aromatic ring of cis-2 also disclosed that para- and
ortho-substitution tended to decrease its potency for the inhibition,
but meta-substituted cis-2 analogues were more likely to be potent.
Development of reliable molecular fluorescent probes requires that
they should show bioactivity corresponding to the original bio-
active compounds. Additionally, in order to avoid false conclusions
on target sites by non-specific binding of the probes to bio-
polymers, control experiments using biologically inactive
fluorescence-labeled analogues are required.17 Based on this con-
cept, we designed the fluorophore-possessing cis-2 analogues as
a molecular probe, and a biologically inactive trans-isomer (trans-2)
as a control probe. Our previous SAR study indicated that the meta-
substitution would be less negatively effective for the bioactivity
described above, thereby suggesting that the meta position of the
aromatic ring would be a suitable connecting position of the linker
having a fluorophore at another terminal (Figs. 2 and 3). Although
m-methoxy and m-ethoxy analogues (cis-3a and cis-3b) showed
similar bioactivity to cis-2, sterically bulky m-alkoxy analogues (cis-
3c and cis-3d) reduced the bioactivity (Table 1, entries 1e4). This
tendency demonstrated that substitutions, even at the meta posi-
tion, were sensitive to steric effects on the bioactivity. We thus
concluded that alkyl ethers were not suitable as a connector be-
tween cis-2 and a dye.
trans-4
>500
a
EC50 values are the effective concentration required to induce a half-maximum
effect against root-growth of lettuce (L. sativa cv.).
Fig. 2. Design of fluorescent probe for cis-2.
Scheme 1. Synthesis of diazo analogues cis-4 and trans-4: (a) ethyl 2-[bis(2-
isopropylphenoxy)phosphoryl]acetate, Triton B, THF, ꢀ78 ꢁC: 83%, (b) SnCl2, THF/
H2O, reflux, 92% for cis-6, 64% for trans-6, (c) NaNO2, concd HCl aq, THF, 0 ꢁC then
phenol, NaOAc, DMF/MeOH, 87% for cis-7, 68% for trans-7, (d) 10% NaOH, EtOH, rt, 56%
for cis-4, 56% for trans-4, (e) ethyl 2-(diethoxyphosphoryl)acetate, Triton B, THF,
ꢀ78 ꢁC, 60%.
modified HornereWadswortheEmmons reagent,18 followed by
reduction to afford cis-6, which in turn was subjected to a diazo-
coupling reaction with phenol to give cis-7. Finally, cis-7 was hy-
drolyzed to provide cis-4. The trans-isomer (trans-4) was also
prepared via the same procedure.
The bioactivity of cis-4 (EC50 20
enough as a probe, because the corresponding trans-4 was inactive
(EC50 >500 M) (Table 1, entries 5 and 6). Since the phenylazo
mM) was expected to be potent
m
compounds (cis- and trans-4) displayed a red color, they were ex-
pected to be visible molecular probes for molecular imaging
without the use of a fluorescent microscope. Figure 4 demonstrates
Fig. 3. meta-Substituted cis-2 analogues.
the lettuce radicles stained by 1000 mM of cis-4 and trans-4 after
incubation for 48 h. The radicles with their growth inhibited by cis-
4 were stained red as a whole, with especially their tips deeply
dyed, while radicles treated with trans-4 grew normally and were
hardly stained. This contrast of staining suggested that cis-4 would
likely be incorporated into the radicles and inhibited their growth
by localizing there; however, trans-4 would be taken up only
After screening of the other functional groups at the meta po-
sition as a connector, we found a azo group as a potential candidate
that could be easily prepared. Azo analogues 4 were prepared as
shown in Scheme 1. m-Nitrobenzaldehyde was olefinated with the

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H. Fukuda et al. / Tetrahedron 72 (2016) 6492e6498
Scheme 2. (a) Ethyl 2-[bis(2-isopropylphenoxy)-phosphoryl]acetate, Triton B, THF,
ꢀ78 ꢁC, 91%, (b) O-benzylhydroxylamine hydrochloride, pyridine, EtOH, reflux, 92%, (c)
10% NaOH aq, EtOH, rt, cis-5a: 93%, cis-5b (n¼1): 66%, cis-5c (n¼6): 85%, cis-5d (n¼10):
53%, (d) NaH, N-hydroxyphthalimide, DMF, 70 ꢁC, cis-10b (n¼1): 89%, cis-10c (n¼6):
92%, cis-10d (n¼10): 85%, (e) NH2NH2$H2O, EtOH, rt, (f) cis-8, EtOH, 50 ꢁC to rt, 89% for
cis-12b (n¼1), 79% for cis-12c (n¼6), 89% for cis-12d (n¼10).
Table 2
Growth inhibitory activities of cis-5
Fig. 4. The lettuce radicles stained by (a) no compound, (b) cis-4, and (c) trans-4 after
incubation for 48 h at 1000 mM of each compounds.
Entry
Compounds
EC50 (m
M)a,b
1
2
3
4
cis-5a
cis-5b
cis-5c
cis-5d
4.0
170
2.4
slightly or would be metabolized rapidly, resulting in no effects on
their growth. These experiments can be regarded as concise mo-
lecular imaging which can be performed without the use of ex-
pensive facilities and can be visualized by the naked eye.
>500
a
EC50 values are the effective concentration required to induce a half-maximum
effect against root-growth of lettuce (L. sativa cv.).
b
All the corresponding trans-5 analogues were inactive (EC50 >500 mM).
2.2. Synthesis of fluorescent probes
cinnamic acids were prepared as shown in Scheme 3. The com-
pounds 13aed were treated with hydrazine for deprotection of
phthalimide moiety, followed by formation of oxime ethers cis-
14aed by addition of the aldehyde cis-8. After hydrolysis of ethyl
ester, the resulting carboxylic acids cis-15aed were deprotected by
treatment with trifluoroacetic acid (TFA) to afford the amino acids,
which were treated with FITC to provide the fluorescent probes cis-
16aed. In the same manner, trans-16c was also prepared as a neg-
ative control as shown in Scheme 4.
In order to detect more precise sites of localization of the in-
hibitors with higher sensitivity and contrasts, we decided to pre-
pare fluorescent probes. Judging from the azo probe experiments
described above, we deduced that sp2 hybridized atoms were im-
portant as a connector unit, without the loss of bioactivity. We thus
adopted oxime ethers as an alternative sp2-hybridized atom con-
nector, because they can be easily constructed.19 As shown in
Scheme 2, isophthalaldehyde was mono-olefinated with the
modified HornereWadswortheEmmons reagent to give cis-8,
which was then condensed with O-benzylhydroxylamine to afford
2.3. Inhibitory activity of fluorescein-labeled cis-cinnamic
acids
an oxime ether cis-5a. The bioactivity of cis-5a was 4.0 mM despite
having sterically hindered benzyl ether (Table 2, entry 1). This may
be owing to the planar sp2 carbon linkage of oxime moiety, which is
less sterically hindered in the vicinity of the meta-position on the
aromatic ring. Encouraged by this result, we further examined the
length of the carbon-chain linker moiety. O-Alkoxyamines (11bed)
were prepared from N-alkoxyphthalimides (10bed), which were
derived from the alkylation of N-hydroxyphthalimide. The
alkoxyamines (11bed) were condensed with cis-8, followed by
hydrolysis, to afford oxime ethers (5bed). Although propyl (cis-5b)
and dodecyl (cis-5d) oxime ethers were not potent, the bioactivity
of octyl oxime ether (cis-5c) was similar to that of cis-2 (Table 2).
These results suggested dependence of bioactivity on the length of
the carbon-chain linker.
We examined the inhibitory activity of the fluorescein-labeled
cis-CA analogues against the root-growth of lettuce (Table 3).
Among the four cis-analogues, cis-16c was seen to be most potent.
Although the EC50 value (58 mM) was less than that of cis-2, it would
be suitable for bioimaging, because trans-16c was observed to have
lesser bioactivity (Table 3, entry 5). Based on these results, we de-
cided to use cis-16c and trans-16c as fluorescent and control probes,
respectively.
2.4. Molecular imaging
We then synthesized various fluorescent probes with different
linker lengths and fluorophores. After fluorophore screening, we
selected fluorescein as a fluorophore.20 The fluorescein-labeled cis-
With the biologically active fluorescent probe and the control
probe, we then examine molecular imaging using lettuce roots for
investigating the distribution of the bioactive compounds in the

H. Fukuda et al. / Tetrahedron 72 (2016) 6492e6498
6495
Table 3
Inhibitory activity of the fluorescent probes on the root-growth of lettuce seedlings
(L. sativa cv.)
Entry
Compounds
N
EC50 (mM)
1
2
3
4
5
cis-16a
cis-16b
cis-16c
cis-16d
trans-16c
1
2
6
10
6
>500
260
58
>500
>500
Scheme 3. Synthesis of fluorescent probes: (a) NH2NH2$H2O, EtOH, rt, (b) cis-8, EtOH,
rt, 90% for cis-14a; 95% for cis-14b; 74% for cis-14c; 84% for cis-14d, (c) 10% NaOH, EtOH,
rt, 87% for cis-15a, 67% for cis-15b, 87% for cis-15c, 98% for cis-15d, (d) TFA, CH2Cl2, rt,
(e) FITC, Et3N, MeOH, reflux, 54% for cis-16a, 43% for cis-16b, 65% for cis-16c, 65% for
cis-16d.
radicles. The probes cis- and trans-16c were dissolved in water at
concentrations of 50 m
M each. Pregerminated seedlings (at 25 ꢁC in
water in the dark) of lettuce (L. sativa cv. Great Lakes 366) were
stained by incubation with these fluorescent-labeled compound
solutions for 48 h at 25 ꢁC in the dark. After being washed with
ethanol for 30 s, the radicles were placed on a glass slide and
monitored by a fluorescent microscope. We focused on the tip of
the radicles, where the images (200ꢂ) showed the fluorescence
emission by cis-16c (Fig. 5B), but no emission was seen by the
unstained control (Fig. 5A) and trans-16c (Fig. 5B). This contrast
obviously indicated the specific emission by cis-16c.
Fig. 5. Images of the tips of radicles. A: control (200ꢂ): (a) bright; (b) fluorescent
image, B: stained by cis-16c and trans-16c (200ꢂ): (a) bright; (b) fluorescent image.
Conditions: 200ꢂ (exposure time: left 1/230, right 1/35).
To obtain more precise images and to determine the sites of
localization, we observed the radicles treated by the fluorescent
probe cis-16c using confocal fluorescence microscopy. As shown in
Figure 6a, the fluorescence was observed at the region of the root
cap. The magnified photo (Fig. 6b) indicated that the fluorescence
could be traced to the granules, the diameter of which was ap-
proximately 2
mm. Judging from these fluorescence images, we
speculated that amyloplasts in the columella cells may be stained
by the fluorescent probe.
2.5. Competition experiments
To confirm that the localizing site of cis-16c in the radicles was
identical to that of cis-2, competitive inhibition between cis-2 and
Scheme 4. Synthesis of trans-16c: (a) NH2NH2$H2O, EtOH, rt, (b) trans-8, EtOH, rt,
quant., (c) 10% NaOH, EtOH, rt, 97%, (d) TFA, CH2Cl2, rt, (e) FITC, Et3N, MeOH, reflux,
Fig. 6. Fluorescent images of a radicle stained by cis-16c with a confocal fluorescence
21%.
microscope.

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H. Fukuda et al. / Tetrahedron 72 (2016) 6492e6498
Fig. 7. Competitive inhibition. Fluorescent images of lettuce radicle treated with (a) a mixture of cis-16c (50
mM) and cis-2 (5e50
mM), (b) a mixture of cis-16c (50
mM) and trans-2
(5e50 M). Fluorescent images of (c) the cis-16c (50 M)-stained lettuce radicle, (d) the cis-16c (50 M)-stained radicle followed by treatment with cis-2 (50 m
m
m
m
M).
cis-16c was carried out. Germinated lettuce seeds were treated
biologically inactive trans-13c did not. If cis-2 works in the colu-
mella cells which are considered to be responsible for gravity
sensing of the root,21 it might affect gravitropism of roots, either
negatively or positively. While the confocal microscopy may only
shed light on the surface layers of the radicle,22 these molecular
imaging studies would be very useful in elucidating the mecha-
nisms of cis-2.
with a mixture of cis-16c (50 mM) and cis-2 (0e50 mM). After 24 h of
incubation at 25 ꢁC, the surfaces of the radicles were washed with
ethanol, and fluorescence was observed. As shown in Figure 7a, the
fluorescence reduced at 25 mM of cis-2 and disappeared with 50 mM
of cis-2, whereas trans-2 did not affect the fluorescence of cis-16c.
For the same purpose, the germinated lettuce seeds were in-
cubated with cis-16c (50
added at a concentration of 50
m
M) for 24 h at 25 ꢁC, and then cis-2 was
mM. After incubation for 2 h, surfaces
4. Conclusions
of radicles were washed with ethanol. Figure 7d shows the disap-
pearance of fluorescence by addition of cis-16c, suggesting that cis-
16c was competitively excluded by cis-2.
We synthesized the first fluorescein-labeled cis-cinnamic acid
analogue possessing lettuce root growth inhibitory activity, and its
trans-isomer with no bioactivity as a control probe. The fluorescent
images of the radicles which were incubated with each of those
molecular probes depicted that the root cap, presumably columella
cells, was fluorescence-stained while those of control radicles
treated with or without trans-isomer fluorescent probes did not
show the fluorescence at the root cap. These contrasts suggested
specific localization of the cis-cinnamate analogue at this site. Al-
though we demonstrated that the columella cells are a possible
target of cis-cinnamic acid, the underlying route of localization is
still unknown. Furthermore, it is not clear if the localization of cis-2
in columella cells is directly related to the growth inhibition of
radicles. For further clarifications regarding the target site and the
inhibitory mechanism, biochemical and molecular biological
studies are additionally required. This chemical probe study thus
provides a useful foundation to further explore the biological
mechanisms of cis-2.
3. Discussion
Fluorescent probes with fluorescein connected by a linker were
synthesized. Their bioactivities were dependent on the carbon-
chain linker length as well as the functional group, which is an
oxime ether in this case, connecting the linker to cinnamic acid.
Among the several fluorescent cis-cinnamic acid analogues pre-
pared, cis-16c with an eight-carbon chain linker showed the
highest bioactivity. A hydrophobic carbon chain linker would in-
crease non-specific binding to proteins, but also improve the up-
take of the compounds into plant tissues. A balance of these effects
would therefore be important. We considered cis-16c to be an ef-
fective fluorescent probe, because the corresponding fluorescent
trans-analogue trans-16c was biologically inactive. The trans-ana-
logue is useful as a control probe, which could enable discrimina-
tion between non-specific binding of the probes. The tip of the
radicles was stained only with cis-16c, and not with trans-16c.
Confocal microscopy revealed the root cap, most likely the colu-
mella cells, to be stained. The competitive inhibition studies clearly
demonstrated that cis-16c competed with cis-2 in the root cap,
but not with trans-2. Consequently, these contrasts suggested that
cis-16c specifically localized in the columella cells, whereas
5. Experimental
5.1. General
1H NMR, 13C NMR were measured in CDCl3 solution using JEOL
JNM-AL-400 spectrometer (1H NMR at 400 MHz, 13C NMR at

H. Fukuda et al. / Tetrahedron 72 (2016) 6492e6498
6497
100 MHz) and JNM-ECA-600 spectrometer (1H NMR at 600 MHz,
13C NMR at 150 MHz) as the referenced standard (1H NMR at
0.00 ppm (TMS), 13C NMR at 77.0 ppm (CDCl3)). Chemical sifts are
reported in ppm. Peak multiplicities are used the following ab-
breviations: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet;
sept, septet; m, multiplet; br, broadend. IR spectra were recorded
on Shimadzu FTIR-8300 and IR Prestige-21 spectrometers. Mass
spectra and high resolution mass spectra were obtained on a JMS-
K9 (GCeMS), JEOL JMS-700 and Shimadzu LCMS-2010EV mass
spectrometers. Melting points were measured with a Yanaco MP-
500D apparatus and are uncorrected. Thin-layer chromatography
(TLC) was performed on precoated plates (0.25 mm, silica gel Merck
60F245). Column chromatography was performed on silica gel
(Kanto Chemical Co., Inc.). Preparative HPLC was performed on
a system utilizing a system utilizing a JASCO PU-2087 Intelligent
Pump with Dynamic Mixer MX-2080-32 and UV detector UV-2075
and RI detector RI-2031. All reactions were performed under an air
atmosphere unless otherwise noted, and dry dichloromethane
(CH2Cl2), diethyl ether (Et2O), and tetrahydrofuran (THF) were
purchased from Kanto Chemical Co., Inc., and other solvents were
distilled. Unless otherwise noted, reagents were obtained from
chemical sources and without further purification.
147.7 (d), 155.9 (s), 165.9 (s); IR (KBr) 3454, 1713, 1631 cmꢀ1; MS
(FAB) m/z 447 ([MþH]þ); HRMS (FAB) m/z calcd for C25H38N2O5
447.2859, found 447.2859 ([MþH]þ).
5.2.4. General procedure of hydrolysis of ethyl cis-cinnamate ana-
logues. To a solution of cis-14c in EtOH was added 10% NaOH at
room temperature. After 12 h, the mixture was adjusted to pH 1.0
with 1 M HCl, and then extracted with EtOAc. The organic layer was
washed with brine, dried over MgSO4, filtered, and concentrated in
vacuo. The residue was purified by recrystallization to afford a cis-
cinnamic acid derivative.
5.2.5. (Z)-3-((E)-((8-(tert-Butoxycarbonylamino)octyloxy)-imino)
methyl)phenylacrylic acid (cis-15c). 87% as colorless needles. Mp
46e50 ꢁC (EtOAc/hexane¼10%); 1H NMR (CD3OD, 600 MHz)
d:
1.32e1.45 (m, 19H), 1.69 (quintet, J¼7.2 Hz, 2H), 3.01 (t, J¼7.2 Hz,
2H), 4.13 (t, J¼7.2 Hz, 2H), 6.00 (d, J¼12.6 Hz,1H), 6.97 (d, J¼12.6 Hz,
1H), 7.34 (t, J¼7.8 Hz, 1H), 7.56 (d, J¼7.8 Hz, 1H), 7.59 (d, J¼7.8 Hz,
1H), 7.76 (s, 1H), 8.07 (s, 1H); 13C NMR (CDCl3, 150 MHz)
d: 26.9 (t),
27.8 (t), 28.8 (q), 30.2 (t), 30.3 (t), 30.4 (t), 30.9 (t), 41.3 (t), 75.3 (t),
79.7 (s), 122.0 (d), 128.0 (d), 129.3 (d), 129.4 (d), 131.8 (d), 133.8 (s),
137.0 (s), 142.5 (d), 149.1 (d), 158.5 (s), 169.6 (s); IR (KBr) 1697,
1631 cmꢀ1; MS (FAB) m/z 419 ([MþH]þ); HRMS (FAB) m/z calcd for
5.2. Synthesis of fluorescein-labeled probes
C
23H35N2O5 419.2546, found 419.2550 ([MþH]þ).
5.2.1. Synthesis of (Z)-ethyl 3-(3-formylphenyl)acrylate (cis-8). To
a solution of isophthalaldehyde (1.0 g, 7.4 mmol) and ethyl 2-[bis(2-
isopropylphenoxy)phosphoryl]acetate (1.0 g, 2.5 mmol) in THF
(140 mL), a solution of Triton B (40% in MeOH, 1.3 mL, 3.2 mmol) in
THF (30 mL) was added dropwise at ꢀ78 ꢁC under an argon at-
mosphere. After 14 h, the mixture was quenched with saturated
aqueous NH4Cl and extracted with EtOAc. The organic layer was
washed with H2O, saturated aqueous NaHCO3 and brine, dried over
MgSO4, filtered, and concentrated in vacuo. The crude product was
purified by silica gel column chromatography (EtOAc/hexane 5%,
then 8%) to give cis-8 (91%) as a colorless oil. 1H NMR (CDCl3,
5.2.6. General procedure for synthesis of fluorescein-labeled probes
(cis-16). To a solution of 15 (0.28 mmol) in CH2Cl2 (8 mL) was
added TFA (2.3 mL, 29.5 mmol) dropwise at room temperature
under an argon atmosphere. After 1.5 h, the solvent was removed in
vacuo. The resultant crude oil (TFA salt) was used in the next re-
action without further purification. To a solution of the crude TFA
salt in CH2Cl2eMeOH (25:1, 42 mL) were added fluorescein iso-
thiocyanate (FITC, 0.28 mmol) and Et3N (4.7 mmol) at room tem-
perature under an argon atmosphere. The mixture was refluxed for
23 h. After being cooled to room temperature, the solvent was re-
moved in vacuo. The residue was purified by reverse-phase HPLC
(Nacalai Tesque COSMOSIL 5C18-AR-II, 250 mmꢂ20 mm) to give cis-
16. In the 13C spectra, several peaks due to quaternary carbons were
missed, probably because fluorescein would be in equilibrium with
the lactone form. cis-16c: 65% as yellow needles (reverse-phase
HPLC, MeCNeH2O containing 0.5% TFA, 55:45). Mp 108e109 ꢁC
400 MHz)
d
: 1.25 (t, J¼7.2 Hz, 3H), 4.18 (q, J¼7.2 Hz, 2H), 6.06 (d,
J¼12.8 Hz, 1H), 7.02 (d, J¼12.8 Hz, 1H), 7.53 (t, J¼7.6 Hz, 1H), 7.84 (d,
J¼7.6 Hz, 1H), 7.86 (d, J¼7.6 Hz, 1H), 8.08 (s, 1H), 10.03 (s, 1H); 13C
NMR (CDCl3, 100 MHz) d: 14.0 (q), 60.4 (t), 121.5 (d), 128.6 (d), 129.7
(d), 131.0 (d), 135.3 (d), 135.7 (s), 136.1 (s), 141.6 (d), 165.6 (s), 191.9
(d); IR (NaCl Neat) 1703 cmꢀ1; MS (EI) m/z 204 (Mþ),175 (Mþ-CHO),
159 (MþꢀOEt, 100%); HRMS (EI) m/z calcd for C12H12O3(Mþ)
204.0786, found 204.0783.
(toluene); 1H NMR (CD3OD, 600 MHz)
d: 1.38e1.45 (m, 8H),
1.66e1.73 (m, 4H), 3.50 (m, 2H), 4.04 (t, J¼7.2 Hz, 2H), 5.90 (d,
J¼12.6 Hz, 1H), 6.45 (d, J¼8.4 Hz, 2H), 6.58 (m, 4H), 6.86 (d,
J¼12.6 Hz, 1H), 7.03 (d, J¼8.4 Hz, 1H), 7.24 (t, J¼7.8 Hz, 1H), 7.45 (d,
J¼7.8 Hz,1H), 7.48 (d, J¼7.8 Hz,1H), 7.65 (m, 2H), 7.97 (s,1H), 8.02 (s,
5.2.2. General procedure of synthesis of cis-14. Hydrazine mono-
hydrate (5.1 mmol) was added to a solution of 13 (1.7 mmol) in
EtOH (9 mL) at room temperature under an argon atmosphere.
After 12 h, the solvent was removed in vacuo, diluted with Et2O,
filtered and concentrated. The resultant oil was submitted to the
next reaction without further purification. A solution of (Z)-ethyl 3-
(3-formylphenyl)acrylate (cis-8) (1.71 mmol) in dry EtOH (3.5 mL)
was slowly added to a solution of the alkoxyamine in dry EtOH
(3.5 mL) at room temperature. After 12 h, the solvent was removed
in vacuo. The crude product was purified by silica gel column
chromatography (EtOAc/hexane, 10:90) to give cis-14aed.
1H); 13C NMR (CD3OD,150 MHz)
d: 26.9 (t), 27.9 (t), 29.9 (t), 30.2 (t),
30.36 (t), 30.42 (t), 49.9 (t), 75.2 (t), 103.5 (d), 111.5 (s), 113.6 (d),
113.7 (d), 120.3 (d), 122.0 (d), 125.6 (s), 128.0 (d), 129.3 (d), 129.4 (d),
130.3 (d), 131.8 (d), 133.8 (s), 137.8 (s), 142.5 (d), 149.1 (d), 150.1 (s),
153.2 (s), 154.2 (s), 171.2 (s), 173.0 (s); IR (KBr) 3448, 1749, 1730,
1608 cmꢀ1; MS (FAB) m/z 706 (M-Hþ); HRMS (FAB) m/z calcd for
C
39H36N3O8S 706.2223, found 706.2234 (M-Hþ).
5.3. Root growth inhibition assay
The assay was performed according to Hiradate’s method3 with
5.2.3. (Z)-Ethyl 3-((E)-((8-(tert-butoxycarbonylamino)-octyloxy)im-
minor modifications. A
dish. Methanol solution of test compounds and growth inhibitors
were added into the dish simultaneously (70
L each, ꢂ10 con-
centration for desired concentration). The solvent was removed
from filter paper under reduced pressure. After addition of 700
distilled water, six pre-germinated Lettuce seedlings (L. sativa cv.
Great Lakes 366) were placed on the filter paper. The pre-
germination was induced with distilled water at 25 ꢁC, 60% rela-
tive humidity for 24 h in a dark. Two dishes were prepared for each
f27 filter paper was placed in a glass Petri
ino)methyl)phenylacrylate (cis-14c). 74% as a colorless oil; 1H NMR
(CDCl3, 400 MHz)
d
: 1.23 (t, J¼7.2 Hz, 3H), 1.32e1.45 (m, 19H),
m
1.67e1.70 (m, 2H), 3.07e3.11 (m, 2H), 4.14e4.19 (m, 4H), 5.98 (d,
J¼12.8 Hz, 1H), 6.94 (d, J¼12.8 Hz, 1H), 7.34 (t, J¼8.0 Hz, 1H), 7.54 (d,
J¼8.0 Hz, 1H), 7.57 (d, J¼8.0 Hz, 1H), 7.73 (s, 1H), 8.07 (s, 1H); 13C
m
L
NMR (CDCl3, 100 MHz) d: 14.0 (q), 25.7 (t), 26.6 (t), 28.4 (q), 29.0 (t),
29.1 (t), 29.3 (t), 30.0 (t), 40.5 (t), 60.3 (t),74.3 (t), 78.8 (s), 120.7 (d),
127.2 (d), 128.0 (d), 128.2 (d), 130.6 (d), 132.2 (s), 135.3 (s), 142.0 (d),

6498
H. Fukuda et al. / Tetrahedron 72 (2016) 6492e6498
concentration (n¼12). The dishes were incubated at 25 ꢁC, 60%
relative humidity for 48 h in a dark. The root growth was evaluated
by measuring the length of each root and compared to that of un-
treated control.
References and notes
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We thank Prof. Y. Fujii, Dr. N. Wasano (Tokyo University of Ag-
riculture and Technology) and Prof. M. Morita (Nagoya University)
for helpful discussions. This work was supported by the Program
for Promotion of Basic and Applied Research for Innovations in the
Bio-oriented Industry (BRAIN) from the Bio-oriented Technology
Research Advancement Institution, Science and Technology Re-
search Promotion Program for Agriculture, Forestry, Fisheries and
Food Industry (No. 25029AB), Japan Society for the Promotion of
Science (JSPS) KAKENHI Grant numbers JP26293004, JP2667003,
JP16H01157 (M.S.), and JP23790131 (K.M.).
Supplementary data
22. To obtain fluorescent images of the inner layers, special techniques of making
the section samples without efflux of the cytoplasm would be required.
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Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.tet.2016.08.060.