Photo-controlled release of fipronil from a coumarin triggered precursor

Article abstract of DOI:10.1016/j.bmcl.2017.03.091

Developing efficient controlled release system of insecticide can facilitate the better use of insecticide. We described here a first example of photo-controlled release of an insecticide by linking fipronil with photoresponsive coumarin covalently. The generated coumarin-fipronil (CF) precursor could undergo cleavage to release free fipronil in the presence of blue light (420?nm) or sunlight. Photophysical studies of CF showed that it exhibited strong fluorescence properties. The CF had no obvious activity against mosquito larvae under dark, but it can be activated by light inside the mosquito larvae. The released Fip from CF by blue light irradiation in vitro retained its activity to armyworm (Mythimna separate) with LC₅₀ value of 24.64?μmol?L^?1. This photocaged molecule provided an alternative delivery method for fipronil.

Full text of DOI:10.1016/j.bmcl.2017.03.091

Accepted Manuscript
Photo-controlled Release of Fipronil from a Coumarin Triggered Precursor
Zhenhong Gao, Pengtao Yuan, Donghui Wang, Zhiping Xu, Zhong Li, Xusheng
Shao
PII:
S0960-894X(17)30353-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.03.091
BMCL 24843
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
4 March 2017
29 March 2017
30 March 2017
Please cite this article as: Gao, Z., Yuan, P., Wang, D., Xu, Z., Li, Z., Shao, X., Photo-controlled Release of Fipronil
from a Coumarin Triggered Precursor, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/
10.1016/j.bmcl.2017.03.091
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Zhenhong Gao, Pengtao Yuan, Donghui Wang, Zhiping Xu, Zhong Li and Xusheng Shao*

Bioorganic & Medicinal Chemistry Letters
Photo-controlled Release of Fipronil from a Coumarin Triggered Precursor
Zhenhong Gao,a Pengtao Yuan,a Donghui Wang,b Zhiping Xu,a Zhong Li,a and Xusheng Shaoa,
aShanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Shanghai, 200237, China
bCollege of Life Sciences, Peking University, Beijing 100871, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Developing efficient controlled release system of insecticide can facilitate the better use of
insecticide. We described here a first example of photo-controlled release of an insecticide by
linking fipronil with photoresponsive coumarin covalently. The generated coumarin-fipronil
(CF) precursor could undergo cleavage to release free fipronil in the presence of blue light (420
nm) or sunlight. Photophysical studies of CF showed that it exhibited strong fluorescence
properties. The CF had no obvious activity against mosquito larvae under dark, but it can be
activated by light inside the mosquito larvae. The released Fip from CF by blue light irradiation
in vitro retained its activity to armyworm (Mythimna separate) with LC50 value of 24.64 μmol L-
1. This photocaged molecule provided an alternative delivery method for fipronil.
Keywords:
Photocaged
Fipronil
Coumarin
Insecticide
Light
2009 Elsevier Ltd. All rights reserved.
———
Corresponding author. Tel: +86-21-64253967; fax: +86-21-64252603; e-mail: shaoxusheng@ecust.edu.cn

Direct delivery of pesticide has many uncontrolled adverse
effects such as high toxicity, large application amount and
decomposition of active ingredients.1 To address these issues,
methods for conditionally controlling release are needed, since
controlled release of pesticides has substantial advantages
including enhanced bioavailability, prolonged length of activity,
improved physiochemical properties, reduced phytotoxicity and
lowering of the environment secondary effects.1 The previously-
developed pesticide-release systems took advantage of
stability, light wavelength, efficiency of desire cleavage reaction,
avoidance of photodamage/photodegradation and the toxicity of
cleavage product of caging group.36 Most importantly, a PPG
must be subtly selected. Various PPGs have been developed in
the past decades. The well-studied and frequently used PPGs
include
o-nitrobenzyl,
coumarin-4-ylmethyl
and
p-
hydroxylphenacyl.36 The coumarin phototriggers attract the most
attentions recently due to their superior features, such as longer
absorption wavelength, large molar coefficients, fast release rates,
improved stability, high biocompatibility and fluorescent
emitting.36 Considering the substituent effects on this cages, 7-
dialkylamino substituted coumarin was selected here since it has
the absorption band at biologically benign region. Thus, the
diethylamino-coumarin-4-ylmethyl caged fipronil was prepared
for our subsequent investigations (Figure 1).
nanotechnology,1,6,7 microencapsulation2-4,8
and
polymer
science.5,9 These methodologies provided slow release of active
ingredients, but the release process cannot be easily regulated at
spatial and temporal resolution. In this context, a more precise
controlled technology is desired to the spatiotemporal control
over the pesticide release.
The recent fast-developing photo-triggered technology
provides possibilities for controlled release which promises better
remote, temporal and spatial control than conventional
methods.10,11 To realize such a photochemical process, a photo-
labile protecting (photocaging) group (PPG) is usually used to
covalently couple with the molecule being released, generating a
caged precursor that can undergo cleavage under light.12 The
advent of photocaged conception creates great opportunities for
spatiotemporal manipulation of a variety of processes in chemical,
biological and material science.10,13 Excellent reviews include
light-triggered catalysts,14 organic surfaces,15 biomedical
materials13 and photocontrol of cellular chemistry16-18 or gene
expression.19,20 Normally, such photoresponsive system has
blocked function in the caged state and can be irreversibly
activated upon irradiation to release the functional ingredients.
Due to the encouraging advances, this technology thus far has
been well applied for the photo-regulated release of bioactive
molecules, such as neurotransmitters, enzyme substrates,
pheromones, lipids, and second messengers.10, 21, 22
Turning attention back to agrochemical science, the
combination of PPG with pesticide provided a promising release
method with pioneering work done by N. D. Pradeep Singh et al.
Example applications included photo-controlled release of 2, 4-D
herbicide,23-25 plant growth regulators,26 sex pheromone27 and
plant hormone salicylic acid.28 However, photo-controlled release
of an insecticide molecule has not yet been described.
Figure 1. Molecular design of photoresponsive coumarin-caged fipronil and
its synthetic route. Reagents and condition: a) 1. SeO2, Ar, p-xylene, reflux,
53 h; 2. NaBH4, CH3OH, r.t., 4 h, 26%. b) p-nitrophenyl chloroformate,
DIPEA, Ar, dry dichloromethane, r. t., 20h, 50%. c) fipronil, DMAP, Ar, dry
dichloromethane, r. t., 48h, 24%.
Fipronil (Fip) is a phenylpyrazole insecticide widely used for
seed-treatment, sanitary pest control and animal health, but its
application was strictly restricted in some districts due to high
toxicity to non-target wildlife.29-32 Fipronil is stable at dark in
mildly acidic to neutral water, but is prone to undergo photolysis
or biological oxidation or reduction in vivo to form desulfinyl,
sulfone, sulfide or amide metabolites.29,33 Its half-life time of
photodegradation is 0.33 day (Florida summer sunlight).34 In a
specific case, the persistence of fipronil reduced significantly
when exposed to sunlight.35 As a consequence, two attempts were
previously made for controlling release of fipronil using
microencapsulation of in situ polymerization or biocompatible
silica nanocapsules. Herein, we demonstrated a photochemical
method that releases insecticidal fipronil using pulses of light,
which enables more precise control and real-time activation. The
fipronil was caged using coumarin as photoremovable protecting
group. The caged compound is stable and would release fipronil
only upon irradiation.
The photoresponsive CF was prepared from a three-steps
route starting from commercially available 7-(diethylamino)-4-
methyl-2H-chromen-2-one 1 (Figure 1). Oxidation of 1 with
Selenium dioxide and the following reduction by Sodium
borohydride (NaBH4) afforded alcohol intermediate 2 (Cou).
Alcohol 2 then condensed with p-nitrophenyl chloroformate to
furnish intermediate 3 under catalysis of DIPEA. Finally, 3
reacted with Fip at presence of DMAP to present the target caged
product CF.
With successful obtaining of coumarin-caged fipronil, its
photophysical properties were studied first. The absorption and
emission maximum wavelength, molar absorption coefficient,
Stokes shift and fluorescence quantum yield of CF were
summarized in Table 1. In UV-Vis spectra, CF features two
obvious absorption bands centered at 390 nm and 245 nm (Figure
2, A), which corresponded to the absorption of coumarin and
fipronil fragments, respectively. The maximum emission
wavelength of CF is about 475 nm and the Stokes shift is 85 nm
(Figure 2, B). CF has high fluorescence quantum yield (Φf = 0.2),
which facilitate its application in imaging study of tested targets.
The existence of an amino group in fipronil provides the
possibility for the installation of PPG. Covalent linking of a PPG
with an active molecule is a straightforward way to generate a
photocaged molecule. Many factors should be taken into account
in designing such a photocaged molecule, such as the solubility,

Table 1.UV-Vis and fluorescence data for CF
degradation pathway under the biological testing conditions, the
hydrolytic and enzymatic stability (see Supplementary Material)
of CF were also investigated. No significant decomposition (<
5%) of CF was observed when CF was kept at dark for two
weeks or was incubated with homogenized mixture of mosquito
larvae, guaranteeing the CF can only be activated using light.
Together, all the above results indicated that our uncaging
strategy have good dark stability and can successfully release
insecticidal Fip upon light irradiation.
UV-Vis
Fluorescence
c
εb(104M
-1cm-1)
λmax
(
stokes
shiftd(nm)
compound
CF
λmaxa(nm)
φfe
nm)
390
1.6
475
85
0.2
aMaximum absorption wavelength. bMolar absorption coefficient (M-1cm-1) at
the wavelength of 390 nm. cMaximum emission wavelength. dDifference
e
between wavelengths of the maximal emission and excitation. Fluorescence
quantum yield.
CN
O
S
CF3
O
Cl
N
N
A
CF
HN
Cl
To measure the efficiency of the light-induced release of Fip
from the cage, we irradiated CF with biologically benign blue
light (420 nm, LED). Significant UV-Vis and fluorescence
spectra changes were observed at different intervals of time under
irradiation (420 nm) (Figure 2, A). The intensity of peak
absorption at 390 nm corresponding to the CF gradually
F3
C
O
O
O
N
CN
OCH3
O
O
Cl
N
N
OH
S
CF3
NH2
F3
C
Cl
N
O
N
O
O
Fip
Cou
decreased
and
maximum
absorption
wavelength
75 min
65
55
45
35
25
15
bathochromically shifted to 395 nm after 50 min irradiation.
Similar emission intensity decrease and bathochromic shift were
also detected in the fluorescence spectra of CF (Figure 2, B),
indicating the chemical reactions occurred upon irradiation. The
shift in the peak absorbance and emission to longer wavelength
was caused by the generation of photolysis product Cou whose
maximum absorption and emission wavelength were 395 nm and
485 nm (Figure 2, D and E), respectively.
5
0
B
CN
CN
0 min
O
S
CF3
O
Cl
N
N
OCH3
OH
O
S
CF3
A
Cl
N
N
B
2.0
1.5
1.0
0.5
0.0
10 min
20 min
30 min
40 min
50 min
60 min
70 min
HN
Cl
0 min
0.5
0.0
F3C
NH2
O
O
F3
C
Cl
N
O
O
10 min
20 min
30 min
40 min
50 min
N
O
O
800
400
0
O
N
400
60 min
400
500
600
200
300
400
500
Wavelenght(nm)
30 min
Wavelenght(nm)
1000
1.0
3
2
1
0
Fluorescence of 2
E
UV-Vis of Fip
D
UV-Vis of 2
C
800
600
400
200
0
0 min
0.5
0.0
400
500
600
nm)
700
200
300
400
500
200
300
400
wavelength
(
Wavelenght(nm)
Wavelenght (nm)
Figure 3. UPLC tracking of photolysis process of CF in CH3OH/H2O (50:50
v/v) under blue light (420 nm, 1 w) (A) and under sunlight (B).
Figure 2. A: UV-Vis absorption of CF at regular intervals of irradiation in
MeOH/H2O (50:50) (5.6×10-5 M). B: Emission spectra of CF at regular
intervals of irradiation in MeOH/H2O (50:50) (5.6×10-6 M). C: UV-Vis
absorption of Fip. D: UV-Vis absorption of Cou. E: Emission spectra of Cou.
100
40
A
off
on
B
80
60
40
20
0
off
on
off
on
off
on
The UPLC analysis was then conducted to monitor the
photoreaction process and identify the photolysis products
(Figure 3). The UPLC chart at regular intervals of irradiation
time clearly showed the photolysis process of CF. The gradual
peak decrease at Rt = 8.0 min and increase at Rt = 7.0 min
demonstrated the photocleavage of CF and generation of Fip,
respectively. Besides Fip, two other photolysis products Cou and
methylation product of Cou were confirmed by matching the
NMR spectra of isolated products with authentic samples. The
methylation reaction occurred between Cou and the solvent
methanol.
20
off
on
0
0
20
40
60
80
0
20
40
60
80
Irradiation time (min)
Irradiation time (min)
Figure 4. Photo-controlled release of Fip irradiated by blue light. A) Time-
dependent release of Fip upon blue light (420 nm) irradiation. B) The release
process can be turned on and off by switching the light on and off.
To evaluate the efficiency of photo-induced release in living
organism and the subsequent toxic effects, the bioassay was
conducted using mosquito larvae (Table 2) because of its
transparent body structure that allows light easy to penetrate. In
the dark, CF alone showed low activity against mosquito larva
suggesting the inefficiency of the caged compound. Upon
exposure to blue light (420 nm), the activity increased
Nearly complete Fip release (95%) was achieved in 80 min
and approximately half of the Fip was released in 11 min of light
irradiation (Figure 4, A). The Fip release can be turned on and off
with the light exposure or not (Figure 4, B), suggesting that the
release proceeded only at the presence of light. To exclude other

dramatically with nearly 25-folds activity enhancement (LC50
=
Time-dependent activity of CF upon blue light (420 nm) irradiation. Each
value given as mean ± SE of three replicates.
0.56 μmol L-1) (Figure 5, A and B). When subjected to sunlight, a
similar activity enhancing trend was observed with a slight
higher activity (LC50 = 0.37 μmol L-1) than that irradiated by blue
light (Figure 5, A and B), indicating that natural sunlight can be
used as a clean light source to release the Fip. As comparison, the
effect of photolysis product Cou or blue light alone were
investigated and no obvious poisoning sign was observed on
these two treatments. The increased activity correlated closely
with the increased time of light exposure (Figure 5, C). Light do
not have any turbulence to the Fip activity as the activities were
almost the same at dark or irradiated by blue light or sunlight.
Slight decrease of Fip potency under sunlight may partly attribute
to its tiny photodecomposition since Fip is sensitive to sunlight.
These results proved that the activity was mainly from the
released Fip and the CF can only be activated by light irradiation.
In order to explore the possibility of releasing Fip in larger
and opaque insect, activity to armyworm (Mythimna separate)
was screened using the above described method. However, the
activity is low due to the fact that blue light cannot penetrate into
the armyworm. Therefore, a modified method was employed
alternatively in which CF was irradiated by blue light prior to be
administrated to armyworm. The released Fip retained its activity
with LC50 value of 24.64 μmol L-1. Such application method has
its own rationale, because part of Fip would be released under
natural sunlight before the caged Fip reaches the target insects in
field application. The CF-light-treated armyworms have the
similar poisoning signs with that of Fip-treated ones (Figure 6),
which suggested that Fip was the main toxic ingredient among
the photolysis products of CF.
Table 2. Insecticidal activity of CF, Fip and Cou against
Aedes albopictus larvae.
LC50a (μmol L-1)
Treatment
Mythimna
separatec
Aedes larvaeb
CF-bluelightd
CF-sunlighte
CF-dark
0.56
0.37
24.64
------
13.73
0.11
> 50
Fip-blue light
Fip-sunlight
Fip-dark
------
0.12
------
0.11
6.82
Figure 6. Poisoning signs of CF-treated armyworms plus light (A), CF-
Cou-blue light
Cou-sunlight
Cou-dark
>50
>100
treated armyworms at dark (B), Fip-treated armyworms (C) and blank control
(D).
>50
------
>50
>100
The blue fluorescence of CF makes it possible for the
visualization study on the treated insects. The fluorescence and
confocal microscopy images were collected as depicted in Figure
7 and 8, respectively. After treatment of mosquito larvae with CF
at dark, CF mainly accumulated in the gastric caeca and guts
lumen37 (Figure 7, A and Figure 8, A). When CF and light (blue
light or sunlight) were co-applied, the release of Fip led to the
death of larvae and the fluorescent CF and Cou dispersed
throughout the whole body (Figure 7, B and C, and Figure 8, B),
indicating the tissue damage caused by Fip. For the Cou-treated
mosquito larvae, the Cou was also mainly located in the digestive
system.
CK-blue light
CK-dark
no activity
no activity
no activity
no activity
aLC50 is the median lethal concentration. bLight was imposed after 1 h
treatment of Aedes larvae with CF. cLight was impose to the CF directly
before administrated to Mythimna separate. dIrradiated under visible light
(420 nm). eIrradiated under sunlight.
>100
B
15
10
5
A
100
50
0
Bluelight (410m)
Sunlight
Dark
fipronil
CF-sunlight
CF-light
CF-dark
In conclusion, fast and efficient light-triggered release of
fipronil was achieved using coumarin as a cage. To our
knowledge, this is the first example of photo-controlled release of
an insecticide molecule. The coumarin-fipronil precursor can
release fipronil in vivo in mosquito larvae upon blue light or
sunlight irradiation, providing the possibility of spatiotemporal
insecticide release. Additionally, due to the dual role of CF being
a photoresponsive molecule and fluorescent dye, imaging-guided
insecticide delivery is an attractive possibility, which might be
useful to the toxicology study of an insecticide. The caged Fip
would facilitate the understanding of action mechanism of
fipronil in living organism with spatiotemporal resolution. The
relative shorter absorption wavelength of the current light-
triggered insecticide make it difficult to achieve the in vivo
release in opaque insects, but this can be conquered by using
auxiliary equipment, such as optical fiber, to pump the light
inside the body. Furthermore, structural modifications are needed
0
Fip
CF
Cou
1
2
3
Concentration (μmol/L)
100
50
0
C
CF 0.5 mg/L
CK
0
2
4
6
8
10
Irradiation time (min)
Figure 5. A: The activity of CF, Fip and Cou with or without irradiation
against Aedes albopictus larvae. B: Mortality of CF at dark and irradiated
under visible light (420 nm) or sunlight against Aedes albopictus larvae. C:

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Acknowledgments
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