Design, synthesis and aphicidal activity of N-terminal modified insect kinin analogs

Article abstract of DOI:10.1016/j.peptides.2014.07.028

The insect kinins are a class of multifunctional insect neuropeptides present in a diverse variety of insects. Insect kinin analogs showed multiple bioactivities, especially, the aphicidal activity. To find a biostable and bioactive insecticide candidate with simplified structure, a series of N-terminal modified insect kinin analogs was designed and synthesized based on the lead compound [Aib]-Phe-Phe-[Aib]-Trp-Gly-NH₂. Their aphicidal activity against the soybean aphid Aphis glycines was evaluated. The results showed that all the analogs maintained the aphicidal activity. In particular, the aphicidal activity of the pentapeptide analog X Phe-Phe-[Aib]-Trp-Gly-NH₂ (LC₅₀ = 0.045 mmol/L) was similar to the lead compound (LC₅₀ = 0.048 mmol/L). This indicated that the N-terminal protective group may not play an important role in the activity and the analogs structure could be simplified to pentapeptide analogs while retaining good aphicidal activity. The core pentapeptide analog X can be used as the lead compound for further chemical modifications to discover potential insecticides.

Full text of DOI:10.1016/j.peptides.2014.07.028

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Contents lists available at ScienceDirect
Peptides
journal homepage: www.elsevier.com/locate/peptides
Design, synthesis and aphicidal activity of N-terminal modified insect
kinin analogs
Chuanliang Zhanga, Yanyan Qub, Xiaoqing Wua, Dunlun Songb,
Yun Linga, Xinling Yanga,∗
a Department of Applied Chemistry, College of Science, China Agricultural University, Beijing 100193, PR China
b Department of Entomology, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The insect kinins are a class of multifunctional insect neuropeptides present in a diverse variety of insects.
Insect kinin analogs showed multiple bioactivities, especially, the aphicidal activity. To find a biostable
and bioactive insecticide candidate with simplified structure, a series of N-terminal modified insect kinin
analogs was designed and synthesized based on the lead compound [Aib]-Phe-Phe-[Aib]-Trp-Gly-NH2.
Their aphicidal activity against the soybean aphid Aphis glycines was evaluated. The results showed
that all the analogs maintained the aphicidal activity. In particular, the aphicidal activity of the pen-
tapeptide analog X Phe-Phe-[Aib]-Trp-Gly-NH2 (LC50 = 0.045 mmol/L) was similar to the lead compound
(LC50 = 0.048 mmol/L). This indicated that the N-terminal protective group may not play an important
role in the activity and the analogs structure could be simplified to pentapeptide analogs while retaining
good aphicidal activity. The core pentapeptide analog X can be used as the lead compound for further
chemical modifications to discover potential insecticides.
Received 1 June 2014
Received in revised form 30 July 2014
Accepted 30 July 2014
Available online xxx
Keywords:
Insect kinins
N-terminal modified
Peptidomimetics
Aphicidal activity
Insecticides
© 2014 Elsevier Inc. All rights reserved.
hydrolysis sites in the core pentapeptide sequence Phe1-Tyr2-Pro3-
Trp4-Gly5-NH2, the primary site is between Pro3 and the Trp4
Introduction
The insect kinins are a class of multifunctional insect neuro-
peptides present in a diverse variety of insects. Insect kinins share
the highly conserved C-terminal pentapeptide sequence Phe-Xaa-
Yaa-Trp-Gly-NH2, where Xaa = His, Asn, Ser, or Tyr and Yaa = Ser,
Pro, or Ala [4,5,8,21,27]. The physiological functions of insects
including regulation of hindgut contraction, diuresis, and modu-
lation of digestive enzyme release are associated with insect kinins
and/or analogs. The C-terminal aldehyde insect kinin analog was
reported to enhance the inhibition of weight gain and lead to sig-
nificant mortality in the corn earworm (Helicoverpa zea) larvae [13].
Recently, insect kinin analogs showed good antifeedant and aphici-
dal activities [23]. Therefore, the active insect kinins and/or analogs
are considered as the leads in the development of novel pest control
agents/insecticides [23].
residue, and is susceptible to cleavage by angiotensin converting
enzyme (ACE) from the housefly and neprilysin (NEP). The sec-
ondary site is N-terminal to the Phe1 residue and is also susceptible
to neprilysin (NEP) [19,28]. The hydrolysis of the insect kinins by
similar peptidases leads to their inactivity.
To overcome these disadvantages, the peptidomimetic
approach (PA), a method widely used in the discovery of peptide-
based pharmaceuticals [7,10] was employed to enhance their
biostability and activity. Blocking or overstimulation of the recep-
tors of insect neuropeptides by peptidomimetic analogs could lead
to a reduction of insect fitness or death and has been proposed to
be a strategy for the design of potential peptide-based pest control
agents/insecticides [6,15]. In the past several decades, the primary
structure–activity relationships of insect kinins and/or analogs via
the peptidomimetic approach have been broadly studied. The C-
terminal core pentapeptide was the minimum sequence required
for full cockroach myotropic and cricket diuretic activity in assays
with tissues [11,14], and for bioluminescence response in CHO-K1
cells expressing kinin receptors [9,20,25]. However, the activity in
these assays and bioluminescence response in receptor expressing
system was completely lost when the C-terminal amide of the
insect kinins was replaced with a negatively charged acid moiety
However, insect kinins, and/or analogs are not suitable for use
as pest control agents or insecticides directly, as a consequence
of their susceptibility to both exo- and endopeptidases in the
hemolymph and on the tissues of insects. There are two susceptible
∗
Corresponding author. Tel.: +86 10 62732223; fax: +86 10 62732223.
E-mail address: yangxl@cau.edu.cn (X. Yang).
http://dx.doi.org/10.1016/j.peptides.2014.07.028
0196-9781/© 2014 Elsevier Inc. All rights reserved.
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O
C
O
C
CH3O
O
C
O
C
H
N
H
C
H
N
H
C
H
N
H
N
H
C
H
N
H
C
H
C
C
NH2
CH2
CH2
CH3
CH2
H
HN
analog X
removing Aib
CH3O
O
O
H
CH3O
O
O
H
N
H
C
H
H
C
H
N
H
C
H
H
C
H2N
C
C
C
N
C
N
C
C
C
N
C
NH2
CH3
Aib
CH2
CH2
CH3
CH2
H
HN
lead: Aib-Phe-Phe-Aib-Trp-Gly-NH2
replacing Aib with different
protective groups
O
O
C
CH3O
O
C
O
H
N
H
C
H
N
H
C
H
N
H
N
H
C
H
N
H
R
C
C
C
C
H
C
NH2
CH2
CH2
CH3
CH2
HN
analog I~IX
Fig. 1. Design of the N-terminal modified insect kinin analogs.
[12,25]. The results of an Ala scanning experiment indicated the
importance of the side chains of Phe1 and Trp4. The replacement
of these two positions with Ala could lead to the complete loss
of activity in both mosquito and tick receptor expression sys-
tems and in myotropic and diuretic assays. The variable position
Xaa tolerated a wide range of chemical characteristics, while
aromatic residues at this position showed the highest potency
in Malpighian tubule fluid secretion assays [22,25]. The analogs
with sterically hindered ␣,␣-disubstituted amino acids such as
␣-amino-isobutyric acid (Aib), anamethyl-phenylalanine residue
(␣-Me Phe) and ␤-amino acids showed enhanced resistance to
the endopeptidases ACE and NEP, enzymes that deactivated the
natural insect kinins. As a result, their biostability and bioavail-
ability markedly increased. Significantly, these analogs matched
and/or exceeded the diuretic and receptor binding activity of
native insect kinins [16,19,24–26,28,29]. Among these analogs, the
double Aib-containing kinin analog (K-Aib-1: [Aib]-Phe-Phe-[Aib]-
Trp-Gly-NH2) exhibited good aphicidal activity against the pea
aphid Acyrthosiphon pisum [23], and thus could be regarded as the
lead compound for the development of novel selective, environ-
mentally friendly pest control agents/insecticides [23]. Herein, we
chose K-Aib-1 as the lead compound, designed and synthesized 10
analogs by removing or replacing the protective group of the sec-
ondary hydrolysis site Aib with (aromatic) saturated/unsaturated
acids. Aib in position Yaa was retained because it was a good
protective group of the primary hydrolysis site [16,18,23,24]. The
design strategy is shown in Fig. 1. Their aphicidal activity against
the Soybean aphid Aphis glycines was evaluated so that we could
determine the primary structure–activity relationship to assess the
importance of protective group in the N-terminal hydrolysis site
and whether insect kinin analogs could be structurally simplified
while retaining good activity.
Materials and methods
Synthesis
Materials
Rink Amide-AM resin (0.56 mmol/g), O-benzotriazole-N,N,Nꢀ,Nꢀ-
tetramethyluronium hexafluorophosphate (HOBt), 1-hydroxy-
benzotriazole anhydrate (HBTU), N,Nꢀ-diisopropylethylamine
(DIEA), N,Nꢀ-diisoprophlcarbodiimide (Dic), trifluoroacetic acid
(TFA), Fmoc-protected amino acids, Fmoc-Gly-OH, Fmoc-l-
Phe-OH, Fmoc-l-Trp(Boc)-OH, Fmoc-l-Aib-OH were purchased
from GL Biochem, Ltd. (Shanghai, China). High-performance
liquid chromatography (HPLC)-grade N,N-dimethylformamide
(DMF), dichloromethane (DCM), methanol were purchased
from Dima Technology, Inc. (Richmond Hill, Ontario, Canada).
Thioanisole, phenol, trans-cinnamic acid, 4-nitrocinnamic acid,
4-methylcinnamic acid, hydrocinnamic acid, acetic acid, pivalic
acid, 2-hydroxyisobutyric acid, acrylic acid and methacrylic acid
were purchased from Alfa Aesar, USA.
General synthetic procedure
FF[Aib]WG with Resin was synthesized from Rink Amide-AM
resin (536 mg, 0.3 mmol) using the standard Fmoc/tBu chemistry
and HBTU/HOBt protocol [3]. Incoming amino acids were acti-
vated with HOBt (164 mg, 1.2 mmol), HBTU (456 mg, 1.2 mmol),
and DIEA (210 ␮L, 1.2 mmol) in DMF (5 mL) for 5 min, and cou-
plings were run for 2 h. Removal of the N-terminal Fmoc group from
the residues was accomplished with 20% piperidine in DMF (5 mL)
for 20 min. The lead compound and analog I–IX were cleaved from
the resin with TFA (9 mL) containing 5% phenol, 2.5% thioanisole
(0.5 mL), and 5% water (0.5 mL) for 2 h after the (amino) acids
(1.2 mmol) were coupled to the FF[Aib]WG resin (0.3 mmol) with
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Fig. 2. Synthesis of the lead compound and analog I ∼ X.
HOBt (164 mg, 1.2 mmol), HBTU (456 mg, 1.2 mmol), and DIEA
(210 ␮L, 1.2 mmol) in DMF (5 mL) for 3 h at room temperature. Ana-
log X was directly cleaved from the FF[Aib]WG resin. Removal of the
Boc group of Trp was also accomplished in the cleavage procedure.
To avoid the generation of OBt esters, several analogs containing a
carboxylic acid group were synthesized using Dic as the coupling
reagent instead of HBTU/HOBt. The general synthetic procedure
was shown in Fig. 2.
Analog II. Yellow solid; purity = 97%; purification condition:
acetonitrile/water (v/v) = 60/40, flow rate = 10 mL/min; retention
time = 10.7 min; HRMS (ESI) m/z calcd for C44H46N8O8 (M+Na)+,
837.3331; found, 837.3325.
Analog III. White solid; purity = 96%; purification condition:
acetonitrile/water (v/v) = 40/60, flow rate = 10 mL/min; retention
time = 9.2 min; HRMS (ESI) m/z calcd for C45H49N7O6 (M+Na)+,
806.3637; found, 806.3644.
Analog IV. White solid; purity = 98%; purification condition:
acetonitrile/water (v/v) = 40/60, flow rate = 10 mL/min; retention
time = 9.7 min; HRMS (ESI) m/z calcd for C44H49N7O6 (M+Na)+,
794.3637; found, 794.3642.
Analog V. White solid; purity = 98%; purification condition:
acetonitrile/water (v/v) = 60/40, flow rate = 10 mL/min; retention
time = 6.7 min; HRMS (ESI) m/z calcd for C37H43N7O6 (M+Na)+,
704.3167; found, 704.3169.
Analog VI. White solid; purity = 97%; purification condition:
acetonitrile/water (v/v) = 50/50, flow rate = 10 mL/min; retention
time = 9.9 min; HRMS (ESI) m/z calcd for C40H49N7O6 (M+Na)+,
746.3637; found, 746.3647.
Analog VII. White solid; purity = 96%; purification condition:
acetonitrile/water (v/v) = 40/60, flow rate = 10 mL/min; retention
time = 16.7 min; HRMS (ESI) m/z calcd for C39H47N7O7 (M+Na)+,
748.3429; found, 748.3427.
Analog VIII. White solid; purity = 98%; purification condition:
acetonitrile/water (v/v) = 35/65, flow rate = 10 mL/min; retention
time = 19.1 min; HRMS (ESI) m/z calcd for C38H43N7O6 (M+Na)+,
716.3167; found, 716.3174.
Analog IX. White solid; purity = 96%; purification condition:
acetonitrile/water (v/v) = 50/50, flow rate = 10 mL/min; retention
time = 12.9 min; White solid; HRMS (ESI) m/z calcd for C39H45N7O6
(M+Na)+, 730.3324; found, 730.3324.
Purification and characterization
The crude compounds were purified on a C18 reversed-phase
preparative column with a flow rate of 10 mL/min using different
ratio of acetonitrile/water (v/v) from 30:70 to 60:40 containing
0.01% TFA as an ion-pairing reagent. The UV detection was at
215 nm. The purity of each purified compound was higher than
95%. The analogs structures were confirmed with high-resolution
mass spectrometry (HRMS) using an Agilent Accurate-Mass-Q-TOF
MS 6520 system equipped with an Electro Spray Ionization (ESI)
source. All the MS experiments were detected in the positive ion-
ization mode. For Q-TOF/MS conditions, fragment and capillary
voltages were kept at 130 and 3500 V, respectively. Nitrogen was
supplied as the nebulizing and drying gas. The temperature of the
drying gas was set at 300 ◦C. The flow rate of the drying gas and the
pressure of the nebulizer were 10 L/min and 25 psi, respectively.
Full-scan spectra were acquired over a scan range of m/z 80–1200 at
1.03 spectra s−1
.
Lead compound. White solid; purity = 97%; purification con-
dition: acetonitrile/water (v/v) = 40/60, flow rate = 10 mL/min;
retention time = 9.6 min; HRMS (ESI) m/z calcd for C39H48N8O6
(M+H)+, 725.3770; found, 725.3771.
Analog I. White solid; purity = 98%; purification condition:
acetonitrile/water (v/v) = 60/40, flow rate = 10 mL/min; retention
time = 9.7 min; HRMS (ESI) m/z calcd for C44H47N7O6 (M+Na)+,
792.3480; found, 792.3480.
Analog X. Gray solid; purity = 96%; purification condition:
acetonitrile/water (v/v) = 40/60, flow rate = 10 mL/min; retention
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time = 6.9 min; HRMS (ESI) m/z calcd for C35H41N7O5 (M+H)+,
640.3242; found, 640.3246.
The standard deviations of the tested aphicidal values were 10%.
The LC50 values were calculated using SAS 9.0 (SAS Institute Inc.,
USA).
Aphicidal bioassay
Results
The aphicidal activity of insect kinin analogs against A. glycines
was evaluated using the reported procedure [2].
The bioassay was carried out using the leaf-dipping method. The
commercial insecticide Pymetrozine was used as the control. The
preliminary result (at a concentration of 200 mg/L) showed that
most analogs had good activity against the soybean aphid. Espe-
cially, the activity of analogs III, V, VIII and V was similar to the lead
compound, but less than the insecticide Pymetrozine. Guy Smagghe
et al. [23] figured that the treatment of pea aphid A. pisum dur-
ing 1 day with the lead compound K-Aib-1 at 0.5 nmol/ml in the
artificial diet caused a remarked reduction in the amounts of hon-
eydew produced by aphid nymphs, as compared to the control after
visualization by using the Ninhydrin test. In our bioassay, we also
observed the similar phenomenon after the honeydew air-dried.
The compounds were dissolved in acetone at a concentration
of 2000 mg/L, then diluted to lower concentrations with 0.05% Tri-
ton X-100. Soybean leaf discs of about 3 cm diameter were dipped
into the test solution for 15 s. The discs dipped into 0.05% Tri-
ton X-100 were set as the negative control. After air-drying, the
treated leaf discs were placed individually into bioassay plates with
1% agar to keep them moist. The discs were then infested with
20 3 3-day old aphids, kept in the culture incubator with constant
temperature (25 1 ◦C) for 48 h. Thereafter, the number of dead
aphids was counted and the mortality rates were corrected using
the Abbott’s formula [1]. Experiments were performed three times.
Table 1
Aphicidal activity of the lead compound, analog I–X and Pymetrozine against soybean aphid. R-Phe-Phe-Aib-Trp-Gly-NH2.
Analogs
Ra
Aphicidal activity (48 h)
Screening mortality (%), 200 mg/L
LC50 (95% FL), mmol/L
0.048 (0.030–0.079)
Lead compd.
78.65 4.91
67.18 4.98
I
0.231 (0.090–1.065)
0.190 (0.099–0.773)
II
70.62 4.15
80.35 1.84
III
0.069 (0.042–0.124)
IV
V
63.11 0.25
71.88 3.59
58.28 3.30
0.260 (0.122–1.419)
0.526 (0.199–1.230)
ND
VI
VII
44.40 1.94
75.88 4.33
ND
VIII
0.131 (0.081–0.268)
IX
X
67.37 2.89
78.53 3.04
0.103 (0.063–0.207)
0.045 (0.026–0.074)
H
Pymetrozine
90.37 2.50
0.034 (0.012–0.083)
ND: LC50 value was not determined when screening mortality was lower than 60% at the concentration of 200 mg/L, 48 h.
95% FL: 95% fiducial interval.
a
The R of Pymetrozine is its chemical structure.
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Subsequently, the aphicidal activity of different concentrations
of the kinin analogs was determined and the LC50 values were cal-
culated using SAS 9.0 software (SAS Institute Inc., USA).
commercial insecticide Pymetrozine. Therefore, the N-terminal
protective group may not play an important role in activity and
the analogs structure could be simplified to pentapeptide analogs
while retaining good aphicidal activity. The core pentapeptide ana-
log X could be used as the lead compound for further chemical
modifications and simplifications to discover potential insecticides.
Discussion
Design and synthesis of analogs
Acknowledgements
Previous studies showed that the insect kinin analog K-Aib-
1, which modified or protected the “active core” pentapeptide,
exhibited significant biological, receptor binding and aphicidal
activity [17,23,24]. This demonstrates that K-Aib-1 could be a good
lead compound. In this study, K-Aib-1 ([Aib]-Phe-Phe-[Aib]-Trp-
Gly-NH2) was synthesized and evaluated as the lead compound.
To determine whether the protective group in the N-terminal
hydrolysis site was necessary for activity and the analog could
be structurally simplified, we designed ten analogs by remov-
ing/replacing the protective group of the secondary hydrolysis site
Aib with (aromatic) saturated/unsaturated acids (Fig. 1). Aib in posi-
tion Yaa was retained because previous studies have demonstrated
that it was a good protective group in the primary hydrolysis site
[16,18,23,24].
We acknowledge the financial support from the National Nat-
ural Science Foundation of China (No. 21372257) and the National
Basic Research Program of China (No. 2010CB126104). We are
grateful to Prof. Stephen Tobe for his kind help on this manuscript
writing.
References
[1] Abbott W. A method of computing the effectiveness of an insecticide. J Econ
Entomol 1925;18:265–7.
[2] Busvine JR. Recommended methods for measurement of pest resistance to
pesticides; 1980.
[3] Chan WC, White PD. Fmoc solid phase peptide synthesis. Oxford University
Press; 2000.
[4] Coast GM, Orchard I, Phillips JE, Schooley DA. Insect diuretic and antidiuretic
hormones. Adv Insect Physiol 2002;29:279–409.
As shown in Fig. 2, the analogs and lead compound were syn-
thesized from Rink Amide-AM resin using solid-phase organic
synthesis employing Fmoc/tBu chemistry and HBTU/HOBt proto-
col [3]. Incoming amino acids were activated with HOBt, HBTU,
and DIEA in DMF. Removal of the N-terminal Fmoc group from the
residues was accomplished with 20% piperidine in DMF. Removal of
the Boc group of Trp was accomplished in the cleavage procedure.
To avoid the generation of OBt esters, the incorporation of cinnamic
acids was accomplished using Dic as the coupling reagent instead
of HBTU/HOBt [3].
[5] Gäde G. Regulation of intermediary metabolism and water balance of insects
by neuropeptides. Annu Rev Entomol 2004;49:93–113.
[6] Gäde G, Goldsworthy GJ. Insect peptide hormones: a selective review of
their physiology and potential application for pest control. Pest Manag Sci
2003;59:1063–75.
[7] Giannis A, Rübsam F. Peptidomimetics in drug design. Adv Drug Res
1997;29:1–78.
[8] Holman GM, Nachman RJ, Coast GM. Isolation, characterization and biolog-
ical activity of a diuretic myokinin neuropeptide from the housefly, Musca
domestica. Peptides (NY) 1998;20:1–10.
[9] Holmes S, Barhoumi R, Nachman R, Pietrantonio P. Functional analysis of a
G protein-coupled receptor from the Southern cattle tick Boophilus microplus
(Acari: Ixodidae) identifies it as the first arthropod myokinin receptor. Insect
Mol Biol 2003;12:27–38.
[10] Hruby VJ, Li G, Haskell-Luevano C, Shenderovich M. Design of peptides, pro-
teins, and peptidomimetics in chi space. Biopolymers 1997;43:219–66.
[11] Nachman RJ, Coast GM, Douat C, Fehrentz JA, Kaczmarek K, Zabrocki J, et al.
A C-terminal aldehyde insect kinin analog enhances inhibition of weight
gain and induces significant mortality in Helicoverpa zea larvae. Peptides
2003;24:1615–21.
[12] Nachman RJ, Coast GM, Holman GM, Beier RC. Diuretic activity of C-terminal
group analogs of the insect kinins in Acheta domesticus. Peptides (Tarrytown,
NY) 1995;16:809–13.
[13] Nachman RJ, Fehrentz J-A, Martinez J, Kaczmarek K, Zabrocki J, Coast GMA. C-
terminal aldehyde analog of the insect kinins inhibits diuresis in the housefly.
Peptides (Amsterdam, Netherlands) 2007;28:146–52.
[14] Nachman RJ, Holman GM. Myotropic insect neuropeptide families from the
cockroach Leucophaea maderae. Structure–activity relationships. ACS Symp Ser
1991;453:194–214.
[15] Nachman RJ, Holman GM, Haddon WF. Leads for insect neuropeptide mimetic
development. Arch Insect Biochem Physiol 1993;22:181–97.
[16] Nachman RJ, Isaac RE, Coast GM, Holman GM. Aib-containing analogues
of the insect kinin neuropeptide family demonstrate resistance to an
insect angiotensin-converting enzyme and potent diuretic activity. Peptides
1997;18:53–7.
[17] Nachman RJ, Pietrantonio PV, Coast GM. Toward the development of novel pest
management agents based upon insect kinin neuropeptide analogues. Ann N Y
Acad Sci 2009;1163:251–61.
Effect of analogs against the soybean aphid
In the aphicidal bioassay, we also observed a reduction in the
amounts of honeydew produced by aphids treated with analogs
compared with the negative control. This further indicates that
insectkininand/oranalogsinduceantifeedanteffectagainstaphids.
Table 1 displays the aphicidal activity LC50 values for the lead
compound, target compounds and Pymetrozine against soybean
aphid. The results indicate that the pentapeptide analog X remov-
ing protective group in the N-terminal from the lead compound was
the most toxic for the soybean aphid. Its LC50 value 0.045 mmol/L
was similar to that of the lead compound, LC50 = 0.048 mmol/L. The
analogs containing cinnamoyl, hydrocinnamoyl group in the N-
terminal did not showed outstanding aphicidal activity with the
exception of analog III containing 4-methyl cinnamoyl, of which
the LC50 was 0.069 mmol/L, close to that of the lead compound.
Analogs V, VI, VII, VIII and IX, consisting of saturated and unsatu-
rated acyl group was also deactivated. The above results suggested
that the protective group in the N-terminal hydrolysis site did not
play an important role in the bioactivity. In contrast, removing the
N-terminal protective group might not only simplify the structure
but also maintain or even increase the aphicidal activity of analogs.
We surmised the reason might be that the removal of the hydrolysis
target peptide bond could enhance higher peptidase-resistance and
bioavailability than replacement of the hydrolysis target peptide
bond. Further structure–activity relationship studies of structure-
simplified analogs are in progress.
[18] Nachman RJ, Smagghe G. Biostable analogs of insect kinin and insectat-
achykinin neuropeptides:
a review of novel classes of antifeedants and
aphicides. Pestycydy 2011;2:3–34.
[19] Nachman RJ, Strey A, Isaac E, Pryor N, Lopez JD, Deng J-G, et al. Enhanced in vivo
activity of peptidase-resistant analogs of the insect kinin neuropeptide family.
Peptides 2002;23:735–45.
[20] Pietrantonio P, Jagge C, Taneja-Bageshwar S, Nachman R, Barhoumi R. The
mosquito Aedes aegypti (L.) leucokinin receptor is a multiligand receptor for
the three Aedes kinins. Insect Mol Biol 2005;14:55–67.
[21] Riehle MA, Garczynski SF, Crim JW, Hill CA, Brown MR. Neuropeptides and
peptide hormones in Anopheles gambiae. Science 2002;298:172–5.
[22] Roberts VA, Nachman RJ, Coast GM, Hariharan M, Chung JS, Holman GM, et al.
Consensus chemistry and ␤-turn conformation of the active core of the insect
kinin neuropeptide family. Chem Biol 1997;4:105–17.
[23] Smagghe G, Mahdian K, Zubrzak P, Nachman RJ. Antifeedant activity and high
mortality in the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae) induced
by biostable insect kinin analogs. Peptides (New York, NY, United States)
2010;31:498–505.
In summary, a series of new insect kinin analogs has been
designed and synthesized. The results of the bioassays showed
that the analogs exhibited considerable aphicidal activity against
the soybean aphid, especially the structurally simplified analog
X, displaying comparable activity to the lead compound and the
Please cite this article in press as: Zhang C, et al. Design, synthesis and aphicidal activity of N-terminal modified insect kinin analogs.
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PEP-69305; No. of Pages6
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[24] Taneja-Bageshwar S, Strey A, Isaac RE, Coast GM, Zubrzak P, Pietrantonio
PV, et al. Biostable agonists that match or exceed activity of native insect
kinins on recombinant arthropod GPCRs. Gen Comp Endocrinol 2009;162:
122–8.
[25] Taneja-Bageshwar S, Strey A, Zubrzak P, Pietrantonio PV, Nachman RJ. Compar-
ative structure–activity analysis of insect kinin core analogs on recombinant
kinin receptors from southern cattle tick Boophilus microplus (Acari: Ixodidae)
and mosquito Aedes aegypti (Diptera: Culicidae). Arch Insect Biochem Physiol
2006;62:128–40.
acid agonists of recombinant insect kinin receptors from the southern cattle
tick Boophilus microplus and mosquito Aedes aegypti. Peptides (Amsterdam,
Netherlands) 2008;29:302–9.
[27] Torfs P, Nieto J, Veelaert D, Boon D, Van de Water G, Waelkens E, et al. The kinin
peptide family in invertebrates. Ann N Y Acad Sci 1999;897:361–73.
[28] Zubrzak P, Williams H, Coast GM, Isaac RE, Reyes-Rangel G, Juaristi E, et al. ␤-
Amino acid analogs of an insect neuropeptide feature potent bioactivity and
resistance to peptidase hydrolysis. Biopolymers 2007;88:76–82.
[29] Zubrzak P, Williams H, Coast GM, Isaac RE, Reyes-Rangel G, Juaristi E, et al.
␤-amino acid analogs of an insect neuropeptide feature potent bioactivity and
resistance to peptidase hydrolysis. Pept Sci 2007;88:76–82.
[26] Taneja-Bageshwar S, Strey A, Zubrzak P, Williams H, Reyes-Rangel G, Juar-
isti E, et al. Identification of selective and non-selective, biostable ␤-amino
Please cite this article in press as: Zhang C, et al. Design, synthesis and aphicidal activity of N-terminal modified insect kinin analogs.
Peptides (2014), http://dx.doi.org/10.1016/j.peptides.2014.07.028