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Design, synthesis and aphicidal activity of N-terminal modified insect kinin analogs
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Design, synthesis and aphicidal activity of N-terminal modified insect kinin analogs Chuanliang Zhang a , Yanyan Qu b , Xiaoqing Wu a , Dunlun Song b , Yun Ling a , Xinling Yang a,∗ a b
Department of Applied Chemistry, College of Science, China Agricultural University, Beijing 100193, PR China 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
Article history: 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
a b s t r a c t 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. © 2014 Elsevier Inc. All rights reserved.
Introduction The insect kinins are a class of multifunctional insect neuropeptides present in a diverse variety of insects. Insect kinins share the highly conserved C-terminal pentapeptide sequence Phe-XaaYaa-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 modulation 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 significant mortality in the corn earworm (Helicoverpa zea) larvae [13]. Recently, insect kinin analogs showed good antifeedant and aphicidal 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]. 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: [email protected] (X. Yang).
hydrolysis sites in the core pentapeptide sequence Phe1 -Tyr2 -Pro3 Trp4 -Gly5 -NH2 , the primary site is between Pro3 and the Trp4 residue, and is susceptible to cleavage by angiotensin converting enzyme (ACE) from the housefly and neprilysin (NEP). The secondary 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 peptidebased pharmaceuticals [7,10] was employed to enhance their biostability and activity. Blocking or overstimulation of the receptors 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 Cterminal 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
http://dx.doi.org/10.1016/j.peptides.2014.07.028 0196-9781/© 2014 Elsevier Inc. All rights reserved.
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CH3O O O O O H H H H H H H H H N C C N C C N C C N C C N C C NH2
H
CH3
CH2
CH2
H
CH2
HN analog X
removing Aib
CH3O H2N
C
H N
C
O
H C
CH3 Aib
H N
C
O
H C
CH3O
H N
C
C
CH2
CH2
H N
C
H C
O C
H N
O C
NH2
H
CH2
CH3
H C
HN lead: Aib-Phe-Phe-Aib-Trp-Gly-NH2 replacing Aib with different protective groups
R
H N
H C
O C
CH2
H N
H C
O C
H N
CH3O C
C
H N
O C
CH2
CH3
CH2
H C
H N
H C
O C
NH2
H
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 systems 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 bioavailability 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, environmentally 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 secondary 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,N ,N tetramethyluronium hexafluorophosphate (HOBt), 1-hydroxybenzotriazole anhydrate (HBTU), N,N -diisopropylethylamine (DIEA), N,N -diisoprophlcarbodiimide (Dic), trifluoroacetic acid (TFA), Fmoc-protected amino acids, Fmoc-Gly-OH, Fmoc-lPhe-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 activated 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 couplings 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. Analog 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. 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 ionization 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 condition: acetonitrile/water (v/v) = 40/60, flow rate = 10 mL/min; retention time = 9.6 min; HRMS (ESI) m/z calcd for C39 H48 N8 O6 (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 C44 H47 N7 O6 (M+Na)+ , 792.3480; found, 792.3480.
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 C44 H46 N8 O8 (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 C45 H49 N7 O6 (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 C44 H49 N7 O6 (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 C37 H43 N7 O6 (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 C40 H49 N7 O6 (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 C39 H47 N7 O7 (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 C38 H43 N7 O6 (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 C39 H45 N7 O6 (M+Na)+ , 730.3324; found, 730.3324. 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 C35 H41 N7 O5 (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 compounds were dissolved in acetone at a concentration of 2000 mg/L, then diluted to lower concentrations with 0.05% Triton 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% Triton 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.
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. Especially, 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 during 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 honeydew 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.
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
Lead compd.
78.65 ± 4.91
0.048 (0.030–0.079)
I
67.18 ± 4.98
0.231 (0.090–1.065)
II
70.62 ± 4.15
0.190 (0.099–0.773)
III
80.35 ± 1.84
0.069 (0.042–0.124)
IV
63.11 ± 0.25
0.260 (0.122–1.419)
V
71.88 ± 3.59
0.526 (0.199–1.230)
VI
58.28 ± 3.30
ND
VII
44.40 ± 1.94
ND
VIII
75.88 ± 4.33
0.131 (0.081–0.268)
IX
67.37 ± 2.89
0.103 (0.063–0.207)
78.53 ± 3.04
0.045 (0.026–0.074)
90.37 ± 2.50
0.034 (0.012–0.083)
X
Pymetrozine
H
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 calculated using SAS 9.0 software (SAS Institute Inc., USA). Discussion Design and synthesis of analogs Previous studies showed that the insect kinin analog K-Aib1, 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]-TrpGly-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 removing/replacing the protective group of the secondary hydrolysis site Aib with (aromatic) saturated/unsaturated acids (Fig. 1). Aib in position Yaa was retained because previous studies have demonstrated that it was a good protective group in the primary hydrolysis site [16,18,23,24]. As shown in Fig. 2, the analogs and lead compound were synthesized from Rink Amide-AM resin using solid-phase organic synthesis employing Fmoc/tBu chemistry and HBTU/HOBt protocol [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]. 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 insect kinin and/or analogs induce antifeedant effect against aphids. 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 removing 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 Nterminal 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 unsaturated 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 structuresimplified analogs are in progress. 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
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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 analog X could be used as the lead compound for further chemical modifications and simplifications to discover potential insecticides. Acknowledgements We acknowledge the financial support from the National Natural 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. [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 biological 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, proteins, 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. Cterminal 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. [18] Nachman RJ, Smagghe G. Biostable analogs of insect kinin and insectatachykinin 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.
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Peptides journal homepage: www.elsevier.com/locate/peptides
Design, synthesis and aphicidal activity of N-terminal modified insect kinin analogs Chuanliang Zhang a , Yanyan Qu b , Xiaoqing Wu a , Dunlun Song b , Yun Ling a , Xinling Yang a,∗ a b
Department of Applied Chemistry, College of Science, China Agricultural University, Beijing 100193, PR China 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
Article history: 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
a b s t r a c t 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. © 2014 Elsevier Inc. All rights reserved.
Introduction The insect kinins are a class of multifunctional insect neuropeptides present in a diverse variety of insects. Insect kinins share the highly conserved C-terminal pentapeptide sequence Phe-XaaYaa-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 modulation 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 significant mortality in the corn earworm (Helicoverpa zea) larvae [13]. Recently, insect kinin analogs showed good antifeedant and aphicidal 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]. 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: [email protected] (X. Yang).
hydrolysis sites in the core pentapeptide sequence Phe1 -Tyr2 -Pro3 Trp4 -Gly5 -NH2 , the primary site is between Pro3 and the Trp4 residue, and is susceptible to cleavage by angiotensin converting enzyme (ACE) from the housefly and neprilysin (NEP). The secondary 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 peptidebased pharmaceuticals [7,10] was employed to enhance their biostability and activity. Blocking or overstimulation of the receptors 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 Cterminal 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
http://dx.doi.org/10.1016/j.peptides.2014.07.028 0196-9781/© 2014 Elsevier Inc. All rights reserved.
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CH3O O O O O H H H H H H H H H N C C N C C N C C N C C N C C NH2
H
CH3
CH2
CH2
H
CH2
HN analog X
removing Aib
CH3O H2N
C
H N
C
O
H C
CH3 Aib
H N
C
O
H C
CH3O
H N
C
C
CH2
CH2
H N
C
H C
O C
H N
O C
NH2
H
CH2
CH3
H C
HN lead: Aib-Phe-Phe-Aib-Trp-Gly-NH2 replacing Aib with different protective groups
R
H N
H C
O C
CH2
H N
H C
O C
H N
CH3O C
C
H N
O C
CH2
CH3
CH2
H C
H N
H C
O C
NH2
H
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 systems 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 bioavailability 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, environmentally 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 secondary 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,N ,N tetramethyluronium hexafluorophosphate (HOBt), 1-hydroxybenzotriazole anhydrate (HBTU), N,N -diisopropylethylamine (DIEA), N,N -diisoprophlcarbodiimide (Dic), trifluoroacetic acid (TFA), Fmoc-protected amino acids, Fmoc-Gly-OH, Fmoc-lPhe-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 activated 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 couplings 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. Analog 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. 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 ionization 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 condition: acetonitrile/water (v/v) = 40/60, flow rate = 10 mL/min; retention time = 9.6 min; HRMS (ESI) m/z calcd for C39 H48 N8 O6 (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 C44 H47 N7 O6 (M+Na)+ , 792.3480; found, 792.3480.
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 C44 H46 N8 O8 (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 C45 H49 N7 O6 (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 C44 H49 N7 O6 (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 C37 H43 N7 O6 (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 C40 H49 N7 O6 (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 C39 H47 N7 O7 (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 C38 H43 N7 O6 (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 C39 H45 N7 O6 (M+Na)+ , 730.3324; found, 730.3324. 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 C35 H41 N7 O5 (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 compounds were dissolved in acetone at a concentration of 2000 mg/L, then diluted to lower concentrations with 0.05% Triton 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% Triton 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.
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. Especially, 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 during 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 honeydew 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.
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
Lead compd.
78.65 ± 4.91
0.048 (0.030–0.079)
I
67.18 ± 4.98
0.231 (0.090–1.065)
II
70.62 ± 4.15
0.190 (0.099–0.773)
III
80.35 ± 1.84
0.069 (0.042–0.124)
IV
63.11 ± 0.25
0.260 (0.122–1.419)
V
71.88 ± 3.59
0.526 (0.199–1.230)
VI
58.28 ± 3.30
ND
VII
44.40 ± 1.94
ND
VIII
75.88 ± 4.33
0.131 (0.081–0.268)
IX
67.37 ± 2.89
0.103 (0.063–0.207)
78.53 ± 3.04
0.045 (0.026–0.074)
90.37 ± 2.50
0.034 (0.012–0.083)
X
Pymetrozine
H
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 calculated using SAS 9.0 software (SAS Institute Inc., USA). Discussion Design and synthesis of analogs Previous studies showed that the insect kinin analog K-Aib1, 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]-TrpGly-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 removing/replacing the protective group of the secondary hydrolysis site Aib with (aromatic) saturated/unsaturated acids (Fig. 1). Aib in position Yaa was retained because previous studies have demonstrated that it was a good protective group in the primary hydrolysis site [16,18,23,24]. As shown in Fig. 2, the analogs and lead compound were synthesized from Rink Amide-AM resin using solid-phase organic synthesis employing Fmoc/tBu chemistry and HBTU/HOBt protocol [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]. 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 insect kinin and/or analogs induce antifeedant effect against aphids. 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 removing 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 Nterminal 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 unsaturated 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 structuresimplified analogs are in progress. 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
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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 analog X could be used as the lead compound for further chemical modifications and simplifications to discover potential insecticides. Acknowledgements We acknowledge the financial support from the National Natural 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. [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 biological 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, proteins, 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. Cterminal 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. [18] Nachman RJ, Smagghe G. Biostable analogs of insect kinin and insectatachykinin 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.
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