Article pubs.acs.org/JAFC

Synthesis, Biological Activity, and Conformational Study of N‑Methylated Allatostatin Analogues Inhibiting Juvenile Hormone Biosynthesis Yong Xie,†,‡,§ Li Zhang,† Chuanliang Zhang,† Xiaoqing Wu,†,‡ Xile Deng,† Xinling Yang,*,† and Stephen S. Tobe‡ †

Department of Applied Chemistry, College of Science, China Agricultural University, Beijing 100193, People’s Republic of China Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, Ontario M5S 3G5, Canada § State Key Laboratory of the Discovery and Development of Novel Pesticide, Shenyang Research Institute of Chemical Industry Company, Limited, Shenyang, Liaoning 110021, People’s Republic of China ‡

ABSTRACT: An allatostatin (AST) neuropeptide mimic (H17) is a potential insect growth regulator, which inhibits the production of juvenile hormone (JH) by the corpora allata. To determine the effect of conformation of novel AST analogues and their ability to inhibit JH biosynthesis, eight insect AST analogues were synthesized using H17 as the lead compound by Nmethylation scanning, which is a common strategy for improving the biological properties of peptides. A bioassay using JH production by corpora allata of the cockroach Diploptera punctata indicated that single N-methylation mimics (analogues 1−4) showed more activity than double N-methylation mimics (analogues 5−8). Especially, analogues 1 and 4 showed roughly equivalent activity to that of H17, with IC50 values of 5.17 × 10−8 and 6.44 × 10−8 M, respectively. Molecular modeling based on nuclear magnetic resonance data showed that the conformation of analogues 1 and 4 seems to be flexible, whereas analogues 2 and 3 showed a type IV β-turn. This flexible linear conformation was hypothesized to be a new important and indispensable structural element beneficial to the activity of AST mimics. KEYWORDS: allatostatins, N-methylated allatostatin analogues, molecular modeling, juvenile hormone biosynthesis, conformation

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INTRODUCTION The FGLamide allatostatins (ASTs) belong to a family of insect neuropeptides originally isolated from the cockroach Diploptera punctata on the basis of their ability to inhibit juvenile hormone (JH) biosynthesis by corpora allata (CA) in vitro in cockroaches,1−4 termites,5,6 and crickets.7 This family of ASTs also appears to have other functions, including serving as neuromodulators, inhibitory regulators of muscle contraction, and regulators of enzyme biosynthesis.4,8,9 However, natural ASTs are readily degraded by peptidases; this issue precludes the use of ASTs themselves in pest management. Earlier structure−activity studies showed that the C-terminal pentapeptide Y/FXFGLa (X = A, N, G, and S), which is shared in all identified AST neuropeptides, was the minimum sequence capable of eliciting inhibition of JH production.10 Hayes et al.11 suggested that the most important side chains of Dippu-AST 5 were L8, followed by F6 and Y4, and a β-turn involving XFGL could represent an “active conformation”. Piulachs et al.12 synthesized ketomethylene and methyleneamino pseudopeptide analogues of Dippu-AST 5 that can inhibit JH biosynthesis and, subsequently, vitellogenin production by the fat body of the cockroach Blattella germanica. Nachman et al.13,14 studied the chemical and conformational features of Dippu-AST 6 by incorporating turn-promoting pseudopeptide moieties in the C-terminal region of the pentapeptide. These authors found that several peptidomimetics incorporating a βturn in the C terminus could inhibit JH biosynthesis and were resistant to peptidase degradation. Banerjee et al.15 found that Dippu-AST 5, one of the most potent inhibitors of JH © 2015 American Chemical Society

production, had a 310 helix involving three of its residues and a γ-turn at the end of its C-terminal motif. In our previous studies,16−19 we suggested that a potent AST analogue should contain an aromatic group, an appropriate length of linker, and a FGLa moiety and reported an AST mimic H17, which had a significant effect on JH biosynthesis by cockroach CA, both in vitro (IC50 value = 12 nM) and in vivo (IC50 value = 33 nM). Recently, we put forward a hypothesis that a flexible conformation could be an important and indispensable structural element for activity based on nuclear magnetic resonance (NMR) spectroscopy and molecular modeling.20 To find more evidence to support the hypothesis, we have designed and synthesized a series of H17 mimics using the N-methylation scanning method, a common strategy for improving the pharmacological properties of peptides, such as bioavailability and conformational rigidity. As shown in Figure 1, every amide bond was replaced sequentially by the corresponding N-methylated moiety. All newly synthesized analogues were assayed for their ability to inhibit JH biosynthesis by CA of the cockroach in vitro, and the conformation of these analogues was studied on the basis of NMR data and molecular modeling. We report here their synthesis, biological activity, and conformational analysis. Received: Revised: Accepted: Published: 2870

December 10, 2014 March 9, 2015 March 9, 2015 March 9, 2015 DOI: 10.1021/acs.jafc.5b00882 J. Agric. Food Chem. 2015, 63, 2870−2876

Article

Journal of Agricultural and Food Chemistry

Figure 1. Structures of N-methylated AST analogues 1−8.

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Ultraviolet (UV) detection was at 225 nM. Peptide fractions determined to be pure by analytical HPLC were combined, concentrated under reduced pressure, and lyophilized. Preparation of Analogue 1. Nα-Me-Leu-Rink amide-AM resin (0.5 mmol) was prepared as described in the general procedure. NαFmoc-Gly (3 mmol, 6 equiv) and Dic (1.5 mmol, 3 equiv) were mixed in CH2Cl2 (5 mL) for 30 min at 0 °C, and then Nα-Me-Leu-Rink amide-AM resin and DIEA (3 mmol, 6 equiv) were added. The reaction mixture was shaken for 2 h, and the procedure was repeated. After removal of the Nα-Fmoc group, Nα-Fmoc-Phe, Nα-Fmoc-Gly, and (E)-3-(4-nitrophenyl)-acrylic acid were coupled to the resin with HBTU, HOBt, and DIEA in DMF. The crude analogue 1 was purified by reversed-phase HPLC with a flow rate of 10 mL/min using acetonitrile/water (50:50, v/v) to yield 100 mg of peptide; HPLC tR, 10.5 min; HRMS, m/z 603.2537 [M + Na]+ (calcd [M + Na]+, 603.2538). Preparation of Analogue 2. Nα-Me-Gly-Leu-Rink amide-AM resin (0.5 mmol) was prepared as described in the general procedure. Nα-Fmoc-Phe (3 mmol, 6 equiv) and Dic (1.5 mmol, 3 equiv) were mixed in CH2Cl2 (5 mL) for 30 min at 0 °C, and then Nα-Me-GlyLeu-Rink amide-AM resin and DIEA (3 mmol, 6 equiv) were added. The reaction mixture was shaken for 2 h. After removal of the NαFmoc group, Nα-Fmoc-Gly and (E)-3-(4-nitrophenyl)-acrylic acid were coupled to the resin with HBTU, HOBt, and DIEA in DMF. The crude analogue 2 was purified by reversed-phase HPLC with a flow rate of 10 mL/min using acetonitrile/water (50:50, v/v) to yield 97 mg of peptide; HPLC tR, 8.7 min; HRMS, m/z 603.2541 [M + Na]+ (calcd [M + Na]+, 603.2538).

MATERIALS AND METHODS α

α

Chemistry. Protected N -Fmoc-amino acids, N -Fmoc-[N-Me]amino acids, Rink amide-AM resin (0.68 mmol/g), 1-hydrozybenzotriazole anhydrous (HBTU), O-benzotriazole-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HOBt), N,N-diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), and N,N′-diisoprophlcarbodiimide (Dic) were purchased from GL Biochem, Ltd. (Shanghai, China). N,N-Dimethylformanide (DMF), dichloromethane (CH2Cl2), and acetonitrile were purchased from DIMA Technology, Inc. (Beijing, China) and were of high-performance liquid chromatography (HPLC)-grade or better. (E)-3-(4-nitrophenyl)-acrylic acid (NAA) and other reagents for chemical synthesis were purchased from Alfa Aesar (Shanghai, China). All N-methylated analogues were synthesized by solid-phase peptide synthetic techniques.21−23 The analogues were prepared using a NαFmoc-protection scheme with Rink amide-AM resin. Individual NαFmoc-amino acids were coupled via HBTU, HOBt, and DIEA in DMF for 3 h. Removal of the Nα-Fmoc group was accomplished with 20% piperidine in CH2Cl2 for 20 min. The resin was then washed in turn with DMF, MeOH, and CH2Cl2 (2× each) and dried under reduced pressure. The peptides were then cleaved from the resin using TFA containing 5% phenol, 2.5% thioanisole, and 5% water for 2.5 h. The peptides were then washed with anhydrous ethyl ether and lyophilized. Crude peptides were dissolved in a mixture of aqueous 0.1% TFA and acetonitrile (exact ratio depending upon the solubility of the peptide) and then purified by preparative reversed-phase HPLC with a flow rate of 10 mL/min using acetonitrile/water (50:50, v/v). 2871

DOI: 10.1021/acs.jafc.5b00882 J. Agric. Food Chem. 2015, 63, 2870−2876

Article

Journal of Agricultural and Food Chemistry Preparation of Analogue 3. Nα-Me-Phe-Gly-Leu-Rink amideAM resin (0.5 mmol) was prepared as described in the general procedure. Nα-Fmoc-Gly (3 mmol, 6 equiv) and Dic (1.5 mmol, 3 equiv) were mixed in CH2Cl2 (5 mL) for 30 min at 0 °C, and then NαMe-Phe-Gly-Leu-Rink amide-AM resin and DIEA (3 mmol, 6 equiv) were added. The reaction mixture was shaken for 2 h. After removal of the Nα-Fmoc group, (E)-3-(4-nitrophenyl)-acrylic acid was coupled to the resin with HBTU, HOBt, and DIEA in DMF. The crude analogue 2 was purified by reversed-phase HPLC with a flow rate of 10 mL/min using acetonitrile/water (50:50, v/v) to yield 104 mg of peptide; HPLC tR, 11.2 min; HRMS, m/z 581.2715 [M + H]+ (calcd [M + H]+, 581.2718). Preparation of Analogue 4. Nα-Me-Gly-Phe-Gly-Leu-Rink amide-AM resin (0.5 mmol) was prepared as described in the general procedure. (E)-3-(4-Nitrophenyl)-acrylic acid (3 mmol, 6 equiv) and Dic (1.5 mmol, 3 equiv) were mixed in CH2Cl2 (5 mL) for 30 min at 0 °C, and then Na-Me-Gly-Phe-Gly-Leu-Rink amide-AM resin and DIEA (3 mmol, 6 equiv) were added. The reaction mixture was shaken for 2 h. The crude analogue 2 was purified by reversed-phase HPLC with a flow rate of 10 mL/min using acetonitrile/water (50:50, v/v) to yield 89 mg of peptide; HPLC tR, 8.8 min; HRMS, m/z 603.2540 [M + Na]+ (calcd [M + Na]+, 603.2538). Preparation of Analogue 5. Nα-Me-Phe-Gly-Leu-Rink amideAM resin (0.5 mmol) was prepared as described in the general procedure. Nα-Fmoc-N-Me-Gly (3 mmol, 6 equiv) and Dic (1.5 mmol, 3 equiv) were mixed in CH2Cl2 (5 mL) for 30 min at 0 °C, and then Nα-Me-Phe-Gly-Leu-Rink amide-AM resin and DIEA (3 mmol, 6 equiv) were added. The reaction mixture was shaken for 2 h. After removal of the Na-Fmoc group, (E)-3-(4-nitrophenyl)-acrylic acid (3 mmol, 6 equiv) and Dic (1.5 mmol, 3 equiv) were mixed in CH2Cl2 (5 mL) for 30 min at 0 °C and then Nα-Me-Gly-N-Me-Phe-Gly-Leu-Rink amide-AM resin and DIEA (3 mmol, 6 equiv) were added. The reaction mixture was shaken for 2 h, and the procedure was repeated. The crude analogue 5 was purified by reversed-phase HPLC with a flow rate of 10 mL/min using acetonitrile/water (50:50, v/v) to yield 85 mg; HPLC tR, 8.7 min; HRMS, m/z 595.2874 [M + H]+ (calcd [M + H]+, 595.2875). Preparation of Analogue 6. Nα-Me-Gly-Leu-Rink amide-AM resin (0.5 mmol) was prepared as described in the general procedure. Nα-Fmoc-N-Me-Phe (3 mmol, 6 equiv) and Dic (1.5 mmol, 3 equiv) were mixed in CH2Cl2 (5 mL) for 30 min at 0 °C, and then Nα-MeGly-Leu-Rink amide-AM resin and DIEA (3 mmol, 6 equiv) were added. The reaction mixture was shaken for 2 h. After removal of the Nα-Fmoc group, Nα-Fmoc-Gly was coupled to the resin using the same method as Nα-Fmoc-N-Me-Phe. (E)-3-(4-Nitrophenyl)-acrylic acid was coupled to the resin with HBTU, HOBt, and DIEA in DMF. The crude analogue 6 was purified by reversed-phase HPLC with a flow rate of 10 mL/min using acetonitrile/water (50:50, v/v) to yield 58 mg of peptide; HPLC tR, 10.9 min; HRMS, m/z 617.2694 [M + Na]+ (calcd [M + Na]+, 617.2694). Preparation of Analogue 7. Nα-Me-Leu-Rink amide-AM resin (0.5 mmol) was prepared as described in the general procedure. NαFmoc-Gly (3 mmol, 6 equiv) and Dic (1.5 mmol, 3 equiv) were mixed in CH2Cl2 (5 mL) for 30 min at 0 °C, and then Nα-Me-Leu-Rink amide-AM resin and DIEA (3 mmol, 6 equiv) were added. The reaction mixture was shaken for 2 h. After removal of the Nα-Fmoc group, Nα-Fmoc-N-Me-Phe, Nα-Fmoc-Gly, and (E)-3-(4-nitrophenyl)acrylic acid were coupled to the resin with HBTU, HOBt, and DIEA in DMF. The crude analogue 7 was purified by reversed-phase HPLC with a flow rate of 10 mL/min using acetonitrile/water (50:50, v/v) to yield 67 mg of peptide; HPLC tR, 12.3 min; HRMS, m/z 617.2698 [M + Na]+ (calcd [M + Na]+, 617.2694). Preparation of Analogue 8. Nα-Me-Leu-Rink amide-AM resin (0.5 mmol) was prepared as described in the general procedure. NαFmoc-Gly (3 mmol, 6 equiv) and Dic (1.5 mmol, 3 equiv) were mixed in CH2Cl2 (5 mL) for 30 min at 0 °C, and then Na-Me-Leu-Rink amide-AM resin and DIEA (3 mmol, 6 equiv) were added. The reaction mixture was shaken for 2 h. After removal of the Nα-Fmoc group, Nα-Fmoc-Phe and Nα-Fmoc-N-Me-Gly were coupled to the resin with HBTU, HOBt, and DIEA in DMF. (E)-3-(4-Nitrophenyl)-

acrylic acid was coupled to the resin using Dic. The crude analogue 8 was purified by reversed-phase HPLC with a flow rate of 10 mL/min using acetonitrile/water (50:50, v/v) to afford 63 mg of peptide; HPLC tR, 7.9 min; HRMS, m/z 617.2692 [M + Na]+ (calcd [M + Na]+, 617.2694). Biological Assay in Vitro. All radiochemical assays for JH biosynthesis were performed using individual CA from day 7 mated females. Compounds were dissolved in medium 199 with Hank’s salts, without L-methionine (custom formulation) (GIBCO, Grand Island, NY) for assay as described previously.24−26 Compounds were used in the bioassay on the same day of sample preparation. The solutions were discarded at the end of each day. Rates of JH release were determined using the modified in vitro radiochemical assay.26 This assay measures the incorporation of radiolabeled methionine into JH III in the final step of biosynthesis by CA maintained in vitro. CA were incubated for 3 h in 100 μL of medium 199 (GIBCO, 1.3 mM Ca2+, 2% Ficoll, methionine-free) containing [14C-S-methyl]-L-methionine (40 μM, specific radioactivity of 1.48−2.03 GBq/mmol) (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). Samples were extracted, and JH release was determined. Each data point on the dose−response figures represents replicate incubations of 10−17 experimental CA compared to control CA (i.e., no analogue added). NMR Experiments. All spectra of analogues 1−4 were obtained using a Bruker AMX 400 spectrometer (Bruker, Beijing, China) located at Beijing Nuclear Magnetic Resonance Center, Peking University, with tetramethylsilane (TMS) as the internal standard. The probe temperature was 298 K. The analogues were dissolved in 6 mM DMSO-d6 in a standard 5 mm NMR tube. Peaks were assigned using total correlation spectroscopy (TOCSY) spectra. TOCSY spectra were recorded with 640 points in F2 and 256 points in F1. Proton distances were calculated using the two-spin approximation from nuclear Overhauser enhancement spectroscopy (NOESY). The mixing times in TOCSY and NOESY spectra were 50 and 500 ms, respectively.13,27,28 Molecular Modeling. All molecular modeling calculations were performed using Sybyl 7.3 (Tripos, Inc., St. Louis, MO) running on a Silicon Graphics Fuel Visual workstation (Silicon Graphics, Inc., Milpitas, CA). Distance ranges of 1.8−2.7, 1.8−3.3, and 1.8−5.0 Å were assigned for strong, medium, and weak nuclear Overhauser enhancement (NOE) interactions, respectively. The dihedral angles of θ (H−N−Cα−Hα) and θ (Hα−Cα−Cβ−Hβ) were calculated according the value of 3JNα and 3Jαβ.29,30 The energy minimization of four analogues (analogues 1−4) used the minimized module of Sybyl. The force field was Merck Molecular Force Field 94 (MMFF94) with an 8.0 Å cutoff for non-bonded interactions, and the atomic point charges were also calculated by MMFF94. The minimizations were implemented with the descent method for the first 100 steps, followed by the Broyden Fletcher Goldfarb Shanno (BFGS) method until the root mean square (RMS) of the gradient was less than 0.005 kcal mol−1 Å−1.28 Then, these sample minimization procedures were selected to study their conformations using the method of the rotatingframe Overhauser enhancement (ROE)-restrained simulated annealing (SA) calculations. The ROE-derived distance constraints were applied as a biharmonic constraining function, and force constants for upper and lower boundaries were initially set to 20 kcal mol−1 Å−2. The atomic velocities were applied following a Boltzmann distribution about the center of mass to obtain a starting temperature of 100 K.

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RESULTS AND DISCUSSION Chemistry. Several methods for N-methylation of amino acids are known in the literature.23,31−33 Following the Fmoc strategy in peptide synthesis, methods of symmetrical anhydrides and esterification are favorable because it results in N-methylated Fmoc-protected amino acids in good overall yields without racemization. To permit N-protected amino acids and (E)-3-(4-nitrophenyl)-acrylic acid coupling to HBTU, their symmetrical anhydrides were used if amino acids or (E)-3-(4-nitrophenyl)2872

DOI: 10.1021/acs.jafc.5b00882 J. Agric. Food Chem. 2015, 63, 2870−2876

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Journal of Agricultural and Food Chemistry

Figure 2. Dose−response curves for analogues 1−8 and H17 with respect to inhibition of JH release by CA of day 7 mated female D. punctata in vitro. The IC50 is the concentration of analogue required to achieve 50% inhibition relative to maximal inhibition of JH production achievable. Each point represents the mean ± standard error of the mean (SEM); N = 10−20.

Table 1. NMR Assignment for AST Analogues analogue

residue

Hα (ppm)

Hβ (ppm)

1

NAA1 a Gly2 Phe3 Gly4 N-Me-Leu5 NAA1 a Gly2 Phe3 N-Me-Gly4 Leu5 NAA1 a Gly2 N-Me-Phe3 Gly4 Leu5 NAA1 a N-Me-Gly2 Phe3 Gly4 Leu5

6.92 3.75, 3.87 4.64 4.06 4.98 6.90 3.75 4.93 3.87 4.22, 4.32 7.05 3.89 4.85 3.56, 3.72 4.27 7.41 4.10 4.56 3.75 4.21

7.54

2

3

4

a

N−H (ppm)

other (ppm) 7.83 Hγ, 8.26 Hδ

8.40 8.21 8.26

2.75, 3.05

7.22 Hγ, 7.15 Hδ, 7.19 Hε 1.38 Hγ, 0.86 Hδ, 2.84 N-CH3, 7.08, 7.29 NH2 7.84 Hγ, 8.20 Hδ

1.61 7.54 8.42 8.36

2.81, 2.96 1.43, 1.48 7.56

7.89

7.25 2.76 1.57 7.85

Hγ, 7.16 Hδ, 7.20 Hε N-CH3 Hγ, 0.85 Hδ, 7.05, 7.35 NH2 Hγ, 8.23 Hδ

8.28 7.30 Hγ, 7.21 Hδ, 7.26 Hε, 2.78 N-CH3

2.96, 3.26 8.14 7.82

1.49 7.52 2.77, 3.05

8.43 8.32 8.01

1.47

1.57 7.93 2.70 7.26

Hγ, 0.84 Hδ, 7.17, 7.48 NH2 Hγ, 8.26 Hδ N-CH3 Hγ, 7.16 Hδ, 7.22 Hε

1.55 Hγ, 0.85 Hδ, 7.01, 7.31 NH2

NAA = (E)-3-(4-nitrophenyl)-acrylic acyl group.

acrylic acid were coupled to the Nα-Me-amino acid-Rink amideAM resin. If incomplete incorporation of the acylating agent was indicated, the amino acid was recoupled until a negative Kaiser test30,34 was obtained. After coupling of (E)-3-(4nitrophenyl)-acrylic acid, the peptide mimics were cleaved from the resin and then the crude peptide mimics were extracted into aqueous solution, concentrated under reduced pressure, and

lyophilized. Purification by reversed-phase high-performance liquid chromatography (RP-HPLC) yielded peptides that were >98% pure. Biological Data in Vitro. The ability of N-methylated AST analogues to inhibit biosynthesis of JH by the CA of D. punctata was evaluated by measuring the incorporation of radiolabeled methionine into JH III. As shown in Figure 2, analogues 1 and 2873

DOI: 10.1021/acs.jafc.5b00882 J. Agric. Food Chem. 2015, 63, 2870−2876

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Journal of Agricultural and Food Chemistry 4 in which C-terminal Leu5 and Gly2 were modified into Nmethylated Leu and N-methylated Gly, respectively, exhibited activity similar to H17 (IC50 = 6.15 × 10−8 M in vitro and IC50 = 1.2 × 10−8 M in vitro19). These results demonstrated that analogues 1 and 4 have a significant effect in vitro when compared to the pentapeptide (IC50 = 1.3 × 10−7 M).18 Single N-methylation of Phe3 and Gly4 produced about 2- and 4-fold lower activity than H17 but still showed significant potency with IC50 values of 1.03 × 10−7 and 2.97 × 10−7 M, respectively. A comparison of the activities elicited by single N-methylation mimics with double N-methylation mimics (to give analogues 5−8) suggested that the introduction of a double Nmethylation could decrease activity. Analogue 5, containing both N-methylated Gly2 and N-methylated Phe, was less active, with an IC50 value of 4.98 × 10−7 M. Analogue 6, containing both N-methylated Phe and N-methylated Gly4, was less active than analogue 2 but still showed activity, with an IC50 of 1.84 × 10−7 M. Analogue 7, which contains N-methylated Phe and Nmethylated Leu, and Analogue 8, which contains N-methylated Gly2 and N-methylated Leu, were found to have the lowest activity, with IC50 values of 3.37 × 10−6 and 2.11 × 10−6 M, respectively. NMR Studies. Conformational investigations of analogues 1−4 were performed using standard one-dimensional (1D) and two-dimensional (2D) NMR spectroscopy in DMSO-d6 at 298 K. A complete assignment of the protons was obtained (Table 1). The aliphatic spin systems and amide protons, besides the amide protons of the first residues, were observed using TOCSY experiments. The protons of Phe and (E)-3-(4nitrophenyl)-acrylic acid as well as the prochiral methylene protons of Gly2 and Gly4 were identified by corresponding 3Jαβ coupling constants and NOE correlations to their respective aromatic protons. Hα of the (E)-3-(4-nitrophenyl)-acrylic acyl group in analogues 1−3 appeared near 7 ppm, and in analogue 4, it appeared as 7.4 ppm. For analogue 1, N-CH3 appeared as a singlet at 2.84 ppm and Hα of N-Me-Leu5 appeared near 4.98 ppm. For analogue 2, N-CH3 appeared as a singlet at 2.76 ppm and Hα of N-Me-Gly4 appeared near 3.87 ppm. For analogue 3, N-CH3 appeared as a singlet at 2.78 ppm and Hα of N-Me-Gly4 appeared near 4.85 ppm. For analogue 4, N-CH3 appeared as a singlet at 2.70 ppm and Hα of N-Me-Gly4 appeared near 4.11 ppm. Important NOE correlations for analogue 1 included strong interactions between Gly4 Hα and Leu5 Hα and (E)-3-(4nitrophenyl)-acrylic acyl group Hγ and Phe3 N−H, a medium interaction between (E)-3-(4-nitrophenyl)-acrylic acyl group Hβ and Phe3 Hγ, and a weak interaction between (E)-3-(4nitrophenyl)-acrylic acyl group Hα and Gly2 N−H. For analogue 2, strong interactions were observed for (E)-3-(4nitrophenyl)-acrylic acyl group Hδ and Leu5 N−H and (E)-3(4-nitrophenyl)-acrylic acyl group Hα and Leu5 CO−NH2, a medium interaction was observed for (E)-3-(4-nitrophenyl)acrylic acyl group Hβ and Leu5 N−H, and a weak interaction was observed for (E)-3-(4-nitrophenyl)-acrylic acyl group Hγ and Leu5 CO−NH2. For analogue 3, an interaction was observed for (E)-3-(4-nitrophenyl)-acrylic acyl group Hβ and Leu5 CO−NH2, a medium interaction was observed for (E)-3(4-nitrophenyl)-acrylic acyl group Hγ and Gly4 N−H, and a weak interaction was observed for (E)-3-(4-nitrophenyl)-acrylic acyl group Hγ and Leu5 N−H. Finally, for analogue 4, a medium interaction occurred between (E)-3-(4-nitrophenyl)acrylic acyl group Hδ and Leu5 N−H, and a weak interaction

occurred between (E)-3-(4-nitrophenyl)-acrylic acyl group Hα and Phe3 Hδ. Molecular Modeling. Because of conformational flexibility, restrained molecular dynamics was applied, to determine conformation in the case of peptides. The 10 lowest energy structures found for the four analogues are shown in Figure 3.

Figure 3. Backbone atom superposition of the low-energy structures obtained for analogues 1−4 by simulated annealing. Only backbone atoms are shown for clarity.

Torsion angles and distance between Cα of different residues of the lowest energy structure are summarized in Tables 2 and 3, respectively. The different turn conformations were assigned by comparing the average Φ (i + 1), Ψ (i + 1), Φ (i + 2), and Ψ (i + 2) values with standard values, allowing for a ±30° deviation from standard values.35 Analogue 2 showed a type IV β-turn around Gly2-Phe3-Gly4-Leu5, and analogue 3 formed a type IV β-turn around NAA1-Gly2-Phe3-Gly4. As shown in Table 3, the distances of Cα(NAA1)−Cα(Leu5) of analogues 1 and 4 were 9.17 and 11.39 Å, respectively, and the distances of Cα(NAA1)−Cα(Leu5) for analogues 2 and 3 were 5.00 and 4.56 Å, respectively. Analogues 1 and 4 did not form a classic turn because the Cα(NAA1)−Cα(Gly4) and Cα(Gly2)−Cα(Leu5) distances were larger than 7.0 Å, the upper limit for turns.35 It seems that the conformation of analogues 1 and 4 was flexible and linear.36,37 In previous studies, the β-turn at the end of the C terminus was predicted in ASTs and AST mimics.11,13,28,38−40 We also found that analogues 2 and 3, which demonstrated significant activity, formed a type IV β-turn. However, analogues 1 and 4, which exhibited more activity, showed a flexible linear conformation. Banerjee et al.15 observed that Dippu-AST 5 did not form a β-turn at the end of its C-terminal motif, using sodium dodecyl sulfate and water as solvent, but rather showed a γ-turn.15 Therefore, we suggest that this kind of flexible linear conformation is a very important and indispensable structural element in AST mimics, and it may be directly involved in the recognition of the AST receptors. However, these ASTs and AST mimics are flexible, which are free to form any number of conformations under different conditions, such as β-turns, helical turns, or other conformations. Particularly, the flexible conformation does not eliminate the fact that the actual active conformation may be a β-turn, so that more data are required to confirm this hypothesis. In summary, eight AST analogues were synthesized using Nmethylation scanning, in an effort to understand the 2874

DOI: 10.1021/acs.jafc.5b00882 J. Agric. Food Chem. 2015, 63, 2870−2876

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Journal of Agricultural and Food Chemistry Table 2. Torsion Angles of the Lowest Energy Structure of Four Analogues by Simulated Annealing Gly2

Phe3

Gly4

Leu5

analogue

Φ (deg)

Ψ (deg)

Φ (deg)

Ψ (deg)

Φ (deg)

Ψ (deg)

Φ (deg)

Ψ (deg)

1 2 3 4

110 175 67 136

−32 −45 24 −71

11 −129 −134 −146

−78 −54 95 −51

122 86 113 −155

−72 88 93 117

−99 −121 −158 88

−86 37 147 −130

Table 3. Average Distance (Å) between Cα of NAA1 and Cα of Leu5, between Cα of NAA1 and Cα of Gly4, and between Cα of Gly2 and Cα of Leu5 in Analogues 1−4 of the Lowest Energy Conformation

a

analogue

Cα(NAA1 a)−Cα(Leu5) distance (Å)

Cα(NAA1 a)−Cα(Gly4) distance (Å)

Cα(Gly2)−Cα(Leu5) distance (Å)

1 2 3 4

9.17 5.00 4.56 11.39

7.82 6.09 4.79 8.00

7.84 6.44 7.38 9.13

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conformation of novel AST analogues and their ability to inhibit JH biosynthesis. The activity of analogues 1 and 4 proved roughly equivalent to that of the lead H17, with IC50 values of 5.17 × 10−8 and 6.44 × 10−8 M, respectively. Single N-methylation mimics (analogues 1−4) showed higher activity than double N-methylation mimics (analogues 5−8). We further investigated the conformational properties of single Nmethylation mimics (analogues 1−4) by 1D and 2D 1H NMR and molecular modeling. Evaluating the torsion angles and distances between Cα of different residues, we found that analogues 1 and 4, which formed a flexible linear conformation, exhibited more activity than analogues 2 and 3 containing a type IV β-turn. On the basis of our present study, these results provide evidence that the flexible linear conformation may be an indispensable structural element in AST mimics and may be involved in the interaction with the AST receptors.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: 86-10-62732223. E-mail: [email protected]. Author Contributions

Yong Xie, Xinling Yang, and Stephen S. Tobe designed experiments. Yong Xie, Chuanliang Zhang, Xiaoqing Wu, and Xile Deng synthesized AST mimics. Yong Xie finished biological assay and NMR studies. Li Zhang investigated the molecular modeling. Yong Xie, Xinling Yang, and Stephen S. Tobe wrote the paper. Funding

Financial support was provided by the National Natural Science Foundation of China (21372257 and 21132003), the National Basic Research Program of China (973 Program) (2010CB126104 and 2010CB126105), and the Natural Sciences and Engineering Research Council of Canada. Notes

The authors declare no competing financial interest.

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REFERENCES

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