X-ray crystal structure, UV–Vis and NMR spectroscopic, and molecular docking studies of pyribencarb isomers

Abstract

The crystal structures of the pyribencarb E and Z stereoisomers were determined using single-crystal X-ray crystallography. The isomers were confirmed a single data respectively by crystal analysis, LC-UVD mass spectrometry, and NMR spectroscopy. Pyribencarb E crystallizes in triclinic P − 1 and the Z isomer in monoclinic P2₁/c, with the crystal structures showing comparable packing motifs. Moreover, molecular docking was carried out with cytochrome bc₁, revealing binding energies in the ranges of − 24.9 to − 17.6 and − 21.6 to − 14.7 kcal/mol for the E and Z isomers, respectively. Through a combined experimental and theoretical approach, this study contributes to our understanding of pesticides.

Graphical Abstract

Introduction

X-ray crystallography has advanced rapidly because X-rays were discovered in 1895 and applied to crystal structure determination for the first time in 1913 [1]. It provides accurate and reliable three-dimensional (3D) structural parameters [2]. With the development of molecular structure technology over the past 30 years, the range of applications and reliability of X-ray crystallography have been significantly improved. And along with nuclear magnetic resonance (NMR) and mass spectrometry (MS), it has emerged as one of the key structural characterization methods for chemical compounds and biological molecules [1].

Pyribencarb is a carbamate fungicide composed of the E (methyl{2-chloro-5-[(1E)-1-(6-methyl-2-pyridylmethoxyimino)ethyl]benzyl} carbamate) and the corresponding Z stereoisomers [3]. It was developed by Kumiai Chemical Industry Co., Ltd. and Ihara Chemical Industry Co., Ltd., and it is effective against pathogenic plant fungi, especially Botrytis cinerea (cucumber gray mold) and Sclerotinia sclerotiorum (stem rot). Pyribencarb is a quinone outside inhibitor (QoI) that interrupts mitochondrial respiration by binding to the Qo site of the cytochrome bc₁ enzyme complex (complex III) of the electron transport system [4]. The property of QoIs to inhibit mitochondrial respiration by binding to the outer quinol-oxidation site of the cytochrome bc₁ enzyme complex is the mechanism of their fungicidal action. The synthesis of ATP is stopped in fungal cells as a result of this inhibition, which prevents electron transmission between cytochrome b and cytochrome c₁ [5].

The E and Z stereoisomers have different toxicokinetic properties, and only pyribencarb E is used in commercial formulations [6]. Because stereochemistry affects crystal packing and photophysical properties, we determined the 3D structure of pyribencarb and carried out molecular docking studies with cytochrome bc₁.

Materials and methods

Chemicals

Sodium chloride, ammonium formate, formic acid, acetone, and methanol were purchased from Merck (Darmstadt, Germany). Deionized water was prepared by reverse osmosis (LaboStar™ TWF UV 7, Siemens, MA, USA). Pyribencarb E and Z were purchased from Chem Service Inc. (West Chester, PA, USA). All chemicals were purchased at the LC–MS grade.

LC–MS/MS and NMR

LC–MS/MS analyses were conducted on a UHPLC system coupled with a triple quadrupole mass spectrometer (Shimadzu LCMS-8040, Kyoto, Japan). UV–Vis spectra were recorded in the range of 190–700 nm, and in-line chromatograph acquisition was conducted using 270 nm. Individual standard solutions of pyribencarb E and Z were separated on a Raptor ARC-18 (2.1 × 10 mm, 2.7 μm, Restek, USA). The column temperature was maintained at 40 °C, and the injection volume was 5 µL. A gradient elution consisting of mobile phases A (0.1% formic acid and 5 mM ammonium formate in water) and B (0.1% formic acid and 5 mM ammonium formate in methanol) was used at a flow rate of 0.2 mL/min that was maintained for the duration of the analysis (15 min). Initially, 5% of mobile phase B was used for 1 min, which was ramped to 70% over 2 min, maintained at 70% for 3 min, ramped to 98% over 1 min, maintained at 98% for 2.5 min, lowered to 5% over 0.5 min, and maintained at 5% for 5 min.

For MS analysis, positive electrospray ionization was applied. In order to generate mass spectra, a range of 100–500 m/z was scanned. The heat block and desolvation line had temperatures of 250 and 400 °C, respectively. Gas flow rates for nebulizing and drying were 3 and 15 L/min, respectively. LabSolutions (version 5.60) was used for data processing. NMR spectra were measured in deuterated chloroform using an AVANCE-600 NMR spectrometer (600 MHz, Bruker, Germany).

X-ray crystallography

Crystals of the E and Z isomers were prepared based on differing solubilities as follows. The isomers (5 mg) were dissolved in dichloromethane (0.2 mL); hexane was slowly added until the solution started turning turbid; finally, 1–2 drops of dichloromethane were added until the solution cleared. After 2–3 days at room temperature (25 °C), crystals sufficient for single-crystal X-ray crystallography were formed. Diffraction data were collected using Mo Kα₁ radiation (RIGAKU R-AXIS RAPID diffractometer).

Design of molecular docking

Molecular docking is used to investigate the binding of ligands (small molecules) by macromolecules (proteins). It can distinguish between the binding modes for different orientations of a ligand within the protein binding site [7]. The crystal structure of the Rhodobacter sphaeroides cytochrome bc₁ complex with azoxystrobin was obtained from the research collaboratory for structural bioinformatics protein data bank (https://www.rcsb.org/, file: ‘6NHH.pdb’) [8].

The binding site was determined based on the LigPlot analysis of the three-dimensional (3D) structure of cytochrome bc₁ deposited in the Protein Data Bank as 6NHH.pdb. As shown in Additional file 1: Fig. S1 obtained from the LigPlot analysis of the crystallographic structure, 6NHH.pdb, 18 residues including M336, F337, F301, I340, M140, M154, I162, F298, I292, P294, V293, G158, F144, S155, V148, A159, Y297, Y147 participated in the hydrophobic interactions with the ligand, Azoxystrobin (named as Azo1003), and E295 formed hydrogen-bond. These 19 residues formed the binding site. Because the Sybyl program adapted the flexible docking method, the docking radius was set to 6.5 Å surrounding the binding site. Thirty iterations of the docking procedure were carried out for the flexible docking, so that 30 complexes between the protein and the ligand were generated. Of 30 complexes with different docking poses, the complex with the low binding energy and good docking pose was selected for analyzing the in silico docking experimental result.

The crystal structure consists of A–G chains and A and E chains that correspond to cytochrome b. The A chain was selected for docking experiments. Docking experiments were performed on an Intel Core 2 Quad Q6600 (2.4 GHz) Linux PC using SYBYL 7.3 (Tripos, St. Louis, MO, USA) [9]. The binding sites were determined using Ligplot [10] and PyMOL 2.0 (Schrödinger, Portland, OR, USA). PyMOL was used for visualizing complex structures, and images were generated for the configurations with the lowest binding energies.

Results and discussion

Spectrometry results

LC–MS/MS analysis parameters were optimized for separating the pyribencarb E and Z isomers. UV–Vis and total ion chromatograms are shown in Fig. 1, indicating clear separation. Figure 2 shows the LC–MS/MS scan spectra of the pyribencarb E and Z. Figure 3 shows that the peak at 270 nm is not affected by the solvent (methanol solvent cut off: 210 nm).

Fig. 1

LC–MS/MS chromatograms of pyribencarb using A UV–Vis (270 nm) and B total ion detection

Fig. 2

LC–MS/MS product ion fragmentation spectra of A the precursor and pyribencarb E (B) and Z (c)

Fig. 3

UV–Vis spectra of pyribencarb E (A) and Z (B)

The stereoisomers are indistinguishable by 1D ¹H NMR spectroscopy; however, the peaks at 7.20 and 2.30 ppm are attributed to C5 and C9, respectively. The ROESY spectrum of pyribencarb E shown in Fig. 4c indicates a correlation between the hydrogens on C5 and C9, which is absent for pyribencarb Z. Therefore, the geometrical differences of the E and Z isomers could be confirmed by 2D ROESY NMR spectroscopy.

Fig. 4

ORTEP diagrams of pyribencarb E (A) and Z (B). C ROESY ¹H NMR spectra of the pyribencarb isomers

Crystal structure analysis

The crystal structures of pyribencarb E and Z were determined using X-ray crystallography (Fig. 5; Table 1; Pyribencarb E: space group = P-1, Z = 2, a = 8.0729(5) Å, b = 10.1590(6) Å, c = 11.4462(6) Å, α = 77.684(2)°, β = 83.711(2)°, γ = 77.840(2)°, volume = 894.50(9) ų; Pyribencarb Z: space group = P2₁/c, Z = 4, a = 8.0092(4) Å, b = 10.9729(4) Å, c = 21.7358(11) Å, α = 90°, β = 100.109(2)°, γ = 90°, volume = 1880.58(15) ų). The lengths of the crystals formed are 58 µm for E (A) and 47 µm for Z (B), respectively. ORTEP diagrams of pyribencarb E and Z are shown in Fig. 4a, b, respectively, and bond lengths and angles are summarized in Tables 2 and 3, respectively. While the bond lengths and most of the angles of the two isomers are comparable, the N(2)–C(8)–C(10) and N(2)–C(8)–C(9) bond angles were approximately opposite at 116.20° and 123.62°, respectively, in pyribencarb E, and 125.39° and 115.82° in pyribencarb Z.

Fig. 5

Photographs of prepared crystals of pyribencarb E (A) and Z (B)

Table 1 Crystal data and structure refinement for the stereoisomers of pyribencarb

Table 2 Bond lengths of pyribencarb E and Z

Table 3 Valence angles of pyribencarb E and Z

Pyribencarb E crystallizes in triclinic P-1 and the Z isomer in monoclinic P2₁/c. Packing diagrams of pyribencarb E and Z are shown in Fig. 6; the methyl groups are facing each other in both crystal structures.

Fig. 6

Packing diagrams of pyribencarb E (A) and Z (B)

From a comparison of the total energies of pyribencarb E and Z, it can be understood why both isomers are detected in crops treated with the commercial fungicide composed of only pyribencarb E. The energies of the molecules were calculated using Tripos in Sybyl to be 4.195 and 6.033 kcal/mol for the E and Z forms, respectively. These results indicate that only 1.838 kcal/mol is required to convert from the E to the Z form. Because the visible region of light comprises photons with energies of 36 to 72 kcal/mol [11], it is sufficient to effect this conversion.

Molecular electrostatic potential map

Molecular electrostatic potential (MEP) maps aid understanding of the electronic distribution of a molecule, locating electrophilic or nucleophilic reactive sites, and indicating possible hydrogen bonding interactions [12, 13]. Incoming electrophiles attack negative regions (red), while the positive regions (blue) are preferred sites for nucleophilic attack [14]. As shown in Fig. 7, electron-deficient regions (red) are associated with chloride and oxygen atoms, and electron-rich regions (blue) are found close to the N atom of the pyridine ring. From their comparable MEP maps, it can be inferred that both stereoisomers of pyribencar undergo similar intermolecular interactions and have similar reactivities.

Fig. 7

MEP maps of pyribencarb E (A) and Z (B)

Molecular docking analysis

Our molecular docking analysis indicated a difference between the binding energies of the stereoisomers to cytochrome b. The target site of pyribencarb is the Qo site of the cytochrome b of complex III in the electron transport system of the respiratory chain [15]. The cytochrome b complex forms the mid-section of the cellular respiratory chain [16].

Pyribencarb was docked into the binding pocket of the Rhodobacter sphaeroides cytochrome bc₁ complex with azoxystrobin [8]. Azoxystrobin has the same mode of action as pyribencarb; therefore, comparable docking results are expected. Pyribencarb docking results are shown in Fig. 8a and listed in Table 4. Overall, pyribencarb E shows a more favorable binding energy (− 24.9 to − 17.6 kcal/mol) than pyribencarb Z (− 21.6 to − 14.7 kcal/mol). Moreover, the E isomer forms 3 hydrogen bonding (R94, A98, N279) and 10 hydrophobic interactions with residues, whereas the Z isomer forms 2 hydrogen bonds (R94, Y297) and 12 hydrophobic interactions. The higher binding efficiency of the E isomer rationalizes its commercial application as a pesticide instead of the Z isomer or a racemate.

Fig. 8

A Pyribencarb bound in the Qo site of cytochrome bc₁. B A detailed view of the binding site

Table 4 Molecular docking results of pyribencarb bound in the Qo site of cytochrome bc₁

We have evaluated the structural features of the pyribencarb E and Z stereoisomers utilizing X-ray crystallography, UV-Vis, LC-MS/MS, NMR, and molecular docking. The isomers were successfully separated using UHPLC (270 nm and MS/MS detection) and distinguished using ROESY NMR. Single-crystal X-ray diffraction analysis of pyribencarb E and Z showed moderate differences in their respective crystal packing. In addition, molecular docking experiments were carried out to evaluate binding efficiencies with the Qo site of cytochrome bc₁, indicating stronger binding by the E isomer.

The detailed structural insights gained of pyribencarb, along with elucidation of its docking mode with cytochrome bc₁, further the understanding of pesticides and inform future investigations.