Crystal structure of diaphorin methanol monosolvate isolated from Diaphorina citri Kuwayama, the insect vector of citrus greening disease

‘Candidatus Profftella armatura’ a bacterial endosymbiont of D. citri, biosynthesizes diaphorin, which is a hybrid polyketide–nonribosomal peptide comprising two highly substituted tetrahydropyran rings joined by an N-acyl aminal bridge. The relative configurations of three out of its nine stereogenic centers, which could not be determined by NMR, were assigned based on the crystal structure.


Chemical context
Huanglongbing (HLB), also known as citrus greening disease, which destroys the marketability of citrus fruit and eventually kills the tree, is a major threat to world citrus production (Wang et al., 2017;Bové, 2006). HLB is associated with plant infection by one of three fastidious bacterial species, 'Candidatus Liberibacter asiaticus', 'Candidatus Liberibacter americanus' or 'Candidatus Liberibacter africanus'. All three bacteria are spread within a grove by psyllids -sap-sucking insects in the order Hemiptera. In North America, 'Ca. L. asiaticus' is transmitted by the invasive citrus pest, the Asian citrus psyllid, Diaphorina citri Kuwayama. A complex community of vertically transmitted endosymbiotic bacteria colonizes D. citri, whether or not the psyllids are infected with 'Ca. L. asiaticus' (Nakabachi et al., 2013). Two of these D. citri endosymbionts, 'Candidatus Profftella armatura', and 'Candidatus Carsonella rudii' are localized to the bacteriome, an organ in the D. citri abdomen (Nakabachi et al., 2013). While 'Ca. C. rudii' is the primary endosymbiont of many psyllid species, 'Ca. P. armatura' is only found in D. citri and ISSN 2056-9890 has been detected in every D. citri population surveyed, worldwide (Nakabachi et al., 2013). Approximately 15% of the 'Ca. P. armatura' genome is composed of a hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) gene and associated tailoring genes dedicated to the biosynthesis of diaphorin. Because 'Ca. P. armatura' is unculturable, diaphorin is extracted directly from its D. citri host (Nakabachi et al., 2013). Diaphorin is a hybrid polyketide-nonribosomal peptide in which two highly functionalized tetrahydropyran rings are joined by an N-acyl aminal bridge. It is a tri-O-desmethyl analog of pederin, a potent cytotoxin deriving from an undetermined Pseudomonas-like endosymbiont of staphylinid beetles in the genus Paederus (Cardani et al., 1967;Mosey & Floreancig, 2012;Cardani et al., 1965;Furusaki et al., 1968;Matsumoto et al., 1968;Piel, 2002). Nakabachi et al. (2013) assigned the relative configuration of six of the nine stereogenic centers in diaphorin, but carbons 7, 10 and 17 remained unspecified. We pursued the crystal structure of diaphorin to complete the assignment of the relative configuration of the molecule.

Structural commentary
The title compound crystallizes in the monoclinic P2 1 space group and features an N-acyl aminal bridge that connects two highly substituted tetrahydropyran rings adopting chair conformations (Fig. 1). Ring A substitutions comprise an equatorial methyl group on C2, an axial methyl group on C3, an exomethylene group on C4, and a methoxy group at C6. Ring A (O1/C2-C6) has a chair conformation with puckering parameters: amplitude Q = 0.541 (4) Å , = 173.0 (5) , ' = 265 (4) . Ring B (O11/C11-C15) substitutions comprise a hydroxyl group at C13, a geminal pair of methyl groups at C14 and a 2,3 dihydroxypropyl group at C15. It also has a chair conformation with puckering parameters: amplitude Q = 0.559 (4) Å , = 8.1 (4) , ' = 258 (3) . The mean planes of rings A and B are inclined to each other at an angle of 80.1 (2) . For the plane including the central amide bond, (C7/O7/C8/O8/ N9/C10), the r.m.s. deviation from the plane for those atoms is 0.045 Å . This planar conformation is likely influenced by a hydrogen bond in which the amide proton H9 is the donor and O7 is the acceptor with an interatomic distance of 2.16 Å between the participants (Fig. 1, Table 1). The chain from C13 through O18, viz. C13-C18/O18, is seen to be approximately planar, with an r.m.s. deviation from the plane of 0.117 Å . This conformation appears to result from crystal-packing interactions and probably has no biological significance. The crystal structure of the title compound assigns the three chiral centers left undetermined by Nakabachi et al. (2013) as 10S*, 13R*, and 17S*, and thus provides the complete relative configuration of diaphorin. The absolute configuration, as depicted in Fig. 1, was inferred by analogy to that of pederin di-pbromobenzoate (Furusaki et al., 1968), which it matches at all stereogenic centers.

Figure 1
A view of the molecular structure of the title compound, with the atom labeling. Displacement ellipsoids are drawn at the 50% probability level. Hydrogen bonds are shown as thin black lines (see Table 1) The combination of these intermolecular interactions leads to the formation of slabs lying parallel to the ab plane.  Corradi et al., 1971). They both have the same skeleton as diaphorin, except for the addition of the two p-bromobenzoate substituents. The structure of diaphorin can be matched to that of pederin by rotations about the following single bonds: C7-C8, N9-C10, C10-C11, and bonds in the C15-O18 moiety.

Isolation and crystallization
Diaphorin was isolated using a liquid-liquid extraction scheme with semi-preparative HPLC with modifications from the published method (Nakabachi et al., 2013). A batch of ca 3000 D. citri was reared on Citrus macrophylla (not infected by 'Ca. L. asiaticus') at the US Horticultural Research Labora-tory, Fort Pierce, FL 34945, USA. Insects were allocated to 2 ml microcentrifuge tubes, then flash frozen in liquid N 2 and cryoground for 3.5 min at 30 Hz using 3 Â 3.2 mm metal beads per tube in a ball mill apparatus (Retsch Mixer Miller MM-400). Ground insects in each tube were then extracted three times in MeOH for 45 min at 298 K. After agitation, the tubes were centrifuged for 2 min at 16,000 g, and the supernatants were pooled, filtered through two layers of Whatman #1 paper and dried in vacuo. The residue was taken up in 90% MeOH and partitioned against cyclohexane. The methanolic phase was then fractionated by repetitive semi-preparative reversed phase HPLC using a Thermo Fluophase1 column (250 Â 10 mm ID, 5 mm particle), eluted at 4 ml min À1 with 20% MeCN, and 1 ml fractions were collected. Following detection by UV absorption at 215 nm, selected fractions were monitored for the presence of diaphorin by syringe pump infusion (5 mL min À1 ) into a Waters-Micromass ZQ single quadrupole mass spectrometer (scan range: m/z 50-1500 in 1 sec with cone and capillary voltages of 25 and 3500 V, respectively). Fractions showing the pseudomolecular ion of diaphorin (M + Na + at m/z 484) were recombined and dried in vacuo to afford ca 4.0 mg of diaphorin. Crystals of the title compound were obtained by slow evaporation from MeOH. A single crystal measuring approximately 0.01 Â 0.02 Â 0.20 mm was harvested using a needle dipped in a drop of oil for adhesion (type A immersion oil, Hampton Research Corp.) and mounted in a small nylon loop (Hampton). The identity and purity of diaphorin was confirmed by comparing 1 H NMR data acquired using the sample that afforded crystals with published data (Nakabachi et al., 2013). Further confirmation was obtained by HPLC with detection by high resolution electrospray mass spectrometry (HRESIMS). Retention time (t R ) and accurate mass estimates were compared with those of authentic diaphorin using a Waters Acquity UPLC system with a Waters C18 BEH column (2.1 Â 50 mm; 1.7 mm), eluted at 0.3 ml min À1 using a gradient formed from 0.1% formic acid (A) and acetonitrile (B) with 0.1% formic acid (90% A 0-1 min, 14 min linear ramp to 80% A, followed by a 1 min ramp to 10% A, a 2 min hold, and a ramp back to 90% A in 1 min). Spectra were acquired on a Waters Xevo G-2 QTOF mass spectrometer operated in positive ion mode scanning the mass range from m/z 50 to 1200 in 0.1 sec with capillary and cone voltages set at 3.5 V and 25 k V, respectively. The spectrometer was calibrated in the range m/z 50-1200 using sodium formate. Spectra were calibrated in real-time using the M + H + of co-infused leucine encephalin (m/z 556.2771) as the reference and were further processed by centering using the proprietary 'automatic peak detection' tool supplied with Waters MassLynx1 4.1 software. A view normal to the ab plane of the crystal packing of the title compound. The methanol solvent molecules are shown in green and the hydrogen bonds as thin red lines (see Table 1

Refinement
Crystal data, data collection and structure refinement details are summarized in Table 2. The hydrogen atoms were fixed geometrically (O-H = 0.84 Å , N-H = 0.86 Å , C-H = 0.98-0.10 Å ) and allowed to ride on their parent atoms with U iso (H) = 1.5U eq (C-methyl, O-hydroxyl) and 1.2U eq (N, C) for other H atoms.
The absolute structure of the molecule in the crystal could not be determined by resonant scattering. It was assigned by analogy to that of pederin di-p-bromobenzoate methanol monosolvate (Furusaki et al., 1968), for which no atomic coordinates are available, and pederin di-p-bromobenzoate ethanol monosolvate (Corradi et al., 1971), for which the absolute configurations were determined by resonant scattering.
X-ray crystallographic data were collected at the Cornell High Energy Synchrotron Source (Ithaca, NY, 14853, USA). The synchrotron beamline available to us (CHESS F1) is normally used for macromolecular data collection. It is a fixedwavelength line and it is not possible (due to interference with equipment including the crystal-mounting robot) to move the area detector (Pilatus 6M) close enough to the sample to record data beyond 0.95 Å (in the corners; only to 1.15 Å at the edges). This explains the lack of high-resolution data, and the large s.u.'s on the cell dimensions, which may also be related to the use of the program XDS, which is typically used for macromolecular data reduction, for refinement of these and other experimental parameters.