Synthesis of Carlactone Derivatives to Develop a Novel Inhibitor of Strigolactone Biosynthesis

Abstract

Strigolactones (SLs), phytohormones that inhibit shoot branching in plants, promote the germination of root-parasitic plants, such as Striga spp. and Orobanche spp., which drastically reduces the crop yield. Therefore, reducing SL production via chemical treatment may increase the crop yield. To design specific inhibitors, it is valid to utilize the substrate structure of the target proteins as lead compounds. In this study, we focused on Os900, a rice enzyme that oxidizes the SL precursor carlactone (CL) to 4-deoxyorobanchol (4DO), and synthesized 10 CL derivatives. The effects of the synthesized CL derivatives on SL biosynthesis were evaluated by the Os900 enzyme assay in vitro and by measuring 4DO levels in rice root exudates. We identified some CL derivatives that inhibited SL biosynthesis in vitro and in vivo.

Figure 1

SL biosynthesis inhibitors. (A) Hydroxamic acid derivatives-type inhibitors. (B) Inhibitors with an azole moiety.

Figure 2

Structures of CL derivatives. (A) CL structure, with focus on four parts. (B) Structures of synthesized CL derivatives.

Scheme 1. Synthesis of KCL1, 2, 3, 5, 8, 9, and 10; (a) (1) C₄H₇ClO₂, Phenothiazine, NaH, Pyridine, −15 to 10 °C, 3.5 h (2) NaOH, MeOH, 0 °C, 1 h (3) NaBH₄, MeOH, 0 °C to rt, Overnight; (b) R–Br or R–COCl, n-BuLi, THF, 0 °C to rt, Overnight; (c) Thiophen-2(5H)-one or tert-Butyl (4-Methylthiazol-2-yl)carbamate, DEAD, PPh₃, Toluene, 0 °C to rt, 2 h or Overnight; (d) (1) Et₃SiH, RhCl(PPh₃)₃, 55 °C, 5 h (2) K₂CO₃, MeOH, 0 °C to rt, Overnight; (e) Ph₃P⁺CH₂OCH₃Cl^–, n-BuLi, THF, −78 to 0 °C, 30 min; rt, 24 h; (f) TsOH, Acetone/H₂O = 3:1, Reflux, 3 h; (g) NaBH₄, MeOH, 0 °C to rt, Overnight

Scheme 2. Synthesis of KCL4, 6, and 7; (a) NaClO, MeOH, 0 °C, 1 h; rt, 24 h; (b) Red-Al, THF, reflux, 1 h; (c) 5-Bromo-3-methyl-2(5H)-furanone, n-BuLi, THF, 0 °C to rt, 3 h; (d) MnO₂, CH₂Cl₂, rt, 1 h; (e) (1) C₄H₇ClO₂, Phenothiazine, NaH, Pyridine, −15 to 10 °C, 3.5 h (2) NaOH, MeOH, 0 °C, 1 h (3) AcOH, 0 °C, 10 min; (f) NaBH₄, EtOH, rt, 2 or 5 h

Figure 3

Effects of CL derivatives on Os900. CLA levels in case of (A) addition of 100 μM and (B) addition of 10–100 μM of each CL derivative to microsomal proteins, including Os900. CLA levels were determined by LC–MS/MS. “n.d.” = not detected. The data are mean ± S.D. (n = 3). * and ** denote statistically significant differences from the level for non-addition of CL derivatives (control) (Dunnett’s test; 0.01 < p < 0.05 and p < 0.01 respectively).

Figure 4

Effects of CL derivatives on the production of 4DO in planta at the concentration of 100 μM. (A) Comparison of KCL2 with KCL4, (B) KCL4 with KCL8 and KCL10. 4DO levels were determined by LC–MS/MS. The data are the means ± S.D. (n = 3 in A, n = 5 in B). ** denotes statistically significant difference from the 4DO level in the control (Dunnett’s test; p < 0.01).