Biologically active Phytophthora mating hormone prepared by catalytic asymmetric total synthesis

Abstract

A Phytophthora mating hormone with an array of 1,5-stereogenic centers has been synthesized by using our recently developed methodology of catalytic enantioselective conjugate addition of Grignard reagents. We applied this methodology in a diastereo- and enantioselective iterative route and obtained two of the 16 possible stereoisomers of Phytophthora hormone alpha1. These synthetic stereoisomers induced the formation of sexual spores (oospores) in A2 mating type strains of three heterothallic Phytophthora species, P. infestans, P. capsici, and P. nicotianae but not in A1 mating type strains. The response was concentration-dependent, and the oospores were viable. These results demonstrate that the biological activity of the synthetic hormone resembles that of the natural hormone alpha1. Mating hormones are essential components in the sexual life cycle of a variety of organisms. For plant pathogens like Phytophthora, sexual reproduction is important as a source of genetic variation. Moreover, the thick-walled oospores are the most durable propagules that can survive harsh environmental conditions. Sexual reproduction can thus greatly affect disease epidemics. The availability of synthetic compounds mimicking the activity of Phytophthora mating hormone will be instrumental for further unravelling sexual reproduction in this important group of plant pathogens.

Fig. 1.

Structure of mating hormone α1 (1) (A) and enantioselective CA of Grignard reagents to α, β-unsaturated thioesters (B).

Fig. 2.

Retrosynthesis of mating hormone α1.

Scheme 1.

Synthesis of dithiane 3. Reagents: (a) TBSCl, Et₃N, and DMAP in CH₂Cl₂ at room temperature. (b) IBX in EtOAc at 70°C. (c) PPh₃CH-COSEt in CH₂Cl₂ at 40°C. (d) MeMgBr and 2 mol% (R, S)-7 in ^tBuOME at −78°C. (e) DIBALH in CH₂Cl₂ at −40°C. (f) TBDPSCl, Et₃N, and DMAP in CH₂Cl₂ at room temperature. (g) AcOH, THF, and H₂O (3/2/2) at 50°C. (h) IBX in EtOAc at 50°C. (i) HS(CH₂)₃SH and BF₃·OEt₂ in CH₂ at −78°C.

Scheme 2.

Synthesis of iodide 4. Reagents: (a) MeMgBr and 1 mol% (R,S)-7 in ^tBuOMe at −78°C. (b) DIBALH in CH₂Cl₂ at −40°C. (c) PPh₃CH-COCH₃ in THF at 66°C. (d) H₂ and Pd/C in EtOAc at room temperature. (e) CH₂CH-CHCHCOTMS(OEt), TBAT, 10 mol% Cu(OTf)₂, and 10 mol% (R)-Tol-BINAP in THF at room temperature. (f) H₂, Pd/C in EtOAc at room temperature. (g) DIBALH in hexane at −78°C. (h) PPh₃CH-COSEt in CH₂Cl₂ at 40°C. (i) TESOTf and 2,6-lutidine in CH₂Cl₂ at 0°C to room temperature. (j) MeMgBr and 2 mol% (S,R)-7 in BuOMe at −78°C. (k) DIBALH in CH₂Cl₂ at −40°C. (l) PPh₃, I₂, and imidazole in CH₂Cl₂ at room temperature.

Scheme 3.

Synthesis of and stereoisomers. Reagents: (a) ^tBuLi in 10% HMPA in THF at −78°C. (b) Mel and CaCO₃ in MeCN, H₂O, and THF (4/1/1) at 50°C. (c) TBAF in THF at room temperature.

Fig. 3.

Growth deformation in Phytophthora infestans induced by synthetic hormone. (A and B) Mycelium grown for 14 days on clarified V8 medium in the absence (0) or presence (800, 1,600) of stereoisomer 1. Seven days after transfer of a mycelial plug to fresh medium, a 10-μl solution of stereoisomer 1 was added to wells placed on both sides of the mycelial plug along the “a” line. (A) A2 strain NL88133. (B) A1 strain NL80029. For further details on the bioassay, see Materials and Methods. (C) Graphical display of change in colony shape induced by the two stereoisomers, 1 and 1′. The y axis shows the ratio a/b in which “a” and “b” are the diameters of the colony measured along the lines marked by “a” and “b” in A, respectively. The graph shows the result of one experiment. The experiment was repeated three times with the same outcome. (D and E) Morphology of hyphae visualized by light microscopy. Strain NL88133 was grown in the absence (D) or presence (E) of stereoisomer 1 (1,600 ng). The arrow points to one of the many bulbed protrusions observed on the hyphae in the presence of the synthetic hormone.

Fig. 4.

Oospore formation in Phytophthora species induced by synthetic hormone. (A) Mycelium of P. capsici strain LT3241 grown in the presence of 1,600 ng of synthetic hormone. The dark area is the border of the well to which stereoisomer 1 was added. Numerous oospores are formed at the border and right of the border, one of which is marked by a white arrow. The black arrow points to a chlamydospore. Chlamydospores are asexual spores that are also found in P. capsici cultures of single strains so independent of the presence of the opposite mating type or synthetic hormone. At higher magnification, oospores and chlamydospores are clearly distinguishable. (B) Oospore obtained by cocultivation of two P. capsici strains of opposite mating type, LT3112 (A1) and LT3241 (A2). (C–F) Oospores induced by synthetic hormone. (C and D) Oospore of P. capsici strain LT3241. In C, the white arrows point to germination tubes emerging from the oospore. (E and F) Oospore of P. nicotianae strain P582 (E) and P. infestans strain CN505502B (F). (Scale bars: A, 100 μm; B–F, 10 μm.)