Mandipropamid as a chemical inducer of proximity for in vivo applications

Direct control of protein interactions by chemically induced protein proximity holds great potential for both cell and synthetic biology as well as therapeutic applications. Low toxicity, orthogonality and excellent cell permeability are important criteria for chemical inducers of proximity (CIPs), in particular for in vivo applications. Here, we present the use of the agrochemical mandipropamid (Mandi) as a highly efficient CIP in cell culture systems and living organisms. Mandi specifically induces complex formation between a sixfold mutant of the plant hormone receptor pyrabactin resistance 1 (PYR1) and abscisic acid insensitive (ABI). It is orthogonal to other plant hormone-based CIPs and rapamycin-based CIP systems. We demonstrate the applicability of the Mandi system for rapid and efficient protein translocation in mammalian cells and zebrafish embryos, protein network shuttling and manipulation of endogenous proteins.

All fragments were ligated by Gibson Assembly and sequenced using standard primers (EGFP-C-F-31).

pYFP-PYR
Here, we present an alternative method to access mandipropamid at the gram scale: the extraction and isolation of mandipropamid from Revus TOP ® , an agrochemical marketed by Syngenta.

Extraction of Mandipropamid (1) from Revus TOP ®
Mandipropamid was isolated from the commercially available suspension concentrate Revus TOP ® , which contains mandipropamid and difenoconazol. 10 g Revus TOP ® was suspended in 50 ml H2O and extracted 10-fold with 50 ml DCM. The yellow extract was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography (SiO2, DCM:EA-9:1) yielding in an off-white solid (2.2 g, 5.34 mmol).

3-(p-Tolyl)prop-2-yn-1-yl 4-methylbenzenesulfonate
According to Omar et al. 7 , tosyl chloride (26.1 mg, 136.8 µmol, 1 eq) and (p-tolyl)prop-2-in-1-ol (20.0 mg, 136.8 µmol, 1 eq) were dissolved in 5 ml diethyl ether and cooled to 0 °C. Freshly grinded KOH (76.8 mg, 1,37 mmol, 10 eq) was slowly added and the reaction was stirred for further 4 h at 0 °C. After TLC showed full conversion, the reaction mixture was quenched with ice water and extracted with diethyl ether. The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure. The product was obtained as a brown oil (29.9 mg, 99.5 µmol, 73%). The measured NMR spectrum was in accordance with literature and the product was directly used without further purification.

ABA-(S)-OH
According to Takeuchi et al. 6 , a flame-dried Schlenk flask was equipped with abscisic acid 3(50 mg, 189.2 µmol, 1 eq) and dissolved in 4 ml dry methanol. The solution was cooled to 0°C and cerium(III) chloride heptahydrate (211.4 mg, 567.5 µmol, 3 eq) and sodium borohydride (15.1 mg, 399.1 μmol, 2.1 eq) were added. The reaction mixture was stirred for further 60 min and quenched with a saturated solution of ammonium chloride. After concentration of the solution in vacuo to remove methanol, the reaction mixture was acidified to pH 2 by addition of aqueous 1 M HCl and extracted with DCM. The combined organic layer was washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography (SiO2, Cy:EA-3:1), yielding in a colorless, cloudy oil (31.3 mg, 117.5 µmol, 62 %).