β-Turn mimetic-based stabilizers of protein–protein interactions for the study of the non-canonical roles of leucyl-tRNA synthetase† †Electronic supplementary information (ESI) available: Detailed procedures for synthesis, bioassay, and biophysical experiments, and full characterization data of all new compounds. See DOI: 10.1039/c5sc03493k

For the systematic perturbation of protein–protein interactions, we designed and synthesized tetra-substituted hexahydro-4H-pyrazino[2,1-c][1,2,4]triazine-4,7(6H)-diones as β-turn mimetics. 5c{3,9} stabilizes the direct interaction between LRS and RagD and activates mTORC1 in living cells.


III. Synthesis and characterization of acid partners 1. General procedure for the preparation of tolyl acid partners
To a stirred suspension of p-tolylhydrazine hydrochloride in tetrahydrofuran (THF) under ice-water external bath, triethylamine was carefully added. Isocyanate (0.5 equiv.) was added dropwise to the solution. The resulting mixture was stirred at 0 o C and warmed up to room temperature for several hours.
After the completion of the reaction monitored by TLC, the solvent was removed under reduced pressure. Ethyl acetate was added and the organic layer was washed with saturated NaCl (aq.) and saturated ammonium chloride (aq.). The organic layer was dried over anhydrous MgSO4 (s) and filtered.
The organic solvent was evaporated under vacuum. The resulting mixture was purified through the recrystallization by ethyl acetate and n-hexane to afford a desired solid. Then, the resulting solid, KHCO3 and tert-butyl bromoacetate were dissolved in DMF and stirred at 80 o C for several hours. After completion of the reaction indicated by TLC, the reaction mixture was washed with saturated NaCl (aq.) and ammonium chloride (aq.) and extracted with ethyl acetate. The combined organic layer was condensed was dried over anhydrous MgSO4 and filtered. The filtrate was condensed under reduced pressure, followed by silica-gel flash column chromatography to afford a desired solid. The resultant was dissolved in 1,4-dioxane and 4N HCl was added slowly. The reaction mixture was stirred at room temperature for several hours. After completion of the reaction indicated by TLC, the solution was concentrated under reduced pressure. The saturated NaHCO3 (aq.) was added and the aqueous layer was washed with ethyl acetate. Concentrated HCl was added dropwise slowly at 0 o C (pH 2-3). The mixture was extracted with ethyl acetate (EA), and the organic layer was dried over anhydrous MgSO4(s) and evaporated. The residue was purified by recrystalization with ethyl acetate and n-hexane to give the desired product A-1 (30~35% overall yields in three steps).

General procedure for the preparation of methyl acid partners
To a stirred suspension of methylhydrazine sulfate in water (200 ml) under ice-water external bath, NaHCO3 was carefully added. Di-tert-butyl dicarbonate in tetrahydrofuran (200 mL) was added to the solution. The resulting mixture was left to stir and allowed to warm to room temperature for overnight.
After completion of the reaction indicated by TLC, the organic layer was extracted with ethyl acetate.
The combined solution was dried over anhydrous MgSO4(s) and filtered, and evaporated in vacuo to afford pale yellow oily compound without further purification. The resultant was dissolved in THF and isocyanate was added dropwise. The resulting mixture was stirred at 0 o C and warmed up to room temperature for several hours. After the completion of the reaction monitored by TLC, the solvent was removed under reduced pressure. The reaction mixture was purified through the recrystallization by ethyl acetate and n-hexane to afford a desired solid. The resulting solid dissolved in 1,4-dioxane and 4N HCl was added slowly. The reaction mixture was stirred at room temperature for several hours.
After completion of the reaction indicated by TLC, the solution was concentrated under reduced pressure. The saturated NaHCO3 (aq.) was added slowly (pH 11-12) under ice-water external bath. The aqueous layer was extracted by ethyl acetate, and it was dried over anhydrous MgSO4(s) and filtered.

Leucine Starvation
For leucine depletion, cells were rinsed with leucine-free DMEM twice, incubated in leucine-free DMEM for 60 min and replaced with and incubated in DMEM.

Western blotting
HEK293T cells were seeded on 6-well plate and incubated in 5% CO2 incubator at 37 °C overnight.
HEK293T cells were starved for leucine for 1 h and treated with compounds in leucine-deprived condition. Cells were washed by PBS and harvested. Cell lysates were obtained by 30 min treatment with RIPA cell lysis buffer containing protease inhibitors and phosphatase inhibitors at ice. After the centrifugation of cell lysates at 15,000 rpm and 4 °C for 30 min, the protein concentration in the supernatant was measured by BCA assay. The resulting proteome were analyzed by SDS-PAGE and transferred into PVDF membrane, followed by 2% BSA blocking in TBST over 1 h. The samples were subjected to western blotting to detect the S6K1, phospho-S6K1 (T389) or GAPDH with specific primary antibodies, e.g. anti-S6K1 and anti-phopho-S6K1 (T389) [Abcam], GAPDH [Cell Signaling Technology] antibodies for overnight at 4 °C, followed by washing with TBST for 1 h. The resulting S95 membrane was exposed into HRP-conjugated secondary antibody for 1 h at room temperature. After washing with TBST, the membrane was developed by ECL prime solution [GE healthcare] and the chemiluminescent signal was measured by ChemiDoc TM MP imaging system.

FRET imaging experiment and analysis
We carried out FRET imaging with DeltaVision