Bioorthogonal pro-metabolites for profiling short chain fatty acylation† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc00247e

A systematically designed panel of biorthogonal pro-metabolites was synthesized and evaluated as agents for tracing cellular short chain fatty acylation.

Experimental workflow for assessing labeling of overexpressed FLAG-EP300 by pro-metabolite 6. Cells were transfected with FLAG-EP300 for 48 h, followed by treatment with 6 (5 mM) for 6 h. Cells were lysed, subject to Cu-catalyzed azidealkyne cycloaddition with biotin alkyne, enriched via FLAG immunoprecipitation kit (Sigma), and analyzed for EP300 capture and 6-labeling by Western blot.
Control experiments were performed in parallel, in which 6 or FLAG-EP300 were

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DCC (687 mg, 3.33 mmol) was added and stirred for 10 minutes. The ice bath was removed and the reaction was stirred overnight at room temperature. The reaction mixture was filtered and DMF was removed in vacuo. The purified compound was isolated using flash chromatography (030% dichloromethane: methanol) to yield product 4 as an oil (79 mg, 23%). 1  were dissolved in 2 mL of water in a 10 mL microwave reaction vessel. The mixture was microwaved at 120 C for 30 minutes with stirring. The reaction was allowed to cool to room temperature, extracted with diethyl ether, dried over Na 2 SO 4 , and solvent removed in vacuo. The purified compound was isolated and by flash chromatography (030% dichloromethane: methanol) to yield product 5 as a colorless oil (99 mg, 86%). 1 13
The reaction mixture was filtered and DMF was removed in vacuo. The purified compound was isolated by flash chromatography (030% CH 2 Cl 2 : MeOH) to yield product 8 as an oil (157 mg, 41%). 1       The reaction mixture was filtered and DMF was removed in vacuo. The purified compound was isolated by HPLC using a mobile phase consisting of a gradient of MeCN in 0.1% trifluoroacetic acid (aqueous). HPLC purification was performed using an Agilent 1250 Infinity HPLC equipped with a semi-preparative Phenomenex Gemini C18 column (150 x 21.2 mm, 10 μm) to yield product 11 as an oil (125 mg, 52%). 1 -1,2,3-triyl tris(pent-4-ynoate) (xii) propane-1,2,3-triyl tris(hex-5-ynoate)  SILEC experiments were performed as previously described. Acyl-CoAs were isolated by acid extraction and analyzed by LC-MS as previously described. 5, 6 3azidopropionyl-CoA was synthesized and purified by standard methods, 2, 7 and used as a standard in LC-MS analyses. Histones were isolated by acid extraction and analyzed by immunoblotting as previously described. 8,9 Toxicity of prometabolites were assessed by sulforhodamine B staining 10

Treatment of cells with pro-metabolites for metabolic labeling analyses
For labeling experiments, cell lines were plated and allowed to adhere overnight.
Cells were then treated with pro-

Mass spectrometry characterization of azidopropionyl-CoA formation
Cellular formation of azidopropionyl-CoA was confirmed by LC-HRMS and LC-MS/HRMS using previously described methods. 12  dppm= -0.27) (Fig. S3a, Fig. S3c). To further confirm this finding, we performed stable isotope labeling by essential nutrients in cell culture (SILEC), treating Hepa1c1c7 murine hepatocellular carcinoma cells with 13 C 3 15 N 1 pantothenate as previously reported. 13 Treatment of cells with 6 (0.1 mM, 1 h) generated 13 C 3 15 N 1 -azidopropionyl-CoA, where the stable isotope labeling is enriched in the region of the CoA backbone generated from pantothenate. A mixture of 1:1 by volume of cell extract from unlabeled and SILEC labeled cells produced a perfectly co-eluting HRMS and MS/HRMS peak, validating our identification of azidopropionyl-CoA as a biochemically produced acyl-CoA metabolite (Fig. S3d).