Tryptophan

Amino acids tyrosine and tryptophan (99% and 97% Acros, Düsseldorf, Germany), (AIBN, 98% Acrōs Germany) 2,2′-azobis(isobutyronitrile) were recrystallized from methanol, and N-isopropylacrylamide (NIPAAm, Düsseldorf) was recrystallized from distilled hexane. Vanillin (99% Düsseldorf, Germany), triethylamine (99% Merck, Darmstadt, Germany), acryloyl chloride (98% Merck, Darmstadt, Germany), formaldehyde (38% Sigma-Aldrich, Darmstadt, Germany), diethyl amine (99% Acros, Düsseldorf, Germany), Dichloromethane, dioxane, tetrahydrofuran (THF), and diethyl ether were distilled over potassium hydroxide. Other chemicals were used as received. For pH 1.68, pH 7 and pH 12.46 buffer solutions were used as purchased from Thermo Fisher (Loughborough, US).

Defined minimal medium contained 6.7 g/L of yeast nitrogen base without amino acids (catalog no. Y0626; Sigma-Aldrich, MO, USA), 20 g/L of glucose, 380 mg/L leucine (catalog no. 172130250; Acros Organics, CA, USA), 76 mg/L uracil (catalog no. 157301000; Acros Organics, CA, USA), and various concentrations of [EMIM][OAc] (>95% purity) (IoLiTec, AL, USA). Synthetic complete medium without leucine and uracil (SC-Leu-Ura) was prepared with 6.7 g/L of yeast nitrogen base without amino acids; 1.46 g/L of yeast synthetic dropout medium supplement without uracil, leucine, and tryptophan (catalog no. Y1771; Sigma-Aldrich, MO, USA); 76 mg/L tryptophan (catalog no. 172110250; Acros Organics, CA, USA), 20 g/L glucose, and various concentrations of [EMIM][OAc]. SC without leucine (SC-Leu) was prepared by adding 76 mg/L uracil to SC-Leu-Ura medium.

An IDO enzymatic activity was performed using a protocol adapted from previously described methods [53 (link), 54 (link)]. Murine samples used in the IDO activity assays were prepared in Dulbecco's Phosphate Buffered Saline (DPBS) (Fisher) containing protease inhibitors followed by sonication. 2X IDO reaction buffer (200 mg/ml catalase from bovine liver (Sigma), 800 mM L-tryptophan (Sigma), 100 mM DPBS, 40 mM L-ascorbic acid (Sigma), 20 mM methylene blue (Fisher), with and without tryptophan, was added to samples that were normalized for total protein by BCA assay. Samples were incubated at 37°C for 30 min and the reaction was stopped with 30% trichloroacetic acid (TCA) (Sigma). Samples were incubated again at 52°C for 30 min followed by centrifugation (5 min, 10,000 rpm, 4°C). Supernatants were used for spectrophotometric analyses by the addition of an equal amount of Ehrlich's Solution (Sigma). Samples were read at an absorbance of 480 nm and values were calculated based on a standard curve of L-Kynurenine (Sigma) from 0-30,000 mM. Final IDO activity values were determined by taking the difference between samples without tryptophan-containing IDO reaction buffer and those receiving tryptophan-containing IDO reaction buffer.