N6-(2,2,2-Trifluoroacetyl)-L-lysine

Sodium (4.6 g; 0.2 mol) was dissolved in 200 ml of ethanol (abs). To this solution, 18.25 g (0.1 mol) of lysine (Sigma-Aldrich) hydrochloride (1) was slowly added with stirring. The mixture was stirred for 1 hour, and the precipitate filtered away. To the remaining solution cooled on ice, 21.3 g (0.15 mol) of ethyltrifluoroacetate was slowly added with intense stirring. The solution was brought up to rt and stirred for another 2 hours. 6 g (0.1 mol) of AcOH was added, and the precipitate formed was washed with EtOH and acetone. Yield: 15 g (62percent). MALDI-TOF, positive mode: 242.455 (Ml + 1 ; 67percent).To a solution of sodium (9.24 g; 402 mmol) in 350 ml anhydrous EtOH, L-lysine x HCl (36.5 g; 200 mmol) was added with stirring. In 1 h, the precipitate of NaCl was filtered off, and reaction mixture was cooled to 5 °C, then ethyltrifluoroacetate (39.76 g; 280 mmol) was added dropwise over 30 min with stirring. The mixture was stirred for 1.5 h at 5 °C and 1 h at rt, then AcOH (12.12 g, 202 mmol) was added and the mixture was stirred for 10 min. The precipitate was collected, washed and dried in vacuo to give N/V-s-trifluoroacetamido-L-lysine (25 g) and sodium bromide (37.2 g) were added to a 3-necked jacketed reaction vessel fitted with an internal thermocouple, mechanical stirrer, and a powder funnel. The solids were subsequently dissolved in water (69 mL) and aqueous HBr solution (20.9 mL, 8.9 M). The powder funnel was removed and an addition funnel charged with sodium nitrite (12.8 g) that had been dissolved in 16.5 mL of water. The addition funnel was fitted with a nitrogen inlet was added to the reaction vessel. The reaction exhaust was passed through a solution of sodium sulfite prior to being vented into the fume hood. The reaction mixture was chilled to < 0 C and then the sodium nitrite solution was slowly added to the reaction mixture at such a rate that the internal reaction temperature was not allowed to exceed 3 C. The addition funnel was removed, and a separate addition funnel preloaded with concentrated sulfuric acid (5.5 ml_) was fitted to the reaction vessel. The sulfuric acid was added to the reaction mixture at such a rate that the internal reaction temperature did not exceed 5 C. Following this addition, the reaction mixture was actively sparged with ishto remove dissolved Br2. After sparging for 20 minutes at room temperature, sparging was discontinued, and the reaction mixture was partitioned against 80 ml_ of methyl -terf-butyl ether (MTBE). The mixture was rapidly stirred for five minutes, and then the phases were allowed to separate. The organic layer was collected, and the aqueous layer was extracted with two additional 70 ml_ portions of MTBE. The combined organic layers were washed with several portions of 5% Na2S03 solution until nearly colorless, and then subsequently washed with brine (100 ml_), dried (MgS04), filtered and concentrated under reduced pressure to provide a pale yellow colored oil which was further dried under high vacuum overnight. The dried material was dissolved in methanol (350 ml_) and p-TsOH monohydrate (0.35 g) was added. The mixture was heated at 65 C under nitrogen, overnight. Following this time, heating was discontinued and the reaction mixture was allowed to cool to room temperature. The reaction mixture was concentrated under reduced pressure to provide a yellow oil that was (0165) subsequently purified by flash chromatography on S1O2 (330 g column, 5 (0166) 50% EtOAc-hexanes over 12 column volumes). The isolated product was a nearly colorless oil 20.3 g, (62%). LCMS: tr = 3.72 min., (M-H)~ 318, 320 (0167) ( Column : Waters Aquity UPLC BEFI Shield RP18 1.7 pm 2.1 x 75 mm; Channel A: 0.1 % formic acid in water; Channel B 0.1 % formic acid in MeCN; Flow: 1.0 mL/min.; Program: 0.0 - 0.5 min., 98:2 A:B; 0.5 -9.0 min. 98:2 A:B 2:98 A:B; 9.0-9.5 min., 2:98 A:B; Gradient: linear)Nu-epsilon-trifluoroacetamido- L-lysine (25 g, 103.2 mmol) and sodium bromide (37.2 g, 361 .2 mmol) were added to a 500 mL, 3-necked jacketed reaction vessel fitted with an internal thermocouple, mechanical stirrer, and a powder funnel. The solids were subsequently dissolved in 69 mL of water and aqueous HBr solution (20.9 mL, 8.9M). The powder funnel was removed and an addition funnel charged with sodium nitrite (12.8 g, 185.8 mmol) that had been predissolved in 16.5 mL of water fitted with a nitrogen inlet was added to the reaction vessel. The reaction exhaust was passed through a solution of sodium sulfite prior to being vented into the fume hood. The internal temperature of the reaction mixture was chilled to separated. The yellow-brown colored organic layer was collected, and the aqueous layer was extracted with two additional 70 mL portions of MTBE. The combined organic layers were washed with several portions of Na2SO3 solution until nearly colorless, and then subsequently washed with brine (100 mL), dried (MgSO4), filtered and concentrated under reduced pressure to provide a pale yellow colored oil. The isolated material was dried under high vacuum overnight, and a sample was subsequently taken for analytical characterization (UPLC-MS demonstrated > 80% conversion to 44 (m/z 306)) The isolated material was weighed, 28.1 g, and the residue was subsequently taken on to the next step without additional purification.When 95% ethanol was added to a 130ml four-necked flask, 11.4 g of 3-benzoyl-acrylate, N6-trifluoroacetyl-lysine -L- 12g, the reaction solution was cooled to 0 deg.] C, was added dropwise to the flask A 1.0 mol/L pre-chilled lithium hydroxide solution (50 ml) was added and stirring was continued for 40 ± 5 minutes. After completion of the reaction, 12 ml of purified hydrochloric acid was added to the reaction mixture, followed by stirring for 20 minutes to obtain a lyo-adduct reaction solution, which was left to stand.

A cysteine conjugate metabolite adduct formation with specific mitochondrial proteins using antibodies raised against halothane metabolite adducts.