(Z)-2-methylbut-2-enoic acid

Conditions: temp:2.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Angelic acid (601 mg, 6.0 mmol) was dissolved in dichloromethane (3.0 mL) under argon. Diisopropylethylamine (1.23 mL, 7.20 mmol) was added at 5-10° C. in a period of 1 min. To this solution was added 2,4,6-trichlorobenzoyl chloride (1.12 mL, 7.20 mmol) at 3-6° C. in a period of 4 min. After the reaction solution had been stirred at 2° C. for 45 min, petroleum ether (9.0 mL) was added. The obtained suspension was purified by flash chromatography (petroleum ether/dichloromethane 3:1), giving the title compound as a white solid (605 mg, 33%). The obtained suspension was purified by flash chromatography (petroleum ether/dichloromethane 3:1)

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A solution of ingenol-5,20-acetonide (100 mg, 0.26 mmol), angelic acid (39 mg, 0.39 mmol, 1.5 mol. equiv.) and DMAP (48 mg, 0.39 mmol, 1.5 mol. equiv.) in toluene (4 ml), was stirred at room temperature for 2 hours and was then filtered through a bed of celite and evaporated. The resulting material was filtered in silica gel (ca. 5 g) and evaporated. The residue was purified by means of silica gel gravity column chromatography (5 g, petroleum ether:EtOAc 85:15) until obtaining over 80 mg of ingenol-3-angelate with a purity>99%. For physical and spectroscopic data, see Hohmann J et al. Planta Medica 2000, 66, 291-294. was then filtered through a bed of celite evaporated The resulting material was filtered in silica gel (ca. 5 g) evaporated The residue was purified by means of silica gel gravity column chromatography (5 g, petroleum ether:EtOAc 85:15)

Conditions: temp:2.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Angelic acid (601 mg, 6.0 mmol) was dissolved in dichloromethane (3.0 mL) under argon. Diisopropylethylamine (1.23 mL, 7.20 mmol) was added at 5-10° C. in a period of 1 min. To this solution was added 2,4,6-trichlorobenzoyl chloride (1.12 mL, 7.20 mmol) at 3-6° C. in a period of 4 min. After the reaction solution had been stirred at 2° C. for 45 min, petroleum ether (9.0 mL) was added. The obtained suspension was purified by flash chromatography (petroleum ether/dichloromethane 3:1), giving the title compound as a white solid (605 mg, 33%). The obtained suspension was purified by flash chromatography (petroleum ether/dichloromethane 3:1)

Conditions: temp:35.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 5.0 mL suspension of 0.500 g (wet cell paste) E. coli SS1001 cells (ATCC PTA-1177) in 50 mM potassium phosphate buffer (pH 7.0) was added to a mixture containing 4.80 mL of 50 mM potassium phosphate buffer (pH 7.0), 80.5 mg of (E)-2-methyl-2-butenenitrile (100 mM final concentration of (E)-2-methyl-2-butenenitrile), and 80.7 mg of (Z)-2-methyl-2-butenenitrile (100 mM final concentration of (Z)-2-methyl-2-butenenitrile), and the resulting suspension stirred at 35° C. Samples (0.100 mL) were mixed with 0.900 mL of 60 mM N-ethylacetamide (HPLC external standard) in 1:1 acetonitrile:methanol, the resulting mixture was mixed, centrifuged, and the supernatant analyzed by HPLC for (E)- and (Z)-2-methyl-2-butenenitrile and (E)- and (Z)-2-methyl-2-butenoic acid. After 6 h, the conversions of (E)-2-methyl-2-butenenitrile and (Z)-2-methyl-2-butenenitrile were 100% and 0%, respectively, and the yields of (E)-2-methyl-2-butenoic acid and (Z)-2-methyl-2-butenoic acid were 99% and 0%, respectively.

Conditions: temp:25.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 5.0 mL suspension of 0.500 g (wet cell paste) E. coli SS1001 cells (ATCC PTA-1177) in 50 mM potassium phosphate buffer (pH 7.0) was added to a mixture containing 4.91 mL of 50 mM potassium phosphate buffer (pH 7.0), 38.4 mg of (E)-2-methyl-2-butenenitrile (47.4 mM final concentration of (E)-2-methyl-2-butenenitrile), and 37.7 mg of (Z)-2-methyl-2-butenenitrile (46.5 mM final concentration of (Z)-2-methyl-2-butenenitrile), and the resulting suspension stirred at 25° C. Samples (0.100 mL) were mixed with 0.900 mL of 60 mM N-ethylacetamide (HPLC external standard) in 1:1 acetonitrile:methanol, the resulting mixture was mixed, centrifuged, and the supernatant analyzed by HPLC for (E)- and (Z)-2-methyl-2-butenenitrile and (E)- and (Z)-2-methyl-2-butenoic acid. After 23 h, the conversions of (E)-2-methyl-2-butenenitrile and (Z)-2-methyl-2-butenenitrile were 100% and 0%, respectively, and the yields of (E)-2-methyl-2-butenoic acid and (Z)-2-methyl-2-butenoic acid were 100% and 0%, respectively.

Conditions: temp:35.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 5.0 mL suspension of 0.500 g (wet cell paste) E. coli SS1001 cells (ATCC PTA-1177) in 50 mM potassium phosphate buffer (pH 7.0) was added to a mixture containing 4.80 mL of 50 mM potassium phosphate buffer (pH 7.0), 80.5 mg of (E)-2-methyl-2-butenenitrile (100 mM final concentration of (E)-2-methyl-2-butenenitrile), and 80.7 mg of (Z)-2-methyl-2-butenenitrile (100 mM final concentration of (Z)-2-methyl-2-butenenitrile), and the resulting suspension stirred at 35° C. Samples (0.100 mL) were mixed with 0.900 mL of 60 mM N-ethylacetamide (HPLC external standard) in 1:1 acetonitrile:methanol, the resulting mixture was mixed, centrifuged, and the supernatant analyzed by HPLC for (E)- and (Z)-2-methyl-2-butenenitrile and (E)- and (Z)-2-methyl-2-butenoic acid. After 6 h, the conversions of (E)-2-methyl-2-butenenitrile and (Z)-2-methyl-2-butenenitrile were 100% and 0%, respectively, and the yields of (E)-2-methyl-2-butenoic acid and (Z)-2-methyl-2-butenoic acid were 99% and 0%, respectively.

Conditions: temp:25.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 5.0 mL suspension of 0.500 g (wet cell paste) E. coli SS1001 cells (ATCC PTA-1177) in 50 mM potassium phosphate buffer (pH 7.0) was added to a mixture containing 4.91 mL of 50 mM potassium phosphate buffer (pH 7.0), 38.4 mg of (E)-2-methyl-2-butenenitrile (47.4 mM final concentration of (E)-2-methyl-2-butenenitrile), and 37.7 mg of (Z)-2-methyl-2-butenenitrile (46.5 mM final concentration of (Z)-2-methyl-2-butenenitrile), and the resulting suspension stirred at 25° C. Samples (0.100 mL) were mixed with 0.900 mL of 60 mM N-ethylacetamide (HPLC external standard) in 1:1 acetonitrile:methanol, the resulting mixture was mixed, centrifuged, and the supernatant analyzed by HPLC for (E)- and (Z)-2-methyl-2-butenenitrile and (E)- and (Z)-2-methyl-2-butenoic acid. After 23 h, the conversions of (E)-2-methyl-2-butenenitrile and (Z)-2-methyl-2-butenenitrile were 100% and 0%, respectively, and the yields of (E)-2-methyl-2-butenoic acid and (Z)-2-methyl-2-butenoic acid were 100% and 0%, respectively.

Conditions: temp:35.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 5.0 mL suspension of 0.500 g (wet cell paste) E. coli SS1001 cells (ATCC PTA-1177) in 50 mM potassium phosphate buffer (pH 7.0) was added to a mixture containing 4.80 mL of 50 mM potassium phosphate buffer (pH 7.0), 80.5 mg of (E)-2-methyl-2-butenenitrile (100 mM final concentration of (E)-2-methyl-2-butenenitrile), and 80.7 mg of (Z)-2-methyl-2-butenenitrile (100 mM final concentration of (Z)-2-methyl-2-butenenitrile), and the resulting suspension stirred at 35° C. Samples (0.100 mL) were mixed with 0.900 mL of 60 mM N-ethylacetamide (HPLC external standard) in 1:1 acetonitrile:methanol, the resulting mixture was mixed, centrifuged, and the supernatant analyzed by HPLC for (E)- and (Z)-2-methyl-2-butenenitrile and (E)- and (Z)-2-methyl-2-butenoic acid. After 6 h, the conversions of (E)-2-methyl-2-butenenitrile and (Z)-2-methyl-2-butenenitrile were 100% and 0%, respectively, and the yields of (E)-2-methyl-2-butenoic acid and (Z)-2-methyl-2-butenoic acid were 99% and 0%, respectively.

Conditions: temp:25.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Reaction of a 1:1 Mixture of (E)- and (Z)2-Methyl-2-Butenenitrile with E. coli SS1001 Cells A 5.0 mL suspension of 0.500 g (wet cell paste) E. coli SS1001 cells (ATCC PTA-1177) in 50 mM potassium phosphate buffer (pH 7.0) was added to a mixture containing 4.91 mL of 50 mM potassium phosphate buffer (pH 7.0), 38.4 mg of (E)-2-methyl-2-butenenitrile (47.4 mM final concentration of (E)-2-methyl-2-butenenitrile), and 37.7 mg of (Z)-2-methyl-2-butenenitrile (46.5 mM final concentration of (Z)-2-methyl-2-butenenitrile), and the resulting suspension stirred at 25° C. Samples (0.100 mL) were mixed with 0.900 mL of 60 mM N-ethylacetamide (HPLC external standard) in 1:1 acetonitrile:methanol, the resulting mixture was mixed, centrifuged, and the supernatant analyzed by HPLC for (E)- and (Z)-2-methyl-2-butenenitrile and (E)- and (Z)-2-methyl-2-butenoic acid. After 23 h, the conversions of (E)-2-methyl-2-butenenitrile and (Z)-2-methyl-2-butenenitrile were 100% and 0%, respectively, and the yields of (E)-2-methyl-2-butenoic acid and (Z)-2-methyl-2-butenoic acid were 100% and 0%, respectively.