3-ethyl-3-methylpyrrolidine-2,5-dione

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

Procedure: To a slurry of 92.1 milligrams (mg) of the HCl salt of N-(4-aminobutyl)2-ethyl-2-methylsuccinimide in 0.6 mL of dry DMF at room temperature under argon was added 107 microliters (μL) of triethylamine. The suspension was stirred 15 min; then a solution of 180.2 mg of carbonyldiimidazole (CDI) in 0.6 mL of DMF was added via syringe in one portion. The resulting suspension was stirred at room temperature for 50 min to complete formation of the imidazole. It was then added dropwise, over 8 min, to a stirring solution of 250 mg of Miles Pentex® crystalline bovine serum albumin (BSA) (Miles Laboratories, Inc., Elkhart, IN, USA) in 108 mL of water at pH 4.5 and 5° C. The pH was maintained at 4.5 during and after the addition by an automatic titrator (HCl). After 18 hrs at pH 4.5 and 5° C., the clear, translucent reaction was adjusted to pH 8 with sodium hydroxide solution, and applied to a 3.0×62 cm column of Sephadex G-25F gel (Pharmacia, Piscataway, NJ, USA) in 50 millimolar (mM) TRIS buffer [tris(hydroxymethyl)aminomethane], pH 8.2. The column was eluted with this buffer at a flow rate of 1 mL/min and 10 mL fractions were collected. The resulting suspension was stirred at room temperature for 50 min The pH was maintained at 4.5 during and After 18 hrs at pH 4.5 and 5° C. the clear, translucent reaction The column was eluted with this buffer at a flow rate of 1 mL/min and 10 mL fractions were collected