Preparation of enantiopure pregabalin intermediate using cross linked enzyme aggregates (CLEAs) in basket reactor

Route to the synthesis of enantiomerically pure ethyl (<i>S</i>)-3-cyano-5-methylhexanoate, (<i>S</i>)-<b>5</b>, a key chiral intermediate for Pregabalin has been improved. The racemic β-cyano diester, <b>3</b> was prepared in 98% purity via gelatine catalysed Knoevenagel condensation of diethylmalonate with isovaleraldehyde followed by hydrocyantion of α,β-unsaturated diester <b>14</b> using acetone cyanohydrin and K₂CO₃. Racemic diethyl 2-(1-cyano-3-methylbutyl)malonate, <i>rac</i>-<b>3</b>, has been resolved using lipase from <i>Thermomyces lanuginosus</i> immobilised in form of crosslinked enzyme aggregates, CLEAs. The CLEAs were made by employing commercial soymilk as an additional protein source and a reaction was carried out in a moving basket reactor. The immobilised enzyme was found to be stable in many organic solvents and temperature up to 50 °C. The resolution reaction was studied in a basket reactor at 50% substrate loading in calcium acetate buffer, pH 7.5 at 30 °C by using 20% w/w enzyme loading. The apparent kinetic parameters were V<i>_max,app</i> = (8.74 ± 0.43) mM/h/g and K<i>_m,app</i> = (1.5 ± 0.07) M (correlation coeff. <i>r</i> = 0.98). The desired ethyl (<i>S</i>)-3-cyano-5-methylhexanoate, (<i>S</i>)-<b>5</b> is obtained in 90-92% theoretical yield and <i>e.e</i> &gt; 99%. The advantages of this improved process are mild reaction conditions; an alternate method for hydrocyanation step avoiding the use of highly toxic potassium cyanide at large scale operation and an immobilised enzyme that can be reused for at least 11 recycles.