Reaction conditions: 5 mM ketone, 100 mM amine, 0.5 mM NADP + , 50 mM glucose, 1 mg mL -1 purified IRED a or 5 mg/m IRED lyophilised cell-free extract b , 0.5 mg mL -1 GDH (CDX-901), 1% (v/v) DMSO, 100 mM Tris buffer pH 9.0, 500 μL reaction volume, 30 °C, 250 rpm, 24 h. Finally, to demonstrate the synthetic applicability of the investigated cascade, a series of preparative-scale syntheses were performed. Attempts to intensify reactions were unsuccessful and consequently, the same conditions used on analytical-scale were applied for the preparative-scale reactions. Using our three-step one-enzyme cascade, six N-alkylated 2,5-disubstituted pyrrolidines were successfully synthesised on a 0.3 mmol scale (Scheme 9). Both diastereoisomers of pyrrolidines 21i were synthesised with excellent enantioselectivities and good yields within 24 h. However, for most examples, preparativescale reactions proceeded at slower rates than on an analyticalscale, requiring up to 72 h for completion, depending on the substrate and enzyme. Typically, full substrate consumption was observed after 24 h, and the enamine intermediate/by-product was observed along with the desired product. Allowing the reaction to proceed for an additional 24-48 h period led to complete conversion of the intermediate to the final product, indicating that the enamine is not a dead-end for this annulation. Scheme 9. Preparative scale biocatalytic diketone annulation.