Using the Enzymatic Toolbox for the Synthesis of Iminocyclitols and High-Throughput Screening of ω-Transaminases in Liquid and Solid-Phase Using a Smart Fluorogenic Chiral Substrate

Iminosugars are of great pharmaceutical interest due to their biological relevance, their chemical resistance and their high water solubility. This thesis reports a one-pot, multi-enzyme telescopic cascade for the stereoselective synthesis of chiral iminocyclitols directly from inexpensive starting materials. N-phenylacetyl (N-PhAc) protected amino alcohols were synthesized and oxidized to their corresponding aldehydes using 2-iodoxybenzoic acid (IBX) or catalytic TEMPO/N-iodosuccinimide (NIS). The generated electrophilic acceptors underwent asymmetric C–C bond formation using specific transketolases and aldolases. The resulting aldol/acyloin adducts were subjected to enzymatic deprotection catalyzed by Penicillin G acylase (PGA), thereby leading to spontaneous cyclic imine formation. Subsequent reduction was mediated either chemically using 2-picoline-borane complex or biocatalytically and stereoselective using imine reductases (IREDs) identified from a high-throughput screening panel generating the desired iminosugars. The multi-enzyme telescopic cascade was successfully established and validated from analytical to preparative scales. The second part of this work addresses a complementary sensitive, high-throughput screening (HTS) assays for Ꞷ-aminotransaminases (Ꞷ-ATAs), which are highly valuable biocatalysts for stereoselective chiral amine synthesis. A fluorogenic "smart" substrate assay based on aminoumbelliferone derivatives was hypothesized to detect both omega-ATA activity and enantioselectivity. Enzymatic deamination of these samples triggers a spontaneous beta-elimination mechanism, releasing the fluorescent reporter umbelliferone. Confirmation of our hypothesis using an achiral model substrate lead to the synthesis of enantiopure (R)- and (S)-7-(3-aminobutoxy)-2H-chromen-2-one compounds. Activity testing revealed unexpected conversion of the (R)-substrate by several (S)-selective ATAs, which due to molecular modeling might be attributed to alternative binding conformations allowed by the flexible alkyl chain and planar coumarin core. Finally, the system was implemented as a solid-phase HTS protocol, enabling direct, visual identification of active ATA-expressing bacterial colonies under 366 nm UV light.