Biocatalytic alkylations are important reactions to obtain chemo‐, regio‐ and stereoselectively alkylated compounds. This can be achieved using S‐adenosyl‐l‐methionine (SAM)‐dependent methyltransferases and SAM analogs. It was recently shown that a halide methyltransferase (HMT) from Chloracidobacterium thermophilum can synthesize SAM from SAH and methyl iodide. We developed an iodide‐based assay for the directed evolution of an HMT from Arabidopsis thaliana and used it to identify a V140T variant that can also accept ethyl‐, propyl‐, and allyl iodide to produce the corresponding SAM analogs (90, 50, and 70 % conversion of 15 mg SAH). The V140T AtHMT was used in one‐pot cascades with O‐methyltransferases (IeOMT or COMT) to achieve the regioselective ethylation of luteolin and allylation of 3,4‐dihydroxybenzaldehyde. While a cascade for the propylation of 3,4‐dihydroxybenzaldehyde gave low conversion, the propyl‐SAH intermediate could be confirmed by NMR spectroscopy.

We engineered the cytochrome P450 monooxygenase CYP107D1 (OleP) from Streptomyces antibioticus for the stereo‐ and regioselective 7β‐hydroxylation of lithocholic acid (LCA) to yield ursodeoxycholic acid (UDCA). OleP was previously shown to hydroxylate testosterone at the 7β‐position but LCA is exclusively hydroxylated at the 6β‐position, forming murideoxycholic acid (MDCA). Structural and 3DM analysis, and molecular docking were used to identify amino acid residues F84, S240, and V291 as specificity‐determining residues. Alanine scanning identified S240A as a UDCA‐producing variant. A synthetic “small but smart” library based on these positions was screened using a colorimetric assay for UDCA. We identified a nearly perfectly regio‐ and stereoselective triple mutant (F84Q/S240A/V291G) that produces 10‐fold higher levels of UDCA than the S240A variant. This biocatalyst opens up new possibilities for the environmentally friendly synthesis of UDCA from the biological waste product LCA.

Methylation of free hydroxyl groups is an important modification for flavonoids. It not only greatly increases absorption and oral bioavailability of flavonoids, but also brings new biological activities. Flavonoid methylation is usually achieved by a specific group of plant O‐methyltransferases (OMTs) which typically exhibit high substrate specificity. Here we investigated the effect of several residues in the binding pocket of the Clarkia breweri isoeugenol OMT on the substrate scope and regioselectivity against flavonoids. The mutation T133M, identified as reported in our previous publication, increased the activity of the enzyme against several flavonoids, namely eriodictyol, naringenin, luteolin, quercetin and even the isoflavonoid genistein, while a reduced set of amino acids at positions 322 and 326 affected both, the activity and the regioselectivity of the methyltranferase. On the basis of this work, methylated flavonoids that are rare in nature were produced in high purity.

The use of light as an external trigger to change ligand shape and as a result its bioactivity, allows the probing of pharmacologically relevant systems with spatiotemporal resolution. A hetero-stilbene lead resulting from the screening of a compound that was originally designed as kinase inhibitor served as a starting point for the design of photoswitchable sirtuin inhibitors. Because the original stilbenoid structure exerted unfavourable photochemical characteristics it was remodelled to its heteroarylic diazeno analogue. By this intramolecular azologization, the shape of the molecule was left unaltered, whereas the photoswitching ability was improved. As anticipated, the highly analogous compound showed similar activity in its thermodynamically stable stretched-out (E)-form. Irradiation of this isomer triggers isomerisation to the long-lived (Z)-configuration with a bent geometry causing a considerably shorter end‐to‐end distance. The resulting affinity shifts are intended to enable real‐time photomodulation of sirtuins in vitro.

Biokatalytische Alkylierungen sind wichtige Reaktionen, um chemo‐, regio‐ und stereoselektiv alkylierte Verbindungen zu erhalten. Dies kann mit S‐Adenosyl‐l‐methionin (SAM)‐abhängigen Methyltransferasen und SAM‐Analoga erreicht werden. Kürzlich wurde gezeigt, dass eine Halogenid‐Methyltransferase (HMT) aus Chloracidobacterium thermophilum SAM ausgehend von SAH und Methyliodid herstellen kann. Wir entwickelten einen Iodid‐basierten Assay für die gerichtete Evolution einer HMT aus Arabidopsis thaliana und nutzten diesen, um eine V140T‐Variante zu identifizieren, die auch Ethyl‐, Propyl‐, und Allyliodid akzeptiert, um die entsprechenden SAM‐Analoga herzustellen (90, 50 und 70 % Umsatz ausgehend von 15 mg SAH). Die V140T‐AtHMT wurde in einer Eintopfkaskade mit O‐Methyltransferasen (IeOMT und COMT) eingesetzt, um die regioselektive Alkylierung von Luteolin und 3,4‐Dihydroxybenzaldehyd zu erzielen. Obwohl eine Kaskade zur Propylierung von 3,4‐Dihydroxybenzaldehyd niedrige Umsätze ergab, konnte das Propyl‐SAH‐Intermediat durch NMR‐Spektroskopie bestätigt werden.

The DNA repair protein PARP-1 emerged as a valuable target in the treatment of tumor entities with deficiencies of BRCA1/2, such as breast cancer. More recently, the application of PARP inhibitors (PARPi) such as olaparib has been expanded to other cancer entities including colorectal cancer (CRC). We previously demonstrated that PARP-1 is overexpressed in human CRC and promotes CRC progression in a mouse model. However, acquired resistance to PARPi and cytotoxicity-mediated adverse effects limit their clinical applicability. Here, we detailed the role of PARP-1 as a therapeutic target in CRC and studied the efficacy of novel PARPi compounds in wildtype (WT) and DNA repair-deficient CRC cell lines together with the chemotherapeutics irinotecan (IT), 5-fluorouracil (5-FU), and oxaliplatin (OXA). Based on the ComPlat molecule archive, we identified novel PARPi candidates by molecular docking experiments in silico, which were then confirmed by in vitro PARP activity measurements. Two promising candidates (X17613 and X17618) also showed potent PARP-1 inhibition in a CRC cell-based assay. In contrast to olaparib, the PARPi candidates caused no PARP-1 trapping and, consistently, were not or only weakly cytotoxic in WT CRC cells and their BRCA2- or ATR-deficient counterparts. Importantly, both PARPi candidates did not affect the viability of nonmalignant human colonic epithelial cells. While both olaparib and veliparib increased the sensitivity of WT CRC cells towards IT, no synergism was observed for X17613 and X17618. Finally, we provided evidence that all PARPi (olaparib > veliparib > X17613 > X17618) synergize with chemotherapeutic drugs (IT > OXA) in a BRCA2-dependent manner in CRC cells, whereas ATR deficiency had only a minor impact. Collectively, our study identified novel lead structures with potent PARP-1 inhibitory activity in CRC cells but low cytotoxicity due to the lack of PARP-1 trapping, which synergized with IT in homologous recombination deficiency.