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 3,4dihydroxybenzaldehyde. 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 7b-hydroxylation of lithocholic acid (LCA) to yield ursodeoxycholic acid (UDCA). OleP was previously shown to hydroxylate testosterone at the 7b-position but LCA is exclusively hydroxylated at the 6b-position, forming murideoxycholic acid (MDCA). Structural and 3DM analysis, and molecular docking were used to identify amino acid residues F84, S240, and V291 as specificitydetermining 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 regioand 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. Ursodeoxycholic acid (UDCA) is a valuable bile acid frequently prescribed for the treatment of cholecystitis as it can solubilize cholesterol gallstones with fewer side effects than chenodeoxycholic acid (CDCA). [1] UDCA also has anti-inflammatory properties [2] and is applied in the therapy of cystic fibrosis [3] and liver diseases like primary biliary cholangitis. [4] The major natural source of UDCA is bear bile, [5] a popular traditional medicine obtained by biliary catheterization of farmed bears. Alternatively, semi-synthetic UDCA can be produced from cholic acid (CA) [6] or CDCA. [7, 8] The synthesis route starting from CA forms CDCA within 5 steps, including a Wolff-Kishner reduction, and an epimerization at C7 to produce UDCA (Scheme 1 a, Scheme S1). [9] The yields of this pathway do not exceed 30 %. To overcome these limitations, a shorter synthesis route based on the biocatalytic epimerization of CDCA to UDCA (Scheme 1 a) has been developed. [7, 10] LCA is an abundant and inexpensive waste product of meat production [11] as this bile acid is found in farmed animals like sheep, [12] cattle, [12] and pigs. [13] Currently, no biotechnological process [14] utilizing LCA originating from these sources is known, making it a desirable starting material for the synthesis of UDCA. A few microbial organisms have been reported to form UDCA from LCA. [15] For example, the fungus Fusarium equiseti converts LCA to a number of products, including UDCA at 35 % yield. [16] However, there is currently no enzyme known to selectively hydroxylate LCA at the 7b-position to form UDCA and its synthesis pathway in microbial organisms, starting from LCA, remains enigmatic. An enzyme for direct 7b-hydroxylation would be a valuable tool for direct conversion of LCA to UDCA without involving the complex metabolism of fungi that invariably [16] produce multiple undesired side products, [15] complicating downstream processing. A major challenge in the enzymatic conversion of LCA to UDCA is the hydrophobicity and thus, extremely low water solubility of LCA co...

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.

Because isoenzymes of the experimentally and therapeutically extremely relevant sirtuin family show high similarity, addressing the unique selectivity pocket of sirtuin 2 is a promising strategy towards selective inhibitors. An unrelated approach towards selective inhibition of isoenzymes with varied tissue distribution is targeted drug delivery or spatiotemporal activation by photochemical activation. Azologization of two nicotinamide‐mimicking lead structures was undertaken to combine both approaches and yielded a set of 33 azobenzenes and azopyridines that have been evaluated for their photochemical behaviour and bioactivity. For some compounds, inhibitory activity reached the sub‐micromolar range in their thermodynamically favoured E form and could be decreased by photoisomerization to the metastable Z form. Besides, derivatization with long‐chain fatty acids yielded potent sirtuin 2 inhibitors, featuring another intriguing aspect of azo‐based photoswitches. In these compounds, switching to the Z isomer increased aqueous solubility and thereby enhanced biological activity by up to a factor of 21. The biological activity of two compounds was confirmed by hyperacetylation of sirtuin specific histone proteins in a cell‐based activity assay.

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.