Affiliation:
1. Department of Biotechnology and Enzyme Catalysis Institute of Biochemistry University of Greifswald Felix-Hausdorff-Str. 4 17487 Greifswald Germany
2. Discovery Sciences BioPharmaceuticals R&D AstraZeneca Pepparedsleden 1 43183 Mölndal Sweden
Abstract
AbstractEnzyme‐catalyzed late‐stage functionalization (LSF), such as methylation of drug molecules and lead structures, enables direct access to more potent active pharmaceutical ingredients (API). S‐adenosyl‐l‐methionine‐dependent methyltransferases (MTs) can play a key role in the development of new APIs, as they catalyze the chemo‐ and regioselective methylation of O‐, N‐, S‐ and C‐atoms, being superior to traditional chemical routes. To identify suitable MTs, we developed a continuous fluorescence‐based, high‐throughput assay for SAM‐dependent methyltransferases, which facilitates screening using E. coli cell lysates. This assay involves two enzymatic steps for the conversion of S‐adenosyl‐l‐homocysteine into H2S to result in a selective fluorescence readout via reduction of an azidocoumarin sulfide probe. Investigation of two O‐MTs and an N‐MT confirmed that this assay is suitable for the determination of methyltransferase activity in E. coli cell lysates.
Funder
Deutsche Forschungsgemeinschaft
Subject
General Chemistry,Catalysis