公开(公告)号：US20240035023A1

公开(公告)日：2024-02-01

申请号：US18340913

申请日：2023-06-26

申请人： Kcat Enzymatic Private Limited

发明人： Pravin Kumar R ,  Gladstone Sigamani G ,  Roopa L ,  Naveen B K ,  Anuj J Shetty ,  Likith M

IPC分类号： C12N15/10 ,  G16C20/64

CPC分类号： C12N15/1089 ,  G16C20/64 ,  C12N15/1058

摘要： The present invention discloses a computer-implemented method for engineering fluorinase enzymes towards the synthesis of fluorophenyl compounds. Limited or no mechanistic details of fluorinase enzymes have hindered progress in understanding their catalytic mechanisms for synthesizing synthetic organofluorine compounds. Through a comprehensive computational screening process, specific methionine-sulfonium phenyl substrates, including [(3S)-3-amino-3-carboxypropyl][2,5-difluoro-4-(4-methoxy-2,4-dioxobutyl)phenyl]methylsulfonium, were designed and optimized using quantum chemical optimization techniques. This methodology uncovers crucial information on F— ion attack conformation and the catalytic mechanism of the substrate, leading to the formation of Methyl 3-oxo-4-(2,4,5-trifluorophenyl)butanoate. Furthermore, a protein sequence and 3D modeling-based enzyme screening process was employed to identify the most suitable enzyme for this substrate. The identified enzyme was then engineered using the mechanistic insights gained from the studies, resulting in improved substrate scope, stability and catalytic efficiency. This computer-based approach offers an efficient and precise alternative to traditional trial-and-error methods, advancing the field towards the successful synthesis fluorophenyl compounds.