title: Quantum Chemical Calculations and Experimental Validation of the Photoclick Reaction for Fluorescent Labeling of the 5' cap of Eukaryotic mRNAs.
abstract: Bioorthogonal click reactions are powerful tools to specifically label biomolecules in living cells. Considerable progress has been made in site-specific labeling of proteins and glycans in complex biological systems, but equivalent methods for mRNAs are rare. We present a chemo-enzymatic approach to label the 5' cap of eukaryotic mRNAs using a bioorthogonal photoclick reaction. Herein, the N7-methylated guanosine of the 5' cap is enzymatically equipped with an allyl group using a variant of the trimethylguanosine synthase 2 from Giardia lamblia (GlaTgs2). To elucidate whether the resulting N (2)-modified 5' cap is a suitable dipolarophile for photoclick reactions, we used Kohn-Sham density functional theory (KS-DFT) and calculated the HOMO and LUMO energies of this molecule and nitrile imines. Our in silico studies suggested that combining enzymatic allylation of the cap with subsequent labeling in a photoclick reaction was feasible. This could be experimentally validated. Our approach generates a turn-on fluorophore site-specifically at the 5' cap and therefore presents an important step towards labeling of eukaryotic mRNAs in a bioorthogonal manner.
authors.affiliation: Institute of Biochemistry, Westfälische Wilhelms-Universität Münster Wilhelm-Klemm-Straße 2, 48149, Münster, Germany ; Cells-in-Motion Cluster of Excellence (EXC 1003-CiM), Westfälische Wilhelms-Universität Münster Wilhelm-Klemm-Straße 2, 48149, Münster, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Stummer
authors.affiliation: Institute of Inorganic Chemistry, University of Hamburg Martin-Luther-King-Platz 6, 20146, Hamburg, Germany.
authors.forename: Carmen
authors.initials: C
authors.lastname: Herrmann
authors.affiliation: Institute of Biochemistry, Westfälische Wilhelms-Universität Münster Wilhelm-Klemm-Straße 2, 48149, Münster, Germany ; Cells-in-Motion Cluster of Excellence (EXC 1003-CiM), Westfälische Wilhelms-Universität Münster Wilhelm-Klemm-Straße 2, 48149, Münster, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Rentmeister
keywords: 5’ cap; Kohn–Sham DFT calculations; bioorthogonal labeling; fluorescent labeling; mRNA; photoclick reactions