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Quantum Chemical Calculations and Experimental Validation of the Photoclick Reaction for Fluorescent Labeling of the 5' cap of Eukaryotic mRNAs.

Stummer D, Herrmann C, Rentmeister A - ChemistryOpen (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Energies of calculated frontier orbitals from nitrile imine 4 a and N2-allyl-m7GTP-H+ (8) as well as isosurface plots of the corresponding molecular orbitals. A) Schematic representation of frontier orbital energies of nitrile imine 4 a as well as N2-allyl-modified m7GTP-H+ (8) calculated by KS-DFT. Based on these calculations, the HOMO–LUMO gap of reactants is 2.1 eV. B) Orbitals with a suitable spatial structure for interaction were chosen as frontier molecular orbitals.
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fig01: Energies of calculated frontier orbitals from nitrile imine 4 a and N2-allyl-m7GTP-H+ (8) as well as isosurface plots of the corresponding molecular orbitals. A) Schematic representation of frontier orbital energies of nitrile imine 4 a as well as N2-allyl-modified m7GTP-H+ (8) calculated by KS-DFT. Based on these calculations, the HOMO–LUMO gap of reactants is 2.1 eV. B) Orbitals with a suitable spatial structure for interaction were chosen as frontier molecular orbitals.

Mentions: The HOMO of the nitrile imine 4 a and the LUMO+2 of N2-allyl-m7GTP-H+8 are shown in Figure 1. They were identified as suitable frontier orbitals because they meet the criteria for symmetry and phases required for the reaction. Although the frontier MOs (FMOs) chosen for 8 are not the overall HOMO and LUMO of the N2-allyl-modified cap, these were selected because they are located at the N2-allyl group involved in the reaction.


Quantum Chemical Calculations and Experimental Validation of the Photoclick Reaction for Fluorescent Labeling of the 5' cap of Eukaryotic mRNAs.

Stummer D, Herrmann C, Rentmeister A - ChemistryOpen (2015)

Energies of calculated frontier orbitals from nitrile imine 4 a and N2-allyl-m7GTP-H+ (8) as well as isosurface plots of the corresponding molecular orbitals. A) Schematic representation of frontier orbital energies of nitrile imine 4 a as well as N2-allyl-modified m7GTP-H+ (8) calculated by KS-DFT. Based on these calculations, the HOMO–LUMO gap of reactants is 2.1 eV. B) Orbitals with a suitable spatial structure for interaction were chosen as frontier molecular orbitals.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4522179&req=5

fig01: Energies of calculated frontier orbitals from nitrile imine 4 a and N2-allyl-m7GTP-H+ (8) as well as isosurface plots of the corresponding molecular orbitals. A) Schematic representation of frontier orbital energies of nitrile imine 4 a as well as N2-allyl-modified m7GTP-H+ (8) calculated by KS-DFT. Based on these calculations, the HOMO–LUMO gap of reactants is 2.1 eV. B) Orbitals with a suitable spatial structure for interaction were chosen as frontier molecular orbitals.
Mentions: The HOMO of the nitrile imine 4 a and the LUMO+2 of N2-allyl-m7GTP-H+8 are shown in Figure 1. They were identified as suitable frontier orbitals because they meet the criteria for symmetry and phases required for the reaction. Although the frontier MOs (FMOs) chosen for 8 are not the overall HOMO and LUMO of the N2-allyl-modified cap, these were selected because they are located at the N2-allyl group involved in the reaction.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.