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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.


Analysis of the photoclick reaction of tetrazole 4 with N2-pentenynyl-m7GpppA (3 c). A) a) The cap-analog m7GpppA (1) was used with the GlaTgs2 variant and cosubstrate (CS) 2 c to give N2-pentenynyl-m7GpppA (3 c), which was subsequently used for photoclick reaction with 4. b) The product 3 c together with tetrazole 4 is irradiated at 254 nm for 5 min and left at 4 °C o/n. to give 13 (yield not determined). B)  Reaction mixtures were separated by PAGE and analyzed for the formation of a fluorescent pyrazoline cycloadduct, visible upon irradiation at 365 nm. A fluorescent band is observed in lane 1 (arrow), which is absent in the control reaction containing denatured enzyme (+*, lane 2).
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fig03: Analysis of the photoclick reaction of tetrazole 4 with N2-pentenynyl-m7GpppA (3 c). A) a) The cap-analog m7GpppA (1) was used with the GlaTgs2 variant and cosubstrate (CS) 2 c to give N2-pentenynyl-m7GpppA (3 c), which was subsequently used for photoclick reaction with 4. b) The product 3 c together with tetrazole 4 is irradiated at 254 nm for 5 min and left at 4 °C o/n. to give 13 (yield not determined). B)  Reaction mixtures were separated by PAGE and analyzed for the formation of a fluorescent pyrazoline cycloadduct, visible upon irradiation at 365 nm. A fluorescent band is observed in lane 1 (arrow), which is absent in the control reaction containing denatured enzyme (+*, lane 2).

Mentions: Calculating the FMO-energies of N2-pentenynyl-m7GTP-H+ (12, structure in Supplementary Figure 1 in the Supporting Information) we found a LUMO+1 energy of only −3.26 eV, concomitant with an energy gap of only 1.32 eV between the HOMO of 4 a and the LUMO of 12. These calculations suggest that a pentenynyl-substituted cap may be a better substrate for the photoclick reaction with 4. We therefore produced N2-pentenynyl-modified m7GpppA 3 c by enzymatic modification of m7GpppA (1) using the GlaTgs2 variant and the cosubstrate 5’-[(R/S)(3S)-3-amino-3-carboxypropyl]pent-2-en-4-ynylsulfonio]-5’-deoxyadenosine (AdoEnYn, 2 c), as described previously (∼25 % yield).30 As expected, 3 c reacted with tetrazole 4 in a photoclick reaction to form a fluorescent product that could be detected by in-gel fluorescence, although the band is rather faint (Figure 3). We presume the low concentration of modified cap 3 c in bioconversions (about 200 μm) to be the limiting factor in this case for the photoclick reaction. A control reaction was carried out under identical conditions, but with denatured enzyme, and did not give a new fluorescent product (Figure 3). These data, together with our calculations, suggest that the expected product 13 was formed, confirming that KS-DFT calculations are useful to predict reactivity between a nitrile imine and a modified cap acting as a dipolarophile.


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)

Analysis of the photoclick reaction of tetrazole 4 with N2-pentenynyl-m7GpppA (3 c). A) a) The cap-analog m7GpppA (1) was used with the GlaTgs2 variant and cosubstrate (CS) 2 c to give N2-pentenynyl-m7GpppA (3 c), which was subsequently used for photoclick reaction with 4. b) The product 3 c together with tetrazole 4 is irradiated at 254 nm for 5 min and left at 4 °C o/n. to give 13 (yield not determined). B)  Reaction mixtures were separated by PAGE and analyzed for the formation of a fluorescent pyrazoline cycloadduct, visible upon irradiation at 365 nm. A fluorescent band is observed in lane 1 (arrow), which is absent in the control reaction containing denatured enzyme (+*, lane 2).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig03: Analysis of the photoclick reaction of tetrazole 4 with N2-pentenynyl-m7GpppA (3 c). A) a) The cap-analog m7GpppA (1) was used with the GlaTgs2 variant and cosubstrate (CS) 2 c to give N2-pentenynyl-m7GpppA (3 c), which was subsequently used for photoclick reaction with 4. b) The product 3 c together with tetrazole 4 is irradiated at 254 nm for 5 min and left at 4 °C o/n. to give 13 (yield not determined). B)  Reaction mixtures were separated by PAGE and analyzed for the formation of a fluorescent pyrazoline cycloadduct, visible upon irradiation at 365 nm. A fluorescent band is observed in lane 1 (arrow), which is absent in the control reaction containing denatured enzyme (+*, lane 2).
Mentions: Calculating the FMO-energies of N2-pentenynyl-m7GTP-H+ (12, structure in Supplementary Figure 1 in the Supporting Information) we found a LUMO+1 energy of only −3.26 eV, concomitant with an energy gap of only 1.32 eV between the HOMO of 4 a and the LUMO of 12. These calculations suggest that a pentenynyl-substituted cap may be a better substrate for the photoclick reaction with 4. We therefore produced N2-pentenynyl-modified m7GpppA 3 c by enzymatic modification of m7GpppA (1) using the GlaTgs2 variant and the cosubstrate 5’-[(R/S)(3S)-3-amino-3-carboxypropyl]pent-2-en-4-ynylsulfonio]-5’-deoxyadenosine (AdoEnYn, 2 c), as described previously (∼25 % yield).30 As expected, 3 c reacted with tetrazole 4 in a photoclick reaction to form a fluorescent product that could be detected by in-gel fluorescence, although the band is rather faint (Figure 3). We presume the low concentration of modified cap 3 c in bioconversions (about 200 μm) to be the limiting factor in this case for the photoclick reaction. A control reaction was carried out under identical conditions, but with denatured enzyme, and did not give a new fluorescent product (Figure 3). These data, together with our calculations, suggest that the expected product 13 was formed, confirming that KS-DFT calculations are useful to predict reactivity between a nitrile imine and a modified cap acting as a dipolarophile.

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.