Novel trinucleotide mRNA capping reagents: improved synthetic route and efficient cotranscriptional incorporation in mRNA

³¹P NMR spectra of (A) a conversion of ^7mG_mDP into imidazolide intermediate using EDC activation (from top to bottom: starting ^7mG_mDP, and formation of ^7mG_mDP-imidazolide after 1, 2, 4, 8, and 24 h, at room temperature); (B) the synthesis of ^7mG_mpppA_mpG (compound 1-2A) by coupling ^7mG_mDP-imidazolide and pA_mpG (from top to bottom: starting mixture of ^7mG_mDP-imidazolide and pA_mpG before MgCl₂ addition, and formation of ^7mG_mpppA_mpG after 1, 2, 4, 8, and 24 h after MgCl₂ addition; all at room temperature).