Post-Synthetic Modification of DNA by Inverse-Electron-Demand Diels−Alder Reaction

Abstract

There is currently a tremendous interest in developing bioorthogonal "click chemistry" methods for the modification of biopolymers. Very recently, inverse-electron-demand Diels-Alder reactions have received attention, but to date they have not been applied to nucleic acids. Here we describe the first example of DNA modification by inverse-electron-demand Diels-Alder reaction. We synthesized four different building blocks for 3'-terminal, 5'-terminal, and internal incorporation of norbornene dienophiles into oligonucleotides. These DNA strands were either directly reacted with suitably derivatized tetrazine dienes or first subjected to enzymatic manipulations. We demonstrate that the inverse-electron-demand Diels-Alder reaction allows efficient site-specific post-synthetic conjugation, often at a 1:1 stoichiometry, without any side reaction. The reaction works in aqueous media at room temperature, and no transition metals are required. Both short chemically synthesized oligonucleotides and long enzymatically amplified DNA strands were successfully conjugated.

Inverse electron demand Diels–Alder (iEDDA)-initiated conjugation: a (high) potential click chemistry scheme

“…5 shows different building blocks which can be used to incorporate norbornenes at both termini (20,23) and internal (21,22) positions in oligonucleotides using phosphoramidite chemistry. 36 It should also be highlighted that, at an equimolar ratio, up to 96% yield could be observed for the iEDDA conjugation (as determined by HPLC). 20 was also used to incorporate norbornene at the 5 0 -terminus of a short RNA sequence using a solid phase synthesis protocol.…”

Norbornenes in Inverse Electron‐Demand Diels–Alder Reactions

“…Among other strained dienophiles, norbornenes stand out because they are synthetically easily available, stable, and comparatively small in size. Indeed, others and us used the unusual reactivity of norbornene [26,27] to label nucleic acids [28][29][30] and proteins. [31][32][33][34] We therefore reasoned that we should investigate the parameters that determine the reactivity of norbornenes and of heteroatom-containing norbornene derivatives [35] with tetrazines to find the best reaction partner in the DAR inv for biomolecule labeling.…”

Efficient Synthesis of DNA Conjugates by Strain‐Promoted Azide–Cyclooctyne Cycloaddition in the Solid Phase

“…Mass analysis was performed with a LASER-TOF LT3 or Applied Biosystem Voyager with 3-hydroxypicolinic acid or 2Ј,4Ј,6Ј-trihydroxyacetophenone as matrix or recorded by Metabion, Germany. NMR spectra were obtained with a Bruker instrument at 25°C ( 1 H at 300 MHz; 13 C at 75 MHz). Chemical shifts are reported in ppm downfield from TMS as standard.…”

“…[6][7][8][9][10][11] Additional, metal-free bioorthogonal conjugation strategies include Diels-Alder cycloadditions and photoinduceable cycloadditions of tetrazines or nitrile imines to alkenes. [12][13][14] More recent advances in this area include strain-promoted azide-alkyne cycloadditions (SPAAC). These reactions, first observed more than 50 years ago, [15] exploit the intrinsic ring strain of cyclooctynes.…”