Fluoride-Cleavable, Fluorescently Labelled Reversible Terminators: Synthesis and Use in Primer Extension

Fluorescent 2′-deoxynucleotides containing a protecting group at the 3′-O-position are reversible terminators that enable array-based DNA sequencing-by-synthesis (SBS) approaches. Herein, we describe the synthesis and full characterisation of four reversible terminators bearing a 3′-blocking moiety and a linker-dye system that is removable under the same fluoride-based treatment. Each nucleotide analogue has a different fluorophore attached to the base through a fluoride-cleavable linker and a 2-cyanoethyl moiety as the 3′-blocking group, which can be removed by using a fluoride treatment as well. Furthermore, we identified a DNA polymerase, namely, RevertAid M-MuLV reverse transcriptase, which can incorporate the four modified reversible terminators. The synthesised nucleotides and the optimised DNA polymerase were used on CodeLink slides spotted with hairpin oligonucleotides to demonstrate their potential in a cyclic reversible terminating approach.

UV spectroscopy was performed on a Jasco V-650 spectrometer using 0.1 cm cuvettes.
Fluorescence spectroscopy was performed on a Hitachi F4500 fluorescence spectrometer using 0.3 cm cuvettes.
Elemental analyses (EA) were recorded on a Foss-Heraeus CHN-O Rapid instrument.
The numbering of the atoms to assign the NMR signals, are not related to the IUPAC numbering or the numbers used in the names of the compounds.
After 2 h another 100 µL (0.86 mmol, 0.4 eq.) of benzoyl chloride were added and the reaction mixture stirred at room temperature for 18 h.

Synthesis of 2-N-(N,N-dimethylaminomethylidenyl)-N 1 -benzoyl-7-deaza-7-iodo-5'-O-(4-monomethoxytrityl)-2'-deoxyguanosine 19
Compound 18 (600 mg, 1.17 mmol, 1.0 eq.) was dissolved in 10 mL of dry pyridine, 13 mg ( In a well dried Erlenmeyer flask, equipped with a big triangle stirring bar and a septum connected to an argon line, the fully protected nucleoside was dissolved in 20 eq. of freshly distilled acrylonitrile and stirred vigorously for 5 min. To the concentrated solution tBuOH (3 mL/mmol to 9 mL/mmol, depending on the solubility of the nucleoside) was added. For the 2'-deoxycytidine derivative 12 DMF was necessary as additional co-solvent (3 mL/mmol). The resulting solution again was stirred vigorously for 5 min before 1 eq. of Cs 2 CO 3 was added. The heterogeneous mixture was stirred vigorously for 2 to 3 h at room temperature, then filtrated over silica gel and the solvents removed under reduced pressure. The crude product was either additionally purified by column chromatography or directly used for the subsequent deprotection. [