Synthesis and stability studies of bicyclo[6.1.0]nonyne scaffolds for automated solid-phase oligonucleotide synthesis

Two novel bicyclo[6.1.0]nonyne (BCN) linker derivatives, which can be directly incorporated into oligonucleotide sequences during standard automated solid-phase synthesis, are reported. Stabilities of BCN-carbinol and two BCN-oligonucleotides are evaluated under acidic conditions. In addition, derivatized BCN linkers (non-acidic and acid treated) are evaluated for strain-promoted alkyne–azide cycloaddition (SPAAC).


General Information
Unless otherwise stated, all reagents and solvents used in chemical synthesis were of commercial grade.
HPLC was performed on UltiMate 3000 HPLC system with UV detection at 254 nm.
Chromatographic separations were performed on CombiFlash® Rf 200 by Teledyne ISCO system using Biotage® normal or reversed-phase (RP) silica columns.
Phosphorothioate oligonucleotide syntheses were performed using an ÄKTA oligopilot 10 synthesizer and conventional RNA/DNA synthesis procedures on 50 μmol ((A)2-T5) or 25 μmol ((B)2-T5) scales.All reagents, solvents and building block solutions were kept under inert (N2) atmosphere.0.1 M anhydrous MeCN solutions of commercial T amidite (Thermo Fisher Scientific) and synthesized BCN linker amidites A and B were used.The ON syntheses were performed using Primer Support™ 5G T 350 solid support.0.3 M 5-benzylthio-1-H-tetrazole (BTT) was used as an activator and 0.1 M xanthane hydride in pyridine as sulfurizing agent.Coupling times were as follows: 2.5 min for DNA phosphoramidites and 12 min for BCN linker phosphoramidites (linker A and linker B).
GC analysis was performed with an Agilent Technologies 6850 GC system.
NMR spectra were recorded using a Bruker AV 500 MHz (500.13 MHz in 1H,125.76 MHz in 13C and 202.47 MHz in 31P,470.56 MHz in 19F) spectrometer using the deuterated solvent signal as an internal standard.Chemical shifts (δ scale) are reported in parts per million (ppm).Coupling constants (J values) are given in Hertz (Hz).

BCN-linker containing oligonucleotides
A column loaded with solid-supported all-P(S)-heptamer (A) 2 -T 5 (50 µmol) or all-P(S)-heptamer (B) 2 -T 5 (25 µmol) was connected to the oligonucleotide synthesizer.Each derivative was exposed to five detritylation cycles (15 column volumes (CV) of 3% DCA in toluene per cycle, linear flow 400 cm/h).After each acidic cycle, 10 mg of solid-supported material was removed for LC-MS analysis.In addition, after 5 acidic cycles including subsequent washing with MeCN, both oligonucleotide derivatives were evaluated again for SPAAC.

S-2.
NMR spectra of linkers and intermediates