Fast and Efficient Postsynthetic DNA Labeling in Cells by Means of Strain-Promoted Sydnone-Alkyne Cycloadditions

Abstract

Sydnones are highly stable mesoionic 1,3-dipoles that react with cyclooctynes through strain-promoted sydnone-alkyne cycloaddition (SPSAC). Although sydnones have been shown to be valuable bioorthogonal chemical reporters for the labeling of proteins and complex glycans, nucleic acids have not yet been tagged by SPSAC. Evaluation of SPSAC kinetics with model substrates showed fast reactions with cyclooctyne probes (up to k=0.59 M^-1 s^-1 ), and two different sydnones were effectively incorporated into both 2'-deoxyuridines at position 5, and 7-deaza-2'-deoxyadenosines at position 7. These modified nucleosides were synthetically incorporated into single-stranded DNAs, which were successfully postsynthetically labeled with cyclooctyne probes both in vitro and in cells. These results show that sydnones are versatile bioorthogonal tags and have the premise to become essential tools for tracking DNA and potentially RNA in living cells.

Keywords: cell biology cyclooctyne; fluorescence; oligonucleotide; strain; sydnone.

Figure 1

Postsynthetic labeling of DNA by iEDDA using tetrazines and triazines as reactive groups in comparison with SPSAC.

Figure 2

Second‐order rate constants of the strain‐promoted sydnone–alkyne cycloadditions (SPSAC) between sydnones PhSydH and PhSydCl with three cyclooctynes (MeO‐DIBO, MeO‐DIBAC and BCN).

Scheme 1

Synthesis of sydnone‐modified 2’‐deoxyuridines 1 and 2 and the 7‐deaza‐2’‐deoxyadenosine 3 and 4 and SPSACs with BCN 11: a) NaClO (12 % act. Cl₂), dioxane/HCl, RT, 20 h, 43 %; b) N‐hydroxysuccinimide, N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide hydrochloride, DMF, RT, 16 h, 5: 78 %, 6: 89 %; c) Et₃N, DMF, RT, 16 h, 1: 29 %, 2: 54 %; d) Et₃N, DMF, RT, 16 h, 3: 29 %, 4: 36 %; e) MeOH, 20 °C, 4 h; f) MeOH, 20 °C, 4 h.

Figure 3

UV/Vis absorbance (solid lines) and fluorescence (dashed lines, λ _ex=315 nm) changes recorded during SPSAC labeling experiments with 25 μM nucleoside 1 (top) or nucleoside 2 (bottom) and 250 μM BCN 11 (10.0 equiv.) in MeOH at 20 °C over 4 h. For experiments with nucleoside 3 and nucleoside 4, see Figures S14 and S15.

Scheme 2

Synthesis of sydnone‐modified DNA3–DNA6: a) DNA synthesizer; b) 25 % NH₄OH, 55 °C, 16 h; c) 7, Et₃N, DMSO, RT, 16 h d) 8, Et₃N, DMSO, RT, 16 h; e) DNA synthesizer; f) 25 % NH₄OH, 55 °C, 16 h; g) 7, Et₃N, DMSO, RT, 16 h; f) 8, Et₃N, DMSO, RT, 16 h.

Figure 4

Confocal laser microscopy images of HeLa cells that were transfected for 26 h with either 0.7 μM of DNA3 or DNA5 and subsequently fixed and labeled by means of SPSAC with sulfoCy3‐DIBAC (7 nM) within 4 h. The cells were washed with 2× PBS, followed by imaging. λ _ex=488 nm, λ _em=550–650 nm. The merged column presents the merger of the fluorescence and the transmission.