The Journal of organic chemistry
Synthesis of Fluorescent Jasplakinolide Analogues for Live-Cell STED Microscopy of Actin
Belov, V. N., Stoldt, S., Rüttger, F., John, M., Seikowski, J., Schimpfhauser, J., & Hell, S. W.
Dyes and pigments, Peptides and proteins, Monomers, Fluorescence, Mixtures
The nanometer thickness of filaments and the dynamic behavior of actin—a protein playing a crucial role in cellular function and motility—make it attractive for observation with super-resolution optical microscopy. We developed the solution-phase synthesis of des-bromo-des-methyl-jasplakinolide-lysine, used as the “recognition unit” (ligand) for F-actin in living cells. The first amino acid—Fmoc-O-TIPS-β-tyrosine—was prepared in 78% yield (two steps in one pot). The new solution-phase synthesis involves 2-phenylisopropyl protection of the carboxyl group and does not require excesses of commercially unavailable amino acids. The overall yield of the target intermediate obtained in nine steps is about 8%. The 2-phenylisopropyl group can be cleaved from carboxyl with 2–3% (v/v) of TFA in acetonitrile (0–10 °C), without affecting TIPS protection of the phenolic hydroxyl in β-tyrosine and N-Boc protection in lysine. Des-bromo-des-methyl-jasplakinolide-lysine was coupled with red-emitting fluorescent dyes 580CP and 610CP (via 6-aminohexanoate linker). Actin in living cells was labeled with 580CP and 610CP probes, and the optical resolution measured as full width at half-maximum of line profiles across actin fibers was found to be 300–400 nm and 100 nm under confocal and STED conditions, respectively. The solution-phase synthesis of des-bromo-des-methyl-jasplakinolide-lysine opens a way to better fluorescent probe perspective for actin imaging.