Synthesis of Fluorescent Jasplakinolide Analogues for Live-Cell STED Microscopy of Actin

Abstract

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.

Rhodamines with a Chloronicotinic Acid Fragment for Live Cell Superresolution STED Microscopy**

“…For staining of actin, we used conjugate of 5 ‐F with the established actin ligand des‐bromo‐des‐methyljasplakinolide (bound via l ‐lysine residue and 6‐aminohexanoic acid linkers with a fluorescent dye) [18, 19] . Actin protein plays a crucial role in cellular function and motility [20] .…”

“…The thickness and dynamic behavior of actin filaments makes them an attractive object for observation with superresolution optical microscopy [6d, 18–20] . Figure 5 shows confocal and STED images of tubulin fibers stained with 565pR ‐Tubu (green) and actin stained with 610CP ‐Actin [6d, 18] (magenta) in a Drosophila melanogaster wild type egg chamber ( ex vivo ). Observing the confocal counterpart, we cannot exclude co‐localization of these structures.…”

Optical Microscopy and the Extracellular Matrix Structure: A Review

“…Much like other super-resolution methods, STED and GSD have been used extensively in the past and have found ongoing use in imaging live cells [ 126 , 127 ] and cellular components, such as actin in live cells [ 128 ], ribonucleic acids (RNA) in fixed cells [ 129 ], and the endoplasmic reticulum of neural cells [ 130 ]; they have found limited use in the characterization of the ECM.…”

See You at the Molecular Scale

“…Furthermore, their outstanding cell permeability and increased fluorogenicity allow wash-free multicolor live-cell labeling at concentrations which are 10 times lower than that required when using conventional live probes. abberior LIVE dyes have been extensively tested in living cells (Figure 3) and tissue (Figure 4) [15][16][17]. With this technique (Figure 5), high-contrast images of living systems at a resolution below 50 nm can be easily acquired.…”