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

Vladimir N Belov,Franziska Rüttger,Jan Seikowski,Stefan Stoldt,Stefan W Hell,Jens Schimpfhauser,Michael John

doi:10.1021/acs.joc.0c00653

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.

Highlights

Actin protein plays a crucial role in cellular function and motility.[1]
It can be present either as a monomer (G-actin; globular) or, upon polymerization, it may form filaments (Factin): flexible fibers with a diameter of 4−7 nm and length of up to several micrometers. Both forms of actin are present in equilibrium; they are essential for the proper mobility and contraction of cells during cell division, cell motility, cytokinesis, vesicle and organelle movement, cell signaling, as well as the establishment and maintenance of cell junctions and cell shapes
We found a shorter route to Fmoc-OTIPS-β-tyrosine, which starts from commercially available Fmoc-β-tyrosine (Scheme 2)

Summary

Introduction

Actin protein plays a crucial role in cellular function and motility.[1]. It can be present either as a monomer (G-actin; globular) or, upon polymerization, it may form filaments (Factin): flexible fibers with a diameter of 4−7 nm and length of up to several micrometers. The fluorescent probes for super-resolution and live imaging of actin[2−5] incorporate the so-called des-bromo-des-methyljasplakinolide-lysine (Figure 1),[6] as the ligand or “recognition unit” for F-actin in living cells. This macrocyclic depsipeptide has a reactive amino group, and its salts can be readily generated from N-tert-butoxycarbonyl derivative (7-H in Scheme 3) which represents the key intermediate and stable precursor of the conjugates with organic dyes. Compound 7-H is commercially unavailable, and the solid-phase synthesis of 7-

Objectives

Results

Conclusion