Synthesis of Small-Molecule Fluorescent Probes for the In Vitro Imaging of Calcium-Activated Potassium Channel KCa 3.1

Abstract

Small-molecule probes for the in vitro imaging of K_Ca 3.1 channel-expressing cells were developed. Senicapoc, showing high affinity and selectivity for the K_Ca 3.1 channels, was chosen as the targeting component. BODIPY dyes 15-20 were synthesized and connected by a Cu^I -catalyzed azide-alkyne [3+2]cycloaddition with propargyl ether senicapoc derivative 8, yielding fluorescently labeled ligands 21-26. The dimethylpyrrole-based imaging probes 25 and 26 allow staining of K_Ca 3.1 channels in NSCLC cells. The specificity was shown by removing the punctate staining pattern by pre-incubation with senicapoc. The density of K_Ca 3.1 channels detected with 25 and by immunostaining was identical. The punctate structure of the labeled channels could also be observed in living cells. Molecular modeling showed binding of the senicapoc-targeting component towards the binding site within the ion channel and orientation of the linker with the dye along the inner surface of the ion channel.

Keywords: KCa3.1 channels; cycloaddition; fluorescent probes; molecular modelling; non-small cell lung cancer.

Figure 1

Chemical structure of senicapoc (1) and its derivative 2.

Scheme 1

Synthesis of the senicapoc derivative 8. Reagents and reaction conditions: a) imidazole, TBDPS‐Cl, DMF, 24 h, rt, 96 %. b) 1. n‐ butyllithium, THF, 1 h, −78 °C; 2. 4‐fluorobenzophenone, THF, 3 h, rt, 58 %. c) 1. InCl₃, trimethylsilyl cyanide, CH₂Cl_2, 1 h, 20–40 °C, 1.5 h, 40–50 °C; 2. KOH, KNa‐tartrate, H₂O, 12 h, 0 °C—rt, 89 %. d) KOH, KNa‐tartrate, 2‐methylbutan‐2‐ol,5 h, 100 °C, 88 %. e) KOH, EtOH, H₂O, 90 °C, 48 h, 23 %, f) propargyl bromide, Cs₂Co₃, DMF, 30 min, rt, 90 %.

Scheme 2

General concept for the synthesis of BODIPY dyes. a) H⁺. b) oxidant, for example, p‐chloranil. c) NEt₃, BF₃⋅OEt₂. (modified according to literature13).

Scheme 3

A) Structures of benzaldehyde derivatives 9–11 and pyrrole derivatives 12–14 used for the BODIPY synthesis. B–D) Synthesis of BODIPY dyes with azide moiety in the side chain. Series (B) is based on the thiophene annulated pyrrole 12, series (C) is based on the ethyldimethylpyrrole 13 and series (D) on the dimethylpyrrole 14. Reagents and reaction conditions: a) 1. TFA, CH₂Cl₂, molecular sieves 3 Å, rt, 24 h; 2. p‐chloranil, CH₂Cl₂, rt, 30 min; 3. NEt₃, CH₂Cl₂, rt, 15 min; 4. BF₃⋅OEt₂, CH₂Cl₂, rt, 12 h. Yields: 15: 7 %. 16: 14 %. 17: 30 %. 18: 22 %. 19: 16 %. 20: 27 %. Yields were calculated over three reaction steps for >90 % purity compounds.

Scheme 4

Synthesis of fluorescently labeled tool compounds. Reagents and reaction conditions: a) CuSO₄, sodium ascorbate, DMF, H₂O, rt, 24 h, 21: 58 %, 22: 57 %, 23: 61 %, 24: 2 %, 25: 19 %, 26: 68 %.

Figure 2

Fluorescently labeled ligands 21–26 designed to image K_Ca3.1 channels.

Figure 3

Setup for the staining of NSCLC cells.

Figure 4

A) NSCLC cells incubated for 10 min with a 10 μm staining solution of imaging probe 25. B) Magnification of (A) (white box). C) NSCLC cells incubated for 10 min with a 10 μm staining solution of imaging probe 26. D) Magnification of (C) (white box). E) NSCLC cells blocked for 5 min with unlabeled senicapoc (30 μm) and subsequently stained for 10 min with a 10 μm staining solution of imaging probe 25. F) NSCLC cells blocked for 5 min with unlabeled senicapoc (30 μm) and subsequently for 10 min with a 10 μm staining solution of imaging probe 26.

Figure 5

Signal analysis with linescans (blue lines in magnification boxes). 1 pixel≈60 nm. A) NSCLC cells incubated for 10 min with a 10 μm staining solution of imaging probe 25 with 50×50 pixel square, magnification of this square with linescan and 16 signals for K_Ca3.1 channels. B) Linescan of (A) Intensity profile [arbitrary units] of signal 1 and signal 2. C) NSCLC cells after performing the indirect immunofluorescence assay with 50×50 pixel square, magnification of this square with linescan and 16 signals for K_Ca3.1 channels. D) Linescan of (C) Intensity profile [arbitrary units] of signal 1.

Figure 6

A model of 25 (green) in the K_Ca3.1 Cryo‐EM structure (pdb 6cn0)18 in the bound state II (light blue) with bound calmodulin (khaki). A,B) Overview of 25 binding in the inner pore. C) Surface representation of 25. D) Molecular representation of 25 and surface representation of ion channel. The given distance of 7.86 Å, indicates the distance between the O‐atoms and thus the length of the linker and the distance between senicapoc moiety and the fluorescent dye.