Boosting Anion Transport Activity of Diamidocarbazoles by Electron Withdrawing Substituents

Abstract

Artificial chloride transporters have been intensely investigated in view of their potential medicinal applications. Recently, we have established 1,8-diamidocarbazoles as a versatile platform for the development of active chloride carriers. In the present contribution, we investigate the influence of various electron-withdrawing substituents in positions 3 and 6 of the carbazole core on the chloride transport activity of these anionophores. Using lucigenin assay and large unilamellar vesicles as models, the 3,6-dicyano- and 3,6-dinitro- substituted receptors were found to be highly active and perfectly deliverable chloride transporters, with EC_50,270s value as low as 22 nM for the Cl^-/NO₃ ^- exchange. Mechanistic studies revealed that diamidocarbazoles form 1:1 complexes with chloride in lipid bilayers and facilitate chloride/nitrate exchange by carrier mechanism. Furthermore, owing to its increased acidity, the 3,6-dinitro- substituted receptor acts as a pH-switchable transporter, with physiologically relevant apparent pK_a of 6.4.

Keywords: LUVs; anion transport; carbazole; chloride transport; liposomes; lucigenin; pH-switchable transport.

FIGURE 1

Examples of chloride transporters from literature: 1 - ref. (Valkenier et al., 2014); 2 - ref. (Dias et al., 2018b); 3 - ref. (Valkenier et al., 2015); 4 - ref. (Dias et al., 2018a), 5 - ref(Bąk et al., 2018); and 6 - ref. (Picci et al., 2020).

FIGURE 2

Diamidocarbazole transporters 7–10 investigated in the present study.

FIGURE 3

X-ray crystal structure of 9×TBACl; the TBA⁺ counter cations were omitted for clarity. Hydrogen bonds between 9 and Cl⁻ are colored into light-blue. a) view along [010] direction; b) packing of 9×TBACl, view along [001] direction.

FIGURE 4

(A) Schematic representation of the lucigenin assay; (B) changes in the relative fluorescence F/F ₀ measured during the transport of Cl⁻ into 200 nm LUVs mediated by receptors 7–10 pre-incorporated in the membrane (7: 400 nM, 8–10: 40 nM).

FIGURE 5

(A) Raw data from the chloride transport studies by 10 (A) pre-incorporated in the membrane, (B) post-incorporated in the membrane. Plots of initial rates I vs transporter:lipid ratio for 10 and corresponding results from half-life times t _1/2 calculations: (C) pre-incorporated anionophore, (D) anionophore added externally as a solution in DMF.

FIGURE 6

Chloride transport with the anionophores either pre-incorporated in the membrane (solid lines) or added externally as a solution in DMF to vesicles without anionophore (dashed lines), mediated by: (A) 8–10 (1:10,000 transporter:lipids ratio); (B) 10 at various transporter:lipids ratios.

FIGURE 7

Relative fluorescence F/F ₀ traces measured as a function of pH for the pH-dependent Cl⁻ transport into 200 nm LUVs with pre-incorporated 10 at 1:10,000 transporter:lipids ratio.