Rhenium (I) Complexes as Probes for Prokaryotic and Fungal Cells by Fluorescence Microscopy: Do Ligands Matter?

Abstract

Re(I) complexes have exposed highly suitable properties for cellular imaging (especially for fluorescent microscopy) such as low cytotoxicity, good cellular uptake, and differential staining. These features can be modulated or tuned by modifying the ligands surrounding the metal core. However, most of Re(I)-based complexes have been tested for non-walled cells, such as epithelial cells. In this context, it has been proposed that Re(I) complexes are inefficient to stain walled cells (i.e., cells protected by a rigid cell wall, such as bacteria and fungi), presumably due to this physical barrier hampering cellular uptake. More recently, a series of studies have been published showing that a suitable combination of ligands is useful for obtaining Re(I)-based complexes able to stain walled cells. This review summarizes the main characteristics of different fluorophores used in bioimage, remarking the advantages of d⁶-based complexes, and focusing on Re(I) complexes. In addition, we explored different structural features of these complexes that allow for obtaining fluorophores especially designed for walled cells (bacteria and fungi), with especial emphasis on the ligand choice. Since many pathogens correspond to bacteria and fungi (yeasts and molds), and considering that these organisms have been increasingly used in several biotechnological applications, development of new tools for their study, such as the design of new fluorophores, is fundamental and attractive.

Keywords: Equatorial ligand; ancillary ligand; bacteria; fungi; molds; rhenium (I) tricarbonyl complexes; yeasts.

Figure 1

Structural scheme of Re(I) tricarbonyl complexes and their different functional groups [i.e., substituted dinitrogenated ligand (N, N); ancillary ligand (L)]. Some examples of (N, N) ligands include (1) 4,4′-dimethyl-2,2′-bpy (dmb); (2) 2,2′-bipyridine (bpy); (3) 1,10-phenanthroline (phen); (4) 5,6-dione-1,10-phenanthroline (dione). Some examples of ancillary ligands (L) include (1) halogens; (2) 3-chloromethylpyridyl; (3) (E)-2-{[(3-aminopyridin-4-yl)imino]-methyl}-4,6-di-tert-butyl-phenol (a pyridine Schiff base harboring an intramolecular hydrogen bond) (Carreño et al., 2014, 2015a,b, 2017a).

Figure 2

Effect of ligands in the use of fac-Re(I)(CO)₃(N,N)L^{(0, +)} complexes in walled cells (yeasts). Fluorescence confocal microscopy images of Candida albicans (yeasts) stained with fac-Re(I)(CO)₃(4,4′-dimethyl-2,2′-bpy)Br (A), fac-Re(I)(CO)₃(4,4′-diethanoate-2,2′-bpy)Br (B), or fac-Re(I)(CO)₃ (4,4′-diethanoate-2,2′-bpy) [(E)-2-((3-amino-pyridin-4-ylimino)-methyl)-4,6-di-tert-butylphenol]⁺ (C) were compared. “Red channel” corresponds to excitation of 405 nm and emission collected in a range of 555 to 625 nm. In all cases, microorganisms were observed fresh, immobilized with 1% agarose, using a 100× objective. DMSO alone was used to set the detection threshold (not shown). White bars represent 5 μm. The complete protocol for staining and other examples of how ligand choice impacts on staining properties of d⁶ complexes were previously reported (Carreño et al., 2016, 2017a, 2019b).