Luminescent protein staining with Re(i) tetrazolato complexes

Abstract

Within the general framework of our past and current studies dealing with the investigation of the photophysical properties and the biological behavior of the family of tetrazolato and tetrazole Re(i) complexes, we have endeavored to investigate their potential in the luminescent staining of proteins purified by acrylamide gel electrophoresis. With the aim to provide the first examples of luminescent Re(i) complexes to be exploited for this specific purpose, we have designed and prepared four new Re(i)-based species with the general formula fac-[Re(CO)3(N^N)(Tph)]2-/0, where Tph is the 5-(phenyl)tetrazolato anion and N^N is in turn represented by bathophenanthroline disulfonate (BPS), bathocuproine disulfonate (BCS) or by the SO3- free bathocuproine (BC). In this latter case, the neutral complex fac-[Re(CO)3(BC)(Tph)] served as a model species for the characterization of the former disulfonate complexes. Its cationic analogue fac-[Re(CO)3(BC)(Tph-Me)]+ was also prepared by a straightforward methylation reaction. All complexes displayed bright phosphorescence in organic media and, relative to their water solubility, the dianionic species fac-[Re(CO)3(BPS)(Tph)]2- and fac-[Re(CO)3(BCS)(Tph)]2- were also highly emissive in aqueous solution. The sulfonate groups played a key role in promoting and significantly enhancing the luminescent staining performances of both the Re(i) complexes fac-[Re(CO)3(BPS)(Tph)]2- and fac-[Re(CO)3(BCS)(Tph)]2- for proteins. Highlighting a response superior to that of Coomassie Blue and comparable to the one obtained by the well-known silver staining method, these dianionic Re(i)-complexes could efficiently detect up to 50 ng of pure Bovine Serum Albumin (BSA), as well as all proteins found in a Standard Protein Marker mix and from a total protein extract. A lower but still good response for luminescent protein staining was surprisingly obtained by employing the -SO3- free neutral and cationic complexes fac-[Re(CO)3(BC)(Tph)] and fac-[Re(CO)3(BC)(Tph-Me)]+, respectively. These preliminary results open up new possibilities for the further widening of the use of Re(i)-based complexes as luminescent protein staining agents.

New Cationic fac-[Re(CO)3(deeb)B2]+ Complex, Where B2 Is a Benzimidazole Derivative, as a Potential New Luminescent Dye for Proteins Separated by SDS-PAGE

“…In that study, the authors explored different equatorial ligands, such as bathophenanthroline disulfonate (BPS) or bathocuproine disulfonate (BC), yielding fac-[Re(CO) 3 (BPS)(Tph)] 2− and fac-[Re(CO) 3 (BC)(Tph)] 2− . As expected, these two anionic complexes successfully marked proteins separated by SDS-PAGE due to disulfonate groups (Fiorini et al, 2018). Although the presence of the disulfonate groups has been widely used to achieve the (dye-SO 3 − / + NH 3 -protein) interaction, as stated above, other strategies can also be used (Jin and Choi, 2004).…”

“…As previously described, rhenium(I) tricarbonyl complexes harboring disulfonated phenanthroline-derivative ligands have been reported as useful to stain proteins separated by SDS-PAGE. Nevertheless, the use of rhenium(I) complexes lacking these ligands has been less studied to this end (Fiorini et al, 2018). In this sense, we explored whether the cationic complex fac-[Re(CO) 3 (deeb)B2] + , its respective precursor fac-Re(CO) 3 (deeb)Br, or the free ligand B2 was able to stain proteins resolved by SDS-PAGE.…”

“…Most protein dyes present an anionic nature, which allows for electrostatic interactions with protonated amino acids (-NH 3 + or -NH + -) (i.e., lysine, arginine, or histidine) (Sundaram et al, 2012). Nevertheless, cationic dyes have also been reported as useful to stain proteins, which can be explained by electrostatic interactions with negatively charged residues (e.g., the presence of -COO − in aspartate and glutamate) (Jin and Choi, 2004;Fiorini et al, 2018). We infer that the presence of the B2 moiety in fac-[Re(CO) 3 (deeb)B2] + could also be contributing to the staining capacity of this complex.…”

“…Regarding the use of Re(I) complexes, it has been reported that Re(I)-tetrazolato complexes fac-[Re(CO) 3 (N,N)(Tph)] 0/+/2− , where Tph is 5-phenyl-tetrazolato, have been analyzed as luminescent dyes for proteins separated by electrophoresis (SDS-PAGE) (Fiorini et al, 2018). In that study, the authors explored different equatorial ligands, such as bathophenanthroline disulfonate (BPS) or bathocuproine disulfonate (BC), yielding fac-[Re(CO) 3 (BPS)(Tph)] 2− and fac-[Re(CO) 3 (BC)(Tph)] 2− .…”

“…In order to improve these kinds of cationic complexes, new ancillary ligands have been reported as useful to modulate different properties. Since hydrophobic interactions, van der Waals forces, and hydrogen bonding can also contribute to the binding of the dye to proteins (Jin and Choi, 2004;Fiorini et al, 2018), the exploration of new ligands could allow obtaining alternative complexes to stain proteins, especially when considering the timesaving synthesis of sulfonated-free complexes (Fiorini et al, 2018). In this context, an ancillary ligand allowing for the formation of hydrophobic interactions, van der Waals forces, and/or hydrogen bonding with proteins could contribute to this development, even with cationic complexes.…”