Replacement of the phosphodiester backbone between canonical nucleosides with a dirhenium carbonyl "click" linker-a new class of luminescent organometallic dinucleoside phosphate mimics.

The first-in-class luminescent dinucleoside phosphate analogs with a [Re2(µ-Cl)2(CO)6(µ-pyridazine)] "click" linker as a replacement for the natural phosphate group are reported together with the synthesis of luminescent adenosine and thymidine derivatives having the [Re2(µ-Cl)2(CO)6(µ-pyridazine)] entity attached to positions 5' and 3', respectively. These compounds were synthesized by applying inverse-electron-demand Diels-Alder and copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition reactions in three or four steps. The obtained compounds exhibited orange emission (λPL ≈ 600 nm, ΦPL ≈ 0.10, and τ = 0.33-0.61 µs) and no toxicity (except for one nucleoside) to human HeLa cervical epithelioid and Ishikawa endometrial adenocarcinoma cancer cells in vitro. Furthermore, the compounds' ability to inhibit the growth of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacterial strains was moderate and only observed at a high concentration of 100 µM. Confocal microscopy imaging revealed that the "dirhenium carbonyl" dinucleosides and nucleosides localized mainly in the membranous structures of HeLa cells and uniformly inside S. aureus and E. coli bacterial cells. An interesting finding was that some of the tested compounds were also found in the nuclei of HeLa cells.

Metallodrug Profiling against SARS-CoV-2 Target Proteins Identifies Highly Potent Inhibitors of the S/ACE2 interaction and the Papain-like Protease PLpro.

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has called for an urgent need for dedicated antiviral therapeutics. Metal complexes are commonly underrepresented in compound libraries that are used for screening in drug discovery campaigns, however, there is growing evidence for their role in medicinal chemistry. Based on previous results, we have selected more than 100 structurally diverse metal complexes for profiling as inhibitors of two relevant SARS-CoV-2 replication mechanisms, namely the interaction of the spike (S) protein with the ACE2 receptor and the papain-like protease PLpro . In addition to many well-established types of mononuclear experimental metallodrugs, the pool of compounds tested was extended to approved metal-based therapeutics such as silver sulfadiazine and thiomersal, as well as polyoxometalates (POMs). Among the mononuclear metal complexes, only a small number of active inhibitors of the S/ACE2 interaction was identified, with titanocene dichloride as the only strong inhibitor. However, among the gold and silver containing complexes many turned out to be very potent inhibitors of PLpro activity. Highly promising activity against both targets was noted for many POMs. Selected complexes were evaluated in antiviral SARS-CoV-2 assays confirming activity for gold complexes with N-heterocyclic carbene (NHC) or dithiocarbamato ligands, a silver NHC complex, titanocene dichloride as well as a POM compound. These studies might provide starting points for the design of metal-based SARS-CoV-2 antiviral agents.

Luminescent pyrenyl-GNA nucleosides: synthesis, photophysics and confocal microscopy studies in cancer HeLa cells.

Glycol nucleic acids (GNA) are synthetic genetic-like polymers with an acyclic three-carbon propylene glycol phosphodiester backbone. Here, synthesis, luminescence properties, circular dichroism (CD) spectra, and confocal microscopy speciation studies of (R,S) and (S,R) pyrenyl-GNA (pyr-GNA) nucleosides are reported in HeLa cells. Enantiomerically pure nucleosides were obtained by a Sharpless asymmetric dihydroxylation reaction followed by semi-preparative high-performance liquid chromatography (HPLC) separation using Amylose-2 as the chiral stationary phase. The enantiomeric relationship between stereoisomers was confirmed by CD spectra, and the absolute configurations were assigned based on experimental and theoretical CD spectra comparisons. The pyr-GNA nucleosides were not cytotoxic against human cervical (HeLa) cancer cells and thus were utilized as luminescent probes in the imaging of these cells with confocal microscopy. Cellular staining patterns were identical for both enantiomers in HeLa cells. Compounds showed no photocytotoxic effect and were localized in the lipid membranes of the mitochondria, in cellular vesicles and in other lipid cellular compartments. The overall distribution of the pyrene and pyrenyl-GNA nucleosides inside the living HeLa cells differed, since the former compound gives a more granular staining pattern and the latter a more diffuse one.

Ferrocenyl GNA Nucleosides: A Bridge between Organic and Organometallic Xeno-nucleic Acids.

The enantioselective synthesis and electrochemistry of the first ferrocenyl GNA nucleosides is reported. These compounds were obtained by a Sharpless asymmetric dihydroxylation reaction of [3-(N1-thyminyl)-1-(ferrocenyl)]propene as S,R and R,S enantiomers in about 70 % yield with enantiomeric excesses of >99 % and 71 %, respectively. The absolute configurations of the chiral carbon atoms in the nucleosides were assigned by single-crystal X-ray diffraction analysis of the methyl derivatives in the solid state. The compounds were also studied with circular dichroism (CD) spectroscopy in solution. The enantiomeric relationship between the S,R and R,S isomers was confirmed by the near-mirror-image CD spectra. The redox properties of the nucleosides and their methylated derivatives were investigated using cyclic voltammetry. The cyclic voltammograms revealed reversible redox processes for the entire series of compounds at potentials of -25 mV (for nonmethylated derivatives) and 75 mV (for methylated derivatives) versus the ferrocene/ferrocenium reference redox couple.

Mechanisms of proton relay and product release by Class A ß-lactamase at ultrahigh resolution.

The ß-lactam antibiotics inhibit penicillin-binding proteins (PBPs) by forming a stable, covalent, acyl-enzyme complex. During the evolution from PBPs to Class A ß-lactamases, the ß-lactamases acquired Glu166 to activate a catalytic water and cleave the acyl-enzyme bond. Here we present three product complex crystal structures of CTX-M-14 Class A ß-lactamase with a ruthenocene-conjugated penicillin-a 0.85 Å resolution structure of E166A mutant complexed with the penilloate product, a 1.30 Å resolution complex structure of the same mutant with the penicilloate product, and a 1.18 Å resolution complex structure of S70G mutant with a penicilloate product epimer-shedding light on the catalytic mechanisms and product inhibition of PBPs and Class A ß-lactamases. The E166A-penilloate complex captured the hydrogen bonding network following the protonation of the leaving group and, for the first time, unambiguously show that the ring nitrogen donates a proton to Ser130, which in turn donates a proton to Lys73. These observations indicate that in the absence of Glu166, the equivalent lysine would be neutral in PBPs and therefore capable of serving as the general base to activate the catalytic serine. Together with previous results, this structure suggests a common proton relay network shared by Class A ß-lactamases and PBPs, from the catalytic serine to the lysine, and ultimately to the ring nitrogen. Additionally, the E166A-penicilloate complex reveals previously unseen conformational changes of key catalytic residues during the release of the product, and is the first structure to capture the hydrolyzed product in the presence of an unmutated catalytic serine. DATABASE: Structural data are available in the PDB database under the accession numbers 5TOP, 5TOY, and 5VLE.

Antibacterial Properties of Metallocenyl-7-ADCA Derivatives and Structure in Complex with CTX-M ß-Lactamase.

A series of six novel metallocenyl-7-ADCA (metallocenyl = ferrocenyl or ruthenocenyl; 7-ADCA = 7-aminodesacetoxycephalosporanic acid) conjugates were synthesized and their antibacterial properties evaluated by biochemical and microbiological assays. The ruthenocene derivatives showed a higher level of inhibition of DD-carboxypeptidase 64-575, a Penicillin Binding Protein (PBP), than the ferrocene derivatives and the reference compound penicillin G. Protein X-ray crystallographic analysis revealed a covalent acyl-enzyme complex of a ruthenocenyl compound with CTX-M ß-lactamase E166A mutant, corresponding to a similar complex with PBPs responsible for the bactericidal activities of these compounds. Most interestingly, an intact compound was captured at the crystal-packing interface, elucidating for the first time the structure of a metallocenyl ß-lactam compound that previously eluded small molecule crystallography. We propose that protein crystals, even from biologically unrelated molecules, can be utilized to determine structures of small molecules.

Mitochondria Targeting with Luminescent Rhenium(I) Complexes.

Two new neutral fac-[Re(CO)3(phen)L] compounds (1,2), with phen = 1,10-phenanthroline and L = O2C(CH2)5CH3 or O2C(CH2)4C≡CH, were synthetized in one-pot procedures from fac-[Re(CO)3(phen)Cl] and the corresponding carboxylic acids, and were fully characterized by IR and UV-Vis absorption spectroscopy, ¹H- and 13C-NMR, mass spectrometry and X-ray crystallography. The compounds, which display orange luminescence, were used as probes for living cancer HeLa cell staining. Confocal microscopy revealed accumulation of both dyes in mitochondria. To investigate the mechanism of mitochondrial staining, a new non-emissive compound, fac-[Re(CO)3(phen)L], with L = O2C(CH2)3((C5H5)Fe(C5H4), i.e., containing a ferrocenyl moiety, was synthetized and characterized (3). 3 shows the same mitochondrial accumulation pattern as 1 and 2. Emission of 3 can only be possible when ferrocene-containing ligand dissociates from the metal center to produce a species containing the luminescent fac-[Re(CO)3(phen)]⁺ core. The release of ligands from the Re center was verified in vitro through the conjugation with model proteins. These findings suggest that the mitochondria accumulation of compounds 1-3 is due to the formation of luminescent fac-[Re(CO)3(phen)]⁺ products, which react with cellular matrix molecules giving secondary products and are uptaken into the negatively charged mitochondrial membranes. Thus, reported compounds feature a rare dissociation-driven mechanism of action with great potential for biological applications.

Antibacterial properties and atomic resolution X-ray complex crystal structure of a ruthenocene conjugated ß-lactam antibiotic.

We have determined a 1.18 Å resolution X-ray crystal structure of a novel ruthenocenyle-6-aminopenicillinic acid in complex with CTX-M ß-lactamase, showing unprecedented details of interactions between ruthenocene and protein. As the first product complex with an intact catalytic serine, the structure also offers insights into ß-lactamase catalysis and inhibitor design.

Ferrocenyl bioconjugates of ampicillin and 6-aminopenicillinic acid--synthesis, electrochemistry and biological activity.

We report on the synthesis of ferrocenyl-ampicillin and ferrocenyl-6-aminopenicillinic acid bioconjugates. Title compounds were characterized by (1)H NMR, IR, MS and elemental analysis. These novel ferrocenyl-antibiotic conjugates were also investigated by cyclic voltammetry (CV). Ferrocenyl-ampicillin complexes revealed reversible uncomplicated oxidation whereas ferrocenyl-6-aminopenicillinic acid derivatives were found to exhibit adsorption waves in cathodic scans. Antibacterial activities of our ferrocenyl-antibiotic conjugates against Gram-positive methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), vancomycin-resistant S. aureus (VRSA) and Staphylococcus epidermidis bacterial strains were determined. Our experiments show significant antibacterial activity of ferrocenyl-6-aminopenicillinic acid bioconjugates against the bacterial strains tested. Contrary to that ferrocenyl-ampicillin derivatives were inactive. The inhibitory effects on the dd-carboxypeptidase 64-575 II exerted by our ferrocenyl-6-aminopenicillinic acid bioconjugates were established in the low nanomolar range. The tumor cell growth inhibition of representative ferrocenyl-ampicillin and ferrocenyl-6-aminopenicillinic acid bioconjugates against MCF-7 breast adenocarcinoma and HT-29 colon carcinoma cell lines were studied in vitro. Similar to the antibacterial activity tests the assays in tumor cells revealed significant antiproliferative effects exerted by ferrocenyl-6-aminopenicillinic acid bioconjugates.