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[This corrects the article DOI: 10.1039/C9SC05586J.].

[This corrects the article DOI: 10.1039/C9SC05586J.].

Herein, we describe the synthesis and characterisation of four new supramolecular cobalt conjugates of antimicrobial peptides functionalised with terpyridine ligands (L). Peptides were chosen based on the well-established arginine-tryptophan (RW)3 motif, with terpyridine-derivatized lysine (Lys(tpy)) added to the sequence, or replacing tryptophan residues. Self-assembly of the antimicrobial peptides with Co(BF4)2·6H2O formed exclusively CoL2 dimers (for peptides with one tpy ligand each) and Co2L4 metallo-macrocycles (for peptides with two tpy ligands for each peptide), which could be 'locked' by oxidation of Co(+II) to Co(+III) with ammonium ceric nitrate. The Co-peptide complexes were characterised by mass spectrometry and in solution by NMR spectroscopy, including 2D diffusion ordered NMR spectroscopy (DOSY) which confirmed the proposed stoichiometries. The antimicrobial activity of the novel peptides and their metallo-supramolecular assemblies was investigated by determination of their minimal inhibitory concentration (MIC) against a panel of Gram-positive and Gram-negative bacteria. Complexation with cobalt increases the activity of the peptides in almost every case. Most of the new metal–peptide conjugates showed good activity against Gram-positive bacteria, including a multi-resistant S. aureus strain and the opportunistic pathogenic yeast C. albicans (down to 7 μmol l−1 for the most active Co2L4 derivate), a value that is increased five-fold compared to the lysine-derivatized peptide ligand alone. Interestingly, conjugates of the CoL2 type also showed decent activity against Gram-negative bacteria including the WHO-flagged problematic A. baumannii strain (down to 18 μmol l−1 for the most active derivative). [ABSTRACT FROM AUTHOR]

The synthesis and characterization of three compounds [Rh(η5‐C5Me5)Cl(N^N)]PF6 (N^N=4,4'‐disubstituted‐2,2'‐bipyridines, 1–3) are described. The cationic complexes contain the bidentate ligands N^N=4,4'‐di‐tert‐butyl‐2,2'‐bipyridine (1), N^N=4,4'‐dinonyl‐2,2'‐bipyridine (2) and N^N=4,4'‐diamino‐2,2'‐bipyridine (3). The complex salts were obtained by the bridge‐splitting reaction from the precursor [{Rh(η5‐C5Me5)(μ‐Cl)Cl}2] and subsequent salt metathesis affording their corresponding hexafluorido phosphate salts. All compounds were characterized by elemental analysis and spectroscopic means. Additionally, the molecular structure of compound 3 in the solid was determined by a single‐crystal X‐ray diffraction study. The cytotoxicity of all three compounds was examined by MTT assay against two cancer cell lines – HT‐29 (colon adenocarcinoma) and MCF‐7 (human breast adenocarcinoma) ‐ and normal human fibroblast cells (GM5657T). Compound 1 has moderate cytotoxicity against both cell lines, while compound 2 is seven to nine times more cytotoxic than cisplatin against MCF‐7 and HT‐29, respectively. In contrast to cisplatin, both compounds are more active against cancer cells than fibroblasts, thus showing some cancer selectivity. [ABSTRACT FROM AUTHOR]

A series of new conjugates comprised from a small synthetic antimicrobial peptide (AMP) and a siderophore‐type vector component was designed and tested for activity on P. aeruginosa PAO1 and several genetically modified strains. As AMP, the well‐established arginine‐tryptophane combination K(RW)3 (P1) was chosen with an added lysine for siderophore attachment. This peptide is easy to prepare, modify, and possesses good anti‐bacterial activity. On the vector part, we examined several moieties: (i) the natural siderophore deferoxamine (DFO); (ii) bidentate iron chelators based on the hydroxamate building block (4 a–c) ; (iii) the non‐siderophore chelators deferasirox (DFX) and deferiprone‐carboxylate (DFP‐COOH). All conjugates were prepared by solid phase synthesis techniques and fully characterized by HPLC and mass spectrometry (including HR‐MS). 55Fe uptake assays indicate a receptor‐mediated uptake for 4 a–c, DFP‐COOH and DFO, which is dependent on the outer membrane transporter FoxA in the case of DFO. All conjugates showed increased antibacterial activity against P. aeruginosa compared to the parent peptide P1 alone when investigated in iron‐depleted medium. MIC values were as low as 2 μM (for P1‐DFP) on wild type P. aeruginosa. The activity of P1‐DFO and P1‐DFP was even better on genetically mutated strains unable to produce siderophores (down to 0.5 μM). Although the DFX vector on its own was not able to transport iron inside the bacterial cell as shown by 55Fe uptake studies, the P1‐DFX conjugate had excellent antibacterial activity compared to P1 (2 μM, and as low as 0.25 μM on a receptor‐deficient strain unable to produce siderophores), suggesting that the conjugates were indeed recognized and internalized by an (unknown) transporter. Control experiments with an equimolar mixture of P1 and DFX confirm that the observed activity is intrinsic to vectorization. This work thus demonstrates the power of linking small AMPs covalently to siderophores for a new class of Trojan Horse antibiotics, with P1‐DFP and P1‐DFX being the most potent conjugates. [ABSTRACT FROM AUTHOR]

The synthesis and characterization of four compounds [M(η5‐C5Me5)(N^N)Cl]PF6 [N^N=4,7‐dichloro‐1,10‐phenanthroline with M=Rh, 1, and M=Ir, 2, and N^N=4'‐(4‐chlorophenyl)‐2,2':6',2''‐terpyridine in the κ2N,N'‐coordination mode with M=Rh, 3, and M=Ir, 4] are described. All compounds were characterized by spectroscopic means and their molecular structures in the crystal were confirmed by single‐crystal X‐ray diffraction studies. The cytotoxicity of all compounds was evaluated by MTT assay against the three cancer cell lines HeLa (cervical carcinoma), HT‐29 (colon adenocarcinoma) and MCF‐7 (human breast adenocarcinoma). The complexes 3 and 4 display promising activity with IC50 values of 1 μM. The rhodium(III) complex 1 also shows highly improved cytotoxicity compared to cisplatin against the cancer cell lines HT‐29 and MCF‐7. In contrast to this, the iridium(III) complex 2 is even less active against the HeLa cell line than cisplatin. [ABSTRACT FROM AUTHOR]

Due to rising resistance, new antibacterial strategies are needed, including methods for targeted antibiotic release. As targeting vectors, chelating molecules called siderophores that are released by bacteria to acquire iron have been investigated for conjugation to antibacterials, leading to the clinically approved drug cefiderocol. The use of small‐molecule catalysts for prodrug activation within cells has shown promise in recent years, and here we investigate siderophore‐linked ruthenium catalysts for the activation of antibacterial prodrugs within cells. Moxifloxacin‐based prodrugs were synthesised, and their catalyst‐mediated activation was demonstrated under anaerobic, biologically relevant conditions. In the absence of catalyst, decreased antibacterial activities were observed compared to moxifloxacin versus Escherichia coli K12 (BW25113). A series of siderophore‐linked ruthenium catalysts were investigated for prodrug activation, all of which displayed a combinative antibacterial effect with the prodrug, whereas a representative example displayed little toxicity against mammalian cell lines. By employing complementary bacterial growth assays, conjugates containing siderophore units based on catechol and azotochelin were found to be most promising for intracellular prodrug activation. [ABSTRACT FROM AUTHOR]

The synthesis and characterization of two new bis‐cyclometalated compounds [Ir(ptpy)2(κ2N‐terpy‐C6H4Cl‐p)]PF6 [terpy‐C6H4Cl‐p=4'‐(4‐chlorophenyl)‐2,2':6',2"‐terpyridine, (1)], and [Ir(ptpy)2(κ2N‐terpy‐Cl)]PF6 [terpy‐Cl=4'‐chloro‐2,2':6',2''‐terpyridine, (2); ptpy=2‐(p‐tolyl)pyridinato)] are described. The molecular structures of compounds 1 and 2 in the crystal were determined by single‐crystal X‐ray diffraction. 1 crystallized from dichloromethane/methanol/iso‐hexane in the monoclinic space group P2/n and 2 from the same mixture of solvents in the triclinic space group P−1. Photophysical investigations on 1 and 2 revealed broad unstructured luminescence in the red spectral region with the emission maxima in dichloromethane at 620 and 630 nm respectively. To explore cytotoxic properties of compounds 1 and 2, a colorimetric assay (MTT assay) against prominent cancer cell lines, MCF‐7 and HT‐29, was performed. The determined IC50 values are in the low micromolar range (2–3 μM). In comparison to cisplatin, the tested complexes 1 and 2 exhibit up to >20‐fold (MCF‐7) and >40‐fold (HT‐29) increase in biological activity. [ABSTRACT FROM AUTHOR]

Bioorthogonal reactions are extremely useful for the chemical modification of biomolecules, and are already well studied in mammalian cells. In contrast, very little attention has been given to the feasibility of such reactions in bacteria. Herein we report modified coumarin dyes for monitoring the internalisation and activity of bioorthogonal catalysts in the Gram-positive bacterial species Bacillus subtilis . Two fluorophores based on 7-aminocoumarin were synthesised and characterised to establish their luminescence properties. The introduction of an allyl carbamate (R 2 N-COOR') group onto the nitrogen atom of two 7-aminocoumarin derivatives with different solubility led to decreased fluorescence emission intensities and remarkable blue-shifts of the emission maxima. Importantly, this allyl carbamate group could be uncaged by the bioorthogonal, organometallic ruthenium catalyst investigated in this work, to yield the fluorescent product under biologically-relevant conditions. The internalisation of this catalyst was confirmed and quantified by ICP-OES analysis. Investigation of the bacterial cytoplasm and extracellular fractions separately, following incubation of the bacteria with the two caged dyes, facilitated their localisation, as well as that of their uncaged form by catalyst addition. In fact, significant differences were observed, as only the more lipophilic dye was located inside the cells and importantly remained there, seemingly avoiding efflux mechanisms. However, the uncaged form of this dye is not retained, and was found predominantly in the extracellular space. Finally, a range of siderophore-conjugated derivatives of the catalyst were investigated for the same transformations. Even though uptake was observed, albeit less significant than for the non-conjugated version, the fact that similar intracellular reaction rates were observed regardless of the iron content of the medium supports the notion that their uptake is independent of the iron transporters utilised by Gram-positive Bacillus subtilis cells., Competing Interests: The authors declare no conflict of interest., (This journal is © The Royal Society of Chemistry.)

Herein, we describe the synthesis and characterisation of four new supramolecular cobalt conjugates of antimicrobial peptides functionalised with terpyridine ligands (L). Peptides were chosen based on the well-established arginine-tryptophan (RW) 3 motif, with terpyridine-derivatized lysine (Lys(tpy)) added to the sequence, or replacing tryptophan residues. Self-assembly of the antimicrobial peptides with Co(BF 4 ) 2 ·6H 2 O formed exclusively CoL 2 dimers (for peptides with one tpy ligand each) and Co 2 L 4 metallo-macrocycles (for peptides with two tpy ligands for each peptide), which could be 'locked' by oxidation of Co(+II) to Co(+III) with ammonium ceric nitrate. The Co-peptide complexes were characterised by mass spectrometry and in solution by NMR spectroscopy, including 2D diffusion ordered NMR spectroscopy (DOSY) which confirmed the proposed stoichiometries. The antimicrobial activity of the novel peptides and their metallo-supramolecular assemblies was investigated by determination of their minimal inhibitory concentration (MIC) against a panel of Gram-positive and Gram-negative bacteria. Complexation with cobalt increases the activity of the peptides in almost every case. Most of the new metal-peptide conjugates showed good activity against Gram-positive bacteria, including a multi-resistant S. aureus strain and the opportunistic pathogenic yeast C. albicans (down to 7 μmol l -1 for the most active Co 2 L 4 derivate), a value that is increased five-fold compared to the lysine-derivatized peptide ligand alone. Interestingly, conjugates of the CoL 2 type also showed decent activity against Gram-negative bacteria including the WHO-flagged problematic A. baumannii strain (down to 18 μmol l -1 for the most active derivative).

A series of new conjugates comprised from a small synthetic antimicrobial peptide (AMP) and a siderophore-type vector component was designed and tested for activity on P. aeruginosa PAO1 and several genetically modified strains. As AMP, the well-established arginine-tryptophane combination K(RW) 3 (P1) was chosen with an added lysine for siderophore attachment. This peptide is easy to prepare, modify, and possesses good anti-bacterial activity. On the vector part, we examined several moieties: (i) the natural siderophore deferoxamine (DFO); (ii) bidentate iron chelators based on the hydroxamate building block (4 a-c) ; (iii) the non-siderophore chelators deferasirox (DFX) and deferiprone-carboxylate (DFP-COOH). All conjugates were prepared by solid phase synthesis techniques and fully characterized by HPLC and mass spectrometry (including HR-MS). 55 Fe uptake assays indicate a receptor-mediated uptake for 4 a-c, DFP-COOH and DFO, which is dependent on the outer membrane transporter FoxA in the case of DFO. All conjugates showed increased antibacterial activity against P. aeruginosa compared to the parent peptide P1 alone when investigated in iron-depleted medium. MIC values were as low as 2 μM (for P1-DFP) on wild type P. aeruginosa. The activity of P1-DFO and P1-DFP was even better on genetically mutated strains unable to produce siderophores (down to 0.5 μM). Although the DFX vector on its own was not able to transport iron inside the bacterial cell as shown by 55 Fe uptake studies, the P1-DFX conjugate had excellent antibacterial activity compared to P1 (2 μM, and as low as 0.25 μM on a receptor-deficient strain unable to produce siderophores), suggesting that the conjugates were indeed recognized and internalized by an (unknown) transporter. Control experiments with an equimolar mixture of P1 and DFX confirm that the observed activity is intrinsic to vectorization. This work thus demonstrates the power of linking small AMPs covalently to siderophores for a new class of Trojan Horse antibiotics, with P1-DFP and P1-DFX being the most potent conjugates., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Due to rising resistance, new antibacterial strategies are needed, including methods for targeted antibiotic release. As targeting vectors, chelating molecules called siderophores that are released by bacteria to acquire iron have been investigated for conjugation to antibacterials, leading to the clinically approved drug cefiderocol. The use of small-molecule catalysts for prodrug activation within cells has shown promise in recent years, and here we investigate siderophore-linked ruthenium catalysts for the activation of antibacterial prodrugs within cells. Moxifloxacin-based prodrugs were synthesised, and their catalyst-mediated activation was demonstrated under anaerobic, biologically relevant conditions. In the absence of catalyst, decreased antibacterial activities were observed compared to moxifloxacin versus Escherichia coli K12 (BW25113). A series of siderophore-linked ruthenium catalysts were investigated for prodrug activation, all of which displayed a combinative antibacterial effect with the prodrug, whereas a representative example displayed little toxicity against mammalian cell lines. By employing complementary bacterial growth assays, conjugates containing siderophore units based on catechol and azotochelin were found to be most promising for intracellular prodrug activation., (© 2022 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

The number of donor atoms available on peptides that can competitively coordinate to metal centers renders the site-selective generation of advanced metal-peptide conjugates in high purity a challenging venture. Herein, we present a transmetalation-based synthetic approach on solid support in which an imidazolium pro-ligand can be used to selectively anchor a range of transition metal half-sandwich complexes onto peptides in the presence of multiple coordinative motifs. Amenable to solid support, a range of N-terminus and/or lysine conjugated metal-peptide conjugates were obtained in high purity after cleavage from the resin. The metalated peptides were evaluated for their anticancer properties against human cancer cell lines. While no cytotoxic activity was observed, this platform has the potential to i) provide a pathway to site-selective peptide labelling, ii) be explored as a biorthogonal handle and/or iii) generate a new strategy for ligand design in transition metal catalysts., (© 2021 Wiley-VCH GmbH.)