Amide Moieties Modulate the Antimicrobial Activities of Conjugated Oligoelectrolytes against Gram-negative Bacteria

Cationic conjugated oligoelectrolytes (COEs) are a class of compounds that can be tailored to achieve relevant in vitro antimicrobial properties with relatively low cytotoxicity against mammalian cells. Three distyrylbenzene-based COEs were designed containing amide functional groups on the side chains. Their properties were compared to two representative COEs with only quaternary ammonium groups. The optimal compound, COE2−3C−C3-Apropyl , has an antimicrobial efficacy against Escherichia coli with an MIC=2 μg mL⁻¹, even in the presence of human serum albumin low cytotoxicity (IC₅₀=740 μg mL⁻¹) and minimal hemolytic activity. Moreover, we find that amide groups increase interactions between COEs and a bacterial lipid mimic based on calcein leakage assay and allow COEs to readily permeabilize the cytoplasmic membrane of E. coli. These findings suggest that hydrogen bond forming moieties can be further applied in the molecular design of antimicrobial COEs to further improve their selectivity towards bacteria.     

Informed Molecular Design of Conjugated Oligoelectrolytes To Increase Cell Affinity and Antimicrobial Activity

Membrane‐intercalating conjugated oligoelectrolytes (COEs) are emerging as potential alternatives to conventional, yet increasingly ineffective, antibiotics. Three readily accessible COEs, belonging to an unreported series containing a stilbene core, namely D4 , D6 , and D8 , were designed and synthesized so that the hydrophobicity increases with increasing side‐chain length. Decreased aqueous solubility correlates with increased uptake by E. coli. The minimum inhibitory concentration (MIC) of D8 is 4 μg mL^?1 against both E. coli and E. faecalis, with an effective uptake of 72 %. In contrast, the MIC value of the shortest COE, D4 , is 128 μg mL^?1 owing to the low cellular uptake of 3 %. These findings demonstrate the application of rational design to generate efficacious antimicrobial COEs that have potential as low‐cost antimicrobial agents.     

Synthetic studies of cystobactamids as antibiotics and bacterial imaging carriers lead to compounds with high in vivo efficacy

There is an alarming scarcity of novel chemical matter with bioactivity against multidrug-resistant Gram-negative bacterial pathogens. Cystobactamids, recently discovered natural products from myxobacteria, are an exception to this trend. Their unusual chemical structure, composed of oligomeric para-aminobenzoic acid moieties, is associated with a high antibiotic activity through the inhibition of gyrase. In this study, structural determinants of cystobactamid''s antibacterial potency were defined at five positions, which were varied using three different synthetic routes to the cystobactamid scaffold. The potency against Acinetobacter baumannii could be increased ten-fold to an MIC (minimum inhibitory concentration) of 0.06 μg mL⁻¹, and the previously identified spectrum gap of Klebsiella pneumoniae could be closed compared to the natural products (MIC of 0.5 μg mL⁻¹). Proteolytic degradation of cystobactamids by the resistance factor AlbD was prevented by an amide-triazole replacement. Conjugation of cystobactamid''s N-terminal tetrapeptide to a Bodipy moiety induced the selective localization of the fluorophore for bacterial imaging purposes. Finally, a first in vivo proof of concept was obtained in an E. coli infection mouse model, where derivative 22 led to the reduction of bacterial loads (cfu, colony-forming units) in muscle, lung and kidneys by five orders of magnitude compared to vehicle-treated mice. These findings qualify cystobactamids as highly promising lead structures against infections caused by Gram-positive and Gram-negative bacterial pathogens.

Structure–activity relationship studies of the natural product cystobactamid at four different positions led to novel imaging probes and analogs with superior antibacterial activities and in vivo efficacy.     

Metabolic Profiling of Inga Species with Antitumor Activity

This work evaluated the metabolic profiling of Inga species with antitumor potential. In addition, we described the antigenotoxicity of polyphenols isolated from I. laurina and a proteomic approach using HepG2 cells after treatment with these metabolites. The in vitro cytotoxic activity against HepG2, HT-29 and T98G cancer cell lines was investigated. The assessment of genotoxic damage was carried out through the comet assay. The ethanolic extract from I. laurina seeds was subjected to bioassay-guided fractionation and the most active fractions were characterized. One bioactive fraction with high cytotoxicity against HT-29 human colon cancer cells (IC₅₀ = 4.0 µg mL⁻¹) was found, and it was characterized as a mixture of p-hydroxybenzoic acid and 4-vinyl-phenol. The I. edulis fruit peel (IC₅₀ = 18.6 µg mL⁻¹) and I. laurina seed (IC₅₀ = 15.2 µg mL⁻¹) extracts had cytotoxic activity against the cell line T98G, and its chemical composition showed a variety of phenolic acids. The chemical composition of this species indicated a wide variety of aromatic acids, flavonoids, tannins, and carotenoids. The high concentration (ranging from 5% to 30%) of these polyphenols in the bioactive extract may be responsible for the antitumor potential. Regarding the proteomic approach, we detected proteins directly related to the elimination of ROS, DNA repair, expression of tumor proteins, and apoptosis.     

Guanidinylated Amphiphilic Polycarbonates with Enhanced Antimicrobial Activity by Extending the Length of the Spacer Arm and Micelle Self‐Assembly

Nine guanidinylated amphiphilic polycarbonates are rationally designed and synthesized. Each polymer has the same biodegradable backbone but different side groups. The influence of the hydrophobic/hydrophilic effect on antimicrobial activities and cytotoxicity is systematically investigated. The results verify that tuning the length of the spacer arm between the cationic guanidine group and the polycarbonate backbone is an efficient design strategy to alter the hydrophobic/hydrophilic balance without changing the cationic charge density. A spacer arm of six methylene units (CH₂)₆ shows the best antimicrobial activity (minimum inhibitory concentration, MIC = 40 µg mL^?1 against Escherichia coli, MIC = 20 µg mL^?1 against Staphylococcus aureus, MIC = 40 µg mL^?1 against Candida albicans) with low hemolytic activity (HC₅₀ > 2560 µg mL^?1). Furthermore, the guanidinylated polycarbonates exhibit the ability to self‐assemble and present micelle‐like nanostructure due to their intrinsic amphiphilic macromolecular structure. Transmission electron microscopy and dynamic light scattering measurements confirm polymer micelle formation in aqueous solution with sizes ranging from 82 to 288 nm.     

Triazole-based osmium(ii) complexes displaying red/near-IR luminescence: antimicrobial activity and super-resolution imaging

Cellular uptake, luminescence imaging and antimicrobial activity against clinically relevant methicillin-resistant S. aureus (MRSA) bacteria are reported. The osmium(ii) complexes [Os(N^N)₃]²⁺ (N^N = 1-benzyl-4-(pyrid-2-yl)-1,2,3-triazole (1²⁺); 1-benzyl-4-(pyrimidin-2-yl)-1,2,3-triazole (2²⁺); 1-benzyl-4-(pyrazin-2-yl)-1,2,3-triazole (3²⁺)) were prepared and isolated as the chloride salts of their meridional and facial isomers. The complexes display prominent spin-forbidden ground state to triplet metal-to-ligand charge transfer (³MLCT) state absorption bands enabling excitation as low as 600 nm for fac/mer-3²⁺ and observation of emission in aqueous solution in the deep-red/near-IR regions of the spectrum. Cellular uptake studies within MRSA cells show antimicrobial activity for 1²⁺ and 2²⁺ with greater toxicity for the meridional isomers in each case and mer-1²⁺ showing the greatest potency (32 μg mL⁻¹ in defined minimal media). Super-resolution imaging experiments demonstrate binding of mer- and fac-1²⁺ to bacterial DNA with high Pearson''s colocalisation coefficients (up to 0.95 using DAPI). Phototoxicity studies showed the complexes exhibited a higher antimicrobial activity upon irradiation with light.

Cellular uptake, luminescence imaging and antimicrobial activity of facial and meridional isomers of Os(ii) triazole-based complexes against methicillin-resistant S. aureus, MRSA.     

Diverse ring-opening reactions of rhodium η4-azaborete complexes

Sequential treatment of [Rh(COE)₂Cl]₂ (COE = cyclooctene) with PⁱPr₃, alkyne derivatives and ^tBuN BMes (Mes = 2,4,6-trimethylphenyl) provided functionalized rhodium η⁴-1,2-azaborete complexes of the form (η⁴-azaborete)RhCl(PⁱPr₃). The scope of this reaction was expanded to encompass alkynes with hydrogen, alkyl, aryl, ferrocenyl, alkynyl, azaborinyl and boronate ester substituents. Treatment of these complexes with PMe₃ led to insertion of the rhodium atom into the B–C bond of the BNC₂ ring, forming 1-rhoda-3,2-azaboroles. Addition of N-heterocyclic carbenes to azaborete complexes led to highly unusual rearrangements to rhodium η²,κ¹-allenylborylamino complexes via deprotonation and C–N bond cleavage. Heating and photolysis of an azaborete complex also led to rupture of the C–N bond followed by subsequent rearrangements, yielding an η⁴-aminoborylallene complex and two isomeric η⁴-butadiene complexes.

Rhodium η⁴-azaborete complexes can be transformed into a variety of species with ring-opened, BN-containing ligands by treatment with Lewis bases.     

Selective Cytotoxicity of Portuguese Propolis Ethyl Acetate Fraction towards Renal Cancer Cells

Renal cell carcinoma is the most lethal cancer of the urological system due to late diagnosis and treatment resistance. Propolis, a beehive product, is a valuable natural source of compounds with bioactivities and may be a beneficial addition to current anticancer treatments. A Portuguese propolis sample, its fractions (n-hexane, ethyl acetate, n-butanol and water) and three subfractions (P1–P3), were tested for their toxicity on A498, 786-O and Caki-2 renal cell carcinoma cell lines and the non-neoplastic HK2 kidney cells. The ethyl acetate fraction showed the strongest toxicity against A498 (IC₅₀ = 0.162 µg mL⁻¹) and 786-O (IC₅₀ = 0.271 µg mL⁻¹) cells. With similar toxicity against 786-O, P1 (IC₅₀ = 3.8 µg mL⁻¹) and P3 (IC₅₀ = 3.1 µg mL⁻¹) exhibited greater effect when combined (IC₅₀ = 2.5 µg mL⁻¹). Results support the potential of propolis and its constituents as promising coadjuvants in renal cell carcinoma treatment.     

A combination therapy strategy for treating antibiotic resistant biofilm infection using a guanidinium derivative and nanoparticulate Ag(0) derived hybrid gel conjugate

Bacteria organized in biofilms show significant tolerance to conventional antibiotics compared to their planktonic counterparts and form the basis for chronic infections. Biofilms are composites of different types of extracellular polymeric substances that help in resisting several host-defense measures, including phagocytosis. These are increasingly being recognized as a passive virulence factor that enables many infectious diseases to proliferate and an essential contributing facet to anti-microbial resistance. Thus, inhibition and dispersion of biofilms are linked to addressing the issues associated with therapeutic challenges imposed by biofilms. This report is to address this complex issue using a self-assembled guanidinium–Ag(0) nanoparticle (AD-L@Ag(0)) hybrid gel composite for executing a combination therapy strategy for six difficult to treat biofilm-forming and multidrug-resistant bacteria. Improved efficacy was achieved primarily through effective biofilm inhibition and dispersion by the cationic guanidinium ion derivative, while Ag(0) contributes to the subsequent bactericidal activity on planktonic bacteria. Minimum Inhibitory Concentration (MIC) of the AD-L@Ag(0) formulation was tested against Acinetobacter baumannii (25 μg mL⁻¹), Pseudomonas aeruginosa (0.78 μg mL⁻¹), Staphylococcus aureus (0.19 μg mL⁻¹), Klebsiella pneumoniae (0.78 μg mL⁻¹), Escherichia coli (clinical isolate (6.25 μg mL⁻¹)), Klebsiella pneumoniae (clinical isolate (50 μg mL⁻¹)), Shigella flexneri (clinical isolate (0.39 μg mL⁻¹)) and Streptococcus pneumoniae (6.25 μg mL⁻¹). Minimum bactericidal concentration, and MBIC₅₀ and MBIC₉₀ (Minimum Biofilm Inhibitory Concentration at 50% and 90% reduction, respectively) were evaluated for these pathogens. All these results confirmed the efficacy of the formulation AD-L@Ag(0). Minimum Biofilm Eradication Concentration (MBEC) for the respective pathogens was examined by following the exopolysaccharide quantification method to establish its potency in inhibition of biofilm formation, as well as eradication of mature biofilms. These effects were attributed to the bactericidal effect of AD-L@Ag(0) on biofilm mass-associated bacteria. The observed efficacy of this non-cytotoxic therapeutic combination (AD-L@Ag(0)) was found to be better than that reported in the existing literature for treating extremely drug-resistant bacterial strains, as well as for reducing the bacterial infection load at a surgical site in a small animal BALB/c model. Thus, AD-L@Ag(0) could be a promising candidate for anti-microbial coatings on surgical instruments, wound dressing, tissue engineering, and medical implants.

Dispersion of biofilms that protect bacteria and its subsequent killing in the planktonic state are effectively achieved by a guanidinium–Ag(0) nanocomposite.     

Improved broad-spectrum antibiotics against Gram-negative pathogens via darobactin biosynthetic pathway engineering

The development of new antibiotics is imperative to fight increasing mortality rates connected to infections caused by multidrug-resistant (MDR) bacteria. In this context, Gram-negative pathogens listed in the WHO priority list are particularly problematic. Darobactin is a ribosomally produced and post-translationally modified bicyclic heptapeptide antibiotic selectively killing Gram-negative bacteria by targeting the outer membrane protein BamA. The native darobactin A producer Photorhabdus khanii HGB1456 shows very limited production under laboratory cultivation conditions. Herein, we present the design and heterologous expression of a synthetically engineered darobactin biosynthetic gene cluster (BGC) in Escherichia coli to reach an average darobactin A production titre of 13.4 mg L⁻¹. Rational design of darA variants, encoding the darobactin precursor peptide with altered core sequences, resulted in the production of 13 new ‘non-natural’ darobactin derivatives and 4 previously hypothetical natural darobactins. One of the non-natural compounds, darobactin 9, was more potent than darobactin A, and showed significantly improved activity especially against Pseudomonas aeruginosa (0.125 μg mL⁻¹) and Acinetobacter baumannii (1–2 μg mL⁻¹). Importantly, it also displayed superior activity against MDR clinical isolates of E. coli (1–2 μg mL⁻¹) and Klebsiella pneumoniae (1–4 μg mL⁻¹). Independent deletions of genes from the darobactin BGC showed that only darA and darE, encoding a radical forming S-adenosyl-l-methionine-dependent enzyme, are required for darobactin formation. Co-expression of two additional genes associated with the BGCs in hypothetical producer strains identified a proteolytic detoxification mechanism as a potential self-resistance strategy in native producers. Taken together, we describe a versatile heterologous darobactin platform allowing the production of unprecedented active derivatives in good yields, and we provide first experimental evidence for darobactin biosynthesis processes.

Heterologous expression of a synthetically engineered darobactin gene cluster in E. coli yields new darobactin derivatives with improved anti-Gram-negative activity. Targeted gene deletions provide first insights into biosynthetic steps.     

Synthesis and mechanistic investigations of pH-responsive cationic poly(aminoester)s

The synthesis and degradation mechanisms of a class of pH-sensitive, rapidly degrading cationic poly(α-aminoester)s are described. These reactive, cationic polymers are stable at low pH in water, but undergo a fast and selective degradation at higher pH to liberate neutral diketopiperazines. Related materials incorporating oligo(α-amino ester)s have been shown to be effective gene delivery agents, as the charge-altering degradative behavior facilitates the delivery and release of mRNA and other nucleic acids in vitro and in vivo. Herein, we report detailed studies of the structural and environmental factors that lead to these rapid and selective degradation processes in aqueous buffers. At neutral pH, poly(α-aminoester)s derived from N-hydroxyethylglycine degrade selectively by a mechanism involving sequential 1,5- and 1,6-O→N acyl shifts to generate bis(N-hydroxyethyl) diketopiperazine. A family of structurally related cationic poly(aminoester)s was generated to study the structural influences on the degradation mechanism, product distribution, and pH dependence of the rate of degradation. The kinetics and mechanism of the pH-induced degradations were investigated by ¹H NMR, model reactions, and kinetic simulations. These results indicate that polyesters bearing α-ammonium groups and appropriately positioned N-hydroxyethyl substituents are readily cleaved (by intramolecular attack) or hydrolyzed, representing dynamic “dual function” materials that are initially polycationic and transform with changing environment to neutral products.

The synthesis and degradation mechanisms of a class of pH-sensitive, rapidly degrading cationic poly(α-aminoester)s are described.     

The Synthesis,Fungicidal Activity,and in Silico Study of Alkoxy Analogues of Natural Precocenes I,II, and III

This study aimed to synthesize, characterize, and explore the eco-friendly and antifungal potential of precocenes and their derivatives. The organic synthesis of the mono-O-alkyl-2,2-dimethyl 2H-1-chromene series, including the natural product precocene I, and the di-O-alkyl 2,2-dimethyl-2H-1-chromene series, including the natural 2H-1-chromenes precocenes II and III, was achieved. The synthetic compounds were subjected to spectroscopic analysis, ¹HNMR,¹³CNMR, and mass characterization. The antifungal activity of synthesized precocenes I, II, and III, as well as their synthetic intermediates, was evaluated by the poison food technique. Precocene II (EC₅₀ 106.8 µg × mL⁻¹ and 4.94 µg mL⁻¹), and its regioisomers 7a (EC₅₀ 97.18 µg × mL⁻¹ and 35.30 µg × mL⁻¹) and 7d (EC₅₀ 170.58 × µg mL⁻¹), exhibited strong fungitoxic activity against Aspergillus niger and Rhizoctonia solani. Some of the novel chromenes, 11a and 11b, which had never been evaluated before, yielded stronger fungitoxic effects. Finally, docking simulations for compounds with promising fungitoxic activity were subjected to structure–activity relationship analyses against the polygalactouronases and voltage-dependent anion channels. Conclusively, precocenes and their regioisomers demonstrated promising fungitoxic activity; such compounds can be subjected to minor structural modifications to yield promising and novel fungicides.     

Chitosan-coated silver nanoparticles promoted antibacterial,antibiofilm, wound-healing of murine macrophages and antiproliferation of human breast cancer MCF 7 cells

In the present study, environmentally benign silver nanoparticles were synthesized using commercially purchased shrimp-shell chitosan as a capping agent. The synthesized chitosan-silver nanoparticles (Ch-AgNPs) were physico-chemically characterized by UV–Vis spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), high-resolution transmission electron microscopy (HR-TEM) along with energy dispersive X-ray analysis (EDX), DLS and Zeta potential analysis. Ch-Ag NPs were crystalline, uniformly dispersed, and spherically shaped, with particle size between 8 and 48 nm. The average size of Ch-AgNPs was 21 nm. In-vitro anti-biofilm activity of Ch-AgNPs was tested against wound infection-causing pathogenic bacteria such as Staphylococcus aureus (Gram-positive) and Pseudomonas aeruginosa (Gram-negative). Ch-AgNPs displayed anti-biofilm activity in a dose-dependent manner. Light and confocal-laser scanning microscopy confirmed the significant inhibition of biofilm growth of S. aureus (85%) and P. aeruginosa (95%) at 100 μg mL⁻¹ of Ch-AgNPs. Moreover, Ch-AgNPs promoted wound healing by increasing the migration of RAW 264.7 murine macrophages cells at 75 and 100 μg mL⁻¹after 24 h. In addition, in vitro cytotoxicity of Ch-AgNPs against MCF 7 (human breast cancer) cells, depicted the greater inhibition of proliferation of cells (64%) at 100 μg mL^−1.     

Efficient alkene hydrosilation with bis(8-quinolyl)phosphine (NPN) nickel catalysts. The dominant role of silyl-over hydrido-nickel catalytic intermediates

A cationic nickel complex of the bis(8-quinolyl)(3,5-di-tert-butylphenoxy)phosphine (NPN) ligand, [(NPN)NiCl]⁺, is a precursor to efficient catalysts for the hydrosilation of alkenes with a variety of hydrosilanes under mild conditions and low catalyst loadings. DFT studies reveal the presence of two coupled catalytic cycles based on [(NPN)NiH]⁺ and [(NPN)NiSiR₃]⁺ active species, with the latter being more efficient for producing the product. The preferred silyl-based catalysis is not due to a more facile insertion of alkene into the Ni–Si (vs. Ni–H) bond, but by consistent and efficient conversions of the hydride to the silyl complex.

A cationic nickel complex of the bis(8-quinolyl)(3,5-di-tert-butylphenoxy)phosphine (NPN) ligand, [(NPN)NiCl]⁺, is a precursor to efficient catalysts for the hydrosilation of alkenes with hydrosilanes under mild conditions and low catalyst loadings.     

Near-infrared absorbing Ru(ii) complexes act as immunoprotective photodynamic therapy (PDT) agents against aggressive melanoma

Mounting evidence over the past 20 years suggests that photodynamic therapy (PDT), an anticancer modality known mostly as a local treatment, has the capacity to invoke a systemic antitumor immune response, leading to protection against tumor recurrence. For aggressive cancers such as melanoma, where chemotherapy and radiotherapy are ineffective, immunomodulating PDT as an adjuvant to surgery is of interest. Towards the development of specialized photosensitizers (PSs) for treating pigmented melanomas, nine new near-infrared (NIR) absorbing PSs based on a Ru(ii) tris-heteroleptic scaffold [Ru(NNN)(NN)(L)]Cl_n, were explored. Compounds 2, 6, and 9 exhibited high potency toward melanoma cells, with visible EC₅₀ values as low as 0.292–0.602 μM and PIs as high as 156–360. Single-micromolar phototoxicity was obtained with NIR-light (733 nm) with PIs up to 71. The common feature of these lead NIR PSs was an accessible low-energy triplet intraligand (³IL) excited state for high singlet oxygen (¹O₂) quantum yields (69–93%), which was only possible when the photosensitizing ³IL states were lower in energy than the lowest triplet metal-to-ligand charge transfer (³MLCT) excited states that typically govern Ru(ii) polypyridyl photophysics. PDT treatment with 2 elicited a pro-inflammatory response alongside immunogenic cell death in mouse B16F10 melanoma cells and proved safe for in vivo administration (maximum tolerated dose = 50 mg kg⁻¹). Female and male mice vaccinated with B16F10 cells that were PDT-treated with 2 and challenged with live B16F10 cells exhibited 80 and 55% protection from tumor growth, respectively, leading to significantly improved survival and excellent hazard ratios of ≤0.2.

Ru(ii) photosensitizers (PSs) destroy aggressive melanoma cells, triggering an immune response that leads to protection against tumor challenge and mouse survival.     

ESI-MS/MS Analysis of Phenolic Compounds from Aeonium arboreum Leaf Extracts and Evaluation of their Antioxidant and Antimicrobial Activities

Aeonium is a genus of succulents belonging to the Crassulaceae family. Their importance in traditional medicine has stimulated both pharmacological and chemical research. In this study, we optimized extraction, separation, and analytical conditions using a high performance liquid chromatographic method coupled with electrospray ionization mass spectrometry by the negative mode (HPLC-ESI-MS) in order to, for the first time, determine thirty-four compounds from Aeonium arboreum leaves. Twenty-one of them are assigned among which are sixteen flavonoids and five phenolic acids. FRAP, TAC, DPPH, and ABTS^•+ radical scavenging were used to evaluate antioxidant activity. The obtained IC₅₀ values ranged from 0.031 to 0.043 mg.mL⁻¹ for DPPH and between 0.048 and 0.09 mg·mL⁻¹ for ABTS^•+. Antimicrobial activity was also assessed. The obtained minimum inhibitory concentrations (MIC) of these extracts ranged from 12.5 to 50 µg·mL⁻¹ against Micrococcus luteus, Listeria ivanovii, Staphylococcus aureus, Salmonella enterica, Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger, and Fusarium oxysporum, and from 25 to 50 µg·mL⁻¹ against Candida albicans. Therefore, these extracts can be considered as a potential source of biological active compounds.     

An engineered biosynthetic–synthetic platform for production of halogenated indolmycin antibiotics

Indolmycin is an antibiotic from Streptomyces griseus ATCC 12648 with activity against Helicobacter pylori, Plasmodium falciparum, and methicillin-resistant Staphylococcus aureus. Here we describe the use of the indolmycin biosynthetic genes in E. coli to make indolmycenic acid, a chiral intermediate in indolmycin biosynthesis, which can then be converted to indolmycin through a three-step synthesis. To expand indolmycin structural diversity, we introduce a promiscuous tryptophanyl-tRNA synthetase gene (trpS) into our E. coli production system and feed halogenated indoles to generate the corresponding indolmycenic acids, ultimately allowing us to access indolmycin derivatives through synthesis. Bioactivity testing against methicillin-resistant Staphylococcus aureus showed modest antibiotic activity for 5-, 6-, and 7-fluoro-indolmycin.

A semi-synthetic system for producing indolmycin, an antibiotic, was developed and used to make indole-substituted, halogenated derivatives of indolmycin, some with modest bioactivity against methicillin-resistant Staphylococcus aureus.

Antibiotic-resistant bacteria pose a great threat to human health,^1–4 and the rates of new antibiotic discoveries and clinical approvals have been in a steep decline since the 1980s.¹ Without the discovery and development of new antibiotics, drug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), will become increasingly prevalent.^2–4 One strategy to increase antibiotic development has been to “rediscover” known, but underdeveloped, antibiotics.¹ One such example is indolmycin, which was originally discovered in 1960 from Streptomyces griseus ATCC 12648⁵ but was not originally developed for clinical use because of its narrow spectrum of activity^6–10 and its interference with tryptophan catabolism in the liver.^9,10 However, reignited interest in this old antibiotic led to the discovery of its activity against Helicobacter pylori,⁶Plasmodium falciparum,¹¹ and MRSA.¹² For MRSA, indolmycin was found to be active against mupirocin- and fuscidic acid-resistant MRSA strains, with strains resistant to indolmycin emerging infrequently and with reduced fitness compared to sensitive strains.¹² In addition, indolmycin has been shown to have minimal activity against common members of the human microbiota, suggesting that its narrow spectrum of activity is an asset.⁶ The first indole-substituted derivatives, 5-hydroxy and 5-methoxyindolmycin, were made by precursor-directed feeding of the indolmycin producer, Streptomyces griseus ATCC 12648, and they showed modest improvements in bioactivity against S. aureus and Escherichia coli.¹³ Two practical synthetic routes to indolmycin and some indole-substituted derivatives have been reported more recently,^14,15 which enabled access to a small variety of indole-substituted derivatives. Additionally, a previous patent has described synthetic methods to produce a variety of derivatives; however, these methods do not appear to offer stereochemical control, and some require tailoring steps specific to each analog.¹⁶ Therefore, further development of indolmycin would benefit from a simpler diversification method that could be applied to produce a wider variety of analogs with stereochemical control.Inspired by early biosynthetic studies,^17,18 our group previously identified the indolmycin gene cluster and elucidated the biosynthetic pathway, demonstrating that indolmycin (1) is assembled from tryptophan, arginine and S-adenosylmethionine (SAM) in a three-part process (Fig. 1).¹⁹ In the first part, l-arginine is oxidized by Ind4 in an oxygen- and PLP-dependent reaction to 4,5-dehydro-2-iminoarginine, which is then enantioselectively reduced by imine reductase Ind5 and its chaperone Ind6 to 4,5-dehydro-d-arginine. In the second part, tryptophan (2) is deaminated by PLP-dependent transaminases, giving indole pyruvate (3). Compound 3 is then methylated by SAM-dependent C-methyltransferase Ind1 to 3-methyl-indolepyruvate (4) which is reduced by NADH-dependent ketone reductase Ind2 to form indolmycenic acid (5). Then, in the third part, 4,5-dehydro-d-arginine and 5 are coupled in an ATP-dependent fashion by Ind3 and Ind6, resulting in an oxazolinone-cyclized molecule, N-desmethyl-indolmycin, which is finally N-methylated by Ind7, a SAM-dependent N-methyltransferase, to form 1.Open in a separate windowFig. 1Indolmycin biosynthesis from Streptomyces griseus ATCC 12648. (a) Indolmycin biosynthetic gene cluster. (b) Indolmycin biosynthetic pathway from Streptomyces griseus ATCC 12648. (c) Semi-synthetic scheme towards indolmycin and derivatives using indolmycin biosynthetic genes. The dashed arrow indicates a predicted side-product based on LC-MS analysis.Armed with the elucidated biosynthetic pathway for 1, we set out to create an in vivo system to make 1 in E. coli. We first cloned all necessary genes into four plasmids and co-expressed the genes in E. coli (Fig. S1^†), a strain which we named E. coli I1234670P5 (Table S1^†). We found that the genes needed to produce indolmycin in E. coli were ind1, ind2, ind3, ind4, ind6, ind7, ind0 and pel5, a homologous gene of ind5 from Paenibacillus elgii B69 showing better production of active protein in E. coli.^20,21 We also relied on the activity of endogenous E. coli aminotransferases to catalyze the initial tryptophan deamination step. However, only a small amount of 1 was produced (∼170 μg L⁻¹ of bacterial culture) and the yield could not be improved despite our best efforts (Fig. 2a). However, we found that this construct produced substantial amounts of 5 ([M + H]⁺ = 220 m/z) at 40–50 mg L⁻¹ of culture, along with a shunt product, C-desmethyl-indolmycenic acid (6; [M + H]⁺ = 206 m/z).Open in a separate windowFig. 2Biosynthetic production of 5 and semi-synthetic production of 1. (a) Extracted ion chromatograms show production of 5 with minimal production of 1 from E. coli I1234670P5. (b) Synthetic scheme to 1 from 5, adapted from literature methods.^14,15 (c) Total ion chromatogram of compound 1 isolated after semi-synthesis and final purification by semi-preparative HPLC. Compounds are indicated with coloured boxes and numbered.Compound 5 itself has been a focus of total synthetic efforts toward 1, as it is the key chiral precursor.^{14,15,22–26} Since production of 5 was much higher than that of 1 from E. coli I1234670P5, we pursued a semi-synthetic method of obtaining 1 using our biosynthetic platform to access 5, combined with a three-step chemical transformation (Fig. 2b). We attempted to remove extraneous genes from our biosynthetic platform by only including ind0, ind1, and ind2, but the changes resulted in reduced amounts of 5 (Fig. S2^†). At this time, it is unclear which of the other genes may be contributing to the production of compound 5. Therefore, we employed the full eight-gene construct toward synthesis of 5. We then adapted the three-step synthesis to make indolmycin,^14,15 in which purified 5 was esterified to make the ethyl ester (7; [M + H]⁺ = 248 m/z; Fig. S3a^†), cyclized to give N-desmethyl-indolmycin (8; [M + H]⁺ = 244 m/z; Fig. S3b and S4a^†), and methylated at the exocyclic nitrogen to give 1 ([M + H]⁺ = 258 m/z; Fig. 2, S4b and Table S4^†).Then, we wanted to make derivatives of 1 from indole derivatives, which are more widely accessible than derivatives of 2. The tryptophan synthase (TrpS) from Salmonella enterica has been previously shown to couple a wide variety of indole derivatives to l-serine to generate derivatives of 2.²⁷ We were able to replace pel5 with trpS in our biosynthetic platform without a reduction in the amount of 5 produced (Fig. S1b^†), and we named the resulting strain E. coli I1234670TS. When we fed 5-fluoroindole to E. coli I1234670TS, we observed increased production of fluorinated metabolites, 5-fluoro-indolmycenic acid (5F-5; [M + H]⁺ = 238 m/z) and 5-fluoro-C-desmethyl-indolmycenic acid (5F-6; [M + H]⁺ = 224 m/z) (Fig. 3a). We then optimized the feeding conditions, finding that 5F-5 amounts were optimal when we fed E. coli I1234670TS with 0.5 mM 5-fluoroindole per day over two days (Fig. S5^†).Open in a separate windowFig. 3Addition of the trpS gene to the biosynthetic platform allows incorporation of substituted indoles into 5. (a) LC-MS analysis of strains with and without trpS (E. coli I1234670TS and E. coli I1234670P5, respectively) when fed 5-fluoroindole. Chemical structures of compounds with the corresponding extracted ion chromatogram are shown to the right of the traces. (b) Indole derivatives tested. Dark blue shows indoles that were incorporated into an analog of 5 (>48% of underivatized 5 by LC-MS analysis; Table S5^†) and further purified; medium blue indicates indoles that were incorporated into an analog of 5 at lower levels (6–22% of underivatized 5 by LC-MS analysis; Table S5^†) but were not further verified by purification; and light blue indicates indoles that did not show detectable incorporation into 5.To determine the scope of indole derivatives accepted by our biosynthetic platform, we fed a variety of indoles to E. coli I1234670TS and monitored the production of 5 and its derivatives by LC-MS. Out of the indoles tested, we found that fluorinated and chlorinated indoles substituted at the 5-, 6- and 7-positions were the best accepted by the biosynthetic platform (Fig. 3b, S6 and S7^†). We predict that lower acceptance of indoles substituted at the 4-position may be due to steric hindrance, as 4-fluoroindole was moderately accepted, while 4-chloroindole was not observed at all. Although we observed LC-MS peaks consistent with conversion of some of the azaindoles and hydroxyindoles into 5 derivatives, further work is required to confirm, optimize and scale up the purification of these compounds (Fig. S7^†). Cultures producing derivatives of 5, substituted at 5-, 6- and 7-positions, were further scaled up for purification of the 5-derivatives and downstream synthesis of 1-derivatives (Fig. S8–S10 and Table S4^†). Each purified derivative of 5 and 1 was characterized by HR-MS and NMR (Table S3 and ESI Methods^†). Overall, cultures fed with the fluorinated indoles produced a higher amount of 5-derivatives than the cultures fed with the chlorinated indoles. 1 and its derivatives were tested against MRSA (Fig. S11^†). While the fluorinated derivatives showed bioactivity, the chlorinated derivatives of 1 did not show bioactivity at the maximum amount tested in the disk diffusion assay (30 μg). We determined MIC₅₀ values for each fluorinated compound (Table 1). The MIC₅₀ values demonstrate that 1 is a more potent inhibitor of MRSA than its derivatives, while 6F-1 showed the most potent inhibition of MRSA compared to any of the derivatives, followed by 7F-1 and 5F-1. The lack of bioactivity of the chlorinated compounds may be due to the bulky chlorinated substituent hindering the compounds'' abilities to bind to the tryptophanyl-tRNA synthetase (TrpRS) target, which is supported by docking studies of the analogs into a bacterial TrpRS structure (Fig. S12^†).MIC₅₀ values determined for 1 and its derivatives against MRSA. Values represent the average of three replicates ± the standard deviation. For 1, 5F-1, 6F-1 and 7F-1, concentrations ranging from 50 μg mL⁻¹ to 0.128 ng mL⁻¹ were tested, and for 5Cl-1, 6Cl-1 and 7Cl-1, concentrations ranging from 200 μg mL⁻¹ to 0.512 ng mL⁻¹ were tested. The reported MIC₅₀ value for indolmycin is 0.5 μg mL⁻¹ (range: 0.125–2 μg mL⁻¹) against MRSA¹²

Chemical Characterization of Plant Extracts and Evaluation of their Nematicidal and Phytotoxic Potential

Nacobbus aberrans ranks among the “top ten” plant-parasitic nematodes of phytosanitary importance. It causes significant losses in commercial interest crops in America and is a potential risk in the European Union. The nematicidal and phytotoxic activities of seven plant extracts against N. aberrans and Solanum lycopersicum were evaluated in vitro, respectively. The chemical nature of three nematicidal extracts (EC_50,48h ≤ 113 µg mL⁻¹) was studied through NMR analysis. Plant extracts showed nematicidal activity on second-stage juveniles (J2): (≥87%) at 1000 µg mL⁻¹ after 72 h, and their EC₅₀ values were 71.4–468.1 and 31.5–299.8 µg mL⁻¹ after 24 and 48 h, respectively. Extracts with the best nematicidal potential (EC_50,48h < 113 µg mL⁻¹) were those from Adenophyllum aurantium, Alloispermum integrifolium, and Tournefortia densiflora, which inhibited L. esculentum seed growth by 100% at 20 µg mL⁻¹. Stigmasterol (1), β-sitosterol (2), and α-terthienyl (3) were identified from A. aurantium, while 1, 2, lutein (4), centaurin (5), patuletin-7-β-O-glucoside (6), pendulin (7), and penduletin (8) were identified from A. integrifolium. From T. densiflora extract, allantoin (9), 9-O-angeloyl-retronecine (10), and its N-oxide (11) were identified. The present research is the first to report the effect of T. densiflora, A. integrifolium, and A. aurantium against N. aberrans and chemically characterized nematicidal extracts that may provide alternative sources of botanical nematicides.     

Characterization of the Trans-Epithelial Transport of Green Tea (C. sinensis) Catechin Extracts with In Vitro Inhibitory Effect against the SARS-CoV-2 Papain-like Protease Activity

This work describes an untargeted analytical approach for the screening, identification, and characterization of the trans-epithelial transport of green tea (Camellia sinensis) catechin extracts with in vitro inhibitory effect against the SARS-CoV-2 papain-like protease (PLpro) activity. After specific catechin extraction, a chromatographic separation obtained six fractions were carried out. The fractions were assessed in vitro against the PLpro target. Fraction 5 showed the highest inhibitory activity against the SARS-CoV-2 PLpro (IC₅₀ of 0.125 μg mL⁻¹). The untargeted characterization revealed that (−)-epicatechin-3-gallate (ECG) was the most abundant compound in the fraction and the primary molecule absorbed by differentiated Caco-2 cells. Results indicated that fraction 5 was approximately 10 times more active than ECG (IC₅₀ value equal to 11.62 ± 0.47 μg mL⁻¹) to inhibit the PLpro target. Overall, our findings highlight the synergistic effects of the various components of the crude extract compared to isolated ECG.