In Vitro Activities of Oxazolidinone Antibiotics Alone and in Combination with C-TEMPO against Methicillin-Resistant Staphylococcus aureus Biofilms.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a global health concern. The propensity of MRSA to form biofilms is a significant contributor to its pathogenicity. Strategies to treat biofilms often involve small molecules that disperse the biofilm into planktonic cells. Linezolid and, by extension, theoxazolidinones have been developed to treat infections caused by Gram-positive bacteria such as MRSA. However, the clinical development of these antibiotics has mainly assessed the susceptibility of planktonic cells to the drug. Previous studies evaluating the anti-biofilm activity of theoxazolidinones have mainly focused on the biofilm inhibition of Enterococcus faecalis and methicillin-sensitive Staphylococcus aureus, with only a few studies investigating the activity of oxazolidinones for eradicating established biofilms for these species. Very little is known about the ability of oxazolidinones to eradicate MRSA biofilms. In this work, five oxazolidinones were assessed against MRSA biofilms using a minimum biofilm eradication concentration (MBEC) assay. All oxazolidinones had inherent antibiofilm activity. However, only ranbezolid could completely eradicate MRSA biofilms at clinically relevant concentrations. The susceptibility of the MRSA biofilms to ranbezolid was synergistically enhanced by coadministration with the nitroxide biofilm dispersal agent C-TEMPO. We presume that ranbezolid acts as a dual warhead drug, which combines the mechanism of action of the oxazolidinones with a nitric oxide donor or cytotoxic drug.

Preclinical Evaluation of Nitroxide-Functionalised Ciprofloxacin as a Novel Antibiofilm Drug Hybrid for Urinary Tract Infections.

Urinary tract infections (UTIs) are the second most common bacterial infection with high recurrence rates and can involve biofilm formation on patient catheters. Biofilms are inherently tolerant to antimicrobials, making them difficult to eradicate. Many antibiofilm agents alone do not have bactericidal activity; therefore, linking them to antibiotics is a promising antibiofilm strategy. However, many of these hybrid agents have not been tested in relevant preclinical settings, limiting their potential for clinical translation. Here, we evaluate a ciprofloxacin di-nitroxide hybrid (CDN11), previously reported to have antibiofilm activity against uropathogenic Escherichia coli (UPEC) strain UTI89 in vitro, as a potential UTI therapeutic using multiple preclinical models that reflect various aspects of UTI pathogenesis. We report improved in vitro activity over the parent drug ciprofloxacin against mature UTI89 biofilms formed inside polyethylene catheters. In bladder cell monolayers infected with UTI89, treatment with CDN11 afforded significant reduction in bacterial titers, including intracellular UPEC. Infected mouse bladders containing biofilm-like intracellular reservoirs of UPEC UTI89 showed decreased bacterial loads after ex vivo bladder treatment with CDN11. Activity for CDN11 was reported across different models of UTI, showcasing nitroxide-antibiotic hybridization as a promising antibiofilm approach. The pipeline we described here could be readily used in testing other new therapeutic compounds, fast-tracking the development of novel antibiofilm therapeutics.

Simple and Efficient Synthesis of 3-Aryl-2-oxazolidinone Scaffolds Enabling Increased Potency toward Biofilms.

Infectious diseases caused by bacterial pathogens are a leading cause of mortality worldwide. In particular, recalcitrant bacterial communities known as biofilms are implicated in persistent and difficult to treat infections. With a diminishing antibiotic pipeline, new treatments are urgently required to combat biofilm infections. An emerging strategy to develop new treatments is the hybridization of antibiotics. The benefit of this approach is the extension of the useful lifetime of existing antibiotics. The oxazolidinones, which include the last resort antibiotic linezolid, are an attractive target for improving antibiofilm efficacy as they present one of the most recently discovered classes of antibiotics. A key step in the synthesis of new 3-aryl-2-oxazolidinone derivatives is the challenging formation of the oxazolidinone ring. Herein we report a direct synthetic route to the piperazinyl functionalized 3-aryl-2-oxazolidinone 17. We also demonstrate an application of these piperazine molecules by functionalizing them with a nitroxide moiety as a strategy to extend the useful lifetime of oxazolidinones and improve their potency against Methicillin-resistant Staphylococcus aureus (MRSA) biofilms. The antimicrobial susceptibility of the linezolid-nitroxide conjugate 11 and its corresponding methoxyamine derivative 12 (a control for biofilm dispersal) was assessed against planktonic cells and biofilms of MRSA. In comparison to linezolid and our lead compound 10 (a piperazinyl oxazolidinone derivative), the linezolid-nitroxide conjugate 11 displayed a minimum inhibitory concentration that was 4-16-fold higher. The opposite effect was seen in biofilms where the linezolid-nitroxide hybrid 11 was >2-fold more effective (160 µg/mL versus >320 µg/mL) in eradicating MRSA biofilms. The methoxyamine derivative 12 performed on par with linezolid. The drug-likeness of the compounds was also assessed, and all compounds were predicted to have good oral bioavailability. Our piperazinyl oxazolidinone derivative 10 was confirmed to be lead-like and would be a good lead candidate for future functionalized oxazolidinones. The modification of antibiotics with a dispersal agent appears to be a promising approach for eradicating MRSA biofilms and overcoming the antibiotic resistance associated with the biofilm mode of growth.

Synergistic or antagonistic antioxidant combinations - a case study exploring flavonoid-nitroxide hybrids.

Neurodegenerative diseases impose a considerable medical and public health burden on populations throughout the world. Oxidative stress, an imbalance in pro-oxidant/antioxidant homeostasis that leads to the generation of reactive oxygen species (ROS), has been implicated in the progression of a number of neurodegenerative diseases. The manipulation of ROS levels may represent a promising treatment option to slow down neurodegeneration, although adequate potency of treatments has not yet been achieved. Using a hybrid pharmacology approach, free radical nitroxide antioxidants were hybridised with a class of natural antioxidants, flavonoids, to form a potential multitargeted antioxidant. Modification of the Baker-Venkataraman reaction achieved the flavonoid-nitroxide hybrids (6-9) in modest yields. Antioxidant evaluation of the hybrids by cyclic voltammetry showed both redox functionalities were still active, with little influence on oxidation potential. Assessment of the peroxyl radical scavenging ability through an ORAC assay showed reduced antioxidant activity of the hybrids compared to their individual components. It was hypothesized that the presence of the phenol in the hybrids creates a more acidic medium which does not favour regeneration of the nitroxide from the corresponding oxammonium cation, disturbing the typical catalytic cycle of peroxyl radical scavenging by nitroxides. This work highlights the potential intricacies involved with drug hybridization as a strategy for new therapeutic development.

Strategies to Improve the Potency of Oxazolidinones towards Bacterial Biofilms.

Biofilms are part of the natural lifecycle of bacteria and are known to cause chronic infections that are difficult to treat. Most antibiotics are developed and tested against bacteria in the planktonic state and are ineffective against bacterial biofilms. The oxazolidinones, including the last resort drug linezolid, are one of the main classes of synthetic antibiotics progressed to clinical use in the last 50 years. They have a unique mechanism of action and only develop low levels of resistance in the clinical setting. With the aim of providing insight into strategies to design more potent antibiotic compounds with activity against bacterial biofilms, we review the biofilm activity of clinically approved oxazolidinones and report on structural modifications to oxazolidinones and their delivery systems which lead to enhanced anti-biofilm activity.

Four component Passerini polymerization of bulky monomers under high shear flow.

We introduce a four component Passerini polymerization utilizing sterically bulky isocyanide monomers. Under typical Passerini conditions, bulky isocyanides do not react within standard Passerini reaction timescales (hours). We overcome this challenge via the unique physiochemical conditions present in a vortex fluidic device, reducing the reaction time to 2 h on average. Under these high-shear thin-film conditions, bulky isocyanides are readily incorporated into the multicomponent polymerization without the need of high-pressure or temperature. Finally, we demonstrate that the four component approach using functional cyclic anhydrides allows for post-polymerization modification.

Dual-Wavelength Gated oxo-Diels-Alder Photoligation.

The control of chemical functionalization with orthogonal light stimuli paves the way toward manipulating materials with unprecedented spatiotemporal resolution. To reach this goal, we herein introduce a photochemical reaction system that enables two-color control of covalent ligation via an oxo-Diels-Alder cycloaddition between two separate light-responsive molecular entities: a UV-activated photocaged diene based on ortho-quinodimethanes and a carbonyl dienophile appended to a diarylethene photoswitch, whose reactivity can be modulated upon illumination with UV and visible light.

Oxidized Hemithioindigo Photoswitches-Influence of Oxidation State on (Photo)physical and Photochemical Properties.

The photophysical and photochemical properties of sulfoxide and sulfone derivatives of hemithioindigo photoswitches are scrutinized and compared to the unoxidized parent chromophores. Oxidation results in significantly blue-shifted absorptions and mostly reduction of photochromism while thermal stabilities of individual isomers remain largely unaltered. Effective photoswitching takes place at shorter wavelengths compared to parent hemithioindigos and high isomeric yields can be obtained reversibly in the respective photostationary states. Reversible solid-state photoswitching is observed for a twisted sulfone derivative accompanied by visible color changes. These results establish oxidized hemithioindigo photoswitches as promising and versatile tools for robust light-control of molecular behavior for a wide range of applications.

It's a Trap: Thiol-Michael Chemistry on a DASA Photoswitch.

Donor-acceptor Stenhouse adducts (DASA) are popular photoswitches capable of toggling between two isomers depending on the light and temperature of the system. The cyclized polar form is accessed by visible-light irradiation, whereas the linear nonpolar form is recovered in the dark. Upon the formation of the cyclized form, the DASA contains a double bond featuring a ß-carbon prone to nucleophilic attack. Here, an isomer selective thiol-Michael reaction between the cyclized DASA and a base-activated thiol is introduced. The thiol-Michael addition was carried out with an alkyl (1-butanethiol) and an aromatic thiol (p-bromothiophenol) as reaction partners, both in the presence of a base. Under optimized conditions, the reaction proceeds preferentially in the presence of light and base. The current study demonstrates that DASAs can be selectively trapped in their cyclized state.

Visible-Light-Induced Passerini Multicomponent Polymerization.

Herein, we introduce an additive-free visible-light-induced Passerini multicomponent polymerization (MCP) for the generation of high molar mass chains. In place of classical aldehydes (or ketones), highly reactive, in situ photogenerated thioaldehydes are exploited along with isocyanides and carboxylic acids. Prone to side reactions, the thioaldehyde moieties create a complex reaction environment which can be tamed by optimizing the synthetic conditions utilizing stochastic reaction path analysis, highlighting the potential of semi-batch procedures. Once the complex MCP environment is understood, step-growth polymers can be synthesized under mild reaction conditions which-after a Mumm rearrangement-result in the incorporation of thioester moieties directly into the polymer backbone, leading to soft matter materials that can be degraded by straightforward aminolysis or chain expanded by thiirane insertion.

Simultaneous complementary photoswitching of hemithioindigo tweezers for dynamic guest relocalization.

Remote control of complex molecular behavior and function is one key problem in modern chemistry. Using light signaling for this purpose has many advantages, however the integration of different photo processes into a wholesome yet complex system is highly challenging. Here we report an alternative approach to increase complexity of light control-simultaneous complementary photoswitching-in which spectral overlap is used as an advantage to drastically reduce the signaling needed for controlling multipart supramolecular assemblies. Two photoswitchable molecular tweezers respond to the same light signals with opposite changes in their binding affinities. In this way the configuration of two host tweezers and ultimately the dynamic relocation of a guest molecule can be trigged by only one signal reversibly in the same solution. This approach should provide a powerful tool for the construction of sophisticated, integrated, and multi-responsive smart molecular systems in any application driven field of chemistry.

Transforming hemithioindigo from a two-way to a one-way molecular photoswitch by isolation in the gas phase.

Hemithioindigo compounds are attractive two-way molecular photoswitches combining stilbene and thioindigo parts connected by a C-C double bond. In solution, these photoswitches have been well studied. This study presents the investigation of a hemithioindigo derivative in the gas phase. Visible absorption spectra, measured by standard (visPD) and helium-tagging visible photodissociation (He-visPD) techniques were used to unravel absorption characteristics at the level of isolated molecules at 3 Kelvin. Comparison between the Z and E isomers shows a quite distinctive behavior upon visible light absorption. The Z isomer readily undergoes Z → E conversion in the gas phase, as evidenced by the changes in the helium-tagging infrared photodissociation (He-IRPD) spectra. Surprisingly, visible light excitation of the E isomer does not lead to efficient E → Z isomerization unlike in solution. Instead, the ions relax back to their ground state. Influencing the microenvironment of the E isomer by complexation with the highly polar betaine zwitterion resulted in absorption changes, albeit without activating the photoswitching process. Hence, isolation in the gas phase transforms hemithioindigo into a one-way molecular photoswitch. Furthermore, the combination of He-visPD and IRPD spectroscopies proved to be an excellent method for studying photochemical processes such as the double-bond isomerization in the gas phase.

The production of tyramine via the selective hydrogenation of 4-hydroxybenzyl cyanide over a carbon-supported palladium catalyst.

The selective production of primary amines is a problem that plagues heterogeneously catalysed nitrile hydrogenation reactions. Whilst the target amine tyramine (HOC6H4CH2CH2NH2) is biochemically available through the action of enzymes, synthetic routes to this species are not widely reported. Here, a heterogeneously catalysed method is proposed that utilises a Pd/C catalyst to effect the selective hydrogenation of 4-hydroxybenzyl cyanide within a three-phase reactor. The aforementioned selectivity issues are overcome by adjustment of various experimental parameters (hydrogen supply, agitation rate, temperature, use of an auxiliary agent) that result in improved catalytic performance, such that the desired tyramine salt (tyramine hydrogen sulphate) can be produced in quantitative yield. Accordingly, through consideration of the interconnectivity of hydrogenation and hydrogenolysis processes, a selective synthetic strategy is achieved with the findings suitable for extension to other substrates of this nature.

Ingredients to TICT Formation in Donor Substituted Hemithioindigo.

Twisted intramolecular charge transfer (TICT) formation in hemithioindigo photoswitches has recently been reported and constitutes a second deexcitation pathway complementary to photoisomerization. Typically, this behavior is not found for this type of photoswitches, and it takes special geometric and electronic conditions to realize it. Here we present a systematic study that identifies the molecular preconditions leading to TICT formation in donor substituted hemithioindigo, which can thus serve as a frame of reference for other photoswitching systems. By varying the substitution pattern and providing an in-depth physical characterization including time-resolved and quantum yield measurements, we found that neither ground-state pretwisting along the rotatable single bond nor the introduction of strong push-pull character across the photoisomerizable double bond alone leads to formation of TICT states. Only the combination of both ingredients produces light-induced TICT behavior in polar solvents.

Bistable Photoswitching of Hemithioindigo with Green and Red Light: Entry Point to Advanced Molecular Digital Information Processing.

Photoswitches reacting to visible light instead of harmful UV irradiation are of very high interest due to the mild and broadly compatible conditions of their operation. Shifting the absorption into the red region of the electromagnetic spectrum usually comes at the cost of losing thermal stability of the metastable state-the switch switches off by itself. Only recently have photoswitches become available that combine visible light responsiveness with high bistability. However, shifting the wavelengths for bistable photoswitching beyond 600 nm is still a great challenge without involving secondary processes such as two-photon absorption or sensitization. We present a simple hemithioindigo photoswitch that can efficiently be photoisomerized using green and red light while maintaining a high thermal barrier of the metastable state. This highly sought after properties allow for selective switching in a mixture of hemithioindigo dyes. In addition, protonation can be used as second independent input altering the light response of the switch and allowing construction of advanced molecular digital information processing devices. This is demonstrated by realizing a broad variety of logical operations covering combinational and sequential logic behavior. By making use of the protonation-induced loss of thermal bistability, a high security keypad lock could be realized, which distinguishes the sequences of three different inputs and additionally erases its unlocked state after a short time.

Twisted Hemithioindigo Photoswitches: Solvent Polarity Determines the Type of Light-Induced Rotations.

Controlling the internal motions of molecules by outside stimuli is a decisive task for the generation of responsive and complex molecular behavior and functionality. Light-induced structural changes of photoswitches are of special high interest due to the ease of signal application and high repeatability. Typically photoswitches use one reaction coordinate in their switching process and change between two more or less-defined states. Here we report on new twisted hemithioindigo photoswitches enabling two different reaction coordinates to be used for the switching process. Depending on the polarity of the solvent, either complete single bond (in DMSO) or double bond (in cyclohexane) rotation can be induced by visible light. This mutually independent switching establishes an unprecedented two-dimensional control of intramolecular rotations in this class of photoswitches. The mechanistic explanation involves formation of highly polar twisted intramolecular charge-transfer species in the excited state and is based on a large body of experimental quantifications, most notably ultrafast spectroscopy and quantum yield measurements in solvents of different polarity. The concept of pre-twisting in the ground state to open new, independent reaction coordinates in the excited state should be transferable to other photoswitching systems.

Making fast photoswitches faster--using Hammett analysis to understand the limit of donor-acceptor approaches for faster hemithioindigo photoswitches.

Hemithioindigo (HTI) photoswitches have a tremendous potential for biological and supramolecular applications due to their absorptions in the visible-light region in conjunction with ultrafast photoisomerization and high thermal bistability. Rational tailoring of the photophysical properties for a specific application is the key to exploit the full potential of HTIs as photoswitching tools. Herein we use time-resolved absorption spectroscopy and Hammett analysis to discover an unexpected principal limit to the photoisomerization rate for donor-substituted HTIs. By using stationary absorption and fluorescence measurements in combination with theoretical investigations, we offer a detailed mechanistic explanation for the observed rate limit. An alternative way of approaching and possibly even exceeding the maximum rate by multiple donor substitution is demonstrated, which give access to the fastest HTI photoswitch reported to date.