Electrochemical Biosensing Platform Based on Toehold-Mediated Strand Displacement Reaction and DSN Enzyme-Assisted Amplification for Two-Target Detection.

Simultaneous detection of multiple targets is of great significance for accurate disease diagnosis. Herein, based on duplex-specific nuclease (DSN) assisted signal amplification and the toehold-mediated strand displacement reaction (TSDR), we constructed an electrochemical biosensor with high sensitivity and high specificity for dual-target detection. MiRNA-141 and miRNA-133a were used as the targets, and ferrocene (Fc) and methylene blue (MB) with significant peak potential differentiation were used as the electrochemical signal probes. The elaborately designed hairpin probe H1, which was fixed on the electrode surface, could be hybridized with the target miRNA-141 to perform signal amplification by the DSN-assisted enzyme cleavage cycle; thus, miRNA-141 could be detected by Fc signal changes at 0.41 V. The hairpin H1 can also combine with the MB-labeled signal probe (SP) output from miRNA-133a-induced TSDR, and the detection of miRNA-133a can be realized according to the response signal generated by MB at -0.26 V. The two sensing lines are independent of each other, and there is no mutual interference in the detection process. Therefore, two independent detection lines could be connected in series, and the simultaneous detection of two targets can be achieved on a single electrode. This novel detection strategy provides a new way to simultaneously detect different biomarkers.

Dual-enzyme inhibiting nanomedicines for enhanced cancer chemodynamic therapy by inducing intratumoral acidosis.

Deficiency of endogenous hydrogen peroxide and insufficient intracellular acidity are usually two important factors limiting chemodynamic therapy (CDT). Here we report a glutathione-responsive nanomedicine that can provide a suitable environment for CDT by inhibiting dual-enzymes simultaneously. The nanomedicine is constructed by encapsulation of a novel hydrogen sulfide donor in nanomicelle assembled by glutathione-responsive amphiphilic polymer. In response to intracellular glutathione, the nanomedicine can efficiently release the active ingredients hydrogen sulfide, carbonic anhydrase inhibitor and ferrocene. The hydrogen sulfide can increase the concentrations of hydrogen peroxide and lactic acid by inhibiting catalase and enhancing glycolysis. The carbonic anhydrase inhibitor can further induce intratumoral acidosis by inhibiting the function of carbonic anhydrase IX. Therefore, the nanomedicine can provide more efficient reaction conditions for the ferrocene-mediated Fenton reaction to generate abundant toxic hydroxyl radicals. In vivo results show that the combination of enhanced CDT and acidosis can effectively inhibit tumor growth. This design of nanomedicine provides a promising dual-enzyme inhibiting strategy to enhance antitumor efficacy of CDT.

Label-Free Mode Based on Ferrocene/PEDOT:PSS-PPy for Molecularly Imprinted Electrochemically Ultrasensitive Detection of Amino Acids.

Generally, molecularly imprinted (MIP) electrochemical sensors for amino acids operate in a "label-like" mode. That is, after an amino acid is specifically recognized by an imprinted cavity at the sensing interface, the amino acid itself provides the sensing signal for quantitative detection. However, poorly electroactive amino acids impede electron transfer at the sensing interface and require high potentials to drive the reaction; thus, more interfering reactions tend to be triggered in practical applications, causing enhanced background noise in the detection. To address these issues, a "label-free" mode of the MIP sensor based on the ferrocene (Fc)/PEDOT:PSS-polypyrrole (PPy) composite was designed for the first time. The Fc/PEDOT:PSS-PPy is drop coated on the electrode surface as a substrate, and MIP polymers with specific recognition ability are immobilized on the substrate via electrostatic adsorption. As a proof of concept, l-tyrosine (l-Tyr) was selected as a model analyte and the "label-free" mode MIP/Fc/PEDOT:PSS-PPy sensor was constructed. The limit of detection (LOD) and linearity range of the MIP/Fc/PEDOT:PSS-PPy sensor were 2.31 × 10-11 M and from 100 pM to 5 mM, respectively. Compared with the label-like mode, the LOD was three orders of magnitude lower, the linear range was increased by three orders of magnitude, and the sensitivity was improved by more than four times. This work provides a universal and effective concept for MIP electrochemical sensing of amino acids.

Self-assembled hyaluronic acid nanomicelle for enhanced cascade cancer chemotherapy via self-sensitized ferroptosis.

The clinical utility of chemotherapy is often compromised by its limited efficacy and significant side effects. Addressing these concerns, we have developed a self-assembled nanomicelle, namely SANTA FE OXA, which consists of hyaluronic acid (HA) conjugated with ferrocene methanol (FC), oxaliplatin prodrug (OXA(IV)) and ethylene glycol-coupled linoleic acid (EG-LA). Targeted delivery is achieved by HA binding to the CD44 receptors that are overexpressed on tumor cells, facilitating drug uptake. Once internalized, hyaluronidase (HAase) catalyzes the digestion of the SANTA FE OXA, releasing FC and reducing OXA(IV) into an active form. The active oxaliplatin (OXA) induces DNA damage and increases intracellular hydrogen peroxide (H2O2) levels via cascade reactions. Simultaneously, FC disrupts the redox balance within tumor cells, inducing ferroptosis. Both in vivo and in vitro experiments confirmed that SANTA FE OXA inhibited tumor growth by combining cascade chemotherapy and self-sensitized ferroptosis, achieving a tumor inhibition rate of up to 76.61 %. Moreover, this SANTA FE OXA significantly mitigates the systemic toxicity commonly associated with platinum-based chemotherapeutics. Our findings represent a compelling advancement in nanomedicine for enhanced cascade cancer therapy.

Ferrocenyl Azoles: Versatile N-Containing Heterocycles and their Anticancer Activities.

The medicinal chemistry of ferrocene has gained its momentum after the discovery of biological activities of ferrocifen and ferroquine. These ferrocenyl drugs have been designed by replacing the aromatic moiety of the organic drugs, tamoxifen and chloroquine respectively, with a ferrocenyl unit. The promising biological activities of these ferrocenyl drugs have paved a path to explore the medicinal applications of several ferrocenyl conjugates. In these conjugates, the ferrocenyl moiety has played a vital role in enhancing or imparting the anticancer activity to the molecule. The ferrocenyl conjugates induce the cytotoxicity by generating reactive oxygen species and thereby damaging the DNA. In medicinal chemistry, the five membered nitrogen heterocycles (azoles) play a significant role due to their rigid ring structure and hydrogen bonding ability with the biomolecules. Several potent drug candidates with azole groups have been in use as chemotherapeutics. Considering the importance of ferrocenyl moiety and azole groups, several ferrocenyl azole conjugates have been synthesized and screened for their biological activities. Hence, in the view of a wide scope in the development of potent drugs based on ferrocenyl azole conjugates, herein we present the details of synthesis and the anticancer activities of ferrocenyl compounds bearing azole groups such as imidazole, triazoles, thiazole and isoxazoles.

Ratiometric electrochemical biosensor based on hybridization chain reaction signal amplification for sensitive microRNA-155 detection.

In this work, a sensitive ratiometric electrochemical biosensor for microRNA-155 (miRNA-155) detection is reported based on a hybridization chain reaction amplifying the electrochemical signal. The biosensor was fabricated using Au NPs as a modified material to assemble capture DNA labeled with ferrocene (Fc) molecules, and a DNA probe labeled with methylene blue (MB) was employed for the signal probe. In the presence of target miRNA-155, it can be dual hybridized with capture and signal probe, especially with signal probe to continuously produce long concatemers containing lots of MB molecules. The electrochemical signal of Fc was used for the internal signal, and the signal from MB was used as an indicator signal. As the concentration of miRNA-155 was altered, the internal reference signal of Fc remained constant, and only the indicator signal changed in a sensitive way. The change in the ratio (IMB/IFc) between the indicator signal of MB and internal reference signal of Fc can be used to monitor the concentration of miRNA-155. Under optimal conditions, the prepared ratiometric biosensor could detect miRNA-155 within a wide linear range from 100 fM to 100 nM with low detection limit of 33 fM (at S/N = 3). Moreover, the biosensor was evaluated with human serum samples, and satisfactory recoveries were obtained, indicating that the ratiometric biosensor can be applied to clinical sample analysis.

Carrageenan-ferrocene-eicosapentaenoic acid composite hydrogel induce ferroptosis and apoptosis for anti-tumor recurrence and metastasis.

The immune-suppressive microenvironment of solid tumors is a key factor limiting the effectiveness of immunotherapy, which seriously threatens human life and health. Ferroptosis and apoptosis are key cell-death pathways implicated in cancers, which can synergistically activate tumor immune responses. Here, we developed a multifunctional composite hydrogel (CE-Fc-Gel) based on the self-assembly of poloxamer 407, cystamine-linked ιota-carrageenan (CA)-eicosapentaenoic acid (EPA), and ferrocene (Fc). CE-Fc-Gel improved targeting in tumor microenvironment due to its disulfide bonds. Moreover, CE-Fc-Gel promoted lipid peroxidation, enhanced reactive oxygen species (ROS) production, and decreased glutathione peroxidase 4 (GPX4), inducing ferroptosis by the synergistic effect of Fc and EPA. CE-Fc-Gel induced apoptosis and immunogenic cell death (ICD), thereby promoting dendritic cells (DCs) maturation and T cell infiltration. As a result, CE-Fc-Gel significantly inhibited primary and metastatic tumors in vivo. Our findings provide a novel strategy for enhancing tumor immunotherapy by combining apoptosis, ferroptosis, and ICD.

An electrochemical ratiometric immunosensor for the detection of NMP22 based on ZIF-8@MWCNTs@Chit@Fc@AuNPs and AuPt-MB.

Nuclear matrix protein 22 (NMP22) is one of the most important tumor markers of bladder cancer and is significantly elevated in the urine of bladder cancer patients. Therefore, in this work, a highly sensitive ratiometric electrochemical immunosensor was constructed to detect NMP22 based on ZIF-8@MWCNTs@Chit@Fc@AuNPs composites. ZIF-8 had a large surface area and good adsorption ability. Multi-Walled Carbon Nanotubes (MWCNTs) can optimize the electrical conductivity of ZIF-8, so that the electrode surface of ferrocene (Fc) obtains a stable and strong electrochemical signal. In addition, AuPt-MB provided another strong detection signal methylene blue (MB) while immobilizing the secondary antibody (Ab2) through Au-N and Pt-N bonds. A ratiometric electrochemical sensor was formed based on ZIF-8@MWCNTs@Chit@Fc@AuNPs and AuPt-MB, which showed a great linear connection between IMB/IFc and the logarithmic concentration of NMP22 with a detection limit of 3.33 fg mL-1 (S/N = 3) under optimized specifications in the concentration interval of 0.01 pg mL-1 to 1000 ng mL-1. In addition, the ratiometric immunosensor showed good selectivity and stability.

Co-crosslinking strategy for dual functionalization of small magnetic nanoparticles with redox probes and biological probes.

Surface functionalization strategy is becoming a crucial bridge from magnetic nanoparticles (MNPs) to their broad bio-application. To realize the multiple functions of MNPs such as magnetic manipulation, target capture, and signal amplification in their use of electrochemical biosensing, co-crosslinking strategy was proposed here to construct dual-functionalized MNPs by combining ultra-sensitive redox moieties and specific biological probes. In this work, MNPs with a TEM size of 10 nm were synthesized by co-precipitation for amination and PEGylation to maintain colloid stability once dispersed in high-ionic-strength buffer (such as phosphate-buffered saline). Then, MNPs@IgG were prepared via the bis(sulfosuccinimidyl) suberate (BS3) cross-linker to conjugate these IgG onto the MNP surface, with a binding efficiency of 73%. To construct dual-functionalized MNPs, these redox probes of ferrocene-NHS (Fc) were co-crosslinked onto the MNP surface, together with IgG, by using BS3. The developed MNPs@Redox@IgG were characterized by SDS‒PAGE to identify IgG binding and by square wave voltammetry (SWV) to validate the redox signal. Additionally, the anti-CD63 antibodies were selected for the development of MNPs@anti-CD63 for use in the bio-testing of exosome sample capture. Therefore, co-crosslinking strategy paved a way to develop dual-functionalized MNPs that can be an aid of their potential utilization in diagnostic assay or electrochemical methods.

Hybrid nanoflower-based electrochemical lateral flow immunoassay for Escherichia coli O157 detection.

An electrochemical biosensor has been developed for detection of Escherichia coli O157 by integrating lateral flow with screen-printed electrodes. The screen-printed electrodes were attached under the lateral flow detection line, and organic-inorganic nanoflowers prepared from E. coli O157-specific antibodies as an organic component were attached to the lateral flow detection line. In the presence of E. coli O157, an organic-inorganic nanoflower-E. coli O157-antimicrobial peptide-labelled ferrocene sandwich structure is formed on the lateral flow detection line. Differential pulse voltammetry is applied using a smartphone-based device to monitor ferrocene on the detection line. The resulting electrochemical biosensor could specifically detect E. coli O157 with a limit of detection of 25 colony-forming units mL-1. Through substitution of antibodies of organic components in organic-inorganic nanoflowers, biosensors have great potential for the detection of other pathogens in biomedical research and clinical diagnosis.

"Rigid-Flexible" Dual-Ferrocene Chimeric Nanonetwork for Simultaneous Tumor-Targeted Tracing and Photothermal/Photodynamic Therapy.

There is an urgent need to develop phototherapeutic agents with imaging capabilities to assess the treatment process and efficacy in real-time during cancer phototherapy for precision cancer therapy. The safe near-infrared (NIR) fluorescent dyes have garnered significant attention and are desirable for theranostics agents. However, until now, achieving excellent photostability and fluorescence (FL) imaging capability in aggregation-caused quenching (ACQ) dyes remains a big challenge. Here, for the only FDA-approved NIR dye, indocyanine green (ICG), we developed a dual-ferrocene (Fc) chimeric nanonetwork ICG@HFFC based on the rigid-flexible strategy through one-step self-assembly, which uses rigid Fc-modified hyaluronic acid (HA) copolymer (HA-Fc) and flexible octadecylamine (ODA) bonded Fc (Fc-C18) as the delivery system. HA-Fc reserved the ability of HA to target the CD44 receptor of the tumor cell surface, and the dual-Fc region provided a rigid space for securely binding ICG through metal-ligand interaction and π-π conjugation, ensuring excellent photostability. Additionally, the alkyl chain provided flexible confinement for the remaining ICG through hydrophobic forces, preserving its FL. Thereby, a balance is achieved between outstanding photostability and FL imaging capability. In vitro studies showed improved photobleaching resistance, enhanced FL stability, and increased singlet oxygen (1O2) production efficiency in ICG@HFFC. Further in vivo results display that ICG@HFFC had good tumor tracing ability and significant tumor inhibition which also exhibited good biocompatibility.. Therefore, ICG@HFFC provides an encouraging strategy to realize simultaneous enhanced tumor tracing and photothermal/photodynamic therapy (PTT/PDT) and offers a novel approach to address the limitations of ACQ dyes.

Reactive oxygen species responsive chitooligosaccharides based nanoplatform for sonodynamic therapy in mammary cancer.

Sonodynamic therapy (SDT) has been extensively studied as a new type of non-invasive treatment for mammary cancer. However, the poor water solubility and defective biocompatibility of sonosensitizers during SDT hinder the sonodynamic efficacy. Herein, a nanoplatform has been developed to achieve high efficient SDT against mammary cancer through the host-guest interaction of ß-cyclodextrin/5-(4-hydroxyphenyl)-10,15,20-triphenylporphyrin (ß-CD-TPP) and ferrocenecarboxylic acid/chitooligosaccharides (FC-COS). Moreover, the glucose oxidase (GOx) was loaded through electrostatic adsorption, which efficiently restricts the energy supply in tumor tissues, thus enhancing the therapeutic efficacy of SDT for tumors. Under optimal conditions, the entire system exhibited favorable water solubility, suitable particle size and viable biocompatibility. This facilitated the integration of the characteristics of starvation therapy and sonodynamic therapy, resulting in efficient inhibition of tumor growth with minimal side effects in vivo. This work may provide new insights into the application of natural oligosaccharides for construct multifunctional nanocarrier systems, which could optimize the design and development of sonodynamic therapy strategies and even combination therapy strategies.

Ferrocene-based metal-organic frameworks with dual synergistic active sites for selectively electrochemical removal of arsenic from contaminated water.

The effective removal of trace levels of the highly toxic arsenite (As(Ⅲ)) from groundwater is crucial to address the threat to drinking water supply. Herein, we developed an electrochemical separation system utilizing redox-active ferrocene-based metal-organic frameworks (termed Fe-DFc) for selective removal of As(III). This system leveraged 1,1'-ferrocenedicarboxylic acid as a ligand coordinated with iron, enabling the highly selective capture and conversion of As(III) from groundwater. The Fe-DFc electrode-based electrochemical system not only effectively removed As(III) even in the presence of a 1250-fold excess of competing electrolytes, but also converted about 96 % of the adsorbed As(III) into the less toxic As(V), surpassing the results of those documented in the current literature. X-ray absorption fine structure analysis and density functional theory calculations demonstrated that the high selectivity of Fe-O6 moiety and the exceptional redox activity of Fc synergistically contributed to the efficient removal of As(III). Moreover, the electrochemical separation system enabled the remediation of arsenic-contaminated groundwater at a low energy cost of 0.033 kWh m-3 during long-term operation, highlighting the application potential of the electrochemical technology for arsenic removal from contaminated water.

Dual-Mode Arginine Assay Based on the Conformation Switch of a Ferrocene-Grafted Polypeptide.

Both controllable regulation of the conformational structure of a polypeptide and specific recognition of an amino acid are still arduous challenges. Here, a novel dual-mode (electrochemical and colorimetric) biosensor was built for arginine (Arg) recognition based on a conformation switch, utilizing controllable and synergistic self-assembly of a ferrocene-grafted hexadecapeptide (P16Fc) with gold nanoparticles (AuNPs). Benefiting from the flexibility and unique topological structure of P16Fc formed nanospheres, the assembly and disassembly can undergo a conformation transition induced by Arg through controlling the distance and number of Fc detached from the gold surface, producing on-off electrical signals. Also, they can induce aggregation and dispersion of AuNPs in solution, causing a color change. The mechanism of Arg recognition with polypeptide conformation regulation was well explored by combining microstructure characterizations with molecular mechanics calculations. The electrochemical and colorimetric assays for Arg were successfully established in sensitive and selective manner, not only obtaining a very low detection limit, but also effectively eliminating the interference from other amino acids and overcoming the limitation of AuNP aggregation. Notably, the conformational change-based assay with the peptide regulated by the target will make a powerful tool for the amino acid biosensing and health diagnosis.

High-Frequency Quartz Crystal Microbalance and Dual-Signaling Electrochemical Ratiometric Assays of PTP1B Activity Based on COF@Au@Fc Hybrids.

The abnormal expression of protein tyrosine phosphatase 1B (PTP1B) is highly related to several serious human diseases. Therefore, an accurate PTP1B activity assay is beneficial to the diagnosis and treatment of these diseases. In this study, a dual-mode biosensing platform that enabled the sensitive and accurate assay of PTP1B activity was constructed based on the high-frequency (100 MHz) quartz crystal microbalance (QCM) and dual-signaling electrochemical (EC) ratiometric strategy. Covalent-organic framework@gold nanoparticles@ferrocene@single-strand DNA (COF@Au@Fc-S0) was introduced onto the QCM Au chip via the chelation between Zr4+ and phosphate groups (phosphate group of the phosphopeptide (P-peptide) on the QCM Au chip and the phosphate group of thiol-labeled single-stranded DNA (S0) on COF@Au@Fc-S0) and used as a signal reporter. When PTP1B was present, the dephosphorylation of the P-peptide led to the release of COF@Au@Fc-S0 from the QCM Au chip, resulting in an increase in the frequency of the QCM. Meanwhile, the released COF@Au@Fc-S0 hybridized with thiol/methylene blue (MB)-labeled hairpin DNA (S1-MB) on the Au NPs-modified indium-tin oxide (ITO) electrode. This caused MB to be far away from the electrode surface and Fc to be close to the electrode, leading to a decrease in the oxidation peak current of MB and an increase in the oxidation peak current of Fc. Thus, PTP1B-induced dephosphorylation of the P-peptide was monitored in real time by QCM, and PTP1B activity was detected sensitively and reliably using this innovative QCM-EC dual-mode sensing platform with an ultralow detection limit. This platform is anticipated to serve as a robust tool for the analysis of protein phosphatase activity and the discovery of drugs targeting protein phosphatase.

Construction of Metal-Organic Framework as a Novel Platform for Ratiometric Determination of Cyanide.

Metal-organic frameworks (MOFs) are frequently utilized as sensing materials. Unfortunately, the low conductivity of MOFs hinder their further application in electrochemical determination. To overcome this limitation, a novel modification strategy for MOFs was proposed, establishing an electrochemical determination method for cyanides in Baijiu. Co and Ni were synergistically used as the metal active centers, with meso-Tetra(4-carboxyphenyl)porphine (TCPP) and Ferrocenecarboxylic acid (Fc-COOH) serving as the main ligands, synthesizing Ni/Co-MOF-TCPP-Fc through a hydrothermal method. The prepared MOF exhibited improved conductivity and stable ratio signals, enabling rapid and sensitive determination of cyanides. The screen-printed carbon electrodes (SPCE) were suitable for in situ and real-time determination of cyanide by electrochemical sensors due to their portability, low cost, and ease of mass production. A logarithmic linear response in the range of 0.196~44 ng/mL was demonstrated by this method, and the limit of detection (LOD) was 0.052 ng/mL. Compared with other methods, the sensor was constructed by a one-step synthesis method, which greatly simplifies the analysis process, and the determination time required was only 4 min. During natural cyanide determinations, recommended readouts match well with GC-MS with less than 5.9% relative error. Moreover, this electrochemical sensor presented a promising method for assessing the safety of cyanides in Baijiu.

A novel dual-signal output strategy for POCT of CEA based on a smartphone electrochemical aptasensing platform.

A smartphone-based electrochemical aptasensing platform was developed for the point-of-care testing (POCT) of carcinoembryonic antigen (CEA) based on the ferrocene (Fc) and PdPt@PCN-224 dual-signal labeled strategy. The prepared PdPt@PCN-224 nanocomposite showed a strong catalytic property for the reduction of H2O2. Phosphate group-labeled aptamer could capture PdPt@PCN-224 by Zr-O-P bonds to form PdPt@PCN-224-P-Apt. Therefore, a dual signal labeled probe was formed by the hybridization between Fc-DNA and PdPt@PCN-224-P-Apt. The presence of CEA forced PdPt@PCN-224-P-Apt to leave the electrode surface due to the specific affinity, leading to the decrease of the reduction current of H2O2. At the same time, the Fc-DNA strand changed to hairpin structure, which made Fc closer to the electrode and resulted in the increase of the oxidation current of Fc. Thus, CEA can be accurately determined through both signals: the decrease of H2O2 reduction current and the increase of Fc oxidation current, which could avoid the false positive signal. Under the optimal conditions, the prepared aptasensor exhibited a wide linear range from 1 pg·mL-1 to 100 ng·mL-1 and low detection limits of 0.98 pg·mL-1 and 0.27 pg·mL-1 with Fc and PdPt@PCN-224 as signal labels, respectively. The aptasensor developed in this study has successfully demonstrated its capability to detect CEA in real human serum samples. These findings suggest that the proposed sensing platform will hold great potential for clinical tumor diagnosis and monitoring.

Multifunctional Organometallic Compounds Active against Infective Trypanosomes: Ru(II) Ferrocenyl Derivatives with Two Different Bioactive Ligands.

Human African trypanosomiasis (HAT, sleeping sickness) and American trypanosomiasis (Chagas disease) are endemic zoonotic diseases caused by genomically related trypanosomatid protozoan parasites (Trypanosoma brucei and Trypanosoma cruzi, respectively). Just a few old drugs are available for their treatment, with most of them sharing poor safety, efficacy, and pharmacokinetic profiles. Only fexinidazole has been recently incorporated into the arsenal for the treatment of HAT. In this work, new multifunctional Ru(II) ferrocenyl compounds were rationally designed as potential agents against these pathogens by including in a single molecule 1,1'-bis(diphenylphosphino)ferrocene (dppf) and two bioactive bidentate ligands: pyridine-2-thiolato-1-oxide ligand (mpo) and polypyridyl ligands (NN). Three [Ru(mpo)(dppf)(NN)](PF6) compounds and their derivatives with chloride as a counterion were synthesized and fully characterized in solid state and solution. They showed in vitro activity on bloodstream T. brucei (EC50 = 31-160 nM) and on T. cruzi trypomastigotes (EC50 = 190-410 nM). Compounds showed the lowest EC50 values on T. brucei when compared to the whole set of metal-based compounds previously developed by us. In addition, several of the Ru compounds showed good selectivity toward the parasites, particularly against the highly proliferative bloodstream form of T. brucei. Interaction with DNA and generation of reactive oxygen species (ROS) were ruled out as potential targets and modes of action of the Ru compounds. Biochemical assays and in silico analysis led to the insight that they are able to inhibit the NADH-dependent fumarate reductase from T. cruzi. One representative hit induced a mild oxidation of low molecular weight thiols in T. brucei. The compounds were stable for at least 72 h in two different media and more lipophilic than both bioactive ligands, mpo and NN. An initial assessment of the therapeutic efficacy of one of the most potent and selective candidates, [Ru(mpo)(dppf)(bipy)]Cl, was performed using a murine infection model of acute African trypanosomiasis. This hit compound lacks acute toxicity when applied to animals in the dose/regimen described, but was unable to control parasite proliferation in vivo, probably because of its rapid clearance or low biodistribution in the extracellular fluids. Future studies should investigate the pharmacokinetics of this compound in vivo and involve further research to gain deeper insight into the mechanism of action of the compounds.

Red Fluorescent Aminoferrocene (Pro)Drugs for in Cellulo and in Vivo Imaging.

Red fluorescent dyes are usually charged, lipophilic molecules with relatively high molecular weight, which tend to localize in specific intracellular locations, e. g., a cyanine dye Cy5 is biased towards mitochondria. They are often used as markers of biomolecules including nucleic acids and proteins. Since the molecular weight of the dyes is much smaller than that of the biomolecules, the labelling has a negligible effect on the properties of the biomolecules. In contrast, conjugation of the dyes to low molecular weight (pro)drugs can dramatically alter their properties. For example, conjugates of Cy5 with lysosome-targeting aminoferrocenes accumulate in mitochondria and exhibit no intracellular effects characteristic for the parent (pro)drugs. Herein we tested several neutral and negatively charged dyes for labelling lysosome-targeting aminoferrocenes 7 and 8 as well as a non-targeted control 3. We found that a BODIPY derivative BDP-TR exhibits the desired unbiased properties: the conjugation does not disturb the intracellular localization of the (pro)drugs, their mode of action, and cancer cell specificity. We used the conjugates to clarify the mechanism of action of the aminoferrocenes. In particular, we identified new intermediates, explained why lysosome-targeting aminoferrocenes are more potent than their non-targeted counterparts, and evaluated their distribution in vivo.

Development of Novel Immobilized Copper-Ligand Complex for Click Chemistry of Biomolecules.

Copper-catalyzed azide-alkyne cycloaddition click (CuAAC) reaction is widely used to synthesize drug candidates and other biomolecule classes. Homogeneous catalysts, which consist of copper coordinated to a ligand framework, have been optimized for high yield and specificity of the CuAAC reaction, but CuAAC reaction with these catalysts requires the addition of a reducing agent and basic conditions, which can complicate some of the desired syntheses. Additionally, removing copper from the synthesized CuAAC-containing biomolecule is necessary for biological applications but inconvenient and requires additional purification steps. We describe here the design and synthesis of a PNN-type pincer ligand complex with copper (I) that stabilizes the copper (I) and, therefore, can act as a CuAAC catalyst without a reducing agent and base under physiologically relevant conditions. This complex was immobilized on two types of resin, and one of the immobilized catalyst forms worked well under aqueous physiological conditions. Minimal copper leaching was observed from the immobilized catalyst, which allowed its use in multiple reaction cycles without the addition of any reducing agent or base and without recharging with copper ion. The mechanism of the catalytic cycle was rationalized by density functional theory (DFT). This catalyst's utility was demonstrated by synthesizing coumarin derivatives of small molecules such as ferrocene and sugar.