Ferrocene tripeptide Gly-Pro-Arg conjugates: Synthesis and inhibitory effects on Alzheimer’s Aβ1–42 fibrillogenesis and Aβ-induced cytotoxicity in vitro

Alzheimer’s disease (AD) is the most common cause of dementia, and currently there is no clinical treatment to cure it or to halt its progression. Aggregation and fibril formation of β-amyloid peptides (Aβ) are central events in the pathogenesis of AD. Many efforts have been spent on the development of effective inhibitors to prevent Aβ fibrillogenesis and cause disaggregation of preformed Aβ fibrils. In this study, the conjugates of ferrocene and Gly-Pro-Arg (GPR) tripeptide, Boc-Gly-Pro-Arg(NO₂)-Fca-OMe (4, GPR–Fca) and Fc-Gly-Pro-Arg-OMe (7, Fc–GPR) (Fc: ferrocene; Fca: ferrocene amino acid) were synthesized by HOBT/HBTU protocol in solution. These ferrocene GPR conjugates were employed to inhibit Aβ_1–42 fibrillogenesis and to disaggregate preformed Aβ fibrils. The inhibitory properties of ferrocene GPR conjugates on Aβ_1–42 fibrillogenesis were evaluated by thioflavin T (ThT) fluorescence assay, and confirmed by atomic force microscopy (AFM) analysis. The interaction between the ferrocene GPR conjugates and Aβ_1–42 was monitored by electrochemical means. Our results showed that both GPR and GPR–Fca can significantly inhibit the fibril formation of Aβ_1–42, and cause disaggregation of the preformed fibrils. As expected, GPR–Fca shows stronger inhibitory effect on Aβ_1–42 fibrillogenesis than that of its parent peptide GPR. In contrast, Fc–GPR shows no inhibitory effect on fibrillogenesis of Aβ_1–42. Furthermore, GPR–Fca demonstrates significantly protection against Aβ-induced cytotoxicity and exhibits high resistance to proteolysis and good lipophilicity.     

A novel Bod-mediator biosensor based on Ferrocene and <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis> yeast cells

The mediators ferrocene, 1,1'-dimethylferrocene, ferrocene carboxaldehyde, ferrocene acetonitrile, neutral red, 2,6-dichlorophenolindophenol, thionine, methyl blue, and potassium ferricyanide were used in combination with Debaryomyces hansenii yeast cells to create a biosensor receptor for biochemical oxygen demand (BOD) assays. In this eukaryote, ferrocene and neutral red were observed to efficiently transfer electrons. The biosensor based on ferrocene was characterized by long-term stability (39 days) and a wide range of substrate specificity. The lower detected concentration boundary was 25.2 mg O₂/dm³. A high correlation (R = 0.9971) was observed between the results obtained with water samples by this approach and the standard method. This is the first attempt to create a combination of yeast cells and a mediator. The biosensor can be employed for further research on the possibilities of its conventional use.     

An immunoblotting technique for the detection of bound and secreted bacterial Fc receptors

A rapid semiquantitative procedure that enables bacteria to be screened for surface or secreted receptors for the Fc region of human IgG is described. Surface Fc receptors were detected by direct transfer of bacterial colonies to nitrocellulose by electroblotting and then probing with ¹²⁵I-labeled human IgG in the presence of a two fold molar excess of unlabeled F(ab′)₂fragments. The blots were exposed to X-ray film and the intensity of the resulting autoradiograph was a measure of surface Fc receptors expression. This procedure reliably distinguished Staphylococcus aureus strains which expressed different levels of surface Fc receptors. When applied to the study of group A streptococci, a number of Fc receptor-positive strains were identified. Unlike the homogeneous Fc receptor expression on individual colonies of the staphylococcal strains, a wide variation in the level of Fc receptor expression was observed within a given streptococcal strain. Group A streptococcal substrains which expressed high and low levels of surface Fc receptors could be isolated from replica plates.Secreted Fc receptors were measured by a simple modification of the blotting procedure in which the nitrocellulose was placed on the opposite side of the agar from the bacterial colonies. Secreted Fc receptors was electroblotted through the agar onto nitrocellulose and probed as described above. This approach readily detected nanogram quantities of secreted type I Fc receptor (protein A) from the Staphylococcus aureus Cowan strain. None of the group A streptococcal strains tested were found to secrete detectable quantities of Fc receptors.     

Preparation of ferrocene-functionalized gold nanoparticles by primer extension reaction on the particle surface

DNA molecules possessing multiple ferrocene (Fc) molecules as a redox active probe were prepared by the primer extension (PEX) reaction using a 2′-deoxyuridine-5′-triphosphate derivative in which Fc was connected to the C5-position of the uridine by a diethylene glycol linker. Gold nanoparticles (AuNP) covered with DNA possessing the Fc molecules were prepared by the PEX reaction on the surface. The AuNP–FcDNA conjugates exhibit a detectable electrochemical signal due to the Fc molecules. Possible application of the PEX reaction on AuNP is demonstrated for the detection of a single nucleotide mutation in the target DNA.     

Ferrocene-assisted stabilization of collagen mimetic triple helices: solid-phase synthesis and structure

A series of ferrocene-containing collagen models Fc-CO-(Pro-Hyp-Gly)n-Cys (n = 4 (1), 6 (2), 7 (3), 8 (4), 9 (5)) were synthesized by solid-phase synthesis. Biophysical studies using circular dichroism (CD) show that these collagen analogues form triple-helical conformations, and the peptides showed a range of thermal stabilities ((T(m)), 38-74 degrees C). Results also indicate that the ferrocene (Fc)-labeled collagen models possesses a higher triple-helical propensity than the unlabeled collagen models as demonstrated by the higher melting temperatures and thermodynamic parameters, and we conclude that the Fc group at the N-terminal position of the peptide strands increases the stability of the triple helix.     

Bis(dialkylaminophosphino)ferrocenes: Reactivity and electrochemistry

New compounds containing the 1,1′-bis(dialkylaminophosphino)ferrocene ligands 1,1′-bis(dimethylaminophosphino)ferrocene (dmapf) and 1,1′-bis(diethylaminophosphino)ferrocene (deapf) were prepared and characterized by NMR. The oxidative electrochemistries of these compounds were examined in methylene chloride. The transition metal compounds M_nCl₂(P-P) (M = Pd or Pt, n = 1, P-P = dmapf or deapf; M = Au, n = 2, P-P = dmapf) displayed one-electron, Nernstian waves. The corresponding selenides were prepared and the oxidative electrochemistries, along with those of the previously prepared 1,1′-bis(alkylaminophosphine sulfide)ferrocene, were examined. The sulfides displayed one-electron, Nernstian oxidations. The selenides displayed electrochemically irreversible two-electron waves. The X-ray structures of the sulfides and selenides as well as that of the transition metal compounds [(AuCl)₂(dmapf)] and [PtCl₂(dmapf)] were determined.     

Conjugates of ferrocene with biological compounds. Coordination to gold complexes and antitumoral properties

Several bioconjugates of ferrocene with biological compounds such as aminoacid esters and related species have been prepared by reaction of chlorocarbonyl ferrocene with the corresponding amino acid ester (histidine methyl ester, tryptophan methyl ester, methionine methyl ester and lysine ethyl ester) or histamine or prolinamide in the presence of NEt₃. The reaction of the tryptophan or prolinamide ferrocene conjugates with [Au(acac)(PR₃)] (acac = acetylacetonate) results in the substitution of the proton of the cyclic NH groups by the fragment AuPR₃⁺ affording the complexes [Au(FcCO-tryptophan-OMe)(PR₃)] or [Au(FcCO-prolinamide)(PR₃)] (Fc = ferrocenyl group). The reaction of FcCO-Met-OMe with [Au(OTf)(PR₃)] (OTF = trifluoromethysulfonate) or [Au(C₆F₅)₃(OEt₂)] yields the gold(I) or gold(III) derivatives [Au(FcCO-Met-OMe)(PR₃)]OTf or [Au(C₆F₅)₃(FcCO-Met-OMe)], respectively. Cytotoxicity studies towards several cancer lines such as MCF-7, HeLa or NIE-115 have been performed. The ferrocene bioconjugates show no activity whereas the gold complexes exhibit antiproliferative effect. Preliminary studies of interaction of compounds with cells were carried out with the goal of increasing our knowledge on the mechanism of action of these potential drugs.     

Use of the Sonogashira coupling reaction for the "two-step" labeling of phenylalanine peptide side chains with organometallic compounds

The Pd-catalyzed Sonogashira coupling of ferrocene alkyne derivatives as metal probes to iodophenylalanine containing peptides is described. 4-Iodophenylalanine was incorporated into dipeptides and the neuropeptide [Leu5]-enkephalin (Enk) by solid phase peptide synthesis, thereby creating a functional group suitable for the Sonogashira coupling. The reaction with two different ferrocene alkynes resulted in the corresponding ferrocene-labeled derivatives, which were obtained in good yield and purity. All new compounds were comprehensively characterized, including elemental analysis, 1D and 2D NMR, EI-, FAB- or ESI-MS, IR and UV-vis spectroscopy, and electrochemistry of the ferrocene label. Unlike well-established conjugation methods for peptide side chains such as lysine and cystein, the phenyl group in Phe is not readily available for derivatization. This work presents a versatile procedure for the regioselective introduction of an organometallic label into biologically relevant peptides as exemplified for enkephalin.     

Direct electrochemistry of hemoglobin based on chitosan–ionic liquid–ferrocene/graphene composite film

This paper described an ingenious approach for the fabrication of a promising biosensor, hemoglobin (Hb)/chitosan (Chit)–ionic liquid (IL)–ferrocene (Fc)/graphene (Gr)/glassy carbon electrode (GCE), that exploited the synergistic beneficial characteristics of Fc, Gr and IL for Hb. The proposed biosensor showed a strong electrocatalytic activity toward the reduction of H₂O₂, which could be attributed to the favored orientation of Hb in the well-confined surface as well as the high electrical conductivity of the resulting Chit–IL–Fc/Gr inorganic hybrid composite. The developed biosensor exhibited a fast amperometric response (2 s), a good linear response toward H₂O₂ over a wide range of concentration from 50 μM to 1200 μM, and a low detection limit of 3.8 μM. The apparent Michaelis–Menten constant (K_m) of Hb on the composite medium was 0.16 mM, showing high bioelectrocatalytic activity of immobilized protein toward H₂O₂ reduction. High sensitivity and stability, technically simple and possibility of preparation at short period of time are of great advantages of the developed biosensors.     

Blank peak current-suppressed electrochemical aptameric sensing platform for highly sensitive signal-on detection of small molecule

In this contribution, an electrochemical aptameric sensing scheme for the sensitive detection of small molecules is proposed using adenosine as a target model. A ferrocene (Fc)-functionalized thiolated aptamer probe is adapted and immobilized onto an electrode surface. Introducing a recognition site for EcoRI into the aptamer sequence not only suppresses the peak current corresponding to blank sample but also provides a signal-on response mechanism. In the absence of adenosine, the aptamer can fold into a hairpin structure and form a cleavable double-stranded region. Fc is capable of being removed from electrode surface by treatment with endonuclease, and almost no peak current is observed. The adenosine/aptamer binding induces the conformational transition of designed aptamer, dissociating the cleavable double-stranded segment. Therefore, the integrated aptamer sequence is maintained when exposing to endonuclease, generating a peak current of Fc. Utilizing the present sensing scheme, adenosine even at a low concentration can give a detectable current signal. Thus, a detection limit of 10⁻¹⁰ M and a linear response range from 3.74 × 10⁻⁹ to 3.74 × 10⁻⁵ M are achieved. The proposed proof-of-principle of a novel electrochemical sensing is expected to extend to establish various aptameric platforms for the analysis of a broad range of target molecules of interest.     

Effect of some redox mediators on FAD fluorescence of glucose oxidase from Penicillium adametzii LF F-2044.1

Glucose oxidase (GOx) of Penicillium adametzii LF F-2044.1 recovered by ultrafiltration, was characterized by spectrophotometric and spectrofluorometric methods. It was shown that spectra of GOx from P. adametzii are typical for flavoproteins. Optimal buffer composition was chosen. It was determined that the GOx is the most efficiently interacting with substrate (glucose) in phosphate buffer at pH 7.0 with k_kat/K_M = 15,217 ± 550 M⁻¹ s⁻¹. P. adametzii GOx fluorescence in the presence of different redox mediators (9,10-phenanthroline-5,6-dione, 9,10-phenanthrenequinone, 1,4-benzoquinone, methylene blue, ferrocene, ferrocenecarboxylic acid, α-methylferrocenemethanol, ferrocenecarboxaldehyde) was evaluated. Maximal differences in fluorescence emission intensity were observed in the presence of ferrocene and 9,10-phenanthrenequinone.     

Electrochemical determination of uric acid at ordered mesoporous carbon functionalized with ferrocenecarboxylic acid-modified electrode

Ordered mesoporous carbon (OMC) functionalized with ferrocenecarboxylic acid (Fc) was used to modify the glassy carbon (GC) electrode. The characterization of OMC–Fc shows that, after anchoring ferrocene on the mesoporous, ordered mesostructure of the material (OMC–Fc) remains intact and Fc is electrochemically accessible. The obtained OMC–Fc-modified electrode was used to investigate the electrochemical behavior of uric acid (UA). UA oxidation is catalyzed by this electrode in aqueous buffer solution (pH 7.3) with a decrease of 200 mV in overpotential compared to GC electrode. The detection and determination of UA in the presence of ascorbic acid (AA), the main interferent, were achieved. The voltammetric signals due to UA and AA were well separated with a potential difference of 308 mV, a separation that can allow the simultaneous determination of UA and AA. With amperometric method, at a constant potential of 375 mV, the catalytic current of UA versus its concentration shows a good linearity in the range 60–390 μM (R = 0.998) with a detection limit of 1.8 μM (S/N = 3). These results are not influenced by the presence of AA in the sample solution. With good stability and reproducibility, the present OMC–Fc-modified electrode was applied in the determination of UA content in urine sample and satisfactory results were obtained.     

Synthesis, in vitro antimalarial activities and cytotoxicities of amino-artemisinin-ferrocene derivatives

Novel derivatives bearing a ferrocene attached via a piperazine linker to C-10 of the artemisinin nucleus were prepared from dihydroartemisinin and screened against chloroquine (CQ) sensitive NF54 and CQ resistant K1 and W2 strains of Plasmodium falciparum (Pf) parasites. The overall aim is to imprint oxidant (from the artemisinin) and redox (from the ferrocene) activities. In a preliminary assessment, these compounds were shown to possess activities in the low nM range with the most active being compound 6 with IC₅₀ values of 2.79?nM against Pf K1 and 3.2?nM against Pf W2. Overall the resistance indices indicate that the compounds have a low potential for cross resistance. Cytotoxicities were determined with Hek293 human embryonic kidney cells and activities against proliferating cells were assessed against A375 human malignant melanoma cells. The selectivity indices of the amino-artemisinin ferrocene derivatives indicate there is overall an appreciably higher selectivity towards the malaria parasite than mammalian cells.     

Cynomolgus and pigtail macaque IgG subclasses: characterization of IGHG genes and computational analysis of IgG/Fc receptor binding affinity

Macaques are the most widely used experimental nonhuman primate (NHP) species. Rhesus (Macaca mulatta, Macmul), cynomolgus (Macaca fascicularis, Macfas), and pigtail (Macaca nemestrina, Macnem) macaques continue to be popular models for vaccine and infectious diseases research, especially HIV infection and AIDS, and for the development of antibody-based therapeutic strategies. Increased understanding of the immune system of these species is necessary for their optimal use as models of human infections and intervention. In the past few years, the antibody/Fc receptor system has been characterized in a stepwise manner in these species. We have continued this characterization by identifying the four IG heavy gamma (IGHG) genes of Macfas and Macnem in this study. Our results show that these genes share a high degree of similarity with those from other NHP species, while presenting consistent differences when compared to human IGHG genes. Furthermore, comparison of Macfas IGHG genes with those described in other studies suggests the existence of polymorphism. Using sequence- and structure-based computational tools, we performed in silico analysis on multiple polymorphic Macfas IgG and their interactions with human IgG Fc receptors (FcγR), thus predicting that Macfas IGHG polymorphisms influence IgG protein stability and/or binding affinity towards FcγR. The presence of macaque IGHG polymorphisms and macaque/human amino acid changes at locations potentially involved in antibody functional properties indicate the need for cautious design and data interpretation of studies in these models, possibly requiring the characterization of antibody/Fc receptor interactions at the individual level.     

Iron-containing metallocenes as active site directed inhibitors of the proteinase that cleaves the NH2-terminal propeptides from type I procollagen

Derivatives of ferrocene (dicyclopentadienyliron) (Fc) were examined as active site directed inhibitors of type I procollagen N-proteinase, the enzyme that cleaves the NH2-terminal propeptides from type I procollagen. The compounds were shown here to be reversible, competitive inhibitors of the enzyme. The effectiveness of the Fc inhibitors varied with modification of the cyclopentadienyl (cp) rings. The monocarboxylic acid (I) and the 1,1'-dicarboxylic acid (II) derivatives of Fc inhibited 50% of the enzymic activity (I50) at concentrations of 1.0 and 0.5 mM, respectively. The Ki values were 0.3 mM for both I and II. Derivatization of the carbonyl alpha to the cp ring of compound I (FcCOCH2CH2COOH, III) increased the inhibitory activity (I50 = 0.100 mM; Ki = 0.065 mM). Removal of the carbonyl alpha to the cp ring of III did not improve inhibitory activity: FcCH2CH2COOH, I50 = 2 mM; FcCH = CHCOOH, I50 = 1.5 mM. The active inhibitory species apparently contained iron in the 3+ valence state since two ferrocenium derivatives were very effective inhibitors: ferrocenium tetrachloroferrate, IV (I50 = 0.030 mM; Ki = 0.004 mM), and carboxyferrocenium hexafluorophosphate, V (I50 less than 0.1 mM; Ki less than 0.05 mM). In addition, reduction of III with ascorbic acid abolished its inhibitory activity. Compounds I and III stabilized the enzyme to heat denaturation in the absence of exogenous calcium; compound IV did not stabilize the enzyme. Further observations indicated that Fc derivatives were specific inhibitors of procollagen N-proteinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

A strategic approach to the synthesis of ferrocene appended chalcone linked triazole allied organosilatranes: Antibacterial,antifungal, antiparasitic and antioxidant studies

A series of ferrocene appended chalcone allied triazole coupled organosilatranes (FCTSa 7–FCTSa 12) were synthesised with the aim of amalgamating the pharmacological action of the constituting moieties into a single molecular scaffold. All the synthesised silatranes were well characterized by various spectroscopic techniques like IR, ¹H NMR, ¹³C NMR and elemental analysis. Organosilatranes were then evaluated for their biological alacrity against bacterial and fungal strains compared with the standard drugs Rifampicin and Amphotericin B respectively. The ferrocene conjugates were found to be only moderately effective against the tested microbes. However, the organosilatranes conceded excellent efficacy against parasite G. lamblia with FCTSa 11 arraying the leading results. On the other hand against another parasite T. vaginalis, FCTSa 8 has emerged as an outstanding composite. Further, Total Antioxidant Assay (TAA) with 2,2′-azino-bis-3-(ethylbenzothiazoline-6-sulphonic acid) revealed FCTSa 10 to be the best claimant for radical scavenging activity. Along these lines, introducing some different substituents in the synthesised hybrids may act as a useful strategy for increasing the biological profile of the drugs.     

Recombinant proteins produced into yolk of genetically manipulated chickens are partly sialylated in N-glycan

The transgenic chicken is a candidate for the production of biopharmaceutical proteins with several economic superiorities. In general, the addition of sialic acid at the terminal of N-glycan is important for the bioactivity of biopharmaceuticals including plasma half-life; however, sialic acid has not been detected in the N-glycan of proteins produced in the egg white of genetically manipulated chickens. In this study, the extracellular domain of the TNF receptor and single chain Fv fused to Fc (referred to as TNFR/Fc and scFv/Fc, respectively) were purified from the egg yolk of genetically manipulated chickens and their sialylation in N-glycan was examined. In contrast to the glycan in egg white, yolk-derived proteins were partly sialylated. Lectin blot showed the existence of α2,6-sialic acid on TNFR/Fc, which disappeared with the removal of N-glycan by PNGase. In scFv/Fc, up to 7 % of N-glycan contained sialic acid. Disialyl glycans, which were detected in serum-derived scFv/Fc in a previous study, were not found in the yolk sample. Ovarian follicular tissue, which surrounds growing yolk, expressed several neuraminidases, suggesting the partial truncation of glycan during the yolk transfer process from the blood.     

Decreased Fc and C3 receptor function in macrophage populations which are refractory to migration inhibitory factor,C3 activators,and immune complex

Guinea pig macrophage populations previously established to be either responsive or refractory to activation by migration inhibitory factor (MIF) and Lotus fucolectin in the macrophage migration inhibition (MMI) assay were further characterized for their MMI response to diverse effectors as correlated with their Fc and C3b receptor function. MIF-refractory populations were found to be uniformly unresponsive to the complement activators: bacterial lipopolysaccharide, cobra venom factor, zymosan, and immune complex. MIF-responsive macrophages were responsive to the same activators. Fc-mediated binding and phagocytosis of IgG-coated sheep erythrocytes (EA) were markedly depressed in freshly harvested refractory macrophages as compared to responsive cells. Fc phagocytosis by refractory populations increased rapidly during 24–28 hr in vitro culture to levels equal to that of responsive cells which corresponded with an increase in their MMI response to MIF. Refractory macrophages also had decreased C3b receptor function as shown by reduced binding and phagocytosis of EAC or serum-coated zymosan and displayed a greater loss in C3b binding capacity than responsive cells during 48 hr in vitro culture. Trypsinization of responsive macrophages rendered them refractory in their MMI response to the various activators and selectively reversed C3b-dependent binding without effect on Fc binding. The plasmin esterase inhibitors, ?-amino-n-caproic acid, tranexamic acid, and l-lysine, previously established to reverse the MMI response to MIF, FBP, and C3 activators were found to inhibit both Fc- and C3-dependent phagocytosis. These results indicate that macrophage populations which are refractory to migration inhibition by MIF and C3 activators also have reduced Fc- and C3b-mediated phagocytic functions as compared to more mature responsive populations.     

Streptococcal Fc receptors. I. Isolation and partial characterization of the receptor from a group C streptococcus

A receptor for the Fc region of immunoglobulin G was extracted from a group C streptococcus, purified and physicochemically characterized. The Fc receptor was extracted in high yield by lysis of the bacteria after infection with bacteriophage. The soluble receptor was purified to functional homogeneity by sequential chromatography on cellulose phosphate, DEAE, and selective elution from a column of immobilized human IgG. Four hundred micrograms of the functionally pure protein was obtained per gram (wet weight) of bacteria extracted. The affinity-purified receptor was functionally homogeneous in binding to the Fc region of human IgG; however, the product was heterogeneous on both non-denaturing and SDS polyacrylamide gels. Four major protein bands were observed, with the predominant form of the Fc receptor having an m.w. of 64,000 daltons. Antibody prepared against the major Fc receptor protein ( FcRc -II) was capable of reacting with all the fractions and completely inhibiting functional activity. The results of the competitive binding studies suggest that the purified Fc receptor behaves as a single receptor, and that the differences in charge and size were probably due to covalently bound cell wall constituents.     

Optimized ferrocene-functionalized ZnO nanorods for signal amplification in electrochemical immunoassay of Escherichia coli

A novel amplified electrochemical immunoassay based on ferrocene (Fc)-functionalized ZnO nanorods (NRs) was developed in the present work. The detection antibody ((d)Ab) and Fc were immobilized onto the surface of ZnO NRs, denoted as {(d)Ab-ZnO-Fc} bioconjugates. The amount of (d)Ab and Fc in the bioconjugates was investigated using the copper reduction/bicinchoninic acid reaction (BCA protein assay) and inductive coupled plasma-atomic emission spectroscopy (ICP-AES), respectively. Greatly amplified signal was achieved in the sandwich-type immunoassay when (d)Ab and Fc linked to ZnO NRs at a proper ratio. Using Escherichia coli (E. coli) as a model antigen, the designed immunoassay showed an excellent analytical performance, and exhibited a wide dynamic response range of E. coli concentration from 10(2) to 10(6)cfu/mL with a detection limit of 50 cfu/mL (S/N=3). By introducing a pre-enrichment step, the detection of 5 cfu/10 mL E. coli in hospital sewage water was realized. This proposed signal amplification strategy was promising and could be easily extended to monitor other biorecognition events.