Differential Effects of Betacyanin and Betaxanthin Pigments on Oxidative Stress and Inflammatory Response in Murine Macrophages.

SCOPE: Betalain pigments are increasingly highlighted for their bioactive and anti-inflammatory properties, although research is lacking to demonstrate contributions of individual betalains. The work herein aimed to compare effects of four main betalains on inflammatory and cell-protective markers and to highlight potential structure-related relationships of the two main subgroups: betacyanins vs betaxanthins. METHODS AND RESULTS: Murine RAW 264.7 macrophages were stimulated with bacterial lipopolysaccharide following incubation with betacyanins (betanin, neobetanin) and betaxanthins (indicaxanthin, vulgaxanthin I) in concentrations from 1 to 100 µM. All betalains suppressed expression of pro-inflammatory markers IL-6, IL-1ß, iNOS, and COX-2 with tendency for stronger effects of betacyanins compared to betaxanthins. In contrast, HO-1 and gGCS showed mixed and only moderate induction, while more emphasized effects were observed for betacyanins. While all betalains suppressed mRNA levels of NADPH oxidase 2 (NOX-2), a superoxide generating enzyme, only betacyanins were able to counteract hydrogen peroxide induced reactive oxygen species (ROS) generation, in alignment with their radical scavenging potential. Furthermore, betaxanthins exerted pro-oxidant properties, elevating ROS production beyond hydrogen peroxide stimulation. CONCLUSION: In summary, all betalains display anti-inflammatory properties, although only betacyanins demonstrate radical scavenging capacities, indicating potential differing responses under oxidative stress conditions, which requires further research.

Uptake and Immunomodulatory Properties of Betanin, Vulgaxanthin I and Indicaxanthin towards Caco-2 Intestinal Cells.

The present study aimed to compare the absorption and transport patterns of three main betalains, betanin, vulgaxanthin I and indicaxanthin, into intestinal epithelial cells and to assess their distinct molecular effects on inflammatory and redox-related cell signalling in association with their radial scavenging potencies. All three betalains showed anti-inflammatory effects (5-80 µM), reflected by attenuated transcription of pro-inflammatory mediators such as cyclooxygenase-2 and inducible NO-synthase. Concomitant increases in antioxidant enzymes such as heme oxygenase-1 were only observed for betanin. Moreover, betanin uniquely demonstrated a potent dose-dependent radical scavenging activity in EPR and cell-based assays. Results also indicated overall low permeability for the three betalains with Papp of 4.2-8.9 × 10-7 cm s-1. Higher absorption intensities of vulgaxanthin and indicaxanthin may be attributed to smaller molecular sizes and greater lipophilicity. In conclusion, betanin, vulgaxanthin I and indicaxanthin have differentially contributed to lowering inflammatory markers and mitigating oxidative stress, implying the potential to ameliorate inflammatory intestinal disease. Compared with two betaxanthins, the greater efficacy of betanin in scavenging radical and promoting antioxidant response might, to some extent, compensate for its poorer absorption efficiency, as demonstrated by the Caco-2 cell model.

Multipurpose made colorimetric materials for amines, pH change and metal ion detection.

Sensors are routinely developed for specific applications, but multipurpose sensors are challenging, due to stability and poor functional design. We report organic materials that operate in solution and gas phase. They show a strong response behaviour to at least three types of environmental changes: pH, amine and metal ion binding/detection. We have confirmed and validated our findings using various analytical and computational methods. We found that the changes in polarity of the solvent and pH not only red shift the tail of the absorption spectra, but also extend the peak optical absorption of these structures by up to 100 nm, with consequential effects on the optical gap and colour changes of the materials. Acid-base response has been studied by spectrophotometric titrations with trifluoroacetic acid (TFA) and triethyl amine (TEA). The experiments show excellent reversibility with greater sensitivity to base than acid for all compounds. Analysis into metal sensing using Zn(ii) and Cu(ii) ions as analytes show that the materials can successfully bind the cations forming stable complexes. Moreover, a strong suppression of signal with copper gives an operative modality to detect the copper ion as low as 2.5 × 10-6 M. The formation of the metal complexes was also confirmed by growing crystals using a slow diffusion method; subsequent single crystal X-ray analysis reveals the ratio of ligand to metal to be 2 to 1. To test sensitivity towards various amine vapours, paper-based sensors have been fabricated. The sensors show a detection capability at 1 ppm of amine concentration. We have employed CIE L*a*b* colour space as the evaluation method, this provides numeric comparison of the samples from different series and allows comparison of small colour differences, which are generally undetectable by the human-eye. It shows that the CIE L*a*b* method can assess both sensitivity to a particular class of analytes and a specificity response to individual amines in this subclass offering an inexpensive and versatile methodology.

Novel approach for purification of major betalains using flash chromatography and comparison of radical scavenging and antioxidant activities.

The present study focused on the development of a new purification protocol suitable for betanin and other major betalains, vulgaxanthin I, indicaxanthin and neobetanin, using flash chromatography which is a convenient and fast method to isolate unstable materials. Following preliminary tests, a gradient procedure using 0-60% acetonitrile, with 0.1% (v/v) formic acid as mobile phase, was selected for the purification. Different fractions were collected based on UV detection at 254 and 280 nm and purities were confirmed by reverse-phase HPLC analysis to be 97%, 95%, 79% and 52% for betanin, indicaxanthin, vulgaxanthin I, and neobetanin, respectively, with pigment yields ranging from 120 to 487 mg per 100 g of powdered raw material. Comparative assessment of antioxidant and radial scavenging properties of individual betalains indicated highest potential for betanin followed by neobetanin, vulgaxanthin I and indicaxanthin.

Classic highlights in porphyrin and porphyrinoid total synthesis and biosynthesis.

Porphyrins feature prominently in nature, be it as enzymatic cofactors, electron and exciton shuffles, as photoactive dyes, or as signaling substances. Their involvement in the generation, storage and use of oxygen is pivotal to life, while their photochemical properties are central to the biochemical functioning of plants. When complexed to metals, porphyrins can engage in a multitude of contemporary applications ranging from solar energy generation to serving as catalysts for important chemical reactions. They are also able to function as useful theranostic agents, and as novel materials for a wide range of applications. As such, they are widely considered to be highly valuable molecules, and it almost goes without saying that synthetic organic chemistry has dramatically underpinned all the key advances made, by providing reliable access to them. In fact, strategies for the synthesis of functionalized porphyrins have now reached a state of refinement where pretty well any desired porphyrin can successfully be synthesized with the approaches that are available, including a cornucopia of related macrocycle-modified porphyrinoids. In this review, we are going to illustrate the development of this exciting field by discussing a number of classic syntheses of porphyrins. Our coverage will encompass the natural protoporphyrins and chlorophylls, while also covering general strategies for the synthesis of unsymmetrical porphyrins and chlorins. Various industrial syntheses of porphyrins will also be discussed, as will other routes of great practical importance, and avenues to key porphyrinoids with modified macrocycles. A range of selected examples of contemporary functionalization reactions will be highlighted. The various key syntheses will be described and analyzed from a traditional mechanistic organic chemistry perspective to help student readers, and those who are new to this area. The aim will be to allow readers to mechanistically appreciate and understand how many of these fascinating ring-systems are built and further functionalized.

Fabrication of Hybrid Materials from Titanium Dioxide and Natural Phenols for Efficient Radical Scavenging against Oxidative Stress.

Oxidative stress caused by free radicals is one of the great threats to inflict intracellular damage. Here, we report a convenient approach to the synthesis, characterization, and evaluation of the radical activity of titanium-based composites. We have investigated the potential of natural antioxidants (curcumin, quercetin, catechin, and vitamin E) as radical scavengers and stabilizers. The titanium oxide composites were prepared via three steps including sol-gel synthesis, carboxylation, and esterification. The characterization of the titanium-phenol composites was carried out by FTIR, PXRD, UV-vis and SEM methods. The radical scavenging ability of the novel materials was evaluated using DPPH and an in vitro LPO assay using isolated rat liver mitochondria. The novel materials exhibit both a higher stability and an antioxidant activity in comparison to bare TiO2. It was found that curcumin and quercetin based composites show the highest antioxidant efficiency among the composites under study followed by catechin and vitamin E based materials. The results from an MTT assay carried out on the Caco-2 cell line indicate that the composites do not contribute to the cytotoxicity in vitro. This study demonstrates that a combination of powerful antioxidants with titanium dioxide can change its functional properties and provide a convenient strategy against oxidative stress.

A photochemical approach for a fast and self-limited covalent modification of surface supported graphene with photoactive dyes.

Herein, we report a simple method for a covalent modification of surface supported graphene with photoactive dyes. Graphene was fabricated on cubic-SiC/Si(001) wafers due to their low cost and suitability for mass-production of continuous graphene fit for electronic applications on millimetre scale. Functionalisation of the graphene surface was carried out in solution via white light induced photochemical generation of phenazine radicals from phenazine diazonium salt. The resulting covalently bonded phenazine-graphene hybrid structure was characterised by scanning tunnelling microscopy (STM) and spectroscopy (STS), Raman spectroscopy and density functional theory (DFT) calculations. It was found that phenazine molecules form an overlayer, which exhibit a short range order with a rectangular unit cell on the graphene surface. DFT calculations based on STM results reveal that molecules are standing up in the overlayer with the maximum coverage of 0.25 molecules per graphene unit cell. Raman spectroscopy and STM results show that the growth is limited to one monolayer of standing molecules. STS reveals that the phenazine-graphene hybrid structure has a band gap of 0.8 eV.

White light induced covalent modification of graphene using a phenazine dye.

Herein, we report a novel strategy for a covalent modification of graphene nanoplatelets with photoactive dyes. The functionalization of the graphene surface was carried out using white light to photochemically generate phenazine radicals and the reaction progress was followed up spectrophotometrically. The characterization of the modified material was carried out using FTIR, XRD, UV-vis absorption, fluorescence, Raman spectroscopy and SEM imaging. The hybrid material has improved solubility, shows an optical band gap of 1.95 eV and is highly emissive in the visible wavelength region.

Lead Structures for Applications in Photodynamic Therapy. 6. Temoporfin Anti-Inflammatory Conjugates to Target the Tumor Microenvironment for In Vitro PDT.

Due to the ongoing development of clinical photodynamic therapy (PDT), the search continues for optimized photosensitizers that can overcome some of the side effects associated with this type of treatment modality. The main protagonists being: post-treatment photosensitivity, due to only limited cellular selectivity and post-treatment tumor regrowth, due to the up-regulation of pro-inflammatory agents within the tumor microenvironment. A photosensitizer that could overcome one or both of these drawbacks would be highly attractive to those engaged in clinical PDT. Certain non-steroidal anti-inflammatory drugs (NSAIDs) when used in combination with PDT have shown to increase the cytotoxicity of the treatment modality by targeting the tumor microenvironment. Temoporfin (m-THPC), the gold standard chlorin-based photosensitizer (PS) since its discovery in the 1980's, has successfully been conjugated to non-steroidal anti-inflammatory compounds, in an attempt to address the issue of post-treatment tumor regrowth. Using a modified Steglich esterification reaction, a library of "iPorphyrins" was successfully synthesized and evaluated for their PDT efficacy.

Lead structures for applications in photodynamic therapy. 5. Synthesis and biological evaluation of water soluble phosphorus(V) 5,10,15,20-tetraalkylporphyrins for PDT.

Improved photosensitizers for use in photomedicine must possess good water-solubility and optimal photophysical properties. Phosphorus(V) porphyrins fulfill these criteria and are a class of porphyrins with significant potential applications in phototherapy. Five phosphorus(V) porphyrins bearing alkyl substituents have been synthesized. Reasonable to good yields were obtained for all P(V) insertions and all compounds underwent biological evaluation for their PDT activity on two esophageal cancer cell lines, OE33 and SKGT-4. Their cellular uptake was investigated using a high content screening method. Notably, three compounds displayed good uptake and using the MTS cell proliferation assay, two were shown to have photocytotoxicity comparable to mTHPC (Temoporfin(®)) with IC50 values of 6.5 and 5.5 µM.

Homolytic cleavage of molecular oxygen by manganese porphyrins supported on Ag(111).

Oxygen binding and cleavage are important for both molecular recognition and catalysis. Mn-based porphyrins in particular are used as catalysts for the epoxidation of alkenes, and in this study the homolytic cleavage of O2 by a surface-supported monolayer of Mn porphyrins on Ag(111) is demonstrated by scanning tunneling microscopy, X-ray absorption, and X-ray photoemission. As deposited, {5,10,15,20-tetraphenylporphyrinato}Mn(III)Cl (MnClTPP) adopts a saddle conformation with the average plane of its macrocycle parallel to the substrate and the axial Cl ligand pointing upward, away from the substrate. The adsorption of MnClTPP on Ag(111) is accompanied by a reduction of the Mn oxidation state from Mn(III) to Mn(II) due to charge transfer between the substrate and the molecule. Annealing the Mn(II)ClTPP monolayer up to 510 K causes the chlorine ligands to desorb from the porphyrins while leaving the monolayer intact. The Mn(II)TPP is stabilized by the surface acting as an axial ligand for the metal center. Exposure of the Mn(II)TPP/Ag(111) system to molecular oxygen results in the dissociation of O2 and forms pairs of Mn(III)OTPP molecules on the surface. Annealing at 445 K reduces the Mn(III)OTPP complex back to Mn(II)TPP/Ag(111). The activation energies for Cl and O removal were found to be 0.35 ± 0.02 eV and 0.26 ± 0.03 eV, respectively.

Ni-Cu ion exchange observed for Ni(II)-porphyrins on Cu(111).

A Ni-Cu ion exchange has been observed for (5,15-dibromo-10,20-diphenylporphyrinato)nickel(II) (NiDBrDPP) and (5,10,15,20-tetrakis(4-bromophenyl)porphyrinato)nickel(II) (NiTBrPP) on Cu(111). The ion exchange proceeds at a faster rate for the NiDBrDPP/Cu(111) system compared to NiTBrPP/Cu(111). This is explained in terms of the macrocycle-substrate distance and the distortions that occur when the molecules are deposited on the Cu(111) surface.

Synthesis and biological evaluation of Foscan® bile acid conjugates to target esophageal cancer cells.

Porphyrins and chlorins such as Foscan® have a natural proclivity to accumulate in cancer cells. This trait has made them good candidates for photosensitizers and as imaging agents in phototherapy. In order to improve on cellular selectivity to lower post-treatment photosensitivity bile acid porphyrin bioconjugates have been prepared and investigated in esophageal cancer cells. Bile acids which are known to selectively bind to, or be readily taken up by cancer cells were chosen as targeting moieties. Synthesis of the conjugates was achieved via selective nucleophilic monofunctionalization of 5,10,15,20-tetrahydroxyphenylporphyrins with propargyl bromide followed by Cu(I) mediated cycloaddition with bile acid azides in good yields. The compounds were readily taken up by esophageal cancer cells but showed no PDT activity.

Correlation between charge-transfer and rotation of C60 on WO2/W(110).

Understanding molecular switching between different charge states is crucial to further progress in molecule-based nano-electronic devices. Herein we have employed scanning tunnelling microscopy to visualize different charge states of a single C60 molecule within a molecular layer grown on the WO2/W(110) surface. The results obtained demonstrate that individual C60 molecules within the layer switch between neutral and negatively charged states in the temperature range of 220-260 K over the time scale of the experiment. The charging of the C60 causes changes in the local density of electron states and consequently a variation in tunnelling current. Using density functional theory calculations, it was found that the charged state corresponds to the negatively charged C60(-), which has accepted an electron. The switching of the molecule into the charged state is triggered continuously by tunnelling electrons when the STM tip is static above an individual C60 molecule with a bias applied. Molecular movement accompanies the molecule's switching between these states.

Synthesis and evaluation of the europium(III) and zinc(II) complexes as luminescent bioprobes in high content cell-imaging analysis.

Novel phenanthroline derivatives and their europium(III) and zinc(II) complexes have been prepared in up to 92%. In contrast to the stable zinc complexes, the europium compounds exhibit a strong luminescence in THF solution. However, quenching of the emission is observed in DMSO indicating complete dissociation of the complexes back to free ligands in this solvent. (1)H NMR studies of the Eu(III)-complexes 5 and 6 also confirmed the existence of different states depending on the solvent used. Moreover, it was found that compound 5 is stable in EtOH-PBS solutions; here a strong signal in the emission spectra corresponding to the europium ion was detected. No spectral changes were observed for the zinc(II) complexes, they were shown to be stable in the media. These metal complexes can be used as fluorescence markers for the diagnosis of oesophageal squamous carcinoma (OE21) cells at low concentrations. Cell images were acquired using the compounds 5, 7-9 as luminescent agents. The first images were taken already after 20 min incubation time at a very low concentration range (0.7-1.6 µM).

Evidence for the formation of an intermediate complex in the direct metalation of tetra(4-bromophenyl)-porphyrin on the Cu(111) surface.

A strong molecule-surface interaction between free-base-tetra(4-bromophenyl)-porphyrin and Cu(111) results in a distortion of both the molecule and the underlying copper surface in the vicinity of the molecule. This in turn leads to the formation of an intermediate complex due to bonding between the iminic nitrogens and surface copper atoms.

Simple but powerful: phenanthroline-based small molecules for cellular imaging and cancer screening.

A two-step synthetic procedure gives highly fluorescent phenanthroline molecular probes. The compounds localize in the endoplasmic reticulum and their potential as bioactive probes was evaluated. The materials are quickly taken up by living cells within 5 min. Preliminary in vitro studies have shown that these compounds are selective to esophageal cancer cells and can be used as selective markers in intracellular cancer diagnostics. The materials show a remarkable cytotoxicity towards cancer cells vs normal as 7-1.

Self-assembled rows of Ni porphyrin dimers on the Ag(111) surface.

The growth and ordering of 5-(10,15,20-triphenylporphyrinatonickel(ii))dimer (NiTPP-dimer) molecules on the Ag(111) surface have been investigated using scanning tunnelling microscopy/spectroscopy (STM/STS) and low-energy electron diffraction (LEED). At one monolayer (ML) coverage the NiTPP-dimer forms a well-ordered close-packed molecular layer in which the porphyrin molecules have a flat orientation with the molecular plane lying parallel to the substrate. STM and LEED data obtained from one monolayer of the NiTPP-dimer on the Ag(111) surface show the formation of three domains which grow along the main crystallographic directions of the substrate. Scanning tunnelling spectroscopy data obtained from the NiTPP-dimer on the Ag(111) surface show good agreement with optical band gap measurements and density functional theory calculations.

Synthesis of stannyl porphyrins and porphyrin dimers via stille coupling and their (119)Sn NMR and fluorescence properties.

Free base stannyl porphyrins and free base porphyrin dimers have been successfully synthesized via copper-free Stille coupling in 21-67% yields. This approach provides an access to stannyl porphyrin synthons that were previously unavailable. Moreover, variation of the reaction conditions selectively provides access to either stannyl porphyrins or porphyrin dimers. Full (119/117)Sn NMR analysis was used for characterization of the stannyl porphyrins and detailed (119)Sn-(1)H-(13)C NMR analyses were carried out on a series of the starting tin reagents and the stannyl porphyrins. These investigations indicate that significant structural information can be gathered by use of commonly known NMR techniques. Photophysical properties of the novel porphyrins prepared including absorption, emission, and fluorescence lifetimes were investigated. The stannyl porphyrins emitted in the visible region, and in all cases large Stokes shifts were observed. The emission intensities of the stannyl porphyrins were 100-fold higher than those of the starting bromoporphyrins. Measured fluorescence lifetime (S(0)-->S(1)) of the stannyl and dimeric porphyrins were in the 7.7-12 ns region.

Synthesis, transformations, and comparative studies of porphyryl acrylic acids and their homologues.

The reactivity of porphyryl acrylates and their homologues was studied systematically and to establish their potential as building blocks for the synthesis of novel tetrapyrroles. A new synthetic approach for multifunctional porphyrins was developed using alpha,beta-unsaturated acyl porphyrins as versatile building blocks with yields of 44-95%. The reaction of acyl chlorides generated in situ with ethyl diazoacetate in the presence of PPh(3) led to the corresponding phosphazine, which was quickly self-transformed into a novel porphyrin beta-keto ester system in up to 78% yield. Comparative studies of the next homologue of acrylic porphyrins, i.e., those bearing an additional CH(2) group next to the double bond of the alpha,beta-unsaturated fragment, showed that these can undergo rearrangement reactions via vinylketenes to yield both regioisomers. Depending on the reaction conditions, this method gives regioselectively access to either a rearrangement product or the product of esterification reactions in yields of 81% or 57%, respectively. Enyne metathesis of novel propargyl esters with allyl porphyrins provided an easy access to 1,3-disubstituted butadienes in up to 76% yield.