Mechanism of Antiradical Activity of Newly Synthesized 4,7-Dihydroxycoumarin Derivatives-Experimental and Kinetic DFT Study

Coumarin derivatives have proven beneficial biological activities, but the mechanism of their radical scavenging potency is not fully understood. In this study, the antiradical capacity of two newly synthesized 4,7-dihydroxycoumarin derivatives: (E)-3-(1-((3-hydroxy-4-methoxyphenyl)amino)-ethylidene)-2,4-dioxochroman-7-yl acetate (A-3OH) and (E)-3-(1-((4-hydroxy-3-methoxyphenyl)amino)ethylidene)-2,4-dioxochroman-7-yl acetate (A-4OH) towards HO center dot were examined by Electron Paramagnetic Resonance (EPR) Spectroscopy and Density Functional Theory (DFT). The compounds were fully characterized by the elemental microanalysis, IR, and NMR spectroscopies. The effect of pH on the acid-base equilibria is separately discussed and the predominant species at the physiological pH were determined. Several common mechanisms (Hydrogen Atom Transfer (HAT), Single-Electron Transfer followed by Proton Transfer (SET-PT), Sequential Proton Loss followed by Electron Transfer (SPLET), Radical Adduct Formation (RAF), and Intramolecular Hydrogen Atom Abstraction (iHAA)) of radical scavenging were investigated based on thermodynamic and kinetic parameters. EPR results indicated that both compounds significantly reduce the amount of present HO center dot. The results of the kinetic DFT study demonstrated that both compounds predominantly exhibit antiradical capacity through HAT and SPLET mechanisms. The estimated overall rate constants (k(overall)) proved that A-4OH shows better antioxidant capacity than A-3OH which is well-correlated with the results obtained by EPR measurement

Uticaj vanadata na vremensko-zavisni metabolički odgovor micelije Laetiporus sulphureus

Laetiporus sulphureus (Bull.) Murrill 1920, (Ph. Basidiomycota, Cl. Agaricomycetes, O. Polyporales, Fam. Polyporaceae) je jestiva gljiva, široko rasprostranjena u Evropi, Aziji i Severnoj Americi. Izolovana micelija autohtone gljive (vrba obala Dunava, Novi Sad) kultivisana je na čvrstoj podlozi i u potopljenom medijumu. Cilj istraživanja bio je da se upotrebom NMR spektroskopije ispita uticaj vanadijuma na metabolizam fosfata. Utvrđeno je da signali pozicionirani oko 4 ppm i 2,6 ppm pripadaju šećernim fosfatima (SP) i da nakon tretmana sa 10 mM vanadatom dolazi do povećanja signala za 78%, odnosno 23%. Nakon ispiranja micelijuma dolazi do postepene transformacije jednog oblika u drugi, tako da posle sat vremena signal SP na 2,6 ppm postaje dominantan u odnosu na signal pozicioniran na 4 ppm. Promene signala SP mogu ukazati na efektivnost inhibitora ili stimulatora energetskog metabolizma. Pored SP, polifosfati (PolyP) prisutni u miceliji gljive, takođe imaju ulogu u skladištenju energije. Primećeno je da se nakon izlaganja nepovoljnjim uslovima signal PolyP smanjio, što je potvrđeno u studijama sa kvascima S. cereviseae1 i N. crassa2. Nasuprot tome, nakon tretmana vanadijumom kod L. sulphureus nije došlo do redukcije signala PolyP, već je zabeležen mali porast signala dugolančanih polifosfata što može ukazati na mehanizam detoksifikacije micelije L. sulphureus kao metaboličkog odgovora micelije na dodavanje vanadata

Biofizički pristup u rasvetljavanju metabolizma vanadijuma kod gljiva

Vanadijum je esencijalni mikroelement za mnoge žive organizme i učesnik je mnogobrojnih bioloških procesa kod njih. načajnost u ispitivanju ovog elementa porasla je naglo u poslednje tri decenije pretpostavkom o njegovom značajnom doprinosu u tretmanu raznih vrsta bolesti. Skoro sve potrebne informacije koje se tiču oksidacionog oblika, koordinacione geometrije, oligomernog stanja vanadijuma kao i kako prisustvo različitih substrata inhibitora enzimskih aktivnosti utiču na njegove karakteristike se mogu dobiti upotrebom i kombinacijom različitih spektroskopskih metoda. Ispitivanje promena strukture vanadijuma kao potencijalno toksičnog ali i metabolički korisnog elementa je našlo veliku primenu u ispitivanju njegovog metabolizma kod gljiva kao vrste organizama koji imaju sposobnost usvajanja i modulacije različitih vrsta elemenata. Različite vrste gljiva usvajaju vanadijum u različitim oblicima, metabolišu ga na različite načine i imaju različite odgovore na njegovo prisustvo. bog složenih fiziko-hemijskih karakteristika i osetljivosti na uslove u kojima se nalazi, ispitivanje vanadijuma zahteva istovremenu primenu više spektroskopskih tehnika. Primena NMR, EPR, Ramanske, IC, optičke i Rendgenske spektroskopije u izučavanju ovog elementa kod gljiva rasvetljava njegovu ulogu u živim sistemima i daje nam informacije o mogućnostima biotransformacije ovog elementa što može imati značajnu ulogu u mnogim granama nauke a pre svega u medicini i zaštiti životne sredine

Struktura adrenalina u DMSO: NMR studija

Adrenalin (Adr) je fiziološki važan kateholamin koji kao hormon, neurotransmiter i lek ima širok spektar dejstava. Konformacija je definisana inter i intramolekulskim interakcijama Adr sa rastvaračem, kao i vodoničnim vezama.1-3 Postoje brojne teorijske studije koje se bave proučavanjem vodoničnih veza i konformacije Adr u različitim rastvaračima, ali nedostaju eksperimentalni podaci. U ovom istraživanju koristili smo 1H NMR, 1H-1H COSY, 1H-15N HSQC i NOESY da se ispita i uporedi struktura Adr u polarnim rastvaračima - dimetil sulfoksidu (DMSO) i vodi. Glavne razlike su dobijene za NH2 i CH2 grupu. Obe grupe pokazuju hemijske neekvivalentne protone u DMSO, koji nisu prisutni u vodi. Efekat povećane temperature i izmene rastvarača u NMR spektru pokazuje da u dimetil sulfoksidu jedan od protona Adr iz NH2 grupe formira jaku intramolekulsku vezu sa alifatičnom OH grupom, koja je donor protona drugoj vodoničnoj vezi koju Adr formira sa samim rastvaračem. U skladu sa tim rezultatima zaključeno je da Adr u dimetil sulfoksidu zauzima "škorpion" konformaciju u kojoj kateholni prsten predstavlja telo, a bočni niz zakrivljeni rep škorpije. U vodi Adr ne gradi intramolekulsku vodoničnu vezu, što govori da njegova struktura u velikoj meri zavisi od same okoline. Ovo je veoma važno za razumevanje transporta i vezivanja za receptore, kao i za razumevanje interakcija kateholamina sa biološkim molekulima, što direktno utiče na biološke efekte

INFLUENCE OF VANADIUM ON THE GROWTH AND METABOLISM OF COPRINELLUS TRUNCORUM FUNGAL MYCELIUM

Fungi could absorb heavy metals, metalloids, or radionuclides, thus fungal species possess great potential in bioremediation. Since fungi absorb the vanadium, in the present study ability of Coprinellus truncorum mycelia for vanadate uptake and its intracellular metabolism were investigated. The submerged cultivated C. truncorum was exposed to a rising concentration of vanadate. 31P NMR spectroscopy was used to investigate phosphate metabolism of the mycelium, while the status of vanadium in the cell was followed by 51V NMR spectroscopy. The mycelium could grow, and overcome vanadate presence, up to the concentration of 1.6 mM in the submerged medium. 31P NMR measurements pointed out that vanadate induced changes in the concentration of the crucial metabolite containing phosphorus, particularly sugar phosphates. The major result of vanadate action is evinced through an appearance of a signal positioned at around 2.8 ppm, and an increased signal of hexosephosphates. Using 51V NMR spectroscopy the presence of vanadate monomer in the mycelia of the fungal cell was confirmed

Biliverdin-copper complex at the physiological pH

Biliverdin (BV) is a degradation product of heme catabolism, which is rapidly converted to bilirubin (BR) by BV reductase 1. Biliverdin and unconjugated BR, commonly named bile pigments, have important function in biochemical processes. The presence of copper and other biological and toxic transitional metals at significant concentrations in bile implies the possibility that metal complexes with bile pigments can be formed 2. Consequently, our interest was to study the complex of BV with copper in physiological conditions – phosphate buffer with pH 7.4. UV-Vis spectrophotometry was applied to investigate formation/degradation of complex of BV with copper ions and to check stoichiometry by titration, showing that BV interacted with Cu2+ in 1:1 stoichiometry. Mass spectroscopy analysis confirmed this – ion at m/z 643.36 was detected. The results of Raman spectroscopy of BV were in good agreement with previous reports 3. Comparing spectra of BV and BV-Cu complex, the following differences were observed: a new band at low wave number is emerged for the complex may be attributed to Cu-N bond vibration; the band which was shifted to lower energies implicates increased stability of BV in the complex; intensity changes imply a more planar structure of BV in the complex, while stronger bands in complex imply higher delocalization of π-electrons and consequently a higher stability of the BV structure. Pertinent to this, it has been proposed that complexes of BV model compounds with Cu2+ may show unusual electronic structures that exhibit a significant ligand radical character. 1H NMR spectrum of BV in phosphate buffer had a poor resolution of signals, which may originate from aggregation, but this was of little relevance here, since the addition of copper ions led to very strong effect - the complete loss of almost all lines. The loss of signals represents the result of strong paramagnetic effects that may come from an unpaired e- that is delocalized in pπ orbitalss of the ring/ligand influencing all protons in the complex. The EPR spectrum of Cu2+ (S = 1/2; I = 3/2) in phosphate buffer shows that Cu2+ is weakly coordinated in an axial symmetry with one gr line and four lines coming from hyperfine coupling along gs. The addition of BV in equimolar concentration led to the loss of Cu2+ signal. The remaining signal in the [BV]/[Cu2+] = 1 system was broad, and did not show hyperfine structure. The g-value of the isotropic signal of BV-Cu complex was significantly lower than the average g-value of Cu2+ in the phosphate buffer indicating delocalization of the spin away from the metal nucleus. Similar EPR signals have been reported previously 4. Parallel-mode EPR showed no signal. Furthermore, the spectra were run over a wide field range and no half field lines were observed, either in parallel or in perpendicular mode. These results are consistent with S = 0 for the copper center. Further, redox properties of the complex were examined. BV showed a well-defined anodic peak. The [BV]/[Cu2+] = 2 system showed two additional oxidation peaks at much lower potentials than BV. The former potential corresponds to the oxidation of Cu1+, as we have shown previously 5. There was a slight consumption of O2 in [BV]/[Cu2+] = 1 system, which may be explained by traces of ‘free’ copper. However, in the presence of an excess of copper ([BV]/[Cu2+] = 0.5), the consumption of O2 was significant. This implies that ‘free’ Cu2+ reacts with the complex and ‘shuttles’ an e- to O2. The complex was susceptible to oxidizing agents but not to reducing agents. Considering the results obtained we conclude that, at physiological pH, BV builds a complex with copper ions in 1:1 stoichiometry. The formation of complex involves the rearrangement of electronic structure which provides increased energetic stability and strong paramagnetic effects. We believe that a complex with a highly delocalized unpaired e- and the formal BV•+-Cu1+ character best suites the outlined properties, but other structures of the complex cannot be completely ruled out. The presented results may shed new light on long-standing issues of BV chemistry and catalysis in biological systems

Biliverdin-copper complex at the physiological pH

Biliverdin (BV) is a degradation product of heme catabolism, which is rapidly converted to bilirubin (BR) by BV reductase 1. Biliverdin and unconjugated BR, commonly named bile pigments, have important function in biochemical processes. The presence of copper and other biological and toxic transitional metals at significant concentrations in bile implies the possibility that metal complexes with bile pigments can be formed 2. Consequently, our interest was to study the complex of BV with copper in physiological conditions – phosphate buffer with pH 7.4. UV-Vis spectrophotometry was applied to investigate formation/degradation of complex of BV with copper ions and to check stoichiometry by titration, showing that BV interacted with Cu2+ in 1:1 stoichiometry. Mass spectroscopy analysis confirmed this – ion at m/z 643.36 was detected. The results of Raman spectroscopy of BV were in good agreement with previous reports 3. Comparing spectra of BV and BV-Cu complex, the following differences were observed: a new band at low wave number is emerged for the complex may be attributed to Cu-N bond vibration; the band which was shifted to lower energies implicates increased stability of BV in the complex; intensity changes imply a more planar structure of BV in the complex, while stronger bands in complex imply higher delocalization of π-electrons and consequently a higher stability of the BV structure. Pertinent to this, it has been proposed that complexes of BV model compounds with Cu2+ may show unusual electronic structures that exhibit a significant ligand radical character. 1H NMR spectrum of BV in phosphate buffer had a poor resolution of signals, which may originate from aggregation, but this was of little relevance here, since the addition of copper ions led to very strong effect - the complete loss of almost all lines. The loss of signals represents the result of strong paramagnetic effects that may come from an unpaired e- that is delocalized in pπ orbitalss of the ring/ligand influencing all protons in the complex. The EPR spectrum of Cu2+ (S = 1/2; I = 3/2) in phosphate buffer shows that Cu2+ is weakly coordinated in an axial symmetry with one gr line and four lines coming from hyperfine coupling along gs. The addition of BV in equimolar concentration led to the loss of Cu2+ signal. The remaining signal in the [BV]/[Cu2+] = 1 system was broad, and did not show hyperfine structure. The g-value of the isotropic signal of BV-Cu complex was significantly lower than the average g-value of Cu2+ in the phosphate buffer indicating delocalization of the spin away from the metal nucleus. Similar EPR signals have been reported previously 4. Parallel-mode EPR showed no signal. Furthermore, the spectra were run over a wide field range and no half field lines were observed, either in parallel or in perpendicular mode. These results are consistent with S = 0 for the copper center. Further, redox properties of the complex were examined. BV showed a well-defined anodic peak. The [BV]/[Cu2+] = 2 system showed two additional oxidation peaks at much lower potentials than BV. The former potential corresponds to the oxidation of Cu1+, as we have shown previously 5. There was a slight consumption of O2 in [BV]/[Cu2+] = 1 system, which may be explained by traces of ‘free’ copper. However, in the presence of an excess of copper ([BV]/[Cu2+] = 0.5), the consumption of O2 was significant. This implies that ‘free’ Cu2+ reacts with the complex and ‘shuttles’ an e- to O2. The complex was susceptible to oxidizing agents but not to reducing agents. Considering the results obtained we conclude that, at physiological pH, BV builds a complex with copper ions in 1:1 stoichiometry. The formation of complex involves the rearrangement of electronic structure which provides increased energetic stability and strong paramagnetic effects. We believe that a complex with a highly delocalized unpaired e- and the formal BV•+-Cu1+ character best suites the outlined properties, but other structures of the complex cannot be completely ruled out. The presented results may shed new light on long-standing issues of BV chemistry and catalysis in biological systems

Supplementary data for article: Dimitrijević, M. S.; Bogdanović Pristov, J.; Žižić, M.; Stanković, D. M.; Bajuk-Bogdanović, D.; Stanić, M.; Spasić, S.; Hagen, W.; Spasojević, I. Biliverdin-Copper Complex at Physiological PH. Dalton Transactions 2019, 48 (18), 6061–6070. https://doi.org/10.1039/c8dt04724c

Supplementary material for: [https://pubs.rsc.org/en/content/articlelanding/2019/DT/C8DT04724C#!divAbstract]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/3066]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/3068

Activities of antioxidant enzymes in mycelium of fungus Phycomyces blakesleeanus

Phycomyces blakesleeanus is a strict aerobic filamentous fungus often used as a model system in studies of physiology, genetics, environmental sensing, and metabolism. As all other aerobic organisms, this fungus faces the toxic effects of oxygen-reactive species, but data about its antioxidative defense systems are scarce. The aim of this research was to examine the activities of three antioxidant enzymes during different phases of growth. The fungus was grown in two ways, in Petri dishes, and on a shaker in Erlenmeyer flasks. The activities of superoxide dismutase (SOD) and peroxidase (POD) were determined spectrophotometrically, while the activity of catalase (CAT) was determined polarographically with a Clark-type oxygen electrode. The highest activities of SOD were noticed in mycelia grown in Erlenmeyer flasks in the stationary phase of growth. In mycelia grown in Petri dishes, the highest activities of POD (0,014U/mg protein) and CAT (20,63 U/mg protein) were noticed in the early exponential phase. The activities of these two enzymes decreased with mycelial growth. In mycelia grown in Erlenmeyer flasks, POD and CAT showed similar behavior, but differences in activities between exponential and stationary phases were smaller and the highest activities were noticed in the mid-exponential to stationary phase of growth (0,011U/mg protein for POD and 17,21U/mg protein for CAT). High activities of these two enzymes indicated increased production of H2O2 and pointed out the importance of this phase for mycelia grown in this way

PRODUCTION AND CHARACTERISATION OF SELENIUM NANOPARTICLES BY MYCELIUM OF FUNGUS PHYCOMYCES BLAKESLEEANUS

In this study, mycelium of fungus Phycomyces blakesleeanus was exposed to soluble toxic form of selenium, selenite (Se+4), to examine its ability to reduce it to nanoparticles. Red coloration appeared after only a few hours of incubation with 10 mM Se+4 indicating formation of selenium nanoparticles (SeNPs). SEM-EDS confirmed pure selenium NPs with an average diameter of 57 nm, which indicates to potentially very good medical, optical and photoelectric characteristics. Raman spectroscopy showed several structural forms of SeNPs formed in the extracellular space with a monoclinic Se8 chain as the most represented, and the other observed forms were trigonal Se polymer chain, Se8 ring and Se6 chain structures