Kiselo-bazna i elektrokemijska svojstva meso(ortho- i meta-N-alkilpiridil)manganoporfirina

Istraživanje oksidativnog stresa stanica i molekula koje mogu umanjiti ili ukloniti oštećenja stanica uzrokovana njime predmetom je velikog interesa suvremenih istraživanja. Molekulne vrste koje uzrokuju oksidativni stres su reaktivne vrste kisika (ROS) i dušika (RNS), poput superoksidnog aniona (O2 •-), peroksinitrita (ONOO-) ili hidroksilnog radikala (OH•). Djelotvornu obranu stanice od O2 •- čine enzimi iz klase superoksid-dismutaza (SOD) koji kataliziraju reakciju 2O2 •- + 2H+ → H2O2 + O2. Potraga za terapijskim sredstvima koja bi mogla neutralizirati O2 •- dovela je do razvoja makrocikličkih kompleksa prijelaznih kovina kao potencijalnih redoks-aktivnih terapeutika. Zahvaljujući iznimnim elektronskim svojstvima, osobito djelotvornima pokazali su se spojevi iz klase manganoporfirina (MnP) koji mogu stvarati redoks cikluse sa staničnim reducensima poput glutationa, tetrahidrobiopterina ili askorbinske kiseline. Pojačana djelotvornost manganoporfirinskih mimetika SOD postignuta je supstitucijom porfirinskog prstena, čime dolazi do promjene redukcijskog potencijala kovinskog iona. Alkilacijom piridilskih prstena meso(orto-tetrapiridil) manganoporfirina postignut je redukcijski potencijal vrlo blizak optimalnom, pri čemu se svojom aktivnošću ističe meso(orto-tetraetilpiridil) manganoporfirin, MnTE-2-PyP. Pored prikladnog formalnog redukcijskog potencijala, za uspješnost djelovanja manganoporfirina in vivo ključna je njihova lipofilnost. Povećanje lipofilnosti može se postići pomicanjem alkilnih supstituenata iz orto- u meta položaje, te su nedavno sintetizirani meso(meta-tetraalkilpiridil) manganoporfirini, uključujući meso(meta-tetraetilpiridil) manganoporfirin, MnTE-3-PyP. U prvom dijelu ovog rada istražena su kiselo-bazna svojstva meso(orto- i meta-tetraetilpiridil) manganoporfirina. U vodenim otopinama manganoporfirini koordiniraju do dvije aksijalno smještene molekule vode čija deprotonacija može imati velik utjecaj na redukcijski potencijal manganoporfirina i time na njihovu citoprotektivnu djelotvornost. U vodenim otopinama kompleksa MnP u rasponu vrijednosti pH 2-13 potvrđeno je postojanje sljedećih specija: (H2O) Mn(II)TE-m-PyP4+, (HO) Mn(II)TE-m-PyP3+, (H2O)2Mn(III)TE-m-PyP5+, (H2O)(HO)Mn(III)TE-m-PyP4+, (O)(H2O)Mn(III)TE-m-PyP3+, (O)(H2O)Mn(IV)TE-m-PyP4+ i (O)(HO)Mn(IV)TE-m-PyP3+ (m =2, 3). Spektrofotometrijskim titracijama određene su sve konstante disocijacije koje povezuju navedene specije u navedenom rasponu vrijednosti pH, kao i termodinamički parametri navedenih konstanata disocijacije. S obzirom da je vrijednost redukcijskog potencijala ključna za njihovo biološko djelovanje, u drugom dijelu ovog rada istražena su elektrokemijska svojstva meso(orto- i meta-tetraetilpiridil) manganoporfirina. Cikličkom voltametrijom određeni su apsolutni formalni redukcijski potencijali i formalni redukcijski potencijali prema standardnoj vodikovoj elektrodi koji povezuju navedene specije u rasponu vrijednosti pH 2-13, kao i termodinamički parametri navedenih formalnih redukcijskih potencijala. Također su određeni elektrokemijski parametri elektronskih prijelaza navedenih specija i potvrđeni računalnim simulacijama mjerenja. Kronokulometrijskim mjerenjima određeni su difuzijski koeficijenti specija MnP i procijenjene veličine njihovih vodenih šupljina. Naposljetku, konstruirana je potpuna shema koja bi mogla opisati ponašanje kompleksa MnP u vodenim otopinama navedenom rasponu vrijednosti pH, s obzirom na navedene disocijacijske i redoks ravnoteže.There has been a great deal of interest in the research of cellular oxidative stress and particularly the molecules which could alleviate the damage it can cause. Molecular species causing oxidative stress are the reactive oxygen (ROS) and nitrogen species (RNS), such as superoxide anion (O2 •-), peroxynitrite (ONOO-) or hydroxyl radical (OH•). A class of enzymes called the superoxide dismutases (SOD), catalyzing the reaction 2O2 •- + 2H+ → H2O2 + O2, constitutes an effective cellular protection from oxidative damage. Macrocyclic complexes of transition metals have been developed as potential redox-active therapeutics able to neutralize O2 •-. The compounds from the class of manganese porphyrins (MnPs) have been shown to be particularly effective, due to their unique electronic properties enabling them to construct redox cycles that involve the consumption of superoxide and NO-derived oxidants at the expense of readily available biochemical reductants like glutathione, tetrahydrobiopterine or ascorbic acid. The increased activity of MnPs has been achieved by attenuating the metal-centered redox potential using various substituents to the porphyrin ring. The alkylation of the meso(ortho-tetrapyridyl) MnP yielded the redox potential close to optimal and one of the most active compounds was found to be the meso(ortho-tetraethylpyridyl) derivative, MnTE-2-PyP. Besides the appropriate redox potential, the efficiency of MnPs as scavengers of reactive species is influenced by their lipophylicity. Changing the position of alkyl substituents from ortho- to meta- positions on the porphyrin ring increases lipophilicity and new meso(meta-tetraalkylpyridyl) derivatives have been synthesized recently, including meso(meta-tetraethylpyridyl) derivative, MnTE-3-PyP. In the first part of this work, acid-base properties of meso(ortho- i meta-tetraethylpyridyl) MnPs have been investigated. In aqueous solutions MnP complexes can coordinate two axial water molecules which can deprotonate and have a significant influence on the redox potential of MnPs and therein their citoprotective activity. The following species have been confirmed to exist in aqueous solutions in the pH range 2-13: (H2O) Mn(II)TE-m-PyP4+, (HO)Mn(II)TE-m-PyP3+, (H2O)2Mn(III)TE-m-PyP5+, (H2O)(HO)Mn(III)TE-m-PyP4+, (O)(H2O)Mn(III)TE-m-PyP3+, (O)(H2O)Mn(IV)TE-m-PyP4+ and (O)(HO)Mn(IV)TE-m-PyP3+ (m =2, 3).. Using the combined potentiometric and spectrophotometric measurements all dissociation constants connecting these species have been determined, as well as the thermodynamic parameters of dissociation reactions. Considering that the redox potential of MnPs is crucial for their biological activity, in the second part of this work the electrochemical properties of meso(ortho- and meta-tetraethylpyridyl) MnPs have been investigated. The absolute formal potentials and the formal potentials vs. standard hydrogen electrode have been determined by cyclic voltammetry in the pH range 2-13 and the thermodynamic parameters of these formal potentials have been determined as well. Additionally, the electrochemical parameters of these electron transitions have been determined and confirmed by computer simulations. Chronocoulometric measurements enabled the determination of the diffusion coefficients of the MnP species and the sizes of their water cavities have been estimated. Finally, a complete scheme describing the behaviour of MnP complexes in aqueous solutions in the pH range 2-13 has been constructed, taking into account the dissociation and redox equilibria

Electrochemistry of redox-active Mn porphyrin-based SOD mimic MnTnBuOE-2-PyP5+ - Study of Redox Species Involved in ROS/RNS Scavenging

Manganese ortho tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, MnTnBuOE-2-PyP5+, is a third-generation redox-active compound currently undergoing preclinical exploration. This work is intended to complement the already extensive research of its chemical and biological properties by a simple electrochemical study. The thermodynamic parameters related to the Mn(IV) porphyrin species of MnTnBuOE-2-PyP5+ determined in this work support its observed reactivity as an efficient scavenger of peroxynitrite. The corresponding driving forces for the possible single-electron or two-electron reductions of ONOO- have been estimated as well

Thermal analysis of N-carbamoyl benzotriazole derivatives

Thermal properties of N-carbamoyl benzotriazole derivatives and N,N',N''-tribenzyloxyisocyanuric acid were investigated using thermogravimetric analysis and differential scanning calorimetry. The results revealed a difference between structural analogs of N-carbamoyl benzotriazole derivatives. They seem to be in agreement with the previously proposed formation of N,N',N''-tribenzyloxyisocyanuric acid from 1-(N-benzyloxycarbamoyl) benzotriazole, via an intermediary N-benzyloxyisocyanate acid, during heating. Substantially different thermal properties were observed for structural analogues, 1-(N-methoxycarbamoyl) benzotriazole and 1-(N-ethoxycarbamoyl) benzotriazole. In contrast to N-benzyloxyisocyanate, no corresponding reactions were observed for their decomposition products, i.e., methoxyisocyanate and ethoxyisocyanate

Spectrophotometric Determination of Malondialdehyde in Urine Suitable for Epidemiological Studies

A reliable method for spectrophotometric determination of urinary malondialdehyde (MDA), according to the thiobarbituric acid (TBA) assay, is described. To account for matrix interference and differences in individual urine composition, standard addition procedure was applied. The method is adequately selective (LoQ = 0.09 μM in the presence of 0.1 M creatinine and 0.5 M urea) and reliable (within-day and between-day variability of less than 5 %). The mean level of urinary MDA was 1.52 ± 0.73 µM that is in good agreement with spectrofluorometric determination (1.20 ± 0.56 μM; p = 0.085) as well as with previous studies that used HPLC. Furthermore, it is demonstrated that MDA is stabile in urine at room temperature for 24 h and when stored at –20 °C for 6 months. The described method enables simple, rapid and cost-effective determination of urinary MDA as a relevant and non-invasive marker of “whole-body” oxidative stress. This work is licensed under a Creative Commons Attribution 4.0 International License

Interaction of α-Melanocortin and Its Pentapeptide Antisense LVKAT : Effects on Hepatoprotection in Male CBA Mice

The genetic code defines nucleotide patterns that code for individual amino acids and their complementary, i.e., antisense, pairs. Peptides specified by the complementary mRNAs often bind to each other with a higher specificity and efficacy. Applications of this genetic code property in biomedicine are related to the modulation of peptide and hormone biological function, selective immunomodulation, modeling of discontinuous and linear epitopes, modeling of mimotopes, paratopes and antibody mimetics, peptide vaccine development, peptidomimetic and drug design. We have investigated sense-antisense peptide interactions and related modulation of the peptide function by modulating the effects of a-MSH on hepatoprotection with its antisense peptide LVKAT. First, transcription of complementary mRNA sequence of a-MSH in 3’→5’ direction was used to design antisense peptide to the central motif that serves as a-MSH pharmacophore for melanocortin receptors. Second, tryptophan spectrofluorometric titration was applied to evaluate the binding of a-MSH and its central pharmacophore motif to the antisense peptide, and it was concluded that this procedure represents a simple and efficient method to evaluate sense-antisense peptide interaction in vitro. Third, we showed that antisense peptide LVKAT abolished potent hepatoprotective effects of a-MSH in vivo

A Simple three-step method for design and affinity testing of new antisense peptides: an example of erythropoietin

Antisense peptide technology is a valuable tool for deriving new biologically active molecules and performing peptide–receptor modulation. It is based on the fact that peptides specified by the complementary (antisense) nucleotide sequences often bind to each other with a higher specificity and efficacy. We tested the validity of this concept on the example of human erythropoietin, a well-characterized and pharmacologically relevant hematopoietic growth factor. The purpose of the work was to present and test simple and efficient three-step procedure for the design of an antisense peptide targeting receptor-binding site of human erythropoietin. Firstly, we selected the carboxyl-terminal receptor binding region of the molecule (epitope) as a template for the antisense peptide modeling ; Secondly, we designed an antisense peptide using mRNA transcription of the epitope sequence in the 3'→5' direction and computational screening of potential paratope structures with BLAST ; Thirdly, we evaluated sense–antisense (epitope–paratope) peptide binding and affinity by means of fluorescence spectroscopy and microscale thermophoresis. Both methods showed similar Kd values of 850 and 816 µM, respectively. The advantages of the methods were: fast screening with a small quantity of the sample needed, and measurements done within the range of physicochemical parameters resembling physiological conditions. Antisense peptides targeting specific erythropoietin region(s) could be used for the development of new immunochemical methods. Selected antisense peptides with optimal affinity are potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines

Speciation of aqueous solutions of iron(III)-nitrilotriacetic acid complexes - simulations and experimental data

<p>Experimental data for a paper entitled <i>Preparation and characterization of iron(III) nitrilotriacetate complex in aqueous solutions for quantitative protein binding experiments</i> (paper in preparation; citation will be updated pending publication).</p><p> </p><p>a) Speciation of aqueous solutions of iron(III)-nitrilotriacetic acid complexes - simulations</p><p>A custom VBA script for Microsoft Excel 365 (Version 2304, build 16.0.16327.20200, 64-bit) was used to solve the 5th-order polynomial for the free ligand NTA3− as a function of all congruent association and dissociation constants and the total concentrations of iron, ligand, and hydrogen ions using the Jenkins-Traub algorithm. [1-4] For comparison, the FeNTA species distribution was also simulated using HySS software [5], which applies the Newton-Raphson algorithm to solve the mass balance equations using known equilibrium constants. [6-7]</p><p>The Excel workbook is provided with the following information:</p><p>Sheet 1: FeNTA-Speciation - contains all relevant equilibrium constants and corresponding equations, expressions for polynomial coefficients, polynomial roots, and equilibrium concentrations of all species. The values are calculated from the analytical concentrations provided to the HySS software and imported to Sheet 2.</p><p>Sheet 2: HySS-Import - contains the values obtained from the HySS software (the HySS model file is also included for reference). The values in the columns headed 'total Fe', 'total NTA' and 'p(H)' are used for the calculation in Sheet 1.</p><p>Sheet 3: Examples - contains plots for the FeNTA species distribution obtained by solving the 5th-order polynomial in Excel and HySS software: Top: Fe:NTA = 0.15 M:0.15 M, Bottom: Fe:NTA = 0.15 M:0.30 M.</p><p>Sheet 4: Polynomial-Procedure - contains the details of using the VBA script in Excel. [1]</p><p> </p><p>b) UV-Vis spectrophotometric titration of human serum transferrin (hTf) with FeNTA - experimental data and results</p><p>Additional files containing the experimental data for the UV-Vis spectrophotometric titration of human serum transferrin (hTf) with FeNTA and the obtained equilibrium constants for binding of the first and second iron(III) ion to hTf, obtained using HypSpec software [8-9], are also included.</p><p>The files contain the HypSpec model and the summary of the results in Excel, including the concentrations of all species for each titration point, the observed and calculated absorbance, and the values and errors for the molar absorption coefficients in the range from 445 to 800 nm.</p><p> </p><p>For more details, please visit: <a href="https://glymech.pharma.hr//GlyMech.html">https://glymech.pharma.hr//GlyMech.html</a>.</p><p> </p><p>References:</p><p>[1] Jenkins D (2014) Solving Quadratic, Cubic, Quartic and higher order equations; examples. In: Newton Excel Bach, not (just) an Excel Blog. <a href="https://newtonexcelbach.wordpress.com/2014/01/14/solving-quadratic-cubic-quartic-and-higher-order-equations-examples/">https://newtonexcelbach.wordpress.com/2014/01/14/solving-quadratic-cubic-quartic-and-higher-order-equations-examples/</a>. Accessed 18 May 2017</p><p>[2] Jenkins MA, Traub JF (1972) Algorithm 419: zeros of a complex polynomial [C2]. Commun ACM 15:97–99. <a href="https://doi.org/10.1145/361254.361262">https://doi.org/10.1145/361254.361262</a></p><p>[3] Jenkins MA (1975) Algorithm 493: Zeros of a Real Polynomial [C2]. ACM Trans Math Softw 1:178–189. <a href="https://doi.org/10.1145/355637.355643">https://doi.org/10.1145/355637.355643</a></p><p>[4] Ralston A, Rabinowitz P (1978) A first course in numerical analysis, 2d ed. McGraw-Hill, New York ISBN: 9780486414546 <a href="https://www.worldcat.org/title/44883559">https://www.worldcat.org/title/44883559</a></p><p>[5] Alderighi L, Gans P, Ienco A, Peters D, Sabatini A, Vacca A (1999) Hyperquad simulation and speciation (HySS): a utility program for the investigation of equilibria involving soluble and partially soluble species. Coordination Chemistry Reviews 184:311–318. <a href="https://doi.org/10.1016/S0010-8545(98)00260-4">https://doi.org/10.1016/S0010-8545(98)00260-4</a></p><p>[6] Motekaitis RJ, Martell AE (1994) The Iron(III) and Iron(II) Complexes of Nitrilotriacetic Acid. Journal of Coordination Chemistry 31:67–78. <a href="https://doi.org/10.1080/00958979408022546">https://doi.org/10.1080/00958979408022546</a></p><p>[7] Hegenauer J, Saltman P, Nace G (1979) Iron(III)-phosphoprotein chelates: stoichiometric equilibrium constant for interaction of iron(III) and phosphorylserine residues of phosvitin and casein. Biochemistry 18:3865–3879. <a href="https://doi.org/10.1021/bi00585a006">https://doi.org/10.1021/bi00585a006</a></p><p>[8] Gans P, Sabatini A, Vacca A (1996) Investigation of equilibria in solution. Determination of equilibrium constants with the HYPERQUAD suite of programs. Talanta 43:1739–1753. <a href="https://doi.org/10.1016/0039-9140(96)01958-3">https://doi.org/10.1016/0039-9140(96)01958-3</a></p><p>[9] Gans P, Sabatini A, Vacca A (1999) Determination of equilibrium constants from spectrophometric data obtained from solutions of known pH: The program pHab. Annali di Chimica 89:45–49.</p><p> </p>This work was supported by funding from the Croatian Science Foundation grant UIP-2017-05-9537 – Glycosylation as a factor in the iron transport mechanism of human serum transferrin (GlyMech). Additional support was provided by the European Regional Development Fund grants for 'Strengthening of Scientific Research and Innovation Capacities of the Faculty of Pharmacy and Biochemistry at the University of Zagreb' (KK.01.1.1.02.0021), 'Development of methods for production and labelling of glycan standards for molecular diagnostics' (KK.01.1.1.07.0055) and 'Scientific center of excellence for personalized health care' (KK.01.1.1.01.0010)

Speciation of aqueous solutions of iron(III)-nitrilotriacetic acid complexes - simulations

Experimental data for a paper entitled Method for preparation and characterization of iron(III) nitrilotriacetic acid solution for quantitative protein binding experiments (paper in preparation; citation will be updated pending publication). A custom VBA script for Microsoft Excel 365 (Version 2304, build 16.0.16327.20200, 64-bit) was used to solve the 5th-order polynomial for the free ligand NTA3− as a function of all congruent association and dissociation constants and the total concentrations of iron, ligand, and hydrogen ions using the Jenkins-Traub algorithm. [1-4] For comparison, the FeNTA species distribution was also simulated using HySS software [5], which applies the Newton-Raphson algorithm to solve the mass balance equations using known equilibrium constants. [6-7] The Excel workbook is provided with the following information: Sheet 1: FeNTA-Speciation - contains all relevant equilibrium constants and corresponding equations, expressions for polynomial coefficients, polynomial roots, and equilibrium concentrations of all species. The values are calculated from the analytical concentrations provided to the HySS software and imported to Sheet 2. Sheet 2: HySS-Import - contains the values obtained from the HySS software (the HySS model file is also included for reference). The values in the columns headed 'total Fe', 'total NTA' and 'p(H)' are used for the calculation in Sheet 1. Sheet 3: Examples - contains plots for the FeNTA species distribution obtained by solving the 5th-order polynomial in Excel and HySS software: Top: Fe:NTA = 0.15 M:0.15 M, Bottom: Fe:NTA = 0.15 M:0.30 M. Sheet 4: Polynomial-Procedure - contains the details of using the VBA script in Excel. [1] For more details, please visit: https://glymech.pharma.hr//GlyMech.html. References: [1] Jenkins D (2014) Solving Quadratic, Cubic, Quartic and higher order equations; examples. In: Newton Excel Bach, not (just) an Excel Blog. https://newtonexcelbach.wordpress.com/2014/01/14/solving-quadratic-cubic-quartic-and-higher-order-equations-examples/. Accessed 18 May 2017 [2] Jenkins MA, Traub JF (1972) Algorithm 419: zeros of a complex polynomial [C2]. Commun ACM 15:97–99. https://doi.org/10.1145/361254.361262 [3] Jenkins MA (1975) Algorithm 493: Zeros of a Real Polynomial [C2]. ACM Trans Math Softw 1:178–189. https://doi.org/10.1145/355637.355643 [4] Ralston A, Rabinowitz P (1978) A first course in numerical analysis, 2d ed. McGraw-Hill, New York ISBN: 9780486414546 https://www.worldcat.org/title/44883559 [5] Alderighi L, Gans P, Ienco A, Peters D, Sabatini A, Vacca A (1999) Hyperquad simulation and speciation (HySS): a utility program for the investigation of equilibria involving soluble and partially soluble species. Coordination Chemistry Reviews 184:311–318. https://doi.org/10.1016/S0010-8545(98)00260-4 [6] Motekaitis RJ, Martell AE (1994) The Iron(III) and Iron(II) Complexes of Nitrilotriacetic Acid. Journal of Coordination Chemistry 31:67–78. https://doi.org/10.1080/00958979408022546 [7] Hegenauer J, Saltman P, Nace G (1979) Iron(III)-phosphoprotein chelates: stoichiometric equilibrium constant for interaction of iron(III) and phosphorylserine residues of phosvitin and casein. Biochemistry 18:3865–3879. https://doi.org/10.1021/bi00585a006This work was supported by funding from the Croatian Science Foundation grant UIP-2017-05-9537 – Glycosylation as a factor in the iron transport mechanism of human serum transferrin (GlyMech). Additional support was provided by the European Regional Development Fund grants for 'Strengthening of Scientific Research and Innovation Capacities of the Faculty of Pharmacy and Biochemistry at the University of Zagreb' (KK.01.1.1.02.0021), 'Development of methods for production and labelling of glycan standards for molecular diagnostics' (KK.01.1.1.07.0055) and 'Scientific center of excellence for personalized health care' (KK.01.1.1.01.0010)

Stability and Structure of Inclusion Complexes of Zaleplon with Natural and Modified Cyclodextrins

The interaction between zaleplon (ZAL) and different cyclodextrins in aqueous solutions was investigated by spectrofluorimetric and phase solubility studies. Stability constants determined by both methods showed that among natural cyclodextrins, β-cyclodextrin (βCD) formed the most stabile complex but its solubilizing efficiency was limited. Among βCD derivatives, the complex stability and solubilisation efficiency decreased in order: randomly methylated-βCD (RAMEB) > sulphobutylether-βCD (SBEβCD ) > hydroxypropyl-βCD (HPβCD). The inclusion complexes of ZAL with βCD and RAMEB were further characterised by 1H-NMR spectroscopy and the inclusion complex formation was confirmed in both cases. ROESY spectra showed two binding modes between ZAL and βCD which exist simultaneously in the solution. The first binding mode occurs by the inclusion of the phenyl ring of ZAL into the βCD central cavity via the wider rim of the cyclodextrin cone and is dominant. The second one is formed by the inclusion of pyrazolo[1,5-a]pyrimidine ring of ZAL.(doi: 10.5562/cca1800