Reducing the Inner Filter Effect in Microplates by Increasing Absorbance? Linear Fluorescence in Highly Concentrated Fluorophore Solutions in the Presence of an Added Absorber.

The fluorescence attenuation caused by the absorption of the excitation and/or emission light is called the Inner Filter Effect (IFE) and can lead to a nonlinear fluorescence concentration response. In this article, we propose the AddAbs (Added Absorber) method, which counterintuitively corrects IFE by increasing the absorbance of the sample. In this method, an equal amount of a highly absorbing chromophore is added to each sample to compensate for the nonuniform quenching caused by different fluorophore concentrations. The AddAbs method was able to provide a linear fluorescence response (R2 > 0.999) for very concentrated fluorophore solutions with extreme IFE over more than 97% of the concentration range with less than 1% deviation in calibration slope. The true limit for the AddAbs method with respect to fluorophore concentration was apparently not reached and could be higher than measured (Aex,1cm > 33.94). The IFE-corrected data are obtained by a single fluorescence measurement per sample without additional mathematical procedures. The method also does not require absorbance measurements, so it can be performed in non-transparent microplates with similar results. In addition, preliminary measurements indicate that the method is also suitable for measurements in standard cuvettes using a fluorimeter with a 90° angle setup.

Potential Clinically Relevant Effects of Sialylation on Human Serum AAG-Drug Interactions Assessed by Isothermal Titration Calorimetry: Insight into Pharmacoglycomics?

Human serum alpha-1 acid glycoprotein is an acute-phase plasma protein involved in the binding and transport of many drugs, especially basic and lipophilic substances. It has been reported that the sialic acid groups that terminate the N-glycan chains of alpha-1 acid glycoprotein change in response to certain health conditions and may have a major impact on drug binding to alpha-1 acid glycoprotein. The interaction between native or desialylated alpha-1 acid glycoprotein and four representative drugs-clindamycin, diltiazem, lidocaine, and warfarin-was quantitatively evaluated using isothermal titration calorimetry. The calorimetry assay used here is a convenient and widely used approach to directly measure the amount of heat released or absorbed during the association processes of biomolecules in solution and to quantitatively estimate the thermodynamics of the interaction. The results showed that the binding of drugs with alpha-1 acid glycoprotein were enthalpy-driven exothermic interactions, and the binding affinity was in the range of 10-5-10-6 M. Desialylated alpha-1 acid glycoprotein showed significantly different binding with diltiazem, lidocaine, and warfarin compared with native alpha-1 acid glycoprotein, whereas clindamycin showed no significant difference. Therefore, a different degree of sialylation may result in different binding affinities, and the clinical significance of changes in sialylation or glycosylation of alpha-1 acid glycoprotein in general should not be neglected.

Inner Filter Effect Correction for Fluorescence Measurements in Microplates Using Variable Vertical Axis Focus.

The inner filter effect (IFE) hinders fluorescence measurements, limiting linear dependence of fluorescence signals to low sample concentrations. Modern microplate readers allow movement of the optical element in the vertical axis, changing the relative position of the focus and thus the sample geometry. The proposed Z-position IFE correction method requires only two fluorescence measurements at different known vertical axis positions (z-positions) of the optical element for the same sample. Samples of quinine sulfate, both pure and in mixtures with potassium dichromate, showed a linear dependence of corrected fluorescence on fluorophore concentration (R2 > 0.999), up to Aex ≈ 2 and Aem ≈ 0.5. The correction extended linear fluorescence response over ≈98% of the concentration range with ≈1% deviation of the calibration slope, effectively eliminating the need for sample dilution or separate absorbance measurements to account for IFE. The companion numerical IFE correction method further eliminates the need for any geometric parameters with similar results. Both methods are available online at https://ninfe.science.

Rational design of superoxide dismutase (SOD) mimics: the evaluation of the therapeutic potential of new cationic Mn porphyrins with linear and cyclic substituents.

Our goal herein has been to gain further insight into the parameters which control porphyrin therapeutic potential. Mn porphyrins (MnTnOct-2-PyP(5+), MnTnHexOE-2-PyP(5+), MnTE-2-PyPhP(5+), and MnTPhE-2-PyP(5+)) that bear the same positive charge and same number of carbon atoms at meso positions of porphyrin core were explored. The carbon atoms of their meso substituents are organized to form either linear or cyclic structures of vastly different redox properties, bulkiness, and lipophilicities. These Mn porphyrins were compared to frequently studied compounds, MnTE-2-PyP(5+), MnTE-3-PyP(5+), and MnTBAP(3-). All Mn(III) porphyrins (MnPs) have metal-centered reduction potential, E1/2 for Mn(III)P/Mn(II)P redox couple, ranging from -194 to +340 mV versus NHE, log kcat(O2(•-)) from 3.16 to 7.92, and log kred(ONOO(-)) from 5.02 to 7.53. The lipophilicity, expressed as partition between n-octanol and water, log POW, was in the range -1.67 to -7.67. The therapeutic potential of MnPs was assessed via: (i) in vitro ability to prevent spontaneous lipid peroxidation in rat brain homogenate as assessed by malondialdehyde levels; (ii) in vivo O2(•-) specific assay to measure the efficacy in protecting the aerobic growth of SOD-deficient Saccharomyces cerevisiae; and (iii) aqueous solution chemistry to measure the reactivity toward major in vivo endogenous antioxidant, ascorbate. Under the conditions of lipid peroxidation assay, the transport across the cellular membranes, and in turn shape and size of molecule, played no significant role. Those MnPs of E1/2 ∼ +300 mV were the most efficacious, significantly inhibiting lipid peroxidation in 0.5-10 µM range. At up to 200 µM, MnTBAP(3-) (E1/2 = -194 mV vs NHE) failed to inhibit lipid peroxidation, while MnTE-2-PyPhP(5+) with 129 mV more positive E1/2 (-65 mV vs NHE) was fully efficacious at 50 µM. The E1/2 of Mn(III)P/Mn(II)P redox couple is proportional to the log kcat(O2(•-)), i.e., the SOD-like activity of MnPs. It is further proportional to kred(ONOO(-)) and the ability of MnPs to prevent lipid peroxidation. In turn, the inhibition of lipid peroxidation by MnPs is also proportional to their SOD-like activity. In an in vivo S. cerevisiae assay, however, while E1/2 predominates, lipophilicity significantly affects the efficacy of MnPs. MnPs of similar log POW and E1/2, that have linear alkyl or alkoxyalkyl pyridyl substituents, distribute more easily within a cell and in turn provide higher protection to S. cerevisiae in comparison to MnP with bulky cyclic substituents. The bell-shape curve, with MnTE-2-PyP(5+) exhibiting the highest ability to catalyze ascorbate oxidation, has been established and discussed. Our data support the notion that the SOD-like activity of MnPs parallels their therapeutic potential, though species other than O2(•-), such as peroxynitrite, H2O2, lipid reactive species, and cellular reductants, may be involved in their mode(s) of action(s).

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.

Hepatoprotective effects of Met-enkephalin on acetaminophen-induced liver lesions in male CBA mice.

Recent histopathological investigations in patients with hepatitis suggested possible involvement of Met-enkephalin and its receptors in the pathophysiology of hepatitis. Consequently, we evaluated the potential hepatoprotective effects of this endogenous opioid pentapeptide in the experimental model of acetaminophen induced hepatotoxicity in male CBA mice. Met-enkephalin exhibited strong hepatoprotective effects in a dose of 7.5 mg/kg, which corresponds to the protective dose reported for several different animal disease models. In this group plasma alanine aminotransferase and aspartate aminotransferase enzyme activities, as well as liver necrosis score were significantly reduced in comparison to control animals treated with physiological saline (p>0.01). The specificity of the peptide hepatoprotection was investigated from the standpoint of the receptor and peptide blockade. It was concluded that Met-enkephalin effects on the liver were mediated via δ and ζ opioid receptors. Genotoxic testing of Met-enkephalin confirmed the safety of the peptide.

Protein sialylation affects the pH-dependent binding of ferric ion to human serum transferrin.

Physiological or pathophysiological changes lead to posttranslational changes in the sialic acid content of human serum transferrin (hTf), an essential mediator of iron transport in the human body, resulting in a significantly increased concentration of desialylated hTf. The intrinsic fluorescence quenching upon binding of iron to hTf was successfully modeled using the binding polynomial for two iron-binding sites, allowing measurements in a high-throughput format. Removal of sialic acid residues resulted in a 3-fold increase in iron binding affinity for both sites of hTf at pH 7.4. The pH-dependence of iron binding showed significant differences in equilibrium constants, resulting in a 10-fold increase in binding affinity for desialylated hTf at pH 5.9. The changes in hTf sialylation apparently result in tuning of the stability of the conformational state, which in turn contributes to the stability of the diferric hTf. The observed differences in the conditional thermodynamic equilibrium constants suggest that the desialylated protein has a higher preference for diferric hTf over monoferric hTf species down to pH 6.5, which may also influence the interaction with transferrin receptors that preferentially bind to diferric hTf. The results suggest a link between changes in hTf glycan structure and alterations in iron binding equilibrium associated with tissue acidosis.

Influence of Desialylation on the Drug Binding Affinity of Human Alpha-1-Acid Glycoprotein Assessed by Microscale Thermophoresis.

Human serum alpha-1-acid glycoprotein (AAG) is an acute-phase plasma protein involved in the binding and transport of many drugs, especially basic and lipophilic substances. The sialic acid groups that terminate the N-glycan chains of AAG have been reported to change in response to numerous health conditions and may have an impact on the binding of drugs to AAG. In this study, we quantified the binding between native and desialylated AAG and seven drugs from different pharmacotherapeutic groups (carvedilol, diltiazem, dipyridamole, imipramine, lidocaine, propranolol, vinblastine) using microscale thermophoresis (MST). This method was chosen due to its robustness and high sensitivity, allowing precise quantification of molecular interactions based on the thermophoretic movement of fluorescent molecules. Detailed glycan analysis of native and desialylated AAG showed over 98% reduction in sialic acid content for the enzymatically desialylated AAG. The MST results indicate that desialylation generally alters the binding affinity between AAG and drugs, leading to either an increase or decrease in Kd values, probably due to conformational changes of AAG caused by the different sialic acid content. This effect is also reflected in an increased denaturation temperature of desialylated AAG. Our findings indicate that desialylation impacts free drug concentrations differently, depending on the binding affinity of the drug with AAG relative to human serum albumin (HSA). For drugs such as dipyridamole, lidocaine, and carvedilol, which have a higher affinity for AAG, desialylation significantly changes free drug concentrations. In contrast, drugs such as propranolol, imipramine, and vinblastine, which have a strong albumin binding, show only minimal changes. It is noteworthy that the free drug concentration of dipyridamole is particularly sensitive to changes in AAG concentration and glycosylation, with a decrease of up to 15% being observed, underscoring the need for dosage adjustments in personalized medicine.

Differential coordination demands in Fe versus Mn water-soluble cationic metalloporphyrins translate into remarkably different aqueous redox chemistry and biology.

The different biological behavior of cationic Fe and Mn pyridylporphyrins in Escherichia coli and mouse studies prompted us to revisit and compare their chemistry. For that purpose, the series of ortho and meta isomers of Fe(III) meso-tetrakis-N-alkylpyridylporphyrins, alkyl being methyl to n-octyl, were synthesized and characterized by elemental analysis, UV/vis spectroscopy, mass spectrometry, lipophilicity, protonation equilibria of axial waters, metal-centered reduction potential, E(1/2) for M(III)P/M(II)P redox couple (M = Fe, Mn, P = porphyrin), kcat for the catalysis of O2(•-) dismutation, stability toward peroxide-driven porphyrin oxidative degradation (produced in the catalysis of ascorbate oxidation by MP), ability to affect growth of SOD-deficient E. coli, and toxicity to mice. Electron-deficiency of the metal site is modulated by the porphyrin ligand, which renders Fe(III) porphyrins ≥5 orders of magnitude more acidic than the analogous Mn(III) porphyrins, as revealed by the pKa1 of axially coordinated waters. The 5 log units difference in the acidity between the Mn and Fe sites in porphyrin translates into the predominance of tetracationic (OH)(H2O)FeP complexes relative to pentacationic (H2O)2MnP species at pH ∼7.8. This is additionally evidenced in large differences in the E(1/2) values of M(III)P/M(II)P redox couples. The presence of hydroxo ligand labilizes trans-axial water which results in higher reactivity of Fe relative to Mn center. The differences in the catalysis of O2(•-) dismutation (log kcat) between Fe and Mn porphyrins is modest, 2.5-5-fold, due to predominantly outer-sphere, with partial inner-sphere character of two reaction steps. However, the rate constant for the inner-sphere H2O2-based porphyrin oxidative degradation is 18-fold larger for (OH)(H2O)FeP than for (H2O)2MnP. The in vivo consequences of the differences between the Fe and Mn porphyrins were best demonstrated in SOD-deficient E. coli growth. On the basis of fairly similar log kcat(O2(•-)) values, a very similar effect on the growth of SOD-deficient E. coli was anticipated by both metalloporphyrins. Yet, while (H2O)2MnTE-2-PyP(5+) was fully efficacious at ≥20 µM, the Fe analogue (OH)(H2O)FeTE-2-PyP(4+) supported SOD-deficient E. coli growth at as much as 200-fold lower doses in the range of 0.1-1 µM. Moreover the pattern of SOD-deficient E. coli growth was different with Mn and Fe porphyrins. Such results suggested a different mode of action of these metalloporphyrins. Further exploration demonstrated that (1) 0.1 µM (OH)(H2O)FeTE-2-PyP(4+) provided similar growth stimulation as the 0.1 µM Fe salt, while the 20 µM Mn salt provides no protection to E. coli; and (2) 1 µM Fe porphyrin is fully degraded by 12 h in E. coli cytosol and growth medium, while Mn porphyrin is not. Stimulation of the aerobic growth of SOD-deficient E. coli by the Fe porphyrin is therefore due to iron acquisition. Our data suggest that in vivo, redox-driven degradation of Fe porphyrins resulting in Fe release plays a major role in their biological action. Possibly, iron reconstitutes enzymes bearing [4Fe-4S] clusters as active sites. Under the same experimental conditions, (OH)(H2O)FePs do not cause mouse arterial hypotension, whereas (H2O)2MnPs do, which greatly limits the application of Mn porphyrins in vivo.

Design, mechanism of action, bioavailability and therapeutic effects of mn porphyrin-based redox modulators.

Based on aqueous redox chemistry and simple in vivo models of oxidative stress, Escherichia coli and Saccharomyces cerevisiae, the cationic Mn(III) N-substituted pyridylporphyrins (MnPs) have been identified as the most potent cellular redox modulators within the porphyrin class of drugs; their efficacy in animal models of diseases that have oxidative stress in common is based on their high ability to catalytically remove superoxide, peroxynitrite, carbonate anion radical, hypochlorite, nitric oxide, lipid peroxyl and alkoxyl radicals, thus suppressing the primary oxidative event. While doing so MnPs could couple with cellular reductants and redox-active proteins. Reactive species are widely accepted as regulators of cellular transcriptional activity: minute, nanomolar levels are essential for normal cell function, while submicromolar or micromolar levels impose oxidative stress, which is evidenced in increased inflammatory and immune responses. By removing reactive species, MnPs affect redox-based cellular transcriptional activity and consequently secondary oxidative stress, and in turn inflammatory processes. The equal ability to reduce and oxidize superoxide during the dismutation process and recently accumulated results suggest that pro-oxidative actions of MnPs may also contribute to their therapeutic effects. All our data identify the superoxide dismutase-like activity, estimated by log k(cat)O2-*), as a good measure for the therapeutic efficacy of MnPs. Their accumulation in mitochondria and their ability to cross the blood-brain barrier contribute to their remarkable efficacy. We summarize herein the therapeutic effects of MnPs in cancer, central nervous system injuries, diabetes, their radioprotective action and potential for imaging. Few of the most potent modulators of cellular redox-based pathways, MnTE2-PyP5+, MnTDE-2-ImP5+, MnTnHex-2-PyP5+ and MnTnBuOE-2-PyP5+, are under preclinical and clinical development.

Glycoproteomics meets thermodynamics: A calorimetric study of the effect of sialylation and synergistic anion on the binding of iron to human serum transferrin.

The thermodynamic parameters for the binding of ferric ions to human serum transferrin (hTf) as the major mediator of iron transport in blood plasma were determined by isothermal titration calorimetry in the presence of carbonate and oxalate as synergistic anions at pH 7.4. The results indicate that the binding of ferric ions to the two binding sites of hTf is driven both enthalpically and entropically in a lobe-dependent manner: binding to the C-site is mainly enthalpically driven, whereas binding to the N-site is mainly entropically driven. Lower sialic acid content of hTf leads to more exothermic apparent binding enthalpies for both lobes, while the increased apparent binding constants for both sites were found in the presence of carbonate. Sialylation also unequally affected the heat change rates for both sites only in the presence of carbonate, but not in the presence of oxalate. Overall, the results suggest that the desialylated hTf has a higher iron sequestering ability, which may have implications for iron metabolism.

Preparation and characterization of iron(III) nitrilotriacetate complex in aqueous solutions for quantitative protein binding experiments.

Various preparations of iron(III) nitrilotriacetate (FeNTA) solution reported in the literature lack a comprehensive method for accurate determination of FeNTA concentration and often result in unstable solutions. A detailed procedure for the preparation of FeNTA solution is presented that includes the standardization of both components of the chelate. The standardization of the components allowed the accurate determination of the molar absorption coefficients for the calculation of the FeNTA concentration in two different buffers at pH 5.6 and 7.4. The variation of pH in this range or ionic strength in the range from 0 M to 3 M (KCl) has little effect on the value of the molar absorption coefficient. The precise concentrations of all species involved in the equilibria between Fe and NTA were determined in the pH range 2-12 using the Jenkins-Traub algorithm to solve the 5th-order polynomial in Microsoft Excel. In view of the experimental observations and the calculated distribution of species, the stability of FeNTA solutions may be affected by the Fe : NTA ratio and the total concentrations, with dilute solutions and those with an excess of NTA over Fe showing higher stability.

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 continuous 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.

Interaction of silver nanoparticles with plasma transport proteins: A systematic study on impacts of particle size, shape and surface functionalization.

Metallic nanoparticles are an important and widely used materials in development of nano-enabled medicine. For that reason, their interaction with biological molecules has to be systematically examined, as use of nanoparticles can lead to altered biological functions. In this study, we evaluated the interaction between silver nanoparticles (AgNPs) and two important plasma transport proteins - albumin and α-1-acid glycoprotein. To investigate comprehensively how different physico-chemical properties impact interaction of proteins with nanosurface, AgNPs of different size, shape and surface coating was prepared. The study was conducted using UV-Vis absorption, fluorescence, inductively coupled plasma mass spectrometry, circular dichroism spectroscopy, transmission electron microscopy, dynamic and electrophoretic light scattering techniques. The results showed significant complexities of the nano-bio interface and binding affinities of proteins onto surface of different AgNPs, which were affected by both AgNPs and protein properties. The most significant role on AgNPs-protein interaction had the coating agents used for AgNPs surface stabilization. Our findings should improve safe-by-design approach to development of the metallic nanomaterials for medical use.

Low-pressure chromatographic separation and UV/Vis spectrophotometric characterization of the native and desialylated human apo-transferrin.

Low-pressure pH gradient ion exchange separation provides a fast, simple and cost-effective method for preparative purification of native and desialylated apo-transferrin. The method enables easy monitoring of the extent of the desialylation reaction and also the efficient separation and purification of protein fractions after desialylation. The N-glycan analysis shows that the modified desialylation protocol successfully reduces the content of the sialylated fractions relative to the native apo-transferrin. In the optimized protocol, the desialylation capacity is increased by 150 %, compared to the original protocol provided by the manufacturer. The molar absorption coefficients in the near-UV region for the native and desialylated apo-transferrin differ by several percent, suggesting a subtle dependence of the glycoprotein absorbance on the variable sialic acid content. The method can easily be modified for other glycoproteins and is particularly appropriate for quick testing of sialic acid content in the protein glycosylation patterns prior to further verification by mass spectrometry.

Solvent effects on the absorption and fluorescence spectra of Zaleplon: Determination of ground and excited state dipole moments.

Solvent effects on the absorption and fluorescence spectra of Zaleplon, a nonbenzodiazepine sedative/hypnotic drug that is mainly used for the short term treatment of insomnia, were investigated in 18 different solvents with diverse polarities. Dipole moments of the ground and excited state (µg and µe) were determined by Lippert-Mataga, Bakhshiev, Reichardt, McRae and Suppan solvatochromic methods. The dipole moment of Zaleplon ground state in the gas phase has been calculated as µg = 10.95 D (TD-DFT) with B3LYP/cc-pVTZ functional. There is a good agreement of theoretical data with Reichardt, McRae, and Suppan correlations, while some dissidence with Lippert-Mataga and Bakhshiev equations is suggesting the occurrence of specific solute-solvent interactions. Additionally, multiple linear regression analysis with Kamlet-Taft and Catalan solvatochromic models was applied to solute-solvent interactions. Dominant property of the solvent that affects the absorption band and Stokes shifts of Zaleplon is polarity of the solvent while the emission band is influenced mainly by solvent basicity.

Acido-base behavior of hydroxamic acids: experimental and ab initio studies on hydroxyureas.

The values of Ka, DeltaSa, and DeltaHa for deprotonation of hydroxyurea (HU) and N-methylhydroxyurea (NMHU), as targeted compounds, and for betainohydroxamic acid, were potentiometrically determined. Although NMHU has two and HU even three deprotonation sites, the measurements confirm that they behave as weak acids with a single pK a approximately 10. Comparison with analogous thermodynamic parameters previously determined for series of monohydroxamic acids reveals deviations from a DeltaSa, vs DeltaHa plot for HU and NMHU, raising the question of the dissociation site of hydroxureas in water. In addition to the deprotonation of the hydroxyl oxygen, ab initio calculations performed at the MP2/6-311++G(d,p) level of theory for these two compounds indicate a notable participation of the nitrogen deprotonation site in HU. The calculations for the isolated, monohydrate, trihydrate, and decahydrate molecular and anionic forms of hydroxyureas support the importance of hydrogen bonding in the gas and aqueous phases. The hydroxylamino nitrogen in HU is the most acidic site in water, contributing approximately 94% to the overall deprotonation process at 25 degrees C. On the contrary, the hydroxylamino oxygen is by far the most favored deprotonation site in NMHU, contributing almost 100% in aqueous medium. The predicted participations of two deprotonation sites in HU, calculated at the MP2/6-311++G(d,p) level of theory, combined with the calculated relative reaction enthalpy and entropy for the deprotonation, satisfactorily explain the observed deviation from linearity of DeltaHa vs DeltaSa, plot. There is no such a simple explanation for acid-base behavior of NMHU.

CNS bioavailability and radiation protection of normal hippocampal neurogenesis by a lipophilic Mn porphyrin-based superoxide dismutase mimic, MnTnBuOE-2-PyP5.

Although radiation therapy can be effective against cancer, potential damage to normal tissues limits the amount that can be safely administered. In central nervous system (CNS), radiation damage to normal tissues is presented, in part, as suppressed hippocampal neurogenesis and impaired cognitive functions. Mn porphyrin (MnP)-based redox active drugs have demonstrated differential effects on cancer and normal tissues in experimental animals that lead to protection of normal tissues and radio- and chemo-sensitization of cancers. To test the efficacy of MnPs in CNS radioprotection, we first examined the tissue levels of three different MnPs - MnTE-2-PyP5+(MnE), MnTnHex-2-PyP5+(MnHex), and MnTnBuOE-2-PyP5+(MnBuOE). Nanomolar concentrations of MnHex and MnBuOE were detected in various brain regions after daily subcutaneous administration, and MnBuOE was well tolerated at a daily dose of 3mg/kg. Administration of MnBuOE for one week before cranial irradiation and continued for one week afterwards supported production and long-term survival of newborn neurons in the hippocampal dentate gyrus. MnP-driven S-glutathionylation in cortex and hippocampus showed differential responses to MnP administration and radiation in these two brain regions. A better understanding of how preserved hippocampal neurogenesis correlates with cognitive functions following cranial irradiation will be helpful in designing better MnP-based radioprotection strategies.

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.

Radioprotection of the brain white matter by Mn(III) n-Butoxyethylpyridylporphyrin-based superoxide dismutase mimic MnTnBuOE-2-PyP5+.

Cranial irradiation is a standard therapy for primary and metastatic brain tumors. A major drawback of radiotherapy (RT), however, is long-term cognitive loss that affects quality of life. Radiation-induced oxidative stress in normal brain tissue is thought to contribute to cognitive decline. We evaluated the effectiveness of a novel mimic of superoxide dismutase enzyme (SOD), MnTnBuOE-2-PyP(5+)(Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin), to provide long-term neuroprotection following 8 Gy of whole brain irradiation. Long-term RT damage can only be assessed by brain imaging and neurocognitive studies. C57BL/6J mice were treated with MnTnBuOE-2-PyP(5+) before and after RT and evaluated three months later. At this time point, drug concentration in the brain was 25 nmol/L. Mice treated with MnTnBuOE-2-PyP(5+)/RT exhibited MRI evidence for myelin preservation in the corpus callosum compared with saline/RT treatment. Corpus callosum histology demonstrated a significant loss of axons in the saline/RT group that was rescued in the MnTnBuOE-2-PyP(5+)/RT group. In addition, the saline/RT groups exhibited deficits in motor proficiency as assessed by the rotorod test and running wheel tests. These deficits were ameliorated in groups treated with MnTnBuOE-2-PyP(5+)/RT. Our data demonstrate that MnTnBuOE-2-PyP(5+) is neuroprotective for oxidative stress damage caused by radiation exposure. In addition, glioblastoma cells were not protected by MnTnBuOE-2-PyP(5+) combination with radiation in vitro. Likewise, the combination of MnTnBuOE-2-PyP(5+) with radiation inhibited tumor growth more than RT alone in flank tumors. In summary, MnTnBuOE-2-PyP(5+) has dual activity as a neuroprotector and a tumor radiosensitizer. Thus, it is an attractive candidate for adjuvant therapy with RT in future studies with patients with brain cancer.