Flow-based bioconjugation of coumarin phosphatidylethanolamine probes: Optimised synthesis and membrane molecular dynamics studies.

A series of phosphatidylethanolamine fluorescent probes head-labelled with 3-carboxycoumarin was prepared by an improved bioconjugation approach through continuous flow synthesis. The established procedure, supported by a design of experiment (DoE) set-up, resulted in a significant reduction in the reaction time compared to the conventional batch method, in addition to a minor yield increase. The characterization of these probes was enhanced by an in-depth molecular dynamics (MD) study of the behaviour of a representative probe of this family, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine labelled with 3-carboxycoumarin (POPE-COUM), in bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (SLPC) 2:1, mimicking the composition of the egg yolk lecithin membranes recently used experimentally by our group to study POPE-COUM as a biomarker of the oxidation state and integrity of large unilamellar vesicles (LUVs). The MD simulations revealed that the coumarin group is oriented towards the bilayer interior, leading to a relatively internal location, in agreement with what is observed in the nitrobenzoxadiazole fluorophore of commercial head-labelled NBD-PE probes. This behaviour is consistent with the previously stated hypothesis that POPE-COUM is entirely located within the LUVs structure. Hence, the delay on the oxidation of the probe in the oxygen radical absorbance capacity (ORAC) assays performed is related with the inaccessibility of the probe until alteration of the LUV structure occurs. Furthermore, our simulations show that POPE-COUM exerts very little global and local perturbation on the host bilayer, as evaluated by key properties of the unlabelled lipids. Together, our findings establish PE-COUM as suitable fluorescent lipid analogue probes.

Exploiting Kinetic Features of ORAC Assay for Evaluation of Radical Scavenging Capacity.

The analysis and interpretation of data retrieved from Oxygen Radical Absorbance Capacity (ORAC) assays represent a challenging task. ORAC indexes originate from different mathematical approaches often lacking correct elucidation of kinetic features concerning radical scavenging reactions by antioxidant compounds. In this work, the expression of ORAC values as area under fluorescein (FL) decay curves (AUC) and lag time are critically compared. This multi-parametric analysis showed the extension of radical scavenging reactions beyond the lag time period for caffeic acid, gallic acid, reduced glutathione and quercetin, extending their antioxidant protection of FL. Ethanol delayed the reaction of both FL and antioxidant compounds with free radical species generated from 2,2'-azobis(2-amidinopropane) dihydrochloride thermolysis. Trolox equivalent values, commonly used to express ORAC values, were more affected by the differences in radical scavenging kinetics between the reference and the tested antioxidant compounds when calculated from AUC than from lag time. These findings stressed the importance of choosing calibrator compounds presenting ORAC kinetics similar to samples to prevent biased estimation of the antioxidant capacity. Additionally, the framework proposed here provides a sustainable analytical method for the evaluation of antioxidant capacity, with an AGREE score of 0.73.

Acetonitrile Adducts of Tranexamic Acid as Sensitive Ions for Quantification at Residue Levels in Human Plasma by UHPLC-MS/MS.

The quantitative analysis of pharmaceuticals in biomatrices by liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is often hampered by adduct formation. The use of the molecular ion resulting from solvent adducts for quantification is uncommon, even if formed in high abundance. In this work, we propose the use of a protonated acetonitrile adduct for the quantitative analysis of tranexamic acid (TXA) by LC-MS/MS. The high abundance of the protonated acetonitrile adduct [M + ACN + H]+ was found to be independent of source-dependent parameters and mobile phase composition. The results obtained for TXA analysis in clinical samples were comparable for both [M + ACN + H]+ and [M + H]+, and no statistically significant differences were observed. The relative stability and structure of the [M + ACN + H]+ ions were also studied by analyzing probable structures from an energetic point of view and by quantum chemical calculations. These findings, and the studied fragmentation pathways, allowed the definition of an acetimidium structure as the best ion to describe the observed acetonitrile protonated adduct of TXA.

Determination of neuropeptide Y Y1 receptor antagonist BIBP 3226 and evaluation of receptor expression based on liquid chromatography coupled with tandem mass spectrometry.

Neuropeptide Y (NPY) is a peptide widely distributed throughout the body that is involved in various physiological processes, including the regulation of feeding behavior and energy homeostasis. 5-Carbamimidamido-2-(2,2-diphenylacetamido)-N-[(4-hydroxyphenyl)methyl]pentanamide (BIBP 3226) is a selective NPY Y1 receptor antagonist with recognized application in bone regeneration studies, requiring quantification at picogram levels. Hence, BIBP 3226 determination is proposed here by a validated HPLC-MS/MS method, based on a reversed-phase Kinetex® core-shell C8 column (2.6 µm, 150 × 2.1 mm) at 30 °C, elution in isocratic mode using a mixture of acetonitrile and water (30:70, v/v), containing 0.1% (v/v) formic acid, at 0.25 mL min-1, detection in positive ionization mode, and data acquisition in selected reaction monitoring mode. Calibration curves were linear for concentrations ranging from 0.25 to 30 ng mL-1 with LOD and LOQ values as low as 0.1 and 0.3 pg in cell extracts and 16 and 48 pg in supernatant culture media, respectively. BIBP 3226 was successfully determined in cell extracts and supernatants obtained from internalization assays. Using similar exposure conditions, the amount of BIBP 3226 found in breast cancer cells (MCF7) was 72 to 657 times higher than that found in bone marrow cells (Wt C57BL/6 mice), providing an indirect indicator of NPY Y1 receptor expression.

Emergent Glycerophospholipid Fluorescent Probes: Synthesis and Applications.

Fluorescent labeling through bioconjugation is the preferred tool for the investigation of biological functions involving lipids, namely for clarifying metabolic pathways and molecular mechanisms of diseases. The lack of functionalized lipid probes with biological and physicochemical properties suitable for these studies is still a major limitation. Moreover, the synthesis of these probes is challenging and strongly dependent on the application envisioned. The objective of this Review is to highlight advances in the application of fluorescent glycerophospholipid probes through innovative approaches in the synthesis reported in the past decade. The reaction pathways, choice of fluorophore, and location of fluorophore in the glycerophospholipid structure are critically addressed. The relevance of these bioconjugates is exemplified with applications using advanced analysis by fluorescence enhancement or quenching to unravel biomembrane structure features and phospholipase activity. Finally, this Review reinforces the need for innovative and more efficient routes for the synthesis of tailored glycerophospholipids fluorescent conjugates.

Chromatographic method for the simultaneous quantification of dapsone and clofazimine in nanoformulations.

The low bioavailability and nonspecific distribution of dapsone and clofazimine, commonly applied in combination for the treatment of leprosy, can produce toxic effects. Nanotechnological approaches enhance the delivery of these drugs. Therefore, a high-performance liquid chromatography method was developed for the simultaneous determination of dapsone and clofazimine loaded in nanoformulations for quality control purposes. Chromatographic separation was achieved on a reversed-phase Kinetex core-shell C18 column, followed by spectrophotometric detection at 280 nm. Considering the different physicochemical properties of dapsone and clofazimine, elution was performed in gradient mode using an aqueous acetate buffer (50 mmol/L, pH 4.8) and an increasing acetonitrile content from 27 to 63% v/v at a flow rate of 1.0 mL/min with retention times of 6.2 and 14.0 min, respectively. The method was validated according to the European Medicines Agency guideline and it was found to be specific, accurate (99.6-114.0%), and precise for intra- (RSD ≤ 1.8%) and interday assays (RSD ≤ 12.5%). Both drugs showed stability after 24 h at room temperature and over three freeze-thaw cycles with recoveries ≥86.2%. Low temperature (4°C) in the autosampler caused the precipitation of clofazimine and must be avoided. The validated method was successfully applied in the quantification of both drugs in nanoformulations.

Gas-phase structural characterization of neuropeptides Y Y1 receptor antagonists using mass spectrometry: Orbitrap vs triple quadrupole.

Collision induced dissociation of triple quadrupole mass spectrometer (CID-QqQ) and high-energy collision dissociation (HCD) of Orbitrap were compared for four neuropeptides Y Y1 (NPY Y1) receptor antagonists and showed similar qualitative fragmentation and structural information. Orbitrap high resolution and high mass accuracy HCD fragmentation spectra allowed unambiguous identification of product ions in the range 0.04-4.25 ppm. Orbitrap mass spectrometry showed abundant analyte-specific product ions also observed on CID-QqQ. These results show the suitability of these product ions for use in quantitative analysis by MRM mode. In addition, it was found that all compounds could be determined at levels >1 µg L-1 using the QqQ instrument and that the detection limits for this analyzer ranged from 0.02 to 0.6 µg L-1. Overall, the results obtained from experiments acquired in QqQ show a good agreement with those acquired from the Orbitrap instrument allowing the use of this relatively inexpensive technique (QqQ) for accurate quantification of these compounds in clinical and academic applications.

Lipid remodelling in human melanoma cells in response to UVA exposure.

Extensive exposure to UVA is thought to increase the risk of malignancy and the progression of melanoma, the most serious type of skin cancer. It is well known that alterations in lipid metabolism represent an early event in carcinogenesis, but the impact of UVA exposure on the lipid composition of cancer cells is still largely unknown. In this study we aimed at investigating lipid remodeling in human melanoma cells in response to UVA exposure. After UVA irradiation, lipid extracts were either immediately collected from SK-MEL-28 cells or collected after a recovery period of 2 h or 24 h. The lipid profiles for each event were determined by liquid chromatography or gas chromatography coupled to mass spectrometry. UVA exposure led to major alterations in both fatty acids (FA) and phospholipid profiles. An increase of monounsaturated FA (MUFA) and FA18:0, as well as a decrease of FA16:0, were observed 24 h after irradiation. Moreover, phosphatidylcholine (PC) decreased and phosphatidylinositol (PI) increased after UVA exposure. Molecular alterations in the PC, lysoPC, PI, phosphatidylethanolamine (PE), ether-linked PE and phosphatidylglycerol (PG) profiles were also observed. The absence of cleaved caspase-3 after 2 h and 24 h of re-incubation is correlated with impairment of apoptosis. Overall, these data showed changes in membrane lipids, which may be associated with lipogenesis after UVA exposure which, in turn, is usually a determinant for cell survival.

Development and validation of a liquid chromatography-MS/MS method for simultaneous quantification of tenofovir and efavirenz in biological tissues and fluids.

Millions of people worldwide live with human immunodeficiency virus (HIV) infection thus justifying the continuous search for new prevention and treatment strategies, including topical microbicide products combining antiretroviral drugs (ARVs) such as tenofovir (TFV) and efavirenz (EFV). Therefore, the aim of this work was to develop and validate a high performance liquid chromatography method coupled to triple quadrupole-tandem mass spectrometry (HPLC-MS/MS) for the quantification of TFV and EFV in biological matrices (mouse vaginal tissue, vaginal lavage and blood plasma). Chromatographic separation was achieved using a reversed phase C18 column (3µm, 100×2.1mm) at 45°C and elution in gradient mode using a combination of 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile at 0.35mLmin-1. Total run time was 9min, with retention time of 2.8 and 4.1min for TFV and EFV, respectively. The MS was operated in positive ionization mode (ESI+) for TFV and in negative ionization mode (ESI-) for EFV detection. Data were acquired in selected reaction monitoring (SRM) mode and deuterated ARVs were employed as internal standards. Calibration curves were linear for ARV concentrations ranging from 4 to 500ngmL-1 with LOD and LOQ for both analytes ≤0.4 and ≤0.7ngmL-1 in sample extracts, respectively. The method was found to be specific, accurate (96.0-106.0% of nominal values) and precise (RSD<2.4%) in all matrices. Both TFV and EFV were found to be stable in all matrices after standing 24h at room temperature (20°C) or in the autosampler, and after three freeze-thawing cycles. Mean recovery values of ARVs spiked in mice tissues or fluids were ≥88.4%. Matrix effects were observed for EFV determination in tissue and plasma extracts but compensated by the use of deuterated internal standards. The proposed methodology was successfully applied to a pharmacokinetic study following intravaginal administration of both ARVs.

Characterization of 2,3-diarylxanthones by electrospray mass spectrometry: gas-phase chemistry versus known antioxidant activity properties.

RATIONALE: Xanthones (XH) are a class of heterocyclic compounds widely distributed in nature that hold numerous noteworthy biological and antioxidant activities. Therefore, it is of utmost importance to achieve relevant detailed structural information to understand and assist prediction of their biological properties. The potential relationship between radical-mediated xanthone chemistry in the gas phase and their promising antioxidant activities has not been previously explored. METHODS: Protonated xanthones XH1-9 were generated in the gas phase by electrospray ionization (ESI) and the main fragmentation pathways of the protonated XH1-9 formed due to collision-induced dissociation (CID) were investigated. RESULTS: In the CID-MS/MS spectra of [M+H](+) ions of XH1, XH2 and XH4 the product ions formed due to H2 O elimination corresponding to the base peak of the spectra. For the remaining six xanthones (XH3, XH5-9), showing the most promising biological profile, the product ion produced with the highest relative abundance (RA) corresponded to the one formed through concomitant loss of H2 O plus CO. Indicative of an inexistent or lower biological activity is the combined loss of CO plus O unique to the CID-MS/MS spectra of XH1, XH2, XH4, and XH5. The product ion formed by loss of 64 Da (concomitant loss of two molecules of H2 O plus CO) is only observed for xanthones containing a catechol unit (XH3 and XH6-9). This product ion has the highest RA for the most potent scavenger of reactive oxygen and nitrogen species XH9 that contains two of these catechol moieties. CONCLUSIONS: A strong relationship between some of the biological activities of the studied 2,3-diarylxanthones and their ESI-MS/MS fragmentation spectra was found. The multivariate statistical analysis results suggest that the selected MS features are related to the important biological features. Copyright © 2016 John Wiley & Sons, Ltd.

Do cinnamylideneacetophenones have antioxidant properties and a protective effect toward the oxidation of phosphatidylcholines?

Cinnamylideneacetophenones (CA) are an important group of α,ß,γ,δ-diunsaturated ketones that have been widely used in a variety of synthetic transformations. Biological studies concerning these compounds are scarce and refer mainly to antiviral and antibacterial evaluations. Curcumin (CR), a natural polyphenol, is a yellow pigment extracted from the plant Curcuma longa, which is one of the major spices used in the Indian culinary. It has been reported that CR has cancer chemopreventive properties in a range of animal models of chemical carcinogenesis, along with antioxidative and anti-inflammatory properties. Inspired by the biological activity shown by CR and their structural resemblance with CA, it was considered to study the ability of the latter molecules to inhibit lipid oxidation induced by the hydroxyl radical (Fenton reaction) by electrospray ionization (ESI) mass spectrometry (MS) using phosphatidylcholine (PC) liposomes as a model of cell membrane. Compound 4, holding a methylated hydroxy group in the position R(2), and CR showed similar effects in inhibiting lipid peroxidation. In the presence of 7, the extension of oxidation was higher than the one verified in all other compounds. Other methodologies, namely DPPH radical scavenging and oxygen radical absorption capacity (ORAC) assays, were performed to complement and clarify the results attained by oxidation of PC monitored by ESI-MS and to evaluate the antioxidant profile of compounds. For both assays, compound 7 showed to be rather efficient due to its specific structure. This derivative can form a quite stable allylic radical by abstraction of a hydrogen atom which accounts for these results.

Evaluation of the photooxidation of galactosyl- and lactosylceramide by electrospray ionization mass spectrometry.

RATIONALE: Glycosphingolipids are important lipid molecules namely as constituents of the plasma membrane organized in lipid rafts, in signal transduction, and cell-cell communication. Although many human diseases are associated with oxidative stress and lipid oxidation, a link between oxidative stress and modification of glycosphingolipids has never been addressed. METHODS: In this study, the structural changes caused by UVA-induced photooxidation of galactosyl- (GalCer) and lactosylceramide (LacCer) molecular species were studied by electrospray ionization mass spectrometry (ESI-MS and MS/MS), using a quadrupole time-of-flight (QTOF) mass spectrometer and high-performance liquid chromatography/tandem mass spectrometry with a C5 stationary phase (C5 HPLC/MS/MS) using a linear ion trap. RESULTS: ESI-MS spectra of GalCer and LacCer after photooxidation showed new ions with a mass shift of +32 Da when compared with the ions of the non-modified glycosphingolipids. These new species were assigned as hydroperoxyl derivatives, confirmed by HPLC/MS/MS and through FOX 2 assay. In the ESI-MS and LC/MS of lactosylceramide a new ion with lower m/z value, assigned as glucosylceramide (GlcCer) + 32 Da, was also detected and proposed to be formed due to oxidative cleavage of lactosyl moieties. ESI-MS/MS of the oxidized species allowed us to infer the presence of isomeric hydroperoxyl derivatives, with the hydroperoxyl moiety either linked to the sphingosine backbone or in the unsaturated acyl chain. Oxidation in the sugar moieties was observed in the case of LacCer, suggesting an oxidation via radical reactive oxygen species that can induce the oxidative cleavage of the lactosyl moiety. CONCLUSIONS: This study shows that glycosphingolipids are prone to oxidation and the identified mass spectrometry fingerprint of oxidized galactosyl- and lactosylceramide species will support their future identification in lipidomic studies of biological samples under oxidative conditions.

Structural effects of the ß-vinyl linker in pyridinium porphyrins: spectroscopic studies in organic solvents and AOT reverse micelles.

Two isomeric ß-vinylpyridinium porphyrins, 2-[2-(2-methylpyridinium)vinyl]-5,10,15,20-tetraphenylporphyrin (1, ortho isomer) and 2-[2-(4-methylpyridinium)vinyl]-5,10,15,20-tetraphenylporphyrin (2, para isomer), which have shown different photodynamic behavior were investigated in organic solvents and sodium 1,4-bis(2-ethylhexyl)sulfosuccinate (AOT) reverse micelles. In organic systems, the absorption spectra present a red-shifted band that is more intense in the para isomer, in addition to the usual Soret band. This new band presents interesting solvatochromic effects which obey the multiparametric Kamlet-Taft equation. In AOT reverse micelles, the ortho isomer exhibits a strong dependence with the parameter ω0 = [H2O]/[AOT] which indicates that the molecule resides at the interface toward the organic phase. By contrast, no evidence was detected for the encapsulation of para isomer 2 in AOT reverse micelles. The hypothesis of two ground state isomers with different contributions of trans and quinoid structures is advanced on the basis of the overall data collected from electronic absorption, steady-state, and transient-state fluorescence emission. A charge transfer state in which an electron is fully transferred from the porphyrin to the pyridinium moiety is associated to a quinoid structure in isomer 2. The trans/quinoid relative proportions may be accounted for by the orientation of the ortho-/para-pyridinium isomers relatively to the porphyrin core.

Photooxidation of glycated and non-glycated phosphatidylethanolamines monitored by mass spectrometry.

Phosphatidylethanolamines (PE) are one of the major components of cells membranes, namely in skin and in retina, that are continuously exposed to solar UV radiation being major targets of photooxidation damage. In addition, due to the presence of the free amine group, PE can also undergo glycation, in hyperglycemic conditions which may increase the susceptibility to oxidation. The aim of this study is to develop a model, based on mass spectrometry (MS) analysis, to identify photooxidative degradation of selected PE (POPE: PE 16:0/18:1, PLPE: PE 16:0/18:2, PAPE: PE 16:0/20:4) and glycated PEs due to UV irradiation. Photooxidation products were analysed by electrospray ionization MS (ESI-MS) and tandem MS (ESI-MS/MS) in positive and negative mode. Emphasis is placed in the influence of glycation in the generation of distinct photooxidation products. ESI-MS spectra of PE after UV photo-irradiation showed mainly hydroperoxy derivatives, due to oxidation of unsaturated fatty acyl chains. Glycated PE gave rise to several new photooxidation products formed due to oxidative cleavages of the glucose moiety, namely between C1 and C2, C2 and C3, and C5 and C6 of this sugar unit. These new products were identified by ESI-MS/MS in positive mode showing distinct neutral loss depending on the different structure of the polar head group. These new identified advanced glycated photooxidation products may have a deleterious role in the etiology of diabetic retinopathy and in diabetic retinal microvascular complications.

Characterisation of (E)-2-styrylchromones by electrospray ionisation mass spectrometry: singular gas-phase formation of benzoxanthenones.

RATIONALE: The electrospray ionisation (ESI) mass spectra (ESI-MS(n) , n = 2 and 3) of six (E)-2-styrylchromones containing electron-donating and electron-withdrawing groups were studied for the first time. The potential application of these compounds as drug candidates brings a need for the development of an analytical method to ensure a detailed knowledge of their structures. METHODS: ESI-MS, CID-MS(2) and MS(3) spectra of all the studied compounds were acquired using a LXQ linear ion trap mass spectrometer with an electrospray source, operating in the positive ion mode. ESI conditions were: nitrogen 8.00 sheath gas flow rate (arb), spray voltage 5 kV, heated capillary temperature 275 °C, capillary voltage 10.99 V and tube lens voltage 75.01 V. CID-MS(2) and MS(3) experiments were performed on mass-selected precursor ions using standard isolation and excitation procedures (activation q value of 0.25, activation time of 30 ms). RESULTS: A comparable pattern of product ions was observed for all the studied compounds. Only the product ion ((1,2)(3,4)) A(+) is common to all the studied compounds. Common to compounds 1 (R(2) = H), 1a (R(2) = OMe) and 1c (R(2) = Cl) are the product ions formed by loss of H(2)O, CO, C(6)H(5)OH, and the product ions ((1,2)(2,3))B(+) and ((1,2)(3,4))B(+). Among all compounds, 2-SC 1b (R(2) = NO(2)) shows an obvious differentiation showing the lowest number of product ions under CID-MS(2) conditions. CONCLUSIONS: The analysed compounds revealed a comparable pattern of product ions formed for each compound only different in what concerns the relative abundance of these formed product ions. A rather interesting and unexpected loss of 2 Da was observed for some compounds. The structure of a benzoxanthenone was proposed for this product ion based on evidence obtained by synthesised benzoxanthenone as a model compound.

Tandem mass spectrometry based investigation of cinnamylideneacetophenone derivatives: valuable tool for the differentiation of positional isomers.

Cinnamylideneacetophenones have been extensively used as versatile starting materials in numerous different transformations. The structural characterization of this type of compounds is, therefore, of crucial importance since it can give information on the chemistry, reactivity and also the potential biological activity of this type of compounds. Thus, 24 derivatives were systematically studied by tandem mass spectrometry (MS(2)) with electrospray ionization (ESI), in positive ion mode. The protonated molecules, [M + H](+), formed under ESI conditions were induced to dissociate and the fragmentation patterns were studied. The information collected provided important structural information on the type of substituents present and constitute an important database concerning this family of compounds. Overall, it was found that the substitution pattern of the cinnamylideneacetophenone derivatives changes the ESI-MS(2) fragmentation considerably. These results indicate that ESI-MS(2) is a useful technique for distinguishing positional isomers of these cinnamylideneacetophenone derivatives.

Chain-dependent photocytotoxicity of tricationic porphyrin conjugates and related mechanisms of cell death in proliferating human skin keratinocytes.

Photodynamic therapy (PDT) is a poor treatment option for nodular basal cell carcinomas and squamous cell carcinomas. As a result, the search for new photosensitizers with better effectiveness is of current interest. The photocytotoxicity of conjugates (P-R) of a water-soluble tri-cationic porphyrin (P-H) having similar efficiency of production of singlet oxygen, the PDT cytotoxin, has been assessed in vitro. Links between uptake, intracellular localization, photooxidative stress, photocytotoxicity and ability to induce programmed cell death are established. Conjugates bearing methyl (P-Me), Di-O-isopropylidene-(-d-galactopyranosyl (P-OGal) or N,N'-dicyclohexylureidooxycarbonyl (P-DDC) chains are efficiently taken-up by proliferating NCTC 2544 keratinocytes. The relative order of photocytotoxicity is P-OGal >P-DDC=P-Me≫P-H. The photocytotoxic potential of P-Me, P-OGal and P-DDC equals that of endogenous protoporphyrin IX induced by δ-aminolevulinic acid or its esters, the pro-drugs currently employed for PDT of skin lesions. Microfluorometry shows that P-Me, P-OGal, and P-DDC localize in endocytotic or pinocytotic vesicles but not in mitochondria or nucleus. Absence of annexin V binding, caspase activation or chromatin condensation suggests that cell photosensitization by P-R does not induce apoptosis. On the other hand, P-OGal photocytotoxicity correlates with appearance of multiple vesicles that have hallmarks of autophagy compartments, being decorated with the marker LC3 in cells transfected with an expression vector encoding GFP-LC3. p38 and JNK phosphorylation and inhibition of ERK1/2 phosphorylation suggest close relationship between mortality of NCTC 2544 keratinocytes and MAPK pathway impairment. Given their potentially easy formulation, water-soluble P-R are promising powerful photosensitizers for PDT of skin lesions.

Tricationic porphyrin conjugates: evidence for chain-structure-dependent relaxation of excited singlet and triplet States.

Conjugates of 5-(4-carboxyphenyl)-10,15,20-tris(4-methylpyridinium-4-yl)porphyrin (P-H) are promising photoactive agents for medical applications. As their ultimate efficacy will depend on the behavior of initial excited states, photophysical parameters have been determined with conventional steady-state absorption and fluorescence as well as time-resolved femto- and nanosecond spectroscopies. The fluorescence quantum yield of P-H and P-H conjugated to uncharged groups increases from approximately 0.03 in pH 7 buffer to approximately 0.05 in Triton X100 micelles (TX100) and in ethanol and to 0.12 in sodium dodecyl sulfate (SDS) micelles. Corresponding (1)S(1) lifetimes are approximately 5-10 ns. In buffer, an equilibrium between P-H monomers and small-size aggregates is observed. Conjugation with poly-S-lysine (P-(Lys)(n)) results in fluorescence quenching in all solvents. Structural reorganization of conjugates bearing a Di-O-isopropylidene-alpha-d-galactopyranosyl or a alpha/beta-d-galactopyranosyl group occurs in ethanol (k approximately 0.15 ps(-1)) after (1)S(1) state solvation (approximately 700 fs). Relaxation of bulky P-(Lys)(n) polypeptide chains takes place on a longer time scale in all solvents (k

Electrospray tandem mass spectrometry of beta-nitroalkenyl meso-tetraphenylporphyrins.

Beta-nitroalkenyl meso-tetraphenylporphyrins [beta-TPPCHC(NO(2))R)], as free-bases and Zn(II) complexes, were studied by electrospray mass spectrometry (ESI-MS). Under this ionisation condition the [M + H](+) ions are formed. The fragmentation pattern of the resulting [M + H](+) ions were studied by electrospray tandem mass spectrometry (ESI-MS/MS). The ESI-MS/MS of beta- nitroalkenylporphyrins, either as free-bases or as Zn(II) complexes, show several interesting features, distinct from the typical behaviour of nitro compounds. For the studied compounds, common main fragmentation patterns are observed, namely characteristic losses of NO(2), HNO(2), 2OH, RNO(2), RCNO, RCNO(2), RCH(2)NO(2), C(6)H(5) plus NO(2) and the formation of the protonated macrocycle, [TPP + H](+) or [ZnTPP + H](+). However, depending on the presence or absence of the metal and the nature of the R substituent, important differences are observed on the relative abundances of the ions formed by the same fragmentation pathway. The presence of bromine in the alkenyl group leads to a peculiar behaviour, since the main fragmentation pattern corresponds to the combined elimination of the bromine atom with the typical nitro group fragments. When R = Br, the loss of the nitro group occurs in low relative abundance (11-16%). However, when R = CH(3), the relative abundance of the ion due to the loss of HNO(2) changes drastically from 100%, observed for the free-base porphyrin, to 29% in the case of the Zn(II) complex. These variations of the relative abundance of the fragment corresponding to the loss of the nitro moiety (typically considered as a diagnostic fragment) can induce to an erroneous interpretation of their MS/MS spectra. Some fragmentations are observed only for the free-base porphyrins, namely the loss of CH(NO(2)R and HNO(2) plus C(2)H(2), while the loss of OH, H(2)O, OH plus H(2)O and RCCH plus H(2)O is observed only for the complexes. Unusual and unexpected fragmentations are also observed, namely the losses of RCNO, RCNO(2) and HNO(2) plus C(2)H(2). This work demonstrates that valuable structural information about the beta-nitroalkenyl substituents linked to meso- tetraarylporphyrins can be achieved using MS/MS. These results can also be useful for the interpretation of the mass spectra of other nitroalkenyl substituted compounds.

Synthesis of neutral and cationic tripyridylporphyrin-D-galactose conjugates and the photoinactivation of HSV-1.

Neutral and cationic tripyridylporphyrin-D-galactose conjugates were synthesized and their antiviral activity against herpes simplex virus type 1 (HSV-1) was evaluated. At non-cytotoxic concentrations the studied compounds show significant antiviral activity after photoactivation. The influence of photoactivation on drug treated cells was also analyzed, at different times of infection with HSV-1, for a neutral (1b) and a cationic glycoporphyrin (3b) derivative. The results show that the inhibition of the viral yield is more dependent on photoactivation for compound 1b than for compound 3b. These two compounds also differ in the inhibitory effect during the viral replicative cycle: while compound 3b inhibits the viral yield at all the addition times assayed, compound 1b is more efficient in later times of infection.