Badanie mechanizmów indukowanych fotochemicznie i radiacyjnie reakcji utleniania peptydów zawierających metioninę

Several factors (radiation, metabolism, pollutants) lead to the generation of oxidizing free radicals in living organisms that damage all biomolecules and especially proteins. One of the protein targets is Methionine (Met). Its oxidation causes highly damaging effects, such as Alzheimer’s or prion disease. The aim of this work was to investigate the transient species and the stable products formed after radiolytic and photolytic oxidation of Met-containing peptides. The reaction of hydroxyl radicals (•OH) and 3-carboxybenzophenone triplet state with Met-residue in peptides was investigated for model compounds (Met-dipeptides) and for longer peptides (e.g. Bradykinin). Laser flash photolysis and pulse radiolysis were used to characterize short-lived transient species, while gamma radiolysis and steady-state photolysis were used for quantitative and qualitative characterization of stable products. The structural modifications induced by oxidation have been characterized by the HPLC coupled with mass spectrometry and Infrared Multi Photon Dissociation Spectroscopy (IRMPD, CLIO Free electron laser). The oxidation of investigated Met-containing compounds by •OH or 3CB* led to the formation of S-centered radical cation >S•+ on Met-residue, that were further stabilized by formation of two-centered three-electron bond (S∴Y)+ or underwent the deprotonation reaction yielding the α-(alkylthio)alkyl radicals (α-S). The oxidation of Met-containing dipeptides by •OH radicals yielded the formation of Met sulfoxide (MetSO) as a main product. Undoubtedly, the identification and characterization of MetSO in deoxygenated solutions containing catalase was a milestone in investigation of stable products. However, in some cases, other products were identified. The stable products of photolysis were adducts with 3-carboxybenzophenone moiety, resulting from radical recombination reaction. Another identified product formed during photolysis was 3CB-3CB benzpinacol photoadduct, which has similar structure to the product of BP irradiations. Identified products (MetSO and the photo-adduct) were formed from the α-S via disproportionation or reaction with 3CBH•/3CBH•⁻. The oxidation of Met-Lys-Bradykinin (MKBR) yielded formation of similar photo-adducts via sensitized reaction with the 3CB*. The •OH induced oxidation of MKBR yielded several products, e.g. the sulfoxide and hydroxylated phenylalanine. In addition, other derivatives of benzophenone (oxybenzone (OXB) and sulisobenzone (SB)) were investigated due. They are widely used in commercial sun-protecting products dp to their unique photophysical properties. However the application of sunscreens awakes controversies because some epidemiological studies indicated an increased risk of malignant melanoma for their users. Photo-instability of sunscreen filters would result in reduced protection and may produce reactive free radicals or mutagens. In addition, the reactions of the sunscreens with oxygen free radicals e.g. hydroxyl radicals are likely to arise and they were not yet sufficiently documented. Finally, the radiolytic and photolytic properties of SB and OXB were investigated using femto-and nanosecond laser flash photolysis. Pulse radiolysis studies of the oxidation of those molecules by •OH radicals were performed. The results obtained for SB and OXB were compared to several other benzophenone derivatives. The results shown the formation of excited singlet state that was deactivated efficiently via the Excited State Intramolecular Proton Transfer (ESIPT). In case of polar solvent, the formation of trace amounts phenoxyl radicals was identified, while for nonpolar media those radicals were not observed. The reactivity of UV-excited sun filters towards simple derivatives of Met was also investigated, however, this topic requires further and more detailed investigations.Plusieurs facteurs conduisent à la génération de radicaux libres oxydants dans les organismes qui endommagent les biomolécules et en particulier les protéines du vivant. L’une des cibles de l’oxydation dans les protéines est la méthionine (Met). Son oxydation provoque des effets très dommageables, comme la maladie d’Alzheimer ou les maladies à prion. Nous avons étudié la réaction des radicaux hydroxyle (•OH) et de l’état triplet de la 3-carboxybenzophénone (3CB*) avec ces peptides. Le but de ce travail était de caractériser les espèces transitoires et les produits stables formés après oxydation radiolytique et photolytique de peptides contenant la Met (les dipeptides contenant de la Met et les pepetides plus longs tels que la bradykinine, une hormone humaine importante impliquée dans la diminution de la pression artérielle). Nous avons utilisé la photolyse éclair laser (LFP) et la radiolyse pulsée (PR) (pour les espèces transitoires de courte durée de vie), tandis que la radiolyse gamma et la photolyse continue (pour obtenir une caractérisation des produits stables). Les modifications structurelles ont été caractérisées par des techniques de HPLC et par couplage de la spectrométrie de masse (MS) et la détection par spectrométrie infrarouge couplée à la MS (IRMPD, CLIO laser à électrons libres). En outre, il nous a paru intéressant d’étudier deux autres dérivés de la benzophénone (BP), l’oxybenzone (OXB) et le sulisobenzone (SB), qui sont largement utilisés dans les produits de protection solaire commerciaux. En effet, l’application d’écrans solaires est controversée car certaines études épidémiologiques ont indiqué un risque accru de mélanome malin pour leurs utilisateurs. L’oxydation de dipeptides contenant Met par les radicaux •OH ou photosensibilisée par la ³(CB)* a conduit à la formation de radicaux cations centrés sur le soufre de la Met (>S•⁺) qui ont été en outre stabilisés par la formation de liaison deux centres à trois électrons (S∴Y)⁺, Y étant un atome possédant un doublet libre, ou qui ont subi une déprotonation donnant les radicaux contrés sur le carbone en α (α-S). L’oxydation des dipeptides par •OH a abouti à la formation de sulfoxyde de Met (MetSO) en tant que produit principal. Sans aucun doute, l’identification et la caractérisation des MetSO en solutions désoxygénées contenant la catalase est une étape importante dans la quête de produits stables. Toutefois, dans certains cas, d’autres produits ont été identifiés. En ce qui concerne, les produits stables de photolyse, ce sont des adduits avec le groupement 3CB, probablement résultant de la réaction de recombinaison radical-radical. Un autre produit formé au cours de la photolyse était 3CB-3CB résultant d’une photo-addition, qui a une structure similaire à celle du produit d’irradiations de la BP. Tous les produits identifiés (MetSO et la photo-adduits) ont été formés à partir des radicaux α-S par l’intermédiaire d’une dismutation ou une réaction avec 3CBH•/3CB•⁻. L’oxydation de la Met-Lys-bradykinine (MKBR) a abouti à la formation de photo-adduits similaires par réaction sensibilisée avec 3CB. L’oxydation induite de MKBR par •OH a abouti à plusieurs produits, en accord avec la non sélectivité des radicaux •OH. L’un des principaux produits est le MetSO et la phénylalanine hydroxylée. Notons que l’arginine n’est pas oxydée. Enfin, la photolyse de SB et OXB a été étudiée à l’aide de photolyse éclair au laser femto-et nanoseconde, ainsi que l’oxydation à un électron de ces molécules par radicaux •OH ont été réalisées en PR. Les résultats obtenus ont été comparés à ceux d’autres dérivés de la BP. L’état singulet excité subit un quenching à 100 % par transfert de proton intraomléculaire à l’état excité (ESIPT) en milieu aprotique et en milieu non polaire. Dans le cas d’un solvant polaire, la formation de radicaux phénoxyles a été identifiée. La réactivité des filtres solaires UV-excité vers dérivés simples de méthionine est également en cours d’étude

Study of the mechanism of radiation- and photo-induced oxidation of methionine containing peptides

Wydział Chemii: Zakład Fizyki ChemicznejW ramach niniejszej pracy doktorskiej przebadano mechanizm reakcji jedno-elektronowego utleniania peptydów zawierających metioninę (np. Met-Lys, Lys-Met, Met-Lys-bradykininę, neurokininę B) wykorzystując metody czasowo-rozdzielczej nanosekundowej laserowej fotolizy błyskowej i radiolizy impulsowej. Scharakteryzowano także produkty trwałe powstałe w wyniku reakcji z foto-sensybilizatorem (3-karboksybenzofenonem, 3CB) i rodnikiem hydroksylowym (●OH) wykorzystując metody chromatografii cieczowej i spektrometrii mas. W oparciu o uzyskane wyniki zaproponowano mechanizm modyfikacji peptydów zawierających Met zachodzących i wykazało. iż pierwsze etapy tych reakcji są podobne dla foto- i radicyjnie indykowanego utleniania. Wykazano także, że głównym produktem trwałym powstałym w wyniku reakcji z rodnikami hydroksylowymi w warunkach beztlenowych jest sulfotlenek metioniny, którego strukturę potwierdzono wykorzystując technikę spektroskopii IRMPD (wielofotonowa fragmentacja w podczerwieni) sprzężoną z spektrometrem mas (FTICR-MS-TOF). Pomimo podobieństw w pierwszych etapach utleniana, produkty otrzymane dla poszczególnych metod utleniania były odmienne. Wykryto tworzący się addukt z wiązaniem kowalencyjnym C-C pomiędzy resztą metioninową i cząsteczką 3CB. W drugiej części pracy przebadano foto-stabilność komercyjnie stosowanych filtrów słonecznych na bazie chromoforu benzofenonu (oksybenzonu, sulisobenzonu) wykazując tworzenie się reaktywnych rodników fenoksylowych pod wpływem naświetlania UVA/UVB.In this thesis the one-electron oxidation of methionine-containing peptides (e.g. Met-Lys, Lys-Met, Met-Lys-bradykinin, neurokinin B) was investigated using the time-resolved nanosecond laser flash photolysis and pulse radiolysis. In addition stable products resulting from the reaction of Met-containing peptide with the photo- sensitizer (3-carboxybenzophenone, 3CB ) and hydroxyl radical (●OH) were characterized using liquid chromatography and mass spectrometry. Based on the obtained results the oxidation mechanism was proposed for the modification of Met-containing peptides and it was shown that the primary steps of oxidation are similar for the photo- and radiation-induced oxidation. It was also presented that the main product from the reaction with ●OH under anaerobic conditions was methionine sulfoxide. Its structure was confirmed using a IRMPD spectroscopy (infrared multiphoton dissociation) coupled to a mass spectrometer (TOF-FTICR-MS). Despite the similarities in the early stages of oxidation for both applied techniques, the stable products were different. The sensitized photo-oxidation yielded the C-C covalently bounded adduct between the methionine residue and 3CB molecule. In the second part of this work, the photo-stability of commercially used sunscreens containing benzophenone chromophore (oxybenzone, sulisobenzone ) was investigated showing that they are able to form reactive phenoxyl radicals under the UVA/UVB radiation

Suchość skóry jako powikłanie nowoczesnych terapii przeciwnowotworowych — patofizjologia i leczenie

W leczeniu systemowym we wspołczesnej onkologii coraz większe znaczenie mają nowoczesne terapie przeciwnowotworowe — leki ukierunkowane molekularnie i immunoterapia. Terapie te wiążą się z toksycznością, ktorej profil rożni się od tradycyjnej chemioterapii i jest dużym wyzwaniem dla klinicystow oraz pacjentow. Do najczęstszych działań niepożądanych należą toksyczności skorne, między innymi suchość skory, ktore mogą wpływać na obniżenie jakości życia pacjentow. Leczone nieskutecznie mogą prowadzić do konieczności modyfikacji dawki bądź zaprzestania leczenia. Suchość skory jest objawem wynikającym z upośledzenia bariery skornej na drodze rożnych patomechanizmow, ktore są zależne od zastosowanego leku. Pacjenci wskazują suchość skory jako objaw niespodziewany i znacznie obniżający jakość życia w trakcie i po zakończeniu terapii. Pomimo tego suchość skory jest powikłaniem, ktorego leczenie jest często zaniedbywane w praktyce klinicznej. Profilaktyka wystąpienia i leczenie suchości skory obejmują unikanie czynnikow drażniących, kąpiele w letniej wodzie i stosowanie emolientow. Wczesne wprowadzenie leczenia zapobiega rozwojowi stanow zapalnych i nadkażeń bakteryjnych

Xerosis as the toxicity of novel anti-cancer therapies — pathophysiology and management

In the systemic treatment of modern oncology, novel anti-cancer therapies are becoming increasingly important. The toxicity profile of these therapies is different from that of standard chemotherapy and has become an emerging challenge for clinicians and patients. Among the most common adverse events are skin toxicities, including xerosis, that might be debilitating and have a negative effect on patients’ quality of life. Untreated or treated ineffectively can necessitate dose modification or treatment withdrawal. Xerosis is a symptom stemming from a skin barrier dysfunction caused by a variety of different mechanisms, which differ depending on the therapy. Patients indicate xerosis as an unexpected symptom that significantly decreases their quality of life. Even so, it is a complication often neglected in clinical practice. Prevention and treatment of xerosis include avoiding irritating factors, bathing in lukewarm water, and applying emollients. Early treatment prevents inflammation and secondary bacterial infections.In the systemic treatment of modern oncology, novel anti-cancer therapies are becoming increasingly important. The toxicity profile of these therapies is different from that of standard chemotherapy and has become an emerging challenge for clinicians and patients. Among the most common adverse events are skin toxicities, including xerosis, that might be debilitating and have a negative effect on patients’ quality of life. Untreated or treated ineffectively can necessitate dose modification or treatment withdrawal. Xerosis is a symptom stemming from a skin barrier dysfunction caused by a variety of different mechanisms, which differ depending on the therapy. Patients indicate xerosis as an unexpected symptom that significantly decreases their quality of life. Even so, it is a complication often neglected in clinical practice. Prevention and treatment of xerosis include avoiding irritating factors, bathing in lukewarm water, and applying emollients. Early treatment prevents inflammation and secondary bacterial infections

Does the presence of ground state complex between a PR-10 protein and a sensitizer affect the mechanism of sensitized photo-oxidation?

The mechanisms of one-electron protein oxidation are complicated and still not well-understood. In this work, we investigated the reaction of sensitized photo-oxidation using carboxybenzophenone (CB) as a sensitizer and a PR-10 protein (MtN13) as a quencher, which is intrinsically complicated due to the complex structure of the protein and multiple possibilities of CB attack.To predict and examine the possible reactions precisely, the 3D structure of the MtN13 protein was taken into account. Our crystallographic studies revealed a specific binding of the CB molecule in the protein's hydrophobic cavity, while mass spectrometry identified the amino acid residues (Met, Tyr, Asp and Phe) creating adducts with the sensitizer, thus indicating the sites of 3CB* quenching. In addition, protein aggregation was also observed.The detailed mechanisms of CB quenching by the MtN13 molecule were elucidated by an analysis of transient products by means of time-resolved spectroscopy. The investigation of the transient and stable products formed during the protein photo-oxidation was based on the data obtained from HPLC-MS analysis of model compounds, single amino acids and dipeptides.Our proposed mechanisms of sensitized protein photo-oxidation emphasize the role of a ground state complex between the protein and the sensitizer and indicate several new and specific products arising as a result of one-electron oxidation. Based on the analysis of the transient and stable products, we have demonstrated the influence of neighboring groups, especially in the case of Tyr oxidation, where the tyrosyl radical can be formed via a direct electron transfer from Tyr to CB* or via an intramolecular electron transfer from Tyr to Met radical cation Met > S●+ or thiyl radical CysS● from neighboring oxidized groups