Università degli Studi di Bari. Laboratorio di Metodologie Chimico Fisiche per sistemi complessi

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1 Università degli Studi di Bari Corso di Laurea in Scienze Chimiche Laboratorio di Metodologie Chimico Fisiche per sistemi complessi Prof. ssa Pinalysa Cosma,

2 Outline Chemical and Physical Methodologies applied to complex systems: 4-Thiothymidine/ Rose Bengal and H 2 O 2 / 4-Thiothymidine A Thermodynamic Exploration of Lysozyme-Eosin Binding. Förster Resonance Energy Transfer and Conformational Stability of Cytochrome c. Use of Alginate Microparticles for dyes removal from wastewater

3 Chemical and Physical Methodologies applied to a complex system: 4-Thiothymidine/ Rose Bengal and H 2 O 2 / 4-Thiothymidine Photo Dynamic Therapy (PDT) and its applications

4 Photo Dynamic Therapy (PDT) and its applications Definitions Photodynamic therapy or PDT is a treatment that use special drugs, called photosenstizing agents. The drugs only work after they have being actived or turned on by certain kinds of light Front. Microbiol., 2012, 3, therapy /realtime-pdt-dosimetry

5 Type I Type II Photochem. Photobiol. Sci., 2007,6,

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7 Photosensitisers

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9 Photo Dynamic Therapy (PDT) and its applications Treatment procedure for topical PDT. A) Skin cancer lesion; B) Cream application (MAL or ALA); C) Occlusion of the lesion; D) Illumination; E) Inflammation and necrosis; F) Curative 5-aminolevulinic acid methyl-aminolevulinate Cintia Teles de Andrade, Natalia Mayumi Inada, Dora Patricia Ramirez, Vanderlei Salvador Bagnato and Cristina KurachiPhotodynamic Therapy for Non-Melanoma Skin Cancer

10 Typical Device Treatment Cycle Device applied at same time as PDT cream The device works during normal daily activities Device disposed

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12 Early Gastric Cancer 3 days after treatment the tumour is undergoing necrosis 2 months after treatment the tumour is healed Lung Cancer

13 Advantages of PDT

14 Rose Bengal Standard for Singlet Oxygen generation with a quantum Yield of 0.8

15 The Chemistry?

16 1 RB 0 1 RB 1 3 RB + The Chemistry? LIGHT 3 RB 1 RB 0 3 RB 3 3 RB RB RB 0 3 RB + 3 RB RB.+ + RB.- Possible ways to quench the RB triplet excited state 3 RB + 1 RB RB 0 3 RB + 1 RB 0 RB.+ + RB.- 3 RB + 3 O 1 2 RB O 2 3 RB + 3 O 2 RB.+ + O.- 2 RB O 1 2 RB 0 + O.- 2 Only at high RB concentration

17 In biological systems the photooxidative quenching of 3 RB 2- by a quencher Q (which may include RB 2- itself) and subsequent electron transfer from Q.- to O 2 is an alternative mechanism for the formation of O 2.- Back electron transfer

18 Photochem. Photobiol. Sci., 2012,11, 14 -Thymidine -Thiothymidine dimer

19 Abs Abs UV Visible : 4-THIOTHYMIDINE ph-related features Thymidine 270 nm * * π-π* Wavelength / nm 337 nm 4-Thiothymidine ph Wavelength / nm ph-related features Vito Rizzi, Ilario Losito,Andrea Ventrella, Paola Fini,Angela Agostiano,Francesco Longobardi and Pinalysa Cosma, RSC Adv., 2014, 4, 48804

20 Log ([S 4 TdR]/[S 4 TdR - ]) n=6, R 2 = pka=9.1± ph 4 [S TdR] log 4 - [S TdR ] pk a - ph Henderson-Hasselbalch equation

21 4-THIOTHYMIDINE ESI MS ANALYSIS LCQ ESI-3D-Ion Trap Mass Spectrometer MS(+) [S 4 TdR+H] + [S 4 TdR+Na] + [2S 4 TdR+Na] + [2S [S 4 TdR+ 4 TdR+ CH 3 OH+Na] + CH 3 OH+Na] + [3S 4 TdR+Na] + Infusion: H 2 O/CH 3 OH, 50:50 (v/v) Interface/Ion optics parameters: sheath gas flow rate, 60 (a.u.); auxiliary gas flow rate, 0 (a.u.); spray voltage, 6 kv; capillary temperature, 190 C; capillary voltage, 15 V; tube lens offset, 10V; octapole 1 offset, 2.5 V; lens voltage, 16 V; octapole 2 offset, 5.5 V; octapole RF amplitude, 400 V p-p

22 4-THIOTHYMIDINE ESI MS ANALYSIS x8 MS/MS 1 m/z units-wide isolation window including the main isotopologue (M+0) of the precursor ion. Vito Rizzi, Ilario Losito,Andrea Ventrella, Paola Fini,Angela Agostiano,Francesco Longobardi and Pinalysa Cosma, RSC Adv., 2014, 4, 48804

23 In the presence of RB? Lamp emission wavelength: nm Power surface density: 60 mw/cm 2 S 4 tdr By-Products? Vito Rizzi, Ilario Losito,Andrea Ventrella, Paola Fini,Aurore Fraix,,Salvatore Sortino, Angela Agostiano,Francesco Longobardi and Pinalysa Cosma Phys.Chem.Chem.Phys., 2015, 17, 26307

24 Relative Abundance 4-THIOTHYMIDINE ESI MS ANALYSIS Relative Abundance h [S 4 TdR+Na] Relative Abundance M M [RB + H] [2S 4 TdR+Na] + [S 4 TdR - H +2 Na] [RB-2H+3Na] [2S 4 TdR 2H +3Na] m/z m/z M M LCQ ESI-3D-Ion Trap Mass Spectrometer x10 [RB + Na] [RB-H+2Na] ph 7 1 h of irradiation h [(S 4 TdR) cd + Na] [S 4 TdR + Na] [TdR + Na] ddr -ddr m/z

25 Relative Abundance h [S 4 TdR+Na] Relative Abundance THIOTHYMIDINE ESI MS ANALYSIS ph 12 1 h of irradiation [2S 4 TdR+Na] m/z LCQ ESI-3D-Ion Trap Mass Spectrometer 1 h [TdR+Na] [S 4 TdR+Na] [S 4 TdR+TdR+Na] [2TdR+Na] [(S 4 TdR) cd + Na] m/z

26 Reaction scheme +RB k Q RB RB.+ +RB. - 0 RB + hv 1 RB φ T I a 3 RB k D RB Physical quenching or or electron transfer 3 3 O O 2 k Q O O 2 k Δ O 2 RB by-products k r RB +RB 1 O 2 k d 3 O 2 ph 7 +S 4 TdR K q S 4 TdR +S 4 TdR - ph 12 K q* S 4 TdR 3 O 2 k Q S 4 TdR - +2S 4 TdR +S 4 TdR +S 4 TdR - +2S 4 TdR - k Q* S 4 TdR - 3 O 2 S-S bridged S 4 TdR dimer k r S 4 TdR k r* S 4 TdR - S-S bridged S4TdR dimer TdR TdR

27 Abs Abs UV-VIS ANALYSIS 1,2 1,0 0,8 ph 7 FAST DEGRADATION 0,6 0,4 1,2 1,0 0,8 0,6 0,4 0,2 0,0 ph Wavelength/nm 0,2 0, Wavelength/nm

28 abs Reaction between S 4 -TdR and H 2 O 2 : UV-Vis investigation Spectroscopic features: Decrease of the absorption band at 337 nm red shift occurring between the 337 and the 363 nm absorption maxima: a clue for the generation of intermediate byproducts of S 4 -TdR TdR oxidation at the level of the C=S chromophore presence of a band at 363 nm, initially increasing and then decreasing at the increase of the band at 270 nm nm Comparison between UV-VIS absorption spectra obtained, at 5 min intervals and over a 100 min time range, for an aqueous solution initially containing 1 x 10-3 M S 4 -TdR and 2 x 10-1 M H 2 O 2

29 Relative Abundance ESI-MS spectra obtained for a 1 x 10-3 M solution of S 4 -TdR 2 min after the addition of 200 x 10-3 M of H 2 O 2 [2 HO-S 4 TdR+2 Na + Cl - ] [HO-S 4 TdR+Na] [2 HO-S 4 TdR 2 H + 3 Na] [2 HO-S 4 TdR -H + 2 Na] [2 HO-S 4 TdR+Na] [S 4 TdR + HO-S 4 TdR+Na] [HO-S 4 TdR H +2 Na] + 40 [HO-S 4 TdR+ 2 Na + Cl - ] [HO-S 4 TdR+H] + [HO-S 4 TdR H + 3 Na + Cl - ] [S 4 TdR+TdR+Na] [TdR+Na] + m/z

30 Relative Abundance ESI-MS spectra obtained for a 1 x 10-3 M solution of S 4 -TdR 30 min after the addition of 200 x 10-3 M of H 2 O [HO-S 4 TdR+Na] [O=C-TdR+Na] [S-S bridged S 4 TdR dimer + Na] [TdR+Na] [O=C-TdR+H] [TdR + O=C-TdR+Na] [O-O bridged TdR dimer + Na] m/z

31 Relative Abundance ESI-MS spectra obtained for a 1 x 10-3 M solution of S 4 -TdR 90 min after the addition of 200 x 10-3 M of H 2 O [TdR+Na] [O=C-TdR+Na] + [TdR + O=C-TdR+Na] O H N H NH O [O=C-TdR+H] + [HO-S 4 TdR+H] [2 TdR+Na] [HO-S 4 TdR+Na] [TdR+H] + [HO-S 4 TdR O=C-TdR+Na] m/z

32 Relative Abundance ESI-MS spectra obtained for a 1 x 10-3 M solution of S 4 -TdR 120 min after the addition of 200 x 10-3 M of H 2 O [O=C-TdR+Na] [S 4 TdR+Na] Still present [HO-S 4 TdR+H] [HO-S 4 TdR+Na] Low [TdR+Na] [S 4 TdR+H] m/z

33 Relative Abundance ESI-MS spectra obtained for a 1 x 10-3 M solution of S 4 -TdR 60 min after the addition of 50 x 10-3 M of H 2 O [O=C-TdR+Na] [HO-S 4 TdR+Na] Hydroxylated forms predominate [HO-S 4 TdR+H] [S-S bridged S 4 TdR dimer + Na] [TdR+Na] [O=C-TdR+H] [O-O bridged TdR dimer + Na] m/z [2S 4 TdR+Na] + Still present

34 O S OH OHC NH N N O N O O O OH Na + OH OH Na + OH