Yeni silisyum ftalosiyaninlerin sentezi ile fotokimyasal ve biyolojik özelliklerinin incelenmesi
Synthesis of new silicon phthalocyanines with the investigation of their photochemical and biological properties
Yazar: CANAN USLAN
Danışman: YRD. DOÇ. DR. B. ŞEBNEM SESALAN
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Ana Bilim Dalı / Kimya Eğitimi Bilim Dalı
Derece: Yüksek Lisans, 2012
Konu: Kimya = Chemistry
Dizin: Fotokimya = Photochemistry ; Fotokimyasal reaksiyonlar = Photochemical reactions ; Ftalosiyaninler = Phthalocyanines ; Singlet oksijen = Singlet oxygen

Özet:
Doğal porfirin halkası ile yapısal olarak benzer olan ftalosiyaninler, 18-? elektron sistemlerine sahip olmalarından dolayı organik fonksiyonel materyallerin en yoğun çalışılan sınıflarından biri olmuştur.Ftalosiyaninlerin kimyası üzerine yapılan önemli araştırmalardan biri de onların çeşitli solventlerde çözünürlüğünü artırmaktır. Bu yüzden agregasyona uğramayan ve suda çözünebilen ftalosiyaninler önemlidir ve birçok uygulama için kullanılabilme potansiyeline sahip maddelerdir.Ftalosiyaninler geleneksel olarak boya ve pigment olarak kullanılagelmiştir. Son zamanlarda yoğun olarak araştırılan ftalosiyaninler, fotokopi cihazlarında fotoiletken, kimyasal sensör, elektrokataliz, elektrokromizm ajanı olarak birçok bilimsel alanda kullanım alanı bulmuştur.Endüstrideki yaygın uygulama alanlarının yanında ftalosiyaninler; fotodinamik terapi (PDT), patolojik bakterilerin ve mantarların fotoinaktivasyonu amacıyla da kullanılırlar. Ftalosiyaninler porfirinlere göre çok daha şiddetli, yaklaşık 680 nm civarında gösterdikleri absorbsiyon ile ve singlet oksijen üretimi ile PDT için umut verici fotoalgılayıcılardır. Ancak ftalosiyaninler sulu çözeltide agrage olabilirler. Bu durum 680 nm civarındaki absorbsiyonlarını söndürdüğü için ftalosiyaninlerin fotoalgılayıcı yeteneklerini de önemli ölçüde azaltır.Son zamanlarda hücre kültürü kullanılarak yapılan kanser araştırmalarının çoğunda, DNA'ya bağlandığı tespit edilen katyonik ftalosiyaninler kullanılmaktadır. Bağlanan ftalosiyaninlerin uyarılmasıyla singlet oksijen oluşumu sağlandığı ve tümör hücresinin parçalandığı görülmektedir. Tıbbi uygulamalar için suda çözünürlük önemli bir faktördür. Ftalosiyaninlere karboksilat, sülfonat ve kuaternize edilmiş amino gibi hidrofilik grupların eklenmesiyle onların su ve DMSO gibi polar çözücülerde çözünmelerini sağladığı bilinmektedir. Genellikle, PDT çalışmalarında singlet oksijen oluşturma kapasitesi yüksek olan silisyum ftalosiyaninler kullanılmaktadır.Bu çalışmada, N-metil pirolidin-2-ol, N-(2-Hidroksietil) hekzametilenimin ve N,N,N-trimetilamonyum metil fenoksi yapılarının, silisyum ftalosiyaninato diklorür ile kuru toluen içerisinde NaH varlığında 24 saat reaksiyona sokulmasıyla eksenel konumları farklı fonksiyonel gruplara sahip silisyum ftalosiyanin bileşikleri elde edilmiştir. Bu yeni silisyum ftalosiyaninler dimetil sülfatın aşırısı kullanılarak kuaternize edilmiştir.Elde edilen bileşiklerin yapıları UV-Vis, FT-IR, 1H NMR, floresans ve kütle (MALDI-TOFF yöntemi ? dihidroksibenzoik asit matriksi kullanılarak) spektrumları ile elementel analiz sonuçları yardımıyla karakterize edilmiştir. Bunlara ilave olarak agaroz jel elektroforez, UV/Vis ve floresans DNA-Pc titrasyon spektrumları, bir DNA boyası olan SYBR Green I ile DNA kompleksinin floresansını sönümlendirme spektrumları, KDNA bağlanma sabitleri ve DNA termal denaturasyon eğrileri kullanılarak DNA'ya bağlandıkları tespit edilirken diğer yandan literatürde yaygın olarak kullanılan ve ticari olarak satılan singlet oksijen tuzakları kullanılarak hem nötr hem de suda çözünür kuaternize ftalosiyaninlerin singlet oksijen ürettikleri ortaya konmuştur.Anahtar Kelimeler: PDT, ftalosiyanin, DNA, singlet oksijen

Abstract:
Phthalocyanines (Pcs) have been one of the most extensively studied classes of organic functional materials because of their aromatic 18-? electron system which is closely related to that of the naturally occurring porphyrin ring. Phthalocyanines have a long-wavelength band with a large extinction coefficient (~105 M-1cm-1) and generally a low dark toxicity. Some are efficient 1O2 generators, and are easily obtainable in pure form. Some phthalocyanines are promising second-generation photosensitizers. Based on the extensive biological research done on photosensitizers, some of the desired properties of second generation photosensitizers have been established. They should be, for example, chemically pure and maintain a constant composition during the treatment; they should exhibit minimal photobleaching and have minimal dark toxicity; they should be preferentially retained by the target tissue (to minimize damage to healthy cells); they should be quickly cleared after PDT treatment; they should have a high quantum yield of long-lived triplet states (to produce a high yield of 1O2); and they should have a large extinction coefficient in the region of 600-800 nm (for good light penetration into tissue). These desired properties have provided major guidelines for designing new photosensitizers, although no photosensiter can be expected to meet all these criteria.One of the important aims of research on the chemistry of phthalocyanines is to enhance their solubility in various solvents. In this case non-aggregated and water-soluble phthalocyanines are important and potentially useful materials for many applications.Phthalocyanines have been used as dyes and pigments traditionally. During the last few years, phthalocyanines (Pcs) have been intensively studied due to their applications in many scientific areas such as photoconducting agents in photocopying devices, chemical sensors, electrocatalyst, electrochromism agents.In addition to their conventional use in industry, phthalocyanines are also used in photoinactivation of bacterias and photodynamic therapy (PDT). Photodynamic therapy (PDT) is a promising cancer treatment utilizing a photosensitizer, visible light and oxygen. Usually PDT is carried out in three stages. First, the PDT drug is administered by an appropriate method, intravenous injection is a common one. Then, a drug-biodistribution interval is allowed. Finally, the tumor is irradiated with visible light. In a key step of PDT, a photochemical reaction generates singlet oxygen, 1O2. This is strongly cytotoxic because of its great oxidizing ability. Phthalocyanines are particularly promising photosensitizer for PDT because of their intense absorptions at about 680 nm (about 2 orders of magnitude larger in intensity than porphyrin long wavelength absorptions) and their ability for generating singlet oxygen. The ground state of the PDT drug is excited to its singlet state with light of suitable wavelength. This state can relax to its ground state by non-radiative decay or by emitting a fluorescence photon, or it can be converted to a triplet state by intersystem crossing. The triplet state can relax to the ground state by emitting a phosphorescence photon, by electron transfer to a surrounding substrate with the generation of hydroxyl or superoxide radicals (a Type-I photoreaction), or by energy transfer to triplet ground-state oxygen with an accompanying production of singlet oxygen (a Type-II photoreaction). Type-II photoreactions dominate during normal PDT. Generally the outcome of PDT is the combined result of three processes, direct cell killing, tumor vascular effects and immune system effects. The direct cell effects involve damage to organelles such as lysosomes and mitochondria, and to cell membranes. These lead to tumor hypoxia and cell death. The immune effects can include inflammatory cell effects and the generation of long-term anti-tumor responses. These too can cause cell death. However, Phthalocyanines are well known for their high tendency to aggregate in aqueous solutions, which can significantly decrease their photosensitizing ability through self-quenching.The central metal ion and peripheral substituents play an important role in the photophysical properties of phthalocyanines. The ligands or the metal in these macromolecules can be varied in an easily controlled manner to facilitate an individual application. When a diamagnetic ion is in the center of the ring (e.g., Zn, Al, Ga), the phthalocyanine generally possesses a high triplet state yield (?T > 0.4) with a long lifetime (?T > 200 ?s) and enough energy (110-126 kJ/mol-1) to generate 1O2 (94.5 kJ/mol-1 is required). The triplet-state lifetimes of an axially substituted silicon phthalocyanine typically vary from 100 to 200 ?s and the yields from 0.2 to 0.5. Some phthalocyanines (e.g., AlPcX, SiPcX2) have one or two axial ligands. The peripheral substituents and axial ligands govern to an important extent the physical and chemical and biological properties of these compounds (e.g., stability, solubility) and make it possible to prepare satisfactorily optimized phthalocyanines for use as photosensitizers.The solubility of Pcs is very important for the investigation of their chemical and physical characteristics. To increase the solubility of Pcs some different kinds of solubility-enhancing substituents like alkyl, alkoxy, phenoxy groups can be added to the peripheral and axial positions of the Pc ring. Amphiphilic phthalocyanines which incorporate polar and non-polar groups at the same time are significant especially in photodynamic theraphy. The sulfonated silicon phthalocyanines were expected to have higher solubility in polar solvents than their unsubstituted phthalocyanine analogues. This property has the potential to make it easier to disperse these phthalocyanines in surfactants where phthalocyanines can serve as photobleaches. They were also expected to provide good candidates for photodynamic therapy studies.It is known that cationic Pcs form complexes with nucleic acids. Recently, cationic phthalocyanines which bind to DNA have been used on cell culture experiments for the purpose of PDT. It was seen that these phthalocyanines produce singlet oxygen and cause the lysis of cell. Water solubility is an important factor for medical applications. It has been known that, phthalocyanines which are substituted with hydrophilic groups such as sulfonates, carboxylates or quaternized amino groups can be soluble in polar solvents such as water or DMSO. Generally silicon phthalocyanines are used in PDT due to their high singlet oxygen quantum yields.In this work, silicon phthalocyanine compounds with different functional groups in axial positions were obtained by reacting N-methyl pyrrolidine-2-ol, N-(2-Hydroxyethyl) hekzamethyleneimine and N,N,N-trimethylamonium methyl fenoxy groups with unsubstituted silicon phthalocyanine in the presence of NaH in dry toluene for 24 h. Thus, they have less capacity to aggregate with their macrocycles close to each other, and more capacity to hydrogen bond. Because of this, they have more potential to be taken up by cells and less to have short triplet lifetimes. Thus, they have the promise of being good PDT agents. The new compounds are quaternized with the use of excess amount of dimethyl sulfate.The structures of these compounds were characterized by using UV-vis, FT-IR, 1H NMR, fluorescence and mass spectroscopic (MALDI-TOFF method- using of dihydroxybenzoic acid matrix) techniques. In addition, while the interaction of these compounds with DNA was evaluated by agarose gel electrophoresis, UV/ Vis DNA titration spectras, fluorescence DNA-Pc titration spectras, quenching spectrums of DNA with SYBR Gren I which is a dye of DNA, KDNA binding constants and DNA thermal denaturation curves on one side, on the other side the generation of singlet oxygen of all species was determined by the using of singlet oxygen quenchers which are using generally in the literature and commercially avaliable. For the evaluation of the results of both UV/vis titrations and gel electrophoresis, it was shown that high water solubility of the complexes makes them suitable for biological investigations under different solution conditions. Not intercalative but electrostatic interactions between pcs and DNA were the main factors for binding. The extended ? system of Pcs will lead to a deeper penetration and make stronger stacking with DNA.