13 research outputs found
Synthesis of novel secondary sulfonamides and their in vitro carbonic anhydrase isozymes inhibition effects
Tez (Doktora) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2017Theses (Ph.D.) -- İstanbul Technical University, Institute of Science and Technology, 2017Karbonik anhidraz enzimleri tüm canlı organizmalarda bulunan ve geri dönüşümlü bir reaksiyon olan karbondioksitin bikarbonata ve protona dönüştüğü reaksiyonu katalizleyen bir metaloenzim ailesidir (EC 4.2.1.1). Karbonik anhidraz enziminin yapısı 260 adet aminoasitten oluşmaktadır ve bu enzim doğal hali kürecik şeklinde olan bir protein zinciridir. Karbondioksitin hidratasyonu haricinde, COS ve CS2 hidratasyonu, siyanamid hidratasyonu, ester hidrolizleri gibi reaksiyonlar da bu enzim süper ailesi tarafından katalizlenmektedir. Bu reaksiyonların katalizlenmeleri diğer tüm karbonik anhidraz katalizleme reaksiyonlarında olduğu gibi metal hidroksitin nükleofil olarak davranması yoluyla gerçekleşir. Karbonik anhidraz enzim aileleri, katalizleme işlemi sırasında aktif bölgede bulundurdukları metal iyonuna göre farklılık göstermektedirler. Enzimin aktif bölgesi koni biçiminde geniş bir alana yayılmış, molekülün merkezine kadar uzanan oyuk şeklindedir ve çinko iyonu oyuğun alt kısmına bitişik durumdadır. Tüm karbonik anhidraz ailelerinde katalitik reaksiyon iki kademeden oluşur. İlk kademede enzimin aktif kısmı olan metal hidroksit kısmı (EM2+-OH-) nötr pH ortamında güçlü bir nükleofil gibi davranarak karbondioksit molekülü ile bikarbonat enzim kompleksi oluşturur ve sonrasında bu bikarbonat molekülü su molekülüyle yer değiştirir. İkinci kademede enzim metal kompleksine bağlı su molekülü (EM2+- H2O) enzimin aktif kısım kalıntısına veya bir proton alıcısına (B) proton transfer ederek enzim metal hidroksit kompleksi oluşturur (EM2+- OH-). α-karbonik anhidraz ailesi insanlarda bulunan türüdür ve şimdiye kadar bu türe ait 16 farklı izoenzim bulunmuştur. Bunların beş tanesi (CA I, II, III, VII ve XIII) sitozolik, dört tanesi (CA IV, IX, XII ve XIV) membrana bağlı, iki tanesi (CA VA ve VB) mitokondriyal, bir tanesi (CA VI) tükürük salgısında ve sütte bulunur. CA XV insanlarda ve günümüz primatlarında gözlenmez fakat kemirgenlerde ve yüksek omurgalılarda gözlenir. Üç adet de katalitik özellik göstermeyen ve karbonik anhidrazla alakalı proteinler olarak adlandırılan türü mevcuttur (CARP-VIII, CARP-X ve CARP-XI). CA I enzimi kırmızı kan hücreleri ve kalın bağırsakta yaygın olarak bulunmasına rağmen plazmada bulunan klorür ve bikarbonat ile inhibe olması fizyolojik fonksiyonlarıyla ilgili soru işaretleri oluşturmaktadır ve genellikle araştırmacıların hedefinde pek yoktur. Fakat yapılan bazı araştırmalar bu enzimin eritrositlerdeki aktivitesinin azalmasının kansızlık ve kronik asidoz ile ilişkili olduğu, ve hücre dışı ortamda yüksek seviyede bulunmasının diabetik maküler ödem ve çoğalan diabetik iltihapsız retina hastalığı gibi durumlara yol açan aşırı retinal damar geçirgenliğiyle ilişkili olduğu yapılan çalışmalarla gösterilmiştir. Tüm bunların ötesinde, CA I'in kendisi CA II ve CA IV ile birlikte, Hippel-Lindau tümörleri, ilerleyen polikistik böbrek hastalığı, acinar-ductal pankreatik karsinom, veya bağışıklık sistemi/idyopatik kronik pankreatitis gibi hastalıklarla ilişkilendirilmiş ama henüz tam olarak fonksiyonları ortaya konulamamıştır. Diğer yandan, CA I'in sadece yüksek seviyelerinin değil bazı durumlarda düşük seviyelerinin de patolojik durumlarla ilişkili olduğu gözlemlenmektedir ve tip II diyabet hastalarının eritrositlerinde CA I seviyelerindeki düşüklük buna örnek olarak verilebilir. CA II enzimi, kırmızı kan hücrelerinde baskın fizyolojik özellikleriyle bulunan izoenzimdir. Bu izoenzim sitozolik bir karbonik anhidraz izoenzimidir ve tüm omurgalılarda birçok hücre, doku ve organda yaygın olarak bulunmaktadır. Çok önemli bazı görevlerin yanında insan CA II izoenziminin (hCA II) görev aldığı, karbondioksitin bikarbonata ve protona dönüştürülmesi gibi spesifik görevleri de vardır. Görev aldığı diğer fonksiyonlar ise, idrarın oluşturulması ve börek kanallarında bikarbonatın geri absorplanması, glikoneojenez, lipojenez ve ürejenez gibi biyosentetik reaksiyonlar, kemik erimesi, kireçlenme ve henüz yeterli olarak anlaşılamamış birçok fizyolojik/patolojik reaksiyon. Bu izoenzimin aktivitesindeki düzensizliklerin önemli patolojik sonuçları olmaktadır, bunlar (i) gözde aşırı göz sıvısı salgılanması sonucu oluşan göz içi basıncın arttığı glokom; (ii) CA II'nin böbrekte diğer izoformlardan bazılarıyla birlikte rol aldığı prosesler sonucu idrarda yeterli miktarda sıvı salgılanmaması veya elimine edilmemesi sonucu vücütta sıvı birikmesiyle oluşan ödem; (iii) epilepsi; (iv) bazı kanser türlerinde, CA II izoenziminin CA IX ve CA XII gibi diğer bazı izoenzimlerle birlikte seviyelerinde gözlenen yükseklikler. Yukarıda bazı örneklerini verdiğimiz ve bu enzimlerin seviyelerinde gözlenen yükseklikle ilişkilendirilen patolojik durumlar dolayısıyla bu enzimlerin inhibisyonları önem arz etmektedir. On yıllarca yıldır yapılan çalışmalar nedeniyle günümüzde artık inhibisyon mekanizmaları birçok durumda detaylı olarak anlaşılmıştır. Şimdiye kadar yapılan çalışmalar doğrultusunda dört farklı karbonik anhidraz inhibitör grubu ortaya konulmuştur: (i) Sülfonamidler ve onların izosterik eşdeğerleri gibi Zn2+ iyonuna bağlananlar, (ii) fenoller, poliaminler, sülfokumarinler gibi çinko iyonuna koordine su molekülüne/hidroksit iyonuna tutunanlar, (iii) kumarinler ve onların izosterik eşdeğerleri gibi enzimin aktif bölgesinin girişini kapatanlar; (iv) ve henüz inhibisyon süreci yeterli olarak anlaşılamamış sekonder/tersiyer sülfonamidler, imatinib, nilotinib ve diğerleri. İlk olarak 1930'larda antibakteriyel özellikleri keşfedilen sülfonamidler aynı zamanda en önemli karbonik anhidraz inhibitör sınıfıdır ve asetazolamid, methazolamid, etoksazolamid, sultiyam, diklorofenamid, dorzolamid, brinzolamid, sulpirid ve zonisamid gibi örnekleri yıllardır klinik olarak idrar söktürücü, glokom veya epilepsi tedavisinde kullanılmaktadırlar. Fenoller hidroksil grubunda bulunan hidrojeni vererek, lipitlerin ve protein, karbonhidrat, nükleikasitler gibi diğer biyomoleküllerin serbest radikallerle okside olmalarını önlerler. Antioksidan özelliklerinin dışında fenollerin, antikanserojen, antimutajen, antibakteriyel, antiviral ve antienflamatuar özelliklerinin olduğu raporlanmıştır. Fenoller yukarıda saydığımız özelliklerinin dışında potansiyel birer karbonik anhidraz enzimi inhibitörüdürler. Fenolün CA II enzimiyle oluşturmuş olduğu kompleksin X ışını kristal yapısı incelendiğinde daha önce öngörülmemiş şekilde hidroksil grubu vasıtasıyla enzimin çinko iyonuna bağlı su/hidroksit iyonuyla ve Thr199'un NH grubuyla bağ kurduğu görülmüştür. Bu çalışmada her biri ayrı ayrı farklı biyolojik özellikler ihtiva eden ve aynı zamanda her biri farklı inhibisyon mekanizmasına sahip karbonik anhidraz inhibitör grubu olan sülfonamidler ve fenoller tek bir molekülde birleştirilerek, yeni sekonder sülfonamid grubu oluşturulmuş ve bunların insan kanındaki eritrosit hücrelerinden Sepharose-4B-L-tirozin-sülfanilamid afinite kromatografisi kullanılarak elde edilen hCA I ve hCA II izoenzimlerinin in vitro inhibisyonu etkileri incelenmiştir. Bu çalışmada kullanılan sülfonamidler, zamanında antibakteriyel ve değişik amaçlarla klinik olarak kullanılmış ve bir kısmı hala aktif olarak kullanılmakta olup, birçok meyve ve bitkide doğal olarak bulunmakta olan ve antioksidan, antikanserojen gibi özellikler göstermekte olan p-hidroksibenzoik asit ve 3,4,5-trihidroksibenzoik asit gibi fenollerle birleştirilerek, üzerlerinde tiyazol, pirimidin, piridin, izosoksazol, tiyadiazol, metizol ve dimetoksin gibi gruplar içeren yeni asetoksi benzamid ve hidroksi benzamid grupları elde edilmiştir. Yapılan in vitro hCA I ve hCA II inhibisyon çalışmalarından elde edilen sonuçlar incelendiğinde genel itibariyle elde edilen yeni moleküllerin başlangıç moleküllerinden daha iyi birer karbonik anhidraz inhibitörü oldukları, ve bazı moleküllerin ise klinik olarak kullanılmakta olan asetazolamid bileşiğinden daha etkin birer karbonik anhidraz inhibitörü olduğu görülmektedir.Carbonic anhydrase isozymes are abundant in all life kingdoms and is a super family of metalloenzyme (EC 4.2.1.1) that catalyze the reversible hydration of carbon dioxide to bicarbonate and proton. It has a globular form and is a protein chain builded up from 260 aminoacids. In addition to hydration of carbon dioxide they also catalyze hydariton of COS and CS2, hydration of cyanamide and hydrolysis of esters. All these processes are achieved in a simple way where, activated metal hydroxide act as a nucleophile. All carbonic anhydrases are metalloenzymes and all six distinct families (α-, β-, γ-, δ-, ζ- and η-CAs) discovered up to now differ in their presence for metal ions used within the active site for performing the catalysis. Active site of the enzyme is widespread in a conical shape cavity reach out way to the center of the enzyme and the zinc ion is next to the underside of this cavity. In all CA classes, the reversible hydration of carbon dioxide to bicarbonate and proton ( CO2 + H2O ↔ HCO3- + H+) follows a two step catalytic mechanism, where an enzyme metal hydroxide adduct (E-M2+-OH-) is the catalytically active species. At neutral pH, this enzyme metal hydroxide adduct act as a strong nucleophile on the carbon dioxide molecule bound in a hydrophobic pocket nearby by with consequent formation of bicarbonate ion, and then it is displaced from the active site by a water molecule. At the second step of the reaction, proton transfer from the water molecule bound to metal ion to an exogenous proton acceptor regenerates the metal hydroxide species again. This second step is the rate-determining step of the catalytic turnover in many enzymes. For some α- and ζ-carbonic anhydrases achieves kcat / KM values bigger than 108 M-1xs-1, making carbonic anhydrases among the most effective catalysts known in the nature. Human carbonic anhydrases (hCAs) belong to the α-class of this super enzyme family; 15 different isoforms out of 16 in this group described in humans so far. 12 of them are catalytically active (CAs I-IV, VA, VB, VI, VII, IX, and XII-XIV) and 3 of them do not exhibit any catalytic acitivity so called CA-related proteins (CARPs). The catalytically active human carbonic anhydrases differ on their subcellular localization and can be grouped in four different subclasses; hCAs I, II, III, VII and XIII are cytosolic, hCAs IV, IX, XII and XIV are membrane-associated, hCAs VA and VB exist in mitochondria, hCA VI is secreted in saliva and milk. These isozymes are involved in various crucial physiological processes and can be found in many tissues and organs through out the body. CA I isozyme is in general not in the target of scientists although it is prevalently expressed in red blood cells and colon. Since it can be inhibited by clorine and bicarbonate ions, there are question marks raising about its physiological functions. However, several evidences demonstrated that changing levels of this isozyme are correlated with several pathological processes. For example, studies showed up that there is a correlation between some types of anemia and chronic acidosis with a decrease of CA I activity in erythrocytes. Another discovery reported by Gao et al. is that the excessive retinal vascular permeability was correlated with a high level of CA I in the extracellular medium. CA I was also associated alone or together with some other CA isozymes, with vasogenic edema, colorectal cancers, prostate cancer, von Hippel-Lindau tumors, progressive kidney disease, acinar-ductal pancreatic carcinomas, or autoimmune/idiopathic chronic pancreatitis. Not only high levels, but also lower levels of CA I is associated to pathological processes, such as low levels of CA I in erythrocytes have been evidenced in type II diabetic patients. CA II is a ubiquitously cyctosolic carbonic anhydrase isoform which is first discovered in the red blood cells with essential physiological functions over than 80 years ago, and it is widespread in a multitude of cells, tissues, and organs in all vertebrates. CA II has crucial and specialized functions, and among them the human carbonic anhydrase (hCA II) is involved in the transport of carbondioxide/bicarbonate from tissues where it is generated to the lungs. Blood of mammals comprise two carbonic anhydrase isoforms, CA I and CA II, and the total concentration can be as high as 0.2 mM. The catalytic activity of CA II is much higher than CA I isozyme. In addition to catalytic acitivity for the conversion of water and carbondioxide to bicarbonate and proton, CA II has several functions include urine formation and bicarbonate reabsorption in the kidney tubules, biosynthetic reactions, such as ureagenesis, lipogenesis and gluconeogenesis, bone resorption and calcification. To regulate the irregular activities of this isoform in one or more tissues is or can be a solution for some pathological processes associated with it, such as, increase in the intraocular pressure due to excessive aqueous humor secreted in the eye (glaucoma), fluid accumulation in the body due to in adequate fluids are secreted/eliminated in the urine (edema) (due to processes in the kidney associated to CA II with other carbonic anhydrase isozymes such as CA IV, XII and XIV), processes associated with CA II with other brain isoforms which causes epilepsy, and some forms of cancers associated to increased quantities of CA II alone or with other cancer related isoforms CA IX and CA XII. In addition to pathologies above, CA II is involved in acute mountain sickness (AMS), atherosclerosis and also osteoporosis. Due to the pathological processes associated with CA activities, inhibition of these enzymes become an essential task. Due to the investigations done for decades on the inhibition of these enzymes, inhibition mechanisms are well known in detail today. If we need to categorize, we can sum the CA inhibitors into four main groups: (i) Inhibitors that bind to metal ions, for example sulfonamides and their isosteres (such as sulfamates, sulfamides and N-hydroxy-sulfonamides), the dithiocarbamates and their isosteres (such as the xanthates), the hydroxamates. (ii) Inhibitors that anchor to the zinc-coordinated water molecule/hydroxide ion, such as phenols, polyamines and sulfocoumarins. (iii) Inhibitors that occlude the entrance of the enzyme active site, such as coumarins and their isosteres. (iv) Inhibitors that bind in an unknown manner such as secondary/tertiary sulfonamides, imatinib and nilotinib. Sulfonamides are best known carbonic anhydrase inhibitors, and since their first discovery in 1930s as antibiotics they become solution for many other pathologies such as glaucoma, epilepsy and diuretic. Today several sulfonamides such as acetazolamide, methazolamide, ethoxazolamide, sulthiame, dichlorophenamide, dorzolamide, brinzolamide, sulpiride, zonisamide, topiramate, saccharin, celecoxib, chlorothiazide, furosamide and bumethanide are still in clinical use as diuretics, antiglaucoma agents and antiepileptics. Phenols prevent biomolecules such as lipids, proteins, carbohydrates and nucleic acids to get oxidized by giving proton of their hydroxyl group. In addition to their antioxidant properties, anticarcinogen, antimutagenic, antibacterial, antiviral and antiinflammatory effects are also reported by various studies. Phenols are also potentially a carbonic anhydrase inhibitors in addition to properties we mentioned above. Phenol is also the first compound reported to inhibit CA by different mechanism than binding to zinc ion. In this group of inhibitors, the inhibitor is not in a direct contact with the metal ion, instead by a presence of anchoring group which is attached to a scaffold that can interact with the two halves of the active side of the enzyme. Further studies on the X-ray crystallographic examinations of adduct formed by CA II enzyme and phenol evidenced that, hydroxyl group of phenol interacts with zinc-coordinated water/hydroxide ion and NH moiety of Thr199 by forming hydrogen bonds. In this work new secondary sulfonamides are synthesized by combining primary sulfonamides with naturally occuring phenols in one molecule. Sulfonamides used in this work are clinically approved for various applications, and phenols (p-hydroxybenzoic acid and 3,4,5-trihydroxybenzoic acid) are naturally occuring in various plants and evidenced that they comprise properties such as antioxidant, antibacterial and anticarcinogen. These new secondary sulfonamides are classified in four groups, (i) acetoxybenzamide, (ii) triacetoxybenzamide, (iii) hydroxybenzamide and (iv) trihydroxybenzamide, all having thiazole, pyrimidine, pyridine, isoxazole and thiadiazole moieties and their inhibitory effects were studied in vitro on two metalloenzymes, i. e. human carbonic anhydrase isozymes (hCA I and II), purified from human erythrocyte cells by Sepharose-4B-L tyrosine-sulfanilamide affinity chromatography. In comparison to starting primary sulfonamides, new compounds in general has better inhibition activity and also some of them showed better activity than acetazolamide which is a CA inhibitor in clinical use.DoktoraPh.D
Determination of sulfonamides in milk by ID-LC-MS/MS
In this study, an ID-LC-MS method was developed and validated for the determination of 14 sulfonamides in milk samples. Recovery results were found to be in the range of 91%-114% for milk samples. The relative measurement uncertainty was between 7.5%-12.7%. Validated method was performed on milk samples obtained from market and street vendors. The amount of sulfonamides in the analyzed samples was found to be below the legal limits (Sulfamethazine: 6.46 +/- 0.76 ng/g and sulfisoxazole: 7.3 +/- 0.71 ng/g)
Peripartum Management of a Pregnant with Glanzmann’s Thrombasthenia: A Case Report
In this study, we aimed to present the case of a patient with Glanzmann’s thrombasthenia (GT) which is a hereditary disorder characterized by defective platelet function. A twenty-six-year old woman with GT who underwent cesarean section due to labor arrest and persisting vaginal bleeding despite receiving 18 unit of random platelet concentrates, 6 units of apheresis platelet concentrates, and 2 units of erythrocyte suspension. Since patients with GT are at high risk for massive bleeding during and after delivery, they should give birth under close monitoring in advanced healthcare centers where experienced hematologists are available.(The Medical Bulletin of Haseki 2014; 52: 208-11
The Ostracoda assemblage of the Eocene-Oligocene transition in northwestern Thrace: Kırklareli–Edirne area (northwestern Turkey)
COVID-19 pandemic and the global perspective of Turkish Thoracic Society
It has been more than 3 months now since the first case of COVID-19 was reported in Turkey. Globally, the number of confirmed cases and deaths reached 9,653,048 and 491,128 respectively, as reported by 216 countries by June 27, 2020. Turkey had 1,396 new cases, 194,511 total cases, and 5,065 deaths by the same date. From the first case until today, the Turkish Thoracic Society (TTS) has been very proactive in educating doctors, increasing public awareness, undertaking academic studies, and assisting with public health policies. In the present report, social, academic, and management perspectives of the pandemic are presented under appropriate subtitles. During this critical public health crisis, TTS has once again demonstrated its readiness and constructive stance by supporting public health, healthcare workers, and the environment. This review summarizes the perspective of TTS on each aspect of the COVID-19 pandemic and casts light on its contributions
CCQM-K131 Low-polarity analytes in a multicomponent organic solution: polycyclic aromatic hydrocarbons (PAHs) in acetonitrile
Solutions of organic analytes of known mass fraction are typically used to calibrate the measurement processes used to determine these compounds in matrix samples. Appropriate value assignments and uncertainty calculations for calibration solutions are critical for accurate measurements. Evidence of successful participation in formal, relevant international comparisons is needed to document measurement capability claims (CMCs) made by national metrology institutes (NMIs) and designated institutes (DIs). To enable NMIs and DIs to update or establish their claims, in 2015 the Organic Analysis Working Group (OAWG) sponsored CCQM-K131 "Low-Polarity Analytes in a Multicomponent Organic Solution: Polycyclic Aromatic Hydrocarbons (PAHs) in Acetonitrile".
Polycyclic aromatic hydrocarbons (PAHs) result from combustion sources and are ubiquitous in environmental samples. The PAH congeners, benz[a]anthracene (BaA), benzo[a]pyrene (BaP), and naphthalene (Nap) were selected as the target analytes for CCQM-K131. These targets span the volatility range of PAHs found in environmental samples and include potentially problematic chromatographic separations. Nineteen NMIs participated in CCQM-K131. The consensus summary mass fractions for the three PAHs are in the range of (5 to 25) μg/g with relative standard deviations of (2.5 to 3.5) %.
Successful participation in CCQM-K131 demonstrates the following measurement capabilities in determining mass fraction of organic compounds of moderate to insignificant volatility, molar mass of 100 g/mol up to 500 g/mol, and polarity pKow < −2 in a multicomponent organic solution ranging in mass fraction from 100 ng/g to 100 μg/g: (1) value assignment of primary reference standards (if in-house purity assessment carried out), (2) value assignment of single and/or multi-component organic solutions, and (3) separation and quantification using gas chromatography or liquid chromatography