Progesteron bileşiğinin aspergıllus glaucus küfü ile biyotransformasyonu

Abstract

Doğal ürünler bulundukları canlılara bazı avantajlar sağlayan ve daha çok diğer canlılara olan etkileri ile dikkat çeken kimyasal maddelerdir. Doğal ürünler genelde terpenoidler, alkaloidler, steroidler, poliketidler, peptidler, fenilpropanoidler, özelleşmiş peptidler, özelleşmiş karbonhidratlar ve yağ asitleri ile yağ asitlerinin türevleri olarak gruplandırılırlar. Enzimlerin doğal substratları olmayan maddeler üzerinde gerçekleştirdikleri kimyasal değişimler biyotransformasyonlar olarak bilinir. Biyotransformasyonlarda yer alan enzimler serbest olarak, sabitlenmiş olarak veya çeşitli biyolojik sistemlerin bünyelerinde etkilerini gösterebilirler. Biyotransformasyonların gerçekleştirilmesi için en çok kullanılan biyolojik sistemler küfler, mayalar ve bakteriler gibi mikroorganizmalardır. Bu mikroorganizmalar ile gerçekleştirilen mikrobiyal biyotransformasyonlar mevcut kimyasal sentez yöntemlerine göre birçok avantajlara sahiptir. Günümüzde küfler ile gerçekleştirilen mikrobiyal steroid biyotransformasyonları küf enzimlerinin yüksek regio ve stereoseçici olmaları nedeni ile ilaçlar gibi çok sayıda bileşiklerin elde edilmesi için yaygın bir şekilde kullanılmaktadır. Bu çalışma progesteron (2) bileşiğinin Aspergillus glaucus MRC 200914 küflünde nasıl metabolize edildiğinin incelenmesini amaçlamıştır. Biyotransformasyon deneyi öncesinde Aspergillus glaucus MRC 200914 için periyodik olarak taze alt kültürler hazırlandı. Sonrasında küf için besiyeri hazırlanıp erlenlere dağıtıldı ve otoklavda sterilize edildi. Bu erlenlere en taze alt kültürdeki küf steril şartlarda inoküle edildikten sonra erlenler 3 gün inkübasyona bırakıldı. Üçüncü günün sonunda erlenlere progesteron (2) steril şartlarda eklenerek 5 gün inkübe edildi. İnkübasyonu takiben besiyeri filtre edildikten sonra besiyerindeki steroidler etil asetat ekstraksiyonu ile organik faza çekildi. Etil asetat ekstraktlarının evaporatörde uçurulması ile elde edilen kalıntıdaki steroidler bir kolon kromatografisi çalışması ile ayrıldı. Steroidlerin yapı tayinleri ise erime noktaları tayini, NMR ve IR spektroskopisi gibi yöntemler ile gerçekleştirildi. Yapı tayinleri sonucunda Aspergillus glaucus MRC 200914 ile progesteron (2) biyotransformasyonunun 11α-hidroksipregn-4-en-3,20-dion (4) ve 11α,15β-dihidroksipregn-4-en-3,20-dion (5) metabolitlerini verdiği anlaşıldı.

Natural products are compounds that are not essential for the reproduction and development of living things. Natural products provide benefits to the living things and attract more attention due to their effects on other living things. Natural products are generally clasisified under groups such as terpenoids, alkaloids, steroids, polyketides, peptides, phenylpropanoids, specialized amino acids, specialized carbohydrates and fatty acids and their derivatives. Biological systems may perform chemical changes on ksenobiotics and these changes are called biotransformations. Biotransformations are carried out by free or immobilised enzymes and biological systems with enzymes. Cell cultures, tissue cultures, organ cultures, microsomes, microorganisms and spores of microorganisms are generally used as common biological systems for biotransformations. Enzymes perform almost all reactions in living organisms by lowering the energy of activation (EA). Although enzymes reduce the time to reach the reaction equilibrium, enzymes are not consumed or changed by the reaction and they do not change the DG and the equilibrium position of the reaction. The International Union of Biochemistry reported more then 3200 enzymes and it is thought that nature might offer 25000 enzymes. Enzymes provide some advantages for their users as they are very effitive catalysts, For example, the reaction rates of enzymatic reactions are accelerated by a factor of 108-1010 and this may even exceed a value of 1012. Enzymes are environmentally acceptable since they are made of amino acids and are totally degradable. Although most other chemical reagents cause environmental problems, enzymes generally act under mild conditions (around pH 7, 30 °C and 1 atm). This minimises some problems such as, isomerisation, racemisation, rearrangements, decomposition. As enzymes are compatible with each other enzymes usually function under the same or similar conditions. Therefore, by using multienyme systems in a one flask several reactions can be carried out. Some enzymes are liable to their natural role although some enzymes exhibit a high substrate telorance. These enzymes might accept a large variety of natural or unnatural compounds. Enzymes might catalyse a broad spectrum of reactions and there is an enzyme-catalysed reaction equivalent to almost every known reaction. Enzymes are chemoselective, regioselective and enantioselective molecules. As enzymes are chemoselective, they usally act on just one single type of functional group and other functionalities remain unchanged. Therefore, enzymatic reactions usually tend to be cleaner. As enzymes are regioselective they can distinguish between functional groups that are chemically located in different regions of the same substrate molecule. Enzymes might carry out this due to their complex three-dimensional structures. Enzymes are enantioselective and are chiral catalysts as they are made from L-amino acids. Therefore, any type of chrality on substrate molecule is recognised by enzymes. A prochiral substrate can be converted into a chiral product and both enantiomers in a racemic substrate usually can react at different rates, giving a kinetic resulotion. However, there are also some disadvantages for using enzymes. Nature provides enzymes as a one type of enantiomer. When the other type of enantiomeric product is required, an enzyme with exactly the opposite stereochemical selectivity is needed. However, this is often impossible. Enzymes need narrow operation parameters. Working under mild conditions sometimes causes problems as elevated temperatures and extreme pH lead to inhibition of enzymes. Although enzymes show their highest catalytic activity in water, water is generally the least suitable solvent for most organic reactions due to its high boiling point and high heat of vaporisation. Furthermore, most organic compounds are hardly soluble in aqueous media. Therefore, shifting enzymatic reaction from an aqueous medium to an organic medium would be higly desired. However, this may cause loss of catalytic activity due to enzyme denaturation. Enzymes are very dependent on their natural cofactors. Although enzymes are extremely flexible for acepting unnatural substrates they are almost exclusively dependent to their natural cofactors such as NADH and NADPH. Unfortunately, these molecules are relatively unstable and too expensive to use in stoichiometric amounts and can not be replaced by their more economical man-made substitues. Enzymes are sensitive to inhibition phenomena. Many enzymatic reactions are sensitive to substrate or product inhibition which forces enzymes to stop performing at higher substrate and/or product concentrations. Some enzymes might also cause allergies. Although enzymes can cause allergic reactions this might be minimised by considering enzymes as chemicals and usng with the same care. Biotransformations are usally performed either by isolated enzyme systems or by intact whole microorganisms. İt is known that there are more than 300 commercially available isolated enzyme systems. Since many enzyme systems that are involved are membrane bound and difficult to isolate, intact whole microorganisms are usually used for biotransformations. Main groups of microorganisms generally used for biotransformations are molds, yeasts, bacteria and microalgae. Microorganisms carry out a number of reactions on both natural and synthetic substrates via their non-specific enzyme systems. Microbial hydroxylations are the most common and are favourite among these reactions. The value of microbial hydroxylation was first noticed in 1952 when it helped to overcome a major problem in the synthesis of cortical steroids. The insertion of an oxygen function at C-11 was a very long difficult and expensive process as the mentioned position was remote from other functional groups. This problem was efficiently solved by Rhizopus arrhizus via microbial hydroxylation. Microbial biotransformations became popular after this microbial hydroxylation. Since then, steroids and a number of other different substrate groups have been genereally used for microbial biotransformations. Microbial steroid biotransformations have found worldwide application for the preparation of some important steroid hormones and drugs due to their high regio- and stereoselectivity. A lot of microbial steroid biotransformations have been described in recent years. There are still tremendous attempts to rise the effictiveness of microbial biotransformations. Since first microbial hydroxylation was reported in 1952, A number of different fungi have routinely been one of the most studied whole-cell systems for biotransformation reactions. Different fungi have been used for the biotransformations of many types of steroids. These biotransformations gave some interesting results, such as microbial hydroxylations, Baeyer-Villiger oxidations and 5α-reduction. In the present study, progesterone (2) has been incubated with Aspergillus glaucus MRC 200914 in order to see how this fungus metabolises the substrate. The medium was prepared for the fungus in 1 L of distilled water. The medium was evenly disrubuted into 10 erlenmeyer flasks of 250 mL and sterilized by an autoclave. These flasks were inoculated by the fungus. The flasks were incubated for 3 days at 25 ºC on a shaker and the substrate in DMF was added aseptically into these flasks. All flasks were further incubated for 5 days. After incubation, the mycellium was separated from the broth by filtration under the vacuum. The mycellium was rinsed with ethyl acetate and the broth was then extracted with ethyl acetate. The extracts were dried over sodium sulfate anhydrous and evaporated in vacuo to give a brown gum that was then chromatographed on silica gel 60. 11α-hydroxypregn-4-ene-3,20-dione (4) and 11α,15β-dihydroxypregn-4-ene-3,20-dione (5) were obtained from the chromatography work. The structures of these compounds were determined by comparing melting points, NMR and IR spectra of the substrate with those of steroids.