Synthesis of potential pharmaceutical active ingredients using omega-transaminase

Transaminases (EC 2.6.1.X) are enzymes which catalyze reversible transfer of amino group from amino acids to α-keto acids by using piridoxal-5ʼ-phosphate as a coenzyme. There is a huge interest for the application of ω-transaminases in industrial production of chiral amines and alkaloids since those compounds are extensively used in pharmaceutical, agricultural, and chemical industries. Application of ω-transaminases in asimetric synthesis of these compounds enables efficient production of biologicaly active amines, due to their catalytic properties for synthesis with a high level of enantioselectivity, supstrate promiscuity (they are capable to aminate keto acids, aldehydes and ketones), high turnover number, no requirement for regeneration of external cofactors, and among other cheaper, simpler and green process of production. We are developing biocatalytic route for the synthesis of amino steroids by using ω- transaminase, (R)-selective, ATA-117 enzyme variant from Arthrobacter sp 3. It can be observed that enzyme expression was done in Echerichia coli BL21 D3 pLysS (Figure 1), and HPLC analysis of enzyme activity and specificity toward 15 structuraly different steroid compounds was performed. (R)-methylbenzylamine was used as amino group donor and pyridoxal-5ʼ-phosphate as cofactor. Activity of the enzyme was measured in bacterial lysate based on the absorbance of acetophenone, that is formed during the transamination reaction of (R)-methylbenzylamine. Figures 2 and 3 are showing chromatograms of acetophenone standard and products of reaction performed with enzyme expressed in E. coli and 16,17-epoxypregnenolone. Reactions were analysed on reversed phase column NucleosilC18. Based on the results, we have selected four steroid compounds for which enzyme showed highest activity and with a potential for biological activity. The next step was optimisation of the reaction conditions with a low cost amino donor isopropylamine, and isolation and characterisation of a pure amino steroid products. Until now we have managed to enzymatically synthesize and purify one amino steroid which should be further analysed by spectral characterization and its biological activity will be determined

Binding of 17-substituted 16-nitrile 16,17-secoestrane Compounds to Estrogen Receptors – „In Vitro“ and „In Silico“ Study

About 75% of breast cancers express estrogen receptors (ERs), which is a good base for an efficient endocrine therapy. This gives the opportunity for the treatment of patients with antiestrogens, compounds that bind to the ERs and thus compete to estradiol (E2), preventing its action in progression of estrogen-depending cancers. Here we present results of testing the effect of the modified steroids, namely 17-substituted 16-nitrile 16,17-secoestrane compounds on the E2-ER complex forming, its stability, nuclear translocation and binding to DNA. Almost all compounds in moderate to high rate induced lower forming of this complex, destabilizing it – they increased Kd of this complex and decreased number of binding sites. Complex formed in the presence of some test secosteroids could pass to the nucleus, while other compounds inhibited translocation. In the presence of some compounds binding of the formed complex E2-ER to DNA was noticed. Docking followed molecular dynamics simulation was performed to reveal binding mode of E2 to ER in the presence of test secosteroids. Amino acids important for binding process and complex stabilization were detected. Analysis of the simulation data allowed identifying key amino acids and type of binding of the secoestrane compounds, important for high affinity binding of the steroidal compounds

Determination of 17α-hydroxylase-C_17,20-lyase (P450_17α) enzyme activities and their inhibition by selected steroidal picolyl and picolinylidene compounds

17α-hydroxylase-C17,20-lyase (P45017α) is a key regulator enzyme of the steroid hormone biosynthesis in both the adrenals and the testes. Inhibition of this enzyme can block androgen synthesis in an early step, and may thereby be useful in the treatment of several androgen-dependent diseases. We developed radio-substrate in vitro incubation methods for the determination of the distinct 17α-hydroxylase and C17,20-lyase activities of the enzyme using rat testicular homogenate as enzyme source. With this method we have studied the inhibiting activity of selected steroidal picolyl and picolinylidene compounds. Tests revealed a substantial inhibitory action of the 17-picolinyliden-androst-4-en-3-one compound

Biological activity of the newly synthesized D-secoand D-homoestratriene derivatives in in vivo and in vitro experiments

Sintetisana su nova jedinjenja, 16- i 17-supstituisani 16,17-sekoestratrienski derivati i D-homoestranski derivati, polazeći od 3-benziloksi-17-hidroksi-16,17-sekoestra-1,3,5(10)-trien-16-nitrila. Ispitana je estrogena i antiestrogena aktivnost u eksperimentima in vivo, antiaromatazna aktivnost in vitro, antioksidantna aktivnost DPPH  i TBA testom, kao i antiproliferativna aktivnost prema ćelijskim linijama MCF-7 ATCC, MDA-MB-231, HT-29 i MRC-5 novosintetisanih jedinjenja.Some new compounds, 16- and 17-substituted 16,17-secoestratriene derivatives, as  well as D-homoestratriene derivatives, were synthesized, starting from 3-benzyloxy-17-hydroxy-16,17-secoestra-1,3,5 (10)-triene-16-nitrile. The newly synthesized compounds were tested for their in vivo estrogenic and antiestrogenic activity,  in vitro antiaromatase activity, antioxidative activity by DPPH and TBA tests, as well as antiproliferative activity against MCF-7 ATCC, MDA-MB-231, HT-29 i MRC-5 cell lines

Synthesis of potential pharmaceutical active ingredients using omega-transaminase

Transaminases (EC 2.6.1.X) are enzymes which catalyze reversible transfer of amino group from amino acids to α-keto acids by using piridoxal-5ʼ-phosphate as a coenzyme. There is a huge interest for the application of ω-transaminases in industrial production of chiral amines and alkaloids since those compounds are extensively used in pharmaceutical, agricultural, and chemical industries. Application of ω-transaminases in asimetric synthesis of these compounds enables efficient production of biologicaly active amines, due to their catalytic properties for synthesis with a high level of enantioselectivity, supstrate promiscuity (they are capable to aminate keto acids, aldehydes and ketones), high turnover number, no requirement for regeneration of external cofactors, and among other cheaper, simpler and green process of production. We are developing biocatalytic route for the synthesis of amino steroids by using ω- transaminase, (R)-selective, ATA-117 enzyme variant from Arthrobacter sp 3. It can be observed that enzyme expression was done in Echerichia coli BL21 D3 pLysS (Figure 1), and HPLC analysis of enzyme activity and specificity toward 15 structuraly different steroid compounds was performed. (R)-methylbenzylamine was used as amino group donor and pyridoxal-5ʼ-phosphate as cofactor. Activity of the enzyme was measured in bacterial lysate based on the absorbance of acetophenone, that is formed during the transamination reaction of (R)-methylbenzylamine. Figures 2 and 3 are showing chromatograms of acetophenone standard and products of reaction performed with enzyme expressed in E. coli and 16,17-epoxypregnenolone. Reactions were analysed on reversed phase column NucleosilC18. Based on the results, we have selected four steroid compounds for which enzyme showed highest activity and with a potential for biological activity. The next step was optimisation of the reaction conditions with a low cost amino donor isopropylamine, and isolation and characterisation of a pure amino steroid products. Until now we have managed to enzymatically synthesize and purify one amino steroid which should be further analysed by spectral characterization and its biological activity will be determined

Synthesis of some D-homo-D-aza estratriene derivatives

In this paper two synthetic routes for obtaining D-homo-D-aza estratriene derivatives were described. Namely, starting from 3-methoxyestra-1,3,5(10)-trien-16-oximino-17-one (1) 3-methoxy-17-aza-D-homoestra-1,3,5(10)-triene (5) was synthesized in two step. Another D-aza derivative was synthesize, starting from 3-methoxy-17-oxo-16,17-secoestra-1,3,5(10)-trien-16-nitrile (6). For that purpose, the seco-cyanoaldehyde was converted into its 17-ethylenacetal 7 followed by subsequent reduction of the nitrile function with sodium borohydride in the presence of cobalt chloride hexahydrate. Finally, under acidic conditions the obtained 16-amino-17-ethylenacetal 8 was transformed into 3-metoxy-17-aza-D-homoestra-1,3,5(10),17(17a)-tetraene (9)

Immobilization of ArRMut11 omega-transaminase for increased operational stability and reusability in the synthesis of 3α-amino-5α-androstan-17β-ol

The aim of this research was to improve the operational stability and enable the reusability of ω-transaminase for synthesis of new enantiopure chiral amines of steroids. Dihydrotestosterone was used to optimize the synthetic procedure of corresponding amino-steroid on a larger scale. The obtained product 3α-amino-5α-androstan-17β-ol was isolated and characterized. The enzyme was immobilized on a methacrylate-based carrier, giving the specific activity of 1.84 U/g of dry polymer. Higher residual activity of the immobilized enzyme in comparison to the soluble form (100 % versus 35%) after 24 h incubation in 35 % dimethylformamide (DMF) was obtained. The soluble enzyme retained 19 % of the initial activity after 2 h incubation in 35 % DMF at 70 °C, while the activity of the immobilized enzyme decreased only to 75 %. Immobilized retained 85 % of initial activity after ten consecutive cycles of 3α-amino-5α-androstan-17β-ol synthesis. We have tested the specificity of the ArRMut11 variant, further increased its stability by immobilization, and used it in several cycles for the synthesis of 3α-amino-5α-androstan-17β-ol. We showed that the enzyme previously evolved for higher stability as the immobilized variant showed more increased stability and high reusability that can more effectively be applied for the biosynthesis of amino steroids

Immobilization of ArRMut11 omega-transaminase for increased operational stability and reusability in the synthesis of 3α-amino-5α-androstan-17β-ol

The aim of this research was to improve the operational stability and enable the reusability of ω-transaminase for synthesis of new enantiopure chiral amines of steroids. Dihydrotestosterone was used to optimize the synthetic procedure of corresponding amino-steroid on a larger scale. The obtained product 3α-amino-5α-androstan-17β-ol was isolated and characterized. The enzyme was immobilized on a methacrylate-based carrier, giving the specific activity of 1.84 U/g of dry polymer. Higher residual activity of the immobilized enzyme in comparison to the soluble form (100 % versus 35%) after 24 h incubation in 35 % dimethylformamide (DMF) was obtained. The soluble enzyme retained 19 % of the initial activity after 2 h incubation in 35 % DMF at 70 °C, while the activity of the immobilized enzyme decreased only to 75 %. Immobilized retained 85 % of initial activity after ten consecutive cycles of 3α-amino-5α-androstan-17β-ol synthesis. We have tested the specificity of the ArRMut11 variant, further increased its stability by immobilization, and used it in several cycles for the synthesis of 3α-amino-5α-androstan-17β-ol. We showed that the enzyme previously evolved for higher stability as the immobilized variant showed more increased stability and high reusability that can more effectively be applied for the biosynthesis of amino steroids