Search

Follicle-stimulating hormone FSH (FSH) receptor expression has been reported in many extra-gonadal tissues, raising the question of non-reproductive effects of FSH. Because of increasing usage of FSH in treatment of male infertility, deeper knowledge of possible harmful off-target effects of FSH is warranted.In total 33 healthy young men (mean age 30 yrs) were included in the study. All received a s.c. injection of gonadotropin-releasing hormone (GnRH) antagonist and n = 16 were randomized to 300 IU recombinant FSH (300 IE 3 times/week) for 5 weeks at first visit (V1) whereas n = 17 served as controls. Blood samples were taken at (V1), after 3 weeks (V2) and after 5 weeks (V3), when the study ended. At V2, all subjects received 1000 mg testosterone undecanoate i.m. A standard set of bio- and inflammatory markers were compared between the groups using the Mann-Whitney test adjusted for multiple testing.As compared to controls, the FSH treated men had higher SHBG and albumin concentrations at V2 (p = 0.024 and p = 0.027, respectively), and lower levels of alanine aminotransferase (p = 0.026) and magnesium (p = 0.028) at V3. However, none of the results remained statistically significant after Bonferroni correction (p0.0011).FSH had no significant effects on non-reproductive organs when given in standard therapeutic doses to young men for five weeks.t. Therefore, FSH treatment can be considered safe in otherwise healthy young men, constituting candidates for infertility treatment with FSH. This article is protected by copyright. All rights reserved.

Although the application of organic solvents in biocatalysis is well explored, in-depth understanding of solvent interactions with proteins, in particular oligomeric ones, is still scant. Understanding of these interactions is essential in tailoring enzymes for industrially relevant catalysis in nonaqueous media. In our study, homotetrameric enzyme halohydrin dehalogenase (HHDH) from Agrobacterium radiobacter AD1 (HheC) was investigated, as a model system, in DMSO-water solvent mixtures. DMSO, the most commonly used co-solvent for biocatalytic transformations, was found to act as a mixed-type inhibitor with a prevalent competitive contribution. Already 5% (v/v) DMSO inhibits the activity of HheC by half. Molecular dynamics (MD) simulations showed that DMSO keeps close to Ser-Tyr catalytic residues forming alternate H-bonds with them. Stability measurements paired with differential scanning calorimetry (DSC), dynamic light scattering (DLS) methods and MD studies revealed that HheC conserves structural integrity in content as high as 30% (v/v) DMSO.

Interleukin 17 (IL-17) cytokines promote inflammatory pathophysiology in many autoimmune diseases, including psoriasis, multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. Such broad involvement of IL-17 in various autoimmune diseases makes it an ideal target for drug discovery. Psoriasis is a chronic inflammatory disease characterized by numerous defective components of the immune system. Significantly higher levels of IL-17A have been noticed in lesions of psoriatic patients, if compared to non-lesion parts. Therefore, this paper is focused on the macrolide inspired macrocycles as potential IL-17A/IL-17RA modulators and covers the molecular design, synthesis, and in vitro profiling. Macrocycles are designed to diversify and enrich chemical space through different ring sizes and a variety of three-dimensional shapes. Inhibitors in the nM range were identified in both target-based and phenotypic assays. In vitro ADME as well as in vivo PK properties are reported.

Based on a nationwide register data we recently reported a link between male infertility and increased risk of early onset prostate cancer. However, mortality due to prostate cancer, which can be regarded as the ultimate proxy for its clinical significance, especially in the context of over-diagnosis and over-treatment, could not be explored in the previous study, since the follow-up period in most cases was too short. Data therefore must be retrieved from other cohorts, with longer follow-up. We sourced data from a population-based prospective cohort including 11,343 men aged over 45 years, enrolled in the 1970s. The results showed that childless men have higher risk for prostate cancer related mortality (HR: 1.49, 95% CI: 1.09–2.03, p = 0.01) compared to men with children, in particular when only married men, who most probably are involuntary childless, were considered (HR 1.54, 95% CI 1.13 − 2.10, p = 0.006). However, the prostate cancer incidence did not differ (HR = 1.04, 95% CI: 0.88–1.24). In conclusion, our results show that childless men are at higher risk for dying from prostate cancer, probably due to a more aggressive form of the disease. (Less)

Cataract is a leading cause of reversible blindness. Secondary cataract is the result of migration, proliferation, and epithelial-mesenchymal transformation (EMT) of lens epithelial cells (LECs), induced by transforming growth factor β (TGFβ). The extracellular signal-regulated kinases 1 and 2 (ERK 1/2) and casein kinase 2 (CK2) are important for fundamental cellular processes. This study aimed to evaluate the role of CK2 in the EMT of LECs. Primary LECs cultures were obtained from age-related and type 2 diabetes-induced cataracts. Diabetic LECs were capable of spontaneous in vitro differentiation. Treatment of LECs with TGFβ2-stimulated ERK1/2 activity, which correlated with the expressed α-smooth muscle actin (α-SMA). siRNA-mediated attenuation of the endogenous catalytic subunit (α) of CK2-inhibited ERK1/2 activity and decreased α-SMA expression. This study shows evidence that the function of ERK1/2 is regulated by the constitutively active heterotetrameric protein kinase CK2 in this cellular phenotype. This study enriches the knowledge on LECs physiology in normal and in pathological conditions.

Despite the big and rapid growth of technologies during the 21st century, there is still an industry that is lagging behind with the optimization and launch of its digital transformation. In fact, this is the agricultural sector. Therefore, in recent years, many attempts have been made to develop and implement optimized processes and technologies, in order to increase production and reduce costs while maintaining product quality. The purpose of this publication is to summarize the current situation on the basis of existing researches and share facts.

Halogenhidrin-dehalogenaza tipa C (HheC iz Agrobacterium radiobacter) zauzima važno mjesto u industrijskoj biokatalizi zahvaljujući visokoj enantioselektivnosti i mogućnosti sinteze raznovrsnih β-supstituiranih alkohola i epoksida [1]. Zbog hidrolitičke nestabilnosti i ograničene topljivosti supstrata epoksida, HheC-katalizirane reakcije mogu se provoditi u alternativnim medijima poput organskih otapala. Pritom otapala mogu imati štetan utjecaj na strukturna i katalitička svojstva enzima. Stoga se operacijska stabilnost enzima može povećati metodama imobilizacije poput kapsuliranja u metalo-organske mreže (engl. metalorganic frameworks – MOFs). MOF su porozni materijali visokog stupnja kristaliničnosti koji mogu zaštititi enzim od nepovoljnih reakcijskih uvjeta. Zeolitne imidazolne mreže (ZIF) moguće je sintetizirati pri biokompatibnilim uvjetima gdje enzim služi kao nukleus kristalizacije [2]. U ovom radu ispitana je mogućnost sinteze HheC@ZIF-8 s ciljem povećanja stabilnosti prilikom biokatalize u organskom mediju. Ovaj rad izrađen je u okviru projekta „Enzimska sinteza fluoriranih kiralnih građevnih blokova“ EnzyFluor (IP- 2018-01-4493) financiranog sredstvima Hrvatske zaklade za znanost te hrvatskoslovenskog bilateralnog projekta „Stabilizacija halogenhidrin-dehalogenaza radi upotrebe u nekovencionalnim medijima“.

Halohydrin dehalogenases (HHDHs) are important and versatile biocatalysts, that can be used for the synthesis of important building blocks and precursors in the pharmaceutical, agrochemical and chemical industries. HHDHs have a wide spectrum of different compounds, such as chiral epoxides, β-substituted alcohols, oxazolidinones and others, that can be synthesized by their action. However, the enzyme was reported to lack stability under operational conditions. Therefore, immobilization of HHDH onto magnetic nanoparticles (MNPs) represents a powerful tool to overcome its limitations and improve its enzyme characteristics as a biocatalyst. HHDH was immobilized onto bare maghemite nanoparticles, chitosan coated MNPs and carboxymethyl dextran (CMD) coated MNPs. All types of MNPs were prepared by co-precipitation method of ferrous and ferric ions. The immobilization protocol was initially optimized using 10% (v/v) cross-linker glutaraldehyde (GA) in order to improve residual activity and immobilization efficiency of immobilized HHDH. The highest immobilization efficiency (91%) of immobilized HHDH was achieved, while using CMD coated MNPs, which were cross-linked with 10% (v/v) GA for 1 hour at 350 rpm and later immobilized with HHDH for 1 hour at 350 rpm. To implement HHDH at industrial scale, initial optimization of HHDH immobilization was performed, which offers room for improvement in order to achieve an effective biocatalyst for various synthetic applications.

Enzymes, receptors, and other binding molecules in biological processes can recognize enantiomers as different molecular entities, consequently, using a single pure enantiomer instead of a racemate can enhance the effectiveness and safety of the treatment [1]. The method for the synthesis of enantiopure or highly enantio-enriched compounds is asymmetric organometallic catalysis, which has been recently replaced by biocatalysis. An important example of enzymatic biocatalysis is the nucleophilic opening of epoxy rings with sodium azide catalysed by halohydrin dehalogenase (HHDHs) [2]. Enantiomerically pure β-azido alcohols are very important building blocks in the synthesis of 1, 2, 3-triazole derivatives [3]. As part of our research on developing multi-target small molecule anticancer agents, we designed, synthesized, and biologically evaluated a series of 66 novel hydroxyl aryl-substituted N-heterocycle derivatives. A series of 50 racemic and 16 optical pure 1, 4-disubstituted 1, 2, 3-triazoles were synthesized by the copper(I)-catalyzed Huisgen 1, 3-dipolar cycloaddition of terminal alkynyl derivatives of purine and purine isosteres and aryl-substituted 1, 2-azido alcohols under environmentally friendly ultrasound irradiation. The synthesis and antitumor effects of all derivatives will be presented.

Enantiopure epichlorohydrin (ECH) is an important building block for the production of chiral pharmaceuticals and agrochemicals such as L- carnitine, β-blockers and pheromones. ECH is also used as an intermediate in the preparation of paper chemicals, synthetic rubbers, epoxy resins, as a starting material in the production of adhesives, surfactants, insecticides, coatings, plasticizers and solvents. Conventional ways of producing ECH have disadvantages such as relatively low reaction yields (around 50 %), high energy consumption and use of a metal catalyst (cobalt-salen complexes) that lead to environmental pollution. Because all mentioned, biocatalytic methods for the preparation of chiral ECH are getting much attention. The advantages of using biocatalytic methods are high enantioselectivity, low production cost, extensive enzyme sources and environmentally friendly conditions. Halohydrin dehalogenases (HHDHs) can be used as biocatalysts for the ring-closure of a haloalcohol to an epoxide (Figure 1). HHDHs belong to the superfamily of the short-chain dehydrogenase/reductase proteins and structurally they are homotetramers. HHDHs are divided into subtypes from letter A to G. These enzymes are easily obtained from different bacterial hosts and can be expressed in recombinant form in Escherichia coli. In this study, enzymes from Corynebacterium sp. N- 1074 (HheB) and modified HHDH from Mycobacterium sp. GP1 (HheB2-T120A), belonging to group B, were utilised for (R)-ECH synthesis (Figure 1). The kinetic characterisation was done with both enzymes and kinetic models for enzymatic synthesis of (R)-ECH from 1, 3-dichloro- 2-propanol (DCP) were developed. The kinetic characterisation is a crucial step in the development of a mathematical model as it offers a better understanding of reaction which leads to the optimization of the reaction conditions. Except kinetic characterization, the stability of enzymes during incubation with different concentrations of (R)- ECH and DCP was assessed. The experiments were done by incubating the enzymes with a different substrate (DCP) or product ((R)-ECH) concentration over 24 hours period. Enzyme activity assay was performed spectrophotometrically through absorption differences between para-nitro-2-bromo- 1- phenylethanol and para-nitrostyrene oxide. It was found that (R)-ECH has no considerable effect on enzyme stability, while DCP decreased activity of both enzymes in concentration-dependent manner. Subsequently, validation of models was performed in batch reactor experiments. As implied by monitoring enzyme stability during incubation, it was confirmed within validation experiments that substrate deactivates the enzyme. The deactivation rate constants were obtained through batch reactor experiments and it was concluded that the deactivation rate is dependent upon the concentration of the substrate. Mathematical modelling is an essential step in the optimization of a process. The model can be used to predict reaction profiles in various reactor types and under different conditions. That information can be used for selection of the appropriate reactor type and process conditions. With this approach, mathematical modelling can also be used for the scale-up of the process. Acknowledgements: Work was supported by CAT PHARMA (KK.01.1.1.04.0013), a project co-financed by the Croatian Government and the European Union through the European Regional Development Fund - the Competitiveness and Cohesion Operational Programme.

Halohydrin dehalogenases (HHDHs) are important enzymes that display excellent selectivity and activity in reversible dehalogenation of vicinal halohydrins. Furthermore, HHDHs may employ wide range of unnatural nucleophiles in ring-opening reactions, extending the possibilities for the synthesis of enantiopure β-alcohols1. On account of these desirable properties, the variant of the HheC enzyme is employed in the synthesis of Lipitor, one of the best-selling pharmaceuticals in the world2. However, insufficient is known about the enzyme kinetic stability that is a prerequisite for further industrial implementations2, 3. In this work, we assessed the stability wild-type HheC and two mutants with enhanced kinetic properties. First, freeze-thaw cycle testing was performed via activity measurements, whereby HheC proved to be very stable, facilitating protein handling procedures. The kinetic stability of different biocatalyst forms (whole cells, cell-free extract, and purified enzyme) in buffer was determined through activity tests, which led us to the conclusion that the cell wall and other proteins in the extract protect the enzyme and keep it active for longer period. Size distribution of purified protein was also investigated by dynamic light scattering methods to establish existence of larger protein particles formed due structural changes and aggregation. The stability of mutants during incubation with substrates and unnatural nucleophiles (azides, cyanides) was monitored to determine whether the components of the reaction mixture have an effect on enzyme activity. It was found that at higher concentrations of fluorinated styrene oxide derivatives, the enzyme loses activity faster, which was later reflected in the results of enzyme operational stability decay in the batch reactor. The co-dependency of operational stability decay rate constant on substrate initial concentration (Figure 1) was mathematically described by Eq 1. Understanding and quantifying the influence of compounds concentrations on enzyme behavior is of a great importance for larger scale processes3. Presented results give a better insight into the limitations of HheC application and help with biocatalysts handling for prolonging its longevity in reactors.

Enzimi nitrilaze, bez stvaranja slobodnih amidnih međuprodukata, kataliziraju reakcije hidrolize nitrila u karboksilne kiseline i amonijak. [1] Stoga se nitrilaza može koristiti kao biokatalizator u reakcijama pretvorbe (R)-4-kloro- 3-hidroksibutironitrila (KHB) u (R)-4-kloro-3- hidroksibutansku kiselinu, pri čemu dolazi do spontane ciklizacije produkta u (R)-3-hidroksi-γ- butirolakton (HBL) (slika 1). HBL je koristan kiralni građevni blok s primjenom u sintezi različitih polimera, otapala i farmaceutika. Postoje brojni kemijski putevi sinteze HBL-a, no većina ima nedostatke poput skupih ili opasnih reaktanata, intenzivnih procesnih uvjeta, te niskih iskorištenja i selektivnosti, što uzrokuje poskupljenje procesa izdvajanja produkta. Nasuprot tome, enzimski katalizirani procesi koriste blage reakcijske uvjete, postižu visoku selektivnost te mogu pružiti ekonomski isplativ postupak sinteze ove vrijedne kemikalije. [2, 3] U ovom radu provedena je evaluacija 24 nitrilaze. Tijekom ispitivanja korišteni su isti početni uvjeti, koncentracija supstrata (KHB) i masena koncentracija nitrilaza, dok se reakcija pratila 6 sati. Enzim s najvećim iskorištenjem na produktu izabran je za sljedeći korak, kinetičko istraživanje. Određeni su kinetički parametri nitrilazom katalizirane sinteze (R)-4-kloro-3- hidroksibutanske kiseline iz (R)-4-kloro-3- hidroksibutironitrila.

International audience; Since the days of Hofstede (1980), cross-cultural comparisons of countries based on societal-level work values have been a norm. This approach has been represented more recently in Ronen and Shenkar’s (2013) 11 clusters of country cultures. However, more contemporary research found within-country heterogeneity of values/behaviors is substantial and growing exponentially across today’s twenty-first century businessworld. We investigated, across a sample of 39 societies, whether work values variance within societies was greater than work values variance across societies, and whether individual work values differences contributed more to predictions of behavioral performance criteria than the society in which the individuals lived. Both sets of analyses addressed how work values conceived at societal-levels are relevant in understanding the twenty-first century businessworld. Our findings revealed first that there was substantial within-society values heterogeneity, which resulted in the failure to replicate Ronen and Shanker’s (2013) societal cluster aggregations. Second, we found individual-level values contributed significantly to the prediction of employees’ behaviors, while societal-level values contributed substantially less. These findings strongly suggest that cross-cultural studies of work values predictive power are most relevant when conducted at the individual-level. Finally, we also make available for future investigators a 51-society database containing 11,780 individual-level records.

Halohydrin dehalogenases (HHDHs) belong to a distinct group of enzymes catalyzing the removal of a halide ion and a proton from a vicinal halohydrin with the formation of an epoxide. Moreover, these enzymes can catalyze the epoxide ring-opening reactions with a range of anionic nucleophiles affording β-substituted alcohols as products. Based on sequence similarities, they are divided into 7 phylogenetic groups: A, B, C, D, E, F and G. Activity, enantioselectivity and enantiopreference are dependent on the type of enzyme and the substrate structure. HheC from Agrobacterium radiobacter is the most studied because of its high enantioselectivity. In order to expand the catalytic relevance of HHDHs we focus our attention to an enzyme from B group on which a small amount of research has been done. There are two similar enzymes in group B, one found in Corynebacterium sp. N1074 (HheB) and the other in Mycobacterium sp. GP1 (HheB2). They share high sequence identity (95%) with only 4 amino acid substitutions HheB/HheB2: F36/I36, T120/A120, C124/Y124, H125/Q125. HheB exhibits higher enantioselectivity than does HheB2. HheB2 has been previously characterized as a non-enantioselective enzyme, and neglected as a biocatalyst. In this work, HheB2 and HheB were investigated in the ring-opening reaction on a set of 21 structurally different aliphatic and aromatic epoxides using sodium azide as a nucleophile. To gain more insight into the difference of their enantioselectivity, mutants HheB2-F36I, HheB2- T120A, HheB2-C124Y, HheB2-H125Q were constructed and also tested. We examined nucleophiles that are accepted in the active site of B-type and used kinetics measurements to determine enzyme affinity and inhibition concentration for all accepted nucleophiles. The screening confirmed low to moderate enantioselectivity of HheB2 towards monosubstituted epoxides, however high activity and enantioselectivity in conversion of 2, 2- disubstituted (E-values up to >200). Enantioselectivity of HheB was slightly higher than HheB2 in the majority of cases. T120A mutation has been shown to be a key for increasing enantioselectivity in aliphatic epoxides. Among all tested substrates, disubstituted benzyl epoxides showed great potential for producing enantiomerically pure β-substituted alcohols. We showed that bicyclic structures as well, can be accepted into the active site of B-type with high E values (>200). Screening of nucleophiles confirmed that azide, cyanide, nitrite, cyanate and thiocyanate are accepted into the active site of the enzyme. A kinetics measurement showed that reaction rates are highest with azide ion and it is the only nucleophile that does not cause enzyme inhibition even at concentrations of 100 mM. Four other nucleophiles (CN-, NO2-, OCN- and SCN-) cause inhibition at concentrations between 5 and 25 mM.

Homotetrameric halohydrin dehalogenase from Agrobacterium radiobacter AD1, HheC is extensively used for industrial green synthesis of enantiopure building blocks. It naturally catalyses reversible dehalogenation of vicinal haloalcohols, but it is utilized with a whole range of unnatural nucleophiles in epoxide ring-opening reactions. In order to increase solubility of lipophilic epoxides and conversion efficiency, addition of various solvent is explored. The results of our study of effects of widely explored solvent DMSO (dimethyl sulfoxide) on catalytic activity of HheC will be presented. Besides determination of kinetic parameters, differential scanning calorimetry (DSC) and dynamic light scattering (DLS), molecular dynamics (MD) is used to elucidate mechanisms of DMSO action on HheC. We carried out MD simulations (GROMACS) on natural tetrameric and hypothetical monomeric HheC in water as well as in 20% and 50% (v/v) DMSO/aqueous environment. The tetramer HheC exhibits remarkable conformational tolerance towards DMSO up to 30% and it instantly aggregates at 50% DMSO, but its catalytic activity exponentially decreases with DMSO addition. 5% DMSO inhibits the HheC activity by half. The MD demonstrates that while subunit conformations slightly changes with DMSO addition, distinct sheering of the main structural motifs between subunits occurs, with changes proceeding from more localized (20%) to more extended and collective (50%). However, no dissociation (up to 300 ns) was observed in accordance with DSC and DLS results, but buried surface area increases and the catalytic site becomes more constrained. DMSO is found to replace H2O molecules in the catalytic site forming alternately H-bonds with the catalytic amino acid residues S132 and Y145, and to form small clusters around the protein (Fig. 1).

Propargylic epoxides and alcohols, owing to the presence of a triple bond, undergo various intermolecular and intramolecular reactions. [1, 2]. Halohydrin dehalogenases can be used to obtain enantiomerically pure starting compounds from racemic propargylic epoxides for triple bond and/or nucleophile transformations. [3] Therefore, the synthesis of mono- and disubstituted propargylic epoxides and subsequent enantioselective ring opening by halohydrin dehalogenases (HheC, HheAN178A and HheB-4x mutant) was described (Figure 1). Internal propargylic epoxides were synthesized from the corresponding terminal acetylenes by introduction and epoxidation of a double bond. Also, p- and m-tolyl derivatives were synthesized in a similar reaction sequence, starting from the corresponding iodotoluene and trimethylsilylacetylene. 2, 2-Disubstituted epoxides were prepared from corresponding methyl ketones, starting with selective α-halogenation, followed by triple bond introduction though a Grignard reaction and Williamson-type cyclisation. Kinetic resolution reactions in the presence of sodium azide were catalyzed by three HHDHs. Reactions of monosubstituted substrates with HheC and HheA-N178A yielded enantiomerically pure secondary azido alcohols (ee > 99%, E > 200). While HheC yielded almost exclusively (R)-β-azido alcohol (up to 99:1), HheA-N178A gave mostly (S)- β-azido alcohol (β : α ratio between 90:10 and 54:46). For disubstituted epoxides, reactions with HheC and HheB-4x yielded tertiary azido alcohols almost regiospecifically (β : α ratio >99:1) with excellent to very good enantioselectivities(ee between >99 and 91%, E between >200 and 60).

Optically pure fluorinated organic nitriles are synthetically valuable building blocks in the pharmaceutical and fine chemical industries. Since direct fluorination is challenging from both economic and environmental perspectives, the development of methods to modify existing fluorinated synthons is a prized alternative. Halohydrin dehalogenases (HHDHs) are synthetically valuable enzymes because they can accept numerous anionic nucleophiles such as cyanide, cyanate, azide and others in epoxide ring-opening reactions. HHDHs exhibit enantioselectivity or enantiopreference in a wide range of reactions and provide the ability to perform kinetic resolution of fluorinated building blocks, resulting in higher value products, i.e., optically pure β-substituted alcohols and unreacted enantiomers of substrates. However, if the enzyme is not completely enantioselective, the optical purity of the final products depends largely on the reaction conditions and reactor design. In this work, the HHDH-catalysed synthesis of (S)-3-(4-fluorophenyl)-3-hydroxypropanenitrile was investigated from a reaction engineering point of view. Since both enantiomers of 2-(4-fluorophenyl)oxirane are accepted in the biocatalytic reaction with cyanide ions, the reaction was kinetically studied in detail starting from the individual enantiomers of the substrate. In addition, the inevitable hydrolysis of the substrate was kinetically characterised. The effect of all concentrations of the reaction components on the enzyme activity was investigated. Differences were found between the enzyme affinities toward (R)- and (S)-2-(4-fluorophenyl)oxirane, as well as the presence of different inhibitions in both biocatalytic reactions. These effects are expressed numerically by Vm, Km and Ki values. In addition to activity, the stability of the enzyme in the presence of compounds from the reaction mixture was also evaluated, and as a result, a co-dependence of the operational stability decay on the initial substrate concentration was found. The mathematical model for the synthesis of (S)-3-(4-fluorophenyl)-3-hydroxypropanenitrile was developed and used as a guide for the detection and minimization of reaction bottlenecks. Through mathematical modelling, it is possible to manipulate the outcome of the reaction in the desired direction within the limitations of the enzyme. Process simulations were used to select suitable conditions for the synthesis of a product with high optical purity and yield.

Halohydrin dehalogenases (HHDHs) are synthetically highly versatile biocatalysts. In this sense, they can be applied for the enantioselective and regioselective syntheses of a wide range of 1, 2-difunctionalised chiral compounds. Enzyme-catalysed ring-opening reaction of epoxides represents an attractive alternative to metal-catalysed reactions, since they take place at ambient temperature in aqueous media at neutral pH and atmospheric pressure. Over the last years, we and others have demonstrated that HHDHs efficiently catalyses kinetic resolution of aliphatic, aromatic and functionalised epoxides, affording enantiomerically pure or highly enantioenriched products. Because of its high enantioselectivity and wide nucleophile tolerance, HheC from Agrobacterium radiobacter has been particularly used for exploring synthetic applicability. In this study, we are focused on the HHDH- catalysed transformation of epoxides bearing fluoroaryl groups. Fluoroaromatic compounds are widely used as starting materials for chemical syntheses, especially pharmaceuticals and agrochemicals. Introduction of fluorine atoms into the molecule usually increases target effectiveness, biological half-life, bioabsorption etc. Such fluorinated organics can be modified in an enantioselective fashion leading to optically active building blocks, for example alcohols, amines and epoxides. While 1, 2-azido alcohols are precursors for aziridines, tetrazoles and 1, 2-amino alcohols, epoxides are highly reactive intermediates that can be modified with many different nucleophiles via ring-opening reactions. An alternative to these methods is the kinetic resolution of epoxides which involves the use of HHDHs and sodium azide as a nucleophile. Synthesis of both chiral epoxide and b-substituted azido alcohol can be achieved this way. In this context, two HHDHs showing opposite stereopreference, HheC (R-selective wild-type enzyme) and HheA-N178A (S-selective mutant), were investigated for the azidolysis of fluorine-substituted styrene-oxide derivatives. In general, kinetic resolutions experiments were performed at analytical scale in order to determine enantioselectivity and regioselectivity of both enzymes. The origin of enantioselectivity is studied using in silico approach. These results lead to further understanding of biocatalytic properties of HHDHs and guide their application to the synthesis of chiral fluoroaryl substituted azido alcohols and epoxides.

INTRODUCTION AND OBJECTIVE:Non-obstructive azoospermia (NOA) is a severe infertility diagnosis. No level-1 evidence supports a specific surgical sperm retrieval procedure. Microdissection testicula...