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Laccase-mediated oxyfunctionalization of 3β-hydroxy-Δ5-steroids
Accepted Manuscript Title: Laccase-mediated oxyfunctionalization of 3-hydroxy-5 -steroids Author: Sergey M. Khomutov Andrey A. Shutov Alexey M. Chernikh Nina M. Myasoedova Ludmila A. Golovleva Marina V. Donova PII: DOI: Reference:
S1381-1177(15)30098-9 http://dx.doi.org/doi:10.1016/j.molcatb.2015.11.004 MOLCAB 3268
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
25-7-2015 26-10-2015 1-11-2015
Please cite this article as: Sergey M.Khomutov, Andrey A.Shutov, Alexey M.Chernikh, Nina M.Myasoedova, Ludmila A.Golovleva, Marina V.Donova, Laccase-mediated oxyfunctionalization of 3rmbeta-hydroxy-Delta5-steroids, Journal of Molecular Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2015.11.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Laccase-mediated oxyfunctionalization of 3β-hydroxy-Δ5-steroids Sergey M. Khomutov* [email protected], Andrey A. Shutov, Alexey M. Chernikh, Nina M. Myasoedova, Ludmila A. Golovleva, Marina V. Donova G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences,Pushchino, Moscow Region, 142290 Russia *
Corresponding author.
Graphical Abstract
Highlights Fungal laccases were tested in bioconversion of non-phenolic steroids. Laccase-mediated systems effectively catalyze oxidation of 3β-hydroxy-Δ5-steroids. The oxidation of 3β-hydroxyandrost-5-en-17-one includes the hydroxylation at positions 7α and 7β. Regiospecific oxidation of the 7(α/β)-alcohols results in 3β-hydroxy-androst-5-en7,17-dione. 3β-Hydroxypregn-5-ene-7,20-dione is an only laccase product from pregnenolone.
Abstract Laccase mediator systems (LMS) were studied as catalysts for steroid oxidation. The fungal laccases from Lentinus strigosus 1566 and Trametes versicolor were used in the work. Among five mediators screened, 1-hydroxybenzotriasol (HBT) excelled in activity. The LMS effectively catalyzed oxidation of 3β-hydroxy-Δ5-steroids like DHEA (3β-hydroxyandrost-5en-17-one) and pregnenolone (3β-hydroxypregn-5-en-20-one), while no activity was observed towards 3-oxo-4-ene-steroids (androstenedione, 9α-hydroxyandrostenedione, testosterone and 20-hydroxymethylpregn-4-en-3-one). The pathway of DHEA oxidation by LMS included the hydroxylation at positions 7α and 7β followed by oxidation of the corresponding 7(α/β)alcohols to form 3β-hydroxyandrost-5-ene-7,17-dione. Regiospecific oxidation of allylic hydroxyl functions by LMS was confirmed using 3β,7α- and 3β,7β-dihydroxyandrost-5-en17-ones as substrates. 3β-Hydroxypregn-5-ene-7,20-dione was produced with LMS as an only product from pregnenolone. The yield of crystalline product
reached 58.3% yield with a
purity of 96%. The results demonstrate that application of LMS may be a promising approach for steroid oxyfunctionalization.
Keywords: laccase; steroid; oxidation; laccase mediator system; dehydroepiandrosterone; pregnenolone; oxyfunctionalization
1. Introduction Laccases
(benzenediol:oxygen
oxidoreductases,
E.C.1.10.3.2)
are
copper-containing
oxidoreductases that specifically catalyze the oxidation of a wide range of organic substrates including phenols, polyphenols, anilines, aryl diamines, methoxy-substituted phenols, hydroxyindoles, benzenethiols and inorganic/organic metal compounds, to the corresponding radicals, using molecular oxygen as the final electron acceptor [1-4]. The reactive radical species rapidly react further to various oxidation products forming water as an only byproduct. The broad substrate specificity makes laccases interesting “eco-friendly” enzymes with high industrial potential [2]. Laccase applications in biotechnology, e.g. for delignification and bleaching of paper pulp, detoxification and decolourization of textile dyes, bioremediation, detoxification of wastewaters, chemical grafting, polymer surface modifications and others, had been reviewed several times [3, 5-7]. Laccase activity has been found in fungi, plants, bacteria, and insects and its significant role was evidenced for synthetic and mineralization processes in nature [3]. Fungal laccases are of particular commercial interest due to their higher redox potential as compared with other enzyme forms isolated from prokaryotes, insects or plants [8]. The main laccase producers are lignolytic basidiomycetes, - white-rot fungi, which secrete the enzymes extracellularly, thus providing further simple enzyme purification [6]. Steroids are terpenoid lipids of specific structure that contain a gonane nucleus of four cycloalkane rings. These compounds fulfill essential physiological functions and are widespread in all living systems. Steroid preparations are widely used in different fields of medicine, veterinary, and agrochemistry. Bioconversion is a powerful tool for the production of valuable steroids compounds [9]. The data on laccase action on steroids mainly concern the dimerization, or polymerization of phenolic steroid hormones, such as estradiol, estrone, ethynilestradiol, and their derivatives [10-13]. These reactions can be exploited for the removal of estrogens, which are the endocrine disrupting steroids, from the wastewaters [14, 15]. The information on laccase activity towards the non-phenolic steroids is scarce [16, 17]. Mechanism of laccase action on phenolic compounds, including estrogens, is often based on the formation of the corresponding phenoxy radicals followed by further condensation. In the case of non-phenolic steroid substrates, the redox potential proves to be insufficient for the formation of steroid radicals [16]. It is known that the substrate range of laccases can be expanded to the compounds with a higher redox potential than the enzymes themselves in the
presence of small natural (e.g. syringaldehyde or coumaric acid which can be found in the lignocellulosic biomass), or artificial molecules, which are capable to act as electron transfer mediators. Due to the specific mediators, laccases became able to enlarge the substrate spectrum and oxidize non-phenolic compounds. The application of the so-called Laccase Mediator Systems (LMS) for the oxidation of different substrates (e.g. alcohols, sugars, esters, alkenes, amides etc.) had been reviewed [2, 4]. LMS composed of Pycnoporus cinnabarinus laccase and 1-hydroxybenzotriazole (HBT) as a mediator was applied for the oxidation of the model lipids representative for main paper pulp lipophilic extractives [16]. The unsaturated lipids including phytosterols were shown to be oxidized by the LMS to the corresponding epoxy- and hydroxy fatty acids from fatty acids, and free and esterified 7-ketosterols and steroid ketones from sterols and sterol esters. The treatment of eucalypt kraft pulp by LMS composed of laccase and HBT resulted in the decrease of pulp sterol content with an increase of 7-oxo-sitosterol and stigmasta-3,5-dien-7one [17]. Detection of 7-ketosterols among the products of phytosterol oxidation indicated the possibility of LMS to catalyze the introduction of an oxygen function at the allylic position of 3β-hydroxy-Δ5-steroids. Allylic moiety oxidation of Δ5-steroids can provide obtainment of valuable 7-keto derivatives with diverse biological properties [18]. Metallic catalysis in combination with hydroperoxides is often used for the reaction. For instance, the allylic oxidation by cobalt (II) alkyl phosphonate modified silica and tert-butyl hydroperoxide resulted in good product yields [19]. The catalyst based on dirhodium caprolactamate was highly effective at the hydroperoxide oxidation of Δ5-steroids [20]. However, chemical catalysis may not provide regioselectivity of the reaction: e.g. undesirable oxidation at C4 was observed at the application of selenic dioxide [21]. Moreover, hazardous reagents such as chrome trioxide [22], or pyridinium chlorochromate [23] are used as oxidants in stoichiometric, or even higher amounts. Noteworthy, chemical protection of the 3β-hydroxyl function by esterification is required for the preservation of the ∆5-double bond in steroid molecule at the allylic peroxide oxidation by chemical catalysis. Of importance are new simple methods which exclude the necessity of 3β-hydroxyl function protection, allow reaction performance under mild conditions in aqueous media, and provide effective environmentally friendly synthesis of the valuable steroids. The specific oxidation of allylic hydroxyls represents another relevant problem [24, 25]. Allylic steroid alcohols may be oxidized chemically to the corresponding aldehydes and ketones using different co-oxidant systems in the presence of saturated alcohols. Bioconversion could provide alternative promising approach.
Herein, we studied the possibility of regioselective oxygen functionalization of 3β-hydroxyΔ5-steroids and the oxidation of the corresponding allylic 7-alcohols using LMS oxidation.
2. Experimental
2.1 Substrates and reagents
Sinapic
acid,
acetosyringone,
syringaldehyde,
1-hydroxybenzotriazole
(HBT),
N-
hydroxyacetanilide, 2,2`-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) [ABTS], (2,2,6,6tetramethylpiperidin-1-yl)oxyl [TEMPO], 3β-hydroxypregn-5-en-20-one (pregnenolone), 3βhydroxyandrost-5-ene-17-one (dehydroepiandrosterone, DHEA), 17β-hydroxyandrost-4-ene3-one (testosterone), 2,6-dimethylphenol, Tween-80, Tween-20 and Trametes versicolor laccase were purchased from Sigma-Aldrich Co (USA). 9α-Hydroxyandrost-4-ene-3-one (9α-OH-AD, purity - 98%, m.p. – 217-220 °C, [E, 240 nm (0.01% in ethyl alcohol) - 14780 M-1 cm-1], 20-hydroxymethylpregn-4-en-3-one (purity - 98%, m.p. 181 - 183 C; [E, 240 nm (0.01% in methyl alcohol) - 16100 M-1 cm-1] were obtained from G.K.Skryabin Institute of Biochemistry and Physiology of Microorganisms (RAS, Russia). 3β,7α-Dihydroxyandrost-5ene-17-one (7α–OH-DHEA) and 3β,7β -dihydroxyandrost-5-ene-17-one (7β–OH-DHEA), 3β-hydroxyandrost-5-ene-7,17-dione (7-keto-DHEA), 3β-hydroxypregn-5-ene-7,20-dione (7keto-pregnenolone) were provided by Steraloids (USA). Other materials and solvents were of analytical grade and purchased from domestic companies (Russia).
2.2 Strain and cultivation
The strain of Lentinus strigosus 1566 was obtained from Basidiomycetes Collection of the Komarov Institute of Botanics (Moscow, Russian Federation), and was grown on a soyaglycerol medium (medium 1) composed of (g/l): NH4NO3 – 0.2; KH2PO4 – 0.2; K2HPO4 – 0.02; MgSO4 x 7 H2O – 0.01; peptone – 0.5; soybean flour – 0.5; glycerol – 2 ml (pH 5.0). The cultivation was carried out in 750 ml rotary flasks containing 100 ml of the Medium 1 for 7 days aerobically, on a rotary shaker at 200 rpm. The biomass obtained was fragmented with glass beads while stirring on a rotary shaker at 200 rpm for 10 min. The fragmented biomass (5 ml) was used for inoculation of 100 ml of Medium 2 composed of (g/l): glucose – 20; yeast extract – 5.0; peptone – 5.0; MgSO4 x 7H2O – 1.0; and polycaproamide fibre – 1.0. The cultivation was carried out aerobically under the same conditions. After 5 days of the growth, 2,6-dimethyl phenol (final concentration - 1 mM) and CuSO4 x 5H2O (2 mM) were added for
the induction of laccase activity. After 18 days of growth, the cultivation broth was separated by filtration and used for the obtainment of the enzyme preparation.
2.3 Enzyme preparation from Lentinus strigosus
The filtered cultivation broth (700 ml) obtained as described above was applied to a column with DE-52 (carrier volume - 350 ml) previously equilibrated with 20 mM Na-acetate buffer (pH 5.0) (Buffer A). The column was washed with an equal volume of the same buffer. Elution was carried out using linear gradient 0 – 0.5 М NaCl in 1800 ml of Buffer A at a rate of 1.5 ml/min. The fractions with laccase activity were combined, desalinated and concentrated in an ultrafiltration cell (Amicon, USA) with membrane UM-10. The laccase preparation obtained was applied on a column with Q-Sepharose (carrier volume of 60 ml) equilibrated with Buffer A. The column was washed with one volume of the same buffer. The elution was carried out using linear gradient 0–0.4 М NaCl in 1000 ml of Buffer A at a rate of 1 ml/min. The fraction volume was 5.6 + 0.2 ml. The active fractions were combined, desalinated and concentrated in an ultrafiltration cell (Amicon, USA) with membrane UM-10. The activity of the enzyme preparation obtained was determined spectrophotometrically with ABTS as substrate [17].
2.4 Laccase activity assay
Laccase activity was determined spectrophotometrically on UV-1700 (Shimadzu, Japan). ABTS was used as a substrate (0.5 mM). It was dissolved in 100 mM acetate buffer, pH 5.0 at 25°C. The enzyme activity was calculated with the absorption coefficient for ABTS ε = 29300 M-1 cm-1 [17] at λ= 420 nm. One unit of laccase activity was defined as the amount of laccase that oxidizes 1 μmol of ABTS per min under the selected assay conditions.
2.5 LMS transformation of steroids
Screening of LMS-catalysed reactions was performed in 50 mL test-tubes containing 10 ml of 0.1 M acetate buffer (pH 5.0). The reaction system contained 1 mg of the mediator and 0.5 U of the enzyme per 1 mg of steroid substrate. Sinapic acid, acetosyringone, syringaldehyde, HBT, N-hydroxyacetanilide, and TEMPO were tested as mediators. Nonionic detergent Tween-80 (0.1 %, v/v) was added to the medium. The reactions were carried out aerobically in a thermostatic rotary shaker at 250 rpm and 40°C. In blanks, steroid substrate were treated
with laccase alone (without mediator), or mediator alone (without enzymes). If not otherwise mentioned, the duration of transformation was 24 h. The samples (1 ml) were taken at 0.5, 2, 3, 6, 22, and 24 h after the reaction start. Steroids were extracted by triple volume of ethyl acetate.
2.6 Steroid analyses
2.6.1 TLC The aliquots of steroid extracts were applied to silica gel plate Sorbfil UV 254 (Russia) and developed in a mixture of benzene : acetone (25 : 15, v/v). The compounds were visualized under UV light (254 nm). Then, the TLC plates were treated by 4% (w/v) solution of phosphomolybdic acid in ethanol, dried, and developed by heating to 100 – 140°C. 3βHydroxy-5-ene-steroids were visualized as glaucous coloured spots; 7-keto and 7hydroxylated 3β-hydroxy-5-ene-steroids appeared as blue-coloured spots.
2.6.2 HPLC The ethyl acetate extracts of the samples were dried and re-dissolved in acetonitrile. An acetonitrile : water mixture (50 : 50, v/v) was used for dissolution of steroid standards and dilution of liquid samples. The analysis was carried out on a Series 1200 system (Agilent, USA) with reverse phase Symmetry C18, 5 μm, 100 Å, 250 x 4.6 mm (Waters, USA); guardcolumn (4.6 x 10 mm) of the same type, using mixture of acetonitrile : deionized water of 45 : 55 (v/v), flow rate of 1 ml/min and UV absorbance detection at 200, or 240 nm. Peaks quantification was performed using area values; steroids were quantified using the corresponding standards.
2.6.3 Mass spectra The electron impact mass spectra (MS) were obtained on a Finnigan MAT INCOS SO (USA) apparatus by direct inlet at 100°C with an ionization energy of 70 eV.
2.7 Preparation of crystalline product from pregnenolone
Pregnenolone (60 mg) was suspended in 200 ml of 0.1 M potassium - acetate buffer (pH 5.0) with 0.1% Tween 80 (final steroid concentration: 1 mM). The bioconversion was carried out in 1L flask by addition of the LMS composed of 6 mg HBT and 30 U of Trametes versicolor laccase. The reaction was carried out aerobically on a rotary shaker at 250 rpm and 40°C. The
bioconversion was completed in approximately 24 h. Then, the broth was extracted with two volumes of ethyl acetate. The combined organic extract was concentrated by rotary evaporation till 30 ml and dried with molecular sieves 4A. After the filtration, the organic solution was evaporated till initiation of crystallization. The solution was cooled to 4°C, and the crystals were recovered by filtration and washed with hexane. After drying 42 mg of the crystalline steroid product was obtained. Characteristics of the product obtained: white crystals, mp 196-199°C; Rf 0.35 and traces of substrate with Rf 0.85 (benzene : acetone= 5:3, v/v); Rt 5.39 min; HPLC purity (200 / 240 нм) corresponded to 96% of 3β-hydroxypregn-5-ene-7,20-dione (7-keto-pregnenolone). Molar yield of the crystalline product consisted 58.3%. Re-crystallization from ethyl alcohol afforded a crystalline product, mp 208-212°C corresponding to those of the authentic standard (212-213°C). Mass spectrum of the product corresponded to C21H30O3. Main fragments, m/z (%): 331 (100, М+), 313 (56, M-H 2 O + ), 295 (38, M-2H 2 O + ), 271 (28, M-H 2 O-COCH 2 + ), 253 (13, M-2H 2 O-COCH 2 + ).
3. Results and discussion
A strain of Lentinus strigosus 1566 was earlier selected was earlier selected as the most active laccase producer among over 220 basidiomycete strains belonging to 104 species [26]. The production of different laccase isoforms by this strain was shown to be regulated by culture conditions [27]. In this study, the prevailing isoform of laccase was isolated from the cultivation broth of L. strigosus 1566 and applied in the experiments. The activity of L. strigosus 1566 laccase preparation was estimated as 103+2 U/mg. In addition, commercially available fungal laccase from Trametes versicolor was investigated for steroid oxidation. The structures of non-phenolic steroids of androstane and pregnane series used as substrates for laccase-mediated oxidation are presented in Fig. 1. The 3β-hydroxy-Δ5-steroids were represented by DHEA, pregnenolone and the stereoisomeric 7-alcohols (7α–OH-DHEA and 7β–OH-DHEA). Among 3-oxo-4-ene-steroids, AD, 9α-OH-AD, testosterone and 20-HMP were tested.
3.1 LMS oxidation of non-phenolic steroids
Preliminary experiments showed that laccase alone did not converted non-phenolic steroids (data not shown). In this study, laccases were tested in combination with mediators of different types.
General scheme of LMS oxidation via radical (A) and ion (B) mechanisms is given in Fig. 2 [28, 29]. We used typical mediators, which may generate oxidation via radical mechanism. Those are HBT, sinapic acid, acetosyringone, syringaldehyde, while TEMPO is known as a mediator of ionic mechanism of non-phenolic compound oxidation. The activity of the LMS with mediators of A-type was studied towards steroids with mobile allylic protons at C-7, i.e., DHEA, pregnenolone, and steroidal allylic 7-alcohols (Table 1). When 3-oxo-4-ene steroids with hydroxyl functions at C- 9 (9α-OH-AD), С-17 (testosterone) or C-22 (20-HMP) were used as substrates, the mediators of both radical and ionic action (Aand B-types) were applied. The latters were shown to be effective at the oxidation of alcohols to corresponding carbonyl compounds [2, 30]. In all cases, reaction was carried out at pH 5 in acetate buffer in order to provide maximal laccase activity, mediator stability, and reaction ability of the generated radicals [31]. The results are summarized in Table 1. As shown in Table 1, steroids with allylic protons or hydroxyls were converted by LMS. No substrate decrease, or any product formation was observed with laccase-alone or mediator-alone system (data not shown). In the case of DHEA oxidation, either Tween-80 or Tween-20 were tested as surfactants, but no significant differences were found in the composition of reaction products and the dynamics of the process (data not shown). This points to the absence of interfering effect of Tween-80 in the steroid oxidation via generation of peroxy-сompounds by LMS oxidation of olefin moiety of surfactant. The results evidence the possibility of the non-phenolic steroids conversion with LMS. Noteworthy, both L. strigosus and T. versicolor laccases were able to oxidize steroids in the presence of mediators of radical types and showed the comparable level of activity. Among the mediators, HBT and acetosyringone provided almost twice higher conversion as compared with sinapic acid and N-hydroxyacetanilide at the conversion of DHEA. No conversion of 3oxo-4-ene steroids of both androstane (AD, 9α-OH-AD, testosterone) and pregnane series (20-HMP) was observed with none of mediators investigated. The results prove that the presence of allylic moiety in steroid molecule is important for laccase-mediated oxidation. With that, the presence of a hydroxyl group at C3 determines to a certain extent the regioselectivity of the LMS oxidation at C7. Similar to a free radical reaction of sterol autooxidation, LMS reaction starts with the abstraction of a reactive allylic hydrogen at C7. An alternative abstraction of a hydrogen atom at C4 is prevented due to steric hindrance and stabilization by hydroxyl group at C3 [32].
3.2 Oxyfunctionalization of DHEA at C-7 by LMS
The TLC/HPLC monitoring of the products during bioconversion of DHEA by LMS showed formation of 3β,7α-dihydroxy-5-ene-17-one (7α-OH-DHEA) and its stereoisomer - 3β,7βdihydroxy-5-ene-17-one (7β-OH-DHEA) with a prevalence of the former (Table 2). Earlier, we reported the obtaining of 7α-OH-DHEA and 7β-OH-DHEA using whole-cell microbial catalysts [33]. The possibility of enzymatic insertion of 7-hydroxyl function using laccasemediated reactions was not reported so far. During reaction course, the content of 7(α/β)-hydroxyderivatives decreased with an increase of the major bioconversion product - 3β-hydroxy-5-ene-7,17-dione (7-keto-DHEA), which reached over 50% conversion after 22 h of incubation of the substrate with LMS. The concentration of residual substrate decreased to 7% for this period, thus indicating over 90% DHEA conversion. The data allowed us to propose that the route of DHEA conversion by LMS includes the formation of the intermediate 7α-OH-DHEA and 7β-OH-DHEA followed by their oxidation to form 7-keto-DHEA (Fig.3). The suggestion was confirmed using 7α-OH-DHEA and 7β-OH-DHEA as substrates for LMS conversion. Both stereoisomers were transformed to 7-keto-DHEA with high selectivity and comparable rate (Table 2). The formation of 7-keto-DHEA as the only product evidenced a selective oxidation of the allylic hydroxyl in the presence of the other hydroxyl group at C-3. This finding is of importance for specific allylic alcohols oxidation without protection / deprotection of 3β-hydroxyl function of the corresponding steroids.
3.3 Pregnenolone to 7-ketopregnenolone oxidation
7-Ketopregnenolone was detected as the only product from pregnenolone, - no any minor products were detected during the reaction. The conversion exceeded 86% after 19 h (Fig.4). The product was isolated, and its structure was confirmed by MS and by the comparison of the values of chromatographic and physico-chemical parameters with the corresponding authentic standard (the data are given above in 2.7). Expectedly, laccase-only system did not catalyze pregnenolone conversion. The results clearly indicate that the presence of mobile protons at C7 of the 3β-ol-Δ5-steroids (DHEA) provides insertion of oxygen functions. It may be explained by the easy formation of the corresponding allylic radicals as shown in [34]. Unlike DHEA, the corresponding 3-oxo4-ene steroid, - AD without allylic moiety was not converted (Table 1).
Similar results were obtained with hydroxylated steroids. The 3-oxo-4-ene steroids with hydroxyls in steroid core (9α-OH-AD), at C17 (testosterone), or in side chain (20-HMP) did not undergo LMS oxidation. Presumably, the redox potential of the LMS tested was insufficient for the oxidation of the inactivated hydroxyls in these compounds, while the presence of the activated allylic hydroxyls as in 7(α/β)-hydroxy DHEA provided effective reaction. The mechanism of LMS oxidation involves the laccase-mediated generation of the relatively stable mediator radicals [35]. In the case of DHEA, this step is probably followed by a nonenzymatic breakaway of the reactive allylic proton at C7 and its reaction with molecular oxygen to form 7-peroxide radicals. The latters are stabilized through the corresponding stereoisomeric 7α- and 7β-OH-DHEA, and further oxidized to 7-keto-DHEA (Fig. 3). Although no intermediates were detected at the LMS oxidation of pregnenolone, one may assume the same reactions resulted in the accumulation of 7-ketoderivative. Similar mechanism was suggested for LMS oxidation of the acyclic compounds and expanded to LMS oxidation of sitosterol [16].
4. Conclusion
In this work, laccase-mediated oxidation of non-phenolic steroids of androstane and pregnane series was investigated. Fungal laccases from two different sources were studied in combination with different type mediators. The insertion of oxygen function into allylic 7 position of DHEA and pregnenolone, using LMS catalysis was revealed for the first time. The oxidation by means of LMS can extend synthetic tools and represents a promising alternative to chemical methods for regioselective inserting of oxygen functional groups in position 7 of 3-hydroxy-Δ5-steroids. No protection/deprotection of hydroxyl function at C3 is required at the LMS application. The retention of 3β-ol-5-ene moiety in steroid molecule with simultaneous insertion of oxygen function at C7 by LMS is important because such steroids intermediates show a promising potential for pharmaceutical applications. For instance 7-keto DHEA provide support of immunomodulatory activities [36], enhancement of cognition, memory storage [37] and retrieval, and weight management [38, 39]. The reaction performance in aqueous solutions under mild conditions corresponds to the principles of “green chemistry” and may be considered as an environmentally friendly way for the preparative scale exploitation in pharmaceutical industry for the production of valuable Δ5-7hydroxy- and Δ5-7-oxo-steroids.
Acknowledgements S.M. Khomutov, A.A. Shutov and M.V. Donova are grateful to Russian Science Foundation (Project No. 142400169) for support of their work.
References
1.
H. Claus, Arch. Microbiol. 179 (2003) 145–150.
2.
S. Riva, Trends Biotechnol. 24 (2006) 219-226.
3.
P. Giardina, V. Faraco, C. Pezzella, A. Piscitelli, S. Vanhulle, G. Sannia, Cell. Mol. Life Sci. 67 (2010), 369-385.
4.
S. Witayakran, A.J. Ragauskas, Adv. Synth. Catal. 351 (2009) 1187-1209.
5.
P. Baldrian, FEMS Microbiol. Rev. 30 (2006) 215–242.
6.
A. Leonowicz, N.S. Cho , J. Luterek, A. Wilkolazka, M. Wojtas-Wasilewska, A. Matuszewska, M. Hofrichter, D. Wesenberg, J. Rogalski, J. Basic Microbiol. 41 (2001) 185-227.
7.
F. Spina, C. Cordero, B. Sgorbini, T. Schiliro, G. Gilli, C. Bicchi, G.C. Varese, Chem. Eng. Trans. 32 (2013) 391-396.
8.
A. Kunamneni, A. Ballesteros, F.J. Plou, M. Alcalde, in: A. Mendez-Vilas (Ed.) Communicating Current Research and Educational Topics and Trends in Applied Microbiology, Formex, Badajoz, Spain, 2007, pp. 233–245.
9.
M.V. Donova, O.V. Egorova, Appl. Microbiol. Biotechnol. 94 (2012) 1423-1447.
10.
G. Lugaro, G. Carrea, P. Cremonesi, M.M. Casellato, E. Antonini, Arch. Biochem. Biophys. 159 (1973) 1–6.
11.
T. Tanaka, T. Tamura, Y. Ishizaki, A. Kawasaki, T. Kawase, M. Teraguchi, M. Taniguchi, J. Environ. Sci. 21 (2009) 731–735.
12.
K. Suzuki, H. Hirai, H. Murata, T. Nishida, Water Res. 37 (2003) 1972–1975.
13.
S. Nicotra, A. Intra, G. Ottolina, S. Riva, D. Bruno, Tetrahedron: Asymmetry, 15 (2004) 2927–2931.
14.
L. Lloret, , G. Eibes, G. Feijoo, M.T. Moreira, J. M. Lema, J. Hazard. Mater. 213– 214 (2012) 175-183.
15.
M. Ueda, K. Shintani, A. Nakanishi-Anjyuin, M. Nakazawa, M. Kusuda, F. Nakatani, T. Kawaguchi, S. Tsujiyama, M. Kawanishi, T. Yagi, K. Miyatake, Enzyme Microb. Technol. 51 (2012) 402-407.
16.
S. Molina,
J. Rencoret, J. Río, A. Lomascolo, E. Record, A. T. Martínez, A.
Gutiérrez, Appl. Microbiol. Biotechnol. 80 (2008) 211–222. 17.
C. Valls, S. Molina, T. Vidal, J. Río, J.F. Colom, A.T. Martínez, A. Gutiérrez, Process Biochem. 44 (2009) 1032-1038.
18.
E.J. Parish, S.A. Kizito, Z. Qiu, Lipids, 39 (2004) 801-804.
19.
M. Jurado-Gonzalez, A.C. Sullivan, J.R.H. Wilson, Tetrahedron Lett. 44 (2003) 4283– 4286.
20.
H. Choi, M. P. Doyle, Org. Lett. 9 (2007) 5349–5352.
21.
D. Crich Y. Zou, Org. Lett. 6 (2004) 775–777.
22.
W.G. Salmond, M.A. Barta, J. L. Havens, J. Org. Chem. 43 (1978) 2057–2059.
23.
E. J. Parish, S. Chitrakorn, T. Wei, Synth. Commun. 16 (1986) 1371–1375.
24.
C.C. Cosner, P.J. Cabrera, K.M. Byrd, A.M. Thomas, P. Helquist, Org. Lett. 13 (2011) 2071-2073.
25.
E.J. Parish, S. Chitrakorn, S. Lowery, Lipids, 19 (1984) 550-552.
26.
N.M. Myasoedova, A.M. Chernykh, N.V. Psurtseva, N.V. Belova, L.A. Golovleva, Appl. Biochem. Microbiol. 44 (2008) 73–77.
27.
N.M. Myasoedova, N.B. Gasanov, A.M. Chernykh, M.P. Kolomytseva, L.A. Golovleva, Prikl. Biokhim. Mikrobiol. 51 (2015 )221-228 [Article in Russian].
28.
A. Wells, M. Teria, T. Eve, Biochem. Soc. Trans. 34 (2006) 304-308.
29.
M. Fabbrini, C. Galli, P. Gentili, J. Mol. Catal. B: Enzym. 16 (2002) 231–240.
30.
F. D’Acunzo, P. Baiocco, M. Fabbrini, C. Galli, P. Gentili , Eur. J. Org. Chem. 24 (2002) 4195–4201.
31.
L. Lloret, G. Eibes, M.T. Moreira, G. Feijoo, J.M. Lema, J. Mol. Catal. B: Enzym. 97 (2013) 233– 242.
32.
L. L. Smith, Lipids, 31 (1996) 453-487.
33.
T.G. Lobastova, S.M. Khomutov, L.L. Vasiljeva, M.A. Lapitskaya, K.K. Pivnitsky, M.V. Donova, Steroids, 74 (2009) 233-237.
34.
E. J. Parish, S. A. Kizito, Z. Qiu, Lipids, 39 (2004) 801-804.
35.
F. Xu, T. Damhus, S. Danielsen, L.H. Østergaard, in: R. D. Schmid, V.B. Urlacher (Eds.), Modern Biooxidation: Enzymes, Reactions and Applications, Wiley-VCH, Weinheim, Germany, 2007, pp. 43-77.
36.
S.C. Castle, Clin. Infect. Dis. 31 (2000) 578-585.
37.
J. Shi, S. Schulze, H.A. Lardy, Steroids, 65 (2000) 124-129.
38.
R. Hampl, L. Starka, L. Jansky Physiol Res. 55 (2006) 123-131.
39.
J. L. Zenk, J. L. Frestedt, M. A. Kuskowski, J. Nutr. Biochem. 18 (2007) 629-634.
Figure Captions Figure 1. Structures of non-phenolic steroids used as substrates for LMS oxidation.
Figure 2. Scheme of LMS oxidation via radical (A) and ion (B) mediator mechanism.
Figure 3. Scheme of LMS oxidation of DHEA.
Figure 4. HPLC chromatogram (λ=240 nm) of pregnenolone to 7-keto-pregnenolone oxidation by LMS based on HBT.
Tables Table 1. Bioconversion of steroid substrates by laccases from Lentinus strigosus 1566 (LLs) and Trametes versicolor (LTv) №
Substrate
Mediator
1
DHEA
HBT**
2
Pregnenolone
LMS bioconversion* LLs LTv ++ ++
N-hydroxyacetanilide
+
+
acetosyringone
++
++
sinapic acid
±
±
HBT
++
++
acetosyringone
++
++
3
AD
HBT
–
–
4
9α-OH-AD
5
20-HMP
TEMPO HBT N-hydroxyacetanilide acetosyringone TEMPO
– – – – –
– – – – –
HBT
–
–
TEMPO
–
–
HBT
–
–
6
Testosterone
7
7α-DHEA
HBT
++
++
8
7β- DHEA
HBT
++
++
*LMS oxidation products determined by HPLC (200/240 nm) at 24 h: (++) ≥ 50%, (+) 10 50%, (±) ≤ 10%, (–) not determined ** Tween-80 and Tween-20 were compared as surfactants in LMS transformation
Table 2. Bioconversion of DHEA and its 7(α/β)-hydroxy derivatives by LMS* №
Compounds presented in the reaction mixture (%) Substrate
1
DHEA
Products
Rt(min)/Rf*
Time of conversion (h)
*
0
-
8.3/0.8
7α-OH-
3.5/0.22
DHEA 7β-OH-DHEA
2
3
6
22
100±
34±3
19±2
7.3±0.
5
25±2
24.3±
7
3.3/0.33
8.7±0.
2
19.8±2
3.9/0.56
8
16.6±
8.1±0.
32±2
1
7
38±3
51±5
7-keto-DHEA 2 7α-OH-DHEA
-
3.5/0.22
100±
88±
77±7
7-keto-DHEA
3.9/0.56
5
5
22±2
11± 1 3 7β-OH-DHEA 7-keto-DHEA
3.3/0.33
100±
84±
73±7
3.9/0.56
5
8
27±2
16± 1 LMS composed of L. strigosus laccase and HBT; initial concentration of substrate 100 mg/L ** Rt - retention time (HPLC), Rf - retardation factor (TLC)
S1381-1177(15)30098-9 http://dx.doi.org/doi:10.1016/j.molcatb.2015.11.004 MOLCAB 3268
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
25-7-2015 26-10-2015 1-11-2015
Please cite this article as: Sergey M.Khomutov, Andrey A.Shutov, Alexey M.Chernikh, Nina M.Myasoedova, Ludmila A.Golovleva, Marina V.Donova, Laccase-mediated oxyfunctionalization of 3rmbeta-hydroxy-Delta5-steroids, Journal of Molecular Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2015.11.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Laccase-mediated oxyfunctionalization of 3β-hydroxy-Δ5-steroids Sergey M. Khomutov* [email protected], Andrey A. Shutov, Alexey M. Chernikh, Nina M. Myasoedova, Ludmila A. Golovleva, Marina V. Donova G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences,Pushchino, Moscow Region, 142290 Russia *
Corresponding author.
Graphical Abstract
Highlights Fungal laccases were tested in bioconversion of non-phenolic steroids. Laccase-mediated systems effectively catalyze oxidation of 3β-hydroxy-Δ5-steroids. The oxidation of 3β-hydroxyandrost-5-en-17-one includes the hydroxylation at positions 7α and 7β. Regiospecific oxidation of the 7(α/β)-alcohols results in 3β-hydroxy-androst-5-en7,17-dione. 3β-Hydroxypregn-5-ene-7,20-dione is an only laccase product from pregnenolone.
Abstract Laccase mediator systems (LMS) were studied as catalysts for steroid oxidation. The fungal laccases from Lentinus strigosus 1566 and Trametes versicolor were used in the work. Among five mediators screened, 1-hydroxybenzotriasol (HBT) excelled in activity. The LMS effectively catalyzed oxidation of 3β-hydroxy-Δ5-steroids like DHEA (3β-hydroxyandrost-5en-17-one) and pregnenolone (3β-hydroxypregn-5-en-20-one), while no activity was observed towards 3-oxo-4-ene-steroids (androstenedione, 9α-hydroxyandrostenedione, testosterone and 20-hydroxymethylpregn-4-en-3-one). The pathway of DHEA oxidation by LMS included the hydroxylation at positions 7α and 7β followed by oxidation of the corresponding 7(α/β)alcohols to form 3β-hydroxyandrost-5-ene-7,17-dione. Regiospecific oxidation of allylic hydroxyl functions by LMS was confirmed using 3β,7α- and 3β,7β-dihydroxyandrost-5-en17-ones as substrates. 3β-Hydroxypregn-5-ene-7,20-dione was produced with LMS as an only product from pregnenolone. The yield of crystalline product
reached 58.3% yield with a
purity of 96%. The results demonstrate that application of LMS may be a promising approach for steroid oxyfunctionalization.
Keywords: laccase; steroid; oxidation; laccase mediator system; dehydroepiandrosterone; pregnenolone; oxyfunctionalization
1. Introduction Laccases
(benzenediol:oxygen
oxidoreductases,
E.C.1.10.3.2)
are
copper-containing
oxidoreductases that specifically catalyze the oxidation of a wide range of organic substrates including phenols, polyphenols, anilines, aryl diamines, methoxy-substituted phenols, hydroxyindoles, benzenethiols and inorganic/organic metal compounds, to the corresponding radicals, using molecular oxygen as the final electron acceptor [1-4]. The reactive radical species rapidly react further to various oxidation products forming water as an only byproduct. The broad substrate specificity makes laccases interesting “eco-friendly” enzymes with high industrial potential [2]. Laccase applications in biotechnology, e.g. for delignification and bleaching of paper pulp, detoxification and decolourization of textile dyes, bioremediation, detoxification of wastewaters, chemical grafting, polymer surface modifications and others, had been reviewed several times [3, 5-7]. Laccase activity has been found in fungi, plants, bacteria, and insects and its significant role was evidenced for synthetic and mineralization processes in nature [3]. Fungal laccases are of particular commercial interest due to their higher redox potential as compared with other enzyme forms isolated from prokaryotes, insects or plants [8]. The main laccase producers are lignolytic basidiomycetes, - white-rot fungi, which secrete the enzymes extracellularly, thus providing further simple enzyme purification [6]. Steroids are terpenoid lipids of specific structure that contain a gonane nucleus of four cycloalkane rings. These compounds fulfill essential physiological functions and are widespread in all living systems. Steroid preparations are widely used in different fields of medicine, veterinary, and agrochemistry. Bioconversion is a powerful tool for the production of valuable steroids compounds [9]. The data on laccase action on steroids mainly concern the dimerization, or polymerization of phenolic steroid hormones, such as estradiol, estrone, ethynilestradiol, and their derivatives [10-13]. These reactions can be exploited for the removal of estrogens, which are the endocrine disrupting steroids, from the wastewaters [14, 15]. The information on laccase activity towards the non-phenolic steroids is scarce [16, 17]. Mechanism of laccase action on phenolic compounds, including estrogens, is often based on the formation of the corresponding phenoxy radicals followed by further condensation. In the case of non-phenolic steroid substrates, the redox potential proves to be insufficient for the formation of steroid radicals [16]. It is known that the substrate range of laccases can be expanded to the compounds with a higher redox potential than the enzymes themselves in the
presence of small natural (e.g. syringaldehyde or coumaric acid which can be found in the lignocellulosic biomass), or artificial molecules, which are capable to act as electron transfer mediators. Due to the specific mediators, laccases became able to enlarge the substrate spectrum and oxidize non-phenolic compounds. The application of the so-called Laccase Mediator Systems (LMS) for the oxidation of different substrates (e.g. alcohols, sugars, esters, alkenes, amides etc.) had been reviewed [2, 4]. LMS composed of Pycnoporus cinnabarinus laccase and 1-hydroxybenzotriazole (HBT) as a mediator was applied for the oxidation of the model lipids representative for main paper pulp lipophilic extractives [16]. The unsaturated lipids including phytosterols were shown to be oxidized by the LMS to the corresponding epoxy- and hydroxy fatty acids from fatty acids, and free and esterified 7-ketosterols and steroid ketones from sterols and sterol esters. The treatment of eucalypt kraft pulp by LMS composed of laccase and HBT resulted in the decrease of pulp sterol content with an increase of 7-oxo-sitosterol and stigmasta-3,5-dien-7one [17]. Detection of 7-ketosterols among the products of phytosterol oxidation indicated the possibility of LMS to catalyze the introduction of an oxygen function at the allylic position of 3β-hydroxy-Δ5-steroids. Allylic moiety oxidation of Δ5-steroids can provide obtainment of valuable 7-keto derivatives with diverse biological properties [18]. Metallic catalysis in combination with hydroperoxides is often used for the reaction. For instance, the allylic oxidation by cobalt (II) alkyl phosphonate modified silica and tert-butyl hydroperoxide resulted in good product yields [19]. The catalyst based on dirhodium caprolactamate was highly effective at the hydroperoxide oxidation of Δ5-steroids [20]. However, chemical catalysis may not provide regioselectivity of the reaction: e.g. undesirable oxidation at C4 was observed at the application of selenic dioxide [21]. Moreover, hazardous reagents such as chrome trioxide [22], or pyridinium chlorochromate [23] are used as oxidants in stoichiometric, or even higher amounts. Noteworthy, chemical protection of the 3β-hydroxyl function by esterification is required for the preservation of the ∆5-double bond in steroid molecule at the allylic peroxide oxidation by chemical catalysis. Of importance are new simple methods which exclude the necessity of 3β-hydroxyl function protection, allow reaction performance under mild conditions in aqueous media, and provide effective environmentally friendly synthesis of the valuable steroids. The specific oxidation of allylic hydroxyls represents another relevant problem [24, 25]. Allylic steroid alcohols may be oxidized chemically to the corresponding aldehydes and ketones using different co-oxidant systems in the presence of saturated alcohols. Bioconversion could provide alternative promising approach.
Herein, we studied the possibility of regioselective oxygen functionalization of 3β-hydroxyΔ5-steroids and the oxidation of the corresponding allylic 7-alcohols using LMS oxidation.
2. Experimental
2.1 Substrates and reagents
Sinapic
acid,
acetosyringone,
syringaldehyde,
1-hydroxybenzotriazole
(HBT),
N-
hydroxyacetanilide, 2,2`-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) [ABTS], (2,2,6,6tetramethylpiperidin-1-yl)oxyl [TEMPO], 3β-hydroxypregn-5-en-20-one (pregnenolone), 3βhydroxyandrost-5-ene-17-one (dehydroepiandrosterone, DHEA), 17β-hydroxyandrost-4-ene3-one (testosterone), 2,6-dimethylphenol, Tween-80, Tween-20 and Trametes versicolor laccase were purchased from Sigma-Aldrich Co (USA). 9α-Hydroxyandrost-4-ene-3-one (9α-OH-AD, purity - 98%, m.p. – 217-220 °C, [E, 240 nm (0.01% in ethyl alcohol) - 14780 M-1 cm-1], 20-hydroxymethylpregn-4-en-3-one (purity - 98%, m.p. 181 - 183 C; [E, 240 nm (0.01% in methyl alcohol) - 16100 M-1 cm-1] were obtained from G.K.Skryabin Institute of Biochemistry and Physiology of Microorganisms (RAS, Russia). 3β,7α-Dihydroxyandrost-5ene-17-one (7α–OH-DHEA) and 3β,7β -dihydroxyandrost-5-ene-17-one (7β–OH-DHEA), 3β-hydroxyandrost-5-ene-7,17-dione (7-keto-DHEA), 3β-hydroxypregn-5-ene-7,20-dione (7keto-pregnenolone) were provided by Steraloids (USA). Other materials and solvents were of analytical grade and purchased from domestic companies (Russia).
2.2 Strain and cultivation
The strain of Lentinus strigosus 1566 was obtained from Basidiomycetes Collection of the Komarov Institute of Botanics (Moscow, Russian Federation), and was grown on a soyaglycerol medium (medium 1) composed of (g/l): NH4NO3 – 0.2; KH2PO4 – 0.2; K2HPO4 – 0.02; MgSO4 x 7 H2O – 0.01; peptone – 0.5; soybean flour – 0.5; glycerol – 2 ml (pH 5.0). The cultivation was carried out in 750 ml rotary flasks containing 100 ml of the Medium 1 for 7 days aerobically, on a rotary shaker at 200 rpm. The biomass obtained was fragmented with glass beads while stirring on a rotary shaker at 200 rpm for 10 min. The fragmented biomass (5 ml) was used for inoculation of 100 ml of Medium 2 composed of (g/l): glucose – 20; yeast extract – 5.0; peptone – 5.0; MgSO4 x 7H2O – 1.0; and polycaproamide fibre – 1.0. The cultivation was carried out aerobically under the same conditions. After 5 days of the growth, 2,6-dimethyl phenol (final concentration - 1 mM) and CuSO4 x 5H2O (2 mM) were added for
the induction of laccase activity. After 18 days of growth, the cultivation broth was separated by filtration and used for the obtainment of the enzyme preparation.
2.3 Enzyme preparation from Lentinus strigosus
The filtered cultivation broth (700 ml) obtained as described above was applied to a column with DE-52 (carrier volume - 350 ml) previously equilibrated with 20 mM Na-acetate buffer (pH 5.0) (Buffer A). The column was washed with an equal volume of the same buffer. Elution was carried out using linear gradient 0 – 0.5 М NaCl in 1800 ml of Buffer A at a rate of 1.5 ml/min. The fractions with laccase activity were combined, desalinated and concentrated in an ultrafiltration cell (Amicon, USA) with membrane UM-10. The laccase preparation obtained was applied on a column with Q-Sepharose (carrier volume of 60 ml) equilibrated with Buffer A. The column was washed with one volume of the same buffer. The elution was carried out using linear gradient 0–0.4 М NaCl in 1000 ml of Buffer A at a rate of 1 ml/min. The fraction volume was 5.6 + 0.2 ml. The active fractions were combined, desalinated and concentrated in an ultrafiltration cell (Amicon, USA) with membrane UM-10. The activity of the enzyme preparation obtained was determined spectrophotometrically with ABTS as substrate [17].
2.4 Laccase activity assay
Laccase activity was determined spectrophotometrically on UV-1700 (Shimadzu, Japan). ABTS was used as a substrate (0.5 mM). It was dissolved in 100 mM acetate buffer, pH 5.0 at 25°C. The enzyme activity was calculated with the absorption coefficient for ABTS ε = 29300 M-1 cm-1 [17] at λ= 420 nm. One unit of laccase activity was defined as the amount of laccase that oxidizes 1 μmol of ABTS per min under the selected assay conditions.
2.5 LMS transformation of steroids
Screening of LMS-catalysed reactions was performed in 50 mL test-tubes containing 10 ml of 0.1 M acetate buffer (pH 5.0). The reaction system contained 1 mg of the mediator and 0.5 U of the enzyme per 1 mg of steroid substrate. Sinapic acid, acetosyringone, syringaldehyde, HBT, N-hydroxyacetanilide, and TEMPO were tested as mediators. Nonionic detergent Tween-80 (0.1 %, v/v) was added to the medium. The reactions were carried out aerobically in a thermostatic rotary shaker at 250 rpm and 40°C. In blanks, steroid substrate were treated
with laccase alone (without mediator), or mediator alone (without enzymes). If not otherwise mentioned, the duration of transformation was 24 h. The samples (1 ml) were taken at 0.5, 2, 3, 6, 22, and 24 h after the reaction start. Steroids were extracted by triple volume of ethyl acetate.
2.6 Steroid analyses
2.6.1 TLC The aliquots of steroid extracts were applied to silica gel plate Sorbfil UV 254 (Russia) and developed in a mixture of benzene : acetone (25 : 15, v/v). The compounds were visualized under UV light (254 nm). Then, the TLC plates were treated by 4% (w/v) solution of phosphomolybdic acid in ethanol, dried, and developed by heating to 100 – 140°C. 3βHydroxy-5-ene-steroids were visualized as glaucous coloured spots; 7-keto and 7hydroxylated 3β-hydroxy-5-ene-steroids appeared as blue-coloured spots.
2.6.2 HPLC The ethyl acetate extracts of the samples were dried and re-dissolved in acetonitrile. An acetonitrile : water mixture (50 : 50, v/v) was used for dissolution of steroid standards and dilution of liquid samples. The analysis was carried out on a Series 1200 system (Agilent, USA) with reverse phase Symmetry C18, 5 μm, 100 Å, 250 x 4.6 mm (Waters, USA); guardcolumn (4.6 x 10 mm) of the same type, using mixture of acetonitrile : deionized water of 45 : 55 (v/v), flow rate of 1 ml/min and UV absorbance detection at 200, or 240 nm. Peaks quantification was performed using area values; steroids were quantified using the corresponding standards.
2.6.3 Mass spectra The electron impact mass spectra (MS) were obtained on a Finnigan MAT INCOS SO (USA) apparatus by direct inlet at 100°C with an ionization energy of 70 eV.
2.7 Preparation of crystalline product from pregnenolone
Pregnenolone (60 mg) was suspended in 200 ml of 0.1 M potassium - acetate buffer (pH 5.0) with 0.1% Tween 80 (final steroid concentration: 1 mM). The bioconversion was carried out in 1L flask by addition of the LMS composed of 6 mg HBT and 30 U of Trametes versicolor laccase. The reaction was carried out aerobically on a rotary shaker at 250 rpm and 40°C. The
bioconversion was completed in approximately 24 h. Then, the broth was extracted with two volumes of ethyl acetate. The combined organic extract was concentrated by rotary evaporation till 30 ml and dried with molecular sieves 4A. After the filtration, the organic solution was evaporated till initiation of crystallization. The solution was cooled to 4°C, and the crystals were recovered by filtration and washed with hexane. After drying 42 mg of the crystalline steroid product was obtained. Characteristics of the product obtained: white crystals, mp 196-199°C; Rf 0.35 and traces of substrate with Rf 0.85 (benzene : acetone= 5:3, v/v); Rt 5.39 min; HPLC purity (200 / 240 нм) corresponded to 96% of 3β-hydroxypregn-5-ene-7,20-dione (7-keto-pregnenolone). Molar yield of the crystalline product consisted 58.3%. Re-crystallization from ethyl alcohol afforded a crystalline product, mp 208-212°C corresponding to those of the authentic standard (212-213°C). Mass spectrum of the product corresponded to C21H30O3. Main fragments, m/z (%): 331 (100, М+), 313 (56, M-H 2 O + ), 295 (38, M-2H 2 O + ), 271 (28, M-H 2 O-COCH 2 + ), 253 (13, M-2H 2 O-COCH 2 + ).
3. Results and discussion
A strain of Lentinus strigosus 1566 was earlier selected was earlier selected as the most active laccase producer among over 220 basidiomycete strains belonging to 104 species [26]. The production of different laccase isoforms by this strain was shown to be regulated by culture conditions [27]. In this study, the prevailing isoform of laccase was isolated from the cultivation broth of L. strigosus 1566 and applied in the experiments. The activity of L. strigosus 1566 laccase preparation was estimated as 103+2 U/mg. In addition, commercially available fungal laccase from Trametes versicolor was investigated for steroid oxidation. The structures of non-phenolic steroids of androstane and pregnane series used as substrates for laccase-mediated oxidation are presented in Fig. 1. The 3β-hydroxy-Δ5-steroids were represented by DHEA, pregnenolone and the stereoisomeric 7-alcohols (7α–OH-DHEA and 7β–OH-DHEA). Among 3-oxo-4-ene-steroids, AD, 9α-OH-AD, testosterone and 20-HMP were tested.
3.1 LMS oxidation of non-phenolic steroids
Preliminary experiments showed that laccase alone did not converted non-phenolic steroids (data not shown). In this study, laccases were tested in combination with mediators of different types.
General scheme of LMS oxidation via radical (A) and ion (B) mechanisms is given in Fig. 2 [28, 29]. We used typical mediators, which may generate oxidation via radical mechanism. Those are HBT, sinapic acid, acetosyringone, syringaldehyde, while TEMPO is known as a mediator of ionic mechanism of non-phenolic compound oxidation. The activity of the LMS with mediators of A-type was studied towards steroids with mobile allylic protons at C-7, i.e., DHEA, pregnenolone, and steroidal allylic 7-alcohols (Table 1). When 3-oxo-4-ene steroids with hydroxyl functions at C- 9 (9α-OH-AD), С-17 (testosterone) or C-22 (20-HMP) were used as substrates, the mediators of both radical and ionic action (Aand B-types) were applied. The latters were shown to be effective at the oxidation of alcohols to corresponding carbonyl compounds [2, 30]. In all cases, reaction was carried out at pH 5 in acetate buffer in order to provide maximal laccase activity, mediator stability, and reaction ability of the generated radicals [31]. The results are summarized in Table 1. As shown in Table 1, steroids with allylic protons or hydroxyls were converted by LMS. No substrate decrease, or any product formation was observed with laccase-alone or mediator-alone system (data not shown). In the case of DHEA oxidation, either Tween-80 or Tween-20 were tested as surfactants, but no significant differences were found in the composition of reaction products and the dynamics of the process (data not shown). This points to the absence of interfering effect of Tween-80 in the steroid oxidation via generation of peroxy-сompounds by LMS oxidation of olefin moiety of surfactant. The results evidence the possibility of the non-phenolic steroids conversion with LMS. Noteworthy, both L. strigosus and T. versicolor laccases were able to oxidize steroids in the presence of mediators of radical types and showed the comparable level of activity. Among the mediators, HBT and acetosyringone provided almost twice higher conversion as compared with sinapic acid and N-hydroxyacetanilide at the conversion of DHEA. No conversion of 3oxo-4-ene steroids of both androstane (AD, 9α-OH-AD, testosterone) and pregnane series (20-HMP) was observed with none of mediators investigated. The results prove that the presence of allylic moiety in steroid molecule is important for laccase-mediated oxidation. With that, the presence of a hydroxyl group at C3 determines to a certain extent the regioselectivity of the LMS oxidation at C7. Similar to a free radical reaction of sterol autooxidation, LMS reaction starts with the abstraction of a reactive allylic hydrogen at C7. An alternative abstraction of a hydrogen atom at C4 is prevented due to steric hindrance and stabilization by hydroxyl group at C3 [32].
3.2 Oxyfunctionalization of DHEA at C-7 by LMS
The TLC/HPLC monitoring of the products during bioconversion of DHEA by LMS showed formation of 3β,7α-dihydroxy-5-ene-17-one (7α-OH-DHEA) and its stereoisomer - 3β,7βdihydroxy-5-ene-17-one (7β-OH-DHEA) with a prevalence of the former (Table 2). Earlier, we reported the obtaining of 7α-OH-DHEA and 7β-OH-DHEA using whole-cell microbial catalysts [33]. The possibility of enzymatic insertion of 7-hydroxyl function using laccasemediated reactions was not reported so far. During reaction course, the content of 7(α/β)-hydroxyderivatives decreased with an increase of the major bioconversion product - 3β-hydroxy-5-ene-7,17-dione (7-keto-DHEA), which reached over 50% conversion after 22 h of incubation of the substrate with LMS. The concentration of residual substrate decreased to 7% for this period, thus indicating over 90% DHEA conversion. The data allowed us to propose that the route of DHEA conversion by LMS includes the formation of the intermediate 7α-OH-DHEA and 7β-OH-DHEA followed by their oxidation to form 7-keto-DHEA (Fig.3). The suggestion was confirmed using 7α-OH-DHEA and 7β-OH-DHEA as substrates for LMS conversion. Both stereoisomers were transformed to 7-keto-DHEA with high selectivity and comparable rate (Table 2). The formation of 7-keto-DHEA as the only product evidenced a selective oxidation of the allylic hydroxyl in the presence of the other hydroxyl group at C-3. This finding is of importance for specific allylic alcohols oxidation without protection / deprotection of 3β-hydroxyl function of the corresponding steroids.
3.3 Pregnenolone to 7-ketopregnenolone oxidation
7-Ketopregnenolone was detected as the only product from pregnenolone, - no any minor products were detected during the reaction. The conversion exceeded 86% after 19 h (Fig.4). The product was isolated, and its structure was confirmed by MS and by the comparison of the values of chromatographic and physico-chemical parameters with the corresponding authentic standard (the data are given above in 2.7). Expectedly, laccase-only system did not catalyze pregnenolone conversion. The results clearly indicate that the presence of mobile protons at C7 of the 3β-ol-Δ5-steroids (DHEA) provides insertion of oxygen functions. It may be explained by the easy formation of the corresponding allylic radicals as shown in [34]. Unlike DHEA, the corresponding 3-oxo4-ene steroid, - AD without allylic moiety was not converted (Table 1).
Similar results were obtained with hydroxylated steroids. The 3-oxo-4-ene steroids with hydroxyls in steroid core (9α-OH-AD), at C17 (testosterone), or in side chain (20-HMP) did not undergo LMS oxidation. Presumably, the redox potential of the LMS tested was insufficient for the oxidation of the inactivated hydroxyls in these compounds, while the presence of the activated allylic hydroxyls as in 7(α/β)-hydroxy DHEA provided effective reaction. The mechanism of LMS oxidation involves the laccase-mediated generation of the relatively stable mediator radicals [35]. In the case of DHEA, this step is probably followed by a nonenzymatic breakaway of the reactive allylic proton at C7 and its reaction with molecular oxygen to form 7-peroxide radicals. The latters are stabilized through the corresponding stereoisomeric 7α- and 7β-OH-DHEA, and further oxidized to 7-keto-DHEA (Fig. 3). Although no intermediates were detected at the LMS oxidation of pregnenolone, one may assume the same reactions resulted in the accumulation of 7-ketoderivative. Similar mechanism was suggested for LMS oxidation of the acyclic compounds and expanded to LMS oxidation of sitosterol [16].
4. Conclusion
In this work, laccase-mediated oxidation of non-phenolic steroids of androstane and pregnane series was investigated. Fungal laccases from two different sources were studied in combination with different type mediators. The insertion of oxygen function into allylic 7 position of DHEA and pregnenolone, using LMS catalysis was revealed for the first time. The oxidation by means of LMS can extend synthetic tools and represents a promising alternative to chemical methods for regioselective inserting of oxygen functional groups in position 7 of 3-hydroxy-Δ5-steroids. No protection/deprotection of hydroxyl function at C3 is required at the LMS application. The retention of 3β-ol-5-ene moiety in steroid molecule with simultaneous insertion of oxygen function at C7 by LMS is important because such steroids intermediates show a promising potential for pharmaceutical applications. For instance 7-keto DHEA provide support of immunomodulatory activities [36], enhancement of cognition, memory storage [37] and retrieval, and weight management [38, 39]. The reaction performance in aqueous solutions under mild conditions corresponds to the principles of “green chemistry” and may be considered as an environmentally friendly way for the preparative scale exploitation in pharmaceutical industry for the production of valuable Δ5-7hydroxy- and Δ5-7-oxo-steroids.
Acknowledgements S.M. Khomutov, A.A. Shutov and M.V. Donova are grateful to Russian Science Foundation (Project No. 142400169) for support of their work.
References
1.
H. Claus, Arch. Microbiol. 179 (2003) 145–150.
2.
S. Riva, Trends Biotechnol. 24 (2006) 219-226.
3.
P. Giardina, V. Faraco, C. Pezzella, A. Piscitelli, S. Vanhulle, G. Sannia, Cell. Mol. Life Sci. 67 (2010), 369-385.
4.
S. Witayakran, A.J. Ragauskas, Adv. Synth. Catal. 351 (2009) 1187-1209.
5.
P. Baldrian, FEMS Microbiol. Rev. 30 (2006) 215–242.
6.
A. Leonowicz, N.S. Cho , J. Luterek, A. Wilkolazka, M. Wojtas-Wasilewska, A. Matuszewska, M. Hofrichter, D. Wesenberg, J. Rogalski, J. Basic Microbiol. 41 (2001) 185-227.
7.
F. Spina, C. Cordero, B. Sgorbini, T. Schiliro, G. Gilli, C. Bicchi, G.C. Varese, Chem. Eng. Trans. 32 (2013) 391-396.
8.
A. Kunamneni, A. Ballesteros, F.J. Plou, M. Alcalde, in: A. Mendez-Vilas (Ed.) Communicating Current Research and Educational Topics and Trends in Applied Microbiology, Formex, Badajoz, Spain, 2007, pp. 233–245.
9.
M.V. Donova, O.V. Egorova, Appl. Microbiol. Biotechnol. 94 (2012) 1423-1447.
10.
G. Lugaro, G. Carrea, P. Cremonesi, M.M. Casellato, E. Antonini, Arch. Biochem. Biophys. 159 (1973) 1–6.
11.
T. Tanaka, T. Tamura, Y. Ishizaki, A. Kawasaki, T. Kawase, M. Teraguchi, M. Taniguchi, J. Environ. Sci. 21 (2009) 731–735.
12.
K. Suzuki, H. Hirai, H. Murata, T. Nishida, Water Res. 37 (2003) 1972–1975.
13.
S. Nicotra, A. Intra, G. Ottolina, S. Riva, D. Bruno, Tetrahedron: Asymmetry, 15 (2004) 2927–2931.
14.
L. Lloret, , G. Eibes, G. Feijoo, M.T. Moreira, J. M. Lema, J. Hazard. Mater. 213– 214 (2012) 175-183.
15.
M. Ueda, K. Shintani, A. Nakanishi-Anjyuin, M. Nakazawa, M. Kusuda, F. Nakatani, T. Kawaguchi, S. Tsujiyama, M. Kawanishi, T. Yagi, K. Miyatake, Enzyme Microb. Technol. 51 (2012) 402-407.
16.
S. Molina,
J. Rencoret, J. Río, A. Lomascolo, E. Record, A. T. Martínez, A.
Gutiérrez, Appl. Microbiol. Biotechnol. 80 (2008) 211–222. 17.
C. Valls, S. Molina, T. Vidal, J. Río, J.F. Colom, A.T. Martínez, A. Gutiérrez, Process Biochem. 44 (2009) 1032-1038.
18.
E.J. Parish, S.A. Kizito, Z. Qiu, Lipids, 39 (2004) 801-804.
19.
M. Jurado-Gonzalez, A.C. Sullivan, J.R.H. Wilson, Tetrahedron Lett. 44 (2003) 4283– 4286.
20.
H. Choi, M. P. Doyle, Org. Lett. 9 (2007) 5349–5352.
21.
D. Crich Y. Zou, Org. Lett. 6 (2004) 775–777.
22.
W.G. Salmond, M.A. Barta, J. L. Havens, J. Org. Chem. 43 (1978) 2057–2059.
23.
E. J. Parish, S. Chitrakorn, T. Wei, Synth. Commun. 16 (1986) 1371–1375.
24.
C.C. Cosner, P.J. Cabrera, K.M. Byrd, A.M. Thomas, P. Helquist, Org. Lett. 13 (2011) 2071-2073.
25.
E.J. Parish, S. Chitrakorn, S. Lowery, Lipids, 19 (1984) 550-552.
26.
N.M. Myasoedova, A.M. Chernykh, N.V. Psurtseva, N.V. Belova, L.A. Golovleva, Appl. Biochem. Microbiol. 44 (2008) 73–77.
27.
N.M. Myasoedova, N.B. Gasanov, A.M. Chernykh, M.P. Kolomytseva, L.A. Golovleva, Prikl. Biokhim. Mikrobiol. 51 (2015 )221-228 [Article in Russian].
28.
A. Wells, M. Teria, T. Eve, Biochem. Soc. Trans. 34 (2006) 304-308.
29.
M. Fabbrini, C. Galli, P. Gentili, J. Mol. Catal. B: Enzym. 16 (2002) 231–240.
30.
F. D’Acunzo, P. Baiocco, M. Fabbrini, C. Galli, P. Gentili , Eur. J. Org. Chem. 24 (2002) 4195–4201.
31.
L. Lloret, G. Eibes, M.T. Moreira, G. Feijoo, J.M. Lema, J. Mol. Catal. B: Enzym. 97 (2013) 233– 242.
32.
L. L. Smith, Lipids, 31 (1996) 453-487.
33.
T.G. Lobastova, S.M. Khomutov, L.L. Vasiljeva, M.A. Lapitskaya, K.K. Pivnitsky, M.V. Donova, Steroids, 74 (2009) 233-237.
34.
E. J. Parish, S. A. Kizito, Z. Qiu, Lipids, 39 (2004) 801-804.
35.
F. Xu, T. Damhus, S. Danielsen, L.H. Østergaard, in: R. D. Schmid, V.B. Urlacher (Eds.), Modern Biooxidation: Enzymes, Reactions and Applications, Wiley-VCH, Weinheim, Germany, 2007, pp. 43-77.
36.
S.C. Castle, Clin. Infect. Dis. 31 (2000) 578-585.
37.
J. Shi, S. Schulze, H.A. Lardy, Steroids, 65 (2000) 124-129.
38.
R. Hampl, L. Starka, L. Jansky Physiol Res. 55 (2006) 123-131.
39.
J. L. Zenk, J. L. Frestedt, M. A. Kuskowski, J. Nutr. Biochem. 18 (2007) 629-634.
Figure Captions Figure 1. Structures of non-phenolic steroids used as substrates for LMS oxidation.
Figure 2. Scheme of LMS oxidation via radical (A) and ion (B) mediator mechanism.
Figure 3. Scheme of LMS oxidation of DHEA.
Figure 4. HPLC chromatogram (λ=240 nm) of pregnenolone to 7-keto-pregnenolone oxidation by LMS based on HBT.
Tables Table 1. Bioconversion of steroid substrates by laccases from Lentinus strigosus 1566 (LLs) and Trametes versicolor (LTv) №
Substrate
Mediator
1
DHEA
HBT**
2
Pregnenolone
LMS bioconversion* LLs LTv ++ ++
N-hydroxyacetanilide
+
+
acetosyringone
++
++
sinapic acid
±
±
HBT
++
++
acetosyringone
++
++
3
AD
HBT
–
–
4
9α-OH-AD
5
20-HMP
TEMPO HBT N-hydroxyacetanilide acetosyringone TEMPO
– – – – –
– – – – –
HBT
–
–
TEMPO
–
–
HBT
–
–
6
Testosterone
7
7α-DHEA
HBT
++
++
8
7β- DHEA
HBT
++
++
*LMS oxidation products determined by HPLC (200/240 nm) at 24 h: (++) ≥ 50%, (+) 10 50%, (±) ≤ 10%, (–) not determined ** Tween-80 and Tween-20 were compared as surfactants in LMS transformation
Table 2. Bioconversion of DHEA and its 7(α/β)-hydroxy derivatives by LMS* №
Compounds presented in the reaction mixture (%) Substrate
1
DHEA
Products
Rt(min)/Rf*
Time of conversion (h)
*
0
-
8.3/0.8
7α-OH-
3.5/0.22
DHEA 7β-OH-DHEA
2
3
6
22
100±
34±3
19±2
7.3±0.
5
25±2
24.3±
7
3.3/0.33
8.7±0.
2
19.8±2
3.9/0.56
8
16.6±
8.1±0.
32±2
1
7
38±3
51±5
7-keto-DHEA 2 7α-OH-DHEA
-
3.5/0.22
100±
88±
77±7
7-keto-DHEA
3.9/0.56
5
5
22±2
11± 1 3 7β-OH-DHEA 7-keto-DHEA
3.3/0.33
100±
84±
73±7
3.9/0.56
5
8
27±2
16± 1 LMS composed of L. strigosus laccase and HBT; initial concentration of substrate 100 mg/L ** Rt - retention time (HPLC), Rf - retardation factor (TLC)