Hydroxy lactones with the gem-dimethylcyclohexane system – Synthesis and antimicrobial activity

1 Introduction

The use of natural catalysts, i.e. whole cells, cell organelles or isolated enzymes, for chemical transformations has been well known for over 100 years (Faber, 2011). Biotransformation is a very good method that allows to obtain molecules identical or similar to natural ones. Microorganisms can also produce compounds which are completely different from these receiving during the chemical synthesis (Pinheiro and Marsaioli, 2007; Serra et al., 2005; Garcı́a-Pajón et al., 2003; de Carvalho and da Fonseca, 2006). Biotechnological methods play an important role in biochemistry and traditional organic synthesis by opening up new possibilities in receiving important, biologically active compounds. The methods used in classical organic synthesis have not been changed, but have been greatly expanded and enriched. This has been evidenced in recent years by the emergence of a number of works based on biocatalysis. Obviously, biochemical methods are not inherently better, but they are definitely an effective tool, complementary to other methods used in modern organic synthesis (Wedge et al., 2000; Borges et al., 2009; Maurs et al., 1999).

In our laboratory we have been investigating syntheses and biotransformations of bicyclic lactones with a methyl-substituted cyclohexane ring and we found that some of the hydroxy lactones obtained during these processes exhibited biological activity, mainly antifeedant activity against different insects (Szczepanik et al., 2000, 2003; Wawrzeńczyk et al., 2003; Dancewicz et al., 2005) and also antimicrobial activity (Grabarczyk et al., 2013). Other researchers also reported that structurally simple hydroxy lactones both natural and obtained during chemical syntheses exhibited biological activity, for example antifeedant (Gonzalez-Coloma et al., 2012), antifungal (Skaltsa et al., 2000; Wedge et al., 2000), antibacterial (Lee et al., 2008), anti-diabetic (Hunyadi et al., 2013) or antioxidant (Yang et al., 2011).

With reference to our ongoing research on the biotransformation of halolactones with a methyl-substituted cyclohexane ring (Gładkowski et al., 2011; Grabarczyk and Białońska, 2010; Grotowska and Wawrzeńczyk, 2002; Grabarczyk, 2012), we hereby present further examples of the biohydroxylation of chloro-, bromo- and iodolactone with the gem-dimethylcyclohexane ring. These lactones were chosen as model compounds to verify whether certain fungal strains had the ability to replace the halogen atom with the hydroxy group.

2 Materials and methods

2.3

2.3 Synthesis of substrates

The starting substrate for the synthesis of three racemic halolactones 3–5 was 4,4-dimethylcyclohexane-1,3-dione, which was converted into 4,4-dimethylcyclohex-2-en-1-ol (Wińska et al., 2010). At the next steps the known γ,δ-unsaturated ester 1 and γ,δ-unsaturated acid 2 (Gładkowski et al., 2011) were obtained. The new chlorolactone 3 and bromolactone 4 were synthesized in tetrahydrofuran by chloro- and bromolactonization, respectively. The already known iodolactone 5 (Gładkowski et al., 2011) was obtained by iodolactonization of acid 2, according to the previously described procedure (Grabarczyk et al., 2005) (Scheme 1).

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Scheme 1 Synthesis of halolactones 3–5.

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In this paper we present the synthesis of new compounds and their spectral data. These data will be useful in studying the changes in the molecules during biotransformation.

2.3.1

2.3.1 2-Chloro-5,5-dimethyl-9-oxabicyclo[4.3.0]nonan-8-one (3)

N-chlorosuccinimide (14.5 mmol) was added to acid 2 (8.9 mmol) dissolved in 30 ml of tetrahydrofuran. This mixture was stirred for 24 h at room temperature and then water was added (30 ml). The product was extracted with diethyl ether (3 × 30 ml). The combined ethereal fractions were washed with saturated NaHCO₃ solution (20 ml), brine (20 ml) and dried with anhydrous magnesium sulfate. The crude product was purified on silica gel (hexane:acetone, 3:1) yielding 7.5 mmol (84%) of chlorolactone 3 with the following physical and spectral data: n_D = 1.4953, ¹H NMR (300 MHz, CDCl₃): 0.96 and 1.08 (two s, 6H, (CH₃)₂C-5), 1.46 (m, 2H, CH₂-4), 1.85 (m, 1H, one of CH₂-3), 2.06 (m, 1H, one of CH₂-3), 2.33 (d, J = 6.8 Hz, 1H, one of CH₂-7), 2.37 (d, J = 1.7 Hz, 1H, one of CH₂-7), 2.50 (m, 1H, H-6), 3.75 (ddd, J = 14.1, 9.0 and 5.1 Hz, 1H, H-2), 4.54 (dd, J = 9.0 and 6.9 Hz, 1H, H-1); ¹³C NMR: 27.45 (C-10), 28.60 (C-9), 29.78 (C-3), 30.26 (C-7), 31.79 (C-5), 33.26 (C-4), 47.56 (C-6), 59.87 (C-2), 83.48 (C-1), 175.47 (C-8), IR (KBr, cm⁻¹): 2961 (s), 1782 (s), 1456 (s), 1151(s), 1020 (s). EI-MS m/z (%): 203 (8), 187 (7), 167 (6), 125 (100), 97 (20), 79 (14), 69 (24), 55 (18), 39 (35).

2.3.2

2.3.2 2-Bromo-5,5-dimethyl-9-oxabicyclo[4.3.0]nonan-8-one (4)

N-bromosuccinimide (7.3 mmol) was added to 7.1 mmol of acid 2 dissolved in 30 ml of tetrahydrofuran and the mixture was stirred for 24 h at room temperature. After this time 30 ml of water was added to the reacting mixture and the product was extracted with diethyl ether (3 × 30 ml). The combined ethereal fractions were washed with saturated NaHCO₃ solution (20 ml), brine (20 ml) and dried with anhydrous magnesium sulfate. After purification of the crude product on silica gel (hexane:acetone, 3:1), 6.1 mmol (87%) of bromolactone 4 was obtained. The physical and spectral data of this product are as follows: n_D = 1.5130, ¹H NMR (300 MHz, CDCl₃): 0.95 and 1.09 (two s, 6H, (CH₃)₂C-5), 1.46 (m, 2H, CH₂-4), 2.02 (m, 1H, one of CH₂-3), 2.16 (m, 1H, one of CH₂-3), 2.34 (d, J = 2.2 Hz, 1H, one of CH₂-7), 2.37 (s, 1H, one of CH₂-7), 2.46 (m, 1H, H-6), 3.83 (ddd, J = 14.4, 9.3 and 5.1 Hz, 1H, H-2), 4.68 (dd, J = 9.3 and 6.6 Hz, 1H, H-1); ¹³C NMR: 27.46 (C-10), 28.63 (C-9), 30.06 (C-7), 30.65 (C-3), 31.79 (C-5), 34.25 (C-4), 47.80 (C-6), 50.77 (C-2), 83.63 (C-1), 175.26 (C-8), IR (KBr, cm⁻¹): 2959 (s), 1791 (s), 1456 (s), 1169(s), 1020 (s). EI-MS m/z (%): 247 (12), 167 (57), 125 (100), 97 (27), 81 (29), 69 (36), 55 (34), 39 (64).

2.3.3

2.3.3 2-Iodo-5,5-dimethyl-9-oxabicyclo[4.3.0]nonan-8-one (5)

Iodolactonization was conducted on 6.0 mmol of acid 1 according to the known procedure (Grabarczyk et al., 2005) giving 4.8 mmol (80.2%) of iodolactone 5 with the following physical and spectral data: m.p. = 58–60 °C, ¹H NMR (300 MHz, CDCl₃): 0.93 and 1.09 (two s, 6H, (CH₃)₂C-5), 1.25 (ddd, J = 14.0, 3.7 and 3.6 Hz, 1H, one of CH₂-4), 1.42 (m, 1H, one of CH₂-4), 2.18 (m, 2H, CH₂-3), 2.37 (m, 3H, CH₂-7 and H-6), 3.86 (ddd, J = 15.0, 9.6 and 5.4 Hz, 1H, H-2), 4.78 (ddd, J = 9.6, 4.4 and 1.7 Hz, 1H, H-1), IR (KBr, cm⁻¹): 2956 (s), 1789 (s), 1453 (s), 1168(s), 1016 (s). EI-MS m/z (%): 295 (8), 167 (100), 139 (36), 121 (21), 107 (44), 95 (17), 81 (10), 69 (8), 55 (11), 39 (24).

2.4

2.4 Biotransformations

2.4.1

2.4.1 Microorganisms

The fungal strains used in all biotransformation reactions came from the collection of the Institute of Biology and Botany, Medical University, Wrocław: Fusarium culmorum AM 10, Fusarium avenaceum AM 11, Fusarium oxysporum AM 13, Fusarium tricinctum AM 16, Fusarium semitectum AM 20, Syncephalastrum racemosum AM 105, Fusarium solani AM 203, Fusarium scirpi AM 199, Cunninghamella japonica AM 472.

2.4.2

2.4.2 Screening procedure

The fungal strains used for biotransformation were cultivated at 25 °C in two Erlenmeyer flasks (capacity of 250 ml) containing 100 ml of a medium comprising 3% of glucose and 1% of bacteriological peptone (Biocorp). After three days 10 mg of the substrate dissolved in 1 ml of acetone was added to each flask with the mature culture. Incubation of the shaken cultures with the substrate continued for 7 days. After 3, 5 and 7 days of incubation the mixture of unreacted substrate, products and mycelium was extracted with dichloromethane (15 ml) and analyzed by GC (DB-17 column).

2.4.3

2.4.3 Preparative biotransformation

A total of 100 mg of halolactone 3–5 was dissolved in 10 ml acetone and dispensed into 10 Erlenmeyer flasks (capacity of 250 ml) with the 3-day cultures of fungal strains prepared as described in the screening procedure. The microorganisms selected during the screening transformations were incubated with substrates for 7 days and then the product mixture was extracted with dichloromethane (3 × 40 ml). The combined organic fractions were dried over anhydrous magnesium sulfate and the solvent was evaporated in vacuo. The pure product was separated from the unreacted substrate and the metabolites of the fungi by column chromatography (silica gel, hexane:acetone 3:1). During all of the preparative biotransformation only one and the same product, hydroxy lactone 6, was obtained in every case (Scheme 2).

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Scheme 2 Biotransformation of halolactones 3–5.

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The physical and spectral data of hydroxy lactone 6 are given below.

2.4.3.1

2.4.3.1 2-Hydroxy-5,5-dimethyl-9-oxabicyclo[4.3.0]nonan-8-one (6)

m.p. = 74–76 °C, ¹H NMR (300 MHz, CDCl₃): 0.95 and 1.00 (two s, 6H, (CH₃)₂C-5), 1.08 (m, 1H, one of CH₂-3), 1.67 (dd, J = 12.6 and 2.5 Hz, 1H, one of CH₂-4), 1.71 (ddd, J = 13.2, 12.6 and 2.5 Hz, 1H, one of CH₂-4), 1.84 (m, 1H, one of CH₂-3), 2.21 (dd, J = 15.9 and 8.4 Hz, 1H, one of CH₂-7), 2.35 (m, 1H, H-6), 2.50 (m, 1H, OH), 2.66 (dd, J = 15.9 and 12.4 Hz, 1H, one of CH₂-7), 4.12 (m, 1H, H-2), 4.58 (dd, J = 6.9 and 6.9 Hz, 1H, H-1); ¹³C NMR: 25.80 (C-3), 27.22 (C-4), 27.77 (C-9), 29.12 (C-10), 31.48 (C-5), 31.89 (C-7), 46.06 (C-6), 66.62 (C-2), 78.94 (C-1), 178.36 (C-8), IR (KBr, cm⁻¹): 3398 (sb), 2963 (s), 1762 (s), 1193 (s), 1028 (s). EI-MS m/z (%): 185 (81), 167 (40), 128 (46), 125 (63), 110 (33), 85 (100), 69 (43), 55 (35), 41 (51), 39 (72).

2.4.4

2.4.4 Synthesis of (1R, 2S, 6R, 13S)-2-(Camphor-10-sulfonyloxy)-5,5-dimethyl-9-oxabicyclo[4.3.0] nonan-8-one (7)

To the solution of hydroxy lactone 6 (0.08 mmol) and pyridine (0.18 mol) in dry diethyl ether (1 ml), (1S)-(+)-(10)-camphorsulfonyl-chloride (0.11 mmol) in diethyl ether (1 ml) was added dropwise. The mixture was stirred at room temperature for 40 min. After that time, the reaction mixture was extracted with diethyl ether (3 × 2 ml). The ethereal solution was washed with a 10% aqueous H₂SO₄, saturated solution NaHCO₃, and brine, dried over MgSO₄ and evaporated in vacuo. Crude product was purified on silica gel (hexane:acetone, 3:1) giving 0.065 mol (80%) of ester 7 (Scheme 3).

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Scheme 3 Synthesis of (1R, 2S, 6R, 13S, 16R)-2-(camphor-10-sulfonyloxy)-5,5-dimethyl-9-oxabicyclo[4.3.0]nonan-8-one (7).

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The physical and spectral data of compound 7 are given below.m.p. = 162–164 °C, ¹H NMR (300 MHz, CDCl₃): 0.94 (s, 3H, (one of (CH₃)₂C-19), 1.03 (s, 3H, (one of (CH₃)₂C-5), 1.06 (s, 3H, (one of (CH₃)₂C-5), 1.12 (s, 3H, (one of (CH₃)₂C-19), 1.08 (m, 1H, one of CH₂-4), 1.46 (m, 1H, one of CH₂-17), 1.76 (m, 1H, one of CH₂-3), 1.79 (m, 2H, one of CH₂-3 and one of CH₂-4), 1.97 (d, J = 18.4 Hz, 1H, H-16), 2.08 (m, 1H, one of CH₂-17), 2.14 (m, 2H, CH₂-18), 2.32 (dd, J = 16.6 and 8.4 Hz, 1H, one of CH₂-7), 2.36 (m, 1H, H-6), 2.44 (m, 2H, CH2–15), 2.59 (dd, J = 16.6 and 16.6 Hz, 1H, one of CH₂-7), 3.17 (d, J = 14.9 Hz, 1H, one of CH₂-12), 3.56 (d, J = 14.9 Hz, 1H, one of CH₂-12), 4.61 (dd, J = 6.8 and 4.7 Hz, 1H, H-1), 5.12 (m, 1H, H-2); ¹³C NMR: 19.63 (C-21), 19.78 (C-20), 25.34 (C-3), 25.73 (C-4), 26.89 (C-9), 27.62 (C-5), 27.88 (C-10), 28.75 (C-17), 31.11 (C-7), 31.35 (C-6), 42.63 (C-16), 43.05 (C-18), 45.78 (C-15), 48.08 (C-19), 48.69 (C-12), 58.02 (C-13), 75.68 (C-1), 76.88 (C-2), 176.46 (C-8), 213.72 (C-14), IR (KBr, cm⁻¹): 2960 (s), 2364 (m), 1777 (s), 1746 (s), 1347 (s), 1146 (s), 1053 (s), 833 (s).

Crystal data for 7: C₂₀H₃₀O₆S, Mr = 398.50, colorless block, crystal dimensions 0.96 × 0.35 × 0.25 mm, monoclinic, space group P2₁, a = 9.975(2), b = 17.966(3), c = 11.249(3) Å, β = 96.55(2)°, V = 2002.8(8) ų, Z = 4, Mo Kα radiation, λ = 0.71073 Å, μ = 0.20 mm⁻¹, Dx = 1.322 g cm⁻³, T = 170(2) K, R_int = 0.045, wR = 0.100, (for 6844 reflections with I > 2σ(I)) for 488 variables. Flack parameter equal to 0.03(7). CCDC No: 1046777.

3 Results

Three racemic halolactones 3–5 were synthesized from the already known γ,δ-unsaturated acid 2. The new chlorolactone 3 and bromolactone 4 were obtained with 84% and 87% yield respectively. The known iodolactone 5 was obtained with 80% yield.

All three lactones 3–5 were subjected to a screening biotransformation using nine fungal strains of local origin. These were: F. culmorum AM10, F. avenaceum AM11, F. oxysporum AM13, Fusarium tricinctum AM16, F. semitectum AM20, F. scirpi AM1199, F. solani AM203, S. racemosum AM105, and C. japonica AM472. These strains were chosen in order to test their ability to conduct hydrolytic dehalogenation of previously synthesized halolactones. The progress of all the screening transformations was monitored by means of standard techniques (TLC and GC). The results of this step are shown in Table 1.

Looking at the Table 1 it can be seen that all Fusarium strains (entries 1–7) and also S. racemosum (entry 8) were capable of transforming bromo-4 and iodolactone 5. When chlorolactone 3 was used as a substrate, only five Fusarium strains (entries 1–3 and 5–6) were capable of converting it. C. japonica strain (entry 9) was unable to transforming all three halolactones. The same single product was obtained during all of the tests. All three substrates were transformed into a product with good yield (over 57%) by four microorganisms: F. culmorum AM10, F. avenaceum AM11, F. oxysporum AM13, and F. scirpi AM199. In the case of iodolactone 5, S. racemosum AM105 and F. solani AM203 were also good biocatalysts. The aim of the screening biotransformation was only to select fungal strains capable of converting substrates to a product with at least 50% effectiveness. The screening procedure assessed only the conversion degree.

For all three halolactones (compounds 3–5) the control of the stability of the substrate was carried out by adding each one of them to a sterile medium containing 3 g glucose and 1 g peptobac in 100 ml of water and shaking for a week. The samples were taken analogously as in the case of the screening procedure. As a result of these experiments, it was found that only the substrate was always in the reaction mixture what means that the hydrolytic dehalogenation of lactones 3–5 does not occur without the presence of the microorganisms.

In the next step, i.e. the preparative scale biotransformations, fungal strains chosen during the screening procedure were used. The results of these transformations are presented in Fig. 1.

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Figure 1 Results (by GC) of preparative biotransformations of lactones 3–5.

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Two microorganisms, F. avenaceum AM11 and F. scirpi AM199, transformed all three substrates with comparable efficacy, and the degree of conversion was approximately 78% for the first species and 85% for the second one. Larger differences were observed for F. culmorum AM10 and F. oxysporum AM13. The first microorganism transformed chlorolactone 3 with the best yield (88%), while the best substrate for the second microorganism was bromolactone 4 (the yield was 82%). Iodolactone 5 was the least effectively transformed by the tested fungal strains. The above data clearly indicate the substrate specificity of the microorganisms that were used for the biotransformation. Table 2 presents the isolated yields of hydroxy lactone 6 obtained during all preparative biotransformation.

One of the aims of our experiments was obtaining a product with high optical purity, and therefore, the enantiomeric excess and optical rotation were checked for every hydroxy lactone received during the preparative biotransformations of chloro- 3, bromo- 4 or iodolactone 5. The enantiomeric excess was determined using GC with the chiral column (Beta Dex, 30 m × 0.25 mm × 0.25 μm). The results of these analyses are shown in Table 3.

4 Discussion

The substrates for the transformation were three racemic halo-γ-lactones (3–5) with a gem-dimethylcyclohexane ring in their structure. The structures of chloro- 3 and bromo-γ-lactone 4 were determined on the basis of their spectral data. The structure of the already known iodo-γ-lactone 5 was confirmed by its ¹H NMR and IR spectra. The absorption bands at 1782, 1791 and 1789 cm⁻¹ on the IR spectra for lactones 3–5, respectively, indicated that the lactone ring present in their molecules was the γ-lactone ring. As we know from previous reports (Gładkowski et al., 2011), iodolactone 5 is in the chair conformation with the trans-diequatorial orientation of the iodine atom and C–O bond of the lactone ring. The similarity between the spectral data of iodolactone 5 and new chloro-3 and bromolactone 5 indicated that their structures were identical. The analysis of ¹H NMR spectra of lactones 3, 4 and 5 showed that the coupling constants of H-1 with H-2 (J = 9.0, 9.3 and 9.6 Hz, respectively), H-1 with H-6 (J = 6.9, 6.6 and 4.4 Hz, respectively), H-2 with H-3 axial (J = 14.1, 14.4 and 15.0 Hz, respectively) and H-2 with H-3 equatorial (J = 5.1, 5.1 and 5.4 Hz, respectively) were similar for all three compounds. This indicates that for lactones 4 and 5 the cyclohexane ring was in slightly twisted chair conformation with H-1 and H-2 protons that were in trans-diaxial positions and H-6 proton was in equatorial one. Lactone ring and H-2 proton were trans-oriented in relation to the axial methyl group at C-5, just as in the case of lactone 3 (Fig. 2).

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Figure 2 Structure of chlorolactone 3.

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The structure of the product obtained during all the biotransformations was established on the basis of its spectral data. The IR spectra showed that the γ-lactone ring was retained in the product during the biotransformation (absorption bands at 1762 cm⁻¹). The presence of a hydroxyl group in the molecule was suggested by a strong and broad band at 3398 cm⁻¹. Earlier analysis of halolactones 3, 4 and 5 structure proved that H-1 and H-2 protons were in trans-diaxial positions, and the halogen atom was in equatorial position and was trans-oriented in relation to the C–O bond and the axial methyl group at C-5.

The analysis of NMR spectra of microbiologically obtained hydroxy lactone 6 and halolactones 3–5 allowed us to determine the differences between these molecules. The signal from H-1 proton was observed between 4.54 ppm (for halolactone 3) and 4.78 ppm (for halolactone 5). The signal from the same proton in hydroxy lactone 6 molecule was shifted to 4.46 ppm, indicating a substantial change in this proton’s chemical surroundings. The signal of H-2 proton was located between 3.75 ppm (for 3) and 3.86 ppm (for 5). Additionally, for halolactones 3–5 one big coupling constant (J = 14.1, 14.4 or 15.0) suggested the axial position of H-2 proton. The signal from H-2 proton for hydroxy lactone 6 moved into 4.12 ppm. Moreover, in this case we could observe a multiplet with a small coupling constant derived from H-2. These observations indicated that H-2 proton in hydroxy lactone 6 was in equatorial position and that OH group occupied an axial position in the cyclohexane ring, and that OH group was also located cis to the C–O bond of the lactone ring (Fig. 3).

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Figure 3 Structure of hydroxy lactone 6.

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These data suggested that the halogen atom present in the substrate molecule was changed into a hydroxy group in the course of hydrolytic dehalogenation similar to the S_N2 mechanism. Such a mechanism was observed earlier when similar halolactones were subjected to hydrolytic dehalogenation by the same microorganisms (Grabarczyk and Białońska, 2010; Grabarczyk, 2012), and also for enzymes from the Absidia cylindrospora culture catalyzing hydrolytic dehalogenation in iodolactones (Gładkowski et al., 2011).

In every case both substrates (halolactones) and products (hydroxy lactones) with the cyclohexane ring existed in chair conformation. During biotransformation we observed changing of conformation of cyclohexane ring (for lactones with trimethyl- and 4.4-dimethylcyclohexane ring) or remaining the cyclohexane ring in the same conformation (for lactones with 4.6-dimethyl-, 5.5-dmethyl- and 4-methylcyclohexane ring).

In case of halolactones with 4.4.6-trimethylcyclohexane ring (Grabarczyk and Białońska, 2010) lactone ring was cis oriented in relation to the axial methyl group. H-1 and H-2 protons were in trans diaxial positions. In hydroxy lactone, lactone ring changed its orientation into trans in relation to the axial methyl group. H-2 proton remains in axial positions, but H-1 changed its position into equatorial.

The same situation was observed for lactones with 4.4-dimethylcyclohexane ring (Grabarczyk et al., 2013).

Analyzing structure of halolactones with 4.6-dimethylcyclohexane ring (Grabarczyk, 2012) and iodolactone with 4-methylcyclohexane ring (Gładkowski et al., 2011) we can also see many similarities. In all halo- and hydroxy lactones, lactone ring was cis oriented in relation to the equatorial methyl group. H-1 protons were always in equatorial position and H-2 proton changed its orientation from equatorial in substrates into axial in products.

During biotransformation of iodolactone with 5.5-dimethylcyclohexane ring by Absidia cylindrospora (Gładkowski et al., 2011) into hydroxy lactone in both compounds lactone ring and one of methyl groups at C-5 were in trans diaxial positions. H-1 and H-2 protons were in axial positions in iodolactone. In hydroxy lactone H-1 proton was in axial position and H-2 proton changed its orientation into equatorial. The same situation was observed when three halolactones with 5.5-dimethylcyclohexane were transformed by Fusarium species.

All hydroxy lactones obtained during the preparative biotransformations were checked for their optical purity. As can be seen in Table 3, when chloro- or bromolactone was used as a substrate the enantiomeric excess was low or moderate, and it did not exceed 30% and 41% for the product from chlorolactone 3 and bromolactone 4, respectively. When iodolactone 5 was used as a substrate the situation was different. Two microorganisms, F. culmorum AM10 and F. scirpi AM199, showed high enantiospecificity. In these cases hydroxy lactone 6 was obtained with high enantiomeric excess (75% and 91.6%, respectively). Other microorganisms used for the biotransformations of iodolactone 5 exhibited lower enantiospecificity (up to 43%). These data suggest high enantiospecificity of the chosen fungal strains in relation to iodolactone 5. Most of the microorganisms preferred the formation of (+) hydroxy lactone isomer, and (−) isomer of hydroxy lactone 6 was formed only during biotransformation of lactones 3 and 4 by F. oxysporum AM13, and lactone 5 by F. solani AM203.

As it was mentioned above hydroxy lactone 6 obtained during biotransformation of iodolactone 5 using F. scirpi AM199 was characterized by high enantiomeric excess equal to 91.6%. Lactone 6 was used to prepare derivative with (1S)-(+)-10-camphorsulfonyl chloride named 7. X-ray diffraction analysis of derivative 7 made it possible to determine the absolute configuration of stereogenic centers present in the molecule of lactone 6. Absolute configuration of stereogenic centers was as follows: C-1 (R), C-2 (S), C-6 (R), C-13 (S), C-16 (R) (see Fig. 4).

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Figure 4 Structure of (1R, 2S, 6R, 13S, 16R)-2-(camphor-10-sulfonyloxy)-5,5-dimethyl-9-oxabicyclo[4.3.0]nonan-8-one (7).

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Based on the crystallographic structure it was possible to assign absolute configuration 1R, 2S, 6R to (+) enantiomer of hydroxy lactone 6. Therefore, it can be concluded that most of the tested microorganisms preferred formation of this hydroxy lactone with predominant 1R, 2S, 6R isomer. Hydroxy lactone 6 with a small predominance of second isomer 1R, 2R, 6R was obtained during biotransformation chloro- 3 or bromolactone 4 by F. oxysporum AM13 and iodolactone 5 by F. solani AM203.

Hydroxy lactone 6 was examined for its biological activity against bacteria, yeasts and filamentous fungi. The results of the bioassay tests are given in Table 4.

Results presented above showed that hydroxy lactone 6 inhibited the growth of six of fourteen tested strains. In the case of bacteria strains this compound showed growth inhibition against two strains: M. flavus (entry 1) and B. cereus (entry 2). Yeast was more vulnerable, from three tested strains and two S. cerevisiae (entry 7) and S. pombe (entry 9) were inhibited by this lactone. In the case of fungal strains hydroxy lactone 6 inhibited growth of only Penicillium sp. (entry 13). The obtained results clearly showed that this compound had the highest antimicrobial activity against S. pombe (entry 9), inhibiting growth of 69% of the culture. The results of the bioassay tests are illustrated on Figs. 5–10.

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Figure 5 Effect of hydroxy lactone 6 on the growth of M. flavus.

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Figure 6 Effect of hydroxy lactone 6 on the growth of B. cereus.

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Figure 7 Effect of hydroxy lactone 6 on the growth of S. cerevisiae.

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Figure 8 Effect of hydroxy lactone 6 on the growth of S. pombe.

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Figure 9 Effect of hydroxy lactone 6 on the growth of R. rubra.

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Figure 10 Effect of hydroxy lactone 6 on the growth of Penicillium sp.

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5 Conclusions

All fungal strains chosen for the biotransformations of chloro-, bromo- and iodolactone transformed them into the same hydroxy lactone. Those microorganisms which converted halolactones into hydroxy lactone showed very high regioselectivity. The OH group was always introduced at C-2 in the axial position due to S_N2 mechanism. Two lactones: chloro- 3 and bromolactone 4, were transformed on the preparative scale by four fungal strains with a good yield (from 69% to 88%). The third substrate, iodolactone 5, was converted with a lower yield (from 58% to 81%) by six fungal strains. Most of the microorganisms formed the (+) isomer of hydroxy lactone, and only two of them were able to create the (−) isomer. The best substrate-microorganism combination considering enantioselectivity was found to be iodolactone 5 and F. scirpi. This microorganism converted iodolactone into hydroxy lactone 6 with a very high enantiomeric excess (91.6%). Hydroxy lactone 6 obtained with ee = 91.6% was used to prepare crystalline derivative using chiral camphorsulfonic chloride. X-ray diffraction analysis of derivative 7 led to determine the absolute configuration of stereogenic centers present in the molecule of lactone 6 as 1R, 2S, 6R. In addition, the biological tests carried out led to the conclusion that hydroxy lactone 6 inhibited the growth of certain microorganism strains.