Biotransformation of Bicyclic Halolactones with a Methyl Group in the Cyclohexane Ring into Hydroxylactones and Their Biological Activity

The aim of this study was the chemical synthesis of a series of halo- and unsaturated lactones, as well as their microbial transformation products. Finally some of their biological activities were assessed. Three bicyclic halolactones with a methyl group in the cyclohexane ring were obtained from the corresponding γ,δ-unsaturated ester during a two-step synthesis. These lactones were subjected to screening biotransformation using twenty two fungal strains. These strains were tested on their ability to transform halolactones into new hydroxylactones. Among the six strains able to catalyze hydrolytic dehalogenation, only two (Fusarium equiseti, AM22 and Yarrowia lipolytica, AM71) gave a product in a high yield. Moreover, one strain (Penicillium wermiculatum, AM30) introduced the hydroxy group on the cyclohexane ring without removing the halogen atom. The biological activity of five of the obtained lactones was tested. Some of these compounds exhibited growth inhibition against bacteria, yeasts and fungi and deterrent activity against peach-potato aphid.


Introduction
Due to their properties, chloro-organic compounds are used in many areas. They are very often applied as antifungal [1,2], antibacterial [3], antifidant [4,5], cytotoxic, and anticancer agents [6]. They are also used as organic solvents, refrigerants and media, dielectrics, pesticides, monomers for the production of plastic materials, and intermediates for organic synthesis [7]. Usually, these compounds are obtained by organic synthesis although they are increasingly being obtained from natural substances too. The compounds are obtained mainly from marine organisms such as red algae, corals or bacteria [8][9][10]. Longifoliosides A and B, which were isolated from Veronica longifolia, exhibit radical-scavenging activity against nitric oxide, superoxide, and 2,2-diphenyl-1-picrylhydrazyl radicals [11]. Halogened compounds could also be obtained by using the biotransformation method. Four antimicrobial halogenated compounds, bromomethyl-chlamydosporols A, B, chlamydosporol and fusarielin A, were isolated from a culture of Fusarium tricintum. These halogenated chlamydosporol analogues were obtained as products of the fermentation of a marine fungus. The transformation process was inducted by the addition of CaBr 2 [12]. All the obtained compounds exhibited low antibacterial activity against Staphylococcus aureus, methicillin-resistant S. aureus, and multidrug-resistant S. aureus.
Isolated from the Australian ascidian Aplidium caelestis brominated natural products were tested against three mammalian cell lines (MCF-7, NFF, and MM96L), but they showed only minor cytotoxicity [13]. The bromotyrosine alkaloid psammaplysin was isolated from the Australian marine sponge Hyattella sp, along with psammaplysin F. These compounds were tested against two different strains of the parasite Plasmodium falciparum (Dd2 and 3D7). The tests showed that one of them, psammaplysin G, inhibited the chloroquine-resistant (Dd2) strain of P. falciparum by 98% [14].
Despite the interesting biological properties of halogenated organic compounds, in the 60s it was found that they may accumulate in soil or water without being broken down. At high concentrations, these compounds may have a negative impact on living organisms, endangering their health or life. For decades, researchers conducted experiments on the effects of waste on living organisms. It has been proven that halogenated organic compounds can cause endocrine system disorders in humans [15]. Therefore, effective methods for disposing of organic halo compounds have been the subject of research since the middle of the last century. One of the most popular methods is to transform them using filamentous fungi utilizing their ability for hydrolytic dehalogenation. Our team has been conducting studies dealing with halolactone transformations since 1998 [16]. In these studies we have obtained new compounds with interesting biological properties.

Results and Discussion
Substrates for biotransformation halolactones 3-5 were obtained during the two-step synthesis from the unsaturated ester 1. Firstly, the ester was hydrolyzed to the γ,δ-unsaturated acid 2. In the second step, this acid was transformed into the new chloro-3, bromo-4, and known iodolactone 5. Iodolactone 5 further reacted with DBU giving the unsaturated lactone 6 (Scheme 1).
Molecules 2016, 21,1453 2 of 13 chlamydosporols A, B, chlamydosporol and fusarielin A, were isolated from a culture of Fusarium tricintum. These halogenated chlamydosporol analogues were obtained as products of the fermentation of a marine fungus. The transformation process was inducted by the addition of CaBr 2 [12]. All the obtained compounds exhibited low antibacterial activity against Staphylococcus aureus, methicillin-resistant S. aureus, and multidrug-resistant S. aureus. Isolated from the Australian ascidian Aplidium caelestis brominated natural products were tested against three mammalian cell lines (MCF-7, NFF, and MM96L), but they showed only minor cytotoxicity [13]. The bromotyrosine alkaloid psammaplysin was isolated from the Australian marine sponge Hyattella sp, along with psammaplysin F. These compounds were tested against two different strains of the parasite Plasmodium falciparum (Dd2 and 3D7). The tests showed that one of them, psammaplysin G, inhibited the chloroquine-resistant (Dd2) strain of P. falciparum by 98% [14].
Despite the interesting biological properties of halogenated organic compounds, in the 60s it was found that they may accumulate in soil or water without being broken down. At high concentrations, these compounds may have a negative impact on living organisms, endangering their health or life. For decades, researchers conducted experiments on the effects of waste on living organi sms. It has been proven that halogenated organic compounds can cause endocrine system disorders in humans [15]. Therefore, effective methods for disposing of organic halo compounds have been the subject of research since the middle of the last century. One of the most popular methods is to transform them using filamentous fungi utilizing their ability for hydrolytic dehalogenation. Our team has been conducting studies dealing with halolactone transformation s since 1998 [16]. In these studies we have obtained new compounds with interesting biological properties.

Results and Discussion
Substrates for biotransformation halolactones 3-5 were obtained during the two-step synthesis from the unsaturated ester 1. Firstly, the ester was hydrolyzed to the γ,δ-unsaturated acid 2. In the second step, this acid was transformed into the new chloro-3, bromo-4, and known iodolactone 5. The structures of these compounds were established on the basis of their spectral data ( 1 H-NMR, 13 C-NMR, COSY, HMQC, and IR) and confirmed by HRMS analysis. In the IR spectra the absorption bands at 1780 (for 3), 1772 (for 4), 1776 (for 5), and 1776 (for 6) cm −1 confirmed the presence of the γ-lactone ring in the halolactone molecules and the unsaturated lactone. Analysis of the 1 H-NMR spectra of halolactones allowed for the determination of the orientations of protons H-1 and H-2. The signal coming from proton H-1 appeared as a narrow singlet in the case of chloralactone 3 and for 4 Scheme 1. Synthesis of lactones 3-6.
The structures of these compounds were established on the basis of their spectral data ( 1 H-NMR, 13 C-NMR, COSY, HMQC, and IR) and confirmed by HRMS analysis. In the IR spectra the absorption bands at 1780 (for 3), 1772 (for 4), 1776 (for 5), and 1776 (for 6) cm −1 confirmed the presence of the γ-lactone ring in the halolactone molecules and the unsaturated lactone. Analysis of the 1 H-NMR spectra of halolactones allowed for the determination of the orientations of protons H-1 and H-2. The signal coming from proton H-1 appeared as a narrow singlet in the case of chloralactone 3 and for 4 and 5 it was a doublet with the small coupling constant (5.2 Hz for 4 and 6.1 Hz for 5). Such shapes for these signals suggest their equatorial orientation and therefore an axial orientation of the C-O bond  Our previous experiences indicated that in most cases halolactones subjected to biotransformation by filamentous fungi strains were converted into hydroxylactones. This reactionhydrolytic dehalogenation-always proceeded according to a mechanism similar to a SN2 substitution. Independently, the structure of the substrate OH group was always introduced in an equatorial position. Taking this into account, it was interesting to see if in this case the fungal strains would be able to conduct the reaction of hydrolytic dehalogenation, giving new hydroxylactone 7 (Scheme 2). The fourth substrate-unsaturated lactone 6 was also tested for its ability to give any product. Screening biotransformation was conducted on twenty two fungal strains. The progress of all of these transformations was monitored using standard techniques (TLC and GC). During the present research, one of the chosen microorganisms (Penicillium vermiculatum AM30) generated a different product than the expected one. Taking this observation into consideration, the effects of the screening biotransformation are presented in Tables 1 and 2.
As it can be seen in the tables, the biotransformed halocompounds were transformed into hydroxycompounds 7-10 by some microorganisms. From the eight Fusarium strains, only Fusarium equiseti AM22 (Table 1, entry 7) proved to be a good catalyst , giving a product in high yield when bromolactone 4 and iodolactone 5 were used as substrates. The second strain which was able to transform these halolactones into hydroxylactone 7 in good yield was Yarrowia lipolytica AM71 (Table 1, entry 18). Some of the other strains (Table 1, entries 8,9,11,12) were able to create only a small percentage of product. Our previous experiences indicated that in most cases halolactones subjected to biotransformation by filamentous fungi strains were converted into hydroxylactones. This reaction-hydrolytic dehalogenation-always proceeded according to a mechanism similar to a S N 2 substitution. Independently, the structure of the substrate OH group was always introduced in an equatorial position. Taking this into account, it was interesting to see if in this case the fungal strains would be able to conduct the reaction of hydrolytic dehalogenation, giving new hydroxylactone 7 (Scheme 2). The fourth substrate-unsaturated lactone 6 was also tested for its ability to give any product.  Our previous experiences indicated that in most cases halolactones subjected to biotransformation by filamentous fungi strains were converted into hydroxylactones. This reactionhydrolytic dehalogenation-always proceeded according to a mechanism similar to a SN2 substitution. Independently, the structure of the substrate OH group was always introduced in an equatorial position. Taking this into account, it was interesting to see if in this case the fungal strains would be able to conduct the reaction of hydrolytic dehalogenation, giving new hydroxylactone 7 (Scheme 2). The fourth substrate-unsaturated lactone 6 was also tested for its ability to give any product. Screening biotransformation was conducted on twenty two fungal strains. The progress of all of these transformations was monitored using standard techniques (TLC and GC). During the present research, one of the chosen microorganisms (Penicillium vermiculatum AM30) generated a different product than the expected one. Taking this observation into consideration, the effects of the screening biotransformation are presented in Tables 1 and 2.
As it can be seen in the tables, the biotransformed halocompounds were transformed into hydroxycompounds 7-10 by some microorganisms. From the eight Fusarium strains, only Fusarium equiseti AM22 (Table 1, entry 7) proved to be a good catalyst , giving a product in high yield when bromolactone 4 and iodolactone 5 were used as substrates. The second strain which was able to transform these halolactones into hydroxylactone 7 in good yield was Yarrowia lipolytica AM71 (Table 1, entry 18). Some of the other strains (Table 1, entries 8,9,11,12) were able to create only a small percentage of product.

Scheme 2. The expected biotransformation product 7 of halolactones 3-5.
Screening biotransformation was conducted on twenty two fungal strains. The progress of all of these transformations was monitored using standard techniques (TLC and GC). During the present research, one of the chosen microorganisms (Penicillium vermiculatum AM30) generated a different product than the expected one. Taking this observation into consideration, the effects of the screening biotransformation are presented in Tables 1 and 2. As it can be seen in the tables, the biotransformed halocompounds were transformed into hydroxycompounds 7-10 by some microorganisms. From the eight Fusarium strains, only Fusarium equiseti AM22 (Table 1, entry 7) proved to be a good catalyst, giving a product in high yield when bromolactone 4 and iodolactone 5 were used as substrates. The second strain which was able to transform these halolactones into hydroxylactone 7 in good yield was Yarrowia lipolytica AM71 (Table 1, entry 18). Some of the other strains (Table 1, entries 8,9,11,12) were able to create only a small percentage of product.  It is worth noticing that Penicillium camembertii AM83 transformed only one of the halolactones. During the screening biotransformation it turned out that Penicillium vermiculatum AM30 strain (Table 2, entry 1) was able to produce different products than the other microorganisms. This was very surprising because till now we had not observed any products when this strain was used for biotransformation. It turned out that P. vermiculatum produced new hydroxyhalolactones 8-10. These lactones were obtained as a product of hydroxylation on carbon C-5 without the removal of the halogen atom from carbon C-2 (Scheme 3).   It is worth noticing that Penicillium camembertii AM83 transformed only one of the halolactones. During the screening biotransformation it turned out that Penicillium vermiculatum AM30 strain (Table 2, entry 1) was able to produce different products than the other microorganisms. This was very surprising because till now we had not observed any products when this strain was used for biotransformation. It turned out that P. vermiculatum produced new hydroxyhalolactones 8-10. These lactones were obtained as a product of hydroxylation on carbon C-5 without the removal of the halogen atom from carbon C-2 (Scheme 3). As mentioned earlier, unsaturated lactone 6 was also checked for its ability to give any product. Unfortunately none of the tested microorganisms were able to convert this substrate. As mentioned earlier, unsaturated lactone 6 was also checked for its ability to give any product. Unfortunately none of the tested microorganisms were able to convert this substrate.
Since the aim of the present study was to obtain and characterize the structures of the biotransformation productsf-hydroxylactone 7 and also hydroxy-halolactones 8-10-preparative scale biotransformations were performed in the next step. The best strains selected during the screening biotransformation were used for this purpose. The results of this are shown in Figure 2. Since the aim of the present study was to obtain and characterize the structures of the biotransformation productsf-hydroxylactone 7 and also hydroxy-halolactones 8-10-preparative scale biotransformations were performed in the next step. The best strains selected during the screening biotransformation were used for this purpose. The results of this are shown in Figure 2. Two strains-F. equiseti and Y. lipolytica-were able to convert lactones 4, 5 into the same product-hydroxylactone 7. The better substrate was bromolactone 4, which was transformed into a product with a very good degree of conversion (87% and 88%). In the case of iodolactone 5 the product was created with a lower conversion (55%-58%). The third strain P. vermiculatum led to hydroxyhalolactones with low efficiency, due to a narrow degree of conversion of the substrates (between 31% and 38%). For this reason it was only possible to check their chemical properties.
Analysis of 1 H-NMR spectra of hydroxylactone 7 proved that the chair conformation of the cyclohexane ring was retained. The narrow signal coming from proton H-1 indicated its equatorial position. It means that the C-O bond of the lactone ring is located in an axial position, as in the substrates. The wide multiplet coming from proton H-2 suggested its axial orientation and consequently an equatorial position of the hydroxy group ( Figure 3). The hydrolytic dehalogenation reaction occurred similarly to a SN2 mechanism in every case. Moreover the hydroxy group was always introduced in the equatorial position in the cyclohexane ring. When a halogen atom occupied an equatorial position the simultaneous change of the conformation of the cyclohexane ring was observ ed during the reaction of hydrolytic dehalogenation [16,17]. When the halogen atoms occupied an axial position and the equatorial position was free, conformational change of the cyclohexane ring was not observed [18]. Because the halogen atom was in an axial position as described in the situation here, the conformation of the cyclohexane ring had not changed. Two strains-F. equiseti and Y. lipolytica-were able to convert lactones 4, 5 into the same product-hydroxylactone 7. The better substrate was bromolactone 4, which was transformed into a product with a very good degree of conversion (87% and 88%). In the case of iodolactone 5 the product was created with a lower conversion (55%-58%). The third strain P. vermiculatum led to hydroxyhalolactones with low efficiency, due to a narrow degree of conversion of the substrates (between 31% and 38%). For this reason it was only possible to check their chemical properties.
Analysis of 1 H-NMR spectra of hydroxylactone 7 proved that the chair conformation of the cyclohexane ring was retained. The narrow signal coming from proton H-1 indicated its equatorial position. It means that the C-O bond of the lactone ring is located in an axial position, as in the substrates. The wide multiplet coming from proton H-2 suggested its axial orientation and consequently an equatorial position of the hydroxy group ( Figure 3).
Since the aim of the present study was to obtain and characterize the structures of the biotransformation productsf-hydroxylactone 7 and also hydroxy-halolactones 8-10-preparative scale biotransformations were performed in the next step. The best strains selected during the screening biotransformation were used for this purpose. The results of this are shown in Figure 2. Two strains-F. equiseti and Y. lipolytica-were able to convert lactones 4, 5 into the same product-hydroxylactone 7. The better substrate was bromolactone 4, which was transformed into a product with a very good degree of conversion (87% and 88%). In the case of iodolactone 5 the product was created with a lower conversion (55%-58%). The third strain P. vermiculatum led to hydroxyhalolactones with low efficiency, due to a narrow degree of conversion of the substrates (between 31% and 38%). For this reason it was only possible to check their chemical properties.
Analysis of 1 H-NMR spectra of hydroxylactone 7 proved that the chair conformation of the cyclohexane ring was retained. The narrow signal coming from proton H-1 indicated its equatorial position. It means that the C-O bond of the lactone ring is located in an axial position, as in the substrates. The wide multiplet coming from proton H-2 suggested its axial orientation and consequently an equatorial position of the hydroxy group ( Figure 3). The hydrolytic dehalogenation reaction occurred similarly to a SN2 mechanism in every case. Moreover the hydroxy group was always introduced in the equatorial position in the cyclohexane ring. When a halogen atom occupied an equatorial position the simultaneous change of the conformation of the cyclohexane ring was observ ed during the reaction of hydrolytic dehalogenation [16,17]. When the halogen atoms occupied an axial position and the equatorial position was free, conformational change of the cyclohexane ring was not observed [18]. Because the halogen atom was in an axial position as described in the situation here, the conformation of the cyclohexane ring had not changed. The hydrolytic dehalogenation reaction occurred similarly to a S N 2 mechanism in every case. Moreover the hydroxy group was always introduced in the equatorial position in the cyclohexane ring. When a halogen atom occupied an equatorial position the simultaneous change of the conformation of the cyclohexane ring was observed during the reaction of hydrolytic dehalogenation [16,17]. When the halogen atoms occupied an axial position and the equatorial position was free, conformational change of the cyclohexane ring was not observed [18]. Because the halogen atom was in an axial position as described in the situation here, the conformation of the cyclohexane ring had not changed.
Analysis of 1 H-NMR spectra of the hydroxyhalolactones 8-10 proved that the halolactone ring structure was retained. Such a conclusion was evidenced by the narrow doublet coming from proton H-1 (J = 3.2 Hz for 8, J = 3.5 Hz for 9, and J = 4.0 Hz for 10) and the wide multiplet coming from proton H-2, indicating their equatorial and axial position, respectively. Also the wide doublet of doublets (J = 10.7 Hz for 8, J = 10.3 Hz for 9, and J = 9.1 Hz for 10) coming from the hydroxy group at carbon C-5 suggested its axial orientation and an equatorial position of the hydroxy group (Figure 4).  In the next step, the isolated yield, enantiospecificity, and optical purity of the hydroxylactone 7 and the hydroxyhalolactones 8-10 obtained during all the preparative biotransformations were determined. The results of shown in Table 3. Optical rotation was me asure d for 589 nm and te mpe rature 20 °C .
In all cases the (-)-isomer of hydroxylactone 7 was formed preferentially. When bromolactone 4 was used as a substrate, the enantiomeric excesses did not exceed 11%. When hydroxylactone 7 was created from iodolactone 5 these values were much better, amounting to 52.9% and 27%. In the case of hydroxyhalolactones 8-10 the (+)-isomer was preferentially created, but the enantiomeric excesses did not exceed 7.6%.
Another aim in this research work was the verification of the biological properties of the obtained compounds. The obtained synthetic halolactones 3-6, as well as the obtained microbiologically formed hydroxylactone 7, were examined for their ability to inhibit the growth of some bacteria, yeast, and fungal strains. Statistical analysis was performed for the results of microbiological tests. Calculations were made for each of the strains separately. Control and further compounds were treated as test variability. The underlying assumption was the null hypothesis that the mean for the control and individual substances do not differ. The hypothesis was verified by a F-Senedecora test at the significance level of p = 0.01. It has been shown that for each strain averages are significantly different. Then Dunnett 's test was checked, in which means are significantly different from the average for the control. The results of this test are presented in Table 4. In the next step, the isolated yield, enantiospecificity, and optical purity of the hydroxylactone 7 and the hydroxyhalolactones 8-10 obtained during all the preparative biotransformations were determined. The results of shown in Table 3. In all cases the (-)-isomer of hydroxylactone 7 was formed preferentially. When bromolactone 4 was used as a substrate, the enantiomeric excesses did not exceed 11%. When hydroxylactone 7 was created from iodolactone 5 these values were much better, amounting to 52.9% and 27%. In the case of hydroxyhalolactones 8-10 the (+)-isomer was preferentially created, but the enantiomeric excesses did not exceed 7.6%.
Another aim in this research work was the verification of the biological properties of the obtained compounds. The obtained synthetic halolactones 3-6, as well as the obtained microbiologically formed hydroxylactone 7, were examined for their ability to inhibit the growth of some bacteria, yeast, and fungal strains. Statistical analysis was performed for the results of microbiological tests. Calculations were made for each of the strains separately. Control and further compounds were treated as test variability. The underlying assumption was the null hypothesis that the mean for the control and individual substances do not differ. The hypothesis was verified by a F-Senedecora test at the significance level of p = 0.01. It has been shown that for each strain averages are significantly different. Then Dunnett's test was checked, in which means are significantly different from the average for the control. The results of this test are presented in Table 4. Table 4. Antimicrobial activity of lactones 3-7. Statistics on a completely randomized design were determined using the one-way analysis of variance (ANOVA) procedure at a level of significance set at p = 0.010. A comparison of average growth OD microorganisms relative to the control by Dunnett's test. Underlined numbers mean the average was significantly different from control. The group of microorganisms exhibiting the greatest growth-inhibition by the test compounds were bacteria. In the case of the Escherichia coli strain, all lactones caused inhibition of the bacterial growth. This is a very interesting result, because so far the compounds which tested by us were relatively inactive to this strain. Lactones 3, 4, and 6 completely inhibited the growth of the Staphylococcus aureus strain. Compounds 5 and 7 also affected the growth of this strain of bacteria causing considerable extension phase adaptive (20-36 h) and a slight increase in the biomass expressed as ∆OD in the range of 0.37-0.62. It is worth noting that in the culture of the Pseudomonas fluorescens strain, conducted in the presence of 5, diauxic growth was observed. The same effect was observed earlier for the E. coli strain and the C. albicans strain [19]. This phenomenon was also reported by other authors [20,21].
In the case of the C. albicans strain, lactone 7 stimulated the growth of these yeasts resulting in the growth of biomass, ∆OD was equal to 1.99, whereas the control yeast culture s ∆OD value was 1.14. A similar effect of the stimulation of growth occurred in the case of a strain of Fusarium avenaceum. In this case compounds 6 and 7 produced an increase in the biomass of 0.62-0.69 compared to the control culture. Lactone 5 inhibited growth in the Fusarium avenaceum strain. Lactone 4 completely inhibited the growth of the Fusarium avenaceum. Other biological tests were conducted to determine the deterrent activity of lactones 3-7 against peach-potato aphid Myzus persicae. The results of this experiment are presented in Table 5. Table 5. Deterrent activity of lactones 3-7. Numbers for C (= control) and T (= treated) represent the mean number (±SE) of aphids that settled on the control or the treated leaf (choice-test) 1 h, 2 h, and 24 h after exposure. p-significance level (Student t-test)-significant differences (p < 0.05) between the number of aphids settled on either leaf are underlined. Among the tested lactones, bromolactone 4 and iodolactone 5 showed high and persistent deterrent properties. The application of these compounds caused the avoidance of plants by aphids as soon as 1 h after exposure and the effect persisted at least until the 24th hour of the experiment. However, the level of activity of 5 tended to subside over the course of time. The other lactones, chlorolactone 3 and the unsaturated lactone 6 also showed deterrent capacity. The relatively strong deterrent effect of 6 ceased before the 24th hour after application. Hydroxylactone 7 initially expressed a tendency to attract aphids, but this tendency reversed over the course of time (Table 5, Figure 5). The changes in the deterrence potency of various low molecular terpenoid lactones are typical for this group of compounds. The switch from attractant to deterrent properties or otherwise was found in the case of piperitone-derived saturated lactones, halolactones and hydroxylactones [22,23]. Literature data [24] suggest that the high enantiomeric excess affected the deterrent activity of the aphids. The halolactones 3-5 presented here were the racemic mixtures. In the case of hydroxylactone 7, ee values were not very high. Because of that, it is impossible to determine any influence of the enantiomeric excesses of these lactones on the deterrent activity of the aphids.

Synthesis of Substrates
Unsaturated ester 1 was hydrolyzed into the known [25] γ,δ-unsaturated acid 2 according to the known method [26]. This acid was then converted into the new chloro-3, bromo-4, and known [25] iodolactone 5 according to the procedure described earlier [27]. Iodolactone 5 was then used to obtain unsaturated lactone 6. Here we present the synthesis and spectral data of compounds 3-6, more Data can be found in the Supplementary Materials. This data will be useful in studying the relationship between the structures of the substrates and their interaction with microorganisms.

Screening Procedure
The fungal strains were cultivated in 300 mL Erlenmayer flasks, containing 100 mL of the medium consisting of glucose (3 g) and peptobac (1 g). After three days when the culture was grown, 10 mg of the substrate dissolved in 1 mL of acetone was added to each flask. The incubation of the shaken cultures with the substrate was continued for 7 days. After 3, 5, and 7 days of incubation, the medium contained the unreacted substrate, and the product and mycelium were extracted with dichloromethane (15 mL) and analyzed by GC (DB-17 column).

Preparative Biotransformation
Reactions were conducted in ten 300 mL Erlenmayer flasks containing 3-day cultures of fungal or yeast strain (prepared similarly as described above). In this case, 100 mg of substrate was dissolved in 10 mL acetone and added to ten flasks. After 7 days the post-reaction mixture was extracted with dichloromethane (3 × 40 mL). The combined organic fractions were dried over anhydrous magnesium sulphate and the solvent was evaporated in vacuo. Pure product was separated from the unreacted substrate and the metabolites of the fungi or yeast by using column chromatography (silica gel, hexane: acetone 3:1). As an effect of these reactions, four products were obtained, more Data can be found in the Supplementary Materials.

Conflicts of Interest:
The authors declare no conflict of interest.