3.3.2. Preparative Biotransformations and Chemical Characterization of Compounds **23a**, **25**, **28**, **35** and **36**

For the main part of the strains tested, the GC-MS analysis of the crude biotransformation mixtures indicated the presence of different compounds whose chemical structures could not be assigned only on the basis of our reference standards. The unidentified peaks taking account of less than 5% of the overall percentage of the compounds obtained by biotransformation were collectively indicated as 'not determined'. Otherwise, compounds **23a**, **25**, **28**, **35** and **36** were isolated from the fermentation broths by extraction and chromatographic separation and then submitted to chemical characterization. Different reasons prompted us to undertake the isolation procedure. First of all, these compounds were a relevant part of the biotransformation mixture and their MS fragmentations clearly indicated a chemical structure deriving from the corresponding starting materials. The compounds **25** was not one of the references standard available from our laboratory. Hence, we identified this compound only after its isolation and chemical characterization. Both the diastereoisomeric forms of the compound **25** have been described in the literature [55] but only low resolution 1H-NMR data was reported. Therefore, we were not able to assign the relative configuration to the compound

obtained by biotransformation. Differently, compound **36** is completely new and its analytic data has not described yet. Concerning compound **28**, we observed that its diastereoisomeric forms (*trans* and *cis* isomers) have the same retention time by GC-MS analysis. As a consequence, the isolation of compound **28** from the biotransformation mixture followed by its NMR analysis was necessary in order to understand what was the main isomer formed. Finally, the case of compound **35** is singular. It is the only compound obtained by biotransformation that was formed through a Baeyer-Villiger oxidation. Consequently, acetate **35** was completely unexpected and the proper reference standard was not synthesized.

Hereafter we reported the procedure for the preparative biotransformation experiments allowing the isolation of compounds **23a**, **25**, **28**, **35** and **36** as well as their main analytic data.

According to the procedure described before for the preparation of the inoculum of spore forming mycelia, *Aspergillus niger*, *Nigrospora oryzae* and *Curvularia lunata*, were inoculated in three 1 L conical pyrex flasks containing 400 mL of MEM. The microorganisms were left to grow at 25 ◦C and 140 rpm for 2 days. Hence the cultures of *Aspergillus niger*, *Nigrospora oryzae* and *Curvularia lunata*, were treated with a solution of 1 g of γ-ionone, 3,4-dehydro-β-ionone and theaspirane, respectively, each one dissolved in 3 mL of DMSO. After 14 days from the substrate injection, using the growing condition described above, the reaction media was filtered through a celite pad, the filter was washed with ethyl acetate and the filtrate was extracted 3 times with the same solvent. The combined organic phases were separated, were washed with brine, dried on Na2SO4 and the solvent was removed under reduced pressure. The residue was then acetylated by treatment with pyridine/acetic anhydride (10 mL of a 2:1 mixture) and catalytic DMAP (10 mg) for 6 h at rt. The acetylating mixture (Py/Ac2O) was then removed under reduced pressure and the resulting oil was purified by chromatography using *n*-hexane/AcOEt mixture as eluent.

The biotransformation of γ-ionone performed using *Aspergillus niger* allowed isolating 0.21 g (18% yield) of compound **23a** and 0.36 g (28% yield) of compound **25** as a single diastereoisomeric form (configuration not determined):

*cis*-2-Hydroxy-γ-ionone acetate (**23a**) = *(1SR,3RS)-2,2-Dimethyl-4-methylene-3-((E)-3-oxobut-1-en-1-yl) cyclohexyl acetate*. 1H-NMR: δ = 6.97 (dd, *J* = 15.8, 9.9 Hz, 1H), 6.10 (d, *J* = 15.8 Hz, 1H), 4.88 (s, 1H), 4.74 (dd, *J* = 9.2, 4.0 Hz, 1H), 4.61 (s, 1H), 2.65 (d, *J* = 9.9 Hz, 1H), 2.41 (dt, *J* = 14.0, 5.4 Hz, 1H), 2.37–2.05 (m, 1H) 2.28 (s, 3H), 2.07 (s, 3H), 1.93–1.83 (m, 1H), 1.72–1.57 (m, 1H), 0.91 (s, 6H). 13C-NMR δ = 197.9 (C), 170.4 (C), 146.0 (C), 145.5 (CH), 132.9 (CH) 111.1 (CH2), 77.8 (CH), 55.9 (CH), 39.0 (C), 31.2 (CH2), 27.6 (CH2), 27.3 (Me), 26.2 (Me), 21.1 (Me), 17.8 (Me). GC-MS (EI): *<sup>m</sup>*/*<sup>z</sup>* (%) = 250 [M+] (12), 235 [M+ − Me] (1), 208 (13), 190 (45), 175 (40), 165 (36), 147 (100), 131 (23), 122 (39), 109 (96), 91 (34), 79 (35), 71 (12).

3-Hydroxy-γ-ionone acetate (**25**) = *(E)-3,3-Dimethyl-5-methylene-4-(3-oxobut-1-en-1-yl)cyclohexyl acetate*. 1H-NMR: δ = 6.82 (dd, *J* = 15.8, 10.1 Hz, 1H), 6.13 (d, *J* = 15.8 Hz, 1H), 4.97 (s, 1H), 4.95–4.86 (m, 1H), 4.63 (s, 1H), 2,73 (dd, *J* = 12.6, 4.9 Hz, 1H), 2.58 (d, *J* = 10.1 Hz, 1H), 2.29 (s, 3H), 2.15–2.01 (m, 1H), 2.03 (s, 3H), 1.85 (dd, *J* = 12.6, 4.5 Hz, 1H), 1.45 (t, *J* = 12.1 Hz, 1H), 0.95 (s, 3H), 0.92 (s, 3H). 13C-NMR: δ = 197.8 (C), 170.3 (C), 145.3 (CH), 144.5 (C), 134.0 (CH), 112.5 (CH2), 69.8 (CH), 56.0 (CH), 45.5 (CH2), 41.1 (CH2), 35.8 (C), 30.4 (Me), 27.3 (Me), 21.4 (Me), 21.3 (Me). GC-MS (EI): *m*/*z* (%) = 250 [M+] (<1), 235 [M<sup>+</sup> − Me] (<1), 190 (28), 175 (29), 157 (14), 147 (100), 131 (32), 119 (20), 105 (39), 91 (27), 79 (15), 69 (14), 55 (8).

The biotransformation of 3,4-dehydro-β-ionone performed using *Nigrospora oryzae* allowed isolating 0.56 g (34% yield) of compound **28** as a 5:1 mixture of *trans*/*cis* isomers:

*trans*-3,4-Dihydroxy-β-ionone diacetate (**28**) = *(1RS,2RS)-3,5,5-Trimethyl-4-((E)-3-oxobut-1-en-1-yl)cyclohex-*3-ene-1,2-diyl diacetate. 1H-NMR: δ = 7.10 (dq, *J* = 16.4, 0.9 Hz, 1H), 6.11 (d, *J* = 16.4 Hz, 1H), 5.51 (d, *J* = 7.8 Hz, 1H), 5.17–5.09 (m, 1H), 2.28 (s, 3H), 2.06 (s, 3H), 2.00 (s, 3H), 1.88–1.66 (m, 2H), 1.62 (br s, 3H), 1.17 (s, 3H), 1.07 (s, 3H). 13C-NMR: δ = 197.8 (C), 170.7 (C), 170.4 (C), 141.0 (CH), 140.2 (C), 133.7 (CH),

128.5 (C), 74.7 (CH), 70.3 (CH), 41.4 (CH2), 36.4 (C), 29.7 (Me), 27.7 (Me), 27.5 (Me), 21.1 (Me), 20.8 (Me), 16.5 (Me). MS (ESI): 331.2 (M<sup>+</sup> + Na).

The biotransformation of theaspirane performed using *Curvularia lunata* allowed isolating 95 mg (7% yield) of compound **35** and 65 mg (5% yield) of compound **36**:

*2-(3-Acetoxy-2,6,6-trimethylcyclohex-1-en-1-yl)ethyl acetate* (**35**). 1H-NMR: δ = 5.13 (t, *J* = 4.6 Hz, 1H), 4.05 (dd, *J* = 9.4, 7.5 Hz, 2H), 2.46–2.37 (m, 2H), 2.06 (br s, 6H), 1.92–1.79 (m, 1H), 1.74–1.52 (m, 2H), 1.66 (s, 3H), 1.44–1.33 (m, 1H), 1.08 (s, 3H), 1.00 (s, 3H). 13C-NMR: δ = 171.0 (C), 171.0 (C), 139.5 (C), 128.2 (C), 72.3 (CH), 63.3 (CH2), 35.1 (C), 34.6 (CH2), 28.2 (Me), 28.0 (CH2), 26.8 (Me), 25.3 (CH2), 21.3 (Me), 21.0 (Me), 16.8 (Me). GC-MS (EI): *<sup>m</sup>*/*<sup>z</sup>* (%) = 268 [M+] (<1), 226 (13), 208 [M+ − AcOH] (16), 166 (40), 148 (17), 133 (100), 120 (28), 110 (36), 91 (16), 79 (8).

*3,6,6-Trimethyl-1,4,5,6,7,8-hexahydro-3H-3,5a-epoxybenzo[c]oxepin-8-yl acetate* (**36**). 1H-NMR: δ = 5.43–5.36 (m, 1H), 5.31 (s, 1H), 4.49 (dt, *J* = 14.2, 2.1 Hz, 1H), 4.20 (d, *J* = 14.2 Hz, 1H), 2.30–1.40 (m, 6H), 2.02 (s, 3H), 1.47 (s, 3H), 1.11 (s, 3H), 1.01 (s, 3H). 13C-NMR: δ = 170.5 (C), 138.7 (C), 116.3 (CH), 105.3 (C), 86.0 (C), 68.4 (CH), 63.7 (CH2), 40.7 (CH2), 35.3 (C), 34.3 (CH2), 30.5 (CH2), 24.6 (Me), 24.4 (Me), 23.3 (Me), 21.2 (Me). GC-MS (EI): *<sup>m</sup>*/*<sup>z</sup>* (%) = 266 [M+] (1), 236 (50), 224 (8), 207 [M+ − AcO] (59), 195 (36), 178 (27), 164 (84), 153 (100), 136 (61), 121 (59), 107 (62), 91 (53), 79 (29).
