**3. Materials and Methods**

#### *3.1. Materials and General Methods*

All air and moisture sensitive reactions were carried out using dry solvents and under a static atmosphere of nitrogen. All solvents and reagents were of commercial quality and were purchased from Sigma-Aldrich (St. Louis, MO, USA). A large number of reference standard compounds were synthesized in our laboratory and were used for the unambiguous identification of the compounds formed in the biotransformation experiments. α-Ionone, γ-ionone and α-damascone were used in racemic form. Commercial theaspirane consists of an equimolar mixture of racemic diastereoisomers. γ-Ionone (**3**) and 3,4-dehydro-β-ionone (**4**) were prepared starting from α-ionone, according to the procedures previously described by us [26–28].

The keto derivatives: 3-keto-α-ionone (**8**), 4-keto-β-ionone (**13**), 3-keto-α-damascone (**37**), 4-ketoβ-damascone (**41**), theaspirone (**31**), 3-keto-α-ionol acetate (**12**) and 4-keto-β-ionol acetate (**15**) were prepared by oxidation of α-ionone, β-ionone, α-damascone, β-damascone, theaspirane, α-ionol acetate (**11**) and β-ionol acetate (**21**), respectively. The oxidation reactions were performed using TBHP/MnO2 as oxidant according to our previously reported procedure [41].

The diastereoisomeric forms of 4,5-epoxy-α-ionone (**10**), 4,5-epoxy-α-damascone (**39**), 4,5-epoxytheaspirane (**33**) as well as 5,6-epoxy-β-ionone and 5,6-epoxy-β-damascone were prepared by epoxidation of α-ionone, α-damascone, theaspirane, β-ionone and β-damascone, respectively, using *m*-chloroperbenzoic acid and CH2Cl2 as solvent.

α-7,8-Dihydroionones, β-7,8-dihydroionone (**22**) and γ-7,8-dihydroionone (**26**) were prepared by reduction of α-, β- and γ-ionone respectively, using hydrogen and Ni Raney as catalyst for α-ionone [42] and Bu3SnH and (Ph3P)2PdCl2 as catalyst for β- and γ-ionone [25,26]. α-8,9-Dihydrodamascone (**40**) was prepared by reduction of α-damascone using NaBH4 in methanol. β-8,9-Dihydrodamascone was prepared by addition of propylmagnesium bromide to β-cyclocitral followed by oxidation of the resulting carbinol using Dess-Martin periodinane [43].

β-Ionol acetate (**21**, racemic), α-ionol acetate (**11**, as a mixture of two racemic diastereoisomers), γ-ionol acetates (**27**, as a mixture of two racemic diastereoisomers) and 3-acetoxy-theaspirane (**32**) (as a mixture of four racemic diastereoisomers) were prepared by chemical acetylation (Ac2O/Py) of the corresponding alcohols, which were in turn obtained through the reduction of α-, β-, γ-ionone and theaspirone, respectively, using NaBH4 in methanol.

*cis*-2-Acetoxy-α-ionone, and *cis*-2-acetoxy-γ-ionone (**23a**) were prepared starting from 2,8,8 trimethyl-6-oxabicyclo[3.2.1]oct-2-en-7-one and 8,8-dimethyl-2-methylene-6-oxabicyclo[3.2.1]octan-7-one (kaharana lactone) respectively, according to the synthetic procedure developed by Audran [44]. The latter lactones were in turn prepared from racemic *cis*-2-hydroxy-α-cyclogeraniol and *cis*-2 hydroxy-γ-cyclogeraniol [45] by oxidation using BAIB and TEMPO as catalyst. In addition, the partial reduction of the two isomeric lactones afforded the corresponding lactols, whose condensation with acetone [46] followed by acetylation (Ac2O/Py) of the crude reaction mixtures, gave the *cis*/*trans* mixtures of acetoxy-α-ionone and acetoxy-γ-ionone, respectively, that were used as GC-MS reference standards for the identification of the corresponding *trans* isomers.

Racemic 2-acetoxy-β-ionone (**18**) was prepared starting from 2-hydroxy-β-cyclogeraniol [45] by selective oxidation of the primary alcohol functional group by MnO2 in CHCl3, condensation with acetone [46] and acetylation (Ac2O/Py) of the obtained hydroxy-ionone derivative.

Samples of 3-acetoxy-α-ionone (**9**) (2:1 *cis*/*trans* mixture), of 4-acetoxy-γ-ionone (**24**) (4:1 *cis*/*trans* mixture) and of 3-acetoxy-β-ionone (**19**) were prepared starting from α-ionone according to the procedure described by Tu [47], by Serra [42] and Khachik [48], respectively. A sample of 3-acetoxy-αdamascone (**38**) (1:1 *cis*/*trans* mixture) was prepared starting from ethyl 3-hydroxy-α-cyclogeraniate according to the procedure described by Takei [49].

4-Acetoxy-β-ionone (**14**) and 4-acetoxy-β-damascone (**42**) were prepared starting from 4,5-epoxyα-ionone and 4,5-epoxy-α-damascone, respectively, by means of NaOMe mediated transposition

followed by chemical acetylation (Ac2O/Py) of the obtained allyl alcohols. A sample of 4-acetoxyβ-ionol acetate (**16**) (1:1 mixture of diastereoisomers) was prepared from 4-keto-β-ionol acetate by reduction with NaBH4 in methanol followed by chemical acetylation (Ac2O/Py).

A sample of 4-acetoxy-β-7,8-dihydroionone acetate (**17**) was prepared from 4-hydroxy-β-ionone by reduction with Ph3SiH followed by chemical acetylation (Ac2O/Py), according to the procedure described by Pascual [50].

3,4-Diacetoxy-β-ionone (**28**) (*cis*/*trans* mixture), was prepared from 3,4-dehydro-β-ionone according to the procedure described by Buschor [51]. The oxidation of 4-keto-β-ionone with IBDA in methanol [52] afforded 3-hydroxy-4-keto-β-ionone that was further oxidated using oxygen in presence of *t*BuOK [53] to give 3,4-diketo-β-ionone. The acetylation (Ac2O/Py) of the latter two compounds afforded 3-acetoxy-4-keto-β-ionone (**29**) and 3-acetoxy-4-keto-2,3-dehydro-β-ionone (**30**). Racemic dihydroactinodiolide (**20**) and 2-hydroxy-2,6,10,10-tetramethyl-1-oxaspiro[4.5]dec-6-en-8-yl acetate (**34**) were prepared as described previously [32,54].

A comprehensive characterization of the above described reference standards is reported in the Supplementary Materials section.
