*3.1. General Analytical Procedures*

Nuclear magnetic resonance (NMR) spectra were recorded in CD3OD on a Varian VNMRS 600 ( 1H: 600 MHz; 13C: 150 MHz; Palo Alto, CA, USA) spectrometer operating at 25 ◦C or in DMSO-d6 on a Varian NMR AS 400 (1H: 400 MHz) spectrometer operating at 25 ◦C. The chemical shifts (δ) were assigned in ppm and the coupling constants (*J*) in Hz. The assignments were based on chemical shifts, integration, homonuclear (COSY), and heteronuclear (HMQC and HMBC) measurements. Optical rotation was measured at 20 ◦C in a Perkin Elmer 343 Polarimeter (Waltham, MA, USA) at 589 nm (sodium *D* line). Analytical HPLC analyses were carried out on a Shimadzu Shim-pack PREP-ODS(H)KIT 5 μm C18 column (4.6 × 250.0 mm, Kyoto, Japan), and the chemical profiles of the

biotransformation and control extracts were obtained using 10% to 100% methanol in water containing 0.01% acetic acid over 30 min with a flow rate of 0.8 mL/min. The crude extracts were analyzed through the injection of 20 μL at 1 mg/mL on a Shimadzu (SIL-20A) multisolvent delivery system, a Shimadzu SPD-M20A, a photodiode array detector, and an Intel Celeron computer for analytical system control, data collection, and processing. The derivatives were isolated by using a chromatographic column (40 × 1.5 cm) containing silica gel (Sigma-Aldrich, 60 Å, Saint Louis, MO, USA). Mixtures of *n*-hexane (Synth) and ethyl acetate (Synth) were employed as a mobile phase.

#### *3.2. Substrates*

Nine synthetic coumarin analogs were submitted to the biotransformation experiments. All coumarins used as substrates were obtained according to known methods through a Pechmann reaction, and all of their spectroscopic data were identical to those previously described for 7-hydroxy-4-methyl-2*H*-2-chromenone (**1**) [32], 5,7-dihydroxy-4-methyl-2*H*-chromen- 2-one (**2**) [33], 7,8-dihydroxy-4-methyl-2*H*-chromen-2-one (**3**) [34], 7-hydroxy-2,3-dihydrocyclopenta[*c*] chromen-4(1*H*)-one (**4**) [33], 7,9-dihydroxy-2,3-dihydrocyclopenta[*c*]chromen-4(1*H*)-one (**5**) [33], 6,7-dihydroxy-2,3-dihydrocyclopenta[*c*]chromen-4(1*H*)-one (**6**) [35], methyl 2-(7,8-dihydroxy-2-oxo-2*H*-chromen-4-yl)acetate (**7**) [36], methyl 2-(5,7-dihydroxy-2-oxo-2*H*-chromen-4-yl)acetate (**8**) [37], and methyl 2-(7-hydroxy-2-oxo-2*H*-chromen-4-yl)acetate (**9**) [38].
