*3.3. Biotransformation Assays*

The biotransformation of all coumarins (**1**–**9**) was done through screening with *Cunninghamella elegans* ATCC 10028b and *Aspergillus brasiliensis* ATCC 16404, which were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The filamentous fungi were maintained in 80% glycerol solution at −20 ◦C.

The fungi were grown in a two-step culture procedure. First, each fungus was grown at 28 ◦C in Petri dishes containing malt agar (malt extract 2.0%, glucose 2.0%, peptone 0.1%, agar 1.8%) for 7 days. Next, an inoculum of five 6-mm disks containing mycelia and agar was added to 125-mL Erlenmeyer flasks, each holding 50 mL of Koch's K1 medium (glucose 0.18%, peptone 0.06%, and yeast extract 0.04%). Coumarins (5 mg) were separately added to each flask as a solution in dimethyl sulfoxide (5 mg dissolved in 200 μL). Control flasks consisted of a culture medium with dimethyl sulfoxide and a fungus (without coumarin), a culture medium with coumarin and dimethyl sulfoxide (without a fungus), and a culture medium by itself. Biotransformation experiments were carried out at 28 ◦C for 72 h with shaking at 120 rpm. Samples were analyzed daily by HPLC. The mycelia were separated by filtration, the fermentation broths were extracted three times with ethyl acetate, and the solvent was evaporated under reduced pressure to yield crude extracts. All experiments were carried out in triplicate.

Biotransformations of two selected coumarins (**4** and **7**) were separately carried out in 10 Erlenmeyer flasks (scale-up biotransformations) using the same aforementioned procedures. According to the yields of the biotransformation assays in the initial screening, the scale-up biotransformations of **4** and **7** were carried out by *C. elegans* and *A. brasiliensis*, respectively. The extraction of the culture broths by ethyl acetate was followed by evaporation of the solvent to yield the crude extracts of the biotransformations of **4** and **7** (41.0 mg and 59.0 mg, respectively).

Additionally, the biotransformation conditions of coumarin **4** were investigated with a view toward increasing its conversion. For this, new assays were designed by using a greater quantity of the fungus *C. elegans* (10 6-mm disks containing mycelia and agar) and by increasing the incubation time (120 and 168 h).
