Preparation of Cinnamic Acids

To investigate the substrate specificity in the formation of pellucidin A in P. pellucida, a series of synthetic ¹³C-labeled cinnamic acids and styrenes were evaluated by in vivo feedings and enzymatic conversion experiments. The cinnamic acids were synthesized by a Knoevenagel condensation between malonic acid and benzaldehyde derivatives. Malonic acid (9.60 mmol) and the benzaldehydes (10.00 mmol) were dissolved in pyridine (3.5 mL), followed by addition of 10 μL of piperidine and 10 μL of aniline, both previously distilled, as described (Katayama et al., 1992). After the reaction was completed, the mixture submitted to work-out yielded colorless crystals of cinnamic acids (Supplementary Figure 9; A1–A16) with a typical yield above 90%. The structures of the cinnamic acids were characterized using ¹H and ¹³C NMR and EIMS data in addition to a comparison with previously published data (Katayama et al., 1992; Mitra and Karchaudhuri, 1999). For the preparation of isotopically labeled versions of cinnamic acids, (2-¹³C)-malonic acid (99%) from Sigma-Aldrich was used and the corresponding cinnamic acids were purified and characterized spectroscopically.