2 13c malonic acid

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 (link)). 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 (link); Mitra and Karchaudhuri, 1999 (link)). 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.

Malonic acid, 2-¹³C-Malonic acid, 4-coumaric acid, 2-methylthiazole-5-carboxylic acid, resveratrol, p-coumaric acid, 7-phenyl heptanoic acid, 6-phenyl hexanoic acid, 5-(4-flurophenyl)valeric acid, 4-biphenylacetic acid, N-acetyl-L-cysteine, benzoic acid adenosine triphosphate (ATP), coenzyme A trilithium salt (CoA), magnesium chloride, dimethyl sulfoxide (DMSO) hexahydrate, phospho(enol) pyruvate potassium salt, PEG 4000, NaCl, potassium and sodium phosphate, IPTG, kanamycin, penicillin, streptomycin, were purchased from Sigma-Aldrich. 2-Chloro-1,3-thiazole-5-carboxylic acid and 4-methylthiazole-5-carboxylic acid were obtained from Matrix Scientific.

Ethyl acetate (EtOAc), hexane, chloroform (CHCl₃) and formic acid were purchased from LabSynth (SP, Brazil). Methanol (MeOH) and acetonitrile (ACN) (HPLC grade) were obtained from J.T. Baker (United States). All solvents were distilled prior to use. The deuterated chloroform used in ¹H NMR analyses and (2-¹³C)-malonic acid and (¹³C)-methane iodine were purchased from Sigma-Aldrich.
All reactions were performed in oven dried glass (temperature ≥100°C) and cooled in a desiccator. Tetrahydrofuran (THF) was refluxed and distilled with sodium/benzophenone under a N₂ atmosphere, and triethylamine and pyridine were refluxed and distilled from calcium hydride under a N₂ atmosphere.
Analytical grade solvents (Merck^®, Tedia^®, and J.T. Baker^®) and water purified using a Milli-Q system (Millipore^®) were used in the chromatographic and spectroscopic analyses. High purity, commercially obtained reagents, were used without prior purification.