Chiral GC-MS Analysis of Methylated Diketide Acids

The concentrated organic extracts of the above-described enzymatic incubations were dissolved in 200 μL of methanol and then treated with 5 μL of TMS-diazomethane (2 M in hexane) for 10 min at room temperature. The derived reduced 2-methyl-diketide acid methyl esters were directly analyzed by chiral GC-MS.^{13 (link)} GC-MS spectra were recorded on a GC-MS Agilent 5977A Series GC-MSD instrument, 70 eV EI in positive ion mode, using a Varian CP-Chirasil-DEX CB capillary column (0.25 mm inside diameter × 25 m length × 0.25 mm film, Agilent Technologies) and a temperature program of (1) initial temp 60 °C for 1 min, (2) increase at rate 0.3 °C/min up to 75 °C, (3) 5 °C/min up to 90 °C, and then 20 °C/min to final temp 200 °C. The extracted ion current (XIC) of the chromatogram was analyzed set on the base peak at m/z 88 for 2-methyl-3-hydroxypentanoic acid methyl ester. All assays were conducted in duplicate. The products were compared directly with authentic standards, as previously described (Figures S11-S15).^{13 (link)}