Unlabeled and [14C]-labeled cyclohexa-1,5-diene-1-carbonyl-CoA and 6-hydroxycyclohex-1-ene-1-carbonyl-CoA. These compounds were synthesized enzymatically from benzoyl-CoA or [phenyl14C]benzoyl-CoA with partially purified benzoyl-CoA reductase from T. aromatica, which contained traces of dienoyl-CoA hydratase . The assay was performed under strictly anaerobic conditions. The 1 mL assay mixture contained 150 mm Mops/KOH, pH 7.3, 10 mm MgCl2, 0.5 mm unlabeled or [phenyl14C]benzoyl-CoA (specific radioactivity: 4.5 GBq mmol−1), 0.75 mm ATP, 1 mm phosphoenolpyruvate, 8 nkat pyruvate kinase, and 8 mm titanium (III)-citrate. The reaction was started by the addition of 1–2 nkat of enriched benzoyl-CoA reductase. After 20 min incubation at 37 °C the reaction was stopped by adding 100 µL of 1 m HClO4. After centrifugation the supernatant was freeze-dried and stored at −20 °C. For large-scale preparation the assay mixture (100 mL) contained 150 mm Mops/KOH, pH 7.3, 10 mm MgCl2, 2 mm unlabeled benzoyl-CoA, 11 MBq of [phenyl14C]benzoyl-CoA, 3 mm ATP, 4 mm phosphoenolpyruvate, 40 nkat of pyruvate kinase and 8 mm titanium (III)-citrate. The reaction was started by the addition of 20 nkat enriched benzoyl-CoA reductase and was stopped with ≈ 8 mL of 2 m formic acid. After removal of precipitated protein by centrifugation the product mixture was applied to a solid-phase extraction column (ICT; end-capped C18-material, 50 g; flow rate, 2 mL·min−1) which had been conditioned with 140 mL methanol and equilibrated with 280 mL of solvent 1 (20 °C). After washing the column with 350 mL of solvent 1, benzoyl-CoA was eluted with 80% (by volume) aqueous methanol. After evaporation of methanol under vacuum at 35 °C, the solution containing cyclohexa-1,5-diene-1-carbonyl-CoA and 6-hydroxycyclohex-1-ene-1-carbonyl-CoA was freeze-dried and stored at −20 °C. This CoA-thioester mixture was used for determination of β-hydroxyacyl-CoA dehydrogenase activity during purification of the enzyme. For separation of these CoA-thioesters the dry powder was dissolved in 6 mL 50 mm potassium phosphate, pH 6.7, and applied in three runs to a preparative HPLC column (Grom; Grom-Sil 120 ODS-4 HE, 11 µm; 250 × 20 mm) with solvent 2 at a flow rate of 8 mL·min−1 (20 °C). Two product fractions were collected and applied for desalting to an Isolute-RP-C18 extraction column (ICT; end-capped C18-material, 1 g; reservoir volume, 6 mL; flow rate, 0.5 mL·min−1). The column had been conditioned with 6 mL of methanol and equilibrated with 12 mL of solvent 1 (20 °C). After washing the column with 12 mL of solvent 1, the CoA-thioester was eluted with 80% (by volume) methanol in water. The samples were freeze-dried. The purified and desalted fraction containing 6-hydroxycyclohex-1-ene-1-carbonyl-CoA was used for determination of specific activity and apparent Km value of alcohol dehydrogenase. The purity and amount of 6-hydroxycyclohex-1-ene-1-carbonyl-CoA was analyzed by comparison of the UV spectra with published data of benzoyl-CoA  under the assumption that the nonaromatic CoA-thioesters had the same absorption coefficient at 260 nm as benzoyl-CoA. The amount of [14C]-labeled CoA-thioester was determined using liquid scintillation counting. For more rigorous control of purity, an analytic HPLC column (Grom; Grom-Sil 120 ODS-4 HE, 5 µm; 120 × 4 mm) was used with solvent 2 (20 °C), or with a linear 2–15% acetonitrile gradient formed from acetonitrile and 50 mm potassium phosphate, pH 6.7, as solvent at a flow rate of 1 mL·min−1. The effluent was monitored using a radioactivity monitoring analyzer with a solid scintillator cell, and by a photodiode array detector. The yield of the CoA-thioester after synthesis and purification was ≈ 30–40%.