The stereochemistry of the enzyme mediated chalcone-flavanone isomerisation has been investigated. Cyclisation of 4,2',4'-trihydroxychalcone catalysed by either one of the two chalconeflavanone isomerases isolated from mung bean seedlings (Phaseolus aureus Roxb.) leads to the (-)(2 5)-7,4'-dihydroxyfiavanone.When 4,2',4'-[a-2H]trihydroxychalcone is the substrate, deuterium is located preferentially in the equatorial position at C-3 of the flavanone as was shown by nuclear magnetic resonance measurements. Conversely, when the cyclisation is carried out in 2H20 with the unlabeled chalcone, deuterium is preferentially located in the axial position at C-3 of the flavanone. The mechanistic implications of these results are discussed. In experiments described in this paper the absolute configuration at (3-2 was determined by circular dichroism measurementswith 7,4'-dihydroxyflavanone which had been obtained with two isomerase enzymes from mung bean seedlings (Phaseolus aureus Roxb.) [3]. We also investigated whether the cyclisation to the 7,4'-dihydroxyflavanone represents a formal cis-or trans-addition to the double bond of the chalcone (I). The idea of this experiment is the following. When a chalcone deuterated at the u-position is cyclised stereospecifically to the flavanone without deuterium exchange, deuterium will appear in either the axial or the equatorial position a t C-3 of the flavanone. Conversely, when the reaction is carried out in 2H20 deuterium must appear in the opposite position at C-3 of the flavanone.
METHODS
Synthesis of 4,2',4'-[a-2H]Trihydroxycha2cone ( I I I )300 mg of 7,4'-[3-2H]bisbenzyloxyflavanone [6] were dissolved in 8 ml of absolute dioxane and stirred with 5°/0 Na02H in 2H,0 for 3 h a t room temperature. At the end of this time thin layer chromatography (on silica gel with benzene-isopropanol-methanol, 96 : 6 : 1, v/v/v) showed 4,4'-bisbenzyloxy-2'-hydroxychalcone (RF = 0.89) to be the only product. After addition of ether to the reaction mixture alkali was removed by washing with 2H,0 and subsequently with H,O. After removal of ether the dried residue was stirred for 2 h at room temperature with 1 g of BBr, and 30 ml of methylene chloride. The solvent and BBr, were removed in wacuo and H,O was added. The reaction product was extracted with ether and after the ether solution had been washed with water to neutrality, it was applied to 16 sheets of Whatman 3MM and chromatographed with 50°/, methanol. The zone with RF = 0.40 was eluted from the paper with methanol and the chalcone recrystallized from methanol-water. [6] were dissolved in a small amount of methanol and treated for 10 min with 2 N NaOH. After acidification with H,SO,, the solution was extracted with