Conformationally constrained analogues of diacylglycerol (DAG) built on a racemic 5(-)[(acyloxy)-methyl]-5-(hydroxymethyl)tetrahydro-2-furanone template were shown previously to have excellent binding affinities for protein kinase C (PK-C). Since the interaction of PK-C with DAG is stereospecific, it was anticipated that PK-C would bind tightly to only one enantiomeric form of the compounds constructed with this new lactone template. Separation of enantiomers by chiral HPLC was discarded due to the ease with which acyl migration occurs in these class of compounds, and a total chiral synthesis was undertaken. Prior to chemical synthesis, the selection of the "correct" enantiomeric template was predicted by a molecular conformational analysis that compared the two enantiomers of DAG in their presumed "active" conformation with the two enantiomeric lactone templates. This presumed "active" conformation for DAG was derived from a previously developed pharmacophore model that uses the molecule of a potent phorbol diester as the ideal rigid template. The results from this analysis indicated that the "correct" lactone template corresponded to the inactive (R)-isomer of DAG. This analysis also predicted that the lactone template corresponding to the active (S)-DAG enantiomer would not fit adequately into the pharmacophore. The chiral syntheses of target compounds 2, 4, and 6, constructed on the selected, and presumably "correct" lactone template, were achieved from a common bicyclic intermediate (5R,8R,9R)-8,9-O-isopropylidene-2-keto-1,7-dioxaspiro[4.4]nonane (10) that was synthesized from commercially available 1,2:3,5-di-O-isopropylidene-alpha-D-threo-apiofuranose (7) by a very effective spirolactonization approach. On the basis of their ability to inhibit the binding of [3H-20]phorbol 12,13-dibutyrate (PDBU) to PK-C alpha, the enantiomeric ligands 2, 4, and 6 were twice as potent as the corresponding racemates. These results confirm that binding of these lactones is stereospecific and consistent with a binding mechanism similar to that of DAG.