Enzymes act as biocatalysts with remarkable selectivity, fixing the three-dimensional arrangement of products. Cornforth inserted isotopes such as deuterium and 14C at defined positions in substrates and compared the stereochemistry of reactants and products. He revealed, for instance, that during cholesterol biosynthesis carbon fragments are incorporated without bond rotation, entering the growing chain as a rigid “face.” These findings guided not only mechanistic enzymology but also the design of asymmetric synthesis. Consequently, chemists could reduce harmful isomer mixtures in drugs, creating large social benefits in the pharmaceutical industry.

Cornforth used NMR and mass spectrometry to analyze site-selectively labeled substrates and to map multi-step stereochemical pathways, such as epoxysqualene ring opening and arachidonic acid oxidation. His “face-to-face” migration model was tested in the biosynthesis of purines, steroids and many other frameworks, and it remains textbook knowledge today. He further provided an electronic-stereochemical rationale for how active-site residues read substrate chirality. The work influenced the design of transition-state analogue inhibitors, the development of enantioselective asymmetric hydrogenation catalysts, and even modern computational enzyme design.

Using 3HF-pyridine generated, site-specific deuterated substrates, Cornforth dissected the (R)-mevalonate → isoprene unit → squalene → lanosterol → cholesterol cascade. Deuterium coupling-constant analysis distinguished anti versus syn hydride transfers and quantified NADPH-dependent redox stereoselectivity. He evaluated stereochemical constraints in enzyme-substrate complexes via a Markov model, showing that transition-state conformational entropy governs enantioselection. These insights laid theoretical foundations for algorithmic chiral-pool exploitation and biocatalytic dynamic kinetic resolution.