A Kinetically Controlled Dynamic Enzymatic Ketone ...

◆The cyclopropanation of 2 with ethyl diazoacetate that was poorly selective and gave 8 isomers

◆Building the tricyclic ring system through an intramolecular enolate alkylation was envisioned.

◆To investigate this synthetic strategy, an efficient synthesis of keto-ester 10 was established. Trimester 7 having one fewer enolizable center and more electrophilic than diester 8.

◆Even though ketone 10 was calculated to have a pKa of 18.5 for the α-hydrogen, we were optimistic that epimerization through an enol pathway could take place at near-neutral pH.

◆The cis diastereomer 11a was favored early (0.8:1 trans/cis at 17% conversion), but as the reaction proceeded, the trans isomer began forming preferentially, ultimately reaching a final ratio of ∼2.4:1 trans/cis. Additional insight came from monitoring the ee of starting material which remained racemic throughout the reaction, indicating epimerization was fast with respect to ketone reduction.

◆The initial slight intrinsic bias for the cis product can be traced to the difference in barrier heights on the red curve (ΔG1‡). As the reaction proceeds, acetone builds up through 2-propanol oxidation and the equilibrium between alcohol products and ketone will become less favorable (reflected by ΔG2) that results in a thermodynamically determined cis/trans ratio (ΔG3).

◆It became apparent that further progress toward developing a highly selective reaction would require two advances: (1) An enzyme with a significant intrinsic bias to generate the trans isomer and (2) preventing reversibility.

◆The first would require an evolved enzyme that provides improved diastereoselectivity. Reversibility could be addressed by removal of acetone with a nitrogen sweep.

◆In partnership with Codexis Inc., six rounds of enzyme evolution delivered KRED-264, a novel variant able to produce 11 with >30:1 dr when a nitrogen sweep is utilized.