The incorporation of fluorine into small molecule pharmaceutical candidates is a powerful method to improve drug properties.[1] Currently, the late-stage introduction of fluorine atoms onto active drug-candidate scaffolds is a significant challenge for synthetic chemists.[2,3] This proposal describes a novel strategy to address the significant challenge of accessing phenols by selective enzymatic aromatic hydroxylation. This enzymatic step will be followed by chemical transformation of the resulting phenols to aryl fluorides using, and improving upon, recently developed chemistry.[4] This strategy will allow chemo-enzymatic aromatic C-H fluorination to be used for the late-stage modification of drug leads. Successful implementation of this strategy will fulfill an existing gap in biocatalytic aromatic hydroxylation, as well as facilitate rapid access to valuable complex fluorinated drugs and drug candidates.
The specific aims of this project are 1) to engineer cytochrome P450 enzymes to conduct aromatic hydroxylation, 2) to scale up enzymatic aromatic hydroxylation and complete the chemical introduction of the aryl fluoride, and 3) to expand the strategy developed in aims 1 and 2 to the drugs warfarin and diclofenac. Using directed evolution, cytochrome P450 BM3 will be developed to carry out aromatic hydroxylation not only in high yield, but also with unprecedented regioselectivity. As ortho, meta, and para hydroxylation are all possible, enzymes with each type of regioselectivity will be engineered. To aid in this engineering effort, a novel high-throughput screening (HTS) protocol will be developed for enzyme activity, with a unique combination of assays that reveal the regioselectivites of the reactions. The enzymes resulting from directed evolution efforts will be used to carry out aromatic hydroxylations on a drug, flurbiprofen, on a gram scale. The complex phenols isolated from these hydroxylations will be transformed to aryl triflates, and finally to the sought-after aryl fluorides using chemistry recently demonstrated on simple aromatic molecules. The aryl fluorides will be assayed to demonstrate improved drug properties. The enzymes evolved for flurbiprofen aryl hydroxylation will be used for the selective hydroxylation of the important drugs warfarin and diclofenac, and the phenolic products will be converted to aryl fluorides and tested for enhanced properties.

Public Health Relevance

The fluorine atom is found in a number of drugs because it can make them much more effective with fewer side-effects. Our proposed strategy will allow pharmaceutical researchers to make drug candidates with fluorine atoms faster by a combined biological and chemical method. This method is expected to shorten the time required to bring new drugs to patients.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1-F04A-B (20))
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Lees, Robert G
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California Institute of Technology
Engineering (All Types)
Schools of Engineering
United States
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Lauchli, Ryan; Pitzer, Julia; Kitto, Rebekah Z et al. (2014) Improved selectivity of an engineered multi-product terpene synthase. Org Biomol Chem 12:4013-20
Lauchli, Ryan; Rabe, Kersten S; Kalbarczyk, Karolina Z et al. (2013) High-throughput screening for terpene-synthase-cyclization activity and directed evolution of a terpene synthase. Angew Chem Int Ed Engl 52:5571-4