A recent publication by Abeles et al. on fluoro ketone inhibitors of hydrolytic enzymes prompted us to report our own observation in the renin inhibitor area. Pepstatin is a naturally occurring pentapeptide that is a general aspartyl protease inhibitor. It was proposed that the statine residue, 4(S)-amino-3(S)-hydroxy-6-methylheptanoic acid, acts as a structural analogue of the tetrahedral species during enzymatic hydrolysis of a peptidic bond. More recently, the ketone analogue (statone) has been prepared by the oxidation of the 3(S)-hydroxyl group of statine in a pepstatin analogue. The ketone carbonyl has been shown by ¹³C NMR spectroscopy to be converted to a tetrahedral acetal upon binding to pepsin via enzyme-catalyzed addition of water. This pseudo substrate is a weaker inhibitor of pepsin than the corresponding statine-containing peptide. The decreased binding constant might be attributed to the poor tendency of the ketone carbonyl to undergo hydration. To facilitate the ease of hydration of the ketone, we proposed to introduce electron-withdrawing fluorine atoms on the methylene carbon adjacent to the carbonyl. In this way, the difluorostatone unit in peptide should exist predominantly in the hydrated form. The greater electrophilicity of the carbonyl of difluorostatone over the nonfluorinated statone should also facilitate the enzyme-catalyzed addition of water to form the tetrahedral acetal upon binding to the active site. We chose to examine this hypothesis on the aspartyl protease renin, which cleaves the protein substrate angiotensinogen into the decapeptide angiotensin I, which in turn is cleaved by converting enzyme into the pressor octapeptide angiotensin II. Highly potent competitive inhibitors of renin have already been reported in which statine was incorporated into renin substrate analogues. Successful renin inhibitors could provide agents for control of cases of renin-associated hypertension. Hallinan and Fried reported the preparation of 2,2-difluoro-3-hydroxy esters by a Reformatsky reaction. Application of this procedure to Boc-L-leucinal afforded the adducts 1a and 1b in good yield, and they were readily separated by chromatography. The esters 1a and 1b were hydrolyzed with 1 equiv of NaOH and the resulting salts lyophilized. These salts were coupled to L-isoleucyl-2-pyridylmethylamide to give the adducts 2a and 2b. Two more amino acids were sequentially added by using diethylphosphoryl cyanide as coupling agent to give the peptides 3a and 3b. Oxidation of the hydroxyl group of 3a and 3b with the Swern reagent afforded the ketones 4e and 4f, respectively. The statine-containing peptides 4a and 4b were also prepared by using the same coupling sequence but with the nonfluorinated statine intermediate. The inhibitory activities of these peptides against human renin, porcine pepsin, bovine cathepsin D, and rabbit ACE were tested. The peptide 4a, which contains the difluorostatine residue, is a potent inhibitor of human renin with an IC₅₀ of 1.4×10⁻⁹ g. The nonfluorinated statine-containing peptide 4b is about 30 times less potent. The ketone analogue 4e, which contains the difluorostatone residue, is slightly less potent than 4a but still 10 times more potent than the nonfluorinated statone-containing peptide 4d. A high degree of enzyme specificity is desirable for a potentially successful therapeutic agent. Pepstatin, for example, is a general aspartyl protease inhibitor and shows poor selectivity. A substrate analogue such as RIP does not discriminate between renin and converting enzyme. The difluoro ketone 4f does exhibit high renin specificity. It is a very poor inhibitor of converting enzyme and is 3-4 orders of magnitude less effective against pepsin and cathepsin D. The transition-state analogue concept remains a viable approach in the design of potent enzyme inhibitors as illustrated in aspartyl proteases. The work presented in this report lends support to the value of an understanding of enzymatic mechanism as an aid to create effective inhibitors of therapeutically important enzymes.