1. Inhibition of chymotrypsin by fluorinated alpha-keto acid derivatives
M F Parisi, R H Abeles Biochemistry. 1992 Oct 6;31(39):9429-35. doi: 10.1021/bi00154a015.
A series of fluorinated alpha-keto acid derivatives [PhCHFCOCO2R,PhCH2CHFCOCO2R,PhCF2-COCO2R, and PhCH2CF2COCO2R (R = H, Me, and Et)] was synthesized. They were inhibitors of chymotrypsin, with Ki values ranging from 4700 to 15 microM. Benzylpyruvic derivatives were generally more potent than the corresponding phenylpyruvic analogs. Esters of the first series were also more potent than their corresponding acids, and potency increased with the number of fluorine atoms. By replacing the ethoxy group of PhCH2CF2COCO2Et (15b) with an amino acid chain (i.e., alanyl-leucyl-arginine methyl ester hydrochloride and alanyl-leucyl-valine ethyl ester), the resultant peptides PhCH2CF2COCO-Ala-Leu-Arg-OMe.HCl.H2O (20) and PhCH2CF2COCO-Ala-Leu-Val-OEt.H2O (23) were found to be slow-binding inhibitors of chymotrypsin with considerably lower Ki values (0.19 and 3.6 microM, respectively). 19F NMR studies indicate, in the case of 20, the presence of an enzyme-inhibitor complex with a hemiketal structure similar to those observed between trifluoromethyl ketones and chymotrypsin. The results illustrate that effective protease inhibitors can be designed by enhancing the electrophilic character of the reactive carbonyl group (with an electron-withdrawing group placed on each side of the carbonyl group). Their potency and/or selectivity can also be improved by taking advantage of binding interactions at S' subsites of the protease.
2. Mild, orthogonal solid-phase peptide synthesis: use of N alpha-dithiasuccinoyl (Dts) amino acids and N-(iso-propyldithio)carbonylproline, together with p-alkoxybenzyl ester anchoring linkages
F Albericio, G Barany Int J Pept Protein Res. 1987 Aug;30(2):177-205. doi: 10.1111/j.1399-3011.1987.tb03327.x.
Several N alpha-dithiasuccinoyl (Dts) amino acids (1) have been esterified without racemization by use of either N,N'-dicyclohexylcarbodiimide or 1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride, each in the presence of 4-dimethylaminopyridine (0.1 equiv.), to 2, 4, 5-trichlorophenyl 4'-hydroxymethylphenoxyacetate (10) or the corresponding propionate (11). The resultant handle derivatives (8,9) were purified and then quantitatively attached onto aminomethyl supports by couplings using as solvent N,N-dimethylformamide containing 1-hydroxybenzotriazole (0.1 M). This methodology facilitates anchoring of Dts-amino acids as p-alkoxybenzyl esters, which can be cleaved in good yields by trifluoroacetic acid-dichloromethane (1:1) at 25 degrees. Model experiments established that quantitative thiolytic removal (greater than 99.8%) of the Dts group occurs with (i) beta-mercaptoethanol (0.5 M)-N,N-diisopropylethylamine (0.5 M) in dichloromethane, 2 X 2 min; (ii) N-methylmercaptoacetamide (0.5 M)-N-methylmorpholine (0.5 M) in dichloromethane, 2 X 2 min; and (iii) N-methylmercaptoacetamide (0.5 M)-1-hydroxybenzotriazole (0.1 M) in N,N-dimethylformamide, 2 X 2 min. The susceptibility of the Dts functionality to nucleophiles was also defined, including demonstration of tertiary amine-catalyzed hydantoin formation from Dts-dipeptidyl units, but side reactions from these processes are entirely avoided under appropriate conditions relevant to peptide synthesis. These observations were exploited to devise efficient, racemization-free solid-phase syntheses of a number of model peptides in high yields and purities, including L-leucyl-L-alanylglycyl-L-valine, H-Gly6-Val-OH, H-Met-Ala-Gly-OH, methionine-enkephalin, and bradykinin.