Enzymatic synthesis of halogen derivatives of L-phenylalanine and phenylpyruvic acid stereoselectively labeled with hydrogen isotopes in the side chain.

Halogenated, labeled with deuterium, tritium or doubly labeled with deuterium and tritium in the 3S position of the side chain isotopomers of L-phenylalanine and phenylpyruvic acid were synthesized. Isotopomers of halogenated L-phenylalanine were obtained by addition of ammonia from isotopically enriched buffer solution to the halogenated derivative of (E)-cinnamic acid catalyzed by phenylalanine ammonia lyase. Isotopomers of halogenated phenylpyruvic acid were obtained enzymatically by conversion of the appropriate isotopomer of halogenated L-phenylalanine in the presence of phenylalanine dehydrogenase. As a source of deuterium was used deuterated water, as a source of tritium was used a solution of highly diluted tritiated water. The labeling takes place in good yields and with high deuterium atom% abundance.

Iron-catalyzed C2-C3 bond cleavage of phenylpyruvate with O2: insight into aliphatic C-C bond-cleaving dioxygenases.

Iron(II)-phenylpyruvate complexes of tetradentate tris(6-methyl-2-pyridylmethyl)amine (6-Me3-TPA) and tridentate benzyl bis(2-quinolinylmethyl)amine (Bn-BQA) were prepared to gain insight into C-C bond cleavage catalyzed by dioxygenase enzymes. The complexes we have prepared and characterized are [Fe(6-Me3-tpa)(prv)][BPh4] (1), [Fe2(6-Me3-tpa)2(pp)][(BPh4)2] (2), and [Fe2(6-Me3-tpa)2(2'-NO2-pp)][(BPh4)2] (3), [Fe(6-Me3-tpa)(pp-Me)][BPh4] (4), [Fe(6-Me3-tpa)(CN-pp-Et)][BPh4] (5), and [Fe(Bn-bqa)(pp)] (8), in which PRV is pyruvate, PP is the enolate form of phenylpyruvate, 2'-NO2-PP is the enolate form of 2'-nitrophenylpyruvate, PP-Me is the enolate form of methyl phenylpyruvate, and CN-PP-Et is the enolate form of ethyl-3-cyanophenylpyruvate. The structures of mononuclear complexes 1 and 5 were determined by single-crystal X-ray diffraction. Both the PRV ligand in 1 and the CN-PP-Et ligand in 5 bind to the iron(II) center in a bidentate manner and form 5-membered chelate rings, but the alpha-keto moiety is in the enolate form in 5 with concomitant loss of a C-H(beta) proton. The PP ligands of 2, 3, 4, and 8 react with dioxygen to form benzaldehyde and oxalate products, which indicates that the C2-C3 PP bond is cleaved, in contrast to cleavage of the C1-C2 bond previously observed for complexes that do not contain alpha-ketocarboxylate ligands in the enolate form. These reactions serve as models for metal-containing dioxygenase enzymes that catalyze the cleavage of aliphatic C-C bonds.

Design and synthesis of 3-fluoro-2-oxo-3-phenylpropionic acid derivatives as potent inhibitors of 4-hydroxyphenylpyruvate dioxygenase from pig liver.

Several rationally designed analogs of 3-fluoro-2-oxo-3-phenylpropionic acid were chemically synthesized, and the reactions of the hydrate form of these compounds with 4-hydroxyphenylpyruvate dioxygenase from pig liver as inhibitors were examined. Compounds 14a and 14b were found to be potent competitive inhibitors of the enzyme with Ki values of 10 and 22 microM, respectively.

Synthesis of isosteres of p-amidinophenylpyruvic acid. Inhibitors of trypsin thrombin, and pancreatic kallikrein.

A series of amino acids, amidino acids, and amidino esters was synthesized and the compounds were evaluated for their inhibitory activity against bovine trypsin, bovine thrombin, and porcine pancreatic kallikrein and as anticoagulants. Among these compounds, ethyl 4-amidino-2-iodophenoxyacetate was found to be the most effective inhibitor of the enzymes in question, with a potency (Ki = 3.16 x 10-6 M vs. trypsin; Ki = 4.8 x 10-5 M vs. thrombin) similar to that of p-amidinophenylpyruvic acid (Ki = 6.0 x 10-6 M vs. trypsin; Ki = 2.0 x 10-5 M vs. thrombin). Ethyl 4-amidino-2-iodophenoxyacetate was also found to be the most effective in blocking the clotting activity of plasma, as indicated by significant prolongation of the partial thromboplastin time. This paper reports the synthetic methods, the enzyme inhibitory activity, and the structure-activity relationships observed.

Metabolism and properties of 3-methoxy-4-hydroxyphenyl-pyruvate; a metabolite of dihydroxyphenylalanine.

The pathway of 3,4-dihydroxyphenylalanine undergoing metabolism via transamination and subsequent oxidative rearrangement to 2,4,5-trihydroxy-phenylacetate was investigated. 3-Methoxytyrosine does not pursue an identical course, since its corresponding keto acid is not subject to action of p-hydroxy-phenylpyruvate hydroxylase. The radiochemical synthesis of 3-methoxy-4-hydroxy[carboxy-14C] pyruvate was accomplished. This metabolite was used to demonstrate that this keto acid does not proceed through oxidative rearrangement both in vitro and in vivo. The keto acid was found to be a competitive inhibitor of the hydroxylase and can help account for some of the metabolites observed in the urine of patients treated with 3,4-dihydroxyphenylalanine.