Xingliang Lu

Inactivation of mitochondrial monoamine oxidase B by methylthio-substituted benzylamines

Mitochondrial monoamine oxidase was inactivated by o-mercaptobenzylamine (1) and o- (2) and p-methylthiobenzylamine (5). Experiments were carried out to provide evidence for possible mechanisms of inactivation. The corresponding o- (3) and p-hydroxybenzylamine (4) are not inactivators. Four radiolabeled analogues of 2 and 5, having radioactivity at either the methyl or benzyl groups, were synthesized, and all were shown to incorporate multiple equivalents of radioactivity into the enzyme. Inactivation in the presence of an electrophile scavenger decreased the number of molecules incorporated, but still multiple molecules became incorporated; catalase did not further reduce the number of inactivator molecules bound. Two inactivation mechanisms are proposed, one involving a nucleophilic aromatic substitution (SNAr) mechanism and the other a dealkylation mechanism. Evidence for both mechanisms is that inactivation leads to reduction of the flavin (oxidation of the inactivator), but upon denaturation the flavin is reoxidized, indicating that attachment is not at the flavin. A cysteine titration indicates the loss of four cysteines after inactivation and denaturation. Support for the SNAr mechanism was obtained by showing that o- and p-chlorobenzylamine also inactivate MAO. Chemical model studies were carried out that also support both SNAr and dealkylation mechanisms.

Inactivation of monoamine oxidase B by benzyl 1-(aminomethyl)cyclopropane-1-carboxylate

Monoamine oxidase (MAO) is a flavoenzyme that catalyzes the oxidation of various biogenic and xenobiotic amines. Benzyl 1-(aminomethyl)cyclopropane-1-carboxylate (1) was designed as a diactivated cyclopropane mechanism-based inactivator of MAO (Silverman, R.B.: Ding, C.Z.; Borrillo, J.L.; Chang, J.T.J. Am. Chem. Soc. 1993, 115, 2982). [1,1-2H2]-1 exhibits a deuterium isotope effect of 4.5 on inactivation, but in D2O the isotope effect is only 2.3. [1-3H]-1 and [1-14C]-1 were synthesized; upon inactivation of MAO, 1.1 and 2.0 equiv of radioactivity, respectively, are incorporated into the enzyme. Tritium as 3H2O, is released during inactivation with [1-3H]-1. The flavin absorption spectrum changes from that of oxidized to that of reduced flavin after inactivation; denaturation of the inactivated enzyme shows a reduced flavin spectrum, suggesting the formation of a modified flavin. Tryptic digestion of the enzyme labeled with [1-3H]-1 or [1-14C]-1, followed by HPLC analysis, monitoring at 310 nm [corrected] (flavin), shows that the radioactivity comigrates with the 310 nm [corrected] absorptions. The metabolites that are generated during inactivation are benzyl 1-formylcyclopropane-1-carboxylate, benzyl alcohol, and 1-formylcyclopropane-1-carboxylic acid; no ring-cleaved products were detected. The partition ratio, as determined from the ratio of nonamines to enzyme, is 110. These results are rationalized in terms of a single-electron transfer mechanism leading to the imine of benzyl 1-formylcyclopropane-1-carboxylate, which alkylates the flavin coenzyme.

Purification and inactivation of 3-hydroxyanthranilic acid 3,4-dioxygenase from beef liver.

3-Hydroxyanthranilic acid 3,4-dioxygenase (EC 1.13.11.6; HADO) was purified to homogeneity from beef liver with the use of two dye columns (Cibacron Blue and Reactive Green 19) and hydroxyapatite. Two active peaks of enzyme were isolated from the hydroxyapatite column or by nondenaturing chromatofocusing of the enzyme prior to hydroxyapatite. The two active forms moved with different electrophoretic mobilities when they were subjected to nondenaturing polyacrylamide gel electrophoresis, regardless of the method of isolation. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), however, these species had apparently identical mobilities and have, therefore, close molecular mass. Analysis by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry gave them a molecular mass of 32566 and 32515 Da, respectively, for the species with apparent pI values of 5.60 and 4.98, respectively, suggesting that they differ only in the presence or absence of the iron cofactor. The N-terminal group appears to be blocked as no amino-terminal sequence was possible from direct Edman degradation. A new inactivator of the enzyme, 6-chloro-3-hydroxyanthranilic acid, was synthesized and was shown to exhibit time-dependent inactivation. A possible mechanism for inactivation is proposed.

Syntheses of amino nitrones. Potential intramolecular traps for radical intermediates in monoamine oxidase-catalyzed reactions.

Monoamine oxidase (MAO) is a flavin-dependent enzyme that catalyzes the oxidative deamination of a variety of amine neurotransmitters and toxic amines. Although there have been several studies that support the intermediacy of an amine radical cation and an alpha-radical during enzyme catalysis, there is no direct, i.e. EPR, evidence for these species as they are formed. Amino nitrones have been designed which, upon radical formation would produce an intermediate that is a resonance structure of the corresponding nitroxyl radical, which should be observable by EPR spectroscopy. Syntheses of seven different amino nitrones, three acyclic, and four cyclic analogues were attempted. The protected amino nitrones were stable, but all three of the acyclic amino nitrones were unstable. One of the cyclic analogues was very stable (39), one was stable only in organic solvents (40), one was stable only in aqueous medium below pH 6.5 (41), and the other (42) was stable for just a short time at room temperature, decomposing to a stable free radical. None of these analogues produced a MAO-catalyzed radical, yet 41 is a poor substrate (Km=0.2mM; k(cat) = 0.034 min-1) and 39 is a mixed inhibitor (Ki = 26.5 mM). Although this approach does not appear to be applicable to amino nitrones, it should be a valuable approach for other enzymes where radical intermediates are suspected and nonamine nitrones can be utilized.

Selective Inhibition of Monoamine Oxidase B by Aminoethyl Substituted Benzyl Ethers.

Aminoethyl 3-chlorobenzyl ether was shown previously (Ding, C.Z. and Silverman, R.B. (1993). Bioorg. Med. Chem. Lett., 3, 2077-2078) to be a potent and selective time-dependent, but reversible inhibitor of monoamine oxidase B (MAO B). Based on this result, a series of novel aminoethyl substituted benzyl ethers was synthesized and the compounds were examined as potential inhibitors of both isozymic forms of MAO. Each compound in the series inhibits both MAO A and MAO B competitively, and IC(50) values for each compound were determined. In general, the B isozyme is much more sensitive to these inhibitors than the A isozyme (except for the o- and p-substituted nitro analogues), in some cases by more than two orders of magnitude. The selectivity in favor of MAO B inhibition is relatively high for all of the meta-substituted analogues and quite low for all of the ortho-substituted analogues. Having the substituent at the ortho-position is most favorable for MAO A inhibition. With MAO B the meta-analogues were, in general, more potent than the corresponding ortho- and para-analogues with respect to their reversible binding constants. The meta-iodo analogue is the most potent analogue.

Anomalous Schmidt reaction products of phenylacetic acid and derivatives

Treatment of carboxylic acids with sodium azide in sulfuric acid normally results in decarboxylation with conversion of the carboxylic acid to an amine (the Schmidt reaction). However, many side reactions have been reported to occur, particularly in the case of α-aryl carboxylic acids, such as sulfonation, direct amination of the phenyl ring, cyclization to a lactam, and elimination of side chains to give aniline. In this study, the reactions of a variety of analogues of phenylacetic acid under given reaction conditions are examined to determine which characteristics are important in the competing side reactions. Some reactions were carried out with TEMPO free radical as a radical scavenger to investigate whether direct amination proceeds by a radical intermediate. Phenylacetic acid is shown to give an ortho-aminated diamine product instead of the para-aminated one expected from direct amination. A mechanism for this side reaction, involving cyclization to a lactam intermediate followed by further cleavage, is proposed; an analogue of the hypothetical intermediate has been isolated for biphenylacetic acids.

Inactivation of monoamine oxidase B by cis- and trans-5-aminomethyl-3-(4-methoxyphenyl)dihydrofuran-2(3H)-ones.

Monoamine oxidase B was previously shown to be inactivated by cis- (3) and trans-5-(aminomethyl)-3-(4-methoxyphenyl)dihydrofuran-2(3H)-one hydrochloride (4) in a time-dependent manner (Ding, Z.; Silverman, R. B. J. Med. Chem. 1992, 35, 885) and to catalyze its oxidative decarboxylation (Silverman, R. B.; Zhou, J.J.P.; Ding, C. Z.; Lu, X. J. Am. Chem. Soc. 1995, 117, 12895). By [14C]-labeling of the aryl methoxyl groups of these two inactivators, it is shown that this is not a mechanism-based inactivation and that multiple enzyme residues are labeled.

The Anti-Ulcer Drug Ranitidine Hydrochloride and its Synthetic Intermediates are Inactivators of Monoamine Oxidase-B

AbstractRanitidine hydrochloride (1) and two of its synthetic precursors (2 and 3) were found to be time-dependent, irreversible inactivators of monoamine oxidase-B from beef liver.