Synthesis of N3-fumaramoyl-L-2,3-diaminopropanoic acid analogues, the irreversible inhibitors of glucosamine synthetase

Several analogues of N3-fumaramoyl-L-2,3-diaminopropanoic acid were synthesized and evaluated for inhibition of glucosamine-6-phosphate synthetase activity. The syntheses were accomplished by acylation reaction of N2-tert.-butoxycarbonyl-L-2,3-diaminopropanoic acid (Boc-A2pr) or N2-tert.-butoxycarbonyl-L-2,4-diaminobutanoic acid (Boc-A2-bu) with the N-succinimidoyl esters of several derivatives of alpha, beta-unsaturated acids 2a-d followed by deprotection of the Boc groups. The obtained compounds were tested for inhibition of glucosamine synthetase isolated from Salmonella typhimurium and Saccharomyces cerevisiae. The results indicated that among the synthesized compounds, N3-4-methoxyfumaroyl-L-2,3-diaminopropanoic acid (FMDP) was the most powerful inhibitor of glucosamine synthetase.     

The influence of L-norvalyl-N3-4-methoxyfumaroyl-L-2,3-diaminopropanoic acid, an antifungal agent, on mammalian cells in tissue culture.

Murine leukemia L1210 cells contain active glucosamine 6-phosphate synthase inhibited by N3-4-methoxyfumaroyl-L-2,3-diaminopropanoic acid (FMDP). However, FMDP-peptides do not exhibit any cytotoxicity against these cells, HeLa S3 human cervical carcinoma cells, and LL2 murine Lewis lung carcinoma cells. It is suggested that the lack of cytotoxicity of FMDP-peptides, in spite of good drug uptake and the presence of target enzyme might be due to the poor rate of peptides cleavage by the intracellular peptidases.     

Interaction of 14C-labelled amphotericin B derivatives with human erythrocytes: relationship between binding and induced K+ leak

Four 14C-labelled amphotericin B (Am B) derivatives with different net electric charges were examined: zwitterionic N-fructosyl Am B, positively charged N-fructosyl Am B methyl ester, negatively charged N-acetyl Am B and neutral N-acetyl Am B methyl ester. The binding of these four derivatives to human red cells and their octanol-water partition coefficients were measured. Simple partitioning between red cells and buffer was found for the four compounds, regardless of concentration, within a range of 10(-8) and 10(-4) M. This indicates the absence of cooperativity and saturability of binding at least in this concentration range. The constant partition coefficients were found to be three to five times higher for the two methyl ester derivatives than for the two non-esterified compounds. All partition coefficients were proportional to those found for the octanol-water system. Efficiency in inducing K+ leak from red cells was measured during the binding experiments. Despite the higher partition coefficients of the two methyl ester derivatives, they were found to have much lower ionophoric efficiency than the two non-esterified compounds. These results are discussed in terms of the mechanism of permeability pathway formation by polyene antibiotics.     

The role of the carboxyl and amino groups of polyene macrolides in their interactions with sterols and their selective toxicity. A P-NMR study

The permeability induced by amphotericin B and vacidin A derivatives in large unilamellar lipidic vesicles containing various sterols has been studied using the proton-cation exchange method and ³¹P-NMR spectroscopy. Derivatives which have a free ionizable carboxyl group induce biphasic ‘all or none’ permeability typical of channel-forming ionophores, whatever the sterol present. In sterol-free membranes, they have no significant activity. Derivatives which lack a free ionizable carboxyl group exhibit this channel-like mode of action only in membranes containing ergosterol or sterols with an alkyl side like that of ergosterol. In membranes containing cholesterol or sterol whose side-chain is alike, a slow and progressive permeability is observed at high concentrations. This activity is observed in sterol-free membranes as well. Derivatives containing sugars with substituted amino groups always have lower ionophoric activity than those which are unsubstituted. The greatest decrease in activity was observed for N-acetyl derivatives. Substitution of the amino groups has no effect on the mode of action. A model of interaction of polyenes with sterols is presented accounting for the data obtained on vesicles and the observed selective toxicity of polyene derivatives in biological membranes.     

Inactivation of glucosamine-6-phosphate synthetase from Salmonella typhimurium LT2 by fumaroyl diaminopropanoic acid derivatives, a novel group of glutamine analogs

A novel group of glutamine analogs, N3-fumaroyl-L-2,3-diaminopropanoic acid (FDP) and its derivatives and analogs including amide (FCDP), methyl ester (FMDP) and its homologue, N4-(4-methoxyfumaroyl)-L-2,4-diaminobutanoic acid, inactivate glucosamine-6-phosphate synthetase (L-glutamine: D-fructose-6-phosphate aminotransferase (hexose-isomerizing), EC 2.6.1.16), isolated from Salmonella typhimurium, by covalent modification. For comparative purposes, selected known glutamine analogs were also examined. Anticapsin, 6-diazo-5-oxo-L-norleucine and, at high concentration, azaserine inactivate the enzyme. The pseudo-first-order rate constants show a hyperbolic dependence on inhibitor concentration for all the above-mentioned inhibitors, suggesting the formation of a reversible complex prior to covalent modification. Dissociation constants for inhibitors were determined and ranged from 10(-4) M for FCDP to 10(-6) M for FMDP. Albizziin, gamma-glutamylhydroxamate and, at low concentration, azaserine inhibit glucosamine synthetase only reversibly. All inhibitors tested are competitive in relation to glutamine. and competitive inhibitors, albizziin and gamma-glutamylhydroxamate protect the enzyme against inactivation. Fructose 6-phosphate accelerates the rate of inactivation. Some analogs of FDP, such as SMDP, CRDP, O-FMSer, MMDP and AADP, are not active against glucosamine-6-phosphate synthetase. The structure-activity relationship of the novel group of glutamine analogs is discussed and structural requirements for the activity of these compounds is established. It is postulated that the compounds examined can be classified as mechanism-based enzyme inactivators.     

Low stimulation of NADH oxidation and oxygen consumption by 5-iminodaunorubicin and its derivatives

NADH oxidation and oxygen consumption mediated by 5-iminodaunorubicin and its derivatives have been studied. Experiments were carried out using two enzyme system: microsomes isolated from rat liver and cytochrome c-reductase. All 5-iminoanthracyclines examined were relatively poor electron transfer mediators. In addition, 5-imino-derivatives of daunorubicin modified at sugar moiety were less effective in stimulating NADH oxidation and oxygen radical production than 5-iminodaunorubicin itself. This may be due to the additive effect of 5-imino structure and the presence of bulky substituents at sugar moiety, which might make the compounds poor enzyme substrates.