Photoreactive derivatives of corticotropin. 1. Preparation and chracterization of 2,4-dinitro-5-azidophenylsulfenyl derivative of corticotropin

A photoaffinity probe for corticotropin (ACTH) receptors was prepared by selective modification of the single tryptophan residue in ACTH. A new photoreactive agent, 2,4-dinitro-5-azidophenylsulfenyl chloride, was synthesized and used for introducing the photoreactive group into ACTH. 2,4-Dinitro-5-azidophenylsulfenyl-Trp9-ACTH (DNAPS-ACTH) was also prepared by thiolysis of 2,4-dinitrophenyl-sulfenyl-Trp9-ACTH to form 2-thiol-Trp9-ACTH and reaction of this with 2,4-dinitro-5-azidofluorobenzene. DNAPS-ACTH was characterized by ultraviolet spectra, peptide mapping, and amino acid analysis. Covalent attachment of DNAPS-ACTH to a pituitary protein fraction FI by photolysis was demonstrated by ultraviolet absorption changes as well as by the use of tritiated DNAPS-ACTH.     

Redox-active dinitrodiphenylthioethers against Leishmania: Synthesis,structure–activity relationships and mechanism of action studies

BTB 06237 (2-[(2,4-dichloro-5-methylphenyl)sulfanyl]-1,3-dinitro-5-(trifluoromethyl) benzene), a compound previously identified through QSAR pharmacophore development and a virtual screen of the Maybridge database, possesses potent and selective activity against Leishmania parasites. In the present study, several analogs of BTB 06237 were synthesized and analyzed for activity against Leishmania axenic amastigotes, their ability to reduce the level of parasitemia in peritoneal macrophages, and their ability to generate reactive oxygen species (ROS) in L. donovani promastigotes. It was found that an aromatic ring must be present in the position occupied by the 2,4-dichloro-5-methylphenyl group in the lead compound, but changing the functional groups generally has little effect on the antileishmanial potency. Alterations to the 1,3-dinitro-5-(trifluoromethyl)benzene ring have more influence on antiparasitic activity with two aromatic nitro groups and a third electron-withdrawing group being required. This structural requirement corresponds with redox potential, the ability to generate ROS in the parasites, and dissipation of the mitochondrial membrane potential. Finally, we used this collection of data to design a new antileishmanial compound with strong activity in vitro and improved properties as an antileishmanial candidate.     

Greenhouse evaluation of chemicals for control of powdery mildews

2,6-Dinitro-4-s-alkylphenols were found to protect apple foliage against powdery mildew more effectively than isomeric 2,4-dinitro-6-s-alkylphenols; regression lines for four para-alkyl compounds were of similar slope but were much steeper than those for two ortho-alkyl compounds (including dinocap phenol). The ED₅₀ and ED₉₅ values of the most active compound studied, 2,6-dinitro-4-(I-ethylhexyl)phenol, were in the ratios to those of dinocap phenol of 1:13 and 1:125, respectively, and the protective action of this compound was greater than the curative, especially at the higher ED values. Protection of barley seedlings against powdery mildew was also greater by I-ethyl- or I-propyl-alkyl compounds than by I-methylalkyl or n-alkyl isomers. For phytotoxic and acaricidal actions, the ortho-alkyl isomers are more effective than para-alkyl. Nevertheless, the acaricidal activity of dinocap phenol is exceeded by that of the isomeric 2,4-dinitro-6-(I-ethylhexyl) phenol. The control of these powdery mildew diseases given by commercial products, supposed to be based on dinocap, cannot be accounted for by the activity shown for dinocap phenol. It can, however, be accounted for by the activities of 2,6-dinitro-4-(I-ethylhexyl)- and -(I-propylpentyl)-phenols, now known to be present in commercial products in larger amounts than dinocap phenol itself. The phytotoxic and acaricidal actions of such products, however, are mainly due to the ortho-octyldinitrophenols present; in view of the small proportion that is dinocap phenol, the acaricidal activity is likely to be due almost entirely to the other ortho-octyl isomers. It is suggested that the common name dinocap be retained for the mixture of dinitrooctylphenols now known to be present in commercial products, and that the two main groups be differentiated as 2,4-dinocap and 2,6-dinocap, respectively. The advantages for powdery mildew control of a product based on 2,6-dinocap are discussed.     

A new arylating agent, 2-carboxy-4,6-dinitrochlorobenzene. Reaction with model compounds and bovine pancreatic ribonuclease.

The reagent 2-carboxy-4,6-dinitrochlorobenzene (CDNCB) reacts with the imino, amino and sulfhydryl groups of model compounds. At pH 8.2, sulfhydryl groups react much faster than do amines. N alpha-Acetylhistidine, N alpha-acetyltyrosine and N alpha-acetyltryptophan do not react. Poly(L-Lysine) and poly(DL-lysine) react about 50 times as fast as does N alpha-acetyllysine. A dichloroanalog, 6-carboxy-2,4-dinitro-1,3-dichlorobenzene, shows stepwise reactivity with amines. With bovine pancreatic ribonuclease, which contains no sulfhydryl, CDNCB reacts preferentially with the epsilon-amino of Lys-41 at 450 times the rate with the epsilon-amino of N alpha-acetyllysine. The preferential reactivity at Lys-41 is discussed in relation to the pK of Ly-41, the cationic character of the active site cleft, and the mechanism of RNAase action on substrates.     

Immobilized enzymes: the catalytic properties of lactate dehydrogenase convalently attached to glass beads

Chick LDH (H₄ and M₄) has been covalently attached to aryl and alkyl amine glass using sodium nitrite and glutaraldehyde respectively. These immobilized enzymes remain active for months at 0°C and exhibit Km values similar to those of the soluble enzyme; however, they have pH-rate profiles that are independent of pH and show decreased substrate inhibition. Disaggregation followed by reassociation indicate the enzymes are bound by all four subunits and the resulting activity restored to the native, aryl amine and glutaraldehyde bound enzyme are 33, 25 and 90% respectively. At a pH of 3.2 and 25°, the soluble and aryl amine glass LDH's are rapidly denatured while the glutaraldehyde bound enzyme shows no loss of activity for at least 35 days.     

Enzymatic dehalogenation of chlorinated nitroaromatic compounds.

4-Chlorobenzoate dehalogenase from Pseudomonas sp. strain CBS3 converted 4-chloro-3,5-dinitrobenzoate to 3,5-dinitro-4-hydroxybenzoate and 1-chloro-2,4-dinitrobenzene to 2,4-dinitrophenol. The activities were 0.13 mU/mg of protein for 4-chloro-3,5-dinitrobenzoate and 0.16 mU/mg of protein for 1-chloro-2,4-dinitrobenzene compared with 0.5 mU/mg of protein for 4-chlorobenzoate.     

Enzymatic dehalogenation of chlorinated nitroaromatic compounds

4-Chlorobenzoate dehalogenase from Pseudomonas sp. strain CBS3 converted 4-chloro-3,5-dinitrobenzoate to 3,5-dinitro-4-hydroxybenzoate and 1-chloro-2,4-dinitrobenzene to 2,4-dinitrophenol. The activities were 0.13 mU/mg of protein for 4-chloro-3,5-dinitrobenzoate and 0.16 mU/mg of protein for 1-chloro-2,4-dinitrobenzene compared with 0.5 mU/mg of protein for 4-chlorobenzoate.     

Screening of new 2,4- and 2,6-dinitroaniline derivates for phytotoxicity and antimitotic activity

Screening of antimitotic activity and phytotoxicity of new 2,4- and 2,6-dinitroaniline derivates has been performed using the Allium-test. All substances that are derivatives of 2,4-dinitroanilines, 2,6-dinitro-(4-fluoromethyl)-aniline, and of (methylsulfonyl) nitrobenzol, proved to change the value of the mitotic index, cause cytogenetic disorders, and have a phytotoxic effect on the roots of Allium cepa seedlings. The results obtained suggest that N-(2,4-dinitrophenyl)-orto-aminobenzoic acid, N, N, -diethyl-2,6-dinitro-4-(trifluoromethyl)-aniline, and 1-methylsulfonyl-3-nitrobenzol are potential herbicides; further studying of their effects is proposed.     

Combinatorial synthesis of SSAO inhibitors using sonogashira coupling: SAR of aryl propargylic amines

The structure-activity relationships for semicarbazide-sensitive amine oxidase (SSAO) inhibitors based on arylpropynylamines was investigated using solution-phase combinatorial Sonogashira coupling. The results suggest that binding to the active site occurs by coordination of the amine to the proximal copper(II) and formation of a pi-complex between topaquinone and the electron-rich aryl group of the inhibitor.     

Cytochrome c interactions with membranes. A photoaffinity-labeled cytochrome c.

The aryl azide, 2,4-dinitro-5-fluorophenylazide, was reacted with horse heart cytochrome c to give a photoaffinity-labeled derivative of this heme protein. The modified cytochrome c, with one to two dinitroazidophenyl groups per mole of the enzyme, has a half-reduction potential the same (± 10 mV) as native cytochrome c. The dissociation constant for the modified cytochrome c from cytochrome c-depleted mitochondrial membranes and the apparent K_m for the reaction with cytochrome c oxidase were each five to six times greater than the values for native cytochrome c. Irradiation of cytochrome c-depleted mitochondrial membranes supplemented with an excess of photoaffinity-labeled cytochrome c resulted in covalent binding of the derivative to the mitochondrial membranes. Fractionation of the irradiated mitochondria in the presence of detergents and salts followed by chromatography on agarose, Bio-Gel A, showed that labeled cytochrome c was bound covalently to cytochrome c oxidase in a 1:1 molar complex. The covalently linked cytochrome c-cytochrome c oxidase complex was active in mediating the electron transfer between N,N,N′,N′-tetramethyl-p-phenylenediamine/ascorbate and the oxidase.     

Reactions of chitosan: 5. The reaction of chitosan with 2,4-dinitrofluorobenzene and determination of the extent of the reaction

The reaction between chitosan and 2,4-dinitrofluorobenzene has been studied and suitable conditions established for hydrolysis of the product prior to determination of the extent of reaction by u.v./visible spectroscopy. The chromophore system in $\text{N-(2,4-}\text{dinitrophenyl}\text{)-2-}\text{amino}\text{-2-}\text{deoxy-}\text{d}\text{-glucose}$, the final product from the acid hydrolysis of N-(2,4-dinitrophenyl)chitosan, is unstable to heating in solution in either water or aqueous acid. The temperature of hydrolysis should therefore not exceed 50°C and at this temperature the u.v./visible absorption spectrum of $\text{N-(2,4-}\text{dinitrophenyl}\text{)-2-}\text{amino}\text{-2-}\text{deoxy-}\text{d}\text{-glucose}$ is constant for up to 50 h. Complete reaction of the amine groups is not achieved under heterogeneous or homogeneous conditions, only approximately 50% of the available amine groups undergoing reaction under homogeneous conditions. This restricted reactivity results from the bulky N-(2,4-dinitrophenyl) residues shielding adjacent unreacted amine groups on the same chain, thereby preventing their reaction with 2,4-dinitrofluorobenzene. Such intramolecular steric hindrance would be expected to increase with increase in the free amine group content of the sample, due to the increase in the fraction of amine groups occurring in sequence length of two or more, and an inverse relationship between the total initial free amine group content and the percentage of these that react with 2,4-dinitrofluorobenzene has been found     

Antigenic reactivity: Molecular delineation of monohaptenic anaphylactogens

Anaphylactogenic, monohaptenic conjugates carrying a 2,4-dinitro-5-carboxyphenyl substituent as haptenic group and either one of two types of auxiliary groups essential for their anaphylactogenicity were studied. The “hydrocarbon auxiliary groups” require the presence of a hydrocarbon structure such as aliphatic chains of discrete length or planar rings such as provided by phenyl- or nicotinoyl residues and become particularly effective in conjunction with a carboxylate group. The benzylpenicilloyl group is an effective auxiliary structure, but the thiazolidine ring as such is not. It appears that the distance between haptenic and hydrocarbon auxiliary groups can be quite large. The “carbohydrate auxiliary group” becomes effective via a different mechanism. It requires disaccharide residues or two closely connected monosaccharides. Single monosaccharides are ineffective. A concept of interest with regard to drug allergy is the possibility that attachment of a single haptenic molecule to a glycoprotein constitutes an anaphylactogen.     

A DFT study of aminonitroimidazoles

Density functional theory (DFT) calculations at the B3LYP/aug-cc-pVDZ level were performed to explore the geometric and electronic structures, band gaps, thermodynamic properties, densities and performances of aminonitroimidazoles. The calculated performance properties, stabilities and sensitivities of the model compounds appear to be promising compared with those of the known explosives 2,4-dinitro-1H-imidazole (2,4-DNI), 1-methyl-2,4,5-trinitroimidazole (MTNI), hexahydro-1,3,5-trinitro-1,3,5-triazinane (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocane (HMX). The position of the NH₂ or the number of NO₂ groups on the diazole presumably determines the structure, heat of formation, stability, sensitivity, density and performance of the compound.     

Determination of the chirality of amino acid residues in the course of subtractive Edman degradation of peptides.

A chiral reagent, 1-fluoro-2,4-dinitro-5-L-alanine, was synthesized for the analysis of enantiomeric mixtures of amino acids after precolumn derivatization. The resulting diastereomers can be separated and quantitated by microbore RP-HPLC. These derivatives are relatively stable under the conditions used for acid hydrolysis of peptide bonds. Thus, this reagent was included in the protocol of a subtractive Edman degradation procedure of peptides to determine the sequence position of amino acid residues with concomitant identification of their chirality at a nanomolar level.     

Specificity of guinea pig liver transglutaminase for amine substrates.

The amine specificity of guinea pig liver transglutaminase, a model enzyme for endo-gamma-glutamine:epsilon-lysin transferases, was explored with the aid of synthetic substrates of high apparent affinities. As exemplified by dansyl- (5-dimethylamino-1-naphthalenesulfonyl), (2,4-dinitrobenzenesulfonyl)-, and (2,4,6-triisopropylbenzenesulfonyl)-cadaverines--each of which showed affinities of approximately 4 x 10(7) M-1--the best amine substrates carried a large hydrophobic substituent attached to an alkylamine side chain of about 7.2 A in length. Altogether, our results point to the importance of a hydrophobic binding region in the enzyme from where the alkyl side chain reaches into a narrow crevice toward the active center and positions the primary amine of the substrate for attacking the carbonyl group of the acyl enzyme intermediate.     

Synthesis and antihyperglycemic evaluation of new 2,4-thiazolidinediones having biodynamic aryl sulfonylurea moieties

New 2,4-thiazolidinediones with aryl sulfonylurea moieties have been synthesized by condensing various substituted sulfonamides and 5-(isocyanatomethyl) thiazolidino-2,4-dione. The isocyanomethyl thiazolidinedione was obtained by using the Curtius rearrangement, starting from known 2,4-dioxo-5-thiazolidineacetic acid. The newly synthesized compounds have been evaluated for the antihyperglycemic activity in normal rats model and among these compounds showed significant antihyperglycemic activity in sucrose loaded rat model.     

Electrophilic amination of a single methionine residue located at the active site of D-amino acid oxidase by O-(2,4-dinitrophenyl)hydroxylamine

O-(2,4-Dinitrophenyl)hydroxylamine is a rapid active-site-directed inhibitor of D-amino acid oxidase: modification results in specific incorporation of an amine group into an accessible nucleophilic residue with concomitant release of 2,4-dinitrophenol. The reaction is prevented by the competitive inhibitor benzoate, indicating an active-site-directed reaction. A stoichiometry of 1-1.5 mol of amine residues per enzyme bound flavin adenine dinucleotide monomer was observed at pH 7.0. Amino acid and sequence analyses show that His-217 is not the target of the modification reaction. Dependence of the modification on pH, model studies on functional groups present on amino acids, and thiolysis studies on aminated enzyme collectively indicate that the modification is located on a methionine residue at or near the active site of the enzyme. Aminated enzyme, although spectrally similar to native enzyme, exhibits a 7-9-nm blue shift in the 455-nm flavin absorption. Benzoate perturbs the spectrum of aminated enzyme, but binding relative to native enzyme is much weaker (Kd ca. 300 times greater at pH 8.0).     

Structural and enzyme activity studies demonstrate that aryl substituted 2,3-butadienamine analogs inactivate Arthrobacter globiformis amine oxidase (AGAO) by chemical derivatization of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor

Copper amine oxidases (CAOs) are a family of redox active enzymes containing a 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor generated from post translational modification of an active site tyrosine residue. The Arthrobacter globiformis amine oxidase (AGAO) has been widely used as a model to guide the design and development of selective inhibitors of CAOs. In this study, two aryl 2,3-butadienamine analogs, racemic 5-phenoxy-2,3-pentadienylamine (POPDA) and racemic 6-phenyl-2,3-hexadienylamine (PHDA), were synthesized and evaluated as mechanism-based inactivators of AGAO. Crystal structures show that both compounds form a covalent adduct with the amino group of the substrate-reduced TPQ, and that the chemical structures of the rac-PHDA and rac-POPDA modified TPQ differ by the allenic carbon that is attached to the cofactor. A chemical mechanism accounting for the formation of the respective TPQ derivative is proposed. Under steady-state conditions, no recovery of enzyme activity is detected when AGAO pre-treated with rac-PHDA or rac-POPDA is diluted with excess amount of the benzylamine substrate (100-fold K(m)). Comparing the IC(50) values further reveals that the phenoxy substituent in POPDA offers an approximately 4-fold increase in inhibition potency, which can be attributed to a favourable binding interaction between the oxygen atom in the phenoxy group and the active site of AGAO as revealed by crystallographic studies. This hypothesis is corroborated by the observed >3-fold higher partition ratio of PHDA compared to POPDA. Taken together, the results presented in this study reveal the mechanism by which aryl 2,3-butadienamines act as mechanism-based inhibitors of AGAO, and the potency of enzyme inactivation could be fine-tuned by optimizing binding interaction between the aryl substituent and the enzyme active site.     

Effect of nucleotide binding on the proximity of the essential sulfhydryl groups of myosin. Chemical probing of movement of residues during conformational transitions.

The reaction of myosin with three bifunctional sulfhydryl reagents of differing cross-linking span is reported. In the absence of nucleotide only p-N,N'-phenylenedimaleimide with a cross-linking span of 12-14 A can bridge between the two essential sulfhydryls of myosin. The other two reagents, 2,4-dinitro-1,5-difluorobenzene and 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone with cross-linking spans of 3-5 and 7-10 A, respectively, react under identical conditions with the SH1 sulfhydryl but do not bridge to the SH2 group. In the presence of MgADP, both p-N,N'-phenylenedimaleimide and 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone bridge across the SH1 and SH2 groups indicating a closer proximity of these two sulfhydryls in the presence of bound nucleotide. These results are discussed in relation to the conformational change induced in myosin by binding of the nucleotide.     

Chemical modification of acetylcholinesterase from eel and basal ganglia: effect on the acetylcholinesterase and aryl acylamidase activities

The effect of chemical modification on the acetylcholinesterase and the aryl acylamidase activities of purified acetylcholinesterase from electric eel and basal ganglia was investigated in the presence and absence of acetylcholine, the substrate of acetylcholinesterase, and 1,5-bis[4-(allyldimethylammonium)phenyl]pentan-3-one dibromide (BW284C51), a reversible competitive inhibitor of acetylcholinesterase. Trinitrobenzenesulfonic acid, pyridoxal phosphate, acetic anhydride, diethyl pyrocarbonate, and 2-hydroxy-5-nitrobenzyl bromide under specified conditions inactivated both acetylcholinesterase and aryl acylamidase in the absence of acetylcholine and BW284C51. Chemical modifications in the presence of acetylcholine and BW284C51 by all the above except diethyl pyrocarbonate selectively prevented the loss of acetylcholinesterase but not aryl acylamidase activity; modification by diethyl pyrocarbonate in the presence of acetylcholine and BW284C51 prevented the loss of both acetylcholinesterase and aryl acylamidase activities. Treatment with N-acetylimidazole resulted in the inactivation of acetylcholinesterase and the activation of aryl acylamidase. These changes in both the activities could be prevented by acetylcholine and BW284C51. Modification by phenylglyoxal, 2,4-pentanedione, or N-ethylmaleimide did not affect the enzyme activities. Indophenylacetate hydrolase activity followed a pattern similar to that of acetylcholinesterase in all the above modification studies. The results suggested essential lysine, tyrosine, tryptophan, and histidine residues for the active center of acetylcholinesterase and essential lysine, histidine, and tryptophan residues for the active center of aryl acylamidase.(ABSTRACT TRUNCATED AT 250 WORDS)