Spectrophotometric determination of hydrazine, hydrazides, and their mixtures with trinitrobenzenesulfonic acid

A method has been developed to measure hydrazine, hydrazides, and their mixtures using a modification of the trinitrobenzenesulfonic acid method [T. Okuyama and K. Satake (1960) J. Biochem. (Tokyo) 47, 654-660]. After incubation of the sample containing hydrazine and hydrazide with trinitrobenzenesulfonate at pH 8.5 at room temperature for 40 min, the reaction mixture was diluted with a Na2CO3-NaHCO3 buffer (0.1 M, pH 10.8) rather than with 0.5 M HCl. Different chromogens were produced from the reaction of hydrazine (lambda max = 570 nm) and hydrazides (lambda max = 385 and 500 nm) with trinitrobenzenesulfonic acid. The method allowed simultaneous determination of hydrazine (5 to 60 nmol) with hydrazide (10 to 120 nmol) in a mixture with a standard deviation of less than 5%. The presence of amino compounds (except for amino sugars) did not interfere with the measurement of hydrazine or hydrazides. Interference by amino sugars in the determination of hydrazine or hydrazides was eliminated by pretreatment of the sample with NaBH4 to reduce the amino sugars to 2-amino-2-deoxy-hexitols.     

Hydrazide oligonucleotides: new chemical modification for chip array attachment and conjugation

We report the synthesis of new phosphoramidite building blocks and their use for the modification of oligonucleotides with hydrazides. The reaction of these hydrazide oligonucleotides with active esters and aldehydes is demonstrated for solution conjugation and immobilization. Compared with the established amino modified oligonucleotides, hydrazides show enhanced reactivity at neutral and acidic buffer conditions. One method to introduce hydrazides is using amidites with preformed, protected hydrazides. A completely novel approach is the generation of the hydrazide functionality during the oligonucleotide cleavage and deprotection with hydrazine. Therefore, building blocks for the introduction of esters as hydrazide precursors are described. For the enhanced attachment on surfaces branched modifier amidites, which introduce up to four reactive groups to the oligonucleotide, are applied. The efficiency of branched hydrazide oligonucleotides compared with standard amino modified oligonucleotides for the immobilization of DNA on active electronic Nanogen chips is demonstrated.     

Spectroscopic studies of the reaction between bovine serum amine oxidase (copper-containing) and some hydrazides and hydrazines.

The carbonyl cofactor of bovine serum amine oxidase, recently identified as pyrroloquinoline quinone [Ameyama, Hayashi, Matsushita, Shinagawa & Adachi (1984) Agric. Biol. Chem. 48, 561-565; Lobenstein-Verbeek, Jongejan, Frank & Duine (1984) FEBS Lett. 170, 305-309], reacts stoichiometrically and irreversibly with hydrazides of phenylacetic acid and of benzoic acid. With the phenylacetic hydrazides a reversible intermediate step was detected by competition with substrate, carbonylic reagents or phenylhydrazine, a typical inhibitor of the enzyme. All hydrazides form an intense broad band with maximum absorbance in a narrow wavelength range (350-360 nm), irrespective of the acyl group, suggesting that the transition is located on the organic cofactor. A different situation is found with some phenylhydrazines, where extended conjugation can occur between the cofactor and the phenyl pi-electron system via the azo group, as shown by the lower energy and higher intensity of the transition. In this case the transition is sensitive to substituents in the phenyl ring. The c.d. spectrum of the adducts is influenced by the type of hydrazide (derived from phenylacetic acid or benzoic acid), by pH and by NN-diethyldithiocarbamate binding to copper, probably as a result of shifts of equilibria between hydrazone-azo tautomers.     

Hydrazide derivatives produce active oxygen species as hydrazine

It is well documented that some hydrazines are quite sensitive to oxidation and may serve as the electron donor for the reduction of oxygen, whereas hydrazides are not believed to react directly with oxygen. Data presented in this paper show that both hydrazides and hydrazines share an N-N moiety, which is assumed to react with atmospheric oxygen and produce oxygen radicals, at various degrees of efficiency. Since spectrometric measurements of hydrazide just after solubilization showed that the molecular mass remains constant in the absence of oxygen, we can conclude that hydrazides do not react with the oxygen through a slow spontaneous hydrolytic release of hydrazine. However, hydrazine is more reactive than hydrazide, which requires hours rather than minutes to produce measurable quantities of radical species. Differences were also apparent for various substituted derivatives. The reaction was significantly enhanced by the presence of metal ions. Data reported here demonstrate that hydrazides cause irreversible damage to the prosthetic group of proteins as well as causing degradation of the polypeptide chain into small fragments.     

New Hydrazides and Hydrazide‐Hydrazones of 2,3‐Dihalogen Substituted Propionic Acids: Synthesis and in vitro Antimicrobial Activity Evaluation

The main aim of this research was the synthesis, spectral identification and in vitro antimicrobial evaluation of new hydrazides and hydrazide‐hydrazones of 2,3‐dihalogen substituted propionic acids. New hydrazides were obtained by the substitution reaction of appropriate ethyl esters of 2,3‐dihalogen substituted propionic acids with hydrazine hydrate. Then obtained hydrazides were subjected to condensation reaction with various aldehydes which yielded with new hydrazide‐hydrazone derivatives. All obtained compounds were identified on the basis of spectral methods (¹H‐NMR, ¹³C‐NMR) and in vitro screened against a panel of bacterial and fungal strains according to EUCAST and CLSI guidelines.     

Fluorescent hydrazides for the high-performance liquid chromatographic determination of biological carbonyls

Methods for the determination of carbonyl compounds of biological origin by high-performance liquid chromatography were improved by the use of new fluorescent derivatizing agents. Eight fluorescent hydrazides were either synthesized or obtained commercially and compared to dansyl hydrazine (1-dimethylaminonaphthalene-5-sulfonylohydrazide). Four of the compounds yielded carbonyl hydrazones with a higher relative fluorescence quantum yield than dansyl hydrazine in acetonitrile:water mixtures. Darpsyl hydrazide [(3-phenylpyrazoline-1-yl)-4-phenylsulfonylohydrazide] and apmayl hydrazide [N-(2-aminophenyl-6-methylbenzthiazole)-acetylohydrazide] both yielded an increase of greater than 20-fold in sensitivity over dansyl hydrazine in determinations of abscisic acid and jasmonic acid from plant tissues. Different hydrazides and derivatizing conditions were found to be optimum for the determination of different carbonyl compounds. Also, a simple method for precolumn purification of the hydrazones of acidic carbonyls was developed to remove contaminants arising during derivatization and from the tissue source.     

Hydrazinolysis of heparin and other glycosaminoglycans.

Heparin, carboxy-group-reduced heparin, several sulphated monosaccharides and disaccharides formed from heparin, and a tetrasaccharide prepared from chondroitin sulphate were treated at 100 degrees C with hydrazine containing 1% hydrazine sulphate for periods sufficient to cause complete N-deacetylation of the N-acetylhexosamine residues. Under these hydrazinolysis conditions both the N-sulphate and the O-sulphate substituents on these compounds were completely stable. However, the uronic acid residues were converted into their hydrazide derivatives at rates that depended on the uronic acid structures. Unsubstituted L-iduronic acid residues reacted much more slowly than did unsubstituted D-glucuronic acid or 2-O-sulphated L-iduronic acid residues. The chemical modification of the carboxy groups resulted in a low rate of C-5 epimerization of the uronic acid residues. The hydrazinolysis reaction also caused a partial depolymerization of heparin but not of carboxy-group-reduced heparin. Treatment of the hydrazinolysis products with HNO2 at either pH 4 or pH 1.5 or with HIO3 converted the uronic acid hydrazides back into uronic acid residues. The use of the hydrazinolysis reaction in studies of the structures of uronic acid-containing polymers and the implications of the uronic acid hydrazide formation are discussed.     

Lipase-catalyzed hydrazinolysis of phenyl benzoate: Kinetic modeling approach

Immobilized lipase-catalyzed synthesis of benzoic acid hydrazide from hydrazine and phenyl benzoate is reported in this work. A series of immobilized lipases such as Candida antarctica lipase B, Mucor miehei lipase and Thermomyces lanuginosus lipase were screened to establish that C. antarctica lipase B was the best lipase for hydrazinolysis. When phenyl benzoate (0.01 mol) and hydrazine (0.02 mol) in toluene (15 ml) were reacted with C. antarctica lipase B (Novozym 435) at 50 °C, 95% of phenyl benzoate was converted to benzoic acid hydrazide after 2 h. The effects of various parameters such as speed of agitation, concentration of the substrates, temperature, enzyme concentration, and reusability of the enzyme were studied to deduce kinetics and mechanism of the reaction. A mechanism based on an ordered bi–bi dead end complex with hydrazine was found to fit the data. Systematic deactivation studies indicated that the enzyme was deactivated due to the hydrazine and phenol, enzyme deactivation obeys first-order series model. The kinetic parameters deduced from these models were used to simulate the lipase activity. There was a very good agreement between the simulated and experimental values.     

Hydrazides of N-blocked amino acids as sole substrates with unique difunctional behavior under papain catalysis

Hydrazides of five N-acylamino acids have been used alone as substrates for papain catalysis to yield N¹,N²-diacylhydrazines. With the exception of N-(benzyloxycarbonyl)(Z)- -alanine hydrazide, they were very effective as both acylating agents of the enzyme and nucleophiles in attacking the enzyme-substrate intermediate. Although Z- -alanine hydrazide was a minimal acylating agent, it was a satisfactory nucleophile. The most favorable reaction involved Z- -alanine hydrazide in producing N¹,N²-bis(Z- -alanyl)hydrazine. When Z- -alanine hydrazide was the substrate, this same chiral diacylhydrazine was formed along with meso N¹-(Z- -alanyl)-N²-(Z- -alanyl)hydrazine. For the acylation step, the enzyme displayed powerful, essentially stereospecific, bias toward the enantiomer. Once the thioester intermediate was formed, little preference was detected for attack by the enantiomers as nucleophiles. The most direct procedure for synthesis of substrates was conversion of Z-amino acids to their esters by means of dry HCl in an absolute alcohol. Treatment with hydrazine produced the hydrazides in excellent yield.     

Effect of hydrazine, isonicotinic acid hydrazide, hydrazine sulfate, and dimethylhydrazine on guanylate cyclase activity.

The chemical carcinogen hydrazine is a potent stimulator of guanylate cyclase. In the present investigation we found that three chemical carcinogens structurally related to hydrazine, isonicotinic acid hydrazide, hydrazine sulfate, and dimethylhydrazine, decreased guanylate cyclase activity. It is of interest that hydrazine has been shown to increase DNA synthesis whereas isonicotinic acid hydrazide, hydrazine sulfate, and dimethylhydrazine decrease DNA synthesis. The relationship, if any, linking the guanylate cyclase-cyclic GMP system to DNA synthesis and carcinogenesis remains to be explored.     

Synthesis, biological evaluation and molecular docking of aryl hydrazines and hydrazides for anticancer activity

Aryl hydrazine and hydrazide analogues were synthesized based on p-tolyl hydrazine, isolated as a breakdown product of a secondary metabolite from the mushroom, Agaricus bisporus, and tested to be highly active molecule than 5-fluorouracil in in vitro anticancer studies. The synthesized analogues were tested for anticancer activity using NCI protocol. Anolgues 12 and 15 emerged as molecules with significant in vitro anticancer activity. Molecular docking study revealed the binding orientations of aryl hydrazines and hydrazides analogues in the active sites of thymidylate synthase.     

Effect of pyridoxal isonicotinoyl hydrazone and other hydrazones on iron release from macrophages, reticulocytes and hepatocytes

A model consisting of 59Fe-labelled macrophages was developed for screening potential iron-chelating drugs. Mouse peritoneal macrophages, induced by previous intraperitoneal injections of 3% thioglycollate, were labelled in vitro by their exposure to immune complexes of 59Fe-transferrin-antitransferrin antibody. Optimal conditions for macrophage labelling and subsequent 59Fe release were established. Sixty-two aromatic hydrazones, the majority of which had iron binding structures similar to pyridoxal isonicotinoyl hydrazone, were synthesized by condensation of aromatic aldehydes (pyridoxal, salicylaldehyde, 2-hydroxy-1-naphthylaldehyde and 2-furaldehyde) with various acid hydrazides prepared by systematic substitutions on the benzene ring. These compounds were examined for their potential to stimulate 59Fe release from 59Fe-labelled macrophages and also from reticulocytes and hepatocytes loaded with non-heme 59Fe. The majority of hydrazones derived from pyridoxal, salicylaldehyde and 2-hydroxy-1-naphthylaldehyde seemed to be equally effective in both the macrophage and reticulocyte testing systems. However, the pyridoxal hydrazones were much more active in hepatocytes than the other groups of hydrazones. Several compounds proved to be very potent in mobilizing 59Fe. These included hydrazones derived from 2-hydroxy-1-naphthylaldehyde and benzoic acid hydrazide, p-hydroxybenzoic acid hydrazide, 2-thiophenecarboxylic acid hydrazide, and also pyridoxal benzoyl hydrazone, pyridoxal m-fluorobenzoyl hydrazone and pyridoxal 2-thiophenecarboxyl hydrazone.     

Stereoselective action of acylated crude papain toward mandelic and atrolactic hydrazides

Acylated crude papain has been shown to exert stereoselective behavior toward racemic hydrazides devoid of an amino acid residue, namely, (RS)-mandelic and (RS)-atrolactic hydrazides. These hydrazides functioned as nucleophiles to yield N¹,N²-diacylhydrazines. Several achiral acylating agents for the enzyme were chosen, including Z-glycine, BOC-glycine, AOC-glycine, and hippuric acid. With the exception of hippuric acid as the acylating agent, the reaction product, in every instance for these achiral hydrazides, consisted of an excess of the (+)-N¹,N²-diacylhydrazine. The relative rates of product formation for the mandelic hydrazides were considerably greater than for corresponding reactions with racemic atrolactic hydrazide. When chiral Z-l-alanine was employed to acylate crude papain, the stereoselective action was most pronounced, with the formation of a mixture of diastereoisomers consisting of 73% N¹-(Z-l-alanyl)-N²-[(R)-mandelyl]hydrazine. The relative reactivities for the electrophiles was Z-l-alanine ? Z-glycine ? hippuric acid ? AOC-glycine > BOC-glycine. The hydrazides of (R)-, (S)-mandelic, and (RS)-atrolactic acids were prepared by conversion of the corresponding acids to their esters by means of a catalytic dehydrating agent and subsequent treatment with a methanolic solution of hydrazine.     

Molecular structure--activity relationship of hydrazides inhibiting glutamic acid decarboxylase, GABA-alpha-oxoglutarate aminotransferase, and monoamine oxidase activities in chick brain.

Several aryl and heteroaryl hydrazides were synthesized and evaluated for their inhibitory effects on glutamic acid decarboxylase (GAD), GABA-alpha-oxoglutarate aminotransferase (GABA-T), and monoamine oxidase (MAO) enzyme systems in chick brain 24 h after their intramuscular administration (0.75 mmol/kg). All compounds produced a reduction in GAD, GABA-T, and MAO activity. Structure-activity relationships indicated that the ring structure had a greater influence on the degree of GAD and GABA-T inhibition than did the N'-terminal group. In contrast, structural requirements for MAO inhibition were much more restrictive. The intramuscular administration of benzoic acid hydrazide to chicks 24 h prior to their being exposed to oxygen at high pressure provided significant protection against the onset of the hyperbaric oxygen-induced seizures.     

Hydrazidase,a Novel Amidase Signature Enzyme That Hydrolyzes Acylhydrazides

The degradation mechanisms of natural and artificial hydrazides have been elucidated. Here we screened and isolated bacteria that utilize the acylhydrazide 4-hydroxybenzoic acid 1-phenylethylidene hydrazide (HBPH) from soils. Physiological and phylogenetic studies identified one bacterium as Microbacterium sp. strain HM58-2, from which we purified intracellular hydrazidase, cloned its gene, and prepared recombinant hydrazidase using an Escherichia coli expression system. The Microbacterium sp. HM58-2 hydrazidase is a 631-amino-acid monomer that was 31% identical to indoleacetamide hydrolase isolated from Bradyrhizobium japonicum. Phylogenetic studies indicated that the Microbacterium sp. HM58-2 hydrazidase constitutes a novel hydrazidase group among amidase signature proteins that are distributed within proteobacteria, actinobacteria, and firmicutes. The hydrazidase stoichiometrically hydrolyzed the acylhydrazide residue of HBPH to the corresponding acid and hydrazine derivative. Steady-state kinetics showed that the enzyme hydrolyzes structurally related 4-hydrozybenzamide to hydroxybenzoic acid at a lower rate than HBPH, indicating that the hydrazidase prefers hydrazide to amide. The hydrazidase contains the catalytic Ser-Ser-Lys motif that is conserved among members of the amidase signature family; it shares a catalytic mechanism with amidases, according to mutagenesis findings, and another hydrazidase-specific mechanism must exist that compensates for the absence of the catalytic Ser residue. The finding that an environmental bacterium produces hydrazidase implies the existence of a novel bacterial mechanism of hydrazide degradation that impacts its ecological role.     

Examination of chiral stability during the preparation of hydrazides and coupling by the azide procedure using a series of model peptides

The peptide hydrazides of N-benzyloxycarbonylglycyl-X where X = alanine, leucine, phenylalanine, valine and isoleucine have been prepared by hydrazinolysis of the corresponding methyl esters and 2,4-distributed-5 (4H)-oxazolones, and by the carbodiimide-mediated reactions of the peptide acids with hydrazine in the presence of 1-hydroxybenzotriazole. The stereochemical purity of the products was examined by coupling them by the azide method with an ester of lysine followed by analysis for the diastereomeric tripeptides by an established procedure. The results concur with and substantiate the generally held notions on the chiral stability associated with hydrazinolysis and the azide procedure, except that an optically pure hydrazide could not be obtained from the oxazolone of the alanyl peptide.     

Synthesis and antituberculosis activity of the derivatives of glycoside steviolbioside from the plant Stevia rebaudiana and diterpenoid isosteviol containing hydrazone, hydrazide and pyridinoyl moieties

Conjugates of antitubercular drug Isoniazid (hydrazide of isonicotinic acid), nicotinic and alpha-picolinic acid hydrazides and glycoside steviolbioside from the plant Stevia rebaudiana as well as the product of its acid hydrolysis, diterpenoid isosteviol, were synthesized. Besides, isosteviol hydrazide and hydrazone derivatives as well as conjugates containing two isosteviol moieties connected by dihydrazide linker were also obtained. Both initial compounds and their synthetic derivatives inhibit the growth of Mycobacterium tuberculosis (H37Rv in vitro). The minimum concentration at which the growth of M. tuberculosis was inhibited by 100% (MIC) for stevioside and steviolbioside equals 7.5 and 3.8 microg/mL, respectively. MIC values for conjugates of the hydrazides of pyridine carbonic acids and steviolbioside as well as isosteviol are in the ranges 5-10 and 10-20 microg/mL, respectively. Maximum inhibitory effect against M. tuberculosis showed the conjugates of isosteviol and adipic acid dihydrazide (MIC values ranged from 1.7 to 3.1 microg/mL). Antitubercular activity of the compounds studied is higher than the activity of antitubercular drug Pyrizanamide (MIC = 12.5-20 microg/mL) but lower than the activity of antitubercular drug Isoniazid (MIC = 0.02-0.04 microg/mL).     

Design of dendritic macromolecules containing folate or methotrexate residues

Polyether dendritic compounds bearing folate residues on their surface were prepared as model drug carriers with potential tumor cell specificity. Starting from ester-terminated polyether dendrimers, hydrazide groups were easily introduced to the surface of the dendrimers by reaction with hydrazine. Folate residues were then conjugated to the hydrazide chain ends of the dendrimers by direct condensation with folic acid in the presence of a condensing agent or by reaction with an active ester derivative of folic acid. Essentially complete functionalization of the terminal hydrazide groups was achieved for both the first and the second generation dendrimers with four and eight hydrazide groups. For the G-2 dendrimer with 16 hydrazide groups, an average number of only 12.6 folate residues were attached to each dendrimer. The conjugates are soluble in aqueous medium above pH 7.4. In addition, a similar conjugation of the antitumor drug methotrexate to the dendrimer was also investigated. Once optimized, these molecules may form the basis for a novel family of multivalent drug carriers.     

Synthesis and antituberculosis activity of derivatives of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> glycoside steviolbioside and diterpenoid isosteviol containing hydrazone,hydrazide, and pyridinoyl moieties

Conjugates of the antituberculosis drug isoniazid (isonicotinyl hydrazine) and isomeric hydrazides of nicotinic and α-picolinic acid with glycoside steviolbioside from the Stevia rebaudiana plant and the product of its acid hydrolysis, diterpenoid isosteviol, were synthesized. In addition, isosteviol hydrazide and hydrazone derivatives as well as conjugates containing two isosteviol moieties joined by a dihydrazide linker were obtained. The parental compounds and their synthetic derivatives were found to inhibit the in vitro growth of Mycobacterium tuberculosis (H₃₇R_V). The measured minimal concentrations of stevio-side and steviolbioside, at which the growth of M. tuberculosis was inhibited by 100% (MIC), were 7.5 and 3.8 μg/ml, respectively. MIC values for steviolbioside and isosteviol conjugates with hydrazides of pyridine carbonic acid were within the ranges of 5–10 and 10–20 μg/ml, respectively. The maximal inhibitory effect against M. tuberculosis was shown by the isosteviol conjugates with adipic acid dihydrazide (MIC 1.7 and 3.1 μg/ml). Antituberculosis activities of the tested compounds were higher than the activity of antituberculosis drug Pyrizanamide (MIC 20 μg/ml) but lower than that of antituberculosis drug isoniazid (MIC 0.02–0.04 μg/ml).     

Synthesis and antiviral activity of hydrazides and substituted benzalhydrazides of betulinic acid and its derivatives

New nitrogen-containing derivatives of betulinic and betulonic acids, hydrazides and N'-benzalhydrazides, were synthesized. Their antiviral activities toward of influenza A virus, herpes simplex type I virus, enterovirus ECHO6, and HIV-1 were studied in vitro. Betulinic acid 3-oxime was found to have the highest activity against the influenza virus. Betulonic acid, betulinic acid 4-chlorobenzalhydrazide, betulonic acid 3-oxime benzalhydrazide, and betulinic acid hydrazide inhibited the replication of herpes simplex type I virus. Betulinic acid hydrazide also showed antiviral activity toward HIV-1. All the derivatives of betulinic acid under study displayed a low antiviral activity toward enterovirus ECHO6.