Hydrazides, detection

There are many reports of the use of mass spectroscopy coupled to chromatography outlets for detection and identification of dmgs and metabolites. An example is compound 126 (99MI2, 99MI3). Carboxylic acids have been converted into hydrazides and hence into 3-substituted [l,2,4]triazolo... 

Isonicotinic acid hydrazide reagent 318 Isoprenaiine 395,396 Isoprenoid compounds 44 Isopulegol 59 Isoquercitrin 279, 280, 323 Isoquinoline alkaloids 46, 66, 262 Isorhamnetin 323 Isothiazolones, microbiocidal 45 Isothiocyanates 75 Isotopes, j8-radiation-emitting 41 -, detection limits 41 Itaconic acid 61... 

An excellent example of PLC applications in the indirect coupling version is provided by the works of Miwa et al. [12]. These researchers separated eight phospholipid standards and platelet phospholipids from the other lipids on a silica gel plate. The mobile phase was composed of methylacetate-propanol-chloro-form-methanol-0.2% (w/v) potassium chloride (25 30 20 10 10, v/v). After detection with iodine vapor (Figure 9.2), each phospholipid class was scraped off and extracted with 5 ml of methanol. The solvent was removed under a stream of nitrogen, and the fatty acids of each phospholipid class were analyzed (as their hydrazides) by HPLC. The aim of this study was to establish a standardized... 

Cyclic hydrazides 411 react with acrolein, crotonaldehyde, and methyl vinyl ketone either by heating in a sealed tube at 150 °C or by refluxing in aqueous ethanol containing a catalytic amount of sodium hydroxide, to provide acceptable yields of the corresponding products 412 (Equation 57). Both possible cyclic tautomers are detectable <2002EJO3447, 2002PCJ598>. 

The creation of hydrazide probes often is based on the derivatization of a detectable molecule with a fezs-hydrazide compound. Although hydrazine itself (in the form of hydrazine hydrate) can be used in a methanolic solution to modify activated carboxylate molecules forming hydrazides, the availability of the bifunctional hydrazides provides a built-in spacer to accommodate greater steric accessibility. 

These techniques have been used to target, detect, or assay glycoproteins in solution or on cell surfaces by using hydrazide-activated enzymes, avidin, or streptavidin (Chapter 23, Section 5) (Bayer and Wilchek, 1990 Bayer et al., 1987a, b, 1990) and to form conjugates with glycoproteins. 

This fluorophore has an excitation maximum at 400 nm and an emission maximum at 420 nm. The extinction coefficient of the molecule in aqueous solution at pH 7 is about 31,000M 1cm 1. Cascade Blue hydrazide and Lucifer Yellow derivatives can be excited simultaneously by light of less than 400 nm, resulting in two-color detection at 420 and 530nm. 

Figure 23.10 Glycoproteins may be oxidized with sodium periodate to generate aldehyde residues. These may be specifically labeled using a hydrazide-streptavidin derivative through hydrazone bond formation. Subsequent detection may be done using biotinylated enzymes.

Hydrazide groups can react with carbonyl groups to form stable hydrazone linkages. Derivatives of proteins formed from the reaction of their carboxylate side chains with adipic acid dihydrazide (Chapter 4, Section 8.1) and the water-soluble carbodiimide EDC (Chapter 3, Section 1.1) create activated proteins that can covalently bind to formyl residues. Hydrazide-modified enzymes prepared in this manner can bind specifically to aldehyde groups formed by mild periodate oxidation of carbohydrates (Chapter 1, Section 4.4). These reagents can be used in assay systems to detect or measure glycoproteins in cells, tissue sections, or blots (Gershoni et al., 1985). 

Other molecules can be used in this type of assay approach. Hydrazide-modified (strept)avidin, lectins, biocytin, fluorescent probes and other detectable molecules can be used to detect specifically glycoconjugates in biological samples (Wilchek and Bayer, 1987). 

Ahn, B., Rhee, S.G., and Stadtman, E.R. (1987) Use of fluorescein hydrazide and fluorescein thiosemicar-bazide reagents for the fluorometric determination of protein carbonyl groups and for the detection of oxidized proteins on polyacrylamide gels. Anal. Biocbem. 161, 245-257. 

Gershoni, J.M., Bayer, E.A., and Wilchek, M. (1985) Blot analysis of glycoconjugates Enzyme-hydrazide— A novel reagent for the detection of aldehydes. Anal. Biochem. 146, 59-63. 

The emission spectra match the fluorescence of the corresponding acid. Methane was detected as a major product in the chemiluminescent oxidation of 57 a and it was suggested that it resulted from the decomposition of methyl-diimine formed after dehydrogenation of the hydrazide 57a ... 

Monomeric dialkylaluminum hydrazides with terminal hydrazido groups (A, Scheme 2) were observed only in those cases in which donor ligands such as ether molecules (28 to 30) or hydride ions (26) saturate the aluminum atoms to yield coordination numbers of four. In the absence of Lewis bases dimers are formed of the monoaluminum and monogallium monohydrazido derivatives, a trimer has never been detected by crystal structure determinations. Three different types of structures were reported ... 

The most common structural motif of aluminum and gallium hydrazides is the centrosymmetric four-membered heterocycle with two exocyclic N—N bonds. The N-N distances of all these compounds fall in a very narrow range of about 145.5 pm on average with the most significant deviation (143.7 pm) detected for the diphenylhydrazido gallium derivative 14. The E2N2 heterocycles are planar with almost ideally equidistant E-N separations. As expected, the Al—N bonds of these compounds (196 to 199 pm) are shorter than the Ga—bonds (199 to 207 pm). 

Carbohydrates are easily oxidized by periodate to aldehydes which react with primary amines or hydrazines. If the formed hydrazide carries a specific ligand, e.g., biotin or digoxigenin, these ligands immobilized via the blotted macromolecule are very sensitively detected by the respective enzyme conjugates. 

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