2.4- Dinitrophenylhydrazone derivatives formation

The metabolism of NPYR is summarized in Figure 1. a-Hy-droxylation (2 or 5.position) leads to the unstable intermediates and decomposition of gives 4-hydroxybutyraldehyde [ ]. The latter, which exists predominantly as the cyclic hemiacetal 1, has been detected as a hepatic microsomal metabolite in rats, hamsters, and humans and from lung microsomes in rats (9-13). The role of 1 and as intermediates in the formation of 6 and 7 is supported by studies of the hydrolysis of 2-acetoxyNPYR and 4-(N-carbethoxy-N-nitrosamino)butanal, which both gave high yields of 7 (9,14). In microsomal incubations, can be readily quantified as its 2,4-dinitrophenylhydrazone derivative (15). The latter has also been detected in the urine of rats treated with NPYR ( ). 

Unfortunately, there are no universal methods to detect all types of protein oxidation, because the products formed can be so diverse in nature. However, some forms of protein oxidation can be assayed using chemical modification (Davies et al., 1999 Shacter, 2000). In particular, the formation of carbonyl groups on proteins can be targeted using the reagent 2,4-dinitrophenyl-hydrazine (DNPH). This compound reacts with aldehydes to form 2,4-dinitrophenylhydrazone derivatives, which create chromogenic modifications that can be detected at high sensitivity in microplate assays or Western blot analysis (Buss et al., 1997 Winterbourn et al., 1999). 

The reaction of an aldehyde or a ketone with 2,4-dinitrophenylhydrazine produces a 2,4-dinitrophenylhydrazone derivative. This reaction is also used as a test for the presence of an aldehyde or ketone. A drop or two of a suspected aldehyde or ketone is added to a solution of 2,4-dinitrophenylhydrazine in ethanol and water. The formation of a precipitate, usually orange or red, of the derivative indicates that the unknown is an aldehyde or ketone ... 

Bell et al. (243) used the formation of the 2,4-dinitrophenylhydrazone derivative of formaldehyde in SSS as the means to quantitate the level of formaldehyde in SSS. 

Periodate ion has been determined by the formation of a chromophore with 2,2 -azinobis(3-ethylbenzothiazoline-6-sulphonic acid) and by means of an iodide-selective electrode. " The 2,4-dinitrophenylhydrazones derived from aldehydic fragments produced on periodate oxidation of carbohydrates have been separated by t.l.c., after which the individual hydrazones could be determined spectrophotometrically. ... 

In the case of compound II-23d, the proposed structure was confirmed by X-ray diffraction via formation of the 2,4-dinitrophenylhydrazone derivative 11-31 (Fig. 3.2). Therefore, the formation of a cyclobutene compound was unambiguously confirmed. 

Imine formation from such reagents as hydroxylamine and 2,4-dinitro-phenylhydrazine is sometimes useful because the products of these reactions— oximes and 2,4-dinitrophenylhydrazones (2,4-DNPs), respectively—are often crystalline and easy to handle. Such crystalline derivatives are occasionally prepared as a means of purifying and characterizing liquid ketones or aldehydes. 

The reaction is quantitative and at gram quantities, the hydrazone precipitates immediately due to its low solubility in the aqueous phase. The removal of the derivative from the aqueous phase shifts the equilibrium toward the formation of more derivate (Esterbauer, 1982). The dinitrophenylhydrazones absorb strongly in the region 360-390 nm. The spectrum of the 2-alkenals is shifted to 455 nm in ethanolic KOH (Fig. 5.9). 

CCIII (R = NO) exists in equilibrium with its open chain form (CCII, R = NO) as evidenced by the slow formation of a conjugated 2,4-dinitrophenylhydrazone. Like haemanthidine, the N-nitroso derivative of haemanthidine underwent alkaline oxidation-reduction to form N-nitrosonortazettine (CCV). Thus, the oxidation-reduction reactions of Cg and Cn in haemanthidine require neither a basic nitrogen nor the 5,10b-ethanophenanthridine ring system. The rearrangement is almost certainly intramolecular and may proceed by hydride transfer either through CCII or CCIII. 

The coloured p-nitro- and 2,4-dinitrophenylhydrazones are ideal derivatives for the separation and characterization of carbonyls by paper, thin-layer and column chromatography. The oximes can easily be revealed on the thin-layer plates by spraying with solutions of copper(Il) chloride or copper(II) acetate (alcoholic) or iron(III) chloride [21,22]. For the visualization of carbonyl compounds by means of fluorogenic labeling by Schiff base formation with reagents such as dansyl-hydrazine, we refer the reader to Chapter 9. 

Formation of 2,4-dinitrophenylhydrazones of carbonyl compounds is a popular method for their derivatization. However, owing to low volatility of these derivatives, their reversed-phase high-performance liquid chromatography (HPLC) analysis seems more preferable than GC analysis. ... 

Purpose. The experiment investigates the use of a polymer-bound chromium trioxide oxidizing agent in the oxidation of a secondary (2°) alcohol to a ketone. An alternative oxidizing agent, sodium hypochlorite, may be used instead. The progress of the reaction is monitored by thin-layer chromatography (TLC). The product ketone can be characterized by formation of its 2,4-dinitrophenylhydrazone (2,4-DNP) derivative. 

Detection may be facilitated by preparation of colored or, more often, fluorescent derivatives prior to spotting and development. Examples include dansylation (reaction with 1-dimethyl aminonaphthalene-5-sulfonyl chloride) of amino acids and formation of 2,5-dinitrophenylhydrazones of amino acids and ketosteroids. Other reactions that have been used include oxidation, reduction, hydrolysis, ha-logenation, enzymatic, nitration, diazotization, esterification, and etherification (Szepesi and Nyiredy, 1996 Kovar and Morlock, 1996). Prechromatographic derivatization sometimes improves separation, stability, and/or quantification of the analytes in addition to providing visualization. 

For example, formation of a 2,4-dinitrophenylhydrazone or a semicarbazone (Fig. 16.50) from an unknown was not only diagnostic for the presence of a carbonyl group, but it yielded a specific derivative that could be compared to a similar sample made from a compound of known structure. Figure 16.50 gives a few of these substituted imines. Be sure to note that all of these reactions are the same— the only difference is the identity of the R group. If you know one, you know them all—but you have to know that one. 

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