2.4- Dinitrophenylhydrazones 2,4-DNP

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. 

Dinitrophenylhydrazone. 2,4-Dinitrophenylhydrazine reacts with aldehydes and ketones to form 2,4-dinitrophenylhydrazones (2,4-DNP). 

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. 

An aldehyde reacts with 2,4-dinitrophenylhydrazine to give a bright, yellow-to-orange crystalline solid called a 2,4-dinitrophenylhydrazone (2,4-DNP). 

The ozonides of choline and ethanolamine phosphatides and triglycerides can be subjected to reduction with triphenylphosphine to yield the corresponding core aldehydes, and further derivatized to the 2,4-dinitrophenylhydrazones (DNP). The core aldehydes and their DNP derivatives can be separated by HPLC and characterized by various techniques, including EI-MS and TS-MS of positive and negative ions . See also Section VHI.E. 

Dinitrophenylhydrazones (DNP) aldehyde determination, 670 core aldehydes, 689 malondialdehyde determination, 669 ozonide reduction, 726... 

These model studies permitted demonstration of metabolic a-hydroxylation of NPy by isolating 2-hydroxytetrahydrofuran as a metabolite of NPy. This was accoitplished by trapping 4-hydroxy-butyraldehyde as its 2,4-dinitrophenylhydrazone (DNP) derivative. For in vitvo studies, NPy-2,5-l C was incubated with rat liver microsomes, 02i and an NADPH generating system. After the incubation was complete, DNP reagent was added to the mixture and the products were extracted and examined by preparative TLC. A radioactive band corresponding to the DNP of 4-hydroxybutyralde-hyde was observed. This band was not present in controls in which NADPH was omitted or in which boiled enzyme was used. The mass spectrum was identical to that of a reference sairple. In addition, a minor metabolite with mass spectrum identical to that of the DNP of 2-butenal was also isolated. These results showed conclusively that NPy underwent metabolic a-hydroxylation in this in vitro system. 

Reductive cleavage of 2,4-dinitrophenylhydrazones. Certain 2,4-dinitrophenyl> hydrazones of 3-ketosteroids can be cleaved under mild conditions if the nitro groups are reduced to amino groups by chromous chloride. Thus when the 2,4-DNP of 4,5a-dihydrocortisone 21-acetate in methylene chloride was shaken in a separatory funnel with chromous chloride and dilute hydrochloric acid for 15 min., workup of the organic layer afforded the ketone in 94% yield. Use of a water-immiscible solvent protects the released ketone an inert atmosphere if required can be provided with dry ice. Cortisone 2,4-DNP was cleaved in only 3-5 hrs. and the yield was 60%. DN P derivatives of A -diene-S-ones were completely resistant to hydrolysis. Contributed by Leon Mandell... 

A procedure developed by Mattox and Kendall for the liberation of cortisone acetate from the C3 2,4-dinitrophenylhydrazone utilizes a mixture of pyruvic acid, acetic acid, chloroform, and hydrogen bromide and affords the parent compound in 80% yield. For comparison. DePuy° prepared a mixture of 1 g. of A -cholestene-S-one 2,4-DNP with 100 ml. of chloroform and 100 ml. of the usual levulinic-hydrochloric acid and heated it under reflux for 3 hrs. Chromatography afforded pure A -cholestene-3-one in high yield. 

Dinitrophenylhydrazones are cleaved by various oxidizing and reducing agents, and by exchange reactions. Some of the methods used for the cleavage of oximes should be applicable for DNP cleavage. 

Cleavage of 2,4-dinitrophenylhydrazones. Carbonyl compounds can be regenerated in high yield (80-95%) from the 2,4-DNP derivatives by treatment in DME with a 20% aqueous solution of TiCls at reflux temperature (N2). McMurry and Sylvestri consider that titanous ion reduces the nitro groups to amino groups and then cleaves the hydrazone N—N bond to generate an imine, which is then hydrolyzed readily to the carbonyl compound. 

Some enzyme reactions can be studied colorimetrically when either the substrate or product can be converted chemically to a coloured product suitable for measurement in a u.v. or visible light spectrophotometer. In the case of alanine aminotransferase, the pyruvate formed in the reaction can be converted to pyruvate-2,4-dinitrophenylhydrazone by the addition of 2,4-dinitrophenylhydrazine (DNP). Addition of sodium hydroxide yields a product with an absorption maximum at 505 nm. Other examples of colorimetric procedures will be found in the last section. Colorimetric procedures are used for enzyme assays in the sampling mode, whereby samples of the reaction mixture are analysed at certain fixed times after starting the reaction. Graphs depicting the reaction rate must then be constructed by plotting amount of substrate transformed against time. 

The separation by TLC of the 2,4-dinitrophenylhydrazone (DNP) of dehydroascorbic acid is extremely specific and has been employed for detection and determination of vitamin C in food and feeding stuffs, fats, fruit juices, wines and bacteria etc. In the method of Stroheckeb et al. [129, 130] the ascorbic acid in the extracts is oxidised with 2,6-dichlorophenol-indophenol (Vuilleumieb and Nobile [139] use bromine for this) the dehydroascorbic acid formed is then reacted for 3 hours at 70 C with 2,4-dinitrophenylhydrazine in the presence of a little trichloroacetic acid and thiourea [131]. After cooling 10 min in ice, the red or red-brown precipitate is collected on a sintered glass filter, washed with water and dissolved in ethyl acetate or acetone the solution is evaporated and the residue taken up in acetone. 0.1—1.0 ml (20 to 50 [xg vitamin C) of this solution is applied as a band to air dried silica gel H layers and chromatographed with chloroform-ethyl acetate (50 + 50) or, better, with chloroform-ethyl acetate-acetic acid (60 + 35 -f 5). The red DNP of the dehydroascorbic acid is thus clearly separated... 

Interaction of formaldehyde with 2,4-dinitrophenylhydrazine in acid media causes 2,4-dinitrophenylhydrazone (DNPhydrazone) formaldehyde formation. Gas-chromatographic analysis of 2,4-DNP-hydrazone formaldehyde toluene extract with an electron holding detector makes it possible to detect it at the level of 0,001 mg/dm. Phenol is detected in the form of tribromphenol yield, the hexane extract of which undergoes chromatography with an electron holding detector which provides the level of phenol detection of 0.001 mg/dm (the limit of quantitative detection). 

When the same reaction is performed with adamantanone dinitrophenylhydrazone (DNP) or adamantanone O-mcthyloxime (OMO), the yields of 2,2-difluoroadamantanc are 65 and 50%, respectively. 4-tert-Butylcyclohexanone is converted via its azine, DNP or OMO into 4-tert-butyl-l.l-difluorocyclohexanc in 65, 75 or 50% yield, respectively. Similar results are obtained with aliphatic straight-chain ketones. Decan-2-one azine and decan-2-one DNP can be transformed into 2,2-difluorodecane in 60 and 80% yield. Deactivated aromatic rings, e.g. in 3-nitroacetophenone, do not undergo bromination with bromine trifluoride and the action of 2 equivalents of bromine trifluoride on 3-nitroacetophenone O-methyloxime results in 90% yield of 1-(1. l-difluorocthylj-S-nitrobenzene.122... 

Dinitrophenylhydrazine is frequently used for making derivatives of ketones and aldehydes because the products (2,4-dinitrophenylhydrazones, called 2,4-DNP derivatives) are even more likely than the phenylhydrazones to be solids with sharp melting points. Propose a mechanism for the reaction of acetone with 2,4-dinitrophenylhydrazine in a mildly acidic solution. 

Both derivatives give hydrazones that tend to be sohds and are rather stable. Therefore, a melting point is easily determined and compared with known derivatives for the purposes of identification. Because of their utihty as an identification agent, they are usually formed in a test tube yields are not always important, although these reactions usually proceed in 3delds >70% and often >90%. A simple example is the reaction of 2,4-dinitrophenylhydrazine (usually abbreviated 2,4-DNP, 106) and cyclooctanone (105), which gives a 2,4-dinitrophenylhydrazone, 107. 

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