Ninhydrin, oxidation

Ninhydrin (2,2-dihydroxy-l,3-indandione) Amax = 570 nm, Amax = 440 nm. This reagent has already been described earlier in this chapter (Sec. III.A. Ion Exchange). Additionally, it should be noted that ninhydrin reagent solutions are susceptible to photodegradation and air oxidation. Older solutions will yield progressively poorer response as the ninhydrin oxidizes. It is an arbitrary decision when to swap a new solution for the old. Representative references include 172-174. 

Reaction with ninhydrin Ninhydrin is a strong oxidizing agent. When a solution of amino acid is boiled with ninhydrin, the amino acid is oxidatively deaminated to produce ammonia and a ketoacid. The keto acid is decarboxylated to produce an aldehyde with one carbon atom less than the parent amino acid. The net reaction is that ninhydrin oxidatively deaminates and decarboxylates a-amino acids to C02, NH3 and an aldehyde. The reduced ninhydrin then reacts with the liberated ammonia and another molecule of intact ninhydrin to produce a purple coloured compound known as Ruhemann s purple. 

Fig. 207. One-dimensional separation in a chamber for continuous development [13] (see p. 76) for detection of leucine and isoleucine in the presence of 18 protein amino acids + j -alanine + y-amino-n-butjrric acid 0.5 Xg of each amino acid applied in a total of 0.5 xl O.IN hydrochloric acid 4.6 h nm detection with ninhydrin oxidation with performic acid [44] is necessary if methionine is present. Identification of leucine and isoleucine is unambiguous if a standard sample of each is chromatographed in a parallel run on the same layer...

Ninhydrin (also named 1 2 3-triketoindane or 1 2 3-triketohydrindene hydrate) is prepared most simply from the inexpensive phthahc anhydride (I). The latter is condensed with acetic anhydride In the presence of potassium acetate to give phthalylacetlc acid (II) reaction of the latter with sodium methoxide in methanol yields 1 3-indanedionecarboxyhc acid, which is decomposed upon warming with dilute hydrochloric or sulphuric acid to indane-1 3-dione (or 1 3-diketohydrindene) (HI). Selenium dioxide oxidation of (III) afibrds indane-1 2 3-trione hydrate (ninhydrin) (IV). 

It is known that not all reactions proceed in the same manner on all adsorbent layers because the material in the layer may promote or retard the reaction. Thus, Ganshirt [209] was able to show that caffeine and codeine phosphate could be detected on aluminium oxide by chlorination and treatment with benzidine, but that there was no reaction with the same reagent on silica gel. Again the detection of amino acids and peptides by ninhydrin is more sensitive on pure cellulose than it is on layers containing fluorescence indicators [210]. The NBP reagent (. v.) cannot be employed on Nano-Sil-Ci8-100-UV2S4 plates because the whole of the plate background becomes colored. 

CK catalyzes the reversible phosphorylation of creatine in the presence of ATP and magnesium. When creatine phosphate is the substrate, the resulting creatine can be measured as the ninhydrin fluorescent compound, as in the continuous flow Auto Analyzer method. Kinetic methods based on coupled enzymatic reactions are also popular. Tanzer and Gilvarg (40) developed a kinetic method using the two exogenous enzymes pyruvate kinase and lactate dehydrogenase to measure the CK rate by following the oxidation of NADH. In this procedure the main reaction is run in a less favorable direction. 

Finally, Droge and coworkers26 showed that the 2-aminoethyl phosphate-substituted KDO group is the lateral KDO unit of the branched trisaccharide (see Fig. 7), as follows. LPS from Salmonella minnesota mR3 was subjected to periodate oxidation. This sample, together with a control that had not been oxidized, was then mildly hydrolyzed with acid (pH = 3.4) during 1 h at 100°. Following removal of lipid A, both samples were analyzed by gel-filtration on Sephadex G-10, and paper electrophoresis. As expected, the ninhydrin-positive material obtained from the control sample was identical with KDO 7-(2-aminoethyl phosphate) (17) as previously identified. This spot was absent from the periodate-treated sample. Instead, an almost neutral, ninhydrin-positive spot was observed. This material (compound 26) was eluted, subjected to reduction with sodium [3H]borohydride, and hydrolyzed under strongly acidic conditions (see Scheme 11). Fol-... 

FIGURE 5.5 Schedule for the solid-phase synthesis of somatostatin, a 14-mer, on 10 g of resin reacted with 5 mequiv of the first amino acid, adapted from J. Rivier, J. Am. Chem. Soc. 96 2986, 1974. Min = time of mixing X2 = two times DCC = dicyclohexylcarbodiimide. Step 4 included 5% of (CH2SH)2 to prevent the oxidation of tryptophan. When the ninhydrin test on an aliquot after step 13 was negative, step 1 followed when positive, steps 9-13 were repeated. 

Liii and Liv absorb at 280 nm, whereas has no tryptophan or tyrosine residues and shows no absorption at 280 nm, but can be detected with ninhydrin after alkaline hydrolyses. This result reveals that ozone causes the oxidation of the methionine residue 105 of lysozyme. 

Cyclobutanecarboxylic acid, 98%, is from Aldrich Chemical Company, Inc., and is vacuum distilled before use. Tetrahydrofuran is freshly distilled from sodium-benzophenone under nitrogen. N-Methyiraorpholine, 99%, is from Aldrich Chemical Company, Inc., and is pre-dried over barium oxide, distilled from ninhydrin, and stored over sodium hydroxide pellets. Ethyl chloroformate, 97%, is from Aldrich Chemical Company, Inc., and is freshly distilled prior to use under a nitrogen atmosphere. Anhydrous ammonia (99.99% min) is from a Matheson lecture bottle. The silica gel used for flash chromatography is... 

Ninhydrin Reaction. A neutral solution of an amino acid will react with ninhydrin (triketohydrindene hydrate) by heating to cause oxidative decarboxylation. The central carbonyl of the tnketone is reduced to an alcohol. This alcohol further reacts with ammonia formed from the amino add and causes a red-purplish color. Since the reaction is quantitative, measurement of the optical density of the color produced is an indication of amino add concentration. Imino acids, such as hydroxyproline and proline, develop a yellow color in the same type of reaction. 

In many kinds of research it is important to have simple and sensitive means for analysis of amino acids, particularly in small quantities. Detection of amino acids can be achieved readily by the ninhydrin color test, whereby an alcoholic solution of the triketone, ninhydrin, is heated with an amino acid and produces an intense blue-violet color. The sensitivity and reliability of this test is such that 0.1 micromole of amino acid gives a color intensity reproducible to a few per cent, provided that a reducing agent such as stannous chloride is present to prevent oxidation of the colored salt by dissolved oxygen. 

Suggest a simple synthesis of 1 and a mechanism for the oxidation of 1 to the ninhydrin. 

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