FDNB

Figure 10.13 The reaction of FDNB with compounds containing a free amino group. The reaction at pH 9.5 between FDNB and amino acids or peptides results in the formation of yellow-coloured dinitrophenyl derivatives.

The amino group of the N-terminal amino acid residue of a peptide will react with the FDNB reagent to form the characteristic yellow DNP derivative, which may be released from the peptide by either acid or enzymic hydrolysis of the peptide bond and subsequently identified. This is of historic interest because Dr F. Sanger first used this reaction in his work on the determination of the primary structure of the polypeptide hormone insulin and the reagent is often referred to as Sanger s reagent. 

Dansyl chloride (dimethylaminonaphthalene-5-sulphonyl chloride) will react with free amino groups in alkaline solution (pH 9.5-10.5) to form strongly fluorescent derivatives (Figure 10.14). This method can also be used in combination with chromatographic procedures for amino acid identification in a similar manner to the FDNB reagent but shows an approximately 100-fold increase in sensitivity. This makes it applicable to less than 1 nmol of material and more amenable for use with very small amounts of amino acids liberated after hydrolysis of peptides. The dansyl amino acids are also very resistant to hydrolysis and they can be located easily after chromatographic separation by viewing under an ultraviolet lamp see Procedure 10.1. 

The main consequence of EDA interactions between solvents of high donicity and nitroarenes is that amines in benzene (or in other similar solvents) compete with the solvents in complexing the nitroarenes253,254. This fact explains the ratio jfcchlorofornyjfcbenzene ] 0 or qlc molecular complex between aniline and FDNB (see Table 1). 

The study of molecular complexation was then extended to other aromatic nitro derivatives125. Although, as was described before, one of the more frequent methods of studying the formation of molecular complexes is by UV-visible spectrophotometry, the author did not observe detectable differences in the UV-visible absorbance spectra between the 2-hydroxypyridine-l-fluoro-2,4-dinitrobenzene (FDNB) mixtures and the sum of their separate components. The author observed that the signals of the 1II NMR spectra of FDNB in apolar solvents were shifted downward by the addition of 2-hydroxypyridine from solutions where [2-hydroxypyridine] [FDNB] he calculated the apparent stability constants, which are shown in Table 13. 

Recently, Forlani129 studied the reactions of fluoro dinitrobenzene (FDNB) with several amines in the presence of some compounds that have been found to catalyse the reaction. The plots of k0bs vs [catalyst] show a linear dependence at low catalyst concentration and then a downward curvature. This behaviour has been previously observed in several related cases the usual interpretation is that the k0bs increases on increasing the [catalyst] value until it reaches a maximum when k- = k + k2 [catalyst]. 

TABLE 14. A, B and C values (see text) for reactions between FDNB and amines in the presence of various catalysts at 25 °C (errors are standard deviations)129. Reproduced by permission of Societa Chimica italiana from Reference 129... 

A new assumption to be discussed in this section is that the fourth-order kinetics in SatAr by amines in aprotic solvents is due to the formation of the substrate-catalyst molecular complex. Since 1982, Forlani and coworkers149 have advocated a model in which the third order in amine is an effect of the substrate-nucleophile interaction on a rapidly established equilibrium preceding the substitution process, as is shown in Scheme 15 for the reaction of 4-fluoro-2,4-dinitrobenzene (FDNB) with aniline (An), where K measures the equilibrium constant for ... 

Scheme 15 could be a reaction pathway parallel to the classical reaction (equation 1), and it was postulated to explain the third order in amine observed in the reactions of FDNB and aromatic amines in benzene and in chloroform184. The K values were calculated from the absorbances of the reaction mixture extrapolated to zero reaction time, in a wavelength range in which the starting materials do not show an appreciable absorbance value. Good agreement was observed between the values of K for the FDNB/aniline complex in chloroform by U.V. and 111-NMR spectroscopy, as well as for the K obtained kinetically (based on Scheme 15) and spectroscopically. 

Catalysis by DABCO in the reactions of FDNB with piperidine, r-butylamine, aniline, p-anisidine and m-anisidine (usually interpreted as base catalysis as in Section B) was also assumed to occur by the formation of a complex between DABCO and the substrate14913. The high (negative) p-value of —4.88 was deemed inappropriate for the usually accepted mechanism of the base-catalysed step (reaction 1). For the reactions with p-chloroaniline, m- and p-anisidines and toluidines in benzene in the presence of DABCO a p-value of —2.86 was found for the observed catalysis by DABCO (fc3DABC0). The results were taken to imply that the transition state of the step catalysed by DABCO and that of the step catalysed by the nucleophile have similar requirements, and in both the nucleophilic (or basicity) power of the nucleophile is involved. This conclusion is in disagreement with the usual interpretation of the base-catalysed step. 

Under the experimental conditions [TNT] [DBU](I. the rate of formation of the second maximum (468 nm) is slow and the authors could make a quantitative evaluation of the first interaction attributed to the formation of a molecular complex (MC). The low reactivity under these conditions was interpreted as due to the fact that the MC has very little tendency to rearrange to the zwitterionic complex, since the amount of DBU complexed by TNB would be unavailable for the nucleophilic attack. Since in this system the base-catalysed step for departure of TIL does not exist, the small increase in k0bs values with the [DBU] was interpreted as evidence of the mechanism shown in Scheme 16. Similarly, the increase in k with [amine] observed in the reactions of FDNB with butylamine in... 

TABLE 24. Reaction of l-fluoro-2,4-dinitrobenzene (FDNB) with cyclohexylamine, and with 1,2-diaminocyclohexane (DACH) in toluene at 5°C ... 

In 1950 an alternative to the Sanger procedure for identifying N-terminal amino acids was reported by Edman—reaction with phenyl-isothiocyanate to give a phenylthiocarbamide labeled peptide. When this was heated in anhydrous HC1 in nitromethane, phenylthiohy-dantoin was split off, releasing the free a-NH2 group of the amino acid in position 2 in the sequence. While initially the FDNB method was probably the more popular, the quantitative precision which could be obtained by the Edman degradation has been successfully adapted to the automatic analysis of peptides in sequenators. 

Lysine is an essential amino acid with an e-amino group on the side chain that can react with various food components. As known, reaction of the e-amine can render lysine nutritionally unavailable reducing the nutritional value of food. While the determination of total lysine is straightforward (it is stable to acid hydrolysis), the determination of available lysine is difficult as lysine adducts are labile to the standard acid hydrolysis. A solution to this problem consists of derivatizing the e-amino group with a chromophore such as l-fluoro-2,4-dinitrobenzene (FDNB) to form a derivate which is stable to optimized hydrolysis conditions [222]. 

Various procedures are used to analyze protein primary structure. Several protocols are available to label and identify the amino-terminal amino acid residue (Fig. 3-25a). Sanger developed the reagent l-fluoro-2,4-dinitrobenzene (FDNB) for this purpose other reagents used to label the amino-terminal residue, dansyl chloride and dabsyl chloride, yield derivatives that are more easily detectable than the dinitrophenyl derivatives. After the amino-terminal residue is labeled with one of these reagents, the polypeptide is hydrolyzed to its constituent amino acids and the labeled amino acid is identified. Because the hydrolysis stage destroys the polypeptide, this procedure cannot be used to sequence a polypeptide beyond its amino-terminal residue. However, it can help determine the number of chemically distinct polypeptides in a protein, provided each has a different amino-terminal residue. For example, two residues—Phe and Gly—would be labeled if insulin (Fig. 3-24) were subjected to this procedure. 

The following abbreviations have been employed FDNB, 2,4-fluorodinitro-benzene F6P, fructose 6-phosphate FDP, fructose 1,6-diphosphate FDPase, fructose-1,6-diphosphatase NEM, iV-ethylmaleimide PFK, phosphofructokinase PLP, pyridoxal phosphate SDP, sedoheptulose 1,7-diphosphate SDS, sodium dodecyl sulfate. 

The original evidence for activation by modification of cysteine residues came from studies with 2,4-fluorodinitrobenzene (15). Incubation of the crystalline enzyme preparations with 4 equivalents of FDNB led to a marked increase in activity in the neutral pH range, together with a small decrease in the activity assayed at alkaline pH (Fig. 4). The modified enzyme showed two broad and nearly equal activity maxima one at pH 7.7 and the other at pH 9.0. When dinitrophenylation was carried out at pH 7.5, this change in catalytic properties was associated with the modification of only 2 of the 20 cysteine residues in the protein (16). These 2 highly reactive cysteine residues were found to be completely protected against the action of FDNB by addition of FDP. 

The activating effects of FDNB were observed only when the enzyme was tested with Mn2+ as the divalent cation. When Mg2+ was substituted for Mn2+, the dinitrophenylated enzyme was less active than the native enzyme throughout the pH range, although it still showed the biphasic pH activity curve (15). 

Other sulfhydryl reagents, such as p-mercuribenzoate and iodoaceta-mide, produced similar activation (44), except that with these compounds increases in activity were also observed at pH 9.1 (Table I). With p-mercuribenzoate maximum activation was observed when 2-4 sulfhydryl groups were titrated, and with excess reagent catalytic activity was almost completely abolished (44)- Similar results were obtained with FDNB (15). The reactive sulfhydryl groups may be located in apolar regions of the enzyme molecule since they were not affected by N-ethylmaleimide or iodoacetic acid. 

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