FDNB, fluoro-2,4-dinitrobenzene

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]. 

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. 

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 ... 

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

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. 

Traditionally, most chemical methods for the direct determination of available lysine depended on the reaction of the e-amine with a chromophoric derivatizing reagent and then spec-trophotometric measurement. The most commonly employed chromophore is 1-fluoro-2,4-dinitrobenzene (FDNB), which was originally employed by Carpenter (102). Since then, numerous articles (103-106) have reported the direct (or indirect by difference) HPLC measurement of the FDNB-derivatized lysine. However, these methods can suffer from the problem that the reaction product of the FDNB-derivatized lysine (dinitrophenyllysine) is not entirely stable during acid hydrolysis (especially in the presence of carbohydrates) and correction factors are recommended (107). 

AAP) 4-aminoantipyrine FDNB l-fluoro-2,4-dinitrobenzene DSA diazotized sulphanilic acid OPA 2-o-phthal-dehyde MCE 2-mercaptoethanol Dns 5-dimethylaminonaphtalene-l-sulphonyl. 

Sanger s reagent, l-fluoro-2,4-dinitrobenzene (FDNB), which was used in the earlier days for the quantitation of primary amino groups by colorimetric determination, can also be used in the identification of amino-terminal residue, but not for sequencing. At the present time, N-terminal analysis is performed on a protein sequencer. 

Treatment with FDNB. FDNB (l-fluoro-2,4-dinitrobenzene) reacted with free amino (but not amido or guanidino) groups in proteins to produce dinitrophenyl (DNP) derivatives of amino acids ... 

The compound l-fluoro-2,4-dinitrobenzene (FDNB) reacts with free amino, imidazole, and phenolic groups at neutral to alkaline pH to yield the corresponding, colored dinitrophenyl (DNP) compounds. Thus, FDNB will react with the free, unprotonated a-amino groups on amino acids, as well as with the side chains of lysine, histidine, and tyrosine (Fig. 6-1). Dansyl chloride is another compound that is known to react with the unprotonated, N-terminal amino groups of peptides. De-rivatization of peptides with this compound yields fluorescent products that provide a very sensitive method of detection of the amino acid derivatives (Fig. 6-2). 

Figure 6-1 Reaction of amino acids with 1-fluoro-2,4-dinitrobenzene (FDNB). DNP = dinitrophenyl HF = hydrofluoric acid...

Besides the common fluorescent probes used for studying protein structure (ANS, TNS, etc.), other labels suitable espa ially for the modification of cell surfaces and the membranes of intracellular structures have been introduced in research. ANDS, for instance, is such a label (3-azido[l, 7]napthalene disulphonate) It is used for nonspecific covalent modification of hydrophilic cell surfaces. Another is FDNB (l-fluoro-2,4-dinitrobenzen) > which is used for identifying membrane proteins associated with glucose transport in human erythrocytes. More detailed information can be found elsewhere... 

In order to investigate this type of binary solvent systems as reaction media, a kinetic study on the reaction between l-fluoro-2,4-dinitrobenzene (FDNB) and the primary amine -butylamine (BU) was performed [32, 33]. The selected mixtures were composed of AN and DMF as molecular solvent and [bmim][BF ] and [bmim] [PF,] as imidazolium-based ILs. It is possible to assume that the reaction proceeds by the classical two-step mechanism as shown in Fig. 13.6. The application of the steady-state approximation affords Eq. 13.3 for the second-order reaction rate k. ... 

Regulation of liver D-fructose 1,6-diphosphatase could involve modification of the sulfhydryl group of the enzyme.397 Cystamine (2,2 -dithiobisethylamine) or homocysteine undergoes a disulfide exchange-reaction with two reactive cysteine residues. This exchange leads to a four-fold increase in catalytic activity.405 Other sulfhydryl reagents, such as l-fluoro-2,4-dinitrobenzene (FDNB), p-mercuri-benzoate (PMB), and 2-iodoacetamide, also activate liver D-fructose... 

Where the method involves cyclization of a nucleoside phosphate to a cyclic phosphate, the following symbols are used for the cyclization reagent DCC = W,W -dicyclohexylcarbodiimide FDNB = l-fluoro-2,4-dinitrobenzene ECF = ethyl chloroformate PC = picryl chloride TFAc = trifluoroacetic acid TFAn = trifluoroacetic anhydride TBO = potassium feri-butoxide in methyl sulfoxide. 

Synonyms Benzene, 2,4-diitro-1-fluoro- Benzene, 1-fluoro-2,4-dinitro- 2,4-Dinitrobenzene fluoride 1,3-Dinitro-4-fluorobenzene 2,4-Dinitro-1-fluorobenzene DNFB 2,4-DNFB DNP-F FDNB 1-Fluoro-2,4-dinitrobenzene... 

Use of DNP-amino acids, formed by condensation of l-fluoro-2,4-dinitrobenzene (FDNB) with the free amino group of an amino acid, was first described by Sanger in 1945 (83). Sanger identified DNP-amino acids by paper chromatography. Since then many modifications in the methods of obtaining derivatives of amino acids for sequence analysis and in identification of such derivatives have been reported, and the use of DNP-amino acids for sequencing purposes is rapidly going out of date. Nevertheless, the importance of DNP-amino acids is not yet lost. In view of the limited applications of DNP-amino acids at present, the methods of preparation of these derivatives from standard amino acids or peptides are not described here. However, the details of those procedures can be obtained from Rosmus and Deyl (88) and Bailey (146). 

As shown in Table 1.5, betaines are widespread in both the animal and plant kingdoms. Derivatization of amino acids by reaction with l-fluoro-2,4-dinitrobenzene (FDNB) yields N-2,4-dinitrophenyl amino acids (DNP-amino acids), which are yellow compounds and crystallize readily. The reaction is important for labeling N-terminal amino acid residues and free 8-amino groups present in peptides and proteins the DNP-amino acids are stable under conditions of acidic hydrolysis (cf. Reaction 1.33). 

---