Derivatization with 2,4-dinitrofluorobenzene

Amines, aliphatic Derivatization with 2,4-dinitrofluorobenzene or benzenesulfonyl chloride, solvent extraction with CH2CI2 GC-MS... 

The analysis of polio RI for VPg did not permit us to decide whether YPg was also linked to polio minus strands, since the quantitation of YPg is too inaccurate and because at 3 5 hr after infection, 90% of the RI structures produce plus strands (32). Evidence that TPg was linked to the 5 "te2 minal poly(u) of minus strands of RI as well as viral double-stranded RNAs (RF) was obtained by an analysis of hybrid double-stranded composed of 52p-labeled minus strands and unlabeled plus strands (I3). (52p ) poly(u) was isolated from these hybrids and had a 5 -terminal pU linked to YPg (13) This result has recently been confirmed by Pettersson al. (30 It has also been extended by Wu al. (15)> who derivatized the YPg of polio RF with dinitrofluorobenzene and subsequently complexed it with anti-DNP IgG. Inspection of these molecules in the electron microscope showed that both ends of RF were protein-tagged. The frequency of tagging was high enough to conclude that both strands of polio RF are protein-linked. 

The reaction of 2,4-dinitrofluorobenzene (DNFB) (Sanger s reagent [10]) with amino acids is another useful technique which is often employed for the analysis of N-terminal amino acids by TLC and column chromatography after derivatization. The reaction involved in product formation is shown in Fig.4.6. The separated derivatives are determined by measuring the quenching of fluorescence on TLC plates or by UV analysis after column chromatography. The generalized absorption curves of dinitrophenyl (DNP)-amino acids in acidic and alkaline solutions are shown in Fig. 4.7. 

Walle [100] carried out the derivatization after prior extraction of an aqueous sample (urine) with benzene. A 3-ml volume of the sample and 1 ml of an aqueous solution of an internal standard were mixed in a 50-ml centrifuge tube, 1 ml of 5 M K2C03 and 30 ml of benzene were added and the mixture was shaken for 10 min. After the centrifugation, the benzene phase was separated and 25 ml of the benzene solution of the amine were mixed with a five-fold excess of 2,4-dinitrofluorobenzene dissolved in 1 ml of benzene. After standing for 5 min at room temperature, the solution was heated at 60°C for 15 min, cooled and 2—5 pi were chromatographed. 

A method for determining the N-terminal amino acid of a peptide. The peptide is treated with 2,4-dinitrofluorobenzene (Sanger s reagent), then completely hydrolyzed. The derivatized amino acid is easily identified, but the rest of the peptide is destroyed in the hydrolysis, (p. 1180)... 

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