[70-34-8]  · C6H3FN2O4  · 1-Fluoro-2,4-dinitrobenzene  · (MW 186.10)

(identification of terminal amino acid residues in peptides;1 amine protecting group;4 deoxygenation of phenols;11,12 thioamide protecting group14)

Alternate Names: dinitrofluorobenzene; DNFB; Sanger's reagent.

Physical Data: mp 27.5-30 °C; bp 178 °C/25 mmHg; d 1.48 g cm-3.

Solubility: sol benzene, Et2O, EtOAc, acetone, DMF, propylene glycol.

Form Supplied in: 99% pale yellow crystals; widely available.

Purification: commercial sample can be recrystallized from diethyl ether.

Handling, Storage, and Precautions: stored at room temperature. Considered as highly toxic and cancer suspect agent. Use in a fume hood.

Peptide Analysis.

Sanger1 developed an efficient method for the identification of the terminal amino acid residues in peptides and proteins using dinitrofluorobenzene. DNFB reacts at room temperature with the terminal amino functionality of the peptides in the presence of sodium bicarbonate and upon acid hydrolysis a dark yellow 2,4-DNP derivative of the terminal amino acid is liberated (eq 1). As the 2,4-DNP-amino acids produced are dark yellow in color, they may be estimated colorimetrically using the corresponding pure 2,4-DNP-amino acid as a standard. Partition chromatography or paper chromatography techniques can be used to separate the components; the DNP-amino acids provide visible yellow bands. DNFB is more widely used than dinitrochlorobenzene as the latter requires higher temperatures to react with amines especially in presence of sodium bicarbonate, which leads to hydrolysis of the protein. DNP derivatives of 4-aminopentanoic acid2 and 2,4-hexadiynylene esters of various amino acids3 have been prepared for characterization purposes.

Amine Protection.

The use of DNFB for protection of amine functionalities in sugars during the synthesis of glycosides was first introduced by Lloyd;4 the unprotected hexosamines gave poor yields in condensation reactions. Wolfrom5 has used DNFB to install N-blocking groups in pyrimidine nucleoside synthesis from 2-amino sugars. DNP-D-glucosamine was prepared by treating 2-amino-2-deoxy-D-glucose hydrochloride with DNFB and sodium bicarbonate. After synthesis of the nucleoside the final deprotection was carried out by simply treating the DNP derivative with barium hydroxide to release the free amine (eq 2). Recently, Valli and co-workers6 have shown that 2,4-DNP derivatives of some amines which are analogs of ranitidine, are good inhibitors of acetylcholinesterase.

Derivatization of Phenols and Alcohols.

DNFB reacts with phenols under mild conditions in the presence of catalytic Triethylamine in acetone to give the corresponding 2,4-dinitrophenyl ethers as solid derivatives in high yields.7 2,4-Dinitrophenyl ether derivatives of alditols and myo-inositol have been prepared at room temperature using DNFB in DMF and triethylamine; the products are usually crystalline with sharp melting points.8 2,4-Dinitrophenyl ethers from neryl and geranyl alcohol were prepared with 1,4-diazabicyclo[2.2.2]octane and DNFB.9

Allyl Alcohols to Allyl Chlorides.

Allyl alcohols are converted to the corresponding allyl chlorides via 2,4-dinitrophenyl ether derivatives.10 The allyl alcohol is treated with DNFB in the presence of triethylamine to yield the corresponding 2,4-dinitrophenyl ether, which in turn is treated with Lithium Chloride in HMPA to give the allyl chloride (eq 3).

Deoxygenation of Phenols.

Pirkle11 reported a method for the deoxygenation of phenols via the corresponding 2,4-dinitrophenyl ethers. The method was employed in their investigation of potential analgesic agents, morphinans and isomorphinans, derived from natural sources.12 Treatment of the phenol derivative with DNFB in toluene-DMF using Sodium Hydride as catalyst gave the 2,4-dinitrophenyl ether in high yields. Catalytic hydrogenation to the 2,4-diaminophenyl ether followed by cleavage with Sodium-Ammonia gave the deoxygenation product in an overall yield of 93% (eq 4).

Degradation of Aldose Sugars.

2,4-DNP derivatives of aldose oximes are used in a Wohl degradation to obtain the next lower aldose sugars.13 For example, D-glucose oxime when treated with DNFB in the presence of sodium bicarbonate provides the oxime aryl ether which decomposes to D-arabinose, 2,4-dinitrophenol, and hydrogen cyanide (eq 5).

Thioamide Protection.

6-Thiodeoxyguanosine upon treatment with DNFB in the presence of triethylamine in acetonitrile give the S-2,4-DNP derivative.14 Deprotection to thiocarbonyl can be achieved simply by treating with 2-Mercaptoethanol at room temperature (eq 6). Such S-2,4-DNP functionalities can also be transformed to free amino, methylamino, methoxy, and oxo groups.

1. (a) Sanger, F. BJ 1945, 39, 507; 1946, 40, 261; 1949, 45, 563. (b) Porter, R. R.; Sanger, F. BJ 1948, 42, 287.
2. Nyquist, H. L.; Davenport, D. A.; Han, P. Y.; Shih, J. G.; Speechly, T. G. JOC 1992, 57, 1449.
3. Bolton, E. C.; Thomson, G. A.; Milburn, G. H. W. JCR(S) 1992, 210.
4. Lloyd, P. F.; Stacey, M. T 1960, 9, 116.
5. (a) Wolfrom, M. L.; Carg, H. G.; Horton, D. JOC 1965, 30, 1556. (b) Wolfrom, M. L.; Bhat, H. B. JOC 1967, 32, 2757.
6. Valli, M. J.; Tang, Y.; Kosh, J. W.; Chapman, Jr., J. M.; Sowell, Sr., W. J. JMC 1992, 35, 3141.
7. Reinheimer, J. D.; Douglass, J. P.; Leister, H. L.; Voelkel, M. B. JOC 1957, 22, 1743.
8. Wolfrom, M. L.; Juliano, B. O.; Toy, M. S.; Chaney, A. JACS 1959, 81, 1446.
9. Astin, K. B.; Musaad, N. S. JOC 1987, 52, 2106.
10. Czernecki, S.; Georgoulis, C. BSF(2) 1975, 405.
11. Pirkle, W. H.; Zabriskie, J. L. JOC 1964, 29, 3124.
12. Pirkle, W. H.; Gates, M. JOC 1965, 30, 1769.
13. Weygand, F.; Löwenfeld, R. CB 1950, 83, 559.
14. Xu, Y.-Z.; Zheng, Q.; Swann, P. F. T 1992, 48, 1729.

Krishnamurthy Nacharaju & Carl R. Johnson

Wayne State University, Detroit, MI, USA

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