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Determination ofD-amino acids. II. Use of a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene

  • Peter Marfey
Article

Abstract

1-fluoro-2,4-dinitrophenyl-5-l-alanine amide has been synthesized in high yield (76%) from 1,5-difluoro-2,4-dinitrobenzene andl-Ala-NH2. This compound contains a reactive fluorine atom which can be used for the reaction with a mixture ofl- andd-amino acids. The resulting diastereomers which are obtained in quantitative yield can be separated and estimated by HPLC. With the five amino acids studied (Ala, Asp, Glu, Met and Phe),l-diastereomers were eluted from the reverse-phase column befored-diastereomers. This behavior can be explained by a stronger intramolecular hydrogen bonding in the latter diastereomer. When artificial mixtures of the five amino acids containing known proportions ofl- andd-isomers were derivatized with the reagent and the reaction products analyzed by HPLC, it was possible to determine the relative content of each isomer in nanomole range.

Keywords

d-amino bifunctional reagent diastereomers 

Abbreviations

DMSO

dimethylsulfoxide

DNDEAP-a a

N-(2,4-dinitro-5-diethylaminophenyl)-amino acid

DNP

2,4-dinitrophenyl

FDAA=F-DNP-l-Ala-NH2

1-fluoro-2,4-dinitrophenyl-5-l-alanine amide

FFDNB

1,5-difluoro-2,4-dinitrobenzene

HO-DAA

1-hydroxy-2,4-dinitrophenyl-5-l-alanine amide

HPLC

high performance liquid chromatography

l-Ala-NH2-HCl

l-Alanine amide hydrochloride

TEAP

triethylammonium phosphate

TLC

thin-layer chromatography

References

  1. 1.
    Manning, J.M. &S. Moore: Determination ofD- andL-amino acids by ion exchange chromatography asL-D andL-L-dipeptides. J. Biol. Chem. 243, 5591–5597 (1968)PubMedGoogle Scholar
  2. 2.
    Marfey, P.S., H. Nowak, M. Uziel &D.A. Yphantis: Reaction of bovine pancreatic ribonuclease A with 1,5-difluoro-2,4-dinitrobenzene. J. Biol. Chem. 240, 3264–3269 (1965)PubMedGoogle Scholar
  3. 3.
    Marfey P.S., M. Uziel &J. Little: Reaction of bovine pancreatic ribonuclease A with 1,5-difluoro-2,4-dinitrobenzene. II. J. Biol. Chem. 240, 3270–3275 (1965)PubMedGoogle Scholar
  4. 4.
    Means, G.E. & R.E. Feeney: Chemical modification of proteins. Holden-Day, Inc. 1971, pp. 39–40Google Scholar
  5. 5.
    Munier, R.L., A.M. Drapier, S. Meunier &P.E. Xuan: New chromogenic reagents of labeling amino groups in peptides. Chromatographia 14, 289–295 (1981)CrossRefGoogle Scholar
  6. 6.
    Wold, F.: Bifunctional reagents. Methods in Enzymology 11, 617–640 (1967)CrossRefGoogle Scholar

Copyright information

© Carlsberg Laboratory 1984

Authors and Affiliations

  • Peter Marfey
    • 1
  1. 1.Department of ChemistryCarlsberg LaboratoryCopenhagen Valby

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