Multistep Enzyme Catalyzed Reactions for Unnatural Amino Acids

  • Paola D’ArrigoEmail author
  • Davide Tessaro
Part of the Methods in Molecular Biology book series (MIMB, volume 794)


The use of unnatural amino acids, particularly synthetic α-amino acids, for modern drug discovery research requires the availability of enantiomerically pure isomers. Starting from a racemate, one single enantiomer can be obtained using a deracemization process. The two more common strategies of deracemization are those obtained by stereoinversion and by dynamic kinetic resolution. Both techniques will be here described using as a substrate the d,l-3-(2-naphthyl)-alanine, a non-natural amino acid: the first one employing a multi-enzymatic redox system, the second one combining an hydrolytic enzyme together with a base-catalyzed substrate racemization. In both cases, the final product, l-3-(2-naphthyl)alanine, is recovered with good yield and excellent enantiomeric excess.

Key words

d-Amino acid oxidase Amino transferase Non-natural amino acid Multi-enzyme reaction Deracemization Dynamic kinetic resolution 



This work was supported by Cost Action CM0701 “CASCAT, Cascade Chemoenzymatic Processes. New synergies between chemistry and biochemistry, WG2 Multistep deracemization of multifunctional compounds”.


  1. 1.
    Senten K, Van der Veken P, De Meester I et al. (2003) Design, synthesis, and SAR of potent and selective dipeptide-derived inhibitors for dipeptidyl peptidases. J Med Chem 46, 5005–5014.PubMedCrossRefGoogle Scholar
  2. 2.
    Wang L and Schultz P G (2005) Expanding the genetic code. Angew Chem Int Ed Engl 44, 34–66.CrossRefGoogle Scholar
  3. 3.
    Sun H, Nikolovska-Coleska Z, Yang C Y et al. (2004) Structure-based design of potent, conformationally constrained Smac mimetics. J Am Chem Soc 126, 16686–16687.PubMedCrossRefGoogle Scholar
  4. 4.
    Ley S V and Priour A (2002) Total synthesis of the cyclic peptide argyrin B. Eur J Org Chem 23, 3995–4004.CrossRefGoogle Scholar
  5. 5.
    Tanaka M (2007) Design and synthesis of chiral alpha,alpha-disubstituted amino acids and conformational study of their oligopeptides. Chem Pharm Bull 55, 349–358.PubMedCrossRefGoogle Scholar
  6. 6.
    Schneider J P and Kelly J W (1995) Templates that induce alpha-helical, beta-sheet, and loop conformations. Chem Rev 95, 2169–2187.CrossRefGoogle Scholar
  7. 7.
    Patel R N (2000) Microbial/enzymatic synthesis of chiral drug intermediates. Adv Appl Microbiol 47, 33–78.PubMedCrossRefGoogle Scholar
  8. 8.
    Leuchtenberger W, Huthmacher K, and Drauz K (2005) Biotechnological production of amino acids and derivatives: current status and prospects. Appl Microbiol Biot 69, 1–8.CrossRefGoogle Scholar
  9. 9.
    Taylor P P, Pantaleone D P, Senkpeil R F et al. (1998) Novel biosynthetic approaches to the production of unnatural amino acids using transaminases. Trends Biotechnol 16, 412–418.PubMedCrossRefGoogle Scholar
  10. 10.
    Gruber C C, Lavandera I, Faber K et al. (2006) From a racemate to a single enantiomer: Deracemization by stereoinversion. Adv Synth Catal 348, 1789–1805.CrossRefGoogle Scholar
  11. 11.
    Curti B, Ronchi S, and Pilone M S (1992) D- and L-amino acid oxidases in: Müller F (ed.) Chemistry and biochemistry of flavoenzymes, CRC Press, Boca Raton, pp. 66–94.Google Scholar
  12. 12.
    Pollegioni L, Sacchi S, Caldinelli L et al. (2007) Engineering the properties of D-amino acid oxidases by a rational and a directed evolution approach. Curr Protein Pept Sci 8, 600–618.PubMedCrossRefGoogle Scholar
  13. 13.
    Pollegioni L, Piubelli L, Sacchi S et al. (2007) Physiological functions of D-amino acid oxidases: from yeast to humans. Cell Mol Life Sci 64, 1373–1394.PubMedCrossRefGoogle Scholar
  14. 14.
    Helaine V, Rossi J, and Bolte J (1999) A new access to alkyl-alpha-ketoglutaric acids, precursors of glutamic acid analogues by enzymatic transamination. Application to the synthesis of (2S,4R)-4-propyl-glutamic acid. Tetrahedron Lett 40, 6577–6580.CrossRefGoogle Scholar
  15. 15.
    Helaine V, Rossi J, Gefflaut T et al. (2001) Synthesis of 4,4-disubstituted L-glutamic acids by enzymatic transamination. Adv Synth Catal 343, 692–697.CrossRefGoogle Scholar
  16. 16.
    Caligiuri A, D’Arrigo P, Gefflaut T et al. (2006) Multistep enzyme catalysed deracemisation of 2-naphthyl alanine. Biocatal Biotransfor 24, 409–413.CrossRefGoogle Scholar
  17. 17.
    Fantinato S, Pollegioni L, and Pilone M S (2001) Engineering, expression and purification of a His-tagged chimeric D-amino acid oxidase from Rhodotorula gracilis. Enzyme Microb Tech 29, 407–412.CrossRefGoogle Scholar
  18. 18.
    Kagamiyama H and Hayashi H (2000) Branched-chain amino-acid aminotransferase of Escherichia coli. Method Enzymol 324, 103–113.CrossRefGoogle Scholar
  19. 19.
    Kamitori S, Hirotsu K, Higuchi T et al. (1987) Overproduction and preliminary X-ray characterization of aspartate aminotransferase from Escherichia coli. J Biochem 101, 813–816.PubMedCrossRefGoogle Scholar
  20. 20.
    Morino Y, Shimada K, and Kagamiyama H (1990) Mammalian aspartate aminotransferase isozymes. From DNA to protein. Ann N Y Acad Sci 585, 32–47.CrossRefGoogle Scholar
  21. 21.
    Berger A, Smolarsky M, Kurn N et al. (1973) A new method for the synthesis of optically active-amino acids and their N derivatives via acylamino malonates. J Org Chem 38, 457–460.PubMedCrossRefGoogle Scholar
  22. 22.
    Audia J E, Evrard D A, Murdoch G R et al. (1996) Potent, selective tetrahydro-beta-carboline antagonists of the serotonin 2B (5HT2B) contractile receptor in the rat stomach fundus. J Med Chem 39, 2773–2780.PubMedCrossRefGoogle Scholar
  23. 23.
    Greenstein J P and Winitz M (1961) Chemistry of Amino Acids (Vol. 2). Wiley, New York.Google Scholar
  24. 24.
    Koeller K M and Wong C H (2001) Enzymes for chemical synthesis. Nature 409, 232–240.PubMedCrossRefGoogle Scholar
  25. 25.
    Kazlauskas R J and Bornscheuer U T (1998) Biotransformations with lipases in: Reem H-J and Reed G (ed.) Biotechnology, Wiley-WCH, Weinheim, 37–191.Google Scholar
  26. 26.
    Williams J M J, Parker R J, and Neri C (2002) Enzymatic kinetic resolution in: Drauz K and Waldmann H (ed.) Enzyme Catalysis in Organic Synthesis, Wiley-VCH, Weinheim 1, 287–310.Google Scholar
  27. 27.
    Pellissier H (2003) Dynamic kinetic resolution. Tetrahedron 59, 8291–8327.CrossRefGoogle Scholar
  28. 28.
    Turner N J (2004) Enzyme catalysed deracemisation and dynamic kinetic resolution reactions. Curr. Opin. Chem. Biol. 8, 114–119.PubMedCrossRefGoogle Scholar
  29. 29.
    Um P J and Drueckhammer D G (1998) Dynamic enzymatic resolution of thioesters. J Am Chem Soc 120, 5605–5610.CrossRefGoogle Scholar
  30. 30.
    Arosio D, Caligiuri A, D’Arrigo P et al. (2007) Chemo-enzymatic dynamic kinetic resolution of amino acid thioesters. Adv Synth Catal 349, 1345–1348.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Dipartimento di ChimicaMateriali ed Ingegneria Chimica “Giulio Natta” Politecnico di Milano, and “The Protein Factory”, Politecnico di Milano and Università degli Studi dell’InsubriaMilanoItaly

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