Synthesis of Amino Acid Phosphoramidate Monoesters via H‐Phosphonate Intermediates

Cindy J. Choy1, Dan P. Drontle1, Carston R. Wagner1

1 University of Minnesota, Minneapolis, Minnesota
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 15.1
DOI:  10.1002/0471142700.nc1501s25
Online Posting Date:  July, 2006
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Abstract

Diphenyl phosphite and bis(N,N-diisopropylamino)chlorophosphine are used as phosphitylating reagents to generate H-phosphonate monoesters. These H-phosphonate intermediates are subsequently oxidized with iodine to generate the 5¢-nucleoside amino acid phosphoramidates.

Keywords: diphenyl phosphite; bis(N,N-diisopropylamino)chlorophosphine; phosphoramidate; AZT; nucleoside monophosphate; oxidation

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Generation of AZT H-Phosphonate with Diphenyl Phosphite and Its Conversion to AZT Amino Acid Phosphoramidate Methyl Amides
  • Alternate Protocol: Formation of H-Phosphonate Monoesters Via Bis(N,N-Diisopropylamino)Chlorophosphine
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol: Generation of AZT H-Phosphonate with Diphenyl Phosphite and Its Conversion to AZT Amino Acid Phosphoramidate Methyl Amides

 Materials
  • 3¢-Azido-3¢-deoxythymidine (S.1; AZT; Toronto Research Chemicals)
  • Argon (or nitrogen) gas
  • Anhydrous pyridine (<50 ppm water; Acros)
  • Diphenyl phosphite (85% pure; Sigma-Aldrich)
  • Triethylamine (TEA, 99.5% pure; Sigma-Aldrich)
  • Dichloromethane (CH2Cl2), ACS reagent grade
  • Silica gel 60 (230 to 400 mesh; EMD)
  • Chloroform (CHCl3), ACS reagent grade
  • Methanol (MeOH), ACS reagent grade
  • Ammonium hydroxide (NH4OH)
  • Amino acid methyl ester hydrochloride for conversion (select one):
    • Glycine methyl ester hydrochloride (a)
    • l-Alanine methyl ester hydrochloride (b)
    • l-Valine methyl ester hydrochloride (c)
    • l-Leucine methyl ester hydrochloride (d)
    • l-Tyrosine methyl ester hydrochloride (e)
    • l-Phenylalanine methyl ester hydrochloride (f)
    • l-Tryptophan methyl ester hydrochloride (g)
    • d-Phenylalanine methyl ester hydrochloride (h)
    • d-Tryptophan methyl ester hydrochloride (i)
  • Trimethylsilyl chloride (TMSCl, 99% pure; Sigma-Aldrich)
  • Iodine, sublimed
  • Amberlite IRP 64 (100 to 400 wet mesh; Sigma-Aldrich)
  • Methylamine solution (see recipe)
  • Vacuum pump
  • 10- and 50-mL pear-shaped flasks
  • Cannula (Teflon tubing)
  • Rotary evaporator equipped with a water aspirator
  • 5 × 17–, 2 × 6–, 3 × 17–, and 1.8 × 8–cm chromatography columns
  • TLC plates: silica-coated aluminum plates with fluorescent indicator (Merck silica gel 60 F254)
  • 254-nm UV lamp
  • 50-mL round-bottom flasks
  • Capped vial
  • Lyophilizer
  • Additional reagents and equipment for column chromatography (appendix 3E) and TLC (appendix 3D)

Alternate Protocol: Formation of H-Phosphonate Monoesters Via Bis(N,N-Diisopropylamino)Chlorophosphine

 Additional Materials (also see Basic Protocol)
  • 2¢-Acetyl-3¢-deoxyadenosine (prepared as described by Zhang et al., 2003)
  • Anhydrous N,N-diisopropylethylamine (DIPEA, 99% pure; Sigma-Aldrich)
  • Bis(N,N-diisopropylamino)chlorophosphine (Acros)
  • Anhydrous dioxane (99% pure; Acros)
  • Glacial acetic acid
  • Dowex 50WX4-50 (20 to 50 mesh; Sigma-Aldrich)
  • Acetone/dry ice bath
  • 2.5 × 15–cm chromatography columns
  • 100-mL round-bottom flasks
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Figures

  •  FigureFigure 15.1.1 Formation of AZT amino acid phosphoramidates. R = amino acid; (a) glycine; (b) l-alanine; (c) l-valine; (d) l-leucine; (e) l-tyrosine; (f) l-phenylalanine; (g) l-tryptophan; (h) d-phenylalanine; (i) d-tryptophan.
    View Image
  •  FigureFigure 15.1.2 Alternate Protocol for the formation of nucleoside H-phosphonates.
    View Image

Videos

Literature Cited

Literature Cited
    Abraham, T.W. and Wagner, C.R. 1994. A phosphoramidite-based synthesis of phosphoramidate amino acid diesters of antiviral nucleosides. Nucleosides Nucleotides 13:1891-1903.
    Abraham, T.W., Kalman, T.I., McIntee, E.J., and Wagner, C.R. 1996. Synthesis and biological activity of aromatic amino acid phosphoramidates of 5-fluoro-2¢-deoxyuridine and 1--arabinofuranosylcytosine: Evidence of phosphoramidase activity. J. Med. Chem. 39:4569-4575.
    Abraham, T.W., McIntee, E.J., Iyer, V.V., Schinazi, R.F., and Wagner, C.R. 1997. Synthesis, biological activity, and decomposition studies of amino acid phosphomonoester amidates of acyclovir. Nucleosides Nucleotides 16:2079-2092.
    Garegg, P.J., Regber, T., Stawinski, J., and Stromberg, R. 1987. Nucleoside phosphonates: Part 7. Studies on the oxidation of nucleoside phosphonate esters. J. Chem. Soc., Perkin Trans. 1 1269-1273.
    Iyer, V.V., Griesgraber, G.W., Radmer, M.R., McIntee, E.J., and Wagner, C.R. 2000. Synthesis, in vitro anti-breast cancer activity, and intracellular decomposition of amino acid methyl ester and alkyl amide phosphoramidate monoesters of 3¢-azido-3¢-deoxythymidine (AZT). J. Med. Chem. 43:2266-2274.
    Jankowska, J., Sobkowski, M., Stawinski, J., and Kraszewski, A. 1994. Studies on aryl H-phosphonates. I. An efficient method for the preparation of deoxyribo- and ribonucleoside 3¢-H-phosphonate monoesters by transesterification of diphenyl H-phosphonate. Tetrahedron Lett. 35:3355-3358.
    Kers, A. and Kraszewski, A. 1998. A new synthetic method for the preparation of nucleoside phosphoramidate analogues with the nitrogen atom in bridging positions of the phosphoramidate linkage. Tetrahedron Lett. 39:1219-1222.
    Marugg, J., Tromp, M., Kuyl-Yeheskiely, E., van der Marel, G.A., and van Boom, J.H. 1986a. A convenient and general approach to the synthesis of properly protected d-nucleoside-3¢-hydrohenphosphonates via phosphite intermediates. Tetrahedron Letters 27:2661-2664.
    Marugg, J., Tromp, M., van der Marel, G.A., and van Boom, J.H. 1986b. A new versatile approach to the preparation of valuable deoxynucleoside 3¢-phosphite intermediates. Tetrahedron Lett. 27:2271-2274.
    Nahum, V., Zundorf, G., Levesque, S.A., Beaudoin, A.R., Reiser, G., and Fischer, B. 2002. Adenosine 5¢-O-(1-boranotriphosphate) derivatives as novel P2Y(1) receptor agonists. J. Med. Chem. 45:5384-5396.
    Zhang, L., Cui, Z., and Zhang, B. 2003. An efficient synthesis of 3¢-amino-3¢-deoxyguanosine from guanosine. Helv. Chim. Acta 86:703-710.
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