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

  • Basic Protocol 1: Generation of AZT H‐Phosphonate with Diphenyl Phosphite and Its Conversion to AZT Amino Acid Phosphoramidate Methyl Amides
  • Alternate Protocol 1: 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 1: 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 (CH 2Cl 2), ACS reagent grade
  • Silica gel 60 (230 to 400 mesh; EMD)
  • Chloroform (CHCl 3), ACS reagent grade
  • Methanol (MeOH), ACS reagent grade
  • Ammonium hydroxide (NH 4OH)
  • 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 F 254)
  • 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 1: Formation of H‐Phosphonate Monoesters Via Bis(N,N‐Diisopropylamino)Chlorophosphine

  • 2′‐Acetyl‐3′‐deoxyadenosine (prepared as described by Zhang et al., )
  • 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

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‐β‐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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