Synthesis of Methylenebis(phosphonate) Analogs of Dinucleotide Pyrophosphates

Krzysztof W. Pankiewicz1, Guangyao Gao1, Steven E. Patterson1

1 The University of Minnesota, Center for Drug Design, Minneapolis, Minnesota
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 13.5
DOI:  10.1002/0471142700.nc1305s24
Online Posting Date:  April, 2006
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Abstract

A facile method is described for preparation of nonsymmetrical P1,P2‐methylenebis(phosphonate) diesters based on the 31P‐NMR‐controlled reaction of methylenebis(phosphonate) monoesters with diisopropylcarbodiimide, resulting in the formation of the intermediate P1,P4‐disubstituted bicyclic trisanhydride. This intermediate, after treatment with an another nucleoside, carbohydrate, or alcohol followed by hydrolysis, is converted into the corresponding methylenebis(phosphonate) diester. An analog of a natural dinucleotide pyrophosphate can be obtained when a nucleoside 5′‐methylenebis(phosphonate) is coupled with another nucleoside. This method is suitable for preparation of metabolically stable (resistant to phosphodiesterase cleavage) analogs of NAD, FAD, and related natural pyrophosphates. The resulting compounds are useful for mechanistic studies of enzymes that use the natural pyrophosphates as cofactors or substrates, and in development of inhibitors that have potential applications as therapeutic agents.

Keywords: NAD analogs; nucleoside 5′‐methylenebis(phosphonate); P1,P4‐disubstituted bicyclic trisanhydrides; MAD analog

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of 2′,3′‐O‐Isopropylidenenucleoside 5′‐Methylenebis(phosphonate)s from 2′,3′‐O‐Isopropylidenenucleosides
  • Alternate Protocol 1: Preparation of Methylenebis(phosphonate) Monoesters from Other Starting Compounds
  • Basic Protocol 2: Preparation of Methylenebis(phosphonate) Analogs of Dinucleoside Pyrophosphates
  • Alternate Protocol 2: Preparation of P1,P2‐Non‐Symmetrically Substituted Methylenebis(phosphonate) Diesters
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of 2′,3′‐O‐Isopropylidenenucleoside 5′‐Methylenebis(phosphonate)s from 2′,3′‐O‐Isopropylidenenucleosides

  Materials
  • 2′,3′‐O‐Isopropylidene nucleoside (e.g., Sigma‐Aldrich or Berry & Associates, http://www.berryassoc.com)
  • Triethyl phosphate (EtO) 3PO, anhydrous
  • Methylenebis(phosphonic acid dichloride) (Sigma‐Aldrich)
  • Solvent A: 0.04 M triethylammonium bicarbonate (TEAB), pH 7.0 to 7.5 (see recipe for 1 M)
  • Solvent B: 70% (v/v) acetonitrile in H 2O
  • 1 M TEAB, pH 7.0 to 7.5 (see recipe)
  • Ethyl acetate (EtOAc)
  • HPLC system with:
    • C18 RP‐HPLC analytical column (Varian Microsorb‐MV 100‐5, 250 × 4.6–mm; 5‐µm particle size, 100 Å pore size)
    • C18 RP‐HPLC preparative column (Varian Microsorb, 250 × 41.4–mm; 8‐µm particle size, 100 Å pore size)
    • UV‐Vis detector
  • Separatory funnel
  • Rotary evaporator
  • Lyophilizer

Alternate Protocol 1: Preparation of Methylenebis(phosphonate) Monoesters from Other Starting Compounds

  • Alcohol derivative (e.g., 7‐O‐benzyl‐C2‐mycophenolic alcohol; Pankiewicz et al., ), carbohydrate, 2′‐ or 3′‐protected nucleoside, or unprotected nucleoside
  • Methanol (MeOH)
  • 10% palladium catalyst on activated carbon (Sigma‐Aldrich)

Basic Protocol 2: Preparation of Methylenebis(phosphonate) Analogs of Dinucleoside Pyrophosphates

  Materials
  • 2′,3′‐O‐Isopropylidenenucleoside 5′‐methylenebis(phosphonate): e.g., 2′,3′‐O‐isopropylideneadenosine 5′‐methylenebis(phosphonate) ( protocol 1)
  • Anhydrous pyridine, HPLC grade
  • Anhydrous pyridine‐d 5
  • Diisopropylcarbodiimide (DIC)
  • Protected nucleoside containing free 5′‐hydroxyl group: e.g., 3‐(2′,3′‐O‐isopropylidene ‐β‐D‐ribofuranos‐5‐yl)benzamide
  • Solvent A: 0.1 M triethylammonium bicarbonate (TEAB), pH 7.0 to 7.5 (see recipe)
  • Solvent B: 70% (v/v) acetonitrile in H 2O
  • Dowex 50WX8‐200, H+ form
  • Dowex 50WX8‐200, Na+ form
  • HPLC system with:
    • C18 HPLC analytical column (Varian Microsorb‐MV 100‐5, 250 × 4.6–mm; 5‐µm particle size, 100 Å pore size)
    • C18 RP‐HPLC preparative column (Varian Microsorb, 250 × 41.4–mm; 8‐µm particle size, 100 Å pore size)
    • UV‐Vis detector
  • Filter paper or Buchner funnel
  • Rotary evaporator
  • Lyophilizer
  • Glass chromatography column

Alternate Protocol 2: Preparation of P1,P2‐Non‐Symmetrically Substituted Methylenebis(phosphonate) Diesters

  • Alcohol 5′‐methylenebis(phosphonate) ( protocol 2)
  • Protected nucleoside containing free 5′‐hydroxyl group: e.g., 2′,3′‐O‐isopropylideneadenosine
  • Trifluoroacetic acid (CF 3COOH)
  • Parr hydrogenation apparatus (Parr Instrument) and hydrogen source
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Figures

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Literature Cited

Literature Cited
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