Synthesis of Acyclic Analogs of Adenosine

Antonin Holý1

1 Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic, Prague
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 14.1
DOI:  10.1002/0471142700.nc1401s22
Online Posting Date:  October, 2005
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Abstract

This unit describes preparation of two groups of chiral acyclic nucleosides that are N‐alkyl derivatives of adenine with hydroxyl group(s) on the alkyl chain. As an example of the neutral group, the synthesis is presented for (S)‐(2,3,dihydroxypropyl)adenine (DHPA), a compound with multiple biological effects in proliferating systems. Its synthesis consists of alkylation of adenine with a suitable chiral synthon based on 1,2‐O‐isopropylideneglycerol. Another group of structurally related biologically active compounds are the 9‐alkyladenines bearing both hydroxyl and carboxyl functions. Large‐scale cyanohydrin reaction of the adenine‐containing acetaldehyde intermediate yields 3‐(adenin‐9‐yl)‐2‐hydroxypropanoic acid (AHPA). Lastly, one of the alternative syntheses of its homolog, the nucleoside antibiotic eritadenine (with a d‐erythro‐2,3‐dihydroxybutanoic acid side‐chain) is described starting from d‐ribose. Procedures for preparation of all intermediates as well as the purification of the resulting crude material are described in detail. All these adenine derivatives inhibit SAM‐dependent methylation reactions via inhibition of SAH hydrolase.

Keywords: acyclic nucleoside analogs; adenine derivatives; methylation inhibitors; DHPA; AHPA; eritadenine

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis of 9‐(S)‐(2,3‐Dihydroxypropyl)Adenine (DHPA)
  • Support Protocol 1: Preparation of (R)‐[2,2‐Dimethyl‐1,3‐Dioxolan‐4‐yl]Methyl p‐Tolylsulfonate
  • Alternate Protocol 1: Synthesis of 9‐(RS)‐(2,3‐Dihydroxypropyl)Adenine
  • Basic Protocol 2: Synthesis of 4‐(Adenin‐9‐yl)‐(2R,3R)‐Dihydroxybutanoic Acid (Eritadenine)
  • Support Protocol 2: Preparation of Methyl 2,3‐O‐Isopropylidene‐5‐O‐(p‐Tolylsulfonyl)‐D‐Ribofuranoside
  • Basic Protocol 3: Synthesis of 3‐(Adenin‐9‐yl)‐2‐Hydroxypropanoic Acid (AHPA)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of 9‐(S)‐(2,3‐Dihydroxypropyl)Adenine (DHPA)

  Materials
  • Sodium hydride, 60% dispersion in paraffin oil
  • Dimethylformamide (DMF), anhydrous
  • Adenine
  • (R)‐2,2‐Dimethyl‐4‐hydroxymethyl‐1,3‐dioxolane p‐tolylsulfonate ( S.2; see protocol 2)
  • Toluene, reaction grade
  • Chloroform, reaction grade
  • Celite
  • Methanol, reaction grade
  • Diethyl ether, reaction grade
  • 0.25 M aqueous sulfuric acid
  • Barium hydroxide octahydrate (or monohydrate), crystalline
  • Saturated aqueous barium hydroxide
  • Ethanol, reaction grade
  • 1‐L round‐bottom flask
  • Magnetic stirrer with heating plate and silicon oil bath
  • Calcium chloride protecting tube
  • Rotary evaporator with diaphragm vacuum pump
  • Reflux condenser
  • 8‐ to 10‐cm‐diameter glass filtration funnel with filter flask
  • 500‐ and 1000‐mL Erlenmeyer flasks
  • 2‐L glass or plastic beaker (flat bottomed)
  • Plastic foil
  • Water aspirator
  • pH detection papers
  • Additional reagents and equipment for TLC ( appendix 3D)

Support Protocol 1: Preparation of (R)‐[2,2‐Dimethyl‐1,3‐Dioxolan‐4‐yl]Methyl p‐Tolylsulfonate

  Materials
  • p‐Tolylsulfonyl chloride
  • Pyridine, anhydrous
  • (S)‐4‐Hydroxymethyl‐2,2‐dimethyl‐1,3‐dioxolane ( S.1; Pfanstiehl Laboratories, Senn Chemicals)
  • Ethanol
  • Ethyl acetate
  • Magnesium sulfate, anhydrous
  • 2‐L round‐bottom flask
  • Rotary evaporator

Alternate Protocol 1: Synthesis of 9‐(RS)‐(2,3‐Dihydroxypropyl)Adenine

  Materials
  • Adenine
  • Potassium carbonate
  • 4‐Chloromethyl‐2,2‐dimethyl‐1,3‐dioxolane ( S.5)
  • Dimethylformamide (DMF), anhydrous
  • Ethanol, reaction grade
  • Diethyl ether, reaction grade
  • Methanol, reaction grade
  • Activated charcoal
  • Celite
  • Chloroform
  • Conc. sulfuric acid, reagent grade
  • Aqueous ammonia
  • Saturated barium hydroxide octahydrate (or monohydrate)
  • 3‐L three‐neck round‐bottom flask
  • Mechanical KPG stirrer
  • Reflux condenser
  • Calcium chloride protecting tube
  • Silicon oil bath
  • Rotary evaporator with diaphragm vacuum pump
  • 2‐L round‐bottom flask
  • 8‐ to 10‐cm‐diameter glass filtration funnel with filter flask
  • Additional reagents and equipment for TLC ( appendix 3D)

Basic Protocol 2: Synthesis of 4‐(Adenin‐9‐yl)‐(2R,3R)‐Dihydroxybutanoic Acid (Eritadenine)

  Materials
  • Sodium hydride, 60% dispersion in paraffin oil
  • Dimethylformamide (DMF), anhydrous
  • Adenine
  • Methyl 2,3‐O‐isopropylidene‐5‐O‐(p‐tolylsulfonyl)‐D‐ribofuranoside ( S.9; see protocol 5)
  • Toluene, reaction grade
  • Chloroform, reaction grade
  • Celite
  • Methanol, reaction grade
  • Ethanol, reaction grade
  • Diethyl ether, reaction grade
  • Conc. and 0.25 M aqueous sulfuric acid
  • Sodium hydroxide (NaOH), reagent grade, pellets and 0.1 mM solution
  • Oxygen
  • 2‐Propanol
  • Conc. aqueous ammonia
  • Dowex 50 × 8, acid form (see recipe)
  • Dowex 1 × 2, acetate form (see recipe)
  • 0.02 M acetic acid
  • Conc. formic acid, reagent grade
  • Amberlite IR 45 resin, OH form (see recipe)
  • Silica gel
  • Conc. hydrochloric acid (HCl), reagent grade
  • Calcium chloride protecting tube
  • 500‐ and 1000‐mL round‐bottom flasks
  • Magnetic stirrer with heating plate and silicon oil bath
  • Calcium chloride protecting tube
  • Rotary evaporator with diaphragm vacuum pump
  • 2‐L Erlenmeyer flask
  • 8‐ to 10‐cm‐diameter glass filtration funnel with filter flask
  • 2‐L glass or plastic beakers (flat bottomed)
  • Glass column: 4‐cm diameter, 40‐cm length
  • Fraction collector (optional)
  • UV detector for continuous‐flow detection (optional)
  • 200‐mL dropping funnel
  • Peristaltic pump (optional)
  • Reflux condenser
  • 250‐, 500‐, and 1000‐mL Erlenmeyer flasks
  • Water aspirator
  • Additional reagents and equipment for TLC ( appendix 3D)

Support Protocol 2: Preparation of Methyl 2,3‐O‐Isopropylidene‐5‐O‐(p‐Tolylsulfonyl)‐D‐Ribofuranoside

  Materials
  • D‐Ribose
  • Methanol
  • Conc. sulfuric acid
  • Fehling solutions (see recipe)
  • Sodium methoxide
  • Triethylamine
  • Toluene, reaction grade
  • Acetone
  • 2,2‐Dimethoxypropene
  • 2‐Methoxypropane
  • Diethyl ether, reaction grade
  • Celite
  • Pyridine anhydrous, reagent grade
  • p‐Tolylsulfonyl chloride
  • Ethyl acetate
  • Diluted (1:20) aqueous sulfuric acid
  • Saturated potassium hydrogen carbonate
  • Chloroform, reaction grade
  • Hexane
  • 2‐L three‐neck round‐bottom flask
  • Calcium chloride protecting tubes
  • Dropping funnels
  • Magnetic stirrer with heating plate and silicon oil bath
  • pH detection papers
  • 8‐ to 10‐cm‐diameter glass filtration funnel with filter flask
  • 1‐ and 2‐L round‐bottom flasks
  • Rotary evaporator with diaphragm vacuum pump
  • Reflux condenser
  • Y connector
  • Distillation apparatus: optionally according to Figure , consisting of a 500‐mL distillation flask on a short condenser, attached to a vacuum oil pump
  • 1‐L separatory funnel
  • 500 and 1000 mL Erlenmeyer flasks
  • Water aspirator
  • Glass column: 4 cm diameter, 40 cm length (optional)
  • Fraction collector (optional)
  • Additional reagents and equipment for TLC ( appendix 3D)

Basic Protocol 3: Synthesis of 3‐(Adenin‐9‐yl)‐2‐Hydroxypropanoic Acid (AHPA)

  Materials
  • Adenine
  • Potassium carbonate
  • Dimethylformamide
  • Bromoacetaldehyde diethylacetal
  • Celite
  • Ethanol
  • Conc. hydrochloric acid (HCl), reagent grade
  • Chloroform
  • Methanol
  • Sodium cyanide
  • Acetic acid
  • Diethyl ether
  • 2.5‐L three‐neck round‐bottomed flask
  • Mechanical KPG stirrer
  • Reflux condenser
  • Calcium chloride protecting tube
  • Dropping funnel
  • Magnetic stirrer with heating element and silicon oil bath
  • 8‐ to 10‐cm‐diameter glass filtration funnel with filter flask
  • Rotary evaporator
  • Additional reagents and equipment for TLC ( appendix 3D)
NOTE: All solvents and chemicals are reagent grade.
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Figures

Videos

Literature Cited

Literature Cited
   De Clercq, E. and Holý, A. 1979. Antiviral activity of aliphatic nucleoside analogs: Structure‐function relationship. J. Med. Chem. 22:510‐513.
   De Clercq, E. and Holý, A. 1985. Alkyl esters of 3‐adenin‐9‐yl‐2‐hydroxypropanoic acid: A new class of broad‐spectrum antiviral agents. J. Med. Chem. 28:282‐287.
   De Clercq, E., Descamps, J., De Somer, P., and Holý, A. 1978. (S)‐9‐(2,3‐Dihydroxy‐propyl)adenine: An aliphatic nucleoside analog with broad‐spectrum antiviral activity. Science 200:563‐564.
   De Clercq, E., Leyten, R., Sobis, H., Matousek, J., Holý, A., and De Somer, P. 1981. Inhibitory effect of a broad‐spectrum antiviral agent, (S)‐9‐(2, 3‐dihydroxypropyl)adenine, on spermatogenesis in mice. Toxicol. Appl. Pharmacol. 59:441‐451.
   Holý, A. 1975. Aliphatic analogs of nucleosides, nucleotides and oligonucleotides. Collect. Czech. Chem. Commun. 40:187‐214.
   Holý, A. 1978a. Synthesis of N‐(2,3‐dihydroxypropyl) derivatives of heterocyclic bases. Collect. Czech. Chem. Commun. 43:2054‐2061.
   Holý, A. 1978b. Synthesis of some 2,3‐dihydroxypropyl derivatives of purine bases. Collect. Czech. Chem. Commun. 43:3103‐3117.
   Holý, A. 1984. Preparation and synthetic utilization of 3‐(adenin‐9‐yl)‐2‐hydroxyalkanoic acids and their derivatives. Collect. Czechoslov. Chem. Commun. 49:2148‐2166.
   Holý, A. and Cihák, A. 1981. Metabolism of 9‐(S)‐(2,3‐dihydroxypropyl)adenine, an antiviral agent, in mice. Biochem. Pharmacol. 30:2359‐2361.
   Holý, A. and Vanecek, M. 1979. Synthesis and pharmacological properties of enantiomeric derivatives of 7‐(2,3‐dihydroxypropyl)theophylline. Collect. Czech. Chem. Commun. 44:2550‐2555.
   Holý, A., Votruba, I., and De Clercq, E. 1982. Synthesis and antiviral properties of stereoisomeric eritadenines. Collect. Czech. Chem. Commun. 47:1392‐1407.
   Holý, A., Votruba, I., and De Clercq, E. 1985a. Structure‐activity studies on open‐chain analogs of nucleosides: Inhibition of S‐adenosyl‐L‐homocysteine hydrolase and antiviral activity. 1. Collect. Czech. Chem. Commun. 50:245‐261.
   Holý, A., Votruba, I., and De Clercq, E. 1985b. Structure‐activity studies on open‐chain analogs of nucleosides: Inhibition of S‐adenosyl‐L‐homocysteine hydrolase and antiviral activity. 2. Collect. Czech. Chem. Commun. 50:262‐279.
   Jelínek, R., Holý, A., and Votruba, V. 1981. Embryotoxicity of 9‐(S)‐(2,3‐dihydroxypropyl)‐adenine. Teratology 24:267‐275.
   Pospisil, P., Pilger, B.D., Marveggio, S., Schelling, P., Wurth, C., Scapozza, L., and Folkers, G. 2002. Synthesis, kinetics, and molecular docking of novel 9‐(2‐hydroxypropyl)purine nucleoside analogs as ligands of herpesviral thymidine kinases. Helv. Chim. Acta 85:3237‐3250.
   Sláma, K., Holý, A., and Votruba, I. 1983. Insect sterility induced by a broad‐spectrum antiviral agent, 9‐(2,3‐dihydroxypropyl)adenine. Entomol. Exp. Appl. 33:9‐14.
   Votruba, I. and Holý, A. 1980. Inhibition of S‐adenosyl‐L‐homocysteine hydrolase by the aliphatic nucleoside analog 9‐(S)‐(2,3‐dihydroxypropyl)adenine. Collect. Czech. Chem. Commun. 45:3039‐3044.
   Votruba, I. and Holý, A. 1982. Eritadenines—novel type of potent inhibitors of S‐adenosyl‐L‐homocysteine hydrolase. Collect. Czech. Chem. Commun. 47:167‐174.
   Votruba, I., Holý, A., and De Clercq, E. 1983. Metabolism of the broad‐spectrum antiviral agent, 9‐(S)‐(2,3‐dihydroxypropyl)adenine, in different cell lines. Acta Virol. 27:273‐276.
   Votruba, I., Hasobe, M., Holý, A., and Borchardt, R.T. 1990. 2‐Methylpropyl ester of 3‐(adenin‐9‐yl)‐2‐hydroxypropanoic acid. Mechanism of antiviral action in vaccinia virus‐infected L929 cells. Biochem. Pharmacol. 39:1573‐1580.
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