Synthesis of 5‐Aminoimidazole‐4‐Carboxamide Riboside (AICAR) and Its Derivatives Using Inosine as Starting Material

Stefano D'Errico1, Giorgia Oliviero1, Nicola Borbone1, Vincenzo Piccialli2, Gennaro Piccialli3

1 Dipartimento di Farmacia, Università degli Studi di Napoli ‘Federico II’, Napoli, 2 Dipartimento di Scienze Chimiche, Università degli Studi di Napoli ‘Federico II’, Napoli, 3 Istituto di Biochimica delle Proteine (IBP), CNR, Napoli
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.35
DOI:  10.1002/0471142700.nc0135s63
Online Posting Date:  December, 2015
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Abstract

This unit contains four basic protocols describing the synthesis of 5‐aminoimidazole‐4‐carboxamide riboside (AICAR), 5‐aminoimidazole‐4‐carboxamide riboside (ZPM), their 4‐N functionalized derivatives, and two sugar‐modified analogs of AICAR. The first and second basic protocols reveal the importance of solid‐phase synthesis to obtain novel AICAR and ZMP imidazole‐modified analogs in a short time, whereas the third and fourth basic protocols allow for the rapid preparation of 5′‐F‐AICAR and D‐ribityl AICA. © 2015 by John Wiley & Sons, Inc.

Keywords: aicar; ZMP; AMPK; acyclic nucleosides; solid‐phase synthesis; base‐modified nucleosides

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of AICAR and Its 4‐N Alkyl/Hydroxyalkyl Derivatives by Solid‐Phase Synthesis
  • Basic Protocol 2: Preparation of ZMP And Its 4‐N Alkyl/Hydroxyalkyl/Aminoalkyl Derivatives by Solid‐Phase Synthesis
  • Basic Protocol 3: Synthesis of 5′‐F‐AICAR Starting from 6‐Chloropurine Riboside
  • Basic Protocol 4: Synthesis of D‐Ribityl AICA Starting from 2′,3′‐O‐Isopropylideneinosine
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of AICAR and Its 4‐N Alkyl/Hydroxyalkyl Derivatives by Solid‐Phase Synthesis

  Materials
  • Inosine (1, Sigma‐Aldrich, >99%)
  • Polystyrenemonomethoxytrityl chloride resin MMTCl (CBL Patras; 1% Divinylbenzene, 200‐400 mesh, f = 1.2 mmol g−1)
  • 4‐Dimethylaminopyridine (DMAP; Sigma‐Aldrich, purity >99.8%)
  • Argon
  • Pyridine (Py; Sigma‐Aldrich, anhydrous, purity >99%)
  • Dichloromethane (DCM; Sigma‐Aldrich, ACS solvent)
  • Methanol (MeOH; Sigma‐Aldrich, ACS solvent)
  • Acetic anhydride (Ac 2O; Sigma‐Aldrich, purity >98%)
  • N,N′‐Dimethylformamide (DMF; Sigma‐Aldrich, anhydrous, purity >99%)
  • Potassium carbonate (K 2CO 3; Sigma‐Aldrich, purity >99%)
  • 1‐Chloro‐2,4‐dinitrobenzene (DNBCl; Sigma‐Aldrich, purity >99%)
  • N,N′‐Dimethylformamide (DMF; Sigma‐Aldrich, ACS solvent)
  • 1,2‐diaminoethane (Sigma‐Aldrich, purified by redistillation, purity >99.5%)
  • Trifluoroacetic acid (TFA; Sigma‐Aldrich, purity 99%)
  • Chloroform (CHCl 3)
  • AICAR, (8; Sigma‐Aldrich, purity >99%)
  • Propylamine (Sigma‐Aldrich, purity >99%)
  • Sodium hydroxide (NaOH; Sigma‐Aldrich, purity 97%)
  • Ethanol (EtOH; Sigma‐Aldrich, absolute)
  • 5‐,10‐ and 25‐mL round‐bottom flasks
  • Stirring bars
  • Rubber septum
  • Magnetic stirrer
  • Isolute single fritted reservoirs (SG), 20 μm PE, equipped with tube caps and luer tip caps (Biotage)
  • Rotary evaporator
  • Vacuum oil pump
  • Multi‐reax vibrating shaker (Heidolph)
  • Oil bath
  • Analytical TLC plates (silica gel 60, F 254, 0.2 mm thick, Merck)
  • Preparative TLC plates (silica gel 60, F 254, 0.5 mm thick, Merck)
  • UV lamp
  • Büchner funnels with fused‐in sintered glass discs of porosity 3
  • Büchner flasks
  • Condenser
  • Additional reagents and equipment for thin‐layer chromatography (TLC; Meyers and Meyers, )

Basic Protocol 2: Preparation of ZMP And Its 4‐N Alkyl/Hydroxyalkyl/Aminoalkyl Derivatives by Solid‐Phase Synthesis

  Materials
  • Inosine (1, Sigma‐Aldrich, >99%)
  • N,N′‐Dimethylformamide (DMF; Sigma‐Aldrich, anhydrous, purity >99%)
  • Imidazole (Sigma‐Aldrich, purity >99%)
  • tert‐Butyl(chloro)diphenylsilane (TBDPSCl; Sigma‐Aldrich, purity >98%)
  • Dichloromethane (DCM; Sigma‐Aldrich, ACS solvent)
  • Methanol (MeOH; Sigma‐Aldrich, ACS solvent)
  • Distilled water
  • Sodium sulfate (Na 2SO 4)
  • Silica gel 60 (0.063‐0.200 μm, Merck)
  • p‐Toluenesulfonic acid monohydrate (p‐TsOH; Sigma‐Aldrich, purity >98.5%)
  • 4‐(Hydroxymethyl)benzaldehyde dimethyl acetal (Sigma‐Aldrich, purity >97%)
  • Ethyl acetate (AcOEt; Sigma‐Aldrich, ACS solvent)
  • Polystyrenemonomethoxytrityl chloride resin MMTCl (CBL Patras, 1% Divinylbenzene, 200‐400 mesh, f = 1.2 mmol g−1)
  • 4‐Dimethylaminopyridine (DMAP; Sigma‐Aldrich, purity >99.8%)
  • Argon
  • Pyridine (Py; Sigma‐Aldrich, anhydrous, purity >99%)
  • Potassium carbonate (K 2CO 3; Sigma‐Aldrich, purity >99%)
  • 1‐Chloro‐2,4‐dinitrobenzene (DNClB; Sigma‐Aldrich, purity >99%)
  • N,N′‐Dimethylformamide (DMF; Sigma‐Aldrich, ACS solvent)
  • Ammonium fluoride (NH 4F, purity >98%)
  • Tetrahydrofuran (THF; Sigma‐Aldrich, anhydrous, purity >99.9%)
  • Bis[2‐(trimethylsilyl)ethyl)diisopropylphosphoramidite (iPr 2NP(OTSE) 2, Iris‐Biotech, 97%]
  • Tetrazole (Sigma‐Aldrich, purity >98%)
  • tert‐Butyl hydroperoxide (tBuOOH; 5.5 M in decane, anhydrous, Sigma‐Aldrich)
  • 1,2‐diaminoethane (Sigma‐Aldrich, purified by redistillation, purity >99.5%)
  • Trifluoroacetic acid (TFA; Sigma‐Aldrich, purity 99%)
  • Isopropanol (iPrOH; Sigma‐Aldrich, HPLC solvent)
  • Ammonium hydroxyde solution (NH 4OH (aq); 30% to 33% NH 3 in H 2O, Sigma‐Aldrich)
  • ZMP (19, Sigma‐Aldrich, purity >99%)
  • Triethylammonium bicarbonate buffer (TEAB; 1 M pH = 8.5, Fluka, HPLC volatile buffer)
  • Acetonitrile (CH 3CN; Sigma‐Aldrich, HPLC solvent)
  • Butylamine (Sigma‐Aldrich, purity >99%)
  • Sodium hydroxide (NaOH; Sigma‐Aldrich, purity 97%)
  • Ethanol (EtOH; Sigma‐Aldrich, absolute)
  • 5‐,10‐, 25‐, and 500‐mL: round‐bottom flasks
  • Analytical TLC plates (silica gel 60, F 254, 0.2 mm thick, Merck)
  • Preparative TLC plates (silica gel 60, F 254, 0.5 mm thick, Merck)
  • UV lamp
  • Rotary evaporator
  • Vacuum oil pump
  • Separatory funnels
  • Glass columns for chromatography
  • Oil bath
  • Stirring bars
  • Rubber septum
  • Magnetic stirrer
  • Isolute single fritted reservoirs (SG) 20 μm PE, equipped with tube caps and luer tip caps (Biotage)
  • Multi‐reax vibrating shaker (Heidolph)
  • HPLC equipped with UV/Vis detector
  • C‐18 reverse phase column (Purosphere STAR, 5 μm, 250 × 10 mm, Merck)
  • Syringe filters for HPLC (25 mm, 0.45 μm GHP membrane, Pall)
  • Lyophilizer
  • Condenser
  • Büchner funnels with fused‐in sintered glass discs of porosity 3
  • Büchner flasks
  • Additional reagents and equipment for thin‐layer chromatography (TLC; Meyers and Meyers, )

Basic Protocol 3: Synthesis of 5′‐F‐AICAR Starting from 6‐Chloropurine Riboside

  Materials
  • 6‐Chloropurine riboside (21, Sigma‐Aldrich, >99%)
  • Acetone (VWR, anhydrous, purity >99,)
  • 2,2‐Dimethoxypropane (DMP; Sigma‐Aldrich, 98%)
  • p‐Toluenesulfonic acid mononhydrate (p‐TsOH; Sigma‐Aldrich, purity >98.5%)
  • Methanol (MeOH; Sigma‐Aldrich, ACS solvent)
  • Chloroform (CHCl 3)
  • Sodium bicarbonate (NaHCO 3, Sigma‐Aldrich, purity >99%)
  • Sodium sulfate (Na 2SO 4)
  • Silica gel 60 (0.063‐0.200 μm, Merck)
  • Tetrahydrofuran (THF; Sigma‐Aldrich, anhydrous, purity >99.9%)
  • Tetrabutylammonium fluoride solution (TBAF; 1 M in THF, Sigma‐Aldrich)
  • p‐Toluensulfonyl fluoride (p‐TsF; Sigma‐Aldrich, purity 98%)
  • n‐Hexane (Sigma‐Aldrich, ACS solvent)
  • Ethyl acetate (AcOEt; Sigma‐Aldrich, ACS solvent)
  • Dichloromethane (CH 2Cl 2; Sigma‐Aldrich, ACS solvent)
  • Dioxane (Sigma‐Aldrich, ACS solvent)
  • Sodium hydroxide (NaOH; Sigma‐Aldrich, purity 97%)
  • Hydrochloric acid (37% aqueous HCl solution, Sigma‐Aldrich, ACS reagent)
  • Trifluoroacetic acid (TFA; Sigma‐Aldrich, purity 99%)
  • Toluene (Sigma‐Aldrich, ACS solvent)
  • Pyridine (Py; Sigma‐Aldrich, anhydrous, purity >99%)
  • Acetic anhydride (Ac 2O; Sigma‐Aldrich, purity >98%)
  • N,N′‐Dimethylformamide (DMF; Sigma‐Aldrich, anhydrous, purity >99%)
  • Potassium carbonate (K 2CO 3; Sigma‐Aldrich, purity >99%)
  • 1‐Chloro‐2,4‐dinitrobenzene (DNClB; Sigma‐Aldrich, purity >99%)
  • N,N′‐Dimethylformamide (DMF; Sigma‐Aldrich, ACS solvent)
  • 1,2‐diaminoethane (Sigma‐Aldrich, purified by redistillation, purity >99.5%)
  • 5‐, 10‐, 25‐, and 50‐mL round‐bottom flasks
  • Analytical TLC plates (silica gel 60, F 254, 0.2 mm thick, Merck)
  • UV lamp
  • Separatory funnels
  • Oil bath
  • Rotary evaporator
  • Vacuum oil pump
  • Glass columns for chromatography
  • Preparative TLC plates (silica gel 60, F 254, 0.5 mm thick, Merck)
  • Büchner funnels with fused‐in sintered glass discs of porosity 3
  • Büchner flasks
  • Stirring bars
  • Condenser
  • Additional reagents and equipment for thin‐layer chromatography (TLC; Meyers and Meyers, )

Basic Protocol 4: Synthesis of D‐Ribityl AICA Starting from 2′,3′‐O‐Isopropylideneinosine

  Materials
  • 2′,3′‐O‐Isopropylideneinosine (29; Sigma‐Aldrich, >99%)
  • Argon
  • Tetrahydrofuran (THF; Sigma‐Aldrich, anhydrous, purity >99.9%)
  • Diisobutylaluminium hydride (DIBAL‐H; 1.0 M toluene solution, Sigma‐Aldrich)
  • Methanol (MeOH; Sigma‐Aldrich, ACS solvent)
  • Chloroform (CHCl 3)
  • Acetic acid (AcOH; Sigma‐Aldrich, purity >99.7 %)
  • Silica gel 60 (0.063‐0.200 μm, Merck)
  • Acetic anhydride (Ac 2O; Sigma‐Aldrich, purity >98%)
  • Pyridine (Py; Sigma‐Aldrich, anhydrous, purity >99%)
  • Toluene (Sigma‐Aldrich, ACS solvent)
  • N,N′‐Dimethylformamide (DMF; Sigma‐Aldrich, anhydrous, purity >99%)
  • 1‐Chloro‐2,4‐dinitrobenzene (DNClB; Sigma‐Aldrich, purity >99%)
  • Potassium carbonate (K 2CO 3; Sigma‐Aldrich, purity >99%)
  • Sodium sulfate (Na 2SO 4)
  • 1,2‐diaminoethane (Sigma‐Aldrich, purified by redistillation, purity >99.5%)
  • Trifluoroacetic acid (TFA; Sigma‐Aldrich, purity 99%)
  • 5‐,10‐, 25‐, 100‐, and 500‐mL round‐bottom flasks
  • Stirring bars
  • Rubber septum
  • Magnetic stirrer
  • Analytical TLC plates (silica gel 60, F 254, 0.2‐mm thick, Merck)
  • UV lamp
  • Rotary evaporator
  • Vacuum oil pump
  • Glass columns for chromatography
  • Oil bath
  • Separatory funnels
  • Preparative TLC plates (silica gel 60, F 254, 0.5‐mm thick, Merck)
  • Büchner funnels with fused‐in sintered glass discs of porosity 3
  • Büchner flasks
  • Additional reagents and equipment for thin‐layer chromatography (TLC; Meyers and Meyers, )
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Figures

Videos

Literature Cited

Literature Cited
  D'Errico, S., Oliviero, G., Borbone, N., Amato, J., Piccialli, V., Varra, M., Mayol, L., and Piccialli, G. 2011. Solid‐phase synthesis of a new diphosphate 5‐aminoimidazole‐4‐carboxamide riboside (AICAR) derivative and studies toward cyclic AICAR diphosphate ribose. Molecules 16:8110‐8118. doi: 10.3390/molecules16098110.
  D'Errico, S., Oliviero, G., Borbone, N., Amato, J., D'Alonzo, D., Piccialli, V., Mayol, L., and Piccialli, G. 2012. A Facile synthesis of 5′‐fluoro‐5′‐deoxyacadesine (5′‐F‐AICAR): A novel non‐phosphorylable AICAR analogue. Molecules 17:13036‐13044. doi: 10.3390/molecules171113036.
  D'Errico, S., Oliviero, G., Borbone, N., Amato, J., Piccialli, V., Varra, M., Mayol, L., and Piccialli, G. 2013. Synthesis of new acadesine (AICA‐riboside) analogues having acyclic D‐ribityl or 4‐hydroxybutyl chains in place of the ribose. Molecules 18:9420‐9431. doi: 10.3390/molecules18089420.
  D'Errico, S., Oliviero, G., Piccialli, V., Amato, J., Borbone, N., D'Atri, V., D'Alessio, F., Di Noto, R., Ruffo, F., Salvatore, F., and Piccialli, G. 2011. Solid‐phase synthesis and pharmacological evaluation of novel nucleoside‐tethered dinuclear platinum(II) complexes. Bioorg. Med. Chem. Lett. 21:5835‐5838. doi: 10.1016/j.bmcl.2011.07.104.
  Meyers, C. and Meyers, D. 2008. Thin‐layer chromatography. Curr. Protoc. Nucl. Acid Chem. 34:A.3D.1‐A.3D.13. doi: 10.1002/0471142700.nca03ds34.
  Oliviero, G., Amato, J., Borbone, N., D'Errico, S., Piccialli, G., and Mayol, L. 2007. Synthesis of N‐1 and ribose modified inosine analogues on solid support. Tetrahedron Lett. 48:397‐400. doi: 10.1016/j.tetlet.2006.11.085.
  Oliviero, G., D'Errico, S., Borbone, N., Amato, J., Piccialli, V., Piccialli, G., and Mayol, L. 2010. Facile solid‐phase synthesis of AICAR 5′‐monophosphate (ZMP) and its 4‐N‐alkyl derivatives. Eur. J. Org. Chem. 1517‐1524. doi: 10.1002/ejoc.200901271.
  Oliviero, G., Amato, J., Borbone, N., D'Errico, S., Piccialli, G., Bucci, E., Piccialli, V., and Mayol, L. 2008. Synthesis of 4‐N‐alkyl and ribose modified analogues on solid support. Tetrahedron 64:6475‐6481. doi: 10.1016/j.tet.2008.04.071.
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