Synthesis of γ‐Phosphate‐Labeled and Doubly Labeled Adenosine Triphosphate Analogs

Stephan M. Hacker1, Moritz Welter1, Andreas Marx1

1 Department of Chemistry and Konstanz Research School Chemical Biology, University of Konstanz, Konstanz
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 13.14
DOI:  10.1002/0471142700.nc1314s60
Online Posting Date:  March, 2015
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Abstract

This unit describes the synthesis of γ‐phosphate‐labeled and doubly labeled adenosine triphosphate (ATP) analogs and their characterization using the phosphodiesterase I from Crotalus adamanteus (snake venom phosphodiesterase; SVPD). In the key step of the synthesis, ATP or an ATP analog, bearing a linker containing a trifluoroacetamide group attached to the nucleoside, are modified with an azide‐containing linker at the terminal phosphate using an alkylation reaction. Subsequently, different labels are introduced to the linkers by transformation of one functional group to an amine and coupling to an N‐hydroxysuccinimide ester. Specifically, the Staudinger reaction of the azide is employed as a straightforward means to obtain an amine in the presence of various labels. Furthermore, the fluorescence characteristics of a fluorogenic, doubly labeled ATP analog are investigated following enzymatic cleavage by SVPD. © 2015 by John Wiley & Sons, Inc.

Keywords: alkylation reaction; Staudinger reaction; modified nucleotides; enzyme probes; Förster resonance energy transfer

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of γ‐Phosphate‐Modified and Doubly Modified ATP Analogs
  • Basic Protocol 2: Coupling of an NHS Ester to Free Amines in the Nucleotide
  • Basic Protocol 3: Analysis of Doubly Labeled ATP Analogs with SVPD
  • Support Protocol 1: Synthesis of 2′‐O‐(6‐Trifluoroacetamidohexyl) Adenosine Triphosphate
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of γ‐Phosphate‐Modified and Doubly Modified ATP Analogs

  Materials
  • Azidoalcohol (MCAT GmbH)
  • p‐Toluenesulfonyl chloride (TsCl)
  • Anhydrous dichloromethane (DCM)
  • Anhydrous pyridine (pyr)
  • 37% Hydrochloric acid
  • Distilled water
  • Dichloromethane (DCM)
  • Magnesium sulfate (MgSO 4)
  • Silica gel
  • n‐Hexane
  • Ethyl acetate (EtOAc)
  • Iodine
  • Chloroform‐d 1 (CDCl 3)
  • 1,6‐Dibromohexane
  • Dimethyl sulfoxide (DMSO)
  • Sodium azide
  • Diethyl ether (Et 2O)
  • Saturated aqueous sodium chloride solution
  • Adenosine triphosphate (ATP)
  • 2′‐O‐(6‐trifluoroacetamidohexyl)adenosine triphosphate (see protocol 4)
  • CHELEX‐100 ion‐exchange resin (sodium form)
  • Tetrabutylammonium bromide
  • Anhydrous dimethylformamide (DMF)
  • Oven‐dried 4 Å molecular sieves (MS, prepared by heating molecular sieves to 160°C under reduced pressure over night)
  • 25% Aqueous ammonia
  • 0.1 M triethylammonium hydrogen carbonate buffer (TEAB; see recipe)
  • 1 M triethylammonium hydrogen carbonate buffer (TEAB; see recipe)
  • Acetonitrile (ACN)
  • 50 mM triethylammonium acetate buffer (TEAA; see recipe)
  • Methanol‐d 4 (CD 3OD)
  • 10‐, 50‐, 100‐, and 250‐mL one‐neck round‐bottom flasks
  • Schlenk line, supplying nitrogen and high vacuum
  • Magnetic stirrer
  • Magnetic stir bar
  • 50‐ and 250‐ mL separatory funnels
  • Folded filter paper
  • Rotary evaporator connected to a vacuum pump
  • Evaporator trap
  • Glass columns (1.5 cm diameter, 20 cm length; and 6 cm diameter, 20 cm length)
  • TLC plates
  • Glass chamber
  • UV lamp
  • Acance III 400 MHz or 600 MHz NMR spectrometer (Bruker)
  • Nanodrop ND‐1000 spectrophotometer (Peqlab)
  • Centrifuge
  • Fast protein liquid chromatography (FPLC) column (see recipe)
  • Äkta purifier (FPLC system, GE Healthcare)
  • Nucleodur RP‐18 HTec column (16 mm diameter, 250 mm length; Macherey‐Nagel)
  • Prominence System with preparative LC‐20AP pumps (preparative HPLC system, Shimadzu)
  • amaZon SL (for electrospray ionization mass spectrometry [ESI‐MS], Bruker)
  • Freeze‐dryer

Basic Protocol 2: Coupling of an NHS Ester to Free Amines in the Nucleotide

  Materials
  • Azide‐containing nucleotide (see protocol 1)
  • Distilled water
  • Methanol (MeOH)
  • Triethylamine (Et 3N)
  • Tris(2‐carboxyethyl)phosphine hydrochloride (TCEP·HCl)
  • Acetonitrile (ACN)
  • 50 mM triethylammonium acetate buffer (TEAA; see recipe)
  • Deuterated water (D 2O)
  • 0.1 M sodium bicarbonate (NaHCO 3; pH 8.7) in distilled water
  • N‐hydroxysuccinimde (NHS) ester of the label (e.g., Sulfo‐Cy3 NHS ester or Sulfo‐Cy5 NHS ester; GE Healthcare)
  • Dimethylformamide (DMF)
  • Methanol‐d 4 (CD 3OD)
  • Magnetic stir bar
  • Magnetic stirrer
  • Rotary evaporator connected to a vacuum pump
  • Evaporator trap
  • Nucleodur RP‐18 HTec column (16 mm diameter, 250 mm length; Macherey‐Nagel)
  • Prominence System with preparative LC‐20AP pumps (preparative HPLC system, Shimadzu)
  • 5‐ and 10‐mL one‐neck round‐bottom flasks
  • Freeze‐dryer
  • amaZon SL (ESI‐MS, Bruker)
  • Acance III 400 MHz NMR spectrometer (Bruker)
  • Nanodrop ND‐1000 spectrophotometer (Peqlab)

Basic Protocol 3: Analysis of Doubly Labeled ATP Analogs with SVPD

  Materials
  • Appropriate doubly labeled ATP analog (see protocol 2)
  • Distilled water
  • Phosphodiesterase I from C. adamanteus (snake venom phosphodiesterase [SVPD]; Worthington)
  • EDTA
  • 10× snake venom phosphodiesterase (SVPD) buffer (see recipe)
  • Acetonitrile (ACN)
  • 50 mM triethylammonium acetate buffer (TEAA; see recipe)
  • 10× PBS (see appendix 2A)
  • Microliter pipets
  • 1.5‐mL Eppendorf tubes
  • Nucleodur RP‐18 HTec column (4 mm diameter, 250 mm length; Macherey‐Nagel)
  • Prominence System with analytical LC‐20AT pumps (analytical HPLC system, Shimadzu)
  • SpeedVac concentrator SPD121P (Thermo Scientific)
  • Freeze‐dryer
  • amaZon SL (ESI‐MS, Bruker)
  • 15‐mL Falcon tubes
  • LS 50 spectrometer (Perkin Elmer)

Support Protocol 1: Synthesis of 2′‐O‐(6‐Trifluoroacetamidohexyl) Adenosine Triphosphate

  Materials
  • Adenosine
  • Anhydrous dimethylformamide (DMF)
  • Dry sodium hydride (Sigma‐Aldrich)
  • 6‐Azido‐1‐bromohexane (see protocol 1)
  • Methanol (MeOH)
  • Silica gel
  • Dichloromethane (DCM)
  • Dimethylsulfoxide‐d 6 (DMSO‐d 6)
  • Tetrahydrofuran (THF)
  • Distilled water
  • Triphenylphosphine (PPh 3)
  • Anhydrous methanol (MeOH)
  • Triethylamine (Et 3N)
  • Ethyl trifluoroacetate (CF 3CO 2Et)
  • 1,8‐Bis(dimethylamino)naphthalene (proton sponge)
  • Trimethyl phosphate, freshly distilled (TMP)
  • Phosphorous oxychloride (POCl 3)
  • Tributylamine (Bu 3N)
  • Bis(tributylammonium)pyrophosphate (0.5 M in DMF, see recipe)
  • 0.1 M triethylammonium hydrogen carbonate buffer (TEAB; see recipe)
  • Ethyl acetate
  • 1 M triethylammonium hydrogen carbonate buffer (TEAB; see recipe)
  • Acetonitrile (ACN)
  • 50 mM triethylammonium acetate buffer (TEAA; see recipe)
  • Deuterated water (D 2O)
  • Oil bath
  • Oven‐dried 25‐, 50‐, and 100‐mL one‐neck round‐bottom flasks
  • Schlenk line supplying nitrogen and high vacuum
  • Magnetic stir bar
  • Magnetic stirrer and heater
  • Rotary evaporator connected to a vacuum pump
  • Fritted evaporator trap
  • Glass columns (5 cm diameter, 20 cm length; 1.5 cm diameter, 20 cm length)
  • TLC plates
  • Glass chamber
  • UV lamp
  • Evaporator trap
  • Avance III 400 MHz NMR spectrometer (Bruker)
  • 50‐mL separatory funnel
  • FPLC column (see recipe)
  • Äkta purifier (FPLC system, GE Healthcare)
  • Nucleodur RP‐18 HTec column (16 mm diameter, 250 mm length, Macherey‐Nagel)
  • Prominence System with preparative LC‐20AP pumps (preparative HPLC system, Shimadzu)
  • Freeze‐dryer
  • amaZon SL (ESI‐MS, Bruker)
  • Nanodrop ND‐1000 spectrophotometer (Peqlab)
CAUTION: Sodium hydride must be stored and handled under inert gas as it will produce gases in contact with water or moisture, which will ignite spontaneously. In case of fire, extiguish with sand or powder.
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Figures

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Literature Cited

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