Syntheses of Specifically 15N‐Labeled Adenosine and Guanosine

Barbara L. Gaffney1, Roger A. Jones1

1 Rutgers University, Piscataway, New Jersey
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.6
DOI:  10.1002/0471142700.nc0106s10
Online Posting Date:  November, 2002
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Abstract

This unit describes the specific incorporation of 15N into the N7 and amino positions of adenosine, and conversion of the adenosine to guanosine labeled at the N1, N7, and amino positions. Two variations of the procedures are also presented that include either 12C or 13C at the C8 position of adenosine, and 13C at either the C8 or C2 position of guanosine. These 13C tags permit the incorporation of two 15N‐labeled nucleosides into an RNA strand while ensuring that their nuclear magnetic resonance (NMR) signals can be distinguished from each other by the presence or absence of C‐N coupling. While the major application of these specifically 15N‐labeled nucleosides is NMR, the additional mass makes them useful in mass spectrometry (MS) as well. The procedures can also be adapted to synthesize the labeled deoxynucleosides. A support protocol describes the synthesis of 7‐methylguanosine.

     
 
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Table of Contents

  • Basic Protocol 1: Syntheses of [7,NH2‐15N2]‐ and [8‐13C‐7,NH2‐15N2]Adenosine
  • Support Protocol 1: Synthesis of 7‐Methylguanosine
  • Basic Protocol 2: Synthesis of [2‐13C‐1,7,NH2‐15N3]‐ and [8‐13C‐1,7,NH2‐15N3]Guanosine
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Syntheses of [7,NH2‐15N2]‐ and [8‐13C‐7,NH2‐15N2]Adenosine

  Materials
  • 4‐Amino‐6‐hydroxy‐2‐mercaptopyrimidine monohydrate, also called 6‐amino‐2‐thioxo‐1,2‐dihydro‐4(3H)‐pyrimidinone (Aldrich)
  • 1 N HCl
  • [15N]Sodium nitrite ([15N] NaNO 2; Isotec or Cambridge Isotope Laboratories)
  • 2:98 to 40:60 (v/v) gradient of acetonitrile/0.1 M triethylammonium acetate (TEAA), pH 6.8
  • 95% (v/v) ethanol, 4°C
  • Acetone, 4°C
  • Phosphorous pentoxide (P 2O 5)
  • Saturated aqueous NaHCO 3
  • Sodium hydrosulfite (Na 2S 2O 4)
  • Glacial acetic acid
  • 96% (v/v) formic acid
  • Nitrogen gas source
  • Dimethylformamide (DMF), anhydrous
  • Diethoxymethyl acetate (DEMA), for 12C synthesis only
  • Acetonitrile, room temperature and 4°C
  • [13C]NaSCSOEt (see recipe), for 13C synthesis only
  • NaOH
  • 50% aqueous Raney 2800 nickel (RaNi) slurry (Aldrich)
  • Dipotassium salt of EDTA
  • Boiling water
  • Phosphorous oxychloride (POCl 3)
  • N,N‐Dimethylaniline
  • 5% (v/v) NH 3, (diluted with water from 30% concentrated aqueous ammonia)
  • Ethyl acetate
  • Ethyl ether
  • 1 M HCl
  • 7‐Methylguanosine (see protocol 2)
  • 0.02 M K 2HPO 4
  • 3 M NaOH
  • Purine nucleoside phosphorylase (Sigma)
  • [15N]Ammonium chloride ([15N]NH 4Cl; Isotec or Cambridge Isotope Laboratories)
  • Dimethylsulfoxide (DMSO), anhydrous
  • KHCO 3, anhydrous
  • 100‐ and 250‐mL round‐bottom flasks
  • Small glass vials
  • 1‐, 3‐, 10‐, and 20‐ml syringes
  • Vacuum desiccator
  • Rubber septa fitted with large‐bore vent needles
  • Condenser
  • Rotary evaporator, connected to water aspirator and a vacuum pump, the latter with a dry‐ice trap
  • Oil bath (silicone oil), 130°C
  • Separatory funnel
  • Continuous extraction apparatus for solvents lighter than water (Aldrich)
  • 30°C oven with shaker
  • 50‐mL bomb with Teflon liner (Parr Instrument)
  • 80°C oven
  • Additional reagents and equipment for analytical and preparative reversed‐phase high‐performance liquid chromatography (HPLC; unit 10.5)

Support Protocol 1: Synthesis of 7‐Methylguanosine

  Materials
  • Guanosine
  • N,N‐Dimethylacetamide
  • Nitrogen gas source
  • Dimethyl sulfate
  • Concentrated aqueous NH 3
  • Acetone, 4°C
  • 95% (v/v) ethanol
  • Ethyl ether
  • 250‐mL round‐bottom flask
  • Rubber septum
  • 10‐mL syringes
CAUTION: Dimethyl sulfate is very dangerous because it is a potent alkylating agent; wear gloves and use caution.

Basic Protocol 2: Synthesis of [2‐13C‐1,7,NH2‐15N3]‐ and [8‐13C‐1,7,NH2‐15N3]Guanosine

  Materials
  • [7,NH 215N 2]Adenosine or [8‐13C‐7,NH 215N 2]adenosine (S.8a or S.8b; see protocol 1)
  • 50% (v/v) methanol
  • 3‐Chloroperoxybenzoic acid (MCPBA), purified (see recipe)
  • Ethyl ether
  • Phosphorous pentoxide (P 2O 5)
  • recipe[13C,15N]Cyanogen bromide or recipe[15N]cyanogen bromide , freshly prepared (see recipe)
  • 0.1 M potassium phosphate (KH 2PO 4), pH 7.5
  • Dimethyl formamide (DMF), anhydrous
  • Acetonitrile
  • Triethylamine
  • Nitrogen gas source
  • Methyl iodide
  • 0.1 M NaOH
  • 1 M HCl
  • 95% (v/v) ethanol
  • Adenosine deaminase (Sigma)
  • 100‐mL round‐bottom flasks
  • Rotary evaporator
  • Vacuum desiccator
  • Oil bath (silicone oil), 60°C
  • Additional reagents and equipment for analytical and preparative reversed‐phase high‐performance liquid chromatography (HPLC; unit 10.5)
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Figures

Videos

Literature Cited

Literature Cited
   Abad, J.‐L., Shallop, A.J., Gaffney, B.L., and Jones, R.A. 1998. Use of 13C tags with specifically 15N‐labeled DNA and RNA. Biopolymers 48:57‐63.
   Gaffney, B.L., Kung, P.‐P., and Jones, R.A. 1990. Nitrogen‐15‐labeled deoxynucleosides. 2. Synthesis of [7‐15N]‐labeled deoxyadenosine,deoxyguanosine, and related deoxynucleosides. J. Am. Chem. Soc. 112:6748‐6749.
   Goswami, B. and Jones, R.A. 1991. Nitrogen‐15‐labeled deoxynucleosides. 4. Synthesis of [115N]‐and [2‐15N]‐labeled 2′‐deoxyguanosines. J. Am. Chem. Soc. 113:644‐647.
   Pagano, A.R., Lajewski, W.M., and Jones, R.A. 1995. Synthesis of [6,7‐15N]‐adenosine, [6,7‐15N]‐2′‐deoxyadenosine, and [7‐15N]‐hypoxanthine. J. Am. Chem. Soc. 117:11669‐11672.
   Pagano, A.R., Zhao, H., Shallop, A., and Jones, R.A. 1998. Synthesis of [1,7‐15N2]‐ and [1,7, NH2‐15N3]‐adenosine and 2′‐deoxyadenosine via an N1‐alkoxy mediated Dimroth rearrangement. J. Org. Chem. 63:3213‐3217.
   Shallop, A.J. and Jones, R.A. 2000. Use of a 13C “indirect tag” to differentiate two 15N7 specifically labeled nucleosides. Middle Atlantic Regional Meeting, ACS, May 15, 2000, Abstr. 33:215‐ORGN.
   Ueda, T., Miura, K., and Kasai, T. 1978. Synthesis of 6‐thioguanine and 2,6‐diaminopurine nucleosides and nucleotides from adenine counterparts via a facile rearrangement in the base portion. Chem. Pharm. Bull. 26:2122‐2127.
   Wang, C., Gao, H., Gaffney, B.L., and Jones, R.A. 1991. Nitrogen‐15‐labeled deoxynucleotides. 3. Protonation of the adenine N1 int he A⋅C and A⋅G mispairs of the duplexes [d[CG(15N1)AGAATTCCCG]}2 and {d[CGGGAATTC(15N1)ACG]}2. J. Am. Chem. Soc. 113:5486‐5488.
   Zhang, X., Gaffney, B.L., and Jones, R.A. 1997. 15N NMR of a specifically labeled RNA fragment containing intrahelical GU wobble pairs. J. Am. Chem. Soc. 119:6432‐6433.
   Zhang, X., Gaffney, B.L., and Jones, R.A. 1998. 15N NMR of RNA fragments containing specifically labeled tandem G⋅A pairs. J. Am. Chem. Soc. 120:6625‐6626.
   Zhao, H., Pagano, A.R., Wang, W., Shallop, A., Gaffney, B.L., and Jones, R.A. 1997. Use of a 13C atom to differentiate two 15N‐labeled nucleosides: syntheses of [15NH2]‐adenosine,[1,NH2‐15N2]‐ and [2‐13C‐1,NH2‐15N2]‐guanosine, and [1,7,NH2‐15N3]‐and [2‐13C‐1,7,NH2‐15N3]‐2′‐deoxyguanosine. J. Org. Chem. 62:7832‐7835.
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