Synthesis of S‐Adenosyl‐L‐Methionine Analogs with Extended Transferable Groups for Methyltransferase‐Directed Labeling of DNA and RNA

Viktoras Masevičius1, Milda Nainytė1, Saulius Klimašauskas2

1 Faculty of Chemistry, Vilnius University, Vilnius, 2 Institute of Biotechnology, Vilnius University, Vilnius
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.36
DOI:  10.1002/0471142700.nc0136s64
Online Posting Date:  March, 2016
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Abstract

S‐Adenosyl‐L‐methionine (AdoMet) is a ubiquitous methyl donor for a variety of biological methylation reactions catalyzed by methyltransferases (MTases). AdoMet analogs with extended propargylic chains replacing the sulfonium‐bound methyl group can serve as surrogate cofactors for many DNA and RNA MTases enabling covalent deposition of these linear chains to their cognate targets sites in DNA or RNA. Here we describe synthetic procedures for the preparation of two representative examples of AdoMet analogs with a transferable hex‐2‐ynyl group carrying a terminal azide or amine functionality. Our approach is based on direct chemoselective alkylation of S‐adenosyl‐L‐homocysteine at sulfur with corresponding nosylates under acidic conditions. We also describe synthetic routes to 6‐substituted hex‐2‐yn‐1‐ols and their conversion to the corresponding nosylates. Using these protocols, synthetic AdoMet analogs can be prepared within 1 to 2 weeks. © 2016 by John Wiley & Sons, Inc.

Keywords: methyltransferase; cofactor engineering; synthetic AdoMet analogs; mTAG labeling; S‐selective alkylation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of 6‐Substituted Hex‐2‐Yn‐1‐Ols
  • Basic Protocol 2: Synthesis of 4‐Nitrobenzenesulfonates
  • Basic Protocol 3: S‐Alkylation of AdoHcy with Nosylates
  • Basic Protocol 4: Purification and Characterization of AdoMet Analogs
  • Reagents And Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of 6‐Substituted Hex‐2‐Yn‐1‐Ols

  Materials
  • 5‐Chloropent‐1‐yne 6
  • Tetrahydrofuran, anhydrous (THF)
  • 2 M n‐butyllithium (in hexane)
  • Paraformaldehyde
  • Ammonium chloride aqueous solution (saturated)
  • Diethyl ether
  • Sodium sulfate (Na 2SO 4)
  • Deuterated chloroform (CDCl 3)
  • Silica gel (60 Å, 230‐400 mesh; for column chromatography)
  • N,N‐Dimethylformamide, anhydrous (DMF)
  • Sodium azide
  • Tetra‐n‐butylammonium bromide
  • Benzene
  • Methylene chloride
  • Potassium phthalimide
  • Methanol
  • 50% to 60% hydrazine hydrate
  • Ethanol
  • Concentrated and 0.5 M HCl
  • Deuterated water (D 2O)
  • Triethylamine
  • Di‐tert‐butyl dicarbonate
  • Brine (saturated solution of NaCl)
  • Chloroform
  • Ethyl acetate
  • 100‐mL double neck round‐bottom flasks
  • Magnetic stir plate and stir bars
  • Freezing equipment or liquid nitrogen
  • 50‐mL separatory funnels
  • Argon balloon
  • 50‐mL syringe
  • Rotary evaporator equipped with a vacuum pump
  • Vacuum distillation apparatus
  • 50‐mL pear‐shaped and round‐bottom flasks, oven dried
  • NMR tube and spectrometer
  • Sand bath
  • Filtration system
  • Ice bath
  • Chromatography column
  • Test tubes

Basic Protocol 2: Synthesis of 4‐Nitrobenzenesulfonates

  Materials
  • Appropriate alcohol (e.g., 6‐azidohex‐2‐yn‐1‐ol 4a or 6‐(N‐Boc‐amino)hex‐2‐yn‐1‐ol 4b; see protocol 1)
  • Tetrahydrofuran, anhydrous (THF)
  • Potassium tert‐butoxide
  • 4‐Nitrobenzenesulfonyl chloride
  • Silica gel (60 Å, 230‐400 mesh; for column chromatography)
  • Methylene chloride, anhydrous
  • Deuterated chloroform (CDCl 3)
  • 10‐mL and 50‐mL round‐bottom flasks, oven dried
  • Drying tube with calcium chloride
  • Magnetic stir plate and stir bar
  • Ice bath
  • Rotary evaporator equipped with a vacuum pump
  • Chromatography column
  • Test tubes
  • NMR tubes and spectrometer

Basic Protocol 3: S‐Alkylation of AdoHcy with Nosylates

  Materials
  • S‐Adenosyl‐L‐homocysteine (AdoHcy, 2)
  • Formic acid
  • Acetic acid
  • 4‐Nitrobenzenesulfonate 5 (see protocol 2)
  • Diethyl ether
  • Boc‐protected cofactor 3b
  • Trifluoroacetic acid
  • Methylene chloride
  • 5‐mL and 50‐mL pear‐shaped flasks, oven dried
  • Magnetic stir plate and stir bars
  • Ice bath
  • Argon balloon
  • 50‐mL separatory funnel
  • Rotary evaporator equipped with a vacuum pump
  • Additional reagents and equipment for HPLC/MS (see unit )

Basic Protocol 4: Purification and Characterization of AdoMet Analogs

  Materials
  • Crude AdoMet analog 3a or 3c obtained from protocol 3, step 8 or 13, respectively
  • 20 mM ammonium formate buffer, pH 3.5 (see recipe)
  • Dowex 1×8, 50‐100 mesh resin (see recipe)
  • 60% methanol
  • 1.8 × 10.8–cm chromatography column
  • 1.5‐mL microcentrifuge tubes
  • UV lamp (254 nm)
  • UV/Vis spectrophotometer
  • Rotary evaporator equipped with a vacuum pump
  • Preparative reversed‐phase HPLC (e.g., Agilent Prep‐C18, 30 × 150, 10 μm; cat. no. PN 413910‐302)
  • Agilent 1100 HPLC system
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Figures

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Literature Cited

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Key Reference
   Lukinavičius, G. , Tomkuvienė, M. , Masevičius, V. , and Klimašauskas, S. 2013. Enhanced chemical stability of AdoMet analogues for improved methyltransferase‐directed labeling of DNA. ACS Chem. Biol. 8:1134‐1139. doi: 10.1021/cb300669x.
  Latest report on key synthetic and analytical procedures.
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