Synthesis of Stable Aminoacyl‐tRNA Analogs

Maryline Chemama1, Matthieu Fonvielle2, Maxime Lecerf2, Dénia Mellal1, Hélène Fief1, Michel Arthur2, Mélanie Etheve‐Quelquejeu1

1 Institut Parisien de Chimie Moléculaire, Université Pierre et Marie Curie, Paris, France, 2 Laboratoire de Recherche Moléculaire sur les Antibiotiques, Centre de Recherche des Cordeliers, Université Pierre et Marie Curie, Paris, France
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.44
DOI:  10.1002/0471142700.nc0444s44
Online Posting Date:  March, 2011
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Abstract

Aminoacyl‐tRNAs have important roles in a variety of biological processes. Here, we describe the synthesis of stable aminoacyl‐tRNA analogs containing 1,4‐substituted 1,2,3‐triazole rings. The procedure involves (i) copper‐catalyzed cycloadditions of 3′‐or 2′‐azido‐adenosine and alkynes, (ii) coupling between the resulting triazole‐deoxyadenosine derivatives and a deoxycytidine phosphoramidite, and (iii) the enzymatic ligation of the 2′‐ or 3′‐triazole‐dinucleotides with a 22‐nt RNA microhelix that mimics the acceptor arm of tRNA. Each nucleoside and nucleotide intermediate was characterized by MS spectrometry and 1H, 31P, and 13C NMR spectroscopy, and the tRNA‐analogs were assayed for inhibition of FemXWv, an alanyl‐transferase essential for the formation of the peptidoglycan network of Gram‐positive bacterial pathogens. The low IC50 values obtained (2 to 4 µM) indicate that the five‐membered triazole rings acted as an isosteres of esters and can be used for the design of stable aminoacyl‐tRNA analogs. Curr. Protoc. Nucleic Acid Chem. 44:4.44.1‐4.44.33. © 2011 by John Wiley & Sons, Inc.

Keywords: aminoacyl‐tRNA; click chemistry; dinucleotides; inhibitors; triazoles; azidoadenosine; transferase

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Triazole 2′‐ or 3′‐Deoxyadenosine
  • Basic Protocol 2: Obtaining the 74‐nt tRNA and 22‐nt Helix
  • Basic Protocol 3: Ligation of the Modified Nucleotide
  • Support Protocol 1: Denaturing Polyacrylamide Gel Electrophoresis for Small RNA Molecules
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Triazole 2′‐ or 3′‐Deoxyadenosine

  Materials
  • 6‐N,N‐dibenzoyl‐2′,5′‐bis‐O‐(tert‐butyldimethylsilyl)‐adenosine ( S.1a)
  • Anhydrous CH 2Cl 2, distilled on CaH 2
  • Argon source
  • Potassium bromide (KBr; BioXtra, ≥99.0%; Sigma‐Aldrich)
  • 5% (w/v) sodium hydrogen carbonate (NaHCO 3), aqueous
  • 2,2,6,6‐tetramethylpiperidinooxy (TEMPO), 98% (Acros Organics)
  • 9% (w/v) sodium hypochlorite (NaOCl)
  • Sodium sulfate (Na 2SO 4) anhydrous
  • Ethyl acetate (EtOAc)
  • Cyclohexane
  • Ethanol (EtOH)
  • Sodium borohydride (NaBH 4; Fluka)
  • Saturated aqueous NaCl
  • 4‐dimethylaminopyridine (DMAP), ≥99% (Sigma‐Aldrich)
  • Trifluoromethanesulfonyl chloride, ≥99.9% (Sigma‐Aldrich)
  • Saturated aqueous sodium hydrogen carbonate (NaHCO 3), ice cold
  • Sodium azide (ReagentPlus, ≥99.5%; Sigma‐Aldrich)
  • Tetrahydrofuran (THF), anhydrous, freshly distilled on sodium/benzophenone
  • Trifluoroacetic acid, 99% (Janssen Chimica)
  • Alkyne: tert‐butyl but‐3‐yn‐2‐ylcarbamate
  • 1 M (+)‐sodium L‐ascorbate, ≥99% (Sigma‐Aldrich), aqueous
  • 7.5% (w/v) CuSO 4, aqueous
  • Ac‐dC‐PCNE phosphoramidite vial (Eurogentec; http://www.eurogentec.com)
  • 0.45 M tetrazole (DNA synthesis grade, Sigma‐Aldrich) in acetonitrile (CH 3CN), filtered through a 1‐µm filter
  • 1 M iodine (I 2) in 2:20:75 H 2O/pyridine/THF
  • Saturated aqueous sodium thiosulfate (Na 2S 2O 3)
  • 0.18 M trichloroacetic acid (TCA, biotech. grade, redistilled, ≥99%;Sigma), aqueous
  • 3‐hydroxypropionitrile (99%, Sigma)
  • Dichloromethane (DCM, purissimum, absolute, dried over molecular sieve (H 2O ≤0.005%, ≥99.5%; Sigma‐Aldrich)
  • N,N‐diisopropylethylamine (DIEA)
  • 2‐cyanoethyldiisopropylchlorophosphoramidite (purum, >97.5%, Fluka)
  • 0.1 M potassium phosphate buffer, pH 7 ( appendix 2A)
  • Diethyl ether
  • Acetonitrile (CH 3CN)
  • 5 M methylamine solution (MeNH 2) in 50% (v/v) ethanol/H 2O: prepare from 40 wt. % methylamine in H 2O (Sigma)
  • Ammonium acetate (NH 4OAc)
  • Hydrochloric acid (HCl)
  • Methanol (MeOH)
  • 10‐, 25‐, 50‐, 100‐, and 250‐mL round‐bottomed flasks
  • Magnetic stir plate and stir bar
  • Rotary evaporator equipped with a vacuum pump
  • Flash chromatography medium: silica gel, 60 Å, 180 to 240 mesh (Merck; also see appendix 3E)
  • TLC plates: silica‐coated aluminum plates with fluorescent indicator (Merck silica gel 60 F 254)
  • 254‐nm UV lamp
  • Separatory funnels
  • Syringe
  • Lyophilizer
  • 15‐cm column for short‐column chromatography
  • HPLC system (also see unit 10.5) with a reversed‐phase C‐18 column (analytic column: 250 × 4.6 mm, HYPERSIL‐100 C18; semi‐preparative column: 250 × 21.2 mm, HYPERSIL HS C18; Thermo Electron Corporation)
  • Additional reagents and equipment for flash chromatography ( appendix 3E) and HPLC (unit 10.5)
NOTE: The experimental part is described for the transformation of the adenosine in 2′position, but the same methodology can be applied in 3′ position. Products are characterized for the modification in 2′ and 3′‐ position.NOTE: Spectra were recorded on Brukers spectrometers ARX 250 for1H (250.13 MHz) and 13C (62.89 MHz), Advance III 500 for1H (500.11 MHz) and 13C (125.75 MHz) and DRX 500 for 31P (202.31 MHz) in CDCl 3 or D 2O. Chemicals shifts (δ) are expressed in ppm relative to residual CHCl 3 (δ7.26) or HDO (δ4.79) for 1H, CDCl 3 (δ77.16) for 13C as internal references, and H 3PO 4 (δ0) for 31P as external references. Signals were attributed based on COSY and DEPT 135 (13C). High‐resolution mass spectroscopy (HRMS) spectra were carried out on a LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific Inc.) in the electrospray ionization (ESI+ or ESI) at the Mass Spectrometry Center of the University Pierre & Marie Curie (Paris).

Support Protocol 1:

  Materials
  • 1,4‐Diazabicyclo(2.2.2)octane, >99% (DABCO; Sigma‐Aldrich)
  • Anhydrous tetrahydrofuran (THF), freshly distilled on sodium/benzophenone
  • Silver nitrate (AgNO 3), ReagentPlus, >99% (Sigma‐Aldrich)
  • Tert‐butyl(chloro)dimethylsilane (TBDMSCl), purum, >95% (Aldrich)
  • Argon source
  • Adenosine
  • Anhydrous CH 2Cl 2, distilled on CaH 2
  • MgSO 4, anhydrous
  • Cyclohexane
  • Ethyl acetate (EtOAc)
  • Pyridine, anhydrous
  • Trimethylsilyl chloride (TMSCl; >98%, Fluka)
  • Benzoyl chloride, ACS reagent, 99% (Sigma‐Aldrich)
  • Sodium hydrogen carbonate (NaHCO 3)
  • Dichloromethane
  • Sodium sulfate (Na 2SO 4) anhydrous
  • Ethanol (EtOH)
  • p‐toluenesulfonic acid
  • Dichloromethane (DCM)
  • 50‐, 250‐mL and 1‐L round‐bottomed flasks
  • Magnetic stir plate and stir bar
  • Rotary evaporator equipped with a vacuum pump
  • Flash chromatography medium: silica gel, 60 Å, 180 to 240 mesh (Merck)
  • TLC plate: silica‐coated aluminum plate with fluorescent indicator (Merck silica gel 60 F 254).
  • 254‐nm UV lamp
  • Additional reagents and equipment for flash chromatography ( appendix 3E)
NOTE: Spectra were recorded on Brukers spectrometers ARX 250 for1H (250.13 MHz) and 13C (62.89 MHz), Advance III 500 for1H (500.11 MHz) and 13C (125.75 MHz) and DRX 500 for 31P (202.31 MHz) in CDCl 3 or D 2O. Chemicals shifts (δ) are expressed in ppm relative to residual CHCl 3 (δ7.26) or HDO (δ4.79) for 1H, CDCl 3 (δ77.16) for 13C as internal references and H 3PO 4 (δ0) for 31P as external references. Signals were attributed based on COSY and DEPT 135 (13C). High resolution mass spectroscopy (HRMS) spectra were carried out on a LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific Inc.) in the electrospray ionization (ESI+ or ESI) at the Mass Spectrometry Center of the University Pierre & Marie Curie (Paris).

Support Protocol 2:

  Materials
  • Adenosine
  • Anhydrous tetrahydrofuran (THF), freshly distilled on sodium/benzophenone
  • Argon source
  • Pyridine, anhydrous
  • Silver nitrate (AgNO 3), ReagentPlus, >99% (Sigma‐Aldrich)
  • Tert‐butyl(chloro)dimethylsilane (TBDMSCl), purum, >95% (Aldrich)
  • Ethyl acetate
  • Cyclohexane
  • Trimethylsilyl chloride (TMSCl; >98%, Fluka)
  • Benzoyl chloride, ACS reagent, 99% (Sigma‐Aldrich)
  • Saturated aqueous NaCl solution
  • MgCl 2, anhydrous
  • 250‐ and 1000‐mL round‐bottomed flasks
  • Magnetic stir plate and stir bar
  • Rotary evaporator equipped with a vacuum pump
  • Flash chromatography medium (also see appendix 3E): silica gel 60 Å, 180‐240 mesh (Merck)
  • TLC plate: silica‐coated aluminum plate with fluorescent indicator (Merck silica gel 60 F 254).
  • 254‐nm UV lamp
  • Additional reagents and equipment for flash chromatography ( appendix 3E)
NOTE: Spectra were recorded on Brukers spectrometers ARX 250 for1H (250.13 MHz) and 13C (62.89 MHz), Advance III 500 for1H (500.11 MHz) and 13C (125.75 MHz) and DRX 500 for 31P (202.31 MHz) in CDCl 3 or D 2O. Chemicals shifts (δ) are expressed in ppm relative to residual CHCl 3 (δ7.26) or HDO (δ4.79) for 1H, CDCl 3 (δ77.16) for 13C as internal references and H 3PO 4 (δ0) for 31P as external references. Signals were attributed based on COSY and DEPT 135 (13C). High resolution mass spectroscopy (HRMS) spectra were carried out on a LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific Inc.) in the electrospray ionization (ESI+ or ESI) at the Mass Spectrometry Center of the University Pierre & Marie Curie (Paris).

Basic Protocol 2: Obtaining the 74‐nt tRNA and 22‐nt Helix

  Materials
  • Genomic DNA from E. faecalis V583
  • 2.5 mM dNTP mix ( appendix 2A)
  • 2000 U/mL Vent R DNA polymerase (New England BioLabs), with provided 10× ThermoPol Reaction Buffer (also see Kramer and Coen, )
  • Custom‐made DNA PCR oligonucleotide primers for amplifying nt 6 to 76 of the tRNAAla sequence (also see Kramer and Coen, ):
    • forward: 5′‐CTT AGC TCA GCT GGG AGA GCG CC‐3′
    • reverse: 5′‐TGG TGG AGC CTA GCG GGA TCG AAC CGC‐3′
  • QIAquick Nucleotide Removal Kit (Qiagen)
  • pUC18 cloning vector (Fermentas)
  • SmaI restriction enzyme (New England BioLabs), with provided NEBuffer 4
  • QIAquick Gel Extraction Kit (Qiagen)
  • T4 DNA ligase (400,000 cohesive end units/mL; New England BioLabs), with provided 10× reaction buffer
  • Frozen electrocompetent E. coli TOP10 strain (Invitrogen)
  • Liquid SOC growth medium (Seidman et al., )
  • Liquid and solid LB growth medium (Elbing and Brent, )
  • Ampicillin disodium salt, 100 mg/mL solution (also see Elbing and Brent, )
  • QIAprep Spin Miniprep Kit (Qiagen)
  • Custom‐made DNA PCR oligonucleotide primers for amplifying DNA matrix of tRNA sequence (also see Kramer and Coen, ):
    • Forward: 5′‐TTT AAT ACG ACT CAC TAT AGG GGC CTT AGC TCA GCT GGG AG‐3′)
    • Reverse: 5′‐GTG GAG CCT AGC GGG ATC G‐3′
  • Agarose, electrophoresis grade (also see Voytas, )
  • 0.7 mg/mL ethidium bromide (also see Voytas, )
  • 10× T7 buffer (see recipe)
  • 25 mM ribonucleotide (NTP) mix (25 mM each ATP, CTP, GTP, and UTP)
  • 1 M MgCl 2
  • Triton X‐100
  • 200 mM guanosine 5′‐monophosphate (GMP)
  • T7 RNA polymerase, purified in the laboratory (alternatively can be bought from a wide range of suppliers)
  • DNase I (RQ1 RNase‐Free DNase; Promega)
  • Water‐saturated phenol, pH 4.5 to 5 (e.g., Eurobio; http://eurobio.free.fr/)
  • 5:1 phenol:chloroform (molecular biology grade; e.g., Sigma)
  • Absolute ethanol (molecular biology grade; e.g., Fisher Scientific), cold
  • 70% ethanol, cold
  • HiLoad 26/60 Superdex 75 prep grad column (GE healthcare)
  • Dex buffer (see recipe)
  • PCR thermal cycler (also see Kramer and Coen, ) and PCR tubes
  • UV spectrophotometer
  • 0.025 µm nitrocellulose membrane filter, 13‐mm diameter (Millipore)
  • Electroporator (also see Seidman et al., )
  • Agarose gel electrophoresis apparatus (also see Voytas, )
  • 1.5‐mL plastic reaction tubes
  • Heat block
  • Refrigerated centrifuge
  • FPLC system, with conductivity and 260‐nm absorbance detection
  • 0.22‐µm syringe filter
  • 15‐mL centrifuge tubes (e.g., BD Falcon)
  • Lyophilizer
  • Molecular membrane tubing, MWCO 12,000 to 14,000 (Spectra/Por)
  • Boiling water bath
  • Additional reagents and equipment for the polymerase chain reaction (Kramer and Coen, ), spectrophotometric determination of DNA concentration (unit 5.2), electroporation (Seidman et al., ), preparation of bacteriological media (Elbing and Brent, ), agarose gel electrophoresis (Voytas, ), denaturing polyacrylamide electrophoresis ( protocol 6), and dialysis (Zumstein, )

Basic Protocol 3: Ligation of the Modified Nucleotide

  Materials
  • Dinucleotide ( S.10; see protocol 1)
  • 74‐nt tRNA (see protocol 4) or 22‐nt helix (see protocol 4 introduction)
  • 10 mM adenosine triphosphate (ATP)
  • Dimethylsulfoxide (DMSO)
  • 1 M HEPES buffer, pH 7.5
  • 200 mM MgCl 2
  • T4 RNA ligase, purified in the laboratory (alternatively, it can be bought from a wide range of suppliers)
  • DNApac PA100 4 × 250 column (Dionex)
  • Buffer A (see recipe)
  • Buffer B (see recipe)
  • FPLC system, with conductivity and 260 nm absorbance detector
  • 22‐µm syringe filter
  • Injection loop
  • Lyophilizer
  • UV spectrophotometer
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (see protocol 6)

Support Protocol 3: Denaturing Polyacrylamide Gel Electrophoresis for Small RNA Molecules

  Materials
  • Urea, DNase and RNase free
  • 29:1 acrylamide:N,N′‐methylbisacrylamide (40%), commercially available
  • 10× TBE buffer ( appendix 2A)
  • N,N,N′,N′‐tetramethylethylenediamine (TEMED)
  • 10% (w/v) ammonium persulfate
  • 0.5 µg/mL ethidium bromide in 1× TBE buffer
  • Protean II xi gel electrophoresis system (BioRad) with 20‐cm cell and appropriate glass plates, spacers, and comb for casting gel
  • 600 V power supply
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Figures

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Literature Cited

Literature Cited
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