Design and Synthesis of Triazolyl‐Donor/Acceptor Unnatural Nucleosides and Oligonucleotide Probes Containing Triazolyl‐Phenanthrene Nucleoside

Subhendu Sekhar Bag1, Sangita Talukdar1, Suman Kalyan Das1

1 Bio‐organic Chemistry Laboratory, Department of Chemistry, Indian Institute of Technology Guwahati, Assam‐781039
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.32
DOI:  10.1002/0471142700.nc0132s58
Online Posting Date:  September, 2014
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Abstract

In the context of abasic DNA or DNA duplex stabilization, several unnatural nucleosidic/non‐nucleosidic base surrogates have been reported. Toward this end, we have designed and synthesized triazolyl‐aromatic donor chomophores as unnatural nucleoside analogs. These modifications display markedly higher thermal stabilization of abasic DNA duplex in comparison to the stabilization offered by other nucleoside/non‐nucleoside base surrogates reported in the literature. The same oligonucleotide probe containing triazolylphenanthrene nucleotide also offers very good stability of the self‐pair duplex via π‐π stacking interaction and hetero‐pair duplex via charge transfer interaction when paired against triazolyl acceptor aromatic nucleoside. Moreover, the probe in the reverse sequence containing triazolylphenanthrene nucleotide has shown FRET efficiency in a chimeric DNA duplex. The triazolyl nucleotides would expectedly show stability toward exonuclease activity. This unit describes protocols for chemical synthesis of unnatural triazolyl nucleosides and one oligonucleotide probe. The unit also provides a summary of various thermal and photophysical applications of triazolylphenantherene‐containing oligonucleotides. Curr. Protoc. Nucleic Acid Chem. 58:1.32.1‐1.32.27. © 2014 by John Wiley & Sons, Inc.

Keywords: unnatural triazolylnucleosides; unnatural oligonucleotide; click chemistry; abasic DNA stabilization; FRET donor nucleotide

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Unnatural β‐Triazolyl Aromatic Donor/Acceptor Nucleosides
  • Basic Protocol 2: Preparation of Phosphoramidite of Triazolyl Unnatural Donor Nucleoside (TPhenBDo‐Amidite)
  • Basic Protocol 3: Preparation of Oligodeoxynucleotides Containing Triazolyl Unnatural Donor Nucleoside
  • Basic Protocol 4: Preparation of Oligodeoxynucleotide Solution for Thermal Denaturation and All Spectroscopic Studies
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Unnatural β‐Triazolyl Aromatic Donor/Acceptor Nucleosides

  Materials
  • Methanol (MeOH), anhydrous
  • Acetyl chloride
  • 2‐deoxy‐α/β‐D‐ribofuranose (1; Sigma‐Aldrich, cat. no. 121649‐25g)
  • Saturated aqueous sodium bicarbonate (NaHCO 3)
  • TLC charring solution: 1.25 mL sulfuric acid (H 2SO 4) plus 34 mL ethanol (EtOH) plus 0.6 mL acetic acid (CH 3COOH) plus 0.95 mL p‐anisaldehyde
  • Chloroform (CHCl 3)
  • Anhydrous acetone
  • p‐toluoyl chloride
  • Triethylamine (Et 3N)
  • Anhydrous dichloromethane (DCM)
  • Brine (saturated aqueous NaCl)
  • Sodium sulfate (Na 2SO 4; anhydrous)
  • Silica gel (60 to 120 mesh size)
  • Hexane
  • Ethyl acetate (EtOAc)
  • Anhydrous diethyl ether
  • Concentrated sulfuric acid (H 2SO 4)
  • Concentrated hydrochloric acid (HCl)
  • Calcium chloride (CaCl 2)
  • Boron trifluoride diethyl etherate (BF 3.Et 2O)
  • Trimethylsilyl azide (TMS‐N 3)
  • Silica gel (230 to 400 mesh size)
  • Cesium azide (CsN 3)
  • Anhydrous dimethylsulfoxide (DMSO)
  • Anhydrous tetrahydrofuran (THF)
  • Aromatic donor/acceptor (Do/Ac) alkynes, e.g.:
    • 9‐ethynylphenantherene
    • 6‐methoxy‐2‐ethynyl naphthalene
    • 1‐ethynyl‐4‐nitrobenzene
    • 1‐ethynyl‐4‐bezonitrile
  • Sodium ascorbate
  • Copper sulfate (CuSO 4)
  • N,N‐Diisopropylethylamine (DIPEA)
  • Saturated aqueous ammonium chloride (NH 4Cl)
  • Sodium methoxide (NaOMe)
  • Ammonium chloride (NH 4Cl), solid
  • 100‐mL and 50‐mL two‐neck round‐ bottom flasks, dry
  • Magnetic stirrer and stir bars
  • 150‐µL Hamilton syringe
  • Rubber septa
  • Nitrogen delivery system: purchased N 2 gas is passed through a chamber of pyrogallol, then through concentrated H 2SO 4, then through dry CaCl 2
  • pH paper
  • Whatman no. 41 filter paper
  • Rotary evaporator connected to vacuum pump, chilled by recirculating methanol (down to −10°C)
  • Silica‐coated aluminum‐backed TLC plates with fluorescent indicator (Silica gel G F 254)
  • 250‐mL separatory funnel
  • 3 × 30–, 2 × 20–, and 2 × 15–cm chromatography columns
  • Dropping funnel appropriate for 100‐mL two‐neck round bottom flasks
  • High‐vacuum pump (1 × 10–3 mbar)
  • Sintered‐glass funnel
  • Reflux condenser
  • Additional reagents and equipment for thin‐layer chromatography ( appendix 3D), column chromatography ( appendix 3E), and HPLC (unit 10.1)

Basic Protocol 2: Preparation of Phosphoramidite of Triazolyl Unnatural Donor Nucleoside (TPhenBDo‐Amidite)

  Materials
  • 2′‐Deoxy‐1′‐β‐triazolyl phenanthrene nucleoside (11; protocol 1, step 53)
  • Anhydrous pyridine
  • Source of dry N 2
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl)
  • Methanol (MeOH)
  • Chloroform (CHCl 3)
  • Triethylamine (Et 3N)
  • Ethyl acetate (EtOAc)
  • Saturated aqueous sodium bicarbonate (NaHCO 3)
  • Brine (saturated aqueous NaCl)
  • Sodium sulfate (Na 2SO 4; anhydrous)
  • Silica gel (60 to 120 mesh size)
  • TLC charring solution: 1.25 mL sulfuric acid (H 2SO 4) plus 34 mL ethanol (EtOH) plus 0.6 mL acetic acid (CH 3COOH) plus 0.95 mL p‐anisaldehyde
  • Anhydrous toluene
  • Anhydrous dichloromethane (DCM), freshly distilled
  • Diisopropylethylamine (DIPEA)
  • 2‐cyanoethyl tetraisopropylphosphorodiamidite (Sigma‐Aldrich, cat. no. 305995‐1g)
  • 25‐mL two‐necked round‐bottom flasks, oven dried
  • 2 × 20–cm chromatography column
  • Rotary evaporator connected to vacuum pump, chilled by recirculating methanol (down to −10°C)
  • Silica‐coated aluminum‐backed TLC plates with fluorescent indicator (Silica gel G F 254)
  • 100‐mL separatory funnel
  • High‐vacuum pump (1 × 10–3 mbar)
  • Short chromatography column: 1.5 × 15–cm
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 3: Preparation of Oligodeoxynucleotides Containing Triazolyl Unnatural Donor Nucleoside

  Materials
  • Modified phosphoramidites: triazolylphenanthrene nucleoside‐phosphoramidite 16 (see protocol 2); 0.10 M in anhydrous MeCN
  • Acetonitrile (MeCN): anhydrous acetonitrile (<10 ppm water content) stored over 4 Å molecular sieves
  • Activator: 0.25 M 4,5‐dicyanoimidazole (DCI) in anhydrous (<10 ppm water) acetonitrile as activator
  • Activated molecular sieves (4 Å)
  • Argon source
  • Controlled pore glass (CPG) supported cartridge containing the desired 3′‐nucleoside (dC for ODN 1 and dG for ODN 7) attached to CPG support
  • Reagents for oligonucleotide synthesis:
    • Detritylation (TCA deblock): 3% dichloroacetic acid in dichloromethane (DCM) as detritylating agent
    • Capping (Cap A): 9:1 (v/v) tetrahydrofuran (THF)/acetic anhydride
    • Capping (Cap B): 10% 1‐methylimidazole in 8:1 (v/v) THF/pyridine
    • Oxidation: iodine (0.02 M) in H 2O/pyridine/THF
    • Washing: Dichloromethane (DCM) and acetonitrile (MeCN) (<30 ppm water content); stored over 4 Å molecular sieves
  • Unmodified phosphoramidites: DMTr‐dABz (0.15 M), DMTr‐dCBz (0.15 M), DMTr‐dGiBu (0.15 M), and DMTr‐T (0.15 M) in anhydrous MeCN
  • 50 mM aqueous ammonia (NH 4OH)
  • 50 mM ammonium formate buffer, pH 7.0: 15.765 g solid ammonium formate (for HPLC; ≥99.0%; Fluka, cat. no. 17843‐250G) dissolved in 5 L Milli‐Q H 2O affords 50 mM ammonium formate stock solution, which is used for HPLC
  • 0.5 M 2,4,6‐trihydroxyacetophenone (Sigma‐Aldrich, cat. no. 91928‐5g) in ethanol
  • 0.1 M diammonium citrate in H 2O
  • Rotary evaporator connected to vacuum pump, chilled by recirculating methanol (down to −10°C)
  • High‐vacuum pump
  • Automated DNA/RNA synthesizer (Applied Biosystems 394 DNA/RNA Synthesizer; also see appendix 3C)
  • 2‐mL microcentrifuge tubes
  • 55°C temperature‐controlled incubator
  • SpeedVac evaporator
  • Whatman 0.2‐µm syringe filter
  • 5‐ODS‐H column (10 × 150 mm; Chemco Scientific)
  • 10‐mL tubes (Eppendorf)
  • Shimadzu UV‐2550 UV‐Visible spectrophotometer with quartz optical 8‐micro cell (120 µL DNA) of 1.0 cm path length and scanning rate of 0.5 nm with wavelength range of 200‐500 nm and slit width of 2 nm
  • UV‐compatible spectrophotometer cuvettes
  • Additional reagents and equipment for synthesis and purification of oligonucleotides ( appendix 3C), MALDI‐TOF mass spectrometry (unit 10.1)

Basic Protocol 4: Preparation of Oligodeoxynucleotide Solution for Thermal Denaturation and All Spectroscopic Studies

  Materials
  • Stock solution of probe DNA ( protocol 3)
  • Stock solution of complementary target natural DNA (Integrated DNA Technologies)
  • Stock solution of complementary target unnatural DNA (obtained via automated DNA synthesis as described under protocol 3)
  • 50 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl in Milli‐Q water
  • 50 mM sodium phosphate buffers (pH 7.0) containing 100 mM sodium chloride and 0.1 mM disodium EDTA
  • Bath sonicator (PCI Analytics)
  • Shimadzu UV‐2550 UV‐visible spectrophotometer with quartz optical 8‐micro cell (120 µL DNA) of 1.0 cm path length and scanning rate of 0.5 nm, with wavelength range of 200 to 500 nm, slit width of 2 nm, and Peltier temperature controller
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Figures

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Literature Cited

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