2‐Pyridinyl Thermolabile Groups as General Protectants for Hydroxyl, Phosphate, and Carboxyl Functions

Jolanta Brzezinska1, Agnieszka Witkowska1, Tomasz P. Kaczyński1, Dominika Krygier1, Tomasz Ratajczak1, Marcin K. Chmielewski1

1 Institute of Bioorganic Chemistry Polish Academy of Sciences, Poznań
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 2.20
DOI:  10.1002/cpnc.26
Online Posting Date:  March, 2017
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Abstract

Application of 2‐pyridinyl thermolabile protecting groups (2‐PyTPGs) for protection of hydroxyl, phosphate, and carboxyl functions is presented in this unit. Their characteristic feature is a unique removal process following the intramolecular cyclization mechanism and induced only by temperature rise. Deprotection rate of 2‐PyTPGs is dependent on certain parameters, such as solvent (aqueous or non‐aqueous medium), pH values, and electron distribution in a pyridine ring. The presented approach pertains not only to protecting groups but also to an advanced system of controlling certain properties of 2‐pyridinyl derivatives. We improved the “chemical switch” method, allowing us to regulate the protecting group stability by inversing the electron distribution in 2‐PyTPG. Together with pH values manipulation, this allows us to regulate the protecting group stability. Moreover, phosphite cyclization to oxazaphospholidine provides a very stable but easily reversible tool for phosphate protection/modifications. For all TPGs we confirmed their utility in a system of protecting groups. This concept can contribute to designing the general protecting group that could be useful in bioorganic chemistry. © 2017 by John Wiley & Sons, Inc.

Keywords: protecting groups; intramolecular cyclisation; “click‐clack”; nucleosides; thermal lability; chemical switch

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Synthesis of Precursors: N‐(2‐Pyridinyl)Aminoethanol (3), 2‐Pyridinyl‐N‐Benzylaminoethanol (5), and 2‐Pyridinyl‐N‐(2,4‐Difluorobenzyl)Aminoethanol (7)
  • Alternate Protocol 1: Synthesis of 4‐Nitro‐2‐Pyridinyl‐N‐Benzylaminoethanol (10) Precursor of TPG
  • Basic Protocol 2: Synthesis of Thermolabile Carbonates
  • Basic Protocol 3: Thermolytic Deprotection of Nucleosidic Carbonates
  • Basic Protocol 4: Carbonate Thermolability Steering Procedure: Synthesis of 3′‐O‐Acetyl‐5′‐O‐[2‐N‐Benzyl‐N‐(4‐Aminopyridin‐2‐YL)]Aminoethyloxycarbonyl Thymidine (21)
  • Basic Protocol 5: Synthesis of Nucelosidic Derivative‐Protected Esters
  • Basic Protocol 6: 2‐Py TPG Thermal Removal from Esters
  • Basic Protocol 7: Preparation of TPG‐Protected 3′‐O‐ Succinate Nucleoside with 5′‐OH by Orthogonal Removal of Acid‐Labile 4, 4′‐Dimethoxytrityl (DMT) Group
  • Basic Protocol 8: Temporary Protection of Thermolabile Properties of 2‐Pyridinyl TPG Groups: “Click‐Clack” Approach
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of Precursors: N‐(2‐Pyridinyl)Aminoethanol (3), 2‐Pyridinyl‐N‐Benzylaminoethanol (5), and 2‐Pyridinyl‐N‐(2,4‐Difluorobenzyl)Aminoethanol (7)

  Materials
  • 2‐Bromopyridine (Sigma‐Aldrich)
  • Ethanolamine (Sigma‐Aldrich)
  • Triethylamine (TEA)
  • Desiccant in a drying tube (blue silica gel, Merck)
  • Dichloromethane (DCM)
  • Methanol
  • Anhydrous tetrahydrofuran (THF; dried over 4 Å molecular sieves)
  • Sodium bicarbonate (saturated aq. NaHCO 3)
  • Sodium sulfate (Na 2SO 4)
  • Silica gel (Merck, 200−630 mesh) (Sigma‐Aldrich)
  • Benzyl bromide (Sigma‐Aldrich)
  • 2,4‐Difluorobenzyl bromide (Sigma‐Aldrich)
  • 10‐mL, 50‐mL, and 100‐mL round‐bottom flasks
  • Microwave reactor (Ertec Poland)
  • 60‐cm Vigreux columns
  • Merck thin‐layer chromatography (TLC) silica plates (Kieselgel 60 F‐254; 0.2‐mm thick)
  • Rotary evaporator
  • 300‐mL separatory funnel (Duran)
  • Stir bar

Alternate Protocol 1: Synthesis of 4‐Nitro‐2‐Pyridinyl‐N‐Benzylaminoethanol (10) Precursor of TPG

  Materials
  • 2‐Bromo‐4‐nitropyridine (8; Sigma‐Aldrich)
  • 2‐Benzylaminoethanol (9; Sigma‐Aldrich)
  • Dimethylformamide (DMF; dried over 3 Å molecular sieves)
  • Diisopropylethylamine (DIPEA; dried over 3 Å molecular sieves)
  • Hexane
  • Ethyl acetate
  • Dichloromethane (DCM)
  • Sodium bicarbonate (saturated aq. NaHCO 3) (ChemPure)
  • Sat. aqueous NaCl (brine)
  • Anhydrous sodium sulfate (Na 2SO 4)
  • Magnetic stir bar
  • Silica gel (Merck, 200−630 mesh)
  • Syringes and needles or automatic pipets
  • 10‐mL, 25‐mL, 50‐mL, and 100‐mL round‐bottom flasks
  • Microwave reactor (Ertec Poland)
  • Merck thin‐layer chromatography (TLC) silica plates (Kieselgel 60 F‐254; 0.2‐mm thick)
  • Chromatography column
  • Rotary evaporator
  • 500‐mL separatory funnel

Basic Protocol 2: Synthesis of Thermolabile Carbonates

  Materials
  • 3′‐O‐Acetyl‐thymidine (Carbosynth)
  • Anhydrous acetonitrile (ACN; dried over 4 Å molecular sieves)
  • 1,1′‐Carbonyldiimidazole (CDI; Sigma‐Aldrich)
  • Ethyl acetate
  • Hexane
  • Aminoalcohols (see protocol 1 or protocol 2Alternate Protocol):
    • N‐(2‐pyridinyl)aminoethanol (3)
    • 2‐pyridinyl‐N‐benzylaminoethanol (5)
    • 2‐pyridinyl‐N‐(2,4‐difluorobenzyl)aminoethanol (7)
    • 2‐[benzyl‐(4‐nitropyridin‐2‐yl)amino]ethanol (10)
  • 1,1,3,3‐Tetramethylguanidine TMG; Sigma‐Aldrich
  • Dichloromethane (DCM)
  • Methanol
  • Sodium bicarbonate (saturated aq. NaHCO 3)
  • Sat. aqueous NaCl (brine)
  • Sodium sulfate (Na 2SO 4)
  • Silica gel (Merck, 200−630 mesh)
  • 25‐mL round‐bottom flasks
  • Thin‐layer chromatography (TLC) silica plates (Kieselgel 60 F‐254; 0.2‐mm thick; Merck)
  • Rotary evaporator
  • PLC silica plates 60 F 254, 1 mm, glass plate 20 × 20 cm (Merck)
  • Chromatography chamber
  • 500‐mL separatory funnel

Basic Protocol 3: Thermolytic Deprotection of Nucleosidic Carbonates

  Materials
  • Carbonates 13‐16 (see protocol 3)
  • Phosphate buffer, pH 7.2
  • 0.01 M Triethylammonium acetate, pH 7.0 (prepared with 1 M)
  • Water (produced using an ultra‐pure water system with 4 microS/cm; Millipore)
  • 1 M Triethylammonium acetate, pH 7.0 (Sigma‐Aldrich)
  • Acetonitrile (CH 3CN) for HPLC, gradient grade (Sigma‐Aldrich)
  • 3‐mL Screw‐cap glass vials
  • Thermomixer Eppendorf
  • Column: LUNA 3u C18(2) 100 A, size:150 × 4.60‐mm, 3 μm Phenomenex
  • RP‐HPLC equipment
  • Vacuum apparatus

Basic Protocol 4: Carbonate Thermolability Steering Procedure: Synthesis of 3′‐O‐Acetyl‐5′‐O‐[2‐N‐Benzyl‐N‐(4‐Aminopyridin‐2‐YL)]Aminoethyloxycarbonyl Thymidine (21)

  Materials
  • Anhydrous tetrahydrofuran (THF; dried over 3 Å molecular sieves)
  • Argon gas
  • Water (Milli‐Q water produced using an ultra‐pure water system with 4 microS‐cm; Millipore)
  • Ice bath
  • Titanium tetrachloride (TiCl 4) (Sigma‐Aldrich)
  • Lithium aluminum hydride (LiAlH 4) (Sigma‐Aldrich)
  • 3′‐O‐Acetyl‐5′‐O‐[2‐N‐benzyl‐N‐(4‐nitropyridin‐2‐yl)]aminoethyloxycarbonylthymidine (16)
  • Isopropanol (iPrOH)
  • Dichloromethane (DCM)
  • Methanol
  • Sat. aqueous NaCl (brine)
  • 25‐mL two‐neck, round‐bottomed flasks
  • Rubber septum
  • Magnetic stir bar
  • Merck thin‐layer chromatography (TLC) silica plates (Kieselgel 60 F‐254; 0.2‐mm thick)
  • Chromatography column
  • Centrifuge
  • Syringes and needles or automatic pipets
  • Rotary evaporator
  • PLC silica plates 60 F 254, 1 mm, 20 × 20 cm glass plate (Merck)
  • UV lamp
  • Razor blades
  • 10‐mL, 50‐mL, and 100‐mL round‐bottom flasks
CAUTION: Titanium tetrachloride and LiAlH 4 are extremely moisture‐sensitive and react violently with air and/or Milli‐Q water. Upon reaction with water, they will also liberate highly toxic gases (HCl gas). They must be handled under inert atmosphere to minimize exposure to both air and moisture. TiCl 4 may also be harmful if absorbed through the skin.

Basic Protocol 5: Synthesis of Nucelosidic Derivative‐Protected Esters

  Materials
  • 5′‐O‐(4,4′‐dimethoxytriphenylmethyl)thymidine‐3′‐O‐succinate (22) (Carbosynth)
  • Anhydrous DCM (dried over 4 Å molecular sieves)
  • Argon gas
  • Ice bath
  • N‐(3‐Dimethylaminopropyl)‐N′‐ethylcarbodiimide hydrochloride (EDC, WSC)
  • 4‐Dimethylaminopyridine (DMAP)
  • 2‐((2,4‐difluorobenzyl)(pyridin‐2‐yl)amino)ethanol (7) (see synthesis in protocol 1)
  • Hexane
  • Ethyl acetate
  • Sodium chloride (NaCl)
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Silica gel (Merck, 200−630 mesh)
  • Sat. aqueous NaCl (brine)
  • 25‐mL two‐neck round‐bottom flask
  • Balloons
  • Rubber septum
  • Merck thin‐layer chromatography (TLC) silica plates (Kieselgel 60 F‐254; 0.2‐mm thick)
  • 150‐mL separatory funnels
  • Rotary evaporator
CAUTION: The reaction must be carried out under dry conditions and an argon atmosphere.

Basic Protocol 6: 2‐Py TPG Thermal Removal from Esters

  Materials
  • Compound 23 (see protocol 6)
  • Phosphate buffer solution (pH, 7.2, > 0.044 M Na 2HPO 4, > 0.022 M NaH 2PO 4, Fluka)
  • Acetonitrile
  • Acetone
  • Water (Milli‐Q water producing using an ultra‐pure water system with 4 microS/cm; Millipore)
  • Chloroform
  • 5‐mL glass bottle with Teflon caps
  • Oven
  • Thin‐layer chromatography (TLC) silica plates (Kieselgel 60 F‐254; 0.2‐mm thick)
  • Rotary evaporator
  • Preparative thin‐layer chromatography (PLC) silica plate (20 × 20)
  • UV light
  • Razor blades
  • Frit (for filtering the band)
  • Chromatographic glass chamber

Basic Protocol 7: Preparation of TPG‐Protected 3′‐O‐ Succinate Nucleoside with 5′‐OH by Orthogonal Removal of Acid‐Labile 4, 4′‐Dimethoxytrityl (DMT) Group

  Materials
  • Compound 23 (see protocol 6)
  • Dichloroacetic acid (DCA)
  • Dichloromethane (DCM)
  • Silica gel (200−630 mesh; Merck)
  • Methanol (MeOH)
  • Sodium bicarbonate (saturated aq. NaHCO 3)
  • Sat. aqueous NaCl (brine)
  • Sodium sulfate (Na 2SO 4), anhydrous (ChemPure)
  • Hexane (POCh)
  • Ethyl acetate
  • 25‐mL round‐bottomed flask
  • Stir bars
  • Merck thin‐layer chromatography (TLC) silica plates (Kieselgel 60 F‐254; 0.2‐mm thick)

Basic Protocol 8: Temporary Protection of Thermolabile Properties of 2‐Pyridinyl TPG Groups: “Click‐Clack” Approach

  Materials
  • N‐(2‐pyridinyl)aminoethanol (3)
  • 3′‐O‐Acetylthymidine (Carbosynth)
  • Phosphorus trichloride (Sigma‐Aldrich)
  • Benzene (distilled and stored over 3 Å molecular sieves), anhydrous
  • Argon gas (inert gas)
  • Ice bath
  • Diisopropylamine (DIPEA; distilled and stored over 3 Å molecular sieves)
  • Silica gel (200−630 mesh; Merck)
  • Triethylamine (Sigma‐Aldrich)
  • Acetonitrile (dried over 4 Å molecular sieves)
  • Benzylthiotetrazole (Sigma‐Aldrich)
  • Dichloromethane (dried over 4 Å molecular sieves)
  • Saturated aqueous sodium bicarbonate (saturated aq. NaHCO 3)
  • Sodium sulfate (Na 2SO 4) (ChemPure)
  • Water (Miili‐Q water produced using an ultra‐pure water system with 4 microS/cm; Millipore)
  • Methanol
  • Anhydrous pyridine (dried over 4 Å molecular sieves)
  • Iodine (I 2) (Sigma‐Aldrich)
  • 2‐Mercaptoethanol (Sigma‐Alrich)
  • 25‐mL round‐bottomed flasks
  • Septum
  • Syringe with long needle
  • NMR probe
  • Rotary evaporator
  • Glass column and filter paper for column chromatography
  • Merck thin‐layer chromatography (TLC) silica plates (Kieselgel 60 F‐254; 0.2‐mm thick)
  • Lyophilizer
  • 10‐mL round‐bottomed flasks
  • Magnetic stirrer
  • Separatory funnel
  • Preparative layer chromatography (PLC) plates (Merck, Kieselgel 60 F‐254)
  • UV light
  • Razor blades
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Figures

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Literature Cited

Literature Cited
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