Non‐Nucleoside Phosphoramidites of Xanthene Dyes (FAM, JOE, and TAMRA) for Oligonucleotide Labeling

Maksim V. Kvach1, Dmitry A. Tsybulsky1, Vadim V. Shmanai1, Igor A. Prokhorenko2, Irina A. Stepanova2, Vladimir A. Korshun2

1 Institute of Physical Organic Chemistry, Minsk, Belarus, 2 Shemyakin‐Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.55
DOI:  10.1002/0471142700.nc0455s52
Online Posting Date:  March, 2013
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Abstract

This unit describes the preparation of 5‐ and 6‐carboxy derivatives of the xanthene fluorescent dyes fluorescein (FAM), 4′,5′‐dichloro‐2′,7′‐dimethoxy‐fluorescein (JOE), and tetramethylrhodamine (TAMRA) as individual isomers, and their conversion to non‐nucleoside phosphoramidite reagents suitable for oligonucleotide labeling. The use of a cyclohexylcarbonyl (Chc) protecting group for blocking of phenolic hydroxyls facilitates the chromatographic separation of isomers of carboxy‐FAM and carboxy‐JOE as pentafluorophenyl esters. Acylation of 3‐dimethylaminophenol with 1,2,4‐benzenetricarboxylic anhydride gave a mixture of 4‐dimethylamino‐2‐hydroxy‐2′,4′(5′)‐dicarboxybenzophenones, easily separable into individual compounds upon fractional crystallization. Individual isomeric benzophenones are precursors of 5‐ or 6‐carboxytetramethylrhodamines. The dyes were converted into 6‐aminohexanol‐ (JOE), 4‐trans‐aminocyclohexanol‐ (FAM and JOE), and hydroxyprolinol‐based (TAMRA) phosphoramidite reagents. Curr. Protoc. Nucleic Acid Chem. 52:4.55.1‐4.55.33. © 2013 by John Wiley & Sons, Inc.

Keywords: xanthene fluorophores; fluorescein; JOE; TAMRA; protecting groups; isomer separation; non‐nucleoside phosphoramidites; linkers; fluorescence quenching; energy transfer

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Pentafluorophenyl Esters of 5‐ and 6‐Carboxy Derivatives of 3′,6′‐O‐Diacyl‐Fluorescein (FAM) and 3′,6′‐O‐Diacyl‐4′,5′‐Dichloro‐2′,7′‐Dimethoxy‐Fluorescein (JOE)
  • Support Protocol 1: Synthesis of Cyclohexanecarboxylic Anhydride
  • Basic Protocol 2: Synthesis of 5‐ and 6‐Carboxytetramethylrhodamine (TAMRA)
  • Basic Protocol 3: Synthesis of Non‐Nucleoside Phosphoramidites of FAM, JOE, and TAMRA
  • Basic Protocol 4: Automated Solid‐Phase Synthesis, Purification, and Characterization of Oligonucleotides Labeled with FAM, JOE, and TAMRA
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of Pentafluorophenyl Esters of 5‐ and 6‐Carboxy Derivatives of 3′,6′‐O‐Diacyl‐Fluorescein (FAM) and 3′,6′‐O‐Diacyl‐4′,5′‐Dichloro‐2′,7′‐Dimethoxy‐Fluorescein (JOE)

  Materials
  • N,N‐dimethylformamide (DMF)
  • 5(6)‐Carboxyfluorescein (4), mixture of isomers (prepare as described by Schreder, ; Drechsler and Smagin, ; Haralambidis et al., ; Ueno et al., ; Lee and Grissom, ; Xu et al., ); available also from Acros and ABCR (http://www.abcr.de)
  • N,N‐Diisopropylethylamine (DIEA)
  • Trimethylacetic anhydride (pivalic anhydride; Sigma‐Aldrich, cat. no. 143502)
  • Dichloromethane (DCM)
  • Ethyl acetate (EtOAc)
  • 1 M sodium phosphate buffer, pH 7.0 ( appendix 2A)
  • Magnesium sulfate (MgSO 4) anhydrous
  • Pentafluorophenol (Sigma‐Aldrich, cat. no. 103799)
  • N,N′‐dicyclohexylcarbodiimide (DCC; Sigma‐Aldrich, cat. no. D80002)
  • Chloroform (stabilized with amylene; Sigma‐Aldrich, cat. no. C2432; Acros, cat. no. 38376; or equivalent can be replaced with DCM stabilized with amylene; Sigma‐Aldrich, cat. no. 270997; Acros, cat. no. 11346; or equivalent)
  • Silica gel
  • Toluene, dry
  • Cyclohexanecarboxylic anhydride ( 3; see protocol 2 for synthesis)
  • Hexane
  • 2‐Chloro‐3‐hydroxy‐4‐methoxybenzaldehyde ( 11; prepare as described by Faulkner and Woodcock, and Lyttle et al., )
  • Selenium dioxide (SeO 2)
  • 30% hydrogen peroxide, aqueous
  • 10% (w/v) NaHSO 3 solution
  • Sodium sulfate, anhydrous
  • Methanol (MeOH)
  • Acetyl chloride
  • Diethyl ether
  • Trimellitic anhydride (Sigma‐Aldrich cot. no. B4600)
  • 25% ammonium hydroxide (aqueous)
  • Methanesulfonic acid
  • Tin (IV) chloride (SnCl 4)
  • Neutral alumina (Fisher A/2374 or Brockman I from Sigma‐Aldrich)
  • 2‐Propanol
  • Triethylamine (Et 3N)
  • Concentrated HCl
  • Pyridine (>99.5 purity)
  • Cyclohexanecarboxylic anhydride ( protocol 2)
  • 0.5 M HCl
  • Brine (saturated NaCl)
  • 100‐mL, 250‐mL, 500‐mL and 1‐L round‐bottom flasks
  • Dropping funnel
  • Calcium chloride drying tubes
  • Rotary evaporator equipped with a vacuum pump
  • 1‐L separatory funnel
  • 1‐L Erlenmeyer flask
  • Magnetic stirrer and Teflon‐coated magnetic stirring bars
  • Vacuum filtration system: 350‐ and 200‐mL sintered‐glass funnels (25‐ to 50‐µm porosity) and vacuum adapters
  • Membrane vacuum pump equipped with a vacuum controller (Vacuubrand MV10NT VARIO, http://www.vacuubrand.com, or similar)
  • Silica‐coated aluminum‐backed TLC plates with fluorescent indicator
  • Glass chromatography columns, 8 × 60–cm, 4 × 60–cm
  • Heat gun
  • Overhead stirrer
  • Oil bath
  • Reflux condensers
  • 1‐L beaker
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Support Protocol 1: Synthesis of Cyclohexanecarboxylic Anhydride

  Materials
  • Cyclohexanecarboxylic acid (Sigma‐Aldrich, cat. no. 101834)
  • Thionyl chloride (Sigma‐Aldrich, cat. no. 320536)
  • Toluene, dry
  • Pyridine (>99.5% purity), dry
  • 100‐mL, 250‐mL, 500‐mL and 1‐L round‐bottom flasks
  • Magnetic stirrer and Teflon‐coated magnetic stirring bars
  • 250‐mL graduated cylinder
  • Reflux condensers
  • Calcium chloride drying tubes
  • Oil bath
  • Gas outlet adapter
  • Membrane vacuum pump equipped with a vacuum controller
  • Vigreux column
  • Claisen adapter
  • Thermometer
  • Liebig condenser
  • 4‐way vacuum adapter
  • 1‐L Erlenmeyer flask
  • Vacuum filtration system (350‐mL sintered‐glass funnel, 25‐ to 50‐µm porosity, and vacuum adapter)
  • Rotary evaporator equipped with a vacuum pump
  • High‐vacuum membrane or an oil vacuum pump

Basic Protocol 2: Synthesis of 5‐ and 6‐Carboxytetramethylrhodamine (TAMRA)

  Materials
  • 3‐Dimethylaminophenol (Sigma‐Aldrich, cat. no. D144002)
  • Trimellitic anhydride (Sigma‐Aldrich, cat. no. B4600)
  • Toluene
  • Methanol (MeOH)
  • Acetic acid, glacial
  • Chloroform (stabilized with amylene, Sigma‐Aldrich, cat. no. C2432; Acros, cat. no. 38376; or equivalent)
  • Phosphorus pentoxide
  • Hexamethyldisiloxane (Sigma‐Aldrich, cat. no. 205389)
  • N,N‐dimethylformamide (DMF)
  • 5% (w/v) NaOH
  • Concentrated (12 M) hydrochloric acid (HCl)
  • Triethylamine (Et 3N)
  • 250‐ and 500‐mL, and 1‐L round‐bottom flasks
  • Magnetic stirrer and Teflon‐coated magnetic stirring bars
  • 500‐mL graduated cylinder
  • Oil bath
  • Calcium chloride drying tubes
  • Reflux condensers
  • Vacuum filtration system (20‐mL sintered‐glass funnel, 25 to 50 µm porosity, and vacuum adapter)
  • Membrane vacuum pump equipped with a vacuum controller (Vacuubrand MV10NT VARIO, http://www.vacuubrand.com, or similar)
  • Rotary evaporator equipped with a vacuum pump
  • Silica‐coated aluminum‐backed TLC plates with fluorescent indicator
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 3: Synthesis of Non‐Nucleoside Phosphoramidites of FAM, JOE, and TAMRA

  Materials
  • 3′,6′‐O‐bis(pivaloyl)‐fluorescein‐5‐carboxylic acid, pentafluorophenyl ester ( 6; protocol 1)
  • 3′,6′‐O‐bis(pivaloyl)‐fluorescein‐6‐carboxylic acid, pentafluorophenyl ester ( 7; protocol 1)
  • 3′,6′‐O‐bis(cyclohexylcarbonyl)‐fluorescein‐5‐carboxylic acid, pentafluorophenyl ester ( 9; protocol 1)
  • 3′,6′‐O‐bis(cyclohexylcarbonyl)‐fluorescein‐6‐carboxylic acid, pentafluorophenyl ester ( 10; protocol 1)
  • Dichloromethane (DCM), dry (freshly distilled over CaH 2), stabilized with amylene
  • trans‐4‐aminocyclohexanol hydrochloride (Sigma‐Aldrich, cat. no. 263761)
  • N,N‐diisopropylethylamine (DIEA)
  • N,N‐diisopropylamino‐2‐cyanoethoxychlorophosphine (Sigma‐Aldrich, cat. no. 302309, also can be prepared as described by Smith et al., )
  • Argon (in steel cylinder)
  • N,N‐dimethylformamide (DMF)
  • Sodium sulfate (Na 2SO 4)
  • Silica gel
  • Methanol (MeOH)
  • Toluene
  • Acetone (Me 2CO)
  • Pyridine (>99.5% purity), dry
  • Saturated NaHCO 3 3′,6′‐O‐bis(cyclohexylcarbonyl)‐JOE‐5‐carboxylic acid, pentafluorophenyl ester ( 16; protocol 1)
  • 3′,6′‐O‐bis(cyclohexylcarbonyl)‐JOE‐6‐carboxylic acid, pentafluorophenyl ester ( 17; protocol 1)
  • Ethyl acetate (EtOAc)
  • 6‐Aminohexanol (Sigma‐Aldrich, cat. no. A56353)
  • N‐Cbz‐hydroxy‐L‐proline (CAS 13504‐85‐3; Sigma‐Aldrich, cat. no. 96310)
  • Concentrated (96%) sulfuric acid
  • Tetrahydrofuran
  • Sodium borohydride (NaBH 4)
  • 5% citric acid
  • Chloroform (stabilized with amylene; Sigma‐Aldrich, cat. no. C2432; Acros, cat. no. 38376; or equivalent)
  • Dimethoxytrityl chloride (Sigma‐Aldrich, cat. no. 38827)
  • 10% Pd on carbon (Sigma‐Aldrich, cat. no. 520888)
  • Hydrogen (in steel cylinder)
  • Triethylamine (Et 3N; >99.5% purity)5‐Carboxy‐TAMRA (20; protocol 3)
  • 6‐Carboxy‐TAMRA ( 21; protocol 3)
  • Benzotriazol‐1‐yloxy‐tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP; Sigma‐Aldrich, cat. no. 377848)
  • MgSO 4, anhydride
  • Diisopropylammonium tetrazolide (prepare as described by Caruthers et al., )
  • Bis(N,N‐diisopropylamino)‐2‐cyanoethoxyphosphine (Sigma‐Aldrich, cat. no. 305995; also can be prepared as described by Caruthers et al., , or by Bannwarth and Trzeciak, )
  • Calcium chloride drying tubes
  • 100‐mL, 250‐mL, 500‐mL, and 1‐L round‐bottom flasks
  • Magnetic stirrer and Teflon‐coated magnetic stirring bars
  • 100‐mL and 500‐mL graduated cylinders
  • 500‐mL and 1‐L separatory funnels
  • 250‐mL and 500‐mL Erlenmeyer flasks
  • Vacuum filtration system (various sintered‐glass funnels and vacuum adapter)
  • Membrane vacuum pump equipped with a vacuum controller (Vacuubrand MV10NT VARIO, http://www.vacuubrand.com, or similar)
  • Rotary evaporator equipped with a vacuum pump
  • 4 × 20–cm and 3 × 20–cm chromatography columns
  • Silica‐coated aluminum‐backed TLC plates with fluorescent indicator
  • Addition funnels
  • 0.2 µm PTFE membrane (Millipore, cat. no. JGWP0470Ø)
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 4: Automated Solid‐Phase Synthesis, Purification, and Characterization of Oligonucleotides Labeled with FAM, JOE, and TAMRA

  Materials
  • Dried phosphoramidites 23, 25, 27, 29, 31, 33, 35, 37, 44, and 46 prepared as described in protocol 4
  • Dry acetonitrile
  • Dichloromethane (DCM)
  • CPG supports dA, dG, dC, dT, BHQ‐1 (Glen Research)
  • Conventional phosphoramidite monomers of dA, dG, dC, and dT (Glen Research)
  • Reagents for oligonucleotide synthesis (also see appendix 3C):
    • 0.45 M 1H‐tetrazole in MeCN (Glen Research)
    • Cap A solution: acetic anhydride in tetrahydrofuran/pyridine (Glen Research)
    • Cap B solution: 1‐methylimidazole in tetrahydrofuran (Glen Research)
    • Deblocking solution: 3% trichloroacetic acid in DCM (Glen Research)
  • 25% (v/v) ammonia
  • t‐Butylamine (tBuNH 2; Sigma‐Aldrich, cat. no. 391433)
  • 0.1 M ammonium acetate ( appendix 2A)
  • Acetonitrile (MeCN)
  • Automated DNA synthesizer (ASM 800 from Biosset, http://www.biosset.com; also see appendix 3C)
  • 2‐mL cryovials
  • Speedvac concentrator (Eppendorf)
  • 5‐µm Discovery C18 HPLC column (25 cm × 4.6 mm; Supelco)
  • 1‐mL quartz cuvettes with Teflon caps
  • UV/Vis spectrophotometer
  • Additional reagents and equipment for automated DNA synthesis ( appendix 3C), denaturing polyacrylamide gel electrophoresis (PAGE; unit 10.4), ethanol precipitation of DNA (Moore and Dowhan, ), column chromatography ( appendix 3E), HPLC purification (unit 10.5). and MALDI‐TOF mass spectrometry (unit 10.1)
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Literature Cited

Literature Cited
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