Vibration Ball Milling for the Synthesis of 5′‐Thioadenosine 5′‐Pyrophosphate (P′→5′) Adenosine (dASppA)

Olga Eguaogie1, Joseph S. Vyle1

1 School of Chemistry and Chemical Engineering, Queen's University Belfast, Belfast
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.41
DOI:  10.1002/cpnc.37
Online Posting Date:  September, 2017
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Abstract

Using vibration ball milling, 5′‐chloro‐5′‐deoxyadenosine (CldA) reacts cleanly with 4‐methoxybenzyl mercaptan (MobSH), under basic conditions, to the corresponding thioether (MobSdA), which is isolated following precipitation and trituration. Under acidic conditions, in a one‐pot, two‐step process, MobSdA is transformed into 5′‐deoxy‐5′‐(5‐nitropyridyl‐2‐disulfanyl)‐adenosine (NPySSdA). Michaelis‐Arbuzov (M‐A) reaction of NPySSdA with tris(trimethylsilyl) phosphite proceeds to completion within 30 min as determined by 31P NMR, and the persilylated M‐A product thus formed can be stored in solution under anhydrous conditions at room temperature for several days (in contrast, the anionic phosphorothiolate monoester is labile to hydrolysis). Following evaporation, mechanochemical mixing of the crude M‐A product with the nucleotide donor adenosine 5′‐monophosphomorpholidate under acidic activation in the presence of additional water gives rapid hydrolytic desilylation and phosphate coupling, so that essentially complete reaction is observed after 90 min and dASppA isolated following C‐18 reversed phase HPLC and desalting (>99% pure as determined by monitoring at 260 nm). © 2017 by John Wiley & Sons, Inc.

Keywords: ball mill; mechanochemistry; Michaelis‐Arbuzov; phosphate coupling; thionucleosides

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of 5′‐S‐(4‐Methoxybenzyl)‐5′‐Thioadenosine (2)
  • Basic Protocol 2: Synthesis of 5′‐Deoxy‐5′‐(5‐Nitropyridyl‐2‐Disulfanyl)‐ Adenosine (3)
  • Basic Protocol 3: Synthesis of 5′‐Thioadenosine 5′‐Pyrophosphate (P′→5′) Adenosine (5)
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of 5′‐S‐(4‐Methoxybenzyl)‐5′‐Thioadenosine (2)

  Materials
  • Argon
  • 5′‐Chloro‐5′‐deoxyadenosine (1; Carbosynth, cat. no. NC05644)
  • 4‐Methoxybenzyl mercaptan (a.k.a. MobSH, 4‐methoxy‐α‐toluenethiol; Sigma‐Aldrich, cat. no. 113158)
  • 1,1,3,3‐Tetramethylguanidine (TMG; Sigma‐Aldrich, cat. no. 241768)
  • Acetonitrile
  • Ethanol
  • 10% (w/v) KH 2PO 4, cooled on ice
  • 2 M HCl, cooled on ice
  • 18.2 MΩ water (purified by reverse osmosis) cooled on ice
  • Phosphorus pentoxide (P 2O 5)
  • Potassium hydroxide (KOH)
  • 5:1 diethyl ether:n‐hexane cooled on ice
  • Balloons for argon application
  • Two zirconia‐lined vessels (25‐mL internal volume; Retsch, cat. no. 01.462.0201)
  • 1 or 2 15‐mm zirconia balls (10.70 g; Retsch, cat. no. 05.368.0094
  • Retsch Mixer Mill MM 400 (Retsch, cat. no. 20.745.0001)
  • Silica gel TLC plate with fluorescent indicator
  • 254‐nm UV lamp
  • 250‐mL conical flask
  • Magnetic stirring bar
  • Magnetic stirrer
  • pH paper
  • Bath sonicator
  • Sintered‐glass funnels (porosity 4)
  • 250‐mL Büchner flasks
  • Vacuum desiccator
  • Vacuum pump (capable of < 10 mm Hg)
  • 100‐mL conical flask with ground glass joint and stopper
  • Additional reagents and equipment for thin‐layer chromatography (TLC, appendix 3D; Meyers & Meyers, )

Basic Protocol 2: Synthesis of 5′‐Deoxy‐5′‐(5‐Nitropyridyl‐2‐Disulfanyl)‐ Adenosine (3)

  Materials
  • 5′‐S‐(4‐Methoxybenzyl)‐5′‐thioadenosine (2; protocol 1
  • 2,2′‐dithiobis(5‐nitropyridine) (Sigma‐Aldrich, cat. no. 158194)
  • Argon
  • Thioanisole
  • Trifluoroacetic acid
  • Methanol
  • Dichloromethane
  • Chloroform [CHCl 3; supplied commercially with stabilizer (either 1% v/v ethanol; Sigma‐Aldrich, cat. no. 132950 or 200 ppm amylene; VWR, cat. no. BDH83626)]
  • Silica gel 60 Å for chromatography (40 to 63 μm) dried at 130°C
  • 25‐mL oven‐dried round‐bottom flask
  • Balloons for argon application
  • Right‐angled ground glass jointed balloon (tubing) adapter (two)
  • Magnetic stirring bar
  • Magnetic stirrer
  • 50‐mL ground glass jointed conical flask
  • Silica gel TLC plate with fluorescent indicator
  • 254‐nm UV lamp
  • Rotary evaporator
  • Vacuum desiccator
  • Vacuum pump (capable of < 10 mm Hg)
  • 2.5 × 35 cm chromatography column
  • Additional reagents and equipment for thin‐layer chromatography (TLC, appendix 3D; Meyers & Meyers, ) and column chromatography ( appendix 3E; Meyers, 2001)

Basic Protocol 3: Synthesis of 5′‐Thioadenosine 5′‐Pyrophosphate (P′→5′) Adenosine (5)

  Materials
  • Chloroform [CHCl 3; supplied commercially with stabilizer (either 1% v/v ethanol; Sigma‐Aldrich, cat. no. 132950 or 200 ppm amylene; VWR, cat. no. BDH83626)]
  • Phosphorus pentoxide
  • Activated basic aluminum oxide (Sigma‐Aldrich, cat. no. 199443)
  • Argon
  • Bis(trimethylsilyl)acetamide (BSA; Sigma‐Aldrich, cat. no. 128910)
  • 5′‐deoxy‐5′‐(5‐nitropyridyl‐2‐disulfanyl)‐adenosine (3; protocol 2)
  • Tris(trimethylsilyl)phosphite [(TMSO) 3P; Tokyo Chemical Industry, cat. no. P1217]
  • Adenosine 5′‐monophosphomorpholidate 4‐morpholine‐N,N′‐dicyclohexylcarboxamidine salt (AMP‐morpholidate; Sigma‐Aldrich, cat. no. A1127)
  • 18.2 MΩ deionized water
  • 1H‐Tetrazole
  • Magnesium chloride hexahydrate (>99%)
  • HPLC eluents:
    • 100 mM triethylammonium acetate (TEAA) pH 6.5 in 100% H 2O
    • 100 mM triethylammonium acetate (TEAA) pH 6.5 in 95:5 H 2O:acetonitrile
    • 100 mM triethylammonium acetate (TEAA) pH 6.5 in 35:65 H 2O:acetonitrile
    • 100 mM triethylammonium bicarbonate (TEAB) pH 7.8 in 100% H 2O
    • 100 mM triethylammonium bicarbonate (TEAB) pH 8.3 in 35:65 H 2O:acetonitrile
  • 65°C drying oven
  • Vacuum desiccator
  • Vacuum pump (capable of < 10 mm Hg)
  • Three oven‐dried ground‐glass‐jointed 50‐mL conical flasks
  • Oven‐dried rubber septa (B14 and as needed for glassware)
  • Oven‐dried balloon adaptors
  • Balloons for application of argon atmosphere
  • 10‐mL disposable syringes (include one with the top 10% of the barrel removed)
  • Lint‐free tissues (e.g., Kimwipes)
  • Oven‐dried 5‐ and 10‐mL round‐bottom flasks
  • Oven‐dried magnetic stirring bar
  • Magnetic stirrer
  • Oven‐dried Hamilton gas‐tight syringes (100 μL, 500 μL ,and 2.5 mL)
  • Oven‐dried 5‐mm‐diameter NMR tubes
  • Two zirconia‐lined vessels (25‐mL internal volume; Retsch, cat. no. 01.462.0201)
  • 1 or 2 15‐mm zirconia balls (10.70 g; Retsch, cat. no. 05.368.0094)
  • Retsch Mixer Mill MM 400 (Retsch, cat. no. 20.745.0001)
  • Bath sonicator
  • 0.45 µm Spin‐X® cellulose acetate membrane filter for 1.5 mL Eppendorf tubes
  • HPLC system capable of pumping at 8 mL/min: the system used to develop this protocol was a ThermoFinnigan SpectraSYSTEM consisting of a P2000 binary gradient pump with UV1000 sample detector; samples were injected manually via a Rheodyne injection valve into a 20‐µL or 5‐mL injection loop
  • 150 × 4.60 mm Phenomenex Clarity 5 µm Oligo‐RP column
  • 250 × 21.2 mm Phenomenex Clarity 5 µm Oligo‐RP column
  • 50‐µL syringe with blunt‐ended needle for HPLC injection
  • pH paper
  • Cold‐finger rotary evaporator (Büchi, cat. no. R‐114; fitted with cold trap, Büchi, cat. no. 32351)
  • Quartz UV cuvette, 1‐cm pathlength, 1.5‐mL capacity
  • UV‐Vis spectrometer
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Figures

Videos

Literature Cited

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Internet Resources
  http://www.retsch.com/products/milling/ball‐mills/mixer‐mill‐mm‐400/
  Retsch Web site (supplier of Mixer Mill MM400).
  https://www.youtube.com/watch?v=ZaepF_‐cXoc
  YouTube videos showing use of Mixer Mill MM400.
  https://www.youtube.com/watch?v=k6mPWPuR8PY
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