Synthesis of Phosphorothioate Oligonucleotides with Stereodefined Phosphorothioate Linkages

Piotr Guga1, Wojciech J. Stec1

1 Polish Academy of Sciences, Lodz
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.17
DOI:  10.1002/0471142700.nc0417s14
Online Posting Date:  October, 2003
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A method for solid‐phase synthesis of stereodefined PS‐oligos via an oxathiaphospholane approach using pure P‐diastereomers of nucleoside oxathiaphospholane monomers is described. The oxathiaphospholane monomers are synthesized by phosphitylation of 5′‐O‐DMTr‐N‐protected deoxyribonucleosides with 2‐chloro‐spiro‐4,4‐pentamethylene‐1,3,2‐oxathiaphospholane followed by sulfurization. The procedure is general and may be applied to other analogs, depending on the aldehyde (or mercaptoalcohol) used. Starting from an 18O‐labeled mercaptoalcohol, the corresponding 18O‐labeled phosphitylating reagent and nucleoside monomers can be obtained and used for synthesis of labeled stereodefined PS‐oligos, which are useful for studying mechanisms of enzymatic reactions. Details are provided for chromatographic separation of the 5′‐O‐DMTr‐N‐protected‐deoxyribonucleoside‐3′‐O‐(2‐thio‐spiro‐4,4‐pentamethylene‐1,3,2‐oxathiaphospholane)s into their P‐diastereomers, and for manual solid‐phase synthesis of PS‐oligos. Oxidation of 5′‐O‐DMTr‐N‐protected‐deoxyribonucleoside‐3′‐O‐(2‐thio‐spiro‐4,4‐pentamethylene‐1,3,2‐oxathiaphospholane)s with selenium dioxide yields their 2‐oxo‐analogs, which are suitable either for elongation of stereodefined PS‐oligos with segments consisting of unmodified nucleotide units possessing phosphate internucleotide linkages, or for generating isotopomeric 18O‐labeled PO‐oligos of predetermined P‐chirality.

Keywords: nucleic acid; analog; oligodeoxyribonucleotide; oligonucleotide probe; P‐chirality; stereocontrolled synthesis; phosphorothioate; oxathiaphospholane

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Table of Contents

  • Basic Protocol 1: Synthesis of Phosphitylating Reagent: 2‐Chloro‐spiro‐4,4‐Pentamethylene‐1,3,2‐Oxathiaphospholane
  • Alternate Protocol 1: Synthesis of 2,2′‐Dithiobis([18O]Cyclohexanecarboxaldehyde)
  • Support Protocol 1: High‐Vacuum Technique for Transfer of Dry Solvents
  • Basic Protocol 2: Synthesis of 5′‐O‐DMTr‐Deoxyribonucleoside‐3′‐O‐(2‐Thio‐4,4‐spiro‐Pentamethylene‐1,3,2‐Oxathiaphospholane)s and Their Separation into P‐Diastereomers
  • Alternate Protocol 2: Synthesis of 5′‐O‐DMTr‐Deoxyribonucleoside‐3′‐O‐(2‐Oxo‐spiro‐4,4‐Pentamethylene‐1,3,2‐Oxathiaphospholane)s
  • Basic Protocol 3: Manual Solid‐Phase Synthesis of Stereodefined Oligo(Nucleoside Phosphorothioate)s
  • Support Protocol 2: Attachment of Nucleoside 3′‐O‐Succinyl Hemiesters to Sarcosinylated Solid‐Phase Support
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Synthesis of Phosphitylating Reagent: 2‐Chloro‐spiro‐4,4‐Pentamethylene‐1,3,2‐Oxathiaphospholane

  • 4 to 5 M and 1.5 M sodium hydroxide (NaOH)
  • Sulfur monochloride (S 2Cl 2), freshly distilled over 2 g elemental sulfur (S 8; dried ≥12 hr under vacuum) per 50 mL S 2Cl 2
  • Argon (or, optionally, nitrogen), dry
  • Cyclohexanecarboxaldehyde (Fluka)
  • Methylene chloride
  • Diethyl ether, anhydrous
  • Sodium borohydride (NaBH 4)
  • Isopropyl alcohol
  • Anti‐bumping granules
  • 20% (w/v) hydrochloric acid
  • Chloroform
  • Magnesium sulfate, anhydrous
  • Hexane
  • Lithium aluminum hydride
  • Ethyl acetate, dry
  • Tetrahydrofuran (THF), with traces of added moisture
  • 10% (v/v) H 2O/THF
  • Phosphorus trichloride (PCl 3)
  • Benzene, anhydrous
  • Pyridine
  • Dry molecular sieves (4A, 4‐ to 6‐µm‐o.d. beads, Aldrich)
  • 250‐mL absorber with safety flask (see Fig. )
  • 250‐mL four‐neck round‐bottom flask
  • Heated oil bath capable of magnetic stirring
  • Thermometer (capable of reading 150°C)
  • 100‐mL dropping funnel
  • Reflux condensers
  • Glass gas inlet adapter (preferred) or syringe needle and rubber septum
  • Rotary evaporator with a water aspirator and a diaphragm vacuum pump (10 to 15 mmHg; optional)
  • 500‐mL Erlenmeyer flask (29/42 joint)
  • 1‐L two‐neck round‐bottom flask (two 29/42 joints)
  • Stopcock, 29/42
  • Flexible adapter (glass M/F joints, 29/42, on corrugated Teflon tubing; optional)
  • 500‐mL separatory funnel
  • Filter funnel and Whatman no.1 filter paper (or equivalent)
  • High‐vacuum fractional distillation apparatus
  • High‐vacuum oil pump (0.01 mmHg)
NOTE: Upon storage, cyclohexanecarboxaldehyde undergoes polymerization. Order only the amount required for use within 2 to 3 weeks.NOTE: Within this unit, evaporation of solvents is performed using a rotary evaporator connected to a water aspirator, unless otherwise specified.

Alternate Protocol 1: Synthesis of 2,2′‐Dithiobis([18O]Cyclohexanecarboxaldehyde)

  • 2,2′‐Dithiobis(cyclohexanecarboxaldehyde) (S.6; see protocol 1, step )
  • Aniline, freshly distilled in inert atmosphere
  • 95:5 (v/v) chloroform/hexane
  • H 2[18O] (95 atom%)
  • Hydrogen chloride, anhydrous
  • Tetrahydrofuran (THF), dried over sodium hydride
  • 250‐mL two‐neck round‐bottom flasks
  • Azeotropic trap (e.g., mini Dean‐Stark trap, Aldrich)
  • 8 × 40–cm chromatography column packed with silica gel 60, 230 to 400 mesh (Merck)
  • Rubber septum
  • TLC silica gel plates with UV indicator (Merck; also see appendix 3D)
  • High vacuum valve (e.g., Rotaflo, Quickfit)
  • Drying tube (8 × 5/8 in. with connectors, Aldrich)
  • 2‐ to 5‐mL gas‐tight syringe
  • Rotary evaporator with water aspirator or membrane pump
  • Buchner funnel with glass frit
  • Additional reagents and equipment for column chromatography ( appendix 3E), thin‐layer chromatography (TLC; appendix 3D), and high‐vacuum transfer of solvent (see protocol 3)

Support Protocol 1: High‐Vacuum Technique for Transfer of Dry Solvents

  • 5′‐O‐DMTr‐N‐protected deoxyribonucleosides (Chemgen):
    • N6‐Benzoyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxyadenosine (5′‐O‐DMTr‐dABz)
    • N2‐Isobutyryl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxyguanosine (5′‐O‐DMTr‐dGi‐Bu)
    • N4‐Benzoyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxycytidine (5′‐O‐DMTr‐dCBz)
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐deoxythymidine (5′‐O‐DMTr‐T)
  • Argon (or, optionally, nitrogen), dry
  • Acetonitrile, anhydrous
  • 2‐Chloro‐spiro‐4,4‐pentamethylene‐1,3,2‐oxathiaphospholane (S.9 or labeled S.12; see protocol 1 or protocol 2)
  • Elemental sulfur, anhydrous (dried overnight at high vacuum)
  • Chloroform (distilled with 1 mL pyridine per L)
  • Toluene, dry
  • Pyridine, anhydrous
  • Diisopropylethylamine (Aldrich), anhydrous
  • Diphenylcarbamoyl chloride (Aldrich)
  • 9:1 (v/v) chloroform/methanol
  • Merck 60H silica gel, particle size 5 to 40 µm
  • Ethyl acetate
  • Butyl acetate
  • Benzene
  • 25‐mL two‐neck round‐bottom flasks
  • High‐vacuum valve (Rotaflo, Quickfit)
  • Rubber septum
  • High‐vacuum oil pump (0.01 mmHg)
  • 2‐mL and 10‐mL gas‐tight syringes
  • Buchner funnel
  • 25 × 3–cm chromatography column packed with 20 g of 230 to 400 mesh silica gel
  • TLC silica gel plates with UV indicator (Merck; also see appendix 3D)
  • Constant temperature water bath
  • 30 × 2–cm chromatography column
  • Filter paper (Whatman no. 1)
  • High‐performance TLC (HP‐TLC) plates (silica gel 60 F 254) with UV indicator (Merck; also see appendix 3D)
  • Additional reagents and equipment for column chromatography ( appendix 3E) and thin‐layer chromatography (TLC; appendix 3D)

Basic Protocol 2: Synthesis of 5′‐O‐DMTr‐Deoxyribonucleoside‐3′‐O‐(2‐Thio‐4,4‐spiro‐Pentamethylene‐1,3,2‐Oxathiaphospholane)s and Their Separation into P‐Diastereomers

  • 5′‐O‐DMTr‐deoxyribonucleoside‐3′‐O‐(2‐thio‐spiro‐4,4‐pentamethylene‐1,3,2‐oxathiaphospholane)s (S.3; see protocol 4)
  • Silica gel 60, 230 to 400 mesh
  • Acetonitrile, anhydrous
  • Argon (or, optionally, nitrogen), dry
  • Selenium dioxide, anhydrous (dried overnight at high vacuum)
  • 95:5 (v/v) chloroform/methanol (distill chloroform with 1 mL pyridine per L)
  • Two‐neck 10‐mL round‐bottom flask
  • High‐vacuum valve (e.g., Rotaflo, Quickfit)
  • Rubber septum
  • High‐vacuum oil pump
  • 10‐µmol‐scale DNA synthesis column
  • 1‐ to 2‐mL polypropylene syringes with luer ends
  • 5‐mL gas‐tight syringe
  • TLC silica gel plates with UV indicator (Merck; also see appendix 3D)
  • 10‐mL gas‐tight syringe
  • Luer male‐to‐male adapter
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D)
CAUTION: Selenium dioxide is toxic.

Alternate Protocol 2: Synthesis of 5′‐O‐DMTr‐Deoxyribonucleoside‐3′‐O‐(2‐Oxo‐spiro‐4,4‐Pentamethylene‐1,3,2‐Oxathiaphospholane)s

  • Deoxyribonucleoside oxathiaphospholane monomers (see protocol 4 and/or protocol 5)
  • Chloroform (optional), distilled with 1 mL pyridine per L
  • Toluene, anhydrous
  • Low‐pressure argon or nitrogen, dried (see recipe)
  • Sarcosinylated solid support functionalized with a nucleoside (first from the 3′ end of sequence to be synthesized) at a concentration ranging from 20 to 30 µmol/g support (see protocol 7)
  • Capping reagent A: 1:1:8 (v/v/v) acetic anhydride/pyridine/tetrahydrofuran (THF)
  • Capping reagent B: 7 g 4‐dimethylaminopyridine/93 mL THF
  • Anhydrous acetonitrile (H 2O < 20 ppm; see recipe) in bottle with rubber septum, with dry gas delivered inside through a line ending in a needle
  • Detritylating reagent: 3.5% (w/v) dichloroacetic acid in methylene chloride
  • Acetonitrile, HPLC grade (Baker)
  • 1:4.5 (v/v) 1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU) in anhydrous acetonitrile (H 2O <20 ppm)
  • Methylene chloride
  • Aqueous ammonia, concentrated (Baker)
  • 10 × 3–cm chromatography column packed with 10 g 230‐ to 400‐mesh silica gel 60 (optional)
  • 4‐mL sample vials with open‐top screw caps with Teflon‐faced rubber septa
  • 19‐ to 22‐G, 1‐ to 1.5‐in. luer‐lock needles
  • Vacuum desiccator (≤0.05 mmHg; provided by high‐vacuum oil pump) with condenser cooled by liquid N 2
  • 1‐, 2‐, and 5‐mL all‐polypropylene luer‐lock syringes
  • 19‐ to 22‐G, 2‐ to 3‐in. luer‐lock needles with blunt 90° tips
  • Columns for DNA synthesis:
    • For 1‐µmol scale: Applied Biosystems DNA synthesis column (cat. no. 400407), empty, 1.0 µmol crimp‐style
    • For 2‐µmol scale: emptied and reassembled Applied Biosystems oligonucleotide purification cartridge (OPC; cat. no. 400771)
  • Column filters (two for each column; Applied Biosystems, cat. no. 400059)
  • Aluminum seals (caps; two for each column; Aldrich cat. no. Z11413‐8; Wheaton aluminum cap, 13 mm, tear‐off)
  • Crimper for aluminum seals (Aldrich, cat. no. z 11423)
  • Polypropylene (or other chemically inert) luer male‐to‐male adapter
  • 100‐ and 500‐µL gas‐tight syringes (Hamilton)
  • Glass drying tube (∼2‐cm i.d., 25‐cm length), with three‐way valve (or two independent valves) at the top and a rubber septum at the opposite side
  • High‐vacuum (0.05 mmHg) oil pump
  • 500‐mL filtering flask capped with a rubber septum pierced with a 3‐ to 4‐mm i.d. hole
  • Water aspirator with a manostat
  • Two waste bottles: one for chlorinated waste (methylene chloride, detritylating reagent) and another for water‐miscible wastes (acetonitrile, capping reagents A and B)
  • 60°C water bath (optional)
  • Speedvac concentrator with vacuum provided by water aspirator
  • 19‐ to 22‐G, 4‐ to 5‐in. luer‐lock needles with blunt 90° tips
  • Additional reagents and equipment for column chromatography (optional; appendix 3E) and purification of oligonucleotides (units 10.3 10.5)
CAUTION: The drying tube must be tested to make sure that it is safe for high‐vacuum usage, i.e., at a pressure lower than 0.05 mmHg.

Basic Protocol 3: Manual Solid‐Phase Synthesis of Stereodefined Oligo(Nucleoside Phosphorothioate)s

  • Long‐chain alkylamine controlled‐pore glass (LCAA‐CPG) beads (80 to 120 mesh, 500 Å; Sigma)
  • 9‐Fluorenylmethoxycarbonyl (Fmoc)–sarcosine monohydrate (Fluka)
  • Argon (or, optionally, nitrogen), dry
  • 1,3‐Dicyclohexylcarbodiimide (DCC; Aldrich)
  • Dimethylformamide (DMF), anhydrous
  • Pyridine, anhydrous
  • 1:1:1 (v/v/v) acetonitrile/methanol (reagent grade)/pyridine
  • Acetonitrile, anhydrous
  • 10% (v/v) piperidine in pyridine
  • 5′‐O‐DMTr‐N‐protected nucleoside‐3′‐O‐succinyl hemiester (Sigma)
  • 25‐mL two‐neck round‐bottom flasks
  • High‐vacuum valve (e.g., Rotaflo, Quickfit)
  • Rubber septum
  • High‐vacuum oil pump
  • 1‐mL and 10‐mL gas‐tight syringes
  • Buchner funnel with glass frit
  • Filter flask
  • Vacuum source (e.g., water aspirator)
  • 50‐mL Erlenmeyer flask with stopcock
CAUTION: 1,3‐Dicyclohexylcarbodiimide can cause skin or eye irritation and allergic reactions. Use appropriate protection.
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Literature Cited

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