Microwave‐Assisted Functionalization of Solid Supports for Rapid Loading of Nucleosides

Radhakrishnan P. Iyer1, Seetharamaiyer Padmanabhan1, John E. Coughlin1

1 Spring Bank Technologies, Milford, Massachusetts
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 3.13
DOI:  10.1002/0471142700.nc0313s23
Online Posting Date:  January, 2006
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Abstract

Ultra‐fast and efficient functionalization of solid supports such as controlled‐pore glass (CPG), amino methyl polystyrene, and Tentagel has been achieved using microwave‐assisted procedures. Both amino‐ and carboxy‐terminated supports are easily prepared within minutes, in a reproducible manner, using microwave‐assisted methodologies. The resulting functionalized supports are efficiently coupled to nucleosides using dimethylformamide as a solvent in conjunction with a specially designed reactor and workstation called LOTUS. Using these improved protocols, CPG with loadings of 75 to 85 µmol/g can be prepared on a large scale within 3 to 4 days starting from native CPG, as opposed to traditional methods that require 10 to 15 days to achieve the same objective. In addition, the methods described here can potentially be employed for rapid functionalization of other solid matrices such as beads, slides, and pins for applications in microarrays or combinatorial chemistry.

Keywords: microwave; nucleosides; loading; LOTUS; solid supports; CPG

     
 
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Table of Contents

  • Basic Protocol 1: Microwave‐Assisted Amination of Controlled‐Pore Glass
  • Basic Protocol 2: Microwave‐Assisted Succinylation of 3‐Aminopropyl‐CPG
  • Basic Protocol 3: Loading of Nucleosides on Succinylated CPG Using LOTUS
  • Alternate Protocol 1: Loading of Nucleosides on Succinylated CPG Using Orbital Shaking
  • Basic Protocol 4: Recovery of Excess Unreacted Nucleoside
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Microwave‐Assisted Amination of Controlled‐Pore Glass

  Materials
  • Native controlled‐pore glass (CPG; 500 Å; Prime Synthesis). Various lots of CPG supplied by the manufacturer have the following range of physical properties:
    • Pore size: 340 to 535 Å
    • Bulk density: 0.25 to 0.35 g/cm3
    • Solid surface area: 101 to 163 m2/g
    • Pore volume: 1.1 cm3/g
    • Particle size: 120 to 200 mesh
    • Loading: 184 µmol/g
  • (3‐Aminopropyl)triethoxysilane (APTES)
  • Toluene, reagent grade
  • Methanol, reagent grade
  • Dichloromethane, reagent grade
  • Hexanes, reagent grade
  • Ninhydrin solution, 0.2% (w/v) in ethanol
  • 500‐mL thick‐walled glass reactor (Spring Bank) fitted with a Teflon screw‐cap stopper and chemically resistant O‐ring (Chemraz)
  • Domestic microwave oven (800 watt, high power setting)
  • Safety shield (3/4‐in. polycarbonate; e.g., VWR)
  • Temperature laser gun (Raytek minitemp)
  • Fritted funnels, medium porosity, small (60 mL) and large (19‐cm height × 16‐cm diameter)
  • Filter flask
  • Vacuum pump
  • Large glass dish (14 × 23 cm; Anchor Hocking Co.)
  • Aluminum foil
  • Master heat gun (Master Appliance Corp.)
  • 8 × 35–mm flat‐bottom sample tubes
  • Vacuum manifold
  • Additional reagents and equipment for amino group estimation by trityl analysis (unit 3.2)
CAUTION: All microwave reactions should be carried out behind a safety shield, and in a specially fabricated thick‐walled glass reactor fitted with a Teflon stopper and chemically resistant O‐ring. The safety shield is used to protect against injuries from minor explosions.

Basic Protocol 2: Microwave‐Assisted Succinylation of 3‐Aminopropyl‐CPG

  Materials
  • 3‐Aminopropyl‐CPG ( S.2; see protocol 1)
  • Succinic anhydride, reagent grade
  • 4‐Dimethylaminopyridine (DMAP), reagent grade
  • N,N‐Dimethylformamide (DMF), reagent grade, water content <15 ppm
  • Methanol, reagent grade
  • Dichloromethane (DCM), reagent grade
  • Hexanes, reagent grade
  • Ethyl acetate (EtOAc), reagent grade
  • 500‐mL thick‐walled glass reactor (Spring Bank) fitted with Teflon screw‐cap stopper and chemically resistant O‐ring (Chemraz)
  • Domestic microwave oven (800 watt, high power setting)
  • Safety shield (3/4‐in. polycarbonate; e.g., VWR)
  • Temperature laser gun (Raytek minitemp)
  • Fritted funnels, medium porosity, small (60 mL) and large (19‐cm height, 16‐cm diameter)
  • Filtration flask
  • 8 × 35–mm flat‐bottom sample tubes
  • Master heat gun (Master Appliance Corp.)
  • Large glass dish (14 × 23 cm; Anchor Hocking Co.)
  • Additional reagents and solutions for carboxyl group estimation (unit 3.2)

Basic Protocol 3: Loading of Nucleosides on Succinylated CPG Using LOTUS

  Materials
  • Succinylated CPG ( S.5; see protocol 2)
  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐N6‐benzoyl‐2′‐deoxyadenosine (DMTr‐dABz) or other 5′‐O‐DMTr‐N‐protected deoxyribonucleoside (Reliable Biopharmaceutical Corporation; ChemGenes)
  • 4‐Dimethylaminopyridine (DMAP), reagent grade
  • Anhydrous N,N‐dimethylformamide (DMF), freshly distilled over CaH 2
  • Argon gas
  • Anhydrous triethylamine (TEA), freshly distilled over CaH 2
  • 1‐[3‐(Dimethylamino)propyl]‐3‐ethylcarbodiimide hydrochloride (EDC), reagent grade
  • Methanol, reagent grade
  • Dichloromethane (DCM), reagent grade
  • Hexanes, reagent grade
  • CAP A and B capping solutions (American International Chemical; ChemGenes)
  • LOTUS reactor workstation (Iyer et al., ; Fig. ; Spring Bank Technologies)
  • Solid addition funnels
  • 1‐L round‐bottom flask
  • Rubber septa and syringe needles
  • Fritted funnels, small size (60 ml), of medium porosity
  • 8 × 35–mm flat‐bottom sample tubes
  • Vacuum manifold
  • Additional reagents and equipment for trityl analysis (unit 3.2)
CAUTION: DMTr‐nucleosides and carbodiimide reagents such as EDC can cause severe skin and eye irritation and allergic reactions. Use appropriate skin and eye protection and wipe all work areas with a wet cloth immediately after use.NOTE: Anhydrous DMF and TEA are prepared by distilling reagent‐grade solvents over calcium hydride and are stored over 4 Å molecular sieves. They may also be purchased from commercial vendors (Aldrich, J.T. Baker). If procured from commercial sources, it is important to use certified solvents that have <5 ppm water.

Alternate Protocol 1: Loading of Nucleosides on Succinylated CPG Using Orbital Shaking

  • 1‐L single‐neck round‐bottom flasks
  • Rubber septum
  • Orbital shaker (e.g., Lab‐Line Orbit Environ‐Shaker)
  • Fritted funnel, large size (19‐cm height × 16‐cm diameter), of medium porosity

Basic Protocol 4: Recovery of Excess Unreacted Nucleoside

  Materials
  • Nucleoside‐containing DMF filtrate (e.g., protocol 3, step )
  • Sodium chloride
  • Chloroform, reagent grade
  • 5% (w/v) aqueous citric acid, reagent grade
  • 5% (w/v) aqueous sodium carbonate, reagent grade
  • Brine (saturated aqueous NaCl)
  • Anhydrous sodium sulfate, reagent grade
  • Separatory funnel with stopper
  • 24‐cm Buchner funnel with Whatman no. 1 filter paper
  • Rotary evaporator
  • Vacuum pump
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Figures

Videos

Literature Cited

Literature Cited
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   Padmanabhan, S., Iyer, R.P. Patent pending.
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