Pyrrole‐Imidazole Polyamides: Manual Solid‐Phase Synthesis

Steven M. Pauff1, Andrew J. Fallows2, Simon P. Mackay3, Wu Su4, Paul M. Cullis2, Glenn A. Burley1

1 Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, 2 Department of Chemistry, University of Leicester, Leicester, 3 Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, 4 Guangdong Key Laboratory of Nanomedicine, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 8.10
DOI:  10.1002/0471142700.nc0810s63
Online Posting Date:  December, 2015
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Abstract

Pyrrole‐imidazole polyamides (PAs) are a family of DNA‐binding peptides that bind in the minor groove of double‐stranded DNA (dsDNA) in a sequence‐selective, programmable fashion. This protocol describes a detailed manual procedure for the solid‐phase synthesis of this family of compounds. The protocol entails solution‐phase synthesis of the Boc‐protected pyrrole (Py) and imidazole (Im) carboxylic acid building blocks. This unit also describes the importance of choosing the appropriate condensing agent to form the amide linkages between each building block. Finally, a monomeric coupling protocol and a fragment‐based approach are described that delivers PAs in 13% to 30% yield in 8 days. © 2015 by John Wiley & Sons, Inc.

Keywords: pyrrole‐imidazole polyamide; DNA; minor groove; gene expression; solid‐phase synthesis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Boc‐Protected Pyrrole Carboxylic Acid Used for Boc‐Assisted Manual Solid‐Phase Peptide Synthesis
  • Basic Protocol 2: Synthesis of Boc‐Protected Imidazole Carboxylic Acid Used for Boc‐Assisted Manual Solid‐Phase Peptide Synthesis
  • Basic Protocol 3: Manual Solid‐Phase Synthesis of Pyrrole‐Imidazole Polyamides Via Monomeric Coupling of N‐Boc‐Protected Pyrrole and Imidazole Carboxylic Acid Units
  • Basic Protocol 4: Convergent Synthesis of Pyrrole‐Imidazole Polyamides
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of Boc‐Protected Pyrrole Carboxylic Acid Used for Boc‐Assisted Manual Solid‐Phase Peptide Synthesis

  Materials
  • Methyl 1‐methyl‐4‐nitro‐1H‐pyrrole‐2‐carboxylate (9)
  • Methanol, HPLC‐grade
  • Boc anhydride (Boc 2O)
  • Nitrogen gas (N 2)
  • Palladium on carbon, 10% by weight (Pd/C)
  • Hydrogen gas (H 2)
  • Filter agent (Celite)
  • Methanol, puriss p.a. (MeOH)
  • n‐Hexane
  • 1 M lithium hydroxide solution (LiOH)
  • 1 M hydrochloric acid solution (HCl)
  • Ethyl acetate
  • Magnesium sulfate
  • 250‐mL reaction vessel for Parr hydrogenator (Parr shaker vessel)
  • Medium pressure Parr shaker apparatus
  • 500‐ and 1000‐mL Erlenmeyer flasks
  • Büchner funnel
  • Filter paper
  • 250‐ and 500‐mL round‐bottom flasks
  • Rotary evaporator
  • Vacuum pump
  • Rubber septa
  • Magnetic stir bar
  • Oil bath
  • Hot plate with magnetic stirrer
  • pH paper
  • 500‐mL separatory funnel

Basic Protocol 2: Synthesis of Boc‐Protected Imidazole Carboxylic Acid Used for Boc‐Assisted Manual Solid‐Phase Peptide Synthesis

  Materials
  • N‐Methylimidazole
  • Triethylamine
  • Acetonitrile
  • Ethyl chloroformate
  • Chloroform
  • Brine
  • Magnesium sulfate
  • Ethyl acetate
  • Silica gel
  • Deuterated dimethyl sulfoxide (DMSO‐d 6)
  • Concentrated sulfuric acid (H 2SO 4)
  • Fuming nitric acid (HNO 3)
  • Dichloromethane
  • Saturated sodium bicarbonate (NaHCO 3)
  • n‐Hexane
  • Methanol, anhydrous
  • Boc anhydride (Boc 2O)
  • Nitrogen gas (N 2)
  • Palladium on carbon, 10% by weight (Pd/C)
  • Hydrogen gas (H 2)
  • Ninhydrin solution (see recipe)
  • Filter agent (Celite)
  • Ethanol
  • 1 M lithium hydroxide solution (LiOH)
  • 4 M hydrochloric acid solution (HCl)
  • pH 3 water (see recipe)
  • Liquid nitrogen
  • Syringes
  • 150‐, 250‐, and 500‐mL round‐bottom flasks
  • Magnetic stir bars
  • Hot plate with magnetic stirrer
  • Pressure equalizing dropping funnel
  • Dewar (for ice bath)
  • Rubber septa
  • Büchner funnels
  • Filter paper
  • 250‐mL, 1‐liter, and 2‐liter separatory funnels
  • 250‐mL, 500‐mL, 1‐liter, and 2‐liter Erlenmeyer flasks
  • Reflux condenser
  • Oil bath
  • 500‐ and 1000‐mL beakers
  • Rotary evaporator
  • Vacuum pump
  • Thermometer
  • 250‐mL reaction vessel for Parr hydrogenator (Parr shaker vessel)
  • Medium pressure Parr shaker apparatus
  • TLC plates
  • 3.8 × 15.2–cm columns
  • Graduated cylinder
  • pH paper
  • 50‐mL centrifuge tubes (Falcon)
  • Centrifuge
  • Sonicator
  • Freeze dryer vessels
  • Lyophilizer

Basic Protocol 3: Manual Solid‐Phase Synthesis of Pyrrole‐Imidazole Polyamides Via Monomeric Coupling of N‐Boc‐Protected Pyrrole and Imidazole Carboxylic Acid Units

  Materials
  • Boc‐β‐alanine bound to PAM resin (Boc‐β‐Ala‐PAM resin, Peptides International, cat. no. RPA‐1451‐PI)
  • N,N‐Dimethylformamide (DMF), peptide synthesis grade
  • Dichloromethane (DCM), peptide synthesis grade
  • TPW solution: 92.5% TFA, 5% phenol, 2.5% water (see recipe)
  • N,N‐Dimethylformamide, anhydrous, amine‐free (DMF anh, Alfa Aesar, cat. no. 43465)
  • Nitrogen gas (N 2)
  • 4‐(tert‐Butoxycarbonylamino)‐1‐methyl‐1H‐pyrrole‐2‐carboxylic acid (Boc‐Py‐OH, 1; see protocol 1)
  • 1‐[Bis(dimethylamino)methylene]‐1H‐1,2,3‐triazolo[4,5‐b]pyridinium 3‐oxid hexafluorophosphate (HATU)
  • N,N‐Diisopropylethylamine (DIPEA)
  • 4‐(tert‐Butoxycarbonylamino)butyric acid (Boc‐GABA‐OH, 3)
  • Dimethylaminopropylamine (DMAPA, 6)
  • Diethyl ether
  • Methanol, puriss p.a. (MeOH)
  • Methanol, HPLC grade
  • 4‐(tert‐Butoxycarbonylamino)‐1‐methyl‐1H‐imidazole‐2‐carboxylic acid (Boc‐Im‐OH, 2, see protocol 2)
  • 1‐Hydroxy‐7‐azabenzotriazole (HOAt)
  • N,N'‐dicyclohexylcarbodiimide (DCC)
  • Filter agent (Celite)
  • Bis(trichloromethyl) carbonate (BTC)
  • Tetrahydrofuran, anhydrous (THF anh)
  • 2,3,5‐Collidine
  • 1‐Methyl‐1H‐imidazole‐2‐carboxylic acid (Im‐OH, 4)
  • HPLC solvent A: 0.1% TFA in water (see recipe)
  • HPLC solvent B: 0.1% TFA in acetonitrile (see recipe)
  • Solid‐phase synthesis vessel
  • Manual solid‐phase synthesizer (PLS Organic Synthesizer, Activotec)
  • Rubber septa
  • 500‐mL round‐bottom flask
  • Vacuum oil pump
  • Pasteur pipets
  • Reaction vials
  • 1‐ and 2‐mL syringes
  • 1.5‐ and 2‐mL microcentrifuge tubes (Eppendorf)
  • Heated shaker (Eppendorf thermomixer)
  • Microcentrifuge
  • Glass wool
  • SpeedVac evaporator
  • 15‐ and 50‐mL centrifuge tubes (Falcon)
  • Centrifuge
  • Analytical HPLC (Ultimate 3000, Dionex)
  • Reversed‐phase HPLC column (C18, 4.6 × 150–mm, Phenomenex, cat. no. 00 G‐4454‐E0)

Basic Protocol 4: Convergent Synthesis of Pyrrole‐Imidazole Polyamides

  Materials
  • 4‐(tert‐Butoxycarbonylamino)‐1‐methyl‐1H‐imidazole‐2‐carboxylic acid (Boc‐Im‐OH, 2; prepared in protocol 2)
  • Ethanol
  • Distilled water
  • Cesium carbonate
  • Nitrogen gas (N 2)
  • Dimethylformamide, anhydrous (DMF dry)
  • Benzyl bromide
  • n‐Hexane, cold
  • Deuterated chloroform (CDCl 3)
  • 4 M hydrogen chloride in dioxane (4 M HCl in dioxane)
  • Liquid nitrogen
  • Deuterated methanol (CD 3OD)
  • 4‐(tert‐Butoxycarbonylamino)‐1‐methyl‐1H‐pyrrole‐2‐carboxylic acid (Boc‐Py‐OH, 1; prepared in protocol 1)
  • N,N‐Dimethylformamide, peptide synthesis grade (DMF)
  • N,N'‐dicyclohexylcarbodiimide (DCC)
  • 1‐Hydroxy‐7‐azabenzotriazole (HOAt)
  • N,N‐Diisopropylethylamine (DIPEA)
  • pH 3 water (see recipe)
  • Ethyl acetate
  • Deuterated dimethyl sulfoxide (DMSO‐d 6)
  • 1‐[Bis(dimethylamino)methylene]‐1H‐1,2,3‐triazolo[4,5‐b]pyridinium 3‐oxid hexafluorophosphate (HATU)
  • 1‐Methyl‐1H‐imidazole‐2‐carboxylic acid (Im‐OH, 4)
  • Methanol, anhydrous
  • Palladium hydroxide on carbon, 20% weight (Pd(OH 2)/C)
  • 1 M hydrochloric acid (HCl)
  • Hydrogen gas (H 2)
  • Filter agent (Celite)
  • N,N‐Dimethylformamide, anhydrous, amine‐free (DMF anh, Alfa Aesar, cat. no. 43465)
  • Boc‐GABA‐Py‐Py‐Py‐Py‐β‐Ala‐PAM (19; prepared in protocol 3)
  • TPW solution: 92.5% TFA, 5% phenol, 2.5% water (see recipe)
  • Dichloromethane, peptide synthesis grade (DCM)
  • Dimethylaminopropylamine (DMAPA)
  • Diethyl ether, cold
  • HPLC solvent A: 0.1% TFA in water (see recipe)
  • HPLC solvent B: 0.1% TFA in acetonitrile (see recipe)
  • 5‐, 10‐, and 25‐mL round‐bottom flasks
  • Pasteur pipets
  • Magnetic stir bars
  • Hot plate with magnetic stirrer
  • Rotary evaporator
  • Vacuum oil pump
  • Rubber septa
  • Syringes
  • Needles
  • Oil bath
  • 15‐ and 50‐mL tubes (Falcon)
  • Centrifuge
  • Freeze dryer vessels
  • Lyophilizer
  • Glass wool
  • Microwave
  • Glass column
  • Reaction vessel for Parr hydrogenator
  • Medium pressure Parr shaker apparatus
  • Büchner funnel
  • Filter paper
  • 1.5‐ and 2‐mL tubes (Eppendorf)
  • Heated shaker
  • Solid‐phase synthesis vessel
  • Manual solid‐phase synthesizer (PLS Organic Synthesizer; Activotec)
  • Analytical HPLC (Ultimate 3000, Dionex)
  • Reversed‐phase HPLC Column (C18, 4.6 × 150–mm, Phenomenex, cat. no. 00 G‐4454‐E0)
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Figures

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Literature Cited

Literature Cited
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  Krpetić, Z., Singh, I., Su, W., Guerrini, L., Faulds, K., Burley, G.A., and Graham, D. 2012. Directed assembly of DNA‐functionalized gold nanoparticles using pyrrole‐imidazole polyamides. J. Am. Chem. Soc. 134:8356‐8359. doi: 10.1021/ja3014924.
  Meier, J.L., Yu, A.S., Korf, I., Segal, D.J., and Dervan, P.B. 2012. Guiding the design of synthetic DNA‐binding molecules with massively parallel sequencing. J. Am. Chem. Soc. 134:17814‐17822. doi: 10.1021/ja308888c.
  Nickols, N.G., Jacobs, C.S., Farkas, M.E., and Dervan, P.B. 2007. Improved nuclear localization of DNA‐binding polyamides. Nucleic Acids Res. 35:363‐370. doi: 10.1093/nar/gkl1042.
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  Singh, I., Wendeln, C., Clark, A.W., Cooper, J.M., Ravoo, B.J., and Burley, G.A. 2013. Sequence‐selective detection of double‐stranded DNA sequences using pyrrole‐imidazole polyamide microarrays. J. Am. Chem. Soc. 135:3449‐3457. doi: 10.1021/ja309677h.
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Key References
  Fallows, A.J., Singh, I., Dondi, R., Cullis, P.M., and Burley, G.A. 2014. Highly efficient synthesis of DNA‐binding polyamides using a convergent fragment‐based approach. Org. Lett. 16:4654‐4657.
  This work outlines the development of the polyamide sythesis strategy described in .
  Su, W., Gray, S.J., Dondi, R., and Burley, G.A. 2009. Highly efficient synthesis of DNA‐binding hairpin polyamides via the use of a new triphosgene coupling strategy. Org. Lett. 11:3910‐3913.
  This work introdudes bis(trichloromethyl) carbonate (BTC) as an efficient reagent for solid phase synthesis and details the benefits of BTC over more commonly employed carboxylic acid activating agents.
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