Pyrrole‐Imidazole Polyamides: Automated Solid‐Phase Synthesis

Lijing Fang1, Zhengyin Pan1, Paul M. Cullis2, Glenn A. Burley3, Wu Su1

1 Guangdong Key Laboratory of Nanomedicine, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong, 2 Department of Chemistry, University of Leicester, Leicester, 3 Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 8.11
DOI:  10.1002/0471142700.nc0811s63
Online Posting Date:  December, 2015
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Abstract

In this unit, the fully automated solid‐phase synthetic strategy of hairpin Py‐Im polyamides is described using triphosgene (BTC) as a coupling agent. This automated methodology is compatible with all the typical building blocks, enabling the facile synthesis of polyamide libraries in 9% to 20% yield in 3 days. © 2015 by John Wiley & Sons, Inc.

Keywords: Py‐Im polyamide; DNA; minor groove; automated synthesis; solid‐phase synthesis; triphosgene

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Automated Synthesis of Py‐Im Polyamides Using Boc Chemistry
  • Alternate Protocol 1: Automated Synthesis of Py‐Im Polyamides Using Fmoc‐Chemistry
  • Support Protocol 1: Preparation of Boc‐Py‐Hydrazine Resin and Fmoc‐Py Hydrazine Resin
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Automated Synthesis of Py‐Im Polyamides Using Boc Chemistry

  Materials
  • Boc‐Py‐hydrazine resin (prepared manually from Boc‐Py‐OH and Fmoc‐hydrazinobenzoyl AM resin, see protocol 3Support Protocol)
  • Dichloromethane (DCM, anhydrous)
  • Trifluoroacetic acid (TFA, anhydrous)
  • 92.5:5:2.5 (v/v/v) TFA/phenol/H 2O (see recipe)
  • N,N‐dimethylformamide (DMF; anhydrous, Sigma‐Aldrich)
  • 10% (v/v) N,N′‐diisopropylethylamine (DIEA) in dry DMF (see recipe)
  • Tetrahydrofuran (THF; anhydrous, Sigma‐Aldrich)
  • 4‐[(tert‐butoxycarbonyl)amino]‐1‐methyl‐1H‐pyrrole‐2‐carboxylic acid (Boc‐Py‐OH, J&K Scientific)
  • Triphosgene (BTC; Aladdin, cat. no. T103041)
  • Nitrogen gas (N 2)
  • 15% 2,4,6‐collidine in dry THF (see recipe)
  • 4‐tert‐butoxycarbonylamino‐1‐methyl‐1H‐imidazole‐2‐carboxylic acid (Boc‐Im‐OH, J&K Scientific)
  • 1‐Hydroxy‐7‐aza‐benzotriazole (HOAt; Aladdin, cat. no. H109328)
  • Fmoc‐(N‐γ‐Boc)‐D‐α, γ‐diaminobutyric acid (Fmoc‐D‐Dab(Boc)‐OH, Alfa Aesar)
  • Im‐CCl 3 (prepared according to Masiukiewicza et al., )
  • 1‐methyl‐1H‐imidazole‐2‐carboxylic acid (Im‐OH, Sigma‐Aldrich)
  • Benzotriazol‐1‐yl‐oxytripyrrolidino‐phosphonium hexafluorophosphate (PyBOP; Aladdin, cat. no. P109336)
  • 20% piperidine in DMF (see recipe)
  • Di‐tert‐butyl dicarbonate (Boc 2O; Aladdin, cat. no. D106159)
  • 3,3′‐diamino‐N‐methyl‐dipropylamine (Aladdin, cat. no. B105353)
  • Methanol (MeOH; HPLC‐grade, J&K Scientific)
  • Diethyl ether (Et 2O, anhydrous)
  • Acetonitrile (MeCN; HPLC‐grade, J&K Scientific)
  • CS Bio 336X peptide synthesizer (http://www.csbio.com)
  • Air compressor
  • Disposable syringe filter (Nylon 66, 0.22 μm; Jinteng)
  • ULTIMAT 3000 Instrument (Dionex)
  • ACE C18 column (10 × 250 mm, 5 μm, 300 Å)
  • Photodiode array detector capable of measuring absorbance
  • Lyophilizer and freeze dryer vessels
  • Centrifuge
  • Shimadzu LC 20 with UV detector SPD‐20 A
  • Inertsil ODS‐SP column (4.6 × 250 mm, 5 μm, 100 Å)
  • Additional reagents and equipment for deprotection procedure ( protocol 2Alternate Protocol) and HPLC (appendix 3b)
NOTE: To increase the solubility in THF, fine powders of Boc‐Im‐OH are obtained by dispersion of the commercial reagent (5 g) in H 2O (300 mL) via ultrasonication at 40°C followed by lyophilization. All other commercial reagents are used as received.

Alternate Protocol 1: Automated Synthesis of Py‐Im Polyamides Using Fmoc‐Chemistry

  Additional Materials (also see protocol 1Basic Protocol)
  • 4‐(Fmoc‐amino)‐1‐methyl‐1H‐imidazole‐2‐carboxylic acid (Fmoc‐Im‐OH, Atomax Chemicals)
  • Fmoc‐4‐amino‐1‐methylpyrrole‐2‐carboxylic acid (Fmoc‐Py‐OH, Atomax Chemicals)
  • 4‐(Fmoc‐amino)butyric acid (Fmoc‐GABA‐OH; Aladdin, cat. no. F122242)
  • Dimethylaminopropylamine (Aladdin, cat. no. D110909)
  • Additional reagents and equipment for monomer activation ( protocol 1Basic Protocol)
NOTE: To increase the solubility in THF, powders of Fmoc‐Py‐OH and Fmoc‐Im‐OH are obtained by dissolving the commercial reagents (2 g) in THF (300 mL), removing the solvents under reduced pressure, then drying in a vacuum oven at 50°C overnight.

Support Protocol 1: Preparation of Boc‐Py‐Hydrazine Resin and Fmoc‐Py Hydrazine Resin

  Additional Materials (also see protocol 1Basic Protocol and protocol 2Alternate Protocol)
  • Fmoc‐hydrazinobenzoyl AM resin (Novabiochem)
  • Acetic anhydride (Ac 2O)
  • 1‐[Bis(dimethylamino)methylene]‐1H‐1,2,3‐triazolo[4,5‐b]pyridinium 3‐oxid hexafluorophosphate (HATU; Aladdin, cat. no. H109327)
  • 2,4,6‐collidine (Aladdin, cat. no. T108942)
  • N,N′‐diisopropylethylamine (DIEA)
  • Solid‐phase synthesis vessel
  • 5‐mL reaction vials (wholesale 5‐mL clear glass vials with orifice reducers and plastic caps; Yanzhou United)
  • Stirring bar and magnetic stirrer
  • Rubber septa
  • Round bottom flask (500 mL)
  • Oil‐type vacuum pump
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Figures

Videos

Literature Cited

Literature Cited
  Blackledge, M.S. and Melander, C. 2013. Programmable DNA‐binding small molecules. Bioorg. Med. Chem. 21:6101‐6114. doi: 10.1016/j.bmc.2013.04.023.
  Falb, E., Yechezkel, T., Salitra, Y., and Gilon, C. 1999. In situ generation of Fmoc‐amino acid chlorides using bis‐(trichloromethyl) carbonate and its utilization for difficult couplings in solid‐phase peptide synthesis. J. Pept. Res. 53:507‐517. doi: 10.1034/j.1399-3011.1999.00049.x.
  Fang, L., Wu, C., Yu, Z., Shang, P., Cheng, Y., Peng, Y., and Su, W. 2014. Triphosgene‐mediated couplings in the solid phase: Total synthesis of Brachystemin A. Eur. J. Org. Chem. 34:7572‐7576. doi: 10.1002/ejoc.201403145.
  Fang, L., Yao, G., Pan, Z., Wu, C., Wang, H., Burley, G.A., and Su, W. 2015. Fully automated synthesis of DNA‐binding Py‐Im polyamides using a triphosgene coupling strategy. Org. Lett. 17:158‐161. doi: 10.1021/ol503388a.
  Masiukiewicza, E., Mrugala, D., and Rzeszotarska, B. 2005. An improved synthesis of 1‐methyl‐2‐trichloroacetylimidazole. Org. Prep. Proced. Int.: New J. Organ. Synth. 37:403‐405. doi: 10.1080/00304940509354973.
  Muzikar, K.A., Nickols, N.G., and Dervan, P.B. 2009. Repression of DNA‐binding dependent glucocorticoid receptor‐mediated gene expression. Proc. Natl. Acad. Sci. U.S.A. 106:16598‐16603. doi: 10.1073/pnas.0909192106.
  Nickols, N.G. and Dervan, P.B. 2007. Suppression of androgen receptor–mediated gene expression by a sequence‐specific DNA‐binding polyamide. Proc. Natl. Acad. Sci. U.S.A. 104:10418‐10423. doi: 10.1073/pnas.0704217104.
  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. doi: 10.1021/ol9015139.
  Thern, B., Rudolph, J., and Jung, G. 2002. Total synthesis of the nematicidal cyclododecapeptide omphalotin A by using racemization‐free triphosgene‐mediated couplings in the solid phase. Angew. Chem., Int. Ed. 41:2307‐2309. doi: 10.1002/1521-3773(20020703)41:13%3c2307::AID-ANIE2307%3e3.0.CO;2-Y.
  Yang, F., Nickols, N.G., Li, B.C., Marinov, G.K., Said, J.W., and Dervan, P.B. 2013. Antitumor activity of a pyrrole‐imidazole polyamide. Proc. Natl. Acad. Sci. U.S.A. 110:1863‐1868. doi: 10.1073/pnas.1222035110.
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