Synthesis of Dimeric 2‐Amino‐1,8‐Naphthyridine and Related DNA‐Binding Molecules

Kazuhiko Nakatani1, Hanping He1, Shin‐nosuke Uno1, Tsuyoshi Yamamoto1, Chikara Dohno1

1 Osaka University, Ibaraki, Japan
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 8.6
DOI:  10.1002/0471142700.nc0806s32
Online Posting Date:  March, 2008
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The synthetic protocols for dimeric 2‐amino‐1,8‐naphthyridine and related compounds are described in this unit. These compounds represent a novel class of compounds that bind selectively to mismatched base pairs. The compounds consist of two main components: the heterocycles and a linker. Connecting two heterocycles by a linker was found to modulate the binding selectivity. This unit describes the synthesis of dimeric 2‐amino‐1,8‐naphthyridine (which binds to the G‐G mismatch), naphthyridine‐azaquinolone (for the G‐A mismatch), N‐alkyl naphthyridine dimer (for the C‐C mismatch), and naphthyridine carbamate dimer (for G‐G mismatches in the (CGG)n trinucleotide repeat). Protocols for connecting the short linker to these molecules providing the compounds suitable for immobilization on solid surface are also described. Curr. Protoc. Nucleic Acid Chem. 32:8.6.1‐8.6.21. © 2008 by John Wiley & Sons, Inc.

Keywords: naphthyridine; DNA binding; mismatch; linker; azaquinolone

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Dimeric 2‐Amino‐1,8‐Naphythyridine
  • Basic Protocol 2: Preparation of Naphthyridine‐Azaquinolone
  • Basic Protocol 3: Preparation of N‐Alkylnaphthyridine Dimer
  • Basic Protocol 4: Preparation of Naphthyridine‐Carbamate Dimer
  • Basic Protocol 5: Preparation of Naphthyridine Dimer and Related Compounds with Extra Tethers
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Preparation of Dimeric 2‐Amino‐1,8‐Naphythyridine

  Materials
  • Acetyl chloride, 98% pure
  • Methanol (MeOH, analytical grade)
  • 3,3′‐Iminobis(propionitrile), 90% (technical grade, Aldrich)
  • Celite (Wako)
  • Chloroform (CHCl 3, analytical grade)
  • Triethylamine
  • Di‐tert‐butyl dicarbonate, 99% pure (Wako)
  • Potassium hydrogen sulfate (KHSO 4), aqueous, saturated
  • Sodium bicarbonate, (NaHCO 3), aqueous, saturated
  • Sodium chloride (NaCl), aqueous, saturated
  • Magnesium sulfate (MgCl 2), anhydrous
  • Tetrahydrofuran (THF, analytical grade)
  • 2 M sodium hydroxide (NaOH)
  • Amberlite IR‐120 PLUS(H) ion‐exchange resin (Wako)
  • N,N‐Dimethylformamide (DMF, analytical grade)
  • N‐Hydroxysuccinimide (Wako)
  • 1‐(3‐Dimethylaminopropyl)‐3‐ethylcarbodiimide hydrochloride (EDCI; available from TCI)
  • Ammonium chloride (NH 4Cl), aqueous, saturated
  • 2‐Amino‐7‐methyl‐1,8‐naphthyridine (Brown, )
  • Silica gel (Wakogel C‐200, 75 to 150 µm; Wako)
  • Ethyl acetate (EtOAc, analytical grade)
  • 4 M HCl in EtOAc (Kokusan Chemical)
  • 28% (v/v) ammonia
  • Drying tube
  • Dropping funnel
  • Rotary evaporator connected to diaphragm pump
  • Separatory funnel
  • Vacuum oil pump (to remove traces of solvent after evaporative concentration with diaphragm pump)
  • 1 × 40‐cm glass chromatography column
  • TLC plates: silica‐coated glass plates with fluorescent indicator (Merck silica gel 60 F 254; also see appendix 3D)
  • 254‐nm UV lamp
  • Additional reagents and equipment for column chromatography ( appendix 3E) and thin‐layer chromatography ( appendix 3D)
NOTE: All reactions are performed at room temperature unless specified otherwise.

Basic Protocol 2: Preparation of Naphthyridine‐Azaquinolone

  Materials
  • N‐(tert‐butoxycarbonyl)imino‐3,3′‐bis(propionic acid) (S.3; see protocol 1, step 20)
  • N,N‐Dimethylformamide (DMF, analytical grade)
  • Pentafluorophenol (Wako)
  • 1‐(3‐Dimethylaminopropyl)‐3‐ethylcarbodiimide hydrochloride (EDCI)
  • Chloroform (CHCl 3, analytical grade)
  • Sodium chloride (NaCl), aqueous, saturated
  • Magnesium sulfate (MgSO 4), anhydrous
  • Silica gel (Wakogel C‐200, 75 to 150 µm; Wako)
  • Hexane
  • 2‐Amino‐7‐methyl‐1,8‐naphthyridine (Brown, )
  • Diisopropylethylamine
  • Ethyl acetate (EtOAc, analytical grade)
  • Methanol (MeOH, analytical grade)
  • 40% (v/v) sulfuric acid (H 2SO 4)
  • Sodium nitrite
  • 28% (v/v) ammonia
  • N‐Bromosuccinimide (NBS)
  • Carbon tetrachloride (CCl 4, analytical grade)
  • N,NAzobisisobutyronitrile (AIBN; Wako)
  • Liquid ammonia
  • 4 M HCl in EtOAc (Kokusan Chemical)
  • Rotary evaporator connecting to diaphragm pump
  • Vacuum oil pump (to remove traces of solvent after evaporative concentration with diaphragm pump)
  • 4.8 × 20–cm, 4 × 20–cm, 4 × 15–cm, and 2 × 20–cm glass chromatography columns
  • TLC plates: silica‐coated glass plates with fluorescent indicator (Merck silica gel 60 F 254; also see appendix 3D)
  • 254‐nm UV lamp
  • Additional reagents and equipment for column chromatography ( appendix 3E) and thin‐layer chromatography ( appendix 3D)
NOTE: All reactions are performed at room temperature unless specified otherwise.

Basic Protocol 3: Preparation of N‐Alkylnaphthyridine Dimer

  Materials
  • 2‐Chloro‐7‐methyl‐1,8‐naphthyridine (Brown, )
  • 1,3‐Diaminopropane (Wako)
  • Chloroform (CHCl 3, analytical grade)
  • 1 M NaOH
  • Sodium chloride (NaCl), aqueous, saturated
  • Magnesium sulfate (MgSO 4), anhydrous
  • Methanol (MeOH)
  • 3‐Aminopropionealdehyde diethyl acetal (available from TCI)
  • Sodium bicarbonate, (NaHCO 3), aqueous, saturated
  • 80% (v/v) Acetic acid (AcOH)
  • Triethylamine
  • Sodium cyanoborohydride (Aldrich)
  • 4 M HCl in EtOAc (Kokusan chemical)
  • Separatory funnels
  • Recycling preparative HPLC system (gel permeation chromatography system): Japan Analytical Industry Corp. (http://www.jai.co.jp) model LC‐928
  • Gel permeation chromatography (GPC) column (JAIGEL GS310, mol. wt. exclusion limit, 40,000, 50 cm long, 2 cm diameter): Japan Analytical Industry Corp. (http://www.jai.co.jp)
  • Rotary evaporator connecting to diaphragm pump
  • Vacuum oil pump (to remove traces of solvent after evaporative concentration with diaphragm pump)
  • TLC plates: silica‐coated glass plates with fluorescent indicator (Merck silica gel 60 F 254)
  • 254‐nm UV lamp
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D)
NOTE: All reactions are performed at room temperature unless specified otherwise.

Basic Protocol 4: Preparation of Naphthyridine‐Carbamate Dimer

  Materials
  • 3‐Amino‐1‐propanol, 98% pure (Wako)
  • 3‐Chloro‐1‐propanol, 97% pure (Wako)
  • Acetonitrile (CH 3CN, analytical grade)
  • Chloroform (CHCl 3)
  • Di‐tert‐butoxycarbonate
  • Triethylamine
  • Sodium bicarbonate (NaHCO 3), aqueous, saturated
  • Sodium chloride (NaCl), aqueous, saturated
  • Magnesium sulfate (MgSO 4), anhydrous
  • Silica gel (Wakogel C‐200, 75 to 150 µm; Wako)
  • Methanol (MeOH, analytical)
  • N,N′‐Disuccinimidyl carbonate (Wako)
  • Ethyl acetate (EtOAc, analytical grade)
  • Dichloromethane (CH 2Cl 2, analytical grade)
  • 2‐Amino‐7‐methyl‐1,8‐naphthyridine (Brown, )
  • 4 M HCl in EtOAc
  • Separatory funnels
  • Rotary evaporator connected to diaphragm pump
  • Vacuum oil pump (to remove traces of solvent after evaporative concentration with diaphragm pump)
  • 2 × 40–cm and 1 × 20–cm glass chromatography columns
  • Additional reagents and equipment for column chromatography ( appendix 3E) and thin‐layer chromatography ( appendix 3D)
NOTE: All reactions are performed at room temperature unless specified otherwise.

Basic Protocol 5: Preparation of Naphthyridine Dimer and Related Compounds with Extra Tethers

  Materials
  • 4‐Aminobutanoic acid
  • 2 M NaOH
  • Tert‐butyl alcohol
  • Di‐tert‐butyl dicarbonate (Wako)
  • Citric acid solution, saturated
  • Chloroform (CHCl 3, analytical grade)
  • Sodium chloride (NaCl), aqueous, saturated
  • Magnesium chloride (MgSO 4)
  • N,N‐Dimethylformamide (DMF)
  • Pentafluorophenol (Wako)
  • 1‐(3‐Dimethylaminopropyl)‐3‐ethylcarbodiimide hydrochloride (EDCI; available from TCI)
  • Toluene
  • Sodium bicarbonate (NaHCO 3), aqueous, saturated
  • Sodium chloride (NaCl), aqueous, saturated
  • Magnesium sulfate (MgSO 4), anhydrous
  • Silica gel (Wakogel C‐200, 75 to 150 µm; Wako)
  • Hexane
  • Tetrahydrofuran (THF, analytical grade)
  • 3‐Aminopropionealdehyde diethyl acetal (available from TCI)
  • Ethyl acetate (EtOAc, analytical grade)
  • 60% (v/v) acetic acid
  • Sodium carbonate (Na 2CO 3), aqueous, saturated
  • Methanol (MeOH), dry
  • 0.1 M NaOH, methanolic
  • Sodium cyanoborohydride (Aldrich)
  • Separatory funnels
  • Rotary evaporator connected to diaphragm pump
  • Vacuum oil pump (to remove traces of solvent after evaporative concentration with diaphragm pump)
  • 2 × 40–cm and 1 × 20–cm glass chromatography columns
  • Additional reagents and equipment for column chromatography ( appendix 3E) and thin‐layer chromatography ( appendix 3D)
NOTE: All reactions are performed at room temperature unless specified otherwise.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Brown, E.V. 1964. 1,8‐Naphthyridines. I. Derivatives of 2‐ and 4‐methyl‐1,8‐naphthyridines. J. Org. Chem. 30: 1607‐1610.
   Hagihara, S., Kumasawa, H., Goto, Y., Hayashi, G., Kobori, A., Saito, I., and Nakatani, K. 2004. Detection of guanine‐adenine mismatches by surface plasmon resonance sensor carrying naphthyridine‐azaquinolone hybrid on the surface. Nucleic Acids Res. 32: 278‐286.
   Kobori, A., Horie, S., Suda, H., Saito, I., and Nakatani, K. 2004. The SPR sensor detecting cytosine‐cytosine mismatches. J. Am. Chem. Soc. 126: 557‐562.
   Nakatani, K., Sando, S., Kumasawa, H., Kikuchi, J., and Saito, I. 2001a. Recognition of guanine‐guanine mismatches by the dimeric form of 2‐amino‐1,8‐naphthyridine. J. Am. Chem. Soc. 123: 12650‐12657.
   Nakatani, K., Sando, S., and Saito, I. 2001b. Scanning of guanine‐guanine mismatches in DNA by synthetic ligands using surface plasmon resonance. Nat. Biotechnol. 19: 51‐55.
   Nakatani, K., Hagihara, S., Goto, Y., Kobori, A., Hagihara, M., Hayashi, G., Kyo, M., Nomura, M., Mishima, M., and Kojima, C. 2005. Small‐molecule ligand induces nucleotide flipping in (CAG)n trinucleotide repeats. Nat. Chem. Biol. 1: 39‐43.
   Peng, T. and Nakatani, K. 2005. Binding of naphthyridine carbamate dimer to the (CGG)n repeat results in the disruption of the G‐C base pairing. Angew. Chem. Int. Ed. 44: 7280‐7283.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library