Synthesis of 1-methyl-7-nitroisatoic anhydride (1M7)

Synthetic Chemistry

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Authors: Kady-Ann Steen, Nathan A. Siegfried & Kevin M. Weeks

Abstract

SHAPE (selective 2’-hydroxyl acylation analyzed by primer extension) chemistry is a powerful approach for single-nucleotide resolution analysis of RNA secondary and tertiary structure. SHAPE uses hydroxyl-selective electrophiles to react with and form covalent adducts at the ribose 2’-hydroxyl group. The most useful and robust reagent for routine SHAPE experiments is 1-methyl-7-nitroisatoic anhydride (1M7). This protocol describes a straightforward synthesis of 1M7 from commercially available precursors. The synthesis of 1-methyl-7-nitroisatoic anhydride was originally described by Mortimer et al. . The reaction scheme is:

Fig 1

Table 1

Amounts of each starting material can be varied using the above Table.

Reagents

  1. 4-nitroisatoic anhydride (4NIA) (AstaTech, cat. no. 69441)
  2. Sodium hydride (NaH) – 60% dispersion in mineral oil (Sigma-Aldrich, cat. no. 452912)
  3. Methyl iodide (MeI) (Sigma-Aldrich, cat. no. 289566)
  4. Anhydrous dimethylfluoride (DMF) (Sigma-Aldrich, cat. no. 227056)
  5. HCl (Fisher Scientific, cat. no. A144)
  6. H2O
  7. Ether (Fisher Scientific, cat. no. E138)

REAGENT SETUP

  • H2O and HCl should be ice cold before use.

Equipment

  1. Round bottom flasks (250 and 500 mL)
  2. Bunsen burner
  3. N2 tank and gas line
  4. Teflon-coated stirring bar
  5. Rubber septa
  6. Disposable syringes (1 mL, 20 mL, 60 mL)
  7. Disposable needles (18 gauge)
  8. Stirring plate
  9. Buchner funnel
  10. Vacuum rotary evaporator
  11. Watch glass
  12. Oven maintained at 90 °C

EQUIPMENT SETUP

  • All glassware used in the described reactions are washed with soap and water, rinsed with water, rinsed with acetone, and flame-dried over Bunsen burner. Rubber septa are immediately placed over flasks, flasks are flushed with N2, and allowed to cool.

Procedure

  1. Dissolve 4NIA in 60 mL DMF in a 250 mL flame-dried round bottom flask under N2.
  2. In a separate 500 mL flame-dried round bottom flask under N2, make a slurry of NaH in 15 mL DMF and stir.
  3. Slowly add 4NIA solution dropwise to slurry of NaH in DMF. Stir for 5 minutes.
  4. Slowly add MeI dropwise and then stir at room temperature for 4 hours.
  5. Pour reaction into 100 mL ice cold 1N HCl.
  6. Filter resulting bright orange precipitate by vacuum filtration in a Buchner funnel.
  7. Rinse precipitate twice with ice cold water, followed by 3 rinses with ether.
  8. Dry overnight in oven on watch glass.

Anticipated Results

Analytical data

Yield ~80%; bright orange powdery solid.

1H NMR [400 MHz, CO(CD3)2]: σ 3.69 (s, 3H, -NCH3-), 8.12 (dd, J=8.8 Hz, 2 Hz, 1 H, ArH), 8.2 (d, J=2hz, 1h, ArH), 8.34 (d, J=8.4 Hz, 1 H, ArH).

Associated Publications

Selective 2′-hydroxyl acylation analyzed by protection from exoribonuclease (RNase-detected SHAPE) for direct analysis of covalent adducts and of nucleotide flexibility in RNA. Kady-Ann Steen, Nathan A Siegfried, and Kevin M Weeks. Nature Protocols 6 (11) 1683 - 1694 doi:10.1038/nprot.2011.373

Author information

Kady-Ann Steen, Nathan A. Siegfried & Kevin M. Weeks, Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-3290

Correspondence to: Kevin M. Weeks ([email protected])

Source: Protocol Exchange (2011) doi:10.1038/protex.2011.255. Originally published online 3 November 2011.

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