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

protocol.md

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:

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

Reagents

1. 4-nitroisatoic anhydride (4NIA) (AstaTech, cat. no. 69441)

• Sodium hydride (NaH) – 60% dispersion in mineral oil (Sigma-Aldrich, cat. no. 452912)
• Methyl iodide (MeI) (Sigma-Aldrich, cat. no. 289566)
• Anhydrous dimethylfluoride (DMF) (Sigma-Aldrich, cat. no. 227056)
• HCl (Fisher Scientific, cat. no. A144)
• H2O
• 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)

• Bunsen burner
• N2 tank and gas line
• Teflon-coated stirring bar
• Rubber septa
• Disposable syringes (1 mL, 20 mL, 60 mL)
• Disposable needles (18 gauge)
• Stirring plate
• Buchner funnel
• Vacuum rotary evaporator
• Watch glass
• 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.

• In a separate 500 mL flame-dried round bottom flask under N2, make a slurry of NaH in 15 mL DMF and stir.
• Slowly add 4NIA solution dropwise to slurry of NaH in DMF. Stir for 5 minutes.
• Slowly add MeI dropwise and then stir at room temperature for 4 hours.
• Pour reaction into 100 mL ice cold 1N HCl.
• Filter resulting bright orange precipitate by vacuum filtration in a Buchner funnel.
• Rinse precipitate twice with ice cold water, followed by 3 rinses with ether.
• 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 (weeks@unc.edu)

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