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

doi:10.1038/protex.2011.255

Method Article

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

https://doi.org/10.1038/protex.2011.255

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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:

Reagents	MW \(g/mol)	Amount \(g)	Amount \(mL)	Moles \(mmol)	ratio
4NIA	208.13	5.000		24.023	1
NaH	24	0.990		24.744	1.03
MeI	141.94		1.499	24.023	1

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

Synthetic chemistry

1-methyl-7-nitroisatoic anhydride

SHAPE

• 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.

• 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.

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.

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).

No Competing Financial Interests

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

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

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Abstract

Figures

Reagents

Equipment

Procedure

Anticipated Results

Additional Declarations

Associated Publications

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