Estimating the reactivity of 2'-hydroxyl groups along an RNA chain of interest aids in the modeling of the folded RNA structure; flexible loops tend to be reactive, whereas duplex regions are generally not. Among the most useful reagents for probing 2'-hydroxyl reactivity is 1-methyl-7-nitroisatoic anhydride (1m7), but the absence of a reliable, inexpensive source has prevented widespread adoption. An existing protocol for the conversion of an inexpensive precursor 4-nitroisatoic anhydride (4NIA) recommends the use of NaH in dimethylformamide (DMF), a reagent combination that most molecular biology labs are not equipped to handle, and that does not scale safely in any case. Here we describe a safer, one-pot method for bulk conversion of 4NIA to 1m7 that reduces costs and bypasses the use of NaH. We show that 1m7 produced by this method is free of side products and can be used to probe RNA structure in vitro.

Backbone N-methylation is common among peptide natural products and has a substantial impact on both the physical properties and the conformational states of cyclic peptides. However, the specific impact of N-methylation on passive membrane diffusion in cyclic peptides has not been investigated systematically. Here we report a method for the selective, on-resin N-methylation of cyclic peptides to generate compounds with drug-like membrane permeability and oral bioavailability. The selectivity and degree of N-methylation of the cyclic peptide was dependent on backbone stereochemistry, suggesting that conformation dictates the regiochemistry of the N-methylation reaction. The permeabilities of the N-methyl variants were corroborated by computational studies on a 1,024-member virtual library of N-methyl cyclic peptides. One of the most permeable compounds, a cyclic hexapeptide (molecular mass = 755 Da) with three N-methyl groups, showed an oral bioavailability of 28% in rat.