Synthesis and Application of Nitro-Substituted Nucleosides and Nucleotides

Abstract

Protocols for the synthesis of C⁷-nitropurine nucleosides and nucleotides are described. Each of these nucleotides can completely replace its naturally occurring counterpart in polymerase chain reaction (PCR), generating amplified duplex DNA containing nitropurines. As predicted, the electron-withdrawing power of the nitro group rendered these modified oligonucleotides susceptible to chemoselective cleavage at the modified bases. Treatment with secondary amines at elevated temperatures produced selective DNA scission at nitropurine sites. Interestingly, this Maxam-Gilbert type of DNA cleavage reaction was facilitated by the addition of tris(2-carboxyethyl)phosphine (TCEP), a strong reducing agent and nucleophile. Mass spectrometry analysis not only confirmed that DNA cleavage occurred at the nitropurines, but also revealed that the fragmented DNA products are appended by a TCEP moiety at its 3′-end. This surprising result provided new insights into the mechanisms of such DNA cleavage reactions.

Key words

cleavage - nucleotides - oligonucleotides - nucleophilic additions - regioselectivity

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1

Present address: B.H. Wang, Genzyme Corp., Waltham, MA 02451, USA.

2

Present address: C. R. Allerson, ISIS Pharmaceuticals, Carlsbad, CA 92008, USA.

26

The spectra were recorded by Dr. Jin Hong, 77 Pine Ridge Drive, Ayer, MA 01432, 978-772-6939, [e-mail].