1,9-Dialkoxyanthracene as a 1O2-Sensitive Linker

We developed a O-1(2)-sensitive linker based on a 9,10-dialkoxyanthracene structure. Its cleavage in the presence of O-1(2) is quick and high-yielding. A phosphoramidite containing this fragment was prepared and coupled to a variety of molecular fragments, including nucleosides, fluorescent dyes, and a cholesteryl derivative. On the basis of this building block we prepared a fluorogenic probe for monitoring O-1(2) in live mammalian cells and visible-light-activated "caged" oligodeoxyribonucleotides. In particular, the fluorogenic O-1(2) probe is a conjugate of 4,7,4',7'-tetrachlorofluorescein and N,N,N',N'-tetramethylrhodamine coupled to each other via the O-1(2)-sensitive linker. Fluorescence of the dyes in this probe is quenched. In the presence of O-1(2), the linker is cleaved with formation of 9,10-anthraquinone and two strongly fluorescent dyes: 4,7,4',7'-tetrachlorofluorescein and N,N,N',N'-tetramethylrhodamine derivatives. We observed that the fluorescence of the probe correlates with the amount of O-1(2) present in solution. The red-light-activated "caged" oligodeoxyribonucleotides are stable duplexes, which consist of an unmodified strand and a blocker strand. The O-1(2)-sensitive linker is introduced in the interior of the blocker strand. Upon exposure of the duplex to red light in the presence of In3+(pyropheophorbide-a) chloride, the linker is cleaved with formation of the unstable duplex structure. This product decomposes spontaneously, releasing the unmodified strand, which can bind to the complementary target nucleic acid. This uncaging reaction is high-yielding. In contrast, previously reported visible-light-activated reagents are uncaged inefficiently due to competing reactions of sulfoxide and disulfide formation.