N-phenyl-carbazole-based two-photon fluorescent probes: Strong sequence dependence of the duplex vs quadruplex selectivity

Herein we report on the synthesis and DNA recognition properties of a series of three N-phenyl carbazole-based light-up probes initially designed for two-photon absorption. The vinylic derivatives (Cbz-2Py, Cbz-3Py) display strong fluorescence enhancement when bound to various duplex- and quadruplex-forming oligonucleotides whereas the oxazole derivative is not fluorescent in DNA. Determination of affinity constants by fluorimetric titrations evidenced that Cbz-2Py has a clear preference for AT-rich duplex structures. Circular Dichroism (CD) measurements confirmed the sequence-dependent binding of this compound and suggest insertion in the minor groove as shown by a strong induced CD (ICD) signal and further supported by molecular modeling. Altogether the data indicate that duplex vs quadruplex selectivity of the dyes is strongly dependent on the sequence of the duplex. Finally, the dyes exhibit high two-photon absorption cross-sections (up to 540 GM in glycerol) and allow a fine and bright staining of nuclear DNA with low background fluorescence as shown by one and two-photon confocal microscopy imaging of fixed cells.

► N-phenylcarbazole derivatives as two-photon excited fluorescent DNA probes.
► High two-photon absorption cross sections and quantum yields.
► Interaction with DNA was studied by fluorimetric titration and circular dichroism.
► Compounds preferentially bind to the minor groove of AT-rich duplexes.
► Selective and bright staining of nuclear DNA visualized by confocal microscopy.