We report on the design and synthesis of two-photon fluorescent triphenylamines bearing two or three vinyl branches terminated by a N-methyl benzimidazolium moiety. The new compounds (TP-2Bzim, TP-3Bzim) are light-up fluorescent DNA probes with a long wavelength emission (>580 nm). Compared to their pyridinium models, the TP-Bzim dyes exhibit a remarkable improvement of both their DNA affinity and fluorescence quantum yield, especially for the two-branch derivative (TP-2Bzim: ΦF = 0.54, Ka = 10(7) M(-1)), resulting in a large fluorescence emission turn-on ratio of up to 140. Concomitantly, the two-photon absorption cross-section of TP-2Bzim is dramatically enhanced upon DNA binding (δ = 1080 vs 110 GM for the free form). This effect of the DNA matrix on the nonlinear absorption is uncovered for the first time. This is attributed to a tight fit of the molecule inside the minor groove of AT-rich DNA which induces geometrical rearrangements in the dye ground state as supported by circular dichroism and molecular modeling data. Consequently, TP-2bzim displays an exceptional two-photon molecular brightness (δ×ΦF = 583 GM), a value unrivalled for a small biofluorophore. These properties enable to image nuclear DNA in fixed cells at submicromolar concentration ([TP-2Bzim] = 100 nM) and to visualize ultrabright foci of centromeric AT-rich chromatin. Finally TP-2Bzim exhibits a high photostability, is live-cell permeant, and does not require RNase treatment. This outstanding combination of optical and biological properties makes TP-2Bzim a bioprobe surpassing the best DNA stainers and paves the way for studying further nonlinear optical processes in DNA.

A series of mono-, bis- and trisvinyl-pyridinium triphenylamines (TP-py) has been synthesised and evaluated for its one- and two-photon absorption (2PA) induced-fluorescence properties under biological conditions. Interestingly, these compounds are only weakly fluorescent in water, whereas their fluorescence emissions are strongly restored (exaltation factors of 20-100) upon binding to double-stranded DNA. Additional measurements in glycerol indicate that the fluorescence increases are the result of immobilisation of the dyes in the DNA matrix, which inhibits rotational de-excitation modes. This particular feature is especially remarkable in the case of the bis and tris derivatives (TP-2 py, TP-3 py), which each display a high affinity (K(d) ~ microM) for dsDNA. TPIF measurements have shown that TP-2 py and TP-3 py each have a large 2PA cross section (delta up to 700 GM) both in glycerol and in the presence of DNA, which ranks them amongst the best 2PA biological fluorophores. Finally, one- and two-photon confocal imaging in cells revealed that these compounds perform red staining (lambda(em)=660-680 nm) of nuclear DNA with excellent contrast. The remarkable optical properties of the TP-py series, combined with their high photostability and their easy synthetic access, make these compounds extremely attractive for use in confocal and 2PA microscopy.

A versatile synthetic strategy to access a set of highly fluorescent pi-conjugated triphenylamines bearing a functional linker at various positions on one phenyl ring is described. These compounds were designed for large two-photon absorption (2PA) and in particular for labeling of biomolecules. The monoderivatized trisformylated or trisiodinated intermediates described herein allow introduction of a large variety of electron-withdrawing groups required for large 2PA as well as a panel of chemical functions suitable for coupling to biomolecules. The monoderivatized three-branched compounds and in particular the benzothiazole (TP-3Bz) series show remarkable linear (high extinction coefficients and high quantum yield) and nonlinear (high 2-photon cross sections) optical properties. Interestingly the presence of functional side chains does not disturb the two-photon absorption. Finally, monoderivatized two-branched derivatives also appear to be valuable candidates. Altogether the good optical properties of the new derivatizable pi-conjugated TPA combined with their small size and their compatibility with bioconjugation protocols suggest that they represent a new chemical class of labels potentially applicable for the tracking of biomolecules using two-photon scanning microscopy.