Guillaume Bordeau

Synthetic Strategies to Derivatizable Triphenylamines Displaying High Two-Photon Absorption

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.

Vinyl-pyridinium triphenylamines : Novel far-red emitters with high photostability and two-photon absorption properties for staining 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 (Kd ∼μM) for dsDNA. TPIF measurements have shown that TP‐2 py and TP‐3 py each have a large 2PA cross section (δ 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 (λem=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.

Trinaphthylamines as Robust Organic Materials for Two-Photon-Induced Fluorescence

The synthesis of a novel pi-conjugated trinaphthylamines series is described. These original push-pull octupolar systems exhibit large two-photon action cross section (sigma phi up to 510 GM) increased by a factor of 2-3 as compared to their triphenylamines analogues. This substantial improvement of the two-photon absorption properties is attributed to the stronger donor character of the trinaphthyl core.

DNA Switches on the Two-Photon Efficiency of an Ultrabright Triphenylamine Fluorescent Probe Specific of AT Regions

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.

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 540GM 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.

Optimisation of fluorescent DNA labels for two-photon microscopy

We have investigated a series of mono-, bis- and trisvinyl-pyridinium TP derivatives (TP-Py) which exhibit interesting properties : good water solubility, reduced size, high photostability, large two-photon absorption cross-sections (δ up to 700 GM), red emission (λem=660-680nm). Most importantly, TP-Py happen to be poorly fluorescent in water whereas their fluorescence is strongly enhanced when binding to various forms of DNA. We showed that this property originates in the spontaneous immobilisation of the dye inside the double-stranded DNA helix. Due to moderate fluorescence quantum yield efficiencies (η), the brightness of TP-Py appears however to be improvable (ηδ=19). For this purpose, a new generation of specifically engineered TP derivatives have been designed and extensively characterized. We showed that switching from a classical TP core to a more electron-rich trinaphtyl (TN) core enables to increase the molecular brightness although reducing drastically the dye-DNA specific interactions.

New triarylamine-based far-red DNA stainers with high two-photon absorption properties.

A series of red emitting vinyl-triphenyamines (TP) have been synthesized and evaluated for their two photon absorption (2PA) properties. These compounds are virtually non fluorescent in the free state but exhibit a bright red fluorescence upon binding to double-stranded DNA, both in one- and two-photon absorption. This feature allows one- and two-photon confocal imaging in cells of nuclear DNA with an excellent contrast. Derivatizable analogues for covalent bioconjugation to oligonucleotides are described and variation on the structure is discussed.