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Fluorogenic dendrons with multiple donor chromophores as bright genetically targeted and activated probes.

ABSTRACT: We have developed a class of dendron-based fluorogenic dyes (termed dyedrons) comprised of multiple cyanine (Cy3) donors coupled to a single malachite green (MG) acceptor that fluoresce only when the MG is noncovalently but specifically bound to a cognate single chain antibody (scFv). These cell-impermeant dyedrons exploit efficient intramolecular energy transfer from Cy3 donors to stoichiometrically amplify the fluorescence of MG chromophores that are activated by binding to the scFv. These chromophore enhancements, coupled with our optimized scFv, can significantly increase fluorescence emission generated by the dyedron/scFv complex to brightness levels several-fold greater than that for single fluorescent proteins and targeted small molecule fluorophores. Efficient intramolecular quenching of free dyedrons enables sensitive homogeneous (no wash) detection under typical tissue culture conditions, with undetectable nonspecific activation.

SUBMITTER: Szent-Gyorgyi C

PROVIDER: S-EPMC2920033 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Fluorogenic dendrons with multiple donor chromophores as bright genetically targeted and activated probes.

Szent-Gyorgyi Christopher C Schmidt Brigitte F BF Fitzpatrick James A J JA Bruchez Marcel P MP

Journal of the American Chemical Society 20100801 32

We have developed a class of dendron-based fluorogenic dyes (termed dyedrons) comprised of multiple cyanine (Cy3) donors coupled to a single malachite green (MG) acceptor that fluoresce only when the MG is noncovalently but specifically bound to a cognate single chain antibody (scFv). These cell-impermeant dyedrons exploit efficient intramolecular energy transfer from Cy3 donors to stoichiometrically amplify the fluorescence of MG chromophores that are activated by binding to the scFv. These chr ...[more]

PMID: 20698676

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Project description:Octopolar D2-symmetric chromophores, based on the MPZnM supermolecular motif in which (porphinato)zinc(II) (PZn) and ruthenium(II) polypyridyl (M) structural units are connected via ethyne linkages, were synthesized. These structures take advantage of electron-rich meso-arylporphyrin or electron-poor meso-(perfluoroalkyl)porphyrin macrocycles, unsubstituted terpyridyl and 4'-pyrrolidinyl-2,2';6',2″-terpyridyl ligands, and modulation of metal(II) polypyridyl-to-(porphinato)zinc connectivity, to probe how electronic and geometric factors impact the measured hyperpolarizability. Transient absorption spectra obtained at early time delays (tdelay < 400 fs) demonstrate fast excited-state relaxation, and formation of a highly polarized T1 excited state; the T1 states of these chromophores display expansive, intense T1 → T n absorption manifolds that dominate the 800-1200 nm region of the NIR, long (μs) triplet-state lifetimes, and unusually large NIR excited absorptive extinction coefficients [ε(T1 → T n ) ∼ 105 M-1 cm-1]. Dynamic hyperpolarizability (βλ) values were determined from hyper-Rayleigh light scattering (HRS) measurements, carried out at multiple incident irradiation wavelengths spanning the 800-1500 nm spectral domain. The measured βHRS value (4600 ± 1200 × 10-30 esu) for one of these complexes, RuPZnRu, is the largest yet reported for any chromophore at a 1500 nm irradiation wavelength, highlighting that appropriate engineering of strong electronic coupling between multiple charge-transfer oscillators provides a critical design strategy to realize octopolar NLO chromophores exhibiting large βHRS values at telecom-relevant wavelengths. Generalized Thomas-Kuhn sum (TKS) rules were utilized to compute the effective excited-state-to-excited-state transition dipole moments from experimental linear-absorption spectra; these data were then utilized to compute hyperpolarizabilities as a function of frequency, that include two- and three-state contributions for both β zzz and β xzx tensor components to the RuPZnRu hyperpolarizability spectrum. This analysis predicts that the β zzz and β xzx tensor contributions to the RuPZnRu hyperpolarizability spectrum maximize near 1550 nm, in agreement with experimental data. The TKS analysis suggests that relative to analogous dipolar chromophores, octopolar supermolecules will be likely characterized by more intricate dependences of the measured hyperpolarizability upon irradiation wavelength due to the interactions among multiple different β tensor components.

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Fluorogenic dendrons with multiple donor chromophores as bright genetically targeted and activated probes.

Publications

Fluorogenic dendrons with multiple donor chromophores as bright genetically targeted and activated probes.

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