Amine-reactive fluorene probes: synthesis, optical characterization, bioconjugation, and two-photon fluorescence imaging

Bioconjug Chem. 2008 Dec;19(12):2559-67. doi: 10.1021/bc800415t.


With the increasing demand for confocal and two-photon fluorescence imaging, the availability of reactive probes that possess high two-photon absorptivity, high fluorescence quantum yield, and high photostability is of paramount importance. To address the demand for better-performing probes, we prepared two-photon absorbing amine-reactive fluorenyl-based probes 2-(9,9-bis(2-(2-methoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)benzothiazole (1) and 2-(4-(2-(9,9-bis(2-(2-ethoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)vinyl)phenyl)benzothiazole (2), incorporating the isothiocyanate as a reactive linker. Probe design was augmented by integrating high optical nonlinearities, increased hydrophilicity, and coupling with reactive functional groups for specific targeting of biomolecules, assuring a better impact on two-photon fluorescence microscopy (2PFM) imaging. The isothiocyanate (NCS) derivatives were conjugated with cyclic peptide RGDfK and Reelin protein. The study of the chemical and photophysical properties of the new labeling reagents, as well as the conjugates, is described. The conjugates displayed high chemical stability and photostability. The NCS derivatives had low fluorescence quantum yields, while their bioconjugates exhibited high fluorescence quantum yields, essentially "lighting up" after conjugation. Conventional and 2PFM imaging and fluorescence lifetime imaging (FLIM) of HeLa, NT2, and H1299 cells, incubated with two-photon absorbing amine-reactive probe (1), RGDfK-dye conjugate (7), and Reelin-dye conjugate (6), was demonstrated.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Absorption
  • Amines / chemistry*
  • Butylamines / chemistry
  • Cell Adhesion Molecules, Neuronal / chemistry
  • Cell Adhesion Molecules, Neuronal / metabolism
  • Cell Line
  • Electrons
  • Extracellular Matrix Proteins / chemistry
  • Extracellular Matrix Proteins / metabolism
  • Fluorenes / chemistry*
  • Fluorescent Dyes / chemical synthesis*
  • Fluorescent Dyes / chemistry
  • Fluorescent Dyes / metabolism*
  • Humans
  • Indicators and Reagents / chemistry
  • Isothiocyanates / chemistry
  • Microscopy, Fluorescence
  • Nerve Tissue Proteins / chemistry
  • Nerve Tissue Proteins / metabolism
  • Peptides, Cyclic / chemistry
  • Peptides, Cyclic / metabolism
  • Photons*
  • Reelin Protein
  • Serine Endopeptidases / chemistry
  • Serine Endopeptidases / metabolism
  • Spectrum Analysis


  • Amines
  • Butylamines
  • Cell Adhesion Molecules, Neuronal
  • Extracellular Matrix Proteins
  • Fluorenes
  • Fluorescent Dyes
  • Indicators and Reagents
  • Isothiocyanates
  • Nerve Tissue Proteins
  • Peptides, Cyclic
  • Reelin Protein
  • cyclic arginine-glycine-aspartic acid peptide
  • isothiocyanic acid
  • fluorene
  • RELN protein, human
  • Serine Endopeptidases