Immobilization procedures, intended
to enable prolonged observation
of single molecules by fluorescence microscopy, may generate heterogeneous
microenvironments, thus inducing heterogeneity in the molecular behavior.
On that account, we propose a straightforward surface preparation
procedure for studying chemical reactions on the single-molecule level.
Sensor fluorophores were developed, which exhibit dual-emissive characteristics
in a homogeneously catalyzed showcase reaction. These molecules undergo
a shift of fluorescence wavelength of about 100 nm upon Pd(0)-induced
deallylation in the Tsuji–Trost reaction, allowing for separate
visualization of the starting material and product. Whereas a simultaneous
immobilization of dye and inert silane leads to strongly polydisperse
reaction kinetics, a consecutive immobilization routine with deposition
of dye molecules as the last step provides substrates underlying the
kinetics of ensemble experiments. Also, the found kinetics are unaffected
by the chemical variation of inert silanes, nearly uniform, and therefore
well reproducible. Additional parameters like photostability, signal-to-noise
ratio, dye-molecule density, and spatial distribution of dye molecules
are, as well, hardly affected by surface modification in the successive
immobilization scheme.
Electrophilic monofluorination with Selectfluor and nucleophilic trifluoromethylation with the Ruppert-Prakesh reagent of dimethyl-, tetramethyl- and pentamethyl-substituted boron dipyrromethenes (BODIPY) are investigated. Monofluorinated dyes are synthesized with low yields (<30%), however trifluoromethyl derivatives are obtained in moderate to high yields (≈40-90%). All compounds are characterized by steady-state and time-resolved fluorescence spectroscopy, the photostability is investigated with fluorescence correlation spectroscopy (FCS) and total internal reflection fluorescence microscopy (TIRF). Monofluorination hardly affects the spectroscopic parameters of the unsubstituted parent compounds, but distinctly enhances the photostability, whereas trifluoromethylation leads to a hypsochromic shift by up to 17 nm in both absorption and emission, slightly enhanced intersystem crossing, and higher photostability. Further development of soft fluorination and trifluoromethylation methods is therefore highly desired.
Single-molecule (SM) chemistry is devoted to unravel reaction steps which are hidden in cuvette experiments. Controversies about the substrate activation during the Tsuji-Trost deallylation motivated us to study, on the single-molecule level, the kinetics of the catalyst precursor Pd(PPh 3 ) 4 with our recently designed two-color fluorescent probes. Photochemical, metal-free bypass reactions were found and taken into account by the combina-tion of spectrally separated single-molecule TIRF-microscopy and state-of-the art analysis procedures. Unselective π-complex formation (K D � 10 3 M À 1 ) precedes the insertion of the active catalyst into the CÀ OR bond (RO À = leaving group), indicated by the lacking immediate change of fluorescence color. The formed intermediate then decomposes on a time scale of � 2 -3 s to the deallylated product.
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