Amidopyrylium-Fluoreszenz-Farbstoffe

The measurement of fluorescence has become a very useful tool in medical and biological
diagnostics as well as in environmental analysis and material sciences due to the high
sensitivity of fluorescence spectroscopy. Many techniques and analytical concepts use
fluorescent dyes for labelling of biological compounds. It is possible to enhance the
sensitivity of detection by using marker dyes that absorb and fluoresce in the red or near
infrared spectral region.
Only few dyes exhibiting intense red fluorescence have been known until recently, e. g.
cyanines, oxazines, carbopyronins and some xanthene derivatives. A new class of fluorescent
dyes for the red region of the visible spectrum is the topic of this thesis. The first synthesis of
the amide-bridged benzopyrylium dyes was published in 1971. A great variety of new dyes
belonging to this class of fluorescent dyes has been prepared. Their spectral properties, i. e.
absorption and fluorescence, have been studied with emphasis on temperature and
environmental (solvent, pH) influences.
The absorption maximum of the amidopyrylium dyes occurs, depending on the molecular
structure, in the region between 590 and 680 nm. A special feature of these dyes is the large
Stokes shift of about 50 nm. From experimental data a natural fluorescence lifetime of about
8 ns can be expected. The quantum yield of fluorescence of the dyes varies, depending on the
molecular structure, between 10 and 50 % at room temperature. The solvent, viscosity and
temperature have a strong influence on the quantum yield.
Amidopyrylium dyes can form a colourless pseudobase in alkaline media. It is possible to
protect the reactive centre by a spacious carboxyphenyl substituent. Such dyes are stable
towards base. Further it was possible to introduce coupling groups, e. g. COOH, without
affecting the optical properties of the dyes. Via an active ester such dyes can be coupled easily
to amino groups of proteins, nucleotides and other analyte molecules and thus can be used as
long-wavelength fluorescent labels.