Fine Tuning of Pyrene Excimer Fluorescence in Molecular Beacons by Alteration of the Monomer Structure

Oligonucleotide
probes labeled with pyrene pairs that form excimers
have a number of applications in hybridization analysis of nucleic
acids. A long excited state lifetime, large Stokes shift, and chemical
stability make pyrene excimer an attractive fluorescent label. Here
we report synthesis of chiral phosphoramidite building blocks based
on (R)-4-amino-2,2-dimethylbutane-1,3-diol, easily
available from an inexpensive d-(−)-pantolactone.
1-Pyreneacetamide, 1-pyrenecarboxamide, and DABCYL derivatives have
been used in preparation of molecular beacon (MB) probes labeled with
one or two pyrenes/quenchers. We observed significant difference in
the excimer emission maxima (475–510 nm; Stokes shifts 125–160
nm or 7520–8960 cm–1) and excimer/monomer
ratio (from 0.5 to 5.9) in fluorescence spectra depending on the structure
and position of monomers in the pyrene pair. The pyrene excimer formed
by two rigid 1-pyrenecarboxamide residues showed the brightest emission.
This is consistent with molecular dynamics data on excimer stability.
Increase of the excimer fluorescence for MBs after hybridization with
DNA was up to 24-fold.