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Oligonucleotide probes labeled with pyrene excimer pairs that form excimers have a number of applications in hybridization methods of 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 robust and reliable synthesis of novel 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 were used for in oligonucleotide solid phase synthesis of molecular beacons (MBs) labeled with one or two pyrenes. We observed significant difference in the excimer emission maxima (475–510 nm; Stokes shifts 125–160 nm) and excimer/monomer ratio (from 0.5 to 5.9) in fluorescence spectra depending on the structure and position of monomer members of in the pyrene pair. The pyrene excimer formed by two more rigid 1-pyrenecarboxamide residues showed the brightest emission that is consistent with molecular dynamics data on the excimer stability. Increase of the excimer fluorescence for MBs after hybridization with DNA was up to 24-fold that exceeds previously described excimer MBs.