SYNTHESIS AND PROPERTIES OF AN OLIGODEOXYNUCLEOTIDE MODIFIED WITH A PYRENE DERIVATIVE AT THE 5'-PHOSPHATE

The synthesis of an oligonucleotide (ODN) modified with pyrene (pyr) on the 5'-phosphate is described. The ODN and pyrene are joined through a linker composed of four methylene groups. Modification of the oligonucleotide was effected via condensation of the 2-cyanoethyl NN-diisopropylphosphoramidite of 4-(1-pyrenyl)butanol (Pyr-m4OPAm, 2) with the 5'-OH of an ODN. This derivative is suitable for incorporation into automated solid-phase DNA synthesis and was attached to the 5' terminus of the DNA chain through a phosphodiester linkage. The properties of the 5'-(pyr-m4)d(T)15 (3) and the duplex it formed with d(A)15 were investigated by fluorescence and absorbance spectroscopy. The pyrene fluorescence in the modified duplex was quenched 96.3 % relative to an identical concentration of free 4-(1-pyrenyl)butanol. The ultraviolet spectrum of the 5'-(pyr-M4)-d(T)15 and 5'-(pyr-m4)-d(T)15.d-(A)15 modified duplex, in the 320-360-nm region, was red-shifted 6 nm relative to the free 4-(1-pyrenyl)-butanol. The T(m) values of the unmodified and modified duplexes at 0.1 M NaCl were 34.9 and 41.9-degrees-C, respectively. The pyrene-induced stabilization corresponds to a free energy change (DELTADELTAG-degrees) of -2.6 kcal/mol.