Cyanine 5 Phosphoramidite - CAS 351186-76-0

Cyanine dyes represent a family of organic fluorophores with widespread utility in biological-based applications ranging from real-time PCR probes to protein labeling. One burgeoning use currently being explored with indodicarbocyanine (Cy5) in particular is that of accessing exciton delocalization in designer DNA dye aggregate structures for potential development of light-harvesting devices and room-temperature quantum computers. Tuning the hydrophilicity/hydrophobicity of Cy5 dyes in such DNA structures should influence the strength of their excitonic coupling; however, the requisite commercial Cy5 derivatives available for direct incorporation into DNA are nonexistent. Here, we prepare a series of Cy5 derivatives that possess different 5,5'-substituents and detail their incorporation into a set of DNA sequences. In addition to varying dye hydrophobicity/hydrophilicity, the 5,5'-substituents, including hexyloxy, triethyleneglycol monomethyl ether, tert-butyl, and chloro groups were chosen so as to vary the inherent electron-donating/withdrawing character while also tuning their resulting absorption and emission properties. Following the synthesis of parent dyes, one of their pendant alkyl chains was functionalized with a monomethoxytrityl protective group with the remaining hydroxyl-terminated N-propyl linker permitting rapid, same-day phosphoramidite conversion and direct internal DNA incorporation into nascent oligonucleotides with moderate to good yields using a 1 μmole scale automated DNA synthesis. Labeled sequences were cleaved from the controlled pore glass matrix, purified by HPLC, and their photophysical properties were characterized. The DNA-labeled Cy5 derivatives displayed spectroscopic properties that paralleled the parent dyes, with either no change or an increase in fluorescence quantum yield depending upon sequence.

5-Fluorouridine (1a) was converted to its N(3)-farnesylated nucleoterpene derivative 8 by direct alkylation with farnesyl bromide (4). Reaction of the cancerostatic 1a with either acetone, heptan-4-one, nonadecan-10-one, or hentriacontan-16-one afforded the 2',3'-O-ketals 2a-2d. Compound 2b was then first farnesylated (→5) and subsequently phosphitylated to give the phosphoramidite 6. The ketal 2c was directly 5'-phosphitylated without farnesylation of the base to give the phosphoramidite 7. Moreover, the recently prepared cyclic 2',3'-O-ketal 11 was 5'-phosphitylated to yield the phosphoramidite 12. The 2',3'-O-isopropylidene derivative 2a proved to be too labile to be converted to a phosphoramidite. All novel derivatives of 1a were unequivocally characterized by NMR and UV spectroscopy and ESI mass spectrometry, as well as by elemental analyses. The lipophilicity of the phosphoramidite precursors were characterized by both their retention times in RP-18 HPLC and by calculated log P values. The phosphoramidites 6, 7, and 12 were exemplarily used for the preparation of four terminally lipophilized oligodeoxynucleotides carrying a cyanine-3 or a cyanine-5 residue at the 5'-(n-1) position (i.e., 14-17). Their incorporation in an artificial lipid bilayer was studied by single-molecule fluorescence spectroscopy and fluorescence microscopy.

A series of six cyanine-5-labeled oligonucleotides (LONs 10-15), each terminally lipophilized with different nucleolipid head groups, were synthesized using the recently prepared phosphoramidites 4b-9b. The insertion of the LONs within an artificial lipid bilayer, composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), was studied by single molecule fluorescence spectroscopy and microscopy with the help of an optically transparent microfluidic sample carrier with perfusion capabilities. The incorporation of the lipo-oligonucleotides into the bilayer was studied with respect to efficiency (maximal bilayer brightness) as well as stability against perfusion (final stable bilayer brightness). Attempts to correlate these parameters with the log P values of the corresponding nucleolipid head groups failed, a result which clearly demonstrates that not only the lipophilicity but mainly the chemical structure and topology of the head group is of decisive importance for the optimal interaction of a lipo-oligonucleotide with an artificial lipid bilayer. Moreover, fluorescence half-live and diffusion time values were measured to determine the diffusion coefficients of the lipo-oligonucleotides.