Metallo-phthalocyanine near-IR fluorophores: oligonucleotide conjugates and their applications in PCR assays.

Water soluble, metallo-pthalocyanine (MPc) near-IR fluorophores were designed, synthesized, and evaluated as highly stable and sensitive reporters for fluorescence assays. Their conjugation to oligonucleotides was achieved via succinimidyl ester-amino coupling chemistry with the conditions for conjugation extensively examined and optimized. In addition, various conjugate purification and isolation techniques were evaluated as well. Results showed that under proper conditions and following purification using reverse-phase ion-pair chromatography, labeling efficiencies near 80% could be achieved using ZnPc (Zn phthalocyanine) as the labeling fluorophore. Absorption and fluorescence spectra accumulated for the conjugates indicated that the intrinsic fluorescence properties of the MPc's were not significantly altered by covalent attachment to oligonucleotides. As an example of the utility of MPc reporters, we used the MPc-oligonucleotide conjugates as primers for PCR (polymerase chain reaction) amplifications with the products sorted via electrophoresis and detected using near-IR fluorescence (lambda ex = 680 nm). The MPc dyes were found to be more chemically stable under typical thermal cycling conditions used for PCR compared to the carbocyanine-based near-IR reporter systems typically used and produced single and narrow bands in the electrophoretic traces, indicative of producing a single PCR product during amplification.

Water soluble metallo-phthalocyanines: the role of the functional groups on the spectral and photophysical properties.

Strategies are reported that produce symmetrical metal-free and metallo-phthalocyanine dyes, Pc and MPc, respectively, containing various numbers of water solubilizing carboxylic acid groups on their periphery that provide a dual role by also serving as functional groups to covalently link primary amine-containing targets to these dyes. In order to induce water compatibility and to minimize the degree of aggregation, the periphery of the macrocycle was decorated with various numbers of water-solubilizing groups and/or altering the identity of the metal center. The influence of the number of solubilizing groups and metal center on the spectral and photophysical properties were evaluated. MPc dyes containing 4, 8, or 16 carboxylic acid groups exhibited similar absorption and emission maxima (677 and 686 nm, respectively) with the molar absorptivity of the Q-band approximately 10(5) M(-1) cm(-1). Results indicated that the fluorescence lifetimes and quantum yields varied as a function of the metal center; the degree of carboxylation did not significantly alter these properties in DMSO, but did mediate the solubility and aggregation states when placed in aqueous solvents. The water solubilizing groups could also serve as labeling moieties for targets bearing primary amines. Results showed that the conjugate, produced by covalently linking an MPc to streptavidin through one of its carboxylate groups, generated a red-shift in the emission maximum with a fluorescence lifetime shorter than that of the native MPc dye.