Synthesis of the DNA-[Ru(tpy)(dppz)(CH3CN)]2+ conjugates and their photo cross-linking studies with the complementary DNA strand

We here report our studies on the conjugation of photoreactive Ru2+ complex to oligonucleoticles (ODNs), which give a stable duplex with the complementary target DNA strand. These functionalized DNA duplexes bearing photoreactive Ru2+ complex can be specifically photolyzed to give the reactive aqua derivative, [Ru(tpy)(dppz)(H2O)](2+)-ODN (tpy = 2,2':6',2"-terpyridine; dppz = dipyrido(3,2-a:2',3'-c]phenazine), in situ, which successfully cross-links to give photoproduct(s) in the duplex form with the target complementary DNA strand. Thus, the stable precursor of the aquaruthenium complex, the monofunctional polypyridyl ruthenium complex [Ru(tpy)(dppz)(CH3CN)](2+), has been site-specifically tethered to ODN, for the first time, by both solid-phase synthesis and postsynthetic modifications. (i) In the first approach, pure 3'-[Ru(tpy)(dppz)(CH3CN)](2+)-ODN conjugate has been obtained in 42% overall yield (from the monomer blocks) by the automated solid-phase synthesis on a support labeled with (Ru(tpy)(dppz)Cl]l complex with subsequent liberation of the crude conjugate from the support under mild conditions and displacement of the Cl- ligand by acetonitrile in the coordination sphere of the Ru2+ label. (ii) In the second approach, the single-modified (3'- or 5'- or middle-modified) or 3',5'-bis-modified Ru2+-ODN conjugates were prepared in 28-50% yield by an amide bond formation between an active ester of the metal complex and the ODNs conjugated with an amino linker. The pure conjugates were characterized unambiguously by ultraviolet-visible (UV-vis) absorption spectroscopy, enzymatic digestion followed by HPLC quantitation, polyacrylamide gel electrophoresis (PAGE), and mass spectrometry (MALDI-TOF as well as by ESI). (Ru(tpy)(dppz)(CH3CN)](2+)-ODNs form highly stabilized ODN(.)DNA duplexes compared to the unlabeled counterpart (DeltaT(m) varies from 8.4 to 23.6 degreesC) as a result of intercalation of the dppz moiety; they undergo clean and selective photodissociation of the CH3CN ligand to give the corresponding aqua complex, (Ru(tpy)(dppz)(H2O)](2+)ODNs (in the aqueous medium), which is evidenced from the change of their UV-vis absorption properties and the detection of the naked Ru2+-ODN ions generated in the course of the matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometric analysis. Thus, when [Ru(tpy)(dppz)(CH3CN)](2+)-ODN conjugate was hybridized to the complementary guanine (G)-rich target strand (T), and photolyzed in a buffer (pH 6.8), the corresponding aqua complex formed in situ immediately reacted with the G residue of the opposite strand, giving the cross-linked product. The highest yield (34%) of the photo cross-linked product obtained was with the ODN carrying two reactive Ru2+ centers at both 3'- and 5'ends. For ODNs carrying only one Ru2+ complex, the yield of the cross-linked adduct in the corresponding duplex is found to decrease in the following order: 3'-Ru2+-ODN (22%) > 5'-Ru2+-ODN (9%) > middle Ru2+-ODN (7%). It was also found that the photo cross-coupling efficiency of the tethered Ru2+ complex with the target T strand decreased as the stabilization of the resulting duplex increased: 3'-Ru2+-ODN ((VIT)-T-.) (DeltaT(m)(b) = 7 degreesC) < 5'-Ru2+-ODN (V.T) (DeltaT(m)(b) = 16 degreesC) < middle-Ru This shows that, with the rigidly packed structure, as in the duplex with middle-Ru2+-ODN, the metal center flexibility is considerably reduced, and consequently the accessibility of target G residue by the aquaruthunium moiety becomes severely restricted, which results in a poor yield in the cross-coupling reaction. The cross-linked product was characterized by PAGE, followed by MALDI-TOF MS.