Lanthanide(III)-macrocyclic complexes as catalysts for RNA cleavage and para-cest agents
Five mononuclear europium(III) complexes of 1,4,7-tris(carbamoylmethyl)- 1,4,7,10-tetrazacyclododecane (1) , 1,4,7-tris[(N-ethyl)carbamoylmethyl]-1,4,7,10- tetraazacyclododecane (2) , 1,4,7-tris[(N-N-diethyl)carbamoylmethyl]-1,4,7,10- tetraazacyclododecane (3) , 1-methyl-,4,7,10-tris(carbamoylmethyl)-1,4,7,10- tetraazacyclododecane (8) , 1-[3'-(N,N-diethylaminomethyl)benzyl]-4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (9) and two novel dinuclear europium(III) complexes of 1,3-bis[1-(4,7,10-tris(carbamoylmethyl)-1,4,7,10- tetraazacyclododecane]- m -xylene (10) , 1,4-bis[1-(4,7,10-tris(carbamoylmethyl)-1,4,7,10- tetraazacyclododecane]-p-xylene (11) have been synthesized. Europium(III) excitation luminescence spectroscopy as a function of pH shows a single major peak for the 7 F 0 [arrow right] 5 D 0 transition from pH 5 to 9, except for Eu (9) , indicating that a single species exists under these conditions. Time resolved luminescence data show that each Eu 3+ ion has two bound waters, consistent with a nine coordinate complex (Eu( L )(H 2 O) 2 for L = ligand). All complexes promote cleavage of the RNA model compound 2-hydroxypropyl-4-nitrophenyl phosphate (HpPNP) at 25°C ( I = 0.10 M NaNO 3 , 20 mM buffer). The Eu(III) complexes show a linear dependence of second-order rate constants for the cleavage of 4-nitrophenyl-2-hydroxyethylphosphate (HpPNP) on pH in the range 6.5 to 8.0 for Eu (3) , 7.0 to 8.5 for Eu (2) and 7.0 to 9.0 for Eu(1), Eu (8) , Eu (10) and Eu (11) . Only one pH (7.0) was studied for Eu (9) due to insolubility of the complex at higher pH. This pH-rate profile is consistent with the Eu(III) complex-substrate complex being converted to the active form by loss of a proton and with Eu(III) water p K a values that are higher than 8.0 for Eu (3) , 8.5 for Eu (2) and 9.0 for Eu (1) , Eu (8) , Eu (10) and Eu (11) . The second order rate constants of the dinuclear Eu(III) complex for the cleavage of HpPNP at pH 7 are more than two hundred fold higher than that of Eu (1) but only 7-fold higher than a monomeric complex with an aryl pendent group Eu (9) . This shows that the aryl pendent group enhances catalysis in both mononuclear and dinuclear complexes. Eu 2 (10) promotes cleavage of a dinucleoside, uridylyl(3 ́,5 ́)uridine (UpU) with a second-order rate constant at pH 7.6 (0.021 M -1 s -1 ) that is 46-fold higher than the mononuclear Eu (1) complex. The strength of binding of the Eu(III) complexes to the methylphosphate dianion correlates roughly to catalytic properties: strongest binding is observed for the most active Eu(III) complexes that contain an aryl pendent group. Inhibition studies show that Eu (1) , Eu (8) , Eu 2 (10) and Eu 2 (11) bind strongly to the dianionic ligand methylphosphate ( K d = 0.28, 0.18, 0.060, 0.084 mM), and more weakly to diethylphosphate ( K d = 7.5, 2.7, 0.43, 1.9 mM), consistent with a catalytic role of the Eu(III) complexes in stabilizing the developing negative charge on the phosphorane transition state. The two Eu(III) centers in Eu 2 (10) are only weakly cooperative as shown by comparison of rate constants for cleavage of phosphate esters and by binding of the two Eu(III) centers to a bridging methylphosphate. A dinuclear Nd(III) complex of ligand 11 whose exchangeable amide protons resonate 14-15 ppm downfield from bulk water protons has been studied as a paramagnetic chemical exchange saturation transfer (PARACEST) agent. Upon saturation, at the resonance frequency of the amide protons, the complex produces 15% decrease in bulk water signal intensity at 5 mM concentration and room temperature. The CEST spectra have an optimal pH of 7.5. Interactions of the complex with DNA but not phosphate diesters gives rise to an increase in the CEST signal. A pronounced CEST effect is observed upon binding of the Nd(III) complex to a DNA hairpin and is sensitive to the structure of the DNA hairpin.