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Four high-spin Fe(III) macrocyclic complexes, including three dinuclear and one mononuclear complex, were prepared toward the development of more effective iron-based magnetic resonance imaging (MRI) contrast agents. All four complexes contain a 1,4,7-triazacyclononane macrocyclic backbone with two hydroxypropyl pendant groups, an ancillary aryl or biphenyl group, and a coordination site for a water ligand. The pH potentiometric titrations support one or two deprotonations of the complexes, most likely deprotonation of hydroxypropyl groups at near-neutral pH. Variable-temperature 17 O NMR studies suggest that the inner-sphere water ligand is slow to exchange with bulk water on the NMR time scale. Water proton T 1 relaxation times measured for solutions of the Fe(III) complexes at pH 7.2 showed that the dinuclear complexes have a 2- to 3-fold increase in r 1 relaxivity in comparison to the mononuclear complex per molecule at field strengths ranging from 1.4 T to 9.4 T. The most effective agent, a dinuclear complex with macrocycles linked through para-substitution of an aryl group (Fe 2 (PARA)), has an r 1 of 6.7 mM -1 s -1 at 37 °C and 4.7 T or 3.3 mM -1 s -1 per iron center in the presence of serum albumin and shows enhanced blood pool and kidney contrast in mice MRI studies.

Four high-spin macrocyclic Co(II) complexes with hydroxypropyl or amide pendants and appended coumarin or carbostyril fluorophores were prepared as CEST (chemical exchange saturation transfer) MRI probes. The complexes were studied in solution as paramagnetic CEST (paraCEST) agents and after loading into Saccharomyces cerevisiae yeast cells as cell-based CEST (cellCEST) agents. The fluorophores attached to the complexes through an amide linkage imparted an unusual pH dependence to the paraCEST properties of all four complexes through of ionization of a group that was attributed to the amide NH linker. The furthest shifted CEST peak for the hydroxypropyl-based complexes changed by ∼90 ppm upon increasing the pH from 5 to 7.5. At acidic pH, the Co(II) complexes exhibited three to four CEST peaks with the most highly shifted CEST peak at 200 ppm. The complexes demonstrated substantial paramagnetic water proton shifts which is a requirement for the development of cellCEST agents. The large shift in the proton resonance was attributed to an inner-sphere water at neutral pH, as shown by variable temperature 17 O NMR spectroscopy studies. Labeling of yeast with one of these paraCEST agents was optimized with fluorescence microscopy and validated by using ICP mass spectrometry quantitation of cobalt. A weak asymmetry in the Z-spectra was observed in the yeast labeled with a Co(II) complex, toward a cellCEST effect, although the Co(II) complexes were toxic to the cells at the concentrations necessary for observation of cellCEST.

Three paramagnetic Co II macrocyclic complexes containing 2-hydroxypropyl pendant groups, 1,1',1'',1'''-(1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrayl)tetrakis- (propan-2-ol) ([Co(L1)] 2+ , 1,1'-(4,11-dibenzyl-1,4,8,11-tetraazacyclotetradecane-1,8-diyl)bis(propan-2-ol) ([Co(L2)] 2+ ), and 1,1'-(4,11-dibenzyl-1,4,8,11-tetraazacyclotetradecane-1,8-diyl)bis(octadecan-2-ol) ([Co(L3)] 2+ ) were synthesized to prepare transition metal liposomal chemical exchange saturation transfer (lipoCEST) agents. In solution, ([Co(L1)] 2+ ) forms two isomers as shown by 1 H NMR spectroscopy. X-ray crystallographic studies show one isomer with 1,8-pendants in cis-configuration and a second isomer with 1,4-pendants in trans-configuration. The [Co(L2)] 2+ complex has 1,8-pendants in a cis-configuration. Remarkably, the paramagnetic-induced shift of water 1 H NMR resonances in the presence of the [Co(L1)] 2+ complex is as large as that observed for one of the most effective Ln III water proton shift agents. Incorporation of [Co(L1)] 2+ into the liposome aqueous core, followed by dialysis against a solution of 300 mOsm L -1 produces a CEST peak at 3.5 ppm. Incorporation of the amphiphilic [Co(L3)] 2+ complex into the liposome bilayer produces a more highly shifted CEST peak at -13 ppm. Taken together, these data demonstrate the feasibility of preparing Co II lipoCEST agents., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Yeast-derived β-glucan particles (GPs) are a class of microcarriers under development for the delivery of drugs and imaging agents to immune-system cells for theranostic approaches. However, the encapsulation of hydrophilic imaging agents in the porous GPs is challenging. Here, we show that the unique coordination chemistry of Fe III -based macrocyclic T 1 MRI contrast agents permits facile encapsulation in GPs. Remarkably, GPs labeled with the simple Fe III complexes are stable under physiologically relevant conditions, despite the absence of amphiphilic groups. In contrast to the free Fe III coordination complex, the labeled Fe III -GPs have lowered T 1 relaxivity and act as a silenced form of the contrast agent. Addition of a fluorescent tag to the Fe III complex produces a bimodal agent to further enable tracking of the nanoparticles and to monitor release. Treatment of the iron-labeled GPs with a maltol chelator or with mildly acidic conditions releases the intact iron complex and restores enhanced T 1 relaxation of the water protons., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Early studies suggested that Fe III complexes cannot compete with Gd III complexes as T 1 MRI contrast agents. Now it is shown that one member of a class of high-spin macrocyclic Fe III complexes produces more intense contrast in mice kidneys and liver at 30 minutes post-injection than does a commercially used Gd III agent and also produces similar T 1 relaxivity in serum phantoms at 4.7 T and 37 °C. Comparison of four different Fe III macrocyclic complexes elucidates the factors that contribute to relaxivity in vivo including solution speciation. Variable-temperature 17 O NMR studies suggest that none of the complexes has a single, integral inner-sphere water that exchanges rapidly on the NMR timescale. MRI studies in mice show large in vivo differences of three of the Fe III complexes that correspond, in part, to their r 1 relaxivity in phantoms. Changes in overall charge of the complex modulate contrast enhancement, especially of the kidneys., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A newly discovered isomer of Co(ii) (1,4,8,11-tetrakis(carbamoylmethyl)-1,4,8,11-tetraazacyclotetradecane = CCRM) produces four highly paramagnetically shifted chemical exchange saturation transfer (CEST) peaks. The 1,8-pendants of the complex are bound in a trans-arrangement to produce a Co(ii) complex of increased kinetic inertness. The isomers have a stabilized Co(ii) center (E1/2 of 540 to 550 mV versus SHE). Both the 1,8 and the 1,4-isomer are excellent pH probes in solution and in tissue homogenate by virtue of their highly paramagnetically shifted amide protons. These isomers produce both a ratiometric pH readout as well as amide proton exchange rate constants that correlate to pH.

Several paramagnetic Co(II) and Fe(II) macrocyclic complexes were prepared with the goal of introducing a bound water ligand to produce paramagnetically shifted water 1 H resonances and for paramagnetic chemical exchange saturation transfer (paraCEST) applications. Three 12-membered macrocycles with amide pendent groups including 1,7-bis(carbamoylmethyl)-1,4,7,10-tetraazacyclodocane (DCMC), 4,7,10-tris(carbamoylmethyl)-,4,7,10-triaza-12-crown-ether (N3OA), and 4,10-bis(carbamoylmethyl)-4,10-diaza-12-crown-ether (NODA) were prepared and their Co(II) complexes were characterized in the solid state and in solution. The crystal structure of [Co(DCMC)]Br 2 featured a six-coordinated Co(II) center with distorted octahedral geometry, while [Co(NODA)(OH 2 )]Cl 2 and [Co(N3OA)](NO 3 ) 2 were seven-coordinated. The analogous Fe(II) complexes of NODA and NO3A were successfully prepared, but the complex of DCMC oxidized rapidly to the Fe(III) form. Similarly, [Fe(NODA)] 2+ oxidized over several days, forming crystals of the Fe(III) complex isolated as the μ-O bridged dimer. Magnetic susceptibility values and paramagnetic NMR spectra of the Fe(II) complexes of NODA and N3OA, as well as Co(II) complexes of DCMC, NODA, and N3OA, were consistent with high spin complexes. CEST peaks ranging from 60 ppm to 70 ppm, attributed to NH groups of the amide pendents, were identified. Variable-temperature 17 O NMR spectra of Co(II) and Fe(II) NODA complexes were consistent with rapid exchange of the water ligand with bulk water. Notably, the Co(II) and Fe(II) complexes presented here produced substantial paramagnetic shifts of bulk water 1 H resonances, independent of having an inner-sphere water.

Two macrocyclic complexes of 1,4,7-triazacyclononane (TACN), one with N-methyl imidazole pendants, [Fe(Mim)] 3+ , and one with unsubstituted NH imidazole pendants, [Fe(Tim)] 3+ , were prepared with a view toward biomedical imaging applications. These low-spin Fe 3+ complexes produce moderately paramagnetically shifted and relatively sharp 1 H NMR resonances for paraSHIFT and paraCEST applications. The [Fe(Tim)] 3+ complex undergoes pH-dependent changes in NMR spectra in solution that are consistent with the consecutive deprotonation of all three imidazole pendant groups at high pH values. N-Methylation of the imidazole pendants in [Fe(Mim)] 3+ produces a complex that dissociates more readily at high pH in comparison to [Fe(Tim)] 3+ , which contains ionizable donor groups. Cyclic voltammetry studies show that the redox potential of [Fe(Mim)] 3+ is invariant with pH ( E 1/2 = 328 ± 3 mV vs NHE) between pH 3.2 and 8.4, unlike the Fe(III) complex of Tim which shows a 590 mV change in redox potential over the pH range of 3.3-12.8. Magnetic susceptibility studies in solution give magnetic moments of 0.91-1.3 cm 3 K mol -1 (μ eff value = 2.7-3.2) for both complexes. Solid-state measurements show that the susceptibility is consistent with a S = 1/2 state over the temperature range of 0 to 300 K, with no crossover to a high-spin state under these conditions. The crystal structure of [Fe(Mim)](OTf) 3 shows a six-coordinate all-nitrogen bound Fe(III) in a distorted octahedral environment. Relativistic ab initio wave function and density functional theory (DFT) calculations on [Fe(Mim)] 3+ , some with spin orbit coupling, were used to predict the ground spin state. Relative energies of the doublet, quartet, and sextet spin states were consistent with the doublet S = 1/2 state being the lowest in energy and suggested that excited states with higher spin multiplicities are not thermally accessible. Calculations were consistent with the magnetic susceptibility determined in the solid state.

High-spin Co(II) complexes are promising for development as paraCEST agents (paraCEST = paramagnetic chemical exchange saturation transfer) for magnetic resonance imaging applications. The first examples of Co(II) paraCEST agents with bound water ligands are presented here. Four Co(II) macrocyclic complexes based on 1,4,7-triazacyclononane and containing either pendent alcohol or pendent amide groups were prepared. Two of the macrocycles encapsulate the Co(II) and contain no water ligands as shown by X-ray crystallographic studies, and two complexes have macrocycles with only five ligand donor groups to leave an open coordination site for bound water. The ionization of alcohol, water, or amide groups in the complexes was characterized by using pH potentiometry. These data show that one of the complexes has a readily deprotonated group with a pK a close to 6, which is assigned as an alcohol pendent. Amide pendents deprotonate at high pH (>8), and the water ligands of the Co(II) complexes are not deprotonated at neutral pH. All complexes produce CEST peaks through either alcohol OH or amide NH proton exchange. The water ligands exchange too rapidly to produce a CEST effect as shown by variable-temperature 17 O NMR spectroscopy studies. The complexes with available coordination sites for inner-sphere water ligands produce large paramagnetic shifts and broadening of the 17 O resonances of bulk water, whereas the encapsulated complexes show much less shifting and broadening of 17 O resonances. All complexes produce substantial paramagnetic shifts of the 1 H resonances of bulk water, which is promising for the development of supramolecular CEST agents.

Several complexes of Co(ii) or Fe(ii) with 1,4,7,10-tetraazacyclododecane (CYCLEN) appended with 1,7-(6-methyl)2-picolyl groups are studied as 1 H NMR paraSHIFT agents (paramagnetic shift agents) for the registration of temperature. Two of the complexes, [Co(BMPC)] 2+ and [Fe(BMPC)] 2+ , contain methyl groups only on the methyl picolyl pendents. Two other complexes, [Co(2MPC)] 2+ and [Fe(2MPC)] 2+ , contain picolyl groups and also methyl groups on the macrocyclic amines. All macrocyclic complexes are in high spin form as shown by solution magnetic moments in the range of 5.0-5.9μ BM and 5.3-5.8μ BM for Co(ii) and Fe(ii) complexes, respectively. The 1 H NMR spectra of both of the Fe(ii) complexes and one of the Co(ii) complexes are consistent with a predominant diastereomeric form in deuterium oxide solutions. The highly shifted methyl proton resonances for [Co(2MPC)] 2+ appear at 164 and -113 ppm for macrocycle and pendent picolyl methyls and show temperature coefficients of -0.58 ppm °C -1 and 0.49 ppm °C -1 , respectively. Fe(ii) complexes have less shifted methyl proton resonances and smaller temperature coefficients. The 1 H resonances of [Fe(2MPC)] 2+ appear at 105 ppm and -46 ppm with corresponding temperature coefficients (CT) of -0.29 ppm °C -1 and 0.22 ppm °C -1 , respectively. The relatively narrow linewidths of [Fe(2MPC)] 2+ , however, produce superior CT/FWHM values of 0.44 and 0.31 °C -1 for the N-methyl and picolyl proton resonances where FWHM is the full width at half maximum of the 1 H resonance. The crystal structure of [Co(BMPC)]Cl 2 shows a six-coordinate Co(ii) bound to the macrocyclic amines and two pendent picolyl groups. The distorted trigonal prismatic geometry of the complex resembles that of an analogous complex containing four 6-methyl-2-picolyl groups, in which only two picolyl pendents are coordinated.

A reversible Fe 3+ /Fe 2+ redox couple of an azamacrocyclic complex is evaluated as an electrolyte with a pH-tunable potential range for aqueous redox-flow batteries (RFBs). The Fe III complex is formed by 1,4,7-triazacyclononane (TACN) appended with three 2-methyl-imidazole donors, denoted as Fe(Tim). This complex exhibits pH-sensitive redox couples that span E 1/2 (Fe 3+ /Fe 2+ )=317 to -270 mV vs. NHE at pH 3.3 and pH 12.8, respectively. The 590 mV shift in potential and kinetic inertness are driven by ionization of the imidazoles at various pH values. The Fe 3+ /Fe 2+ redox is proton-coupled at alkaline conditions, and bulk electrolysis is non-destructive. The electrolyte demonstrates high charge/discharge capacities at both acidic and alkaline conditions throughout 100 cycles. Given its tunable redox, fast electrochemical kinetics, exceptional stability/cyclability, this complex is promising for the design of aqueous RFB catholytes and anolytes that utilize the earth-abundant element iron., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Fe(II) , Co(II) and Ni(II) complexes of two tetraazamacrocycles (1,4,8,11-tetrakis(carbamoylmethyl)-1,4,8,11-tetraazacyclotetradecane (L1) and 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (L2) show promise as paraCEST agents for registration of temperature (paraCEST=paramagnetic chemical exchange saturation transfer). The Fe(II) , Co(II) and Ni(II) complexes of L1 show up to four CEST peaks shifted ≤112 ppm, whereas analogous complexes of L2 show only a single CEST peak at ≤69 ppm. Comparison of the temperature coefficients (CT ) of the CEST peaks of [Co(L2)](2+) , [Fe(L2)](2+) , [Ni(L1)](2+) and [Co(L1)](2+) showed that a CEST peak of [Co(L1)](2+) gave the largest CT (-0.66 ppm (o) C(-1) at 4.7 T). NMR spectral and CEST properties of these complexes correspond to coordination complex symmetry as shown by structural data. The [Ni(L1)](2+) and [Co(L1)](2+) complexes have a six-coordinate metal ion bound to the 1-, 4-amide oxygen atoms and four nitrogen atoms of the tetraazamacrocycle. The [Fe(L2)](2+) complex has an unusual eight-coordinate Fe(II) bound to four amide oxygen atoms and four macrocyclic nitrogen atoms. For [Co(L2)](2+) , one structure has seven-coordinate Co(II) with three bound amide pendents and a second structure has a six-coordinate Co(II) with two bound amide pendents., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Transition-metal-ion-based paramagnetic chemical exchange saturation transfer (paraCEST) agents are a promising new class of compounds for magnetic resonance imaging (MRI) contrast. Members in this class of compounds include paramagnetic complexes of Fe(II), Co(II), and Ni(II). The development of the coordination chemistry for these paraCEST agents is presented with an emphasis on the choice of the azamacrocycle backbone and pendent groups with the goals of controlling the oxidation state, spin state, and stability of the complexes. Chemical exchange saturation transfer spectra and images are compared for different macrocyclic complexes containing amide or heterocyclic pendent groups. The potential of paraCEST agents that function as pH- and redox-activated MRI probes is discussed.

Substitution of a nucleobase pair with a pair of 1,2-hydroxypyridinone (1,2-HOPO) ligands in the center of a 10-base-pair peptide nucleic acid (PNA) duplex provides a strong binding site for Eu(III) as evidenced by UV thermal melting curves, UV titrations, and luminescence spectroscopy. Eu(III) excitation spectra and luminescence lifetime data are consistent with Eu(III) bound to both 1,2 HOPO ligands in a PNA-HOPO duplex as the major species present in solution.

Luminescent lanthanide (Ln(III)) ions are valuable spectroscopic probes for metal ion binding sites in nucleic acids. In this chapter, we briefly review Ln(III) luminescence and the information available from these experiments. An emphasis is placed on direct excitation Eu(III) spectroscopy as a tool. Eu(III) excitation spectroscopy is used to show that solutions containing micromolar Eu(III), 100 mM NaCl, and 20 mM MES buffer contain predominantly a mononuclear Eu(III) aqua complex and an Eu(III) hydroxide complexes. The binding of these species to various RNA and DNA sequences are monitored by using Eu(III) excitation spectroscopy. Eu(III) luminescence lifetime data shows that the Eu(III) ion typically loses 1-3 water molecules to form innersphere complexes with RNA and DNA that contain tandem base pair mismatches or hairpin loops. In addition, early studies that used nucleobase-sensitized Eu(III) or Tb(III) luminescence within transfer RNA or in the hammerhead ribozyme are presented. Luminescence resonance energy transfer studies are shown to be useful for determining distances between bound Ln(III) ion and organic fluorophores or between two different Ln(III) ions. To supplement luminescence data, the binding sites of paramagnetic Ln(III) ions are determined by monitoring the chemical shifts of nucleotide protons. Binding sites are identified by following the protons that are influenced by the Ln(III) pseudo-contact shift.

Dinuclear europium(III) complexes of the macrocycles 1,3-bis[1-(4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane]-m-xylene (1), 1,4-bis[1-(4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane]-p-xylene (2), and mononuclear europium(III) complexes of macrocycles 1-methyl-,4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (3), 1-[3'-(N,N-diethylaminomethyl)benzyl]-4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (4), and 1,4,7-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (5) were prepared. Studies using direct excitation ((7)F0 --> (5)D0) europium(III) luminescence spectroscopy show that each Eu(III) center in the mononuclear and dinuclear complexes has two water ligands at pH 7.0, I = 0.10 M (NaNO3) and that there are no water ligand ionizations over the pH range of 7-9. All complexes promote cleavage of the RNA analogue 2-hydroxypropyl-4-nitrophenyl phosphate (HpPNP) at 25 degrees C (I = 0.10 M (NaNO3), 20 mM buffer). Second-order rate constants for the cleavage of HpPNP by the catalysts increase linearly with pH in the pH range of 7-9. The second-order rate constant for HpPNP cleavage by the dinuclear Eu(III) complex (Eu2(1)) at pH 7 is 200 and 23-fold higher than that of Eu(5) and Eu(3), respectively, but only 7-fold higher than the mononuclear complex with an aryl pendent group, Eu(4). This shows that the macrocycle substituent modulates the efficiency of the Eu(III) catalysts. Eu2(1) 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 that of the mononuclear Eu(5) complex. Methyl phosphate binding to the Eu(III) complexes is energetically most favorable for the best catalysts, and this supports an important role for the catalyst in stabilization of the developing negative charge on the phosphorane transition state. Despite the formation of a bridging phosphate ester between the two Eu(III) centers in Eu2(1) as shown by luminescence spectroscopy, the two metal ion centers are only weakly cooperative in cleavage of RNA and RNA analogues.

Catalysis is an important process in chemistry and enzymology. The rate acceleration for any catalyzed reaction is the difference between the activation barriers for the uncatalyzed (Delta G(HO)(#)) and catalyzed (Delta G(Me)(#)) reactions, which corresponds to the binding energy (Delta G(S)(#) = Delta G(Me)(#)-Delta G(HO)(#)) for transfer of the reaction transition state from solution to the catalyst. This transition state binding energy is a fundamental descriptor of catalyzed reactions, and its evaluation is necessary for an understanding of any and all catalytic processes. We have evaluated the transition state binding energies obtained from interactions between low molecular weight metal ion complexes or high molecular weight protein catalysts and the phosphate group of bound substrate. Work on catalysis by small molecules is exemplified by studies on the mechanism of action of Zn2(1)(H2O). A binding energy of Delta G(S)(#) = -9.6 kcal/mol was determined for Zn2(1)(H2O)-catalyzed cleavage of the RNA analogue HpPNP. The pH-rate profile for this cleavage reaction showed that there is optimal catalytic activity at high pH, where the catalyst is in the basic form [Zn2(1)(HO-)]. However, it was also shown that the active form of the catalyst is Zn2(1)(H2O) and that this recognizes the C2-oxygen-ionized substrate in the cleavage reaction. The active catalyst Zn2(1)(H2O) shows a high affinity for oxyphosphorane transition state dianions and a stable methyl phosphate transition state analogue, compared with the affinity for phosphate monoanion substrates. The transition state binding energies, Delta G(S)(#), for cleavage of HpPNP catalyzed by a variety of Zn2+ and Eu3+ metal ion complexes reflect the increase in the catalytic activity with increasing total positive charge at the catalyst. These values of Delta G(S)(#) are affected by interactions between the metal ion and its ligands, but these effects are small in comparison with Delta G(S)(#) observed for catalysis by free metal ions, where the ligands are water. Enzymes are unique in having evolved mechanisms to effectively utilize binding interactions with nonreacting fragments of the substrate in stabilization of the reaction transition state. Orotidine 5'-monophosphate decarboxylase, alpha-glycerol phosphate dehydrogenase, and triosephosphate isomerase catalyze dissimilar decarboxylation, hydride transfer, and proton transfer reactions, respectively. Each enzyme derives ca. 12 kcal/mol of transition state stabilization from protein interactions with the nonreacting phosphate group, which is larger than the highest approximately 10 kcal/mol transition state stabilization that we have determined for small-molecule catalysis of phosphate diester cleavage in water. Each of these enzymes catalyze the slow reaction of a truncated substrate that lacks the phosphate group, and in each case, the reaction of the truncated substrate is strongly activated by the allosteric binding of the second substrate "piece" phosphite dianion, HPO3(2-). We propose a modular design for these enzymes with a classical active site that recognizes the reactive substrate fragment and a separate phosphodianion binding site. The second site is created, in part, by flexible protein loops that wrap around the substrate phosphodianion group and bury the substrate in an environment with an optimal local dielectric constant for the catalyzed reaction and with the most favorable positioning of the catalytic side chains. This design is easily generalized to a wide variety of enzyme-catalyzed reactions.

The complex between Eu(III) and 1,7-diaza-4,10,13-trioxacyclopentadecane-N,N'-diacetic acid (L4) was characterized by pH potentiometric titration and 1H NMR spectroscopy. The conversion of the monomer to a dimeric complex is observed as the pH is increased from 7 to 10 in a reaction that releases one mol/HO- per dimer formed. The dimeric complex undergoes a further ionization with a pKa of 10.7. Kinetic parameters are reported for the cleavage of the simple phosphodiester 2-hydroxypropyl-4-nitrophenyl phosphate catalyzed by both the monomeric and the dimeric Eu(III) complexes. These data show that the monomer and dimer stabilize their bound reaction transition states with similar free energies of 7.1 and 7.6 kcal/mol, respectively. Clearly, a bridging hydroxide is not an optimal linker to promote cooperative catalysis between Eu(III) centers in macrocycles with multiple polyaminocarboxylate pendent groups.

Two macrocycles (1 and 2) are prepared that incorporate pendent groups in macrocycle 3 (3=1-oxa-4,7,10-triazacyclododecane) with the goal of studying the effect of these pendent groups on metal ion complexation, solution chemistry and catalysis. Zn(1) contains a macrocyclic ligand with a pendent acridine group and Zn(2) has an appended methyl group. Water ligand pK(a) values for Zn(1) (6.7) and Zn(2) (7.3) are lower than that of Zn(3) (7.7). Zn(II) complexes of 1 and 2 are studied as catalysts for the cleavage of 2-hydroxypropyl 4-nitrophenylphosphate (HpPNP), an RNA analog. Zn(2) has a lower catalytic activity over the pH range 7-10 for cleavage of HpPNP compared to the parent macrocyclic complex, Zn(3). In contrast, Zn(1) has a threefold larger rate constant at pH 7.0 compared to Zn(2), attributed to the presence of a catalytic species which has a protonated acridine amino group. The binding constant of 1.5mM at pH 8.0 for formation of the Zn(2)-uridine adduct is similar to that for Zn(3), suggesting that N-alkylation of the macrocyclic ligand does not interfere with binding of the Zn(II) complex to uridine groups. Binding of cytidine to Zn(2) was not detectable under similar conditions up to 25mM nucleoside. Binding experiments under similar conditions could not be carried out for adenosine or guanosine due to their low solubility.

Zn(II) complexes of 1-oxa-4,7,10-triazacyclododecane (12[ane]N3O), 1,5,9-triazacyclododecane (12[ane]N3), and 1-hydroxyethyl-1,4,7-triazacyclononane (9[ane]N3OH) promote cleavage of the RNA analogue, 2-hydroxypropyl-4-nitrophenyl phosphate (HpPNP) at pH 8.0, I=0.10 M (NaCl), 25 degrees C with second-order rate constants of 8.9x10(-3), 9.0x10(-3), and 3.3x10(-3) M-1 s-1, respectively. Cleavage of HpPNP by these catalysts is inhibited by uridine with inhibition constants (Ki) of 1.2, 0.46, and 45 mM, respectively, under these conditions. Binding constants derived from these inhibition constants are 2-200-fold larger than those for binding of related Zn(II) complexes to phosphate diesters under similar conditions, suggesting that uridine sequences in RNA will inhibit Zn(II)-catalyzed cleavage by competing with phosphate diester binding sites. Further studies are carried out that utilize pH-potentiometric titrations to monitor uridine binding to five Zn(II) macrocyclic complexes in aqueous solution at 25 degrees C, I=0.10 M (NaCl). The data are consistent with binding of the Zn(II) complexes to the N3-deprotonated form of uridine to give log KU.-values of 5.29, 4.57, 4.56, 3.47, and 2.65 for the Zn(II) complexes of 12[ane]N3, 12[ane]N4, 12[ane]N3O, 15[ane]N3O2, and 9[ane]N3OH, respectively (12[ane]N4=1,4,7,10-tetraazacyclododecane, 15[ane]N3O2=1,4-dioxa-7,10,13-triazacyclopentadecane). For the five Zn(II) complexes studied, there is a linear relationship between uridine anion binding constants and hydroxide binding constants.

Synthetic metallonucleases are versatile metal ion catalysts that use multiple catalytic strategies for the cleavage of RNA. Recent work in the design of more active metallonucleases combines a single metal ion with functional groups that interact with RNA, including amino acid fragments or additional metal ions. Rate enhancements by multifunctional catalysts for cleavage of simple model substrates with good leaving groups are as high as 10(6) but somewhat lower (10(5)) for real RNA. However, cleavage of RNA substrates is complicated by different binding modes and steric interactions that can interfere with catalysis. Antisense oligonucleotides, peptides and small molecules that act as RNA recognition agents increase the strength of substrate binding, but not necessarily the catalytic rate constant. In general, catalytic strategies used by synthetic metallonucleases are probably not optimized. A better grasp of the mechanism of RNA cleavage by metal ions and more effort on positioning the metal ion complex with respect to the cleavage site may lead to improved catalysts.

A dinuclear metal ion complex Zn(2)()(L2O) and its mononuclear analogue Zn(L1OH) were synthesized and studied as catalysts of the cleavage of the phosphate diester 2-hydroxypropyl-4-nitrophenyl phosphate (HPNP). X-ray crystal structure data, potentiometric titrations, and (1)H NMR spectra obtained over a wide range of pH values provide strong evidence that the alcohol linker in the complex Zn(2)()(L2O) is ionized below pH 6.0, while the alcohol group in the complex Zn(L1OH) remains protonated even at high pH. The ionizations observed at high pH correspond to the formation of the monohydroxo complexes, Zn(2)(L2O)(OH) and Zn(L1OH)(OH), with pK(a)'s of 8.0 and 9.2, respectively. The pH-rate profiles of second-order rate constants for metal-ion complex-catalyzed cleavage of HPNP are reported. These show downward curvature centered at the pK(a)'s for the respective zinc-bound waters, and limiting second-order rate constants at high pH of k(c) = 0.71 M(-)(1) s(-)(1) for Zn(2)()(L2O) and 0.061 M(-)(1) s(-)(1) for Zn(L1OH). The larger catalytic activity of Zn(2)()(L2O) compared with Zn(L1OH) is due to the cooperative role of the metal ions in facilitating the formation of the ionized zinc-bound water at close to neutral pH and in providing additional stabilization of the rate-limiting transition state for phosphodiester cleavage. Zn(2)()(L2O) complex (1 M) at pH 7.6 stabilizes the transition state for the uncatalyzed reaction by 9.3 kcal/mol. Assuming that the dissociation constant determined for a diethyl phosphate inhibitor is similar to that for substrate, then ca. 2.4 kcal/mol of these stabilizing interactions are expressed in the ground-state Michaelis complex, while the bulk of these interactions are only expressed as the reaction approaches the transition state for phosphodiester cleavage.