Pavel B. Tsitovich

Iron(II) PARACEST MRI Contrast Agents

The first examples of Fe(II) PARACEST magnetic resonance contrast agents are reported (PARACEST = paramagnetic chemical exchange saturation transfer). The iron(II) complexes contain a macrocyclic ligand, either 1,4,7-tris(carbamoylmethyl)-1,4,7-triazacyclononane (L1) or 1,4,7-tris[(5-amino-6-methyl-2-pyridyl)methyl]-1,4,7-triazacyclononane (L2). The macrocycles bind Fe(II) in aqueous solution with formation constants of log K = 13.5 and 19.2, respectively, and maintain the Fe(II) state in the presence of air. These complexes each contain six exchangeable protons for CEST which are amide protons in [Fe(L1)](2+) or amino protons in [Fe(L2)](2+). The CEST peak for the [Fe(L1)](2+) amide protons is at 69 ppm downfield of the bulk water resonance whereas the CEST peak for the [Fe(L2)](2+) amine protons is at 6 ppm downfield of bulk water. CEST imaging using a MRI scanner shows that the CEST effect can be observed in solutions containing low millimolar concentrations of complex at neutral pH, 100 mM NaCl, 20 mM buffer at 25 °C or 37 °C.

A Redox‐Activated MRI Contrast Agent that Switches Between Paramagnetic and Diamagnetic States

The design of molecular switches for the production of responsive or “smart” imaging agents is a major challenge. Of particular interest are agents that respond to biological redox environment to map those disease states that involve redox imbalances.[1] Redox imbalances may be triggered in many ways, including very low oxygen pressure or hypoxia in the case of solid tumors.[2] Importantly, the development of probes to map oxygen levels and corresponding redox status of tumor tissue may guide the development of tumor-selective drugs.[3]

Redox-activated MRI contrast agents based on lanthanide and transition metal ions

The reduction/oxidation (redox) potential of tissue is tightly regulated in order to maintain normal physiological processes, but is disrupted in disease states. Thus, the development of new tools to map tissue redox potential may be clinically important for the diagnosis of diseases that lead to redox imbalances. One promising area of chemical research is the development of redox-activated probes for mapping tissue through magnetic resonance imaging (MRI). In this review, we summarize several strategies for the design of redox-responsive MRI contrast agents. Our emphasis is on both lanthanide(III) and transition metal(II/III) ion complexes that provide contrast either as T1 relaxivity MRI contrast agents or as paramagnetic chemical exchange saturation transfer (PARACEST) contrast agents. These agents are redox-triggered by a variety of chemical reactions or switches including redox-activated thiol groups, and heterocyclic groups that interact with the metal ion or influence properties of other ancillary ligands. Metal ion centered redox is an approach which is ripe for development by coordination chemists. Redox-triggered metal ion approaches have great potential for creating large differences in magnetic properties that lead to changes in contrast. An attractive feature of these agents is the ease of fine-tuning the metal ion redox potential over a biologically relevant range.

Comparison of Divalent Transition Metal Ion ParaCEST MRI Contrast Agents

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 FeII, CoII, and NiII. 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.

The reactivity of macrocyclic Fe(II) paraCEST MRI contrast agents towards biologically relevant anions, cations, oxygen or peroxide.

The reactivity of four macrocyclic Fe(II) complexes (L1-L4) is studied with the goal of developing paramagnetic chemical exchange saturation transfer (paraCEST) magnetic resonance imaging (MRI) contrast agents for in vivo studies. (L1 = 1,4,7-tris(carbamoylmethyl)-1,4,7-triazacyclononane; L2 = 1,4,7-tris[(5-methyl-2-pyridyl)methyl]-1,4,7-triazacyclononane; L3 = 1,4,7-tris[(2-pyridyl)methyl]-1,4,7-triazacyclononane; L4 = 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane). The Fe(II) complexes remain intact in the presence of 25 mM carbonate, 0.40 mM phosphate and 100mM NaCl for 12h at 37 °C, consistent with their moderately high formation constants (log K=13.5, 19.2, 7.50 for [Fe(L1)](2+), [Fe(L3)](2+) and [Fe(L4)](2+), respectively). [Fe(L4)](2+), [Fe(L2)](2+) and [Fe(L3)](2+) do not dissociate over 12h in the presence of excess Cu(II) at 37 °C. None of the complexes show appreciable redox cycling as measured by consumption of ascorbate in the presence of oxygen, corresponding to their highly stabilized Fe(II) oxidation state (E(o)=860, 930, 970, and 800 mV versus NHE for [Fe(L1)](2+), [ [Fe(L2)](2+), [Fe(L3)](2+) and [Fe(L4)](2+). None of the Fe(II) complexes produce appreciable amounts of hydroxyl radical in the presence of peroxide and ascorbate as shown by limited hydroxylation of benzoate. Fe(II) complexes of L1, L2, and L3 show 25-28% cleavage of supercoiled plasmid DNA in the presence of peroxide and ascorbate over 2h at 37 °C while [Fe(L4)](2+) shows 6% cleavage.

Using modularly assembled ligands to bind RNA internal loops separated by different distances.

Many studies have identified RNA drug or probe targets present in genomic sequence, however, most of these RNAs remain underexploited due to the difficulties in designing high affinity and specific RNA binders. Modular assembly and multivalency provide unique opportunities to tune the affinity and specificity of ligands for a target biomolecule. Herein, studies to investigate how far ligand modules should be spaced apart to bind RNA internal loops separated by different numbers of base pairs are reported. Results show that about four spacing submonomers between bis-benzimidazole modules displayed on a peptoid scaffold is optimal to span two base pairs. These results have general implications for the rational and modular design of ligands that specifically target RNA.

Six-coordinate Iron(II) and Cobalt(II) paraSHIFT Agents for Measuring Temperature by Magnetic Resonance Spectroscopy

Paramagnetic Fe(II) and Co(II) complexes are utilized as the first transition metal examples of (1)H NMR shift agents (paraSHIFT) for thermometry applications using Magnetic Resonance Spectroscopy (MRS). The coordinating ligands consist of TACN (1,4,7-triazacyclononane) and CYCLEN (1,4,7,10-tetraazacyclododecane) azamacrocycles appended with 6-methyl-2-picolyl groups, denoted as MPT and TMPC, respectively. (1)H NMR spectra of the MPT- and TMPC-based Fe(II) and Co(II) complexes demonstrate narrow and highly shifted resonances that are dispersed as broadly as 440 ppm. The six-coordinate complex cations, [M(MPT)](2+) and [M(TMPC)](2+), vary from distorted octahedral to distorted trigonal prismatic geometries, respectively, and also demonstrate that 6-methyl-2-picolyl pendents control the rigidity of these complexes. Analyses of the (1)H NMR chemical shifts, integrated intensities, line widths, the distances obtained from X-ray diffraction measurements, and longitudinal relaxation time (T1) values allow for the partial assignment of proton resonances of the [M(MPT)](2+) complexes. Nine and six equivalent methyl protons of [M(MPT)](2+) and [M(TMPC)](2+), respectively, produce 3-fold higher (1)H NMR intensities compared to other paramagnetically shifted proton resonances. Among all four complexes, the methyl proton resonances of [Fe(TMPC)](2+) and [Co(TMPC)](2+) at -49.3 ppm and -113.7 ppm (37 °C) demonstrate the greatest temperature dependent coefficients (CT) of 0.23 ppm/°C and 0.52 ppm/°C, respectively. The methyl groups of these two complexes both produce normalized values of |CT|/fwhm = 0.30 °C(-1), where fwhm is full width at half-maximum (Hz) of proton resonances. The T1 values of the highly shifted methyl protons are in the range of 0.37-2.4 ms, allowing rapid acquisition of spectroscopic data. These complexes are kinetically inert over a wide range of pH values (5.6-8.6), as well as in the presence of serum albumin and biologically relevant cations and anions. The combination of large hyperfine shifts, large temperature sensitivity, increased signal-to-noise ratio, and short T1 values suggests that these complexes, in particular the TMPC-based complexes, show promise as paraSHIFT agents for thermometry.

A Chemoenzymatic Route to Diversify Aminolgycosides Enables a Microarray‐Based Method to Probe Acetyltransferase Activity

Specific modification of functional groups in aminoglycosides poses a significant synthetic challenge. In this report, a chemoenzymatic route for modification of aminoglycosides is disclosed. The critical feature of this approach is the discovery that the aminoglycoside 3-N-acetyltransferase AAC(3)-IV from Escherichia coli [1] accepts azido acetyl coenzyme A (AzAcCoA) as a substrate similarly as the natural substrate, acetyl coenzyme A (AcCoA). After enzymatic delivery of an azido acetyl group, it can be chemically modified via a Huisgen dipolar cycloaddition reaction (HDCR)[2] enabling further diversification. Thus, this method accelerates access to modified compounds with diversity beyond that which can be installed directly via AAC(3) and a modified CoA thioester. The approach was further developed to study modification of aminoglycosides by AAC(3), which causes broad-scale aminoglycoside inactivation, using a fluorescence-based microarray platform. This platform is a useful analytical tool for the facile identification of both protein and carbohydrate substrates for acetyltransferases, which play critical roles in a multitude of cellular processes.[3]

Low-Spin Fe(III) Macrocyclic Complexes of Imidazole-Appended 1,4,7-Triazacyclononane as Paramagnetic Probes

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 Fe3+ complexes produce moderately paramagnetically shifted and relatively sharp 1H 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 ( E1/2 = 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 cm3 K mol-1 (μeff 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.

An FeIII Azamacrocyclic Complex as a pH-Tunable Catholyte and Anolyte for Redox-Flow Battery Applications

A reversible Fe3+ /Fe2+ redox couple of an azamacrocyclic complex is evaluated as an electrolyte with a pH-tunable potential range for aqueous redox-flow batteries (RFBs). The FeIII 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 E1/2 (Fe3+ /Fe2+ )=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 Fe3+ /Fe2+ 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.