Dynamics of Coordinated Phosphonate Group Directly Observed by 17O-NMR in Lanthanide(iii) Complexes of a Mono(ethyl phosphonate) DOTA Analogue.

Biological phosphates can coordinate metal ions and their complexes are common in living systems. Dynamics of mutual oxygen atom exchange in the tetrahedral group in complexes has not been investigated. Here, we present a direct experimental proof of exchange ("phosphonate rotation") in model Ln(III) complexes of monophosphonate H4dota analogue which alters phosphorus atom chirality of coordinated phosphonate monoester. Combination of macrocycle-based isomerism with P-based chirality leads to several diastereoisomers. (Non)-coordinated oxygen atoms were distinguished through 17O-labelled phosphonate group and their mutual exchange was followed by various NMR techniques and DFT calculations. The process is sterically demanding and occurs through bulky bidentate (κ2-PO2)- coordination and was observed only in twisted-square antiprism (TSA) diastereoisomer of large Ln(III) ions. Its energy demands increase for smaller Ln(III) ions (298ΔG≠(exp./DFT)=51.8/52.1 and 61.0/71.5 kJ mol-1 for La(III) and Eu(III), respectively). These results are helpful in design of such complexes as MRI CA and for protein paramagnetic NMR probes. It demonstrates usefulness of 17O NMR to study solution dynamics in complexes involving phosphorus acid derivatives and it may inspire use of this method to study dynamics of phosphoric acid derivatives (as e. g. phosphorus acid-based inhibitors of metalloenzymes) in different areas of chemistry.

A tutorial on potentiometric data processing. Analysis of software for optimization of protonation constants.

Defining the distribution of the chemical species in a multicomponent system is a task of great importance with applications in many fields. To clarify the identity and the abundance of the species that can be formed by the interaction of the components of a solution, it is fundamental to know the formation constants of those species. The determination of equilibrium constants is mainly performed through the analysis of experimental data obtained by different instrumental techniques. Among them, potentiometry is the elective technique for this purpose. As such, a survey was run within the NECTAR COST Action - Network for Equilibria and Chemical Thermodynamics Advanced Research, to identify the most used software for the analysis of potentiometric data and to highlight their strengths and weaknesses. The features and the calculation processes of each software were analyzed and rationalized, and a simulated titration dataset of a hypothetic hexaprotic acid was processed by each software to compare and discuss the optimized protonation constants. Moreover, further data analysis was also carried out on the original dataset including some systematic errors from different sources, as some calibration parameters, the total analytical concentration of reagents and ionic strength variations during titrations, to evaluate their impact on the refined parameters. Results showed that differences on the protonation constants estimated by the tested software are not significant, while some of the considered systematic errors affect results. Overall, it emerged that software commonly used suffer from many limitations, highlighting the urgency of new dedicated and modern tools. In this context, some guidelines for data generation and treatment are also given.

Transition metal complexes of the (2,2,2-trifluoroethyl)phosphinate NOTA analogue as potential contrast agents for 19F magnetic resonance imaging.

A new hexadentate 1,4,7-triazacyclononane-based ligand bearing three coordinating methylene-(2,2,2-trifluoroethyl)phosphinate pendant arms was synthesized and its coordination behaviour towards selected divalent (Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+) and trivalent (Cr3+, Fe3+, Co3+) transition metal ions was studied. The ligand forms stable complexes with late divalent transition metal ions (from Co2+ to Zn2+) and the complexes of these metal ions are formed above pH ∼3. A number of complexes with divalent metal ions were structurally characterized by means of single-crystal X-ray diffraction. The complex of the larger Mn2+ ion adopts a twisted trigonally antiprismatic geometry with a larger coordination cavity and smaller torsion of the pendant arms, whereas the smaller ions Ni2+, Cu2+ and Zn2+ form octahedral species with a smaller cavity and larger pendant arm torsion. In the case of the Co2+ complexes, both coordination arrangements were observed. The complexes with paramagnetic metal ions were studied from the point of view of potential utilization in 19F magnetic resonance imaging. A significant shortening of the 19F NMR longitudinal relaxation times was observed: a sub-millisecond range for complexes of Cr3+, Mn2+ and Fe3+ with symmetric electronic states (t2g3 and HS-d5), the millisecond range for the Ni2+ and Cu2+ complexes and tens of milliseconds for the Co2+ complex. Such short relaxation times are consistent with a short distance between the paramagnetic metal ion and the fluorine atoms (∼5.5-6.5 Å). Among the redox-active complexes (Mn3+/Mn2+, Fe3+/Fe2+, Co3+/Co2+, Cu2+/Cu+), the cobalt complexes show sufficient stability and a paramagnetic-diamagnetic changeover with the redox potential lying in a physiologically relevant range. Thus, the Co3+/Co2+ complex pair can be potentially used as a smart redox-responsive contrast agent for 19F MRI.

Inorganic Chemistry of the Tripodal Picolinate Ligand Tpaa with Gallium(III) and Radiolabeling with Gallium-68.

We report here the improved synthesis of the tripodal picolinate chelator Tpaa, with an overall yield of 41% over five steps, in comparison to the previously reported 6% yield. Tpaa was investigated for its coordination chemistry with Ga(III) and radiolabeling properties with gallium-68 (68Ga). The obtained crystal structure for [Ga(Tpaa)] shows that the three picolinate arms coordinate to the Ga(III) ion, fully occupying the octahedral coordination geometry. This is supported by 1H NMR which shows that the three arms are symmetrical when coordinated to Ga(III). Assessment of the thermodynamic stability through potentiometry gives log KGa-Tpaa = 21.32, with a single species being produced across the range of pH 3.5-7.5. Tpaa achieved >99% radiochemical conversion with 68Ga under mild conditions ([Tpaa] = 6.6 µM, pH 7.4, 37 °C) with a molar activity of 3.1 GBq µmol-1. The resulting complex, [68Ga][Ga(Tpaa)], showed improved stability over the previously reported [68Ga][Ga(Dpaa)(H2O)] in a serum challenge, with 32% of [68Ga][Ga(Tpaa)] remaining intact after 30 min of incubation with fetal bovine serum.

Transition metal complexes of cyclam with two 2,2,2-trifluoroethylphosphinate pendant arms as probes for 19F magnetic resonance imaging.

A new cyclam-based ligand bearing two methylene(2,2,2-trifluoroethyl)phosphinate pendant arms was synthesized and its coordination behaviour towards selected divalent transition metal ions [Co(II), Ni(II), Cu(II), Zn(II)] was studied. The ligand was found to be very selective for the Cu(II) ion according to the common Williams-Irving trend. Complexes with all the studied metal ions were structurally characterized. The Cu(II) ion forms two isomeric complexes; the pentacoordinated isomer pc-[Cu(L)] is the kinetic product and the octahedral trans-O,O'-[Cu(L)] isomer is the final (thermodynamic) product of the complexation reaction. Other studied metal ions form octahedral cis-O,O'-[M(L)] complexes. The complexes with paramagnetic metal ions showed a significant shortening of 19F NMR longitudinal relaxation times (T1) to the millisecond range [Ni(II) and Cu(II) complexes] or tens of milliseconds [Co(II) complex] at the temperature and magnetic field relevant for 19F magnetic resonance imaging (MRI). Such a short T1 results from a short distance between the paramagnetic metal ion and the fluorine atoms (∼6.1-6.4 Å). The complexes show high kinetic inertness towards acid-assisted dissociation; in particular, the trans-O,O'-[Cu(L)] complex was found to be extremely inert with a dissociation half-time of 2.8 h in 1 M HCl at 90 °C. Together with the short relaxation time, it potentially enables in vitro/in vivo utilization of the complexes as efficient contrast agents for 19F MRI.

Copper(II) complexes of cyclams with N-(2,2,2-trifluoroethyl)-aminoalkyl pendant arms as potential probes for 19F magnetic resonance imaging.

A series of Cu(II) complexes with cyclam-based ligands containing two N-(2,2,2-trifluoroethyl)-aminoalkyl pendant arms in 1,8-positions (L1: 1,2-ethylene spacer, L2: 1,3-propylene spacer; L3: 1,4-butylene spacer) was studied in respect to potential use as contrast agents for 19F magnetic resonance imaging (MRI). A number of structures of the complexes as well as of several organic precursors were determined by single-crystal X-ray diffraction analysis. Geometric parameters (especially distances between fluorine atoms and the central metal ion) were determined for each complex and the identity of isomeric complex species present in solution was established. The NMR longitudinal relaxation times (T1) of 19F nuclei in the ligands at clinically relevant fields and temperatures (1-2 s) were significantly shortened upon Cu(II) binding to 7-10 ms for [Cu(L1)]2+, 20-30 ms for [Cu(L2)]2+ and 20-50 ms for [Cu(L3)]2+. The trend of the relaxation time shortening is in accordance with the distance and number of chemical bonds between fluorine atoms and the Cu(II) ion. The signals show promising T2*/T1 ratios in the range 0.25-0.55, assuring their good applicability to 19F NMR/MRI. The results show that even the Cu(II) ion, with a small magnetic moment, causes significant relaxation enhancement with a long-range effect and can be considered as a highly suitable metal ion for efficient 19F MRI contrast agents.

Bn2DT3A, a Chelator for 68Ga Positron Emission Tomography: Hydroxide Coordination Increases Biological Stability of [68Ga][Ga(Bn2DT3A)(OH)].

The chelator Bn2DT3A was used to produce a novel 68Ga complex for positron emission tomography (PET). Unusually, this system is stabilized by a coordinated hydroxide in aqueous solutions above pH 5, which confers sufficient stability for it to be used for PET. Bn2DT3A complexes Ga3+ in a hexadentate manner, forming a mer-mer complex with log K([Ga(Bn2DT3A)]) = 18.25. Above pH 5, the hydroxide ion coordinates the Ga3+ ion following dissociation of a coordinated amine. Bn2DT3A radiolabeling displayed a pH-dependent speciation, with [68Ga][Ga(Bn2DT3A)(OH)]- being formed above pH 5 and efficiently radiolabeled at pH 7.4. Surprisingly, [68Ga][Ga(Bn2DT3A)(OH)]- was found to show an increased stability in vitro (for over 2 h in fetal bovine serum) compared to [68Ga][Ga(Bn2DT3A)]. The biodistribution of [68Ga][Ga(Bn2DT3A)(OH)]- in healthy rats showed rapid clearance and excretion via the kidneys, with no uptake seen in the lungs or bones.

Cyclam with a phosphinate-bis(phosphonate) pendant arm is a bone-targeting carrier of copper radionuclides.

Ligands combining a bis(phosphonate) group with a macrocycle function as metal isotope carriers for radionuclide-based imaging and for treating bone metastases associated with several cancers. However, bis(phosphonate) pendant arms often slow down complex formation and decrease radiochemical yields. Nevertheless, their negative effect on complexation rates may be mitigated by using a suitable spacer between bis(phosphonate) and the macrocycle. To demonstrate the potential of bis(phosphonate) bearing macrocyclic ligands as a copper radioisotope carrier, we report the synthesis of a new cyclam derivative bearing a phosphinate-bis(phosphonate) pendant (H5te1PBP). The ligand showed a high selectivity to CuII over ZnII and NiII ions, and the bis(phosphonate) group was not coordinated in the CuII complex, strongly interacting with other metal ions in solution. The CuII complex formed quickly, in 1 s, at pH 5 and at a millimolar scale. The complexation rates significantly differed under a ligand or metal ion excess due to the formation of reaction intermediates differing in their metal-to-ligand ratio and protonation state, respectively. The CuII-te1PBP complex also showed a high resistance to acid-assisted hydrolysis (t1/2 2.7 h; 1 M HClO4, 25 °C) and was effectively adsorbed on the hydroxyapatite surface. H5te1PBP radiolabeling with [64Cu]CuCl2 was fast and efficient, with specific activities of approximately 30 GBq 64Cu per 1 µmol of ligand (pH 5.5, room temperature, 30 min). In a pilot experiment, we further demonstrated the excellent suitability of [64Cu]CuII-te1PBP for imaging active bone compartments by dedicated small animal PET/CT in healthy mice and subsequently in a rat femoral defect model, in direct comparison with [18F]fluoride. Moreover, [64Cu]CuII-te1PBP showed a higher uptake in critical bone defect regions. Therefore, our study highlights the potential of [64Cu]CuII-te1PBP as a PET radiotracer for evaluating bone healing in preclinical and clinical settings with a diagnostic value similar to that of [18F]fluoride, albeit with a longer half-life (12.7 h) than 18F (1.8 h), thereby enabling extended observation times.

Al(III)-NTA-fluoride: a simple model system for Al-F binding with interesting thermodynamics.

Al(iii) complexes are extensively studied as [18F]fluoride carriers in positron emission tomography. However, our limited knowledge on their thermodynamic and kinetic properties has hindered efforts to easily prepare radiochemically pure compounds while simultaneously reducing the overall labeling time. Thus, to improve our understanding of fluoride binding to coordinatively unsaturated Al(iii) complexes, we investigated the ternary system Al(iii)-H3NTA-F- (H3NTA = nitrilo-triacetic acid) by NMR, potentiometry and X-ray diffraction. Our results show that the [Al(NTA)] complex binds two water molecules, which are replaced by fluorides. Individual species and isomers show separate 19F NMR signals and different stability constants. The two available positions on the [Al(NTA)] complex feature significantly different properties in terms of basicity of the coordinated water molecules and preferential binding of fluoride anions. Fluorides are effectively bound in weakly acidic or neutral solutions, whereas hydroxido species are preferentially formed in alkaline solutions. Our experimental observations were rationalized by theoretical calculations: predictions of the energy ordering of complexes and isomers, interpretation of 19F NMR chemical shifts, and natural bonding orbital analysis. Radiolabeling of [Al(NTA)] with [18F]fluoride gave low yields that confirmed a high affinity of the complex for hydroxide anions.

Paramagnetic Cobalt(II) Complexes with Cyclam Derivatives: Toward 19F MRI Contrast Agents.

In order to develop novel, more efficient, and/or selective contrast agents for magnetic resonance imaging (MRI), different modi operandi are explored as alternatives to water-relaxation enhancement. In this work, cobalt(II/III) complexes of bis(N-trifluoroethyl)cyclam derivatives with two acetate or two phosphonate pendant arms, H2te2f2a and H4te2f2p, were prepared and investigated. X-ray diffraction structures confirmed octahedral coordination with a very stable trans-III cyclam conformation and with fluorine atoms located about 5.3 Å from the metal center. The Co(II) complexes are kinetically inert, decomposing slowly even in 1 M aqueous HCl at 80 °C. The Co(II) complexes exhibited well-resolved paramagnetically shifted NMR spectra. These were interpreted with the help of quantum chemistry calculations. The 13C NMR shifts of the trans-[CoII(te2f2p)]2- complex were successfully assigned based on spin density delocalization within the ligand molecule. The obtained spin density also helps to describe d-metal-induced NMR relaxation properties of 19F nuclei, including the contribution of a Fermi contact relaxation mechanism. The paramagnetic complexes show convenient relaxation properties to be used as 19F MRI contrast agents.

Cross-Bridged Cyclam with Phosphonate and Phosphinate Pendant Arms: Chelators for Copper Radioisotopes with Fast Complexation.

Cross-bridged cyclam derivatives bearing two phosphonate (H4L1), bis(phosphinate) (H4L2), or phosphinate (H2L3) pendant arms were synthesized and studied with respect to their application as copper radioisotope carriers in nuclear medicine. The ligands show high macrocycle basicity (pK1 > 14) and high Cu(II) complex stability (log K = 20-24). The complexation and dissociation kinetics of the Cu(II) complexes were studied by ultraviolet-visible spectroscopy. Phosphonate Cu(II)-H4L1 and bis(phosphinate) Cu(II)-H4L2 complexes form very quickly, reaching quantitative formation within 1 s at pH ∼6 and millimolar concentrations. Conversely, the formation of the phosphinate complex Cu(II)-H2L3 is much slower (9 min at pH ∼6) due to the low stability of the out-of-cage reaction intermediate. All studied complexes are highly kinetically inert, showing half-lives of 120, 11, and 111 h for Cu(II)-H4L1, Cu(II)-H4L2, and Cu(II)-H2L3 complexes, respectively, in 1 M HClO4 at 90 °C. The high thermodynamic stability, fast formation, and extreme kinetic inertness of Cu(II) complexes indicate that phosphonate and bis(phosphinate) derivatives are promising ligands for nuclear medicine.

The solid-state structures and ligand cavity evaluation of lanthanide(iii) complexes of a DOTA analogue with a (dibenzylamino)methylphosphinate pendant arm.

A series of lanthanide(iii) complexes of a monophosphinate analogue of H4dota, 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic-10-methyl[(N,N-dibenzylamino)methyl]phosphinic acid (H4do3apDBAm = H4L1), were prepared and their solid-state structures were studied using single-crystal X-ray diffraction. In all structures, the ligand anion was octadentately coordinated to the Ln(iii) or Sc(iii) ions similarly to other DOTA-like ligands, i.e. forming parallel N4- and O4-planes. The lighter lanthanide(iii) complexes (till dysprosium) were nonacoordinated in the twisted square-antiprismatic (TSA) configuration with the apical coordination of water molecules or oxygen atoms from the neighbouring complex unit. The heavier lanthanide(iii) complexes (from terbium) were found as the "anhydrous" octacoordinated twisted square-antiprismatic (TSA') isomer. For the terbium(iii) ion, both forms were structurally characterized. The structural data of the Ln(iii)-H4L1 complexes and complexes of several related DOTA-like ligands were analysed. It clearly showed that the structural parameters for the square-antiprismatic (SA) isomers were clustered in a small range while those for the TSA/TSA' isomers were significantly more spread. The analysis also gave useful information about the influence of various pendant arms on the structure of the complexes of the DOTA-like ligands. The twist angle (torsion) of the chelate ring containing a larger phosphorus atom was similar to those of the remaining three acetate pendants. It led to a larger separation of the N4O4 planes and to smaller trans-O-Ln-O angles than the parameters found in the complexes of H4dota and its tetraamide derivatives dotam(R). It resulted in a relatively long bond between the metal ion and the coordinated water molecule. It led, together with the negative charge of the oxygen atoms forming the O4-plane, to an extremely fast water exchange rate reported for the Gd(iii)-H4L1 complex and, generally, to a fast water exchange of Gd(iii) complexes with the monophosphorus acid analogues of H4dota, H5do3ap/H4do3apR.

Selective and clean synthesis of aminoalkyl-H-phosphinic acids from hypophosphorous acid by phospha-Mannich reaction.

Aminoalkyl-H-phosphinic acids, also called aminoalkylphosphonous acids, are investigated as biologically active analogues of carboxylic amino acids and/or as valuable intermediates for synthesis of other aminoalkylphosphorus acids. Their synthesis has been mostly accomplished by phospha-Mannich reaction of a P-H precursor, an aldehyde and an amine. The reaction is rarely clean and high-yielding. Here, reaction of H3PO2 with secondary amines and formaldehyde in wet AcOH led to aminomethyl-H-phosphinic acids in nearly quantitative yields and with almost no by-products. Surprisingly, the reaction outcome depended on the basicity of the amines. Amines with pK a > 7-8 gave the desired products. For less basic amines, reductive N-methylation coupled with oxidation of H3PO2 to H3PO3 became a relevant side reaction. Primary amines reacted less clearly and amino-bis(methyl-H-phosphinic acids) were obtained only for very basic amines. Reaction yields with higher aldehydes were lower. Unique carboxylic-phosphinic-phosphonic acids as well as poly(H-phosphinic acids) derived from polyamines were obtained. Synthetic usefulness of the aminoalkyl-H-phosphinic was illustrated in P-H bond oxidation and its addition to double bonds, and in selective amine deprotection. Compounds with an ethylene-diamine fragment, e.g. most common polyazamacrocycles, are not suitable substrates. The X-ray solid-state structures of seventeen aminoalkyl-phosphinic acids were determined. In the reaction mechanism, N-hydroxyalkyl species R2NCH2OH and [R2N(CH2OH)2]+, probably stabilized as acetate esters, are suggested as the reactive intermediates. This mechanism is an alternative one to the known phospha-Mannich reaction mechanisms. The conditions can be utilized in syntheses of various aminoalkylphosphorus compounds.

Coordination Behavior of 1,4-Disubstituted Cyclen Endowed with Phosphonate, Phosphonate Monoethylester, and H-Phosphinate Pendant Arms.

Three 1,4,7,10-tetraazacyclododecane-based ligands disubstituted in 1,4-positions with phosphonic acid, phosphonate monoethyl-ester, and H-phosphinic acid pendant arms, 1,4-H4do2p, 1,4-H2do2pOEt, and 1,4-H2Bn2do2pH, were synthesized and their coordination to selected metal ions, Mg(II), Ca(II), Mn(II), Zn(II), Cu(II), Eu(III), Gd(III), and Tb(III), was investigated. The solid-state structure of the phosphonate ligand, 1,4-H4do2p, was determined by single-crystal X-ray diffraction. Protonation constants of the ligands and stability constants of their complexes were obtained by potentiometry, and their values are comparable to those of previously studied analogous 1,7-disubstitued cyclen derivatives. The Gd(III) complex of 1,4-H4do2p is ~1 order of magnitude more stable than the Gd(III) complex of the 1,7-analogue, probably due to the disubstituted ethylenediamine-like structural motif in 1,4-H4do2p enabling more efficient wrapping of the metal ion. Stability of Gd(III)-1,4-H2do2pOEt and Gd(III)-H2Bn2do2pH complexes is low and the constants cannot be determined due to precipitation of the metal hydroxide. Protonations of the Cu(II), Zn(II), and Gd(III) complexes probably takes place on the coordinated phosphonate groups. Complexes of Mn(II) and alkali-earth metal ions are significantly less stable and are not formed in acidic solutions. Potential presence of water molecule(s) in the coordination spheres of the Mn(II) and Ln(III) complexes was studied by variable-temperature NMR experiments. The Mn(II) complexes of the ligands are not hydrated. The Gd(III)-1,4-H4do2p complex undergoes hydration equilibrium between mono- and bis-hydrated species. Presence of two-species equilibrium was confirmed by UV-Vis spectroscopy of the Eu(III)-1,4-H4do2p complex and hydration states were also determined by luminescence measurements of the Eu(III)/Tb(III)-1,4-H4do2p complexes.

Lanthanide Complexes of DO3A-(Dibenzylamino)methylphosphinate: Effect of Protonation of the Dibenzylamino Group on the Water-Exchange Rate and the Binding of Human Serum Albumin.

Protonation of a distant, noncoordinated group of metal-based magnetic resonance imaging contrast agents potentially changes their relaxivity. The effect of a positive charge of the drug on the human serum albumin (HSA)-drug interaction remains poorly understood as well. Accordingly, a (dibenzylamino)methylphosphinate derivative of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was efficiently synthesized using pyridine as the solvent for a Mannich-type reaction of tBu3DO3A, formaldehyde, and Bn2NCH2PO2H2 ethyl ester. The ligand protonation and metal ion (Gd3+, Cu2+, and Zn2+) stability constants were similar to those of the parent DOTA, whereas the basicity of the side-chain amino group of the complexes (log KA = 5.8) was 1 order of magnitude lower than that of the free ligand (log KA = 6.8). The presence of one bound water molecule in both deprotonated and protonated forms of the gadolinium(III) complex was deduced from the solid-state X-ray diffraction data [gadolinium(III) and dysprosium(III)], from the square antiprism/twisted square antiprism (SA/TSA) isomer ratio along the lanthanide series, from the fluorescence data of the europium(III) complex, and from the 17O NMR measurements of the dysprosium(III) and gadolinium(III) complexes. In the gadolinium(III) complex with the deprotonated amino group, water exchange is extremely fast (τM = 6 ns at 25 °C), most likely thanks to the high abundance of the TSA isomer and to the presence of a proximate protonable group, which assists the water-exchange process. The interaction between lanthanide(III) complexes and HSA is pH-dependent, and the deprotonated form is bound much more efficaciously (∼13% and ∼70% bound complex at pH = 4 and 7, respectively). The relaxivities of the complex and its HSA adduct are also pH-dependent, and the latter is approximately 2-3 times increased at pH = 4-7. The relaxivity for the supramolecular HSA-complex adduct ( r1b) is as high as 52 mM-1 s-1 at neutral pH (at 20 MHz and 25 °C). The findings of this study stand as a proof-of-concept, showing the ability to manipulate an albumin-drug interaction, and thus the blood pool residence time of the drug, by introducing a positive charge in a side-chain amino group.

Low-molecular-weight paramagnetic 19F contrast agents for fluorine magnetic resonance imaging.

OBJECTIVE: 19F MRI requires biocompatible and non-toxic soluble contrast agents with high fluorine content and with suitable 19F relaxation times. Probes based on a DOTP chelate with 12 magnetically equivalent fluorine atoms (DOTP-tfe) and a lanthanide(III) ion shortening the relaxation times were prepared and tested. METHODS: Complexes of DOTP-tfe with trivalent paramagnetic Ce, Dy, Ho, Tm, and Yb ions were synthetized and characterized. 19F relaxation times were determined and compared to those of the La complex and of the empty ligand. In vitro and in vivo 19F MRI was performed at 4.7 T. RESULTS: 19F relaxation times strongly depended on the chelated lanthanide(III) ion. T1 ranged from 6.5 to 287 ms, T2 from 3.9 to 124.4 ms, and T2* from 1.1 to 3.1 ms. All complexes in combination with optimized sequences provided sufficient signal in vitro under conditions mimicking experiments in vivo (concentrations 1.25 mM, 15-min scanning time). As a proof of concept, two contrast agents were injected into the rat muscle; 19F MRI in vivo confirmed the in vivo applicability of the probe. CONCLUSION: DOTP-based 19F probes showed suitable properties for in vitro and in vivo visualization and biological applications. The lanthanide(III) ions enabled us to shorten the relaxation times and to trim the probes according to the actual needs. Similar to the clinically approved Gd3+ chelates, this customized probe design ensures consistent biochemical properties and similar safety profiles.

Improved Conjugation, 64-Cu Radiolabeling, in Vivo Stability, and Imaging Using Nonprotected Bifunctional Macrocyclic Ligands: Bis(Phosphinate) Cyclam (BPC) Chelators.

Bifunctional derivatives of bis(phosphinate)-bearing cyclam (BPC) chelators bearing a carboxylate, amine, isothiocyanate, azide, or cyclooctyne in the BP side chain were synthesized. Conjugations required no protection of phosphinate or ring secondary amine groups. The ring amines were not reactive (proton protected) at pH < ∼8. For isothiocyanate coupling, oligopeptide N-terminal α-amines were more suitable than alkyl amines, e.g., Lys ω-amine (p Ka ∼7.5-8.5 and ∼10-11, respectively) due to lower basicity. The Cu-64 labeling was efficient at room temperature (specific activity ∼100 GBq/µmol; 25 °C, pH 6.2, ∼100 ligand equiv, 10 min). A representative Cu-64-BPC was tested in vivo showing fast clearance and no nonspecific radioactivity deposition. The monoclonal anti-PSCA antibody 7F5 conjugates with thiocyanate BPC derivative or NODAGA were radiolabeled and studied in PC3-PSCA tumor bearing mice by PET. The radiolabeled BPC conjugate was accumulated in the prostate tumor with a low off-target uptake, unlike Cu-64-labeled NODAGA-antibody conjugate. The BPC chelators have a great potential for theranostic applications of the Cu-64/Cu-67 matched pair.

Lanthanide(iii) complexes of monophosphinate/monophosphonate DOTA-analogues: effects of the substituents on the formation rate and radiolabelling yield.

H4dota and its analogues are routinely used for complexation of lanthanide radioisotopes in nuclear medicine. Many of the radioisotopes have short half-lives and, thus, the complexation rate plays an important role. Notwithstanding that, the relationship between ligand structures and complexation rates is not well understood. Here we report a complexation study of H4dota and its analogues bearing one phosphonate or phosphinate pendant arm. The substituents on the phosphinate group were non-coordinating (-H) or contained another coordinating group (-CH2N(CH2COOH)2, -CH2PO2H2 or -CH2NH2). The basicity of ligands, stability of reaction intermediates, formation rates of CeIII complexes, and 177LuIII radiolabelling were studied. The complexation rates and labelling yields do not show any correlation with ligand basicity. In contrast, the additional chelating group attached to the pendant arm plays an important role. A decreased complexation rate and lower labelling yield were found for compounds bearing an additional amino group, whereas improved properties were found for the compound bearing a geminal bis(phosphinate) pendant arm. It indicates that the introduction of chelating pendant arms with acidic coordinating groups might be a promising strategy to improve radiolabelling of macrocyclic carriers with metal radioisotopes.

Efficient formation of inert Bi-213 chelates by tetraphosphorus acid analogues of DOTA: towards improved alpha-therapeutics.

BACKGROUND: The recently growing interest in targeted alpha-therapy (TAT) calls for improvement of the labelling chemistry of the corresponding radionuclides. 213BiIII is a short-lived alpha emitter which emits only one alpha particle in its decay chain. Hence, it might be safer in application than other respective nuclides, such as 223Ra or 225Ac, because no alpha-emitting daughters are released upon recoil. We investigated cyclen derivatives with phosphorus-containing pendant arms regarding their suitability for 213Bi labelling. RESULTS: The concentration dependency of 213Bi labelling at 25 °C and 95 °C was determined for DOTP, DOTPH, DOTPEt, and DOTPI, as well as for DOTA and CHX-A"-DTPA for comparison. The labelling efficiency of the phosphorus-containing ligands was at least comparable to CHX-A"-DTPA and exceeded that of DOTA. DOTP was most efficient, requiring chelator concentrations for labelling which were approx. two orders of magnitude lower than those required for CHX-A"-DTPA, both at 25 °C and 95 °C. The 213Bi complexes of phosphorus ligands furthermore showed a higher stability against demetallation (> 96% of intact complex after 120-min incubation in plasma were found for DOTP, DOTPH, and DOTPEt, compared to 85% for DOTA and 76% for CHX-A"-DTPA). CONCLUSION: Cyclen derivatives bearing four N-methylenephosphonic or -phosphinic acid substituents, e.g., DOTP, are capable of complexing the alpha-emitting radionuclide 213BiIII with higher efficiency and in-vitro stability than the current gold standards DOTA and CHX-A"-DTPA.

NOTA Complexes with Copper(II) and Divalent Metal Ions: Kinetic and Thermodynamic Studies.

H3nota derivatives are among the most studied macrocyclic ligands and are widely used for metal ion binding in biology and medicine. Despite more than 40 years of chemical research on H3nota, the comprehensive study of its solution chemistry has been overlooked. Thus, the coordination behavior of H3nota with several divalent metal ions was studied in detail with respect to its application as a chelator for copper radioisotopes in medical imaging and therapy. In the solid-state structure of the free ligand in zwitterionic form, one proton is bound in the macrocyclic cavity through a strong intramolecular hydrogen-bond system supporting the high basicity of the ring amine groups (log Ka = 13.17). The high stability of the [Cu(nota)]- complex (log KML = 23.33) results in quantitative complex formation, even at pH <1.5. The ligand is moderately selective for Cu(II) over other metal ions (e.g., log KML(Zn) = 22.32 and log KML(Ni) = 19.24). This ligand forms a more stable complex with Mg(II) than with Ca(II) and forms surprisingly stable complexes with alkali-metal ions (stability order Li(I) > Na(I) > K(I)). Thus, H3nota shows high selectivity for small metal ions. The [Cu(nota)]- complex is hexacoordinated at neutral pH, and the equatorial N2O2 interaction is strengthened by complex protonation. Detailed kinetic studies showed that the Cu(II) complex is formed quickly (millisecond time scale at cCu ≈ 0.1 mM) through an out-of-cage intermediate. Conversely, conductivity measurements revealed that the Zn(II) complex is formed much more slowly than the Cu(II) complex. The Cu(II) complex has medium kinetic inertness (τ1/2 46 s; pH 0, 25 °C) and is less resistant to acid-assisted decomplexation than Cu(II) complexes with H4dota and H4teta. Surprisingly, [Cu(nota)]- decomplexation is decelerated in the presence of Zn(II) ions due to the formation of a stable dinuclear complex. In conclusion, H3nota is a good carrier of copper radionuclides because the [Cu(nota)]- complex is predominantly formed over complexes with common impurities in radiochemical formulations, Zn(II) and Ni(II), for thermodynamic and, primarily, for kinetic reasons. Furthermore, the in vivo stability of the [Cu(nota)]- complex may be increased due to the formation of dinuclear complexes when it interacts with biometals.