Innovative smart probes for molecular imaging applications

Programa Oficial de Doutoramento en Química Ambiental e Fundamental. 5031V01[Abstract] The main purpose of the present doctoral thesis is the development of new systems based on metal complexes for their application as contrast agents in Magnetic Resonance Imaging (MRI). To achieve this aim, this study encompasses the design, synthesis and characterisation of new metal complexes of the lanthanide ions or transition metal ions (Mn2+ or Ni2+), as well as a detailed investigation of their structures and physicochemical properties, both in solution and in the solid state. In addition, in vitro studies have also been carried out to assess their potential as contrast agents in MRI applications. Theoretical studies were performed using density functional theory (DFT) providing structural information of the complexes in solution, as well as information at the molecular level on the mechanisms that provide MRI response. The complexes presented in this work provide response at different frequencies (1H or 19F), either following the classical mechanism of longitudinal relaxation enhancement (Mn2+, Gd3+), or through the chemical exchange saturation transfer mechanism. This project was executed within the framework of the European COST Action CA15209 ("European Network on NMR Relaxometry"), funded by the European Science Foundation. Furthermore, it has been funded by the Research Projects “New Generation of Contrast Agents for Magnetic Resonance Imaging (MRI) Alternative to Gadolinium Derivatives” (CTQ2016-76756-P), “Innovative Smart Probes for Molecular Imaging Applications” (CTQ2013-43243-P) and the grant for predoctoral contracts for doctoral training associated to this Project (FPI grant, BES-2014-068399), funded by the Ministerio de Economía y Competitividad (Ministry of Economy and Competitiveness). The results achieved during this project lays the foundations for providing new products for medical application with competitive advantages over the current state-of-art.[Resumen] El objetivo principal de la presente tesis doctoral es el desarrollo de nuevos sistemas basados en complejos metálicos para su aplicación como agentes de contraste en la técnica de imagen por resonancia magética (IRM). Para alcanzar este objetivo, este estudio engloba el diseño, síntesis y caracterización de nuevos complejos metálicos de los iones lantánidos o iones de transición (Mn2+ o Ni2+), así como un estudio detallado de sus estructuras y propiedades fisicoquímicas, tanto en disolución como en estado sólido. Además, también se ha llevado a cabo el estudio in vitro de su capacidad para actuar como agentes de contraste. Los estudios teóricos realizados mediante la teoría del funcional de densidad (DFT) proporcionaron información estructural de las sustancias en disolución, así como información a nivel molecular, sobre los mecanismos que proporcionan respuesta mediante IRM. Los compuestos estudiados en este trabajo proporcionan respuesta a frecuencias diferentes (1H o 19F), bien mediante el mecanismo clásico de relajación longitudinal (Mn2+, Gd3+), o mediante el mecanismo de transferencia de saturación por intercambio químico. Este proyecto fue realizado en el marco de la acción europea COST CA15209 ("European Network on NMR Relaxometry"), financiada por la Fundación Europea de la Ciencia, y ha sido subvencionada con cargo a los proyectos de investigación “Nueva Generación de Agentes de Contraste Para Imagen por Resonancia Magnética (IRM) Alternativos a los Derivados de Gadolinio” (CTQ2016-76756-P), “Nuevas Sondas Inteligentes para Aplicación en Imagen Molecular” (CTQ2013-43243-P) y la ayuda para contratos predoctorales para la formación de doctores asociada a este proyecto (Ayuda FPI, BES-2014-068399), financiadas por el Ministerio de Economía y Competitividad. El trabajo realizado durante este proyecto sienta las bases para la obtención de nuevos productos de aplicación médica con ventajas competitivas respecto a los compuestos utilizados en la actualidad.[Resumo] O obxectivo principal da presente tese de doutoramento é o desenvolvemento de novos sistemas baseados en complexos metálicos para a súa aplicación como axentes de contraste na técnica de imaxe por resonancia magnética (IRM). Para acadar este obxectivo, o estudio engloba o deseño, síntese e caracterización de novos complexos metálicos dos ións lantánidos ou ións de transición (Mn2+ ou Ni2+), así como un estudo detallado das súas estruturas e propiedades fisicoquímicas, tanto en disolución coma no estado sólido. Ademais, tamén se levou a cabo o estudo in vitro da súa capacidade para actuar como axentes de contraste. Os estudos teóricos realizados coa teoría do funcional de densidade (DFT) proveron información estrutural dos complexos en disolución, así como información a nivel molecular, dos mecanismos que proporcionan resposta mediante IRM. Os compostos estudados neste traballo proporcionan resposta a frecuencias diferentes (1H ou 19F), ben mediante o mecanismo clásico de relaxación lonxitudinal (Mn2+, Gd3+), ou mediante o mecanismo de transferencia de saturación por intercambio químico. Este proxecto realizouse no marco da acción europea COST CA15209 (“European Network on NMR Relaxometry”), financiada pola Fundación Europea da Ciencia, e foi subvencionada con cargo aos proxectos de investigación “Nova Xeración de Axentes de Contraste Para Imaxe por Resonancia Magnética (IRM) Alternativos aos Derivados de Gadolinio” (CTQ2016-76756-P), “Novas Sondas Intelixentes para Aplicación en Imaxe Molecular” (CTQ2013-43243-P) e a axuda para contratos predoutorais para a formación de doutores asociada a este proxecto (Axuda FPI, BES-2014-068399), financiadas polo Ministerio de Economía e Competitividade. O traballo realizado durante este proxecto senta as bases para a obtención de novos produtos de aplicación médica con vantaxes competitivas respecto os compostos utilizados na actualidade

Preparación y caracterización de nanopartículas magnéticas biocompatible

[Resumen]En este Trabajo de Fin de Grado (TFG) se ha llevado a cabo la preparación y caracterización morfológica y estructural de nanopartículas de magnetita, Fe3O4. Las nanopartículas de Fe3O4 se han preparado mediante métodos de síntesis en disolución no acuosa, y se ha hecho uso de agentes surfactantes para modificar su morfología y crecimiento. Además, se ha realizado el recubrimiento de éstas con un material biocompatible, sílice. Para su caracterización se han utilizado técnicas de difracción de rayos X de polvo cristalino (DRX), espectroscopia infrarroja (IR), análisis termogravimétrico (ATG), microscopia electrónica de transmisión (MET) y dispersión de luz dinámica (DLS).[Abstract] In this TFG we have carried out the preparation and morphological and structural characterization of magnetite nanoparticles, Fe3O4. Fe3O4 nanoparticles were synthesized by a nonaqueous method, and we have used capping agents to modify their growth and final morphology. Furthermore, nanoparticles have been coated with biocompatible SiO2. For their characterization, we have used Powder X-Ray Diffraction (XRD), Infrared Spectroscopy (IR), Thermogravimetric Analysis (TGA), Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS).[Resumo] Neste Traballo de Fin de Grao leváronse a cabo a preparación e caracterización morfolóxica e estrutural de nanopartículas de magnetita, Fe3O4. As nanopartículas de Fe3O4 preparáronse mediante métodos de síntese en disolución non acuosa, e usáronse axentes surfactantes para modificar a súa morfoloxía y crecemento. Ademais, realizouse o recubrimento destas cun material biocompatible, sílice. Para a súa caracterización, empregáronse as técnicas de difracción de raios X de po cristalino (DRX), espectroscopia infravermella (IR), análise termogravimétrico (ATG), microscopia electrónica de transmisión (MET) e dispersión de luz dinámica (DLS).Traballo fin de grao (UDC.CIE). Química. Curso 2012/201

Controlling Water Exchange Rates in Potential Mn²⁺-Based MRI Agents Derived From NO2A²⁻

[Abstract] We report a series of pentadentate ligands based on a 1,4,7-triazacyclononane-1,4-diacetic acid (H2NO2A) containing different substituents attached to the third nitrogen atom of the macrocyclic unit. Detailed 1H Nuclear Magnetic Relaxation Dispersion (NMRD) characterisation of the corresponding Mn2+ complexes suggests the formation of six-coordinate species in solution containing an inner-sphere water molecule. This was confirmed by recording the transverse 17O relaxation time and chemical shift measurements. The water exchange rate of the coordinated water molecule was found to be strongly influenced by the nature of the substituent R at position 7 of the triazacyclononane unit (R = Me, k298ex = 62.6 × 107 s−1; R = Bz, k298ex = 4.4 × 107 s−1; R = 1-phenylethyl, k298ex = 2.6 × 107 s−1). The decreasing exchange rates are explained by the increasing bulkiness of the substituent, which hinders the approach of the entering water molecule in an associatively activated water exchange mechanism. This is supported by DFT calculations (M062X/TZVP), which confirm the associative nature of the water exchange reaction. A potentially decadentate ligand containing two NO2A units linked by a xylenyl spacer in the meta position was also synthesised. The corresponding binuclear Mn2+ complex contains two metal ions with different hydration numbers, as evidenced by 1H NMRD and 17O NMR measurements. DFT calculations show that this is related to the presence of a bridging bidentate μ–η1-carboxylate group connecting the two metal centers. The results reported in this work provide a straightforward strategy to control the exchange rate of the coordinated water molecule in this family of MRI contrast agent candidates.Authors R. P.-P., I. B., E. I., C. P.-I. and D. E.-G. thank Ministerio de Economía y Competitividad (CTQ2016-76756-P) and Xunta de Galicia (ED431B 2017/59 and ED431D 2017/01) for generous financial support and Centro de Supercomputación of Galicia (CESGA) for providing the computer facilities. R. P.-P. thanks Ministerio de Economía y Competitividad for a PhD FPI grant (BES-2014-068399) and a fellowship for a short term stay in Alessandria (EEBB-I-18-13075). M. B. and F. C. are grateful to Università del Piemonte Orientale for financial support (Ricerca locale 2016). This work was carried out within the framework of the COST CA15209 Action "European Network on NMR Relaxometry"Xunta de Galicia; ED431B 2017/59Xunta de Galicia; ED431D 2017/0

Water exchange rates and mechanisms in tetrahedral [Be(H2O)4]2+ and [Li(H2O)4]+ complexes using DFT methods and cluster‐continuum models

[Abstract] The water exchange reactions in aquated Li+ and Be2+ ions were investigated with density functional theory calculations performed using the [Li(H2O)4]+·14H2O and [Be(H2O)4]2+·8H2O systems and a cluster‐continuum approach. A range of commonly used functionals predict water exchange rates several orders of magnitude lower than the experimental ones. This effect is attributed to the overstabilization of coordination number four by these functionals with respect to the five‐coordinated transition states responsible for the associative (A) or associative interchange (Ia) water exchange mechanisms. However, the M06 and M062X functionals provide results in good agreement with the experimental data: M062X/TZVP calculations yield a concerted Iamechanism for the water exchange in [Be(H2O)4]2+·8H2O that gives an average residence time of water molecules in the first coordination sphere of 260 μs. For [Li(H2O)4]+·14H2O the water exchange reaction is predicted to follow an A mechanism with a residence time of inner‐sphere water molecules of 25 ps.Ministerio de Economía y Competitividad; CTQ2013‐43243‐PMinisterio de Economía y Competitividad; CTQ2015‐71211‐RED

Developing the family of picolinate ligands for Mn2+ complexation

[Abstract] We have reported here a series of ligands containing pentadentate 6,6′-(azanediylbis(methylene))dipicolinic acid units that differ in the substituent present at the amine nitrogen atom (acetate: H3DPAAA; phenyl: H2DPAPhA; dodecyl: H2DPAC12A; 4-hexylphenyl: H2DPAC6PhA). The protonation constants of the hexadentate DPAAA3− and pentadentate DPAPhA2−ligands and the stability constants of their Mn2+ complexes were determined using pH-potentiometry (25 °C, 0.15 M NaCl). The mono-hydrated [Mn(DPAAA)]− complex (logKMnL = 13.19(5)) was found to be considerably more stable than the bis-hydrated [Mn(DPAPhA)] analogue (logKMnL = 9.55(1)). A detailed 1H and 17O NMR relaxometric study was carried out to determine the parameters that govern the proton relaxivities of these complexes. The [Mn(DPAC12A)] complex, which contains a dodecyl lipophilic chain, forms micelles in solution characterized by a critical micellar concentration (cmc) of 96(9) μM. The lipophilic [Mn(DPAC6PhA)] and [Mn(DPAC12A)] derivatives form rather strong adducts with Human Serum Albumin (HSA) with association constants of 7.1 ± 0.1 × 103 and 1.3 ± 0.4 × 105 M−1, respectively. The X-ray structure of the complex {K(H2O)4}{[Mn(DPAAA)(H2O)]}2 shows that the Mn2+ ion in [Mn(DPAAA)]− is coordinated to the six donor atoms of the ligand, a coordinated water molecule completing the pentagonal bipyramidal coordination environment.Ministerio de Economía y Competitividad; CTQ2015-71211-REDTMinisterio de Economía y Competitividad; CTQ2016-76756-

Reinforced Ni(ɪɪ)-Cyclam Derivatives as Dual ¹H/¹⁹F MRI Probes

[Abstract] Reinforced cross-bridged Ni²⁺-cyclam complexes were functionalised with pendant arms containing both amide protons and CF₃ groups that lead to a dual ¹H/¹⁹F response. The resulting complexes possess very high inertness favourable for MRI applications. The paramagnetism of the Ni²⁺ ion shifts the amide resonance 56 ppm away from bulk water favouring the chemical exchange saturation transfer (CEST) effect and shortening the acquisition times in ¹⁹F magnetic resonance imaging (MRI) experiments, thus enhancing the signal-to-noise ratios compared to the fluorinated diamagnetic reference.Authors R. P.-P., D. E.-G. and C. P.-I. thank Ministerio de Economía y Competitividad (CTQ2016-76756-P) and Xunta de Galicia (ED431B 2017/59 and ED431D 2017/01) for generous financial support. R. P.-P. thanks Ministerio de Economía y Competitividad for a PhD FPI grant (BES-2014-068399) and a fellowship for a short term stay in Tuebingen (EEBB-I-17-12213). T. S. thanks the German Academic Exchange Service (DAAD) for the PhD fellowshipXunta de Galicia; ED431B 2017/59Xunta de Galicia; ED431D 2017/0

Lanthanide complexes with 1 H paraCEST and 19 F response for magnetic resonance imaging applications

We present a detailed study of the lanthanide(III) complexes with cyclen-based ligands containing phenylacetamide pen-dants that incorporate CF3 group(s) at different distances from the metal ion. The complexes exhibit square antiprismatic (SAP) coordination in solution, as demonstrated by the analysis of the Yb3+ induced paramagnetic shifts and the X ray structure of the [YbL3] complex. Luminescence lifetime measurements and a detailed 1H and 17O relaxometric characteriza-tion confirmed the presence of an inner-sphere water molecule. The Tm3+ complexes provide Chemical Exchange Satura-tion Transfer (CEST) response upon saturation at the frequency of the amide protons. A 19F relaxation study provided accu-rate estimates of the Ln···F distances that were used to rationalize the efficiency of the complexes as 19F MRI probes, which was tested in vitro using MRI phantom studies.Agencia Estatal de Investigación | Ref. CTQ2016-76756-PXunta de Galicia | Ref. Ref. ED431B 2017/59Xunta de Galicia | Ref. ED431D 2017/01Ministerio de Economía y Competitividad | Ref. BES-2014-068399Ministerio de Economía y Competitividad | Ref. EEBB-I-17-12213Ministerio de Economía y Competitividad | Ref. EEBB-I-18-13075Universitá degli Studi del Piemonte Oriental

A Pentadentate Member of the Picolinate Family for Mn(ɪɪ) Complexation and an Amphiphilic Derivative

[Abstract] We report a pentadentate ligand containing a 2,2′-azanediyldiacetic acid moiety functionalized with a picolinate group at the nitrogen atom (H3paada), as well as a lipophylic derivative functionalized with a dodecyloxy group at position 4 of the pyridyl ring (H3C12Opaada). The protonation constants of the paada3− ligand and the stability constant of the Mn(II) complex were determined using a combination of potentiometric and spectrophotometric titrations (25 °C, 0.15 M NaCl). A detailed relaxometric characterisation was accomplished by recording 1H Nuclear Magnetic Relaxation Dispersion (NMRD) profiles and 17O chemical shifts and relaxation rates. These studies provide detailed information on the microscopic parameters that control their efficiency as relaxation agents in vitro. For the sake of completeness and to facilitate comparison, we also characterised the related [Mn(nta)]− complex (nta = nitrilotriacetate). Both the [Mn(paada)]− and [Mn(nta)]− complexes turned out to contain two inner-sphere water molecules in aqueous solution. The exchange rate of these coordinated water molecules was slower in [Mn(paada)]− (k298ex = 90 × 107 s−1) than in [Mn(nta)]− (k298ex = 280 × 107 s−1). The complexes were also characterised using both DFT (TPSSh/def2–TZVP) and ab initio CAS(5,5) calculations. The lipophylic [Mn(C12Opaada)]− complex forms micelles in solution characterised by a critical micellar concentration (cmc) of 0.31 ± 0.01 mM. This complex also forms a rather strong adduct with Bovine Serum Albumin (BSA) with an association constant of 5.5 × 104 M−1 at 25 °C. The enthalpy and entropy changes obtained for the formation of the adduct indicate that the binding event is driven by hydrophobic interactions.Authors R. P.-P., I. B., E. I., C. P.-I. and D. E.-G. thank Ministerio de Economía y Competitividad (CTQ2016-76756-P) and Xunta de Galicia (ED431B 2017/59 and ED431D 2017/01) for generous financial support and Centro de Supercomputación of Galicia (CESGA) for providing the computer facilities. R. P.-P. thanks Ministerio de Economía y Competitividad for a PhD FPI grant (BES-2014-068399) and a fellowship for a short term stay in Alessandria (EEBB-I-18-13075). R. U.-V. thanks Xunta de Galicia for a PhD grant (ED481A-2018/314). M.B. and F.C. are grateful to Università del Piemonte Orientale for financial support (Ricerca locale 2016). This work was carried out within the framework of the COST CA15209 Action “European Network on NMR Relaxometry”Xunta de Galicia; ED431B 2017/59Xunta de Galicia; ED431D 2017/01Xunta de Galicia; ED481A-2018/31

Synthesis of Organosilicon Ligands for Europium (III) and Gadolinium (III) as Potential Imaging Agents

The relaxivity of MRI contrast agents can be increased by increasing the size of the contrast agent and by increasing concentration of the bound gadolinium. Large multi-site ligands able to coordinate several metal centres show increased relaxivity as a result. In this paper, an “aza-type Michael” reaction is used to prepare cyclen derivatives that can be attached to organosilicon frameworks via hydrosilylation reactions. A range of organosilicon frameworks were tested including silsesquioxane cages and dimethylsilylbenzene derivatives. Michael donors with strong electron withdrawing groups could be used to alkylate cyclen on three amine centres in a single step. Hydrosilylation successfully attached these to mono-, di-, and tri-dimethylsilyl-substituted benzene derivatives. The europium and gadolinium complexes were formed and studied using luminescence spectroscopy and relaxometry. This showed the complexes to contain two bound water moles per lanthanide centre and T1 relaxation time measurements demonstrated an increase in relaxivity had been achieved, in particular for the trisubstituted scaffold 1,3,5-tris((pentane-sDO3A)dimethylsilyl)benzene-Gd3. This showed a marked increase in the relaxivity (13.1 r1p/mM−1s−1)