Final Report Summary - PHIPSPIN (Towards long-lived hyperpolarised spin state) Summary description of the project objectives Hyperpolarisation techniques such as para-hydrogen-induced polarisation (PHIP) produce very strong signal enhancements of magnetic resonance signals in nuclear magnetic resonance (NMR) or magnetic resonance imaging (MRI). They open up new application areas in chemistry, biochemistry or medicine. In the case of PHIP this signal enhancement is produced by a chemical reaction. The PHIPSPIN project concentrates on how the molecular structure and the physical state of the sample affect the lifetime of the hyperpolarised spin state. In particular, the question is raised to what extend the molecular structure, molecular size and isotopic composition influence the relaxation time of the hyperpolarised spin state in solids and liquids. To answers these questions, a detailed investigation of the relevant relaxation mechanisms operating in liquid and solid hyperpolarised samples are planned for different molecular systems and different catalysts (including also heterogenised ones). The knowledge of these relaxation mechanisms can be employed as a guideline for designing long lifetime PHIP active molecules which are applicable as magnetic markers in MRI. The project was intended to conclude with a range of applications, publications as well as international conferences and other scientific meetings. Eventually, the PHIPSPIN project was purposed to train research fellow towards scientific maturity that will allow him to be an independent researcher. Description of the work performed since the beginning of the project Training At the beginning of the project the fellow was trained in number of NMR methods to gain the expertise, necessary both for the realisation of the PHIPSPIN project and for further development of the scientific career of the researcher. This research training included both theoretical and experimental aspects of modern NMR spectroscopy and technology. The theoretical training involved the mathematical tools for a detailed description of the PHIP phenomenon in Hilbert and Liouville space formalisms employing state-of-the-art computational methods. During this training special attention was devoted to NMR pulse influence on the PHIP spin states and to the PHIP line shape analysis. In particular, the fellow became familiar with advanced PHIP NMR simulations employing the MatLab environment. During the experimental training the researcher got familiar with the methodology of para-hydrogen generation, hyperpolarisation of appropriate molecular system, running of the PHIP experiment (special attention has been paid to polarisation transfer methodology and development of the new PHIP-pulse sequence). Moreover, the professional skills of the fellow have been enriched with techniques of preparation and NMR-characterisation of the PHIP active catalysts immobilised on the surface of mesoporous materials, the PHIP techniques in biphasic systems and the PHIP in gas phase. Furthermore, the fellow learned other NMR methods that are not directly concerned with the PHIPSPIN project. In particular, the researcher gained knowledge about time-domain benchtop NMR instruments employed for product quality control and rotational-echo double-resonance (REDOR) employed for structure determination of peptides. Eventually, the researcher had also the opportunity to become familiar with other physic-chemical characterisation techniques available in the host institute as for example high-performance liquid chromatography (HPLC), gas chromatography (GC), ultraviolet / visual (UV / VIS) etc. New technique / methodology development The project began with the preparation of appropriate molecular models for relaxation measurements. Basically, this model consists of two fragments - the 'PHIP active fragment' (where the hydrogenation process takes place) and the part which is changed or functionalised by introducing an appropriate functional group (also fully or partially deuterated). A model with such a kind of framework was realised by phenyl propiolic acid (PPA) esters in which the 'PHIP active centre' is a phenyl ring - triple bond - carboxyl group, where the ester group as an alkyl chain is modified by changing the length or isotopic composition. In the next step a series of relaxation experiments have been done in liquid and solid state on these models. While the PHIP experiments in the liquid state experiments showed strong signal enhancements, the PHIP experiments in solid state gave no visible enhancements. The relaxation experiments from the liquid state suggested that the isotopic composition of the ester fragment has no influence on the relaxation time, and the PHIP effect can be observed even for long ester chains (thus large molecular system). Encouraged by these promising results we decided to check the PHIP activity of a larger molecular system with central biological importance. As system we chose the protease inhibitor SFTI-1, which inhibits the cancer related enzyme matriptase. We succeeded to insert propargylglycine as PHIP active non-native amino-acid into the peptide sequence of the SFTI-1. The PHIP activity of SFTI-1 with different catalyst and solvents was tested. The modified SFTI-1 turned out to be PHIP active and the best PHIP signal was observed for (1,4-Bis(diphenylphosphino)butane)(1,5-cyclooctadiene)rhodium(I) tetrafluoroborate as a catalyst and methanol as a solvent. An important problem in the application of PHIP enhanced NMR is that fairly expensive catalysts are necessary to perform the hydrogenation reaction. In our studies of the PHIP reactions of immobilised catalysts, we discovered that even simple metal salts can work as a PHIP catalyst. Thus we took a closer look at this inorganic system and we examined the PHIP catalytic activity of RhCl3.xH2O and IrCl3.xH2O. As a model reaction the hydrogenation of styrene in different solvents like methanol-d4 and acetone-d6 was chosen. To prove that the results for RhCl3.xH2O are also transferable to other transition metal salts, in a second step IrCl3.xH2O as typical example of an iridium metal salt was tested for PHIP experiments. For both catalyst systems the PHIP effect was investigated as a function of temperature, solvent and substituent at the styrene. Experiments were carried out in methanol-d4 and acetone-d6 at room-temperature and at the boiling point of the solution. As substrates styrene and its para-substituted derivatives 4-methylstyrene and 4-chlorostyrene were chosen. For comparison of the signal enhancement for the different systems and conditions an effective enhancement factor was defined. Such enhancement factor could be used in the future also as a measure for the sensitivity enhancement by PHIP in MRI applications. Description of the main results achieved so far The main results of the project are: (1) It has been shown that also propiolic acid ester derivatives with long alkyl chain are easily hydrogenable and very intensive PHIP signals may be observed. It has been shown that the alkyl chain length in PPA ester derivatives has no essential influence for relaxation of the PHIP signal. Moreover, it has been evidenced that protons from chain are not taking part in dipol-dipol relaxation with the protons streaming from para-H2. (2) For the first time a protease inhibitor has been equipped with a PHIP-active site. Modified SFTI-1 protease inhibitor displays the PHIP activity. Thus our methodology may be applied for low-invasive PHIP labelling of biomolecular systems. Consequently, the structural and functional versatility of peptides provides an access to a variety of target molecules with regard to NMR and MRI PHIP applications. (3) Simple metal catalyst like RhCl3.xH2O and IrCl3.xH2O were found to be PHIP active. This result could be of high interest for the applicability of this technique in technical processes from the economic point of view since these metal catalysts are much cheaper than the expensive metal-organic complexes, which are commonly applied in the PHIP protocol. Finally it has a high potential to study new reaction pathways of simple transition metal catalysts. Expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far) We strongly believe that the new hyperpolarised molecular system would be of central importance for widening the scope of possible NMR and MRI applications. Consequently, the structural and functional versatility of large peptides may provide an access to a variety of target molecules with regard to in vivo PHIP applications. Finally, a simple metal catalyst may open up an access to increase the sensitivity of NMR and MRT by PHIP enhancement using cost-effective catalysts and will be essential for further mechanistic studies of simple transition metal systems. During the whole stay at TU Darmstadt the researcher had regular discussions with professor Buntkowsky in order to control the progress of the research project. The researcher has also participated in the number of scientific conferences, meetings and transferable skills trainings. The researcher was involved in a preparation of a number of grant applications.
