WO2021156063A1

WO2021156063A1 - Method for the preparation of a sample comprising highly polarized nuclear spins and uses and devices for such a method

Info

Publication number: WO2021156063A1
Application number: PCT/EP2021/051265
Authority: WO
Inventors: Jonas MILANI; Mika TAMSKI; Jean-Philippe Ansermet; Christophe Roussel
Original assignee: Ecole Polytechnique Federale de Lausanne EPFL
Current assignee: Ecole Polytechnique Federale de Lausanne EPFL
Priority date: 2020-02-07
Filing date: 2021-01-21
Publication date: 2021-08-12
Anticipated expiration: 2022-08-07

Abstract

Method for the preparation of a sample comprising highly polarized nuclear spins, comprising at least the following steps: a) bringing a polarization system, which using an electrochemical reaction can be converted into an EPR-active radical form thereof, in contact or close proximity with said sample; b) in situ electrochemically forming an EPR-active radical form of said polarization system; c) applying dynamic nuclear polarization techniques in the presence of a magnetic field by applying electromagnetic irradiation with a frequency adapted to transfer spin polarization from the electrons of the polarization system to the nuclear spins of the sample leading to a highly polarized state thereof. d) in situ electrochemically converting said polarization system, which is paramagnetic and comprises an EPR-active radical into a diamagnetic form without an EPR-active radical.

Description

TITLE

METHOD FOR THE PREPARATION OF A SAMPLE COMPRISING HIGHLY POLARIZED NUCLEAR SPINS AND USES AND DEVICES FOR SUCH A METHOD

TECHNICAL FIELD

The present invention relates to a method for the preparation of a sample comprising highly polarized nuclear spins, as well as uses of such a method and devices for carrying out such a method.

PRIOR ART

Dynamic Nuclear Polarization (DNP) designates an array of techniques that seek to enhance Nuclear Magnetic Resonance (NMR) signals by transferring electron’s spin polarization to nuclei thanks to the irradiation of electromagnetic waves around the Electron Paramagnetic Resonance (EPR). Overhauser identified a possible mechanism that he considered applicable for electron and nuclear spins in metals. His prediction was verified and extended to liquids with radicals in solution. The Overhauser Effect (OE) can be obtained in liquids when working with two dipolar-coupled nuclei that have very different gyromagnetic ratios for example between electron’s spin and other nuclear spins. OE designates a mechanism of hyperpolarization when electromagnetic waves are applied at the frequency of EPR. Solid Effect (SE), Cross Effect (CE) and Thermal Mixing (TM) designate other mechanisms of hyperpolarization when electromagnetic waves are applied at the difference or the sum of frequency between EPR and NMR or between these two values. DNP techniques designate all hyperpolarization techniques using electromagnetic wave irradiation to transfer polarization from electrons to nuclei, so it includes the Overhauser effect with electron spins. The polarization is the difference of particle population between the energy level of the spin and is the basis for the NMR signal. A sample polarized or normally polarized is when its polarization is at the Boltzmann equilibrium or thermal equilibrium at a given temperature and a given magnetic field. A sample is considered highly polarized or hyperpolarized when its polarization is higher than the Boltzmann equilibrium or thermal equilibrium at a given temperature and a given magnetic field.

After the demonstration of dissolution DNP in 2003, DNP regained interest and was adapted to the high fields used in modern spectrometers. At high fields, the Overhauser effect requires high microwave powers to achieve saturation, causing the sample solutions to overheat unless great care is taken to avoid this. The Overhauser effect is not only a means to enhance signal. It has been used to study hydration shell, for example.

Hyperpolarization by dissolution dynamic nuclear polarization (d-DNP) is a method to enhance the nuclear magnetic resonance signals by several orders of magnitude, typically >10'000 times. The presence of a radical is required during the hyperpolarization process. However, radicals become unwanted after the process to preserve the hyperpolarized sample, as they induce high relaxation and so a significant loss of the polarization over time. Ji, X., et al. (Nat Commun 8, 13975, 2017) report, that nuclear spin hyperpolarization of relabelled metabolites by dissolution dynamic nuclear polarization can enhance the NMR signals of metabolites by several orders of magnitude, which has enabled in vivo metabolic imaging by MRI. However, because of the short lifetime of the hyperpolarized magnetization (typically <1 min), the polarization process must be carried out close to the point of use. They introduce a concept that markedly extends hyperpolarization lifetimes and enables the transportation of hyperpolarized metabolites. The hyperpolarized sample can thus be removed from the polarizer and stored or transported for use at remote MRI or NMR sites. They show that hyperpoiarization in alanine and glycine survives 16 h storage and transport, maintaining overall polarization enhancements of up to three orders of magnitude. This method is used and limited for nano-crystalline suspensions in toluene. It is based on a two- phase system, one phase for a molecule of interest like alanine and glycine and another phase for the polarizing agent. This second phase limits the application for in vivo application because of the toxicity of the radical and solvent. This method was not investigated with a biocompatible solvent. In this publication the way to store polarization is to use a fast polarizing spin (like ¹H) to hyperpolarize the entire sample by spin diffusion, followed by transfer to a slow polarizing nuclei (as such ¹³C or ¹⁵N). A first difference with the invention presented is that the radical is not removed from the sample. A second difference is the mandatory use of cross polarization coil in their process. The invention presented here is free from the use of any coils. Coils can be used for NMR detection but this is not mandatory.

Gajan et al. (PNAS October 14, 2014 111 (41) 14693-14697) report that hyperpoiarization of substrates for magnetic resonance spectroscopy (MRS) and imaging (MRI) by dissolution dynamic nuclear polarization (d-DNP) usually involves saturating the ESR transitions of polarizing agents (PAs; e.g., persistent radicals embedded in frozen glassy matrices). This approach has shown enormous potential to achieve greatly enhanced nuclear spin polarization, but the presence of PAs and/or glassing agents in the sample after dissolution can raise concerns for in vivo MRI applications, such as perturbing molecular interactions, and may induce the erosion of hyperpoiarization in spectroscopy and MRI. They show that d-DNP can be performed efficiently with hybrid polarizing solids (HYPSOs) with 2, 2, 6, 6- tetramethyl-piperidine-1-oxyl radicals incorporated in a mesostructured silica material and homogeneously distributed along its pore channels. The powder is wetted with a solution containing molecules of interest (for example, metabolites for MRS or MRI) to fill the pore channels (incipient wetness impregnation), and DNP is performed at low temperatures in a very efficient manner. This approach allows high polarization without the need for glass forming agents and is applicable to a broad range of substrates, including peptides and metabolites. During dissolution, HYPSO is physically retained by simple filtration in the cryostat of the DNP polarizer, and a pure hyperpolarized solution is collected within a few seconds. The resulting solution contains the pure substrate, is free from any paramagnetic or other pollutants, and is ready for in vivo infusion. The material presented in this publication is a porous silicate linked with permanent radicals. With it, it is possible to get pure hyperpolarized sample in liquid (and only in liquid). However, the critical point is during the fast dissolution, especially during the few milliseconds when the sample is heated by water but still in contact with electrons. This process leads to a significant loss of hyperpolarized signal. It is also not possible to use this material to store the hyperpolarized sample. The method presented here allows the removal of the radical, and thus storage of the sample.

US2016033590 discloses a method for the preparation of a sample comprising highly polarized nuclear spins, comprising at least the following steps: a) provision of molecules with 1 ,2-dione structural units and/or molecules with 2,5-diene- 1 ,4-dione structural units in the solid state; b) generation of radicals from these molecules by photo induced electron transfer by a first electromagnetic irradiation in the visible or ultraviolet frequency range in the solid state; c) dynamic nuclear polarization in the presence of a magnetic field in the solid state by applying a second electromagnetic irradiation with a frequency adapted to transfer spin polarization from the electrons to the nuclear spins leading to a highly polarized state thereof. Furthermore, uses of correspondingly prepared samples for NMR, MRS and MRI experiments are proposed. This document describes the way to create radical by applied light for example on molecules with 1 ,2-dione structural units and/or molecules with 2,5- diene-1 ,4-dione structural units to create radicals inside the sample. As suggested in the patent, and later shown by Capozzi et. al. (Nat Commun. 8, 15757, 2017) with this method the radicals can be quenched after the polarization transfer by increasing the sample temperature from 77 K to 190 K. In this work the polarization was transferred to ¹³C nuclei, and the polarization was preserved for several days at 4.2 K. The problem with this technique is the necessity to bring the sample temperature to 190 K for quenching of the radical. This heating step reduces the amount of sample polarization down to a few percent. With our proposed method, the sample temperature can be maintained low during the removal of the radicals from the sample, and thus maximal polarization can be preserved for long periods of time.

US2004049108 relates to devices and method for melting solid polarized sample while retaining a high level of polarization. In an embodiment of this invention a sample is polarized in a sample-retaining cup in a strong magnetic field in a polarizing means in a cryostat and then melted inside the cryostat by melting means such as a laser connected by an optical fibre to the interior of the cryostat.

SUMMARY OF THE INVENTION

In this invention, we open up a new application of the hyperpolarization effect (including using OE) by electro-generating and/or removing radicals in solutions or solids from an electroactive redox mediator. Enhancements greater than 20-fold at low fields and room temperature are shown. This value is in the same range as a standard radical like TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl) for OE in the same conditions (at low field and room temperature). For this reason, a similar effect is available at high field and low temperature whenever DNP techniques apply which rely on radicals.

Electrochemical reactions change the oxidation state of a redox mediator at an electrode either by reduction or oxidation reactions. This change in oxidation state can be used to generate or remove a paramagnetic species with at least one unpaired electron, which can be studied by Electron Paramagnetic Resonance (EPR) spectroscopy at low fields, or more recently, at high fields.

The well-known sensitivity issue of NMR renders its application to surface studies tedious. A minimum of 10¹⁷ nuclei is required to begin to envisage surface NMR, whereas a typical surface atom density is around 10¹⁵ cm ². Thus, NMR of electrode surfaces calls for either a large surface area electrode (>100 cm²), where accurate electrochemical potential control may not be possible, or signal enhancement by DNP.

Considering the importance of being able to conduct NMR analysis of electrolytes in confined geometries, and possibly of species chemisorbed on electrodes, here we show how electrochemistry (EC) can serve as a source of radical species for OE experiments and/or for removal of radical species having been used for OE experiments (EC-OE). Hyperpolarized proton NMR is shown, obtained using radicals, which are electrochemica!ly generated in-situ.

The creation/removal of radicals by means of electrochemistry gives an opportunity to create and to annihilate them as required. The proposed invention comprises methods to create radicals by electrochemistry to perform dynamic nuclear polarization and/or to remove them in situ. This includes the creation/annihilation of radicals at electrodes with different surface areas and morphologies. The chemical species responsible for generating or providing the radicals may or may not be bonded to the electrode surface with covalent bonds, or be present as a part of a polymer layer deposited to the electrode surface. With radicals bonded to the electrode surface and polymers, it is possible to use this method in solid, liquid or gas/vapor phases in stop flow or continuous flow experiments. The process proposed by our invention comprises typically the following three steps:

1. Creation of radical with electrochemistry;

2. Hyperpolarization process, this step can include DNP;

3. Remove of the radical from the sample by electrochemistry.

More generally, the process proposed by our invention can comprise the following three steps:

1. Providing a polarization system, which using an electrochemical reaction can be converted into an EPR-active radical form thereof, and converting it into an EPR-active radical form thereof with electrochemistry;

2. Hyperpolarization process, this step can include DNP;

3. Removal of the radical from the sample by electrochemistry or by other means.

The process proposed by our invention can also comprise the following three steps

1. Providing a radical;

2. Hyperpolarization process, this step this step can include DNP;

3. Removal of the radical from the sample by electrochemistry.

Preferably, the process is combined involving generation, DNP and subsequent removal of the radical by electrochemistry in situ.

Then, there is two preferred options to use the sample: a) If the system is in liquid state, the sample can directly be used for analysis in the same probe or with a continuous flow setup in another spatial location. b) If the system is in a frozen state, the sample can then be transferred in an external low temperature storage device such as a dewar with a low magnetic field. Thus, it will be possible to store the sample and use it after transportation in another location.

Dynamic nuclear polarization (DNP) will give a significant advantage on nuclear magnetic resonance (NMR) analysis or Magnetic Resonance Imaging (MRI) scanning. Basically, it increases the signal by a factor > 10Ό00 times for the technique of dissolution-DNP (d- DNP) which is used to rapidly dissolve the frozen sample with hot water or deuterated water and inject the solution in a standard NMR spectrometer. This technique can give enhancement up to 50’000-fold in the condition of final spectrometer in liquid state. The proposed invention gives the opportunity to create and store long lived hyperpolarized samples for NMR or MRI detection. It is a way to provide cheap hyperpolarization for final users (labs or hospitals). This becomes feasible because one is able to remove the radical from the sample after the polarization transfer, significantly extending the lifetime of the sample.

Currently it is very difficult to obtain hyperpolarized samples for NMR or MRI. The cost of a commercial DNP polarizer from GE Healthcare is estimated at US$ 2 Million. This figure does not include maintenance expenses, but gives an idea of the cost structure for any laboratory attempting to acquire DNP polarization capable facilities.

With the development of long-lived, transportable hyperpolarized samples, as described in this patent, if any research laboratory wants to decrease the measurement times related to drug screening, or to study specimens too small or diluted to be detected with the typical sensitivity of the NMR spectrometer, the laboratory can now order the hyperpolarized sample from a specialized company, and then in their own laboratory transfer the hyperpolarization from the purchased sample into their own. This final transfer step can be done routinely with any commercial NMR setup, and thus does not represent unusual complications, but avoids the need to acquire the expensive instrumentation and technical skills required to produce the long-lived, hyperpolarized samples.

DNP helps NMR analysis and MRI scanning by different ways. Here we list three main consequences of the invention:

Drug screening: The complexity of new drugs from biotechnology needs powerful tools for development and quality control purposes. NMR provides a wealth of information and is commonly used. The signal to noise ratio (S/N) can be increased by averaging several scans, and increases as a square root of the no. of scans. However, because of its intrinsically low sensitivity, sometimes days or weeks are needed to record a satisfactory spectrum. DNP offers an opportunity to reduce this measurement time to hours or even minutes. For example, in a situation where averaging of 10Ό00 scans would be needed for an acceptable signal, an increase of 100 in the S/N by other means, such as DNP, would allow the measurement to be performed with a single scan. This is a significant improvement for the entire pharmaceutical industry and will lead to significantly reduced R&D costs. Contrast agent: Within the MRI community, Gadolinium (Gd) is a commonly used contrast agent. Although usually well tolerated by the subjects, in cases such as spleen disease the side effects can vary from mild to severe. Also, a study in 2019 showed significant risks of fetal exposure during pregnancy, and the safety of this contrasting agent remains to be established. Gd is also known to accumulate in the brain and cause long-term damage. DNP hyperpolarized solutions can also act like a contrast agents, introducing a non-invasive option for Gd. The big advantage is that it is possible to choose the target molecule to be polarized, for example water for angiography, as shown by Denysenkov et. al. (Sci Rep 7, 44010, 2017). The method proposed here will remove the radical before the injection in a continuous flow setup and so prevent the toxicity related to radical species.

Alternative to PET scan: The detection of metastasis is essential to significantly enhance the prospects of successful cancer treatment. A common method today is to use PET scan, a powerful and precise method to detect metastasis. The process consists of injection of a radioactive isotope, which according to CHUV documents exposes the patient to equivalent of 7.8 years of natural irradiation. For this reason, PET scan is never applied to a healthy subject, and is highly inadvisable during pregnancy. DNP has provided very promising results for metastasis detection with the additional benefit of being non-invasive. Thus, DNP can contribute to the detection of very early stage cancer in healthy subjects, preventing and reducing the cost of expensive treatments.

Today, DNP equipment are rather expensive for a lab or a hospital. The proposed invention can significantly decrease the cost for the use of DNP, thus democratizing the technique. Previously electrochemistry has been combined with NMR and Electron Paramagnetic Resonance (EPR) spectroscopies to study for example reaction intermediates and products, or kinetics related to electrode processes. However, electrochemical methods have never been used in the context of DNP as an in-situ source of radical species, although many electrochemical reactions intrinsically involve the generation of radical intermediates and/or products.

The feasibility of combining electrochemistry with in-situ NMR and EPR spectroscopies is not obvious. The presence of metal electrodes within an EPR resonator, or subjecting the electrode to strong rf pulses tends to be detrimental for electrochemical experiments, or compromise the NMR performance.

Here it is shown that it is possible to couple these techniques together and handle the emerging challenges. A setup has been assembled comprising an EPR resonator and an NMR coil to combine microwave irradiation to NMR experiment. Also, we have developed and experimented with electrochemical cells that can be assembled to a standard NMR tubes and inserted to our EPR/NMR/DNP setup.

We have generated methyl viologen radical in-situ within the EPR/NMR/DNP spectrometer and detected the effect of the presence of the radical to proton relaxation times. We have also managed to transfer the polarization from electrons of methyl viologen radical to solvent protons (acetonitrile). The enhancement thus far is 23-fold at low field. We have also observed that we can remove the radical from our sample solutions by reversing the electrode potential, showing that the concepts of this proposal work in practice.

We have also successfully functionalized gold and semiconductor surfaces utilizing thiol- chemistry and looked at different redox mediators and spin labels to identify candidates that can serve in our DNP experiments. Also, polymer layers can be electrodeposited to an electrode surface.

In the context of dissolution-DNP, the polarization decreases with Ti. It is well known that radicals induce a high magnetic relaxation, especially at low field.

More generally/alternatively speaking, the present invention relates to the following subject matter:

A method for the preparation of a sample comprising highly polarized nuclear spins using at least one polarization system, which using an electrochemical reaction can be converted from a diamagnetic form without an EPR-active radical into a paramagnetic EPR-active radical form thereof, and/or which is paramagnetic and comprises an EPR-active radical and using an electrochemical reaction can be converted into a diamagnetic form without an EPR-active radical. In other words, the polarization system can be a system containing for example a stable spin label which, without being influenced by an external potential, is in a paramagnetic state and is therefore EPR-active and can be used for DNP, but which can be converted by applying an external potential electrochemically into a system which is diamagnetic and will therefore not disturb the system for example in a subsequent NMR experiment or lead to excessive relaxation. The polarization system can also be one which is diamagnetic without being influenced by an external potential, but which can be converted into a paramagnetic state by applying an electrochemical potential. According to a preferred variant, such a polarization system can be reconverted into the diamagnetic form by applying a correspondingly different electrochemical potential.

When talking about "highly polarized nuclear spins", that means the nuclear spins are polarized more than they are at thermal equilibrium or Boltzmann equilibrium in a given field and at a given temperature, due to the transfer of polarization from the electrons, which have a higher equilibrium spin polarization in the given field and at the given temperature. The method as proposed is involving at least one step of in situ electrochemically forming an EPR-active radical form of a diamagnetic polarization system and/or of in situ electrochemically converting a paramagnetic polarization system comprising an EPR-active radical into a diamagnetic form without an EPR-active radical.

The basic idea is therefore to use an electrochemical reaction for the in-situ generation and/or for the removal of the paramagnetic species used for the DNP process.

When the radical is created, an electric potential is applied to a working electrode and by a redox reaction, a radical is created at the electrode. The radical then diffuses to the bulk solution in liquid state, or stays at the electrode surface if a covalently linked or a polymer- based polarization system is used. In practice, a high surface area electrode with covalent linked radical on its surface, or a polymer based radical, is preferably used for a frozen sample. Even if the radical is fixed, the hyperpolarization diffuses by spin diffusion into the bulk, as well. By applying an opposite electric potential, one is able to oxidize or reduce the radical to quench it.

According to a preferred variant, the method for the preparation of a sample comprising highly polarized nuclear spins, is comprising at least the following steps: a) bringing a polarization system, which using an electrochemical reaction can be converted into an EPR-active radical form thereof, in contact or close proximity with said sample; b) in situ electrochemically forming an EPR-active radical form of said polarization system; c) applying DNP techniques in the presence of a magnetic field by applying electromagnetic irradiation with a frequency adapted to transfer spin polarization from the electrons of the polarization system to the nuclear spins of the sample leading to a highly polarized state thereof.

According to another preferred variant, the method comprises at least the following steps: x) bringing a polarization system, which is paramagnetic and comprises an EPR-active radical, in contact or close proximity with said sample; c) applying dynamic nuclear polarization techniques in the presence of a magnetic field by applying electromagnetic irradiation with a frequency adapted to transfer spin polarization from the electrons of the polarization system to the nuclear spins of the sample leading to a highly polarized state thereof d) in situ electrochemically converting said polarization system, which is paramagnetic and comprises an EPR-active radical into a diamagnetic form without an EPR-active radical. According to this preferred variant step x) can be provided by the above-mentioned sequence a) and b), while c) is the same as the above-mentioned step c), so the paramagnetic property is basically switched on for the DNP process and then switched off for subsequent handling of the sample comprising highly polarized nuclear spins, in particular for subsequent nuclear magnetic resonance measurements or imaging measurements.

According to a preferred embodiment, steps a), b), x), c) and d) are carried out in frozen/solid state, liquid state or gaseous/vapour state of the sample. If the state is liquid state or gaseous/vapour state, the system can be used in a continuous flow mode. According to yet another preferred embodiment, step c) is followed by d) in situ electrochemically re-converting the EPR-active radical form of the polarization system into a non-radical form of the polarization system.

Step c) or step d) as given above can be followed by e) spatially separating the polarization system and the highly polarized sample, preferably in that either the polarization system is immobilized on a carrier and the sample is in a mobile (liquid or gaseous) state and separated from the carrier, or in that the sample is immobilized on a carrier and the polarization system is in a liquid state and separated from the carrier. Or the polarization system including the highly polarized sample in frozen/solid state is transferred into an external low temperature storage device such as a dewar with a magnetic field. The sample is stored for a desired time. Then, as before, separating the polarization system and the highly polarized sample (separation is also possible before transfer to the storage device), preferably in that either the polarization system is immobilized on a carrier and the sample is in a mobile (liquid or gaseous) state and separated from the carrier, or in that the sample is immobilized on a carrier and the polarization system is in a liquid state and separated from the carrier The electromagnetic irradiation in step c) can be preferably applied with a frequency essentially equal to the difference or the sum of the Larmor frequencies of the electron spins and the nuclear spins, respectively, or at the Larmor frequencies of the electron spins in the presence of the applied magnetic field, and/or wherein the magnetic field is a static, preferably essentially homogeneous magnetic field with a strength of at least 0.1 T or of 0.2 T or of at least 0.3 T. Possible are also higher fields such as 1 - 10T or 5 - 9.5T.

The (preferably covalently linked) polarization system is preferably one selected from the following group: polycyclic heteroaromatic systems, preferably molecules based on substituted or unsubstituted imidazole, pyrazole, triazole, tetrazole, pyridine, diazine, triazine, tetrazine, pentazine, azepine, diazepine, preferably dimeric or trimeric forms thereof, preferably substituted and unsubstituted bipyridine, most preferably N,N'-dimethyl-4,4'-bipyridinium, or a member of the quinone family, especially for non-aqueous work.

If the covalently linked polarization system is already in a radical EPR active form as described above in step x) then the radical can be selected for example from TEMPOL (4- hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl) and its derivatives: PROXYL,

Spirobisnitroxide, Arylnitroxide, nitronylnitroxyl, Galvinoxyl, Arylnitroxide or other radicals such as but not limited to poly(2,2,6,6-tetramethylpiperidinyloxy methacrylate) and nitronyl nitroxide radicals.

The electrodes containing a polymer layer can be based on conducting backbone such as but not limited to poly(aniline), poly(pyrrole), poly(thiophene), poly(p-phenylene), poly(vinylcarbazole), Poly(phenylthiophene), Poly(phenylene vinylene), Poly(o- aminophenol) and poly(1,8-diaminonaphthalene), or non-conducting backbone such as Poly(methyl acrylate), Poly(vinyl ether), Polyethylene glycol), Poly(vinylidene fluoride) or polystyrene. The polymers above, conducting or non-conducting, can be carrying a redox probe as part of the polymer back bone or as a pendant molecule (redox polymer) such as but not limited to N,N’-diphenyl-2,3,5,6-tetraketopiperazine (PHP), N,N’-diailyl-2,3,5,6-tetraketopiperazine (AP), N,N’-di-n-propyl-2,3,5,6-tetraketopiperazine (PRP), or a polyimide-based erylene- 3,4,9, 10-tetracarboxylic dianhydride(PTCDA).

Instead of carrying a redox probe the polymers above, conducting or non-conducting, can be in EPR active form carrying a radical spin label as part of their backbone or as a pendant molecule (radical polymer) such as but not limited to TEMPOL (4-hydroxy-2, 2,6,6- tetramethylpiperidin-1-oxyl) and its derivatives: PROXYL, Spirobisnitroxide, Arylnitroxide, nitronylnitroxyl, Galvinoxyl, Arylnitroxide or other radicals such as but not limited to poly(2,2,6,6-tetramethylpiperidinyloxy methacrylate) and nitronyl nitroxide radicals.

The polymer membranes can be grown to the electrode surface by electropolymerization, condensation polymerization, step-growth polymerization, or any other suitable method. Subsequent to step c), and/or subsequent to step d) as given above and/or subsequent to step e) as given above the highly polarized nuclear spins are preferably measured in a nuclear magnetic resonance (NMR) or in a magnetic resonance imaging (MRI) experiment, wherein preferably protons, 13C and/or 15N nuclei or a combination thereof is measured. The steps a)-c), and if used at least one of steps d) and e), as well as the nuclear magnetic resonance (NMR) or magnetic resonance imaging (MRI) experiment can but are not necessarily carried out in one same probe head, but the analysis can be done in a commercial spectrometer or MRI scanner.

The molecule carrying the electrons responsible for providing the hyperpolarization can be grafted to a Working Electrode (WE) surface. This can be achieved for example by 1): grafting an electrochemical mediator or a stable radical to the WE surface via covalent bonding, or 2) depositing a polymer layer with conducting electrons, redox mediators, or stable radicals to the WE surface.

In the first case, if a negative enough potential is initially applied to the WE to force the electrochemical mediator to go through an electro-reduction (producing a radical mediator with an unpaired electron), then the unpaired electron can be removed for the NMR experiment by applying a positive enough potential to force the mediator to go through an electro-oxidation, which removes the unpaired electron. An opposite situation exists if the radical mediator was initially produced via electro-oxidation (applying a positive enough potential). In this case the radical is removed by applying a negative enough potential.

In the second case, the unpaired electrons are intrinsically present in the polymer membrane, and the membrane is part of the WE itself. In this case, the electrons can be removed from the electrode surface by applying a positive enough potential to the WE. In this case, the WE is analogous to a positive plate of a parallel plate capacitor, which is depleted from electrons relative to the negative plate.

If a redox polymer is used, the redox mediator can be used to produce a radical mediator according to step b) above, followed by the annihilation of the radical after the polarization transfer according to step d) above. If a radical polymer is used as described in step x) above, after the polarization transfer the radical is annihilated according to step d) above. The potentials are applied with respect to a Reference/Counter electrode, depending on the type of the setup. The principle is the same whether we work with liquid or frozen samples. The electrode material to which the redox mediators or radicals are linked can be for example a noble metal such as Au, Ag or Pt, but also ferro- or ferrimagnetic materials can be incorporated.

According to a preferred embodiment therefore, said polarization system, which using an electrochemical reaction can be converted form a diamagnetic form without an EPR-active radical into a paramagnetic EPR-active radical form thereof and/or which is paramagnetic and comprises an EPR-active radical and using an electrochemical reaction can be converted into a diamagnetic form without an EPR-active radical, is chemically and/or physically attached to a working electrode or incorporated into a coating on the working electrode used for the electrochemical formation, and/or as part of a polymer, coated or forming part of the working electrode surface.

Furthermore, the present invention relates to a Nuclear magnetic resonance (NMR), magnetic resonance spectroscopy (MRS) or magnetic resonance imaging (MRI) experiment, preferably in vitro or in vivo, wherein in a first step a hyperpolarized sample is prepared using a method as detailed above, and the hyperpolarized sample is used in the experiment, preferably for enhancing the signal-to-noise ratio.

Also the present invention relates to the use of a method according as detailed above for diagnostic purposes (e.g. MRI), in vivo, in vitro, for electrochemical systems, for surface analysis, for the monitoring of electrochemical processes and their mechanisms and kinetics and/or identification of species generated during electrochemical processes, for the analysis of devices for storing energy, for the analysis of catalytic processes, in particular taking place on the surface

Furthermore, the present invention relates to a combined EPR/NMR device or EPR/MRI device, suitable and adapted to apply, in the measurement area, not only a static magnetic field as well as the electromagnetic irradiation required for EPR and NMR, but also an electrochemical potential for the in situ generation of a radical from a polarization system, which using an electrochemical reaction can be converted into an EPR-active radical form thereof, in particular a device suitable and adapted to carry out a method as detailed above. Further embodiments of the invention are laid down in the dependent claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,

Fig. 1 shows a schematic presentation of the experimental setup;

Fig. 2 shows in a) cyclic voltammogram of a 1 mM solution of MV²⁺ in acetonitrile with

200 mM TBAP as supporting electrolyte, recorded at a 3mm diameter gold electrode against a Pt pseudo reference at 50 mV/s; inset: The chemical structure of MV²⁺; the electrode processes are marked to the voltammogram; in b) an EPR spectrum of ca. 100 mM of MV^+' in acetonitrile with 200 mM TBAP; Fig. 3 shows as follows: Before EC: NMR spectrum without and with applied microwaves before electrogeneration of the radical species; after EC: NMR spectrum without and with applied microwaves after electrogeneration of the radical species; the MV⁺' concentration for “After EC” was 3.7 mM; starting conditions: 50 mM of MV²⁺, 50 mM of TBAHP as supporting electrolyte in 20:80 MeOH:ACN (w/w); the "Before EC” spectrum is offset by -50 arbitrary units. DESCRIPTION OF PREFERRED EMBODIMENTS

Electrochemical Overhauser Effect DNP (EC-OE-DNP):

The Overhauser effect for two ½-spins system can be described by considering the evolution of the populations of the four energy levels. For an intermolecular Overhauser effect involving radicals as a solute and nuclei in the solution, we refer to the model proposed by Armstrong and al. (J. Chem. Phys. 2007, 127 (10), 104508) The enhancement e is given by :

, iKsrl e — 1 — pfs -

Yi where The coupling factor p is a function of the rates wo, wi and wz, which are the ¹H dipolar relaxation rate factors for the two ½-spin system and on wo which is the ¹H relaxation rate factor in the absence of radical. In the second expression, the leakage factor f depends of Tio and T which are respectively the relaxation rate constant of solvent without and with free radical presence, k is the relaxivity constant and C is the concentration of free radical. The saturation factor s is a function of the power P, the number of hyperfine lines n and a, a factor related to the electron Ti.

We cannot consider our system as homogenous because the radicals are generated locally at the electrode surface and subsequently distributed in the whole solution by the mass transport. The mass transport is expressed by the Nernst-Planck equation, which describes the total mass density flux / as the sum of the diffusion, electrostatic migration and convection,

where v is the fluid velocity, z the ionic species valence, e the elementary charge, k_B the Boltzmann constant, T the temperature. Because of this mass flux, the distribution of radicals across the sample volume depends on the time and distance from the radical- producing electrode. The electrostatic potential f is negligible because of the screening effect of the electrolyte. The diffusion constant D in the system was determined to be 1.3x10 ⁵ cnr²/s at a 10 pm diameter Pt ultra-micro-electrode. If we consider that the concentration decreases from 10¹⁵ cm ³ to zero in 0.1 cm the diffusion contribution to / is of about 10⁹ cm²/s. Therefore, the slightest convection leads to a far greater J in our electrochemical cell than diffusion does. Indeed, after a couple of seconds of electrolysis, the flux of the bright blue methyl viologen radical (MV^+') became visibly turbulent. This means that, during the DNP experiment, the radical was not homogeneously distributed throughout the sample volume, and the observed DNP enhancement is a spatial average over different local concentrations.

Experimental: Chemicals: methylviologen dichloride (MV²⁺), tetrabutylammonium perchlorate (TBAP), tetrabutylammonium hexafluorophosphate (TBAHP) and anhydrous acetonitrile (ACN) were purchased from Sigma-Aldrich. Anhydrous methanol was from ABCR. All the chemicals used were of highest available purity. The sample solutions were made of a 20:80 MeOH:ACN (w/w) mixture with 50 mM of MV²⁺ and 50 mM of TBAHP.

Figure 1 displays a schematic representation of the experimental setup. The electrochemical cell was assembled into a 4 mm inner-diameter NMR tube. The working electrode (WE a in Fig. 1) was a 250 pm diameter coiled silver wire with ca. 0.5 cm² of surface area. A Pt wire tip was used as a pseudo reference (RE, not shown) and a Pt coil as counter electrode (CE b in Fig. 1). A cotton wool plug (c in Fig. 1) was placed between the WE and the CE to prevent the convection of methyl viologen radicals (MV^+‘), thus confining the electrochemically generated radical ions to the sensitive part of the NMR measurement, i.e. in the volume occupied by the WE. A uniform magnetic field B of 0.3322 T is applied perpendicular to the tube.

The setup is made with a working electrode (WE) and counter electrode (CE). A reference electrode (RE) can be added to the setup for a better potential control. The polarizable sample is in contact with the WE. The sample can be solid, liquid or vapor/gas. The working electrode can be fabricated out of metal, alloy or semiconductor. It can be flat, porous or composed of nanowires. The working electrode can be functionalized with stable spin labels (radicals), redox mediators capable of generating a stable radical, membranes, or a polymer layer can be deposited to the electrode surface. Microwave source (MW) irradiates the sample and can be completed with or without wave-guide, horn, resonator or cavity. The entire setup is in a magnetic field, created by resistive coils, superconducting coil or permanent magnet. The NMR detection can be performed with radiofrequency coils, but is not necessary for the DNP process.

The electrochemical Overhauser (EC-OE)-DNP experiment was conducted within a Varian electromagnet in a magnetic field B of 0.33 T. A coupling loop (d in Fig. 1) provided continuous microwave irradiation, at the EPR resonance frequency of the MV^+‘ (9.3 GHz), to a sapphire resonator (e in Fig. 1). An NMR coil was placed in the vicinity of the WE for a simultaneous 14 MHz excitation/detection using a spin-echo technique. By applying a potential negative enough, MV^+' was produced in-situ in controlled quantities, and was used to transfer the polarization from the unpaired electrons to the nuclei of ACN protons.

The EPR spectrum of MV^+' was measured with a Magnettech MiniScope MS 400 benchtop EPR spectrometer inside a ca. 1 mm inner diameter borosilicate tube.

Results and Discussion:

Figure 2a shows a cyclic voltammogram of a 1 mM solution of MV²⁺ at a gold electrode in acetonitrile with 200 mM of TBAP as supporting electrolyte against a Pt pseudo RE. During the forward scan the two visible redox processes are the reduction of MV²⁺ into MV^+' at around -0.158 V, and the consecutive reduction of MV^+' into MV° at around -0.559 V. Both processes are electrochemically reversible. The corresponding processes are depicted on Fig. 2a and the structure of the MV²⁺ is shown in the inset. MV^+' can also be generated by stepping the electrode potential beyond -0.559 V to produce MV° which then comproportionates in the vicinity of the electrode surface according to: MV°+ MV²⁺ 2 MV^+' with an equilibrium constant of 1 ^c 10⁷ in ACN.

Figure 2b shows an X-band EPR spectrum of MV^+' recorded at a radical concentration of about 100 mM in acetonitrile with 200 mM of TBAP as supporting electrolyte. The spectrum shows a hyperfine splitting of ca. 0.137 mT, typical for the ring protons of MV^+' although in acetonitrile, the larger line width masks most of the hyperfine couplings visible in aqueous samples.

Figure 3 presents the result before and after (average of 20 scans) electro-generating the radicals. After the radical production, the much shorter Ti of the protons led to a slightly higher MW-off signal. The OE-DNP signal of the ACN protons is 23-fold enhanced.

The concentration of generated radicals was estimated by determining the Ti of the sample protons, which in liquid phase is sensitive to the concentration of radical species, before and after applying the EC potential. The calculated concentration was 3.7 mM. The Ti were measured by saturation recovery.

In conclusion, we have demonstrated the possibility to provide an Overhauser enhanced proton signal by means of electrochemically generated radicals. Based on these results, other redox mediators are possible and the setup can be optimized in order to get larger enhancements. Also, the possibility of using CISS (chiral induced spin selectivity) effect is given as a method for hyperpolarizing the electron, i.e. to induce an enhanced polarization for electron before DNP process. For example, at a ¹H NMR frequency of 400 MHz the unpaired electron EPR frequency is of about 260 GHz, thus requiring a very special electrochemical cell design to satisfy all the requirements of electrochemistry, NMR and EPR.

It is thus possible to create, perform DNP and remove radicals in liquid state by Overhauser mechanisms. In a sample composed of 50 mM of methyl viologen in acetonitrile we applied a negative voltage to create radicals and subsequently applied microwaves with an X-band setup. We detected a hyperpolarized NMR signal, which prove that electrochemistry can be used as a way to get hyperpolarized samples.

Fields of applicability:

NMR market: Agriculture and Food Industry; Medical and Pharmaceutical Industry; Academic

MRI market: Hospitals (medical imaging centers); Academic

LIST OF REFERENCE SIGNS

ACN acetonitrile WE working electrode

DNP dynamic nuclear polarization TBAP tetrabutylammonium d-DNP dissolution dynamic nuclear perchlorate polarization TBAHP tetrabutylammonium

CE counter electrode hexafluorophosphate

EC electrochemistry TEMPOL 4-hydroxy-2, 2,6,6-

EC-OE electrochemistry driven OE tetramethylpiperidin-1 -oxyl experiments a working electrode (WE)

EPR electron paramagnetic b counter electrode (CE) resonance c cotton wool plug

MV methyl viologen d coupling loop for microwave

MW microwave irradiation (9.3 GHz)

NMR nuclear magnetic resonance e sapphire resonator

NOE nuclear Overhauser effect f NMR coil (14 MHz)

OE Overhauser effect

Claims

1. Method for the preparation of a sample comprising highly polarized nuclear spins using at least one polarization system, which using an electrochemical reaction can be converted from a diamagnetic form without an EPR-active radical into a paramagnetic EPR-active radical form thereof, and/or which is paramagnetic and comprises an EPR- active radical and using an electrochemical reaction can be converted into a diamagnetic form without an EPR-active radical, said method involving at least one step of in situ electrochemically forming an EPR- active radical form of a diamagnetic polarization system and/or of in situ electrochemically converting a paramagnetic polarization system comprising an EPR-active radical into a diamagnetic form without an EPR-active radical.

2. Method according to claim 1 , comprising at least the following steps: a) bringing a polarization system, which using an electrochemical reaction can be converted into an EPR-active radical form thereof, in contact or close proximity with said sample; b) in situ electrochemically forming an EPR-active radical form of said polarization system; c) applying dynamic nuclear polarization techniques in the presence of a magnetic field by applying electromagnetic irradiation with a frequency adapted to transfer spin polarization from the electrons of the polarization system to the nuclear spins of the sample leading to a highly polarized state thereof.

3. Method according to claim 1 or 2, comprising at least the following steps: x) bringing a polarization system, which is paramagnetic and comprises an EPR- active radical, in contact or close proximity with said sample; c) applying dynamic nuclear polarization techniques in the presence of a magnetic field by applying electromagnetic irradiation with a frequency adapted to transfer spin polarization from the electrons of the polarization system to the nuclear spins of the sample leading to a highly polarized state thereof d) in situ electrochemically converting said polarization system, which is paramagnetic and comprises an EPR-active radical into a diamagnetic form without an EPR-active radical.

4. Method according to any of the preceding claims, wherein step c) is carried out in frozen/solid state, liquid state or gaseous/vapour state, preferably in a frozen state.

5. Method according to any of the preceding claims, wherein step c) is followed by d) in situ electrochemically converting or re-converting the EPR-active radical form of the polarization system into a non-radical form of the polarization system.

6. Method according to any of the preceding claims, wherein step c) or step d) according to claim 5 is followed by e) spatially separating the polarization system and the highly polarized sample, preferably in that either the polarization system is immobilised on a carrier and the sample is in a mobile (liquid or gaseous) state and separated from the carrier, or in that the sample is immobilised on a carrier and the polarization system is in a liquid state and separated from the carrier, or the polarization system including the highly polarized sample in frozen/solid state is transferred into an external storage device such as a low temperature dewar with a magnetic field, where the sample can be stored for a desired time, and then separating the polarization system and the highly polarized sample, preferably in that either the polarization system is immobilised on a carrier and the sample is in a mobile (liquid or gaseous) state and separated from the carrier, or in that the sample is immobilised on a carrier and the polarization system is in a liquid state and separated from the carrier

7. Method according to any of the preceding claims, wherein the electromagnetic irradiation in step c) is applied with a frequency essentially equal to the difference or the sum of the Larmor frequencies of the electron spins and the nuclear spins, respectively, or at the Larmor frequencies of the electron spins in the presence of the applied magnetic field, and/or wherein the magnetic field is a static, preferably essentially homogeneous magnetic field with a strength of at least 0.1 T or of 0.2 T or of at least 0.3 T, or fields with a strength of 0.1 - 10T or 0.5 - 9.5T.

8. Method according to any of the preceding claims, wherein the polarization system is at least one selected from the following group: polycyclic heteroaromatic systems, preferably molecules based on substituted or unsubstituted imidazole, pyrazole, triazole, tetrazole, pyridine, diazine, triazine, tetrazine, pentazine, azepine, diazepine, preferably dimeric or trimeric forms thereof, preferably substituted and unsubstituted bipyridine, most preferably N,N'-dimethyl-4,4'-bipyridinium, or a member of the quinone family, especially for non-aqueous work or, preferably if the covalently linked polarization system is already in a radical EPR active then the radical can be selected from TEMPOL (4-hydroxy-2, 2,6,6- tetramethylpiperidin-1-oxyl) and its derivatives including: PROXYL, Spirobisnitroxide, Arylnitroxide, nitronylnitroxyl, Galvinoxyl, Arylnitroxide or other radicals such as but not limited to poly(2,2,6,6-tetramethylpiperidinyloxy methacrylate) and nitronyl nitroxide radicals or electrodes containing a polymer layer can be based on conducting backbone such as but not limited to poly(aniline), poly (pyrrole), poly(thiophene), poly(p-phenylene), poly(vinylcarbazole), Poly(phenylthiophene), Poly(phenylene vinylene), Poly(o- aminophenol) and poly(1,8-diaminonaphthalene), or non-conducting backbone such as Poly(methyl acrylate), Poly(vinyl ether), Poly(ethylene glycol), Poly(vinylidene fluoride) or polystyrene, or preferably the polymers, conducting or non-conducting, can be carrying a redox probe as part of the polymer back bone or as a pendant molecule (redox polymer) such as but not limited to N,N’-diphenyl-2,3,5,6-tetraketopiperazine (PHP), N,N’-diallyl-2, 3,5,6- tetraketopiperazine (AP), N,N’-di-n-propyl-2,3,5,6-tetraketopiperazine (PRP), or a polyimide-based erylene-3,4,9, 10-tetracarboxylic dianhydride(PTCDA) or, instead of carrying a redox probe the polymers, conducting or non-conducting, can be in EPR active form carrying a radical spin label as part of their backbone or as a pendant molecule (radical polymer) such as but not limited to TEMPOL (4-hydroxy-2, 2,6,6- tetramethylpiperidin-1-oxyl) and its derivatives: PROXYL, Spirobisnitroxide, Arylnitroxide, nitronylnitroxyl, Galvinoxyl, Arylnitroxide or other radicals such as but not limited to poly(2,2,6,6-tetramethylpiperidinyloxy methacrylate) and nitronyl nitroxide radicals, and/or wherein preferably the polymer membranes are grown to an electrode surface by electropolymerization, condensation polymerization, step-growth polymerization, or any other suitable method, and/or wherein electrode material to which the redox mediators or radicals are linked is a noble metal including Au, Ag or Pt, and/or one or more of ferro- or ferrimagnetic materials.

9. Method according to any of the preceding claims, wherein subsequent to step c), and/or subsequent to step d) according to claim 3 or 5 and/or subsequent to step e) according to claim 6 the highly polarized nuclear spins are measured in a nuclear magnetic resonance (NMR) or in a magnetic resonance imaging (MRI) experiment, wherein preferably protons, ¹³C and/or ¹⁵N nuclei or a combination thereof is measured.

10. Method according to claim 9, wherein the steps a)-c), and if used at least one of steps d) and e), as well as the nuclear magnetic resonance (NMR) or magnetic resonance imaging (MRI) experiment be carried out in one same probe head, or the analysis can be done in a different spectrometer or MRI scanner.

11. Method according to any of the preceding claims, wherein said polarization system, which using an electrochemical reaction can be converted from a diamagnetic form without an EPR-active radical into a paramagnetic EPR-active radical form thereof and/or which is paramagnetic and comprises an EPR-active radical and using an electrochemical reaction can be converted into a diamagnetic form without an EPR-active radical, is chemically and/or physically attached, preferably by at least one of covalent, electrostatic or hydrogen bonding, to a working electrode or incorporated into a coating on the working electrode used for the electrochemical formation, and/or as part of a polymer, coated or forming part of the working electrode surface.

12. Nuclear magnetic resonance (NMR), magnetic resonance spectroscopy (MRS) or magnetic resonance imaging (MRI) experiment, preferably in vitro or in vivo, wherein in a first step a hyperpolarized sample is prepared using a method according to any of the preceding claims, and the hyperpolarized sample is used in the experiment, preferably for enhancing the signal-to-noise ratio and/or as a contrasting agent.

13. Use of a method according to any of the preceding claims for diagnostic purposes, in vivo, in vitro, for electrochemical systems, for surface analysis, for the monitoring of electrochemical processes and their mechanisms and kinetics and/or identification of species generated during electrochemical processes, for the analysis of devices for storing energy, for the analysis of catalytic processes, in particular taking place on the surface.

14. Combined EPR/NMR device or EPR/MRI device, suitable and adapted to apply, in the measurement area, not only a static magnetic field as well as the electromagnetic irradiation required for EPR and NMR, but also an electrochemical potential for the in situ generation of a radical from a polarization system, which using an electrochemical reaction can be converted into an EPR-active radical form thereof, in particular a device suitable and adapted to carry out a method according to any of the preceding claims.