WO2008149118A1

WO2008149118A1 - Sample preparation method for enhancement of nmr sensitivity

Info

Publication number: WO2008149118A1
Application number: PCT/GB2008/001964
Authority: WO
Inventors: Ulrich Günther
Original assignee: Oxford Instruments Molecular Biotools Limited
Priority date: 2007-06-08
Filing date: 2008-06-06
Publication date: 2008-12-11
Also published as: EP2156205A1

Abstract

A method of preparing a sample for NMR analysis comprises adding to the sample a co-polarisation agent and optionally one or more solvents. The co-polarisation agent should have at least one spin-active nucleus and be added in a molar excess relative to a target molecule in the sample. The method further comprises irradiating the sample with microwave radiation to cause polarisation of the spin-active nucleus of the co- polarisation agent. The co-polarisation agent is then able to transfer this polarisation to the target molecule in order to improve the sensitivity of the NMR analysis.

Description

SAMPLE PREPARATION METHOD FOR ENHANCEMENT QF NMR

SENSITIVITY

The present invention is concerned with a method of preparing samples for analysis by nuclear magnetic resonance, to enhance the sensitivity of that analysis. More specifically, the present invention concerns the use of a co-polarisation agent to enhance the effectiveness of dynamic nuclear polarisation of the sample.

Nuclear magnetic resonance (NMR) spectroscopy is widely used as an analytical tool in chemical and biochemical sciences, as well as in medical applications, where the technique is used for the more commonly known magnetic resonance imaging (MRI). The technique relies on the presence of atomic nuclei having a plurality of quantum spin states. By placing the nuclei in a strong magnetic field, the energy levels corresponding to those spin states are separated. Irradiation of the nuclei with electromagnetic radiation of the correct wavelength (i.e. having a radiofrequency with an energy corresponding to the energy gap between the spin states) allows some of the nuclei to transfer from one energy level to another. Resulting changes in precession of the magnetic moment of spin-active nuclei within the sample are detected and analysed to determine the chemical environment of those nuclei.

One major problem with some types of NMR spectroscopy relates to the requirement for the nucleus to be spin-active, i.e. to have a plurality of spin states. For some elements, there is a high natural abundance of spin-active isotopes, such as for example ¹H and ¹⁹F. However, in relation to the elements carbon (C) and nitrogen (N), the predominant isotopes (¹²C and ¹⁴N respectively) are entirely spin-inactive, and the spin- active isotopes ¹³C and ¹⁵N occur naturally at only very low concentrations. As a result, only a small proportion of these nuclei in each chemical environment (e.g. in a particular position within the molecular structure) will return a signal in NMR spectroscopy, leading to a relatively low sensitivity. Since both elements are commonly found in organic chemistry and in biological molecules (such as proteins), and hence are of great interest in spectroscopy, it is desirable to improve the sensitivity of NMR in detection of these (and other) elements.

A number of methods are known for improving this sensitivity. In particular, it is possible to 'label' molecules with spin-active isotopes of elements for which the natural abundance of those isotopes is low. This is done by using starting materials which have been artificially enriched with these isotopes in chemical synthesis of the required compound. For example, it is possible to purchase amino acids in which the amino group nitrogen atom is present to a significant excess (e.g up to 98%) in the form of the spin-active ¹⁵N isotope. However, the increased cost of such isotopically-labelled materials makes this approach prohibitively expensive for all but the smallest molecules, and the technique cannot usefully be applied where there is no synthetic control of the sample molecules, such as for example in analysing the metabolic products of a living organism.

An alternative method of enhancing sensitivity involves the use of cryogenically-cooled probes in which the electronic components of the spectrometer (such as the receiver coil and preamplifiers) are cooled to temperatures below 30 K to reduce noise. These probes can provide a factor of 3-4 in increased sensitivity, and hence are now widely used in many applications of NMR, despite the relatively high cost.

A still further known method for enhancing the sensitivity of NMR analysis is known as Dynamic Nuclear Polarisation (DNP). In this technique, a sample is placed within a magnetic field and irradiated with microwave radiation, which excites unpaired electron spins in a molecule within the sample (such as those found in organic radicals) to a higher energy level. As the electrons relax back to their ground state, they are able to transfer energy to spin-active nuclei within the same or a nearby molecule, exciting the nuclei to a higher spin state. This results in an increased proportion of spin-active nuclei populating the higher-energy spin states, and hence to an increased NMR signal for those nuclei. Where the target molecule under analysis does not have the required unpaired electron spin, it is known to add polarisation agents such as organic free radicals to the sample in order to enable this technique. DNP is able to produce an increase in sensitivity of the order of several thousand.

One problem with DNP is that the polarisation of electrons is rapidly lost, and electron- nucleus spin transfer is inefficient at room temperature in the liquid phase within the high magnetic fields found in NMR spectrometers. As a result, the polarisation is preferably earned out using a lower magnetic field strength, on low temperature, frozen glassy samples, in which electron spin relaxation is reduced. NMR spectra must therefore be recorded in the solid state at low temperature, or the sample must be rapidly melted before taking a liquid-state spectrum. If it is desired to record the NMR spectra at higher field strength (as is common), the sample must also be transferred to the appropriate NMR spectrometer without substantial loss of polarisation.

It is therefore desirable to have a method of increasing the sensitivity of a sample to NMR analysis, wherein the increased sensitivity is sufficiently long-lived to allow transfer of the sample between magnetic environments. The present invention has been conceived to at least in part address this problem.

According to the present invention, there is provided a method of preparing for NMR analysis a sample containing at least one target molecule, comprising adding to the sample a molar excess of a co-polarisation agent having at least one spin-active nucleus, and optionally one or more solvents, and irradiating the sample with microwave radiation and thereby causing polarisation of the spin-active nucleus of the co- polarisation agent.

The phrase 'target molecule' is intended to mean a chemical compound for which it is desired to record an NMR spectrum.

The phrase 'molar excess of a co-polarisation agent having at least one spin-active nucleus' is intended to mean that the number of molecules of the co-polarisation agent having at least one spin-active nucleus is in excess of the number of target molecules. This may be achieved, for example, by isotopically-enriching the co-polarisation agent, or by adding a significant excess of the co-polarisation agent having a natural abundance of the spin-active nucleus.

In one embodiment the co-polarisation agent having at least one spin-active nucleus is present in at least a 10-fold molar excess, relative to the target molecule.

It will be understood that 'irradiating the sample with microwave radiation and thereby causing polarisation of the spin-active nucleus of the co-polarisation agent' is analogous to the known technique of Dynamic Nuclear Polarisation (DNP) and requires the sample to be placed within a magnetic field in the usual manner. Unless specified otherwise, any other conditions required for this polarisation of the spin-active nucleus of the co-polarisation agent may be readily adapted by the skilled man from those known for DNP.

In one embodiment, the spin-active nucleus in the co-polarisation agent has a T₁ relaxation time (such as that measured in a field of 11.74 T, in solution at room temperature) of at least 5 seconds. In a further embodiment, the Tj relaxation time is at least 15 seconds, or at least 30 seconds. The factors affecting the Ti relaxation time are varied, and will be well understood by the man skilled in the art. In particular, the presence of hydrogen atoms attached to a carbon or nitrogen nucleus will reduce the relaxation time of that nucleus. Thus, for example, quaternary carbon nuclei (those without any attached hydrogen atoms, such as ketone carbonyl atoms) have particularly long Ti relaxation times.

Similarly, the replacement of ¹H nuclei with deuterium (²H) can increase Tj relaxation times and improve spin diffusion in the relaxation matrix. Thus, in some embodiments, the co-polarisation agent may be partially or fully deuterated (i.e. have some or all of any H atoms present replaced with ²H).

In one embodiment, the spin-active nucleus is ¹³C or ¹⁵N. In a further embodiment, the co-polarisation agent has been isotopically-enriched with ¹³C or ¹⁵N. Alternatively, it will be appreciated that other spin-active nuclei may be used, such as for example ³¹P. In one embodiment, the method includes the step of cooling the sample to below 100 K to produce a glassy solid before irradiation with microwaves, and maintaining the sample at that temperature during microwave irradiation. In a further embodiment, the sample is cooled to below 70 K. In a still further embodiment, the sample is cooled to below 50 K, or to below 30 K.

In all embodiments of the present invention, the long-lived spin-active nucleus must be able to transfer polarisation to at least one spin-active nucleus within the target molecule, such as for example by the nuclear Overhauser effect (nθe), the solid state effect, the cross effect or thermal mixing. In one embodiment, the co-polarisation agent forms a non-covalent bond with the target molecule. For example, the co-polarisation agent may form a hydrogen bond or other polar interaction with the target molecule. In one embodiment, the co-polarisation agent does not react chemically with the target molecule to form a covalent bond.

In one embodiment, the spin-active nucleus in the co-polarisation agent is a quaternary carbon atom. In a further embodiment, the spin-active nucleus is a carbonyl carbon nucleus, the co-polarisation agent conveniently being a ketone such as acetone, methyl ethylketone or diethyl ketone. In a still further embodiment, the co-polarisation agent is acetone. Conveniently the co-polarisation agent may serve as the solvent, or one of the solvents, for the sample.

Alternative co-polarisation agents may include DMSO (particularly J₆-DMSO), pyruvate, f-butanol (2-methylpropan-2-ol), isopropanol (propan-2-ol), CO₂, and CO, any of which may also be isotopically enriched with ¹³C. One advantage of using CO₂ or CO (including their ¹³C-enriched forms) is ease of removal, since they are both gaseous at room temperature and so will simply evaporate.

In one embodiment, the spin-active nucleus in the co-polarisation agent is a quaternary

N atom. Suitable N-based co-polarisation agents include urea, pyridine, pyridazine, pyrimidine, pyrazine, and choline. In each case, the co-polarisation agent may be isotopically enriched with N in one position or (where applicable) in both positions. The co-polarisation agent may also be deuterated (have attached hydrogens replaced with deuterium (²H) to increase T₁). Preferred embodiments may include ¹⁵N₂-urea and ¹⁵N₂,£/₄-urea.

In those embodiments in which the sample has been cooled to produce a glassy solid, the method may further include heating the sample following polarisation to melt the glassy solid and thereby obtain the sample in a liquid state. In a further embodiment, this heating takes less than 5 seconds. In a further embodiment still, this heating takes place in less than 3 seconds, or less than 1 second. Without wishing to be bound by theory, it is believed that polarisation transfer may continue after heating.

In some cases, it may be undesirable to include an organic radical in the sample mixture for DNP processing. For example, where the sample includes delicate metabolite products, the presence of a free radical may lead to uncontrolled chemical reaction. Therefore, whereas some embodiments contemplate the addition of a free radical to the sample, other embodiments of the present invention include methods in which no radical is added to the sample before microwave irradiation. In particular, where the co- polarisation agent includes methyl groups (such as for example acetone), it may be possible to achieve DNP excitation without the requirement for a radical.

After preparation, the sample may be transferred to an NMR spectrometer for NMR analysis. Alternatively, the sample may be injected into a living creature for in vivo MRI or MRS analysis (if the sample has previously been cooled, this will usually be after a heating step). In either case, the sample may undergo further processing before analysis, such as for example to remove solvent from the sample.

The invention will now be described by way of example, with reference to the accompanying drawings, in which: Figure 1 shows a series of ¹³C NMR spectra of niethoxyphenol, using standard prior art DNP techniques, and polarisation in the presence of natural abundance and ¹³C-enriched acetone;

Figure 2 shows ¹³C NMR spectra of citrate after polarisation in the presence of natural abundance and C-enriched acetone;

Figure 3 shows ¹³C NMR spectra of oxaloacetate after polarisation in a variety of conditions; and

Figure 4 shows ¹³C NMR spectra of glucose after polarisation in a variety of conditions.

Referring to Figure 1, natural abundance niethoxyphenol (IM) was dissolved in a mixture of 50 μl of methanol and 50 μl of DMSO, together with a 'Finland' organic radical (as supplied by Oxfords Instruments Molecular Biotools Ltd, Abingdon UK )(15 mM). The mixture was irradiated with microwaves at 94 GHz in a 3.35 T magnet according to standard DNP procedure. The polarisation was carried out for 1.5 hours at 1.5 K. The sample was then melted with 4 ml MeOH and subjected to a temperature of 180 ⁰C (to give a sample temperature of 30 ⁰C), and transferred to a 500 MHz (1 1.75 T) NMR magnet for recordal of the 13C NMR spectrum. Melting and transfer were completed in 4 seconds. The spectrum is shown as spectrum (A) in Figure 1.

Similar samples were prepared in which the methanol was replaced by acetone (natural ¹³C abundance) and [2-¹³C]-acetone, all other conditions being identical. The NMR spectra are shown as the spectra (B) and (C) respectively, scaled according to the noise level. It can be seen that increasing concentrations of ¹³C-containing acetone produce increasingly stronger NMR spectra of the methoxyphenol sample. For natural abundance acetone, the signals are enhanced by a factor of approximately 5, whilst for ¹³C-labelled acetone, the enhancement is at least 50-fold.

Referring to Figure 2, IM citrate was dissolved in a mixture of acetone (natural C abundance) or [2-¹³C]-acetone (33 μl), together with 33 μl DMSO, 33 ml water and 0X63 radical (as supplied by Oxfords Instruments Molecular Biotools Ltd, Abingdon UK) (15 mM). The mixture was irradiated with microwaves (94 GHz) at 1.5 K for 1.5 hours, as above, and subsequently melted with 4ml of water (containing EDTA). The sample was then subjected to a temperature of 200 ⁰C (to give a sample temperature of approximately 30 °C) and transferred to a 500 MHz (11.75 T) NMR magnet. Melting and transfer were completed in 4 seconds. The ¹³C NMR spectra were recorded and are shown as spectra (A) and (B) respectively. Again, it can be seen that the increase in ¹³C acetone concentration caused by using the labelled acetone has led to an increase in NMR signal strength of signals corresponding to citrate, including the appearance of the previously unseen signal for the quaternary citrate carbon at a chemical shift of 72 ppm.

Referring to Figure 3, IM oxaloacetate was dissolved in 100 μl of the following solvents: for spectra A and B, equal amounts of natural abundance acetone and DMSO; for spectra C and D, equal amounts of deuterated (d₆) acetone and DMSO; and for spectra E and F, equal amounts of [2-¹³C]-acetone and deuterated DMSO. 0X63^' radical (as supplied by Oxford Instruments Molecular Biotools Limited, Abingdon UK) (15 mM) was added to the samples.

The samples were then irradiated with microwaves at 1.5 K for 1.5 hours. For spectra B, D and F, polarisation was carried out using a frequency of ω_e + CC>_N (where ω_e is the microwave frequency of 94 GHz, and CO_N is the nuclear magnetic resonance frequency of the NMR nucleus, ¹³C)₅ whereas a polarisation frequency of ω_e - CO_N was used for the samples for spectra A, C and E (J Am. Chem. Soc, 2008, 230, p. 6014-6915).

In all cases, the sample was then melted with 4 ml of water subjected to a temperature of 200 ⁰C (to give a sample temperature of approximately 30 ⁰C) (containing EDTA) and transferred to a 500 MHz (11.75 T) NMR magnet, where the ¹³C spectra were recorded. It can be seen that some benefit is obtained from the use of deuterated additives, although a more significant benefit is obtained in this case from use of [2- ¹³C]-acetone. Referring to Figure 4, IM glucose was dissolved in 100 μl of the following solvents: for spectra A and C, equal amounts of unlabelled DMSO and H₂O; and for spectrum B, equal amounts of J₆-DMSO and H₂O. 0X63 (as above) (15 niM) was added to each sample.

The samples were then irradiated with microwaves at 1.5 K for 1.5 hours. For spectra A and B, irradiation was carried out at a frequency of ω_e - CO_N, whereas a polarisation frequency of ω_e + CQ_N was used for spectrum C.

In all cases, the sample was then melted with 4 ml of water subjected to a temperature of 200 °C (to give a sample temperature of approximately 30 ⁰C) (containing EDTA) and transferred to a 500 MHz (11.75 T) NMR magnet, where the ¹³C spectra were recorded. It can be seen that significant benefit could be obtained from using deuterated DMSO.

Claims

CLAIMS:

1. A method of preparing for NMR analysis a sample containing at least one target molecule, comprising adding to the sample a molar excess of a co-polarisation agent having at least one spin-active nucleus, and optionally one or more solvents, and irradiating the sample with microwave radiation to cause polarisation of the spin-active nucleus of the co-polarisation agent.

2. A method as claimed in claim 1, wherein adding to the sample a molar excess of a co-polarisation agent having at least one spin-active nucleus comprises adding to the sample a 10-fold molar excess of the co-polarisation agent.

3. A method as claimed in claim 1 or claim 2, wherein the spin-active nucleus in the co-polarisation agent has a Tj relaxation time of at least 5 seconds.

4. A method as claimed in any preceding claim, wherein the spin-active nucleus is 1³C or ¹⁵N.

5. A method as claimed in claim 4, wherein the co-polarisation agent is isotopically- enriched with at least one of ¹³C and ¹⁵N.

6. A method as claimed in any preceding claim, further comprising cooling the sample to a temperature below 100 K to produce a glassy solid before irradiation with microwaves, and maintaining the sample at that temperature during microwave irradiation.

7. A method as claimed in any preceding claim, further comprising adding a radical to the sample before irradiation with microwaves.