WO2005101044A1

WO2005101044A1 - A solid state nmr involving relaxor with rapidly rotating molecular section and protective cage

Info

Publication number: WO2005101044A1
Application number: PCT/GB2005/001367
Authority: WO
Inventors: Malcolm Harris Levitt; Marina Carravetta
Original assignee: University Of Southampton
Priority date: 2004-04-16
Filing date: 2005-04-08
Publication date: 2005-10-27
Also published as: GB0408564D0; GB2413182A

Abstract

A solid state NMR method for analysing a sample, the method comprising: introducing the sample to relaxors to produce a microscopic mixture, reducing the temperature of said mixture; and performing an NMR measurement on said mixture, wherein said relaxors comprise two sections: a rapidly rotating molecular section; and a cage such as a fullerene which protects said molecular section and is configured to be chemically stable with said sample. Preferably, the rapidly rotating molecular section has a short T1 on the order of less than 100 ms at a temperature of 50K or less and acts as a relaxation sink for the magnetization of the sample. Therefore, T1 of the sample is reduced and the time required for build up of the nuclear polarization is shortened. Preferably, the nuclear polarization is further enhanced via the Haupt effect.

Description

SOLID STATE NMR INVOLVING RELAXOR WITH RAPIDLY ROTATING MOLECULAR SECTION AND PROTECTIVE CAGE

The present invention relates to the field of NMR. More specifically, the present invention relates to a so-called solid state NMR method which is primarily performed at low temperatures.

Nuclear magnetic resonance (NMR) is a powerful and well established tool for studying samples and sample interactions. It can also be used for imaging, for example, Magnetic Resonance Imaging (MRI). Solid-state NMR is an important method for determining molecular structure, and the chemical and physical composition of a sample.

In NMR, the spin and magnetism of atomic nuclei are exploited to provide information about the chemical composition, spatial distribution, or molecular motion of the molecules or atoms.

One of the limitations of NMR is the low intrinsic signal strength. So-called solid-state NMR which is performed at temperatures at or below 77K has great potential advantages in terms of signal strength and noise reduction. For example, the NMR signal at 4K is 60 times larger than at room temperature, allowing a 60 fold reduction in the amount of sample required. However, the time required to establish this large magnetization usually increases dramatically at low temperature and can be hours or longer at 4K.

The time required to establish large magnetisations is governed by the relaxation time constant or as the spin lattice relaxation time constant Ti. Ti is the time constant which is used to characterise the relaxation of the nuclear spins back to thermal equilibrium and is determined by molecular motion.

Currently, there are two main methods for investigating large molecules in the cryogenic solid state regime. The sensitivity issue is particularly critical for large molecules, since the number of nuclei giving rise to each peak is small. Most cryogenic

NMR experiments have been performed so far on small molecules, in which case it's not so important to have large signal strength.

One method is to wait until the large magnetisation is established. This is usually not feasible at very low temperature since experiment times become too long.

The other method is to dope the substance with a paramagnetic species and apply microwaves (or far infra-red radiation, depending on the magnetic field) at the electron Larmor frequency. This technique is known as DNP (dynamic nuclear polarisation): an example of its use is given in D. A. Hall et al., Science 276, 930 (1997). The problem with this technique is that it is complex and very expensive (equipment in excess of £0.5M) and become even more complex and expensive at high magnetic fields.

Recently, Tomaselli et al. (Tomaselli et al. J. Chem. Phys. 118, 8559-8562 (2003) and Tomaselli et al. 120, 4051-4054 (2004)) have reported observations of the Haupt Effect on 4-methylpyridine (also known as γ-picoline). The Haupt Effect is the existence of large dynamic polarization or ordering of the proton spins with respect to a local dipole field after a sudden change in temperature of the solid. The Haupt effect requires a molecular system containing a freely rotating molecular group, typically a -CH₃ group, which is unhindered by interactions with its immediate environment. The crystal structure of 4-methylpyridine provides such an environment for the methyl group. Tomaselli et al. have reported that the Haupt enhanced polarisation of 4-methylpyridine may be transferred to another small molecule dissolved in it in a solid solution at low concentration. They exemplify transfer to the Zeeman order of ¹³C and ¹⁵N-labelled guest molecules and also, in the later paper, show low and zero field transfer of spin angular momentum of CH₃ groups.

However, in the above work, the "target molecule" is in a dilute solid solution of γ- picoline so as not to disrupt its crystal structure significantly. Thus, the work by Tomaselli et al. does not provide a general solution to the above outlined problems of solid state NMR as it will always require a large excess of the rotor system (γ-picoline) compared to the guest or target molecules. In particular, this is not a feasible scenario for studying large biomolecules.

A recent publication by Caravetta et al, J. Am. Chem. Soc; 2004; 126(13) pp 4092 - 4093 describes NMR analysis of molecular hydrogen trapped within an open-cage fullerene. Caravetta et al. demonstrate that the rapidly rotating molecular hydrogen relaxes rapidly and that the molecular hydrogen also acts as a relaxation sink for hydrogen nuclei located on the outside of the cage.

In a first aspect, the present invention provides an NMR method for analysing a sample, the method comprising: mixing the sample with relaxors to produce a microscopic mixture, reducing the temperature of said mixture; and performing an NMR measurement on said mixture, wherein said relaxors comprise two sections: a rapidly rotating molecular section; and a cage which protects said molecular section and is not disrupted by said sample.

Those skilled in the art appreciate that a microscopic mixture is a mixture where the molecules of the two different species of the mixture are in intimate contact.

Exceptionally high molecular mobility at low temperature provided by the rapidly rotating molecular section allows a relaxation mechanism causing relatively rapid thermal polarization of both the relaxor and the sample in contact with the relaxor in close proximity to the relaxor. Typically, by close proximity, a distance of less than 1 nm is meant.

In some systems, exceptionally high molecular mobility can also lead to a Haupt effect, in which a very large nuclear spin polarization is achieved temporarily after a temperature jump.

Both of the above effects require the mobile part of the system to be isolated from the environment otherwise rotation of the rapidly rotating part of the molecule will be hindered. Thus, the cage is provided to isolate the rapidly rotating part from its environment. In addition, the cage can contribute to the chemical and physical stability of the relaxor complex.

Thus, the relaxor has both (a) a rapidly rotating part and (b) a protective cage, where (a) allows rapid thermal polarization and possible Haupt effect, while (b) allows the molecules to be mixed freely with a sample of interest without disturbing the freely- rotating part.

In more detail, the rapidly rotating molecular section works as a relaxation sink at cryogenic temperatures and has a short Ti. Typically, Ti for the rapidly rotating molecular section is less than 100 milliseconds at a temperature of 50 Kelvin or less, more preferably 20 milliseconds at a temperature of 100 Kelvin or less. The rapidly rotating part acts as a relaxation sink which allows the whole sample to come into thermal equilibrium at low temperatures and hence to acquire a large nuclear spin polarisation within a reasonable time. In some circumstances, the rapidly rotating part may also display a Haupt effect allowing much larger nuclear spin polarization to be achieved temporarily after a temperature jump.

The relaxor may be a single molecule comprising both a rapidly spinning part and a cage or may be separate molecules, e.g. a rapidly spinning molecule trapped in a molecular cage.

Said rapidly rotating molecular section preferably has a Ti of less than 100 milliseconds at temperatures of less than 50 Kelvin. Typical examples of rapidly rotating molecules, or parts of molecules, are: H₂ molecules, D₂ molecules, -CH₃ groups, -NH₃ groups,

The cage part of the molecule protects the rapidly rotating part of the molecule and hence gives the molecule robustness which means that the relaxor behaviour of the molecule is not disrupted by contact with other molecules. The cage may comprise a single molecule, for example a simple or modified fullerene cage, derived from compounds such as C₆₀ or C₇₀. Alternatively, the cage may comprise a set of individual molecules designed to self-assemble into a stable cage structure, for example artificial fragments of the 4-methylpyridine crystal structure. The cage is configured so that it is not disrupted when brought into contact with the sample.

In a preferred embodiment of the invention, the cage comprises one or more functional groups which allow the cage to target a specific molecule of interest. For example, the cage may be configured to target a specific part of a biomolecule of interest, allowing enhanced solid-state NMR spectra from that particular region. The functional group could be, for example, an inhibitor to a protein which binds strongly to the active site.

In an embodiment of the invention, the relaxors are supplied in a concentration such that approximately, one relaxor is provided for each 1000 protons. For example, in a preferred embodiment a 40kDa protein will be in contact with approximately 2 relaxor molecules since it is estimated that there are about 2500 protons in a 40kDa protein. However, if the protein was 20kDa, it would contain only around 1000 protons, so only 1 relaxor molecule per protein may be required.

Similarly, if the protein were 80kDa, one may need 4 relaxor molecules per protein. Furthermore, the proteins could be deuterated (the proton nuclei are replaced in part by deuterons). For example, if the 40kDa protein were 50% deuterated, only 1 relaxor molecule per protein may be required, and so on. In addition, if functionalization were used to "tow" the relaxor towards a specific site, only one relaxor may be needed to "light up" the neighbourhood, independent of how large the molecule was. To further complicate matters, there is a separate issue as to how much time is needed for the thermal (or Haupt) polarization to "diffuse" into the biomolecule, which also depends on the proton concentration and which is not fully understood at present at cryogenic temperatures.

As previously mentioned, the present invention is of particular use in studying large molecules. Thus, preferably the molecules which are to be studied of the sample are of at least 10 kDa. The method is expected to be most useful for molecules in the size range lOkDa to 200 kDa. If the molecules are too large, it may take too long for polarization generated on the surface to diffuse into the interior of the molecules of interest. For small molecules, forming a solid solution in gamma-picoline may be an acceptable alternative. However, one should be aware that even larger molecules may be accessible to the described method, if particular locations of interest are targeted by specific binding properties, as indicated above. In addition, the study of small molecules using specifically-designed chemically-inert cryorelaxors may be preferable to using an excess of gamma-picoline, which is a pungent, oily liquid.

In a second aspect, the present invention provides the use of a relaxor in an NMR measurement of a sample, said relaxor comprising two sections: a rapidly rotating molecular section; and a cage which protects said molecular section and is configured to be chemically stable with said sample.

The present invention will now be described with reference to the following preferred non-limiting embodiments in which:

Figure 1 is a flow diagram of a method in accordance with a preferred embodiment of the present invention; and

Figure 2 is a schematic of a typical relaxor for use with the present invention.

The flow diagram of figure 1 schematically illustrates some of the basic steps. In Step S101, a sample which is to be analysed by NMR is identified. In this particular embodiment, a water soluble biomolecule is used, but any molecule, providing it is capable of NMR analysis may be used.

The sample is then mixed in solution with a suitable relaxor in step SI 03. A suitable relaxor will have a rapidly rotating section which is protected by a cage. The cage will be selected so that it is not disrupted by the sample.

In this particular embodiment, the mixture is frozen in step SI 05 as this will bring the relaxors in contact with the outside of the biomolecule. In other examples, the relaxor may be attracted to the biomolecule by electrostatic properties (for example, for negatively-charged DNA, a positively-charged relaxor molecule can be constructed). In other cases, specific binding functions can be added to the cage which allow the relaxor to target the biomolecule or specific parts of the biomolecule. In some cases, the relaxor and biomolecule may form an intimate microscopic mixture without an additional solvent being present. The necessary feature of the sample preparation is that the sample molecules and relaxor molecules or complexes are brought into intimate microscopic contact (within 1 nm) in the solid state.

An NMR measurement may then be performed on the mixture in step SI 07. The NMR measurement may be a standard NMR measurement or possibly a so-called Magic Angle Sp-i-ning (MAS) measurement in which the sample is rotated rapidly at the "magic angle" (arctan> 2 = 54.74 degrees) to the static magnetic field, in order to average out anisotropic nuclear spin interactions which can broaden the NMR features of solid state samples and thus obscure features of the spectra. A large number of solid- state NMR experiments could be performed, as described in standard texts, for example K. Schmidt-Rohr and H. W. Spiess, "Multidimensional Solid-State NMR and Polymers." (Academic, London, 1994) and M. J. Duer, "Solid-State NMR. Principles and Applications." (Blackwell Science, 2002).

The sample may also be subjected to the experimental conditions necessary to observe the Haupt effect. For example, the procedures described in the literature (Tomaselli et al.) could be used.

Figure 2 is a schematic of a possible relaxor. The relaxor comprises H₂ molecules trapped within a azo-thia-open-cage fullerene (H₂@ATOCF). The rapidly rotating H₂ molecules trapped within the cage are protected by the cage so that the H₂@ATOCF is both chemically and physically stable.

In the above molecule Ti of the hydrogens within the cage has been measured to be 25ms at 20 K. If this relaxor is mixed with a protein of size 40 kDa, the relaxor and protein concentrations being selected such that each protein molecule is in contact with 2 relaxor molecules, i.e. the sample is made up so that the molar ratio of protein to relaxor is 1:2, the estimated Ti of the whole system will be about 16 seconds, which is experimentally comfortable.

Claims

CLAIMS:

1. An NMR method for analysing a sample, the method comprising: introducing the sample to relaxors to produce a microscopic mixture, reducing the temperature of said mixture; and performing an NMR measurement on said mixture, wherein said relaxors comprise two sections: a rapidly rotating molecular section; and a cage which protects said molecular section and is configured to be chemically stable with said sample.

2. An NMR method according to claim 1, wherein said rapidly rotating molecular section has a Ti of less than 100 milliseconds at temperatures of less than 50 Kelvin.

3. An NMR method according to any preceding claim, wherein said cage is a single molecule.

4. An NMR method according to claim 3, wherein said cage is a fullerene or modified fullerene.

5. An NMR method according to any preceding claim, wherein said cage comprises a set of individual molecules designed to self-assemble into a stable cage structure.

6. An NMR method according to any preceding claim, wherein a functional group configured to target at least a part of said sample, is provided on said cage.

7. An NMR method according to any preceding claim, wherein said relaxors are water soluble.

8. An NMR method according to any preceding claim, wherein said sample is introduced to said relaxors in a solution.

9. An NMR method according to any preceding claim, wherein the ratio of protons to relaxors is approximately 1000 protons to one relaxor.

10. An NMR method according to any preceding claim, wherein said temperature is reduced to at most 50 Kelvin.

11. An NMR method according to any preceding claim, wherein the sample comprises molecules which are to be analysed having a size of at least 10 kDa.

12. Use of a relaxor in an NMR measurement of a sample, said relaxor comprising two sections: a rapidly rotating molecular section; and a cage which protects said molecular section and is configured to be chemically stable with said sample.

13. An NMR method as substantially hereinbefore described with reference to any of the accompanying figures.

14. Use of a relaxor as substantially hereinbefore described with reference to any of the accompanying figures.