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WO2008149118A1 - Méthode de préparation d'un échantillon améliorant la sensibilité de la rmn - Google Patents

Méthode de préparation d'un échantillon améliorant la sensibilité de la rmn Download PDF

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Publication number
WO2008149118A1
WO2008149118A1 PCT/GB2008/001964 GB2008001964W WO2008149118A1 WO 2008149118 A1 WO2008149118 A1 WO 2008149118A1 GB 2008001964 W GB2008001964 W GB 2008001964W WO 2008149118 A1 WO2008149118 A1 WO 2008149118A1
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WO
WIPO (PCT)
Prior art keywords
sample
polarisation
spin
agent
nmr
Prior art date
Application number
PCT/GB2008/001964
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English (en)
Inventor
Ulrich Günther
Original Assignee
Oxford Instruments Molecular Biotools Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Oxford Instruments Molecular Biotools Limited filed Critical Oxford Instruments Molecular Biotools Limited
Priority to EP08762305A priority Critical patent/EP2156205A1/fr
Publication of WO2008149118A1 publication Critical patent/WO2008149118A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/44—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using nuclear magnetic resonance [NMR]
    • G01R33/46—NMR spectroscopy
    • G01R33/4608—RF excitation sequences for enhanced detection, e.g. NOE, polarisation transfer, selection of a coherence transfer pathway

Definitions

  • 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.
  • NMR spectroscopy relates to the requirement for the nucleus to be spin-active, i.e. to have a plurality of spin states.
  • spin-active isotopes such as for example 1 H and 19 F.
  • the predominant isotopes 12 C and 14 N respectively
  • the spin- active isotopes 13 C and 15 N occur naturally at only very low concentrations.
  • only a small proportion of these nuclei in each chemical environment e.g.
  • 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.
  • DNP Dynamic Nuclear Polarisation
  • DNP Differential nucleus spin transfer
  • 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.
  • 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.
  • 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.
  • 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.
  • the spin-active nucleus in the co-polarisation agent has a T 1 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.
  • the presence of hydrogen atoms attached to a carbon or nitrogen nucleus will reduce the relaxation time of that nucleus.
  • quaternary carbon nuclei (those without any attached hydrogen atoms, such as ketone carbonyl atoms) have particularly long Ti relaxation times.
  • the replacement of 1 H nuclei with deuterium ( 2 H) can increase Tj relaxation times and improve spin diffusion in the relaxation matrix.
  • the co-polarisation agent may be partially or fully deuterated (i.e. have some or all of any H atoms present replaced with 2 H).
  • the spin-active nucleus is 13 C or 15 N.
  • the co-polarisation agent has been isotopically-enriched with 13 C or 15 N.
  • other spin-active nuclei may be used, such as for example 31 P.
  • 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.
  • the sample is cooled to below 70 K.
  • the sample is cooled to below 50 K, or to below 30 K.
  • 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 (n ⁇ e), the solid state effect, the cross effect or thermal mixing.
  • the co-polarisation agent forms a non-covalent bond with the target molecule.
  • the co-polarisation agent may form a hydrogen bond or other polar interaction with the target molecule.
  • the co-polarisation agent does not react chemically with the target molecule to form a covalent bond.
  • the spin-active nucleus in the co-polarisation agent is a quaternary carbon atom.
  • 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.
  • the co-polarisation agent is acetone.
  • 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 J 6 -DMSO), pyruvate, f-butanol (2-methylpropan-2-ol), isopropanol (propan-2-ol), CO 2 , and CO, any of which may also be isotopically enriched with 13 C.
  • DMSO particularly J 6 -DMSO
  • pyruvate particularly pyruvate
  • f-butanol (2-methylpropan-2-ol
  • CO 2 and CO, any of which may also be isotopically enriched with 13 C.
  • CO 2 or CO including their 13 C-enriched forms
  • the spin-active nucleus in the co-polarisation agent is a quaternary
  • N-based co-polarisation agents include urea, pyridine, pyridazine, pyrimidine, pyrazine, and choline.
  • 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 ( 2 H) to increase T 1 ).
  • Preferred embodiments may include 15 N 2 -urea and 15 N 2 ,£/ 4 -urea.
  • 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.
  • an organic radical in the sample mixture for DNP processing.
  • 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.
  • 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.
  • the sample may be transferred to an NMR spectrometer for NMR analysis.
  • 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).
  • the sample may undergo further processing before analysis, such as for example to remove solvent from the sample.
  • Figure 1 shows a series of 13 C NMR spectra of niethoxyphenol, using standard prior art DNP techniques, and polarisation in the presence of natural abundance and 13 C-enriched acetone;
  • Figure 2 shows 13 C NMR spectra of citrate after polarisation in the presence of natural abundance and C-enriched acetone
  • Figure 3 shows 13 C NMR spectra of oxaloacetate after polarisation in a variety of conditions
  • Figure 4 shows 13 C NMR spectra of glucose after polarisation in a variety of conditions.
  • IM citrate was dissolved in a mixture of acetone (natural C abundance) or [2- 13 C]-acetone (33 ⁇ l), together with 33 ⁇ l 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 0 C (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.
  • IM oxaloacetate was dissolved in 100 ⁇ l 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 (d 6 ) acetone and DMSO; and for spectra E and F, equal amounts of [2- 13 C]-acetone and deuterated DMSO.
  • 0X63 ' radical (as supplied by Oxford Instruments Molecular Biotools Limited, Abingdon UK) (15 mM) was added to the samples.
  • 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 0 C) (containing EDTA) and transferred to a 500 MHz (11.75 T) NMR magnet, where the 13 C spectra were recorded. It can be seen that significant benefit could be obtained from using deuterated DMSO.

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  • Physics & Mathematics (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • High Energy & Nuclear Physics (AREA)
  • Condensed Matter Physics & Semiconductors (AREA)
  • General Physics & Mathematics (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention porte sur une méthode de préparation d'un échantillon pour une analyse par RMN consistant: à ajouter à l'échantillon un agent de co-polarisation et facultativement un ou plusieurs solvants. L'agent de co-polarisation devrait avoir au moins un noyau à spin actif et être ajouté avec un excès molaire par rapport à une molécule cible de l'échantillon. La méthode consiste en outre à irradier l'échantillon par des micro-ondes pour provoquer la polarisation du noyau à spin actif de l'agent de co-polarisation. L'agent de co-polarisation peut alors transférer sa polarisation à la molécule cible, ce qui améliore la sensibilité de l'analyse par RMN.
PCT/GB2008/001964 2007-06-08 2008-06-06 Méthode de préparation d'un échantillon améliorant la sensibilité de la rmn WO2008149118A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP08762305A EP2156205A1 (fr) 2007-06-08 2008-06-06 Méthode de préparation d'un échantillon améliorant la sensibilité de la rmn

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0711048 2007-06-08
GB0711048.9 2007-06-08

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WO2008149118A1 true WO2008149118A1 (fr) 2008-12-11

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2348327A1 (fr) * 2010-01-18 2011-07-27 Bruker BioSpin AG Procédé de mesure par RMN utilisant la polarisation nucléaire dynamique de dissolution avec élimination des radicaux libres

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006079702A2 (fr) * 2005-01-27 2006-08-03 Commissariat A L'energie Atomique Procede pour accoitre le signal rmn d ' une solution liquide en utilisant le champ dipolaire longue distance
US7205764B1 (en) * 2006-04-11 2007-04-17 Varian, Inc. Method and apparatus for increasing the detection sensitivity in a high resolution NMR analysis
WO2007136439A2 (fr) * 2006-02-21 2007-11-29 Avrum Belzer Procédés, systèmes et compositions d'hyperporalisation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006079702A2 (fr) * 2005-01-27 2006-08-03 Commissariat A L'energie Atomique Procede pour accoitre le signal rmn d ' une solution liquide en utilisant le champ dipolaire longue distance
WO2007136439A2 (fr) * 2006-02-21 2007-11-29 Avrum Belzer Procédés, systèmes et compositions d'hyperporalisation
US7205764B1 (en) * 2006-04-11 2007-04-17 Varian, Inc. Method and apparatus for increasing the detection sensitivity in a high resolution NMR analysis

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
A.CHERUBINI ET AL.: "Hyperpolarising 13C for NMR studies using laser-polarised 129Xe: SPINOE vs thermal mixing", CHEMICAL PHYSICS LETTERS, vol. 371, 2003, pages 640 - 644, XP002494330 *
C.-G. JOO ET AL.: "In Situ Temperature Jump High-Frequency Dynamic Nuclear Polarization Experiments: Enhanced Sensitivity in Liquid-State NMR Spectroscopy", J.AM.CHEM.SOC., vol. 128, 2006, pages 9428 - 9432, XP002494329 *
HALL D A ET AL: "POLARIZATION-ENHANCED NMR SPECTROSCOPY OF BIOMOLECULES IN FROZEN SOLUTION", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, WASHINGTON, DC, vol. 276, no. 5314, 9 May 1997 (1997-05-09), pages 930 - 932, XP000882848, ISSN: 0036-8075 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2348327A1 (fr) * 2010-01-18 2011-07-27 Bruker BioSpin AG Procédé de mesure par RMN utilisant la polarisation nucléaire dynamique de dissolution avec élimination des radicaux libres
US8564288B2 (en) 2010-01-18 2013-10-22 Bruker Biospin Ag Method for NMR spectroscopy or MRI measurements using dissolution dynamic nuclear polarization (DNP) with scavenging of free radicals

Also Published As

Publication number Publication date
EP2156205A1 (fr) 2010-02-24

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