Patent Application
20090311189
The last decade has witnessed a renaissance in the development of approaches to prepare samples with high nuclear spin polarizations with the goal of increasing signal intensities in NMR spectra of solids and liquids. These approaches have included high frequency, microwave driven dynamic nuclear polarization (DNP)1-9, para hydrogen induced polarization (PHIP)10,11, polarization of noble gases such as He, Xe12-14 and more recently Kr15, and optically pumped nuclear polarization of semiconductors16-18 and photosynthetic reaction centers and other proteins19-22. Dynamic nuclear polarization is an approach in which the large spin polarization in an electron spin system is transferred to a nuclear spin reservoir via microwave irradiation of the electron paramagnetic resonance (EPR) spectrum. All of these approaches successfully yield highly polarized spins, and are studied to elucidate features of the polarization methods or of the material being polarized. However, one of the most appealing aspects of high polarization methods is the possibility of transferring the polarization from the source to a surrounding medium such as a solvent and to subsequently distribute the polarization to chemically, physically or biologically interesting solutes. For this to occur it is necessary that the polarizing agent be strongly coupled to the lattice of nuclear spins, and in this regard paramagnetic polarizing agents are appealing since the large magnetic moment of the electron spin couples effectively to its surrounding nuclei. Accordingly, high frequency microwave (≧100 GHz) driven DNP experiments using stable free radicals as polarizing agents2, 3 are currently used successfully to polarize a variety of systems including solid polymers4, 23-27, frozen solutions of small molecules3, amino acides5,6, virus particles7, soluble and membrane proteins8 and amyloid nanocrystals9 achieving enhancements in the range of 50-400, depending on the details of the experiment. Superscript numbers refer to the attached reference list. The contents of all of these references are incorporated herein by reference.
In addition to polarizing solid samples, there is considerable interest in using high frequency DNP to enhance the sensitivity of liquid state nuclear magnetic resonance (NMR) experiments. However, the polarization mechanisms operative in dielectric solids at high fields—the solid effect28, 29, the cross effect2, 30 and thermal mixing28—are not applicable to liquids. Instead, the Overhauser effect (OE)31, 32 is the dominant polarization mechanism, and it is efficient only at low magnetic fields. In particular, for small molecules, the rotational or translational correlation times are˜10−12s and at low magnetic fields the condition ωsτc≦1 is satisfied (where ωs, is the electron Larmor frequency and τc the correlation time), and the Overhauser effect is effective in transferring polarization. However, in the high field regime commonly employed in contemporary NMR experiments, ωs is large, the rotational and translational spectral densities are vanishingly small, and the Overhauser enhancements decrease significantly33. Thus, to enhance the polarization of liquid samples in high field experiments, an alternative method is required.
In one aspect, the present invention provides a method for enhancing the sensitivity of liquid-state NMR or MRI experiments. In general, the method involves (a) providing a frozen sample in a magnetic field, wherein the frozen sample includes a polarizing agent with at least one unpaired electron and an analyte with at least one spin half nucleus; (b) polarizing the at least one spin half nucleus of the analyte by irradiating the frozen sample with radiation having a frequency that excites electron spin transitions in the at least one unpaired electron of the polarizing agent; (c) melting the frozen sample to produce a molten sample; and (d) detecting nuclear spin transitions in the at least one spin half nucleus of the analyte in the molten sample. In certain embodiments, the methods further comprise a step of freezing a sample in a magnetic field to provide the frozen sample in a magnetic field. In one such embodiment, the freezing, polarizing, melting and detecting steps are repeated at least once.
In general, the methods may be used to polarize any analyte. Without limitation, the analyte may be a protein or nucleic acid. Numerous liquid-state NMR methods have been developed to study the structures of these biomolecules, e.g., one dimensional techniques, multi-dimensional techniques, including without limitation techniques that rely on NOESY, ROESY, TOCSY, HSQC, HMQC, etc. type polarization transfers and combinations thereof. Any of these techniques and variants thereof may benefit from the enhanced NMR signals that can be provided by the inventive methods. The inventive methods may also be advantageously used to improve the detection of analytes (e.g., metabolites) that are present in a sample at low concentrations. Currently, the lower limit of detection by convention liquid-state NMR is on the order of about 10 μM. Since the DNP enhancement provided by the present invention may range from 2 to 10,000 or more (depending on the temperature, magnetic field, etc.) the inventive methods enable the detection of less than 1 μM, less than 100 nM, less than 10 nM or even less than 1 nM of a metabolite or other analyte of interest. When the analyte is being used as an imaging agent for an MRI experiment then it will preferably include at least one spin half nucleus with a long T1 relaxation time. This will ensure that the enhancement is not lost by relaxation in between the polarizing and detecting steps. For example, U.S. Pat. No. 6,311,086 (the contents of which are incorporated herein by reference) describes imaging agents that include spin half nuclei with T1 relaxation times of at least 6 seconds at 310 K in D2O in a magnetic field of 7 T. It will be appreciated that any of the imaging agents that are described in U.S. Pat. No. 6,311,086 may be used as an analyte in an inventive method. It is also to be understood that any known MRI technique may be used to image the spatial distribution of a polarized analyte once administered to a subject (e.g., see MRI in Practice Ed. by Westerbrook et al., Blackwell Publishing, Oxford, UK, 2005, the contents of which are incorporated herein by reference).
Any spin half nucleus within the analyte may be polarized according to the inventive methods. In one embodiment, the spin half nucleus is a 1H nucleus. In one embodiment, the spin half nucleus is a 13C nucleus. In one embodiment, the spin half nucleus is a 15N nucleus. In one embodiment, the spin half nucleus is a 19F nucleus. The spin half nucleus may be present in the analyte at natural abundance levels. Alternatively, stronger signals may be obtained if the spin half nucleus (e.g., 13C, 15N, 19F, etc.) is enriched at one or more positions within the analyte. A variety of methods are known in the art for enriching one or more sites of an analyte (e.g., a protein, nucleic acid, metabolite, imaging agent, etc.). When the at least one spin half nucleus has a γ-value smaller than that of 1H (e.g., 13C, 15N, 19F, etc.) then in certain embodiments, the step of polarizing may further involve irradiating the frozen sample with radiation having a frequency that causes cross-polarization between a 1H nucleus present in the sample (e.g., without limitation from 1H2O) and the at least one spin half nucleus of the analyte.
The inventive methods may be performed under any magnetic field strength. In one embodiment the field may have a strength in the range of about 0. 1 T to about 30 T. For example, some of the experiments that are described herein were performed at 5 T. The radiation for exciting electron spin transitions in the unpaired electron(s) of the polarizing agent at these fields will be in the range of about 2.8 GHz to about 840 GHz. For examples, the radiation in the experiments that are described herein was from a 140 GHz gyrotron. In certain embodiments, the polarization step may take less than about 2 minutes, e.g., less than about 90 seconds or less than about 1 minute.
In certain embodiments, the sample may be recycled by freezing the sample, repolarizing the at least one spin half nucleus of the analyte by irradiating the frozen sample with radiation having a frequency that excites electron spin transitions in the biradical, remelting the frozen sample to produce a molten sample, and redetecting nuclear spin transitions in the at least one spin half nucleus of the analyte in the molten sample. This method can be repeated for as many cycles as needed. This can be used, e.g., to signal average NMR signals and thereby further enhance the sensitivity of a liquid-state NMR experiment. The freezing step can generally be achieved by cooling the sample until it reaches a solid state. In certain embodiments, the sample can be cooled to a temperature of less than about 200 K. For example, the sample may be cooled to a temperature in the range of about 1 K to about 100 K. Some of the experiments that are described herein involved cooling the sample to a temperature of about 90 K. In one embodiment, the freezing step may be completed in less than about 2 minutes, e.g., less than about 90 seconds, or less than about 1 minute.
In general, once a frozen sample has been polarized according to the present invention it can be melted using any suitable method. In certain embodiments, this is achieved by exposing the frozen sample to radiation having a wavelength of less than about 100 μm, e.g., in the range of about 0.5 μm and about 50 μm. In one embodiment, the radiation may come from a laser, e.g., a CO2 laser. In another embodiment, the radiation may come from a lamp, e.g., an infra-red lamp. The frozen sample can be exposed to the radiation using an optical fiber. This will typically involve coupling the radiation (e.g., from a laser or lamp) to one end of the fiber, e.g., using a lens. In one embodiment, the sample is within a cylindrical rotor. Advantageously, the rotor can be made of quartz which allows both microwave radiation (e.g., the 140 GHz radiation from a gyrotron) and infra-red radiation (e.g., from a CO2 laser) to reach the sample. We have also found that a quartz rotor does not crack when exposed to multiple freeze-thaw cycles. Finally, the use of a cylindrical rotor enables the sample to be spun during the melting step (and optionally during other steps including the detecting step) which we have found to significantly improve melting homogeneity and time. In the experiments that are described herein we were able to melt samples in less than about 1 second.
Once melted, the molten sample may be analyzed by liquid-state NMR. Any liquid-state NMR technique can be used to detect the polarized nucleus or nuclei, e.g., one dimensional techniques, multi-dimensional techniques, including without limitation techniques that rely on NOESY, ROESY, TOCSY, HSQC, HMQC, etc. type polarization transfers and combinations thereof. The detected NMR signals may be from any spin half nucleus of the analyte, e.g., 1H, 13C, 15N etc. In certain embodiments it may prove advantageous to decouple the polarized nucleus or nuclei from 1H nuclei present in the sample.
Alternatively, in certain embodiments, at least a portion of the molten sample may be administered to a subject and then imaged by MRI. According to this last embodiment, the administered portion of the molten sample includes an amount of the polarized analyte. In certain embodiments (e.g., when the biradical is toxic) the polarized analyte may be separated from the biradical prior to administration. U.S. Pat. No. 6,311,086 (the contents of which are incorporated herein by reference) describes several methods for achieving such a separation (e.g., physical and chemical separation or extraction techniques). The polarized analyte may be administered to a subject using any known route of administration (e.g., by injection, ingestion, inhalation, etc.).
In general, any polarizing agent with an unpaired electron may be used according to the inventive methods. In certain embodiments, the polarizing agent is a monoradical, e.g., any one of the nitrogen oxide radicals (e.g., TEMPO based radicals) and trityl radicals that have been described in the art. In other embodiments, the polarizing agent is a biradical as further described herein and in U.S. Patent Publication No. 20050107696, the entire contents of which are incorporated herein by reference. Without limitation, in one embodiment, the polarizing agent is bis-TEMPO-2-ethyleneglycol (BT2E). In another embodiment, the polarizing agent is a biradical described in a PCT patent application that we filed on May 10, 2007 entitled “Biradical Polarizing Agents for Dynamic Nuclear Polarization”, the contents of which are incorporated herein by reference. In one embodiment, the polarizing agent is 1-(TEMPO-4-oxy)-3-(TEMPO-4-amino)-propan-2-ol (TOTAPOL).
In another aspect, the present invention also provides systems for performing these methods. Thus in one aspect a system is provided that comprises an NMR spectrometer, an NMR magnet including a probe for coupling radiofrequency radiation with a sample (e.g., a 5 T magnet), a microwave gyrotron (e.g., a 140 GHz source), a source of infra-red radiation (e.g., a CO2 laser) and a quartz rotor for holding a sample. The system may further comprise an optical waveguide for delivering the microwave radiation to the quartz rotor. The system may also comprise an optical fiber for delivering the infra-red radiation to the quartz rotor.
The inventive methods have been used to generate enhancements in the range of 140-280 in NMR spectra of low-γ spins such as 13C and 15N. In these experiments, we polarized the 1H spins in various samples at low temperatures (˜90 K) using biradical polarizing agents2, 34. The polarization was transferred to low-γ spins with cross polarization, the sample was melted with an infrared laser pulse, and the enhanced signal observed in the presence of decoupling. If the polarization step were to be performed at a lower temperature (e.g., 10 K), then an even larger enhancement factor would be observed.
These experiments were carried out with the apparatus shown in
Samples for the experiments consisted of solutions containing high concentrations of 13C labeled small molecules to facilitate observation of signals in the absence of DNP. In particular, the high concentration facilitated observation of the signal intensity in the absence of microwave irradiation and therefore measurement of the enhancement. For example, in the experiments below we used 2 M 13C-urea in a solvent of composition 60% 2H8-glycerol and 40% water (80% 2H2O/20% H2O). The solution was prepared with 3-5 mM TOTAPOL34 as the biradical polarizing agent. About 9 μL of sample was placed in a 2.5 mm OD quartz capillary and NMR measurements were conducted in a custom designed probe in a 5 T magnet (211 MHz for 1H and 53.31 MHz for 13C). Continuous microwave irradiation was generated with a 140 GHz gyrotron35. The sample was maintained at 90 K by circulating cold N2 gas during the experimental cycle. Typically the equilibrium polarization buildup required 15-30 s (the 1H T1 is typically 5-10 s), and the enhancement in the solid state spectra was 165 at this temperature and magnetic field. The rapid temperature jump (TJ) was performed by irradiating the sample with 10.6 μm radiation from the CO2 laser transmitted to the sample through a multimode hollow optic fiber. Haw and coworkers36, 37 used a similar approach in TJ experiments on polymers with the exception that the sample was larger (5 mm diameter rotors) and required higher laser power. Thus, it was necessary to use lenses rather than an optic fiber to irradiate the sample. After melting, the solution NMR spectrum was recorded in the presence of decoupling, and the sample was refrozen and repolarized for another experimental cycle. Typically the freezing required 60 s, and the melting <1 s.
Enhancements, ε†, (vide infra) were determined by comparing the signal intensities of the DNP enhanced 13C signal intensities obtained in the melting experiment with those obtained from room temperature solution NMR experiments. The room temperature liquid state spectra were directly detected and typically acquired by averaging 1024 scans with a long recycle delay (60-120 s) to ensure that we reached the equilibrium Boltzmann polarization. Note that in generating the 13C DNP enhanced signals, we transferred polarization from electrons to 1H and then to 13C via cross-polarization (CP) since this is the most time efficient manner to move polarization from the electrons to the 13C. In principle we could have polarized 13C directly but the method is slower since spin diffusion in the 13C reservoir is slow. It will also be appreciated that the 13C signals could be indirectly detected via observation of 'H as is customary in many solution NMR experiments38.
We noted above that we have labeled the enhancements as ε†, rather than ε as is common in solid state MAS experiments2-9. Thus, there are two conventions in use to report the size of enhancements that deserve explanation.
(1) In solid state magic angle spinning (MAS) experiments it is usual to compare the signal intensity in the presence and absence of microwave irradiation at the temperature where the DNP enhancement is performed. This ratio of signal intensities yields the enhancement ε due to the microwave irradiation. The data and enhancements reported in several other publications from our laboratory at T≧90 K use this convention and are due to the microwave driven enhancement alone 1-9.
(2) In the case of liquids, however, the relevant enhancement, that we define as ε†, is determined by the intensity of the DNP enhanced signal relative to the signal due to the Boltzmann polarization recorded at 300 K. Since the polarization is generated at low temperature, for example 90 K, there is an additional factor of (Tobs/Tμwave)˜(300 K/100 K)=3 included in the calculation of the enhancement ε†. When the polarization is performed at 1.2 K and the observation is at 300 K, this number increases to 250. Thus, enhancements reported in the literature for solid state and liquids experiments differ by the factor (Tobs/Tμwave), which can be substantial. For example, by polarizing at 1.2 K Ardenkjaer-Larsen and coworkers 3 reported ε†=44,400 which corresponds to ε=178 if we take (Tobs/Tμwave)=250. Accordingly, in this specification we quote two enhancements ε† and ε that are related by ε†=ε (Tobs/Tμwave) where Tobs and Tμwave are the temperatures where the signal observation and microwave irradiation occur. Note that, ε†=ε in the limit where Tobs=Tμwave.
In addition, there are several features of the experiments described here that differ in significant ways from the experiments described by Ardenkjaer-Larsen, et al39. In particular, while we performed an in situ TJ melting experiment, they in contrast utilized an approach involving polarization at low field, “dissolution” of the sample, and transfer to a higher field for observation. The difference in the experimental details is as follows. First, in the dissolution experiment the polarization step was performed at 1.2 K rather than 90 K. Second, it was performed in a 3.35 T field using a 200 mW, 94 GHz microwave source to drive the DNP method. Third, the triphenylmethyl based trityl radical40 was the polarizing agent, and the 13C spins in the sample were polarized directly (ε˜178) rather than through the 1H's. Because of the low temperature, the low microwave power, the long T1e of the trityl radical and the fact that they polarized 13C directly, their polarization times were ˜80 minutes. In contrast, we are able to achieve enhancements ε ˜290 in 40 s at 90 K at 5 T using our 140 GHz microwave source and biradical polarizing agents. Finally, in the “dissolution” experiment the sample, consisting of 40-50 mg of frozen polarized pellets, is melted and dissolved in 7 ml of hot water, diluting it by a factor of 150. If the polarized solute is used in imaging experiments, then dilution of the sample with solvent may not be a concern. However, for analytical NMR experiments it is clearly undesirable. Following dissolution, the sample was manually shuttled to a 400 MHz liquids spectrometer where solution NMR spectrum was recorded. Because of the requirement of shuttling to a second magnet, it is not possible to rapidly repolarize the sample. In the results illustrated in
We also mentioned above that in the spectra of the samples that normally contain protons, the 1H's were substituted with 2H. The reason for this is that in the liquid phase, the glassy glycerol mixtures used to polarize the samples are very viscous. Consequently, the 1H relaxation times are very short (milliseconds)41 and the short 1H T1 leads to relaxation of the 13C and loss of the 13C signal. However, preliminary experiments with solvent systems that exhibit lower viscosity in the liquid phase, and still form low temperature glasses that disperse the biradical, suggest that it should be possible to employ protonated molecules in the TJ-DNP experiments.
The results shown in
Our purpose here was to demonstrate the feasibility of using TJ-DNP for observing spectra of liquids with enhanced sensitivity. It will be appreciated that a number of variations on the exemplified methods can be envisaged. For example, one could readily perform the polarization at lower temperatures, improve the efficiency of the melting method, and/or perform the experiments in glassy solvents that have a lower viscosity at room temperature. For example, the spectra displayed in
We have demonstrated the possibility of observing sensitivity enhanced 13C spectra of small molecules by first polarizing the sample and then melting it with laser radiation followed by observation of the solution NMR spectrum. Currently, we utilize biradical polarizing agents and gyrotron microwave sources for the DNP method. The latter enables the experiment to be performed in situ and to be recycled for signal averaging as is customary in conventional time domain NMR spectroscopy. The sensitivity enhancements at room temperature where the spectra are observed are presently 250, but they could be further enhanced by performing the polarization step at even lower temperature.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.
While the present invention has been described in conjunction with various embodiments and examples, it is not intended that the present invention be limited to such embodiments or examples. On the contrary, the present invention encompasses various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.
While the present invention has been particularly shown and described with reference to specific illustrative embodiments, it should be understood that various changes in form and detail may be made without departing from the spirit and scope of the present invention. Therefore, all embodiments that come within the scope and spirit of the present invention, and equivalents thereto, are intended to be claimed.
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This application claims priority to U.S. Provisional Application Ser. No. 60/747,098 filed May 12, 2006, the contents of which are incorporated herein by reference.
The inventions described herein were made with support from funding from the National Institutes of Health, Grant No. EB-002804. The U.S. Government therefore has certain rights in these invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US07/68654 | 5/10/2007 | WO | 00 | 4/15/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60747098 | May 2006 | US |
