Friday, October 30, 2015

Rational design of dinitroxide biradicals for efficient cross-effect dynamic nuclear polarization

Kubicki, D.J., et al., Rational design of dinitroxide biradicals for efficient cross-effect dynamic nuclear polarization. Chem. Sci., 2015.

A series of 37 dinitroxide biradicals have been prepared and their performance studied as polarizing agents in cross effect DNP NMR experiments at 9.4 T and 100 K in 1,1,2,2-tetrachloroethane (TCE). We observe that in this regime the DNP performance is strongly correlated with the substituents on the polarizing agents, and electron and nuclear spin relaxation times, with longer relaxation times leading to better enhancements. We also observe that deuteration of the radicals generally leads to better DNP enhancement but with longer buildup time. One of the new radicals introduced here provides the best performance obtained so far under these conditions.

Thursday, October 29, 2015

[NMR] 2016 Winter School on Biomolecular Solid-State NMR: Application deadline is Oct 30, 2015

From the Ampere Magnetic Resonance List
The 4th U.S.-Canada Winter School on Biomolecular Solid-State NMR
Stowe, Vermont
January 10-15, 2016

Organizers: Mei Hong (MIT), Chris Jaroniec (Ohio State) and Bob Griffin (MIT)

Dear Colleagues,

We invite you to encourage your students, postdocs, and senior associates to attend the 4th Winter School on Biomolecular Solid-State NMR, which will be held on January 10-15, 2016, in Stowe, Vermont. Similar to the three previous highly successful Winter Schools, this pedagogical meeting is aimed at students and postdocs in solid-state NMR as well as more senior scientists in related fields who are interested in entering this vibrant field. Our goals are to provide a focused week of teaching of the core concepts and practices in the increasingly multifaceted and complex field of biological solid-state NMR spectroscopy, and to encourage information sharing among different laboratories. Topics to be covered in the 4th Winter School include:

  • Basics of solid-state NMR: orientation-dependent NMR frequencies, MAS, tensors and rotations, density operator and its time evolution, dipolar recoupling, and average Hamiltonian theory.
  • Multidimensional correlation spectroscopy, resonance assignment and protein structure determination 
  • Theory of dipolar decoupling and polarization transfer
  • Pushing the sensitivity envelope: dynamic nuclear polarization and 1H detection
  • Effective "sensitivity enhancement" by non-uniform sampling
  • Solid-state NMR techniques for measuring molecular motion
  • Solid-state NMR techniques for measuring membrane protein orientation 
  • Beyond spin 1/2: NMR of quadrupolar nuclei 
  • Beating the 800-pound gorilla: NMR of membrane proteins 
  • XPLOR-NIH for structure calculation 
In addition to lectures, problem sets and their discussion sessions will be given at the meeting.

Speakers: The following people have agreed to give lectures:
Tim Cross (Florida State)
Philip Grandinetti (Ohio State)
Bob Griffin (MIT)
Mei Hong (MIT)
Chris Jaroniec (Ohio State)
Vladimir Ladizhansky (Guelph)
Len Mueller (UC Riverside)
Stanley Opella (UC San Diego)
Bernd Reif (Tech Univ Munich)
David Roynyak (Bucknell)
Charles Schwieters (NIH)
Robert Tycko (NIH)

Venue and transportation: The meeting will be held at the beautiful and historical Trapp Family Lodge in Stowe, Vermont. Stowe is accessible from airports in Burlington, VT, Manchester, NH, and Boston, MA. A block of rooms has been reserved at the TFL. We anticipate space for ~70 attendees.

Cost: Room and board will be free for attendees. The registration fee is $425 for academic attendees and $600 for industrial attendees. 

Application: Interested students and postdocs should send the following application material as a single PDF file to The application material includes 1) a CV, 2) publication list, and 3) a 1-page description of your current research and your statement of interest in attending the Winter School. Please indicate your gender in the CV for the purpose of hotel room assignment. Please name this application file as ProfessorLastName_YourLastName_WS2016app.pdf. For example, the filename can be  “Mueller_ Smith_WS2016app.pdf”.

Application deadline: Friday, Oct 30, 2015. Given the space limitation, applications received after the above date may not be possible to be accommodated. 

Please distribute this announcement to members of your research group as well as to colleagues who may be interested in attending or sending their group members.

With kind regards,
Mei and Chris

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Wednesday, October 28, 2015

Polymorphs of Theophylline Characterized by DNP Enhanced Solid-State NMR

Pinon, A.C., et al., Polymorphs of Theophylline Characterized by DNP Enhanced Solid-State NMR. Mol Pharm, 2015.

We show how dynamic nuclear polarization (DNP) enhanced solid-state NMR spectroscopy can be used to characterize polymorphs and solvates of organic solids. We applied DNP to three polymorphs and one hydrated form of the asthma drug molecule theophylline. For some forms of theophylline, sample grinding and impregnation with the radical-containing solution, which are necessary to prepare the samples for DNP, were found to induce polymorphic transitions or desolvation between some forms. We present protocols for sample preparation for solid-state magic-angle spinning (MAS) DNP experiments that avoid the polymorphic phase transitions in theophylline. These protocols include cryogrinding, grinding under inert atmosphere, and the appropriate choice of the impregnating liquid. By applying these procedures, we subsequently demonstrate that two-dimensional correlation experiments, such as 1H-13C and 1H-15N HETCOR or 13C-13C INADEQUATE, can be obtained at natural isotopic abundance in reasonable times, thus enabling more advanced structural characterization of polymorphs.

Monday, October 26, 2015

The effect of Gd on trityl-based dynamic nuclear polarisation in solids

Ravera, E., et al., The effect of Gd on trityl-based dynamic nuclear polarisation in solids. Phys Chem Chem Phys, 2015. 17(40): p. 26969-78.

In dynamic nuclear polarisation (DNP) experiments performed under static conditions at 1.4 K we show that the presence of 1 mM Gd(iii)-DOTAREM increases the (13)C polarisation and decreases the (13)C polarisation buildup time of (13)C-urea dissolved in samples containing water/DMSO mixtures with trityl radical (OX063) concentrations of 10 mM or higher. To account for these observations further measurements were carried out at 6.5 K, using a combined EPR and NMR spectrometer. At this temperature, frequency swept DNP spectra of samples with 5 or 10 mM OX063 were measured, with and without 1 mM Gd-DOTA, and again a (13)C enhancement gain was observed due to the presence of Gd-DOTA. These measurements were complemented by electron-electron double resonance (ELDOR) measurements to quantitate the effect of electron spectral diffusion (eSD) on the DNP enhancements and lineshapes. Simulations of the ELDOR spectra were done using the following parameters: (i) a parameter defining the rate of the eSD process, (ii) an "effective electron-proton anisotropic hyperfine interaction parameter", and (iii) the transverse electron spin relaxation time of OX063. These parameters, together with the longitudinal electron spin relaxation time, measured by EPR, were used to calculate the frequency profile of electron polarisation. This, in turn, was used to calculate two basic solid effect (SE) and indirect cross effect (iCE) DNP spectra. A properly weighted combination of these two normalized DNP spectra provided a very good fit of the experimental DNP spectra. The best fit simulation parameters reveal that the addition of Gd(iii)-DOTA causes an increase in both the SE and the iCE contributions by similar amounts, and that the increase in the overall DNP enhancements is a result of narrowing of the ELDOR spectra (increased electron polarisation gradient across the EPR line). These changes in the electron depolarisation profile are a combined result of shortening of the longitudinal and transverse electron spin relaxation times, as well as an increase in the eSD rate and in the effective electron-proton anisotropic hyperfine interaction parameter.

Friday, October 23, 2015

High-resolution NMR of hydrogen in organic solids by DNP enhanced natural abundance deuterium spectroscopy

Rossini, A.J., et al., High-resolution NMR of hydrogen in organic solids by DNP enhanced natural abundance deuterium spectroscopy. J. Magn. Reson., 2015. 259: p. 192-198.

We demonstrate that high field (9.4 T) dynamic nuclear polarization (DNP) at cryogenic (∼100 K) sample temperatures enables the rapid acquisition of natural abundance 1H–2H cross-polarization magic angle spinning (CPMAS) solid-state NMR spectra of organic solids. Spectra were obtained by impregnating substrates with a solution of the stable DNP polarizing agent TEKPol in tetrachloroethane. Tetrachloroethane is a non-solvent for the solids, and the unmodified substrates are then polarized through spin diffusion. High quality natural abundance 2H CPMAS spectra of histidine hydrochloride monohydrate, glycylglycine and theophylline were acquired in less than 2 h, providing direct access to hydrogen chemical shifts and quadrupolar couplings. The spectral resolution of the 2H solid-state NMR spectra is comparable to that of 1H spectra obtained with state of the art homonuclear decoupling techniques.

Wednesday, October 21, 2015

Hyperpolarized choline as an MR imaging molecular probe: feasibility of in vivo imaging in a rat model

Friesen-Waldner, L.J., et al., Hyperpolarized choline as an MR imaging molecular probe: feasibility of in vivo imaging in a rat model. J Magn Reson Imaging, 2015. 41(4): p. 917-23.

PURPOSE: To assess the feasibility of choline MRI using a new choline molecular probe for dynamic nuclear polarization (DNP) hyperpolarized MRI. MATERIALS AND METHODS: Male Sprague-Dawley rats with an average weight of 400 +/- 20 g (n = 5), were anesthetized and injection tubing was placed in the tail vein. [1,1,2,2-D4 , 1-(13) C]choline chloride (CMP1) was hyperpolarized by DNP and injected into rats at doses ranging from 12.6 to 50.0 mg/kg. Coronal projection (13) C imaging was performed on a 3 Tesla clinical MRI scanner (bore size 60 cm) using a variable flip angle gradient echo sequence. Images were acquired 15 to 45 s after the start of bolus injection. Signal intensities in regions of interest were determined at each time point and compared. RESULTS: (13) C MRI images of hyperpolarized CMP1 at a 50 mg/kg dose showed time-dependent organ distribution patterns. At 15 s, high intensities were observed in the inferior vena cava, heart, aorta, and kidneys. At 30 s, most of the signal intensity was localized to the kidneys. These distribution patterns were reproduced using 12.6 and 25 mg/kg doses. At 45 s, only signal in the kidneys was detected. CONCLUSION: Hyperpolarized choline imaging with MRI is feasible using a stable-isotope labeled choline analog (CMP1). Nonradioactive imaging of choline accumulation may provide a new investigatory dimension for kidney physiology. J. Magn. Reson. Imaging 2015;41:917-923. (c) 2014 Wiley Periodicals, Inc.

Friday, October 16, 2015

Direct dynamic measurement of intracellular and extracellular lactate in small-volume cell suspensions with (13)C hyperpolarised NMR

Breukels, V., et al., Direct dynamic measurement of intracellular and extracellular lactate in small-volume cell suspensions with (13)C hyperpolarised NMR. NMR Biomed, 2015. 28(8): p. 1040-8.

Hyperpolarised (HP) (13)C NMR allows enzymatic activity to be probed in real time in live biological systems. The use of in vitro models gives excellent control of the cellular environment, crucial in the understanding of enzyme kinetics. The increased conversion of pyruvate to lactate in cancer cells has been well studied with HP (13)C NMR. Unfortunately, the equally important metabolic step of lactate transport out of the cell remains undetected, because intracellular and extracellular lactate are measured as a single resonance. Furthermore, typical experiments must be performed using tens of millions of cells, a large amount which can lead to a costly and sometimes highly challenging growing procedure. We present a relatively simple set-up that requires as little as two million cells with the spectral resolution to separate the intracellular and extracellular lactate resonances. The set-up is tested with suspensions of prostate cancer carcinoma cells (PC3) in combination with HP [1-(13)C]pyruvate. We obtained reproducible pyruvate to lactate label fluxes of 1.2 and 1.7 nmol/s per million cells at 2.5 and 5.0 mM pyruvate concentrations. The existence of a 3-Hz chemical shift difference between intracellular and extracellular lactate enabled us to determine the lactate transport rates in PC3. We deduced a lactate export rate of 0.3 s(-1) and observed a decrease in lactate transport on addition of the lactate transport inhibitor alpha-cyano-4-hydroxycinnamic acid.

Wednesday, October 14, 2015

Pushing NMR sensitivity limits using dynamic nuclear polarization with closed-loop cryogenic helium sample spinning

Bouleau, E., et al., Pushing NMR sensitivity limits using dynamic nuclear polarization with closed-loop cryogenic helium sample spinning. Chemical Science, 2015.

We report a strategy to push the limits of solid-state NMR sensitivity far beyond its current state-of-the-art. The approach relies on the use of dynamic nuclear polarization and demonstrates unprecedented DNP enhancement factors for experiments performed at sample temperatures much lower than 100 K, and can translate into 6 orders of magnitude of experimental time-savings. This leap-forward was made possible thanks to the employment of cryogenic helium as the gas to power magic angle sample spinning (MAS) for dynamic nuclear polarization (DNP) enhanced NMR experiments. These experimental conditions far exceed what is currently possible and allows currently reaching sample temperatures down to 30 K while conducting experiments with improved resolution (thanks to faster spinning frequencies, up to 25 kHz) and highly polarized nuclear spins. The impressive associated gains were used to hyperpolarize the surface of an industrial catalyst as well as to hyperpolarize organic nano-assemblies (self-assembling peptides in our case), for whom structures cannot be solved using diffraction techniques. Sustainable cryogenic helium sample spinning significantly enlarges the realm and possibilities of the MAS-DNP technique and is the route to transform NMR into a versatile but also sensitive atomic-level characterization tool.

Friday, October 9, 2015

Metabolic response of prostate cancer to nicotinamide phophoribosyltransferase inhibition in a hyperpolarized MR/PET compatible bioreactor

Keshari, K.R., et al., Metabolic response of prostate cancer to nicotinamide phophoribosyltransferase inhibition in a hyperpolarized MR/PET compatible bioreactor. Prostate, 2015. 75(14): p. 1601-9.

BACKGROUND: Metabolic shifts in disease are of great interest for the development of novel therapeutics. In cancer treatment, these therapies exploit the metabolic phenotype associated with oncogenesis and cancer progression. One recent strategy involves the depletion of the cofactors needed to maintain the high rate of glycolysis seen with the Warburg effect. Specifically, blocking nicotinamide adenine dinucleotide (NAD) biosynthesis via nicotinamide phosphoribosyltransferase (NAMPT) inhibition depletes cancer cells of the NAD needed for glycolysis. To characterize this metabolic phenotype in vivo and describe changes in flux with treatment, non-invasive biomarkers are necessary. One such biomarker is hyperpolarized (HP) [1-(13) C] pyruvate, a clinically translatable probe that allows real-time assessment of metabolism. METHODS: We therefore developed a cell perfusion system compatible with HP magnetic resonance (MR) and positron emission tomography (PET) to develop translatable biomarkers of response to NAMPT inhibition in reduced volume cell cultures. RESULTS: Using this platform, we observed a reduction in pyruvate flux through lactate dehydrogenase with NAMPT inhibition in prostate cancer cells, and showed that both HP lactate and 2-[(18) F] fluoro-2-deoxy-D-glucose (FDG) can be used as biomarkers for treatment response of such targeted agents. Moreover, we observed dynamic flux changes whereby HP pyruvate was re-routed to alanine, providing both positive and negative indicators of treatment response. CONCLUSIONS: This study demonstrated the feasibility of a MR/PET compatible bioreactor approach to efficiently explore cell and tissue metabolism, the understanding of which is critical for developing clinically translatable biomarkers of disease states and responses to therapeutics. Prostate 75:1601-1609, 2015. (c) 2015 Wiley Periodicals, Inc.

Wednesday, October 7, 2015

Affinity screening using competitive binding with fluorine-19 hyperpolarized ligands

Kim, Y. and C. Hilty, Affinity screening using competitive binding with fluorine-19 hyperpolarized ligands. Angew Chem Int Ed Engl, 2015. 54(16): p. 4941-4.

Fluorine-19 NMR and hyperpolarization form a powerful combination for drug screening. Under a competitive equilibrium with a selected fluorinated reporter ligand, the dissociation constant (K(D)) of other ligands of interest is measurable using a single-scan Carr-Purcell-Meiboom-Gill (CPMG) experiment, without the need for a titration. This method is demonstrated by characterizing the binding of three ligands with different affinities for the serine protease trypsin. Monte Carlo simulations show that the highest accuracy is obtained when about one-half of the bound reporter ligand is displaced in the binding competition. Such conditions can be achieved over a wide range of affinities, allowing for rapid screening of non-fluorinated compounds when a single fluorinated ligand for the binding pocket of interest is known.

Monday, October 5, 2015

Polarization Transfer from Ligands Hyperpolarized by Dissolution Dynamic Nuclear Polarization for Screening in Drug Discovery

Min, H., G. Sekar, and C. Hilty, Polarization Transfer from Ligands Hyperpolarized by Dissolution Dynamic Nuclear Polarization for Screening in Drug Discovery. ChemMedChem, 2015. 10(9): p. 1559-63.

Nuclear magnetic resonance (NMR) spectroscopy is a valuable technique for ligand screening, because it exhibits high specificity toward chemical structure and interactions. Dissolution dynamic nuclear polarization (DNP) is a recent advance in NMR methodology that enables the creation of non-equilibrium spin states, which can dramatically increase NMR sensitivity. Here, the transfer of such spin polarization from hyperpolarized ligand to protein is observed. Mixing hyperpolarized benzamidine with the serine protease trypsin, a "fingerprint" of enhanced protein signals is observed, which shows a different intensity profile than the equilibrium NMR spectrum of the protein, but coincides closely to the frequency profile of a saturation transfer difference (STD) NMR experiment. The DNP experiment benefits from hyperpolarization and enables observation of all frequencies in a single, rapid experiment. Based on these merits, it is an interesting alternative to the widely used STD experiment for identification of protein-ligand interactions.

Friday, October 2, 2015

Quantitative Structural Constraints for Organic Powders at Natural Isotopic Abundance Using Dynamic Nuclear Polarization Solid-State NMR Spectroscopy

Mollica, G., et al., Quantitative Structural Constraints for Organic Powders at Natural Isotopic Abundance Using Dynamic Nuclear Polarization Solid-State NMR Spectroscopy. Angewandte Chemie, 2015. 127(20): p. 6126-6129.

A straightforward method is reported to quantitatively relate structural constraints based on 13C–13C double-quantum build-up curves obtained by dynamic nuclear polarization (DNP) solid-state NMR to the crystal structure of organic powders at natural isotopic abundance. This method relies on the significant gain in NMR sensitivity provided by DNP (approximately 50-fold, lowering the experimental time from a few years to a few days), and is sensitive to the molecular conformation and crystal packing of the studied powder sample (in this case theophylline). This method allows trial crystal structures to be rapidly and effectively discriminated, and paves the way to three-dimensional structure elucidation of powders through combination with powder X-ray diffraction, crystal-structure prediction, and density functional theory computation of NMR chemical shifts.