Subscriber access provided by ECOLE NORMALE SUPERIEURE LYON Article Dynamic Nuclear Polarization of Amyloidogenic Peptide Nanocrystals: GNNQQNY, a Core Segment of the Yeast Prion Protein Sup35p iMR Advanced Training Workshop I Patrick C. A. van der Wel, Kan-Nian Hu, Jzef Lewandowski, and Robert G. Griffin J. Am. Chem. Soc., 2006, 128 (33), 10840-10846• DOI: 10.1021/ja0626685 • Publication Date (Web): 02 August 2006 ApplicationsDownloaded of fromDynamic http://pubs.acs.org Nuclear on April 27, 2009 Polarisation

DNP no DNP

More About This Article Józef Lewandowski Additional resources and features associated with this article are available within the HTML version: April 10 2013 • Supporting Information • Links to the 13 articles that cite this article, as of the time of this article download

• Wednesday,Access 10 April to 13 high resolution figures • Links to articles and content related to this article • Copyright permission to reproduce figures and/or text from this article

Journal of the American Chemical Society is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 2/2/2011

2/2/2011 Why to do DNP? Why bother? MR3 Advanced Topics in MR Lectures 5 and 6: Dynamic Nuclear Polarisation 13C image DNP 13C-lactate

.. Walter Kockenberger,Dissolution DNP [email protected] pressure cooker solvent (H O) 100 – 140°C 2 2 – 10 bar

chase gas (Helium) (injected) (newly synthesised) 2 – 5 bar • more signal Klaes Golman et al • polarizer faster acquisition • new, previously not possible applications,sample cup now within the reach

Wednesday, 10 April 13 Why bother? What’s on the plate? • Sensitivity and spin polarisation • Relevant spin interactions • The spin Hamiltonian for liquid state • Energy levels and populations I • The Solomon equations T. Maly/ R. Griffin, MIT • The Overhauser effect • The spin Hamiltonian for solid state • A small perturbation Things can be more12.6s complicated... • Mixing of states 7.2s • The probability for ‘forbidden’ transitions cross effect thermal mixing Enhancement ε+: ~10000 • Energy levels and populationsHardware II requirements e δ à ωn H 14.2sH en • Dynamics of the solid effect e en gly e n DNP e • Hee n Things can be more complicated... Hee n e n • Experimental strategies |ωe1-ωe2| = ωn e Waldemar Senczenko, UoNn 3 spin model liquids solids Multi-spin model SSSSII SSSSI I I++I++-›-› electron electron nuclear Zeeman dipolar Zeeman II reservoir reservoir reservoir dissolution experiment ++++++›› ωωωe1 sample shuttling I+-I+--›-› Lattice low field high field low field high field I+-I+-++›› I-+I-+-›-› Thermodynamical description I-+I-+++›› temperature jump I--I---›-› I--I--++›› positive enhancement ∆ω∆ω∆ωe = ωωωn 48 1

12 What do you need for DNP?

1. source of large polarisation

2. way to transfer it to the species you care about (relaxation or coherent process) ? 3. detect the high signal for your hyperpolarised species (they have to live long enough)

Wednesday, 10 April 13 Hyperpolarisation techniques

• laser polarised noble gases (3He and 129Xe)

• chemical induced dynamic nuclear polarization (CIDNP)

• parahydrogen induced polarization (PHIP)

• microwave driven dynamic nuclear polarization (DNP)

Wednesday, 10 April 13 Laser polarised noble gases (3He and 129Xe)

3He and 129Xe hyperpolarised using spin-exchange optical pumping (SEOP).

SEOP: Circularly polarised infrared laser light excites e- in Cs or Rb ↓ The angular momentum is transferred from metal e- to noble gas nuclei through collisions.

Wednesday, 10 April 13 Chemical induced dynamic nuclear polarization (CIDNP)

• Using a thermal or photochemical reaction to create hyperpolarisation • Powerful but not general

example: photo-CIDNP effect in entire cells of cyanobacteria

Janssen, G. J.; Daviso, E.; van Son, M.; de Groot, H. J. M.; Alia, a; Matysik, J. Photosynthesis research 2010, 104, 275–82.

Wednesday, 10 April 13 Parahydrogen induced polarization (PHIP)

• Using parahydrogen in hydrogenation reactions to create hyperpolarisation • Also known as PASADENA (Parahydrogen and Synthesis Allow Dramatically Enhanced Nuclear Alignment)

Wednesday, 10 April 13 Subscriber access provided by ECOLE NORMALE SUPERIEURE LYON Article Mine is bigger than yours. DynamicEnhancements Nuclear Polarization of Amyloidogenic Peptide Nanocrystals: GNNQQNY, a Core Segment of the Yeast Prion Protein Sup35p Patrick C. A. van der Wel, Kan-Nian Hu, Jzef Lewandowski, and Robert G. Griffin J. Am. Chem. Soc., 2006, 128 (33), 10840-10846• DOI: 10.1021/ja0626685 • Publication Date (Web): 02 August 2006 Downloadedenhancement from http://pubs.acs.org on April 27, 2009(ε)?

More About This Article van der Wel, P. C. a, Hu, K.-N., Lewandowski, J. R. & Griffin, R. G. J. Am. Chem. Soc. 128, 10840–6 (2006).

AdditionalWednesday, resources 10 April and13 features associated with this article are available within the HTML version: • Supporting Information • Links to the 13 articles that cite this article, as of the time of this article download • Access to high resolution figures • Links to articles and content related to this article • Copyright permission to reproduce figures and/or text from this article

Journal of the American Chemical Society is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Mine is bigger than yours. Enhancements

DNP enhancement; same sample at the same temperature ε → SNR microwave on/SNR microwave off

DNP enhancement; same sample at the room temperature ε†→ ε×Boltzmann factor

sensitivity enhancement; compared to a non-DNP sample at the same temperature Σ → ε×θ×√(κ) ratio of T1 for dry material and impregnated with radical solution paramagnetic quenching sensitivity enhancement; compared to a non-DNP sample at the room temperature Σ†→ ε×θ×√(κ)×Boltzmann factor

Wednesday, 10 April 13 What do you need for microwave driven DNP?

polarizing agent/ microwave source unpaired electrons - e +

NMR/MRI set up

oh, and a sample, off course...

Wednesday, 10 April 13 Implementations of microwave driven dynamic nuclear polarization (DNP)

improve the resolution of images of cells HF-liquid DNP Shuttleand other DNP biological systems; Pomplun and Glaser (DOI: 10.1039/c003751f) high frequency liquid DNP: in situ liquiddiscuss sample theoretical is rapidly methods moved for optimizing microwave excitation at the NMR fromtime the domainlow field, DNP where experiments, the an area that has thus far received little attention. detection field polarization is performed, to a All of these approaches are potentially highapplicable field region to a wide for NMR range of important NMRdetection experiments in biology, chemistry, physics and medicine, and their success- ful development will have an enormous impact on the field. Accordingly, a number of academic and industrial Dissolutionresearch groups have DNP recently initiated High Field efforts to overcome the current limita- tions of the techniques. Technical MAS DNP advances in the area of high-frequency the samplemicrowave is polarized sources in andthe components, solid and gyrotron as a microwave state atof very various low DNP temperatures approaches (Fig. 1), will source, solid sample (typicallybe 1–4 of vital K) and importance magnetic for the further irradiated with fields of development3–7 T, rapidly of dissolved, the DNP method, microwaves at the NMR and finallyespecially transferred at the to highest either magnetic a fields available for NMR ( 20 T). In addition, detection field high resolution NMR spectrometero implementationor a MR imager of microwave time domain experiments should open many Fig. 1 Typical experimental approaches for dynamic nuclear polarization spectrometers. new areas of application, just as rf time domain experiments did for high resolu- process, and resolution in low tempera- Applications of this method range from tion solid state and solution NMR. ture MAS experiments, is addressed here MR imaging of metabolites to studies of Other avenues, such as the optimization Downloaded on 07/04/2013 07:54:53. by Barnes et al. (DOI: 10.1039/c003763j). chemical reaction mechanisms (Bowen of polarizing agents, the development Wednesday, 10 April 13 Recently a commercial MAS DNP spec- and Hilty, DOI: 10.1039/c002316g; of new types of polarization transfer

Published on 19 May 2010 http://pubs.rsc.org | doi:10.1039/C0CP90019B trometer became available that is Ludwig et al., DOI: 10.1039/c002700f; methods, and the design of new experi- described in this issue together with some Panek et al., DOI: 10.1039/c002710n; ments focusing on selectivity, contrast recent results obtained with the instru- Cudalbu et al., DOI: 10.1039/c002309b). and additional structural restraints, are ment (Rosay et al., DOI: 10.1039/ An essential ingredient of every DNP ripe for investigation. Thus, collaborative c003685b; Debelouchina et al., DOI: experiment is a stable polarizing agent, efforts among researchers from chemistry, 10.1039/c003661g). Also in this issue, and for the first 50 years of DNP these physics, biology, medicine, and the direct transfers to low-g nuclei (2H, 13C, consisted of readily available monomeric engineering disciplines will be required etc.) are discussed, and the enhance- paramagnetic centers such as a metal, or to optimize DNP for applications in ments, field profiles, and preferred organic radicals like BDPA or TEMPO. high-field NMR and MRI. We foresee polarizing agents are shown to be system More recently, several new polarizing a very bright and expansive future for dependent (Maly et al., DOI: 10.1039/ agents have been introduced that are this field, well into the 21st century. c003705b).40 In Dissolution DNP (lower more efficient in that they produce larger right) the sample is polarized in the solid enhancements at lower concentrations.41–44 state at very low temperatures (typically Four articles describe these new agents: References 1–4 K) and magnetic fields of 3–7 T, narrow line trityl radicals, biradicals and 1 R. R. Ernst and W. A. Anderson, Rev. Sci. rapidly dissolved, and finally transferred spin labeled polymers that separate Instrum., 1966, 37, 93–102. to either a high resolution NMR spectro- at higher temperatures and therefore 2 P. Styles, N. Soffe, C. Scott, D. Cragg, meter or a MR imager (Leggett et al., preserve resolution (Paniagua et al., F. Row, D. White and P. White, J. Magn. DOI: 10.1039/c002566f; Bowen and DOI: 10.1039/c003291n; Dollmann et al., Reson., 1984, 60, 397–404. 3 S. R. Hartmann and E. L. Hahn, Phys. Hilty, DOI: 10.1039/c002316g). Very DOI: 10.1039/c003349a; Ysacco et al., Rev., 1962, 128, 2042–2053. high enhancements (relative to room DOI: 10.1039/c002591g; Macholl et al., 4 A. Pines, M. G. Gibby and J. S. Waugh, temperature) for 13C can be retained DOI: 10.1039/c002699a). J. Chem. Phys., 1972, 56, 1776. 5 G. Morris and R. Freeman, J. Am. Chem. during the dissolution and transfer Finally, two other important and Soc., 1979, 101, 760–762. process, arising from the product of exciting topics are discussed in contri- 6 G. Bodenhausen and D. J. Ruben, Chem. DNP enhancement (B250) and Boltzmann butions to this volume: Thurber and Phys. Lett., 1980, 69, 185–189. polarization (B250). This issue intro- Tycko (DOI: 10.1039/c0cp00157k) con- 7 D. Rugar, R. Budakian, H. J. Mamin and B. W. Chui, Nature, 2004, 329, 430–432. duces several new approaches and sider the possibility of using DNP 8 S. H. J. Mamin, T. H. Oosterkamp, improvements to the experiment. enhancements in solid state imaging to M. Poggio, C. L. Degen, C. T. Rettner

c This journal is the Owner Societies 2010 Phys. Chem. Chem. Phys., 2010, 12,5737–5740 | 5739 304 K.R. Thurber et al. / Journal of Magnetic Resonance 204 (2010) 303–313

[5]. Off-center irradiation of the EPR line creates a polarization of from the same synthesis by chromatography. ESI TOF mass spec- the electron dipolar system, which can then be transferred to the trometry indicated a mass of 628.49 Da for the triradical, in good nuclei. Recent developments in microwave sources and biradical agreement with the proposed structure (C33H63N4O7, theoretical dopants has led to renewed interest in high-field DNP for NMR average mass = 627.87 Da). For TOTAPOL (C21H41N3O4, theoretical spectroscopy [22–30]. average mass = 399.57 Da), the measured mass was 400.32 Da. In this paper, we report the results of DNP experiments at 9.4 T Differences in the electron–electron interactions for the biradi- using a compact and relatively inexpensive (but relatively low- cal and the triradical are clearly seen in the low-field (10 GHz) EPR power) solid state microwave source, in which we observe 1H spectra for 4-hydroxy-TEMPO, TOTAPOL, and DOTOPA-TEMPO NMR signals from frozen glycerol/water solutions that are doped shown in Fig. 1. The low-field EPR spectra in solution are domi- with several nitroxide radical compounds. Glycerol/water solu- nated by hyperfine interactions with 14N nuclei. 4-hydroxy-TEMPO tions are of particular interest because of their relevance to our shows the three lines corresponding to the three spin states of a studies of proteins and protein complexes in frozen solutions single 14N nucleus. If electrons exchange among the nitroxide sites [31–37]. We present data regarding the dependencies of 1H NMR within a single molecule, the EPR lines of the biradical and trirad- signals, nuclear spin relaxation times, and DNP build-up times on ical reflect an average over the multiple 14N sites. In the fast-ex- dopant, dopant concentration, temperature (7–80 K range), and change limit, this results in five EPR lines for the biradical (total microwave power. We introduce a new triradical dopant, DOTOPA- z-component of 14N spin from 2 to +2) and seven EPR lines for À TEMPO, that produces the largest sensitivity enhancements in our the triradical (total z-component of 14N spin from 3 to +3) À experiments. We also show that DNP enhancements can be in- [38,39]. Although the EPR spectrum of the triradical is not fully re- creased substantially by application of magnetic field modulation solved, seven peaks in the derivative spectrum are seen (Fig. 1c). during microwave irradiation, and we discuss possible mecha- Strong features in the derivative spectrum at the edges and in nisms for this effect. The primary conclusion of this work is that the center are characteristic of exchange-broadened spectra quite large enhancements of NMR sensitivity can be achieved in [18,38,40], as EPR lines that correspond to 14N spin states that high fields with relatively low microwave powers, provided that are the same on all nitroxide moieties (i.e., the |+1, +1, +1 , i low temperatures are also employed. Relative to signals at 80 K |0, 0, 0 , and | 1, 1, 1 nuclear spin states) remain sharp in the i À À À i with thermal equilibrium nuclear spin polarization and taking into presence of exchange. These lines occur at the same positions as account the temperature dependence of the DNP build-up time, the hyperfine-split lines of 4-hydroxy-TEMPO (Fig. 1a). Similarly, the sensitivity of 1H NMR measurements is increased by factors the EPR spectrum of TOTAPOL shows three sharp lines of equal greater than 10, 50, 180, and 400 at 80 K, 35 K, 16 K, and 7 K, intensity and broad, unresolved features between these lines Where do the unpaired electrons come from? respectively, in our experiments with 30 mM DOTOPA-TEMPO as (Fig. 1b). This is consistent with narrow EPR lines for the |+1, +1 , i the dopant. |0, 0 , and | 1, 1 14N spin states and broad lines for other 14N i À À i spin states [18]. Nitroxide concentrations- were verified by titration with ascorbic 2. Materials and methods • naturally occurring in someacid samples[41], monitored e by the UV–visible absorption spectrum. The 2.1. Nitroxide dopants UV–visible spectrum has a strong absorbance below 300 nm and (e.g. paramagentic proteins)a weak absorbance at longer wavelength (peaked at 435 nm for 4-Amino-TEMPO and 4-hydroxy-TEMPO were used as pur- DOTOPA-TEMPO) that produces a yellow-orange color, both in the chased from Sigma–Aldrich. The biradical TOTAPOL and the trirad- solid form and in solution. Reduction with ascorbic acid attenuates the 435 nm absorption (see Fig. S1 of Supplementary Material). ical DOTOPA-TEMPO• (4-[N,N-di-(2-hydroxy-3-(TEMPO-4but most of the time 0you-oxy)- have to add it to propyl)]-amino-TEMPO) werethe synthesised sample, based e.g. on the dissolve procedure it Basedin a solution on titration of the 435 nm absorption, our DOTOPA- given in Song et al. for TOTAPOL [17]. Chemical structures are TEMPO had 109 ± 15% of the expected radical concentration shown in Fig. 1. The synthesis was modifiedincluding by reacting the 4-ami- sample(measured at 20 mM in 25/75 mol% glycerol/water). The titration no-TEMPO and 4-(2,3-epoxypropoxy)-TEMPO in 1:1.7 ratio, rather procedure was validated on commercial 4-hydroxy-TEMPO than a 1:1 ratio. Both the biradical and triradical were purified (75 mM in glycerol/water). For TOTAPOL, we measured 85 ± 15% Radicals Transition Metals

Mn2+, Gd3+

Wednesday, 10 April 13

Fig. 1. X-band EPR spectra and chemical structures of 4-hydroxy-TEMPO (a), TOTAPOL (b), and DOTOPA-TEMPO (c). All samples are 0.5 mM solutions in 95/5 vol.% ethanol/ water at room temperature. Considerations for choosing a radical

What mechanism is used for DNP? (This will affect the efficiency of the polarisation enhancement. Need to consider the magnetic field and temperature.)

Does it dissolve in the solvent I want to use? (Well, it has to somehow get near the sample that you try to enhance.)

Wednesday, 10 April 13 Some mechanisms for DNP: enhancement vs. magnetic field

Liquids

Overhauser effect (OE)

2 ε~1/B0

Solids

OE, solid effect (SE), cross effect (CE), thermal mixing (TM)

2 1 ε~1/B0 ε~1/B0

Wednesday, 10 April 13 304 K.R. Thurber et al. / Journal of Magnetic Resonance 204 (2010) 303–313

[5]. Off-center irradiation of the EPR line creates a polarization of from the same synthesis by chromatography. ESI TOF mass spec- the electron dipolar system, which can then be transferred to the trometry indicated a mass of 628.49 Da for the triradical, in good nuclei. Recent developments in microwave sources and biradical agreement with the proposed structure (C33H63N4O7, theoretical dopants has led to renewed interest in high-field DNP for NMR average mass = 627.87 Da). For TOTAPOL (C21H41N3O4, theoretical spectroscopy [22–30]. average mass = 399.57 Da), the measured mass was 400.32 Da. In this paper, we report the results of DNP experiments at 9.4 T Differences in the electron–electron interactions for the biradi- using a compact and relatively inexpensive (but relatively low- cal and the triradical are clearly seen in the low-field (10 GHz) EPR power) solid state microwave source, in which we observe 1H spectra for 4-hydroxy-TEMPO, TOTAPOL, and DOTOPA-TEMPO NMR signals from frozen glycerol/water solutions that are doped shown in Fig. 1. The low-field EPR spectra in solution are domi- with several nitroxide radical compounds. Glycerol/water solu- nated by hyperfine interactions with 14N nuclei. 4-hydroxy-TEMPO tions are of particular interest because of their relevance to our shows the three lines corresponding to the three spin states of a studies of proteins and protein complexes in frozen solutions single 14N nucleus. If electrons exchange among the nitroxide sites [31–37]. We present data regarding the dependencies of 1H NMR within a single molecule, the EPR lines of the biradical and trirad- signals, nuclear spin relaxation times, and DNP build-up times on ical reflect an average over the multiple 14N sites. In the fast-ex- dopant, dopant concentration, temperature (7–80 K range), and change limit, this results in five EPR lines for the biradical (total microwave power. We introduce a new triradical dopant, DOTOPA- z-component of 14N spin from 2 to +2) and seven EPR lines for À TEMPO, that produces the largest sensitivity enhancements in our the triradical (total z-component of 14N spin from 3 to +3) À experiments. We also show that DNP enhancements can be in- [38,39]. Although the EPR spectrum of the triradical is not fully re- creased substantially by application of magnetic field modulation solved, seven peaks in the derivative spectrum are seen (Fig. 1c). during microwave irradiation, and we discuss possible mecha- Strong features in the derivative spectrum at the edges and in nisms for this effect. The primary conclusion of this work is that the center are characteristic of exchange-broadened spectra quite large enhancements of NMR sensitivity can be achieved in [18,38,40], as EPR lines that correspond to 14N spin states that high fields with relatively low microwave powers, provided that are the same on all nitroxide moieties (i.e., the |+1, +1, +1 , i low temperatures are also employed. Relative to signals at 80 K |0, 0, 0 , and | 1, 1, 1 nuclear spin states) remain sharp in the i À À À i with thermal equilibrium nuclear spin polarization and taking into presence of exchange. These lines occur at the same positions as account the temperature dependence of the DNP build-up time, the hyperfine-split lines of 4-hydroxy-TEMPO (Fig. 1a). Similarly, the sensitivity of 1H NMR measurements is increased by factors the EPR spectrum of TOTAPOL shows three sharp lines of equal greater than 10, 50, 180, and 400 at 80 K, 35 K, 16 K, and 7 K, intensity and broad, unresolved features between these lines respectively, in our experiments with 30 mM DOTOPA-TEMPO as (Fig. 1b). This is consistent with narrow EPR lines for the |+1, +1 , i the dopant. |0, 0 , and | 1, 1 14N spin states and broad lines for other 14N i À À i spin states [18]. 2. Materials and methods Nitroxide concentrations were verified by titration with ascorbic acid [41], monitored by the UV–visible absorption spectrum. The 2.1. Nitroxide dopants UV–visible spectrum has a strong absorbance below 300 nm and a weak absorbance at longer wavelength (peaked at 435 nm for 4-Amino-TEMPO and 4-hydroxy-TEMPO were used as pur- DOTOPA-TEMPO) that produces a yellow-orange color, both in the chased from Sigma–Aldrich. The biradical TOTAPOL and the trirad- solid form and in solution. Reduction with ascorbic acid attenuates ical DOTOPA-TEMPO (4-[N,N-di-(2-hydroxy-3-(TEMPO-40-oxy)- the 435 nm absorption (see Fig. S1 of Supplementary Material). propyl)]-amino-TEMPO) were synthesised based on the procedure Based on titration of the 435 nm absorption, our DOTOPA- given in Song et al. for TOTAPOL [17]. Chemical structures are TEMPO had 109 ± 15% of the expected radical concentration shown in Fig. 1. The synthesisMonoradicals was modified by reacting 4-ami- (measured at 20 mM in 25/75 mol% glycerol/water). The titration no-TEMPO and 4-(2,3-epoxypropoxy)-TEMPO in 1:1.7 ratio, rather procedure was validated on commercial 4-hydroxy-TEMPO than a 1:1 ratio. Both the biradical and triradical052211-8 Maly wereet al. purified (75 mM in glycerol/water). For TOTAPOL,J. Chem. Phys. we128 measured,052211͑2008͒ 85 ± 15%

SE DNP experiments. TEMPO is a nitroxide based radical, which leads to the TM mechanism at sufficiently high con- centrations, whereas its biradical derivatives, bis-TEMPO- n-ethylene oxide ͑BTnE͒ and 1-͑TEMPO-4-oxy͒-3- ͑TEMPO-4-amino͒-propan-2-ol ͑TOTAPOL͒ induce the CE DNP mechanism. These and similar nitroxide based free radicals/biradicals have been used for both liquid- and solid- state bio-DNP since they are stable, unreactive, and soluble trityl radical BDPA in a wide variety of solvents. For example, TOTAPOL is 4-hydoxy-TEMPO soluble in glycerol/water solutions and is currently the polar- izing agent of choice for experiments involving proteins. In addition to these gϳ2 species, very successful DNP experi- ments have been performed with paramagnetic chromium ͑V͒ based compounds, which were used in the preparation of polarized targets for nuclear scattering experiments.87 • DNP in solids based on solid effect (SE); two spin process • irradiating a forbidden transition at ωn+ωe or ωn+ωe A. Polarizing agents for the SE 2 • enhancement, ε ~ 1/B0 The resolved SE requires that the homogeneous line- width, ␦ of the radical is less than the nuclear Larmor fre- • requires polarising agent with relatively narrow EPR linewidthquency, ␻0I. This requirement ensures that the zero- and FIG. 7. Chemical structures of three mono- and two biradical polarizing • (ωn >> EPR line width) double-quantum forbidden transitions are not excited simul- agents used in high-field DNP experiments. taneously and there is no cancellation of the positive and negative enhancement. Currently, only two radicals satisfy system͒ or, in principle, endogeneous ͑for example, a stable this property at high magnetic fields, BDPA ͑Ref. 88͒ and radical present in a protein͒. There are many examples of the trityl.89,90 BDPA has an inhomogeneous linewidth of ⌬ use of exogeneous radicals as polarizing agents, and, to date, ϳ20 MHz at 211 MHz 1H Larmor frequency and it was first Wednesday, 10 April 13 the optimal enhancements in MAS experiments have been used to study the SE in a polystyrene matrix.11,12,56,91 How- achieved with biradicals dispersed in a glassy matrix.59,61 In ever, its utility for biological work is greatly limited by its Fig. 1. X-band EPR spectra and chemical structures ofcontrast 4-hydroxy-TEMPO only photo-CIDNP (a), TOTAPOL experiments (b), have and utilized DOTOPA-TEMPO endo- insolubility (c). All in samples aqueous are solutions. 0.5 mM In contrast, solutions the in trityl 95/5 radi- vol.% ethanol/ water at room temperature. geneous polarizing agents in solids.82,83 In solution DNP, the cal, developed by Nycomed/Amersham was originally de- polarizing agent can be dissolved in the solvent,50 attached to signed for low field OE imaging experiments.90,92 It is very a support while the solution flows past it,24,84,85 or connected soluble in aqueous media, and because of its symmetry and to a bulky particle such Si and suspended in solution.86 the absence of large 1H hyperfine couplings, it has a narrow Five of the molecules that have been frequently used for EPR spectrum that supports the SE. Furthermore, trityl has polarizing agents in high-field DNP experiments are illus- been successfully used together with the HyperSense ͑Ox- trated in Fig. 7. Two of these, trityl and bis- ford Instruments, UK͒ to polarize aqueous solutions.25 ␣,␥-diphenylene-␤-phenyl-allyl ͑BDPA͒, have either three- Figure 8͑a͒ shows the 140 GHz EPR spectrum and the fold or approximate threefold symmetry and yield EPR spec- field dependent enhancement profile of trityl ͑⌬=42 MHz͒. tra that are relatively narrow. This makes them suitable for The experimental data were obtained in a MAS experiment

FIG. 8. 140 GHz EPR spectra and DNP field profiles for ͑a͒ trityl, ͑c͒ TEMPO, and ͑c͒ a mixture of trityl and TEMPO. The EPR spectra were recorded in 2 2 2 2 H6-DMSO/ H2O 60:40 w/w ͑1mM͒ at 20 K. The DNP samples were prepared in H6-DMSO/ H2O/H2O 60:34:6 w/w/w with a total radical concentration of 40 mM ͑90 K͒. The solid line represents a simulation of the experimental data. Figure taken from Hu et al. ͑Ref. 60͒.

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Dissolution DNP

Developing DNP/Solid-State NMR Spectroscopy 93 pressure cooker solvent (H2O) The development of solid-state NMR spectroscopy is hampered by the inherently100 – 140°C 2 – 10 bar low sensitivity of NMR. This problem is exaggerated in solid samples because the line widths are usually greater than in solution NMR. Furthermore, the dynamic properties of polypeptides in lipid environments and the mosaic spread of the chase gas (Helium) (injected) (newly synthesised) sample cause additional line broadening effects in oriented membrane samples. 2 – 5 bar Consequently, recording a one-dimensional spectrum can take many hours, even Klaes Golman et al days [22]. Clearly, the range of membrane proteins that could be investigated by solid-state NMR would be broadened if the sensitivity problems were addressed. polarizer This is where dynamic nuclear polarization (DNP)/solid-state NMR spectroscopy has an enormous potential. One solution to improve sensitivity is to combine high-field solid-state NMR spectroscopy with DNP. For this approach, samples are doped with stable radicals (Fig. 1). The polarization of unpaired electron spins is *660 times than of 1H spins and can be transferred onto the nuclei [23]. Various approaches exist to exploit such DNP signal enhancement in solution or in the solid state, each situation having to sample cup deal with its own particular technical problems [23]. Here, we focus on the application of DNP to membrane polypeptides. The large size of the phospholipid protein complexes implies that we follow the protocols that have so far been established for biomolecules in the solid or semi-solid states [24, 25] where microwave irradiation is used to transfer the electron spin polarization to the 1H nuclear spins by various mechanisms [26–28]. The use of DNP has demonstrated impressive solid-state NMR signal enhance- ments of up to 120 times (at 90 K) for biomolecular samples [29] such as amyloid fibers [30], purple membranes [25], protein-associated ligands [31, 32], bacterio- phages [33], and lipid-bilayer inserted peptidesBiradicals [34]. Enhancement factors are even Robert G. Griffin Recipient of the Hu, K.-N., Yu, H., Swager, T. M. & Griffin, R. G. J. Am. Chem.2007 Soc. 126 Laukien, 10844–5 (2004). Prize

Fig. 1 TEMPOL and Z Y dinitroxides currently used for O O DNP/solid-state NMR Golman’s group (2003) X N O X spectroscopy. For bTbK and O N g O O g GE HealthCare, Malmo bCTbK, the relative orientation Y 1 2 of the g-tensors is almost Z Dissolution DNP orthogonal. Whereas in this bTbK paper, NMR experiments with OH TOTAPOL are presented, bTbK has been used in previous work Paul Tordo “forTim his Swagersuccessful Bobdevelopment Griffin of high-field DNP on membranes [34] and bCTbK O O Things canfor besensitivity more enhancementcomplicated... in solid-state MAS NMR” promises to be an even better O N N O N biradical for measurements in Oxford Instruments Ltd. O O O hydrophobic environments [41]. cross effect thermal mixing Therefore, the stability and bCTbK TEMPOL Bruker Biospin Corp. Hardware requirements e octanol–water partitioning of all δ à ωn H H en four compounds are described in e en e n DNP this work e OH H Hee n Hee n O N e |ωe1-ωe2| = ωn n e n N N 3 spin model liquids solids O O Multi-spin model TOTAPOL water solubleSSSSII SSSSI I I++I++-›-› electron electron nuclear Zeeman dipolar Zeeman I++I++++›› reservoir reservoir reservoir dissolution experiment • DNP based on cross-effect 123(CE) and thermal mixing ωωω(TM);e1 ≥3 spin spin processes sample shuttling • ωe1+ωe2 ≈ ωn Lattice 1 I+-I+--›-› low field high field low field high field • enhancement, ε ~ 1/B0 I+-I+-++›› I-+I-+-›-› The HyperSense Solid-StateThermodynamical DNP-NMR description • (ωn < EPR lineI-+I-+++› ›width)

Wednesday, 10 April 13 temperature jump I--I---›-› I--I--++›› positive enhancement ∆ω∆ω∆ωe = ωωωn 48

12 304 K.R. Thurber et al. / Journal of Magnetic Resonance 204 (2010) 303–313

[5]. Off-center irradiation of the EPR line creates a polarization of from the same synthesis by chromatography. ESI TOF mass spec- the electron dipolar system, which can then be transferred to the trometry indicated a mass of 628.49 Da for the triradical, in good nuclei. Recent developments in microwave sources and biradical agreement with the proposed structure (C33H63N4O7, theoretical dopants has led to renewed interest in high-field DNP for NMR average mass = 627.87 Da). For TOTAPOL (C21H41N3O4, theoretical spectroscopy [22–30]. average mass = 399.57 Da), the measured mass was 400.32 Da. In this paper, we report the results of DNP experiments at 9.4 T Differences in the electron–electron interactions for the biradi- using a compact and relatively inexpensive (but relatively low- cal and the triradical are clearly seen in the low-field (10 GHz) EPR power) solid state microwave source, in which we observe 1H spectra for 4-hydroxy-TEMPO, TOTAPOL, and DOTOPA-TEMPO NMR signals from frozen glycerol/water solutions that are doped shown in Fig. 1. The low-field EPR spectra in solution are domi- with several nitroxide radical compounds. Glycerol/water solu- nated by hyperfine interactions with 14N nuclei. 4-hydroxy-TEMPO tions are of particular interest because of their relevance to our shows the three lines corresponding to the three spin states of a studies of proteins and protein complexes in frozen solutions single 14N nucleus. If electrons exchange among the nitroxide sites [31–37]. We present data regarding the dependencies of 1H NMR within a single molecule, the EPR lines of the biradical and trirad- signals, nuclear spin relaxation times, and DNP build-up times on ical reflect an average over the multiple 14N sites. In the fast-ex- dopant, dopant concentration, temperature (7–80 K range), and change limit, this results in five EPR lines for the biradical (total microwave power. We introduce a new triradical dopant, DOTOPA- z-component of 14N spin from 2 to +2) and seven EPR lines for À TEMPO, that produces the largest sensitivity enhancements in our the triradical (total z-component of 14N spin from 3 to +3) À experiments. We also show that DNP enhancements can be in- [38,39]. Although the EPR spectrum of the triradical is not fully re- creased substantially by application of magnetic field modulation solved, seven peaks in the derivative spectrum are seen (Fig. 1c). during microwave irradiation, and we discuss possible mecha- Strong features in the derivative spectrum at the edges and in nisms for this effect. The primary conclusion of this work is that the center are characteristic of exchange-broadened spectra quite large enhancements of NMR sensitivity can be achieved in [18,38,40], as EPR lines that correspond to 14N spin states that high fields with relatively low microwave powers, provided that are the same on all nitroxide moieties (i.e., the |+1, +1, +1 , i low temperatures are also employed. Relative to signals at 80 K |0, 0, 0 , and | 1, 1, 1 nuclear spin states) remain sharp in the i À À À i with thermal equilibrium nuclear spin polarization and taking into presence of exchange. These lines occur at the same positions as account the temperature dependence of the DNP build-up time, the hyperfine-split lines of 4-hydroxy-TEMPO (Fig. 1a). Similarly, the sensitivity of 1H NMR measurements is increased by factors the EPR spectrum of TOTAPOL shows three sharp lines of equal greater than 10, 50, 180, and 400 at 80 K, 35 K, 16 K, and 7 K, intensity and broad, unresolved features between these lines respectively, in our experiments with 30 mM DOTOPA-TEMPO as (Fig. 1b). This is consistent with narrow EPR lines for the |+1, +1 , i the dopant. |0, 0 , and | 1, 1 14N spin states and broad lines for other 14N i À À i spin states [18]. 2. Materials and methods Nitroxide concentrations were verified by titration with ascorbic acid [41], monitored by the UV–visible absorption spectrum. The 2.1. Nitroxide dopants UV–visible spectrum has a strong absorbance below 300 nm and a weak absorbance at longer wavelength (peaked at 435 nm for 4-Amino-TEMPO and 4-hydroxy-TEMPO were used as pur- DOTOPA-TEMPO) that produces a yellow-orange color, both in the chased from Sigma–Aldrich. The biradical TOTAPOL and the trirad- solid308 form and in solution.K.R. Reduction Thurber et al. / Journal with of Magnetic ascorbic Resonance acid 204 (2010) attenuates 303–313 ical DOTOPA-TEMPO (4-[N,N-di-(2-hydroxy-3-(TEMPO-40-oxy)- the 435 nm absorption (see Fig. S1 of Supplementary Material). propyl)]-amino-TEMPO) were synthesised based on the procedure Based on titration of the 435 nm absorption, our DOTOPA- given in Song et al. for TOTAPOL [17]. Chemical structures are TEMPO had 109Triradicals? ± 15% of the Why expected not... radical concentration shown in Fig. 1. The synthesis was modified by reacting 4-ami- (measured at 20 mM in 25/75 mol% glycerol/water). The titration no-TEMPO and 4-(2,3-epoxypropoxy)-TEMPO in 1:1.7 ratio, rather procedure was validated on commercial 4-hydroxy-TEMPO Thurber, K. R., Yau, W.-M. & Tycko, R. J. Magn. Reson. 204, 303–13 (2010). than a 1:1 ratio. Both the biradical and triradical were purified (75 mM in glycerol/water). For TOTAPOL, we measured 85 ± 15%

Fig. 5. Build-up of 1H spin polarization after saturation with DNP (h) and without DNP (d) for 160 mM 4-amino-TEMPO at 35 K (a) and 40 mM 4-amino-TEMPO at 16 K (b). Lines are fits to exponential recovery curves, with an offset from zero to account for background signal or imperfect saturation. Time constants are 2.4 ± 0.2 s with DNP and 3.5 ± 0.8 s without DNP in panel a, and 126 ± 6 s with DNP and 115 ± 30 s without DNP in panel b.

Table 1 Summary of 1H DNP measurements on glycerol/water with various nitroxide dopants. Uncertainties in DNP signal enhancements are roughly ±30%, estimated from repeated measurements on several samples and attributed to variations in microwave alignment. Uncertainties in T values are ±10% or less, unless otherwise noted. DNP Rob Tycko Temperature (K) Dopant Dopant DNP signal DNP-enhanced TDNP (s) DNP-enhanced Ratio of DNP-enhanced a b c 1/2 concentration (mM) enhancement signal (arb. units) signal (T )À signals with and without  DNP /rob tee-ko/ field modulationd 80 DOTOPA-TEMPO 20 12 18 2.7 ± 0.7 11 n.d.  DOTOPA-TEMPO 30 10 17 1.4 14 n.d.  TOTAPOL 20 6 12 8.3 4.1 n.d.  4-Amino-TEMPO 40 11 12 50 ± 15 1.8 n.d.  4-Amino-TEMPO 80 13 17 7.0 6.5 n.d.  4-Amino-TEMPO 160 15 9.4 1.2 ± 0.2 8.5 n.d.  4-Amino-TEMPO 320 9 3.4 <1 n.d.  35 DOTOPA-TEMPO 20 34 114 5.6 48 1.5  DOTOPA-TEMPO 30 26 114 2.4 73 1.2  TOTAPOL 20 11 57 18.5 13 1.5  4-Amino-TEMPO 40 13 33 90 ± 30 3.6 2.4  4-Amino-TEMPO 80 41 118 14.1 31 n.d.  4-Amino-TEMPO 160 34 65 2.4 43 1.1  16 DOTOPA-TEMPO 20 75 578 10.6 177 1.3  DOTOPA-TEMPO 30 59 563 4.5 265 n.d.  TOTAPOL 20 26 333 41 52 1.5  4-Amino-TEMPO 40 29 168 126 15 1.7  4-Amino-TEMPO 80 82 579 27 111 n.d.  Fig. 1. X-band EPR spectra and chemical structures of 4-hydroxy-TEMPO (a), TOTAPOL (b), and DOTOPA-TEMPO4-Amino-TEMPO (c). All 160 samples are 0.554 mM solutions245 in 95/5 vol.%3.4 ethanol/ 133 n.d.  4-Amino-TEMPO 320 15 57 0.8 ± 0.2 63 n.d. water at room temperature.  7 DOTOPA-TEMPO 30 81 1666 8.2 580 1.1  4-Amino-TEMPO 40 48 232 >200 n.d.  a All samples are 10 ll of 25/75 mol% glycerol/water. The 30 mM DOTOPA-TEMPO sample has 0.22 M acetate buffer, pH 3. b 1 Ratio of H NMR spin echo integral with DNP to the integral without DNP, for the same sample and temperature, using 30 mW at 264.0 GHz, s =2TDNP, and no field modulation. For 40 mM 4-amino-TEMPO, s = 170 s. c Spin echo integral with DNP, using 30 mW at 264.0 GHz, s =2TDNP, and no field modulation. For 40 mM 4-amino-TEMPO, s = 170 s. d Spin echo integral with DNP, using 30 mW at 264.0 GHz, s =2TDNP, and 10 G rms field modulation during s, with 6 kHz modulation frequency. n.d. = not determined. Wednesday, 10 April 13

roughly 70, 350, and 1000 at 35 K, 16 K, and 7 K, respectively. Sen- and temperatures. In all cases, field modulation at 2–6 kHz with a sitivity enhancements relative to measurements at 80 K without root-mean-squared (rms) amplitude of 10–40 G (28–112 MHz rms DNP may not be as large (depending on the paramagnetic dopant modulation of the EPR frequency) produces significant increases in

conditions at 80 K, which determine the value of T1n), but are still nT. Field modulation effects are also summarized in Table 1. The much greater than 100 at the lower temperatures. These low- results from various samples indicate that the effect is generally temperature results compare favorably with DNP enhancements stronger at lower doping concentrations, and slightly stronger at obtained at higher temperatures with higher microwave powers higher temperatures. As seen in Fig. 6b, the dependence on modula- [3,17,18,46]. tion frequency does not vary strongly with microwave power level, modulation amplitude, or temperature. The only factor that pro- 3.2. Effect of field modulation during DNP duces clear differences in the frequency dependence is the identity of the nitroxide dopant, as seen in Fig. 6d. For the dopant concentra- Fig. 6 shows the effect of magnetic field modulation during DNP tions measured, the required modulation frequency increases as the

on the observed signal enhancements for various dopant conditions number of nitroxide moieties in the dopant increases. TDNP has the Developing DNP/Solid-State NMR Spectroscopy 93

The development of solid-state NMR spectroscopy is hampered by the inherently low sensitivity of NMR. This problem is exaggerated in solid samples because the line widths are usually greater than in solution NMR. Furthermore, the dynamic properties of polypeptides in lipid environments and the mosaic spread of the Angewandte sample cause additional line broadening effects in oriented membrane samples. Chemie Consequently, recording a one-dimensional spectrum can take many hours, even TEMPO moieties in a desirable relative orientation should maximum positive (DNP(+)) and negative (DNP( )) DNP À days [22]. Clearly, the range of membranefurther increase proteins the enhancement that could obtained be from investigated the polariz- enhancements. by (The pulse sequence used is given in Figure S3 solid-state NMR would be broadeneding agent. if the sensitivity problems were addressed.in the Supporting Information). The 1H polarization was Herein, we demonstrate the utility of bis-TEMPO-biske- monitored by transferring the DNP-enhanced 1H polarization This is where dynamic nuclear polarizationtal (bTbk; Scheme (DNP)/solid-state 1, top), a biradical NMR consisting spectroscopy of two to the 13C nuclei of the urea molecule in a cross-polarization has an enormous potential. TEMPO moieties connected with a rigid bisketal tether. This step.[14, 15] At a temperature of 93 K, the buildup time constant + biradical was originally used to inhibit radical polymeri- tB is 7 s, and a steady-state enhancement of e = 250 was One solution to improve sensitivityzation. is[13] In to particular, combine we compare high-field the performance solid-state of bTbk NMRobserved (Figure 1) at a microwave power of approximately spectroscopy with DNP. For this approach,and TOTAPOL samples in DNP are experiments doped under with similar stable exper- radicals2.5 W (estimated power at the sample position). This (Fig. 1). The polarization of unpairedimental electron conditions. spins is 660 times than of 1H spinsenhancement is larger than that observed with TOTAPOL Earlier studies of a series* of TEMPO-based biradicals as (e+ = 180) under identical experimental conditions (solvent, and can be transferred onto the nucleipolarizing [23]. Various agents in high-field approaches DNP experiments exist to suggested exploit suchtemperature, mw power). DNP signal enhancement in solutionthat or a conformation in the solid in which state, the two eachgzz (or situationgyy) tensor axes having of toFor a more reliable comparison of the two polarizing the TEMPO moieties have a dihedral angle of 908 should agents, the enhancement was extrapolated to infinite micro- deal with its own particular technicalexhibit problemsimproved performance [23]. as Here, a result we of efficient focus fre- onwave the power eÆ . This value can be obtained from the 1 application of DNP to membrane polypeptides.quency matching. The[6] This large requirement size is of approximately the phospholipid ful- dependence of the DNP enhancement on the microwave filled (see below) for bTbk (Scheme 1, top) as a result of the power according to Equation (1): Communication Journalprotein complexes of the American implies that Chemical weodd follow number the of Society spiro protocols junctions between that have the two so TEMPO far been Journal of the American Chemical Society Communication moieties. Specifically, the crystal structure of bTbk shows a 1 1 1 established for biomolecules in the solid or semi-solid states [24, 25] where 1 1 dihedral angleScheme between 1. Synthesis the TEMPO of bTbtk-py moieties of 828. Fur- eÆ ¼ eÆ þ aP ð Þ 1 1  Schememicrowave 1. irradiationSynthesis is of used bTbtk-py to transferthermore, the a calculated electron average spin electron–electron polarization distance to of the H nuclear spins by various mechanisms1.18 [26 nm– (see28]. Figure S1 in the Supporting Information) was in which eÆ is the steady-state enhancement factor, P is the confirmed by pulsed electron–electron double resonance microwave power, and a is the saturation parameter, which The use of DNP has demonstrated(PELDOR) impressive measurements solid-state (see Figure NMR S2 in the signal Supporting enhance-depends on the microwave transmission efficiency and EPR relaxation properties.[6] Different instrumental conditions, ments of up to 120 times (at 90 K) forInformation). biomolecular Thebis-TEMPO-bisketal dihedral samples angle restrains [29] the(bTbk) suchgzz (or asg amyloidyy) tensor components of the two TEMPO moieties in a such as the microwave transmission efficiency or EPR [6] fibers [30], purple membranes [25],perpendicular protein-associated conformation ligands to ensure a[31 large, 32 frequency], bacterio-relaxation properties, affect eÆ and a, but not eÆ . Never- 1 Kiesewetter,separation M. K., Corzilius, for molecular B., Smith, A. a, orientations Griffin, R. G. & Swager, along T. M. these J. Am. axes.Chem. Soc. At 134, 4537–40theless, (2012). the experiments must be performed at similar phages [33], and lipid-bilayer insertedMatsuki, peptidesY. et al. Angewandte [ Chemie34]. 121 Enhancement, 5096–5100 (2009). factors are even the same time, the short distance provides the large electron– temperatures for an accurate comparison. The effective electron resonance frequency can depend electron dipolar coupling required for an efficient DNP Figure 2 shows the power dependence of the DNP strongly on the molecular orientation with respect to the process. CE: ωe1+ωe2 ≈ ωn enhancementsexternal magnetic for bTbk field, and TOTAPOL. and in a biradical From the this matching relation- Initial DNP experiments with bTbk are described in ship, econditionÆ can be is calculated controlled byby usingthe relative Equation orientations (1). For of bTbk the 13 1 1 Figure 1. A typical C-detected buildup curve of the H-bulk and TOTAPOL,electron g tensors. the eÆ values are 325 15 and 227 10, 1 Æ Æ Fig. 1 TEMPOL and polarizationZ is shown together with spectra recorded with andY dinitroxides currently used for without microwavebonds. irradiation TheO X-band at EPR fields spectrumO corresponding is typical for to a nitroxidethe radical; the broad 1:1:1 triplet due to hyperfine coupling to a DNP/solid-state NMR 14 X X O N nitrogen, aiso( N) = 41.8 MHz, is consistentN withO a biradical Figure 1. DNP-enhanced polarization was measured under microwave spectroscopy. For bTbK and featuring a short, rigid tether with a weak electron−electron J- irradiation using a gyrotron beam current of 34 mA, which 13 g1 coupling (exchangeO integral),OJ < 10·a(14N).23,30 The biradicalg2 corresponds to ∼12 W microwave power. (Top) CNMR bCTbK, the relative orientation Y 13 exhibited excellent solubility in the desired 60/40Z glycerol/ (CPMAS) of 1 M urea- C in 60/30/10 glycerol-d8/D2O/H2O (v/ of the g-tensors is almost v/v) with 10 mM of the appropriate biradical acquired with and water mixture (10.9bTbK mM in glycerol/water (60/40); 3.0 mM in without microwave irradiation. (Bottom) Buildup curves were orthogonal. Whereas in this D2O). recorded at the respective field, yielding the maximum enhancement 13 13 Thenot DNP-enhanced water Csoluble−CPMAS NMR of C-urea (1 M in for each radical (i.e., 4.982 T for bTbtk-py, 4.980 T for TOTAPOL; paper, NMR experiments with 60/40 glycerol/water) showed ε =230versusthermalOH see Figure 3) and subsequently scaled in order to normalize the off TOTAPOL are presented, bTbK polarization. Experimentally, 13C-urea and bTbtk-py in the signal intensities at t = ∞. Proton longitudinal relaxation time glassy glycerol/water matrix is continuously irradiated with constants were determined as T1I = 3.9 s for bTbtk-py and T1I = 4.5 s has been used in previous work microwaves under cryogenic (T ≈ 82 K) MAS conditions to for TOTAPOL. Note that aside from the radical used and the 1 ’ 13 magnetic field strength, the experimental conditions are the same for on membranes [34] and bCTbK Opolarize the HOs, and this polarization is transferred to C for each trace. promises to be an even better detection via a cross-polarization step. In the absence ofFigure 2. Power dependence of the steady-state DNP enhancement O N microwaves, the thermal equilibriumN O polarization develops.N In + biradical for measurements in (e ) for bTbk and TOTAPOL. The experiments with the two polarizing Oeither event,O the signal intensity as measured after aagents were carried out under similar conditions. hydrophobic environments [41]. presaturation sequence versus recovery time showsO a monoexponential polarization buildup, Figure 1. The enhance- Therefore, the stability and ment,bCTbKε, is given by the ratio of signal intensityTEMPOL with the microwaves on versus microwaves off. Interrogated underrespectively. Values of eÆ for a series of biradicals previously octanol–water partitioning of all 1 four compounds are described in identical conditions, TOTAPOL shows a lower enhancementexamined in DNP experiments are compared in Table 1. factor, ε = 191. The direct comparison with an externalAmong the polarizing agents investigated to date, bTbk yields this work standard, TOTAPOL in this case, is crucial in order to determine the relativeOH performanceH of different polarizingthe largest DNP enhancement. The enhancement is approx- Figure 1. Buildup curve recorded at a magnetic-field position corre- agents. Absolute enhancements are greatly influenced byimately 1.4 times larger than that of TOTAPOL. sponding to DNP(+) for 1H bulk polarization with bTbk. The 1H instrumentalwaterO (e.g., soluble rotor size,N microwave coupling to the The DNP process is most efficient if the sample and the polarization is detected indirectly from the 13C signal of urea through a sample) and experimental (e.g., MAS frequency, samplepolarizing agent are dispersed in a matrix that forms a rigid cross-polarizationtemperature, step. The insets optimum show the magnetic mw on field) and off parameters, signals thus bonds. The X-band EPRWednesday, spectrum 10 April 13 N is typical for a nitroxideN glassFigure at cryogenic 2. Power dependence temperatures. of the steady-state For example, DNP enhancement mixtures of recorded at field positionsimpeding correspondingcomparison between to DNP( studies+) performed and DNP( under); varying ε ε ε O ÀO ∞ for bTbtk-py and TOTAPOL given by: 1/ = 1/ ∞(1 + 1/aP). T = 94 K, wr/2p = 4.8conditions. kHz. The DNP enhancement derived from bTbtk-pydimethylThe experiments sulfoxide with (DMSO)/water the two polarizing agents (60:40 were carried w/w) out or at the glycerol/ radical; the broad 1:1:1 triplet due toconstitutes hyperfine theTOTAPOL largest signal coupling enhancement observed to so a far for field position giving the optimal enhancement for each. Otherwise, the any biradical in a biologically relevant glycerol/water mixture experimental conditions were identical (see Supporting Information). 14 Angew. Chem. 2009, 121, 5096 –5100  2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.de 5097 nitrogen, aiso( N) = 41.8 MHz, is consistentunder given conditions. with31−34 a biradical Figure 1. DNP-enhanced polarization was measured under microwave Another metric of the efficiency of a polarizing agent is given The enhancement field profile of a polarizing agent shows by measuring ε as a function of microwave power and theirradiation NMR signal intensity as a using function of magnetic a gyrotron field and, beam current of 34 mA, which featuring a short, rigid tether with a weak electron−electronε J- hence, the electron Larmor frequency and provides the field for extrapolating the enhancement to infinite power, ∞ in Figure 13 23,30ε 123optimum enhancement. The breadth and intensity of the 2.14 This factor, ∞, is independent of microwave power, Pmw, corresponds to ∼12 W microwave power. (Top) CNMR coupling (exchange integral), J < 10·a(and theN). saturation parameter,Thea, which biradical depend on microwave enhancement profile of bTbtk-py versus TOTAPOL (Figure 3) 23,24 13 transmission efficiency and EPR relaxation properties. suggest(CPMAS) that the nitroxide of moieties 1 Mhave slightly urea- differentC in 60/30/10 glycerol-d8/D2O/H2O (v/ exhibited excellent solubility in the desiredHowever, care must 60/40 be taken to control glycerol/ sample temperature relative orientation and electron−electron coupling in the two and rotor size. biradicals.v/v) Nevertheless, with the 10 shape of mM the enhancement of the profile appropriate biradical acquired with and water mixture (10.9 mM in glycerol/water (60/40); 3.0 mM in 4538 withoutdx.doi.org/10.1021/ja212054e microwave| J. Am. Chem. Soc. 2012, irradiation. 134, 4537−4540 (Bottom) Buildup curves were D2O). recorded at the respective field, yielding the maximum enhancement 13 13 The DNP-enhanced C−CPMAS NMR of C-urea (1 M in for each radical (i.e., 4.982 T for bTbtk-py, 4.980 T for TOTAPOL; 60/40 glycerol/water) showed ε =230versusthermal see Figure 3) and subsequently scaled in order to normalize the off polarization. Experimentally, 13C-urea and bTbtk-py in the signal intensities at t = ∞. Proton longitudinal relaxation time glassy glycerol/water matrix is continuously irradiated with constants were determined as T1I = 3.9 s for bTbtk-py and T1I = 4.5 s microwaves under cryogenic (T ≈ 82 K) MAS conditions to for TOTAPOL. Note that aside from the radical used and the 1 ’ 13 magnetic field strength, the experimental conditions are the same for polarize the H s, and this polarization is transferred to C for each trace. detection via a cross-polarization step. In the absence of microwaves, the thermal equilibrium polarization develops. In either event, the signal intensity as measured after a presaturation sequence versus recovery time shows a monoexponential polarization buildup, Figure 1. The enhance- ment, ε, is given by the ratio of signal intensity with the microwaves on versus microwaves off. Interrogated under identical conditions, TOTAPOL shows a lower enhancement factor, ε = 191. The direct comparison with an external standard, TOTAPOL in this case, is crucial in order to determine the relative performance of different polarizing agents. Absolute enhancements are greatly influenced by instrumental (e.g., rotor size, microwave coupling to the sample) and experimental (e.g., MAS frequency, sample temperature, optimum magnetic field) parameters, thus Figure 2. Power dependence of the steady-state DNP enhancement impeding comparison between studies performed under varying ε∞ for bTbtk-py and TOTAPOL given by: 1/ε = 1/ε∞(1 + 1/aP). conditions. The DNP enhancement derived from bTbtk-py The experiments with the two polarizing agents were carried out at the constitutes the largest signal enhancement observed so far for field position giving the optimal enhancement for each. Otherwise, the any biradical in a biologically relevant glycerol/water mixture experimental conditions were identical (see Supporting Information). under given conditions.31−34 Another metric of the efficiency of a polarizing agent is given The enhancement field profile of a polarizing agent shows by measuring ε as a function of microwave power and the NMR signal intensity as a function of magnetic field and, extrapolating the enhancement to infinite power, ε∞ in Figure hence, the electron Larmor frequency and provides the field for 2. This factor, ε∞, is independent of microwave power, Pmw, optimum enhancement. The breadth and intensity of the and the saturation parameter, a, which depend on microwave enhancement profile of bTbtk-py versus TOTAPOL (Figure 3) transmission efficiency and EPR relaxation properties.23,24 suggest that the nitroxide moieties have slightly different However, care must be taken to control sample temperature relative orientation and electron−electron coupling in the two and rotor size. biradicals. Nevertheless, the shape of the enhancement profile

4538 dx.doi.org/10.1021/ja212054e | J. Am. Chem. Soc. 2012, 134, 4537−4540 Journal of the American Chemical Society Article

Journal of the American Chemical Society Article currently the most efficient mechanism for DNP at high fields.20 The CE relies upon having a three-spin system consisting of two dipolar coupled electrons and a nucleus. For polarization Can we improve on bTbk? 29 transfer to occur from the electrons to the nucleus upon Figure 2. (a) Proton DNP (εH) and silicon DNP enhancement (εSi,CP). (b) The Si quenching factor (θSi) shown as a function of bCTbK † saturation of one of the EPR frequencies, the two unpaired concentration in 1,1,2,2-tetrachloroethane. (c) Overall (ΣSi,CP and Σ Si,CP) sensitivity enhancements. Note that εH quantifies the enhancement of the ε Zagdoun, A.; Casano, G.; Ouari, O.; Lapadula, G.; Rossini, A. J.; Lelli, M.; Baffert, M.; Gajan, D.; Veyre, L.; Maas, W. E.; Rosay, M. M.; Weber, R. T.; electrons should have a significant dipolar coupling, and their solvent and Si,CP quantifies that of the surface. Experimental details and definition of the enhancement factors are given in the Supporting Information. Thieuleux, C.; Coperet, C.; Lesage, A.; Tordo, P.; Emsley, L. J. Am. Chem. Soc. 2012, 134, 2284–91. EPR frequencies must differ by the nuclear Larmor frequency 18 (here proton). Table 1. DNP Efficiency Comparison and Electron Spin−Lattice Relaxation Times for the TOTAPOL, bTbK, and bCTbK Initial CE high-field DNP experiments were performed with Biradicals under Optimal Conditions solutions doped with high concentrations of monoradical a,b a,c a,c 13 b,d e e 21 biradical solvent εH εSi,CP ΣSi,CP IIm( C) T1e (μs) T2e (μs) species, such as TEMPO. Much effort has been recently f devoted to the design and synthesis of more efficient radicals TOTAPOL water − 31(3) 15(4) 0.75(3) 554(4) 1.6(1) 21−24 bTbK tetrachloroethane 26(3) 21(3) 12(5) 0.51(2) 453(4) 1.5(1) for DNP. Griffin and co-workers demonstrated that bCTbK tetrachloroethane 105(3) 52(3) 38 (7) 1.00(4) 762(4) 2.8(1) biradical polarizing agents yielded significant improvements in aMeasured on Mat-PhOH impregnated with a 16 mM biradical solution. Estimated errors are given in parentheses. bRecorded on a triple resonance the DNP process, and therefore in the sensitivity enhancement CPMAS probe at 99 K. The difference of DNP efficiency between the two probes can be attributed to differences of the hardware (e.g., waveguide 19 factors. The success of biradicals is primarily attributed to the efficiency and alignment) and the lower temperature that could be reached with the triple resonance probe. See the Supporting Information for a uniformly short distance between the two unpaired electrons in more detailed characterization of the DNP efficiency dependence on the biradical concentration on this probe. This explains the discrepancy ε ε c d a biradical, which guarantees that all unpaired electrons are between H and Si for bCTbK in tetrachloroethane. Recorded on longer a double resonance electron CPMAS probe at 102spin-lattice K. In this case, it is not possible to quantify the overall sensitivity enhancement for carbon• due to the fact that it is impossible to obtain a MW off carbon spectrum with high enough dipolar coupled to at least one other electron. With nitroxide- S/N ratio to calculate reliable integrals in a reasonable experimentalrelaxation time. However, time it is possible helps to compare theto relative get efficiency higher of the differ ent based biradicals, the EPR frequency difference is provided by g- biradicals for enhancing the carbon resonances by comparing the integrated intensities per unit of mass (IIm). Here, the IIm values of the aromatic anisotropy, and the difference in EPR frequencies can match resonances of Mat-PhOH impregnated with the various biradical containing solutions are displayed. The concentration of the bTbK and TOTAPOL 1 solutions was 12 mM, and the bCTbK one was a 16 mM solution (concentration givingenhancements optimal sensitivity for each biradical). The IIm of Mat-PhOH the H Larmor frequency when the gxx (or gyy) component of e the g tensor of one of the unpaired electrons is parallel to the impregnated with a 16 mM solution of bCTbK in tetrachloroethane was taken as a reference. Electron spin−lattice (T1e) and spin−spin (T2e) 25,26 23 relaxation times were measured on the edge of the EPR line in a frozen bulk solution at 90 K with 8 mM biradical concentration at an EPR resonance f 1 gyy (gzz) component of the other. Recently, triradical, frequency of 94 GHz. For TOTAPOL, glycerol was added to water to form a glass (60%/40% glycerol/H2O by volume). The H resonance of heterogeneous biradical species or mixtures of radical water is too broad to calculate a reliable proton DNP enhancement. species,27,28 and metal ions29 have been proposed as alternatives to dinitroxide polarizing agents. cyclohexyl-substituted piperidin-4-one 2 (see the Supporting In- DNP enhancement maximum of TOTAPOL (263.334 GHz), with an 22 formation) was prepared according to the method described by estimated 4 W power of the MW beam at the output of the probe The water-soluble biradical TOTAPOL (Figure 1d) is 37 Yamada and Utsumi and was then reacted with pentaerythritol to waveguide. 29Si spectra and 1H spectra of Mat-PhOH impregnated today the most commonly employed exogenous polarizing afford the spirodiamino precursor of bCTbK. Subsequent oxidation of with radical containing solutions were recorded using a double agent in DNP solid-state NMR, in particular for biological the spirodiamino precursor by hydrogen peroxide in the presence of resonance low-temperature CPMAS probe with a sample spinning applications.30 TOTAPOL was shown, however, to have a Figure 1. (a) Transmissionsodium tungstatenot electron water provided microscopy soluble the dinitroxide image bCTbK of the (see hybrid the Supporting frequency of 8 kHz and a sample temperature of 102 K. 1H, 13C, and relatively flexible structure,25 and as a result the frequency mesostructured silicaInformation material for studied further here. experimental (b) Schematic details). representa- tion of the pores. TheSample blue Preparation circle represents for NMR the organic Experiments. moietiesSamples were 15N spectra were recorded using a triple resonance low-temperature matching condition for the CE mechanism is only properly present on the mesoporousprepared by surface. impregnating The 8.9 shaded−9.8 mg gray of areadry powder represents with ca. 17LyndonμL of EmsleyCPMAS probeMelanie with a sampleRosay temperatureAnne of 99 Lesage K and sample a radical containing solution.36 The total mass of impregnated material fulfilled for the fraction of the biradicals that adopts the correct the bulk silica. (c) Structure of Mat-PhOH. Different types of silicon spinning frequencies of 12 kHz for the 13C spectra and 8 kHz for the was determined, and the sample was mixed using a glass-stirring rod to conformation. Another step forward was achieved by Griffin, atoms present at the surface are labeled here. (d−f) Structures of 15 38 26 obtain a homogeneous distribution of the radical containing solution N spectra. SPINAL-64 heteronuclear decoupling was applied Tordo, and co-workers who introduced bTbK (Figure 1e), a TOTAPOL,Wednesday, bTbK, and 10 April bCTbK. 13 in the powder. The impregnated powder was then packed into a 3.2 during acquisition (ω1H/2π = 89 kHz). Additional experimental details binitroxide radical in which the two TEMPO molecules are mm sapphire NMR rotor to maximize MW penetration into the are provided in the Supporting Information. Pulse programs are linked by a rigid tether. The rigid tether constrains the show that thissample. biradical The yields mass of proton impregnated and silicon-29 material inside cross- the rotor was available on request. Processing of the spectra was done using the determined, and a tight polyfluoroethylene plug was inserted to orientation of the TEMPO moieties, such that the gzz polarization (CP) DNP enhancement factors that are between Topspin software package, and fitting of NMR relaxation measure- components of the two g-tensors are roughly orthogonal. It prevent any leakage of the solvent during spinning. The rotor was 2.5 and 4 timescapped larger with a thanzirconia bTbK. drive tip The and quickly reduction inserted in into the DNP ments was done using Kaleidagraph. was demonstrated that bTbK provides DNP enhancement experimental timesspectrometer. provided See by http://pubs.a DNP experimentscs.org/JACSbeta/scivee/index. with EPR Spectroscopy. All measurements were conducted at W band factors, which are 1.4 times larger than TOTAPOL under bCTbK furtherhtml pushes#video2 back for a the video limits demonstration of solid-state of the sample NMR preparation (94 GHz EPR frequency) on a Bruker Elexsys E680 EPR 26 procedure. similar conditions. We have more recently shown that this spectroscopy in terms of sensitivity, notably for the structural spectrometer, with sample temperatures of 90 K. T1e was measured Solid-State NMR Spectroscopy. All spectra were acquired on a biradical may be readily combined with organic solvents for the investigation of dilute surface species. This polarizing mixture is using inversion−recovery sequences, and the curves were fitted with 31 Bruker Avance III 400 MHz DNP NMR spectrometer equipped with a investigation of water incompatible materials. directly applicable to study a wide range of problems.5 We show biexponential functions to obtain T1e. The slow component of the fit is 263 GHz gyrotron microwave system (B0 = 9.4 T, ωH/2π = 400 μ The electron spin−lattice relaxation time T1e is another here that withMHz, the highωC/2π sensitivity= 100 MHz, providedωSi/2π = 79.5 by MHz). DNP The with field sweep coil identified as T1e, while the fast component (<90 s) is attributed to factor that impacts the DNP process. It is known that a longer bCTbK, it becomesof the NMR possible magnet to was expediently set so that MW monitor irradiation the occurred at the spectral diffusion and is not reported here. 21,32 T1e facilitates saturation of the EPR transition. This effect stepwise functionalization at the surface of hybrid materials 2286 dx.doi.org/10.1021/ja210177v | J. Am. Chem.Soc. 2012, 134, 2284−2291 partially accounts for the improved DNP at lower sample by 1H, 13C, 29Si, as well as 15N solid-state NMR spectroscopy at temperature,5,33 because electron relaxation rates slow sig- natural isotopic abundance. Notably, high-quality one-dimen- nificantly with temperature.34,35 However, this factor has not sional 15N solid-state NMR spectra can be acquired with been so far considered directly in the design of biradicals for experiment times on the order of 1 h, and two-dimensional DNP. 1H−15N HETCOR spectra can be acquired in less than 12 h. In this Article, we introduce a new biradical, bis-cyclohexyl- TEMPO-bisketal (bCTbK, Figure 1f), a bulky derivative of ■ EXPERIMENTAL SECTION bTbK, in which the geminal dimethyl groups have been replaced by spirocyclohexyl moieties. In agreement with Synthesis and characterization of the hybrid mesostructured silica materials are described in the Supporting Information. The biradical previous relaxation rate measurements on spirocyclohexyl 35 TOTAPOL was purchased in crystalline form from DyNuPol Inc., functionalized nitroxide radicals, we show that, in 1,1,2,2- MA, and used without further purification. The solvents were tetrachloroethane (“tetrachloroethane”) at 100 K, bCTbK has a purchased from Sigma-Aldrich and used without further purification. T1e value that is almost twice as long as that for bTbK. On a Synthesis of bCTbK. bCTbK was prepared in a four-step model hybrid mesostructured silica-based material,14,36 we sequence starting from 2,2,6,6-tetramethylpiperidin-4-one. The 2,6-

2285 dx.doi.org/10.1021/ja210177v | J. Am. Chem.Soc. 2012, 134, 2284−2291 Journal of the American Chemical Society COMMUNICATION COMMUNICATION Table 1. Experimental DNP Parameters pubs.acs.org/JACS sample enhancement integral enhancement buildup time (s)

a High-Field Dynamic Nuclear Polarization with High-Spin TransitionGdDOTA 12.8 0.98 6.6 High Spin Transition Metals GdDTPAa 3.5 0.70 5.2 Metal Ions MnDOTAb 1.9 0.89 5.6 Bj€orn Corzilius,† Albert A. Smith,† Alexander B. Barnes,† ClaudioCorzilius, Luchinat,B. et al. J. Am.‡ Chem.Ivano Soc. Bertini, 133, 5648–51‡ and (2011). a At 6 W microwave power. b At 5 W microwave power. For further Robert G. Griffin*,† details, see the text. †Department of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States ‡Magnetic Resonance Center and Department of Chemistry, University of Florence, Via L. Sacconi 6, 50019 Sesto Fiorentino (FI), Italy Björn Corzilius polarising agents bS Supporting Information

ABSTRACT: We report the dynamic nuclear polarization of 1Hspinsinmagic-angle-spinningspectrarecordedat5Tand 2 3 84 K via the solid effect using Mn þ and Gd þ complexes as polarizing agents. We show that the magnitude of the en- fi hancements can be directly related toFigure the effective 4. Field-dependent line width of DNP enhancement pro les of MnDOTA 1 1 3 the central (m = / f / )EPRtransition.UsingaGdrecorded at 86 K usingþ a microwave frequency of 139.65 GHz and a S À 2 þ 2 complex with a narrow central transitionmicrowave EPR line power width of of 29 5 W. ForFigure further 1. Chemicalcomplexes details, structures see Figure with of the 2.Mn (protonated)2+ and Gd octadentate3+ chelat- Journal of the AmericanMHz, Chemical we observed Society a maximum enhancement of 13, which is ing ligands DOTA and DTPA. COMMUNICATION comparable to previous results onthenarrow-line-widthtrityl∼ radical. microwave power. However,process. this neglects Besides that, the many contribution inorganic samples of the often contain or Table 1. Experimental DNP Parameters higher-mS transitions to paramagneticcan be doped relaxation. with paramagnetic Additionally, metal ions. the This opens the complex relationshipsample between enhancementpossibility electronic of integral applying relaxation, enhancement DNP tonuclear a buildupvariety relaxa- time of materials (s) science- fi related problems. For these reasons, we initiated investigations tion, hyper neGdDOTA coupling,a andof12.8 DNPdouble- enhancements and zero-quantum 0.98 with high-spin transition transition-metal6.6 ions, fl ffi 3 2 pplications of magic-angle-spinningmoments NMR and spectroscopyGdDTPA their respectivea and3.5 in weuence report on the DNP 0.70 initial e resultsciency for remains Gd 5.2þ and Mn þ in this (MAS NMR) are often limited by the low Boltzmann A to be elucidated theoretib callycommunication. and experimentally. polarization of nuclear spins and therefore the lowMnDOTA inherent 1.9 0.89 5.6 fi a The twob metals werefi used in the form of complexes with the The eld-dependentAt 6 W microwave enhancement power. At 5 W pro microwavele of power. MnDOTA For further sensitivity. Thus, in samples such as large biomolecules or com- octadentate chelating ligandsfl 1,4,7,10-tetraazacyclododecane- pounds with a low abundance of magnetic(Figure nuclei, 4) the hasdetails, experimental a more see the complicated text.1,4,7,10-tetraacetic structure acid that (DOTA) re ects and the diethylenetriaminepentaacetic overlap acquisition time can be prohibitively long.of two The hyper same isfine true sextets even withacid amplitudes (DTPA) (Figure of opposite 1). These sign ligands that form are extremely stable when uniformly 13C/15N-labeled samples are employed and multi-1 complexes and are usedasmagneticresonance imaging contrast shifted by twice the HLarmorfrequencywithrespecttoone3 Figure 5. (a c) Comparison of the microwave-on and -off signals dimensional techniques are required to perform assignments and agents in clinical applications. Furthermore, Gd þ complexes with 1 13 13 13 15 13 13 another. Because1 of4 the narrow linewidthoftheSEtransitionsand À measure C Nand C Cdistancesandtorsionangles. À In DOTA and DTPA exhibit significantly different electricdetectedfield after transfer of the H polarization to C (in 1 M C-urea) via 1 21 fi 3 such cases, high-frequencyÀ dynamÀ ic nuclearthe polarization mismatch (DNP) between• can the gradientsHLarmorfrequencyandthehyper at the metalmodest site, allowing enhancements us to investigatene the incross-polarization.fluence achievable Both ( Gdupþ tocomplexes ε~13) were measured at 84 K and be used to significantly boost the sensitivitycoupling of (212 MAS NMR vs 254• byenhancement MHz),of allthe 12magnitude DNP of peaks therelated zero- arefield clearly splittingto resolved.the (ZFS) e onffective the efficiency6 W line microwaveof width power; of the MnDOTA central was measured EPR transition at 86 K and 5 W. transferring the relatively large polarization of electron spins to the the DNP process. Experimental details about both the DNP/NMR(d) Buildup of polarization for the DNP-enhanced signals under the 5 10 However, the maximum enhancement observed at the second nuclei. À Accordingly, DNP has beenoutermostsuccessfully appliedpeak on to the the high-andfield EPR side experiments is only are1.9 available at a in microwave the Supporting Information.same experimental conditions used for the enhancement measurements. investigation of functional membrane proteins in their native lipid The DNP mechanism∼ that governs the polarization transfer in the 11,12 fi 13power16 of 5 W. We ascribe thiscompounds small enhancement used in this study to is the the soliddistribution effect (SE).22 This is a two- Figure 4. Field-dependentenvironments DNP enhancementand pro amyloidfiles ofbrils MnDOTAÀ without compromising 17 spin process infi which microwave irradiation at ω = ω ( ωfi recorded at 86 K using a microwavethe spectral frequency resolution. ofThese 139.65 experiments GHzof and the would aWednesday, DNP not beconditions feasible 10 April in 13 over all six hyper ne lines, which leads to anmw 0high-S 0eldI peaks that were clearly resolved and well-separated from 1 (where ω0S and ω0I are the electron and nuclear Larmor frequencies, microwave power of 5 W. ForareasonableamountoftimewithconventionalMASNMR. further details, see Figure 2. even smaller fraction ( /18)ofspinsinvolvedintheDNPprocessin any negative DNP condition and then multiplied the result by 3. The The polarizing agents used in contemporary DNP experiments 3 respectively) excites forbidden electron nucleus transitions that comparison with the Gd þ case, in which 70% of the nuclearÀ spins results of this simple analysis are summarized in Table 1. Despite the have primarily been persistent g 2 organic radicals [e.g., trityl, 1,3- become partiallyfi∼ allowed through mixing of adjacent states. The ff microwave power. However,bis(diphenylene)-2-phenylallyl this neglects the contribution (BDPA),≈ oflack and the nitroxide-based a magnetic momentmono- andHamiltonian the hyper relevantne to coupling this system to is the mag- very di erent maximum enhancements obtained from the three 18 20 fi higher-mS transitions to paramagneticand biradicals]. relaxation.À In principle, Additionally, however,netically the paramagnetic active metal nuclei ions is too small^ to have^ a signi ^cant impact^ ^ on^ the^ ^ ^compounds, the integrated enhancements, which represent excita- can also be used as a source of polarization, and they offer at least two H ω0SSZ ω0IIZ2 ASZIZ 5 CSZI C SZI complex relationship between electronic relaxation, nuclear relaxa-line width. In addition, the lower spin¼ stateÀ of Mn þ (S = þ/2)andþ þ Ã tionÀ of all transitions yielding positive enhancement, are very similar. tion, hyperfine coupling, andpotential double- advantages. and zero-quantum First, many transition metalloproteins contain para- fi 3 3 ffi the lower transition momentwhere of th Ae is the central secular EPR hyper transitionne coupling (3 constant, timesC = /2(GdDTPAγSγI/r ) performed 30% less e ciently than the corresponding magneticfl metals or can beffi doped with a paramagnetic substitute, 1 ij À moments and their respective in uence on DNP e ciency remains θ θ À ∼ thus providing an intrinsic source of polarizationlarger than that that could for leadS = sin/2)mightresultinaslightlylowercos e is the usual term in the van Vleck formDOTA of the complex, which might be caused by the onset of the DSE to be elucidated theoretically andffi experimentally. 3 electron nuclear dipole Hamiltonian, and ^S and^I are spin operators fi to e cient enhancementfi of proteinperformance resonances. Second, than theyfor Gd þ. À 23 condition. Although there should be little overlap of double- and The eld-dependento enhancementffer the possibility pro of locallyle of polarizing MnDOTA the nuclei adjacent to the for electrons and nuclei, respectively.ff Double- or zero-quantum (Figure 4) has a more complicated structure that reflects the overlapfi To compare the relative performances of the di erent polarizing zero-quantum transitions at the centers of the respective peaks, there fi metal and thus could yield site-speci c structural details for the fi ff of two hyper ne sextets withactive amplitudes sites of metalloproteins. of opposite sign In addition, thatagents are this quantitatively, local character of weReceived: evaluatedDecember the areas 4, 2010 under the eld- should be some cancellation e ects, especially closer to the center of shifted by twice the 1HLarmorfrequencywithrespecttoonedependent polarization profiles (ε 1). We emphasize thatff the the enhancement profile. However, since the use of a monochro- the polarization could provide important details aboutFigure the5. DNP(a c) ComparisonPublished: ÀMarch of the 29, microwave-on 2011 and -o signals another. Because of the narrow linewidthoftheSEtransitionsandshape anddetected width after ofÀ the transfer enhancement of the 1H polarization profile to peak13C (in are 1 M 13 neitherC-urea) via matic microwave source did not allow us to utilize this integral 1 fi 3 1 the mismatch between the HLarmorfrequencyandthehyperr 2011 Americanin Chemicalflneuenced Societycross-polarization. by nor similar5648 in Both line Gd widthþ complexes todx.doi.org/10.1021/ja1109002 the were enhanced measured|HNMRJ. Am. at Chem.84 K Soc. and2011, 133,enhancement, 5648–5651 these results show that the effect of spectral dilution coupling (212 vs 254 MHz), all 12 DNP peaks are clearly resolved.signal. Rather,6 W they microwave primarily power; reflect MnDOTA the line was width measured of the at EPR 86 K central and 5 W. due to ZFS or hyperfine coupling dominatestheactualenhance- However, the maximum enhancement observed at the secondtransition; in(d) fact, Buildup the fi ofeld-swept polarization DNP for the pro DNP-enhancedfile can be considered signals under as the ment obtained under experimental DNP conditions. Since the outermost peak on the high-field side is only 1.9 at a microwave same experimental conditions used for the enhancement1 measurements. ∼ an EPR spectrum being detected indirectly via the Henhancement. magnitude of the second-order ZFS that broadens the central power of 5 W. We ascribe this small enhancement to the distribution fi of the DNP conditions over all six hyperfine lines, which leadsThus, to an the integralhigh-field over peaks the that DNP were peak clearly should resolved allow and us well-separated to compare from transition scales inversely as the external magnetic eld, these 1 ff fi even smaller fraction ( /18)ofspinsinvolvedintheDNPprocessinthe di erentany paramagnetic negative DNP species condition independent and then multiplied of the linethe result widths by 3. of The compounds at yet higher elds might potentially outperform organic 3 comparison with the Gd þ case, in which 70% of the nucleartheir spins EPR centralresults of transitions. this simple Theanalysis intended are summarized integration in Table was 1. straight- Despite the radicals like trityl, whose EPR resonances are mostly broadened by g ∼ 3 28 lack a magnetic moment and the hyperfine coupling to theforward mag- invery the case different of the maximum two Gd enhancementsþ compounds, obtained since from we could the three anisotropy under these conditions. netically active nuclei is too small to have a significant impact onintegrate the overcompounds, the complete the integrated positiveenhance enhancementments, which peak. Inrepresent contrast, excita- The dynamics of polarization buildup did not differ significantly 2 5 2 line width. In addition, the lower spin state of Mn þ (S = /2the)and full integrationtion of all was transitions not possible yielding in positive the case enhancement, of the Mn þ arecomplex very similar. among the three compounds investigated, as can be seen in Figure 5. the lower transition moment of the central EPR transition (3 times GdDTPA performed 30% lessfi efficiently than the corresponding 1 because of the overlap of the∼ hyper ne lines of the positive and The polarization buildup times are listed in Table 1 and are larger than that for S = /2)mightresultinaslightlylower DOTA complex, which might be caused by the onset of the DSE 17 3 negative DNP peaks. Therefore, we integrated over only the two comparable to those using biradical polarizing agents. The fact performance than for Gd þ. condition. Although there should be little overlap of double- and To compare the relative performances of the different polarizing zero-quantum transitions at the centers of the respective peaks, there agents quantitatively, we evaluated the areas under the field- should be some cancellation effects, especially closer to the center of5650 dx.doi.org/10.1021/ja1109002 |J. Am. Chem. Soc. 2011, 133, 5648–5651 dependent polarization profiles (ε 1). We emphasize that the the enhancement profile. However, since the use of a monochro- shape and width of the enhancementÀ profile peak are neither matic microwave source did not allow us to utilize this integral influenced by nor similar in line width to the enhanced 1HNMR enhancement, these results show that the effect of spectral dilution signal. Rather, they primarily reflect the line width of the EPR central due to ZFS or hyperfine coupling dominatestheactualenhance- transition; in fact, the field-swept DNP profile can be considered as ment obtained under experimental DNP conditions. Since the an EPR spectrum being detected indirectly via the 1Henhancement. magnitude of the second-order ZFS that broadens the central Thus, the integral over the DNP peak should allow us to compare transition scales inversely as the external magnetic field, these the different paramagnetic species independent of the line widths of compounds at yet higher fields might potentially outperform organic their EPR central transitions. The intended integration was straight- radicals like trityl, whose EPR resonances are mostly broadened by g 3 28 forward in the case of the two Gd þ compounds, since we could anisotropy under these conditions. integrate over the complete positive enhancement peak. In contrast, The dynamics of polarization buildup did not differ significantly 2 the full integration was not possible in the case of the Mn þ complex among the three compounds investigated, as can be seen in Figure 5. because of the overlap of the hyperfine lines of the positive and The polarization buildup times are listed in Table 1 and are negative DNP peaks. Therefore, we integrated over only the two comparable to those using biradical polarizing agents.17 The fact

5650 dx.doi.org/10.1021/ja1109002 |J. Am. Chem. Soc. 2011, 133, 5648–5651 Implementations of microwave driven dynamic nuclear polarization (DNP)

improve the resolution of images of cells HF-liquid DNP Shuttleand other DNP biological systems; Pomplun and Glaser (DOI: 10.1039/c003751f) high frequency liquid DNP: in situ liquiddiscuss sample theoretical is rapidly methods moved for optimizing microwave excitation at the NMR fromtime the domainlow field, DNP where experiments, the an area that has thus far received little attention. detection field polarization is performed, to a All of these approaches are potentially highapplicable field region to a wide for NMR range of important NMRdetection experiments in biology, chemistry, physics and medicine, and their success- ful development will have an enormous impact on the field. Accordingly, a number of academic and industrial Dissolutionresearch groups have DNP recently initiated High Field efforts to overcome the current limita- tions of the techniques. Technical MAS DNP advances in the area of high-frequency the samplemicrowave is polarized sources in andthe components, solid and gyrotron as a microwave state atof very various low DNP temperatures approaches (Fig. 1), will source, solid sample (typicallybe 1–4 of vital K) and importance magnetic for the further irradiated with fields of development3–7 T, rapidly of dissolved, the DNP method, microwaves at the NMR and finallyespecially transferred at the to highest either magnetic a fields available for NMR ( 20 T). In addition, detection field high resolution NMR spectrometero implementationor a MR imager of microwave time domain experiments should open many Fig. 1 Typical experimental approaches for dynamic nuclear polarization spectrometers. new areas of application, just as rf time domain experiments did for high resolu- process, and resolution in low tempera- Applications of this method range from tion solid state and solution NMR. ture MAS experiments, is addressed here MR imaging of metabolites to studies of Other avenues, such as the optimization Downloaded on 07/04/2013 07:54:53. by Barnes et al. (DOI: 10.1039/c003763j). chemical reaction mechanisms (Bowen of polarizing agents, the development Wednesday, 10 April 13 Recently a commercial MAS DNP spec- and Hilty, DOI: 10.1039/c002316g; of new types of polarization transfer

Published on 19 May 2010 http://pubs.rsc.org | doi:10.1039/C0CP90019B trometer became available that is Ludwig et al., DOI: 10.1039/c002700f; methods, and the design of new experi- described in this issue together with some Panek et al., DOI: 10.1039/c002710n; ments focusing on selectivity, contrast recent results obtained with the instru- Cudalbu et al., DOI: 10.1039/c002309b). and additional structural restraints, are ment (Rosay et al., DOI: 10.1039/ An essential ingredient of every DNP ripe for investigation. Thus, collaborative c003685b; Debelouchina et al., DOI: experiment is a stable polarizing agent, efforts among researchers from chemistry, 10.1039/c003661g). Also in this issue, and for the first 50 years of DNP these physics, biology, medicine, and the direct transfers to low-g nuclei (2H, 13C, consisted of readily available monomeric engineering disciplines will be required etc.) are discussed, and the enhance- paramagnetic centers such as a metal, or to optimize DNP for applications in ments, field profiles, and preferred organic radicals like BDPA or TEMPO. high-field NMR and MRI. We foresee polarizing agents are shown to be system More recently, several new polarizing a very bright and expansive future for dependent (Maly et al., DOI: 10.1039/ agents have been introduced that are this field, well into the 21st century. c003705b).40 In Dissolution DNP (lower more efficient in that they produce larger right) the sample is polarized in the solid enhancements at lower concentrations.41–44 state at very low temperatures (typically Four articles describe these new agents: References 1–4 K) and magnetic fields of 3–7 T, narrow line trityl radicals, biradicals and 1 R. R. Ernst and W. A. Anderson, Rev. Sci. rapidly dissolved, and finally transferred spin labeled polymers that separate Instrum., 1966, 37, 93–102. to either a high resolution NMR spectro- at higher temperatures and therefore 2 P. Styles, N. Soffe, C. Scott, D. Cragg, meter or a MR imager (Leggett et al., preserve resolution (Paniagua et al., F. Row, D. White and P. White, J. Magn. DOI: 10.1039/c002566f; Bowen and DOI: 10.1039/c003291n; Dollmann et al., Reson., 1984, 60, 397–404. 3 S. R. Hartmann and E. L. Hahn, Phys. Hilty, DOI: 10.1039/c002316g). Very DOI: 10.1039/c003349a; Ysacco et al., Rev., 1962, 128, 2042–2053. high enhancements (relative to room DOI: 10.1039/c002591g; Macholl et al., 4 A. Pines, M. G. Gibby and J. S. Waugh, temperature) for 13C can be retained DOI: 10.1039/c002699a). J. Chem. Phys., 1972, 56, 1776. 5 G. Morris and R. Freeman, J. Am. Chem. during the dissolution and transfer Finally, two other important and Soc., 1979, 101, 760–762. process, arising from the product of exciting topics are discussed in contri- 6 G. Bodenhausen and D. J. Ruben, Chem. DNP enhancement (B250) and Boltzmann butions to this volume: Thurber and Phys. Lett., 1980, 69, 185–189. polarization (B250). This issue intro- Tycko (DOI: 10.1039/c0cp00157k) con- 7 D. Rugar, R. Budakian, H. J. Mamin and B. W. Chui, Nature, 2004, 329, 430–432. duces several new approaches and sider the possibility of using DNP 8 S. H. J. Mamin, T. H. Oosterkamp, improvements to the experiment. enhancements in solid state imaging to M. Poggio, C. L. Degen, C. T. Rettner

c This journal is the Owner Societies 2010 Phys. Chem. Chem. Phys., 2010, 12,5737–5740 | 5739 Where do I put the radical? Types of samples.

• frozen solution (aqueous and non-aqueous) • frozen solution containing macroscopic particles and aggregates • sedimented solutes • porous materials with non-aqueous solvents • crystals?

Wednesday, 10 April 13 Frozen solutions

An aqueous solvent mixture consisting of deuterated glycerol, D2O, water, and a soluble radical polarizing agent provides a glass-forming matrix that distributes the polarizing agents uniformly and at the same time acts as a cryoprotectant.

Wednesday, 10 April 13 What’s the deal with the mixture of deuterated and protonated solvent?

➡ enhancements in deuterated/protonated solvent mixtures are higher because relaxation is reduced and spin diffusion preserved

• fast nuclear relaxation destroys the enhanced signal (Why do they always start with proline and not alanine? How about the temperature?)

• spin diffusion transfers polarisation from the directly polarized protons to the protons that are too far from the polarising agent to experience direct enhancement of polarisation (Do you know what spin diffusion rate depends on?)

➡ typical conditions: [D6]DMSO or [D8]glycerol/ D2O/H2O in 6:3:1 ratio

Wednesday, 10 April 13 stable trityl radical and its derivatives42,43 and 1,3-bis- Bulk-polarization build-up and maximum enhancement (diphenylene)-2-phenylallyl (BDPA).44 Here we choose the Communications During the DNP process, the high thermal electron polariza- trityl radical OX063 (see Fig. 1) as the polarizing agent, tion is transferred to the surrounding nuclei resulting in a because of its copious solubility in aqueous media.45 The bulk-polarization build-up curve that can be modeled by an 140 GHz EPR spectrum of OX063 is shown in Fig. 2 (top). excitation, is responsible for the further increase in e. exponential process with a characteristic bulk-polarization The spectrum is essentially symmetric with a spectral breadth 13 15 build-up time constant tB. Fig. 4 illustrates a C-detected Supporting this hypothesis is the fact thatof the55 direct MHz (FWHM)N as determined from the EPR D E bulk-polarization build-up curve for 2H DNP using OX063 as spectrum. As a consequence, the enhancement profile of DNP enhancement is e = 207 for deuterated SH3 with the polarizing agent. Here the DNP-enhanced 2H polarization OX063 for direct 2H-DNP shown in Fig. 2 is symmetric with protons at 50% of the exchangeable sites. To quantify the is transferred to the proline 13C nuclei for detection via a the maximum positive enhancement occurring at 4983.0 mT 49 13 subsequent cross-polarization (CP) step. This allows an accurate signal per unit time, we recorded a C spectrum(DNP(+)) with and the direct maximum negative enhancement occurring determination of the signal enhancement, because the 13C at 4980.7 mT (DNP( )). excitation and a short relaxation delay of 2 s for a deuteratedÀ spectrum is much narrower compared to the direct-detected A direct comparison of these two enhancement profiles can 2Hspectrum.Atatemperatureof90K,thesteady-statepolariza- protein with 50% protons at the exchangeablebe used to sites illustrate (Fig- another important fact for high-field tion is reached after approximately 100 s of microwave irradiation ure 1E). The intensity at the CO and CglycerolDNP.signal At 5 is T reduced the separation between the optimum field 2 yielding a build-up time constant of tB( H) = 21 s. positions for 1H-DNP using TOTAPOL (DNP(+)) and compared to the CP spectrum of the fully protonated SH3 The absolute enhancement is calculated from the microwave 2H-DNP (or 13C) is approximately 4 mT, corresponding to on and off spectra, recorded under identical experimental sample (Figure 1C), whereas, the intensity112 in MHz the electronaliphatic Larmor frequency. The separation is B conditions (see Fig. 4, inset). For the off signal, 40 times more region is slightly increased. 14 mT between DNP( ) for TOTAPOL and DNP(+) for À scans were averaged to provide sufficient signal-to-noise due to OX063, corresponding to 400 MHz for electrons. To be able Figure 2A shows the dependence of the DNP enhance- the small 2H signal intensity without DNP enhancement and a to study different polarizing agents and to cover the complete ment on the exchangeable proton content in the protein and steady-state 2H DNP enhancement of e Z 700 was observed. field range, the DNP spectrometer has to be equipped either 1 Theoretically, the maximum enhancement that can be achieved buffer. The enhancements obtained for variouswith a sweep nuclei coil or ( theH, gyrotron needs to be tunable over a in a DNP experiment is given by the ratio of the gyromagnetic 13 15 range of > 0.5 GHz.46–48 Note that the sweep/tuning range C, and N) and by using different experimental approaches ratios of the electron and the nucleus that is polarized, here Perdeuterationwill increase may at higher be fields. also good for the2 sample2 itself (MAS and CP-MAS), depend strongly on the exchangeable H(g(eÀ)/g( H)). This gives a theoretical maximum enhance- Acomparisonofthe2H-DNP performance for TOTAPOL and 13 15 ment of 4300 for 2H-DNP. proton content. A gradual increase of theOX063C and is shownN in DNP Fig. 3 and approximately a factor of 4 larger In Fig. 5 two direct 13C-DNP enhanced spectra of proline Akbey, Ü.; Franks, W. T.; enhancementLinden, A.; Lange, is observed S.; Griffin, for OX0 R.63 G.; under van similar Rossum, experimental B.-J.; Oschkinat, H.; Rossum, B. Van Angew. Chem. 2010, 49, 1–5. enhancement is observed by increasing the exchangeable are shown, one spectrum taken without decoupling (A) and conditions. This improvement is due to the much narrower EPR proton content from 15% to approximately 50%. For all one with 83 kHz high-power 2H TPPM decoupling (B).50 As Maly, T.; Andreas, L. B.; Smith,spectrum A. of A.; OX063 Griffin, ( (TOTAPOL)/ R. G. Phys. Chem.(OX063) Chem.11) Phys. allowing 2010 a , 12, 5872–8. D D E expected, no significant difference in resolution was detected types of experiments, the fully protonatedlarger SH3 fraction has of lower the electron spins to be excited by the Downloaded by University of Warwick on 09/04/2013 23:07:24. between the two acquisition schemes. Therefore, the following Published on 11 May 2010 http://pubs.rsc.org | doi:10.1039/C003705B microwave radiation. Note that at the same electron con- centration TEMPO-based biradicals give a factor of 4 larger polarising 2H enhancements compared to monomeric TEMPO,23 and we therefore expect that further improvements could be made using biradicals based on OX063. Due to the much better performance of OX063 over TOTAPOL, the following DNP experiments were all performed using OX063 as the polarizing agent.

Fig. 4 2H bulk-polarization build-up curve recorded at a magnetic field position corresponding to DNP(+) using OX063. The 2H polarization is detected indirectly from the total 13C signal of 2 13 U-[ H7, C5]-proline through a ramped cross-polarization step (1.5 ms), Fig. 3 Comparison of the steady-state 2H signal intensity for 16 scans averaged. The inset shows the mw-on and off signal. The it nevertheless adds a contribution to the line broadening. TOTAPOL (A) and OX063 (B). Both spectra were recorded back- DNP enhanced spectrum was recorded at a field position corresponding 13 There is also the possibility that small inhomogeneities in the Figure 1. The pulse sequences used to record the DNP enhanced C to-back under identical experimental conditions. Due to the insuffi- to DNP(+) with a DNP build-up time of tmw = 20 s. For the mw-on spectra with A) CP-MAS and B) direct 13C excitation, at approximately cient excitation bandwidth of 83 kHz, the magnitude spectrum is signal 32 transients were averaged while for the mw-off signal in total magnetic field at the sample caused additional line broadening. 13 shown. T = 90 K, or/2p = 5.882 kHz. The spectra were recorded 1280 transients were averaged. T = 90 K, or/2p = 5.882 kHz. Spinning 98 K and approximately 9 kHz MAS. C) C CP-MAS spectrum of using a rotor-synchronized quadrupole echo sequence. side bands are marked by asterisks. protonated SH3 with DNP. DNP enhanced D) 13C CP-MAS and

E),F) MAS spectra of the deuterated-SH3 with 50% exchangeable c Sensitivity gain through DNP 5874 | Phys. Chem. Chem. Phys., 2010, 12,5872–5878 This journal is the Owner Societies 2010 proton content. A relaxation delay (RD) of 2 s (C–E) and 12 s (F) were 2 used. Continuous-wave microwave irradiation was used while acquiring Acquisition of a H dimension offers several advantages over 1 the DNP enhanced spectra. For calculation of the DNP enhancement, a H dimension. The deuterium spin system has a lower the spectra were recorded with microwave irradiation and compared to gyromagnetic ratio, and therefore does not suffer from the the spectra recorded without microwave irradiation under exactly same homogenous broadening observed for high concentrations of experimental conditions. The spectra are plotted with the same noise Figure 2. The dependence of the A) DNP enhancement and B) T protons in solids. Spins of interest can be perdeuterated level, to allow direct comparison. 1 relaxation time (seconds) on the overall 1H content. For comparison, a Fig. 9 Two-dimensional DNP-enhanced 2H-DQ-13C correla- without deuteration of solvents, crystallization agents and 2 13 fully protonated (at exchangeable and non-exchangeable sites) and tion spectrum of U-[ H7, C5]-proline recorded at 90 K, oR/2p = cofactors. Comparable sensitivity should also be achievable three different deuterated SH3 (at non-exchangeable sites) proteins 5.882 kHz, sampling time in the indirect dimension Dt1 = 170 ms, DQ with deuterium detection. For example, methyl–methyl con- opposite side of the profile. Nevertheless, in the case of SH3, were➡ used. establish The proton content balance at the exchangeable between sites of the protein reduction of relaxation and retention of spin diffusion excitation and reconversion time t =1ms, D =3ms, tmw = 25 s, the 13C T times are short and in the MAS DNP experiment was tuned by recrystallization of SH3 in buffers containing different tacts are often important for determination of protein struc- 1 64 scans per t1 point, B10 h of total acquisition time. we observe e 148 (Figure 1E). This enhancement is signifi- H2O/D2O ratios. The H2O contents were set around 15, 25, 50 and ture, and in cases where a CD2H labeling is used to reduce  8 cantly larger than the enhancement obtained from the CP 100% to be able to cover the full range of exchangeable proton proton couplings, perdeuteration (B97%) is employed. At Wednesday, 10 April 13 spectrum shows four resolved cross-peaks, corresponding to 13 content. The spectra were recorded at a temperature of approximately experiment (Figure 1D). However, the C DNP enhance- 13 3% protonation, methyl groups are B9% CD2H spin systems 98 K, an MAS frequency of approximately 9 kHz, and with approxi- correlations between the C proline atoms and the covalently ment observed for the fully protonated SH3 sample recorded mately 5 W microwave irradiation in zirconium rotors. The experimen- 2 to first order, with minimal (B0.3%) CDH2 and CH3 labeling. 13 attached H nuclei. 13 with direct C excitation is e 8. These observations strongly tal data points are connected with lines as a guide to the eye, the data This avoids broadening in the C dimension due to the shift in  suggest that protein deuteration, as well as direct 13C points are discontinuous. the isotropic resonance between CH and CD which results in Spectral linewidths different isotropic shifts for CH3, CH2D, CHD2 and CD3 7804 www.angewandte.org  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2010, 49, 7803 –7806 Under the current experimental conditions linewidths of groups. Since 10% labeling is often found to be necessary approximately 10 ppm and 8 ppm were observed for 2H and for optimized relaxation characteristics of amide protons,8 13C, respectively. These linewidths are larger than those observed perdeuteration will be used as a point of comparison, but previously for perdeuterated proteins.8,11 However, the source may need to be adjusted to by a factor of B3 if higher of the increased linewidth is not of a general nature. In protonation is found to be optimal. particular the main contribution arises from the fact that In a perdeuterated sample (B97%) 2H NMR should have a proline is a small molecule embedded in a frozen (90 K) glassy factor of B8.6 higher sensitivity compared to 1H detection, solvent matrix (glycerol/water). DNP samples are typically and a factor of B2.5 was experimentally observed by Agarwal prepared in a glass-forming solvent, which serves as a cryo- et al.11 If CH or CH groups are of primary interest, or if a Downloaded by University of Warwick on 09/04/2013 23:07:24. 2

Published on 11 May 2010 http://pubs.rsc.org | doi:10.1039/C003705B protectant to ensure that the polarizing agent is homogeneously higher proton concentration is found to be optimal, this dispersed throughout the sample and protects proteins from analysis needs to be adjusted. This gain in sensitivity is mainly cold degradation caused by thermal cycling of the sample. This due to the short longitudinal relaxation of the 2H nuclei, a is known to induce conformational distributions, which in direct consequence of the large quadrupolar coupling. There- turn can cause inhomogeneous broadening.53 However, this fore, at room temperature the recycle delay in the NMR factor becomes unimportant for larger systems such as bio- experiment can be short. Furthermore, sample heating is not macromolecules or (nano) crystals. For example in contribu- an issue due to the much lower decoupling power needed for tions by Barnes et al. and Debelouchina et al. (same issue) 13C deuterium. Nevertheless recycle delays between 1.25 and 3 s linewidths of 1–2 ppm are observed for the membrane protein were reported for previous work on biological samples.11 bacteriorhodopsin (bR) and GNNQQNY nanocrystals. This advantage no longer exists at 90 K because the DNP The paramagnetic polarizing agent has only minor effects on build-up time constant is 21 s. Therefore, to run the DNP the linewidth in such systems. For example in DNP-enhanced experiment at the optimum repetition rate one needs to wait MAS-NMR experiments on amyloid nanocrystals GNNQQNY54 21*1.25 s = 26 s between shots and the sensitivity for low- or the membrane protein bacteriorhodopsin (bR) the radical temperature 2H MAS-NMR spectroscopy would be decreased does not penetrate into the protein or nanocrystals. In the case by a factor of 3 to 5 depending on the actual recycle delay used of bR even electron concentrations of up to 100 mM did in the experiment compared to experiments performed at not show any effect on the linewidth of the retinal, which is 300 K. However, the observed DNP signal enhancement of buried inside the protein.55 All experiments described here are e Z 700 leads to an overall sensitivity of a low-temperature performed at a magnetic field strength of 5 T (212 MHz for 1H), 2H-DNP enhanced MAS-NMR experiment that is a factor of which is rather low for contemporary MAS-NMR spectro- 140 to 240 larger than at room temperature. This does not scopy and second order quadrupole effects could have a include the additional factor of B3 in sensitivity due to the contribution to the observed linewidth. Another minor con- lower temperature (300 K/90 K). tribution to the linewidth is of a technical nature. It is rather To compare the overall efficiency of 2H-DNP with 1H-DNP difficult to accurately set the magic angle at cryogenic tempera- the degree of nuclear polarization can be compared. In the tures for this particular probe, since it is not equipped with a case of 2H-DNP this is 16% of the theoretical maximum, and cryogenic sample-eject system16 or a Hall effect sensor.56 for 1H-DNP typically 27% (175/660) is observed at a magnetic Although a misadjusted magic angle has only minor effects field of 5 T.23,38 Therefore, overall 1H-DNP currently performs on the linewidth for double-quantum filtered 2Hexperiments11,14 more efficiently than 2H-DNP. However, with further advances

c 5876 | Phys. Chem. Chem. Phys., 2010, 12,5872–5878 This journal is the Owner Societies 2010 Subscriber access provided by ECOLE NORMALE SUPERIEURE LYON Article Dynamic Nuclear Polarization of Amyloidogenic Peptide Nanocrystals: Crystals/aggregatesGNNQQNY, in a glassy a Core matrix Segment of the Yeast Prion Protein Sup35p

van der Wel, P. C. a, Hu, K.-N., Lewandowski,Patrick J. R. &C. Griffin, A. vanR. G. J. der Am. Chem. Wel, Soc. Kan-Nian 128, 10840–6 (2006). Hu, Jzef Lewandowski, and Robert G. Griffin J. Am. Chem. Soc., 2006, 128 (33), 10840-10846• DOI: 10.1021/ja0626685 • Publication Date (Web): 02 August 2006 Downloaded from http://pubs.acs.org on April 27, 2009

ARTICLES van der Wel et al.

results from microwave irradiation and diffuses into the nano- crystals through the crystal surface. Within the crystal, the enhanced polarization, !0, diffuses into the core, following a process that is assumed to be dominated by 1D nuclear spin diffusion along the crystal x axis24 and described by Fick’s law:19,38

∂P ∂2P P ) - D 2 (1) ∂t ∂x T1n Figure 2. (a) Model for an individual peptide crystallite, showing polarization transfer into the core along the narrowest crystal dimension x. where P(x,t) is the polarization, after subtraction of the Boltz- (b) Predicted enhanced polarization profile along the dimension x of the mann polarization, at a time t and a distance x from the center nanocrystal. of the crystal; D is the diffusion constant, and T1n is nuclear More About This Article spin-lattice relaxation time. In the steady-state ∂P/∂t ) 0, and increase in the applied external magnetic field tends to reduce we obtain the efficiency of the polarization,Additional the extent resources of this reduction and features is associated with this article are available within the HTML version: dependent on the type of polarization mechanism involved. In ∂2P P D ) (2) the case of the CE and TM, the impact• of higherSupporting magnetic Information fields 2 ➡ we can also use spin diffusion to transfer∂x Tpolarisation1n on the polarization transfer efficiency• canLinks be compensated to the 13 articles by that cite this article, as of the time of this article download optimizinginto of the the EPR spectralparticles parameters,• Access that for exampleto high are resolution the notIf figures wepenetrated assume that the nuclei by in the the glassy solventradicals maxtrix have electron-electron dipole interaction• andLinks the EPR to articles frequency and contentreached related a steady-state to this article and that their enhanced polarization is separation of the paramagnetic species,• 17 Copyrightfor the desired permission field to reproduceevenly distributed, figures thenand/or we text have from the boundarythis article condition for the strength. surfaces of the crystals: Wednesday,The resulting 10 April 13 locally enhanced nuclear polarization is dis- tributed to the bulk nuclei via 1H nuclear spin diffusion. In a w w P ) P - ) !0P0 (3) homogeneous sample, the efficiency of this process depends (2) ( 2) on the density and, possibly, on orientations of nuclear spins. where w is the crystal width along the x axis, ! is the steady- However, the presence of a diffusion barrier,33,34 for instance 0 state enhancement factor for the solvent nuclear polarization, resulting from the proximity of a paramagnetic center or two and P is the nuclear Boltzmann polarization at thermal domains characterized by large differences in nuclear spin 0 equilibrium. As illustrated in Figure 2b, the solution to eq 2 in characteristics, can reduce the efficiency of spin diffusion. The the region -w/2 e x e w/2 with the boundary condition in eq nuclear spin diffusion barrier near a paramagnetic species arises 3 is from a strong electron-nuclear dipolar field at the nucleus that isolates the surrounding nuclei in terms of resonance frequency. -1 w x Similarly, a diffusion barrier can be caused by gaps in resonance P(x) ) !0P0 cosh cosh (4) 2 DT DT frequency between two domains that have different magnetic ( 1n) ( 1n) 35-37 ! ! susceptibilities in response to the external magnetic field. Experimentally one observes the average polarization across the This might be a concern for dehydrated nanocrystals embedded whole crystal (-w/2 e x eJournalw/2), of as the given American by Chemical Society is published by the American Chemical in a frozen aqueous solvent matrix. Note that the latter boundary Society. 1155 Sixteenth Street N.W., Washington, DC 20036 is less intrusive when the domain size is smaller (e.g., in 1 w/2 2 DT1n w nanometer range). P(x)dx ) ! P tanh (5) w -w/2 !w 0 0 For simplicity of our discussion, we assume the bulk solvent ∫ (2 DT1n) nuclear spins surrounding the crystals are uniformly polarized ! which dictates that the measured enhancement factor ! of the with an enhancement factor !0. The magnitude of this factor is characteristic of the bulk solvent/radical composition and the crystals is experimental and instrumental details.17,18 In our analysis we 2 DT1n w assume that penetration of enhanced nuclear polarization into ! ) ! tanh (6) a nanocrystal is dominated by uniform nuclear spin diffusion 0 !w (2 DT1n) along the smallest dimension of the crystal. This pseudo-one- ! dimensional (1D) spin diffusion depends on the width of the The spin diffusion constant D in a proton-rich solid can be 2 smallest crystal dimension, the nuclear T1, and the nuclear spin estimated as D ) λ πBL, where BL is the average dipolar diffusion constant, D, of the nanocrystal. interaction at a characteristic 1H-1H distance λ. At room To illustrate our description of the polarization transfer into temperature, the average dipolar interaction depends on mo- uniformly sized crystals, we show the model depicted in Figure lecular dynamics affecting the proton coordinates.19 These 2a. A steady-state enhanced polarization of the glass matrix dynamics are mostly quenched at cryogenic temperatures, except for the three-fold hopping of methyl groups. Nonetheless, the (33) Blumberg, W. E. Phys. ReV. 1960, 119, 79-84. application of sample rotation in solid-state MAS/NMR to the (34) Furman, G. B.; Goren, S. D. J. Phys.: Condens. Matter 2002, 14, 873- 881. frozen sample can significantly modulate the dipolar interaction. (35) Hurlimann, M. D. J. Magn. Reson. 1998, 131, 232-240. 1 1 (36) Sen, P. N.; Axelrod, S. J. Appl. Phys. 1999, 86, 4548-4554. Once the H- H dipolar coupling constant ωd is smaller than (37) Vasenkov, S.; Galvosas, P.; Geier, O.; Nestle, N.; Stallmach, F.; Karger, J. J. Magn. Reson. 2001, 149, 228-233. (38) Lowe, I. J.; Tse, D. Phys. ReV. 1968, 166, 279-291.

10842 J. AM. CHEM. SOC. 9 VOL. 128, NO. 33, 2006 Journal of the American Chemical Society Communication

experiment the sediment is largely segregated from the bulk signal strength as compared to the thermal (Boltzmann) solvent and consists of a highly concentrated protein solution polarization signal acquired without microwave irradiation (off- 13 Journal of the American Chemical Society with a reproducible protein and waterCommunication content (≤700 mg/mL). signal). Direct polarization of C was observed via a Bloch This solution has a high viscosity due to self-crowding30,31 and decay; the enhancement factor was determined to be 22. Due radical molecule per 50 ferritin monomers (i.e., about 1 per 2 13 the water that is contained therein is likely to be bound or to the absence of C in the D2O/H2O matrix, spin polarization ferritin cages). In cases like these, one should32−34 then optimize the biradicalinteracting concentration with as the is customary protein. in a DNPConsequently, experiment. the frozen has to be transferred directly and cannot be transported Thesediment enhancement is not from as susceptible DNP allowed to the ice acquisition formation of within the bulk through the matrix via spin diffusion. Therefore, the protein multidimensionalsolvent as is spectra a homogeneous (Figure 3) of frozen sedimented solution. samples This suggests the must be in proximity to TOTAPOL, limiting the distance possibility that the sedimented protein could exhibit glass like between the unpaired electron spins and the uniformly 13C- behavior and be suitable for DNP experiments in the absence of labeled protein. a glass forming agent such as glycerol. In contrast, the frozen solution (ii) provides very poor 1 13 To investigate this possibility, we studied three samples: (i) enhancements (ε ≈ 2) for both H and C polarization due to ApoF sedimented by MAS at room temperature from an the inability to form a glass and phase separation of water, ff aqueous solution containing TOTAPOL and then frozen; (ii) protein, and TOTAPOL, inhibiting e ective electron−nuclear aqueous solution of ApoF and TOTAPOL frozen sans spin polarization (Figure 1). This shows that sedimentation sedimentation; and (iii) same as (i) but without the addition provides a layer of glassy-like protein on the wall of the of TOTAPOL. All samples were prepared from solutions with sapphire rotor, which enables the biradical to be homoge- an initial protein concentration of 30 mg/mL in 90/10 (v/v) neously dispersed throughout the sediment, providing glass-like properties and efficient e−→1H(13C) polarization transfer. D2O/H2O in 3 mM tris(hydroxymethyl)aminomethane (Tris) Figure 2. Comparison of DNP-enhanced signals from a cryoprotected buffer. Samples (i) and (ii) also contain 2 mM TOTAPOL. The We measured the polarization buildup time constants (TB) ApoF sample (12 mg/mL) in d8-glycerol/D2O/H2O (60/36/4 v/v) 1 and found them to be unusually short for the sedimented with 1H polarization buildup time constants for 2 (A) and 15 mM (B) reduction of the H concentration in the matrix to ∼10% is TOTAPOL concentration under otherwise identical experimental known to yield optimal conditions for 1H DNP.8 sample (i), suggesting direct protein−TOTAPOL interactions conditions. DNP-enhanced spectra (on-signals) are given in blue, 13 15 (vide infra). In order to assess those potential interactions, two while thermal polarization spectra (off-signals) are given in red. FigureFor 3. Representative preparation DNP-enhanced of samples 2D(i)13 andC−13 (iii),C correlation U- C, N-ApoF was ff d8-glycerol/D2O/H2O (60/36/4 v/v) solutions were prepared Spectra are also scaled by a factor of 5 for better visualization (given in spectrumspun (proton-driven at ω /2π = 10spin kHz, di usion, andτmix cross-polarization= 20 ms) of ApoF (CP) was used r with 2 and 15 mM TOTAPOL, respectively. Using a glass- light red color). Non-enhanced background signals from the Vespel sedimentedto monitor from an the D2 sedimentationO/H2O (90/10 v/v)in situsolution, typically at an initial over a period of a spacers are marked with asterisks. concentration of 60 mM ApoFmonomercontaining5mM forming agent to disperse the polarizing agent in a TOTAPOL.few hours. The acquisition The sedimented period was ∼5 sample h. was subsequently frozen while spinning using cooled N gas (avoiding sediment homogeneous solvent is a common approach in many DNP qualitative picture of the TOTAPOL preferentially binding to 2 NMR experiments, and often provides the optimal enhance- the surface of the ApoF 24-mer, and illustrates the importance dispersion) to perform MAS DNP measurements. The ments and protects the protein from cold denaturation at of using low radical concentration (≤5 mM) for SedDNP spinning frequency was reduced to 4.8 kHz at cryogenic studies of proteins. Radical binding to proteins was recently within hours using ∼1.8 mg of a ∼0.5 MDa protein complex. cryogenic temperatures. The bulk 1H concentration of 4% was 35 Recalltemperatures that this was (T recorded< 90 K). at Figureω /2π 1= illustrates 211 MHz signi and ficant gains in reported in other cases. We note that biradicals were 0I chosen in order to slow homonuclear spin diffusion, and thus to specifically developed to function at lower e− concentrations thereforesignal does intensity not permit from resolution DNP of under individual microwave cross peaks irradiation (on- enable us to partially discriminate between polarization than monomeric polarizingSedimented agents. Solutes from a 20 kDa protein, but it does illustrate that standard 2D 1 13 1 signal) for sample (i), indicating the incorporation of the radical ff A summary of the H and C buildup times and H MAS experiments are feasible on1 a sedimented sample doped transported from remote TOTAPOL to ApoF via spin di usion withinto TOTAPOL. the sediment. The H polarization and buildup time was polarization enhancementsRavera, E. for et allal. Dynamic samples nuclear is provided polarization in Tableof sedimented solutes. J. Am. Chem. Soc. 135, 1641–4 (2013).13 versus direct transfer of polarization by bound TOTAPOL. 1. Ininvestigated summary, we by have CP shown to C that and sedimentation yielded a of 42-fold the increase in protein enables significant DNP enhancements without the Thus, samples with the appropriate TOTAPOL concentration 13 15 Table 1. Summary of DNP Enhancements and 1H, 13C addition of a glass-forming material such as glycerol, resulting in were dissolved, and a fraction of the U- C, N-ApoF was Polarization Buildup Time Constants for All Samples an ApoF/TOTAPOL glass at the wall of the rotor, leaving in dissolved and rapidly frozen in a MAS rotor inside the DNP the center of the rotor a pool of bulk water which undergoes 13 NMR spectrometer. C TB ε crystallization upon freezing. The results reported here 1 1 13 sample H TB (s) (s) ( H/ C) represent an important step toward DNP of proteins The enhancements observed from the 2 and 15 mM solution frozen sediment, 2 mM TOTAPOL (i) 1.2 12.4 42/22 sedimented into an MAS rotor by ultracentrifugation samples were 70 and 100 (Figure 2), and biphasic buildup frozen solution, 2 mM TOTAPOL (ii) 2.1 13.4 2.1/1.6 experiments that are currently underway. Enhancements are a times on the order of 20 and 5 s, respectively, were found for frozen sediment sans TOTAPOL (iii) 2.1a 12.1a −b/−b factor of ∼2 lower compared to the “standard approach”, which cryoprotected 2 mM TOTAPOL (A) 20.4/1.1c −d 70/−d the slow component. The fast component appeared with time may be attributed to short T1 relaxation of nuclei, being cryoprotected 15 mM TOTAPOL (B) 5.0/0.6c 3.5 100/∼10 induced by the high concentration of protons in the sediment, constants of 1.1 and 0.6 s, respectively. The biphasic nature of a b TB equals nuclear T1 for non-DNP enhanced signals. ε = 1 by or by the increased content of paramagnetic polarizing agent the buildup can be explained by the existence of two distinct definition for non-DNP-enhanced signals. cSlow and fast component within the sediment itself. The shorter T associated with the ff d B polarization transfer mechanisms, for example, spin-di usion of biphasic buildup. Not determined. sedimented samples is useful in shortening the experimental via bulk and direct transfer from protein-bound polarizing acquisition time, resulting in almost identical sensitivity Radical binding to the protein is not a feature of the between SedDNP and DNP of homogeneously dispersed agent. This interpretation is further supported by an increase of SedNMR or SedDNP, but rather an aspect of the protein protein in glycerol/water. In practice these experiments will be the amplitude ratio between the slow and the fast components chemistry. Thus, we can expect that different proteins will performed by direct centrifugation of the sample into the rotor. ff ff from 4.9:1 to 6.1:1 upon increasing the TOTAPOL interact di erently with di erent biradical polarizing agents. concentration. Saturation of the binding sites obviously leads Although we cannot predict the behavior a priori, one would ■ ASSOCIATED CONTENT expect that, if the radical were not interacting with the protein, to a larger contribution of the bulk polarization transfer its concentration in the sediment layer would be lowered. *S Supporting Information mechanism at higher TOTAPOL concentrations. 34 Assuming a 33% water content in the sediment, and a non- Experimental details for sample preparation and DNP NMR For the sedimented samples, the 1H spin-polarization interacting biradical, the concentration of the radical would be experiments. This material is available free of charge via the 0.66 mM, as compared to the 33 mM ferritin monomer, i.e., 1 Internet at http://pubs.acs.org. buildup time constants were found to be of the order of 1.2 (i) and 2.1 s (ii and iii). As suggested (vide supra), the 1643 Figure 1. dx.doi.org/10.1021/ja312553bComparison of| DNP-enhancedJ. Am. Chem. Soc. 2013, 135, signals1641−1644 from a frozen difference in the observed polarization times could indicate an sedimented sample (i) and for a frozen solution (ii) using cross- 13 1 13 increased TOTAPOL concentration in the sediment with polarization ( C− H) and direct detection ( C) under otherwise respect to the bulk solution. Using the glycerol data and identical experimental conditions. DNP-enhanced spectra (on-signals) ➡ sedimented solutes - “microcrystallineare given in blue, while thermal polarization glass” spectra (off-signals) are relating these to the sedimented samples provides evidence that given in red. Spectra are also scaled by a factor of 5 for better the TOTAPOL concentration is in fact higher in the ➡ use low (< 5mM) concentrationvisualization (given in of light TOTALPOL red or blue color). Resonances marked sedimented samples. Buildup time constants and data from with an asterisk arise from Vespel spacer material in the rotor which model systems suggest that the effective biradical concentration was used for the frozen solution. with respect to the protein is ∼10−20 mM. This provides a

Wednesday, 10 April 13 1642 dx.doi.org/10.1021/ja312553b | J. Am. Chem. Soc. 2013, 135, 1641−1644 View Online

can be compared to the enhancement observed for the same

material with a 9.2 mM solution of TOTAPOL in 90 : 10 D2O/ 2 H2O(eSi = 21). For no obvious reason, the solvents in category 2 yield slightly lower enhancements. The presence of (NMR active) fluorine nuclei may be detrimental, as may the presence of (mobile) methyl groups (vide infra). Further investigation into these effects, which is out of the scope of the present work, is under way. THF in category 3 yields poor enhancements. The 100 K 1H spectrum of I impregnated with THF is relatively narrow (see ESIw, Fig. S1), showing that large-scale molecular motions are present at low temperature. All the solvents in category 4 have only methyl protons (except for one HCQO proton in DMF). Again, these moieties will be undergoing fast rotation at 100 K, leading to shorter T and Fig. 1 (a) Structure of the model hybrid silica material (I). 1H slower spin diffusion, possibly yielding low DNP enhancements. (b) Structure of bTbK. (c) 1H direct detection and (d) 29Si CP MAS spectra acquired with microwaves on (red) and off (blue) of I Finally, dichloromethane is too volatile to allow easy sample impregnated with a 10 mM solution of bTbK in tetrachloroethane. preparation, and gave low enhancements, as expected. Experimental details are given in ESI.w It appears that the best DNP enhancements are obtained with halogenated solvents. This can probably be attributed to displays the observed proton and silicon DNP enhancements several factors: (i) halogen atoms increase the molecular mass, obtained for I impregnated with bTbK (Fig. 1b) solutions in generally leading to higher freezing points and lower vapor 20 different solvents. bTbK is more soluble in organic solvents pressures. (ii) The halogen atoms reduce the 1H densities of the than the popularly used TOTAPOL, and has been reported to organic solvents, allowing the polarization to be distributed yield better enhancements in a DMSO/water mixture than 1 7 5 over a smaller bath of H nuclei. This is for example believed TOTAPOL at the same concentration. Large differences in eH to be the reason why high deuteration levels produce large 1H and eSi are observed in this set of solvents, with eSi ranging enhancements in frozen water/glycerol solutions. The 1H from 1 for THF and MTBE to 18 (1,1,2,2-tetrachloroethane density of fully protonated tetrachloroethane is similar to that and 1,2-dichloroethane). Note that similar enhancements are 1 of 90 : 10 D2O:H2O (Table S4, ESIw). Note that nitroxide observed for the H resonances of the solvent in pure frozen radicals have also been reported to form non-covalent bonds solutions (see ESIw). These polarizing solutions are thus of with halogenated molecules via electron donor–acceptor inter- general DNP use, and not limited to SENS. Overall, the actions, which might affect both electron spin transfer and solvents can be divided into 5 categories (Table 1) based on a relaxation properties.8 A more detailed investigation of the phenomenological correlation between their DNP performance influence of spin diffusion and the role of halogens on DNP Downloaded by University of Warwick on 29 May 2012 and chemical structures. All the solvents in category 1 entirely efficiency is currently under way. From these results, tetra- fulfil the criteria above, and they lead to large enhancements.

Published on 28 October 2011 http://pubs.rsc.org | doi:10.1039/C1CC15242D chloroethane in combination with bTbK appears to be a good The least effiDoescient, solventtrans-dichloroethene, have to be yields alwayseSi of 8, water-based? which is non-aqueous mixture for SENS. The optimal conditions for comparable to the typical SENS efficiency observed with Zagdoun, A. et2 al. Chem. Comm. 48, 654–6 (2012). this solvent/biradical combination are determined below. TEMPO in water, and the best, 1,1,2,2-tetrachloroethane, The effect of introducing a paramagnet, which quenches part of the signal, is not contained in e but can be determined Table 1 Proton and silicon-29 enhancements measured for I impreg- nated with a 10 mM solution of bTbK in different solvents by comparison with the spectrum of I impregnated with pure solvent (no radical). This comparison yields the overall sensitivity a b b w View Online Category Solvent eSi eH gainhybridS silicawhich material also takes into account the Boltzmann factor of 1 Chloroform 10 16 3 gained by doing the experiment at 100 K instead of 298 K good candidate 1,1,2,2-Tetrachloroethane 18 21 and the change in the relaxation properties of thecan sample be compared to the enhancement observed for the same material with a 9.2 mM solution of TOTAPOL in 90 : 10 D O/ trans-Dichloroethene 8 9 induced by the presence of the radical.9 Sw reflects the overall 2 1,2-Dichlorobenzene 16 19 H O( = 21).2 For no obvious reason, the solvents in gain of using SENS rather than conventional room tempera-2 eSi 1,2-Dichloroethane 18 23 category 2 yield slightly lower enhancements. The presence 9 w w 1,2-Dibromoethane 11 11 ture NMR. Fig. 2a shows e, S and S (S withoutof (NMR the active) fluorine nuclei may be detrimental, as may 1,3-Dibromobutane 12 19 Boltzmann factor) as a function of the radical concentrationthe presence of (mobile) methyl groups (vide infra). Further 1,1,2,2-Tetrabromoethane 14 12 for bTbK in tetrachloroethane. Fig. 2b shows the quenching 2 Hexafluoropropan-2-ol 9 6 investigation into these effects, which is out of the scope of the 2,2,2-Trifluoroethanol 7 5 factor (y), also as a function of the radical concentration.present work, is under way. THF in category 3 yields poor Isopropanol 7 6 As expected, and as observed for aqueous biradical solutions,enhancements. The 100 K 1H spectrum of I impregnated with Mesitylene 5 6 y decreases with increasing radical concentration.9,10 TheTHF optimal is relatively narrow (see ESIw, Fig. S1), showing that 3 Tetrahydrofuran 1 4 large-scale molecular motions are present at low temperature. 4 Methyl-ter butylether (MTBE) 1 3 concentration is found to be 14.8 mM for e and 10.8 mM for w All the solvents in category 4 have only methyl protons (except Dimethylformamide (DMF) 3 3 S and S .Thequenchingeffect causes the discrepancy in the for one HCQO proton in DMF). Again, these moieties will be Dimethylsulfoxide (DMSO) 4 4 optimal radical concentration between e and S.Attheoptimal undergoing fast rotation at 100 K, leading to shorter T1H and Acetonitrile 7 5Fig. 1 radical(a) Structure concentration of the modelS =12,whichtranslatesinto hybrid silica material (I). Sw =36. 1,1,1-Trichloroethane 3 3 1 29 slower spin diffusion, possibly yielding low DNP enhancements. (b) StructureThis of corresponds bTbK. (c) H to direct an detection overall acceleration and (d) Si CP in MAS the acquisition 5 Dichloroethane 3 9spectra acquired with microwaves on (red) and off (blue) of I Finally, dichloromethane is too volatile to allow easy sample a b time by more than a factor of 1000 with respect to experimentspreparation, and gave low enhancements, as expected. See text for discussion. Sample preparation and definition of e are in ESI.impregnated with a 10 mM solution of bTbK in tetrachloroethane. Experimentalrun at details room are temperature given in ESI.w on a dry sample. It appears that the best DNP enhancements are obtained with halogenated solvents. This can probably be attributed to displays the observed proton and silicon DNP enhancements This journal is c The Royal Society of Chemistry 2012 Chem. Commun., 2012, 48,654–656several655 factors: (i) halogen atoms increase the molecular mass, obtained for I impregnated with bTbK (Fig. 1b) solutions in generally leading to higher freezing points and lower vapor ➡ a lot of inorganic systems require20 different non-aqueous solvents. bTbK is more soluble solvents in organic solvents pressures. (ii) The halogen atoms reduce the 1H densities of the than the popularly used TOTAPOL, and has been reported to organic solvents, allowing the polarization to be distributed yield better enhancements in a DMSO/water mixture than 1 7 5 over a smaller bath of H nuclei. This is for example believed TOTAPOL at the same concentration. Large differences in 1 Wednesday, 10 April 13 eH to be the reason why high deuteration levels produce large H and eSi are observed in this set of solvents, with eSi ranging enhancements in frozen water/glycerol solutions. The 1H from 1 for THF and MTBE to 18 (1,1,2,2-tetrachloroethane density of fully protonated tetrachloroethane is similar to that and 1,2-dichloroethane). Note that similar enhancements are 1 of 90 : 10 D2O:H2O (Table S4, ESIw). Note that nitroxide observed for the H resonances of the solvent in pure frozen radicals have also been reported to form non-covalent bonds solutions (see ESIw). These polarizing solutions are thus of with halogenated molecules via electron donor–acceptor inter- general DNP use, and not limited to SENS. Overall, the actions, which might affect both electron spin transfer and solvents can be divided into 5 categories (Table 1) based on a relaxation properties.8 A more detailed investigation of the phenomenological correlation between their DNP performance influence of spin diffusion and the role of halogens on DNP Downloaded by University of Warwick on 29 May 2012 and chemical structures. All the solvents in category 1 entirely efficiency is currently under way. From these results, tetra- fulfil the criteria above, and they lead to large enhancements.

Published on 28 October 2011 http://pubs.rsc.org | doi:10.1039/C1CC15242D chloroethane in combination with bTbK appears to be a good The least efficient, trans-dichloroethene, yields eSi of 8, which is non-aqueous mixture for SENS. The optimal conditions for comparable to the typical SENS efficiency observed with 2 this solvent/biradical combination are determined below. TEMPO in water, and the best, 1,1,2,2-tetrachloroethane, The effect of introducing a paramagnet, which quenches part of the signal, is not contained in e but can be determined Table 1 Proton and silicon-29 enhancements measured for I impreg- nated with a 10 mM solution of bTbK in different solvents by comparison with the spectrum of I impregnated with pure solvent (no radical). This comparison yields the overall sensitivity a b b w Category Solvent eSi eH gain S which also takes into account the Boltzmann factor of 1 Chloroform 10 16 3 gained by doing the experiment at 100 K instead of 298 K 1,1,2,2-Tetrachloroethane 18 21 and the change in the relaxation properties of the sample trans-Dichloroethene 8 9 induced by the presence of the radical.9 Sw reflects the overall 1,2-Dichlorobenzene 16 19 1,2-Dichloroethane 18 23 gain of using SENS rather than conventional room tempera- 9 1,2-Dibromoethane 11 11 ture NMR. Fig. 2a shows e, Sw and S (Sw without the 1,3-Dibromobutane 12 19 Boltzmann factor) as a function of the radical concentration 1,1,2,2-Tetrabromoethane 14 12 for bTbK in tetrachloroethane. Fig. 2b shows the quenching 2 Hexafluoropropan-2-ol 9 6 2,2,2-Trifluoroethanol 7 5 factor (y), also as a function of the radical concentration. Isopropanol 7 6 As expected, and as observed for aqueous biradical solutions, Mesitylene 5 6 y decreases with increasing radical concentration.9,10 The optimal 3 Tetrahydrofuran 1 4 4 Methyl-ter butylether (MTBE) 1 3 concentration is found to be 14.8 mM for e and 10.8 mM for w Dimethylformamide (DMF) 3 3 S and S .Thequenchingeffect causes the discrepancy in the Dimethylsulfoxide (DMSO) 4 4 optimal radical concentration between e and S.Attheoptimal Acetonitrile 7 5 radical concentration S =12,whichtranslatesintoSw =36. 1,1,1-Trichloroethane 3 3 5 Dichloroethane 3 9 This corresponds to an overall acceleration in the acquisition time by more than a factor of 1000 with respect to experiments a See text for discussion. b Sample preparation and definition of e are in ESI. run at room temperature on a dry sample.

This journal is c The Royal Society of Chemistry 2012 Chem. Commun., 2012, 48,654–656 655 Article

pubs.acs.org/JPCB

Solvent-Free Dynamic Nuclear Polarization of Amorphous and Crystalline ortho-Terphenyl Ta-Chung Ong,†,‡ Melody L. Mak-Jurkauskas,§ Joseph J. Walish,‡,∥ Vladimir K. Michaelis,†,‡ § § Björn Corzilius,†,‡ Albert A. Smith,†,‡ Andrew M. Clausen, Janet C. Cheetham, Timothy M. Swager,‡ and Robert G. Griffin*,†,‡ †Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States ‡Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States §Amgen Inc., 360 Binney Street, Cambridge, Massachusetts 02142, United States ∥ DyNuPol Inc., Newton, Massachusetts 02458, United States Do we have to have solvent?

Ong, T.-C.; Mak-Jurkauskas, M. L.; Walish, J. J.; Michaelis, V. K.; Corzilius, B.; Smith, A. a; Clausen, A. M.; Cheetham, J. C.; Swager, T. M.; Griffin, R. G. S * Supporting Information J. Phys. Chem. B 2013, 117, 3040–6.

ABSTRACT: Dynamic nuclear polarization (DNP) of amorphous and crystalline ortho-terphenyl (OTP) in the absence of glass forming agents is presented in order to gauge the feasibility of applying DNP to pharmaceutical solid-state nuclear magnetic resonance experiments and to study the effect of intermolecular structure, or lack thereof, on the DNP enhancement. By way of 1H−13C cross-polarization, we obtained a DNP enhancement (ε) of 58 for 95% deuterated OTP in the amorphous state using the biradical bis-TEMPO terephthalate (bTtereph) and ε of 36 in the 1 crystalline state. Measurements of the H T1 and electron paramagnetic resonance experiments showed the crystallization process led to phase separation of the polarization agent, creating an inhomogeneous distribution of radicals within the sample. Consequently, the effective radical concentration was decreased in the bulk OTP phase, and long-range 1H−1H spin diffusion was the main polarization propagation mechanism. Preliminary➡ having DNP glass experiments helps but with DNP canthe provide glass-forming large enhancements anti- in dry, fl solvent-free crystalline systems, while maintaining excellent spectral in ammation drug, indomethacin, showed promising results, and furtherresolution studies are (in spite underway of clustering to prepare of radicals) DNP samples using pharmaceutical techniques.

Wednesday, 10 April 13

■ INTRODUCTION Although informative, NMR is intrinsically an insensitive Solid-state properties of active pharmaceutical ingredients analytical technique as a result of the inherently low nuclear spin polarization. This problem can be further compounded by (APIs) and drug formulations directly impact the safety and 13 15 17 efficacy of a drug.1−3 For example, a drug that is poorly soluble low natural abundance NMR active nuclei (e.g., C, N, O, as a crystal oftentimes is more soluble if it can be prepared in an etc.). These challenges cause drug formulation screening by amorphous form, increasing its bioavailability. Solid-state solid-state NMR to be a time-consuming process. Dynamic nuclear magnetic resonance (NMR) is a uniquely informative nuclear polarization (DNP) at cryogenic temperatures has been shown to provide significant enhancements for NMR signals. and versatile analytical technique in pharmaceutical research ff that includes analysis of the final solid form of the drug, The gains in sensitivity a orded by DNP are typically one to quantification of amorphous content, excipient interactions, two orders-of-magnitude and reduce the need for lengthy signal-averaging thereby dramatically reducing the data and salt and polymorph screening. In practice, solid-state NMR 6−14 is performed alongside other analytical techniques such as acquisition time. As a result, DNP has found utility in ff NMR applied to membrane proteins,15,16 amyloid fibrils,17 electron microscopy, X-ray di raction, IR and Raman spectros- 18 19 copies, differential scanning calorimetry (DSC), and thermog- inorganic complexes, silicon surface functional groups, metabolomics,20 and medical magnetic resonance imaging ravimetric analysis (TGA) to produce a complete character- 21,22 ization of the API and its formulation. Compared to other (MRI). Extension of microwave-driven DNP to analyze APIs and pharmaceutical formulations will potentially lead to techniques, solid-state NMR has the advantage of being an fi inherently quantitative method that nondestructively inter- signi cant savings in cost and time. rogates the whole sample. Depending on the system of interest, Currently, most DNP samples are prepared in a glass- it can be used, for example, to identify and quantify forming medium such as glycerol/water or DMSO/water, polymorphs, detect the number of molecules in a unit cell, which functions as a cryoprotectant to protect biological and elucidate the presence of a hydrate and/or solvate, investigate structural and dynamic properties over a wide Received: November 13, 2012 range of time scales, monitor stability against degradation over Revised: February 18, 2013 time, and analyze crystalline and amorphous environments.4,5 Published: February 19, 2013

© 2013 American Chemical Society 3040 dx.doi.org/10.1021/jp311237d | J. Phys. Chem. B 2013, 117, 3040−3046 A Few Applications

Biomolecules Materials Angewandte. Zuschriften

overlap of spinning sidebands with isotropic resonances, a nrot of 12 kHz was employed for all subsequent experiments. Finally, DNP 1H-13C CPMAS solid-state NMR spectra of the same sample of 1 impregnated with a 16 mm bTbK solution were periodically acquired after the sample was allowed to rest on the bench top inside the rotor for times of 5 min, 1 h and 25 h in between acquisitions. Both e and S were observed to steadily increase from 7 to 13 and 3.0 to 3.6, respectively, with longer sample resting times (Figure S3). This suggests the biradical species slowly diffuse through the relatively small (1.6 nm) pores of the material. Spectra of 1 and 2 were recorded after 25 and 0.5 h of resting time, respectively. For compound 3, however, the enhancement factors were inde- pendent of sample resting time and spectra were recorded 5 min after rotor packing. Note that the enhancements for the

EtCl4 resonance (eS > 20 in all cases) is always larger than that for the MOF materials, suggesting that the biradical mole- cules are partly excluded from the pores for all three types of the materials (see below).[19] Under these optimal experimental conditions, for MOF samples 1 and 2, e values of 13 and 16 were observed, respectively. Significantly lower values of S were measured, corresponding to the fact that the biradicals inside of the material cause a reduction in the signal intensity through various paramagnetic effects.[17] On the other hand, for 3, similar values of e and S are observed (4.8 and 5.8, respectively). This strongly suggests that the bTbK molecules are excluded from the pores of 3, leading to a low value for e, and a relatively high value for S. This suggests that the whole material is contributing to the NMR signal under DNP Scheme 1. A) The crystal structure of MOF (In)-MIL-68 illustrating the conditions. This is in agreement with the fact that for 3, e does three-dimensional structures of the MOFs that are formed with indium not increase with additional sample resting times, and that a Wednesday, 10 April 13 octahedra and terephthalate ligands as bridging linkers.[4] B–D) Sche- matic structures of the three N-functionalized MOFs which are much larger value of e is observed for the solvent resonances (e = 26). It is likely that for 3, the proline groups block the isostructural to MIL-68. B) (In)-MIL-68-NH2 (1), C) the partially N- S functionalized (In)-MIL-68 material obtained with a terephthalate:2- one-dimensional pores of the material and hinder the aminoterephthalate ratio of 80:20 (2), and D) the 10% proline- relatively large bTbK radical from diffusing into the material. functionalized derivative of (1), (In)-MIL-68-NH-Pro (3). All MOFs TEM images of the as prepared 3 reveal that the average display one-dimensional rod-shaped structures, composed by hexahe- crystal size is less than 300 nm (Figure S4). Griffin and co- dral and triangular channels with an aperture of 1.6 and 0.6 nm, workers have previously applied DNP to needle-like nano- respectively. E) Schematic structure of the biradical polarizing agent bTbK.[5] The approximate size of the molecule is 1.5 nm ”0.6 nm. crystalline peptides which possessed an average crystal width of 150 nm.[19] In their system it was not possible for the radical to enter the lattice of the crystal and larger e values were here could not be impregnated with an aqueous solution (due obtained for the solvent resonances than the resonances of to a chemical reaction with water), solutions of the biradical nuclei inside the crystals, as observed here. In a similar way, [5] bTbK (Scheme 1) and 1,1,2,2-tetrachloroethane (EtCl4) we postulate that for 3, the bTbK molecules reside on or near were utilized. Incipient wetness impregnation was used to the surface of the crystallites, and that the enhanced polar- impregnate the dry materials with a minimal amount of ization is distributed into the crystal by spin diffusion. radical containing solution. All details about sample prepa- For all three samples, we estimate that the overall ration and optimization of the experimental conditions are sensitivity gain with respect to a dry sample at room given in the Supporting Information. In particular the temperature[17] (S+ 3.0S) is between 10 and 30, correspond-  influence of radical concentration, spinning frequency (nrot), ing to a reduction in experimental time by a factor between and the amount of time the impregnated samples were 100 and 900. This allows high quality one-dimensional 13C allowed to rest before solid-state NMR experiments were CPMAS spectra of 1–3 to be acquired in less than 5 min at attempted, were optimized on 1. natural isotopic abundance. For comparison, a 1H-13C It was found that 16 mm bTbK solutions provided both the CPMAS spectrum of 1 at room temperature with a similar highest e and S (Supporting Information, Figure S1). signal-to-noise ratio has been recorded in 2.3 h using standard Although e was observed to be higher with a sample spinning instrumentation (Figure S5). However, it should be noted that 13 frequency nrot of 8 kHz, the absolute signal of the spectra was the full width at half height (L) of the C resonances of all

similar with a nrot of 12 kHz (Figure S2). In order to reduce DNP solid-state NMR spectra of 1 are around 2.8 ppm, while

128 www.angewandte.de  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. 2012, 124, 127 –131 Angewandte. Communications

DOI: 10.1002/anie.201206102 NMR Spectroscopy Rapid Natural-Abundance 2D 13C–13C Correlation Spectroscopy Using Dynamic Nuclear Polarization Enhanced Solid-State NMR and Matrix- Free Sample Preparation** Hiroki Takahashi, Daniel Lee, Lionel Dubois, Michel Bardet, Sabine Hediger, and Gal De Pape*

Solid-state nuclear magnetic resonance (SSNMR) has been The relevance of performing DNP experiments has so far extensively used to characterize molecular structures at mainly been judged by comparing the signal-to-noise ratio (S/ atomic scales.[1] Concerning biomolecular applications, struc- N) with and without microwave (MW) irradiation. This “DNP 13 15 tural studies are commonly performed on C and/or N enhancement” (eDNP) has shown factors of up to 200 at 9.4 T enriched samples to compensate for their low natural and 100 K,[5,13] but in most applications, a factor of 10 to 20 is abundance (1.11% for 13C and 0.37% for 15N). However, obtained. However, we demonstrate here that the effective this strategy is mainly restricted to biomolecules that can be sensitivity gain in DNP experiments cannot be simply easily isotopically enriched and has proven difficult to expand evaluated by measuring eDNP. Instead, we propose using the to other types of systems. absolute sensitivity ratio (ASR) to evaluate the relevance of To date, only a few examples of natural abundance (NA) DNP by comparing the S/N per unit time obtained under 2D 13C–13C correlation experiments in solids have been optimized DNP conditions with the one obtained under reported using pulse sequences that rely on through-bond standard NMR conditions (potentially using for example polarization transfer.[2] This type of experiment provides one- larger sample volumes and higher magnetic fields). Previ- bond connections and is limited to small crystalline molecules, ously, Rossini et al. and Vitzthum et al. introduced an overall as it requires 2 to 10 days of experimental time. Owing to the sensitivity factor[14] and a global DNP factor,[15] respectively, low abundance of 13C nuclei, cross-peak intensities are about which take into account some of the important parameters. Angewandte Chemie four orders of magnitude smaller for experiments performed These factors are reduced forms of the ASR and are discussed on NA systems compared to their labeled equivalents. in more detail in the Supporting Information, S4. Recently, dynamic nuclear polarization (DNP) performed One of the main differences betweencorrelations conventional are observed and with good resolution and sensitivity with a high-power high-frequency microwave source (gyro- DNP-enhanced SSNMR experimentsusing is the the effective POST-C7 sample[18] recoupling sequence with a total mixing tron), a low-temperature (LT) magic-angle spinning (MAS) volume. For DNP experiments, samplestime of of interest 1 ms. In are this usually case, the 20 min experimental time is not probe, and a suitable polarizing agent has emerged as an dissolved[3, 5] or suspended[7,8] in glassylimited chemical by sensitivity matrices but to rather by the number of repetitions appropriate answer to the sensitivity limitation of SSNMR achieve uniform radical distribution,required which to complete significantly the phase cycling for DQ selection, the even at high magnetic fields.[3–5] This work by Griffin and co- reduces the effective amount of samplenumber (for of example, points necessary 45 mL in the indirect dimension for workers has triggered a strong interest in the science of 0.1m glycine solution contains 0.34sufficient mg of glycine). resolution, Impreg- and the probe duty cycle. An experiment 13 community and high-field MAS-DNP has been used on nation of DNP matrices into porouson materials a fully reducesC-labeled this system would require the same many different types of systems ranging from biological sample volume reduction problem.[9]experimentalAnother approach duration. was systems[6–8] to materials.[9–11] Herein we will show that the reported using a solvent-free methodBy that increasing utilizes a the spin- recoupling mixing time to 3.5 ms, we sensitivity enhancement obtained with DNP can be signifi- labeling technique where the biradicalwere able TOTAPOL to obtain[16] a spectrumis in only 2 h (see Figure 2b) cant enough to obtain 2D 13C-13C NMR correlation spectra on covalently attached to a peptide. Enhancementcontaining all factors two-bond of up correlations in NA cellulose (C1–C3, inter-unit C1–C4, C1–C5, C2–C4, C3–C5, and C4–C6). As NA microcrystalline solids in 20 min,[12] that is, within an to four were measured on a decapeptide.[15] The use of dipolar truncation is negligible for 13C–13C polarization trans- experimental time comparable to experiments routinely solvents also causes spectral line-broadening owing to the fer in NA systems, long-range correlations are easier to performed on isotopically labeled systems. conformational distribution in frozen solutions.[17] measure than on fully 13C-labeled systems. Furthermore, in To optimize the sample volume and spectral resolution for 13 samples at 1% natural abundance, no relayed mechanism is Figure 1. C-CPMAS spectra ofMAS-DNP NA microcrystalline experiments, cellulose (a–c) we developed and a new sample prep- [*] Dr. H. Takahashi, Dr. D. Lee, Dr. L. Dubois,[2-13 Dr.C]glycine M. Bardet, (d–f) recorded on a dried powder for conventional NMR involved in two-bond polarization transfers. This will enable Dr. S. Hediger, Dr. G. De Pape experiments at RT (a, d), a MFaration sample (b, procedure c), or a frozen that solution is based (e, onthe aperformance matrix-free of (MF) long-range distance measurements with Laboratoire de Chimie Inorganique et Biologique,f) for DNP UMR-E3 experiments CEA/UJF at 105approach K with MW off where (b, e) the and polarizing MW on (c, f). agenthigh is uniformly accuracy. distributedA DNP-enhanced 2D SQ-SQ 13C homonuclear & CNRS, Institut Nanosciences et CryogØThenie MAS (CEA/DSM) frequency was 8 kHzaround for all experiments. the microcrystals. A DNP enhance- This methoddipolar maximizes correlation the effec- experiment using DARR[19] recoupling ment factor e of 20 was observed with a zirconium rotor, although 38054 Grenoble (France) DNP tive quantity of material observed andwas preserves also performed its potential (Supporting Information, S3). E-mail: [email protected] it is 30% less efficient comparedcrystallinity, to a sapphire which rotor according then leads to DNP to narrower spectral lines at LT. experiments on 13C urea (data not shown). Finally, we would like to discuss the essential concept of [**] This work was supported by the ANR (ANR08-CEXC-003-01 and This approach is demonstrated on asensitivity sample of enhancement microcrystal- in the context of DNP-enhanced ANR-08-BLAN-0306-02) and funding from the RTB. G.D.P. was line cellulose using the TOTAPOL polarizingNMR experiments. agent. To evaluate the true sensitivity gain of supported by EU Marie Curie (PIEF-GA-2009-237646) for part of the 13 work. D.L. was supported by CEA-EUROTALENTSspectra with (PCOFUND-GA- MW on and MW off,In a Figure similar 1, DNP we compare enhancement 1D Cperforming CPMAS spectra DNP experiments, of NA we have introduced above the 13 2008-228664). Dr. M. Giffard is acknowledgedof 20 for is the obtained synthesis on of bothmicrocrystalline cellulose and glycine. cellulose However, (a–c) andexperimental [2- C]glycine quality (d–f). factor, For dubbed ASR. All the different the TOTAPOL radical. when the DNP experimentDNP is compared experiments, to the the conventional cellulose samplecontributions was prepared to with the the effective enhancement have to be Supporting information for this article isNMR available experiment on the WWW at roomMF temperature approach, (RT) while on the powders, standard the frozen-solutionconsidered to preparation rationalize the measured ASR. We separate under http://dx.doi.org/10.1002/anie.201206102.ASR on glycine (0.021)method is more was than used three for orders the glycine of them sample. into Comparing eight factors the (Supporting Information, S4): 13 13 magnitude smaller than on cellulose (47). Notably, the S/N 1) eDNP : the standard DNP enhancementNatural factor abundance (MW on/off); C- C of the cellulose C2 site (theAngewandte biggest. peak) using DNP reaches 2) e : the gain obtained at LT, whose origin is twofold: the 11766  2012 Wiley-VCH Verlag GmbH & Co. KGaA, WeinheimCommunications Angew. Chem. Int.T Ed. 2012, 51, 11766 –11769 correlation spectra 747 for a recycle delay of 13 s (5 times T1) and four scans, increaseDOI: of 10.1002/anie.201206102 magnetization owing to the Boltzmann dis- NMR Spectroscopy whereas it was 15 for the same number of scans with tribution and the reductionsee also: of thermalRossini, A. J.; noiseZagdoun, atA.; Hegner, LT; F.; Schwarzwälder, M.; Gajan, D.; 13 13 Copéret, C.; Lesage, A.; Emsley, L. J. Am. Chem. Soc. 2012, 134, 16899–908. conventional NMR at RT (recycleRapid delay Natural-Abundance of 11.5 s). 2D C– C Correlation3) h : takes Spectroscopy into account Using the different repetition times that Dynamic Nuclear Polarization Enhanced Solid-StateT1 NMR and Matrix- The linewidths of the celluloseFree Sample sample Preparation** remain almost can be used in the two experiments. It depends on the unchanged because it keeps itsHiroki crystalline Takahashi, Daniel structure Lee, Lionel with Dubois, the Michel Bardet,longitudinal Sabine Hediger, and relaxation time constant T1 for the RT NMR MF approach. On the other hand,Gal De the Pa glycinepe* peak is strongly broadened atSolid-state low nuclear temper- magnetic resonance (SSNMR) has been The relevance of performing DNP experiments has so far extensively used to characterize molecular structures at mainly been judged by comparing the signal-to-noise ratio (S/ ature, as the dissolved moleculeatomic is scales. frozen[1] Concerning in biomolecular applications, struc- N) with and without microwave (MW) irradiation. This “DNP 13 15 tural studies are commonly performed on C and/or N enhancement” (eDNP) has shown factors of up to 200 at 9.4 T many different conformations.enriched Furthermore, samples to compensate for their low natural and 100 K,[5,13] but in most applications, a factor of 10 to 20 is abundance (1.11% for 13C and 0.37% for 15N). However, obtained. However, we demonstrate here that the effective the strong solvent peaks presentthis strategy is mainly in restricted the to biomolecules that can be sensitivity gain in DNP experiments cannot be simply easily isotopically enriched and has proven difficult to expand evaluated by measuring eDNP. Instead, we propose using the to other types of systems. absolute sensitivity ratio (ASR) to evaluate the relevance of frozen-solution spectrum of glycineTo date, only a few (Fig- examples of natural abundance (NA) DNP by comparing the S/N per unit time obtained under 2D 13C–13C correlation experiments in solids have been optimized DNP conditions with the one obtained under ure 1 f) do not appear in thereported case using pulse of sequences MF that rely on through-bond standard NMR conditions (potentially using for example polarization transfer.[2] This type of experiment provides one- larger sample volumes and higher magnetic fields). Previ- cellulose (Figure 1c). This is becausebond connections the and is MF limited to small crystalline molecules, ously, Rossini et al. and Vitzthum et al. introduced an overall as it requires 2 to 10 days of experimental time. Owing to the sensitivity factor[14] and a global DNP factor,[15] respectively, approach does not require thelow use abundance of of 13 aC nuclei, cry- cross-peak intensities are about which take into account some of the important parameters. four orders of magnitude smaller for experiments performed These factors are reduced forms of the ASR and are discussed oprotectant (such as glycerol,on NA DMSO) systems compared to their labeled equivalents. in more detail in the Supporting Information, S4. Recently, dynamic nuclear polarization (DNP) performed One of the main differences between conventional and form glassy matrices at LTwith and a high-power uniformly high-frequency microwave source (gyro- DNP-enhanced SSNMR experiments is the effective sample tron), a low-temperature (LT) magic-angle spinning (MAS) volume. For DNP experiments, samples of interest are usually distribute the polarizing agent.probe, This and a suitable is very polarizing agent has emerged as an dissolved[3, 5] or suspended[7,8] in glassy chemical matrices to appropriate answer to the sensitivity limitation of SSNMR achieve uniform radical distribution, which significantly important for NA experimentseven becauseat high magnetic fields. sig-[3–5] This work by Griffin and co- reduces the effective amount of sample (for example, 45 mL workers has triggered a strong interest in the science of 0.1m glycine solution contains 0.34 mg of glycine). Impreg- nals of interest are often buriedcommunity under and high-field large MAS-DNP has been used on nation of DNP matrices into porous materials reduces this many different types of systems ranging from biological sample volume reduction problem.[9] Another approach was solvent peaks. systems[6–8] to materials.[9–11] Herein we will show that the reported using a solvent-free method that utilizes a spin- sensitivity enhancement obtained with DNP can be signifi- labeling technique where the biradical TOTAPOL[16] is The substantial effectivecant enhancement enough to obtain 2D 13C-13C NMR correlation spectra on covalently attached to a peptide. Enhancement factors of up NA microcrystalline solids in 20 min,[12] that is, within an to four were measured on a decapeptide.[15] The use of experimental time comparable to experiments routinely solvents also causes spectral line-broadening owing to the obtained on the MF NA cellulose allowed [17] performed on isotopically labeled systems.Figure 2. DNP-enhancedconformational distribution 2D DQ–SQ in frozen solutions.13C–13C POST-C7 dipolar correlation spectra of MF us to acquire a DNP-enhanced 2D double- To optimize the sample volume and spectral resolution for MAS-DNP experiments, we developed a new sample prep- [*] Dr. H. Takahashi, Dr. D.13 Lee, Dr. L. Dubois,NA Dr. M. microcrystalline Bardet, cellulose recorded at 105 K. MAS frequency and the number of scans quantum–single-quantum (DQ–SQ)Dr. S. Hediger, Dr. G. De PaCpe aration procedure that is based on a matrix-free (MF) Laboratoire de Chimie Inorganique et Biologique,are 8 UMR-E3 kHz CEA/UJF andapproach 16, respectively. where the polarizing a) agent The is uniformly spectrum distributed is obtained in 20 min with a recycle delay homonuclear dipolar correlation& experiment.CNRS, Institut Nanosciences et CryogØnie (CEA/DSM) around the microcrystals. This method maximizes the effec- 38054 Grenoble (France) of 0.9 s, 1.4 mstive evolution quantity of material time, observed and and 1 preserves ms POST-C7 its potential mixing. b) The experimental time is E-mail: [email protected] crystallinity, which then leads to narrower spectral lines at LT. The 2D spectrum, which was obtained[**] This work was supportedin only by the ANR (ANR08-CEXC-003-012 h with a and recycleThis approach delay is of demonstrated 3.4 s, 2.3 on a sample ms evolutionof microcrystal- time, and 3.5 ms of POST-C7 mixing. ANR-08-BLAN-0306-02) and funding from the RTB. G.D.P. was line cellulose using the TOTAPOL polarizing agent. supported by EU Marie Curie (PIEF-GA-2009-237646) for part of the 20 min, is shown in Figure 2a. All one-bond Projections are shown on the top.13 work. D.L. was supported by CEA-EUROTALENTS (PCOFUND-GA- In Figure 1, we compare 1D C CPMAS spectra of NA 13 2008-228664). Dr. M. Giffard is acknowledged for the synthesis of microcrystalline cellulose (a–c) and [2- C]glycine (d–f). For the TOTAPOL radical. DNP experiments, the cellulose sample was prepared with the Supporting information for this article is available on the WWW MF approach, while the standard frozen-solution preparation Angew. Chem. Int. Ed. 2012, 51, 11766 –11769under http://dx.doi.org/10.1002/anie.201206102. 2012 Wiley-VCHmethod Verlag was usedGmbH for the & glycine Co. KGaA, sample. Comparing Weinheim the www.angewandte.org 11767 Wednesday, 10 April 13 11766  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2012, 51, 11766 –11769 Functional and shunt states of bacteriorhodopsin resolved by 250 GHz dynamic nuclear polarization–enhanced solid-state NMR Vikram S. Bajaja,1, Melody L. Mak-Jurkauskasa,b, Marina Belenkyb, Judith Herzfeldb, and Robert G. Griffina,2

aDepartment of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139; and bDepartment of Chemistry, Brandeis University, Waltham, MA 02454

Edited by Brian M. Hoffman, Northwestern University, Evanston, IL, and approved April 13, 2009 (received for review January 27, 2009) Observation and structural studies of reaction intermediates of In bacteriorhodopsin (bR), 7 transmembrane helices surround proteins are challenging because of the mixtures of states usually atransportchannelinwhichtheSchiffbase(SB)formed present at low concentrations. Here, we use a 250 GHz gyrotron between retinal and lysine 216 (Fig. 1) provides the binding site C15 C12 C14 (cyclotron resonance maser) and cryogenic temperatures to per- for a labile proton.A The counterionC13 of the protonated SB K216-Cε C20 form high-frequency dynamic nuclear polarization (DNP) NMR comprises a hydrogen-bondedbR568 complex (9, 10) of polar side experiments that enhance sensitivity in magic-angle spinning NMR chains and several waterbR555 molecules (11). spectra of cryo-trapped photocycle intermediates of bacteriorho- Absorption of a visiblebR555b photon by the retinylidene chro- K216-Cε C20 C15 C11 C12 C14 dopsin (bR) by a factor of Ϸ90. Multidimensional spectroscopy of mophore initiates the photocycleC15 U-13C,15N-labeled samples resolved coexisting states and allowed ϩ chemical shift assignments in the retinylidene chromophore for 150 h␯ Ϫ H bR555 e bR568 O¡ J6253K5903 L550 O¡ Mo several intermediates not observed previously. The correlation 160 B C11 412 spectra reveal unexpected heterogeneity in dark-adapted bR, dis- bR568 170 ε tortion in the K state, and, most importantly, 4 discrete L substates. C14 K216-C C20 ϩ Hϩ C13 C15 C12 Thermal relaxation of the mixture of L’s showed that 3 of these 180 3 Mn O¡ N5203O6403bR568 [1] substates revert to bR568 and that only the 1 substate with both the 412190 strongest counterion and a fully relaxed 13-cis bond is functional. These definitive observations of functional and shunt states in the 150 C12 C14 K216-Cε in which the vectoriality ofK the pump is effected by a switch in bR photocycle provide a preview of the mechanistic insights that connectivity of the160 SBC from the extracellularC15 side to the cyto- bR will be accessible in membrane proteins via sensitivity-enhanced plasmic side between170 the early568 and late M states, here designated DNP NMR. These observations would have not been possible C13 C14 K216-Cε Mo and Mn.BecauseisomerizationofthechromophoreoccursC15 C12 C20 absent the signal enhancement available from DNP. 180 at the beginning of the photocycle (bR568 to J) but the chro- mophore connectivity190 does not change until the middle of the magic-angle spinning ͉ photocycle intermediate ͉ retinal protein ͉ photocycle, the details of the structural rearrangements occur- N Chemical Shift (ppm) 150 ion transport ͉ DNP C12 ε ring in the early15 photocycleD C13 intermediates (namely K and L) are C14 K216-C C20 fundamental to the160 control of vectoriality.C15 bR568 ultidimensional magic-angle spinning (MAS) solid-state To apply MAS NMR to bR photocycle intermediates, a C14 C20 170 L174 C15 C12 C14 K216-Cε C20 NMR is a general tool in structural studies of membrane sample of bR568 is irradiated in situ with laser light, andC12 the M 180 L181 K216-Cε proteins that are inaccessible to crystallography and solution- resulting mixture of states isC15 cryostabilized for data acquisition. L state NMR, as demonstrated by recent successful applications The population190 of a desired186 intermediateC15 can be favored byC12 C14 K216-Cε C20 (1–3). But the sensitivity of these experiments is low, which manipulating the temperature and wavelength during irradia- becomes a significant problem when multidimensional experi- tion; however, the290 resultE is generally a mixture of bR568 together ments are needed to characterize systems of higher molecular with various intermediates. In such cases, a distinct advantage of NMR over diffraction300 is that signals from multiple species weight. The sensitivity deficit is even more acute when NMR C20 C11 C12 K216-Cε signals are further divided among multiple states, as is often the usually can be distinguished,310 because the chemical shifts depend C14 M case for trapped reaction intermediates. Consequently, a 1–2 on the local conformation320 O and interactions. This is particularly order of magnitude enhancement of NMR sensitivity is essential so when there is sufficient signalC15 to acquire multidimensional spectra. 330 Functional and shunt states of bacteriorhodopsinfor applications of multidimensional MAS NMR methods to 175 170 165 160 155 140 135 130 125 120 115 110 105 60 55 50 25 20 15 10 studies of reaction intermediates of membrane proteins. Here, we report DNP-enhanced 2D MAS NMR spectra of bR 13 15 13 resolved by 250 GHz dynamic nuclearOne approach to improving the sensitivity of NMR is dynamic intermediates acquired from U- C, N-labeled samples.C Chemical The Shift (ppm) nuclear polarization (DNP), in which the Ϸ660-fold greater spin resolution afforded by chemical shift dispersion in the 2D MAS polarization of unpaired electrons in a paramagneticallyFig. 3. 15 dopedN␨-13C15-spectra13Cx correlation of the cryogenically spectra that trapped trace photocycle the connectivity intermediates of resonances in the retinylidene chromophore of bR in the dark-adapted state (A), the polarization–enhanced solid-stateglassy NMR matrix is transferred to nuclei before an NMR experiment allows us to identify cross-peaks belonging to unreacted bR568, light-adapted stateas (B well), the as K several state with other residual components. bR568 ( ThereC), the was L states evidence with of residual bR568 (D), and the Mo state with its deprotonated SB (E). Conditions are as (4). Here, we demonstrate that high-frequencyspecified DNP by using for Fig. a 2. Vikram S. Bajaja,1, Melody L. Mak-Jurkauskasa,b, Marina Belenkyb, Judithstable, Herzfeld high-powerb, and Robert 250 GHz G. Griffin microwavea,2 source (5) and an

efficient, nonperturbing biradical polarizing agent (6, 7), is a Author contributions: J.H. and R.G.G. designed research; V.S.B., M.L.M.-J., and M.B. per- aDepartment of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139; potentially general approach for biological MAS NMR. A formed research; V.S.B., M.L.M.-J., J.H., and R.G.G. analyzed data; and V.S.B., M.L.M.-J., J.H., and bDepartment of Chemistry, Brandeis University, Waltham, MA 02454 43-fold signal enhancement from DNP, combinedtaneous with opera- presenceand R.G.G. of multiple wrote the paper. photocycle intermediates, minor all photocycle intermediates of bR, because of the large signal TheMagic authors declare Angle no conflict Spinning of interest. DNP on Edited by Brian M. Hoffman, Northwestern University, Evanston, IL, and approved AprilFunctional 13, 2009tion (received at 90 and K, for yields shunt review an January overall states 27, 90-fold of 2009) bacteriorhodopsin signal enhancementcomponents over of the mixture are further diluted by a factor of enhancement provided by low-temperature DNP. Thus, we resolvedprevious by experiments 250 GHz at dynamic 183 K (8). nuclear The resulting Ϸ8,100-fold This articlea is a membrane PNAS Direct Submission. protein Observation and structural studies of reaction intermediates of In bacteriorhodopsinsavings in acquisition (bR), 7 timetransmembrane permits 2-dimensional helices2–20. surround (2D) Nevertheless, resolu- 1Present address: 2D spectra Department of can Chemistry, be recorded University of California, in 12–48 Berkeley, CA h 94720. for detect single cross-peaks that correspond to single sites at an proteins are challenging because of the mixtures of states usually atransportchannelinwhichtheSchiffbase(SB)formedpolarization–enhancedtion of signals from mixtures solid-state of reaction NMR intermediates that 2To whom correspondence should be addressed. E-mail: [email protected]. effective molecular weight of Ϸ660 kDa (see Discussion). Vikram S. Bajaja,1, Melody L. Mak-Jurkauskasa,b, Marina Belenkyb, Judith Herzfeldb, and Robert G. Griffina,2 15 present at low concentrations. Here, we use a 250 GHz gyrotron between retinalwould and be impossible lysine 216 to (Fig. observe 1) provides absent the the enhancement binding site avail- This article contains supporting information online at www.pnas.org/cgi/content/full/ As shown in Figs. 2, 3, and 4, the NSBchemicalshiftis aDepartment of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139; b able from DNP. 0900908106/DCSupplemental. (cyclotron resonance maser) and cryogenic temperatures to per- for aand labileDepartment of proton. Chemistry, Brandeis The University, Waltham, counterion MA 02454 of the protonated SB exquisitely sensitive to its environment (19) and permits reso- form high-frequency dynamic nuclear polarization (DNP) NMR comprisesEdited by Brian a M. hydrogen-bonded Hoffman, Northwestern University, Evanston, complex IL, and approved April (9, 13, 2009 10) (received of for review polar January 27, side 2009) 150 13 15 Observation and structural studies of reaction intermediates of In bacteriorhodopsin (bR), 7 transmembrane helices surround C ε C δ C γ lution of C- Ncross-peaksarisingfromthevariousinterme- experiments that enhance sensitivity in magic-angle spinning NMR chainsproteins and are9244–9249 several challenging because water͉ PNAS of the mixtures molecules͉ June of states 9, usually2009 (11).͉atransportchannelinwhichtheSchiffbase(SB)formedvol. 106 ͉ no. 23160 bR 568 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900908106 present at low concentrations. Here, we use a 250 GHz gyrotron between retinal and lysine 216 (Fig. 1) provides the binding site diates. Additional homonuclear mixing, via either C15 or C␧, spectra of cryo-trapped photocycle intermediates of bacteriorho- Absorption(cyclotron resonance of maser) a and visible cryogenic temperatures photon to per- byfor the a labile retinylidene proton. The counterion chro- of the protonated SB 170 formFunctional high-frequency dynamic nuclear polarization and (DNP) NMR shuntcomprises a hydrogen-bonded states complex of (9, 10) of bacteriorhodopsin polar side bR dopsin (bR) by a factor of Ϸ90. Multidimensional spectroscopy of mophoreexperiments initiates that enhance sensitivitythe photocycle in magic-angle spinning NMR chains and several water molecules (11). 555 ε γ allows the observation of further heteronuclear correlations. Absorption of a visible photon by the retinylidene chro- 180 C δ C 13 15 spectra of cryo-trapped photocycle intermediates of bacteriorho- C U- C, N-labeled samples resolved coexisting states and allowed dopsin (bR) by a factor of Ϸ90. Multidimensional spectroscopy of mophore initiates the photocycle Here, we obtain 9 or 10 chemical shift assignments for each resolved13 15 by 250 GHz dynamicϩ nuclear U- C, N-labeled samples resolved coexisting␯ states and allowed Ϫ 190 h H ϩ chemical shift assignments in the retinylidene chromophore for chemical shift assignments in the retinylidene chromophore for h␯ Ϫ H A 13 bR e bR O J 3K 3 L O M intermediate (as tabulated in Tables S1 and S2). The C several intermediates not observed previously. The correlation 555 568 ¡ 625 590 550 ¡ o 412

bR e bR O¡ J 3K 3 L O¡ M several intermediates not observed previously. The correlation spectra reveal555 unexpected heterogeneity568 in dark-adapted625 bR, dis-590 550 o 412 polarization–enhanced solid-state NMR) C δ Cγ tortion in the K state, and, most importantly, 4 discrete L substates. 150 Cε assignments are based on the relative intensities of the cross- ϩ Hϩ K spectra reveal unexpected heterogeneity in dark-adapted bR, dis- Thermal relaxation of the mixture of L’s showed that 3 of these m a,1 3a,bMn O¡ N5203O6403bR568b [1] b a,2 substatesVikram revert S. to bR Bajaj568 and that, only Melody the 1 substate L. withMak-Jurkauskas both the ,412 Marina Belenky , Judith Herzfeld160, and Robert G. Griffin p peaks and the expected chemical shift range for each site. Note tortion in the K state, and, most importantly, 4 discrete L substates. strongest counterion and a fully relaxed 13-cis bond is functional.

ϩ p bR These definitive observationsϩ of functional and shunt states in the 568 aDepartment of ChemistryH and Francis Bitter Magnetin which Laboratory, the vectoriality Massachusetts of the pump is Institute effected by of aTechnology, switch in ( 170 Cambridge, MA 02139; that the cross-peak intensities are reduced when the polarization Thermal relaxation of the mixture of L’s showed that 3 of these bR photocycle provide a preview of the mechanistic insights that and bDepartment of Chemistry, Brandeis University,connectivity Waltham, of theMA SB 02454 from the extracellular side to the cyto- t δ C γ will be3 accessibleM in membraneO proteinsN via sensitivity-enhanced3O 3bR [1] f α β C 13 n ¡ 520 640 plasmic568 side between the early and late M states, here designated i C ε C DNP NMR. These observations412 would have not been possible 180 C of the SB nitrogen is dispersed to more C’s (compare Figs. 3 substates revert to bR568 and that only the 1 substate with both the Mo and Mn.Becauseisomerizationofthechromophoreoccurs absent the signal enhancement available from DNP. h Edited by Brian M. Hoffman, Northwestern University,at the Evanston, beginning of IL, the and photocycle approved (bR568 Aprilto J) 13, but 2009 the chro- (received for review January 27, 2009) strongest counterion and a fully relaxed 13-cis bond is functional. mophore connectivity does not change until the middle of the S 190 and 4 vs. Fig. 2). magic-angle spinning ͉ photocycle intermediate ͉ retinal protein ͉

l B Observation and structural studies of reactionphotocycle, intermediates the details of the ofstructural rearrangementsIn bacteriorhodopsin occur- (bR), 7 transmembrane helices surround These definitive observations of functional and shunt states in the ion transport ͉ DNP ring in the early photocycle intermediates (namely K and L) are a in whichproteins the vectoriality are challenging of because the pumpof the mixtures isfundamental effected of states to the bycontrol usually a of switch vectoriality.atransportchannelinwhichtheSchiffbase(SB)formed in c 150 bR photocycle provide a preview of the mechanistic insights that i connectivitypresentultidimensional ofat low the magic-angle concentrations. SB spinningfrom (MAS) the Here, solid-state extracellular we useTo a 250apply GHz MAS side NMR gyrotron to to the bR photocycle cyto-between intermediates, retinal a and lysine 216 (Fig. 1)C ε provides the bindingCβ C δ siteC γ Discussion MNMR is a general tool in structural studies of membrane sample of bR568 is irradiated in situ with laser light, and the bR C m 568 will be accessible in membrane proteins via sensitivity-enhanced proteins(cyclotron that are inaccessibleresonance to crystallography maser) and and cryogenic solution- resulting temperatures mixture of states to is per- cryostabilizedfor for a data labile acquisition. proton.160 The counterion of the protonated SB plasmicstate side NMR, as between demonstrated by the recent early successful and applications late MThe states, population hereof a desired designated intermediate can be favored by e Multiplicity in Photocycle Intermediates. Examining the spectra in form high-frequency dynamic nuclear polarization (DNP) NMR comprises a hydrogen-bondedL complexC (9, ε 10) of polar side DNP NMR. These observations would have not been possible (1–3). But the sensitivity of these experiments is low, which manipulating the temperature and wavelength during irradia- h 170 174 Mo and Mn.Becauseisomerizationofthechromophoreoccurs 15 13 becomesexperiments a significant that problem enhance when multidimensional sensitivity experi- in magic-angletion; however, spinning the result is NMR generally a mixturechains of bR and568 together several water moleculesC ε (11). ε C δ

C δ with various intermediates. In such cases, a distinct advantage of L C δ C detail, we find single N- Ccross-peaksforeachcarbonatom absent the signal enhancement available from DNP. ments are needed to characterize systems of higher molecular 180 181 C at theweight.spectra beginning The sensitivity of cryo-trapped deficitof the is even photocycle photocycle more acute when intermediates NMR (bRNMR568 overto of diffraction bacteriorho-J) but is that the signals chro- fromAbsorption multiple species of a visible photon by the retinylidene chro- signals are further divided among multiple states, as is often the usually can be distinguished, because the chemical shifts depend N L 186 in the light-adapted (Fig. 3B), K (Fig. 3C), and Mo (Fig. 3E)

mophore initiates5 the photocycle mophoredopsin connectivity (bR) by a factor does of Ϸ90. not Multidimensional changeon until the local spectroscopy the conformation middle and of interactions. of the This is particularly case for trapped reaction intermediates. Consequently, a 1–2 1 190 β γ 13 15 C ε C C δ C magic-angle spinning ͉ photocycle intermediate ͉ retinal protein ͉ orderU- ofC, magnitudeN-labeled enhancement samples of NMR resolved sensitivity is essential coexistingso when states there and is sufficient allowed signal to acquire multidimensional states. In contrast, the spectra of the dark-adapted (Figs. 2A and photocycle, the details of the structural rearrangements occur- ϩ forchemical applications shift of multidimensional assignments MAS in NMR the methods retinylidene to spectra. chromophore for h␯ Ϫ H ion transport ͉ DNP studies of reaction intermediates of membrane proteins. Here, we report DNP-enhanced 2D MAS NMR spectra of bR ring in the early photocycle intermediates (namely K and13 L)15 are bR e bR O¡ J 3K 3 L O¡ M 3A)andL(Figs.2C and 3D)statesshowheterogeneity.Dark severalOne approach intermediates to improving the sensitivity not observed of NMR is dynamic previously.intermediates The acquired correlation from U- C, N-labeled samples.555 The 290568 625 590 550 o 412 fundamentalnuclear polarization to (DNP), the in control which the Ϸ660-fold of vectoriality. greater spin resolution afforded by chemical shift dispersion in the 2D MAS D polarizationspectra of reveal unpaired unexpected electrons in a paramagnetically heterogeneity doped inspectra dark-adapted of the cryogenically bR, dis- trapped photocycle intermediates adaptation is generally considered to involve the equilibration glassytortion matrix isin transferred the K state, to nuclei and, before most an NMR importantly, experiment allows 4 discrete us to identify L substates. cross-peaks belonging to unreacted bR568, 300 ultidimensional magic-angle spinning (MAS) solid-state To apply MAS NMR to bR photocycleas well as several intermediates, other components. There a was evidence of ϩ (4). Here, we demonstrate that high-frequency DNP by using a ϩ H trans anti cis syn Thermal relaxation of the mixture of L’s showed that 3 of these between all- ,15- bR568 and 13- ,15- bR555,andin NMR is a general tool in structural studies of membrane samplestable, of high-power bR568 250is GHz irradiated microwave source in (5) andsitu an with laser light, and the 310 C ε 3 Mn 412 O¡ N5203O6403bR568 [1] 15 M efficient, nonperturbing biradical polarizing agent (6, 7), is a Author contributions: J.H. and R.G.G. designed research; V.S.B., M.L.M.-J., and M.B. per- substates revert to bR568 and that only the 1 substate with both the 1D Nspectraweobserve2linesinthe40:60intensityratio(19, proteins that are inaccessible to crystallography and solution- resultingpotentially mixture general approach of states for biological is cryostabilized MAS NMR. A formed research; for V.S.B., data M.L.M.-J., J.H., acquisition. and R.G.G. analyzed data; and V.S.B., M.L.M.-J., J.H., 320 M O 43-foldstrongest signal enhancement counterion from andDNP, acombined fully relaxed with opera- 13-andcis R.G.G.bond wrote the is paper. functional. 20) generally assigned to these species, with 15Nshiftsthatreflect state NMR, as demonstrated by recent successful applications The populationtionThese at 90 K, definitive yields an of overall observations a 90-fold desired signal enhancement of intermediate functional over andThe authors shunt declare can no states conflict be of interest.in favored the by previous experiments at 183 K (8). The resulting Ϸ8,100-fold This article is a PNAS Direct Submission. in which the vectoriality330 of the pump is effected by a switch in bR photocycle provide a preview of the mechanistic1 insights that (1–3). But the sensitivity of these experiments is low, which manipulatingsavings in acquisition the time temperature permits 2-dimensional (2D) and resolu- wavelengthPresent address: Department during of Chemistry, University irradia- ofconnectivity California, Berkeley, CA 94720. of the SB from60 the55 extracellular 50 45 40 side 35 to the 30 cyto- 25 20 2distinctenvironmentsfortheSB.However,thestrongersignals tionwill of signals be accessible from mixtures in of membrane reaction intermediates proteins that via2Tosensitivity-enhanced whom correspondence should be addressed. E-mail: [email protected]. 15 13 becomes a significant problem when multidimensional experi- tion; however,would be impossible the to observe result absent is the generally enhancement avail- a mixtureThis article contains of supporting bR information568 together online atplasmicwww.pnas.org/cgi/content/full/ side between the early and late M states, here designated shown in Fig. 2A clearly indicate multiple N- Ccross-peaks ableDNP from NMR. DNP. These observations would have0900908106/DCSupplemental not been. possible 13 Mo and Mn.BecauseisomerizationofthechromophoreoccursC Chemical Shift (ppm) 13 ments are needed to characterize systems of higher molecular with variousabsent the intermediates. signal enhancement In such available cases, from a DNP. distinct advantage of at the beginning of the photocycle (bR568 to J) but the chro- for each species in the Cdimension.Fortheless-shieldedSB weight. The sensitivity deficit is even more acute when NMR NMR9244–9249 over͉ PNAS diffraction͉ June 9, 2009 ͉ vol. 106 is͉ no. that 23 signals from multiple www.pnas.org species͞cgi͞doi͞10.1073͞pnas.0900908106 15 13 Fig. 4.mophore15N␨- connectivity13C␧ correlation does not experiments change until the in middle the dark-adapted of the state (A), the (bR555), the additional N␨- C15 splitting is particularly evident signals are further divided among multiple states, as is often the usuallymagic-angle can be distinguished, spinning ͉ photocycle because intermediate the͉ retinal chemical protein ͉ shifts dependphotocycle, the details of the structural rearrangements occur- Wednesday,on the 10ion localApril transport 13 conformation͉ DNP and interactions. This is particularlyK intermediatering in the early with photocycle residual intermediates bR568 (B), (namely the L intermediate K and L) are with residual bR568 and indicates a well defined chromophore isomerization with an case for trapped reaction intermediates. Consequently, a 1–2 fundamental to the control of vectoriality. so when there is sufficient signal to acquire multidimensional(C), and the Mo state (D). Conditions are as specified for Fig. 2. intensity ratio of Ϸ2:1 (5). We have not previously observed order of magnitude enhancement of NMR sensitivity is essential ultidimensional magic-angle spinning (MAS) solid-state To apply MAS NMR to bR photocycle intermediates, a spectra. for applications of multidimensional MAS NMR methods to MNMR is a general tool in structural studies of membrane sample of bR568 is irradiated in situ with laser light, and the studies of reaction intermediates of membrane proteins. Here,proteins we report that are DNP-enhanced inaccessible to crystallography 2D MAS NMR and solution- spectra ofresulting bR mixture of states is cryostabilized for data acquisition. state NMR, as demonstrated by recent13 15 successful applications9246 Thewww.pnas.org population of a desiredcgi doi intermediate10.1073 pnas.0900908106 can be favored by Bajaj et al. One approach to improving the sensitivity of NMR is dynamic intermediates acquired from U- C, N-labeled samples. The͉ ͞ ͞ ͞ ͞ resolution(1–3). afforded But the sensitivity by chemical of these shift experiments dispersion is low, in the which 2D MASmanipulating the temperature and wavelength during irradia- nuclear polarization (DNP), in which the Ϸ660-fold greater spin becomes a significant problem when multidimensional experi- tion; however, the result is generally a mixture of bR568 together polarization of unpaired electrons in a paramagnetically doped spectraments of the are cryogenicallyneeded to characterize trapped systems photocycle of higher molecular intermediateswith various intermediates. In such cases, a distinct advantage of NMR over diffraction is that signals from multiple species glassy matrix is transferred to nuclei before an NMR experiment allowsweight. us to identify The sensitivity cross-peaks deficit is even belonging more acute to unreacted when NMR bR568, as wellsignals as several are further other divided components. among multiple There states, as was is often evidence the usually of can be distinguished, because the chemical shifts depend (4). Here, we demonstrate that high-frequency DNP by using a case for trapped reaction intermediates. Consequently, a 1–2 on the local conformation and interactions. This is particularly stable, high-power 250 GHz microwave source (5) and an order of magnitude enhancement of NMR sensitivity is essential so when there is sufficient signal to acquire multidimensional for applications of multidimensional MAS NMR methods to spectra. efficient, nonperturbing biradical polarizing agent (6, 7), is a Author contributions: J.H. and R.G.G. designed research; V.S.B., M.L.M.-J., and M.B. per-Here, we report DNP-enhanced 2D MAS NMR spectra of bR studies of reaction intermediates of membrane proteins. 13 15 potentially general approach for biological MAS NMR. A formed research;One V.S.B., approach M.L.M.-J., to improving J.H., and theR.G.G. sensitivity analyzed of data; NMR and is V.S.B., dynamic M.L.M.-J.,intermediates J.H., acquired from U- C, N-labeled samples. The 43-fold signal enhancement from DNP, combined with opera- and R.G.G.nuclear wrote the polarization paper. (DNP), in which the Ϸ660-fold greater spin resolution afforded by chemical shift dispersion in the 2D MAS spectra of the cryogenically trapped photocycle intermediates tion at 90 K, yields an overall 90-fold signal enhancement over The authorspolarization declare no of conflict unpaired of interest. electrons in a paramagnetically doped glassy matrix is transferred to nuclei before an NMR experiment allows us to identify cross-peaks belonging to unreacted bR568, previous experiments at 183 K (8). The resulting Ϸ8,100-fold This article is a PNAS Direct Submission. as well as several other components. There was evidence of (4). Here, we demonstrate that high-frequency DNP by using a savings in acquisition time permits 2-dimensional (2D) resolu- 1Present address:stable, Department high-power of 250 Chemistry, GHz microwave University of source California, (5) Berkeley, and an CA 94720. tion of signals from mixtures of reaction intermediates that 2To whomefficient, correspondence nonperturbing should be biradical addressed. polarizing E-mail: [email protected]. agent (6, 7), is a Author contributions: J.H. and R.G.G. designed research; V.S.B., M.L.M.-J., and M.B. per- would be impossible to observe absent the enhancement avail- potentially general approach for biological MAS NMR. A formed research; V.S.B., M.L.M.-J., J.H., and R.G.G. analyzed data; and V.S.B., M.L.M.-J., J.H., This article43-fold contains signal supporting enhancement information from DNP,online combined at www.pnas.org/cgi/content/full/ with opera- and R.G.G. wrote the paper. 0900908106/DCSupplemental. able from DNP. tion at 90 K, yields an overall 90-fold signal enhancement over The authors declare no conflict of interest. previous experiments at 183 K (8). The resulting Ϸ8,100-fold This article is a PNAS Direct Submission. savings in acquisition time permits 2-dimensional (2D) resolu- 1Present address: Department of Chemistry, University of California, Berkeley, CA 94720. 9244–9249 PNAS June 9, 2009 vol. 106 no. 23 www.pnas.org cgi doi 10.1073 pnas.0900908106 ͉ ͉ ͉ ͉ tion of signals from mixtures of reaction͞ ͞ intermediates͞ ͞ that 2To whom correspondence should be addressed. E-mail: [email protected]. would be impossible to observe absent the enhancement avail- This article contains supporting information online at www.pnas.org/cgi/content/full/ able from DNP. 0900908106/DCSupplemental.

9244–9249 ͉ PNAS ͉ June 9, 2009 ͉ vol. 106 ͉ no. 23 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900908106 DNP on oriented Applied Appl Magn Reson (2012) 43:91–106 membrane proteins DOI 10.1007/s00723-012-0338-5 Magnetic Resonance

Salnikov, E. S.; Ouari, O.; Koers, E.; Sarrouj, H.; Franks, T.; Rosay, M. M.; Pawsey, S.; Reiter, C.; Bandara, P.; Oschkinat, H.; Tordo, P.; Engelke, F.; Bechinger, B. Applied Magnetic Resonance 2012, 43, 91–106. Developing DNP/Solid-State NMR Spectroscopy of Oriented Membranes

If you are interested in what is going Evgeniy S. Salnikov • Olivier Ouari • Eline Koers • Hiba Sarrouj • Trent Franks • Melanie Rosay • Shane Pawsey • Christian Reiter • on in this area check this article. Priyanga Bandara • Hartmut Oschkinat • Paul Tordo • Frank Engelke • Burkhard Bechinger

Developing DNP/Solid-State NMR Spectroscopyvesicles bilayers 97 Received: 11 February 2012 / Revised: 3 April 2012 / Published online: 4 May 2012 Ó Springer-Verlag 2012

Abstract Dynamic nuclear polarization (DNP)/solid-state nuclear magnetic reso- nance (NMR) spectroscopy bears great potential for the investigation of membrane- associated polypeptides which can often be produced only in small amounts and which need to be ‘diluted’ in lipid bilayer environments to adopt or maintain their functional structure. Here we present investigations using biradicals, such as TOTAPOL and bTbK, for solid-state NMR signal enhancement using DNP in the context of lipid membranes. By transferring polarization from electron to nuclear spins using

Special issue on DNP.

E. S. Salnikov H. Sarrouj B. Bechinger (&) Institut de Chimie,Á UMR7177,Á CNRS, Universite´ de Strasbourg, 4, rue Blaise Pascal, 67070 Strasbourg, France e-mail: [email protected] Oschkinat Burkhard Bechinger

O. Ouari P. Tordo Faculte´ desÁ Sciences, Institut de Chimie Radicalaire, UMR 7273, Aix-Marseille University, 13397 Marseille Cedex 20, France

E. Koers M. Rosay (&) S. Pawsey Bruker BioSpinÁ Corporation,Á 15 Fortune Drive, Billerica, MA 01821, USA e-mail: [email protected] Fig. 3 Proton-decoupled 31P solid-state NMR spectra of phospholipid bilayers at 248 K. a POPC-d31 H. Sarrouj C. Reiter P. Bandara F. Engelke (&) membrane in the presence of hU19W (6/1 w/w) and 0.2 wt% TOTAPOL oriented along the surface of a Á Á Á HDPE plastic film and investigated by MAOSS solid-state NMR spectroscopy at 5 kHz spinning Bruker BioSpin, Silberstreifen, 76287 Rheinstetten, Germany frequency. b POPC vesicles (red line) and non-oriented POPC-d31/TOTAPOL film (black line) under e-mail: [email protected] 5 kHz MAS. c POPC bilayers mechanically oriented on glass plates (black) and the static non-oriented spectrum (gray). The simulations of the experimental spectra in a–c are shown in the row below. T. Franks H. Oschkinat Á d During the calculations, a Gaussian distribution of 23° and Lorentzian line broadening of 2.3 kHz was Leibniz-Institut fu¨r Molekulare Pharmakologie, Robert-Ro¨ssle-Straße 10, 13125 Berlin, Germany Wednesday,applied. 10e AprilSimulation 13 using a line broadening of 2 kHz (black) and 0.6 kHz (red line), and f a Gaussian distribution of 12° and a line broadening of 1.5 kHz. The MAS experiments (a, b) were performed at Present Address: 11.2 T using a 31P–1H doubly tuned 3.2 mm MAS probe. The experiment shown in c (black solid line) E. Koers was performed at 9.4 T as described previously [37] Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands acquisitions at room temperature from such a sample (typically 20,000 transients at 3 s recycle delay), in agreement with previous investigations of a similar system [42]. By decreasing the temperature to 100 K, electronic noise and movements that interfere 123 with cross-polarization are reduced [18, 44]. Furthermore, the Zeemann magnetization is also enhanced by a factor of 3 (Boltzmann distribution) and at 100 K, a small signal could be detected already after about 20 min (not shown). Upon irradiation of the sample with l-waves of 5 W at 263 GHz, the 15N spectrum was enhanced up to 12-fold in the presence of TOTAPOL (Fig. 1) which allowed us to record MAOSS 15N spectra also at lower spinning speeds when the total signal intensity is spread into several side bands (Fig. 2c). The observed signal enhancement of the proton-decoupled 15N CPMAS solid-state NMR spectra arises from the transfer of electron magnetization to the 1H nuclei by way of the DNP cross effect which is then cross-polarized onto the dilute heteronuclei.

123 Magic Angle Spinning DNP on

2011, 133, 20208–17. a virus ARTICLE Journal of the American Chemical Society ARTICLE pubs.acs.org/JACS

Chemical Shifts for the Unusual DNA Structure in Pf1 Bacteriophage from Dynamic-Nuclear-Polarization-Enhanced Solid-State NMR Spectroscopy Ivan V. Sergeyev,† Loren A. Day,‡ Amir Goldbourt,§ and Ann E. McDermott*,† †Department of Chemistry, Columbia University, 3000 Broadway, New York, New York 10027, United States ‡The Public Health Research Institute of the University of Medicine and Dentistry of New Jersey, 225 Warren Street, Newark, New Jersey 07103, United States §School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Ramat Aviv 69978, Tel Aviv, Israel Journal of the American Chemical Society bS Supporting Information ARTICLE

ABSTRACT: Solid-state NMR spectra, including dynamic nuclear polar- protein residues, however, we observe one peak per atomization at enhanced 400observed MHz spectra chemical acquired at 100 shift K, as patterns. well as non-DNPFurther, it is consistent with the spectra at a variety of field strengths and at temperatures in the range resolutions well below 1 ppm, indicating that all copies of213 the243 K, have1PFI allowed the model, assignment which of the 13C places and 15N resonances Y40 within of interaction range of the 52,26 À 13 coat protein share the same overall conformation. Surpris-the unusual DNADNA, structure in and the Pf1 supports virion. The C the chemical hypothesized shifts of C30 critical role of Y40 at the and C50, considered to be key reporters of deoxyribose conformation, fall ingly, order parameters observed for the side chains of residuesnear or beyond theprotein edges of theirDNA respective interface. ranges in available databases. The 13C and 15N chemical shiftsÀ of the DNA bases have above-average R44 and K45 in the C-terminal DNA-binding region werevalues low, for AC4, AC5, CC5, TC2, and TC5, and below average values for on par with those of residues in the N-terminal region exposedAC8, to GC8, and GN2,’ pointing to an absence of Watson Crick hydrogen Ann McDermott bonding, yet the presenceCONCLUSIONS of some type of aromatic ring interaction.À Crosspeaks between Tyr40 of the coat protein and several DNA solvent, and in distinct contrast to high values of order param-atoms suggest that Tyr40 is involved in this ring interaction. In addition, these crosspeak resonances and several deoxyribose The mere presence offf the DNA peaks,ff and especially of those eters for other parts of the coat protein, suggesting thatresonances the are multiply split, presumably through the e ects of ordered but di ering interactions between capsid protein subunits and each type of nucleotidecorresponding in each of the two to DNA the strands. bases, Overall, indicates these observations a characterize well-ordered and support DNA the DNA model DNA protein interface is inherently dynamic on a submicrose-proposed by Liu and Day and refined by Tsuboi et al., which calls for the most highly stretched and twisted naturally occurring DNA yet encountered. structure. A number of DNA peaks are split into multiple condÀ time scale,26 and that this may play a role in the observed resonances, consistent with the idea that each nucleotide type peak splitting. Qualitative dynamics observations from 1H 15N À in each strand has an environment shared by all nucleotides of polarization inversion spin exchange at the magic angle (PISEMA)’ INTRODUCTION Pf1 structure has become one of the paradigms for filamentous Pf1, a 36 MDa thatfilamentous type circular in single-stranded that strand. DNA Thesephages unique in that its capsid environments symmetry of approximately share 27 protein experiments also suggest that the coat protein backbone is mostly fi subunits in five helical turns is common to many inoviruses, as is bacteriophage specicommonc to strain K of features,Pseudomonas namely aeruginosa 20-endo/gauche deoxyribose ring13 static, with the notable exceptions of residues at the C-(PAK), and1 is one of the innumerable filamentous phages compris- the small size and high α-helicity of these subunits. However, ing the genus Inovirusconformations, many of which are integrative and andanti someglycosidic of Pf1 has bond an unusual orientations, 1:1 ratio of nucleotides corrobor- to major capsid N-termini and a “hinge region” which was not observed in subseq-which carry virulence factors that convert their bacterial hosts subunits, rather than a ratio23 between 2.0 and 2.5 as in most other 26,50,51 ating conclusively2 6 results frominoviruses. other methods. Of its 7349 nucleotides,Extreme7300 interact DNA with an equal uent work. In summary, it seems clear from our presentfrom work nonpathogens into pathogens. À Although Pf1 itself is ∼ 11,14 17 nonintegrative and carrieschemical no virulence shift factors, values, phages with in Pf1- somenumber cases of gene falling 8 product outside (major coat of protein) BMRB subunits. À On the basis of this 1:1 stoichiometry, and an electron density and previous studies that the overall heterogeneity in the structurelike structural is genes are present as integrated prophages in other 18 fi Ps. aeruginosa strains,chemical some found in shift clinical ranges, isolates from indicate cystic map an of unusual a low temperature DNA form structure. of Pf1, as wellThe as extensive low, but that in the DNA protein interface a signi cant degree of 7 9 spectroscopic data, a model has been developed in which the fibrosis patients. À Pf1 and Pf1-like virions are 7 nm in diameter, À DNA lacks hydrogen bonding,helical also symmetries corroborating of DNA and capsid previous were matched (PDB: static heterogeneity or dynamic disorder is present. like virions of all inoviruses,fi but they11,14,70 are much longer than others 12,19 21 of similar genome sizendings, due to the unusual, stretchedyet conforma- there is some1PFI). sortÀ The of positions stacking of OPO present, groups of the DNA The above observations are relevant to an important feature of (phosphorus plus nondiester oxygens)19 for the model as deduced tion of the packaged circular single-stranded DNA. For example, 12 18 the Pf1 genome of 7.3consistent kb10 is packed in with a virion predicted about 2 μm long, baseby Liutyrosine and Day interactionsfrom the electron densityand map thecorresponded the 1PFI model of the virion: the everted conformation of the Àperfectly with prior electrostatic calculations,20 and placed anti- 12,21 whereas the well-knownpresence phages of ofthe Ff Y40-DNA group (f1, fd, M13) (base and sugar) crosspeaks. Overall, DNA. In this model, nucleotides from the up-strandhave are 6.4 kb genomes packed in 0.9 μm long virions.11,12 The parallel sugar phosphate chains at the center, with the phos- phorus atomsÀ at 2.5 Å radius and phosphorus phosphorus ff circular genomes inthese such virions initial have two high-resolution non-complementary NMR results support the Pf1 DNAÀ positioned quite di erently with respect to their coat proteinDNA strands running in opposite directions from end to end, distances of 7.5 Å within strands andfi 5.2 Å between strands. model put forth by Liu and DayAlso, theand model subsequently has the nondiester re OPOned planes by uniformly Figure 2. DNP-enhanced SSNMRsubunits spectra of Pf1 showing than (a) those the assignments from of the the dC/dT down-strand, base resonances, (b) andthe dA/dG therefore base resonances,with have andthe nature of strand strand and strand12,21capsid interactions (c) the sugar spin systems. Data were collected using a 400 MHz wide-borefi AVANCE III spectrometer at cryogenic temperaturesff (100 K), withvarying a mixing markedly amongTsuboiÀ the diff eterent al. species.À Thus, the struc- time of 22 ms. See Table S1 for additionaldistinct acquisition sets and of processingrst parameters. shell contacts as well as di erent magnetictures of Pf1 and of other filamentous phages are of biomedical Received: May 10, 2011 environments, as illustrated in Figure 6. This arises from alland coat fundamental biophysical interest. Published: August 22, 2011 tyrosineWednesday, peaks from 10 the April coat 13 protein and to closely spaced be obscured by more intense peaks and require additional ’ MATERIALS AND METHODS chemical shifts. Resonances with low signal-to-noise may thus enhancement to be resolvable. Some crosspeaks that were protein subunits being aligned in the same direction, while the r 2011 American Chemical Society 20208 dx.doi.org/10.1021/ja2043062 | J. Am. Chem. Soc. 2011, 133, 20208–20217 13 15 two strands of ssDNA20210 run in oppositedx.doi.org/10.1021/ja2043062 directions. |J. Am. Chem. The Soc. 2011, 133, resulting20208–20217 Uniformly C- and N-Labeled Pf1 Phage. The phage was difference in relative nucleotide position is expected to give rise 52 ff prepared as previously described in Goldbourt et al. The purified virus to di ering chemical and magnetic environments, potentially had a UV absorbance spectrum with a maximum at 270 nm, a minimum splitting the peak for each base atom into a doublet because of its at 245 nm, and an OD270/OD245 ratio of 1.20 ( 0.01. On the basis of an ff 1 2 24 di ering up- and down-strand environment. Some base atoms, extinction coefficient of 2.1 mgÀ cm , and a molecular weight of 36 due to their positions, will almost certainly be more sensitive to MDa, approximately 40% ( 20% of the Pf1 physical particles gave this effect than others, resulting in larger splittings and explaining plaques in assays of infectivity on its specific host PAK. why the relatively low resolution afforded by our DNP spectra DNA 13C,15N-Labeled Pf1 Phage. See Supporting Information. was sufficient to see some splittings but not others. DNP-NMR Sample Preparation. PEG-8000 precipitate (6 mg) On the basis of the Liu and Day12 and Tsuboi et al.21 models, of Pf1 was resuspended in a solution that consisted of 200 μL of DMSO- the same splitting effects would be expected to hold true for the d6 (Cambridge Isotope), 250 μL of D2O (Cambridge Isotope), 50 μL of DNA sugar peaks, because each of the four bases assumes a 10 mM Tris buffer (pH 8.4) [40:50:10 mixture by volume], and 20% 21 slightly different N-glycoside torsion angle, which could affect w/v PEG-8000. The solution was made 5 mM MgCl2 by the addition of the sugar resonances differently. Therefore, in principle, it is not 1 M stock solution, and precipitated. Pelleting was accomplished by unreasonable to expect up to 8 resonances for each sugar carbon centrifugation. The supernatant was removed and the pellet (with a of the four nucleotides in each of the two strands. DFT calculations volume of approximately 16 μL) transferred by centrifugation into a specially designed Bruker 3.2 mm sapphire rotor for DNP. A 220 μM show that deoxyribose magnetic shielding anisotropy, and, by 58 extension, chemical shifts are indeed sensitive to the glycosidic stock solution of the biradical TOTAPOL was added to the sample in 76 the rotor to make it 20 μM TOTAPOL, after which the rotor was sealed. torsion angle. SSNMR data on RNA nucleosides and nucleotides 13 15 44 ff NMR Sample Preparation. For both uniformly- C, N-labeled also supports this view. Nearest-neighbor e ects are highly 13 15 unlikely in Pf1 DNA due to the distances involved (N9/N1 atoms and DNA- C, N-labeled Pf1, 12 mg of the respectively labeled phage was precipitated from 100 mM Tris buffer (pH 8.4) using 8% w/v PEG- of neighboring nucleotides are approximately 11.5 12.1 Å apart 8000 and 5 mM MgCl . Pelleting was accomplished by 20 min in the same strand and 9.4 10.4 Å apart in oppositeÀ strands); 2 À centrifugation at 3700g. The supernatant was removed and the pellet therefore, any splittings must beduetointeractionswiththe (with a volume of approximately 40 μL) transferred by centrifugation coat protein. into a standard 3.2 mm or 4 mm (Bruker or Varian) SSNMR rotor. Protein DNA Interactions. The spectra presented in Figure 3 DNP-NMR Experiments. NMR experiments were conducted at and FiguresÀ S2 and S3 contain previously undetected protein À Bruker Billerica laboratories on an AVANCE III 263 GHz solid-state DNA crosspeaks, namely the Y40CG-AC5 and Y40CE-AC5 DNP-NMR spectrometer (ω1H = 400 MHz, 263 GHz/25 W/9.7 T protein base crosspeaks as well as Y40CA-C1 /C4 ,Y40CA- À 0 0 gyrotron). Spectroscopy was performed by IVS, AEM, and Drs. Ansgar C30,andY40CE-C10/C40 protein sugar crosspeaks at longer 13 À Siemer and Melanie Rosay. C magnetization was prepared using a (200 ms) mixing times. At shorter (20 ms) mixing times, only ramped cross-polarization (CP) process.77 13C 13C magnetization À the Y40CE-AC5 and Y40CZ-C10 crosspeaks are observed; transfer during the mixing time was accomplished using the dipolar these are therefore presumed to be the shortest-range of the assisted rotational resonance (DARR) condition:73 13C magnetization protein DNA contacts. The presence of these contacts was stored parallel to the direction of the magnetic field while 1H RF suggestsÀ a mechanism for aromatic stacking as suggested by irradiation of a power matching the spinning frequency was applied.

20215 dx.doi.org/10.1021/ja2043062 |J. Am. Chem. Soc. 2011, 133, 20208–20217 Article

pubs.acs.org/JACS

Solid-State NMR on Bacterial Cells: Selective Cell Wall Signal Enhancement and Resolution Improvement using Dynamic Nuclear

Article MAS DNP on a cell walls in Polarization pubs.acs.org/JACS whole cells and extracts

† ‡ †Solid-State NMR on Bacterial Cells: Selective† Cell Wall Signal ‡ Hiroki Takahashi, Isabel Ayala, Michel Bardet, EnhancementGael̈ Deand Resolution Paepe,̈ ImprovementJean-Pierre using Dynamic Nuclear Simorre, ,† Polarization Hiroki Takahashi,† Isabel Ayala,‡ Michel Bardet,† Gael̈ De Paepe,̈ † Jean-Pierre Simorre,‡ and Sabine Hediger* and Sabine Hediger*,† †Laboratoire de Chimie Inorganique et Biologique, UMR-E3 (CEA/UJF) and CNRS, Institut Nanosciences et Cryogenie,́ CEA, 38054 Grenoble, France † ‡Institut de Biologie Structurale, UMR5075 (CEA/CNRS/UJF), 38027 Grenoble, France Laboratoire de Chimie Inorganique et Biologique, UMR-E3 (CEA/UJF)*S Supporting Information and CNRS, Institut Nanosciences et Cryogenie,́ CEA,

ABSTRACT: Dynamic nuclear polarization (DNP) enhanced 38054 Grenoble, France solid-state nuclear magnetic resonance (NMR) has recently emerged as a powerful technique for the study of material ‡ surfaces. In this study, we demonstrate its potential to investigate cell surface in intact cells. Using Bacillus subtilis • DNP used for selective Institut de Biologie Structurale, UMR5075 (CEA/CNRS/UJF),bacterial cells 38027 as an example, it Grenoble, is shown that the polarizing France agent 1-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-ol (TOTAPOL) has a strong binding affinity to cell wall polymers (peptidoglycan). This particular interaction is enhancement of the cell walls thoroughly investigated with a systematic study on extracted cell wall materials, disrupted cells, and entire cells, which proved S that TOTAPOL is mainly accumulating in the cell wall. This property is used on one hand to selectively enhance or suppress cell * Supporting Information ff wall signals by controlling radical concentrations and on the other hand to improve spectral resolution by means of a di erence spectrum. Comparing DNP-enhanced and conventional solid-state NMR, an absolute sensitivity ratio of 24 was obtained on the entire cell sample. This importantSabine increase in sensitivity Hediger together with the possibility of enhancing specifically cell wall signals and Gaëlimproving de resolution Paëpe really opens new avenues for the use of DNP-enhanced solid-state NMR as an on-cell investigation tool.

■ INTRODUCTION specifically DNP-enhanced SSNMR to cell extracts or entire ABSTRACT: Dynamic nuclear polarization (DNP) enhancedIn microbiology, many molecular mechanisms, such as host- cells has been demonstrated recently in the context of 12,13 pathogen recognition, cell adhesion, or regulation of cell overexpressed membrane proteins and their study. Using here the bacterial cell as a model, we investigate the pertinence activity, involve interactions with the cell surface. Structural fi solid-state nuclear magnetic resonance (NMR) has recentlycharacterization of cell surface is thus of crucial importance to of using DNP-enhanced SSNMR for the speci c study of cell surface or cell envelope in entire cells. The bacterial cell wall is understand the cell life and interaction with its environment. In 14 addition to phospholipid membranes, most biological cells have composed mainly of peptidoglycan which surrounds the a shell of glycoconjugates at their surface, which plays a crucial cytoplasmic membrane and has a direct impact on the virulence emerged as a powerful technique for the study of material and adherence of the bacteria. It plays a key role as a target of role in all recognition processes. They build a complex 15,16 polymorphic network around the cell and are as such difficult the immune response and of antibacterial treatment. to characterize by classical techniques. Solid-state nuclear Besides its own interest, the bacterial cell serves here as a surfaces. In this study, we demonstrate its potentialmagnetic to resonance (SSNMR) has proven to be useful for their model system to illustrate the potential of DNP-enhanced structural study, with examples on extracted plant and bacterial SSNMR for on-cell applications. For this purpose, essential cell walls.1−5 However, the inherent low sensitivity of NMR still aspects, such as optimal sample preparation, distribution of investigate cell surface in intact cells. Using Bacillus subtilisoften remains a limitation to answer relevant biochemical polarizing agent in the sample, and line broadening, will be here considered. We will show that under certain conditions, the questions, especially in cases where the entire cell has to be fi taken into account. sensitivity enhancement of DNP can be speci cally applied to bacterial cells as an example, it is shown that the polarizingAmong the different existing hyperpolarizing techniques, the cell surface, opening a new investigation tool for on-cell high-field dynamic nuclear polarization (DNP) has recently studies. emerged as a viable technique to significantly enhance the For DNP experiments typically performed at ∼100 K, agent 1-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-olsensitivity of high-resolution SSNMR.6−9 In particular, it has samples are usually suspended in a DNP matrix containing been proven to be useful for the study of catalytic surface solvents, a cryoprotectant, such as d8-glycerol or d6-dimethyl material.10,11 Similarly, DNP-enhanced SSNMR may be an ffi interesting technique to investigate the structure of cell Received: December 21, 2012 (TOTAPOL) has a strong binding a nity to cell wallinterface. The feasibility of applying SSNMR and more polymers (peptidoglycan). This particular interaction is © XXXX American Chemical Society A dx.doi.org/10.1021/ja312501d | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX thoroughly investigated with a systematic study on extractedWednesday, cell 10 April 13 wall materials, disrupted cells, and entire cells, which proved that TOTAPOL is mainly accumulating in the cell wall. This property is used on one hand to selectively enhance or suppress cell wall signals by controlling radical concentrations and on the other hand to improve spectral resolution by means of a difference spectrum. Comparing DNP-enhanced and conventional solid-state NMR, an absolute sensitivity ratio of 24 was obtained on the entire cell sample. This important increase in sensitivity together with the possibility of enhancing specifically cell wall signals and improving resolution really opens new avenues for the use of DNP-enhanced solid-state NMR as an on-cell investigation tool.

■ INTRODUCTION specifically DNP-enhanced SSNMR to cell extracts or entire cells has been demonstrated recently in the context of In microbiology, many molecular mechanisms, such as host- 12,13 pathogen recognition, cell adhesion, or regulation of cell overexpressed membrane proteins and their study. Using here the bacterial cell as a model, we investigate the pertinence activity, involve interactions with the cell surface. Structural fi characterization of cell surface is thus of crucial importance to of using DNP-enhanced SSNMR for the speci c study of cell surface or cell envelope in entire cells. The bacterial cell wall is understand the cell life and interaction with its environment. In 14 addition to phospholipid membranes, most biological cells have composed mainly of peptidoglycan which surrounds the a shell of glycoconjugates at their surface, which plays a crucial cytoplasmic membrane and has a direct impact on the virulence and adherence of the bacteria. It plays a key role as a target of role in all recognition processes. They build a complex 15,16 polymorphic network around the cell and are as such difficult the immune response and of antibacterial treatment. to characterize by classical techniques. Solid-state nuclear Besides its own interest, the bacterial cell serves here as a magnetic resonance (SSNMR) has proven to be useful for their model system to illustrate the potential of DNP-enhanced structural study, with examples on extracted plant and bacterial SSNMR for on-cell applications. For this purpose, essential cell walls.1−5 However, the inherent low sensitivity of NMR still aspects, such as optimal sample preparation, distribution of often remains a limitation to answer relevant biochemical polarizing agent in the sample, and line broadening, will be here considered. We will show that under certain conditions, the questions, especially in cases where the entire cell has to be fi taken into account. sensitivity enhancement of DNP can be speci cally applied to Among the different existing hyperpolarizing techniques, the cell surface, opening a new investigation tool for on-cell high-field dynamic nuclear polarization (DNP) has recently studies. emerged as a viable technique to significantly enhance the For DNP experiments typically performed at ∼100 K, sensitivity of high-resolution SSNMR.6−9 In particular, it has samples are usually suspended in a DNP matrix containing been proven to be useful for the study of catalytic surface solvents, a cryoprotectant, such as d8-glycerol or d6-dimethyl material.10,11 Similarly, DNP-enhanced SSNMR may be an interesting technique to investigate the structure of cell Received: December 21, 2012 interface. The feasibility of applying SSNMR and more

© XXXX American Chemical Society A dx.doi.org/10.1021/ja312501d | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX Angewandte. Communications

DOI: 10.1002/anie.201105984 NMR Spectroscopy Solid-State NMR Spectroscopy on Cellular Preparations Enhanced by Dynamic Nuclear Polarization** Marie Renault, Shane Pawsey, Martine P. Bos, Eline J. Koers, Deepak Nand, Ria Tommassen- van Boxtel, Melanie Rosay, Jan Tommassen, Werner E. Maas, and Marc Baldus*

Solid-state NMR (ssNMR) spectroscopy offers increasing Figure 1, we compared 13C and 15N CP-MAS spectra of possibilities to study complex biomolecules at the atomic uniformly 13C,15N-labeled WC with the CE isolated from level.[1] An important target area concerns membrane-asso- PagL-overproducing E. coli cells, recorded in the presence ciated proteins, which can be investigated by ssNMR methods and absence of microwave irradiation. At higher temper- after reconstitution in synthetic bilayers. While such prepa- atures (271 K), ssNMR spectra of the E. coli CE had rations allow examination of functional aspects of the protein previously revealed atomic details of PagL as well as of interest, the influence of the native cellular environment on endogenous membrane-associated macromolecules, including protein structure and function cannot be monitored. Very the major lipoprotein Lpp and non-proteinaceous compo- recently, we introduced a general approach aimed at deter- nents such as lipopolysaccharides (LPS), peptidoglycans mining complex molecular structures, including integral (PG), and phospholipids.[3] Under low-temperature (LT) membrane proteins, in their native cellular environment by DNP conditions, we observed significant DNP enhancement ssNMR under magic-angle-spinning (MAS) conditions.[2,3] factors for both preparations in spectral regions characteristic Angewandte. Using dedicated sample-preparation routes, we demonstrated for protein signals (aliphatic 13C resonances: d = 50–55 ppm, Communications that high-resolution ssNMR spectra can be obtained on Magicamide 15N Angle backbone Spinning and side-chain DNP resonances on at about 120 13 15 13 DOI: 10.1002/anie.201105984 NMR Spectroscopy uniformly C, N-labeled preparations of Escherichia coli aand whole 80–30 ppm) cells as welland as cell for Cextracts signals of endogenous whole cells (WC) and cell envelopes (CE). Both CE and WC Solid-State NMRmorphology Spectroscopy are on preserved Cellular Preparations under standard Enhanced ssNMR experi-by Dynamic Nuclearmental Polarization** conditions and the corresponding 13C and 15N cross- Marie Renault, Shane Pawsey,polarization Martine P. (CP-MAS) Bos, Eline J. Koers, spectra Deepak are Nand, invariant Ria Tommassen- over time. van Boxtel, Melanie Rosay,However, Jan Tommassen, with increasing Werner E. Maas, levels and Marc of molecular Baldus* complexity, Solid-state NMR (ssNMR) spectroscopyespecially offers in increasing the caseFigure 1, of we WC compared preparations,13C and 15N CP-MAS spectroscopic spectra of possibilities to study complex biomolecules at the atomic uniformly 13C,15N-labeled WC with the CE isolated from level.[1] An important target areasensitivity concerns membrane-asso- becomesPagL-overproducing a critical factor.E. coli cells, recorded in the presence ciated proteins, which can be investigatedIn by recent ssNMR methods years,and dynamic absence of nuclear microwave polarization irradiation. At higher (DNP) temper- has after reconstitution in synthetic bilayers. While such prepa- atures (271 K), ssNMR spectra of the E. coli CE had rations allow examination of functionaldeveloped aspects of the into protein a routinepreviously tool revealed to atomic increase details of the PagL sensitivity as well as of of interest, the influence of the native cellular environment on endogenous[4] membrane-associated macromolecules, including protein structure and functionmultidimensional cannot be monitored. Very ssNMR.the major lipoproteinDNP enhancements Lpp and non-proteinaceous of up compo- to 148- recently,. we introduced a generalfold approach have aimed been at deter- obtainednents such on as micro/nanocrystalline lipopolysaccharides (LPS), peptidoglycans biomolec- Angewandtemining complex molecular structures, including integral (PG), and phospholipids.[3] Under low-temperature (LT) membrane proteins,Communications in their nativeular cellular samples, environment including by DNP conditions, an amyloidogenic we observed significant peptide DNP enhancement and a deu- [2,3] ssNMR under magic-angle-spinningMarc (MAS) Baldus conditions.[5,6] factors for both preparations in spectral regions characteristic Using dedicated sample-preparationterated routes, we protein, demonstrated whilefor protein enhancements signals (aliphatic 13C resonances: betweend = 18-50–55 andppm, 46- that high-resolution ssNMR spectrafold havecan be obtained beenreported on amide 15N for backbone membrane-embedded and side-chain resonances at about polypep- 120 uniformly 13C,15N-labeled preparations of Escherichia coli and 80–30 ppm) as well as for 13C signals of endogenous enhanced[7,8] by a similar factor. Further signal enhancements whole cells (WC) and cell envelopestides, (CE). purple Both CE and membrane WC preparations, and bacteriophages. morphology are preserved under standard ssNMR experi- should be possible by optimization of the sample preparation mental conditions and the correspondingHere, we13C and investigated15N cross- the use of DNP to conduct ssNMR polarization (CP-MAS) spectrastudies are invariant on over13C, time.15N-labeled preparations of E. coli WCprocedures over- including molecular deuteration and the choice of However, with increasing levels of molecular complexity, [9] especially in the case of WCproducing preparations, spectroscopic the integral outer membrane protein PagL.theIn radical. DNP-enhanced ssNMR spectroscopy as shown sensitivity becomes a critical factor. In recent years, dynamic nuclear polarization (DNP) has here provides a novel approach to study diverse cellular developed into a routine tool to increase the sensitivity of multidimensional ssNMR.[4] DNP[*] enhancements Dr. M. Renault, of up to 148- E. J. Koers, Dr. D. Nand, Prof. M. Baldus components and their involvement in fundamental biological fold have been obtained on micro/nanocrystallineBijvoet Center biomolec- for Biomolecular Research, Utrecht Universityprocesses ranging from molecular signal transduction to ular samples, including an amyloidogenic peptide and a deu- [5,6] Padualaan 8, 3584 CH Utrecht (The Netherlands) terated protein, while enhancements between 18- and 46- protein (mis)folding,[11] at atomic resolution with exquisite fold have been reported for membrane-embeddedE-mail: [email protected] polypep- tides, purple membrane preparations, and bacteriophages.[7,8] sensitivity. Here, we investigated the use of DNPDr. to M. conduct P. Bos, ssNMR R. Tommassen-van Boxtel, Prof. J. Tommassen studies on 13C,15N-labeled preparationsDepartment of E. coli WC over- of Molecular Microbiology, Utrecht University producing the integral outer membrane protein PagL.[9] In Padualaan 8, 3584 CH Utrecht (The Netherlands) Figure 1. Comparison of 13C (left) and 15N (right) CP-MAS spectra of [*] Dr. M. Renault, E. J. Koers, Dr. D. Nand, Prof. M. Baldus Dr. S. Pawsey, Dr. M. Rosay, Dr. W. E. Maas 13 15 Bijvoet Center for Biomolecular Research, Utrecht University Experimental(U- C, N)-labeled Section CE (a) and WC (b) with (top trace, “on”) and Padualaan 8, 3584 CH Utrecht (The Netherlands)Bruker BioSpin Corp., Billerica, MA 01821 (USA) 13 15 E-mail: [email protected] UniformlywithoutC, N-labeled (bottom trace, preparations “off”) DNP, ofusingE. a coli microwavewhole irradiation cells (WC), time Dr. M. P. Bos, R. Tommassen-van[**] Boxtel, This Prof. J. study Tommassen was presented in part at the 52nd ENC conference,cell envelopesof 10 (CE), s. Significant and PagL-containing differences in the spectraproteoliposomes of the two preparations (PL) were 15 13 Department of Molecular Microbiology,Asilomar, Utrecht University CA, April 10–15, 2011. Funding by NWO (grants [3] FigurePadualaan 3. 8, 3584Overlay CH Utrecht of (The the Netherlands) DNP-enhanced 2D ( N, C) NCA correlation are denoted by arrows. Asterisks indicate MAS side bands. Assign- Figure 1. Comparison of 13C (left) and 15N (right) CP-MAS spectra ofobtained by following the procedure described elsewhere. Pellets of Dr. S. Pawsey, Dr. M. Rosay,13 Dr.15 W. E. Maas700.26.121 and 815.02.012) and the European Community’s spectra of (U- C, N)-labeled WC (at 100(U-13C, K,15N)-labeled 8 kHz CE MAS) (a) and WC (black) (b) with (top and trace, “on”) and ments of major E. coli molecular components are, if available, anno- Bruker BioSpin Corp., Billerica, MA 01821 (USA) exponentially growing WC were washed three times in a H2O/D2O/ 13 15Seventh Frameworkwithout Program (bottom trace, [FP7/2007-2013] “off”) DNP, using a microwave under irradiation Grant time Agree- tated and signal enhancements are given. Natural abundance 13C [**]the This 2D study wasNCA presented of in(U- part atC, the 52ndN)-labeled ENC conference, CE isolatedof 10 s. Significant from differences noninduced in the spectra of the two preparations[D ]glycerol matrix (10/30/60wt%) containing 60 mm TOTAPOL by Asilomar, CA, April 10–15, 2011. Fundingment by NWO no. (grants 211800 is gratefully acknowledged.13 15 8 13 (green) or PagL-overexpressing (blue) cellsare denoted and by arrows. purified Asterisks (U- indicateC, MAS sideN)- bands. Assign- signals from glycerol (d C 73 and 63[12] ppm) present in the glassy 700.26.121 and 815.02.012) and the European Community’s ments of major E. coli molecular components are, if available, anno-resuspension/centrifugation cycles at 400013 g. The CE were placed in Seventh Framework Program [FP7/2007-2013]Supporting under Grant Agree- informationtated and for signal this enhancements article are is given. available Natural abundance on the13C WWW matrix and from the silicon plug (d C 4 ppm) are visible in the PagL reconstituted in proteoliposomes (red) obtained13 at 271 K and ment no. 211800 is gratefully acknowledged. signals from glycerol (d C 73 and 63 ppm) present in the glassy the same matrix13 with 60 mm TOTAPOL and pelleted at 95000g. WC under http://dx.doi.org/10.1002/anie.201105984. C CP spectra of the WC and CE preparations. Supporting information for this article is available on the WWW matrix and from the silicon plug (d13C 4 ppm) are visible in the 13 kHz MAS). Upper row: region I, lower row: region II.  under http://dx.doi.org/10.1002/anie.201105984. 13C CP spectra of the WC and CE preparations. and CE samples were subsequently transferred into 3.2 mm Bruker sapphire rotors with a (silicon) top spacer using a tabletop centrifuge 2998 2998  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim  2012 Wiley-VCHAngew. Chem. Int. Verlag Ed. 2012, 51 GmbH, 2998 –3001 & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2012, 51, 2998 –3001 Wednesday, 10 April 13 (14000 ” g). Samples were precooled at 208C prior to DNP ssNMR À lipoprotein Lpp, which belongs to the most abundant CE- measurements. DNP experiments were performed on a Bruker 263 GHz Solids DNP spectrometer, consisting of a 263 GHz contin- associated proteins in exponentially growing cells and lacks uous-wave gyrotron source, microwave transmission line, 3.2 mm low- [3] Gly in its amino acid sequence. Thus, regions I and II in temperature MAS probe, gas cooling supply, and 400 MHz AVANCE Figure 3 strongly suggest that both PagL (I) as well as III wide-bore NMR system.[13] NMR spectra were processed using endogenous Lpp (II) can be detected in WC preparations Topspin 3.0 (Bruker BioSpin, Germany) and analyzed with Sparky.[14] under DNP conditions. Notably, with an average 13C linewidth 13C and 1H resonances were calibrated using adamantane as an 15 of d = 0.8–0.9 ppm, the low-temperature ssNMR spectra external reference, and N chemical shifts were referenced indirectly to liquid ammonia.[15,16] Further information about ssNMR exper- compared favorably to ssNMR spectra recorded at 271 K. imental parameters is given in the Supporting Information. Taken together, these results show that DNP can be readily combined with ssNMR approaches in cells. Our Received: August 24, 2011 results suggest that the application of LT-DNP not only Revised: November 2, 2011 increases spectroscopic sensitivity by at least an order of Published online: February 1, 2012 magnitude but also permits the detection of molecular components that are not associated with specific compart- Keywords: analytical methods · dynamic nuclear polarization · ments. Compared to standard ssNMR spectroscopy, DNP at .membrane proteins · RNA · solid-state NMR spectroscopy low temperatures and the use of free radicals can complicate sample preparation and can potentially give rise to reduced spectral resolution. However, using the preparations de- scribed herein, our spectra are of comparable resolution to [1] a) E. Brunner, C. Groger, K. Lutz, P. Richthammer, K. Spinde, those obtained in previous studies at higher temperature. M. Sumper, Appl. Microbiol. Biotechnol. 2009, 84, 607 – 616; ssNMR spectroscopy in cells as described here hence permits b) C. Wasmer, L. Benkemoun, R. Sabate, M. O. Steinmetz, B. Coulary-Salin, L. Wang, R. Riek, S. J. Saupe, B. H. Meier, the tracking of entire cellular compartments similar to cryo- Angew. Chem. 2009, 121, 4952 – 4954; Angew. Chem. Int. Ed. electron tomography but further work will be needed to 2009, 48, 4858 – 4860; c) M. Renault, A. Cukkemane, M. Baldus, conduct such experiments under in vivo conditions. Initial Angew. Chem. 2010, 122, 8524 – 8535; Angew. Chem. Int. Ed. studies in our laboratory suggest that low temperatures and 2010, 49, 8346 – 8357. cryo-protection with 15–40% glycerol are beneficial for WC [2] E. R. Andrew, A. Bradbury, R. G. Eades, Nature 1958, 182, 1659. sample stability and viability. In our experiments, 13C and 15N [3] M. Renault, R. Tommassen-van Boxtel, M. P. Bos, J. A. Post, J. Tommassen, M. Baldus, Proc. Natl. Acad. Sci. USA 2012, in signal enhancements are similar in magnitude to those in press. previous studies that used TOTAPOL as a radical in [4] a) A. B. Barnes, G. D. Paepe, P. C. van der Wel, K. N. Hu, C. G. (protonated) purple-membrane preparations or bTbK on Joo, V. S. Bajaj, M. L. Mak-Jurkauskas, J. R. Sirigiri, J. Herzfeld, peptides in deuterated POPC bilayers.[8,7] Typical E. coli cell R. J. Temkin, R. G. Griffin, Appl. Magn. Reson. 2008, 34, 237 – dimensions of 0.5 mm ” 2 mm are comparable to those in 263; b) T. R. Carver, C. P. Slichter, Phys. Rev. 1956, 102, 975 – earlier successful DNP studies on amyloid nanocrystals.[5] Our 980; c) A. Lesage, M. Lelli, D. Gajan, M. A. Caporini, V. study revealed that both CE-associated molecular compo- Vitzthum, P. Mieville, J. Alauzun, A. Roussey, C. Thieuleux, A. Mehdi, G. Bodenhausen, C. Coperet, L. Emsley, J. Am. Chem. nents, including integral membrane proteins, lipoproteins, Soc. 2010, 132, 15459 – 15461; d) T. Maly, G. T. Debelouchina, lipids, and periplasmic PG, and cytoplasmic macromolecules V. S. Bajaj, K. N. Hu, C. G. Joo, M. L. Mak-Jurkauskas, J. R. such as nucleic acids are readily visible. Unlike the case of the Sirigiri, P. C. van der Wel, J. Herzfeld, R. J. Temkin, R. G. crystals, solute E. coli particles and solvent molecules were Griffin, J. Chem. Phys. 2008, 128, 052211.

3000 www.angewandte.org  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2012, 51, 2998 –3001 Challenges

Can you see any problem/challenge in these biomolecular applications?

Lines generally broaden at low temperatures (getting resolution may be a problem)

Wednesday, 10 April 13 The origins of line broadening at low temperatures?

Inhomogeneous Relaxation? broadening?

Wednesday, 10 April 13 The origins of line broadening at low temperatures?

Linden, A. H.; Franks, W. T.; Akbey, Ü.; Lange, S.; van Rossum, B.-J.; Oschkinat, H. Journal of biomolecular NMR 2011. SH3 J Biomol NMR J Biomol NMR

Fig. 4 a Three spectra of SH3 at 293 K (top) and three at 98 K (bottom). a long pulse of 10 ms at 42.25 dB power (471 Hz) was used to ‘‘burn holes’’ at three different carrier frequencies, colored with orange (293 K) or blue (98 K). b Detailed view of the carbonyl region

hole burning experiments show that the broadening is of inhomogenous nature

Fig. 2 2D 13C–13C PDSD spectra of SH3 recorded at indicated discussed in the text or shown in Fig. 3 (a Thr Cb–Ca; b: Thr Cb–Cc; temperatures. All spectra were recorded with 2 ms CP and 11.25 ms c: Ser Cb–Ca; d: Pro Ca–Cd; e: Ala Ca–Cb; f: Ile Cd1, Cd1–Cc2, PDSD mixing (50 ms for the deuterated sample). Marked regions are Cd1–Cc2). The base level of all spectra is set to 6r of the noise ➡ The line broadening at lower temperatures is of behavior of individual resonances. Generally, the spectra This in an additional indicator for inhomogeneous broad- broaden until each resonance reaches its individual coa- ening. Furthermore, cooling the sample faster by immer- lescence temperature, and then it splits into multiple sig- sion in liquidinhomogenous nitrogen did not change the 1D and 2D nature nals. At low temperatures (153–96 K, depending on the spectra recorded at 95 K substantially. individual residue) the single peaks have a similar line- Signal multiplication leads to poor apparent resolution Wednesday,width as 10 the April motionally 13 averaged peaks at 293 K. The in much of the spectrum. To avoid interferences due to autocorrelation signals are not greatly broadened at low signal overlap, we analyzed the spectral regions with well temperatures as measured perpendicular to the diagonal. resolved peaks at room temperature such as: threonine

123

Fig. 5 a The crystal structure of SH3, surrounded by 14 molecules of orange; The four close-up views show T32 (b), T37 (c), P20 (d), and a simulated crystal (rendered as surfaces; RCSB PDB: 1U06; P54 (e) with their direct intermolecular environment (neighboring Chevelkov et al. 2005). Residues are color-coded according to their SH3 molecules: green surfaces), including crystal associated water NMR characteristics at different temperatures; residues for which the molecules. The primary sequence of SH3 is plotted at the bottom. NMR signals remain between 293 and 213 K are colored blue; Arrows indicate b-strands; the same color-coding is used as in the residues for which the cross peaks disappear in this range are colored structure

1–1.5 A˚ ; B-value 9.81; compare Fig. 5c), is in a loop but backbone NH and not to the backbone CO. This might also in a crystal contact. It is located in a cleft in the first hinder the side chain motions and lead to a lower coales- hydration shell of an adjacent SH3 molecule. Another cence temperature. If multiple conformations are the difference between T37 and the other two observed thre- source of the spectral broadening, it is reasonable to onines in the v1 angle of ?59° (T32: -5°; T24: -66°; out assume that the signals of T24 coalesce at a higher tem- of the 1.49 A˚ crystal structure at 100 K; Chevelkov et al. perature than the signals of T32 and T37. Indeed the 2005) Therefore, the methyl group of T37 is close to the observed order of coalescence temperatures is: T37

123 A Few Applications

Biomolecules Materials Angewandte. Zuschriften

overlap of spinning sidebands with isotropic resonances, a nrot of 12 kHz was employed for all subsequent experiments. Finally, DNP 1H-13C CPMAS solid-state NMR spectra of the same sample of 1 impregnated with a 16 mm bTbK solution were periodically acquired after the sample was allowed to rest on the bench top inside the rotor for times of 5 min, 1 h and 25 h in between acquisitions. Both e and S were observed to steadily increase from 7 to 13 and 3.0 to 3.6, respectively, with longer sample resting times (Figure S3). This suggests the biradical species slowly diffuse through the relatively small (1.6 nm) pores of the material. Spectra of 1 and 2 were recorded after 25 and 0.5 h of resting time, respectively. For compound 3, however, the enhancement factors were inde- pendent of sample resting time and spectra were recorded 5 min after rotor packing. Note that the enhancements for the

EtCl4 resonance (eS > 20 in all cases) is always larger than that for the MOF materials, suggesting that the biradical mole- cules are partly excluded from the pores for all three types of the materials (see below).[19] Under these optimal experimental conditions, for MOF samples 1 and 2, e values of 13 and 16 were observed, respectively. Significantly lower values of S were measured, corresponding to the fact that the biradicals inside of the material cause a reduction in the signal intensity through various paramagnetic effects.[17] On the other hand, for 3, similar values of e and S are observed (4.8 and 5.8, respectively). This strongly suggests that the bTbK molecules are excluded from the pores of 3, leading to a low value for e, and a relatively high value for S. This suggests that the whole material is contributing to the NMR signal under DNP Scheme 1. A) The crystal structure of MOF (In)-MIL-68 illustrating the conditions. This is in agreement with the fact that for 3, e does three-dimensional structures of the MOFs that are formed with indium not increase with additional sample resting times, and that a Wednesday, 10 April 13 octahedra and terephthalate ligands as bridging linkers.[4] B–D) Sche- matic structures of the three N-functionalized MOFs which are much larger value of e is observed for the solvent resonances (e = 26). It is likely that for 3, the proline groups block the isostructural to MIL-68. B) (In)-MIL-68-NH2 (1), C) the partially N- S functionalized (In)-MIL-68 material obtained with a terephthalate:2- one-dimensional pores of the material and hinder the aminoterephthalate ratio of 80:20 (2), and D) the 10% proline- relatively large bTbK radical from diffusing into the material. functionalized derivative of (1), (In)-MIL-68-NH-Pro (3). All MOFs TEM images of the as prepared 3 reveal that the average display one-dimensional rod-shaped structures, composed by hexahe- crystal size is less than 300 nm (Figure S4). Griffin and co- dral and triangular channels with an aperture of 1.6 and 0.6 nm, workers have previously applied DNP to needle-like nano- respectively. E) Schematic structure of the biradical polarizing agent bTbK.[5] The approximate size of the molecule is 1.5 nm ”0.6 nm. crystalline peptides which possessed an average crystal width of 150 nm.[19] In their system it was not possible for the radical to enter the lattice of the crystal and larger e values were here could not be impregnated with an aqueous solution (due obtained for the solvent resonances than the resonances of to a chemical reaction with water), solutions of the biradical nuclei inside the crystals, as observed here. In a similar way, [5] bTbK (Scheme 1) and 1,1,2,2-tetrachloroethane (EtCl4) we postulate that for 3, the bTbK molecules reside on or near were utilized. Incipient wetness impregnation was used to the surface of the crystallites, and that the enhanced polar- impregnate the dry materials with a minimal amount of ization is distributed into the crystal by spin diffusion. radical containing solution. All details about sample prepa- For all three samples, we estimate that the overall ration and optimization of the experimental conditions are sensitivity gain with respect to a dry sample at room given in the Supporting Information. In particular the temperature[17] (S+ 3.0S) is between 10 and 30, correspond-  influence of radical concentration, spinning frequency (nrot), ing to a reduction in experimental time by a factor between and the amount of time the impregnated samples were 100 and 900. This allows high quality one-dimensional 13C allowed to rest before solid-state NMR experiments were CPMAS spectra of 1–3 to be acquired in less than 5 min at attempted, were optimized on 1. natural isotopic abundance. For comparison, a 1H-13C It was found that 16 mm bTbK solutions provided both the CPMAS spectrum of 1 at room temperature with a similar highest e and S (Supporting Information, Figure S1). signal-to-noise ratio has been recorded in 2.3 h using standard Although e was observed to be higher with a sample spinning instrumentation (Figure S5). However, it should be noted that 13 frequency nrot of 8 kHz, the absolute signal of the spectra was the full width at half height (L) of the C resonances of all

similar with a nrot of 12 kHz (Figure S2). In order to reduce DNP solid-state NMR spectra of 1 are around 2.8 ppm, while

128 www.angewandte.de  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. 2012, 124, 127 –131 Dynamic Nuclear Polarization Surface Enhanced NMR Spectroscopy AARON J. ROSSINI,† ALEXANDRE ZAGDOUN,† MORENO LELLI,† ANNE LESAGE,† CHRISTOPHE COPERET, ‡ AND LYNDON EMSLEY*, † †Centre de RMN a Tres Hauts Champs, Universite de Lyon (CNRS/ENS Lyon/UCB Lyon 1), 69100 Villeurbanne, France, and ‡Department of Chemistry, Laboratory of Inorganic Chemistry, ETH Zurich, CH-8093, Zurich, Switzerland

RECEIVED ON DECEMBER 5, 2012 “Surface EnhancedDNP Surface Enhanced NMR DNP”Rossini et al. -SENS CONSPECTUS 1. Rossini, A. J. et al. Acc. Chem. Res. (2013)selectively 10.1021/ar300322x. polarize surface nuclei. However, nuclei too close to radicals will not be observed due to paramagnetic any of the functions and applications of advanced materials quenching (vide infra). This impregnation DNP technique M result from their interfacial structures and properties. How- has the advantage that it is extremely simple, and there is usually no dilution of the amount of solid material in the ever, the difficulty in characterizing the surface structure of these rotor (often the powdered sample is slightly compacted). materials at an atomic level can often slow their further development. Initial DNP SENS experiments focused on phenol function- Solid-state NMR can probe surface structure and complement alized mesoporous silica having ca. 0.5 μmol phenol established surface science techniques, but its low sensitivity often moieties/mg (MatPhOH, Figure 4B).33 With conventional 13 limits its application. Many materials have low surface areas and/or NMR, acquisition of 1D C CPMAS spectra at natural isotopic abundance typically requires a half day. DNP SENS yielded low concentrations of active/surface sites. Dynamic nuclear polariza- ε 25 (defined as the ratio of signal intensities of spectra H ∼ tion (DNP) is one intriguing method to enhance the sensitivity of solid- acquired with and without microwave irradiation) and the state NMR experiments by several orders of magnitude. In a DNP proton DNP enhancement was then transferred to the experiment, the large polarization of unpaired electrons is transferred surface carbon nuclei by CP. This enabled the acquisition of natural abundance 13C CPMAS spectra in only 0.5 h. This to surrounding nuclei, which provides a maximum theoretical DNP allowed for the observation of all the aromatic resonances enhancement of 658 for 1HNMR.InthisAccount,wediscussthe ∼ of the phenol groups as well as byproducts and remaining application of DNP to enhance surface NMR signals, an approach templating surfactant. The signal enhancement from DNP known as DNP surface enhanced NMR spectroscopy (DNP SENS). SENS also enabled the rapid acquisition (4 h) of 2D dipolar 1H 13C HETCOR spectra of MatPhOH. Enabling DNP for these systems requires bringing an exogene- À 29Si DNP SENS CPMAS spectra of a variety of function- ous radical solution into contact with surfaces without diluting the alized mesoporous and nanoparticulate silica materials sample. We proposed the incipient wetness impregnation technique were subsequently obtained (Figure 4D)35 in only 0.5 h with (IWI), a well-known method in materials science, to impregnate porous and particulate materialshigh enough with just sensitivity enough radical to allow containing for observation of the surface functionalized silicon T sites (see below). 1H 29Si solution to fill the porous volume. IWI offers several advantages: it isextremelysimple,providesauniformwettingofthesurface,and À • hybrid materialsdipolar HETCOR spectra were also acquired in minutes does not increase the sample volume or substantially reduce the concentration of the sample.demonstrating the sensitivity enhancement provided by This Account describes the basic principles behind DNP SENS• through organometallic results obtainedDNP for SENS. mesoporous Lafon etspecies and al. subsequently nanoparticulate demonstrated that samples impregnated with radical solutions. We also discuss the quantification of the overallimpregnation sensitivity in conjunction enhancements with obtaineddirect 29Si DNP enables 36 with DNP SENS and compare that with ordinary room temperature• metal-organic NMR spectroscopy.observation We then of review bulk frameworks silica the sites. development of DNP SENS was also demonstrated for quadrupolar nuclei.37 radicals and solvents that give the best possible enhancements today. With the best polarizing mixtures, DNP27 SENS enhances • enhancementsFigure 4E shows the upAl DNPto SENS 100 CPMAS spectra of sensitivity by a factor of up to 100, which decreases acquisition time by five orders of magnitude.γ-alumina, Such with enhancementε 20. Since enables magnetization the transfer by Al CP ∼ CP, necessary for surface selectivity, is usually very inefficient FIGURE 4. (A) CP pulse sequence used to acquire DNP SENS spectra with detailed and expedient atomic level characterization of the surfaces of complex materials at natural isotopic abundance and opens microwave irradiation to drive DNP. (B) Schematic structures of the 14 new avenues for NMR. To illustrate these improvements,Wednesday, 10 we April describe 13 the successful applicationfor quadrupolar of DNP nuclei, SENSlow to temperature characterize DNP is especially hybrid mesoporous silica material (MatPhOH) and a chemical structure beneficial here. DNP SENS enabled the rapid acquisition of a of the surface (right) with the assignment of the relevant carbon and hybrid materials, organometallic surface species, and metal organic frameworks. 13 29 2D CP multiple quantum magic angle spinning (MQMAS) silicon resonances. (C) C and (D) Si CPMAS spectra of MatPhOH À impregnated with TOTAPOL in D O/H O 90:10 solution acquired with spectrum. The same approach has recently been applied 2 2 (upper spectra) and without (lower spectra) microwave irradiation. (E) 38 27 to the characterization of mesoporous alumina. Al DNP SENS spectra of γ-alumina impregnated with TOTAPOL D2O/

H2O 90:10 solution. Adapted from refs 33, 35, and 37. Quantifying Sensitivity Enhancements Introduction chemistry we have today. However, when the species of ε is of direct importance in evaluating the DNP process, but it by 100 K DNP SENS experiments, as compared to room The ability to determine three-dimensional molecular struc- interest is at a surface,is not necessarily the problem relevant to of assess structure the sensitivity elucidation enhance- temperature experiments, the 29Si CPMAS signal per unit tures from single crystals by diffraction methods has trans- becomes much morement provided challenging. with respect to a conventional NMR mass of MatPhOH impregnated with TOTAPOL solutions experiment.39 It is this “overall sensitivity enhancement” of varying concentration was measured (Figure 5).40 The 29Si formed molecular and materials science over the past 100 The rational development(Σ) that is of real importance of new when materials evaluating for the applica-technique CP DNP enhancement (εSi CP) was found to peak at a value for the chemist. To quantify the gain in sensitivity provided of 40 for a TOTAPOL concentration of 16 mM. However, years, leading to the structure-based understanding of tions as diverse as conversion/capture of atmospheric CO2, www.pubs.acs.org/accounts Vol. XXX, No. XX ’ XXXXD ’ ACCOUNTS’ 000–000 OF CHEMICAL’ ACCOUNTS RESEARCH OF’ 000 CHEMICAL–000 ’ XXXX ’ Vol. RESEARCH XXX, No. XX ’ A 10.1021/ar300322x & XXXX American Chemical Society Quadrupoles 14 Journal of Magnetic Resonance 205 (2010) 177–179 N DNP Contents lists available at ScienceDirect 178 V. Vitzthum et al. / Journal of Magnetic Resonance 205 (2010) 177–179 Journal of Magnetic Resonance

nation with DNP begin withjournal cross-polarization homepage: www.else fromvier.com/locate/jmr the DNP (a) Proline Cβ Cγ H enhanced protons to carbons and then proceed as previously de- scribedCommunication[6]. Cα OH A 2D spectrum of proline using DNP at 110 K with 15.625 kHz Cδ Solid-state nitrogen-14 nuclear magnetic resonance enhanced by dynamic MAS at 9.4 T (400 MHz for protons) is shown in Fig. 1a. With a re- N nuclear polarization using a gyrotron H N t O cycle delay of 8 s and 128 scans for each of =16 1 increments Veronika Vitzthum a, Marc A.max Caporini a,*, Geoffrey Bodenhausen a,b (the signal has decayed at t1 = 1.024 ms), we were able to acquire a α δ a 2DInstitut spectrum des Sciences et Ingénierie within Chimiques, a few Ecole Polytechnique hours. When Fédérale de Lausanne, including EPFL, Batochime, the 1015optimiza- Lausanne, Switzerland C C b Département de Chimie, associé au CNRS, Ecole Normale Supérieure, 24 Rue Lhomond, 75231, Paris Cedex 05, France (b) tion of the coherence transfer steps, the experiment can be com- pleted in less than a day. At 9.4 T (400 MHz for protons) and article info abstract 15.625 kHz, we could not obtain any meaningful spectrum of pro- -3 HMQC + REDOR + DNP 14 lineArticle without history: DNP. The DNP-enhancedBy combining indirectspectrum detection ( ofFig.N with 1a) dynamic may nuclear be polarization (DNP) using a gyrotron, the sig- -2 @ 110 K, 9.4 T Received 25 January 2010 nal-to-noise ratio can be dramatically improved and the recovery delay between subsequent experiments comparedRevised 23 April with 2010 the spectrum ofcan a be poly-(but shortened. Spectra not of micro-) glassy samples crystalline of the amino acid proline doped with the stable bi-radical -1 Available online 27 April 2010 0 TOTAPOL rotating at 15.625 kHz at 110 K were obtained in a 400 MHz solid-state NMR spectrometer sample of proline acquired withoutequipped DNP with a in gyrotron 7 days for microwave (Fig. 1b). irradiation The at lat- 263 GHz. DNP enhancement factors on the order 1 Keywords: of e 40 were achieved. The recovery delays can be reduced from 60 s without radicals at 300 K to 6 s terNitrogen-14 non-enhanced NMR spectrum was recorded at 18.8 T (800 MHz for with radicals at 110 K. In the absence of radicals at room temperature, the proton relaxation in proline 2 Dynamic nuclear polarization is inefficient due to the absence of rotating methyl groups and other heat sinks, thus making long recov- protons)Indirect detection and 31.25 kHz, i.e., at twice the magnetic field and twice

(kHz) 3 13 14 Low-temperature MAS ery delays mandatory. DNP allows one to reduce the acquisition times of C-detected N spectra from π 4 theGyrotron spinning microwave frequencysource as theseveral DNP-enhanced days to a few hours. spectrum. The line-

/2 13 14 Ó 2010 Elsevier Inc. All rights reserved. 1 5 widths in both C and N dimensions are broadened in the DNP ω 6 spectrum. Thisε~40 broadening is believed not to be due to paramag- 7 netic1. Introduction effects, since the linewidths do not change(DNP) with with increasing the indirect detection of nitrogen-14 can improve both 8 14 Geoffrey Bodenhausen 9 radical concentration. As expected, the linewidth inthe the signal-to-noiseN dimen- for each scan and reduce the recovery delays, 14N-SQC The high natural abundance of 14N makes it an attractive target because the proton magnetization (which is transferred to car- 10 sionto characterize is roughly nitrogen-containing twice as broad compounds at 9.4[1,2] T than. However, at 18.8bon-13 T because by cross-polarization) of the builds up with DNP on a short the detection of 14N is a difficult task because of its quadrupolar time-scale T 6 s at 110 K. second-order quadrupole interaction, which is inverselyDNP propor- coupling and its spin I = 1. Although quadrupolar couplings can tionalbe a rich to source the of static structural field. and dynamic This information, problem their can mag- only be overcome by 2. Results and discussion usingnitude DNP can lead at to 526 wide 14 GHzN spectra in spanning a field several of 18.8 MHz, T. which In the 13C dimension, are difficult to detect directly. Significant advances have recently (c) The gain in signal-to-noise achieved through DNP [13,14] is thebeen broadening made in 14N NMR may in solids be using due two-dimensional to static disorder (2D) meth- in the frozen glass accomplished through the transfer of polarization from a highly ods based on indirect detection of 14N via spy nuclei (1Hor13C) -3 in which the small molecules are embedded orpolarized to the spin, lackusually ofthe unpaired electron spin of a stable radi- with spin S = 1/2 [1,2]. These methods tend to be rather time-con- HMQC + SFAM + no DNP 13 cal such as TEMPO13 or TOTAPOL [15–17], to nuclear spins such as -2 dynamicssuming because on of a low suitable signal-to-noise time ratios scale. and long It recoveryis possible de- that the C line- protons. Dynamic nuclear polarization can be combined with ma- -1 @ 300 K, 18.8 T lays, particularly if the proton relaxationC-detected times are long. The widths can be improved by optimizing the preparationgic angle of spinning the sam- (MAS) at low temperatures (ca. 100 K), using a coherence of the spy nucleus can be transferred to the quadrupolar 0 high power microwave source (usually a gyrotron), and suitable ple,nucleus which using was residual not dipolar microcrystalline. splittings [2,3] or ‘recoupled’ It is heter- known that proteins in 1 glass-forming solvents [18–21]. Our experiments are performed onuclear dipolar interactions [4–12], and finally transferred back to glassy matrices13 can exhibit14 lines that are significantlyusing narrower. a solid-state DNP-NMR In- spectrometer designed by Bruker 2 the spy nucleus for detection. These indirect detection methods re- deed, Bajaj etC- al. [24] haveN shown narrow2D linewidths correlationsBiospin in[22] the. In center addition to a 400 MHz wide-bore spectrometer,

(kHz) 3 quire several parameters to be optimized empirically in order to the system includes a low-temperature MAS probe capable of spin- π achieve the most efficient coherence transfer. These experiments 4 of a large protein where the structure is less perturbedning 3.2 by mm the rotors glass at frequencies up to 17 kHz at temperatures

/2 often suffer from poor signal-to-noise ratios. In solid samples with 1 5 1 around 100 K, and a 263 GHz gyrotron providing 5 W microwave forminglong proton solvent. relaxation times,In conclusion, such as proline ( weT1( H) have60 s shownat room that DNP can dra-  ω  irradiation to the sample. This microwave power is high enough to 6 temperature), the duration of 2D experiments for indirect 14N matically reduce the experimental time for indirectsaturate detection the DNP enhancement of in typical samples used and has a detection can be on the order of days. As we shall show in the Com- 7 sufficient stability for long term 2D spectroscopy (several days). nitrogen-14munication, the signals combination in of solid-state dynamic nuclear NMR. polarization With further improve- 8 Acquisition times are not limited by the duty cycle or stability of 9 ments in DNP efficiency and faster magic anglethe spinning gyrotron, which at canlow operate continuously for an indefinite per- 14 10 N-SQC temperatures* Corresponding author. it Fax: may +41 21 be 693 94possible 35. to extract importantiod. The spinning structural frequency of 15.625 kHz was stable to ±2 Hz for E-mail address: marcanthony.caporini@epfl.ch (M.A. Caporini). the duration of the experiments and is comparable to a standard 1 14 7075605 65505540453 parameters from unlabeled biomolecules through H– N correla- δ (ppm) 1090-7807/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.1 2 tions.doi:10.1016/j.jmr.2010.04.014 Currently, the resolution in the H spectra is limited by sam-

13 a 13 d 14 ple preparation conditions and insufficient magic angle spinning Fig. 1. (a) Chemical structure of proline with the C , C , and N shown in bold. 13 14 Wednesday,(b) 13C– 1014N April correlation 13 spectrum of U–13C-enriched proline with 20 mM TOTAPOL frequencies, thus we are limited to C– N correlations where 13 in a glass-forming mixture, obtained in a few hours with the HMQC method [6] the C resolution is better. Alternatively, one could use selectively using DNP, leading to an enhancement e 40 after cross-polarization from protons labelled 13C in large biomolecules to determine targeted quadrupo- 13  to C in a 400 MHz solid-state DNP-NMR spectrometer with a 3.2 mm sapphire lar coupling parameters. rotor spinning at 15.625 kHz at 110 K. For recoupling the REDOR sequence [25] was used to transfer the coherences from 13Cto14N and back. (c) 13C–14N correlation spectrum of a polycrystalline powder of pure 13C-enriched proline without any radicals, also recorded with the HMQC method [6], but without DNP in 7 days at Acknowledgments 800 MHz with a 2.5 mm zirconium rotor spinning at 31.25 kHz at 300 K. For 13 recoupling the SFAM sequence [26,27] was used to transfer coherences from Cto The authors are greatly indebted to Melanie Rosay, Frank 14 N and back, because it is more efficient than REDOR [25] at high spinning Engelke, Fabien Aussenac, and Werner Maas of Bruker Biospin. frequencies. Simone Cavadini, Simone Ulzega, Pascal Miéville, Paul Vasos, and Martial Rey helped with the experiments. This work was supported 13 state-of-the-art MAS probe. Fully C-enriched L-proline was dis- by the Swiss National Science Foundation (Grant 200020-124694

solved in D8-glycerol:D2O:H2O (60:30:10 v/v) [23] which gives a to G. Bodenhausen and P. Vasos, and grant CRSI20-122708 to J.-P. glassy matrix at temperatures near 100 K. Three samples were pre- Ansermet, S. Alberti, G. Bodenhausen, J.-P. Hogge, and M.Q. Tran), pared with the bi-radical TOTAPOL [17] at concentrations of 5, 10, the Swiss Federal Institute of Technology in Lausanne (EPFL), the and 20 mM. These concentrations resulted in DNP enhancement Swiss Commission for Technology and Innovation (CTI Grant factors e = 20, 26 and 40. These enhancement factors are defined 9991.1 PFIW-IW to G. Bodenhausen, P. Dyson, J.-P. Ansermet, and as the ratios of 13C signal amplitudes observed after cross-polariza- P. Vasos), and the French CNRS. tion from protons with and without microwaves. At 100 K, the three samples with increasing biradical concentrations had much reduced proton longitudinal relaxation times that were very close References 1 to the time constants of the DNP process, i.e., T1( H) TDNP 14, 8,   [1] Z.H. Gan, Measuring amide nitrogen quadrupolar coupling by high-resolution and 6 s for 5, 10, and 20 mM TOTAPOL, respectively. Two-dimen- 14N/13C NMR correlation under magic-angle spinning, J. Am. Chem. Soc. 128 14 13 sional experiments with indirect detection of N via C in combi- (2006) 6040–6041. Communication

pubs.acs.org/JACS

Dynamic Nuclear Polarization Enhanced Natural Abundance 17O Spectroscopy § § Fredé rić Blanc,*,†,‡ Luke Sperrin,† David A. Jefferson,† Shane Pawsey, Melanie Rosay, and Clare P. Grey†,⊥ †Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom ‡Department of Chemistry, Stephenson Institute for Renewable Energy, University of Liverpool, Crown Street, Liverpool L69 7ZD, United Kingdom §Bruker BioSpin Corporation, 15 Fortune Drive, Billerica, Massachusetts 01821, United States ⊥Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, United States *S Supporting Information with poor signal-to-noise (S/N) ratios even when a ABSTRACT: We show that natural abundance oxygen-17 combination of very high fields, large sample volume, and/or NMR of solids could be obtained in minutes at a moderate extended acquisition times are used.2d,h Isotopic enrichment in magnetic field strength by using dynamic nuclear polar- 17O is thus required, and samples have to be prepared from ization (DNP). Electron spin polarization could be 17 17 extremely expensive O-labeled chemicals such as O2 and transferred either directly to O spins or indirectly via H O, with often extensive and challenging synthesis proce- 1 2 H spins in inorganic oxides and hydroxides using an dures, undoubtedly limiting the use of 17O NMR. oxygen-free solution containing a biradical polarization High-field dynamic nuclear polarization (DNP)4 is currently agent (bTbK). The results open up a powerful method for revolutionizing solid-state NMR by enhancing NMR sensitivity rapidly acquiring high signal-to-noise ratio solid-state by multiple orders of magnitude, using a microwave-induced 17 NMR spectra of O nuclear spins and to probe sites on transfer of polarization from electron to nuclear spins at or near the surface, without the need for isotope labeling. cryogenic temperatures (4−110 K).4c,5 Stable radicals such as TEMPO6 or biradicals, TOTAPOL,7 bTbK8 or tCTbK9 are used as a source of the electrons under aqueous4c,6 or s one of the most abundant elements on Earth and in the nonaqueous conditions.10 Saturation of the electron para- A human body, oxygen is ubiquitous in minerals and is a magnetic resonance (EPR) transitions of the radicals results in major component in biologically relevant compounds such as either polarization enhancement of the abundant 1H spins, organic molecules, peptides and biomolecules. Oxygen-based which is subsequently transferred to lower γ nuclei such as 13C materials and molecules are of considerable importance in a or 15N by cross-polarization (a process called indirect DNP)11 wide range of technologically relevant areas including ceramics, or direct polarization of the heteronuclei (direct DNP).12 catalysts and polymers, being intimately involved in bonding Following the pioneering work of Griffin et al.,4c DNP NMR and controlling structure and physical properties. As such, the has been successfully applied to biologically relevant mole- development of methods for their characterization is of cules13 and more recently in materials science14 to an fundamental importance. increasingly large range of nuclei,4c,12a,14b,c,15,16 with 17O Solid-state nuclear magnetic resonance (NMR) spectroscopy indirect DNP having only been successfully performed very 17 16 is one of most powerful analytical techniques available today to recently on the simple case of O-labeled H2O. Here we investigate the local structure of powdered solids due to its show that natural abundance 17O NMR spectra of inorganic sensitivity at the atomic lengthscale through the interactions of solids can be easily obtained in minutes using either indirect or the nuclear spins with their surroundings.1 Commonly studied direct DNP enhanced NMR, solving the intrinsically poor nuclei in solid-state NMR include spin I = 1/2 nuclei such as sensitivity of 17O nuclear spins. 1H, 13C, 15N, 29Si, 31P but also nuclei having a spin larger than The experimental conditions required to observe DNP 1/2 (quadrupolar nuclei) such as 2H, 14N or 27Al. Oxygen enhancement are essential to the increase in sensitivity, and we 14a,b NMR investigations are, however, relatively scarce, because the have used an already well-established and robust approach 8 only NMR-active isotope of oxygen, 17O, is also the least for sample preparation using a solution of biradical bTbK in 16 abundant (0.037%) of all of the three isotopes ( O - 99.76%, water-free and oxygen-free 1,1,2,2-tetrachloroethane (C2Cl4H2) 10 18O - 0.2%), making 17O one of the more challenging nuclei to solvent to wet the inorganic solids (see the Supporting observe by NMR spectroscopy.2 The quadrupolar nature of this Information [SI]) for details of all samples preparation. Figure 1a shows the remarkable effect of DNP on the 1H 17O nucleus (I = 5/2) also adds to the complexity, as the well- 17 17 established magic angle spinning (MAS) technique fails to selective CP MAS spectra of Mg( OH)2 where we observed completely remove the second-order quadrupolar broadening,3 leading to low-resolution spectra. Natural abundance solid-state Received: January 14, 2013 17 NMR spectra of O are therefore rare and are often associated Published: February 5, 2013 View Online / Journal Homepage / Table of Contents for this issue ChemComm Dynamic Article Links Quadrupoles © 2013 American Chemical Society 2975 dx.doi.org/10.1021/ja4004377 | J. Am. Chem. Soc. 2013, 135, 2975−2978 Cite this: Chem. Commun., 2012, 48,1988–1990 27 www.rsc.org/chemcomm COMMUNICATION DNP of AlView Online Dynamic nuclear polarization of quadrupolar nuclei using cross polarizationmatrices from protons: at 100 K17 surface-enhanced. Like in our recent aluminium-27 DNP studies NMR of 13Cw and 29 15,16 Veronika Vitzthum,Sia Pascal in nanoporous Mie´ ville,*a Diego materials, Carnevale,a Marc A.we Caporini, useda David water Gajan,withoutb b c c c c Christophe Copeglycerol´ ret, Moreno as Lelli, a solventAlexandre in Zagdoun, this work,Aaron where J. Rossini,g-aluminaAnne Lesage, powder Lyndon Emsleyc and Geoffrey Bodenhausenadef was wetted with a H2O/D2O mixture containing TOTAPOL. Received 22nd September 2011, Accepted 7th December 2011 DOI: 10.1039/c2cc15905hFor g-alumina, it appears that only three parameters are relevant for the DNP enhancement factor : (i) the mole The surface of c-alumina nanoparticles can be characterized by between signals from theeDNP surface and the bulk. This can be 1 27 dynamic nuclear polarizationfraction (DNP)x of surface-enhanced H2O in NMR the of H2O/Dachieved2O by cross-polarization mixture (typically (CP) from Hto 0.1Alo as protons 27Al. DNP is combined with cross-polarization and MQ-MAS can only be found near the surface of aluminas, either in hydroxyl to determine localx symmetrieso 0.2) of 27 (ii)Al sites the at the TOTAPOL surface. concentrationgroups or in solvent moleculesc in close the to the H surface.2O/D11 While2O CP is efficient for I =1/2nucleisuchas13Cor29Si, it is less popular for mixture (typically 5 o c o 15 mM), and (iii)27 the extent of Aluminas and in particular g-alumina are some of the most quadrupolar nuclei such as Al because spin-locking is difficult widely used high-surface-areawetting, expressed oxides; they are in applied terms in ofunder the MAS ratio as a resultr of the time-dependencewhich is defined of the quadrupolar 12 solvent1 27 13 industry either directly as catalysts or as supports for single-site splitting. Since CP from Hto Al is not very efficient it has or nanoparticle-basedas catalysts. the mass1 In particular, of the the surface doped is key to solventbeen used divided primarily to by establish the atomic total connectivities, mass rather of than the formation of reactive sites for supported catalysts in various to increase the sensitivity. the sample (typically 0.05 rsolvent 0.45). For incipient applications such as alkene hydrogenation,2 metathesis3 and o With the recento development of Dynamic Nuclear Polarization 4 18 14 alkene polymerization.wetnessSurface sites impregnation are also involved in the (IWI), low (DNP)rsolvent combined= withrIWI MAS,isNMR such sensitivity that can any be greatly temperature activation of hydrogen and alkanes,5 in numerous enhanced and experiments that were hitherto limited by low catalytic dehydrationfurther processes,1 addedand probably solvent in the stabilization will notsignal-to-noise be absorbed; ratios become if feasibler > in a reasonablerIWI, the time frame. of supported metal nanoparticles.6 Despite recent advances,6,7 a Recently, we have shown that it is possible to enhance the signals sample has the appearance of a13 viscous paste. The dry powder15 detailed understanding of the surface structure of g-alumina at a of Coforganicmoleculesgraftedonsurfaces. We have also molecular level stillobtained requires refinement. for r r is to bedemonstrated preferred, that one since can selectively it is enhance easier the to signals of

Downloaded by Massachusetts Institute of Technology on 24 February 2012 o IWI 29 16 Published on 11 January 2012 http://pubs.rsc.org | doi:10.1039/C2CC15905H Solid-state NMR spectroscopy is one of the most powerful Si sites on the surfaces of mesoporous and non-porous silicates. methods to probe atomic-levelpack into surface the structures NMR without rotor. requiring AsTo expected, obtain good DNP the signal ratio enhancementrIWI = we 0.45 prepared the long-range order,8 forparticularly-alumina for carbon-containing corresponds adsorbed or tosamples a fully by ‘incipient impregnated wetness impregnation’ sample with and an aqueous chemisorbed species, or forg silicon-containing materials. Surface solution of stable biradicals such as TOTAPOL. Under MAS sites can be readilygives identified thethrough best differences results in chemical (Fig. shifts, S1aat temperatures in ESIw). of 100 K, saturating the EPR transitions of provided the 13Cor29Si spins are sufficiently concentrated or the radical with ca. 5Wcontinuous-wavemicrowaveirradiation isotopically enriched inThe a speci radicalfic manner. Aluminium-27concentration magic cleadshas to enhanced a pronounced polarization of e theffect solvent on protons, the which 1 13 angle spinning (MAS) solid-state NMR (27Al has a spin I =5/2 is then transferred by CP from the Hnucleitothe Cor DNP enhancement e (Fig.9 S1b29Si nuclei in onESI thew surface.). For g-alumina e is and a quadrupolar coupling constant CQ between 0 and 16 MHz) can also be used to probe the bulk structure of aluminas and Here, we show that a similar strategy can be used to maximised at relatively low concentrations (c =4mM,27 r =0.45). aluminosilicates.10 To obtain structural information about the selectively enhance the signals of Al sites at the surface of surface of these materialsNote one that requires the a method optimal to discriminatec of TOTAPOLg-alumina. First, is we lower optimize here the sample than preparation, in our which is 15,16the key to good DNP enhancements. Large variations of DNP work on mesoporous silicas. enhancements are observed for different sample preparations; in a Institut des Sciences et Inge´nierie Chimiques, Ecole Polytechnique 27 Finally, the mole fraction ourx of hands, H theO best inAl the enhancement solvent achieved mixture was eDNP E 20. Fe´de´rale de Lausanne, EPFL, Batochime, 1015 Lausanne, The enhancement2 reached on protons is the same as that for Switzerland. E-mail: pascal.mieville@epfl.ch 19 b 1 27 27 Department of Chemistry,was ETH optimized. Zu¨rich, CH-8093 Earlier Zu¨rich, DNPaluminium. studies The haveH– Al shown CP efficiency that is notx affcanected by the Fig. 1 Al spectra of g-alumina (r = 0.45, c = 4 mM and x = 0.10): Switzerland εCP O~20 polarization state of the protons. This corresponds to a 400-fold c Centre de RMN a` haveTres Hauts a Champs, significant Universite´ de Lyoneffect on DNPacceleration enhancements, of signal acquisition, andwith as a result,optimal we were able (a) excited directly with a single rf pulse (signals from both surface and (CNRS/ENS Lyon/UCB Lyon 1), 69100 Villeurbanne, France d De´partement de Chimie,values Ecole betweenNormale Supe´rieure, 0.05 and 0.10. Interestingly,to record a DNP surface-enhanced in the ranges CP-MQMAS 0.025 spectrumo of bulk); (b) excited by CP from protons with microwave saturation of the 24 Rue Lhomond, 75231, Paris Cedex 05, France g-alumina in less than 24 hours. This would have required a e Universite´ Pierre-et-Marie Curie, Paris, France full year without DNP. EPR transitions of the TOTAPOL radical (surface enhanced signals); f x o 0.25 and 2 o c o 20 mM there is little effect on the DNP CNRS, UMR 7203, Paris, France Most successful DNP experiments on small molecules and w Electronic supplementary information (ESI) available: Detailed and (c) CP without microwave irradiation (signals from surface only). experimental proceduresenhancements and additional data here (Fig. S1 (Fig. and S2). S1c See inlarge ESI biomoleculesw), in contrast have relied on to dissolving the ratio or suspendingr, the DOI: 10.1039/c2cc15905hfor which a much stronger dependencetarget molecules in is glycerol/water observed. mixtures, The which DNP form glassy 1988 Chem. Commun.,enhancements2012, 48,1988–1990 varied in the range 0.2Thiso journale/eopt is c oThe Royal1.0 Society for 0.05 of Chemistryr 2012 CP experiments, proving that the DNP-enhanced SQ coherence of r r 0.5. 27Al can be converted into TQ coherence without significant losses. Wednesday, 10 April 13 Fig. 1 compares the 27Al spectra obtained via direct excitation Fig. 2 shows a DNP surface-enhanced 2D-CP-MQMAS

Downloaded by Massachusetts Institute of Technology on 24 February 2012 27 Published on 11 January 2012 http://pubs.rsc.org | doi:10.1039/C2CC15905H without DNP, CP without DNP, and CP with DNP. The spectrum of g-alumina. Two distinct Al resonances associated reduced signal intensity of the CP spectra compared to direct with tetrahedral Al(IV)(diso = 67 ppm) and octahedral Al(VI) excitation reflects the selectivity of CP for surface sites. The DNP (diso = 8 ppm) alumina species are visible. A penta-coordinated enhancement here is e E 20. In cases where only the surface is of aluminium species could not be identified in our spectra. It is interest, the DNP–CP approach leads to the suppression of bulk worth bearing in mind that, in order to achieve a DNP 27Al sites as 1Hnucleiareonlylocatednearthesurfaceofthe enhancement, our wetted, glassy and frozen sample is observed material and affords a reduction in experimental time on the under conditions that greatly differ from a dry powder at room order of 400. temperature. This explains the absence of penta-coordinated

Multiple-quantum magic-angle spinning (MQMAS) experi- surface sites that may be coordinated by H2O or TOTAPOL in ments20 offer a means to remove second-order quadrupolar contrast to tetrahedral and octahedral sites. Hydrophobic broadening and, hence, obtain higher resolution NMR spectra preparation methods for DNP25 should help to resolve water of half-integer quadrupolar nuclei. The combination of CP sensitive sites in the future. with triple quantum TQ-MAS experiments has previously been In conclusion, we have shown that DNP can be used accomplished by two different approaches: (i) by transferring effectively to enhance 27Al signals on surfaces with signal polarization directly from protons to the TQ coherence21, or enhancements of up to a factor e E 20, corresponding to time (ii) by transferring magnetization from 1Hto27Al single-quantum savings of a factor 400, and gave guidelines for preparing suitable (SQ) coherence via CP, then converting the SQ coherence to samples by incipient wetness impregnation. We illustrated the longitudinal magnetization of 27Al, followed by conversion to approach with samples of g-alumina, which offer good models TQ coherence.22 These techniques tend to be rather inefficient, for a large palette of materials. The approach could be extended and their use to observe surface signals is challenging. Here, to other quadrupolar nuclei and to solvents other than water, we used the second method in combination with DNP surface which would be of great interest for materials used in hetero- enhancement. We modified the phase-modulated z-filtered geneous catalysis. Non-aqueous glass-forming solvents containing 22 experiment to obtain a phase-modulated shifted-echo split-t1 controlled concentrations of protons and radicals that can wet version,23,24 as shown in Fig. S2 (ESIw). One-dimensional the surface of porous materials should lead to effective DNP experiments yielded similar enhancement factors as ordinary enhancements.

This journal is c The Royal Society of Chemistry 2012 Chem. Commun., 2012, 48,1988–1990 1989 Communication

pubs.acs.org/JACS

Dynamic Nuclear Polarization Enhanced Natural Abundance 17O Spectroscopy § § Fredé rić Blanc,*,†,‡ Luke Sperrin,† David A. Jefferson,† Shane Pawsey, Melanie Rosay, and Clare P. Grey†,⊥ †Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom ‡Department of Chemistry, Stephenson Institute for Renewable Energy, University of Liverpool, Crown Street, Liverpool L69 7ZD, United Kingdom §Bruker BioSpin Corporation, 15 Fortune Drive, Billerica, Massachusetts 01821, United States ⊥Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, United States *S Supporting Information with poor signal-to-noise (S/N) ratios even when a ABSTRACT: We show that natural abundance oxygen-17 combination of very high fields, large sample volume, and/or NMR of solids could be obtained in minutes at a moderate extended acquisition times are used.2d,h Isotopic enrichment in magnetic field strength by using dynamic nuclear polar- 17O is thus required, and samples have to be prepared from ization (DNP). Electron spin polarization could be 17 17 extremely expensive O-labeled chemicals such as O2 and transferred either directly to O spins or indirectly via H O, with often extensive and challenging synthesis proce- 1 2 H spins in inorganic oxides and hydroxides using an dures, undoubtedly limiting the use of 17O NMR. oxygen-free solution containing a biradical polarization High-field dynamic nuclear polarization (DNP)4 is currently agent (bTbK). The results open up a powerful method for revolutionizing solid-state NMR by enhancing NMR sensitivity rapidly acquiring high signal-to-noise ratio solid-state by multiple orders of magnitude, using a microwave-induced 17 NMR spectra of O nuclear spins and to probe sites on transfer of polarization from electron to nuclear spins at or near the surface, without the need for isotope labeling. cryogenic temperatures (4−110 K).4c,5 Stable radicals such as TEMPO6 or biradicals, TOTAPOL,7 bTbK8 or tCTbK9 are used as a source of the electrons under aqueous4c,6 or s one of the most abundant elements on Earth and in the nonaqueous conditions.10 Saturation of the electron para- A human body, oxygen is ubiquitous in minerals and is a magnetic resonance (EPR) transitions of the radicals results in major component in biologically relevant compounds such as either polarization enhancement of the abundant 1H spins, organic molecules, peptides and biomolecules. Oxygen-based which is subsequently transferred to lower γ nuclei such as 13C materials and molecules are of considerable importance in a or 15N by cross-polarization (a process called indirect DNP)11 wide range of technologically relevant areas including ceramics, or direct polarization of the heteronuclei (direct DNP).12 catalysts and polymers, being intimately involved in bonding Following the pioneering work of Griffin et al.,4c DNP NMR and controlling structure and physical properties. As such, the has been successfully applied to biologically relevant mole- development of methods for their characterization is of cules13 and more recently in materials science14 to an fundamental importance. increasingly large range of nuclei,4c,12a,14b,c,15,16 with 17O Solid-state nuclear magnetic resonance (NMR) spectroscopy indirect DNP having only been successfully performed very 17 16 is one of most powerful analytical techniques available today to recently on the simple case of O-labeled H2O. Here we 17 investigate the local structure ofJournal powdered of the American solids due Chemical to its Society show that natural abundance O NMR spectraCommunication of inorganic sensitivity at the atomic lengthscale through the interactions of solids can be easily obtained in minutes using either indirect or 1 kHz). It was recently demonstrated that the combination of the the nuclear spins with their surroundings. Commonly studied direct DNP enhancedCPMG pulse NMR, sequence solving with DNP the yields intrinsically dramatic further poor 17improvements of sensitivity.18 CPMG sequences involving nuclei in solid-state NMR include spin I = 1/2 nuclei such as sensitivity of O nuclear spins. 19 1 13 15 29 31 The experimentalquadrupolar conditions nuclei (QCPMG) requiredare routinely to observe used in solids DNP H, C, N, Si, P but also nuclei having a spin larger than and make use of the much longer T2′ observed for the typically 1/2 (quadrupolar nuclei) such as 2H, 14N or 27Al. Oxygen enhancement arebroad essential NMR resonances to the of increase nuclear spins in greater sensitivity, than 1/2. and Here, we 14a,b NMR investigations are, however, relatively scarce, because the have used an alreadya further signal well-established enhancement of and a factor robust≈ 160 approach is obtained 17 (Figure 1a), showing that this is a powerful way to detect the8 only NMR-active isotope of oxygen, O, is also the least for sample preparationbroad signals using of insensitive a solution nuclei of such biradical as 17O at bTbK natural in 16 16 abundant (0.037%) of all of the three isotopes ( O - 99.76%, water-free andabundance. oxygen-free Under 1,1,2,2-tetrachloroethane static conditions, dramatic (C similar2Cl4H2) 18 17 solvent10 to wetenhancements the inorganic were also solids observed (see (Figure the S5 Supportingin SI), an 17 O - 0.2%), making O one of the more challenging nuclei to important finding for quadrupolar nuclei which often give rise Communication DNP of n.a. O 2 pubs.acs.org/JACS observe by NMR spectroscopy. The quadrupolar nature of this Information [SI])to spectra for details whose line of width all samples frequently preparation. exceeds the available 0.038% Figure 1a showsMAS frequency. the remarkable effect of DNP on the 1H 17O Journal of the American Chemical Society Communication nucleus (I = 5/2) also adds to the complexity, as the well- 17 17 17 DynamicThe sensitivity Nuclear of PolarizationO indirect17 Enhanced DNP observed Natural Abundance on O 17O selective CP MAS spectra of Mg( OH)2 where we17 observed kHz). It was recently demonstrated that the combination of the established magic angle spinning (MAS) technique fails to enrichedSpectroscopy materials clearly opens the way to collect O spectra CPMG pulse sequence with DNP yields dramatic further 3 ,†,‡ † † §1 17 § atFre naturaldé rić Blanc, abundance.* Luke Sperrin, FiguresDavid A.1b Jeff anderson, cShane show Pawsey, the MelanieH O Rosay, CP 18 completely remove the second-order quadrupolar broadening, †,⊥ improvements of sensitivity. CPMG sequences involving and Clare P. Grey 19 and QCPMG MAS spectra of both Mg(OH)2 and Ca(OH)2 at quadrupolar nuclei (QCPMG) are routinely used in solids leading to low-resolution spectra. Natural abundance solid-state Received: January†Department 14, of Chemistry, 2013 University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United17 Kingdom natural‡ abundance, prepared in a similar manner to Mg( OH)2 and make use of the much longer T2′ observed for the typically 17 Department of Chemistry, Stephenson Institute for Renewable Energy, University of Liverpool, Crown Street, Liverpool L69 7ZD, NMR spectra of O are therefore rare and are often associated Published: FebruarydiscussedUnited Kingdom 5, above. 2013 Each DNP spectrum was acquired in only 10 broad NMR resonances of nuclear spins greater than 1/2. Here, min§Bruker with BioSpin 64 Corporation, scans 15and Fortune a recycle Drive, Billerica, delay Massachusetts of 10 01821, s. Although United States 1H 17O a further signal enhancement of a factor ≈ 160 is obtained ⊥Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, United States (Figure 1a), showing that this is a powerful way to detect the CPS MAS spectra acquired under microwave irradiation yield 17 © 2013 American Chemical Society 2975 dx.doi.org/10.1021/ja4004377* Supporting Information17 | J. Am. Chem. Soc. 2013, 135, 2975−2978 broad signals of insensitive nuclei such as O at natural barely visible O signals (which are not visible above the noise 16 with poor signal-to-noise (S/N) ratios even when a abundance. Under static conditions, dramatic similar withoutABSTRACT: DNP),We show the that natural combination abundance oxygen-17 with QCPMGcombination of produces very high fields, large very sample volume, and/or NMR of solids could be obtained in minutes17 at a moderate extended acquisition times are used.2d,h Isotopic enrichment in enhancements were also observed (Figure S5 in SI), an intensemagnetic naturalfield strength abundance by using dynamic nuclearO signals. polar- 17O is thus required, and samples have to be prepared from fi ization (DNP). Electron spin polarization could be 1 17 17 important nding for quadrupolar nuclei which often give rise 17 extremely expensive O-labeled chemicals such as O2 and Thetransferred fast either acquisition directly to O spins of or indirect indirectly via DNPH O,H withO often CP extensive QCPMG and challenging synthesis proce- 1 2 to spectra whose line width frequently exceeds the available H spins in inorganic oxides and hydroxidesεCP using an O~17dures, undoubtedly limiting the use of 17O NMR. spectra allows us to collect multidimensional correlation 4 oxygen-free solution containing a biradical polarization High-field dynamic nuclear polarization (DNP) is currently MAS frequency. spectroscopyagent (bTbK). The resultsspectra open up and a powerful establish method for nuclearrevolutionizing spin solid-state connectivities. NMR by enhancing NMR sensitivity The sensitivity of 17O indirect DNP observed on 17O rapidly acquiring high signal-to-noise ratio solid-state by multiple orders of magnitude, using a microwave-induced 17 1 17 17 FigureNMR spectra 2 shows of O nuclear the spins 2D and toH probe sitesO on CP HETCORtransfer of polarization spectrum from electron of to nuclear spins at enriched materials clearly opens the way to collect O spectra or near the surface, withoutε the need for isotope labeling. ~3400 4c,5 CPCPMG Ocryogenic temperatures (4−110 K). Stable radicals such as 1 17 TEMPO6 or biradicals, TOTAPOL,7 bTbK8 or tCTbK9 are at natural abundance. Figures 1b and c show the H O CP 4c,6 used as a source of the electrons under aqueous or and QCPMG MAS spectra of both Mg(OH)2 and Ca(OH)2 at s one of the most abundant elements on Earth and in the nonaqueous conditions.10 Saturation of the electron para- 17 A human body, oxygen is ubiquitous in minerals and is a magnetic resonance (EPR) transitions of the radicals results in natural abundance, prepared in a similar manner to Mg( OH)2 1 17 17 19 major component in biologically relevant compounds such as either polarization enhancement of the abundant 1H spins, 13 discussed above. Each DNP spectrum was acquired in only 10 Figure 1. H O selective CP (full black lines) and CP QCPMG organic molecules, peptides and biomolecules. Oxygen-based which is subsequently transferred to lower γ nuclei such as C 1 17 17 17 materials and molecules are of considerable importance in a or 15N by cross-polarization (a process called indirect DNP)11 min with 64 scans and a recycle delay of 10 s. Although H O (green lines) MAS spectra of (a) O-enriched Mg( OH)2, (b) wide range of technologically relevant areas including ceramics, or direct polarization of the heteronuclei (direct DNP).12 catalysts and polymers, being intimately involved in bonding Following the pioneering work of Griffin et al.,4c DNP NMR CP MAS spectra acquired under microwave irradiation yield natural abundance Mg(OH)2 and (c) natural abundance Ca(OH)2 17 − 1 and controlling structure and physical properties. As such, the has been successfully applied to biologically relevant mole- barely visible O signals (which are not visible above the noise recorded with microwave irradiation at ν0(e ) = 263.7 GHz. The H development of methods for their characterization is of cules13 and more recently in materials science14 to an 17O selective CP MAS spectrum of 17O-enriched Mg(17OH) recorded fundamental importance. increasingly large range of nuclei,4c,12a,14b,c,15,16 with 17O without DNP), the combination with QCPMG produces very 2 Solid-state nuclear magnetic resonance (NMR) spectroscopy indirect DNP having only been successfully performed very 17 17 16 intense natural abundance O signals. without microwave irradiation is shown in red. All the spectra were is one of most powerful analytical techniques available today to recently on the simple case of O-labeled H2O. Here we 1 17 1 17 investigate the local structure of powdered solids due to its show that natural abundance 17O NMR spectra of inorganic The fast acquisition of indirect DNP H O CP QCPMG recorded at ∼105 K with ν0( H) = 400.430 MHz and ν0( O) = sensitivity at the atomic lengthscale through the interactions of solids can be easily obtained in minutes using either indirect or 1 the nuclear spins with their surroundings.1 Commonly studied direct DNP enhanced NMR, solving the intrinsically poor spectra allows us to collect multidimensional correlation 54.280 MHz under νrot = 12.5 kHz and SPINAL-64 H heteronuclear 17 21 1 nuclei in solid-state NMR include spin I = 1/2 nuclei such as sensitivity of O nuclear spins. spectroscopy spectra and establish nuclear spin connectivities. decoupling with ν1( H) = 100 kHz. Sixty-four scans with a 10 s 1H, 13C, 15N, 29Si, 31P but also nuclei having a spin larger than The experimental conditions required to observe DNP 1 17 1/2 (quadrupolar nuclei) such as 2H, 14N or 27Al. Oxygen enhancement are essential to the increase in sensitivity, and we Figure 2 shows the 2D H O CP HETCOR spectrum of 14a,b recycle delay were averaged (experimental time = 10 min). The NMR investigations are, however, relatively scarce, because the have used an already well-established and robust approach 8 QCPMG spectra shown were processed by first adding all the 60 only NMR-active isotope of oxygen, 17O, is also the least for sample preparation using a solution of biradical bTbK in 16 abundant (0.037%) of all of the three isotopes ( O - 99.76%, water-free and oxygen-free 1,1,2,2-tetrachloroethane (C2Cl4H2) 10 echoes together followed by Fourier transformation (see SI), and the 18O - 0.2%), making 17O one of the more challenging nuclei to solvent to wet the inorganic solids (see the Supporting observe by NMR spectroscopy.2 The quadrupolar nature of this Information [SI]) for details of all samples preparation. intensities are scaled as indicated. Samples were wet with 20 mM 1 17 1 17 17 19 Figure 1a shows the remarkable effect of DNP on the H O Figure 1. H O selective CP (full black lines) and CP QCPMG 8 10 nucleus (I = 5/2) also adds to the complexity, as the well- 17 17 bTbK C Cl H solution. ε , ε and ε were established magic angle spinning (MAS) technique fails to selective CP MAS spectra of Mg( OH)2 where we observed 17 17 2 4 2 H OCP OCPQCPMG (green lines) MAS spectra of (a) O-enriched Mg( OH)2, (b) determined on the 17O labeled samples and were 24, 21, 3400 for completely remove the second-order quadrupolar broadening,3 1 17 Figureleading to 2.low-resolutionContour spectra. plot Natural of abundance the two-dimensional solid-state Received: January (2D) 14, 2013H O CP natural abundance Mg(OH)2 and (c) natural abundance Ca(OH)2 17 17 17 − 1 Mg( OH)2 and 17, 17, 4000 for Ca( OH)2 (see SI), respectively. QCPMGNMR spectra HETCOR of O are therefore spectra rare and are of often Mg(OH) associated 2 atPublished: naturalFebruary abundance 5, 2013 under recorded with microwave irradiation at ν0(e ) = 263.7 GHz. The H 2g 2e Wednesday, 10 April 13 − 5a 17 17 17 Central transition simulations for Mg(OH)2 and Ca(OH)2 are microwave irradiation© 2013 American at Chemicalν0(e Society) = 263.72975 GHz. dx.doi.org/10.1021/ja4004377All the others| J. Am. Chem. Soc. 2013, 135, 2975−2978 O selective CP MAS spectrum of O-enriched Mg( OH)2 recorded shown in black dashed lines (see Figure S3 in SI). acquisition parameters and sample details are identical to those of without microwave irradiation is shown in red. All the spectra were 1 17 Figure 1. A total of 9 t increments of 80 μs with 16 scans and a 10 s recorded at ∼105 K with ν0( H) = 400.430 MHz and ν0( O) = 1 1 1 54.280 MHz under νrot = 12.5 kHz and SPINAL-64 H heteronuclear recycle delay were averaged (experimental time = 24 min). (a) H 21 1 17 1 decoupling with ν1( H) = 100 kHz. Sixty-four scans with a 10 s an O signal DNP enhancement factor of εO CP = 21 MAS spectrum. (b) 2D spectrum. Near left: HF1 sum projection of the 2D spectrum. Top: 17OF sum projection. recycle delay were averaged (experimental time = 10 min). The (determined as ε =(IDNP − IBoltzmann)/IBoltzmann where IDNP 2 QCPMG spectra shown were processed by first adding all the 60 and IBoltzmann are the signal intensities with and without echoes together followed by Fourier transformation (see SI), and the intensities are scaled as indicated. Samples were wet with 20 mM microwave irradiation, respectively), demonstrating that the 8 10 1 bTbK C2Cl4H2 solution. εH, εOCP and εOCPQCPMG were direct H polarization enhancement εH = 24 (see SI) is Mg(OH)2 at natural abundance (with a 20 mM solution of 17 1 17 ffi 8 10 determined on the O labeled samples and were 24, 21, 3400 for Figure 2. Contour plot of the two-dimensional (2D) H O CP e ciently transferred from the solvent to the hydroxide groups bTbK in C2Cl4H2 ) using QCPMG trains during signal 17 17 Mg( OH)2 and 17, 17, 4000 for Ca( OH)2 (see SI), respectively. QCPMG HETCOR spectra of Mg(OH)2 at natural abundance under 17 2g 2e − 5a of the solid. The observed O second-order quadrupolar line acquisitions and under microwave irradiation. This spectrum Central transition simulations for Mg(OH)2 and Ca(OH)2 are microwave irradiation at ν0(e ) = 263.7 GHz. All the others shape can be fitted with reasonable agreement to the previously was acquired in less than 30 min with 16 scans and clearly shown in black dashed lines (see Figure S3 in SI). acquisition parameters and sample details are identical to those of 2g 17 1 Figure 1. A total of 9 t increments of 80 μs with 16 scans and a 10 s determined quadrupolar parameters of Mg(OH)2. We also shows that the O site in Mg(OH)2 is connected with a H 1 recycle delay were averaged (experimental time = 24 min). (a) 1H note that the quadrupolar coupling constant in Mg(OH)2 (CQ signal at about 1 ppm in agreement with the expected shift for 17 1 17 2a − 20 an O signal DNP enhancement factor of εO CP = 21 MAS spectrum. (b) 2D spectrum. Near left: HF1 sum projection of = 7.0(2) MHz) is among the largest for O, demonstrating an isolated OH group. This peak was also observed as a 17 (determined as ε =(I − I )/I where I the 2D spectrum. Top: OF2 sum projection. that 17O indirect DNP could be widely applied with the shoulder in the 1H MAS spectrum (Figure 2a), the more DNP Boltzmann Boltzmann DNP and IBoltzmann are the signal intensities with and without experimental conditions used here (B0 = 9.4 T and νrot = 12.5 intense signal being attributed to the C2Cl4H2 solvent. microwave irradiation, respectively), demonstrating that the 1 direct H polarization enhancement εH = 24 (see SI) is Mg(OH)2 at natural abundance (with a 20 mM solution of 2976 dx.doi.org/10.1021/ja4004377 | J. Am. Chem. Soc. 2013, 135, 2975−2978 ffi 8 10 e ciently transferred from the solvent to the hydroxide groups bTbK in C2Cl4H2 ) using QCPMG trains during signal of the solid. The observed 17O second-order quadrupolar line acquisitions and under microwave irradiation. This spectrum shape can be fitted with reasonable agreement to the previously was acquired in less than 30 min with 16 scans and clearly 2g 17 1 determined quadrupolar parameters of Mg(OH)2. We also shows that the O site in Mg(OH)2 is connected with a H note that the quadrupolar coupling constant in Mg(OH)2 (CQ signal at about 1 ppm in agreement with the expected shift for = 7.0(2) MHz) is among the largest for 17O,2a demonstrating an isolated OH− group.20 This peak was also observed as a that 17O indirect DNP could be widely applied with the shoulder in the 1H MAS spectrum (Figure 2a), the more experimental conditions used here (B0 = 9.4 T and νrot = 12.5 intense signal being attributed to the C2Cl4H2 solvent.

2976 dx.doi.org/10.1021/ja4004377 | J. Am. Chem. Soc. 2013, 135, 2975−2978