Determination of Intermolecular Interactions Using Polarization

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Determination of Intermolecular Interactions using Polarization Compensated Heteronuclear Overhauser Effect of Hyperpolarized Spins Jihyun Kim, Mengxiao Liu, Hsueh-Ying Chen, and Christian Hilty Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02934 • Publication Date (Web): 01 Oct 2015 Downloaded from http://pubs.acs.org on October 6, 2015

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Analytical Chemistry

Determination of Intermolecular Interactions using Polarization Compensated Heteronuclear Overhauser Effect of Hyperpolarized Spins Jihyun Kim†, Mengxiao Liu†, Hsueh-Ying Chen†,‡ and Christian Hilty†* †

Department of Chemistry, Texas A&M University, College Station, TX 77843-3255, USA.

‡

present address: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and

Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520, USA. *Corresponding author: e-mail: [email protected]

Abstract The nuclear Overhauser effect (NOE) has long been used as a selective indicator for intermolecular interactions. Due to relatively small changes of signal intensity, often on the order of several percent, quantitative NOE measurements can be challenging. Hyperpolarization of nuclear spins can dramatically increase the NOE intensity by increasing population differences, but poses its own challenge in quantifying the original polarization level. Here, we demonstrate a method for the accurate measurement of intermolecular heteronuclear cross-relaxation rates by simultaneous acquisition of signals from both nuclei. Using this method, we measure cross-relaxation rates between water protons and

19

F of trifluoroacetic acid at concentrations ranging from 23 mM to 72

mM. A concentration independent value of 2.46·10-4 ± 1.02·10-5 s-1M-1 is obtained at a temperature of 301 K, and validated using a non-hyperpolarized measurement. In a broader context, accurate measurement of heteronuclear cross-relaxation rates may enable the study of intermolecular interactions including those involving macromolecules where

19

F atoms can be introduced as site

selective labels.

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Keywords Nuclear Magnetic Resonance, Nuclear Overhauser Effect, Hyperpolarization, Intermolecular Interaction Introduction The nuclear Overhauser effect (NOE) in nuclear magnetic resonance (NMR) is highly dependent on the distance between interacting spins, as well as on molecular motions.1 The NOE therefore has been used as a sensitive indicator of molecular conformation, forming the basis for example for the determination of the structure of biological macromolecules.2 For the same favorable properties, the NOE has been widely used for the determination of intermolecular interactions, involving both small and large molecules. For proteins, the hydration shell has been characterized by observing NOEs originating from water.3,4 For the determination of specific ligand-protein interactions, a large suite of NOE based NMR experiments has further been developed.5 A majority of these applications are based on the interaction between like spins. Heteronuclear NOEs, on the other hand, were the signal of one type of nucleus, such as 1H, is affected by perturbation of the spin polarization of another type, are interesting for various reasons, including the ability to measure signals free from background.6 Heteronuclear cross-relaxation rates have for example been used to determine solvent-solute interaction and hydration phenomena. To this end, Canet et al. used 13C-1H NOEs to investigate the interaction between water and detergent micelles.7 Using

19

F, contacts

between water and fluorinated proteins as well as surfactants were examined.8,9 The interaction between water and small molecules, including perfluorooctanoate and trifluoroacetate, was also studied by determining cross-relaxation rates.10,11 NOE based experiments in general, however, are limited by a relatively small effect, amounting to as little as several percent change in signal intensity of a target spin upon saturation or inversion of a source spin.10 Therefore, in many NOE experiments, long measurement times comprising averaging to acquire one or multidimensional spectra is required. Since the NOE effect directly depends on the deviation from equilibrium of the source spin, non-equilibrium spin systems generated by hyperpolarization can enhance NOE effects 2


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by several orders of magnitude. The spin polarization-induced nuclear Overhauser effect (SPINOE),12 originating from hyperpolarized xenon has been shown by Landon et al. to identify hydrophobic pockets in proteins.13 For NMR in the liquid state, dynamic nuclear polarization (DNP), another hyperpolarization technique capable of polarizing various nuclei in different molecules, has recently been introduced.14 Using

13

C formate hyperpolarized by dissolution DNP,

Merrit et al. have demonstrated the transfer of large signals from

13

C to 1H, with possible

application for metabolic studies.15 Marco-Rius et al. have further proposed to use such transferred signal for tracking the passage of a bolus of hyperpolarized 13C fumarate in-vivo.16 Authors of this manuscript have demonstrated the measurement of intramolecular homonuclear 1H-1H NOEs,17 as well as the use of intermolecular 1H-1H NOEs originating from DNP polarized ligands for the determination of protein-ligand interactions.18,19 For quantitative measurements of cross-relaxation rates using hyperpolarized spin systems, a challenge lies in the accurate determination of the polarization level, which may vary between experiments.15 Here, we introduce a method for accurate measurements of heteronuclear cross-relaxation rates using hyperpolarization by dissolution DNP. We then demonstrate the use of this method to investigate the interaction between a fluorinated solute in a protonated solvent and discuss the accuracy and concentration dependence of the result. Materials and Methods Hyperpolarized NMR. For the DNP assisted NOE experiment, a total 100 uL of H2O/d6-DMSO (v/v 1:1) mixture consisting of 15mM 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl radical (TEMPOL; Sigma Aldrich, St. Louis, MO) was polarized in a HyperSense DNP polarizer (Oxford Instruments, Abingdon, UK), by irradiating with microwaves (100 mW power, 94.005 GHz frequency) at a temperature of 1.4 K, to create proton hyperpolarization of water. After 30 min, the hyperpolarized sample was dissolved by pre-heated D2O and automatically transferred to a 5 mm NMR tube that was pre-installed in a 9.4 T NMR magnet (Bruker Biospin, Billerica, MA), using a

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sample injector described elsewhere.20 The sample was injected with nitrogen gas at 1862 kPa, against 1034 kPa back pressure. In the NMR tube, 25 µl of a solution of trifluoroacetic acid (EMD Millipore, Billerica, MA) in D2O was pre-loaded and mixed with the hyperpolarized water. Dissolution resulted in a 12 – 15 fold dilution, yielding a free radical concentration in the NMR sample of approximately 1 mM. The NMR probe was a 500 MHz inverse broadband observe (BBO) probe (Bruker Biospin), which was modified to tune the inner coil to the frequency of

19

F

(376.47 MHz), and the outer coil to that of 1H (400.13 MHz) in the magnet used, by the addition of non-magnetic capacitors (Johanson Technology, Camarillo CA) in the tuning circuit. NMR data was acquired on a home-built spectrometer with two excitation and two detection channels configured for

19

F and 1H nuclei, using RadioProcessor and PulseBlaster boards (SpinCore

Technologies, Gainesville, FL).

Figure 1. a) The pulse sequence for DNP assisted NOE. A total number of n = 25 transients were acquired. At the time between a and b, sample injection was carried out during τi = 400 ms and acquisition started after stabilization time, τs = 500 ms. The recycle time tr = 1.151 s, which includes the s. The

19

F acquisition time of 1.089

1

H acquisition time was 4.096 s. b) The corresponding experiment for conventional NOE

measurement. The mixing time τm was varied from 0 s to 16 s. c) Reference experiment for determining the saturation factor b in the conventional NOE experiment. In all panels, narrow black bars represent hard rf pulses with x-phase and 90° flip angle, unless denoted as flip angle of α = 40° or β = 1°. Pulses were applied with γB1 = 6.46 kHz and 6.25 kHz on

19

1

F and H, respectively. In (b) and (c), wide black boxes are saturation

pulses applied at a field strength γB1 = 3.39 kHz for a duration of 10 ms (x-phase) and 5 ms (y-phase), respectively.

4


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The pulse sequence for the DNP assisted NOE experiment is shown in Figure 1a. After sample injection and stabilization, the change in 19F peak intensity was monitored over the time course of 30 s through a series of small flip angle excitations. To obtain the intermolecular cross-relaxation rate, a control measurement was needed (see Results and Discussion). For this purpose, the same pulse sequence was applied to the same sample but at thermal equilibrium after decay of the hyperpolarization. To determine the polarization level of water, 1H signal was detected by the second receiver of the spectrometer with a small flip angle excitation in the first scan. After the DNP experiment, the spin-lattice relaxation rates of 1H and 19F were independently measured using an inversion recovery experiment. All of the measurements were carried out at a temperature of 301 K. At the end of the measurements, the concentrations of the two spins,

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F and 1H, were

determined by comparing the intensities of the respective peaks with that of reference samples containing 120 mM trifluoroacetic acid (TFA) and pure water, respectively. Conventional NMR Spectroscopy. Solutions of TFA were prepared in degassed water at a concentration of 120 mM, with 1 mM TEMPOL. NOE transfer from water protons to 19F of TFA was measured with a similar procedure as described in ref. 10. The pulse sequence used for the conventional NOE measurement is shown in Figure 1b. At each mixing time period τm, 19F signal was averaged in an interlaced manner with and without saturation of water protons (the scan without saturation used irradiation at 100 kHz off-resonance). The degree of 1H saturation was determined in a separate experiment as shown in Figure 1c, by comparing the signals with on- and off-resonance saturation. Data Analysis. Data was analyzed using Matlab (MathWorks, Natick, MA). Each individual transient from the DNP measurements was apodized with an exponential window function (10 Hz line broadening) and Fourier transformed. A baseline correction was performed by fitting a polynominal of order 2 to the region in the spectrum excluding the peak (see supporting Figure S1), followed by peak integration for total signal intensity. A correction for non-linearity of signal 5



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intensity of the spectrometer was performed based on an experimental calibration curve. The DNP enhancement factor ε was determined by comparing the peak integral from the 1H spectrum acquired in the first scan of the dissolution DNP experiment with the integral from a conventionally acquired spectrum. Cross-relaxation rates were determined following the Solomon equations21

(1)

(2) In the present experiments, σHF and σFH are not identical when referencing the

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F and 1H signal

intensities to the respective signals from thermal polarization in the same sample, because the signals depend on the concentrations of the respective species. In this case, the cross-relaxation parameter

(3) can be defined, which takes on the same value irrespective of the concentrations in the sample. The 19

F signal intensities were modeled during each time period free of rf pulses with analytical

solutions of these equations with arbitrarily selectable initial signal intensities for both spins:

(4) and

(5) with 6


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(6) Here, IH and IF are signal intensities, IH,0 and IF,0 the initial signal intensities at the beginning of the time period under consideration, IH,eq and IF,eq the signal intensities at thermal equilibrium, rF and rH the spin-lattice relaxation rate constants, and cF and cH the spin concentrations for the

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F and 1H

spins, respectively. The effect of rf pulses was accounted for by .

(7)

Equation 7 reflects the fact that during the measurement, a series of hard pulses with a fixed small flip angle α was used to acquire NMR spectra at fixed time interval, thereby converting a portion of the longitudinal magnetization into the transverse magnetization. Consequently, only a part of the longitudinal magnetization is preserved for the following scans. This preserved magnetization can be use as the new initial value of 19F spin for the next scan. The time evolution of signals over the entire experiment was therefore calculated by repeated successive application of Equations 4 – 6, and Equation 7, for both the experiment with hyperpolarized sample and the experiment carried out after hyperpolarization had decayed. Signal intensities at the start of the experiment were hyperpolarized case, and

and

and

in the

in the non-hyperpolarized case.

Since the first scan in the DNP experiment occurs a short time after mixing, the measured IH,0 is not exactly representative of the initial hyperpolarization of 1H. In order to account for the difference, it was assumed that the NOE transfer started after two thirds of the injection time period in the pulse program, which corresponds to a time point during the arrival of the hyperpolarized sample in the NMR tube. The original 1H polarization enhancement at the starting point was estimated by back-extrapolation from the first acquired data point using a single exponential decay with time constant rH, which is justified under the conditions that injection time is short compared to the time scale of the cross-relaxation measurement. For fitting the model to the experimental 7



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data, the sum of residual squares between the calculated and measured data points was minimized simultaneously for the experiments measured with hyperpolarization and after hyperpolarization had decayed. The cross-relaxation rate σ was the only optimization parameter. The data obtained from conventional NMR spectroscopy was analyzed by processing the timedomain NMR data and integrating using the same procedure as described for the DNP experiment. Signal intensities were then fitted using Equations 4 – 6 as a function of τm, with

and

The b parameter, which indicates the residual proton signal after saturation, was obtained from integration of the 1H spectra measured with the pulse sequence in Figure 1c. The optimization parameter was the cross-relaxation rate σ. Results and Discussion By comparing the signal intensity obtained from hyperpolarized water with that from thermally polarized water, Figure 2 illustrates both the potential benefit and the main challenge of obtaining accurate measurements of intermolecular cross-relaxation rates using hyperpolarization by dissolution DNP. On one hand, a signal integral that is three orders of magnitude larger in the hyperpolarized case indicates that a corresponding enhancement in the otherwise weak NOE signal should be expected. On the other hand, for the same reason any error in the knowledge of the ε parameter, i.e. signal enhancement due to hyperpolarization, will directly be reflected as an error in the calculated cross-relaxation rate. Experimentally, because of variations in hyperpolarization and sample injection, we found that the determination of ε using a separate hyperpolarized sample can give rise to substantial variations in the calculated values. Here, we therefore propose to use the simultaneous acquisition of an NOE buildup curve together with a signal from the originating nucleus as an initial calibration point, for the accurate determination of cross-relaxation rates from a dissolution DNP experiment. Using this approach ensures not only that the two signals are measured from the same batch of sample, but also that the injection time and path, as well as mixing ratio is the same for both measurements. For a heteronuclear NOE, which originates from 1H and 8


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transfers to

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F, this measurement is achieved using a NMR probe and spectrometer that allows

simultaneous data acquisition on both nuclei, as described in the Methods section. In Figure 2, it can also be seen that the peak from hyperpolarized water is broadened, in part due to radiation damping that occurs after applying a radio-frequency (rf) pulse.

Figure 2. Comparison between the hyperpolarized 1H spectrum of water and thermally polarized spectrum of the same sample. The data shown is from trial 1 in Table 1, obtained with 1º flip angle.

A series of

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F measurements performed on a single sample of hyperpolarized water mixed

with TFA, representing a heteronuclear NOE build-up curve, is shown in the stacked plot of Figure 3a. Due to the rapid tumbling motions of the small molecules involved, as expected the 19F signal of TFA transiently is reduced, before approaching the positive thermal equilibrium value at long time. Other than in conventional transient NOE experiments, however, the deviation from equilibrium is large, here amounting to approximately twice the equilibrium magnetization. As demonstrated in the Methods section, additionally the DNP experiment requires accounting for the depletion of 19F signal intensity due to the successive application of rf pulses, which results in the need for an iterative numerical evaluation of signal intensities in each data point. This effect can readily be seen in the reduction in signal intensity in the measurement starting from a thermally

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polarized spin system, shown in Figure 3b. In this measurement, signal intensity initially depletes rapidly until reaching a steady state after application of a large number of pulses. The result of the numerical calculation, for the best fit parameters to the data in Figure 3a and b, is shown in Figure 3c. It can be seen that these parameters give rise to curves that at the same time closely reproduce both measurements. Experimentally, we found that a varying temperature during the experiment, which occurs if the temperature of the injected sample does not match the set temperature of the NMR probe, gives rise to signal intensities that poorly fit the model equations. This effect occurs presumable due to the temperature dependence of relaxation rates. The quality of the data from these experiments depended on the ability to obtain a reproducible sample temperature in the dissolution process.

Figure 3. a) Stacked plots of the successively acquired

19

F spectra of TFA after mixing with hyperpolarized

water using the pulse sequence in Figure 1a. b) Stacked plots of the successively acquired hyperpolarization had decayed. c) The NOE build up curves of integrated

19

F spectra after

19

F signal as a function of time.

Data points shown as circles were obtained in presence of hyperpolarized water, whereas data points shown as squares stem from the measurement after hyperpolarization had decayed. The solid lines represent the -4

-1

-1

fitted intensities using Equations 4 – 7. Best fit parameter is σ = 2.3710 s M for the data set shown here. The data shown represents trial 1 from Table 1. d) The

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F NOE build up curve of the conventional

measurement without hyperpolarization. This data was acquired using 2 x 32 scans per point, including two experiments each for on- and off-resonance saturation, with a total experiment time of 10 h. Best fit

10


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parameter is σ = 2.6110-4 s-1M-1 for the data set shown here.

At a given temperature, the normalized cross-relaxation rate σ should be a constant value, independent of the concentrations of the spins involved in the NOE transfer. This independence on concentration was used to validate the proposed approach of determining cross-relaxation rates, by obtaining data sets at different concentrations of TFA and hyperpolarized water. The resulting values are summarized in Table 1, and fitted curves are shown in Figure S2 (supporting information). It can be seen that while the σFH parameter depends the molar concentration of the 1H spin, σ is nearly constant among the samples, with an average value of 2.46·10-4 ± 1.02·10-5 s-1M-1. A plot of the measured cross-relaxation rates further shows that the σ values are independent of the concentration within measurement error (Figure 4). Figure 4 also indicates the 95 % confidence intervals calculated from the Jacobian matrix resulting from the fitting procedure. The confidence intervals are on the order of, or narrower than, the variations among the individual data points. This observation indicates that the fit parameter is well determined, likely in part due to the fact that it was possible to model the data using only a single parameter, i.e. σ, as the unknown fit parameter. For comparison, cross-relaxation rates were also measured using a conventional pulse sequence (Figure 1b and c). A representative data set is shown in Figure 3d. Based on three independent measurements taken on one sample, a cross-relaxation rate of σ = 2.49 ·10-4 ± 1.42 ·10-5 s-1M-1 was obtained. In the conventional experiment, the change in signal intensity due to the NOE reaches a maximum of only 3 %, which stands in contrast to the 200 % change that is observed in the experiment with hyperpolarization. Nevertheless, the obtained cross-relaxation rates are in good agreement when comparing the experiments with DNP and the conventional measurement, with a difference that is smaller than the measurement error. Furthermore, from Nordstierna et al.,10 a value of σ = 3.40·10-4 s-1M-1 can be calculated for the same solvent / solute combination, albeit based on measurements at a different field strength, lower temperature of 293 K and without free

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radicals that are present in the DNP experiment. The concentrations explored with the data shown here range from 23 mM to 72 mM of TFA. This range was accessible in the present experiment by diluting 25 µL of TFA solution with hyperpolarized water to reach an NMR sample volume of 450 µL. Higher concentrations would be possible by using a different provision for sample mixing. Using sample injection into a flow cell driven by a liquid, it would for example be possible to achieve mixing ratios of 1:1 or higher, albeit possibly at the expense of a longer stabilization time.22 This option may be interesting for applications of the heteronuclear NOE measurement in cases, where the

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F spin concentration is

low.

Table 1. Experimental parameters and fitted cross-relaxation rates obtained from the DNP assisted NOE experiment at 301 K.

rF [s-1]

rH [s-1]

cF [M]

cH [M]

ε

σ [s-1M-1]

σFH [s-1]

Trial 1

0.400

0.188

0.22

2.42

2454

2.37·10-4

5.73·10-4

Trial 2

0.417

0.203

0.20

2.31

2323

2.32·10-4

5.36·10-4

Trial 3

0.401

0.195

0.18

2.40

2226

2.51·10-4

6.04·10-4

Trial 4

0.417

0.200

0.13

2.04

2421

2.35·10-4

4.80·10-4

Trial 5

0.401

0.183

0.16

2.19

2103

2.60·10-4

5.69·10-4

Trial 6

0.400

0.184

0.13

2.47

1984

2.45·10-4

6.05·10-4

Trial 7

0.381

0.187

0.07

2.53

2093

2.50·10-4

6.32·10-4

Trial 8

0.421

0.202

0.07

2.29

2011

2.45·10-4

5.60·10-4

Trial 9

0.423

0.216

0.07

2.96

1829

2.60·10-4

7.68·10-4

Accurate measurements of 1H – 19F cross-relaxation rates are interesting for the elucidation of molecular interactions in various chemical and biochemical systems. In small, fluorinated molecules, such as used here, the NOE arises due to molecular contacts or solvation, with a temperature dependence that is due to changes in molecular motion associated with temperature. The same observation is true in molecular aggregates or in macromolecules, including micelles, 12


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polymers, proteins or dendrimers. In macromolecules, a determination of the site specificity of molecular interactions is further often of interest. This question can be addressed with the heteronuclear NOE experiment by selective incorporation of 19F at a site of interest, using chemical or biochemical synthesis strategies. The several orders of magnitude enhancement in sensitivity in the detection of the NOE would be particularly beneficial for the study of such molecular constructs with their typically lower solubility. Even in the measurement of the heteronuclear NOE with small molecules shown here, the conventional (non-hyperpolarized) experiment used 2 x 32 scans each for 20 data points, acquired over a time of 10 h, and further reductions in signal strength could cause the experiment time to become prohibitive. The experiment with dissolution DNP used a polarization time of 30 min, after which the transient build-up curve was measured in 25 acquisitions over the time course of 30 s. In both the conventional and the hyperpolarized approach, independent measurements of the 19F and 1H spin-lattice relaxation rates, as well as knowledge of sample concentrations, are still needed for data analysis. However, these measurements can be carried out on the time scale of < 30 min, i.e. comparable to the time required for the DNP experiment and much more rapidly than the conventional NOE experiment. In general, the heteronuclear NOE experiment presents several advantages for the determination of molecular interactions, including the ability to measure a signal free from background because of the absence of non-participating 19F spins in the sample, as well as the possibility to obtain complete NOE buildup curves without the need for multi dimensional NMR spectroscopy.

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Figure 4. Intermolecular cross-relaxation rate σ between the water and TFA with the different concentration of TFA samples. Data points shown as circles stem from DNP assisted NOE experiment. Error bars in these data points represent the 95 % confidence intervals calculated from the fit. A dashed line and gray band indicates the average value and standard deviation of the DNP measurements, which is 2.4610-4 ± 1.0210-5 -1

-1

s M . The data point shown as a square is obtained from an average of three independent conventional measurements of a single sample. The error bar for this point represents the standard deviation of the three measurements.

Conclusions In summary, we have demonstrated a method for the accurate determination of heteronuclear, intermolecular cross-relaxation rates using dissolution DNP. The hyperpolarization in these experiments affords a sensitivity enhancement of three orders of magnitude, enabling the measurement of transient NOE buildup curves in a single, rapid experiment. A main challenge in the calculation of a numerical value for the cross-relaxation rate, the determination of the signal enhancement through hyperpolarization, is solved by simultaneous acquisition of signals from 1H and 19F. In general, the use of heteronuclear, DNP assisted NOE may be of interest for the study of various intermolecular interactions, including those involving a macromolecule, where fluorine atoms can be introduced as site selective labels.

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Author Contributions CH designed experiments. JK, ML and HC carried out experiments and analyzed data. CH, JK, ML and HC wrote manuscript.

Acknowledgments Financial support from the National Science Foundation (Grant CHE-1362691) and the Welch Foundation (A-1658) is gratefully acknowledged.

Supporting Information Supporting Information Available: Figures of baseline correction and fitted curves for DNP assisted NOE measurements. This material is available free of charge via the Internet at http://pubs.acs.org.

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References (1) Overhauser, A. W. Phys. Rev. 1953, 92, 411–415. (2) Wüthrich, K. NMR of Proteins and Nucleic Acids; Wiley, 1986. (3) Otting, G.; Liepinsh, E.; Wüthrich, K. Science 1991, 254, 974–980. (4) Otting, G. Prog. Nucl. Magn. Reson. Spectrosc. 1997, 31, 259–285. (5) Meyer, B.; Peters, T. Angew. Chem. Int. Ed. 2003, 42, 864–890. (6) Dalvit, C. Prog. Nucl. Magn. Reson. Spectrosc. 2007, 51, 243–271. (7) Canet, D.; Mahieu, N.; Tekely, P. J. Am. Chem. Soc. 1992, 114, 6190–6194. (8) Cistola, D. P.; Hall, K. B. J. Biomol. NMR 1995, 5, 415–419. (9) Raulet, R.; Furó, I.; Brondeau, J.; Diter, B.; Canet, D. J. Magn. Reson. 1998, 133, 324–329. (10) Nordstierna, L.; Yushmanov, P. V.; Furó, I. J. Chem. Phys. 2006, 125, 074704. (11) Nordstierna, L.; Yushmanov, P. V.; Furó, I. J. Phys. Chem. B 2006, 110, 25775–25781. (12) Navon, G.; Song, Y.-Q.; Rõõm, T.; Appelt, S.; Taylor, R. E.; Pines, A. Science 1996, 271, 1848–1851. (13) Landon, C.; Berthault, P.; Vovelle, F.; Desvaux, H. Protein Sci. 2001, 10, 762–770. (14) Ardenkjær-Larsen, J. H.; Fridlund, B.; Gram, A.; Hansson, G.; Hansson, L.; Lerche, M. H.; Servin, R.; Thaning, M.; Golman, K. Proc. Natl. Acad. Sci. 2003, 100, 10158–10163. (15) Merritt, M. E.; Harrison, C.; Mander, W.; Malloy, C. R.; Dean Sherry, A. J. Magn. Reson. 2007, 189, 280–285. (16) Marco-Rius, I.; Bohndiek, S. E.; Kettunen, M. I.; Larkin, T. J.; Basharat, M.; Seeley, C.; Brindle, K. M. Contrast Media Mol. Imaging 2014, 9, 182–186. (17) Zeng, H.; Lee, Y.; Hilty, C. Anal. Chem. 2010, 82, 8897–8902. (18) Lee, Y.; Zeng, H.; Mazur, A.; Wegstroth, M.; Carlomagno, T.; Reese, M.; Lee, D.; Becker, S.; Griesinger, C.; Hilty, C. Angew. Chem. Int. Ed. 2012, 51, 5179–5182. (19) Min, H.; Sekar, G.; Hilty, C. ChemMedChem 2015, 10, 1559–1563.

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(20) Bowen, S.; Hilty, C. Phys. Chem. Chem. Phys. 2010, 12, 5766–5770. (21) Solomon, I. Phys. Rev. 1955, 99, 559–565. (22) Chen, H.-Y.; Hilty, C. ChemPhysChem 2015, 16, 2646–2652.

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Figure 1. a) The pulse sequence for DNP assisted NOE. A total number of n = 25 transients were acquired. At the time between a and b, sample injection was carried out during τi = 400 ms and acquisition started after stabilization time, τs = 500 ms. The recycle time tr = 1.151 s, which includes the 19F acquisition time of 1.089 s. The 1H acquisition time was 4.096 s. b) The corresponding experiment for conventional NOE measurement. The mixing time τm was varied from 0 s to 16 s. c) Reference experiment for determining the saturation factor b in the conventional NOE experiment. In all panels, narrow black bars represent hard rf pulses with x-phase and 90° flip angle, unless denoted as flip angle of α = 40° or β = 1°. Pulses were applied with γB1 = 6.46 kHz and 6.25 kHz on 19F and 1H, respectively. In (b) and (c), wide black boxes are saturation pulses applied at a field strength γB1 = 3.39 kHz for a duration of 10 ms (x-phase) and 5 ms (yphase), respectively. 53x16mm (600 x 600 DPI)



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Figure 2. Comparison between the hyperpolarized 1H spectrum of water and thermally polarized spectrum of the same sample. The data shown is from trial 1 in Table 1, obtained with 1º flip angle. 71x62mm (600 x 600 DPI)


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Figure 3. a) Stacked plots of the successively acquired 19F spectra of TFA after mixing with hyperpolarized water using the pulse sequence in Figure 1a. b) Stacked plots of the successively acquired 19F spectra after hyperpolarization had decayed. c) The NOE build up curves of integrated 19F signal as a function of time. Data points shown as circles were obtained in presence of hyperpolarized water, whereas data points shown as squares stem from the measurement after hyperpolarization had decayed. The solid lines represent the fitted intensities using Equations 4 – 7. Best fit parameter is σ = 2.37·10-4 s-1M-1 for the data set shown here. The data shown represents trial 1 from Table 1. d) The 19F NOE build up curve of the conventional measurement without hyperpolarization. This data was acquired using 2 x 32 scans per point, including two experiments each for on- and off-resonance saturation, with a total experiment time of 10 h. Best fit parameter is σ = 2.61·10-4 s-1M-1 for the data set shown here. 84x39mm (600 x 600 DPI)



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Figure 4. Intermolecular cross-relaxation rate σ between the water and TFA with the different concentration of TFA samples. Data points shown as circles stem from DNP assisted NOE experiment. Error bars in these data points represent the 95 % confidence intervals calculated from the fit. A dashed line and gray band indicates the average value and standard deviation of the DNP measurements, which is 2.46·10-4 ± 1.02·10-5 s-1M-1. The data point shown as a square is obtained from an average of three independent conventional measurements of a single sample. The error bar for this point represents the standard deviation of the three measurements. 67x54mm (600 x 600 DPI)


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For TOC only 44x34mm (600 x 600 DPI)


Determination of Intermolecular Interactions Using Polarization

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