Simultaneous Imaging of an Enantiomer Pair by Electron

Dec 18, 2012 - Department of Arts and Sciences, Center for Medical Education, Sapporo Medical University, South 1, West 17, Chuo-ku, Sapporo. 060-8556...
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Simultaneous Imaging of an Enantiomer Pair by Electron Paramagnetic Resonance Using Isotopic Nitrogen Labeling Yusuke Miyake,† Xiaolei Wang,‡ Mitsuo Amasaka,† Kaori Itto,‡ Shu Xu,‡ Hirokazu Arimoto,‡ Hirotada Fujii,§ and Hiroshi Hirata*,† †

Division of Bioengineering and Bioinformatics, Graduate School of Information Science and Technology, Hokkaido University, North 14, West 9, Kita-ku, Sapporo, 060-0814, Japan ‡ Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan § Department of Arts and Sciences, Center for Medical Education, Sapporo Medical University, South 1, West 17, Chuo-ku, Sapporo 060-8556, Japan S Supporting Information *

ABSTRACT: This Article describes the simultaneous imaging of chiral nitroxyl radicals using electron paramagnetic resonance (EPR). Chiral nitroxyl radicals could be simultaneously visualized with the labeling of isotopic nitrogen. Chiral nitroxyl radicals, hydroxylmethyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl, were visualized using the method of simultaneous EPR imaging, which refers to the visualization of two kinds of molecules with unpaired electrons in a single image scan. EPR spectra of a racemic mixture of chiral nitroxyl radicals and those of the respective R and S configurations confirmed labeling by isotopic nitrogen. 1H nuclear magnetic resonance (NMR) imaging and simultaneous imaging of solutions of chiral nitroxyl radicals were performed. The advantages and limitations of simultaneous imaging using EPR are also discussed. Simultaneous imaging with chiral-labeled nitroxyl radicals is a new application of EPR imaging and may be useful for biological studies involving biologically active chiral molecules.

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suitable for 3D imaging of samples with low transparency to light, such as animals. Radioactive isotopes can be used for the molecular imaging of small animals and humans.6−8 In positron emission tomography (PET), enantiomers can be labeled with radioactive elements.9−13 However, each labeled enantiomer is injected into a subject animal separately. This is because the radioactive elements in different enantiomers cannot be discriminated by PET. Fluorescent molecules can also be used to label target molecules.14−16 However, fluorophores with different emission wavelengths have different molecular structures and may alter the chemical properties of the target molecules.17,18 Stevenson et al. observed different electron paramagnetic resonance (EPR) line-shapes for R- and S-anion radicals in chiral solvents.19 This work by Stevenson et al. represents truly enantioselective discrimination with EPR spectroscopy for a pair of enantiomers. Since visualization using the enantioselectivity of target chiral molecules is generally not easy, we sought to trace a pair of target chiral molecules with isotopic

hiral biological molecules play important roles in living organisms, since enantiomers have different activities in biological systems.1,2 To understand the interaction between enantiomers and other molecules in biological systems, enantioselective imaging methods would be important tools for use in biological and biomedical studies. However, it is not easy to distinguish between enantiomers based on their chemical and physical properties. Therefore, enantioselective imaging is not an easy task in in vitro and in vivo experiments. If we could selectively visualize chiral molecules in a single image scan for three-dimensional (3D) subjects, we would be able to investigate the distributions of and temporal changes in chiral molecules in animals. Such observations would give us insights into the pharmacokinetics of chiral molecules and the differences in chirality when enantiomers exist in biological systems. For such selective imaging, we would require a proven imaging method that can be applied to 3D biological subjects. While several molecular imaging methods are available in practice, these methods can not be directly applied to enantioselective imaging. One potential approach involves the use of optical activity, which is the ability of chiral molecules to rotate the plane of linearly polarized light.3−5 While circular dichroism (CD) spectroscopy can be used for the measurement of enantiomers, CD signals are weak, and, thus this is not © 2012 American Chemical Society

Received: September 18, 2012 Accepted: December 18, 2012 Published: December 18, 2012 985

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do not affect the chemical properties of nitroxyl radical molecules, but the EPR absorption peaks are drastically changed. We can label chiral nitroxyl radicals using isotopic nitrogen, 14N and 15N, as shown in Figure 1b. As a result, labeled chiral nitroxyl radicals can be visualized in a single image scan with the method of simultaneous EPR imaging.20 Labeled nitroxyl radicals can also be visualized with Overhauser-enhanced magnetic resonance imaging (OMRI).26 Synthesis of (S)-HMP-14N (1) and (R)-HMP-15N (2). Compounds 1 and 2 were prepared as shown in Scheme 1, from racemic 3-carboxyl-2,2,5,5-tetramethylpyrrolidin-1-oxyl (3-carboxyl-PROXYL, purchased from Kanto Chemical Co., Inc.) and 15N-labeled 3-carboxyl-PROXYL (synthesized as described in the literature27). After resolution with chiral 1phenylethylamine28,29 and reduction with LiAlH4,30 compounds 1 and 2 were obtained with an enantiomeric excess of 95% and 96%, respectively, as determined by 19F-NMR of their Mosher esters.31 Sample Preparation for Imaging Experiments. Four quartz tubes (inner diameter of 3.0 mm and outer diameter of 5.0 mm) were held with a cylindrical sleeve (22 mm in diameter and 25 mm long) made of cross-linked polystyrene, Rexolite 1422. Two of the quartz tubes were filled with 2 mM (S)-(−)-HMP-14N aqueous solution, and one was filled with 2 mM (R)-(+)-HMP-15N aqueous solution. The remaining tube was filled with distilled water. Each quartz tube contained 110 μL of the respective solutions (15 mm in tubes). We did not remove O2 from the solution. Figure 2a shows a photograph of the four tubes held with the cylindrical sleeve. Figure 2b shows how the tubes are arranged for the simultaneous imaging experiments. These tubes were used as a phantom for imaging experiments. EPR Spectrometer and Imager. A laboratory-built 750MHz continuous-wave (CW) EPR spectrometer/imager was used for the experiments. The details of our CW-EPR imager have been reported elsewhere,32−34 and thus, we provide only a brief outline here. The main magnet (X-5253, NEOMAX Company, part of Hitachi Metals, Tokyo, Japan) had a magnetic field of 27 mT, and its magnetic circuit was formed with permanent magnets. The main magnet was used with three pairs of coils for field gradients and a single pair of coils for magnetic field scanning. A multicoil parallel-gap resonator was used in the reflection-type microwave bridge.35 The sample space of the resonator was 22 mm in diameter and 30 mm in length. To control data-acquisition in EPR spectra and imaging, we used a LabVIEW-based program (LabVIEW 8.5, National Instruments Corp., Austin, TX) on MacOS 10.5 and an Apple MacPro computer. EPR Spectroscopy. First-derivative EPR absorption spectra were measured from racemic HMP (purchased from Toronto Research Chemicals Inc., North York, Canada) and chiral 14Nand 15N-labeled HMP (concentration 2 mM, 110 μL) in quartz tubes; however, each sample was set in the center hole of another cylindrical sleeve made of Rexolite 1422 (22 mm in diameter). We used a commercially available racemic HMP radical with 14N to compare chiral HMP radicals. The measurement parameters of EPR spectroscopy for each sample were as follows: microwave power, 2.2 mW; field scanning, 5.0 mT; magnetic field modulation, 0.10 mT; duration of field scanning, 1.0 s; time constant of lock-in amplifier, 1 ms; number of data points, 512; number of averages, 100. The RF magnetic field B1 in our EPR imager was 2.0 μT at an incident microwave power of 2.2 mW. This was calculated from the RF

nitrogen labeling. We considered that it may be possible to discriminate between enantiomers using isotopic nitrogen in a nitroxyl radical. A difference of a single neutron in nitrogen that is close to an unpaired electron would allow us to distinguish between the two kinds of molecules. A method for EPR imaging that uses isotopic nitrogen in the nitroxyl radical was recently reported for the simultaneous visualization of two kinds of nitroxyl radicals and is called “simultaneous EPR imaging”.20 The visualization method reported in ref 20 was based on the spatial imaging protocol using image reconstruction by filtered back-projection. Regarding the visualization of two paramagnetic species with different hyperfine structures, the method of spectral-spatial EPR imaging was reported by Eaton et al.21−23 Spectral-spatial EPR imaging can be used to obtain data for one spectral and one, two, or three spatial dimensions.24 To obtain three-dimensional spatial images of two paramagnetic species with different hyperfine structures, four-dimensional EPR imaging with one spectral and three spatial dimensions should be performed. However, more time is required to obtain projection data in comparison with threedimensional spatial EPR imaging. This may be a limitation in animal imaging because of the reduction of EPR signals for nitroxyl radicals in a subject animal. The purpose of this study was to achieve the simultaneous imaging of R and S configurations of chiral nitroxyl radicals using simultaneous EPR imaging in a 3D manner. In this Article, we describe the simultaneous visualization of chiral hydroxylmethyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (HMP) labeled with isotopic nitrogen, 14N and 15N. HMP is a commonly used blood−brain barrier (BBB)-permeable nitroxyl radical25 that has a chiral carbon in its molecular structure. Thus, we used HMP as a chiral molecule to be labeled with isotopic nitrogen in our experiments. This is a new application of EPR imaging to simultaneous imaging, which may be useful for investigating the pharmacokinetics of enantiomers in small animals.

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EXPERIMENTAL SECTION Approach to Simultaneous Imaging. If enantiomers have different EPR absorption spectra, we should be able to discriminate between chiral molecules using the difference between their EPR spectra. The hyperfine coupling structure of the EPR absorption spectrum for nitroxyl radicals that contain isotopic nitrogen 15N, as shown in Figure 1a, is different from that of nitroxyl radicals with 14N. If we replace 14N with 15N, we

Figure 1. Concept of simultaneous molecular imaging using 14N- and 15 N-labeled nitroxyl radicals. (a) Labeling of isotopic nitroxyl radical (R: tertiary-alkyl bulky group) and (b) (S)-(−)-HMP-14N and (R)(+)-HMP-15N molecules. 986

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Scheme 1. Synthesis of (S)-HMP-14N (1) and (R)-HMP-15N (2)

determined so as to obtain projection data that were distributed uniformly on a sphere. The details of the method for setting the projection angles have been reported elsewhere.34 In brief, the projection angles were calculated from the vertices of polyhedrons that have congruent faces generated from an icosahedron. Each face of an icosahedron was tessellated regularly onto the circumscribed sphere. Surface-rendered images were drawn using IDL 7.1 data visualization software (ITT Visual Information Solution, Boulder, CO). For simultaneous EPR imaging, the measurement parameters were as follows: microwave power, 2.2 mW; field scanning, 3.0 mT; magnetic field modulation, 0.10 mT; field gradient, 30 mT/m; duration of field scanning, 0.5 s; time constant of lockin amplifier, 1 ms; number of projections, 181; number of data points, 512; number of averages, 5; field-of-view (FOV), 50 × 50 × 50 mm. The total acquisition time was 9 min 27 s. 1 H NMR Imaging. To visualize the spatial distribution of paramagnetic spices, i.e., nitroxyl radical molecules, 1H NMR imaging was performed using a 7-T magnetic resonance imaging (MRI) scanner (Varian Unity INOVA 300/183, Varian, Palo Alto, California, USA). This imaging provided additional data regarding the distribution of unpaired electrons in the solutions being visualized. We used a spin echo pulse sequence to visualize solutions in quartz tubes as a 2D sliceselective image. The measurement parameters in T1-weighted NMR imaging were as follows: repetition time TR, 500 ms; echo time TE, 20 ms; matrix size, 256 × 256; number of excitations, NEX 5; FOV, 30 × 30 mm; slice thickness, 5 mm. The total acquisition time was 5 min 23 s.

Figure 2. Sample tubes of 14N- and 15N-labeled enantiomers. (a) Photograph of the sample tubes. Four quartz tubes (3.0 mm i.d. and 5 mm o.d.) were filled with 2 mM (S)-(−)-HMP-14N and (R)(+)-HMP-15N solutions, as well as distilled water (110 μL each, which corresponds to a length of 15 mm in the tubes). A cylindrical sleeve made of cross-linked polystyrene held four quartz tubes. (b) Arrangement of the solutions of 14N- and 15N-labeled enantiomers and distilled water in the sample tubes.

magnetic field generation efficiency (42 μT/W1/2) of the resonator used. For a racemic mixture of 1 mM HMP solution, the EPR signal intensity response is given as a function of the RF magnetic field B1 in the Supporting Information (Figure S1). The signal intensity response indicates that the EPR measurements reported here were carried out in the linearresponse region of the spin system. Simultaneous Visualization of Chiral Nitroxyl Radicals. Three-dimensional visualization of 14N- and 15N-labeled chiral HMP was performed using simultaneous EPR imaging.20 14 N- and 15N-labeled nitroxyl radicals have three-line and twoline EPR absorption spectra, respectively. When no magnetic field gradient was applied, each absorption peak was clearly separated. However, the overlapping of EPR absorption peaks that occurs in the presence of magnetic field gradients requires spectral separation for simultaneous EPR imaging of 14N- and 15 N-labeled nitroxyl radicals. We measured EPR spectra for a 3.0 mT scanning field to contain the lower and center peaks of (S)-(−)-HMP-14N and the lower peak of (R)-(+)-HMP-15N. EPR spectra measured with and without magnetic field gradients were divided into lower- and higher-field parts. EPR spectra for (R)-(+)-HMP-15N in overlapping peaks were recovered by subtracting the signal of (S)-(−)-HMP-14N with a shifting theorem of Fourier transform (details are provided in the electronic Supporting Information of ref 20). We used filtered-back projection for image reconstruction with a Hamming window function. The projection angles were

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RESULTS EPR Spectra of Labeled Chiral Nitroxyl Radicals. Before performing the simultaneous imaging of labeled molecules, we measured the first-derivative EPR absorption spectra of racemic HMP-14N and chiral HMP radicals. Figure 3a,b,c shows the EPR spectra of racemic HMP-14N, (S)-(−)-HMP-14N, and (R)(+)-HMP-15N, respectively. The spectra of racemic HMP-14N and (S)-(−)-HMP-14N show a triplet hyperfine structure due to nuclear spin in 14N. Racemic HMP-14N and (S)-(−)-HMP-14N measured under the same conditions thus displayed similar EPR spectra, as shown in Figure 3a,b. In contrast, the spectrum of (R)-(+)-HMP-15N shows a doublet hyperfine structure due to nuclear spin in 15N, as shown in Figure 3c. 1 H NMR Imaging. Figure 4 shows a T1-weighted 1H NMR image of sample tubes containing (S)-(−)-HMP-14N solution, (R)-(+)-HMP-15N solution, and distilled water. HMP nitroxyl radical solutions clearly exhibit signal enhancement compared 987

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Figure 3. First-derivative EPR absorption spectra of the nitroxyl radicals being investigated. (a) EPR spectrum of a 2 mM racemic mixture of HMP solution, (b) EPR spectrum of a 2 mM (S)(−)-HMP-14N solution, and (c) EPR spectrum of a 2 mM (R)(+)-HMP-15N solution. Quartz tubes (3.0 mm i.d.) were filled with 110 μL of each solution and placed in the center of the resonator of a 750-MHz EPR spectrometer.

Figure 5. Simultaneously obtained images of chiral HMP molecules. (a) Surface-rendered image of the distribution of (S)-(−)-HMP-14N, (b) surface-rendered image of the distribution of (R)-(+)-HMP-15N, (c) slice-selective image of the distribution of (S)-(−)-HMP-14N generated from panel a, and (d) slice-selective image of the distribution of (R)-(+)-HMP-15N generated from panel b. The fieldof-view (FOV) for the 3D images, (a and b), was 30 × 30 × 30 mm (128 × 128 × 128 voxels). These images were clipped from the original images with a FOV of 50 × 50 × 50 mm.

5c,d. These images were generated from 3D images shown in Figure 5a,b. While signals below 50% of the maximum intensity in the image data were not visualized in Figure 5a,b, sliceselective images in Figure 5c,d had no threshold for visualization. These images can be compared to the NMR image in Figure 4 to verify the results of simultaneous imaging of chiral nitroxyl radicals.

Figure 4. Slice-selective 1H NMR image of sample tubes containing chiral nitroxyl radicals and distilled water. The arrangement of the tubes was similar to that in Figure 2a. Pulse sequence of a T1-weighted image was used.

to the sample with distilled water. Since electron spin in HMP nitroxyl radical solutions shortens the T1 relaxation time of 1H nuclear spin, the image intensities of samples containing HMP radical were enhanced for tube numbers 1, 2, and 4. Simultaneous Imaging of Chiral Nitroxyl Radicals. Figure 5a,b shows surface-rendered images of the phantom to show the 3D distributions of chiral HMP solutions. The threshold for generating surface-rendered images was set to 50% of the maximum signal intensity in 3D image data. We visualized the sample tubes that contained both (S)(−)-HMP-14N and (R)-(+)-HMP-15N at the same time using the method of simultaneous EPR imaging. After spectral separation and image reconstruction, we could successfully obtain images of (S)-(−)-HMP-14N and (R)-(+)-HMP-15N. Figure 5a shows the surface-rendered image of (S)(−)-HMP-14N, which agreed with the distribution of (S)(−)-HMP-14N solution in two tubes. The (R)-(+)-HMP-15N solution was contained in a sample tube in the phantom and is visualized in Figure 5b. Distilled water was not visualized in EPR imaging, since it did not contain unpaired electrons. For a comparison of images of labeled nitroxyl radicals to the 1H NMR image in Figure 4, slice-selective images of (S)(−)-HMP-14N and (R)-(+)-HMP-15N are shown in Figure

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DISCUSSION Simultaneous imaging of (S)-(−)-HMP- 14 N and (R)(+)-HMP-15N was successfully demonstrated in Figure 5. This selectivity was based on the difference between the spectra of (S)-(−)-HMP-14N and (R)-(+)-HMP-15N, as shown in Figure 3. The hyperfine coupling structures of EPR spectra were not affected by the chirality of HMP radicals. Thus, isotopic nitrogen labeling was useful for performing simultaneous imaging. In the absence of isotopic nitrogen labeling, it was impossible to discriminate between enantiomers, like racemic and (S)-chiral radicals, as shown in Figure 3. In contrast, (S)-(−)-HMP-14N and (R)-(+)-HMP-15N were not discriminated by 1H NMR imaging, as shown in Figure 4, since there was no difference in the image contrast effects in NMR signals between 14N- and 15N-labeled nitroxyl radicals. The signal-to-noise ratios for two-dimensional EPR images in Figure 5c,d were 63 and 83, respectively. Since the maximum image intensities were normalized in two-dimensional images, there is no difference in the signal intensities in Figure 5c,d. However, the EPR images for 15N-labeled nitroxyl radicals generally have 988

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a better signal-to-noise ratio than those of 14N-labeled nitroxyl radicals. This is because (R)-(+)-HMP-15N has an approximately 1.5-fold higher signal intensity than (S)-(−)-HMP-14N due to the hyperfine structure with two-line absorption peaks. If the level of background noise is homogeneous over the images, the signal-to-noise ratio of the reconstructed image is proportional to the signal intensity of EPR spectra. The higher signal-to-noise ratio in Figure 5d should reflect the tendency of the signal intensities for (S)-(−)-HMP- 14 N and (R)(+)-HMP-15N. The present method for separating the spectra in EPR imaging is limited by the magnetic field gradient. To successfully recover the absorption peak of 15N-labeled nitroxyl radical, we have to have a nonoverlapping line shape of the center absorption peak of 14N-labeled nitroxyl radical. The field gradient and the object size give guidelines that we can use to avoid overlapping of the spectra. In our experiments, the separation Bs between the center absorption peak of the 14Nlabeled nitroxyl radical and the lower absorption peak of the 15 N-labeled nitroxyl radical was 1.09 mT (Figure 3), and the farthest distance of those electron spins in the object was 18.4 mm (the distance between the ends of the tubes placed in parallel). When we applied a field gradient G of 30 mT/m to the object, a distance L of 18.4 mm corresponds to the difference in the magnetic field, GL = 0.552 mT. This value should be less than the separation of the absorption peaks Bs, if we assume the ideal case in which we can neglect the linewidths of EPR absorption peaks. For a given object size L, the maximum field gradient Gmax can be calculated as Gmax = Bs/L. For the tube phantom in Figure 2, the maximum field gradient was calculated to be 59 mT/m. Since the field gradient in our study was set at 30 mT/m, it was sufficiently below the maximum field gradient. In reality, the EPR absorption peak has a finite value for the line width (the peak-to-peak line width was 0.13 mT in Figure 3a). Thus, we have to have a margin to the calculated maximum field gradient. If the present method is used to visualize a larger subject, we would need to pay attention to the relation between the magnetic field gradient and the object size. Simultaneous EPR imaging has unique features and offers several advantages: (i) 14N- and 15N-labeling are not radioactive. PET is a frequently used method for molecular imaging, but labeling with radioactive isotopes requires safety precautions. (ii) The present method enables us to measure chiral molecules simultaneously. In contrast, if enantiomers are visualized with 18F labeling, different enantiomers cannot be discriminated in a single image scan. (iii) Simultaneous EPR imaging has less impact on the target molecules, since the labeled molecules differ only by a single neutron. In contrast, labeled molecules could have different properties in multiwavelength fluorescence imaging, since a target molecule is labeled with different fluorophores to generate fluorescence of different emission wavelengths. These advantages are potentially useful for studies of chiral molecules in small animals. The present simultaneous imaging has several limitations: (i) EPR imaging has a low spatial resolution, and spatial resolution mainly depends on the spectral line width. Since the line width of EPR spectra is essentially broader than that for NMR, the spatial resolution for EPR imaging is lower than that of NMR imaging. (ii) The reduction reaction of nitroxyl radicals in small animals decreases the concentration of radical molecules as a function of time. Consequently, the acquisition time of EPR imaging is limited to a few to 20 min in small animals.36−39

These problems regarding the spatial resolution and the acquisition time are common technical challenges in in vivo EPR imaging and are not related to the chirality of molecules. The postprocessing of resolution recovery in EPR imaging may address the limitation of spatial resolution.40−42 Fast 3D EPR image acquisition techniques can be used in simultaneous EPR imaging.32,33 Technical advancements in these areas may lead to improvements in the present method of simultaneous imaging. Labeling of nitroxyl radicals for an anticancer drug (Lomustin) was reported by Zhelev et al., who replaced the cyclohexyl part of the drug molecule with TEMPO radical.43 This modification did not affect the anticancer effect of the drug. This work with the labeling of drug molecules suggests that target molecules can be visualized in conjunction with the labeling of nitroxyl radicals and EPR imaging. While Lomustin does not have a chiral carbon in its molecular structure, many biologically active molecules are chiral. To visualize biologically active chiral molecules in animals, a method is needed to label enantiomers. This approach to labeling using isotopic nitroxyl radicals and simultaneous EPR imaging can be used for the visualization of chiral molecules. When nitroxyl radical molecules are attached to larger target molecules and are not in a fast-motion regime, the intensities of the three EPR absorption peaks of 14N-labeled nitroxyl radicals are not similar to each other. Under this circumstance, the lower-field peak cannot be successfully subtracted by the present method. To solve this problem, the appropriate line width and intensity of the absorption peak at the lower field should be used in a process of spectral separation. These adjustments have to be included in the process of spectral separation for larger molecules labeled with isotopic nitroxyl radicals. In our experiments, the line width of EPR lineshapes did not need to be adjusted because the spin system in the phantom was in the fast-motion regime and had no line shape distortion. To visualize chiral nitroxyl radicals, one can use Overhauserenhanced MRI, also called proton−electron double resonance imaging, as well as EPR imaging. If the motion of nitroxyl radical molecules is somehow constrained, the line shape of EPR absorption will be distorted and will affect the spatial resolution of the reconstructed image. In contrast, Overhauserenhanced MRI is less influenced by a change in the line shape of EPR absorption, since electron spins are visualized through proton nuclear spins and dynamic nuclear polarization. Since 14 N- and 15N-labeled nitroxyl radicals can be visualized simultaneously with Overhauser-enhanced MRI,26 Overhauser-enhanced MRI, like EPR imaging reported here, can be used for the visualization of chiral nitroxyl radicals using isotopic nitrogen labeling. In this study, 14N- and 15N-labeled chiral HMP radicals were successfully synthesized. Since HMP has a hydroxyl group, it might be easy to enable the labeling of single enantiomers with carboxyl groups by esterification with HMP. When target chiral molecules are labeled with EPR-detectable agents, it is desirable for these agents to not have a chiral center, since single enantiomers might become diastereomers with the labeling of chiral HMP radicals. However, since 14N- and 15N-labeled chiral HMP radicals can be synthesized separately, chiral HMP radicals with an R or S configuration can be selected and connected to single enantiomers to avoid the synthesis of diastereomers. This means that 14N- and 15N-labeled chiral HMP radicals can be used as labeling agents for optically and biologically active compounds in the future. 989

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(19) Stevenson, C. D.; Wilham, A. L.; Brown, E. C. J. Phys. Chem. A 1998, 102, 2999. (20) Pawlak, A.; Ito, R.; Fujii, H.; Hirata, H. Chem. Commun. 2011, 47, 3245. (21) Maltempo, M. M. J. Magn. Reson. 1986, 69, 156. (22) Maltempo, M. M.; Eaton, S. S.; Eaton, G. R. J. Magn. Reson. 1987, 72, 449. (23) Eaton, S. S.; Maltempo, M. M.; Stemp, E. D. A.; Eaton, G. R. Chem. Phys. Lett. 1987, 142, 567. (24) Ahn, K. H.; Halpern, H. J. J. Magn. Reson. 2007, 185, 152. (25) Yokoyama, H.; Itoh, O.; Aoyama, M.; Obara, H.; Ohya, H.; Kamada, H. Magn. Reson. Imaging 2002, 20, 277. (26) Utsumi, H.; Yamada, K.; Ichikawa, K.; Sakai, K.; Kinoshita, Y.; Matsumoto, S.; Nagai, M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 1463. (27) Kao, J. P. Y.; Burks, S. R.; Rosen, G. R. Targeted Delivery of Imaging Probes for In Vivo Cellular Imaging. U.S. Patent Application 20110165087, Jul 7, 2011. (28) Flohr, K.; Paton, R. M.; Kaiser, E. T. J. Am. Chem. Soc. 1975, 97, 1209. (29) Chion, B.; Lajzerowlcz, J.; Bordeaux, D.; Collet, A.; Jacques, J. J. Phys. Chem. 1978, 82, 2682. (30) Itoh, O.; Obara, H.; Aoyama, M.; Ohya, H.; Kamada, H. Anal. Sci. 2001, 17, i1515. (31) Bálint, J.; Kiss, V.; Egri, G.; Kálai, T.; Demeter, Á .; Balog, M.; Fogassy, E.; Hideg, K. Tetrahedron: Asymmetry 2004, 15, 671. (32) Sato-Akaba, H.; Kuwahara, Y.; Fujii, H.; Hirata, H. Anal. Chem. 2009, 81, 7501. (33) Sato-Akaba, H.; Fujii, H.; Hirata, H. Rev. Sci. Instrum. 2008, 79, 123701. (34) Sato-Akaba, H.; Fujii, H.; Hirata, H. J. Magn. Reson. 2008, 193, 191. (35) Kawada, Y.; Ito; Hirata, H.; Fujii, F. J. Magn. Reson. 2007, 184, 29. (36) Fujii, H.; Sato-Akaba, H.; Kawanishi, K.; Hirata, H. Magn. Reson. Med. 2011, 65, 295. (37) Hyodo, F.; Matsumoto, S.; Devasahayam, N.; Dharmaraj, C.; Subramanian, S.; Mitchell, J. B.; Krishna, M. C. J. Magn. Reson. 2009, 197, 181. (38) Miyake, M.; Shen, J.; Liu, S.; Shi, H.; Liu, W.; Yuan, Z.; Pritchard, A.; Kao, J. P. Y.; Liu, K. J.; Rosen, G. M. J. Pharmacol. Exp. Ther. 2006, 318, 1187. (39) Yamato, M.; Shiba, T.; Yamada, K.; Watanabe, T.; Utsumi, H. J. Cereb. Blood Flow Metab. 2009, 29, 1655. (40) Ikebata, Y.; Sato-Akaba, H.; Aoyama, T.; Fujii, H.; Itoh, K.; Hirata, H. Magn. Reson. Med. 2009, 62, 788. (41) Ahmad, R.; Clymer, B.; Vikram, D. S.; Deng, Y.; Hirata, H.; Zweier, J. L.; Kuppusamy, P. J. Magn. Reson. 2007, 184, 246. (42) Hirata, H.; Wakana, M.; Susaki, H. Appl. Phys. Lett. 2006, 88, 254103. (43) Zhelev, Z.; Bakalova, R.; Aoki, I.; Matsumoto, K.; Gadjeva, V.; Anzai, K.; Kanno, I. Chem. Commun. 2009, 53.

CONCLUSION The present experimental results suggest that isotopic nitroxyl radicals can be used as labels for chiral molecules. If biologically important enantiomers could be visualized in animals, this would represent a new approach to molecular imaging in biological studies. Further studies will be needed to determine the effects of labeling of isotopic nitroxyl radicals on the biological activities of the target enantiomers.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone/Fax: +81-11-706-6762. E-mail: [email protected]. jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank Dr. Miho Emoto, Sapporo Medical University, for her assistance in testing the synthesized compounds. This work was supported by a grant from the Japan Society for the Promotion of Science (NEXT Program Grant LR002 to H.H.).

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dx.doi.org/10.1021/ac302710m | Anal. Chem. 2013, 85, 985−990