DOI: 10.1002/chem.201501552
Communication
& Peptidomimetics
NMR Signal Enhancement by Effective SABRE Labeling of Oligopeptides Tomasz Ratajczyk,*[a] Torsten Gutmann,[b] Piotr Bernatowicz,[a] Gerd Buntkowsky,[b] Jaroslaw Frydel,[c] and Bartlomiej Fedorczyk[d] Abstract: Signal amplification by reversible exchange (SABRE) can enhance nuclear magnetic resonance signals by several orders of magnitude. However, until now this was limited to a small number of model target molecules. Here, a new convenient method for SABRE activation applicable to a variety of synthetic model oligopeptides is demonstrated. For the first time, a highly SABRE-active pyridine-based biocompatible molecular framework is incorporated into synthetic oligopeptides. The SABRE activity is preserved, demonstrating the importance of such earmarking. Finally, a crucial exchange process responsible for SABRE activity is identified and discussed.
Nuclear magnetic resonance (NMR) methods suffer strongly from low sensitivity. Therefore, since the early days of NMR, many efforts have been made to overcome this obstacle. One of the most commonly applied methodologies that can improve the NMR signal is hyperpolarization, which allows the generation of a high non-Boltzmann nuclear spin polarization. The most prominent hyperpolarization approaches are noble gas hyperpolarization,[1] dynamic nuclear polarization (DNP),[2] and parahydrogen-induced polarization (PHIP).[3] The latter can induce, in theory, a significant signal enhancement up to 105.[3] This enhancement can be obtained by two main chemical routes. Considering the first one, hyperpolarization of target molecules is achieved through a hydrogenation reaction with parahydrogen (p-H2) of a molecular system containing C¢C double- or triple bonds.[3a–d] During this process, the high spin order is transferred from p-H2 to the hydrogenated product. In [a] Dr. T. Ratajczyk, Dr. P. Bernatowicz Institute of Physical Chemistry, Polish Academy of Sciences Kasprzaka 44/52, 01-224 Warsaw (Poland) E-mail: [email protected]
the second route, the NMR signal is amplified by reversible exchange (SABRE).[3e,f] SABRE employs a reversible interaction where a labile complex containing p-H2, the ligand, and an iridium-based catalyst is formed. In such a complex, high spin polarization is transferred from p-H2 to the ligand via the catalyst.[3e,f] Finally, the complex splits up; however, the released ligand is hyperpolarized. Both of these PHIP variants open up an easy and inexpensive route for generation of hyperpolarized molecular systems, which renders PHIP very attractive for biomedical research and clinical applications. Unfortunately, a large number of molecules cannot be hyperpolarized, owing to the lack of appropriate hyperpolarizable units in their structures. Therefore, a PHIP activation of non-hyperpolarizable structures is at the center of scientific interest. One feasible route, which broadens the variety of hyperpolarizable systems, involves the integration of potently hyperpolarizable unsaturated moieties with non-hyperpolarizable molecules. Only recently, this approach has been demonstrated for important natural products such as barbituric acid, glucose, and simple alkyne-bearing oligopeptides.[4] Furthermore, the high potential for such procedures has also been demonstrated on relevant biological systems such as thiostrepton and fully functional sunflower trypsin inhibitor (SFTI-1).[5] Here, for the first time, we suggest a novel SABRE activation strategy that will further expand the set of hyperpolarizable molecular systems (see Figure 1 a). In particular, we show that the hyperpolarization is accessible through labeling of inactive oligopeptides with a SABRE-active molecular unit. Recently, SABRE was employed for amino acids and dipeptides hyperpolarization that theoretically may open up a path for NMR signal enhancement of larger peptide-based systems.[6] However, SABRE activity of most amino acids is low.
[b] Dr. T. Gutmann, Prof. G. Buntkowsky Technische Universitt Darmstadt Eduard Zintl Institut fìr Anorganische und Physikalische Chemie Alarich-Weiss-Str. 8, 64287 Darmstadt (Germany) [c] Dr. J. Frydel Venitur Sp. z o.o., Wawozowa 34 B, 31-752 Krakow (Poland) [d] B. Fedorczyk University of Warsaw, Faculty of Chemistry Pasteura 1, 02-093 Warsaw (Poland) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201501552, including full experimental details. Chem. Eur. J. 2015, 21, 12616 – 12619
Figure 1. a) General concept of the SABRE activation route. b) SABRE labeling of SABRE-inactive oligopeptides through the SPPS route.
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Communication Therefore, this methodology is not feasible for efficient hyperpolarization of selected parts of peptide-based molecules that would be of particular interest, for example, groups in the active center of an enzyme or enzyme inhibitor. The labeling approach presented in the current work ensures both selectivity and high SABRE activity, which is a key advantage of our approach. A pyridine-based molecular framework is a perfect candidate for labeling because pyridine and some simple pyridine-framework-based molecules exhibit high SABRE activity.[3e,f, 13] In particular, pyridine derivatives such as nicotinic and isonicotinic acids were attached to oligopeptides by standard solid-phase peptide synthesis (SPPS; see Figure 1 b). Both of these markers are biocompatible. Nicotinic acid is one of the ingredients of vitamin B3, whereas isonicotinic acid is used in the synthesis of antituberculosis drugs.[8] Furthermore, after incorporation of nicotinic or isonicotinic acid into the oligopeptide, the SABRE label resembles the nicotinamide or isonicotinamide molecular motif. Thus, based on toxicology research, metabolites of such systems should also be nontoxic.[9] Moreover, similar to our SABRE label, 18F-fluorinated pyridine-based molecular units are used as contrast agents in positron emission tomography (PET).[10] As target oligopeptide fragments, we chose tripeptides containing a combination of the two amino acids: glycine and alanine (see Figure 2). However, other peptide sequences are also feasible, due to the versatility and facility of the synthetic protocol. The standards to compare SABRE activity were nicotinic and isonicotinic amides (see Figure 2). As a catalyst we chose an Ir-metal complex with an electron-donating N-heterocyclic carbene (NHC) ligand (see Figure 2).[11]
Figure 3. Red line: single scan 1H NMR of hyperpolarized a) 4PyGAG, b) 3PyAAG immediately after sample introduction to the magnet; black line: single scan 1H NMR 300 s after the hyperpolarization (i.e., relaxed spectrum). Solvent = [D4]MeOH.
For all investigated oligopeptides no signal enhancement was observed on protons from the peptide unit, that is, ¢CH¢, ¢CH2¢, and ¢CH3 groups from glycine and alanine. For example, the aliphatic region of the hyperpolarized and fully relaxed spectra of 4PyGAG shows no significant difference (see Figure 4). The tiny discrepancies between the aliphatic part of
Figure 2. Investigated models and the catalyst.
Following the preparation and purification of all compounds, the SABRE effect was investigated (see the Supporting Information for experimental details). In all cases a characteristic SABRE 1H NMR pattern was observed for the pyridine label. For 4-substituted pyridine derivatives the signal of the two protons at position 2 has negative amplitude, whereas two protons at position 3 induce a signal with positive amplitude (see Figure 3 a). For 3-subsitituted pyridine derivatives the SABRE 1 H NMR signal from protons at positions 2, 2’, and 4 are of negative amplitude, whereas the signal from the proton at position 3 is positive (see Figure 3 b). Chem. Eur. J. 2015, 21, 12616 – 12619
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Figure 4. Red line: single scan 1H NMR of hyperpolarized 4PyGAG immediately after sample introduction; black line: single scan 1H NMR 300 s after the hyperpolarization (i.e., relaxed spectrum). The signals from ortho-hydrogen (o-H2) and HD gas (*) are also present. Solvent = [D4]MeOH.
the enhanced and the relaxed spectra are due to the lack of time required to build up magnetization immediately after sample introduction rather than the hyperpolarization effect. This hypothesis is supported by the fact that similar discrepancies were observed when a non-hyperpolarized sample was transported to the spectrometer and a 1H NMR spectrum was recorded immediately.
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Communication Table 1. Calculated e values for the investigated compounds. Note: for the e calculation details, see the Supporting Information. Solvent
Entry
e
Entry
e
[D4]MeOH
4Py 4PyGAG 4PyAAG 4Py 4PyGAG 4PyAAG
25.4 13.5 14.7 3.9 1.5 1.4
3Py 3PyGAG 3PyAAG 3Py 3PyGAG 3PyAAG
31.2 15.3 15.1 3.2 1.9 2.1
[D4]MeOH/D2O (v/v 1:1)
The enhancement factors (e) for the investigated model systems are summarized in Table 1. In a series of subsequent experiments for the same system, the value of e varied by approximately 10 %. Comparing the e values for pure [D4]MeOH and [D4]MeOH/D2O mixture, a lower e value was observed in the latter case. This observation relates to the lower p-H2 gas solubility in the binary mixture with water.[12] It was also observed that, with [D4]MeOH as a solvent, the averaged e values of 4PyGAG, 4PyAAG, 3PyGAG, and 3PyAAG varied by approximately 25 %. The e values for 3Py and 4Py standards were approximately two times higher than the e for the investigated oligopeptides. This may be explained by the shorter longitudinal relaxation time (T1) of pyridine-labeled oligopeptides compared to 3Py and 4Py (see Tables S2 and S3 in the Supporting Information). Furthermore, the comparison of the e values between our models and standards requires a deeper understanding of the first stage of the activation processes,[13] that is, catalyst–ligand interaction. To reveal how the catalyst interacts with the model compounds, a series of 1H NMR spectra of each of the oligopeptides dissolved in [D4]MeOH was recorded. In the next step, the catalyst was added to these samples. An exemplary 1 H NMR spectrum for 4PyGAG is presented in Figure 5. It is clearly visible that after adding the catalyst to the solution of 4PyGAG in [D4]MeOH new signals 2’, 3’, a’, and b’ occurred in the aromatic region. These signals result from a newly created complex (for the structure see Figure 5). However, in spite of having 4PyGAG in excess, not all of the free catalyst was involved in the complex formation. Integration of the single-scan 1H NMR spectrum reveals that, in the case of 4PyGAG, 4PyAAG, 3PyGAG, and 3PyAAG, approximately 31– 35 % (see Table S4 in the Supporting Information) of the catalyst was involved in the complex formation, whereas the rest of the catalyst remained free. Interestingly, there is an exchange process operating in the system. The spin-exchange spectroscopy (EXSY) spectrum presented in Figure 6 reveals crosspeaks related to the exchanging couples of signals (2, 2’), (3, 3’), (a, a’), and (b, b’), which confirms the reversibility of the process presented in Figure 5. Finally, as has been presented previously, p-H2 administration to the system seems to result in permanent deactivation of the free catalyst.[13] Therefore, it is assumed that in the case of investigated oligopeptides, a part of the loaded catalyst that had not been bound to the ligand was deactivated after p-H2 administration. This may explain the lower oligopeptide enhancement than in the case of 3Py and 4Py standards for which approximately 88 and 89 % of the catChem. Eur. J. 2015, 21, 12616 – 12619
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Figure 5. 1H NMR spectrum of the 4PyGAG and the catalyst in [D4]MeOH.
Figure 6. NMR 2D EXSY spectrum for the system presented in Figure 5.
alyst is engaged in complex formation, respectively (see Table S4 in the Supporting Information). To conclude, to the best of our knowledge, for the first time hyperpolarization was successfully induced in synthetic oligopeptides bearing a pyridine unit as an efficient SABRE label. Only the pyridine label was hyperpolarized, whereas the remaining parts of all oligopeptides were SABRE-inactive. The hyperpolarization effect was observed in both methanol and a methanol/water mixture. In methanol/water the evaluated enhancement factors are lower than in pure methanol, showing that further optimization of the methodology is still required to increase the hyperpolarization efficiency in biologically relevant systems. In particular, the design of a new watersoluble catalyst may facilitate hyperpolarization of oligopep-
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Communication tides under physiological conditions. Finally, for the first time it was noticed that, during the first step of catalyst activation, there is a slow exchange process between free and catalystbound ligands.
Acknowledgements
[5]
T.R. thanks the Polish National Science Center for financial support (SONATA-2011/03/D/ST4/02345). This research was supported by the Deutsche Forschungsgemeinschaft under contract Bu-911-22-1. Model compounds were analyzed with the use of CePT infrastructure financed by the European Union/European Regional Development Fund within the Operational Programme “Innovative economy” for 2007–2013. Keywords: hyperpolarization · NMR spectroscopy · peptides · signal amplification by reversible exchange · signal enhancement [1] a) B. M. Goodson, J. Magn. Reson. 2002, 155, 157 – 216; b) M. L. Lilburn, G. E. Pavlovskaya, T. Meersmann, J. Magn. Reson. 2013, 229, 173 – 186; c) S. Colombo Serra, M. Karlsson, G. B. Giovenzana, C. Cavallotti, F. Tedoldi, S. Aime, Contrast Media Mol. Imaging 2012, 7, 469 – 477. [2] a) Q. Z. Ni, E. Daviso, T. V. Can, E. Markhasin, S. K. Jawla, T. M. Swager, R. J. Temkin, J. Herzfeld, R. G. Griffin, Acc. Chem. Res. 2013, 46, 1933 – 1946; b) R. G. Griffin, T. F. Prisner, Phys. Chem. Chem. Phys. 2010, 12, 5737 – 5740. [3] a) C. R. Bowers, D. P. Weitekamp, J. Am. Chem. Soc. 1987, 109, 5541 – 5542; b) C. R. Bowers, D. P. Weitekamp, Phys. Rev. Lett. 1986, 57, 2645 – 2648; c) T. C. Eisenschmid, R. U. Kirss, P. P. Deutsch, S. I. Hommeltoft, R. Eisenberg, J. Bargon, R. G. Lawler, A. L. Balch, J. Am. Chem. Soc. 1987, 109, 8089 – 8091; d) M. G. Pravica, D. P. Weitekamp, Chem. Phys. Lett. 1988, 145, 255 – 258; e) R. W. Adams, J. A. Aguilar, K. D. Atkinson, M. J. Cowley, P. I. P. Elliott, S. B. Duckett, G. G. R. Green, I. G. Khazal, J. LopezSerrano, D. C. Williamson, Science 2009, 323, 1708 – 1711; f) K. D. Atkinson, M. J. Cowley, P. I. P. Elliott, S. B. Duckett, G. G. R. Green, J. Lopez-Serrano, A. C. Whitwood, J. Am. Chem. Soc. 2009, 131, 13362 – 13368. [4] a) M. Roth, A. Koch, P. Kindervater, J. Bargon, H. W. Spiess, K. Mìnnemann, J. Magn. Reson. 2010, 204, 50 – 55; b) F. Reineri, D. Santelia, A.
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Viale, E. Cerutti, L. Poggi, T. Tichy, S. S. D. Premkumar, R. Gobetto, S. Aime, J. Am. Chem. Soc. 2010, 132, 7186 – 7193; c) M. Kçrner, G. Sauer, A. Heil, D. Nasu, M. Empting, D. Tietze, S. Voigt, H. Weidler, T. Gutmann, O. Avrutina, H. Kolmar, T. Ratajczyk, G. Buntkowsky, Chem. Commun. 2013, 49, 7839 – 7841; d) T. Trantzschel, M. Plaumann, J. Bernarding, D. Lego, T. Ratajczyk, S. Dillenberger, G. Buntkowsky, J. Bargon, U. Bommerich, Appl. Magn. Reson. 2013, 44, 267 – 278. a) F. Gruppi, X. Xu, B. Zhang, J. A. Tang, A. Jerschow, J. W. Canary, Angew. Chem. Int. Ed. 2012, 51, 11787 – 11790; Angew. Chem. 2012, 124, 11 957 – 11 960; b) G. Sauer, D. Nasu, D. Tietze, T. Gutmann, S. Englert, O. Avrutina, H. Kolmar, G. Buntkowsky, Angew. Chem. Int. Ed. 2014, 53, 12941 – 12945; Angew. Chem. 2014, 126, 13 155 – 13 159. S. Glçggler, R. Mìller, J. Colell, M. Emondts, M. Dabrowski, B. Blìmich, S. Appelt, Phys. Chem. Chem. Phys. 2011, 13, 13759 – 13764. T. Theis, M. Truong, A. M. Coffey, E. Y. Chekmenev, W. S. Warren, J. Magn. Reson. 2014, 248, 23 – 26. a) G. V. Tsarichenko, V. I. Bobrov, M. V. Smarkov, Pharm. Chem. J. 1977, 11, 481 – 483; b) J. B. Bass, L. S. Farer, P. C. Hopewell, R. Obrien, R. F. Jacobs, F. Ruben, D. E. Snider, G. Thornton, Am. J. Respir. Crit. Care Med. 1994, 149, 1359 – 1374. P. Preziosi, Curr. Drug Metab. 2007, 8, 839 – 851. a) D. E. Olberg, J. M. Arukwe, D. Grace, O. K. Hjelstuen, M. Solbakken, G. M. Kindberg, A. Cuthbertson, J. Med. Chem. 2010, 53, 1732 – 1740; b) F. Basuli, C. Li, B. Xu, M. Williams, K. Wong, V. L. Coble, O. Vasalatiy, J. Seidel, M. V. Green, G. L. Griffiths, P. L. Choyke, E. M. Jagoda, Nucl. Med. Biol. 2015, 42, 219 – 225. a) M. J. Cowley, R. W. Adams, K. D. Atkinson, M. C. R. Cockett, S. B. Duckett, G. G. R. Green, J. A. B. Lohman, R. Kerssebaum, D. Kilgour, R. E. Mewis, J. Am. Chem. Soc. 2011, 133, 6134 – 6137; b) B. J. A. van Weerdenburg, S. Glçggler, N. Eshuis, A. H. J. Engwerda, J. M. M. Smits, R. de Gelder, S. Appelt, S. S. Wymenga, M. Tessari, M. C. Feiters, B. Blìmich, F. P. J. T. Rutjes, Chem. Commun. 2013, 49, 7388 – 7390. a) M. Herskowitz, J. Wisniak, L. Skladman, J. Chem. Eng. Data 1983, 28, 164 – 166; b) E. Brunner, J. Chem. Eng. Data 1985, 30, 269 – 273; c) P. Purwanto, R. M. Deshpande, R. V. Chaudhari, H. Delmas, J. Chem. Eng. Data 1996, 41, 1414 – 1417. M. L. Truong, F. Shi, P. He, B. Yuan, K. N. Plunkett, A. M. Coffey, R. V. Shchepin, D. A. Barskiy, K. V. Kovtunov, I. V. Koptyug, K. W. Waddell, B. M. Goodson, E. Y. Chekmenev, J. Phys. Chem. B 2014, 118, 13882 – 13889.
Received: April 21, 2015 Published online on July 17, 2015
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Communication
& Peptidomimetics
NMR Signal Enhancement by Effective SABRE Labeling of Oligopeptides Tomasz Ratajczyk,*[a] Torsten Gutmann,[b] Piotr Bernatowicz,[a] Gerd Buntkowsky,[b] Jaroslaw Frydel,[c] and Bartlomiej Fedorczyk[d] Abstract: Signal amplification by reversible exchange (SABRE) can enhance nuclear magnetic resonance signals by several orders of magnitude. However, until now this was limited to a small number of model target molecules. Here, a new convenient method for SABRE activation applicable to a variety of synthetic model oligopeptides is demonstrated. For the first time, a highly SABRE-active pyridine-based biocompatible molecular framework is incorporated into synthetic oligopeptides. The SABRE activity is preserved, demonstrating the importance of such earmarking. Finally, a crucial exchange process responsible for SABRE activity is identified and discussed.
Nuclear magnetic resonance (NMR) methods suffer strongly from low sensitivity. Therefore, since the early days of NMR, many efforts have been made to overcome this obstacle. One of the most commonly applied methodologies that can improve the NMR signal is hyperpolarization, which allows the generation of a high non-Boltzmann nuclear spin polarization. The most prominent hyperpolarization approaches are noble gas hyperpolarization,[1] dynamic nuclear polarization (DNP),[2] and parahydrogen-induced polarization (PHIP).[3] The latter can induce, in theory, a significant signal enhancement up to 105.[3] This enhancement can be obtained by two main chemical routes. Considering the first one, hyperpolarization of target molecules is achieved through a hydrogenation reaction with parahydrogen (p-H2) of a molecular system containing C¢C double- or triple bonds.[3a–d] During this process, the high spin order is transferred from p-H2 to the hydrogenated product. In [a] Dr. T. Ratajczyk, Dr. P. Bernatowicz Institute of Physical Chemistry, Polish Academy of Sciences Kasprzaka 44/52, 01-224 Warsaw (Poland) E-mail: [email protected]
the second route, the NMR signal is amplified by reversible exchange (SABRE).[3e,f] SABRE employs a reversible interaction where a labile complex containing p-H2, the ligand, and an iridium-based catalyst is formed. In such a complex, high spin polarization is transferred from p-H2 to the ligand via the catalyst.[3e,f] Finally, the complex splits up; however, the released ligand is hyperpolarized. Both of these PHIP variants open up an easy and inexpensive route for generation of hyperpolarized molecular systems, which renders PHIP very attractive for biomedical research and clinical applications. Unfortunately, a large number of molecules cannot be hyperpolarized, owing to the lack of appropriate hyperpolarizable units in their structures. Therefore, a PHIP activation of non-hyperpolarizable structures is at the center of scientific interest. One feasible route, which broadens the variety of hyperpolarizable systems, involves the integration of potently hyperpolarizable unsaturated moieties with non-hyperpolarizable molecules. Only recently, this approach has been demonstrated for important natural products such as barbituric acid, glucose, and simple alkyne-bearing oligopeptides.[4] Furthermore, the high potential for such procedures has also been demonstrated on relevant biological systems such as thiostrepton and fully functional sunflower trypsin inhibitor (SFTI-1).[5] Here, for the first time, we suggest a novel SABRE activation strategy that will further expand the set of hyperpolarizable molecular systems (see Figure 1 a). In particular, we show that the hyperpolarization is accessible through labeling of inactive oligopeptides with a SABRE-active molecular unit. Recently, SABRE was employed for amino acids and dipeptides hyperpolarization that theoretically may open up a path for NMR signal enhancement of larger peptide-based systems.[6] However, SABRE activity of most amino acids is low.
[b] Dr. T. Gutmann, Prof. G. Buntkowsky Technische Universitt Darmstadt Eduard Zintl Institut fìr Anorganische und Physikalische Chemie Alarich-Weiss-Str. 8, 64287 Darmstadt (Germany) [c] Dr. J. Frydel Venitur Sp. z o.o., Wawozowa 34 B, 31-752 Krakow (Poland) [d] B. Fedorczyk University of Warsaw, Faculty of Chemistry Pasteura 1, 02-093 Warsaw (Poland) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201501552, including full experimental details. Chem. Eur. J. 2015, 21, 12616 – 12619
Figure 1. a) General concept of the SABRE activation route. b) SABRE labeling of SABRE-inactive oligopeptides through the SPPS route.
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Communication Therefore, this methodology is not feasible for efficient hyperpolarization of selected parts of peptide-based molecules that would be of particular interest, for example, groups in the active center of an enzyme or enzyme inhibitor. The labeling approach presented in the current work ensures both selectivity and high SABRE activity, which is a key advantage of our approach. A pyridine-based molecular framework is a perfect candidate for labeling because pyridine and some simple pyridine-framework-based molecules exhibit high SABRE activity.[3e,f, 13] In particular, pyridine derivatives such as nicotinic and isonicotinic acids were attached to oligopeptides by standard solid-phase peptide synthesis (SPPS; see Figure 1 b). Both of these markers are biocompatible. Nicotinic acid is one of the ingredients of vitamin B3, whereas isonicotinic acid is used in the synthesis of antituberculosis drugs.[8] Furthermore, after incorporation of nicotinic or isonicotinic acid into the oligopeptide, the SABRE label resembles the nicotinamide or isonicotinamide molecular motif. Thus, based on toxicology research, metabolites of such systems should also be nontoxic.[9] Moreover, similar to our SABRE label, 18F-fluorinated pyridine-based molecular units are used as contrast agents in positron emission tomography (PET).[10] As target oligopeptide fragments, we chose tripeptides containing a combination of the two amino acids: glycine and alanine (see Figure 2). However, other peptide sequences are also feasible, due to the versatility and facility of the synthetic protocol. The standards to compare SABRE activity were nicotinic and isonicotinic amides (see Figure 2). As a catalyst we chose an Ir-metal complex with an electron-donating N-heterocyclic carbene (NHC) ligand (see Figure 2).[11]
Figure 3. Red line: single scan 1H NMR of hyperpolarized a) 4PyGAG, b) 3PyAAG immediately after sample introduction to the magnet; black line: single scan 1H NMR 300 s after the hyperpolarization (i.e., relaxed spectrum). Solvent = [D4]MeOH.
For all investigated oligopeptides no signal enhancement was observed on protons from the peptide unit, that is, ¢CH¢, ¢CH2¢, and ¢CH3 groups from glycine and alanine. For example, the aliphatic region of the hyperpolarized and fully relaxed spectra of 4PyGAG shows no significant difference (see Figure 4). The tiny discrepancies between the aliphatic part of
Figure 2. Investigated models and the catalyst.
Following the preparation and purification of all compounds, the SABRE effect was investigated (see the Supporting Information for experimental details). In all cases a characteristic SABRE 1H NMR pattern was observed for the pyridine label. For 4-substituted pyridine derivatives the signal of the two protons at position 2 has negative amplitude, whereas two protons at position 3 induce a signal with positive amplitude (see Figure 3 a). For 3-subsitituted pyridine derivatives the SABRE 1 H NMR signal from protons at positions 2, 2’, and 4 are of negative amplitude, whereas the signal from the proton at position 3 is positive (see Figure 3 b). Chem. Eur. J. 2015, 21, 12616 – 12619
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Figure 4. Red line: single scan 1H NMR of hyperpolarized 4PyGAG immediately after sample introduction; black line: single scan 1H NMR 300 s after the hyperpolarization (i.e., relaxed spectrum). The signals from ortho-hydrogen (o-H2) and HD gas (*) are also present. Solvent = [D4]MeOH.
the enhanced and the relaxed spectra are due to the lack of time required to build up magnetization immediately after sample introduction rather than the hyperpolarization effect. This hypothesis is supported by the fact that similar discrepancies were observed when a non-hyperpolarized sample was transported to the spectrometer and a 1H NMR spectrum was recorded immediately.
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Communication Table 1. Calculated e values for the investigated compounds. Note: for the e calculation details, see the Supporting Information. Solvent
Entry
e
Entry
e
[D4]MeOH
4Py 4PyGAG 4PyAAG 4Py 4PyGAG 4PyAAG
25.4 13.5 14.7 3.9 1.5 1.4
3Py 3PyGAG 3PyAAG 3Py 3PyGAG 3PyAAG
31.2 15.3 15.1 3.2 1.9 2.1
[D4]MeOH/D2O (v/v 1:1)
The enhancement factors (e) for the investigated model systems are summarized in Table 1. In a series of subsequent experiments for the same system, the value of e varied by approximately 10 %. Comparing the e values for pure [D4]MeOH and [D4]MeOH/D2O mixture, a lower e value was observed in the latter case. This observation relates to the lower p-H2 gas solubility in the binary mixture with water.[12] It was also observed that, with [D4]MeOH as a solvent, the averaged e values of 4PyGAG, 4PyAAG, 3PyGAG, and 3PyAAG varied by approximately 25 %. The e values for 3Py and 4Py standards were approximately two times higher than the e for the investigated oligopeptides. This may be explained by the shorter longitudinal relaxation time (T1) of pyridine-labeled oligopeptides compared to 3Py and 4Py (see Tables S2 and S3 in the Supporting Information). Furthermore, the comparison of the e values between our models and standards requires a deeper understanding of the first stage of the activation processes,[13] that is, catalyst–ligand interaction. To reveal how the catalyst interacts with the model compounds, a series of 1H NMR spectra of each of the oligopeptides dissolved in [D4]MeOH was recorded. In the next step, the catalyst was added to these samples. An exemplary 1 H NMR spectrum for 4PyGAG is presented in Figure 5. It is clearly visible that after adding the catalyst to the solution of 4PyGAG in [D4]MeOH new signals 2’, 3’, a’, and b’ occurred in the aromatic region. These signals result from a newly created complex (for the structure see Figure 5). However, in spite of having 4PyGAG in excess, not all of the free catalyst was involved in the complex formation. Integration of the single-scan 1H NMR spectrum reveals that, in the case of 4PyGAG, 4PyAAG, 3PyGAG, and 3PyAAG, approximately 31– 35 % (see Table S4 in the Supporting Information) of the catalyst was involved in the complex formation, whereas the rest of the catalyst remained free. Interestingly, there is an exchange process operating in the system. The spin-exchange spectroscopy (EXSY) spectrum presented in Figure 6 reveals crosspeaks related to the exchanging couples of signals (2, 2’), (3, 3’), (a, a’), and (b, b’), which confirms the reversibility of the process presented in Figure 5. Finally, as has been presented previously, p-H2 administration to the system seems to result in permanent deactivation of the free catalyst.[13] Therefore, it is assumed that in the case of investigated oligopeptides, a part of the loaded catalyst that had not been bound to the ligand was deactivated after p-H2 administration. This may explain the lower oligopeptide enhancement than in the case of 3Py and 4Py standards for which approximately 88 and 89 % of the catChem. Eur. J. 2015, 21, 12616 – 12619
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Figure 5. 1H NMR spectrum of the 4PyGAG and the catalyst in [D4]MeOH.
Figure 6. NMR 2D EXSY spectrum for the system presented in Figure 5.
alyst is engaged in complex formation, respectively (see Table S4 in the Supporting Information). To conclude, to the best of our knowledge, for the first time hyperpolarization was successfully induced in synthetic oligopeptides bearing a pyridine unit as an efficient SABRE label. Only the pyridine label was hyperpolarized, whereas the remaining parts of all oligopeptides were SABRE-inactive. The hyperpolarization effect was observed in both methanol and a methanol/water mixture. In methanol/water the evaluated enhancement factors are lower than in pure methanol, showing that further optimization of the methodology is still required to increase the hyperpolarization efficiency in biologically relevant systems. In particular, the design of a new watersoluble catalyst may facilitate hyperpolarization of oligopep-
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Communication tides under physiological conditions. Finally, for the first time it was noticed that, during the first step of catalyst activation, there is a slow exchange process between free and catalystbound ligands.
Acknowledgements
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Received: April 21, 2015 Published online on July 17, 2015
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