{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,20]],"date-time":"2026-07-20T04:21:10Z","timestamp":1784521270600,"version":"3.55.0"},"reference-count":46,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2024,1,26]],"date-time":"2024-01-26T00:00:00Z","timestamp":1706227200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Russian Science Foundation","award":["RSF 23-13-00178"],"award-info":[{"award-number":["RSF 23-13-00178"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Highly resistant to reduction nitroxides open new opportunities for structural studies of biological macromolecules in their native environment inside living cells and for functional imaging of pH and thiols, enzymatic activity and redox status in living animals. 3,4-Disubstituted nitroxides of 2,2,5,5-tetraethylpyrrolidine and pyrroline series with a functional group for binding to biomolecules and a polar moiety for higher solubility in water and for more rigid attachment via additional coordination to polar sites were designed and synthesized. The EPR spectra, lipophilicities, kinetics of the reduction in ascorbate-containing systems and the decay rates in liver homogenates were measured. The EPR spectra of all 3,4-disubstituted pyrrolidine nitroxides showed additional large splitting on methylene hydrogens of the ethyl groups, while the spectra of similar pyrroline nitroxides were represented with a simple triplet with narrow lines and hyperfine structure of the nitrogen manifolds resolved in oxygen-free conditions. Both pyrrolidine and pyrroline nitroxides demonstrated low rates of reduction with ascorbate, pyrrolidines being a bit more stable than similar pyrrolines. The decay of positively charged nitroxides in the rat liver homogenate was faster than that of neutral and negatively charged radicals, with lipophilicity, rate of reduction with ascorbate and the ring type playing minor role. The EPR spectra of N,N-dimethyl-3,4-bis-(aminomethyl)-2,2,5,5-tetraethylpyrrolidine-1-oxyl showed dependence on pH with pKa = 3, \u0394aN = 0.055 mT and \u0394aH = 0.075 mT.<\/jats:p>","DOI":"10.3390\/ijms25031550","type":"journal-article","created":{"date-parts":[[2024,1,26]],"date-time":"2024-01-26T08:56:01Z","timestamp":1706259361000},"page":"1550","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Hydrophilic Reduction-Resistant Spin Labels of Pyrrolidine and Pyrroline Series from 3,4-Bis-hydroxymethyl-2,2,5,5-tetraethylpyrrolidine-1-oxyl"],"prefix":"10.3390","volume":"25","author":[{"given":"Mikhail S.","family":"Usatov","sequence":"first","affiliation":[{"name":"N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry SB RAS, Lavrentiev Ave. 9, Novosibirsk 630090, Russia"},{"name":"Department of Natural Sciences, Novosibirsk State University, Pirogova Str. 1, Novosibirsk 630090, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sergey A.","family":"Dobrynin","sequence":"additional","affiliation":[{"name":"N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry SB RAS, Lavrentiev Ave. 9, Novosibirsk 630090, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1770-1968","authenticated-orcid":false,"given":"Yuliya F.","family":"Polienko","sequence":"additional","affiliation":[{"name":"N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry SB RAS, Lavrentiev Ave. 9, Novosibirsk 630090, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2403-2843","authenticated-orcid":false,"given":"Denis A.","family":"Morozov","sequence":"additional","affiliation":[{"name":"N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry SB RAS, Lavrentiev Ave. 9, Novosibirsk 630090, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yurii I.","family":"Glazachev","sequence":"additional","affiliation":[{"name":"Voevodsky Institute of Chemical Kinetics and Combustion SB RAS, Institutskaya 3, Novosibirsk 630090, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sergey V.","family":"An\u2019kov","sequence":"additional","affiliation":[{"name":"N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry SB RAS, Lavrentiev Ave. 9, Novosibirsk 630090, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tatiana G.","family":"Tolstikova","sequence":"additional","affiliation":[{"name":"N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry SB RAS, Lavrentiev Ave. 9, Novosibirsk 630090, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuri V.","family":"Gatilov","sequence":"additional","affiliation":[{"name":"N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry SB RAS, Lavrentiev Ave. 9, Novosibirsk 630090, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Irina Yu.","family":"Bagryanskaya","sequence":"additional","affiliation":[{"name":"N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry SB RAS, Lavrentiev Ave. 9, Novosibirsk 630090, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arthur E.","family":"Raizvikh","sequence":"additional","affiliation":[{"name":"N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry SB RAS, Lavrentiev Ave. 9, Novosibirsk 630090, Russia"},{"name":"Department of Physics, Novosibirsk State University, Pirogova Str. 1, Novosibirsk 630090, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0057-383X","authenticated-orcid":false,"given":"Elena G.","family":"Bagryanskaya","sequence":"additional","affiliation":[{"name":"N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry SB RAS, Lavrentiev Ave. 9, Novosibirsk 630090, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6033-0368","authenticated-orcid":false,"given":"Igor A.","family":"Kirilyuk","sequence":"additional","affiliation":[{"name":"N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry SB RAS, Lavrentiev Ave. 9, Novosibirsk 630090, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,1,26]]},"reference":[{"key":"ref_1","first-page":"1","article-title":"Electron paramagnetic resonance spectroscopy in structural-dynamic studies of large protein complexes","volume":"Volume 134\u2013135","author":"Neuhaus","year":"2023","journal-title":"Progress in Nuclear Magnetic Resonance Spectroscopy"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1002\/cbic.201900291","article-title":"In-Cell EPR: Progress towards Structural Studies Inside Cells","volume":"21","author":"Bonucci","year":"2020","journal-title":"ChemBioChem"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Ouari, O., and Gigmes, D. (2021). Nitroxides: Synthesis, Properties and Applications, The Royal Society of Chemistry. Chapter 10.","DOI":"10.1039\/9781788019651"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Ouari, O., and Gigmes, D. (2021). Nitroxides: Synthesis, Properties and Applications, The Royal Society of Chemistry. Chapter 4.","DOI":"10.1039\/9781788019651"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Likhtenshtein, G.I., Yamauchi, J., Nakatsuji, S., Smirnov, A., and Tamura, R. (2008). Nitroxides: Applications in Chemistry, Biomedicine and Material Science, Wiley\u2014VCH Verlag, GmbH & Co. KGaA. Chapter 7.","DOI":"10.1002\/9783527621743"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1039\/9781849730877-00071","article-title":"Spin labels and spin probes for measurements of local pH and electrostatics by EPR","volume":"Volume 22","author":"Gilbert","year":"2010","journal-title":"Electron Paramagnetic Resonance"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"6120","DOI":"10.1039\/D3SC01079A","article-title":"Polarizing agents for efficient high field DNP solid-state NMR spectroscopy under magic-angle spinning: From design principles to formulation strategies","volume":"14","author":"Menzildjian","year":"2023","journal-title":"Chem. Sci."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1016\/j.jsb.2018.09.011","article-title":"DNP NMR of biomolecular assemblies","volume":"206","author":"Jaudzems","year":"2019","journal-title":"J. Struct. Biol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1004","DOI":"10.3109\/10715762.2012.670874","article-title":"Imaging in vivo redox status in high spatial resolution with OMRI","volume":"46","author":"Ichikawa","year":"2012","journal-title":"Free Radic. Res."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Boudries, D., Massot, P., Parzy, E., Seren, S., Mellet, P., Franconi, J.-M., Miraux, S., Bezan\u00e7on, E., Marque, S.R.A., and Audran, G. (2023). A system for in vivo on-demand ultra-low field Overhauser-enhanced 3D-Magnetic resonance imaging. J. Magn. Reson., 348.","DOI":"10.1016\/j.jmr.2023.107383"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1274","DOI":"10.1039\/D2CC05907J","article-title":"Dance with Spins: Site-Directed Spin Labeling Coupled to Electron Paramagnetic Resonance Spectroscopy Directly inside Cells","volume":"59","author":"Pierro","year":"2023","journal-title":"Chem. Commun."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"3940","DOI":"10.1021\/acs.analchem.2c03194","article-title":"Electron Paramagnetic Resonance Implemented with Multiple Harmonic Detections Successfully Maps Extracellular pH In Vivo","volume":"95","author":"Nakaoka","year":"2023","journal-title":"Anal. Chem."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"13379","DOI":"10.1002\/anie.201506267","article-title":"Enzymatically Shifting Nitroxides for EPR Spectroscopy and Overhauser-Enhanced Magnetic Resonance Imaging","volume":"54","author":"Audran","year":"2015","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"328","DOI":"10.1016\/j.freeradbiomed.2012.04.026","article-title":"Whole-body kinetic image of a redox probe in mice using Overhauser-enhanced MRI","volume":"53","author":"Kosem","year":"2012","journal-title":"Free Rad. Biol. Med."},{"key":"ref_15","unstructured":"Kocherginsky, N., and Swartz, H. (1995). Nitroxide Spin Labels\u2014Reactions in Biology and Chemistry, CRC Press, Inc."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"5322","DOI":"10.1021\/ol302506f","article-title":"Synthesis and Reduction Kinetics of Sterically Shielded Pyrrolidine Nitroxides","volume":"14","author":"Paletta","year":"2012","journal-title":"Org. Lett."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"5298","DOI":"10.1021\/ol502449r","article-title":"Synthesis of unnatural amino acids functionalized with sterically shielded pyrroline nitroxides","volume":"16","author":"Wang","year":"2014","journal-title":"Org. Lett."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"78","DOI":"10.3109\/10715762.2014.979409","article-title":"Sterically Shielded Spin Labels for In-Cell EPR Spectroscopy: Analysis of Stability in Reducing Environment","volume":"49","author":"Jagtap","year":"2015","journal-title":"Free Radic. Res."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Ketter, S., Dajka, M., Rogozhnikova, O., Dobrynin, S.A., Tormyshev, V.M., Bagryanskaya, E.G., and Joseph, B. (2022). In situ distance measurements in a membrane transporter using maleimide functionalized orthogonal spin labels and 5-pulse electron-electron double resonance spectroscopy. J. Magn. Reson. Open, 10.","DOI":"10.1016\/j.jmro.2022.100041"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Bleicken, S., Assafa, T.E., Zhang, H., Elsner, C., Ritsch, I., Pink, M., Rajca, S., Jeschke, G., Rajca, A., and Bordignon, E. (2019). gem-Diethyl Pyrroline Nitroxide Spin Labels: Synthesis, EPR Characterization, Rotamer Libraries and Biocompatibility. ChemistryOpen, 8.","DOI":"10.1002\/open.201900232"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1366","DOI":"10.1002\/anie.201710184","article-title":"Bioresistant Nitroxide Spin Label for In-Cell EPR Spectroscopy: In Vitro and In Oocytes Protein Structural Dynamics Studies","volume":"57","author":"Karthikeyan","year":"2018","journal-title":"Angew. Chem. Int. Ed. Engl."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Dobrynin, S.A., Usatov, M.S., Zhurko, I.F., Morozov, D.A., Polienko, Y.F., Glazachev, Y.I., Parkhomenko, D.A., Tyumentsev, M.A., Gatilov, Y.V., and Chernyak, E.I. (2021). A simple method of synthesis of 3-carboxy-2,2,5,5-tetraethylpyrrolidine-1-oxyl and preparation of reduction-resistant spin labels and probes of pyrrolidine series. Molecules, 26.","DOI":"10.3390\/molecules26195761"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Ovcherenko, S.S., Chinak, O.A., Chechushkov, A.V., Dobrynin, S.A., Kirilyuk, I.A., Krumkacheva, O.A., Richter, V.A., and Bagryanskaya, E.G. (2021). Uptake of Cell-Penetrating Peptide RL2 by Human Lung Cancer Cells: Monitoring by Electron Paramagnetic Resonance and Confocal Laser Scanning Microscopy. Molecules, 26.","DOI":"10.3390\/molecules26185442"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Ran\u010di\u0107, A., Babi\u0107, N., Orio, M., and Peyrot, F. (2023). Structural features governing the metabolic stability of tetraethyl-substituted nitroxides in rat liver microsomes. Antioxidants, 12.","DOI":"10.3390\/antiox12020402"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"144","DOI":"10.1016\/j.freeradbiomed.2020.05.021","article-title":"Unexpected rapid aerobic transformation of 2,2,6,6-tetraethyl-4-oxo(piperidin-1-yloxyl) radical by cytochrome P450 in the presence of NADPH: Evidence against a simple reduction of the nitroxide moiety to the hydroxylamine","volume":"156","author":"Orio","year":"2020","journal-title":"Free Radic. Biol. Med."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"5392","DOI":"10.1021\/acs.joc.8b00085","article-title":"Synthesis of 3,4-bis(hydroxymethyl)-2,2,5,5-tetraethylpyrrolidin-1-oxyl via 1,3-dipolar cycloaddition of azomethine ylide to activated alkene","volume":"83","author":"Dobrynin","year":"2018","journal-title":"J. Org. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Dobrynin, S.A., Gulman, M.M., Morozov, D.A., Zhurko, I.F., Taratayko, A.I., Sotnikova, Y.S., Glazachev, Y.I., Gatilov, Y.V., and Kirilyuk, I.A. (2022). Synthesis of Sterically Shielded Nitroxides Using the Reaction of Nitrones with Alkynylmagnesium Bromides. Molecules, 27.","DOI":"10.3390\/molecules27217626"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Dobrynin, S., Kutseikin, S., Morozov, D., Krumkacheva, O., Spitsyna, A., Gatilov, Y., Silnikov, V., Angelovski, G., Bowman, M.K., and Kirilyuk, I. (2020). Human serum albumin labelled with sterically-hindered nitroxides as potential MRI contrast agents. Molecules, 25.","DOI":"10.3390\/molecules25071709"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1080\/10715760701787701","article-title":"Spin labelling of Bacillus anthracis endospores: A model for in vivo tracking by EPR imaging","volume":"42","author":"Tsai","year":"2008","journal-title":"Free Radic. Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"390","DOI":"10.1055\/s-1993-25870","article-title":"Synthesis of new 3,4-disubstituted 2,5-dihydro-1H-pyrrol-1-yloxyl spin label reagents via allylic rearrangements","volume":"1993","author":"Hideg","year":"1993","journal-title":"Synthesis"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"339","DOI":"10.1080\/10715762.2017.1390744","article-title":"Cellular accumulation and antioxidant activity of acetoxymethoxycarbonyl pyrrolidine nitroxides","volume":"52","author":"Dikalov","year":"2018","journal-title":"Free Radic. Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"3655","DOI":"10.1055\/s-0032-1317516","article-title":"Synthesis of nitroxide-annulated carbocycles and heteroicycles","volume":"44","author":"Zsolnai","year":"2012","journal-title":"Synthesis"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1007\/s10858-011-9545-x","article-title":"A rigid disulfide-linked nitroxide side chain simplifies the quantitative analysis of PRE data","volume":"51","author":"Fawzi","year":"2011","journal-title":"J. Biomol. NMR."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"3828","DOI":"10.1021\/bi002645h","article-title":"Molecular motion of spin labeled side chains in alpha-helices: Analysis by variation of side chain structure","volume":"40","author":"Columbus","year":"2001","journal-title":"Biochemistry"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"578","DOI":"10.1021\/bc980138v","article-title":"Synthesis and application of novel bifunctional spin labels","volume":"10","author":"Philipp","year":"1999","journal-title":"Bioconjug. Chem."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"4875","DOI":"10.1039\/C9CP05584C","article-title":"Combining site-directed spin labeling in vivo and in-cell EPR distance determination","volume":"22","author":"Widder","year":"2020","journal-title":"Phys. Chem. Chem. Phys."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1821","DOI":"10.1073\/pnas.0911116107","article-title":"Copper-free click chemistry in living animals","volume":"107","author":"Chang","year":"2010","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"406","DOI":"10.1021\/bc0497463","article-title":"1-[3-(2-[18F]fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione: Design, synthesis, and radiosynthesis of a new [18F]fluoropyridine-based maleimide reagent for the labeling of peptides and proteins","volume":"16","author":"Kuhnast","year":"2005","journal-title":"Bioconjug. Chem."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"531","DOI":"10.1002\/hlca.19750580224","article-title":"Preparation and some properties of maleimido acids and maleoyl derivatives of peptides","volume":"58","author":"Keller","year":"1975","journal-title":"Helv. Chim. Acta"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1336","DOI":"10.1055\/s-0028-1088018","article-title":"Click Reactions with Nitroxides","volume":"2009","author":"Hubbell","year":"2009","journal-title":"Synthesis"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"38723","DOI":"10.1021\/acsomega.3c06090","article-title":"Origin of Long-Range Hyperfine Couplings in the EPR Spectra of 2,2,5,5-Tetraethylpyrrolidine-1-oxyls","volume":"8","author":"Polienko","year":"2023","journal-title":"ACS Omega"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1719","DOI":"10.1021\/jp9103678","article-title":"Inclusion Complexes of Nitroxides of Pyrrolidine and Imidazoline Series with Cucurbit[7]uril","volume":"114","author":"Kirilyuk","year":"2010","journal-title":"J. Phys. Chem. B"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1269","DOI":"10.1039\/B418707E","article-title":"Nitroxides with two pK values\u2013Useful spin probes for pH monitoring within a broad range","volume":"3","author":"Kirilyuk","year":"2005","journal-title":"Org. Biomol. Chem."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1445","DOI":"10.4155\/bio.09.128","article-title":"Mechanisms of amine accumulation in, and egress from, lysosomes","volume":"1","author":"Goldman","year":"2009","journal-title":"Bioanalysis"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"780","DOI":"10.1016\/j.freeradbiomed.2011.05.019","article-title":"A novel nitroxide is an effective brain redox imaging contrast agent and in vivo radioprotector","volume":"51","author":"Davis","year":"2011","journal-title":"Free Radic. Biol. Med."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"973","DOI":"10.1055\/s-1999-3502","article-title":"Synthesis and Reactions of a Symmetric Paramagnetic Pyrrolidine Diene","volume":"1999","author":"Balog","year":"1999","journal-title":"Synthesis"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/3\/1550\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T13:49:55Z","timestamp":1760104195000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/3\/1550"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,1,26]]},"references-count":46,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2024,2]]}},"alternative-id":["ijms25031550"],"URL":"https:\/\/doi.org\/10.3390\/ijms25031550","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,1,26]]}}}