{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T20:26:20Z","timestamp":1760300780316,"version":"3.41.2"},"reference-count":24,"publisher":"Wiley","issue":"10","license":[{"start":{"date-parts":[[2022,10,6]],"date-time":"2022-10-06T00:00:00Z","timestamp":1665014400000},"content-version":"vor","delay-in-days":5,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000038","name":"Natural Sciences and Engineering Research Council of Canada","doi-asserted-by":"publisher","award":["RGPIN\u20102020\u201005388"],"award-info":[{"award-number":["RGPIN\u20102020\u201005388"]}],"id":[{"id":"10.13039\/501100000038","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001804","name":"Canada Research Chairs","doi-asserted-by":"publisher","award":["950\u2010231665"],"award-info":[{"award-number":["950\u2010231665"]}],"id":[{"id":"10.13039\/501100001804","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2022,10]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>The reversible oxidation of cysteine thiol groups to sulfenic acid by reactive oxygen species (ROS) such as hydrogen peroxide can impact protein function, activity, and localization. As a consequence, ROS have profound effects on cell functions including proliferation, differentiation, and survival. Furthermore, there are clear associations between the effects of ROS on cells and the etiology of several diseases including cancer and neurodegeneration. In spite of the importance of cysteine sulfenylation as a validated post\u2010translational modification, its labile nature impedes efficient and reproducible detection of proteins with cysteine sulfenic acid residues. To overcome this challenge, we developed a novel cell\u2010permeable bifunctional reagent, consisting of two linked bicyclo[6.1.0]nonyne (BCN) moieties coupled with a short ethylenediamine\u2010derived linker (BCN\u2010E\u2010BCN) that enables the detection of sulfenylated proteins <jats:italic>in vitro<\/jats:italic> and in intact cells. The two symmetrical BCN groups allow protein sulfenic acids to be selectively tagged with a BCN at one end while allowing for copper\u2010free click chemistry with azide\u2010tagged reagents of the opposite BCN. In this protocol, the synthesis of BCN\u2010E\u2010BCN and its use to detect cysteine sulfenic acids will be detailed. \u00a9 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Copper\u2010mediated cyclopropanation of 1,5\u2010cyclooctadiene<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Synthesis of <jats:italic>endo\u2010<\/jats:italic> and <jats:italic>exo\u2010<\/jats:italic>bicyclononyne<\/jats:p><jats:p><jats:bold>Basic Protocol 3<\/jats:bold>: Synthesis of <jats:italic>endo\u2010<\/jats:italic>BCN\u2010E\u2010BCN<\/jats:p><jats:p><jats:bold>Basic Protocol 4<\/jats:bold>: BCN\u2010E\u2010BCN treatment of wild\u2010type and cysteine\u2010deficient mutant recombinant cofilin protein<\/jats:p><jats:p><jats:bold>Basic Protocol 5<\/jats:bold>: BCN\u2010E\u2010BCN labeling in live cells<\/jats:p><jats:p><jats:bold>Basic Protocol 6<\/jats:bold>: Western blotting and visualization of BCN\u2010E\u2010BCN\u2010labeled samples<\/jats:p>","DOI":"10.1002\/cpz1.559","type":"journal-article","created":{"date-parts":[[2022,10,6]],"date-time":"2022-10-06T13:28:43Z","timestamp":1665062923000},"source":"Crossref","is-referenced-by-count":4,"title":["Synthesis and Use of the Bifunctional Sulfenic Acid Probe BCN\u2010E\u2010BCN for <i>In Vitro<\/i> and Cell\u2010Based Assays of Protein Oxidation"],"prefix":"10.1002","volume":"2","author":[{"given":"Katarina","family":"Micovic","sequence":"first","affiliation":[{"name":"Department of Chemistry and Biology Toronto Metropolitan University  Toronto Ontario Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Thershan","family":"Satkunarajah","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biology Toronto Metropolitan University  Toronto Ontario Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alexandre","family":"Carnet","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biology Toronto Metropolitan University  Toronto Ontario Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mackenzie","family":"Hurst","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biology Toronto Metropolitan University  Toronto Ontario Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Russell","family":"Viirre","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biology Toronto Metropolitan University  Toronto Ontario Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael F.","family":"Olson","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biology Toronto Metropolitan University  Toronto Ontario Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2022,10,6]]},"reference":[{"key":"e_1_2_12_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0021\u20109258(19)42244\u20108"},{"key":"e_1_2_12_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cub.2015.04.020"},{"key":"e_1_2_12_4_1","doi-asserted-by":"publisher","DOI":"10.1161\/HYPERTENSIONAHA.118.10824"},{"key":"e_1_2_12_5_1","doi-asserted-by":"publisher","DOI":"10.1093\/cvr\/cvab171"},{"key":"e_1_2_12_6_1","doi-asserted-by":"publisher","DOI":"10.1074\/mcp.M700065\u2010MCP200"},{"key":"e_1_2_12_7_1","doi-asserted-by":"publisher","DOI":"10.1155\/2017\/2467940"},{"key":"e_1_2_12_8_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi973035t"},{"key":"e_1_2_12_9_1","doi-asserted-by":"publisher","DOI":"10.1002\/anie.201003761"},{"key":"e_1_2_12_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.abb.2016.09.013"},{"key":"e_1_2_12_11_1","doi-asserted-by":"publisher","DOI":"10.3389\/fnmol.2017.00167"},{"key":"e_1_2_12_12_1","doi-asserted-by":"publisher","DOI":"10.3390\/12102427"},{"key":"e_1_2_12_13_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467\u2010018\u201006790\u20101"},{"key":"e_1_2_12_14_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1156906"},{"key":"e_1_2_12_15_1","doi-asserted-by":"publisher","DOI":"10.1158\/0008\u20105472.CAN\u201005\u20103640"},{"key":"e_1_2_12_16_1","doi-asserted-by":"publisher","DOI":"10.1039\/C7RA08136G"},{"key":"e_1_2_12_17_1","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.6b00742"},{"key":"e_1_2_12_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0076\u20106879(10)73004\u20104"},{"key":"e_1_2_12_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chembiol.2009.01.003"},{"key":"e_1_2_12_20_1","doi-asserted-by":"publisher","DOI":"10.1021\/ja500364r"},{"key":"e_1_2_12_21_1","doi-asserted-by":"publisher","DOI":"10.1039\/c2cc17868k"},{"key":"e_1_2_12_22_1","doi-asserted-by":"publisher","DOI":"10.3109\/10715762.2014.884278"},{"key":"e_1_2_12_23_1","doi-asserted-by":"publisher","DOI":"10.1038\/emm.1999.9"},{"key":"e_1_2_12_24_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00395\u2010013\u20100385\u20106"},{"key":"e_1_2_12_25_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467\u2010018\u201004003\u20103"}],"container-title":["Current 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