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The pyrrolo[2,1\u2010<jats:italic>c<\/jats:italic>][1,4]benzodiazepines (PBDs) have been established as potent DNA binders that can achieve inter\u2010 and intrastrand DNA cross\u2010links when dimerized, and have been used as cytotoxic payloads in multiple antibody\u2010drug conjugates (ADCs). In this study, we explore the potential of click chemistry to obtain PBD heterodimers. The heterodimers <jats:bold>D1<\/jats:bold>\u2013<jats:bold>D4<\/jats:bold> were prepared by copper catalyzed or strain\u2010promoted azide\u2010alkyne cycloadditions (SPAACs) from the corresponding monomers <jats:bold>M1<\/jats:bold> \u2013 <jats:bold>M4<\/jats:bold> and <jats:bold>MbA<\/jats:bold>. The interactions of monomers and dimers with DNA were evaluated by DNA thermal denaturation analysis, a newly developed mass spectrometry\u2010based method for the detection of single stand and double strand modifications, and by DNase I footprinting. All methods demonstrated the DNA binding and cross\u2010linking capabilities of <jats:bold>D1<\/jats:bold>. Testing for cytotoxicity in three cell lines revealed that the monomers <jats:bold>M1<\/jats:bold> and <jats:bold>M4<\/jats:bold> and the dimers <jats:bold>D1<\/jats:bold> and <jats:bold>D4<\/jats:bold> were the most potent. The click chemistry approach opens an easy access to larger libraries of PBD\u2010based DNA binders, including the option for dimerization in live cells.<\/jats:p>","DOI":"10.1002\/chem.202501797","type":"journal-article","created":{"date-parts":[[2025,8,23]],"date-time":"2025-08-23T06:02:12Z","timestamp":1755928932000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Synthesis and Evaluation of DNA Cross\u2010linkers by Click Chemistry\u2010Mediated Heterodimerization of Nor\u2010Tomaymycins"],"prefix":"10.1002","volume":"31","author":[{"given":"Julia","family":"Friederich","sequence":"first","affiliation":[{"name":"Department of Chemical Biology Helmholtz Centre for Infection Research  Inhoffenstra\u00dfe 7 38124 Braunschweig Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Katharina","family":"Rox","sequence":"additional","affiliation":[{"name":"Department of Chemical Biology Helmholtz Centre for Infection Research  Inhoffenstra\u00dfe 7 38124 Braunschweig Germany"},{"name":"German Center for Infection Research (DZIF) Site Hannover\u2010Braunschweig  38124 Braunschweig Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3283-001X","authenticated-orcid":false,"given":"Hazel L. 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Mahbub","family":"Hasan","sequence":"additional","affiliation":[{"name":"Department of Genetic Engineering and Biotechnology Faculty of Biological Sciences University of Chittagong, Chittagong  Chattogram 4331 Bangladesh"},{"name":"Institute of Pharmaceutical Science School of Cancer &amp; Pharmaceutical Sciences King's College London Franklin\u2010Wilkins Building  150 Stamford Street London UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Patrick","family":"Raunft","sequence":"additional","affiliation":[{"name":"Department of Chemical Biology Helmholtz Centre for Infection Research  Inhoffenstra\u00dfe 7 38124 Braunschweig Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David E.","family":"Thurston","sequence":"additional","affiliation":[{"name":"Institute of Pharmaceutical Science School of Cancer &amp; Pharmaceutical Sciences King's College London Franklin\u2010Wilkins Building  150 Stamford Street London UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Keith R.","family":"Fox","sequence":"additional","affiliation":[{"name":"School of Biological Sciences, Life Sciences Building 85 University of Southampton  Southampton SO17 1BJ UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Khondaker Miraz","family":"Rahman","sequence":"additional","affiliation":[{"name":"Institute of Pharmaceutical Science School of Cancer &amp; Pharmaceutical Sciences King's College London Franklin\u2010Wilkins Building  150 Stamford Street London UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8971-7045","authenticated-orcid":false,"given":"Mark","family":"Br\u00f6nstrup","sequence":"additional","affiliation":[{"name":"Department of Chemical Biology Helmholtz Centre for Infection Research  Inhoffenstra\u00dfe 7 38124 Braunschweig Germany"},{"name":"German Center for Infection Research (DZIF) Site Hannover\u2010Braunschweig  38124 Braunschweig Germany"},{"name":"Institute of Organic Chemistry Leibniz University Hannover  30167 Hannover Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2025,8,22]]},"reference":[{"key":"e_1_2_9_1_1","doi-asserted-by":"publisher","DOI":"10.1002\/anie.201510610"},{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1021\/ja00952a050"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1021\/cr100120f"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1517\/13543784.2011.573477"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.7164\/antibiotics.30.349"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/0005-2787(77)90067-3"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.7164\/antibiotics.25.660"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1021\/jm301882a"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jmedchem.8b01849"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jmedchem.0c00328"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1021\/acsinfecdis.7b00130"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1021\/ja902986x"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ab.2007.11.018"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.1021\/jm001064n"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.1158\/0008-5472.CAN-03-2941"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi0479813"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.1080\/14712598.2020.1776255"},{"key":"e_1_2_9_18_1","doi-asserted-by":"publisher","DOI":"10.3390\/molecules26102943"},{"key":"e_1_2_9_19_1","unstructured":"R. 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