{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T00:46:55Z","timestamp":1792025215589,"version":"4.4.0"},"reference-count":26,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2022,12,2]],"date-time":"2022-12-02T00:00:00Z","timestamp":1669939200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Croatian Science Foundation","award":["IP-2018-01-8098"],"award-info":[{"award-number":["IP-2018-01-8098"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antibiotics"],"abstract":"<jats:p>A well-known class of antibacterials, 14- and 15-membered macrolides are widely prescribed to treat upper and lower respiratory tract infections. Azithromycin is a 15-membered macrolide antibiotic possessing a broad spectrum of antibacterial potency and favorable pharmacokinetics. Bacterial resistance to marketed antibiotics is growing rapidly and represents one of the major global hazards to human health. Today, there is a high need for discovery of new anti-infective agents to combat resistance. Recently discovered conjugates of azithromycin and thiosemicarbazones, the macrozones, represent one such class that exhibits promising activities against resistant pathogens. In this paper, we employed an approach which combined LC-SPE\/cryo NMR, MS\/MS and molecular modeling for rapid separation, identification and characterization of bioactive macrozones and their diastereomers. Multitrapping of the chromatographic peaks on SPE cartridges enabled sufficient sample quantities for structure elucidation and biological testing. Furthermore, two-dimensional NOESY NMR data and molecular dynamics simulations revealed stereogenic centers with inversion of chirality. Differences in biological activities among diastereomers were detected. These results should be considered in the process of designing new macrolide compounds with bioactivity. We have shown that this methodology can be used for a fast screening and identification of the macrolide reaction components, including stereoisomers, which can serve as a source of new antibacterials.<\/jats:p>","DOI":"10.3390\/antibiotics11121738","type":"journal-article","created":{"date-parts":[[2022,12,2]],"date-time":"2022-12-02T02:18:06Z","timestamp":1669947486000},"page":"1738","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Screening of Novel Antimicrobial Diastereomers of Azithromycin\u2013Thiosemicarbazone Conjugates: A Combined LC-SPE\/Cryo NMR, MS\/MS and Molecular Modeling Approach"],"prefix":"10.3390","volume":"11","author":[{"given":"Iva","family":"Habinovec","sequence":"first","affiliation":[{"name":"Department of Chemistry, Faculty of Natural Sciences, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ivana","family":"Mikulandra","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Faculty of Natural Sciences, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paula","family":"Pranji\u0107","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Faculty of Natural Sciences, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sa\u0161a","family":"Kazazi\u0107","sequence":"additional","affiliation":[{"name":"Ru\u0111er Bo\u0161kovi\u0107 Institute, Bijeni\u010dka Cesta 54, HR-10000 Zagreb, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8837-3156","authenticated-orcid":false,"given":"Hana \u010cip\u010di\u0107","family":"Paljetak","sequence":"additional","affiliation":[{"name":"Center for Translational and Clinical Research, School of Medicine, University of Zagreb, \u0160alata 3, HR-10000 Zagreb, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Antun","family":"Bari\u0161i\u0107","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Faculty of Natural Sciences, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4540-5648","authenticated-orcid":false,"given":"Branimir","family":"Berto\u0161a","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Faculty of Natural Sciences, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1035-7981","authenticated-orcid":false,"given":"Mirjana","family":"Bukvi\u0107","sequence":"additional","affiliation":[{"name":"Selvita, Prilaz baruna Filipovi\u0107a 29, HR-10000 Zagreb, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Predrag","family":"Novak","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Faculty of Natural Sciences, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,12,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Arsic, B., Novak, P., Rimoli, M.G., Barber, J., Kragol, G., and Sodano, F. (2018). Macrolides Properties, Synthesys and Applications, De Gruyter.","DOI":"10.1515\/9783110515756"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1016\/j.pharmthera.2014.03.003","article-title":"Azithromycin: Mechanism of action and their relevance for clinical applications","volume":"143","author":"Parnham","year":"2014","journal-title":"Pharm. Therap."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"814","DOI":"10.1038\/35101544","article-title":"Structural basis for the interaction of antibiotics with the peptidyl transferase centre in eubacteria","volume":"413","author":"Zarivach","year":"2001","journal-title":"Nature"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1016\/S1097-2765(02)00570-1","article-title":"The structures of four macrolide antibiotics bound to the large ribosomal subunit","volume":"10","author":"Hansen","year":"2002","journal-title":"Mol. Cell"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"17152","DOI":"10.1073\/pnas.1007988107","article-title":"Structures of the Escherichia coli ribosome with antibiotics bound near the peptidyl transferase center explain spectra of drug action","volume":"107","author":"Dunkle","year":"2010","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1038\/nrmicro3155","article-title":"Ribosome-targeting antibiotics and mechanisms of bacterial resistance","volume":"12","author":"Wilson","year":"2014","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"106147","DOI":"10.1016\/j.ijantimicag.2020.106147","article-title":"Discovery of Macrozones, new antimicrobial thiosemicarbazone-based azithromycin conjugates: Design, synthesis and in vitro biological evaluation","volume":"56","author":"Mikulandra","year":"2020","journal-title":"Int. J. Antimicrob. Agents"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Mikulandra, I., Jedna\u010dak, T., Berto\u0161a, B., Parlov-Vukovi\u0107, J., Ku\u0161ec, I., and Novak, P. (2021). Interactions of aminopropyl-azithromycin derivatives, precursors in the synthesis of bioactive macrozones, with E. coli ribosome: NMR and molecular docking studies. Materials, 14.","DOI":"10.3390\/ma14195561"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1016\/j.jpba.2012.03.044","article-title":"A critical review on the use of modern sophisticated hyphenated tools in the characterization of impurities and degradation products","volume":"69","author":"Singh","year":"2012","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1016\/j.trac.2017.09.012","article-title":"Critical review of reports on impurity and degradation product profiling in the last decade","volume":"101","year":"2018","journal-title":"Trends Anal. Chem."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1016\/j.chroma.2012.05.032","article-title":"Liquid chromatography\u2013nuclear magnetic resonance coupling as alternative to liquid chromatography\u2013mass spectrometry hyphenations: Curious option or powerful and complementary routine tool?","volume":"1259","author":"Sturm","year":"2012","journal-title":"J. Chromatogr. A"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1002\/pca.2393","article-title":"Coupling of a high-resolution monoamine oxidase-A inhibitor assay and HPLC-SPE-NMR for advanced bioactivity profiling of plant extracts","volume":"2","author":"Grosso","year":"2013","journal-title":"Phytochem. Anal."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1016\/j.chroma.2004.01.055","article-title":"Combined use of liquid chromatography-nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry for the characterisation of an acarbose degradation product","volume":"1033","author":"Novak","year":"2004","journal-title":"J. Chromatogr. A"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"68","DOI":"10.1016\/j.jpba.2009.03.017","article-title":"LC-NMR and LC-MS identification of an impurity in a novel antifungal drug icofungipen","volume":"50","author":"Novak","year":"2009","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1268","DOI":"10.1016\/j.jpba.2005.09.002","article-title":"The Application of LC-NMR and LC-MS for the Separation and Rapid Structure elucidation of an Unknown Impurity in drug 5-Aminosalicylic Acid","volume":"40","author":"Novak","year":"2006","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Habinovec, I., Mikulandra, I., Sekula, L.E., Ga\u0161perov, J., Kazazi\u0107, S., and Novak, P. (2021). Rapid structure determination of bioactive 4\u2033-tetrahydrofurfuryl macrozone reaction mixture components by LC-SPE\/cryo NMR and MS. Molecules, 26.","DOI":"10.3390\/molecules26206316"},{"key":"ref_17","first-page":"543","article-title":"Rapid Identification of Unknown Impurities in 3-Bromo-5-(trifluoromethyl)aniline by LC-SPE\/NMR","volume":"89","author":"Habinovec","year":"2016","journal-title":"Croat. Chem. Acta"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"82","DOI":"10.1016\/j.chroma.2006.09.058","article-title":"Reaction product analysis by high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance Application to the absolute configuration determination of naturally occurring polyyne alcohols","volume":"1136","author":"Seger","year":"2006","journal-title":"J. Chromatogr. A"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"5857","DOI":"10.1016\/j.bmc.2009.07.013","article-title":"Free and bound state structures of 6-O-methyl homoerythromycins and epitope mapping of their interactions with ribosomes","volume":"17","author":"Novak","year":"2009","journal-title":"Bioorg. Med. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"4495","DOI":"10.1016\/j.bmcl.2004.06.045","article-title":"Synthesis and antibacterial activity of C-6 carbamate ketolides, a novel series of orally active ketolide antibiotics","volume":"14","author":"Henninger","year":"2004","journal-title":"Bioorg. Med. Chem. Lett."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1701","DOI":"10.1002\/jcc.20291","article-title":"GROMACS: Fast, flexible, and free","volume":"26","author":"Lindahl","year":"2005","journal-title":"J. Comput. Chem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"3684","DOI":"10.1063\/1.448118","article-title":"Molecular dynamics with coupling to an external bath","volume":"81","author":"Berendsen","year":"1984","journal-title":"J. Chem. Phys."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1463","DOI":"10.1002\/(SICI)1096-987X(199709)18:12<1463::AID-JCC4>3.0.CO;2-H","article-title":"LINCS: A linear constraint solver for molecular simulations","volume":"18","author":"Hess","year":"1997","journal-title":"J. Comput. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1695","DOI":"10.1103\/PhysRevA.31.1695","article-title":"Canonical dynamics: Equilibrium phase-space distributions","volume":"31","author":"Hoover","year":"1985","journal-title":"Phys. Rev. A"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1117","DOI":"10.1080\/00268979600100761","article-title":"Explicit reversible integrators for extended systems dynamics","volume":"87","author":"Martyna","year":"1996","journal-title":"Mol. Phys."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/0263-7855(96)00018-5","article-title":"VMD: Visual molecular dynamics","volume":"14","author":"Humphrey","year":"1996","journal-title":"J. Mol. 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