Synthesis of Lysozyme–Metallacarborane Conjugates and the Effect of Boron Cluster Modification on Protein Structure and Function Konrad Kowalski,* [a] Tomasz Goszczyn ´ ski, [a] Zbigniew J. Les ´nikowski,* [b] and Janusz Boratyn ´ ski [a] Introduction Contemporary technologies exploit knowledge from different fields of science. Today, the crossroads of biology, materials en- gineering, biochemistry, and inorganic chemistry offer fruitful interconnections and yield new pharmaceuticals, diagnostic methods, and novel materials, such as biological/nonbiological constructs. [1–3] Polyhedral boron hydrides are nonplanar, three- dimensional, “pseudo-aromatic” compounds characterized by a cage-like cluster structure. Because of their unique proper- ties, boron clusters have been tested in an amazingly broad spectrum of applications, ranging from catalysis, optoelectron- ics, and new materials to medical chemistry and drug design. [4, 5] One of the important features of a boron cluster system such as the dicarbaborate anion (nido-7,8-C 2 B 9 H 12 2 ) is the ability to serve as a ligand to form stable complexes with metals: metallacarboranes. [6, 7] The best-known example of this type of complex is 3-cobalt bis(dicarbollide) (Co(C 2 B 9 H 11 ) 2 2 ), which contains one cobalt anion and 18 boron atoms (two boron clusters). Since its discovery in 1965, [8] 3-cobalt bis(dicar- bollide) has become the most-studied metallacarborane. Its re- markable features include thermal and chemical stability, rigidi- ty, nearly ellipsoidal structure, and an amphiphilic character. [9] One of the earliest studies on the medical applications of 3- cobalt bis(dicarbollide) examined its use as a radioactive cobalt ( 57 Co) carrier in the design of agents for radioimmunodetection and radioimmunotherapy. [10] Conjugation of radiometallacar- borane with tumor-specific monoclonal antibodies has also been studied. [10, 11] Here, we propose a new method for protein modification with metallacarboranes that is based on cyclic ether ring opening of oxonium-type adducts of the metallacar- borane. [12, 13] The cyclic ether ring opening in boron cluster- cyclic ether adducts with nitrogen, oxygen and, more recently, sulfur and halogen nucleophiles is widely used in the chemis- try and bio-organic chemistry of boron. [12–17] However, the ap- plication of this methodology for complex biological molecules such as proteins has not been pursued. Two different cyclic oxonium adducts of cobalt bis(dicarbol- lide), bearing tetrahydropyran (1) or 1,4-dioxane (2 ; Scheme 1), were used to bind metallacarborane covalently to the protein. Two complementary methods, “in solution” and “in solid state”, for the synthesis of lysozyme modified with metallacar- borane (cobalt bis(dicarbollide), Co(C 2 B 9 H 11 ) 2 2 ) were devel- oped. As metallacarborane donors, oxonium adducts of cobalt bis(dicarbollide) and 1,4-dioxane or tetrahydropyran were used. The physicochemical and biochemical properties of the obtained lysozyme–metallacarborane conjugates were studied for changes in secondary and tertiary structure, aggregation behavior, and biological activity. Only minor changes in pri- mary, secondary, and tertiary protein structure were observed, caused by the single substitution of metallacarborane on lyso- zyme. However, the modification produced significant changes in lysozyme enzymatic activity and a tendency toward time- and temperature-dependent aggregation. Scheme 1. Metallacarborane oxonium adducts 1 and 2 used in the conjuga- tion reaction with lysozyme. [a] K. Kowalski, Dr. T. Goszczyn ´ski, Prof. J. Boratyn ´ski “Neolek” Laboratory of Biomedical Chemistry Department of Experimental Oncology Institute of Immunology and Experimental Therapy Polish Academy of Science 12 Rudolf Weigl Street, 53-114 Wrocław (Poland) E-mail : konrad.kowalski@iitd.pan.wroc.pl [b] Prof. Z.J. Les´nikowski Laboratory of Molecular Virology and Biological Chemistry Institute of Medical Biology, Polish Academy of Science 106 Lodowa Street, 93-232 Łódz´ (Poland) E-mail : zlesnikowski@cbm.pan.pl Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201402611. ChemBioChem 0000, 00,0–0 # 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 & These are not the final page numbers! ÞÞ These are not the final page numbers! ÞÞ Full Papers DOI: 10.1002/cbic.201402611