Division of Analytical Chemistry

Various studies are planned to be performed at the Division of Analytical Chemistry for the purpose of developing sensitive and selective analytical methods. Analytical atomic spectrometry (AAS, ICP-AES, ICP-MS) will be applied for development of new methods for quantitative metal content determination. The methods will be guided in order to upgrade sample preparation steps and improve spectral control of matrix effects. Special focus will be payed on methods of spectrometric quantification of elemental and isotopic composition of biomaterials and nanomaterials. Developed analytical methodology will strength multidisciplinary researches and it will be implemented through existing and planned joint- projects with economic sector.

Structural analysis of inorganic, organic and biological compounds will be performed by using MS, NMR, UV-Vis, IR and Raman spectroscopies. Structure, interactions and binding modes of bioactive molecules will be investigated by spectroscopic, computational and other physico-chemical methods. Special attention will be devoted to design of novel bioactive compounds and drugs. Hydrogen bonds and their impact on stability, structure and reactivity of studied systems will be studied as well. Process analytical approaches to monitoring chemical and physical processes will be developed and implemented.

IR and Raman spectroscopy will be used for analysis of various synthetic and real samples. Within the vibrational spectroscopic methods, surface-enhanced Raman scattering (SERS) techniques will be developed, which will be applied for structural analysis as well as for study of intermolecular interactions and binding modes of small organic molecules with biomacromolecules. Development of SERS sensors for detection and quantification of various chemical species will include preparation and characterization of nanostructured metallic surfaces and spectra analysis by chemometric methods.

The HPLC and/or UHPLC chromatographic methods will be applied for analysis of real samples (food, dyes, farmaceuticals, etc.). Modern analytical procedures based on hyphenated methods such as LC-SPE-NMR and LC-MS for purity profiling of bioactive compounds, drugs and natural products will be developed. In that respect already established collaboration with industry will be extended. Complex mixtures and aggregation processes in petroleum samples and derivatives will be evaluated.

It is worth highlighting the successful collaboration of scientists of the Division of Analytical Chemistry with several research groups from abroad.

Division of Biochemistry

The accuracy of protein synthesis. The research aims in this area are to understand the molecular basis and the biological importance of cellular mechanisms that ensure the accuracy of protein biosynthesis (translation). Erroneously synthesized proteins affect cell viability and function and are therefore associated with cellular aging and the development of neurodegenerative diseases. Using biophysical, proteomic, biochemical, computational and genetic analysis of structure/function relationship of aminoacyl-tRNA synthetases (AARSs), the key proteins that control the accuracy of protein biosynthesis, together with analysis of kinetic models of AARSs catalytic functions, we shed more light on the mechanistic details, evolution and biological relevance of proofreading reactions of these enzymes, which are developed to ensure the appropriate level of translational accuracy. To understand the impact of the inaccurate protein biosynthesis on protein structure/function we will produce the mistranslated model proteins. Understanding of these processes provides a platform for the synthesis of inhibitors with pharmacological activity, additional components for multicomponent antibiotics, and the treatment of diseases associated with mistranslated proteins. Our research is highly competitive and it is conducted in collaboration with prominent international groups that have complementary expertise (structural biology, laboratory-directed protein evolution, bioinformatics). Research in the field of the accuracy in protein biosynthesis will also include proteomic research in order to analyze and understand the relevance or permissibility of translational accuracy on a global scale. The long-term goal is to include proteomic studies based on mass spectrometry (LC-MS / MS) and to obtain the required instrumentation at the Department of Biochemistry since modern biochemical research is increasingly dependent on this aspect.

Design of Proteins with New Properties. Research goals are to create proteins with novel/improved properties that have superior biotechnological or biomedical applications using methods of protein engineering. Moreover, protein redesign has important application in biochemical, biophysical and cellular-biological studies as it enables the creation of specific properties that can be used to analyze the structure and function of the protein and to track their localization in the cell. Research is focused on the incorporation of synthetic (artificial) amino acids into proteins to effectively extend the standard amino acid alphabet. Interesting candidates to expand Nature's alphabet are fluorinated amino acids because fluorinated therapeutic peptides can easily pass through the membrane and thus have a better pharmacological effect. On the other hand, an attractive biotechnological line of research may include redesigning the existing protein biosynthesis system to prevent incorporation of non-proteinogenic amino acids that accumulate in the cell. This problem is recognized in the process of production of therapeutic proteins in bacterial cells and as such poses a serious problem in the pharmaceutical industry. The research is international, multidisciplinary and is at the intersection of biochemistry, synthetic biology and biotechnology.

Aminoacyl-tRNA-synthetases as targets for antibiotics. The research goals in the field of antibiotic mechanisms of action are to shed light on the correlation between the structure/function of aminoacyl-tRNA synthetases (AARS) and their role of a target in the production of natural antibiotics and in the development of synthetic drugs. Emphasis is on the research related to antibiotic resistance, which is one of the biggest global health problem. Mupirocin is an antibiotic that inhibits protein biosynthesis by inhibition of isoleucyl-tRNA-synthetase (IleRS). Mupirocin resistance is a common problem among the hospital strains of the pathogenic Staphylococcus aureus. Resistance in hospital strains, but also in nature, develops through the evolution of IleRS that is less sensitive to mupirocin or through the acceptance of an additional IleRS, which is insensitive to mupirocin. Using biochemical, kinetic, computational and genetic research in collaboration with groups dealing with biochemical evolution and structural biology, the goal is to address the key issues related to the mechanism of action and resistance to antibiotics as well as the connection of this process with the accuracy of the translation.

Novel noncanonical functions of aminoacyl-tRNA synthetase. Aminoacyl-tRNA synthetases often have additional functions in the cell and participate in a variety of cellular processes that are not directly related to protein biosynthesis. Noncanonical function studies will focus on poorly explored plant aminoacyl-tRNA synthetases, and will examine their role in cell response to abiotic stress using biochemical, molecular biology and genetic methods. At the same time, the protein interactors and the biological role of macromolecular complexes involving plant aminoacyl-tRNA synthetases and their influence on the growth and development of plants will be investigated. The subject of the study will be also amino acid:[carrier protein] ligases, bacterial homologues of aminoacyl-tRNA synthetases that evolved a new and unexpected substrate specificity towards aminoacylation of the carrier proteins instead of tRNAs. Their biological role is unknown, they do not participate in protein biosynthesis, but most likely in the biosynthesis of secondary metabolites or antibiotics.

Interactions of proteins with other biomolecules. Specific interactions between molecules are fundamental to all biological processes. To understand complex interacting systems in the cell, protein-protein interactions, interactions between protein and nucleic acids or with small ligands will be monitored. Various techniques and methods for analyzing macromolecular interactions will be used, with emphasis on quantitative determination of the binding free energy (thermophoresis, fluorescence spectrometry, isothermal titration calorimetry). Research has potential applications in biotechnology and biomedicine since many drugs act as inhibitors and modulators of biomacromolecules and their complexes.

Computational methods in biochemical research. Modern (bio)chemical research implies the use of computational methods in everyday work. Computational methods are used to study the structure, dynamics and energetics at the atomic level of biological macromolecules such as proteins, nucleic acids, and their complexes as well as their complexes with small compounds. In the absence of experimentally solved structures, computational methods are also used for predicting protein structures based on their amino acid sequence. Prediction of protein structures has been advanced in recent years because of methodological development and the exponentially growing number of experimentally solved structures that are used as structural templates. Therefore, computational methods complement experimental research and can significantly contribute to the following objectives: structure and function of erroneously synthesized model proteins and proteins with incorporated synthetic amino acids, modeling of aminoacyl-tRNA synthetases (AARS) to understand the mupirocin resistance. In addition to AARS-related research, computational methods will be used for modeling structure, function, dynamics, studying protein interaction networks and the catalytic mechanism of different enzymes involved in various metabolic pathways. Computational methods of different complexity such as molecular mechanics, molecular dynamics, quantum mechanical methods as well as their mutual combination and empirical valence bond (EVB) simulations will be used in the research.

Division of Physical Chemistry

Scientific work in the Division of physical chemistry is an inseparable part of the teaching process and includes the research in the fields of theoretical and computational chemistry, thermodynamics, chemical kinetics, electrochemistry, colloid and interface chemistry, macromolecular chemistry, chemometrics and education.

In theoretical chemistry, quantum-chemical methods are used to calculate potential energy surfaces and dipole moment surfaces, which enables highly accurate determination of spectroscopic properties of molecules and reaction mechanisms. Interactions of biological macromolecules, as well as their structural and dynamical properties, are studied using force field based computational methods with purpose of understanding biochemical processes on molecular level. Quantitative structure-activity relationship models are generated for investigating biological activity of different classes of compounds.

Thermodynamic investigations involve equilibria of ion association and complex forming reactions in solutions and on the surface. Structures of complexes and relevant thermodynamic parameters are determined by means of experimental and computational chemistry methods. Parallel kinetic investigations provide an insight into the reaction mechanisms. The continuation and extension of already ongoing successful collaborations with pharmaceutical industry related to physico-chemical characterization and synthesis of pharmaceutically active compounds is planned.

Investigations in the field of colloid and interface chemistry deal with the development of theoretical models and experimental techniques for the characterization of interfaces. Aggregation, adsorption and electrical interfacial layer at the solid/liquid interface will be studied. The above mentioned investigations will be performed in collaboration with several research groups from Croatia and abroad.

In physical chemistry of macromolecules, properties of polyelectrolytes and proteins in solution are studied, as well as their adsorption on solid substrates. Formation and properties of polyelectrolyte complexes and multilayers are also investigated. Special emphasis in these investigations will be given on the study of their antibacterial properties and on the specific aspects of ionic condensation on polyions. For that purpose already fruitful collaboration with the researchers from the Faculty of Health Sciences and from the Faculty of Chemistry and Chemical Engineering, University of Ljubljana will be strengthen.

Chemometric methods are developed and applied to interpretation of complex experimental data and their reduction to significant parameters. Use is made of modern computer methods, chemometrics, spectrometry, (micro)calorimetry, potentiometry, conductometry, optical reflectometry, electrokinetics and acoustophoresis. Scientific work in the field of chemistry education is dedicated to developing a quantitative approach to chemical problems, based on clearly defined notions and their interrelations.

Division of General and Inorganic Chemistry

The strategy of the Division of general and inorganic chemistry is based on the results and experience achieved and attained during the last decade of research through domestic and international research projects. It is planned to continue research in the field of new organic and coordination compounds, solid state chemistry, supramolecular chemistry and protein chemistry (H. Pylori proteins and insulin derivatives) and other biologically active compounds. The research will encompass design, preparation and detailed structural spectroscopic and thermal characterisation of the prepared compounds. Research on proteins will include cloning, purification, crystallisation, and structural characterisation. A variety of experimental methods shall be employed in order to study inter- and intramolecular interactions and their influence on molecular structure and properties. The main objectives of the above research are:

• Fundamental research in the field of development of new, environmentfriendly, methods of preparation of organic, bioorganic and organometallic compounds;
• Potential application of novel methods of synthesis and materials with designer properties (e.g. optical, thermal or magnetic) in industry;
• New insight into the influence of study inter and intramolecular interactions on the structure of solids;
• Structural characterisation of proteins for the purpose of obtaining new insight into the relationship between protein structure and function;
• Transfer of the obtained knowledge and experience into education an all levels from undergraduate to postgraduate study;
• Intensifying international collaboration though participation in European and bilateral research projects.

Along with the above scientific research one should point out also professional work of chemical synthesis as well as physical and chemical characterisation of pharmaceutically active solids which is performed as a collaboration with partners from industry.

Division of Organic Chemistry

Scientific activities in organic chemistry are focused in two directions: organic synthesis and physical-organic chemistry. Synthetic organic chemistry is oriented towards design of new bioactive compounds containing heterocyclic aromatic and nonaromatic substructures as well as investigation of their interactions with enzymes, primarily cholinesterases. Research of pyridone derivatives is directed towards synthesis of compounds with antitumor effects, and synthesis of pyridone manozides with application in antiadhesion therapy. Medicinal chemistry studies include design of new molecular conjugates of modified immunomodulating peptides, comprising molecular modeling approach. 

Physical-organic chemistry studies are focused towards development of new conceptual frameworks for explaining mechanisms of thermal organic reactions in condensed phases. Reaction mechanisms in solution are investigated with computational chemistry approach, and methodology of solid-state reactions investigations is based on study of aromatic C-nitroso compounds dimerization reactions. Since these systems show photo/thermochromic effects, corresponding molecular aggregates could in principle possess externally controlled dynamical properties allowing their use in field of molecular electronics. These molecules are also investigated as potential building blocks for self-assembling mono- and multilayers, as well as three-dimensional supramolecular systems.

Future activities in field of organic synthesis will be directed towards design and synthesis of new aromatic and supramolecular systems, their experimental and theoretical study, as well as possible applications (biosensors, molecular electronics, "smart" drugs, new materials, etc.). The second part of synthetic research will comprise design and synthesis of bioactive molecules, especially heterocyclic systems, glycoconjugates and peptides with possible antiproliferative, antibacterial, antitumor, immunomodulating, antioxidative and different inhibitory effects, using conventional as well as new, faster and ecologically more acceptable methods.

Investigation of thermal and photochemical organic reactions mechanisms in condensed phases will be conducted simultaneously with theoretical and experimental approach. Computational results will help to better understand the intermolecular interactions in different solvents and polycrystalline systems. The study of solvation effects will be continued using newly developed computational approach. Experimental studies will be focused towards detailed investigations of kinetics and thermodynamics of organic reactions in solid phase, with emphasis to reactions in cryogenic conditions. New equipment will facilitate opening of new research directions based on mono- and multilayers formed on ordered surfaces.