Znanstveni radovi

Tuning the coordination properties of chiral pseudopeptide bis(2-picolyl)amine and iminodiacetamide ligands in Zn(II) and Cu(II) complexes
Natalija Pantalon Juraj,* Tana Tandarić, Vanja Tadić, Berislav Perić, Dominik Moreth, Ulrich Schatzschneider, Anamaria Brozović, Robert Vianello* and Srećko I. Kirin*
Dalton Transactions 2022, 51, 17008–17021

Seven bis(2-picolyl)amine (bpa) and five iminodiacetamide (imda) ligands were prepared with different modifications in their side chain structure. The coordination properties of the ligands (L) were influenced by changes in the aliphatic linker length (C1, C2, or C3), amide group isomers and type of chiral terminal group. Complexation with Cu(II) afforded two polymorphs of a ML complex which features tetradentate coordination of a ligand with C2 linkers, while crystal structures of three trans fac ML2 complexes with Cu(II) and Ni(II) show tridentate coordination of ligands with a C3 linker. The stoichiometry and stereochemistry of Zn(II) and Cu(II) complexes was further studied in solution by NMR and UV-Vis spectroscopy. DFT calculations gave an insight into the relative stability of isomers, as well as potential hydrogen bonding between two ligands in a ML2 complex. Furthermore, ML complexes of Cu(II) exhibited DNA cleavage activity.

Enzyme reaction engineering as a tool to investigate the potential application of enzyme reaction systems
Nevena Milčić, Ivana Ćevid, Mehmet Mervan Çakar, Martina Sudar and Zvjezdana Findrik Blažević*
Hungarian Journal of Industry and Chemistry 2022, 50, 45–55

It is widely recognized and accepted that although biocatalysis is an exquisite tool to synthesize natural and unnatural compounds under mild process conditions, much can be done to better understand these processes as well as detect resulting bottlenecks and help to resolve them. This is the precise purpose of enzyme reaction engineering, a scientific discipline that focuses on investigating enzyme reactions with the goal of facilitating their implementation on an industrial scale. Even though reaction schemes of enzyme reactions often seem simple, in practice, the interdependence of different variables is unknown, very complex and may prevent further applications. Therefore, in this work, important aspects of the implementation of enzyme reactions are discussed using simple and complex examples, along with principles of mathematical modelling that provide explanations for why some reactions do not proceed as planned.



Deep eutectic solvents as a stabilising medium for NAD coenzyme: unravelling the mechanism behind coenzyme stabilisation effect
Mia Radović, Lucija Hok, Manuela Panić, Marina Cvjetko Bubalo,* Robert Vianello,* Marijana Vinković and Ivana Radojčić Redovniković
Green Chemistry 2022, 24, 7661–7674

Nicotinamide adenine dinucleotide (NAD) coenzyme is a vital part of numerous enzymatic reactions that are involved in all major biological processes from energy metabolism to cell survival. Accordingly, it is used in a great number of biocatalytic reactions, as analytical biosensors, and in test kits for diagnostic and analytical purposes. This coenzyme is unstable in aqueous solutions, meaning that the minimization of its degradation during storage, assays, and enzyme-catalysed oxidoreductive reactions is of high importance. Herein, we report on the stabilisation of NAD (NAD+ as oxidised, and NADH as its reduced form) by deep eutectic solvents (DES), an emerging class of solvents that offer numerous remarkable advantages such as high tunability, and capability to stabilise a wide range of commercially important compounds of natural origin. Preliminary DES screening revealed that out of 7 candidates, choline chloride:urea (ChCl:U) shows the best ability to stabilise both NAD forms. Computational analysis (quantum chemical calculations and molecular dynamics simulations) provided a deeper insight into possible mechanisms behind the observed stabilisation, by identifying geometries and the solvation structure of NAD coenzymes in these solvents, and analysing their possible degradation pathways. Finally, prolonged NAD stability (up to 50 days) in ChCl:U is detected and the system is further confirmed as a stable working NAD solution in a model enzymatic assay.

Tandem amide coupling and hydroamination:unexpected benzotriazole oxide addition to thepropiolic acid triple bond
Saša Opačak,* Berislav Perić, Tomislav Gojšić, Ana Čikoš, Dražen Vikić-Topić and Srećko I. Kirin*
New Journal of Chemistry 2022, 46, 13275–13285

Amides are common molecules which are most often prepared using benzotriazole oxide coupling reagents. Such amides can be derivatised with compounds that contain a triple carbon–carbon bond in order to facilitate easy introduction of the amide fragment into a target molecule via cycloaddition reactions. In this paper, a previously unknown tandem reaction consisting of amide coupling and hydroamination of a triple C–C bond is described. The reaction occurs when the TBTU/HOBt coupling reagent pair is used for coupling of propiolic acid with mono-Boc-diaminocyclohexane. The hydroamination can be circumvented by using COMU as the coupling reagent. The addition reaction was further explored by using methyl propiolate with TBTU/HOBt which yielded a plethora of different products including O- and N-addition products and also a product of formal methyl propiolate dimerization. Due to high symmetry and low number of non-equivalent protons and carbons, most of the products were difficult to characterise by NMR spectroscopy, so it was necessary to obtain X-ray single crystal structures for most of them. In addition, the identification of the products was further supported by very good agreement of NMR spectra collected experimentally with NMR spectra calculated by DFT.

Expanding the Scope of Enantioselective Halohydrin Dehalogenases – Group B
Emina Mehić, Lucija Hok, Qian Wang, Irena Dokli, Marina Svetec Miklenić, Zvjezdana Findrik Blažević, Lixia Tang, Robert Vianello* and Maja Majerić Elenkov*
Advanced Synthesis & Catalysis 2022, 364, 2576–2588, rad na naslovnici.

Halohydrin dehalogenases (HHDHs) possess an unnatural activity of introducing functionalities such as N3, CN, NO2 etc., into a molecule through the ring-opening reaction of epoxides. The enantioselectivity of HHDHs is substrate-dependent and not always high enough for synthetic applications. B-group of HHDHs has been neglected in the past, due to observed low enantioselectivity based on performance on a relatively limited number of substrates. Extensive screening of substrates on HheB2 from Mycobacterium sp. GP1 and HheB from Corynebacterium sp. N-1074 was performed. Several highly enantioselective reactions were discovered (E > 200), with HheB showing higher enantioselectivity and activity toward larger panel of substrates compared to HheB2. Enzymes HheB and HheB2 are highly homologous; they differ by only 4 residues. By using site-directed mutagenesis, residues 120 and 125 were found to be responsible for higher enantioselectivity of HheB compared to HheB2. Computational analysis supported experiments and provided evidence that kinetic and thermodynamic parameters of reactions within HheB enzymes are crucial in determining the observed enantioselectivities. Due to remarkable activity and enantioselectivity, B-group HHDHs emerged as a catalyst of choice for the synthesis of bulky tertiary alcohols, as shown in this work.

COVID-19 infection and neurodegeneration: Computational evidence for interactions between the SARS-CoV-2 spike protein and monoamine oxidase enzymes
Lucija Hok, Hrvoje Rimac, Janez Mavri and Robert Vianello*
Computational and Structural Biotechnology Journal 2022, 20, 1254–1263

Although COVID-19 has been primarily associated with pneumonia, recent data show that its causative agent, the SARS-CoV-2 coronavirus, can infect many vital organs beyond the lungs, including the heart, kidneys and the brain. The literature agrees that COVID-19 is likely to have long-term mental health effects on infected individuals, which signifies a need to understand the role of the virus in the pathophysiology of brain disorders that is currently unknown and widely debated. Our docking and molecular dynamics simulations show that the affinity of the spike protein from the wild type (WT) and the South African B.1.351 (SA) variant towards MAO enzymes is comparable to that for its ACE2 receptor. This allows for the WT/SA∙∙∙MAO complex formation, which changes MAO affinities for their neurotransmitter substrates, thereby impacting their metabolic conversion and misbalancing their levels. Knowing that this fine regulation is strongly linked with the etiology of various brain pathologies, these results are the first to highlight the possibility that the interference with the brain MAO catalytic activity is responsible for the increased neurodegenerative illnesses following a COVID-19 infection, thus placing a neurobiological link between these two conditions in the spotlight. Since the obtained insight suggests that a more contagious SA variant causes even larger disturbances, and with new and more problematic strains likely emerging in the near future, we firmly advise that the presented prospect of the SARS-CoV-2 induced neurological complications should not be ignored, but rather requires further clinical investigations to achieve an early diagnosis and timely therapeutic interventions.

Why monoamine oxidase B preferably metabolizes N-methylhistamine over histamine: evidence from the multiscale simulation of the rate-limiting step
Aleksandra Maršavelski, Janez Mavri, Robert Vianello* and Jernej Stare*
International Journal of Molecular Sciences 2022, 23, 1910.

Histamine levels in the human brain are controlled by rather peculiar metabolic pathways. In the first step, histamine is enzymatically methylated at its imidazole Nτ atom, and the produced N-methylhistamine undergoes an oxidative deamination catalyzed by monoamine oxidase B (MAO-B), as is common with other monoaminergic neurotransmitters and neuromodulators of the central nervous system. The fact that histamine requires such a conversion prior to oxidative deamination is intriguing since MAO-B is known to be relatively promiscuous towards monoaminergic substrates; its in-vitro oxidation of N-methylhistamine is about 10 times faster than that for histamine, yet this rather subtle difference appears to be governing the decomposition pathway. This work clarifies the MAO-B selectivity toward histamine and N-methylhistamine by multiscale simulations of the rate-limiting hydride abstraction step for both compounds in the gas phase, in aqueous solution, and in the enzyme, using the established empirical valence bond methodology, assisted by gas-phase density functional theory (DFT) calculations. The computed barriers are in very good agreement with experimental kinetic data, especially for relative trends among systems, thereby reproducing the observed MAO-B selectivity. Simulations clearly demonstrate that solvation effects govern the reactivity, both in aqueous solution as well as in the enzyme although with an opposing effect on the free energy barrier. In the aqueous solution, the transition-state structure involving histamine is better solvated than its methylated analog, leading to a lower barrier for histamine oxidation. In the enzyme, the higher hydrophobicity of N-methylhistamine results in a decreased number of water molecules at the active side, leading to decreased dielectric shielding of the preorganized catalytic electrostatic environment provided by the enzyme. This renders the catalytic environment more efficient for N-methylhistamine, giving rise to a lower barrier relative to histamine. In addition, the transition state involving N-methylhistamine appears to be stabilized by the surrounding nonpolar residues to a larger extent than with unsubstituted histamine, contributing to a lower barrier with the former.

Ferrocene conjugates linked by 1,2,3-triazole and their Zn(II) and Cu(II) complexes: Synthesis, characterization and biological activity
Silvio Jakopec, Natalija Pantalon Juraj*, Anamaria Brozović*, Dijana Jadreško, Berislav Perić, Srećko I. Kirin and Silvana Raić-Malić*
Applied Organometallic Chemistry 2021, 35, e6575.

Ferrocene derivatives with mono- (8a–c) and bis-1,2,3-triazolyl (9 and 10a13c) chelating groups were synthesized by regioselective copper(I)-catalysed 1,3-dipolar cycloaddition of terminal alkynes with ferrocene azides. Metal complexes of the ligands were prepared with Cu(II) and Zn(II) salts. Crystal structures of ligands 9 and 11a were determined, as well as the structures of complexes [Cu(8a)2](CF3SO3)2 (8aCu) and [Cu(8c)2(CH3OH)2](BF4)2 (8cCu). In addition to NMR and UV–Vis spectroscopy, the metal complexes were characterized by cyclic voltammetry. The cytotoxic effect of ferrocene conjugates and their Zn(II) and Cu(II) complexes was explored, and cell cycle analysis was performed. The complex [Cu(8c)2](CF3SO3)2 showed the most prominent and selective cytotoxicity on cervical carcinoma (HeLa), ovarian cancer (MES-OV), non-small cell lung cancer (A549) and breast carcinoma (MDA-MB-231) cells. This complex increased cell population in the S and G2/M phase of the cell cycle, which was accompanied by an increase of the cells present in the sub-G0/G1 fraction.

Inovativna rješenja u katalitičkim proizvodnim procesima za potrebe farmaceutske industrije (CAT PHARMA)
Robert Vianello* and Srećko Kirin*
Kemija u industriji: časopis kemičara i tehnologa Hrvatske 2021, 70, 756–757.

Inorganic stereochemistry: Geometric isomerism in bis-tridentate ligand complexes
Natalija Pantalon Juraj and Srećko Kirin*
Coordination Chemistry Reviews 2021, 445, 214051.

In this review, we analyze the stereochemistry of hexacoordinated metal complexes with flexible tridentate ligands. Unlike rigid ligands, which can adopt only specific coordination modes, flexible ligands open more possibilities to fine-tuning the system for a specific application. Bis-tridentate [M(A-B-A)2] complexes of flexible ligands can form three geometric isomers: mer, trans-fac, and cis-fac. The analysis of crystallographic data for 844 structures found in the Cambridge Structural Database (CSD) elucidates influences on the formation of different isomers, such as steric and electronic properties of the ligand and metal ion, type and substitution of donor atoms, and the possibility of non-covalent interactions.


Synthesis and stereoselective catalytic transformations of 3-hydroxyisoindolinones
Nikola Topolovčan and Matija Gredičak*
Organic & Biomolecular Chemistry 2021, 19, 4637–4651.

This review focuses on the synthesis of 3-hydroxyisoindolinones, and their application as substrates in stereoselective catalytic transformations reported from 2010 to date. These compounds have attracted much attention among synthetic chemists, as they are integral structural parts of a number of natural products and biologically active compounds. The first part of this review covers methods based on electrochemical, photochemical, and thermal reactions for the synthesis of 3-hydroxyisoindolinones. The second part focuses on their employment as substrates in transition metal-catalyzed and organocatalyzed stereoselective transformations for the preparation of chiral 3-substituted isoindolinone derivatives.


Comparison of nonheme manganese- and iron-containing flavone synthase mimics
Dóra Lakk-Bogáth, Natalija Pantalon Juraj, Bashdar I. Meena, Berislav Perić, Srećko I. Kirin and József Kaizer*
Molecules 2021, 26, 3220.

Heme and nonheme-type flavone synthase enzymes, FS I and FS II are responsible for the synthesis of flavones, which play an important role in various biological processes, and have a wide range of biomedicinal properties including antitumor, antimalarial, and antioxidant activities. To get more insight into the mechanism of this curious enzyme reaction, nonheme structural and functional models were carried out by the use of mononuclear iron, [FeII(CDA-BPA*)]2+ (6) [CDA-BPA = N,N,N’,N’-tetrakis-(2-pyridylmethyl)-cyclohexanediamine], [FeII(CDA-BQA*)]2+ (5) [CDA-BQA = N,N,N’,N’-tetrakis-(2-quinolilmethyl)-cyclohexanediamine], [FeII(Bn-TPEN)(CH3CN)]2+ (3) [Bn-TPEN = N-benzyl-N,N’,N’-tris(2-pyridylmethyl)-1,2-diaminoethane], [FeIV(O)(Bn-TPEN)]2+ (9), and manganese, [MnII(N4Py*)(CH3CN)]2+ (2) [N4Py* = N,N-bis(2-pyridylmethyl)-1,2-di(2-pyridyl)ethylamine)], [MnII(Bn-TPEN)(CH3CN)]2+ (4) complexes as catalysts, where the possible reactive intermediates, high-valent FeIV(O) and MnIV(O) are known and well characterised. The results of the catalytic and stoichiometric reactions showed that the ligand framework and the nature of the metal cofactor significantly influenced the reactivity of the catalyst and its intermediate. Comparing the reactions of [FeIV(O)(Bn-TPEN)]2+ (9) and [MnIV(O)(Bn-TPEN)]2+ (10) towards flavanone under the same conditions, a 3.5-fold difference in reaction rate was observed in favor of iron, and this value is three orders of magnitude higher than was observed for the previously published [FeIV(O)(N2Py2Q*)]2+ [N,N-bis(2-quinolylmethyl)-1,2-di(2-pyridyl)ethylamine] species.

Direct metal-free transformation of alkynes to nitriles: Computational evidence for the precise reaction mechanism
Lucija Hok and Robert Vianello*
International Journal of Molecular Sciences 2021, 22, 3193.

Density functional theory calculations elucidated the precise reaction mechanism for the conversion of diphenylacetylenes into benzonitriles involving the cleavage of the triple C≡C bond, with N-iodosuccinimide (NIS) as an oxidant and trimethylsilyl azide (TMSN3) as a nitrogen donor. The reaction requires six steps with the activation barrier ΔG = 33.5 kcal mol−1 and a highly exergonic reaction free-energy ΔG= −191.9 kcal mol−1 in MeCN. Reaction profiles agree with several experimental observations, offering evidence for the formation of molecular I2, interpreting the necessity to increase the temperature to finalize the reaction, and revealing thermodynamic aspects allowing higher yields for alkynes with para-electron-donating groups. In addition, the proposed mechanism indicates usefulness of this concept for both internal and terminal alkynes, eliminates the option to replace NIS by its Cl- or Br-analogues, and strongly promotes NaN3 as an alternative to TMSN3. Lastly, our results advise increasing the solvent polarity as another route to advance this metal-free strategy towards more efficient processes.

A ferrocene-based pseudopeptide chiroptical switch
Saša Opačak, Darko Babić, Berislav Perić, Željko Marinić, Vilko Smrečki, Barbara Pem, Ivana Vinković Vrček and Srećko I. Kirin*
Dalton Transactions 2021, 50, 4504-4511.

We present a double-stranded ferrocene pseudopeptide 2b which exhibits stimuli responsive chirality inversion triggered by solvent exchange or acid addition. Compound 2b exists as a mixture of self-assembled fast exchanging oligomers which macroscopically behave as a chiroptical switch with two stable states. The ferrocene group inversion results in a distinct CD signal in the visible part of the spectrum. The inversion is accomplished through a conformational change due to a rearrangement of hydrogen bonding forcing the rotation of ferrocene rings.



Halohydrin dehalogenases and their potential in industrial application – A viewpoint of enzyme reaction engineering
Zvjezdana Findrik Blažević,* Nevena Milčić, Martina Sudar and Maja Majerić Elenkov
Advanced Synthesis & Catalysis 2021, 363, 388-410.

At the moment, there are approx. 100 published papers investigating halohydrin dehalogenases from different aspects; enzymology, molecular biology and reactions they can catalyse. Unquestionably, these enzymes are of great importance and hold an immense potential due to the wide spectrum of different compounds that can be synthesized by their action. These compounds, such as chiral epoxides, β‐substituted alcohols, oxazolidinones etc., significantly enrich the chemist’s toolbox and, moreover, open the possibility for the synthesis of even more complex compounds. Still, there are many unknowns, and it is the purpose of this work to demonstrate the possibilities and bottlenecks, in scientific sense, that could further help in broadening the applicative potential of these fascinating enzymes.



Synthesis and biological evaluations of mono- and bis-ferrocene uracil derivatives
Senka Djaković, Ljubica Glavaš-Obrovac, Jasmina Lapić, Silvija Maračić, Juraj Kirchofer, Marija Knežević, Marijana Jukić
and Silvana Raić-Malić*
Applied Organometallic Chemistry 2021, 35, e6052.

Mono- (3a–3e and 4a–4e) and bis-ferrocene (5a–5e and 6a–6e) conjugated 5-substituted uracil derivatives that are bridged by 1,2,3-triazole linker were synthesized. The impact of ferrocene unit and spacer between ferrocene and triazole on radical scavenging potency was observed. Bis-ferrocenyl uracil derivatives exhibited better antiproliferative activities than their monoferrocenyl analogs. Bis-ferrocenyl methyl- (5b) and halogen-substituted (5e, 6c, and 6d) uracil derivatives showed pronounced and selective cytostatic activities on colon adenocarcinoma (CaCo-2) and Burkitt lymphoma (Raji) cells, with higher potency and selectivity than the reference drug 5-fluorouracil. Generation of reactive oxygen species (ROS) in CaCo-2 and Raji cells when treated with compounds 5b, 5e, and 6d was observed. Bisferrocenyl 5-chlorouracil 6c induced significant disruption in mitochondrial membrane potential that is accompanied by activation of apoptosis in CaCo-2, Raji, and acute lymphoblastic leukemia (CCRF-CEM) cells, while 6d caused mitochondrial dysfunction and apoptosis induction in CaCo-2 and Raji cells. Potent antiproliferative activity of 6c and 6d could be associated with mitochondrial membrane potential disruption accompanied by apoptosis induction. Our findings highlighted 6c and 6d with potent and selective antiproliferative activity on CaCo-2, Raji, and CCRF-CEM cells that may be associated with targeting cancer cell mitochondria, as a molecular target.