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Polyhydroxyoctanoate (PHO) is a biocompatible microbially produced polyester with elastomeric properties currently limited by its high production cost and poor biodegradability in open environments. Enzymatic recycling of post­consumer PHO offers a bio­cyclable route to PHO utilization and poses a solution from both ecological and economical aspects. Enzyme engineering enables industrial efficiency in polymer degradation. Therefore, we have created a mutant library for PHO depolymerase from Pseudomonas fluorescens GK13 (PfPHOase) using error­prone PCR. To advance the speed of identification of PfPHOase variants with improved PHO degradation rates high­throughput screening was performed spectroscopically with in­house synthesized p­nitrophenyl esters of 3­hydroxyalkanoate monomer (3­ HA monomer) and 3­hydroxyalkanoic acid dimer (3­HA dimer)1. Further, the degradation of PHO polymer for best­performing PfPHOase variants was assessed on commonly employed emulsified PHO­agarose plates. The obtained results indicate the positive correlation between the degradation of novel PHO model compounds with degradation of PHO polymer. Novel model compounds have been successfully employed for the identification of beneficial mutations for the advancement of enzymatic PHO degradation.

Polyhydroxyalkanoates (PHA) represent a sustainable alternative to conventional plastics due to their microbial biological origin, biodegradability, biocompatibility, and structural diversity. The primary challenge in the industrial enzymatic recycling of PHA lies in the relatively slow biodegradation rates, spanning several months to years, depending on environmental conditions and the characteristics of both PHA and the PHA-degrading enzymes. This study involves experimental and computational testing of monomeric and dimeric units of polyhydroxy octanoate (PHO) as suitable substrates for continuous spectrophotometric monitoring of enzymatic reactions. Additionally, we explore the specificity of these novel model substrates for polyhydroxyalkanoate depolymerases (PHOase) in comparison to other esterases (lipase and cutinase). The experimental findings are complemented by computational evaluations. We have modeled the most active enzyme, poly(3-hydroxyoctanoate) depolymerase from Pseudomonas fluorescens, followed by cutinase LCC, which exhibits lower activity on tested substrates, and lipase CalB that doesn't show detectable activity. Our computational insights provide a rationale for these experimental observations.

In this study,we conducted a comprehensive investigation of newly synthesized fullerene derivatives developed for potential application in perovskite solar cells (PSCs). We explored three novel dihydrofuran-fused C60 fullerene derivatives (13, 14, and 15) that we respecifically designed to enhance solubility and interaction with the substrate, fluorine-dopedtinoxide (FTO). A comparative analysis was performed, with reference to the widely used phenyl-C61-butyric acid methyl ester (PCBM) and compound 12, from which 13, 14, and 15 are derived, to assess the impact of sugar units on materials properties.The synthesized compounds demonstrated significant solubility in common organic solvents, a critical factor in their potential application in wet-processed PSCs. Our investigation included electrochemical property analysis, thin film deposition, surface characterization, and electrochemical impedance spectroscopy (EIS). EIS measurements unveiled key in sights into charge transfer properties at the electrode/electrolyte interface, making the compounds attractive candidates for electron transport layers (ETLs) in PSCs.

The discovery and engineering of novel biocatalysts capable of depolymerizing polyethylene terephthalate (PET) have gained significant attention since the need for green technologies to combat plastic pollution has become increasingly urgent. This study focuses on the development of novel substrates that can indicate enzymes with PET hydrolytic activity, streamlining the process of enzyme evaluation and selection. Four novel substrates, mimicking the structure of PET, were chemically synthesized and labeled with fluorogenic or chromogenic moieties, enabling the direct analysis of candidate enzymes without complex preparatory or analysis steps. The fluorogenic substrates, mUPET1, mUPET2, and mUPET3, not only identify enzymes capable of PET breakdown but also differentiate those with exceptional performance on the polymer, such as the benchmark PETase, LCCICCG. Among the substrates, the chromogenic p-NPhPET3 stands out as a reliable tool for screening both pure and crude enzymes, offering advantages over fluorogenic substrates such as ease of assay using UV–vis spectroscopy and compatibility with crude enzyme samples. However, ferulic acid esterases and mono-(2-hydroxyethyl) terephthalate esterases (MHETases), which exhibit remarkably high affinity for PET oligomers, also show high catalytic activity on these substrates. The substrates introduced in this study hold significant value in the function-based screening and characterization of enzymes that degrade PET, as well as the the potential to be used in screening mutant libraries derived from directed evolution experiments. Following this approach, a rapid and dependable assay method can be carried out using basic laboratory infrastructure, eliminating the necessity for intricate preparatory procedures before analysis.

PHAs are naturally made microbial polyesters that have been commercialized as biodegradable plastics. However, it has been shown that these materials are not so easily biodegraded in natural environments [1]. PHA depolymerases are key PHA degrading enzymes and their identification and characterization is of great interest and importance. Currently, screening is done on polymeric substrates using techniques such as clear zone assays on agar or weight loss measurements. Results obtained using these different methods cannot be directly compared, since they depend highly on the polymer used, PHA granules preparation and assay conditions [2]. In order to design a more specific test for the determination of PHA depolymerase activity, we synthesized 3-hyoxyalkanoate monomers (3-HA monomer) and 3-hyoxyalkanoic acid dimers (3-HA dimer) and their respective p-nitrophenyl esters, allowing for spectrophotometric determination of their activity [3]. Compounds were characterized using N and FTIR. Para-nitrophenyl labeled substrates were then used in the enzymatic activity assay with the benchmark polyhyoxyoctanoate (PHO) depolymerase from Pseudomonas fluorescens GK13 expressed in Escherichia coli CodonPlus-RIPL hosts. This activity was compared to recombinantly expressed leaf-branch compost cutinase (LCC cutinase) and polyethyleneterephtalate (PET) hyolyzing esterase from Ideonella sakaiensis (IsPETase). Our initial results revealed increased specificity of PHO depolymerase towards newly synthetized substrates, suggesting their suitability for specific screens and isolation of new mcl-PHA depolymerases, as well as in high throughput screening assays designed for guiding their directed evolution.

In this study, we conducted a comprehensive investigation of newly synthesized fullerene derivatives developed for potential application in perovskite solar cells (PSCs). We explored three novel dihydrofuran-fused C60 fullerene derivatives (13, 14, and 15) that were specifically designed to enhance solubility and interaction with the substrate, fluorine-doped tin oxide (FTO). A comparative analysis was performed, with reference to the widely used phenyl-C61-butyric acid methyl ester (PCBM) and compound 12, from which 13, 14, and 15 are derived, to assess the impact of sugar units on materials properties. The synthesized compounds demonstrated significant solubility in common organic solvents, a critical factor in their potential application in wet-processed PSCs. Our investigation included electrochemical property analysis, thin film deposition, surface characterization, and electrochemical impedance spectroscopy (EIS). EIS measurements unveiled key insights into charge transfer properties at the electrode/electrolyte interface, making the compounds attractive candidates for electron transport layers (ETLs) in PSCs.

The synthesis and characterization of four dumbbell-shaped fullerene molecules connected by isosorbide and isomannide moieties is presented. Additionally, their electrochemical behavior and their ability to form complexes with [10]cycloparaphenylene ([10]CPP) were investigated. The cyclic voltammetry (CV) results of the fullerene dumbbells demonstrate a high electron affinity, indicating their strong interaction with electron-donating counterparts such as carbon nanorings, which possess complementary charge and shape properties. To study the thermodynamic and kinetic parameters of complexation, isothermal titration calorimetry (ITC) was employed. NMR titration experiments provided further insights into the binding stoichiometries. Two distinct approaches were utilized to create bridged structures: one based on cyclopropane and the other based on furan. Regardless of the type of linker used, all derivatives formed conventional 2 : 1 complexes denoted as [10]CPP2 ⊃C60derivative . However, the methano-dumbbell molecules exhibited distinct binding behavior, resulting in the formation of mono- and bis-pseudorotaxanes, as well as oligomers (polymers). The formation of linear polymers holds significant potential for applications in solar energy conversion processes.

We present a novel approach for the enzymatic functionalization of graphene, utilizing horseradish peroxidase (HPO) and laccase (LC) from Trametes versicolor. This study demonstrates, for the first time, the covalent modification of non-homogeneous graphene with a low surface-to-volume ratio, both in solution and on solid support. Through thermogravimetry analysis, we estimate the degree of functionalization to be 11% with HPO and 4% with LC, attributed to the varying redox potentials of the enzymes. This work highlights the potential of enzymatic reactions for tailored functionalization of graphene under mild conditions.

Polyurethanes (PUs) are an exceedingly heterogeneous group of plastic polymers, widely used in a variety of industries from construction to medical implants. In the past decades, we have witnessed the accumulation of PU waste and its detrimental environmental impacts. PUs have been identified as one of the most toxic polymers leaching hazardous compounds derived both from the polymer itself and the additives used in production. Further environmental impact assessment, identification and characterization of substances derived from PU materials and establishing efficient degradation strategies are crucial. Thus, a selection of eight synthetic model compounds which represent partial PU hydrolysis products were synthesized and characterized both in terms of toxicity and suitability to be used as substrates for the identification of novel biocatalysts for PU biodegradation. Overall, the compounds exhibited low in vitro cytotoxicity against a healthy human fibroblast cell line and virtually no toxic effect on the nematode Caenorhabditis elegans up to 500 µg mL−1, and two of the substrates showed moderate aquatic ecotoxicity with EC50 values 53 µg mL−1 and 45 µg mL−1, respectively, on Aliivibrio fischeri. The compounds were successfully applied to study the mechanism of ester and urethane bond cleaving preference of known plastic-degrading enzymes and were used to single out a novel PU-degrading biocatalyst, Amycolatopsis mediterranei ISP5501, among 220 microbial strains. A. mediterranei ISP5501 can also degrade commercially available polyether and polyester PU materials, reducing the average molecular number of the polymer up to 13.5%. This study uncovered a biocatalyst capable of degrading different types of PUs and identified potential enzymes responsible as a key step in developing biotechnological process for PU waste treatment options.

Polyurethanes (PUs) are an exceedingly heterogeneous group of plastic polymers, widely used in a variety of industries from construction to medical implants. In the past decades, we have witnessed the accumulation of PU waste and its detrimental environmental impacts. PUs have been identified as one of the most toxic polymers leaching hazardous compounds derived both from the polymer itself and the additives used in production. Further environmental impact assessment, identification and characterization of substances derived from PU materials and establishing efficient degradation strategies are crucial. Thus, a selection of eight synthetic model compounds which represent partial PU hydrolysis products were synthesized and characterized both in terms of toxicity and suitability to be used as substrates for the identification of novel biocatalysts for PU biodegradation. Overall, the compounds exhibited low in vitro cytotoxicity against a healthy human fibroblast cell line and virtually no toxic effect on the nematode Caenorhabditis elegans up to 500 µg mL−1, and two of the substrates showed moderate aquatic ecotoxicity with EC50 values 53 µg mL−1 and 45 µg mL−1, respectively, on Aliivibrio fischeri. The compounds were successfully applied to study the mechanism of ester and urethane bond cleaving preference of known plastic-degrading enzymes and were used to single out a novel PU-degrading biocatalyst, Amycolatopsis mediterranei ISP5501, among 220 microbial strains. A. mediterranei ISP5501 can also degrade commercially available polyether and polyester PU materials, reducing the average molecular number of the polymer up to 13.5%. This study uncovered a biocatalyst capable of degrading different types of PUs and identified potential enzymes responsible as a key step in developing biotechnological process for PU waste treatment options.

Size distributions (by intensity percentage) of NPs washed suspended in different dispersants, water,BSA (0.05%),SDS (0.1 %). Distributions data from different combined fractions: from NP-10 to NP-30; from NP-40 to NP-60 and from NP-70 to NP-90. Output from the Malvern zetasizer Nano-ZS ZEN 3600 (Malvern Panalytical, Malvern, UK).

The presence of the ionic surfactant sodium dodecyl sulfate (SDS) was characterized by 1H NMR, where the relative ratio of NP/surfactant was monitored on the basis of the chemical shifts characteristic of PET and SDS. 1H NMR spectra of the NPs preparation before and during all the washing steps. Determination of SDS Level in Corona of PET NPs by 1H NMR. Output from the Varian/Agilent NMR 400 MHz. NMR spectra were processed in Mnova software.

Determination of hydrodynamic diameter of PET NPs washed and dispersed in water, BSA (0.05%), and SDS (0.1%), and NPs unwashed and dispersed in water. Zeta potential, mobility, and conductivity determination in PET NPs at different stages of purification. Output from the Malvern zetasizer Nano-ZS ZEN 3600 (Malvern Panalytical, Malvern, UK).

Percentage of hemolysis of RBC in the presence of washed PET NPs and unwashed NPs (with SDS) determined by incubation of human red blood cells (RBCs) from three healthy donors. Output from the Shimadzu UV/ViS 1800 (Kyoto, Japan) nspectrometer - raw spectral data and the calculation of percentages of hemolysis (procesesd data).

A photochemically promoted intramolecular cyclization of aryl-, vinyl-, and alkyliodo allenes has been developed. The optimal conditions employed [Ir(ppy)2(dtbbpy)]PF6 (1 mol%) as catalyst affording products with high exo selectivity in moderate to good yields. Chiral substrates showed diastereoselectivity of up to 95/5 favoring trans product.

Polyethylene terephthalate (PET) is widely used material and as such became highly enriched in nature. It is generally considered inert and safe plastic, but due to the recent increased efforts to break-down PET using biotechnological approaches, we realized the scarcity of information about structural analysis of possible degradation products and their ecotoxicological assessment. Therefore, in this study, 11 compounds belonging to the group of PET precursors and possible degradation products have been comprehensively characterized. Seven of these compounds including 1-(2-hydroxyethyl)-4-methylterephthalate, ethylene glycol bis(methyl terephthalate), methyl bis(2-hydroxyethyl terephtahalate), 1,4-benzenedicarboxylic acid, 1,4-bis[2-[[4-(methoxycarbonyl)benzoyl]oxy]ethyl] ester and methyl tris(2-hydroxyethyl terephthalate) corresponding to mono-, 1.5-, di-, 2,5- and trimer of PET were synthetized and structurally characterized for the first time. In-silico druglikeness and physico-chemical properties of these compounds were predicted using variety of platforms. No antimicrobial properties were detected even at 1000 μg/mL. Ecotoxicological impact of the compounds against marine bacteria Allivibrio fischeri proved that the 6 out of 11 tested PET-associated compounds may be classified as harmful to aquatic microorganisms, with PET trimer being one of the most toxic. In comparison, most of the compounds were not toxic on human lung fibroblasts (MRC-5) at 200 μg/mL with inhibiting concentration (IC50) values of 30 μg/mL and 50 μg/mL determined for PET dimer and trimer. Only three of these compounds including PET monomer were toxic to nematode Caenorhabditis elegans at high concentration of 500 μg/mL. In terms of the applicative potential, PET dimer can be used as suitable substrate for the screening, identification and characterization of novel PET-depolymerizing enzymes.

In this study, β-keto esters as readily available bio-based building blocks were used to decorate the C60 sphere. Generally, cyclopropanated fullerene derivatives are obtained by the standard Bingel–Hirsch procedure. Herein, omitting the iodine from the reaction mixture and adding TEMPO afforded dihydrofuran fused C60 fullerene derivatives. The mechanism of the reaction shifted from nucleophilic aliphatic substitution to oxidative [3 + 2] cycloaddition via fullerenyl cations as an intermediate. This mechanism is proposed based on a series of control experiments with radical scavengers. Therefore, dihydrofuran-fused C60 derivatives were selectively obtained in good yields and their structures were established based on UV-Vis, IR, NMR spectroscopy and mass spectrometry. The electrochemical properties of the synthesized compounds were investigated by cyclic voltammetry. DFT calculations were performed in order to investigate the difference in stability, electronic properties and π-electron delocalization between methano and furano fullerenes.

U okviru ove disertacije dizajnirano je 30 anilidopipredina, analoga jakog -opioidnog agonista i kliničkog analgetika fentanila. Razvijen je efikasan sintetički put za njihovo dobijanje, a sintetisana jedinjenja mogu se podeliti u dve serije. U okviru prve serije sintetisano je 13 novih analoga fentanila sa različitim azotnim grupama u položaju C3 piperidinskog prstena. Takođe je optimizovana sinteza dva prethodno sintetisana biciklična analoga fentanila. Jedinjenja prve serije predstavljaju prve poznate analoge fentanila sa azotnim grupama u položaju C3. U okviru druge serije sintetisano je 15 novih analoga fentanila, koji su u položaju N1 povezani sa pojedinim N-arilpiperazinima preko linearnih alkil mostova različitih dužina. Jedinjenja druge serije dizajnirana su da budu potencijalni heterobivalenti ligandi, jer sadrže anilidopiperidinsko jezgro fentanila kao -opioidnu agonističku farmakoforu, a N-arilpiperazin kao D2-dopaminergičku, agonističku farmakoforu.Analgetička aktivnost svih 30 jedinjenja ispitana je in vivo. Samo četiri jedinjenja, sva iz prve serije, pokazala su analgetičku aktivnost. Najaktivnije jedinjenje, oko 1,8 puta manje aktivno od fentanila, karakterišu brzo i kratko dejstvo analgetičkog efekta, pa se stoga može potencijalno iskoristiti u različitim farmacutskim formulacijama za tretman bola. Afinitet vezivanja jedinjenja druge serije za dopaminergčke D2 receptore ispitan je in vitro testovima kompeticije koristeći 3H spiperon kao radioaktivni ligand. Odsustvo analgetičke aktivnosti i slab afinitet vezivanja za dopaminergičke D2 receptore, ukazuje da jedinjenja druge serije ne mogu poslužiti kao heterobivalentni ligandi.Preliminarna doking analiza urađena je za jedinjenja koja su pokazala farmakološku aktivnost., This PhD thesis encompasses the design of 30 anilidopiperidines, as analogues of potent -opioid agonist and clinical analgesic, fentanyl. All compounds were prepared by the novel and effcient synthetic routes, and can be divided in two series. In the first series, 13 new analogues of fentanyl with various nitrogen groups in the C3 position of the piperidine ring were synthesized. In addition, the synthesis of two bicyclic analogues of fentanyl, previously synthesized by our group, was optimized. The compounds of the first series represent the first known fentanyl analogues possessing any nitrogen substituent at C3 position of the piperidine ring. The second series involved preparation of 15 new compounds consisting of fentanyl moiety and several arylpiperazine groups joined by the respective secondary nitrogens, via the variable-length alkyl linkers. The compounds were designed to be potential heterobivalent ligands, since they include both the fentanyl core, as the -opioid agonist pharmacophore, and N-arylpiperazine moiety as the D2-dopaminergic agonist pharmacophore.The analgesic activity of 30 compounds was tested in vivo. Only four compounds, all from the first series, showed analgesic activity. The most potent of them was 1.8 times less potent analgesic than fentanyl, with fast onset and short duration of analgesic effect, which makes this compound potentially suitable for different pharmacological formulation in the pain treatment. Binding affinity of the compounds of the second series towards dopaminergic D2 receptors was determined by in vitro competitive assay, using 3H spiperon as radioactive ligand. Compounds of the second series were found to be inactive as analgesics and with low affinity towards dopaminergic D2 receptors. Therefore, the compounds do not represent heterobivalent ligands.Preliminary docking analysis was performed for the pharmacologically active compounds.

In the present work, we describe the chemical synthesis of 3-HFAs as prominent derivatives of fatty acids and assess if they could be applied as phase change materials (PCM). In addition, 3-HFAs were obtained by depolymerization of a bacterial biopolymeric material, polyhydroxyalkanoate. Thermal properties of 3-hydoxyoctanoic, decanoic, and dodecanoic acids are reported for the first time. These materials showed the potential to be applied as PCM in temperature range from 33°C to 66°C. In order to expand the temperature range for application of 3-HFAs as PCM, eutectic mass ratios of three kinds of binary mixtures of 3-HFAs were calculated, and their properties were predicted using the Schröder-van Laar equation. Thermal properties of these mixtures were validated by differential scanning calorimetry (DSC) analysis. These results showed that eutectics considerably expanded the scope of applications of 3-HFAs as PCMs. 3-HFAs originating from biotechnologically obtained polyhydroxyalkanoates also showed potential to be applied in development of PCMs.