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Influence of the type of the reactants and reaction parameters on the synthesis of biodiesel fuels from higher alcohols
No full textIstraživanje i razvoj naprednih biogoriva motiviran je smanjenjem negativnog utjecaja na okoliš uzrokovanog višestoljetnom primjenom fosilnih goriva, a potaknut i od strane zakonskih regulativa Europske Unije koje potiču energetsku diverzifikaciju, dekarbonizaciju prometnog sektora te načela kružnog gospodarstva. Jedno od takvih biogoriva je i biodizel, koji se proizvodi transesterifikacijom triglicerida alkoholom, u prisutnosti katalizatora. Nakon pročišćavanja, moguće ga je namiješati s fosilnim gorivom te, ukoliko zadovolji norme za dizelsko (HRN EN 590) i biodizelsko (HRN EN 14214) gorivo, koristiti kao obnovljivi izvor energije u dizel motorima. U Republici Hrvatskoj dopušteno je namiješavanje biodizelskog s dizelskim gorivom do volumnog udjela od 7 % da bi se njihova mješavina i dalje deklarirala dizelskim gorivom. Biodizel je moguće s dizelskim gorivom namiješati i u višim volumnih udjelima, no u tom se slučaju radi o mješavinama dizelskog i biodizelskog goriva. S obzirom da se komercijalni biodizel u Europi najčešće proizvodi iz djelomično obnovljivog metanola, cilj ovog doktorskog rada bio je ispitati utjecaj vrste reaktanata i reakcijskih uvjeta na proces sinteze biodizela iz izomera različitih viših alkohola (od propanola do oktanola), koje je moguće kao bioalkohole fermentacijom u potpunosti proizvesti iz biomase. U prvom dijelu ovog doktorskog rada praćen je utjecaj reakcijskih uvjeta (temperature, vremena reakcije, molarnog omjera reaktanata te masenog udjela katalizatora) na iskorištenje reakcije transesterifikacije. Primjenom Box-Behnkenovog dizajna eksperimenta i metodologije odzivnih površina, analizirani su i optimirani sustavi u kojima je biodizel sintetiziran iz suncokretovog ulja i alkohola 1-propanola ili 1-oktanola, odnosno otpadnog ulja i sekundarnog alkohola 2-propanola, 2-butanola, 2-pentanola ili 2-oktanola, u prisutnosti kalijeva hidroksida (KOH), kao katalizatora. Dodatno, praćena je i ovisnost iskorištenja sinteze biodizela o spomenutim reakcijskim uvjetima, kemijskoj strukturi te molekulskoj masi reaktanata, u prisutnosti KOH, pri čemu su različite biosirovine bile suncokretovo, repičino, kokosovo, otpadno ulje te životinjska (svinjska) mast, koje su reagirane s 1-propanolom,
1-butanolom, izobutanolom, 1-pentanolom, izopentanolom, 1-heksanolom ili 2-heksanolom.
U drugom dijelu rada, veće šarže biodizela (metilnih, propilnih, butilnih, izobutilnih, pentilnih, heksilnih i oktilnih estera masnih kiselina) sintetizirane su iz otpadnog ulja u prisutnosti KOH, pri uvjetima najviših eksperimentalnih iskorištenja; pročišćene te namiješane u dvokomponentne i trokomponentne mješavine s neaditiviranim mineralnim dizelom i/ili odgovarajućim alkoholom. Provedena je analiza primjenskih svojstava priređenih uzoraka prema odgovarajućim normiranim metodama (gustoće, kinematičke viskoznosti, maglišta, točke filtrabilnosti, tecišta, mazivosti, simulirane destilacije), čije su vrijednosti uspoređene s onima propisanima normom za dizelsko gorivo HRN EN 590. Dodatno su uzorci analizirani metodama: diferencijalna pretražna kalorimetrija, mjerenje napetosti površine goniometrom i mjerenje koeficijenta trenja. Glavni rezultati su pokazali da su za iskorištenje ovih reakcijskih sustava značajni reakcijski uvjeti molarni omjer reaktanata i maseni udio katalizatora KOH, odnosno da se u promatranim sustavima, generalno najviša iskorištenja dobivaju korištenjem kokosovog ulja, a najniža korištenjem životinjske masti, kao biosirovine. Iskorištenja biodizela sintetiziranih iz ravnolančanih alkohola generalno su bila viša od onih sintetiziranih iz granatih, a porast molekulske mase granatih alkohola dovodi do daljnjeg sniženja iskorištenja. Karakterizacijom uzoraka utvrđeno je da dodatak biodizelskih goriva do 20 vol.% u mješavine s dizelom i/ili pripadajućim alkoholom rezultira porastom njihove gustoće, kinematičke viskoznosti i površinske napetosti, dok dodatak viših alkohola do 15 vol.% u odgovarajuće mješavine s dizelom i biodizelom rezultira smanjenjem njihove gustoće, kinematičke viskoznosti i površinske napetosti. Vrijednosti gustoća i kinematičkih viskoznosti za sve dvokomponentne mješavine dizel-biodizel (s do 20 vol.% biodizela) te trokomponentne mješavine dizel-biodizel-pripadajući alkohol (s do 15 vol.% biodizela) nalaze se unutar granica propisanih normom za dizelsko gorivo HRN EN 590. Nadalje, namiješavanjem biodizelskih goriva i/ili odgovarajućih alkohola s mineralnim dizelom nije došlo do narušavanja njegovih nisko-temperaturnih svojstava (maglišta, točke filtrabilnosti, tecišta). Reološkim mjerenjima utvrđeno je da dodatak heksilnih estera masnih kiselina neaditiviranom mineralnog dizelu u količini od 2,5 vol.% značajno poboljšava njegovu mazivost, dok promjena udjela ili vrste biodizela namiješanog s mineralnim dizelom nema značajnog utjecaja na svojstva podmazivanja dizelskog goriva. Naposljetku, uočeno je da su za destilaciju istog postotka predestiliranog, dodatkom pentilnih estera masnih kiselina od
20 vol.% mineralnom dizelu, potrebne više temperature nego u slučaju dodatka istog biodizela u udjelu od 10 vol.%. Diferencijalna pretražna kalorimetrija pokazala je da dvokomponentne i trokomponentne mješavine svih biodizela s mineralnim dizelom i/ili pripadajućim alkoholom imaju temperature početke i kraja kristalizacije slične vrijednostima onih neaditiviranog mineralnog dizela. Navedeno može poslužiti kako dodatni pokazatelj izostanka njihova negativnog djelovanja na niskotemperaturna svojstva dizelskog goriva.The research and development of advanced biofuels is motivated by the reduction of negative environmental impact caused with the use of fossil fuels throughout the centuries, and encouraged with the legal regulations of European Union, that promote energy diversification, decarbonization of the traffic sector, as well as the principles of circular economy. One of such biofuels is biodiesel, that is produced via alcohol transesterification of a certain feedstock, in the presence of a catalyst. Post purification, it is possible to blend it with fossil fuel, if it fulfills the requirements set by the diesel (HRN EN 590) and biodiesel (HRN EN 14214) standard, and use as a renewable source of energy in diesel engines.
In the Republic of Croatia it is allowed to blend up to 7 vol% of biodiesel with diesel fuel for their blend to still be declared as diesel fuel. It is possible to blend higher volume fractions of biodiesel fuel with diesel fuel, as well, however these are referred to as the diesel-biodiesel blends. Since the commercial European biodiesel is most commonly produced from partly renewable methanol, the goal of this doctoral thesis was to investigate the influence of the reactant type and the reaction conditions on the biodiesel synthesis from isomers of selected higher alcohols (from propanol to octanol), which can, as bioalcohols, be completely produced via fermentation from biomass. In the first part of this doctoral thesis, the influence of reaction conditions (temperature, reaction time, molar ratio of the reactants, and mass fraction of the catalyst) on the reaction conversion of the biodiesel systems, was studied. With the use of Design of Experiments by Box-Behnken and Response Surface Methodology, the biodiesel synthesis from either sunflower oil and alcohols 1-propanol or 1-octanol, or waste cooking oil and 2-propanol, 2-butanol, 2-pentanol, or 2-octanol, was analyzed and optimized, in the presence of a catalyst potassium hydroxide. Additionally, the dependence of the reaction conversion on the mentioned reaction conditions, the chemical structure, and the molar mass of the reactants, in the presence of potassium hydroxide, was studied, with the selected feedstock being sunflower oil, rapeseed oil, coconut oil, waste cooking oil, or animal fat, that reacted with 1-propanol, 1-butanol, isobutanol, 1-pentanol, isopentanol,
1-hexanol, or 2-hexanol. In the second part of the thesis, larger amounts of biodiesels (fatty acid methyl, propyl, butyl, isobutyl, pentyl, hexyl, and octyl esters) were synthesized from waste cooking oil, in the presence of KOH, at the conditions of the highest experimental conversions; purified, and blended into the binary and trinary blends with non-additivated mineral diesel and/or corresponding alcohol. The prepared samples underwent the application property analysis via corresponding standardized methods (density, kinematic viscosity, cloud point, cold filter plugging point, pour point, lubricity, simulated distillation),
the results of which were compared with the ones prescribed by the standard for diesel fuel HRN EN 590. Additionallly, samples were analyzed via instrumental analytical methods: differential scanning calorimetry, surface tension measurements via goniometer, and friction coefficient measurements. The main results showed that the most significant reaction conditions in the given systems were the molar ratio of the reactants, and the mass fraction of the catalyst KOH, and that in the selected systems experimentally the highest conversions were obtained using coconut oil, while the lowest using animal fat, as a feedstock.
The reaction conversions in the synthesis using straight-chain alcohols were generally higher than those with branched alcohols, and the increase in the molecular weight of branched alcohols further led to a decrease in the conversion. Sample characterization determined the addition of selected biodiesel fuels up to 20 vol% into the blends with mineral diesel and/or corresponding alcohol led to the increase in their density, kinematic viscosity, and surface tension, while the addition of the selected higher alcohol up to 15 vol% in the blends with diesel and biodiesel resulted in the reduction of their density, kinematic viscosity, and surface tension. Density and kinematic viscosity values of all the binary diesel-biodiesel (with up to 20 vol% of biodiesel), and trinary diesel-biodiesel-corresponding alcohol blends (with up to 15 vol% of biodiesel) were within the limits required by the standard for diesel fuel HRN EN 590. Furthermore, blending of selected biodiesels and/or corresponding alcohols with mineral diesel did not aggravate its analyzed low-temperature properties (cloud point, cold filter plugging point, pour point). Rheological measurements determined that the addition of fatty acid hexyl esters to non-additivated mineral diesel of just 2.5 vol% significantly improved its lubricity, whereas the change in the volume fraction or the type of biodiesel blended with mineral diesel did not have a significant influence on the diesel’s lubricating properties. Finally, it was observed that for the distillation of the same percentage of the sample, the higher temperatures were required with the addition of fatty acid pentyl esters of 20 vol% to mineral diesel, than with the addition of the same biodiesel in the volume fraction of 10 vol%. On the other hand, differential scanning calorimetry showed that the binary and trinary blends of all biodiesels with mineral diesel and/or corresponding alcohol had onset and peak crystallization temperature values close to the ones of non-additivated mineral diesel. This could serve as an additional indicator for the lack of their negative impact on the diesel’s low-temperature properties
Evaluation of halohydrin dehalogenase ancestors as biocatalysts in the synthesis of novel compounds
No full textS obzirom na sva dosadašnja postignuća u području biokatalize, proteinskog inženjerstva i računalnog modeliranja, posljednjih 20 godina halogenhidrin-dehalogenaze (HHDH) su doživjele su nagli razvoj i sve širu primjenu u industriji. Jedna od najznačajnijih je farmaceutska industrija, u kojoj su HHDH svojom visokom kemo-, regio- i stereoselektivnošću napravile veliki iskorak. U reakcijama dehalogeniranja halogenalkohola i nukleofilnog otvaranja epoksida ovi enzimi pokazali su visoku aktivnost te visoku enantioselektivnost, pogotovo C-tip HHDH iz Agrobacterium radiobacter AD1 (HheC). Iz tog razloga su se u ovom diplomskom radu ispitivali preci HheC enzima dobiveni rekonstrukcijom filogenetskog stabla velikog novootkrivenog broja HHDH enzima. U radu su ispitani direktni predak N122, daljnji predak N124 (zajednički predak C- i A-skupina HHDH) i N134 koji je direktni predak svih HHDH enzima. Svi enzimi su sintetizirani u rekombinantnim stanicama E. coli, izolirani i pročišćeni, ali nisu svi pokazali očekivane rezultate. Točnije, preci N122, N124 i N134 nisu pokazali ni približno dobre rezultate kao ranije provedeno istraživanje s istim enzimima. Katalitička aktivnost im je u reakcijama dehalogeniranja para-nitro-2-brom-1-feniletanola (PNSHH) bila čak 20 puta (N134), 55 puta (N124) odnosno 220 puta (N122) manja u odnosu na HheC. Ovi rezultati nisu u skladu s prethodnim istraživanjima gdje su ovi preci pokazali značajno višu aktivnost u odnosu na HheC. Zato je potrebno provesti daljnja istraživanja da bi se ustanovilo što se tijekom vremena dogodilo unutar rekombinantne kulture E. coli što je moglo uzrokovati ovakav gubitak aktivnosti. U reakciji otvaranja epoksida 2-benzil-oksirana ovi enzimi pokazali su vrlo nisku (N134) do nepostojeću aktivnost (N122 i N134), te time i enantioselektivnost, što je dodatno potvrdilo gubitak aktivnosti ovih enzima. Uz pretke HheC, ispitivao se i predak skupine D HHDH – N81 koji je direktni predak HheD12 iz arktičke morske bakterije Halopseudomonas pelagia. On je, s druge strane, pokazao izvanredno dobru katalitičku aktivnost u obje ispitivane reakcije. U reakciji otvaranja PNSHH pokazao je specifičnu aktivnost od čak 1,73 U mg⁻¹ što je čak 15 puta više od N134, 50 puta više od N124 i 171 puta više od N134. Većina enzima iz skupine D nije do sad pokazivalo veliku aktivnost prema ovom tipu supstrata, izuzev HheD (iz Dechloromonas aromatice) i HheD2 (iz gammaproteobacterium HTCC2207 soja) čiji je N81 direktni predak. U otvaranju epoksida N81 je pokazao visoku konverziju od čak 88% što je dvostruko više od divljeg tipa HheC s konverzijom od 41%. Ovaj predak pokazao je u ovoj reakciji osrednju enantioselektivnost, s evs vrijednosti u kinetičkoj rezoluciji epoksida od čak 78% i evp vrijednosti prilikom sinteze (R)-1-azido-fenil-2-propanola od 20%. Ovi rezultati su, iako osrednji, bolji od do sad dobivenih rezultata za divlje D-tipove, što ukazuje na dodatne prilike u istraživanju ovog pretka. Ovo istraživanje upućuje na to da je potrebno provesti daljnja istraživanja sa širim rasponom supstrata, ali tek nakon što se ustanovi razlog gubitka aktivnosti predaka HheC, odnosno pripreme enzimi koristeći svježe pripremljene plazmide. Također, veću pažnju treba dati enzimima skupine D i njihovim precima zbog njihove visoke katalitičke aktivnosti.Given the significant advancements in the fields of biocatalysis, protein engineering, and computational modeling over the past 20 years, halohydrin dehalogenases (HHDHs) have experienced rapid development and increasingly widespread application in industry. One of the most significant areas is the pharmaceutical industry, where HHDHs have made significant strides due to their high chemo-, regio-, and stereoselectivity. In dehalogenation and nucleophilic epoxide ring-opening reactions, these enzymes have demonstrated high activity and enantioselectivity, especially the C-type HHDH from Agrobacterium radiobacter AD1 (HheC). This is why, in this study, ancestral enzymes of HheC, obtained by reconstructing the phylogenetic tree of a large number of newly discovered HHDH enzymes, were examined. The study examined the direct ancestor N122, the further ancestor N124 (the common ancestor of the C- and A-group HHDHs), and N134, which is the ancestor of all HHDH enzymes. All enzymes were synthesized in recombinant E. coli cells, isolated, and purified, but not all showed the expected results. Specifically, the ancestors N122, N124, and N134 did not show nearly as good results as previously conducted research with the same ancestral HheC cells. Their catalytic activity in the dehalogenation reactions of
para-nitro-2-bromo-1-phenylethanol (PNSHH) was up to 20 times (N134), 55 times (N124), and 220 times (N122) lower compared to the wild-type HheC. These results do not align with previous studies where these ancestors showed significantly higher activity compared to HheC. Therefore, further research is needed to determine what might have happened within the recombinant E. coli culture over time that could have caused such a loss of activity. In the epoxide ring-opening reaction of 2-benzyloxirane, these enzymes exhibited very low (N134) to non-existent activity (N122 and N134), and therefore enantioselectivity, further confirming the loss of activity in these enzymes. In addition to HheC ancestors, the ancestor of a large number of D-type HHDHs—N81, the direct ancestor of HheD12 from the Arctic marine bacterium Halopseudomonas pelagia—was also studied. This enzyme, on the other hand, demonstrated exceptionally good catalytic activity in both tested reactions. In the PNSHH opening reaction, it showed a specific activity of as much as 1.73 U mg⁻¹, which is 15 times higher than N134, 50 times higher than N124, and 171 times higher than N134. Most D-types have not shown significant activity towards this type of substrate so far, except for HheD (from Dechloromonas aromatica) and HheD2 (from Gammaproteobacterium HTCC2207 strain), of which N81 is the direct ancestor. In the epoxide opening, N81 showed a high conversion rate of as much as 88%, which is double that of the wild-type HheC with a conversion of 41%. This ancestor demonstrated moderate enantioselectivity in this reaction, with ees values in the kinetic resolution of the epoxide of up to 78% and eep values during the synthesis of (R)-1-azido-3-phenyl-2-propanol of 20%. These results, though moderate, are better than those obtained so far for wild D-types, indicating further opportunities for research into this ancestor. This research suggests that further studies with a broader range of substrates are needed, but only after determining the cause of the activity loss in HheC ancestors and preparing the enzymes using freshly prepared plasmids. Additionally, more attention should be given to D-types of HHDHs and their ancestors due to their high catalytic activity
Novel imidazo[4,5-b]pyridine conjugates: synthesis, structural characterization and biological activity
No full textU okviru doktorskog rada opisana je sinteza, strukturna karakterizacija i biološka aktivnost novih derivata imidazo[4,5-b]piridina. Za sintezu novih spojeva korištene su klasične metode organske sinteze, sinteza potpomognuta mikrovalovima, paralelna sinteza i organometalna sinteza. Akrilonitrilni derivati 21–40 i 58–89 priređeni su aldolnom kondenzacijom 2-cijanometilimidazo[4,5-b]piridina s različitim benzaldehidima. Ispitan je utjecaj supstitucije u položaju N3 imidazo[4,5-b]piridinske jezgre, broj i položaj metoksi i hidroksi skupina, te prisutnost N,N-dialkilnog supstituenta u para-položaju fenilne jezgre na biološku aktivnost i spektroskopske karakteristike. Nadalje, reakcijom ciklokondenzacije
2-cijanometilimidazo[4,5-b]piridina i salicilbenzaldehida priređeni su iminokumarinski derivati 97–119 supstituirani u položajima 6 i 7 iminokumarinske jezgre, te na N3 dušikovom atomu imidazo[4,5-b]piridinske jezgre. 2-fenilamidino-supstituirani derivati imidazo[4,5-b]piridina 126–133 priređeni su iz cijano-supstituiranih prekursora 122–125, dok su reakcijom metiliranja priređeni derivati 134–136. Pinnerovom reakcijom u dva stupnja, priređeni su svi derivati supstituirani cikličkim amidinima, a one-pot reakcijom s reagensom LiHMDS derivati supstituirani nesupstituiranim amidinom. Suzukijevom reakcijom priređeni su derivati 142–146 i 149–154 supstituirani u položaju 6 imidazo[4,5-b]piridinske jezgre, a ovisno o korištenoj bornoj kiselini, u para-položaju fenilnog prstena supstituirani su različitim skupinama. N3-metil-supstituirani amidini imidazo[4,5-b]piridina 163–171 priređeni su iz cijano-supstituiranih prekursora 152, 161 i 162 Pinnerovom reakcijom. Serija konjugata imidazo[4,5-b]piridina i amidino-supstituiranih benzazola 182–190 priređena je kondenzacijom amidino-supstituiranih prekursora 173–181, odnosno
4-amidino-1,2-fenilendiamin-hidroklorida ili 5-amidino-2-aminobenzentiola s
imidazo[4,5-b]piridin-6-karbaldehidom 172. Schiffove baze imidazo[4,5-b]piridina 194–197 priređene su iz imidazo[4,5-b]piridina-karbaldehida 172 s anilinima, a Schiffove baze 202–214 iz 2-aminofenil-supstituiranih imidazo[4,5-b]piridina s benzaldehidima. Priređene su i dvije klase amidnih derivata imidazo[4,5-b]piridina koje se međusobno razlikuju prema položaju amidne veze. Derivati 217, 218, 230, 231 i 234 priređeni su iz benzoilnog klorida i amino-supstituiranih derivata, dok je derivat 222 priređen katalitički. Amidino-supstituirani derivati 219, 220, 232 i 233 priređeni su Pinnerovom reakcijom u dva stupnja.
Amino-supstituirani imidazo[4,5-b]piridini 102, 111, 147, 155, 233 i 235 priređeni su redukcijom iz odgovarajućih nitro-supstituiranih derivata. Radi bolje topljivosti derivati 103, 112, 119, 148, 156, 224 i 236 su pripravljeni kao amonijeve hidrokloridne soli. Strukture svih priređenih ciljanih spojeva i prekursora potvrđene su 1H i 13C NMR spektroskopijom, a za pojedine spojeve i dodatno masenom spektrometrijom. Detaljna spektroskopska karakterizacija u svrhu pronalaženja novih potencijalnih optičkih pH-senzora, provedena je za odabrane spojeve iz klasa akrilonitrila, iminokumarina i Schiffovih baza, a pKa vrijednosti određene su eksperimentalno i računalnim metodama. Svim ciljanim novopriređenim spojevima ispitana je antiproliferativna aktivnost in vitro na nekoliko staničnih linija humanih karcinoma. Za najaktivnije derivate istražen je mehanizam biološkog djelovanja koji je dodatno potvrđen, za neke derivate, i računalnom analizom. Antioksidativna aktivnost in vitro ispitana je spektroskopskim metodama DPPH, FRAP i ABTS, te elektrokemijski, dok je mehanizam antioksidativnog djelovanja potvrđen i računalnom analizom. Najaktivnijim amidinskim derivatima ispitana je interakcija s ct-DNK. Odabranim derivatima ispitana je antibakterijska aktivnost in vitro na nekoliko sojeva Gram-pozitivnih i Gram-negativnih bakterija, te antivirusna aktivnost in vitro prema odabranim vrstama virusa.Within this work, the synthesis, structural characterization and biological activity of novel imidazo[4,5-b]pyridine derivatives was described. For the synthesis of targeted compounds, classical methods of organic chemistry, parallel synthesis, and microwave assisted synthesis as well as organometal chemistry were used. Acrylonitrile derivatives 21–40 and 58–89 were prepared in the aldol condensation from 2-cyanomethylimidazo[4,5-b]pyridines and various benzaldehydes. Influence of N3 substitution on imidazo[4,5-b]pyridine core, the number and the position of hydroxy and methoxy groups as well as presence of N,N-dialkyl substituent placed at para-position of the phenyl ring on biological activity and spectroscopic characteristics was investigated. In the cyclocondensation reaction from 2-cyanomethylimidazo[4,5-b]pyridines and salicyl benzaldehydes, iminocoumarin derivatives 97–119 substituted at positions 6 and 7 of iminocoumarin core as well as at the position N3 of imidazo[4,5-b]pyridine nucleus were prepared. 2-phenylamidino-substituted
imidazo[4,5-b]pyridine derivatives 126–133 were prepared from cyano-substituted precursors 122–125, while derivatives 134–136 were prepared in the reaction of methylation. Two step Pinner reaction was used to prepare compounds substituted with cyclic amidines, while one pot reaction with LiHMDS was used to prepare derivatives with unsubstituted amidines. Suzuki coupling was used to prepare derivatives 142–146 and 149–154 substituted at the postition 6 of imidazo[4,5-b]pyridine core, depending on used boronic acid, substituted at the p–position of the phenyl ring with different substituents.
N3-methyl-substituted amidine derivatives 163–171 were prepared from cyano-substituted precursors 152, 161 and 162 in Pinner reaction and one pot reaction with LiHMDS. Benzazole conjugates of imidazo[4,5-b]pyridine with amidino-substituted benzazoles 182–190 were synthesized by condensation amidino-substituted precursors 173–181, respectively from 4-amidino-1,2-phenylenediamines hydrochlorides or 5-amidino-2-amino-substituted benzenthiolates with imidazo[4,5-b]pyridine-6-carbaldehyde 172. Imidazo[4,5-b]pyridine derived Schiff bases 194–197 were prepared from imidazo[4,5-b]pyridine-6-carbaldehyde 172 with various anilines, while Schiff bases 202–214 obtained from
2-aminophenyl-substituted imidazo[4,5-b]pyridines with benzaldehydes. Two series of amide derivatives were prepared which differ from position of amide bond. Derivatives 217, 218, 230, 231 and 234 were prepared by reactions between benzoil chlorides and
amino-substituted derivatives, while derivative 222 was prepared catalytically.
Amidino-substituted derivatives 219, 220, 232 and 233 were prepared by Pinner reaction.
All amino-substituted imidazo[4,5-b]pyridines 102, 111, 147, 155, 233 i 235 were prepared by reduction of corresponding nitro-substituted compounds. Some derivatives 103, 112, 119, 148, 156, 224 and 236 were prepared as ammonium hydrochloride salts in order to achieve better solubility. Structures of targeted compounds and precursors were analysed by means of 1H and 13C NMR spectroscopy, and were further confirmed by mass spectrometry for selected compounds. Thorough spectroscopic characterization was conducted for selected acrylonitrile, iminocoumarin and Schiff base derivatives, while pKa values were determined experimentally and computationally. All targeted compounds were evaluated for their antiproliferative activity in vitro on several human cancer cell lines. Mechanism of action was further investigated for the most active compounds and for some chosen derivatives confirmed also by computational analysis. Antioxidative activity in vitro was determined by using spectroscopic methods DPPH, FRAP and ABTS as well as electrochemically, while their mechanism of antioxidative action was explained by computational analysis. The most active amidine compounds were chosen for studying their interaction with ct-DNA. Selected compounds were tested also for their antibacterial activity in vitro against some Gram positive and Gram negative bacterial strains, as well as antiviral activity in vitro against selected viruses
Bioremediation of leachate from biowaste
No full textPovećanje broja stanovnika i rastuća industrijalizacija rezultirali su znatnim količinama otpada i onečišćenja okoliša diljem svijeta. Sa sve većim izazovima u održivom gospodarenju otpadom, problematika otpada postaje sve izraženija. Procjedne vode, koje nastaju kao tekući nusproizvodi prolaskom kroz otpadne materijale, posebno su zabrinjavajuće zbog svoje složenosti i visokog sadržaja organskih tvari. Njihovo ispuštanje u prirodne vodotokove može štetno utjecati na okoliš. Biološka obrada procjednih voda postaje odlučujući čimbenik u njihovom upravljanju, pri čemu se mikroorganizmi koriste za uklanjanje onečišćujućih tvari i poboljšanje kvalitete vode. Među različitim postupcima biološke obrade, bioremedijacija se ističe kao učinkovit pristup zbog svoje održive prakse i ekološke prihvatljivosti. U ovom radu provedena je bioremedijacija procjedne vode iz biootpada u šaržnim i submerznim uvjetima. Fizikalno-kemijskim i mikroskopskim analizama dobiven je uvid u tijek procesa biorazgradnje prisutnih organskih onečišćujućih tvari. Svjetlosnom mikroskopijom dobiven je brzi uvid u promjene sustava tijekom procesa bioremedijacije. Učinkovitost bioremedijacije procjedne vode iz biootpada iznosila je 89 %.Population growth and increasing industrialization have led to considerable amounts of waste and environmental pollution in all regions around the world. With the increasing challenges of sustainable waste management, waste-related problems are becoming more and more apparent. Leachates, the liquid by-products of the passage of waste materials, are particularly problematic due to their complexity and high organic content. Their discharge into natural watercourses can have harmful effects on the environment. Biological treatment of leachates is becoming increasingly important in their management, using microorganisms to remove pollutants and improve water quality. Among the various biological treatment methods, bioremediation stands out as an effective approach due to its sustainable practices and ecological acceptability. In this study, the bioremediation of leachate from biowaste was carried out under batch and submerged conditions. Physico-chemical and microscopic analyzes provided information on the course of the biodegradation process of the organic pollutants present. Light microscopy provided a quick insight into the system changes during the biodegradation process. The efficiency of bioremediation of leachate from biowaste was 89 %
Coumarin derivatives of 1,2,4-triazole - synthesis and biological activity related to plant protection application
No full textU okviru doktorskog rada sintetizirani su kumarinski 1,2,4-triazoli s mogućim višestrukim biološkim učincima vezanim za primjenu u zaštiti bilja. Sinteza je provedena kao one-pot reakcija kumarinskih hidrazida (1 – 3) i različito supstituiranih izotiocijanata u niskotemperaturnom eutektičkom otapalu kolin-klorid : urea (molni omjer 1 : 2), miješanjem na magnetskoj miješalici pri 80 °C. Rezultat su tri serije spojeva: prva, u kojoj je
1,2,4-triazolni prsten povezan -CH2- poveznicom na položaju 4, a hidroksilna je skupina na položaju 7 kumarina (1a – k), te druga serija derivata s 1,2,4-triazolnim prstenom povezanim eterskom poveznicom na položaju 7 kumarina i metilnom skupinom na položaju 4 kumarina (2a – o). Treća, dodatna serija derivata od druge se razlikuje po nedostatku metilne skupine na položaju 4 kumarina (3a – h). Korištenje niskotemperaturnog eutektičkog otapala omogućilo je provođenje reakcije u jednom stupnju, bez primjene katalizatora i organskih otapala štetnih za okoliš. Zbog pojednostavljene izolacije i pročišćavanja produkata, skraćeno je vrijeme trajanja postupka u odnosu na konvencionalnu i druge objavljene metode. Derivati kumarinskih 1,2,4-triazola su dobiveni u prinosima od 25 do 91 %, a njihove su strukture potvrđene masenom spektrometrijom te 1H i 13C NMR spektroskopijom. Svim priređenim spojevima računalno je procijenjena sličnost s pesticidima te toksičnost za čovjeka i okoliš. Utvrđeno je kako većina spojeva ima povoljan toksikološki profil u odnosu na komercijalne triazolne pesticide. Antifungalno djelovanje in vitro ispitano je na četiri fitopatogene gljive, a antibakterijsko djelovanje na dvije fitopatogene i dvije zemljišno-korisne bakterije. Kumarinski 1,2,4-triazoli uspješno su inhibirali rast micelija gljiva, s izraženim antifungalnim djelovanjem druge serije derivata na Sclerotinia sclerotiorum i Fusarium oxysporum. Sintetizirani spojevi nisu imali antibakterijski učinak ni na patogene ni na zemljišno-korisne bakterije. Na temelju rezultata antifungalnog djelovanja provedena je analiza kvantitativnog odnosa strukture i aktivnosti (QSAR) spojeva. Model za djelovanje na S. sclerotiorum razvijen je pomoću tri deskriptora (nR=Ct, RDF095m i HATS1e) i može objasniti 79 % inhibitornog djelovanja kumarinskih 1,2,4-triazola. Model za djelovanje na F. oxysporum razvijen je pomoću četiri deskriptora (R4u+, nAROR, RDF080e i Mor11u), a može objasniti 77 % inhibitornog djelovanja spojeva. Pouzdanost razvijenih modela potvrđena je metodama unutarnje i vanjske procjene valjanosti. Potencijalni mehanizam antifungalnog djelovanja istražen je metodom molekulskog uklapanja spojeva u enzime važne za rast micelija (sterol 14α-demetilazu, hitinaze A i B, N-miristoil transferazu) te enzime potrebne za razaranje stanične stijenke domaćina (proteinazu K, endoglukanazu I te endopoligalakturonaze I i II). Spojevi su se dobro uklopili u aktivna mjesta demetilaze, hitinaza A i B, proteinaze K te endopoligalakturonaza I i II. Unatoč dobrim energijama uklapanja, spojevi nisu uspjeli ostvariti interakcije u aktivnim mjestima N-miristoiltransferaze i endoglukanaze. Iz dobivenih rezultata može se zaključiti kako su derivati druge serije kumarinskih triazola potencijalni inhibitori demetilaze, obje hitinaze ili proteinaze K, dok su derivati prve serije spojeva inhibitori endopoligalakturonaza. Spoj 2j ističe se svojim antifungalnim djelovanjem i procjenjenom niskom toksičnosti, što ga čini dobrim kandidatom za razvoj novog aktivnog sastojka sredstava za zaštitu bilja.In this doctoral thesis, coumarin 1,2,4-triazoles with potential multiple biological effects related to application in plant protection were synthesized. The synthesis was carried out as a one-pot reaction of coumarin hydrazides (1 – 3) and differently substituted isothiocyanates in a deep eutectic solvent, choline chloride : urea (molar ratio 1 : 2), by stirring with a magnetic stirrer at 80 °C. The result is three series of compounds: the first in which the 1,2,4-triazole ring is connected by a -CH2- linker at position 4 and the hydroxyl group is at position 7 of the coumarin (1a – k), and the second series of derivatives with a 1,2,4-triazole ring connected by an ether linker at position 7 of the coumarin and a methyl group at position 4 of the coumarin (2a – o). The third, additional series of derivatives, differs from the second by the absence of a methyl group at position 4 of coumarin (3a – h). The use of a deep eutectic solvent enabled to carry out the reaction in one step without the need to use catalysts and environmentally harmful organic solvents. The simplified isolation and purification of the products shortened the duration of the process compared to conventional and other published methods. The derivatives of coumarin 1,2,4-triazoles were obtained in yields of 25 to 91 %, and their structures were confirmed by mass spectrometry and 1H and 13C NMR spectroscopy. All prepared compounds were evaluated in silico for their similarity to pesticides and their toxicity to humans and the environment. Most of the compounds were found to have a favorable toxicological profile compared to commercial triazole pesticides. The antifungal activity was tested in vitro on four phytopathogenic fungi, and the antibacterial activity on two phytopathogenic and two soil-beneficial bacteria. Coumarin 1,2,4-triazoles successfully inhibited the growth of fungal mycelia, with the second series of derivatives showing pronounced antifungal activity on Sclerotinia sclerotiorum and Fusarium oxysporum. The synthesized compounds did not show antibacterial activity, neither on the pathogens nor on the soil-beneficial bacteria. Based on the results of antifungal activity, a quantitative structure-activity relationship (QSAR) analysis of the compounds was performed. The model for the effect on S. sclerotiorum was developed using three descriptors (nR=Ct, RDF095m and HATS1e) and can explain 79 % of the inhibitory effect of coumarin 1,2,4-triazoles. The model for the effect on F. oxysporum was developed using four descriptors (R4u+, nAROR, RDF080e and Mor11u) and can explain 77 % of the inhibitory effect of the compounds. The reliability of the developed models was confirmed by internal and external validation methods. The potential mechanism of antifungal activity was investigated by means of molecular docking of the compounds to the enzymes important for mycelial growth (sterol 14α-demethylase, chitinases A and B, N-myristoyltransferase) and enzymes necessary for host cell wall destruction (proteinase K, endoglucanase I and endopolygalacturonase I and II). The compounds fit well into the active sites of demethylase, chitinases A and B, proteinase K, and endopolygalacturonase I and II. Despite good fitting energies, the compounds failed to achieve interactions in the active sites of N-myristoyltransferase and endoglucanase. From the obtained results, it can be concluded that the derivatives of the second series of coumarin triazoles are potential inhibitors of demethylase, chitinases or proteinase K, while the derivatives of the first series of compounds are inhibitors of endopolygalacturonases. Compound 2j stands out for its antifungal activity and its estimated low toxicity, which emphasized it as a good candidate for the development of a new active ingredient for plant protection products
Development and validation of UHPLC method for purity determination of active pharmaceutical substance of an antirheumatic drug
No full textKvaliteta i sigurnost lijekova ovise o preciznoj kontroli njihovih sastavnica, osobito farmaceutika. Antireumatski lijekovi koji se koriste u terapiji reumatoidnog artritisa zahtijevaju razvoj analitičkih metoda za pouzdano određivanje njihove čistoće i prisutnosti poznatih onečišćenja. U cilju osiguranja kvalitete i sigurnosti primjene lijeka, nužno je razviti selektivnu i osjetljivu analitičku metodu za pouzdano određivanje poznatih onečišćenja prisutnih u farmaceutiku. U ovom radu razvijena je i validirana UHPLC metoda za određivanje čistoće farmaceutika. Validacija je provedena u skladu s ICH smjernicama i obuhvatila je ispitivanje specifičnosti, linearnosti, preciznosti (ponovljivosti), točnosti, granice detekcije, granice kvantifikacije, radnog područja, stabilnosti otopina te robusnosti. Rezultati su pokazali da je metoda pouzdana, osjetljiva i prikladna za rutinsku primjenu u kontroli kvalitete djelatne tvari.The quality and safety of drugs depend on precise control of their components, particularly the pharmaceutical substances. Antirheumatic drugs used in the treatment of rheumatoid arthritis require the development of analytical methods for the reliable determination of their purity and the presence of known impurities. To ensure drug quality and safety, it is essential to develop a selective and sensitive analytical method for the reliable determination of known impurities present in the pharmaceutical substance. In this study, a UHPLC method was developed and validated for the determination of the pharmaceutical's purity. The validation has been conducted in accordance with ICH guidelines and included evaluation of specificity, linearity, precision (repeatability), accuracy, detection limit, quantification limit, working range, solution stability, and robustness. The results demonstrated that the method is reliable, sensitive, and suitable for routine application in quality control of the active pharmaceutical ingredient
Synthesis of new isoxazole and amidoxime purine bioisosteres with antitrypanosomal activity
No full textU ovom radu opisana je sinteza novog amidoksim-supstituiranog derivata benzotiazola 5 te novih hibrida benzimidazola i izoksazola 19a–e. Derivat 5 dobiven je višestupanjskom sintezom iz 4-aminobenzonitrila. Utvrđeno je da derivat 5 pokazuje antitripanosomsku aktivnost s IC50 vrijednosti od 0.92 μM, uz indeks selektivnosti 18. Zbog izražene biološke aktivnosti i visoke selektivnosti, derivat je korišten kao ligand za sintezu metalnih kompleksa renija(I) i mangana(I). N-propargilirani derivat benzimidazola 12 dobiven je reakcijom alkiliranja dok su odgovarajući oksimi 18a–e, potrebni za sintezu izoksazolnih derivata priređeni reakcijom kloriranja odgovarajućih aldoksima. Ciljani derivati 19a–e priređeni su bazno-kataliziranom 1,3-dipolarnom cikloadicijom iz N-propargiliranog derivata 12 i kloriranih oksima 18a–e. Strukture novosintetiziranih spojeva potvrđene su spektroskopijom 1H i 13C NMR. Naknadno će se provesti antitripanosomska ispitivanja ostalih novosintetiziranih spojeva.In this work, the synthesis of novel amidoxime derivatives of benzothiazole 5 and novel benzimidazole derivatives linked via an isoxazole spacer (19a–e) is described. Derivative 5 was obtained from 4-aminobenzothiazole by a multi-step synthesis. Derivative 5 was evaluated for its antitrypanosomal activity and gave an IC50 value of 0.92 μM against T. brucei, with a selectivity index of 18. Due to its significant biological activity and high selectivity, the derivative was used as a ligand for the synthesis of rhenium(I) and manganese(I) metal complexes. N-Propargylated benzimidazole derivative 12 was prepared by alkylation reaction. The corresponding oximes 18a–e for the synthesis of isoxazole derivatives were synthesized by the chlorination reaction of corresponding aldoximes.
The targer isoxazole derivatives 19a–e were prepared by base-catalyzed 1,3-dipolar cycloaddition of N-propargylated benzimidazole derivative 12 and chlorinated oximes 18a–e. The structures of all compounds were determined by 1H NMR and 13C NMR spectroscopy. Antitrypanosomal evaluations of these compounds will be performed
Synthesis and structural characterization of new quinoline-isoxazole conjugates
No full textCilj ovog rada bio je sintetizirati nove konjugate kinolina i izoksazola koji imaju potencijalno antitumorsko djelovanje. U tu svrhu, iz anilina te β-ketoestera pripremljen je 4-hidroksi-kinolin (1) koji je reakcijom propargiliranja preveden u O-propargilirani derivat (2). U sljedećem stupnju provedeno je kloriranje aldoksima (3−6). Reakcijom 1,3-dipolarne cikloadicije O-propargiliranog kinolina (2) i kloriranog aldoksima (7−11) dobivena su četiri konjugata kinolina i izoksazola (12−15). Strukture novosintetiziranih konjugata potvrđene su spektroskopijom 1H i 13C NMR. Biološka ispitivanja pripremljenih spojeva su u tijeku.The aim of this work was to synthesize new quinoline and isoxazole conjugates with potential antitumor activity. For this purpose, 4-hydroxy quinoline (1) was prepared from aniline and β-ketoester, which was converted into O-propargylated quinoline (2) by a propargylation reaction. Chlorinated aldoxime (7−11) were prepared by the chlorination reaction of the corresponding aldoxime (3−6). Four conjugates of quinoline and isoxazole (12−15) were obtained by the 1,3-dipolar cycloaddition reaction of O-propargylated quinoline (2) and chlorinated aldoxime (7−11). The structures of the newly synthesized conjugates were determined by 1H and 13C NMR spectroscopy. Biological evalutions of the prepared compounds are in progress
Potential of environmental bacterial culture for the biodegradation of xenobiotics
No full textSvijet se sve više urbanizira. Rezultat sve veće urbanizacije je rast raznih industrija, poput farmaceutske. Upravo ovakvi spojevi koji se prirodno ne nalaze u okolišu, ali zbog antropogenih aktivnosti dospijevaju u njega, nazivaju se ksenobiotici. Potencijal okolišnih bakterijskih kultura za biorazgradnju ksenobiotika obećavajući je pristup u rješavanju nastalih ekoloških izazova povezanih s postojanošću kemijskih onečišćivača u okolišu. Iskorištavanjem metaboličke aktivnosti bakterijskih kultura iz okoliša, ovaj pristup nudi isplativu i učinkovitu alternativu konvencionalnim postupcima remedijacije i ima potencijal za ublažavanje negativnih učinaka ksenobiotika na zdravlje ljudi i okoliš. U ovom radu procesom biorazgradnje provedeno je uklanjanje farmaceutika amoksicilina pomoću bakterijske kulture Pseudomonas aeruginosa izolirane iz okoliša. Pokus je proveden u šaržnom reaktoru i aerobnim uvjetima u periodu od 12 dana s početnim koncentracijama amoksicilina od
278 mg L^-1 (Pa-1) i 658 mg L^-1 (Pa-2). Prema provedenim testovima osjetljivosti
P. aeruginosa je pokazala otpornost na ispitivani ksenobiotik i nastale produkte.
Rezultati istraživanja pokazuju učinkovitost procesa od 49,6 % za Pa-1, te 36,2 % za Pa-2.The world is becoming increasingly urbanized. The result of increasing urbanization is the growth of various industries, such as the pharmaceutical industry, that produce various compounds. These compounds, which do not occur naturally in the environment but enter the environment due to anthropogenic activities, are known as xenobiotics. The potential of bacterial cultures in the environment to biodegrade xenobiotics is a promising approach to solving environmental problems related to the persistence of chemicals in the environment. By utilizing the metabolic activity of bacterial cultures in the environment, this approach offers a cost-effective and efficient alternative to remediation methods and has the potential to mitigate the harmful effects of xenobiotics on human health and the environment. In this work, the biodegradation process was used to remove amoxicillin using a bacterial culture of Pseudomonas aeruginosa isolated from the environment. The experiment was carried out in a batch reactor and under aerobic conditions for a period of 12 days with initial amoxicillin concentrations of 278 mg L^-1 (Pa-1) and 658 mg L^-1 (Pa-2). According to the sensitivity tests performed, Pseudomonas aeruginosa showed resistance to the xenobiotics tested and the resulting products. The research results showed a process efficiency of 49.6 % for Pa-1 and 36.2 % for Pa-2
Influence of the synthesis parameters on the morphology and application of materials derived with different drying methods
No full textGelovi, aerogelovi i kserogelovi su tri različita stanja materijala, svako od kojih posjeduje jedinstvena svojstva i primjenu. Aerogel je vrsta posebno poroznog materijala, izuzetno niske gustoće te velike poroznosti. Ističe se po svojim iznimnim izolacijskim svojstvima te velikim specifičnim površinama pa se njegova primjena očituje u području optoelektronike, zrakoplovne industrije, svemirske opreme te u mnogim drugim područjima. Celuloza, prirodni biopolimer koji se nalazi u stjenkama biljnih stanica, ističe se kao izvrstan materijal za sintezu aerogelova. Zbog svoje obnovljivosti, biorazgradivosti te biokompatibilnosti celulozni aerogelovi privlače sve više pažnje, pogotovo u području biomedicine gdje se očituje njihov značajan potencijal. Ovaj diplomski rad daje pregled sinteze i karakterizacije kserogelova i aerogelova na bazi celuloze modificiranih ureom, natrijevim kloridom i magnezijevim kloridom. Karakterizacija ovih modificiranih materijala provedena je raznim analitičnim tehnikama, uključujući pretražnu elektronsku mikroskopiju (SEM), energijski disperzivnu spektrometriju (EDS) te rendgensku difrakcijsku analizu (XRD). Primarni cilj je bio istražiti utjecaj ovih modifikacija kao i metode pripreme gela na veličinu pora, poroznost i skupljanje volumena kako bi se dobio uvid u njihovu potencijalnu primjenu u različitim područjima.Gels, aerogels, and xerogels are three distinct states of materials, each with unique properties and applications. Aerogel is a type of particularly porous material, with extremely low density and high porosity. It stands out for its exceptional insulating properties and high specific surface area, so its application is evident in the field of optoelectronics, the aviation industry, space equipment and many other areas. Cellulose, a natural biopolymer found in plant cell walls, is an excellent material for aerogel production. Due to their renewability, biodegradability and biocompatibility, cellulose aerogels are attracting more attention, especially in the field of biomedicine, where they have a significant potential. This thesis provides an overview of the synthesis and characterization of cellulose-based xerogels and aerogels modified with urea, sodium chloride and magnesium chloride. Characterization of these modified materials was performed using various analytical techniques, including scanning electron microscopy (SEM), energy dispersive spectrometry (EDS), and X-ray diffraction analysis (XRD). The primary objective was to investigate the influence of these modifications as well as the gel preparation method on pore size, porosity and volume shrinkage in order to gain insight into their potential application in different fields