PN-III-P2-2.1-PED-2019-3414
Funded by a grant of the Romanian Ministry of
Education and Research, CCCDI - UEFISCDI, project number PN-III-P2-
2.1-PED-2019-3414, within PNCDI III, contract number 367PED
PROJECT CODE: PN-III-P2-2.1-PED-2019-3414, contract number 367PED ⁄ 2020
PROJECT TITLE:
Continuous-flow system bioreactor for the enzymatic kinetic resolution of novel chiral secondary heterocyclic alcohols
Bioreactor pentru rezoluția cinetică enzimatică a unor noi alcooli heterociclici secundari chirali in sistem continuu
ACRONYM: biorezol
PROJECT COORDINATOR: Politehnica University Timişoara
PROJECT DIRECTOR: Lecturer Dr. Eng. Valentin BADEA
PROJECT LAYOUT
Resolution (chiral separation) is an essential undertaking throughout discovery and development of biological active substances, with enantiomeric forms often possessing different biological effects. A comparison between chemical and enzymatic catalysis shows that in many cases biocatalysts are far superior in the production of chiral compounds due to their stereo-, regio- and chemoselectivity
The aim of this project is to develop, at laboratory scale, a continuous-flow system bioreactor for quantitative kinetic enzymatic resolution of racemic mixtures of novel chiral secondary heterocyclic alcohols, with potential biological activity, which will be synthesized within this project. After the synthesis of the new secondary heterocyclic alcohols (R,S)-1-(aryl/methyl)-2-[(4,5-diaryl-4H-1,2,4-triazol-3-yl)thio]ethan-1-ols as racemate, optimal conditions of enzymatic kinetic resolution will be established for each substrate, using selected microbial lipases and various reaction media. The realization of the enzymatic kinetic resolution will be done using a continuous flow bioreactor followed by the isolation and purification of the products with high enantiomeric purity. The realization of the enzymatic kinetic resolution will be done using a continuous flow bioreactor followed by the isolation and purification of the products with high enantiomeric purity. The validation of the experimental system will be accomplished through the effective obtaining of the enantiomers, whose biological activity, after assigning their absolute configuration, will be evaluated and compared with that of the racemic mixture where they come from.
PROJECT TEAM:
Project leader: Lecturer Dr. Eng. Valentin Badea
Reasearchers:
Professor Dr. Eng. Francisc Peter
Lecturer Dr. Eng. Anamaria Todea
Lecturer Dr. Eng. Iulia Maria Păușescu
Lecturer Dr. Eng. Valentin Laurențiu Ordodi
Teaching Assistant Dr. Chem. Diana Maria Aparaschivei
Research Assistant Eng. Ionuț Bîtcan
OBJECTIVE: The laboratory scale development of quantitative continuous-flow system bioreactor for the enzymatic kinetic resolution of novel chiral secondary heterocyclic alcohols racemate with potential biological activity which will be synthesized within this project, as well as choosing the enzymes with the best catalytic efficiency for this specific kinetic resolution.
The major cause of environmental pollution is due to emissions generated by burning of fossil fuels. The known crude oil reserves are going to disappear in short time and the oil crisis in recent years, together with the rising of air pollution levels has shown the need for the replacement of fossil fuels with cleaner biofuels, obtained by processing a range of organic renewable raw materials. The first step in conversion of lignocellulosic biomass to bioethanol is the pretreatment for the release of cellulose from the network formed with lignin and to increase the yield of fermentable sugars. There are many methods of pretreatment, but they are energy consumable and pollute the environment.
In this sense the project proposes an innovative approach on studies regarding biomass pretreatment and enzymatic hydrolysis of cellulose in an integrated system that can improve the exploitation of biomass components and the reuse of the biocatalyst. It is desired to provide novel biocatalysts, immobilized cellulases customized by new sol-gel entrapment techniques, used in the hydrolysis of certain types of lignocellulosic biomass. By immobilization, stability and reusability of cellulases are significantly improved, a key issue for increasing the amount of fermentable sugars and to reduce process costs.
STAGES
Stage 1 (2020, 2 months)- Synthesis, purification and spectroscopic characterization of intermediates used to obtain new chiral heterocyclic secondary alcohols.
Stage 2 (2021, 12 months)- Synthesis, purification and spectroscopic characterization of new heterocyclic secondary alcohols. Development of the enzymatic kinetic resolution process and elaboration of a continuous process for obtaining enantiomerically pure compounds using immobilized enzymes.
Stage 3 (2022, 10 months)- Assigning the absolute configuration by NMR spectroscopy and testing the biological activity for heterocyclic secondary alcohols.
DELIVERABLES:
Stage 1
Experimental synthesis protocol for N-(aryl)hydrazinecarbothioamide in gram amounts and spectroscopic characterization;
Experimental synthesis protocol for benzoyl(acyl)-N-arylhydrazine-1-carbothioamide in gram amounts and spectroscopic characterization;
Experimental synthesis protocol for 4-aryl-5-phenyl(alkyl)-4H-1,2,4-triazol-3-thiol in gram amounts and spectroscopic characterization;
Experimental synthesis protocol for 1-(aryl)-2-[(4-aryl-5-aryl(alkyl)-4H-1,2,4-triazol-3-yl)thio]ethan-1-one in gram amounts and spectroscopic characterization.
Stage 1 report.
Stage 2
Experimental synthesis protocol for N-(aryl)hydrazinecarbothioamide in gram amounts and spectroscopic characterization;
Experimental synthesis protocol for benzoyl(acyl)-N-arylhydrazine-1-carbothioamide in gram amounts and spectroscopic characterization;
Experimental synthesis protocol for 4-aryl-5-phenyl(alkyl)-4H-1,2,4-triazol-3-thiol in gram amounts and spectroscopic characterization;
Experimental synthesis protocol for 1-(aryl)-2-[(4-aryl-5-aryl(alkyl)-4H-1,2,4-triazol-3-yl)thio]ethan-1-one in gram amounts and spectroscopic characterization;
Experimental synthesis protocol for (R,S)-1-(aryl/methyl)-2-[(4-aryl-5-aryl(alkyl)-4H-1,2,4-triazol-3-yl) thio]ethan-1-ols in gram quantities and spectroscopic characterization;
Experimental protocol for enzymatic kinetic resolution;
Quantities of the order of grams of the immobilized enzyme with the highest activity;
Immobilized enzyme data sheet with optimal characteristics;
Laboratory methodology for the resolution of enzymatic kinetics in a continuous flow bioreactor;
Obtaining a heterocyclic secondary alcohol in gram amounts;
A paper submitted for publication in a journal;
Abstracts for two presentations at scientific conferences in the field.
Stage 2 report.
Stage 3
Obtaining a heterocyclic secondary alcohol in gram amounts;
Experimental study for assigning the absolute configuration;
Experimental protocol for assigning the absolute configuration;
Experimental protocol for the evaluation of biological activity;
A paper submitted for publication in a journal;
Stage 3 report
Final report.
RESULTS
Stage 1 (2020, 2 months) - Synthesis, purification and spectroscopic characterization of intermediates used to obtain new chiral heterocyclic secondary alcohols.
Abstract stage 1 report
A number of four compounds have been synthesized in good yields ranging from 53-87% after purification by recrystallization, the last one is not mentioned in the scientific literature. All compounds were preliminarily characterized by melting point and thin layer chromatography (TLC) and then by IR, 1H-NMR, 13C-NMR and 15N-NMR spectroscopy.
The synthesized compounds are: N-(4-methylphenyl)hydrazinecarbotioamide, 2-benzoyl-N-(4-methylphenyl)hydrazin-1-carbotioamide, 5-phenyl-4-(4-methylphenyl)-4H-1,2,4-triazole-3-thiol and 2-{[4-(4-methylphenyl)-5-phenyl-4H-1,2,4-triazol-3-yl]thio}-1-phenyl-1-ethanone.
All the objectives of this stage were fulfilled.
Stage 2(2021, 12 months) - Synthesis, purification and spectroscopic characterization of new heterocyclic secondary alcohols. Development of the enzymatic kinetic resolution process and elaboration of a continuous process for obtaining enantiomerically pure compounds using immobilized enzymes.
Abstract stage 2 report
Three N-(aryl)hydrazinocarbothioamides were synthesized: N-(4-bromophenyl)hydrazincarbothioamide, N-(3-methoxyphenyl)hydrazincarbothioamide, N-(4-methoxyphenyl)hydrazincarbothioamide.
Three 2-benzoyl(acyl)-N-arylhydrazin-1-carbotioamide were synthesized: 2-benzoyl-N-(4-bromophenyl)hydrazin-1-carbotioamide, 2-benzoyl-N-(3-methoxyphenyl)hydrazin-1-carbotioamide, 2-benzoyl-N-(3-methoxyphenyl)hydrazin-1-carbotioamide.
Three 4-aryl-5-phenyl(alkyl)-4H-1,2,4-triazol-3-thiols were synthesized: 5-phenyl-4-(3-methoxyphenyl)-4H-1,2,4-triazol-3-thiol, 5-phenyl-4-(4-methoxyphenyl)-4H-1,2,4-triazol-3-thiol, 5-phenyl-4-(4-bromophenyl)-4H-1,2,4-triazol-3-thiol.
Three novel 1-(aryl)-2-[(4-aryl-5-aryl(alkyl)-4H-1,2,4-triazol-3-yl)thio]ethan-1-one, not yet mentioned in the scientific literature, have been synthesized: 2-{[4-(3-methoxyphenyl)-5-phenyl-4H-1,2,4-triazol-3-yl]thio}-1-phenylethan-1-one, 2-{[4-(4-methoxyphenyl)-5-phenyl-4H-1,2,4-triazol-3-yl]thio}-1-phenylethan-1-one, 2-{[4-(4-bromophenyl)-5-phenyl-4H-1,2,4-triazol-3-yl]thio}-1-phenylethan-1-one. Four novel chiral heterocyclic (R,S)-1-(aryl/methyl)-2-[(4-aryl-5-aryl(alkyl)-4H-1,2,4-triazol-3-yl)thio]ethan-1-ols, not yet mentioned in the scientific literature, were synthesized: 1-phenyl-2-{[4-(4-methylphenyl)-5-phenyl-4H-1,2,4-triazol-3-yl]thio}ethan-1-ol, 1-phenyl-2-{[4-(3-methoxylphenyl)-5-phenyl-4H-1,2,4-triazol-3-yl]thio}ethan-1-ol, 1-phenyl-2-{[4-(4-methoxylphenyl)-5-phenyl-4H-1,2,4-triazol-3-yl]thio}ethan-1-ol, 1-phenyl-2-{[4-(4-bromophenyl)-5-phenyl-4H-1,2,4-triazol-3-yl]thio}ethan-1-ol.
All compounds were obtained in good yields ranging from 50-80% after purification by recrystallization. All synthesized compounds were preliminarily characterized by melting points, thin layer chromatography (TLC) and then by IR, 1H-NMR, 13C-NMR and 15N-NMR spectroscopy. Methods for the chromatographic separation of aromatic substrates by high-performance liquid chromatography and chiral columns were further developed. Several lipases from different microbial sources were tested in different solvents and subsequently the lipase from Burkholderia cepacia was immobilized by entrapment in sol-gel matrices. The obtained enzyme preparations were successfully used for aromatic racemic substrates. The operational stability of the immobilized enzymes in several reaction cycles was determined. Continuous enzyme kinetic resolution was successfully tested for an aromatic substrate. Good substrate conversions were obtained at the minimum flow rates tested. The biocatalyst used exhibited excellent operational stability and will be used to establish optimal continuous reaction conditions for enzymatic kinetic resolution of synthesized racemic substrates.
The results were disseminated through the publication of 4 ISI scientific articles and 2 posters presented at 2 international conferences.
In conclusion, all the objectives of this stage were fulfilled.
Stage 3 (2022, 10 months)- Assigning the absolute configuration by NMR spectroscopy and testing the biological activity for heterocyclic secondary alcohols.
Abstract stage 3 report
During the third phase, studies initiated in the second phase regarding: (i) establishing the optimal conditions of enzymatic kinetic resolution for different substrates, using selected microbial lipases; (ii) isolation and purification of products with high enantiomeric purity; (iii) selection of the appropriate method for assigning the absolute configuration of the enantiomers of the synthesized secondary heterocyclic alcohols, and (iv) biological activity testing for the secondary heterocyclic alcohols were continued.
Regarding the establishment of optimal conditions for enzymatic kinetic resolution for different substrates, using selected microbial lipases the best separation resolution was achieved in the chromatographic separation of enantiomers of compounds AR7, AR8, AR10, DK8, DK9, and of the reference compound 2,2'-furoin using a Phenomenex Lux® 5µm i-Amylose-3 chiral column, the stationary phase consisting of amylose grafted with units (3-chloro-5-methylphenyl carbamate) units. The enantioselective acylation of the compound AR10 and of 2,2’-furoin catalyzed by immobilized lipases was demonstrated for the first time. The entrapment of lipases in a sol-gel matrix proved effective for the enantioselective conversion of some aromatic substrates for which the selectivity of lipases is generally lower. Among the tested lipases, the highest conversion and enantiomeric excess values were obtained with the lipase from Pseudomonas stutzeri entrapped in sol-gel matrices using the mixture of precursor silanes 3GOPrTMOS:TMOS in a 1:1 molar ratio, where 21,7% of the biocatalyst activity was maintained after 5 cycles of use.
The method chosen for the NMR assignment of the absolute configuration of the synthesized secondary alcohols with a chiral center was esterification with α-methoxy-α-trifluoromethylphenyl acetic acid chloride (2-phenyl-3,3,3-trifluoro-2-methoxypropanoic acid chloride) also called Mosher reagent (MTPA-Cl). The determination of the absolute configuration with Mosher's reagent was developed for the secondary alcohol with chiral center 2-((4-(4-methoxyphenyl)-5-phenyl-4H-1,2,4-triazol-3-yl)thio)-2- propanol which was previously synthesized but is applicable to the entire series of synthesized secondary alcohols.
The dissemination of the results was done by participation with 2 oral presentations and 2 posters at prestigious specialized conferences, organized with physical presence.
Therefore, we consider that the objectives of this stage have been met.
Publications:
- Burcă, I.; Badea, V.; Deleanu, C.; Bercean, V.-N. 5-((8-Hydroxyquinolin-5-yl)diazenyl)-3-methyl-1H-pyrazole-4-carboxylic Acid. Molbank, 2021, 2021 (2), M1238, https://doi.org/10.3390/M1238
- Vorga, M.M.; Badea, V. (±)-2-{[4-(4-Bromophenyl)-5-phenyl-4H-1,2,4-triazol-3-yl]sulfanyl}-1-phenyl-1-ethanol. Molbank, 2021, 2021(3), M1268, https://doi.org/10.3390/M1268
- Valicsek, V.-S.; Badea, V. (R,S)-2-{[4-(4-Methylphenyl)-5-phenyl-4H-1,2,4-triazol-3-yl]thio}-1-phenyl-1-ethanol. Molbank 2021, 2021(3), M1241, https://doi.org/10.3390/M1241
- Wurfer, F.-G.; Badea, V. (R,S)-2-{[4-(4-Methoxyphenyl)-5-phenyl-4H-1,2,4-triazol-3-yl] thio}-1-phenyl-1-ethanol. Molbank 2021, 2021, M1231(2), https://doi.org/10.3390/M1231
Conferences:
- Burcă, I; Badea V. Synthesis and characterization of new racemic secondary alcohol with 5-benzyl-4-(2,4-dimethoxyphenyl)-3-sulfanyl-1,2,4-triazole structure, Young Researchers’ International Conference on Chemistry and Chemical Engineering (YRICCCE III), Cluj-Napoca, 04 – 05 iunie, 2021, poster P8, link: http://www.chem.ubbcluj.ro/~schr/yriccce2020/pdf/program_2021.pdf
- Burcă, I; Valicsek, V.-S; Badea, V; Bercean, V.-N Synthesis and characterization of new racemic secondary alcohol with 5-benzyl-4-(4-methylphenyl)-3-sulfanyl-1,2,4-triazole structure, New trends and strategies in the chemistry of advanced materials with relevance in biological systems, technique and environmental protection (13th edition), Timisoara, 07 – 08 octombrie, 2021, poster nr. 35, link: https://ntcr-2021.ro/docs/ntcr2021-book-of-abstracts.pdf
- Ion Burcă, Valentin Badea, Ioan Bîtcan, Anamaria Todea, Francisc Peter Synthesis, enzymatic kinetic resolution and assigning the absolute configuration by NMR of novel racemic secondary (R,S)-2-[(4,5-diaryl-4h-1,2,4-triazol-3-yl)sulfanyl]-1-aryl ethanols, New trends and strategies in the chemistry of advanced materials with relevance in biological systems, technique and environmental protection, 14th Edition, October 20-21, 2022, Timisoara, Romania, https://www.newtrends-timisoara.ro/docs/Book-of-abstracts-new-trends-2022.pdf (oral presentation).
- Valentin Badea, Ion Burcă, Ioan Bîtcan, Anamaria Todea, Flavius-Gabriel Wurfer, Francisc Peter Synthesis and enantiomeric separation of novel racemic secondary 2-[(4,5-diaryl-4H-1,2,4-triazol-3-yl)sulfanyl]-1-aryl ethanols and assigning the absolute configuration by nmr using (R) and (S) Mosher’s chlorides, Conferința Națională de Chimie, ediția XXXVI, October 4-7, 2022, Călimănești-Căciulata, Romania, Book of Abstracts, https://www.schr.ro/administrare/content/doc/evenimente/2022/10/04/1/volum-de-rezumate/doc.pdf (oral presentation, in section conference).
- Ion Burca, Dineș Karina-Bianca, Răzvan-Florin Ardelean, Valentin Badea, Synthesis of new racemic secondary alcohols, 2-((5-(4-methoxyphenyl)-4-aryl-4h-1,2,4-triazol-3-yl)sulfanyl)-1-aryl (alkyl)ethan-1-ol, Conferința Națională de Chimie, ediția XXXVI, October 4-7, 2022, Călimănești-Căciulata, Romania, Book of Abstracts, https://www.schr.ro/administrare/content/doc/evenimente/2022/10/04/1/volum-de-rezumate/doc.pdf (poster)
- Alexandra-Mihaela Diaconescu, Ion Burcă, Valentin Badea Synthesis of new diazen compounds containing the pyrazole and triazole moiety, Conferința Națională de Chimie, ediția XXXVI, October 4-7, 2022, Călimănești-Căciulata, Romania, Book of Abstracts,https://www.schr.ro/administrare/content/doc/evenimente/2022/10/04/1/volum-de-rezumate/doc.pdf (poster)
Prezentarea succintă a rezultatelor obținute în cadrul proiectului
Obiectivul principal al proiectului "Bioreactor pentru rezoluția cinetică enzimatică a unor noi alcooli heterociclici secundari chirali in sistem continuu" a fost realizarea unui model demonstrativ pentru sinteza și separarea pe cale enzimatică în enantiomeri a unor noi alcooli secundari heterociclici aromatici. Utilizarea biocatalizatorilor reprezintă una dintre posibilitățile de a implementa principiile chimiei "verzi" (ecologice) în sinteza organică, aceștia fiind catalizatori biodegradabili obținuți prin cultivarea unor microorganisme, care acționează în condiții de reacție blânde comparativ cu catalizatorii chimici rezultând un consum de energie și grad de poluare a mediului mai reduse. Studiile experimentale au fost realizate în cadrul Centrului de Cercetări în Chimia și Ingineria Compușilor Organici, Macromoleculari și Naturali din Universitatea Politehnica Timișoara.
A fost sintetizată o serie de 10 compuși noi cu structură de alcooli secundari heterociclici aromatici, care au fost caracterizați prin tehnici de Rezonanță Magnetică Nucleară (RMN) și evaluați în vederea separării lor în enantiomeri prin acilare selectivă catalizată de enzime din categoria lipazelor. Asemenea alcooli cu puritate optică ridicată pot avea întrebuințări importante ca intermediari pentru obținerea de compuși cu activitate farmaceutică. S-a determinat că una dintre lipaze, cea obținută din microorganismul Pseudomonas stutzeri, manifestă enantioselectivitate ridicată în reacția studiată și poate fi utilizată pentru obținerea esterilor corespunzători cu puritate optică de 100%. Imobilizarea lipazei prin incluziune într-o matrice de sol-gel permite exploatarea sa în regim continuu, folosind un bioreactor de tip coloană cu strat fix de catalizator. Instalația experimentală realizată a permis stabilirea condițiilor optime pentru un asemenea proces, deschizând calea unor viitoare dezvoltări în acest domeniu. Spectrometria RMN este o metodă versatilă cu ajutorul căreia se poate realiza atribuirea configurației sterice absolute a compușilor chirali de tipul alcoolilor secundari, facilitând în acest mod caracterizarea exhaustivă a acestora.
(a) (b)
Figura 1. Schema procesului de rezoluție cinetică enzimatică folosind enzimă imobilizată (a) și bioreactorul tip coloană utilizat (b)