JOINT APPLIED RESEARCH PROJECT, PARTNERSHIP PROGRAMME (PCCA) founded by

NATIONAL RESEARCH COUNCIL- EXECUTIVE AGENCY FOR HIGHER EDUCATION, RESEARCH, DEVELOPMENT AND INNOVATION FUNDING OF ROMANIA (CNDI– UEFISCDI)

BIOCATALYST- CLICK CHEMISTRY DOWNSTREAMING TANDEM BASED INNOVATIVE KIT FOR OPTICALLY PURE FINE CHEMICALS SYNTHESIS

KIT INOVATIV ÎN SINTEZA COMPUŞILOR CHIMICI OPTIC PURI PRIN COMBINAREA BIOCATALIZEI CU PROCESAREA PRIN REACŢII TIP CLICK

CODE: PN-II-PT-PCCA-2013-4-0734, contract 206/2014

Acronym: BIOCLICK

Project coordinator: Politehnica University of Timişoara

Project director: Prof. Dr. Eng. Francisc PETER

Budget:

 - public budget: 1.250.000 RON

 - cofinnancing: 187.500 RON

Start time: 01.07.2014

End time: 30.09.2017

ABSTRACT:

Biocatalysis is an important tool to implement new, efficient, selective, cost effective and greener technologies, defining a new strategy in the industry of the future. For industrial applications, the stability and reusability of the biocatalysts are important requirements which can be achieved by immobilization, improving also their activity and selectivity. Optimization of the biocatalytic function, as well as the biocatalytic process design became essential topics in industrial biotechnology.

In the forthcoming period, a strong impact of industrial biotechnology can be expected in the fine chemicals sector. As lipases demonstrated the highest application capability among industrial enzymes, the efforts to improve their operational stability and catalytic efficiency led to a remarkable development of the immobilization methods. Certainly, the manufacturing of high value optically active compounds represents the main large-scale process where biocatalysis with lipases will replace the presently employed procedures. Enzymatic kinetic resolution (EKR) of the racemic mixtures represents the most efficient way to obtain high optical purity compounds. However, in large scale EKR an important challenge remains the isolation and purification of the products, which generally involves expensive and laborious physical procedures, decreasing the global process yields and the optical purities of the isolated compounds.

To the best of our knowledge the use of click chemistry involving large carriers, as a tool for easy EKR product separation is still unknown and it could be a practical solution for the efficient large scale isolation and purification of the enzymatic resolution products. Performing the click reaction between a preactivated polymer and one of the appropriate functionalized reaction products in the enzyme free reaction mixture obtained by EKR, would circumvent the tedious isolation and purification procedures.

The project main objective is to develop an innovative kit for efficient and cost-effective sequential continuous flow large-scale (multigram) preparation of optically pure chiral building blocks useful for synthesis of pharmaceutical compounds and agricultural chemicals, based on the tailor-made immobilized lipases mediated kinetic resolution of various racemic substrates and a subsequent click chemistry like efficient downstreaming of the reaction mixture. Such an innovative approach of coupling kinetic resolution of a broad range of racemic substrates with click chemistry type downstreaming was not yet carried out. One of the main technical barriers of large-scale utilization of biocatalysts is the lack of generic catalysts and procedures, as usually an immobilized biocatalyst is designed only for a certain application. Implementation of this project will allow surmounting this bottleneck by developing a solid-phase catalytic block with high activity and selectivity, needing only fine tuning of the process parameters, together with a downstreaming procedure and click chemistry carrier that will allow the efficient separation of the optically active product. The immobilized biocatalysts can be used in continuous flow bioreactors and under harsher reaction conditions.

The use of immobilized biocatalysts-click chemistry doawnstream process tandem will permit to design easily scaled-up continuous flow procedures for industrial manufacturing of the target compounds: (hetero)aril-ethanols, amines, hydroxy- and amino acids, underlining the economic relevance of the project proposal. Our products will significantly decrease the production costs of newly developed enantiopure building blocks due to the achievable high enantiomer selectivity (E) values and yields.

Performing the click reaction between a preactivated polymer and one of the properly functionalized reaction product (BR) in the enzyme free reaction mixture obtained by EKR, would circumvent the tedious isolation and purification procedures generally involving the use of preparative flash-chromatography.

CONSORTIUM STRUCTURE:

Consortium leader:

Politehnica University of Timisoara

Director: Prof. Dr. Eng. Francisc PETER

 Members:

  • Dr. Eng. Cristina Paul
  • Dr. Eng. Valentin Badea
  • Dr. Eng. Emese Biro
  • Eng. Anamaria Todea
  • Eng. Adinela Cimporescu
  • Eng. Claudiu Marcu
  • Eng. Ioana Brăzdău
  • Eng. Paula Aurelia Borza
  • Eng. Horațiu Moldovan

Partner P1:

University “Babes-Bolyai” Cluj Napoca

Responsible: Assoc. Prof. Dr. Eng. Monica Ioana Toşa

Members:

  • Prof. Dr. Eng. Florin Dan Irimie
  • Prof. Dr. Eng. Csaba Paizs
  • Dr. Eng. Csaba Bencze
  • Botond Nagy
  • David Bedo
  • Elena Mădălina Moisa
  • Timea Anita Hull-Szabo

Partner P2:

Natural INGREDIENTS R&D S.R.L

Responsible: Eng. Petru Antin Bârzan

 Members:

  • Dr. Rosenberg Ladislau   
  • Bârzan Carmen Aurelia
  • Eng. Soaita Silviu
  • Eng. Bârzan Gabriela Aurora
  • David Lucian Comaniciu

Objectives:

The project main objective is to develop an innovative kit for efficient and cost-effective sequential continuous flow large-scale (multigram) preparation of optically pure chiral building blocks useful for synthesis of pharmaceutical compounds and agricultural chemicals, based on the tailor-made immobilized lipases mediated kinetic resolution of various racemic substrates followed by the subsequent click chemistry like efficient reactive down streaming.

1. Preparation of various precursors: (hetero)aryl- ethanols, amines, hydroxy- and amino acids and the synthesis of various propargylic esters as O- and N-acylating agents used in EKR will also be performed.

2. Development of optimal EKR and click-chemistry type down streaming procedures.

3. Immobilization of lipases.

4. Development of the continuous flow procedure

 

STAGES

2014: Stage I

1. Chemical synthesis and analytical scale enantiomeric separation of racemic substrates

2015: Stage II

Development of optimal enzymatic kinetic resolution (EKR) and click-chemistry type down streaming procedures. Tailor-made immobilized lipases with high operational stability

2016: Stage III

Continuous flow procedure for synthesis of enantiopure products using immobilized enzymes

2017: Stage IV

Click-chemistry type down streaming of products with high enantiomeric purity

 WORKPLAN:

Deliverable:

1.  Multi-gram amounts of various racemic compounds and various propargylic esters as acyl donors for the EKR

2.  Enantiomeric separation protocol for previously synthesized racemates, chromatographic protocols for testing the enantioselectivity of the enzymatic reactions

3. Scientific article submitted to an ISI quoted journal

4.  Scientific presentation, published in the abstract book of an international conference 

5.  Experimental protocol of down streaming procedures

6.  Immobilization protocols and analysis procedures for tailor-made immobilized lipases

7.   Integrated EKR-click-chemistry type down streaming procedure

 Dissaminated results:

A. Articles in journals indexed in Web of Science database (ISI)

1. A. Todea, P. Borza, A. Cimporescu, C. Paul, F. Peter, Continuous kinetic resolution of aliphatic and aromatic secondary alcohols by sol-gel entrapped lipases in packed bed bioreactors, Catalysis Today, 2017, https://doi.org/10.1016/j.cattod.2017.02.042.

2. B. Nagy, M. E. Moisӑ, A. Filip, L. C. Bencze, M. I. Toșa: Lipase catalyzed parallel kinetic resolution of ibuprofen, Studia Universitas Babeş-Bolyai - Chemia, 2016, 51(4), 255-266.

3. M. E. Moisă, C. G. Spelmezan, C. Paul, H. J. Bartha-Vári, L. C. Bencze, F. D. Irimie, C. Paizs, F. Péter, M. I. Toșa: Tailored sol-gel immobilized lipase preparates for the enzymatic kinetic resolution of heteroaromatic alcohols in batch and continuous flow systems, 2017, în curs de evaluare la RSC Advances.

4. Adinela Cimporescu, Anamaria Todea, Valentin Badea, Cristina Paul, Francisc Peter, Efficient kinetic resolution of 1,5-dihydroxy-1,2,3,4-tetrahydronaphthalene catalyzed by immobilized Burkholderia cepacia lipase in batch and continuous-flow system, Process Biochemistry, 2016, 51(12), 2076-2083.

5. Diana Aparaschivei, Anamaria Todea, Iulia Păuşescu, Valentin Badea, Mihai Medeleanu, Eugen Şişu, Maria Puiu, Adela Chiriţă-Emandi, Francisc Peter, Synthesis, characterization and enzymatic degradation of copolymers of ε-caprolactone and hydroxy-fatty acids, Pure and Applied Chemistry, 2016, 88 (12), 1191-1201.

6. Cristina Paul, P. Borza, Anca Marcu, Gerlinde Rusu, Mihaela Bîrdeanu, Simona Marc Zarcula, Francisc Péter, Influence of the physico-chemical characteristics of the hybrid matrix on the catalytic properties of sol-gel entrapped Pseudomonas fluorescens lipase, Nanomaterials and Nanotechnology, 2016, 6:0, doi: 10.5772/62194.

7. Emese Biro, Daniel Budugan, Anamaria Todea, Francisc Peter, Szilvia Klebert, Tivadar Feczko, Recyclable solid-phase biocatalyst with improved stability by sol-gel entrapment of β-D-galactosidase, Journal of Molecular Catalysis B: Enzymatic, 2016, 123, 81-90.

B. Articles in journals indexed in international databases (BDI)

1. Cristina Paul, Paula Borza, Francisc Péter, High thermal stability of sol-gel entrapped lipases, Journal of Agroalimentary Processes and Technologies, 2015, 21(2), 173-180; ISSN: 2069-0053 (print), ISSN (online) 2068-9551.

2. Paula Borza, Francisc Péter, Iosif Hulka, Cristina Paul, Long-term exposure stability of sol-gel immobilized lipases in organic solvents, Chem. Bull. "POLITEHNICA" Univ. (Timisoara), 2015, 60(74), 25-30,   ISSN (online) 2069-6310.

3. Adinela Cimporescu, Anamaria Todea, V. Badea, Cristina Paul, Francisc Peter, Enzymatic acylation of 1-(isopropylamino)-3-[4-(2-methoxyethyl)phenoxy]propan-2-ol by sol-gel entrapped lipases, Chem. Bull. "POLITEHNICA" Univ. (Timisoara), 2015, 60(74), 2, 7-12,  ISSN (online) 2069-6310.

C. Papers presented at international conferences

1. M. E. Moisă, L. Poppe, F. D. Irimie, M. I. Toşa,  C. Paizs, Click Reaction Strategy for the Efficient Separation of EKR Products, 13th International Symposium on Biocatalysis and Biotransformation,  Biotrans 2017, 9-13 Iulie, Budapesta, Ungaria.

2. Mădălina Elena Moisă, Csaba Paizs, László Poppe, Florin Dan Irimie, Monica Ioana Toşa: Click Reaction Strategy Applied in the Enzymatic Kinetic Resolution Processes, „Systems Biocatalysis” COST Training School, Siena, Italy, 27th-30th April 2016, poster.

3. Mădălina Elena Moisă, Csaba Paizs, László Poppe, Florin Dan Irimie, Monica Ioana Toşa: Click Reactions for Efficient Enzymatic Kinetic Resolution Processing ,3Ird Symposium „Biotransformations 2016”, Warsaw, Poland, 30th June-2nd July 2016, prezentare orală.

4. Anamaria Todea, Diana Aparaschivei, Carmen G. Boeriu, Francisc Peter, A green chemistry route to novel copolyesters, OR 57, 16th International Conference on Polymers and Organic Chemistry, Hersonissos, Crete, Greece, 13–16 June 2016 Book of Abstracts p 45, prezentare orală.

5. Anamaria Todea, Diana Aparaschivei, Adinela Cimporescu, Valentin Badea, Carmen G. Boeriu, Francisc Peter, Efficient biocatalytic route towards new ε-caprolactone copolyesters using microbial lipases, PS 44, 16th International Conference on Polymers and Organic Chemistry, Hersonissos, Crete, Greece, 13–16 June 2016 Book of Abstracts p 139, poster.

6. Cristina Paul, Paula Borza, Anamaria Todea, Francisc Péter, Sol-gel immobilized biocatalyst for continuous-flow enantioselective acylation, The 17th European Congress on Biotechnology, 3–6 Iulie 2016, Krakow, Poland, Abstracts / New Biotechnology  2016, 33S, S104, P8-32, ISSN 1871-6784, DOI: http://dx.doi.org/10.1016/j.nbt.2016.06.1086, poster.

7. Emese Biro, Gianina Bratiloveanu, Anamaria Todea, Francisc Peter, Synthesis of galacto-oligosaccharides by immobilized galactosidase using alkyl or aryl substituted silane precursors for efficient sol-gel entrapment, The 17th European Congress on Biotechnology, 3–6 Iulie 2016, Krakow, Poland, Abstracts / New Biotechnology  2016, 33S,  S104, P8-30, ISSN 1871-6784, DOI: http://dx.doi.org/10.1016/j.nbt.2016.06.1084,  poster.

7. Valentin Badea, Vasile Bercean, Alina Mihaela Muscoi Mindruleanu, Synthesis and characterization by 2D NMR spectroscopy of a new 1-(1H-4-ethoxycarbonyl-3-methyl-pyrazol-5-yl)-azo-2,7-dihydroxynaphthalene and its cyclized derivatives of 8-hydroxy-2-methyl-naphtho[2,1-e]pyrazolo[5,1-c][1,2,4]triazine, 18th Central and Eastern European NMR Symposium & Bruker Users’ Meeting CEUM, 18-20 September 2016, Sofia, Bulgaria,  p 43, P 15, poster.

8. Cristina Paul, Paula Borza, Francisc Péter, Thermal stability of immobilized biocatalysts by sol-gel techniques, 11th International Conference on Renewable Resources & Biorefineries RRB-11, York, Anglia, 3-5 Iunie 2015, Abstract book, pp. 71-72, http://www.rrbconference.com/rrb-11-program.

9. Francisc Péter, Cristina Paul, E. Biro, Paula Borza, Anamaria Todea, Enhancing activity and selectivity of biocatalysts by tailored sol-gel entrapment, XVIII International Sol-gel Conference, Kyoto, September 6-11, 2015, http://kuchem.kyoto-u.ac.jp/solgel2015/program/

10. Valentin Badea, Adinela Cimporescu, Anamaria Todea, Cristina Paul, Francisc Peter, Stereochemical purity determination by NMR analysis of bioactive precursors  from enzymatic kinetic resolution, European Congress on Magnetic Resonance Euromar 2015, 5-10 Iulie 2015, Praga, Cehia, Abstract book pp. 436-437, http://www.euromar2015.org/programme-abstract-book.htm, ISBN 978-80-210-7890-1.

11. Paula Borza, Cristina Paul, Francisc Péter, Enhanced stability of new sol-gel entrapped biocatalysts in organic solvents, 3rd European Congress of Applied Biotechnology - ECAB3, Nice 27 Septembrie - 1 Octombrie 2015, Abstract book, pp. 923, ISBN: 978-2-910239-82-4, http://www.ecce2015.eu/program/synopsis-program.  

12. Adinela Cimporescu, Anamaria Todea, Cristina Paul, Francisc Péter, Lipase - mediated enantioselective kinetic resolution of 1,5 – dihydroxi – 1,2,3,4 tetrahydro – naphthalene, 3rd  European Congress of Applied Biotechnology - ECAB3, Nice 27 Septembrie - 1 Octombrie 2015, Abstract book, pp. 930, http://www.ecce2015.eu/program/synopsis-program, ISBN: 978-2-910239-82-4.

13. Mădălina Elena Moisă, Csaba Paizs, Florin Dan Irimie, Francisc Péter, Monica Ioana Toşa, Novel immobilized lipases preparations for the heteroaromatic ethanols resolution by EKR, 3rd  European Congress of Applied Biotechnology - ECAB 3, 27 Sept.-1 Oct. 2015, Nice, France, Abstract book pp. 932, http://www.ecce2015.eu/program/synopsis-program, ISBN: 978-2-910239-82-4.  

14. Mădălina Elena Moisă, Csaba Paizs, Florin Dan Irimie, Francisc Péter, Monica Ioana Toşa: Activity and stereoselectivity studies of some novel immobilized lipases preparations on heteroaromatic ethanols and β-hydroxy esters, 15th International Symposium and Summer School on Bioanalysis, 13-18 July 2015, Tg. Mures, România.

15. Cristina Paul, Paula Borza, Francisc Peter, Vinyl silane precursors system for combined sol-gel entrapment and adsorption of Candida antarctica B lipase, 7th International Congress on Biocatalysis, Hamburg 31 August - 4 September 2014.
16. Adinela Cimporescu, Anamaria Todea, Francisc Peter, Stereoselective acylation of metoprolol catalyzed by lipases, 7th International Congress on Biocatalysis, Hamburg 31 August - 4 September 2014.


D. Papers presented at national conferences

1. M. E. Moisă: Sinteza stereoselectivă a unor alcooli secundari heteroaromatici prin rezoluţie cinetică enzimatică în sistem continuu, Conferinţa interdisciplinară a doctoranzilor Universităţii Babeş-Bolyai, 23-25 Iunie 2017, Baru-Mare, România - prezentare orală.


2. Mădălina Elena Moisă, Csaba Paizs, Florin Dan Irimie, Gábor Hornyánszkiy, Monica IoanaToşa: Click chemistry - an efficient tool in biocatalysis, Young Researchers’ International Conference on Chemistry and Chemical Engineering (YRICCCE I), Cluj-Napoca, Romania, 12th-14th May 2016, poster.

3. Botond Nagy, Zsolt Galla, Csaba Paizs, Enikő Forró, Ferenc Fülöp: Synthesis and enzymatic kinetic resolution of novel 3-amino-3-(5-phenylfuran-2-yl)propanoic acid ethyl esters, Young Researchers’ International Conference on Chemistry and Chemical Engineering (YRICCCE I), Cluj-Napoca, Romania, 12th-14th May 2016, poster.

4. Mădălina Elena Moisă, Csaba Paizs, Florin Dan Irimie, Francisc Péter, Monica Ioana Toşa: Studii de activitate, selectivitate şi stabilitate ale unor preparate noi de lipaze imobilizate pentru ekr a unor etanoli heteroaromatici, ”QUALITY AND EXCELLENCE IN DOCTORAL RESEARCH” Conference organised in the frame of POSDRU/187/1.5/S/155383 project, Băile Felix, Romania, 16th-20th December 2015, prezentare orală.

5. Gianina Bratiloveanu, Francisc Péter, Emese Biró, Anamaria Todea, Improvement of β-galactosidase properties by immobilization in sol-gel matrices using substituted silane precursors, The 9th edition of the Symposium with International Participation New trends and strategies in the chemistry of advanced materials with relevance in biological systems, technique and environmental protection, Timișoara, România, 9-10th June 2016, P6, poster.

6. Francisc Peter, Anamaria Todea, Diana Aparaschivei, Valentin Badea, Carmen G. Boeriu, Lipases: powerful enzymes for biomaterials synthesis starting from renewable sources, XXXIVth Romanian Chemistry Conference, Călimăneşti-Căciulata, România, October 4-7, 2016, P 1, C.S.IV.-1, prezentare orală.

7. Anamaria Todea Francisc Peter, Linda G. Otten, Isabel W.C.E. Arends, Carmen G. Boeriu, One-pot system for synthesis of estolides by using a combination of two enzymes, XXXIVth Romanian Chemistry Conference, Călimăneşti-Căciulata, October 4-7, 2016, P 17, C.S.I. - 17, prezentare orală.

8. Valentin Badea, Vasile Nicolae Bercean, Alina Mihaela Muscoi Mîndruleanu, Ana Maria Todea, Synthesis of azaheterocyclic compounds or their precursors and 1H-15N 2D NMR spectroscopy as a useful tool for structure determination, XXXIVth Romanian Chemistry Conference, Călimăneşti-Căciulata, October 4-7, 2016, P 10, CP 10, conferinta plenară.

9. Paula Borza, Adinela Cimporescu, Anamaria Todea, Cristina Paul, Francisc Peter,  Continuous kinetic resolution of racemic aromatic alcohols in packed bed bioreactors, XXXIVth Romanian Chemistry Conference, Călimăneşti-Căciulata, October 4-7, 2016, P.S.I.-35, CP 10, poster.

10. Diana Aparaschivei, Anamaria Todea, Iulia Păușescu, Valentin Badea, Mihai Medeleanu, Eugen Șișu, Maria Puiu, Adela Chiriță-Emandi, Francisc Peter, Enzymatic degradation of new copolymers synthesized from e-caprolactone and hydroxy-fatty acids, The XVIIIth Symposium Young People and Multidisciplinary Research, Timișoara, Romania, November 10-11, 2016, p 22, B2, prezentare orală.

1. Francisc Peter, Cristina Paul, Emese Biró, Anamaria Todea, Enzyme immobilization, a possible way towards industrial biocatalysts, The XXXIII-rd Romanian Chemistry Conference, Călimăneşti-Căciulata 1-3 October 2014 (conferinţă în secţiunea I).
2. Cristina Paul, Paula Borza, Francisc Peter, Kinetic resolution of secondary alcohols by a  nanoscale biocatalyst system using Pseudomonas fluorescens lipase, The XXXIII-rd Romanian Chemistry Conference, Călimăneşti-Căciulata 1-3 October 2014 (poster).

 

RESULTS

2014: Stage 1. Chemical synthesis and analytical scale enantiomeric separation of racemic substrates

SCIENTIFIC REPORT ON STAGE I

2015: Stage 2. Development and optimization of the enzymatic kinetic resolution and of an efficient enzyme immobilization procedure

SCIENTIFIC REPORT ON STAGE II

2016: Stage 3. Continuous flow procedure for synthesis of enantiopure products using immobilized enzymes

SCIENTIFIC REPORT ON STAGE III

2017: Stage 4. Click-chemistry type down streaming of products with high enantiomeric purity

SCIENTIFIC REPORT ON STAGE IV