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: 30.06.2016

 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 (B_R) 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

 Partner P1:

University “Babes-Bolyai” Cluj Napoca

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

Members: Prof. Dr. Eng. Florin Dan Irimie

                   Assoc. Prof. Dr. Eng. Csaba Paizs

                   Dr. Eng. Csaba Bencze

                    Botond Nagy

                    David Bedo

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

                   Eng. Ioana Brăzdău

 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

 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

RESULTS

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

SCIENTIFIC REPORT ON STAGE I