BUDAPEST UNIVERSITY OF TECHNOLOGY AND ECONOMICS FACULTY OF CHEMICAL AND BIOENGINEERING
GEORGEN OLAH DOCTORAL SCHOOL
A PPLICATION OF CONTINUOUS - FLOW SYSTEMS IN THE SYNTHESIS AND ENZYME CATALYSED RESOLUTIONS OF
RACEMIC ALCOHOLS , AMINES AND AMINO ACIDS
PhD Thesis
Author: Péter Falus Supervisor: Dr. József Nagy Consultant: Dr. László Poppe
DEPARTMENT OF ORGANIC CHEMISTRY AND TECHNOLOGY
2015
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1. Introduction
The economical synthesis of optically active compounds is one of the greatest challenges of the 21st century in organic chemistry. Beside pharmaceutical industry, the synthesis and application of enantioenriched compounds is in the spotlight in pesticide, plastic, cosmetic and food industry.1,2 The presence of the unwanted enantiomer could cause considerable problems due to the fact that although enantiomers have the same physical and chemical properties but different biological effects.3 One of the most infamous cases was the Contergan scandal. (R)-Thalidomide which was used against nausea and to alleviate morning sickness in pregnant women was not separated from its enantiomeric pair, the teratogenic (S)-Thalidomide.4 The reason why enantiomers have different biological effects is often unclear. Contergan was available between 1957 and 1961 and during this period about 12 000 children was born dead or with the malformation of the limbs. However, the teratogenic effect of (S)-Thalidomide was only unfolded recently.5
A modern, environmentally friendly method for synthesise optically active amines, alcohols and amino acids that could be highly valuable building blocks of numerous drugs is enzyme catalysed kinetic resolution in continuous-flow mode. The importance of biocatalysis is indicated by the industrial application of enzymes. For instance, BASF produces more than 3000 tons/year of chiral amines in continuous-flow system employing lipase catalysis.6
In this dissertation the synthesis of racemic amines and kinetic and dynamic kinetic resolutions of racemic amines, alcohols and amino acids were investigated in continuous-flow systems.
1. Reductive amination of ketones in batch and continuous-flow modes. New and generalizable methods were developed for the Zn dust and Pd/C catalysed synthesis of
1 L. Poppe, L. Novák, „Selective Biocatalysis: A Synthetic Approach”, Wiley-VCH, Weinheim, 1992.
2 K. Faber, „Biotransformations in Organic Chemistry (4th edition)”, Springer, Berlin, 2004.
3 R. T. Coutts, G. B. Baker, Chirality, 1989, 1, 99.
4 H. Nishimura, T. Tanimura, „Clinical Aspects of The Teratogenicity of Drugs”, NY: American Elsevier Publishing Company, New York, 1976.
5 T. Ito, H. Ando, T. Suzuki, T. Ogura, K. Hotta, Y. Imamura, Y. Yamaguchi, H. Handa, Science, 2010, 327, 1345.
6 T. C. Nugent, „Chiral Amine Synthesis: Methods, Developments and Applications”, Wiley-VCH, Weinheim, 2010.
3 racemic amines by the modification of the original Leucart7 and Leucart-Wallach8 reactions. The reactions promoted by Pd/C were carried out in continuous-flow system as well.
2. Kinetic resolution of racemic amines in continuous-flow systems. The kinetic resolutions were realized in the N-acetylation reaction of racemic amines with differently immobilized lipase preparations. The temperature dependence of enantiomeric selectivity and productivity were investigated in the continuous-flow mode kinetic resolutions. It was also investigated whether the mode of enzyme immobilization and the quality of the substrate have any effect on the selectivity and productivity.
3. Kinetic resolution of secondary alcohols in continuous-flow systems. Novel and barely known secondary alcohols bearing indole skeleton were synthesised and then their O-acetylations were carried out in the presence of lipase biocatalysts. The temperature dependence of productivity and selectivity were investigated in continuous-flow mode. Furthermore, also in continuous-flow mode, preparative scale O-acetylations were carried out and enantioenriched (R)- and (S)-alcohols and (R)- acetates were obtained in good yields.
4. Dynamic kinetic resolution of racemic N-Boc protected phenylalanine thioethyl eseter in continuous-flow system. The dynamic kinetic resolution was carried out in the presence of protease immobilized onto solid support, benzylamine as amidation agent and 1,8-diazabicyclo[5.4.0]undec-7-ene as base catalyst. First, the temperature optima of the kinetic resolution and the racemization steps was searched, and then the dynamic kinetic resolution was realized in continuous-flow system by combining the two elementary reactions of DKR.
2. Experimental methods
Reactions were analyzed by gas chromatography on an Agilent 5890 equipment using Hydrodex β-TBDAc column (Machery-Nagel; 25 m×0.25 mm×0.25 μm, heptakis-(2,3-di-O-acetyl-6-O-t-butyl-dimethylsilyl)-β-cyclodextrin) and Hydrodex-β- 6TBDM column (25 m×0.25 mm×0.25 μm, heptakis-(2,3-di-O-methyl-6-O-t-
7 R. Leuckart, Berichte der Deutschen Chemischen Gesellschaft, 1885, 18, 2341.
8 O. Wallach, Berichte der Deutschen Chemischen Gesellschaft, 1891, 24, 3992.
4 butyldimethylsilyl)-β-cyclodextrine; Macherey&Nagel) using H2 carrier gas (injector:
250°C, FID detector: 250°C, head pressure: 12 psi, 50:1 split ratio). The samples were taken from directly the reaction mixture and diluted with dichloromethane to the desired concentration (1–2 mg/mL).
Reactions were analyzed by HPLC on a Hewlett Packard 1090 Series II equipment equipped with a Chiralpack® IB column [Daicel, 150×2.1 mm, 5 μm, cellulose tris(3,5- dimethylphenyl)carbamate] using hexane/isopropanol 98/2 (v/v) as eluent at a flow rate of 0.25 mL/min at 25°C and diode array detection (DAAD) at λ=220 nm. The samples were taken from directly the reaction mixture and diluted with hexane/isopropanol 98/2 to the desired concentration (1–1,5 mg/mL).
Continuous-flow kinetic resolutions were performed in a laboratory flow reactor system or in X-CubeTM laboratory flow reactor (X-CubeTM - trademark of ThalesNano, Inc.; Ser. No.: 002/2006). Catalysts were packed into stainless steel CatCartTM columns according to the filling process of ThalesNano Inc. The columns were sealed by silver metal filter membranes (Sterlitech Silver Membrane Filter from Sigma–Aldrich, Z623237, pore size 0.45 μm; pure metallic silver, 99.97% with no extractable or detectable contaminants) due to the known benefits of Ag (bacteriostatic). The sealings were made of PTFE.
3. New scientific results
3.1. One-step reductive amination of ketones
The two-step production of oximes from ketones and their reductive transformation into amines are well known. First, these two steps were combined in a one pot reaction with ammonium formate via oxime intermediate. It was revealed during the optimization process that the reaction proceeds in one step, in the absence of hydroxylamine hydrochloride. Different catalyst and temperature should be applied in the reaction depending on the position of the carbonyl functional group of ketones. Zn dust promoted reactions and high temperature gave amines from ketones with a carbonyl group at the benzylic position of an aromatic side chain, and 10% Pd/C catalysis and room temperature were more suitable for the conversion of aliphatic and cycloaliphatic ketones into the corresponding amines (Scheme 1).
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Scheme 1. Reductive amination of ketones
Thus, a novel and easily generalizable method was developed for the one step and one pot reductive amination of ketones.
3.2. Reductive amination of ketones in continuous-flow system
In those cases when 10% Pd/C was applied for reductive amination the reaction could be realized in continuous-flow mode as well (1a-d). The reaction mixture – the corresponding ketone and ammonium formate dissolved in methanol – was pumped through the 10% Pd/C filled column termostated at 40°C and the desired amine was gained in good yield (Scheme 2.).
Scheme 2. Reductive amination of ketones in continuous-flow system
This is the first realization of one step reductive amination of aliphatic and cycloaliphatic ketones in the presence of ammonium formate in concinuous-flow mode.
3.3. Lipase catalysed kinetic resolution of racemic amines and secondary alcohols
The N- and O-acetylations with ethyl acetate and vinyl acetate of racemic amines (rac-2a, c, e, g) and secondary alcohols (rac-5a-c) employing different lipase catalysts were investigated comprehensively in both batch and contiuous-flow mode (Scheme 3.).
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Scheme 3. Lipase catalysed kinetic resolution of different racemic amines and secondary alcohols in batch and continuous-flow modes
It was revealed that beside the acetylating agent, the solvent and the quality of the enzyme the mode of its immobilization greatly affect both the productivity and the selectivity. The changes of the temperature dependent properties of the enzyme were investigated in wide temperature range (0–70°C). Furthermore, it was shown that the productivity in a given kinetic resolution reaction performed in batch mode was always surpassed by the corresponding continuous-flow mode reaction.
3.4. Preparative scale lipase catalysed kinetic resolution of racemic amines and secondary alcohols in continuous-flow mode
The preparative scale lipase catalysed N- and O-acetylations of several racemic amines (rac-2a, c, e, g) and secondary alcohols (rac-5a-c) were realised at optimized temperature in continuous-flow systems. Flow through mode was applied for racemic amines and the enantioenriched (R)-acetamides ((R)-3a, c, e, g) were obtained with high yields (›40%) and excellent enantiomeric excess values (98-99%). For secondary alcohols recirculation-flow mode was applied and the enantioenriched (R)-acetates ((R)-6a-c) were obtained with high yields (42-45%) and excellent enantiomeric excess values (›99%).
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Scheme 4. Lipase catalysed preparative scale kinetic resolutions of racemic amines and secondary alcohols in continuous-flow systems
3.5. Dynamic kinetic resolution of racemic β-amino acid derivatives in continuous-flow mode
The dynamic kinetic resolution of racemic N-Boc protected phenylalanine thioethyl eseter (rac-9) was realized in continuous-flow mode (Scheme 5.). The temperature optima of the two elementary parts of DKR were studied separately. The amidation reaction with benzylamine in the presence of protease (Subtilisin A) immobilized onto ethyl-modified silica-gel was carried out at 50°C and the base catalysed (1,8- diazabicyclo[5.4.0]undec-7-ene (DBU)) racemization was realized at 150°C. The reaction mixture of rac-9, benzylamine and DBU dissolved in tert-amyl alcohol was pumped through 11 columns (6 columns filled with biocatalyst and 5 column filled with silica-gel (for racemization) were connected in alternating series) for the realization of continuous-flow DKR. Thus, the two elementary parts of the DKR were spaced apart and carried out at different temperature.
Scheme 5. Dynamic kinetic resolution of racemic N-Boc protected phenylalanine thioethyl ester (rac-9) in continuous-flow system
8 The continuous-flow DKR of other amino acid derivatives or racemic compounds could be easily realized employing this method due to its generalizability.
3.6. Immobilization of a protease (Subtilisin A) onto surface modified silica-gels
It is widely known that proteases gradually lose their activity as a result of self- hydrolysis. The commercially available aqueous solution of Subtilisin A (Alcalase) loses significant part of its activity after six month which is one of the most serious limitations of its applicability as biocatalyst.
Several enzyme preparations were developed by adsorption of protease onto surface modified silica-gels. The shelf-time of the preparations was extended: a significant part of their productivity was retained after a 12 month storing period while the preparations remained highly selective. These biocatalytic properties were thoroughly investigated in the kinetic resolution of racemic N-Boc protected phenylalanine thioethyl ester (rac- 9, Scheme 6.) and it was found that some one-year-old preparations catalysed the amidation reaction with high conversion values (35-40%) and with ›95% ee after 24 hours of reaction time in batch mode.
Scheme 6. The batch mode amidation of racemic N-Boc protected phenylalanine thioethyl eseter with benzylamine in the presence of protease immobilized onto different surface modified silica gels
4. Theses
1. We developed a novel and generalizable method for the synthesis of amines by a one-step reductive amination of ketones in the presence of ammonium formate.
Different catalyst and reaction temperature were used depending on the position of the carbonyl functional group. Zn dust promoted reactions and high temperature gave amines from ketones with a carbonyl group at the benzylic position of an aromatic side chain, and 10% Pd/C catalysis and room temperature were more suitable for the conversion of aliphatic and cycloaliphatic ketones into the corresponding amines. [2]
9 2. The 10% Pd/C catalysed one-step reductive amination of aliphatic and cycloaliphatic ketones – with ammonium formate – were realized for the first time in continuous-flow system. [2]
3. The lipase catalysed N- and O-acetylation of several racemic amines and secondary alcohols were carried out in continuous-flow system. The temperature dependent biocatalytic properties (productivity, selectivity) were investigated in wide temperature range and it was proved that the productivity is always higher when continuous-flow system is applied than the value achieved in batch mode. [3, 4]
4. The preparative scale N- and O-acetylation of several racemic amines and secondary alcohols were realized in continuous-flow system employing Candida antarctica lipase B as catalyst. The corresponding (S)- and (R)-enantiomers were obtained with high enantiomeric excess. [1, 4]
5. We developed a novel and generalizable method for the dynamic kinetic resolution of β-amino acid thioethyl ester derivatives with benzyl amine in continuous-flow system in the presence of protease immobilized onto surface modified silica gel as biocatalyst and 1,8-diazabicyclo[5.4.0]undec-7-ene as base catalyst. The two elementary parts of dynamic kinetic resolution were spaced apart allowing separate optimization for both kinetic resolution and racemization. This is the first realization of dynamic kinetic resolution of amino acid derivatives using a cascade system of alternating kinetic resolution and racemization reaction units. [16]
6. Several stable, active and selective protease preparations from Subtilisin A immobilized by adsorption onto surface modified silica gel were developed. Our preparations have longer shelf-life than the commercially available aqueous solution of protease and catalyse with high enantiomer excess and productivity the amidation reaction of certain racemic amino acid derivatives with benzyl amine. [16]
5. Application possibilities
The general method of reductive amination of ketones could be extended to other
10 ketones and depending on the position of the carbonyl functional group Zn dust or 10%
Pd/C catalyst should be used either in batch or continuous-flow mode. Our continuous- flow mode method for the preparation of amines could be realized in industrial-scale since the scale-up process in continuous-flow mode is easily feasible.
Production of optically active intermediates is an ever expanding area of the pharmaceutical and fine chemical industry. As an important tool for enantioselective syntheses, biocatalysis has become a widely used technology. Recently attention turned to the kinetic resolution of racemic compounds. The dynamic kinetic and kinetic resolutions we described – both in batch and continuous-flow mode – are easily applicable for the resolution of other racemic amines, alcohols and amino acids.
The produced enantioenriched compounds are potentially bioactives or could be highly valuable building blocks of numerous drugs.
6. Publications
6.1. Full scientific publications related to the PhD Thesis
[1] P. Falus, Z. Boros, G. Hornyánszky, J. Nagy, F. Darvas, L. Ürge, L. Poppe:
Synthesis and Lipase catalysed kinetic resolution of racemic amines, Studia Universitatis Babeş-Bolyai Seria Chemia, 2010, 55, 289. [IF: 0,231, FP: 95%]
[2] P. Falus, Z. Boros, G. Hornyánszky, J. Nagy, F. Darvas, L. Ürge, L. Poppe:
Reductive amination of ketones: novel one-step transfer hydrogenations in batch and continuous-flow mode, Tetrahedron Letters, 2011, 52, 1310. [IF: 2,683, FP:
95%, C: 9]
[3] Z. Boros, P. Falus, M. Márkus, D. Weiser, M. Oláh, G. Hornyánszky, J. Nagy, L.
Poppe: How the mode of Candida antarctica lipase B immobilization affects the continuous-flow kinetic resolution of racemic amines at various temperatures, Journal of Molecular Catalysis B: Enzymatic, 2013, 85-86, 119. [IF: 2,823, FP:
10%, C: 7]
[4] P. Falus, Z. Boros, P. Kovács, L. Poppe, J. Nagy: Lipase-Catalyzed Kinetic Resolution of 1-(2-Hydroxycyclohexyl)Indoles in Batch and Continuous-Flow Systems, Journal of Flow Chemistry, 2014, 4, 125. [IF: 1,878, FP: 95%, C: 1]
11 6.2. Oral presentations related to the PhD Thesis
[5] P. Falus, Z. Boros, G. Hornyánszky, J. Nagy, L. Ürge, F. Darvas, L. Poppe:
Ketonok új típusú fémkatalizált reduktív aminálásai szakaszos és folyamatos reaktorban, XXXIII. Kémiai Előadói Napok, 25–27 October 2010. Szeged, Hungary.
[6] P. Falus, Z. Boros, G. Hornyánszky, J. Nagy, L. Ürge, F. Darvas, L. Poppe:
Synthesis of chiral amides in chemo-enzymatic cascade system, XVI. Nemzetközi Vegyészkonferencia, 11–14 November 2010. Cluj-Napoca, Romania.
[7] G. Hornyánszky, P. Falus, Z. Boros, J. Nagy, L. Poppe: Királis savamidok előállítási és funkcionalizálási lehetőségeinek vizsgálata kemo-enzimatikus kaszkád rendszerben, MKE 1. Nemzeti Konferencia, 22–25 May 2011. Sopron, Hungary.
[8] Z. Boros, P. Falus, G. Hornyánszky, J. Nagy, L. Ürge, F. Darvas, L. Poppe:
Continuous-flow systems for synthesis, kinetic resolution and dynamic kinetic resolution of amines, Biotrans, 10th International Symposium on Biocatalysis, 2–6 October 2011. Giardini Naxos, Sicily, Italy.
[9] P. Falus, Z. Boros, L. Poppe: Királis savamidok előállítási lehetőségeinek vizsgálata kemo-enzimatikus kaszkád rendszerben, XXXIV. Kémiai Előadói Napok Tudományos Szimpózium, 2–4 November 2011. Szeged, Hungary.
[10] G. Hornyánszky, Z. Boros, P. Csuka, P. Falus, M. Márkus, D. Weiser, M. Oláh, J.
Nagy, L. Poppe: Optikailag aktív savamidok enzimatikus előállítása és átalakítása biológiailag aktív vegyületekké, XVIII. Nemzetközi Vegyészkonferencia, 22–25 November 2012. Băile Felix, Romania.
[11] Z. Boros, E. Abaháziová, M. Oláh, L. Nagy-Győr, P. Falus, V. Bódai, P.
Sátorhelyi, B. Erdélyi, Gy. Szakács, L. Poppe: Átfolyásos reaktorokban végzett biotranszformációkhoz alkalmazható lipázok tisztítása szelektív adszorpcióval, MKE Vegyészkonferencia, 26–28 June 2013. Hajdúszoboszló, Hungary.
12 6.3. Poster presentations related to the PhD Thesis
[12] Z. Boros, P. Falus, D. Weiser, K. Kovács, G. Hellner, M. Márkus, E. Abaháziová, M. Oláh, B. G. Vértessy, L. Poppe: Silica-based enzyme immobilization methods for lipase-catalyzed kinetic resolutions of racemic amines and alcohols in continuous-flow bioreactors, biocat2012 – 6th International Congress on Biocatalysis, 2–6 September 2012. Hamburg, Germany.
[13] P. Falus, Z. Boros, M. Oláh, V. Bódai, P. Sátorhelyi, B. Erdélyi, P. Kovács, Gy.
Szakács, J. Nagy, L. Poppe: Indolvázat tartalmazó heterociklusos szekunder alkoholok előállítása, és lipáz katalizált kinetikus rezolválása szakaszos és folyamatos reaktorokban, MKE Vegyészkonferencia, 26–28 June 2013.
Hajdúszoboszló, Hungary.
[14] Z. Boros, E. Abaháziová, M. Oláh, L. Nagy-Győr, P. Falus, V. Bódai, P.
Sátorhelyi, B. Erdélyi, L. Poppe: Novel surface-functionalized silica-based supports for selective adsorption of enzymes, Biotrans, 11th International Symposium on Biocatalysis, 21–25 July 2013. Manchester, United Kingdom.
[15] P. Falus, Z. Boros, M. Oláh, V. Bódai, P. Sátorhelyi, B. Erdélyi, P. Kovács, Gy.
Szakács, J. Nagy, L. Poppe: Preparation and kinetic resolution of indole- containing heterocyclic secunder alcohols in batch and continuous-flow systems, Biotrans, 11th International Symposium on Biocatalysis, 21–25 July 2013.
Manchester, United Kingdom.
[16] S. Servi, P. Falus, Z. Boros, L. Cerioli, G. Bajnóczi, D. Weiser, J. Nagy J, D.
Tessaro, L. Poppe: A continuous-flow methodology for the amidation of rac-N- Boc amino acid thioesters in DKR conditions, Transam 2.0 - Chiral Amines Through (Bio)Catalysis, 4–6 March 2015. Greifswald, Germany.
