Asymmetric Synthesis and Application of Î±-Amino Acids - American

Chapter 20

Chemo-Enzymatic Synthesis of Unnatural Amino Acids Ian V. Archer, S. Alison Arnold, Reuben Carr, Ian G.Fotheringham ,Robert E. Speight, and Paul P. Taylor

Downloaded by EAST CAROLINA UNIV on September 18, 2015 | http://pubs.acs.org Publication Date: June 14, 2009 | doi: 10.1021/bk-2009-1009.ch020

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Ingenza Ltd., Wallace Building, Roslin BioCentre, Roslin, Midlothian, EH25 9PP, Scotland, United Kingdom

A general chemo-enzymatic process has been developed to prepare enantiomerically pure L- and D-amino acids in high yield by deracemisation of racemic starting materials. The method has been developed from initial academic studies to be a robust, scalable industrial process. Unnatural amino acids, in high optical purity, are a rapidly growing class of intermediates required for pharmaceuticals, agrochemicals and other fine chemical applications. However, no single method has proven sufficiently adaptable to prepare these compounds generally at large scale. Our approach uses an enantioselective oxidase biocatalyst and a non-selective chemical reducing agent to effect the stereoinversion of one enantiomer and can result in an enantiomeric excess of > 99 % from a starting racemate, and product yields over 90 %. The current approach compares very favourably to resolution methods which have a maximum single pass yield of 50 %. Efficient methods have been developed to adapt the biocatalyst used in this process towards new target compounds and to optimise key factors which improve the process efficiency and offer competitive economics at scale.

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© 2009 American Chemical Society

In Asymmetric Synthesis and Application of -Amino Acids; Soloshonok, V., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2009.

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Introduction The market for chiral intermediates is undergoing double-digit annual growth and is expected to reach at least $30 billion by the year 2010. This rapidly increasing demand for these compounds is mainly to support the development of new, single enantiomer pharmaceuticals and agrochemicals. In parallel, the emergence of biocatalysis as a viable approach to manufacture chiral compounds at large scale is also increasing significantly. The high enantio- and regioselectivity of biocatalysts is ideally suited to chiral organic synthesis and the continuing advances of methods in gene isolation and expression, microbial strain engineering, enzyme evolution and bioinformatics are broadening the availability of new biocatalysts for industry. However, the complexities of biocatalyst production and bioprocess optimisation almost always pose significant hurdles for process implementation, particularly in the rapid time frames required by industry. It is therefore essential to co-develop, along with the appropriate synthetic activities, the enabling technologies required for biocatalyst production, improvement and formulation. It is also highly advantageous if a biocatalytic transformation or process can be readily adapted towards a family of target molecules. Such a "platform" approach reduces the development time for each subsequent application and mitigates against the loss of individual opportunities through the failure of specific products. Ingenza, an Edinburgh, UK based biocatalyst and bioprocess development SME, has addressed each of these key criteria in developing a deracemisation bioprocess platform, which is adaptable to new amino acid targets required at large scale by the pharmaceutical and agrochemical industries. Unnatural amino acids are playing an increasingly significant role in pharmaceutical development. Ingenza's biocatalytic routes to these compounds now include the water-based deracemisation of racemic mixtures of amino acids by the stereo-inversion of the undesired enantiomer, achieved through the concerted use of an oxidase biocatalyst and a chemical reducing catalyst. This process yields a single enantiomer of the amino acid, in very high optical purity and near quantitative yield.


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Background to the Amino Acid Deracemisation Process The deracemisation process, which is now being commercialised by Ingenza, derives from earlier academic studies in the United States, Japan, and notably in the biological chemistry laboratory of Professor Nicholas Turner at the School of Chemistry at Edinburgh University. The method employs the concerted use of a highly enantioselective amino acid oxidase biocatalyst and a non-enantioselective chemical reducing agent or catalyst (Figure 1). The imine,



324 or in some cases the keto acid, which is generated exclusively from one enantiomer of the target compound by the oxidase, can be converted in equal proportions to both enantiomers by the reductant. The progression of this reaction ultimately results in the near complete depletion of the enantiomer which is a substrate for the oxidase, with a concomitant increase of the opposite enantiomer. The process involves no substrate recycling and results in the stereoinversion of one enantiomer to yield the desired enantiomer, typically in > 99% e.e. and conversions which can approach 100%. The major advantages of the technology lie in the co-ordinated action of proven industrial oxidase biocatalysts and supported metal catalysts and high-throughput screening methods to identify and adapt suitable oxidase biocatalyst for each new target.

Oxidase biocatalyst

COOH Non-selective reductant

Figure 1. Principle of chemo-enzymatic amino acid deracemisation showing enantioselective oxidation and nonselective reduction, resulting in the stereoinversion of one enantiomer.

Chemo-enzymatic deracemisation of amino acids was first described in 1971 when Hafher and Wellner reported the production of L-alanine and Lleucine from their corresponding D-enantiomers, through the combined use of Damino acid oxidase (D-AAO) from porcine kidney and NaBH . Subsequently Soda et al carried out a more efficient demonstration of the principle, producing L-proline and L-pipecolic acid in > 98% ee by deracemisation of racemic mixtures, again using D-AAO and NaBH However, the first attempts to develop a practical deracemisation process were carried out by the Turner group, which carried out a programme of process improvement, particularly by introducing more appropriate reducing agents, achieving a 99% yield and 99% ee of Lproline from DL-proline using pig kidney DAAO and only three molar equivalents of NaCNBH . Similar results were observed in the deracemisation of DL-piperazine-2-carboxylic acid, which could be converted to the L-enantiomer in 86% yield and 99% ee using DAAO and NaCNBH There then followed in 1999, a collaboration between the Turner research group and NSC Technologies, 1

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325 a US fine chemical manufacturer, which further significantly enhanced the versatility, efficiency and economic potential of the deracemisation process. In this collaboration, amine-boranes and catalytic transfer hydrogénation using Pd/C were introduced to the process, and proved much more effective reducing agents for the deracemisation of cyclic and acyclic amino acids. Amineboranes, while extremely effective in this chemistry - demonstrating superb activity and selectivity -are unfortunately very expensive. Additionally, the optimal amine-borane (NH -BH ) is not available at the scale required for commercial manufacture. Accordingly, the use of supported metal catalysts in transfer hydrogénation, which was introduced by Dr Scott Laneman of NSC Technologies, has proved to be a particularly versatile and economical approach and provided one basis for the development of the current process. Since 2003, when Ingenza began to develop amino acid deracemisation towards commercialisation, the process development has focused entirely upon the use of catalytic transfer hydrogénation, because of the extensive knowledge base available regarding the industrial use, recovery and recycling of the metal catalysts and the highly favourable large-scale process economics offered by this approach. A second critical improvement to the performance and versatility of this technology derived from the introduction of engineered microbes to produce the biocatalyst required for the process. Engineered strains of Escherichia coli which express enzymes enantioselective for either L- or D-amino acids facilitate the deracemisation of DL-amino acids to yield either D- or L-amino acids as products of the deracemisation process. The initial recombinant bacterial strains, which were constructed by NSC Technologies for this application, did not express an amino acid oxidase enzyme but rather a cloned gene encoding Lamino deaminase ( L - A A D ) The L-AAD enzyme originates from Proteus myxofaciens and carries out amino acid oxidation by a different catalytic mechanism to that of oxidases, and does not produce detectable levels of hydrogen peroxide. Not producing the hydrogen peroxide by-product is a limitation of this L-AAD with respect to the directed evolution studies described below. However the broad range of L-amino acids which can be converted to their corresponding keto acids by the native L-AAD enzyme nevertheless renders it very appropriate for this industrial application/" The deracemisation of DLleucine to D-leucine in a 98% yield and 99% ee. was then demonstrated using recombinant cells which expressed L-AAO and 20 equivalents of ammonia borane as the reductant. The time course of this deracemisation is shown in Figure 2. vn


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Recently a number of D- and L- amino acid oxidase encoding genes, including the D-AAO of Trigonopsis variabilis and the L-AAO of Synechococcus sp., have been cloned by Ingenza and expressed in recombinant microbes for application in this general process.



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Figure 2. Deracemisation of DL-leucine using L-AAO and 20 equiv. ofNH -BH (red. J) or t-BuNH -BH (red.2) 3

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Production and Laboratory Evolution of Oxidase Biocatalysts Importantly, the use of cloned genes which encode various oxidases, permits methods of enzyme evolution to be used efficiently to identify mutated variants of the biocatalysts which display improved or adapted properties. Directed evolution methods can be used to generate biocatalyst variants which show activity towards natural and unnatural amino acids and amines that are not good substrates for the native enzymes, thereby enabling the adaptation of the process towards industrial targets. Useful variants may also show improvements in properties suitable for industrial biocatalysts, such as greater stability or resistance to product inhibition under process conditions. The methods used to improve and adapt the oxidase biocatalysts are described below. By coupling random gene mutation with a powerful and very high-throughput in vitro and in situ selection, highly process suitable amino acid or amine oxidases have been evolved from the wild type enzymes. The high enantioselectivity of the native enzymes is typically retained following laboratory evolution. The screening procedure takes advantage of the fact that members of the oxidase family produce hydrogen peroxide as a reaction by-product. The presence of hydrogen peroxide (and therefore oxidase activity) can be detected colorimetrically by the addition of peroxidase and a substrate that yields a coloured product as shown schematically in Figure 3. x


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Figure 3. Solid phase colony and liquid phase micro-titre plate colorimetric screening for improved oxidase activity.

This screen can be carried out in a solid phase format using plated bacterial colonies which carry randomly mutated oxidase isolates. Ingenza has now optimised this approach to enable 10 -10 isolates to be rapidly and economically screened for activity directly towards the substrate of interest, in a single experiment. The appearance of darker coloured colonies in the solid phase screen indicates improved activity of an oxidase variant towards a specific target substrate. The activity of the variant towards a range of target substrates can then be characterised kinetically in a micro-titre plate based assay. Increases in activity, substrate range and biocatalyst stability can be achieved by tailoring the assay conditions to the desired trait over multiple cycles of this process. The laboratory evolution approach was used to alter the substrate range of the amine oxidase from Aspergillus niger (MAO-N). In this case, the wild-type enzyme displayed a narrow substrate specificity with activity observable only towards simple achiral amines, such as 1-pentylamine and benzylamine. Libraries of randomly mutated variants of MAO-N were screened for activity towards ct-methylbenzylamine. A single residue change in the enzyme (Ser336Asn) was found to significantly expand the substrate specificity of MAO-N so that α-methylbenzylamine and other larger, chiral substrates were accepted. Although α-methylbenzylamine was the substrate used in the screen, the variant displayed higher activity towards other amines, such as amethylcyclohexylamine. In each case, the variant was found to display high (S)enantioselectivity, enabling its use in deracemisation reactions/ Subsequent work has further enhanced and improved the activity of MAO-N variants towards different amines, such as (5)-l,2,3,4-tetrahydro-l-methylisoquinoline by incorporating additional mutations, through successive rounds of laboratory evolution/ 5

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328 In a recent example, Ingenza has improved the activity of Trigonopsis variabilis D-AAO (TvDAAO) over one hundred-fold towards a commercially important unnatural amino acid, using ten rounds of laboratory evolution. The evolved variant contains eleven distinct mutations (3.1% of the protein) that were individually shown to be beneficial at each round of evolution. The mutations were selected from randomly generated error-prone PCR libraries, as well as targeted libraries, in which individual amino acids or small groups were selected for saturation mutagenesis. Regions for saturation mutagenesis were rationally selected using sequence alignment and structural homology modelling of closely related oxidases, for which actual resolved structures were available. Additionally, mutated positions in variants selected from error-prone PCR libraries were then targeted for saturation mutagenesis to further optimise the amino acid substitution at these important positions in the protein. The high throughput colony-based colorimetric screen was then used to both improve the activity of the TvDAAO enzyme towards the commercially important unnatural amino acid and also, by adapting the assay conditions, to screen for variants that displayed increased thermal stability. The wild-type TvDAAO enzyme displays a high K (> 300 mM) towards the target substrate but in the first round of mutation and selection, two amino acid substitutions were identified that reduced the K to < 2 mM. Subsequent rounds of laboratory evolution were then used to improve the turnover rate of the enzyme towards the substrate, whilst retaining the low K , by increasing the stringency of the screening step and by using both random and targeted mutation strategies. Despite the activity of the evolved variants towards the substrate being improved to industrially acceptable levels, the operational stability of the enzyme was then found to be low. By exposing the colonies containing the libraries of randomly mutated enzymes to increased temperature (60°C for one hour) prior to colorimetric screening with the substrate, more stable variants could be selected. The increased stability was confirmed by a time course assay of the residual activity of TvDAAO variants following exposure of the protein to various temperatures. A specific variant, named WT1 TvDAAO, was produced that contained only stabilising mutations and therefore retained the substrate specificity of the wild-type enzyme. The stability of this variant was compared to the wild-type enzyme at various temperatures. Figures 4a and 4b compare the residual activity (initial reaction rate at 10 mM substrate concentration, expressed in milli optical density units (mOD) per minute) for the wild-type and variant WT1 TvDAAO enzymes. Figure 4 demonstrates that whereas the wild-type enzyme is almost completely inactivated after ten minutes at 55.8°C, the WT1 variant retains activity, even at 65.6°C and is significantly more stable at lower temperatures. It was found that the WT1 variant was also more stable to oxidation by either hydrogen peroxide or oxygen gas, indicating a possible common mechanism of denaturation under different sources of stress. Improvements in the level of oxidase biocatalysts produced by recombinant strains can also be readily detected using this colorimetric screen. Such M

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Figure 4 a. Wild type Tv. D-AAO heat treatment assay.



Figure 4b. Variant WTl Tv. D-AAO heat treatment assay.


331 improvements are typically due to alterations to regulatory regions which control gene expression or codon changes within the oxidase genes which improve the efficiency of transcription or translational of the gene. Further rounds of directed evolution and the combination of individual beneficial mutations, generates the industrially appropriate biocatalysts required for the deracemisation process.


Enabling Technologies To fully exploit the potential of any bioprocess, many critical enabling technologies are required to bring the laboratory scale discoveries, such as those outlined above, to full manufacturing scale, in areas of significant market opportunity. This aspect is often understated in the development of new biocatalytic methods. Key enabling technologies include the means to isolate and improve relatively fragile natural enzymes to generate robust industrial biocatalysts; an understanding of the major cost factors in biocatalyst production and formulation; rapid bioprocess optimisation using statistical experimental analysis, and the ability to adapt successful bioprocesses into platform technologies to manufacture multiple targets. Ingenza therefore, has acquired considerable expertise in molecular biology, high-throughput screening, fermentation, bioprocess development and synthetic chemistry for its overall bioprocess development. Each of these areas has been addressed and optimised, to establish an efficient and cost-effective operating philosophy for the deracemisation process.

Fermentation Optimisation Initial lab scale fermentation of oxidase producing strains was carried out using complex growth medium, chemical inducers and antibiotics to maintain the plasmid borne oxidase genes within the host bacterium. The resulting cost contribution of the biocatalyst to the overall bioprocess is in excess of $100 per kg of amino acid product. This was clearly an unacceptable cost basis, for all but a few very high-value products and so each of these aspects required significant enhancement. A high cell density fed-batch fermentation was developed to culture the recombinant E.coli strains required to produce the native and evolved oxidases. This fermentation resulted in a microbial biomass in excess of 90 gL* compared to the earlier 5 gL" . Plasmid vectors were also modified to become completely segregationally stable, thereby eliminating the need for antibiotics to be included in the culture medium. Finally, the induction of enzyme production was regulated by a simple temperature shift during the fermentation, eliminating the need for more costly chemical induction. Therefore when an oxidase biocatalyst is adapted towards a specific substrate of interest, it can then be produced routinely and efficiently under a standard fermentation protocol. 1

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332 Biomass produced in fermentation is harvested, broken by mechanical lysis and clarified by centrifugation with the assistance of flocculants. The resulting lysate can be used directly in the deracemisation reaction.


Deracemisation Process Optimisation Successful industrial bioprocesses must meet aggressive cost targets and, for broad acceptance, must be sufficiently robust to enable routine manufacture. Ideally, processes must be compatible with existing equipment and manufacturing practices, while opportunities for process intensification may be sought. In order to establish deracemisation as a competitive manufacturing route for unnatural amino acids, many process conditions required optimisation in addition to the successful biocatalyst production and directed evolution described above. Ingenza has concentrated upon the optimisation of the operating parameters for the process. Principally these parameters included substrate and catalyst loading, reaction conditions such as temperature, pH, aeration and agitation, catalyst formulation (free or immobilised enzyme and reducing catalyst), catalyst recycling, process scale-up (mass-transfer issues) and product recovery and purification. One early example, the deracemisation of Lpipecolic acid, exemplifies this approach. A detailed screen of catalysts under various process conditions resulted in a pleasing deracemisation reaction yielding L-pipecolic acid in > 99% ee and > 80|% yield. Figure 5a shows a timecourse for this reaction run in triplicate. It is apparent from the timecourse that early in the reaction D-pipecolic acid (D-pip) is consumed at the same rate as the L-enantiomer (L-pip) is formed. However in the latter stages D-pipecolic acid is consumed but not converted to its enantiomer. A detailed examination of the reaction mixture allowed the identification of an impurity (imp) which began to accumulate at around the same time and appeared to account for this loss of yield. An adjustment to the addition of the reductant, ammonium formate, (Figure 5b) allowed us to eliminate both the yield loss and impurity to produce L-pipecolic acid in >99% enantiomeric excess and >95% yield. In a further example, deracemisation was used to prepare L-2-aminobutyric acid from DL-2-aminobutyric acid as the starting material. Statistical design of the experimental procedures enabled a rapid screen of over 40 supported metal catalysts to be conducted with varying levels of ammonium formate and biocatalyst. These experiments established the ideal metal catalyst for the reaction as well as the appropriate balance of chemical and biocatalysts. At substrate loading of 2M, the L-2-aminobutyric acid can be recovered at > 99 % ee in 95 % isolated yield. Through catalyst re-use and efficient reprocessing, the metal catalyst contributes |< $10 kg" to the product cost. It is worthwhile comparing the cost contribution of metal catalysis (< $10 kg" ) with that of the amine-boranes used in the earlier deracemisation process, which would contribute » $1,000 kg" of product. 1

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Asymmetric Synthesis and Application of Î±-Amino Acids - American

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