High-Yield Biocatalytic Amination Reactions in Organic Synthesis

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Current Organic Chemistry, 2010, 14, 0000-0000

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High-Yield Biocatalytic Amination Reactions in Organic Synthesis John Ward1 and Roland Wohlgemuth2,* 1

Institute of Structural and Molecular Biology, Darwin Building, Division of Biosciences, University College, Gower Street, London WC1E 6BT, UK

2

Research Specialties, Sigma-Aldrich, Industriestrasse 25, CH-9470 Buchs, Switzerland Abstract: The amino functionality gives important biological activity in pharmaceutical com-pounds. The formation of chiral amines and amino acids can be accomplished by seve-ral chemical routes but enzymatic formation of amines offers many advantages in pre-paring chiral amino compounds or amination of fragile compounds compared to stoi-chiometric or catalytic chemical transformations. Biocatalytic routes to amines primarily use enzymes of the transaminase class (also known as aminotransferases) which transfer the amino function from a donor organic compound to a ketone or aldehyde acceptor. Although known since 1937, the transamination reaction experiences re-newed interest due to the advances in biochemistry and molecular biology and the excellent selectivity of biocatalysts. Other enzymes that have been used to synthesize chiral amines are from the phenylalanine ammonia lyase class that use ammonia as the amino source. The use of recombinant enzymes for the biocatalytic preparation of amines is expanding at a great rate and the range of enzymes revealed in DNA se-quence databases is of the order of tens of thousands. Since a large number of ketone substrates like ketones, hydroxyketones and ketoacids can be made by chemical syn-thesis, the growing toolbox of - , -, -, -, - and -transaminases enable the synthe-sis of various new chemical entities by biocatalytic amination reactions. In order to simplify the isolation and purification of the product, it is useful to drive the amination reaction to completion. The biocatalytic processes that have been developed show different strategies of overcoming the kinetic limitations of the transaminase reactions and show how some enzymes have been used in processes to make large quantities of chiral compounds with amino functionalities.

Keywords: Biocatalytic Transamination, transaminase, aminotransferase, enzyme, biocatalysis, biocatalytic process, phenylalanine ammonia lyase, amino acid, screening, bioprocess, aldehyde, ketone, biotransformation. 1. INTRODUCTION Many interesting natural and synthetic compounds contain one or more amino functio-nal groups on a central carbon scaffold and in defined spatial positioning to various other functionalities. An interesting feature of the amino group, which is heavily proto-nated under normal physiological conditions, is the capability of distinct electrostatic and hydrogen bond interactions to other cellular biomolecules. The chemical as well as biological synthesis can therefore make use of these and other properties like the che-mical reactivity of the amino group and its various substitution patterns. From a synthe-tic point of view the transfer of amines to carbon or heteroatoms is central, but the route and the number of reaction steps may depend on the tools and ingredients available. Since the construction of many different aldehydes and ketones can be performed by carbon-carbon bond formation reactions, the various formal conversions of the carbonyl group to an amine functionality substantially widens access to a large chemical space. This formal conversion can be achieved by transamination, which is a favoured process concerning redox economy, or by reductive amination. In this latter conversion an imine, which is formed as a Schiff base from a carbonyl compound and an amine, is subsequently reduced to a saturated imine. The ACS Green Chemistry Institute Pharmaceutical Roundtable has highlighted that apart from the elimination of extra steps to prepare and isolate the imine substrates, the direct reductive amination of ketones could have major advantages in terms of overall efficiency and environmental impact (Constable et al. 2007).

*Address correspondence to this author at the Research Specialties, Sigma-Aldrich, Industriestrasse 25, CH-9470 Buchs, Switzerland; Tel. ++41-81-7552640; Fax ++4181-7552584; E-mail [email protected] 1385-2728/10 $55.00+.00

Considering redox economy and selectivity of the carbonyl group-to-amine conversion, a simple and selective amino group transfer reaction from an amino-donor to the carbo-nyl carbon has been of much interest. The intermolecular amino group transfer reaction from an amino acid to a keto acid has been discovered by Braunstein in collaboration with Kritzmann in 1937 (Braunstein & Kritzmann 1937, Braunstein 1939, Braunstein & Shemyakin 1953). The name transamination has been introduced for their newly discovered enzyme-catalyzed reaction. This transamination reaction has been also of inte-rest to chemistry and can not only occur with carbonyl carbon atoms, but also with other carbon atoms and even with heteroatoms like nitrogen. Stoichiometric chemical transamination reactions have been achieved with dialkylaluminium amides as amino group transfer reagents. Stability problems of these transfer reagents require that these chemicals have to be prepared in situ from the corresponding trialkyl-aluminium and the amine or amine hydrochloride. The direct conversion of esters to amides by the use of trimethylaluminium and N,O-dimethylhydroxylamine hydrochloride has the advan-tage of low temperature and high yields, but suffers from the use of the aluminium rea-gents in large excess (Basha et al. 1977, Levin et al. 1982). Titanocarborane amide has been found to catalyze transamination of guanidines, with a broad substrate scope of primary, secondary, heterocyclic, aliphatic, and aromatic amines, using 510 mol % of the catalyst (Shen and Xie 2008). Reductive amination is a powerful, versatile and often used reaction in organic synthe-sis, which allows to create new C-N bonds by coupling diverse ketone- and amine-con-taining fragments and catalytic asymmetric versions continue to be of scientific interest (Jacobsen et al. 2004). Imines are commonly reduced by catalytic hydrogenation or by NaCNBH3. An important and longstanding © 2010 Bentham Science Publishers Ltd.

2 Current Organic Chemistry, 2010, Vol. 14, No. 17

Ward and Wohlgemuth

L-specific alphaAminoacid Transaminase

NH3+

Ketocoproduct NH3+ COO-

COO-

R

COO-

R

D-specific alphaAminoacid Transaminase

O

S-specific betaAminoacid Transaminase

O COO-

R

Ketocoproduct R-specific betaAminoacid Transaminase

NH3+

Aryl

S-specific AmineTransaminase

Ketocoproduct

R

COO-

R

Ketocoproduct

D-specific Aminoacid Dehydrogenase

O

NH3

NH3+ Aryl

AminoDonor

R

COO-

Ketocoproduct

R

AminoDonor

L-specific Aminoacid Dehydrogenase

COO-

R-specific AmineTransaminase

O

Aryl

R

NH3+

AminoDonor

AminoDonor

NH3+ R

R Ketocoproduct

COO-

R

AminoDonor

AminoDonor

NH3+

NH3+ R

COO-

NH3

Fig. (1). Overview of biocatalytic amination reactions.

problem concerns the transformation of pro-chiral ketones into chiral amines. Chiral Rhodium-diphosphine catalysts have been developed for the one-pot preparation of -amino acids in up to 98% ee (Tararov and Börner 2005). Attempts to mimic enzymatic transaminations with various organocata-lysts like chiral Lewis acids or primary amine nitrogen sources have led to moderate yields and low enantioselectivities of < 50% ee (Bachmann et al. 2004). Other organo-catalysts consisting of chiral hydrogen-bond catalysts and Hantsch esters have been found for the simple coupling of a wide range of ketones with aryl and heterocyclic amines (Storer et al. 2006) as well as for the coupling of aromatic aldehydes with amines (Hoffmann et al. 2006). A combined method with a Brönsted acid and a transi-tion metal complex has been used for the direct reductive amination of ketones with anilines (Li et al. 2009). An interesting chemoenzymatic approach has been developed for the preparation of all four diastereo- and enantiomerically pure optical isomers of the 2-methyl-3-amino-4,4,4-trifluorobutanoic acid. This has been achieved by combi-ning a chemical and an enzymatic reaction step. The first reaction has been the biomi-metic transamination of ethyl 2-methyl-3-keto-4,4,4trifluoro-butyrate with benzylamine and the second reaction the penicillin acylase-catalyzed resolution of the correspon-ding diastereomerically pure N-phenyl-acetyl derivatives (Soloshonok et al. 1998). Nature also uses the two approaches of transamination and reductive amination to con-struct chiral amino acids and amines. Enzymatic transamination is thereby the major way that amino acids and other amino containing compounds are aminated in cells. The stereospecific removal of the 4-pro-R-hydrogen of -aminobutyrate (GABA) has been demonstrated with a bacterial -amino acid :

pyruvate aminotransferase, while stereospecific removal of the 4pro-S-hydrogen has been demonstrated with -oxoglu-tarate requiring bacterial GABA transaminase (Burnett et al. 1979). L-Glutamic acid dehydrogenase catalyzes the reductive amination of ketoglutarate with ammonia using NADPH as stereospecific reducing agent to synthesize L-glutamic acid. Trans-aminases (also commonly called aminotransferases) catalyze transamination reactions (Christen and Metzler 1985) to interconvert amino acids and intracellular amino com-pounds. In mammals these are important in the nitrogen balance of cells and catalyse both the biosynthesis of amino acids as well as the catabolism back to L-glutamic acid. The nitrogen in L-glutamic acid is then formally removed by glutamate dehydrogenase in the mitochondria, thereby releasing free ammonia. The versatile metabolite carba-moylphosphate, which can enter the urea cycle, can then be synthesized using ammo-nia, carbon dioxide and ATP through the action of carbamoyl phosphate synthetase. All organisms have several transaminase genes, which cover the many different roles that cells need for both catabolism and synthesis of nitrogen containing compounds. They are used in the interconversion of amino acids, the synthesis of certain vitamins, the synthesis of amino-sugars in bacterial cell wall structures, the synthesis of amino-sugars and other amino compounds in many antibiotic pathways and the catabolism of a myriad of different nitrogenous compounds taken up by cells from the environment. Thus there is a diverse range of transaminases in the biosphere covering a huge range of specificities. The increasing need for chiral amines is driven by the requirement for single enantio-mer drugs. Indeed over 70% of all pharmaceuticals are derivatives of chiral amines and this will require an

High-Yield Biocatalytic Amination Reactions in Organic Synthesis

Current Organic Chemistry, 2010, Vol. 14, No. 17 3

increasingly versatile toolbox for the synthesis of chiral amino compounds. Because biocatalytic asymmetric aminations have been shown to be useful for the synthesis of chiral amines (Zhu and Hua 2009) and because of the difficulties in the chemical methods for making chiral amines, despite the tremendous advances in reactions like C-N cross-coupling and asymmetric aminohydroxylation, biocatalytic methods have a great potential for the future in this area. An overview of biocatalytic aminations is shown in Fig. (1). 2. BIOCATALYSTS FOR AMINATION REACTIONS Transaminases catalyse the transfer of an amino group from a donor, usually an amino acid, to the oxo/carbonyl group of a ketoacid. The Enzyme Commission has categori-zed enzymes by reaction type, groups reacted and reactant. This nomenclature groups the transaminases (or aminotransferases) in the Enzyme Commission list as the EC numbers 2.6.1.x , with 4th place numbers x spanning 1 to 86 (defining the different transaminase types) with a further thirteen EC 2.6.1 enzymes that are as yet un-num-bered. The 99 enzyme reaction types each have many hundreds and sometimes thou-sands of different individual enzyme examples amongst the organisms and genomes that have now been characterised. A different way of classifying enzymes is to align the linear sequence of their amino acid sequences and use alignment programs such as CLUSTAL to score the homo-logies of the enzymes and thus cluster enzymes into related groups. Mehta et al. (1993) aligned the protein sequences of 32 transaminases that had been classified into 14 groups based on some of the enzymology of their initial characterisation. The 32 pro-tein sequences could be grouped into four classes. As the number of genomes sequen-ces has risen explosively since 1993 many thousands of transaminases can now be identified in the DNA sequences of fully sequenced bacterial Transaminase

Enzyme Nameb

Positional

genomes and other orga-nisms by searching for homology of the protein sequences to known transaminases. This initial classification was revisited by using the available protein sequences of the 2005 Pfam database http://pfam.sanger.ac.uk/family/browse? browse =a (Hwang et al., 2005). Of the original four classes, class I has now been subdivided into two (class I and II), creating 5 groups listed as ‘classes’ and a further group listed as ‘sugar aminotransferases’. 36,897 protein sequences are listed under ‘transaminase’ [http://www. uniprot.org/uniprot/?query=transaminase+ &sort=score] and 50,010 as ‘aminotransfera-se in the Uniprot database [http://www.uniprot.org/uniprot/?query=aminotransferase+& sort=score] 36,140 [http://www.uniprot.org/uniprot/?query= aminotransferase+AND+ transaminase&sort=score] entries have both terms, so searches for transaminases/ aminotransferases in databases should probably use the term ‘aminotransferase’. The 6 groups of transaminases (as shown in the series of Figs. 2A-2D) are based on the alignment of the amino acid sequences of the enzymes and then grouping sequences which have the most homology with each other. The 6 transaminase groups have been given the nomenclature class I to VI. Figures 2A – 2D: Biocatalyst classes I to VI for amination reactions a Also known as the DegT,DnrJ,EryC1 group. TDP = Thymidine diphosphate. b Unless otherwise stated, all amino acids are the L form and the abbreviation for the transaminase is given after the full name, c Tam, transaminases also known as aminotransferase (AT). d Position of acceptor keto or aldehyde group relative to carboxylic acid or other major gouping e.g. phosphate for HisPTAm Main Donor

Main Acceptor

Specificityd

Class

O

HO I + II

Aspartate TAmc, AspTAm

alpha

OH O

O

HO

OH O

NH2

O

O

O Alanine TAm, AlaTAm

OH

OH

alpha

O

NH2

O Aromatic TAmc, AroTAm

O

OH

alpha

O

OH

Histidinol phosphate TAm, HisPTAm

Fig. (2A). Transaminase Class I and II Biocatalysts.

alpha

N

O NH2

OH

HO

NH2

N

O

P O

O

OH

O OH

HO O

4 Current Organic Chemistry, 2010, Vol. 14, No. 17

Transaminase

Enzyme Nameb

Ward and Wohlgemuth

Positional

Main Donor

Main Acceptor

Specificityd

Class

O III

Acetyl ornitihine TAm, AcornTAm

omega

O

NH2

HO O

NH

O OH

HO O

O Ornithine TAm, OrnTAm

omega

O NH2

HO

O OH

HO

NH2

O O OH

omega-amino acid TAm, omega-TAm omega

NH2 O

NH2 beta-aminocarboxylic acid TAm,

O

O

OH

OH

beta

ArobA TAms

O O

O

H2N Gamma aminobutyrate TAm

OH

gamma

O OH

omega

DapaTAm

OH

HO O

NH2 Diaminopelargonate TAm,

O

S-Adenosyl-4-methylthio-2-oxo-butanoate

NH2

Fig. (2B). Transaminase Class III Biocatalysts.

The sixth group contains the sugar aminotransferases (SAT) from anti-biotic biosynthetic operons such as streptomycin (Ahlert et al., 1997), erythromycin (Burgie et al., 2007), daunorubicin (Madduri and Hutchinson, 1995) and bacterial cell wall aminosugar biosynthesis (Hwang et al., 2004) and is often listed as the DegT/ DnrJ/EryC1 group from the names of the first genes to be grouped into this class. Originally it was thought that some of these proteins were transcriptional controlling proteins but they are now known to be transaminases (Thorsen et al., 1993). Of the six classes, four of these (class I, II, IV and V) catalyse the transfer of an amino group to a keto acid and are largely involved in amino acid catabolism, synthesis and reassort-ment. The class III enzymes have a broader specificity and, depending on the particular enzyme, can transfer an amino group to aldehydes and ketones of many different types and an important group within the class III enzymes are the -transaminases and the aminobutyrate transaminases, which don’t have a requirement for the 2-ketocarboxy-late grouping. The class IV group contains the D-alanine-D-glutamate transaminases which have the positions of the large and small binding pockets (Fig. 3) reversed compared with the other transaminases. The SAT group, class VI, largely use L-glutamate as the amino donor and transfer the amino group to a nucleotide activated ketohexose sugar or scyllo-inosose (Thorsen et al., 1993; Burgie et al., 2007).

Enzymes that use PLP are a larger group than just the transaminases and include de-hydratases, decarboxylases, racemases, tryptophan synthase and several other reac-tion types (Christen and Mehta, 2001). PLP utilising enzymes are classified into four families, the , , D-alanine aminotransferase and the alanine racemase family. All transaminases except the class IV enzymes, are in the large  family of PLP utilising enzymes, which contains the majority of all the PLP requiring enzymes. This places the class IV transaminases (the branched chain transaminases and the D-amino acid transaminases) in a PLP grouping of their own and they are a more distant evolutionary group from the other transaminases. Enzyme Mechanism These five classes of transaminase all use pyridoxal 5’phosphate (PLP) as a cofactor and illustrate the diversity of reactions which PLP-dependent enzyme can catalyze (Eliot & Kirsch 2004). PLP is a derivative of vitamin B6 and is covalently bound in the enzyme active sites during the catalytic cycle. Transaminases are all dimers with two active sites and two molecules of PLP. In the catalytic cycle the PLP (see Fig. 3) is first bound via an imine linkage between an aldehyde group on PLP and the -amino group of an active site lysine which is conserved in all transaminases (Kirsch et al. 1984; Mehta et al. 1993; Hwang et al. 2005). This covalent intermediate is attacked by the donor amino compound which is usually an amino acid although many other amino

High-Yield Biocatalytic Amination Reactions in Organic Synthesis

Transaminase

Enzyme Nameb

Current Organic Chemistry, 2010, Vol. 14, No. 17 5

Positional

Main Donor

Main Acceptor

Specificityd

Class

O IV

D-Alanine TAm

alpha

O

HO

OH

HO

NH2

O

O

O

HO Branched Chain TAm

Serine TAm

O

HO

alpha

OH O

O V

OH

OH NH2

O

O

OH

HO Phosphoserine TAm

alpha

O

HO

NH2

alpha

O

O

P

NH2

O

O

OH

OH

HO

O

O

Fig. (2C). Transaminase Class IV and V Biocatalysts. Transaminase

Enzyme Nameb

Positional

Main Donor

Main Acceptor

Specificityd

Class

NH2 O

O VI

TDP-4-amino-4,6-dideoxyD-glucose TAm

OH

HO

HO

O

OH O

NH2

TDP

OH HO

L-Glutamine:scyllo-inosine

O

O

HO

aminotrasnferase

O OH

HO OH

O OH

HO TDP-3-keto-6-D-hexose aminotransferase

O

O

OH

HO

H2N

O

OH O

Fig. (2D). Transaminase Class VI Biocatalysts.

O

TDP

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Ward and Wohlgemuth

Fig. (3). Transaminase enzyme mechanism. The second half cycle of the transaminase catalytic cycle. The diagram shows the removal of the amino group of the donor molecule, often an amino acid and at the bottom of the diagram the difussion ot of the enzyme of the keto acid co-product. Also shown at the bottom is the acceptor which can be a new alphaketo-acid (R2 = COOH), as in the case of the -transaminases, a keto group in -position to a carboxylic acid (R2 = CH2 COOH) or a keto group several carbons away from a carboxylic acid, as in the case of -transaminases. When the carbonyl is an aldehyde (R1 = H) the enzyme is an -transaminase. The two circles labelled Small and Large denote the active site pocket which in most transaminases is positioned such that the large pocket accomodates the carbo-xylic acid part of the keto acid. In D-amino acid transaminases and the Branced chain transaminases the sizes of the pockets is reversed and in the class III and VI transaminases the relative sizes of the pocket is not always known and must acco-modate the known donors and acceptors.

compounds such as isopropylamine, -alanine and (S)-methylbenzylamine (MBA) can be donors and these donor compounds have important roles in transaminase biocatalytic processes. The keto or alde-hyde product from the donor diffuses out of the active site reforming the -amino group of the active site lysine and creating pyridoxamine 5’-phosphate (PMP), the amino derivative of PLP. The second part of the catalytic cycle is the binding of the acceptor ketone or aldehyde and the PMP now acts as the amine donor to aminate the acceptor re-forming PLP again, which is subsequently covalently bound again by the active site lysine (Shin & Kim 2002). This cycling between a covalently bound PLP and a non-covalently bound PMP allows some transaminases to release their PMP (Schell et al., 2009) whilst others bind PMP very tightly. The catalytic cycle thus needs one binding pocket for the donor amine and another one for the acceptor ketone or aldehyde. From the X-ray structures of several transamina-ses (Okamoto et al. 1998, Ko et al. 1999) which catalyze the formation of -amino acids, the binding pockets for the donor and acceptor can be seen

and some of the amino acid side chains that determine specificity can be deduced (Shin & Kim, 2002). In most of the crystal structures of transaminases there is a large capacity binding pocket and a smaller capacity binding pocket. Most have the si face of the pyridoxal ring of the PLP exposed to the incoming substrate, whilst the re is closest to the pro-tein. In the class IV enzymes this is reversed and the si face is buried close to the pro-tein leaving the re face to interact with the substrate. The Damino acid transaminases are in the class IV group and as well as having the re face of the PLP to interact with the substrate, the Damino acid transaminases also have a reversal of the orientation of the large and small binding pockets such that they add and abstract the proton from the opposite side of the substrate thus making them specific for certain D-amino acids. Alpha Transaminases Of the 6 classes/groups of transaminases (Figs. 2A-2D) the enzymes of class I, II, IV and V can be defined as -transaminases in

High-Yield Biocatalytic Amination Reactions in Organic Synthesis

that they transfer the amino group to and from the position to a carboxylic acid (or phosphate ester in the case of the class II enzyme L-histidinol-phosphate transaminase). A recent addition to the class II en-zymes (based on the protein amino acid sequence alignment) is from the bacterium Mezorhizobium sp. Strain LUK (Kim et al., 2007) has the substrate specificity of a -transaminase and prefers D--aminocarboxylic acids. This demonstrates that the classification by alignment of the enzymes amino acid sequence reflects evolutionary relatedness of the entire protein and does not always correlate with the positional specificity (e.g. , ) of the class II enzymes. The enzymes of class III have several members that are transaminases and others that are defined as ,  or transaminases. The -transaminases are those that use -alanine, such as the -alanine pyruvate transaminase. The  or 4aminobutyrate transaminase (GABA transaminases) group of enzymes have members that have ra-ther relaxed substrate specificity and can transfer the amino group to ketones and alde-hydes. GABA is a neurotransmitter in the brains of mammals and is also found in bac-teria and plants, where it may have roles as a stress adaptation and signalling molecule (Shelp et al., 1999). A common GABA transaminase is the butyric acid L-glutamate transaminase, which transfers the amino group from GABA to 2-ketoglutarate forming L-glutamate and succinate semialdehyde (Akihiro et al., 2008). Because the back reaction of amine transfer to 3-oxopropanoate (for the -alanine transaminases) and to succinate semialdehyde (for the GABA transaminases), these  and aminotransfe-rases often have a wider acceptance of the aldehyde acceptor and can be versatile enzymes for biocatalysis (Kaulmann et al., 2007). Omega Transaminases Class III aminotransferases also contain the -transaminases, which have recently gained a lot of attention due to their relaxed substrate specificity for the acceptor in the aminotransferase reaction. The -transaminase from Vibrio fluvialis JS17 (Shin and Kim, 2001) was isolated from a screen using arylic amines and has been cloned (Shin et al., 2003).Its activity to many different aldehydes, ketones and amine donors has been characterized (Shin and Kim, 2001; Shin and Kim, 2002). The enzyme is very ver-satile and one of its best amine donors is (S)--methylbenzylamine, but limitations exist due to the unfavorable equilibrium and the enzymes sensitivity to the co-product aceto-phenone (Shin and Kim, 1998). Strategies to overcome this limitation utilize membrane reactors to remove the volatile acetophenone (Shin et al., 2001) and biphasic systems to partition the inhibitory components (Shin and Kim, 1997; 1998; 2009). For the opposite reaction direction addition of lactate dehydrogenase or whole cells pushed the reaction to 90 and 92% yields using 25 mM benzylacetone and 30 mM acetophenone, respectively, and whole cells expressing the recombinant V. fluvialis enzyme have been successful (Shin and Kim, 1999). An enzyme with 38% homology to the V. fluvialis transaminase was cloned by Kaulmann (Kaulmann et al., 2007). This enzyme has a versatile substrate spectrum including aromatic aldehydes (benzaldehyde, cinna-maldehyde) and aliphatic aldehydes (propanal, butanal, hexanal and nonanal). Phenylalanine Ammonia Lyase All plants, some fungi and bacteria have the enzyme phenylalanine ammonia lyase (PAL), which, in planta, catalyzes the formal removal of the nitrogen from phenylalanine to form trans-cinnamic acid, which is a precursor for the phenylpropanoids lignin,

Current Organic Chemistry, 2010, Vol. 14, No. 17 7

flavanoids and coumarins. There are also the closely related enzymes, tyrosine ammo-nia lyase (TAL) and histidine ammonia lyase (HAL), which occur in plants, some fungi and many bacteria and catalyse the removal of ammonia from tyrosine and histidine respectively. PAL, TAL and HAL use a novel prosthetic group that is formed within the enzyme active site from three consecutive amino acids, alanine, serine and glycine of the protein (Schuster and Retey. 1995). These form the active electrophilic prosthetic group 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO). Gloge et al. (2000) have used PAL to synthesize various L-arylalanines by utilizing high concentrations of ammonia (5 M) buffered to pH 10 and several fluoro- and chloro-trans-cinnamic acids were converted to the corresponding L-phenylalanine derivatives in yields from 3799% with an ee of over 99%. The narrow substrate range and strict regioselectivity of ammonia lyases for ammonia addition to the position has recently been enlarged by the use of phenylalanine mutase (PAM) in the biocatalytic amination of cinnamic acid derivati-ves (Wu et al. 2009). 3. AMINATION BIOPROCESSES Transaminases (Aminotransferases) have been studied over many years as bio-catalysts for the production of a wide variety of -amino acids (Crump & Rozzell 1992; Onuffer et al. 1995; Hwang et al. 2003, Cho et al. 2003,2004,2006). If the amino group is not in the -position with respect to the carboxy group, the situation is different and the study of - , - , - , - and -transaminases is the subject of much current research. A large number of ketone substrates like ketones, hydroxyketones and keto-acids can be made by chemical synthesis. The biocatalytic transamination is highly enantio-selective and transaminases are available for converting the keto functional group to amino functional group in both the R- and Sconfiguration. Interfacing the chemical syn-thesis with the biocatalytic transamination is therefore attractive because of a high atom and step economy towards only the desired enantiomer. Proteinogenic and non-proteinogenic L- and D--amino acids as well as enantiomeri-cally pure -amino acid derivatives play an important role in nutrition, health and indus-trial applications. The corresponding bioprocesses for their production are well established (Leuchtenberger et al. 2005, Ager et al. 2001, Bommarius et al. 1998, Lidstroem 2007, Fotheringham & Oswald 2008). In the production of the proteinogenic -amino acids L-alanine, L-leucine, L-isoleucine, L-valine and L-methionine, but also the synthe-sis of a non-proteinogenic -amino acid like L-tert-leucine, the biocatalytic reductive amination process of the corresponding -oxo-acids has been scaled up to production scale. This was mainly due to the general availability of the specific amino acid dehy-drogenases, the easy access to the -oxoacids and the cost savings on the required cofactor NADH, which was recycled in a continuously operated membrane reactor. Using -ketoisocaproate as starting material, Lleucine was obtained with >99% conversion and a space-time yield of 324 mmol per L and day (Wichmann et al.l 1981), using Lleucine dehydrogenase for the reductive amination and formate dehydrogena-se for the regeneration of NADH. Recombinant whole-cell biocatalysts have been deve-loped for the synthesis of Ltert-leucine and L-neopentylglycine by reductive amination (Menzel et al. 2004, Gröger et al. 2006). D-amino acids have been synthesized by the action of D-amino acid transaminase (Galkin et al. 1997) and via reductive amination of the corresponding 2-ketoacids with ammonia using a highly stereoselective D-amino acid dehydrogenase (Vedha-Peters et al. 2006). Since D-amino acid dehydrogenases are not as ubiquitous in

8 Current Organic Chemistry, 2010, Vol. 14, No. 17

nature as the L-amino acid dehydrogenases, the highly Denantioselective enzyme was created by directed evolution using both rational and random mutagenesis, starting from the enzy-me meso-2,6-D-diaminopimelic acid dehydrogenase (Vedha-Peters et al. 2006). Alternatively, branched-chain aliphatic -amino acids like Lleucine and L-valine can be produced by the -transaminasecatalyzed transfer of an amino group from glutamic acid to the oxoacid. A number of efficient bioprocesses for the -transaminasecata-lyzed production of unnatural amino acids have been developed due to the wide substrate tolerance, high turnover number and no cofactor recycling requirements for transaminases (Li et al. 2002). A series of 3- and 4-substituted glutamic acid analogues have been synthesized from the corresponding 2-keto-glutaric acids using the enzymes aspartate aminotransferase from pig heart or Escherichia coli or branched chain amino-transferase from Escherichia coli (Alaux et al. 2005, Xian et al. 2007). L--Phenylalanine has been produced from the same raw material phenylpyruvic acid via two different routes. The biocatalytic reductive amination of the -oxoacid phenyl-pyruvate with ammonia can be catalyzed by L-phenylalanine dehydrogenase from Brevibacterium or Rhodococcus species. The biocatalytic transamination of phenyl-pyruvate with L-glutamic or L-aspartic acid has been developed with the isolated trans-aminase being in the soluble or immobilized form or with the transaminase contained in supended or immobilized whole cells. L--Phenylalanine was produced continuously from L--aspartate and phenylpyruvate with an isolated -transaminase from Pseudo-monas putida in an enzyme membrane reactor and with immobilized whole cells in a stirred tank reactor, respectively (Ziehr et al. 1986). The productivity of the bioprocess with the isolated enzyme in homogeneous phase was about three times higher than the productivity achieved with immobilized cells. The ability of various aliphatic and aroma-tic amino acids like valine, leucine, phenylalanine and phenylglycine to replace L-glutamate in the transaminase-catalyzed amination of 4-hydroxy-3methyl-2-keto-pentanoic acid to 4-hydroxy-isoleucine is of special interest when byproducts can also be utilized as amino donors (Ogawa et al. 2007). The four stereoisomers of L-2-(2carboxycyclo-butyl)glycine have been synthesized from the corresponding cis and trans-2-oxalyl-cyclobutanecarboxylic acids in good yields and with high ee, using aspartate amino-transferase and branched chain aminotransferase from Escherichia coli (Faure et al. 2006). The -transaminase from Mesorhizobium sp. showed similar reactivities in the case of the amino acceptors 2-ketoglutarate, pyruvate and its methyl- or ethylesters, while oxaloacetate showed a slightly lower reactivity (Kim et al. 2007). 3-Amino-3-phenyl-propionic acid, 3-Amino-5-methylhexanoic acid and 3-Aminobutyric acid have been found to be the best amino donors. Although transaminases show in general a clear prefe-rence for either pyruvate or 2-ketoglutarate and oxaloacetate, this -transaminase from Mesorhizobium has no special amino acceptor preference. 3Aminobutyric acid is a promising amino donor for asymmetric synthesis using this transaminase, because it is transformed into acetoacetic acid. This makes the reaction irreversible, because aceto-acetic acid is decomposed spontaneously into acetone and carbon dioxide, leading to a simple product isolation from the reaction mixture at the end of the reaction. The high stereoselectivity of a transaminase from Alcaligenes denitrificans was used for the resolution of racemic -amino-n-butyric acid to give optically pure D--amino-n-butyric acid with >99% enantiomeric excess (Yun et

Ward and Wohlgemuth

al. 2004). Phosphonoacetaldehyde and L-alanine have been formed from 2-aminoethylphosphonate and pyruvate in a stereo-specific pro-S hydrogen removal at the prochiral C2 atom by 2aminoethylphosphonate aminotransferase from Pseudomonas aeruginosa (Lacoste et al. 1993). Bacterial and mammalian -transaminases also remove exclusively the pro-S hydrogen from the prochiral -carbon atom of amino donors (Burnett et al. 1979; Bouclier et al. 1979). The -transaminase from Bacillus sphaericus was highly specific for L-ornithine and -ketoglutarate and catalyzed transamination between them to produce L-glutamate and Lglutamate -semialdehyde, which was spontaneously converted to 1-pyrroline-5-carboxylate exclusively with no 1-pyrroline-2carboxylate formed (Yasuda et al. 1981). The enzyme L-lysine -aminotransferase from Sphingomonas paucimobilis catalyzed the oxidation of the -amino group of lysine in the dipeptide dimer N2-[N-[phenyl-metho-xy)-carbonyl]-Lhomocysteinyl]-L-lysine)1,1-disulphide requiring -ketoglutarate as the amino acceptor (Patel et al. 2000). -transaminases have been used for the resolution or synthesis of enantiomerically pure amines (Stirling et al. 1990; Stirling 1992; Shin & Kim 2002;Hanson et al. 2008). The mode of substrate inhibition of -transaminase from Vibrio fluvialis JS17 has been found to depend on the chirality of the substrate (Shin & Kim 2002). A (S)-transaminase from Bacillus megaterium was used to resolve 1cyclopropylethyl-amine and sec-butyl-amine to the (R)-amines with >99% ee (Hanson et al. 2008). The rapid resolution at a high concentration of the racemic sec-butyl-amine of >0.6 M and the simple work-up procedure by distillation of the amine and crystallization as the hemisulfate salt makes this a very practical bioprocess. While many bacterial -transaminases are specific for the S-enantiomer, an R-specific -transaminase has also been described (Iwasaki et al. 2003, 2006). Racemic -chiral amines were deracemized within 48 hours through kinetic resolution and subsequent stereoselective amination catalyzed by two enantio-complementary transaminases (Koszelewski et al. 2009). By this two-step cascade reaction -chiral primary amines could be obtained with excellent enantioselectivity and up to quantitative conversion. The application of this one-pot two-step procedure to the deracemization of mexiletine yielded the (S) – as well as the (R)-mexiletine in up to >99% ee and 97% conversion (Koszelewski et al. 2009). The order of the applied transaminases thereby allows to select the absolute configuration of the product. The use of transaminases for the asymmetric synthesis of amines by starting from a prochiral ketone is interesting for interfacing the transamination reaction with the chemi-cal ketone synthesis and different approaches have been described (Höhne et al. 2008; Höhne and Bornscheuer 2009; Yun et al. 2006; Iwasaki et al. 2006; Koszelewski et al. 2008; Smithies et al. 2009). High activity and stability of the transaminase towards the amination of Lerythrulose have been identified as key factors for high-efficiency trans-amination (Ingram et al. 2007). The selected examples of amination bioprocesses show that the knowledge about this reaction platform is rapidly growing and amination is becoming an key reaction in industrial biotransformations (Ghisalba et al. 2009). 4. SCALE-UP AND DOWNSTREAM PROCESSING The key bottleneck of transamination bioprocesses is usually the thermodynamics. In transamination reactions involving amino

High-Yield Biocatalytic Amination Reactions in Organic Synthesis

Current Organic Chemistry, 2010, Vol. 14, No. 17 9

A) Removal of Transamination Coproduct O

NH2 Transaminase

R1

R1

R2 Amino Donor

Keto-form of Amino Donor

Coproduct Removal Technologies

B) Removal of Transamination Product

Subsequent Reaction Step

O NH2

Transaminase R1

R2

R2

Desired Product

R1

R2

C) Excess Substrate Feeding O

NH2

Transaminase R1

R1

R2

R2

Non-inhibiting Keto-form of Amino Donor

Inexpensive Amino Donor in excess

D) Better Bioreaction Path O Transaminase * R 1

* R2 Amino Donor

NH2

* * R 1

*

* Keto-form of Amino Donor

R2*

Fig. (4). Engineering concepts for complete conversion transaminations.

acids the equilibrium constant is near unity because an amino acid reacts with a 2-keto acid to give another 2-keto acid and amino acid as products. Various techniques have been developed to drive transamination reactions towards completion and an overview of concepts is shown in Fig. (4). Concept A uses lactate dehydrogenase to remove the inhibitory keto-form of the amino donor, formed as co-product, from the reaction in order to drive the reaction equilibrium towards the desired amine product. The second concept B is a single enzyme system and uses a large excess of isopropylamine to drive the transamination. Concept C requires only a catalytic amount of amine donor, as an amino acid dehydrogenase is employed to regenerate the amine donor in situ using ammonia. All three systems have been demonstrated for the production of optically pure methylbenzylamine

from aceto-phenone. An enantiomeric excess of >99% was achieved for both the R- and S-methyl-benzylamine products (Truppo et al. 2009). Alternatively, the concept D of improving the bioreaction by substrate, amino donor and enzyme engineering can be of interest. Transaminase-catalyzed reactions have been scaled up by Cambrex to the multi-ton scale and applications for the synthesis of chiral amines as well as for an efficient reso-lution of racemic amines have been in industrial use for many years (Scarlato 2009). The ee of the desired amine is increased by converting the unwanted enantiomer com-pletely to the corresponding prochiral ketone, thus achieving excellent enantiomeric purity by eliminating any residual unwanted enantiomer. Cambrex has used transaminases with typical activities of 20-30 U/g as whole-cell spray-dried

10 Current Organic Chemistry, 2010, Vol. 14, No. 17

powder instead of highly purified enzymes in order to save production costs. Racemic para-methoxy-benzylamine was completely resolved by an R-specific transaminase to 50% (S)-paramethoxybenzyl-amine. Pyruvic acid has been used as exclusive amino acceptor in the reaction mixture to the minor paramethoxybenzylamine enantiomer, which was then converted to the para-methoxyphenylmethylketone. Downstream processing is there-fore simple, but requires a readily available and inexpensive amine, because this pro-cess has similar limitations like classical resolutions with a maximum theoretical yield of only 50%. Methods for recycling the ketone will reduce waste and increase yields, but require also more equipment and process time. The asymmetric synthesis of chiral amines from their corresponding prochiral ketones is more elegant and D-amphetamine and (S)-7-methoxy-2-aminotetralin have been manufactured at the multi-ton scale by Cambrex (Scarlato 2009). Amino donor concentrations of > 1.5 M isopropylamine are utilized to drive the equilibrium between the star-ting compound phenylacetone and the product D-amphetamine to near completion. The tightly controlled and regulated liquid product is distilled from the biomass and isolated in good yields. The same amino donor has been used in the transformation of prochiral 7-methoxy-2-tetralone to the chiral intermediate (S)-7-methoxy-2-aminotetra-lin, but the reaction has been performed at about 55° C with a more thermostable trans-aminase. After the use of distillation and extraction processes in downstream pro-cessing, the final product has been obtained in 70% yield. Another approach to drive transamination reactions towards completion is the selective evaporation of an inhibitory product. This was performed in the kinetic resolution of (R,S)-secbutylamine by -transaminase from Vibrio fluvialis JS17, whereby 2-buta-none was evaporated under reduced pressure (Yun et al. 2004). Since the substrate sec-butylamine has a higher evaporation rate at higher pH above 7.0, the optimum pH for this bioprocess has to be lowered from optimum pH of 9.0 for the transaminase reaction. At a substrate concentration of 400 mM racemic secbutylamine the trans-aminase-catalyzed resolution gave the remaining (R)-sec.butylamine in 98% ee in 53% conversion at 150 Torr and pH 7.0 (Yun et al. 2004). The process space-time yields, the subsequent recovery yields of the products and raw material costs are decisive for the selection of a biocatalytic single-step route as well as for a complete fermentation process at large scale. 5 .OUTLOOK Biocatalyst Availability The availability of transaminases with different specificities is currently somewhat limi-ted. Ideally, a researcher carrying out bench scale exploratory research needs to have easy access to different transaminases in order to test which one might be optimal in his/her work for the kinds of compounds they are interested in. They would also need to know that, if a particular transaminase was found to be useful, they could gain access to larger quantities of the enzyme for scale up. This latter fact is crucial for an enzyme to be considered in a process to make any pharmaceutical intermediate, since the process that is fixed in the early stages of development and clinical trials should ideally be scalable. To encourage the use and uptake of transaminase biocatalysis by organic chemists in the exploratory stage of compound synthesis, a larger range of transaminases needs to be available and as easy to use as conventional reagents at the bench. Some com-panies

Ward and Wohlgemuth

have started to make screening kits of transaminases and/or have research pro-grams to discover new transaminases. Companies such as LibraGen, Cambrex, Almac, ChiralVision, Alphamerics, Nagase and others offer transaminases for both screening and in several cases, the same enzymes at large scale. New transaminase enzymes can be derived from screening microorganisms for novel activities and the sources of new bacteria can be varied. For example Yun et al. (2004) screened soil samples from ’various domestic sites’ to find a new -transaminase. New ways of accessing the myriad of transaminases in the biosphere such as metagenomics will come to the fore in the future and allow access to enzymes from novel sources. These include the marine environment, deep-sea vents and muds, extreme environ-ments such as soda lakes, hypersaline waters, hydrothermal environments and psychrophilic environ-ments such as Arctic and Antarctic sources. As the fully sequen-ced genome databases continue to grow at a fast pace, sophisticated ways of inter-rogating the derived protein sequences from these DNA databases will allow rapid cloning by PCR or total gene synthesis of any transaminase gene identified in these databases. As of mid 2009 there are nearly 1,000 fully sequenced bacterial genomes and 17 fungi which can be searched for transaminase genes. There are 41 flowering plant genomes at various stages of DNA sequence completion and these will add to the >50,000 aminotransferase enzymes currently listed (October 2009) listed in the Uniprot database. An example of how a single fully sequenced bacterium can be searched for transaminases is that of Corynebacterium glutamicum (McHardy et al., 2003). 19 trans-aminase genes have been found and characterized in this well known producer of amino acids. In addition to increasing the number and availability of transaminases at both the bench scale for investigation of new routes to chiral amines and at large scale for manu-facturing, there is the need to prepare or optimize present transaminases (and other enzymes for biocatalysis) in a form that allows ease of use at the early stages of deve-loping a synthetic route. This will facilitate the coupling of new transaminase activity discovery (Chen et al., 2008) with the investigations on the enzyme enantioselectivity as well as the optimization of reaction conditions to overcome thermodynamic barriers. The transaminases with their broad substrate specificity and the amino acid or amine dehydrogenases with their inexpensive amino donor are promising approaches towards the asymmetric synthesis of optically pure amines (Zhu and Hua, 2009). Biocatalyst Screening Screening for new transaminase enzymes or activities is necessary if new enzymes are being cloned from organisms which may contain several transaminase activities and from metagenomic libraries which have the potential to contain hundreds of transaminases but diluted within huge numbers (106 – 109) of other clones. Screening methods are also needed during mutagenesis, gene shuffling and directed evolution procedures. We are starting to see groups beginning to address this problem (Truppo et al., 2009) with methods that can be used at small scales compatible with microtitre plates and thus could be used in a high throughput screen of recombinant gene libraries. Other, more novel screening methods such as NMR techniques that assess binding of com-pounds to proteins (Dalvit et al., 2003) may also be a useful adjunct to other methods and NMR binding screening can have advantages for screening many substrates for one enzyme.

High-Yield Biocatalytic Amination Reactions in Organic Synthesis

Current Organic Chemistry, 2010, Vol. 14, No. 17 11

H3C

O

HO H2N

OH Aminosugars OdTDP

NH2

H2N

COOH

R1

H R2

Chiral Amines Aromatic Amino Acids O

NH2

OH

R COOH Aliphatic and beta-Heterocyclic Amino Acids

R1

R2

COOH NH2 Aromatic beta-Amino Acids

NH2 COOH COOH

OH

NH2

D-Amino Acids

Aliphatic beta-Amino Acids NH2 Aminoalcohols

Fig. (5). Transamination reactions in retrosynthetic analysis.

There is also the need of new substrates for screening and testing. Although there are many aldehydes and ketones commercially available, there are still gaps in the homo-logous series of many types of compounds and some compound types are not readily available. Biocatalysis itself can contribute to fill the gaps (Richter et al., 2009) and provide the required compounds for screening. Alteration of Enzymes – Mutagenesis Once an enzyme has been cloned and characterized it may be deemed necessary to start to alter the enzyme to change its substrate specificity, remove inhibition by substrate or product, alter (increase) the ee or to create an enzyme that is more robust towards the biocatalysis conditions whilst keeping the specificity of the enzyme. There are now many methods to alter enzymes by sitedirected mutagenesis, saturation mutagenesis, directed evolution and making chimeras between related enzymes. The development of rapid and sensitive methods for activity screening and selection are thereby key for success (Schätzle et al. 2009). An interesting screening approach was the search for a -transaminase which is overcoming product inhibition by aliphatic ke-tones (Yun et al. 2005). An enzyme library generated by error-prone PCR was enriched on minimal medium containing 2-aminoheptanone as the sole nitrogen source and with increasing amounts of 2-butanone as the inhibitory ketone. It was interesting that the improved transaminase had only two amino acid substitutions (Yun et al. 2005). The alteration of enzymes will become a very important strategy for the transaminase family of biocatalysis and will prompt a need for high resolution x-ray structures (Sayer et al. 2007; de la Torre et al.

2009). Many good x-ray structures exist for transaminases, but these are biased towards the -transaminases. Amination Reactions and Organic Synthesis The growing number of biocatalysts available for amination reactions offer tremendous new opportunities for the development of new synthetic routes with high atom and step economy. In order to take full advantage of the capabilities of these biocatalysts, the the interface between biocatalysis and organic synthesis needs to be explored (Wohlgemuth 2007). This requires the synthesis of new substrates, the development of new analytical methods as well as the discovery of new biocatalytic amination reactions. The power and selectivity of nature’s privileged catalysts will however make this endea-vour a worthwhile effort and will open new synthetic routes to densely functionalized small molecules, where bulky protecting groups may lead to steric crowding and non-selective transformations to product mixtures which are challenging to purify. Both chiral and non-chiral compounds, which are not easily accessible by chemical synthe-sis or would not pass the safety, health and environment filter for industrial large-scale production, are thereby of interest for biocatalytic amination. Fig. (5) shows examples of transamination reactions in retrosynthetic analysis. The presence of amino groups in a number of diverse metabolites and the availability of closely related compounds is another opportunity for the development of novel short amination routes to valuable metabolites. As many of such metabolites are not available

12 Current Organic Chemistry, 2010, Vol. 14, No. 17

as pure product-in-the-bottle, the interaction of biocatalytic amination reactions with organic synthesis can also provide new research reagents and tools (Wohlgemuth 2009). ACKNOWLEDGEMENTS John Ward acknowledges funding from the UK research councils BBSRC (BB/G014426/1) and EPSRC (GR/S62505/01; EP/G005834/1) and from the 12 industrial partners supporting the Bioconversion Integrated with Chemistry and Engineering (BiCE) programme. ABBREVIATIONS Tam

=

Transaminase (or Aminotransferase)

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