Matthias Höhne

Biocatalytic Routes to Optically Active Amines

Optically active amines and amino acids play an important role in the pharmaceutical, agrochemical, and chemical industries. They are frequently used as synthons for the preparation of various pharmaceutically active substances and agrochemicals, but also as resolving agents to obtain chiral carboxylic acids. Consequently, there is a need for efficient methods to obtain the desired enantiomer of a given target structure in optically pure form. Beside a range of chemical methods using for example, asymmetric synthesis with transition metal catalysts, enzymes represent a useful alternative to access this important class of compounds. This review covers biocatalytic approaches using hydrolases (i.e. lipases, amidases), monoamine oxidase and other enzymes. Special focus is given on the application of ω‐transaminases with emphasis on concepts to allow efficient asymmetric synthesis starting from prostereogenic ketones.

Rational assignment of key motifs for function guides in silico enzyme identification

Biocatalysis has emerged as a powerful alternative to traditional chemistry, especially for asymmetric synthesis. One key requirement during process development is the discovery of a biocatalyst with an appropriate enantiopreference and enantioselectivity, which can be achieved, for instance, by protein engineering or screening of metagenome libraries. We have developed an in silico strategy for a sequence-based prediction of substrate specificity and enantiopreference. First, we used rational protein design to predict key amino acid substitutions that indicate the desired activity. Then, we searched protein databases for proteins already carrying these mutations instead of constructing the corresponding mutants in the laboratory. This methodology exploits the fact that naturally evolved proteins have undergone selection over millions of years, which has resulted in highly optimized catalysts. Using this in silico approach, we have discovered 17 (R)-selective amine transaminases, which catalyzed the synthesis of several (R)-amines with excellent optical purity up to >99% enantiomeric excess.

Efficient Asymmetric Synthesis of Chiral Amines by Combining Transaminase and Pyruvate Decarboxylase

Chiral amines and amino acids play an important role in the pharmaceutical, agrochemical and chemical industry. They are frequently used as synthons for the preparation of various pharmaceutically active substances and agrochemicals, or as resolving agents for chiral acids. Consequently, there is a need for efficient methods to obtain the desired R or S enantiomer in an optically pure form. The most frequently used enzymatic method for the production of optically active amines is the kinetic resolution of racemic starting material by enantioselective hydrolysis of, for ACHTUNGTRENNUNGexample, N-acyl amides by peptidases, amidases or lipases. lternatively, transaminases can be used in kinetic resolution (Scheme 1A). The maximum yield in all of these processes is limited to 50% unless a racemization step is included to

Recent achievements in developing the biocatalytic toolbox for chiral amine synthesis.

Novel enzyme activities and chemoenzymatic reaction concepts have considerably expanded the biocatalytic toolbox for chiral amine synthesis. Creating new activities or extending the scope of existing enzymes by protein engineering is a common trend in biocatalysis and in chiral amine synthesis specifically. For instance, an amine dehydrogenase that allows for the direct asymmetric amination of ketones with ammonia was created by mutagenesis of an l-amino acid dehydrogenase. Another trend in chiral amine chemistry is the development of strategies allowing for the synthesis of secondary amines. For example the smart choice of substrates for amine transaminases provided access to secondary amines by chemoenzymatic reactions. Furthermore novel biocatalysts for the synthesis of secondary amines such as imine reductases and Pictet-Spenglerases have been identified and applied. Recent examples showed that the biocatalytic amine synthesis is emerging from simple model reactions towards industrial scale preparation of pharmaceutical relevant substances, for instance, as shown in the synthesis of a Janus kinase 2 inhibitor using an amine transaminase. A comparison of important process parameters such as turnover number and space-time yield demonstrates that biocatalytic strategies for asymmetric reductive amination are maturing and can already compete with established chemical methods.

Rapid and Sensitive Kinetic Assay for Characterization of ω-Transaminases

For the biocatalytic preparation of optically active amines, omega-transaminases (omega-TA) are of special interest since they allow the asymmetric synthesis starting from prostereogenic ketones with 100% yield. To facilitate the purification and characterization of novel omega-TA, a fast kinetic assay was developed based on the conversion of the widely used model substrate alpha-methylbenzylamine, which is commonly accepted by most of the known omega-TAs. The product from this reaction, acetophenone, can be detected spectrophotometrically at 245 nm with high sensitivity (epsilon = 12 mM(-1) cm(-1)), since the other reactants show only a low absorbance. Besides the standard substrate pyruvate, all low-absorbing ketones, aldehydes, or keto acids can be used as cosubstrates, and thus the amino acceptor specificity of a given omega-TA can be obtained quickly. Furthermore, the assay allows the fast investigation of enzymatic properties like pH and temperature optimum and stability. This method was used for the characterization of a novel omega-TA cloned from Rhodobacter sphaeroides, and the data obtained were in excellent accordance with a standard capillary electrophoresis assay.

Bioinformatic analysis of a PLP-dependent enzyme superfamily suitable for biocatalytic applications

In this review we analyse structure/sequence-function relationships for the superfamily of PLP-dependent enzymes with special emphasis on class III transaminases. Amine transaminases are highly important for applications in biocatalysis in the synthesis of chiral amines. In addition, other enzyme activities such as racemases or decarboxylases are also discussed. The substrate scope and the ability to accept chemically different types of substrates are shown to be reflected in conserved patterns of amino acids around the active site. These findings are condensed in a sequence-function matrix, which facilitates annotation and identification of biocatalytically relevant enzymes and protein engineering thereof.

Revealing the Structural Basis of Promiscuous Amine Transaminase Activity

Enzymes are nature’s tool to speed up biochemical reactions that would otherwise hardly proceed. As a result of the complexity of the metabolic network of living cells, enzymes have to fulfill partially competing demands. On the one hand, a high precision in terms of reaction and substrate specificity is often required. This is achieved, for example, by a fine-tuned interaction between the active site structure and the substrate, which forces a defined positioning of the substrates’ reactive groups relative to the catalytic residues. On the other hand, some enzymes have evolved with a relaxed substrate specificity, which is useful and economic in several metabolic tasks: For example, a single enzyme can thus break down various chemically similar nutrients or transform different poisonous compounds for their detoxification. The ability to catalyze more than one chemical transformation within a single active site, called promiscuity, is not only an important property of some enzymes, but it is also recognized as a key mechanism of evolution and thus facilitates adaptation of enzymes to new metabolic needs. Furthermore, the enzymes’ ability to take over more than one physiological function renders metabolism more plastic, even if the promiscuous “underground activities” are very weak. Substrate d] and reaction promiscuity are also often the reasons why enzymes can be applied in biocatalysis. Many fine chemicals that are synthesized by biocatalytic transformations are not produced in nature, but nevertheless, enzymes that can be applied in their syntheses have been identified. For example, pyruvate decarboxylase can be used for the production of (R)-phenylacetylcarbinol. Esterases and lipases are successfully used in organic synthesis for the preparation of various optically active alcohols, acids, and amines. Their natural function, however, might be totally different, as shown for pig liver esterase, which plays a role in signal transduction by ester cleavage of methylated and prenylated proteins. Still, for most enzymes their promiscuous potential has not yet been revealed. One reason is that, after the main activity and possible function has been identified, further in-depth characterization is often not performed. Furthermore, if the promiscuous activity is unrelated to the natural one, a general activity screen is very laborious. Therefore, the understanding of the molecular principles of promiscuity is a challenging frontier in biocatalysis, as the knowledge obtained can be used as a starting point for protein engineering or for the discovery of other promiscuous enzymes. In a recent study, we identified four crystal structures [protein database (PDB) codes: 3HMU, 3I5T, 3FCR, and 3GJU] of transaminases with unknown function, and we could show that these enzymes interconvert small amino acids, for example, alanine (1) and succinic semialdehyde (2, Scheme 1). Additionally, chiral amines can be transaminated, but with very different efficiencies that range from 0.4 to 80 % relative activity, compared with the transamination of 1 + 2 (see values given in Scheme 1). From these observations, we concluded that the conversion of amines is a type of substrate promiscuity, which is pronounced to varying degrees in these four enzymes. Transaminases showing this type of catalytic activity are designated amine transaminases (ATAs): In contrast to the ubiquitous a-amino acid transaminases, they accept substrates lack-

Connecting Unexplored Protein Crystal Structures to Enzymatic Function

Biocatalysis has emerged as an important alternative to traditional chemical synthesis for the preparation of fine chemicals, as recently demonstrated by the biocatalytic manufacture of the drug sitagliptin by using an (R)-amine transaminase (ATA) created by intensive protein engineering. This process was superior with respect to optical purity, yield, and waste generation to the already established transition-metal-catalyzed production of sitagliptin. Process development, identification, and optimization of an appropriate enzyme represent inportant requirements to obtain a successful and efficient enzyme-catalyzed process. Nature has a rich reservoir from which a suitable enzyme can be found as a starting point. In contrast to classical approaches such as screening of strain collections, modern developments in the area of metagenomics offer enormous potential to screen for activity within nonculturable biodiversity. A major advancement was the development of next-generation sequencing techniques, which led to a substantial increase in the genetic information deposited in public databases; currently >20 million protein sequences are waiting to be explored. This development also led to the accumulation of protein sequences with unknown (or wrongly annotated) function, and thus a large part of this rich resource cannot be used reliably. We recently took advantage of this information and developed an in silico enzyme discovery strategy and identified 17 novel and unique (R)-selective ATAs in >5000 sequences deposited in public databases, although no gene or protein sequence was described in the literature ahead of our work. Furthermore, we could show that these (R)ATAs are synthetically useful, as demonstrated recently in the asymmetric synthesis of a set of 12 chiral amines by using 7 out of the 17 enzymes. Modern protein engineering methods rely on high-quality structural information as obtained by X-ray crystallography, which is then the basis for focused, directed evolution to improve the properties of the enzyme. Similar to developments in the genomics area, in the last years automated highthroughput methods were developed for crystallization and determination of crystal structures, which has resulted in a rapid increase in the number of solved protein structures. Still, for many enzymes no structure is available, as for example, (R)and (S)-selective ATAs, which have become very popular in the last years. Hence, structural information is highly desired to understand experimental results and to guide protein engineering. Interestingly, a trend similar to that pointed out above for the discovery of sequences exists: there is a growing number of crystal structures of enzymes that were never characterized with respect to substrate scope, enantioselectivity, or reaction specificity, and their physiological functions are also often unknown. Similar to the lack of experimental validation of annotated proteins in sequence databases, crystallized proteins with unknown function are only rarely characterized. Thus, many structures remain unexplored: For example, from 104 crystal structures of different transaminases found in the Brookhaven protein database (PDB), 46 structures do not have a literature citation that provides a description of the structure or the characterization data of the protein. Connecting the enzymatic function to unexplored proteins in the PDB could provide information that would be of diverse interest. These include protein engineering in the context of biotechnology or biochemical studies to explore enzyme mechanism. Herein we explore the crystal structures with unknown functions in the cluster of “ornithine-aminotransferase (OAT)-like proteins” (cd00610 of the NCBI conserved domain database) deposited in the PDB database. OAT are pyridoxal-5’-phosphate (PLP) dependent enzymes; they belong to PLP fold class I, which represent a very large and diverse superfamily. In the OAT subfamily, eight different enzyme activities are known (Table S1, Supporting Information). All 58 available 3D structures of this cluster show considerable similarity, but they are different in important residues in the active site that are obviously involved in substrate recognition. This search identified four structures (PDB codes: 3HMU, 3I5T, 3FCR, 3GJU), for which we could not find any information associated with their structures or functions. Additionally, these structures differed in im[a] F. Steffen-Munsberg, Dr. C. Vickers, A. Thontowi, Dr. S. Sch tzle, T. Tumlirsch, Prof. Dr. U. T. Bornscheuer, Prof. Dr. M. Hçhne Institute of Biochemistry Felix-Hausdorff-Str. 4, 17487 Greifswald (Germany) Fax: (+ 49) 3834-86-794367 E-mail : matthias.hoehne@uni-greifswald.de uwe.bornscheuer@uni-greifswald.de [b] Dr. M. Svedendahl Humble Dept. of Organic Chemistry Arrhenius Laboratories Stockholm University, SE-106 91 Stockholm (Sweden) [c] H. Land, Prof. Dr. P. Berglund School of Biotechnology KTH Royal Institute of Technology AlbaNova University Center, 106 91 Stockholm (Sweden) Supporting information for this article contains the full experimental details and is available on the WWW under http://dx.doi.org/10.1002/ cctc.201200544.

Engineering the Active Site of the Amine Transaminase from Vibrio fluvialis for the Asymmetric Synthesis of Aryl–Alkyl Amines and Amino Alcohols

Although the amine transaminase from Vibrio fluvialis has often been applied as a catalyst for the biocatalytic preparation of various chiral primary amines, it is not suitable for the transamination of α‐hydroxy ketones and aryl‐alkyl ketones bearing an alkyl substituent larger than a methyl group. We addressed this problem through a systematic mutagenesis study of active site residues to expand its substrate scope towards two bulky ketones. We identified two mutants (F85L/V153A and Y150F/V153A) showing 30‐fold increased activity in the conversion of (S)‐phenylbutylamine and (R)‐phenylglycinol, respectively. Notably, they facilitated asymmetric synthesis of these amines with excellent enantiomeric purities of 98 % ee.

Asymmetric Reductive Amination of Ketones Catalyzed by Imine Reductases

Biocatalysis employing imine reductases is a promising approach for the one‐step generation of chiral amines from ketones. The enzymes reported for this process suffer from low activity and moderate stereoselectivity. We identified a set of enzymes that facilitate this reaction with high to quantitative conversions from a library of 28 imine reductases. This enabled the conversion of ketones with ammonia, methylamine, or butylamine into the corresponding amines. Most importantly, we performed preparative (>100 mg) scale syntheses of amines such as (1S,3R)‐N,3‐dimethylcyclohexylamine and (R)‐N‐methyl‐2‐aminohexane with excellent stereochemical purities (98 % de, 96 % ee) in good yields.