Amine Transaminases in Multi-Step One-Pot Reactions

Mattias Anderson

KTH Royal Institute of Technology

School of Biotechnology

Stockholm 2017

Stockholm 2017 KTH Royal Institute of Technology School of Biotechnology Division of Industrial Biotechnology AlbaNova University Center SE-106 91 Stockholm Sweden

Paper I: © 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Paper II: © 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Paper IV: Reprinted with permission from ACS Catal. 2016, 6, 3932−3940. © 2016 American Chemical Society.

Printed by Universitetsservice US-AB Drottning Kristinas väg 53B SE-114 28 Stockholm Sweden

ISBN 978-91-7729-254-8 TRITA-BIO Report 2017:3 ISSN 1654-2312

Front Cover: Graphic interpretation of how amine transaminases (ATAs) can be combined with other enzymes and metal catalysts in one reaction pot to perform multi-step reactions. In this thesis, the combination of a palladium catalyst, an amine transaminase and a lipase was used for the synthesis of capsaicinoids, spicy compounds found in chili peppers.

Abstract

Amine transaminases are enzymes that catalyze the mild and selective formation of primary amines, which are useful building blocks for biologically active compounds and natural products. In order to make the production of these kinds of compounds more efficient from both a practical and an environmental point of view, amine transaminases were incorporated into multi-step one-pot reactions. With this kind of methodology there is no need for isolation of intermediates, and thus unnecessary work-up steps can be omitted and formation of waste is prevented. Amine transaminases were successfully combined with other enzymes for multi-step synthesis of valuable products: With ketoreductases all four diastereomers of a 1,3-amino alcohol could be obtained, and the use of a lipase allowed for the synthesis of natural products in the form of capsaicinoids. Amine transaminases were also successfully combined with metal catalysts based on palladium or copper. This methodology allowed for the amination of alcohols and the synthesis of chiral amines such as the pharmaceutical compound Rivastigmine. These examples show that the use of amine transaminases in multi-step one-pot reactions is possible, and hopefully this concept can be further developed and applied to make industrial processes more sustainable and efficient in the future.

Keywords Biocatalysis, enzyme, amine transaminase, ω-transaminase, amination, primary amine, chiral amine, chemoenzymatic, green chemistry, synthesis, cascade

Populärvetenskaplig sammanfattning

Aminer är kemiska föreningar som fyller många viktiga funktioner i naturen. Denna typ av molekyl kan även framställas syntetiskt och användas som exempelvis läkemedel eller byggsten till läkemedel. Aminer framställs normalt vid närvaro av en katalysator, vars uppgift är att få reaktionen att gå snabbare och mer kontrollerat. Dessa katalysatorer är ofta tungmetaller, men ett alternativ är att istället använda naturens egna katalysatorer, enzymer. Enzymer är proteiner och är således ofarliga och biologiskt nedbrytbara. En annan fördel med enzymer är att de vanligen fungerar bäst i vatten vid nära rumstemperatur, till skillnad från metallkatalysatorer som ofta behöver högre temperaturer, högre tryck samt organiska lösningsmedel. Amintransaminaser är enzymer som återfinns naturligt i exempelvis bakterier, där de katalyserar framställning och nedbrytning av aminer. Amintransaminaser kan utvinnas ur bakterier som odlats på laboratorium, och kan sedan användas till syntetisk framställning av aminer.

Intressanta kemiska produkter såsom läkemedel framställs ofta i många steg. Vanligtvis kräver de olika stegen olika katalysatorer, reagenser och lösningsmedel. Detta innebär att efter varje steg krävs en upparbetning för att få bort de oönskade komponenterna, vilket resulterar i mer arbete och mer kemiskt avfall. För att kringgå detta problem kan man utföra flera steg i samma reaktionskärl utan någon upparbetning mellan stegen. Denna metod kräver dock att komponenterna i de olika stegen är kompatibla med varandra.

Arbetet i denna avhandling har fokuserat på att använda amintransaminaser för flerstegsreaktioner där alla stegen utförs i samma reaktionskärl. Denna typ av reaktion kan förhoppningsvis på sikt bidra till mer effektiv, miljövänlig och hållbar produktion av exempelvis läkemedel och naturprodukter. I detta syfte har våran forskningsgrupp lyckats kombinera amintransaminaser med andra enzymer såsom ketoreduktaser och lipaser. Med dessa metoder kunde vi framställa värdefulla kemiska byggstenar i form av kirala aminoalkoholer. Dessutom kunde vi framställa capsaicinoider, kryddiga ämnen från chilipeppar som kan användas i exempelvis smärstillande salvor. Vi har även framgångsrikt kombinerat amintransaminaser med metallkatalysatorer baserade på palladium eller koppar, och på så sätt lyckats aminera alkoholer och framställa kirala

iii

aminer såsom läkemedelsföreningen Rivastigmin, som används för behandling av Alzheimers och Parkinsons sjukdomar. Med dessa exempel har vi visat att det är möjligt att effektivt använda amintransaminaser för flerstegsreaktioner i ett reaktionskärl, och förhoppningsvis kan detta koncept vidareutvecklas och bidra till effektivare och mer hållbar kemisk industri i framtiden.

List of appended papers

Paper I Kohls H., Anderson M., Dickerhoff J., Weisz K., Córdova A., Berglund P., Brundiek H., Bornscheuer U. T., Höhne M. “Selective Access to All Four Diastereomers of a 1,3-Amino Alcohol by Combination of a Keto Reductase- and an Amine Transaminase” Adv. Synth. Catal. 2015, 357, 1808-1814.

Paper II Anderson M., Afewerki S., Berglund P., Córdova A. “Total Synthesis of Capsaicin Analogues from Lignin-Derived Compounds by Combined Heterogeneous Metal, Organocatalytic and Enzymatic Cascades in One Pot” Adv. Synth. Catal. 2014, 356, 2113-2118.

Paper III Anderson M., Afewerki S., Berglund P., Córdova A. “Chemoenzymatic amination of alcohols by combining oxidation catalysts with transaminases in one pot” Manuscript.

Paper IV Palo-Nieto C., Afewerki S., Anderson M., Tai C.-W., Berglund P., Córdova A. “Integrated Heterogeneous Metal /Enzymatic Multiple Relay Catalysis for Eco-Friendly and Asymmetric Synthesis” ACS Catal. 2016, 6, 3932-3940.

Contributions to appended papers

Paper I Designed and performed the initial screening of amine transaminases for acceptance of the hydroxy ketones BAHK and VAHK. Contributed to the writing of the manuscript.

Paper II Designed and performed the amination experiments. Performed most of the writing.

Paper III Designed and performed the amination experiments, starting from oxidation products. Performed most of the writing.

Paper IV Designed and performed the kinetic resolution experiments where an amine transaminase was involved, starting from racemic amine intermediates.

Papers not included in this thesis

Scheidt T., Land H., Anderson M., Chen Y., Berglund P., Yi D., Fessner W.-D. “Fluorescence-Based Kinetic Assay for High-Throughput Discovery and Engineering of Stereoselective ω-Transaminases” Adv. Synth. Catal. 2015, 357, 1721-1731.

vii

List of abbreviations

ATA Amine transaminase BAHK Benzaldehyde-acetone hydroxy ketone Cv-ATA Chromobacterium violaceum amine transaminase ee Enantiomeric excess DABCO 1,4-Diazabicyclo[2.2.2]octane FDH Formate dehydrogenase GDH Glucose dehydrogenase HIV Human immunodeficiency virus KRED Ketoreductase L-ADH L-Alanine dehydrogenase LDH Lactate dehydrogenase NAD+ Nicotinamide adenine dinucleotide, oxidized form NADH Nicotinamide adenine dinucleotide, reduced form PAHK Phenylacetaldehyde-acetone hydroxy ketone Pd(0)-CPG Palladium(0) on controlled pore glass Pd(0)-MCF Palladium(0) on mesocellular foam PDC Pyruvate decarboxylase PLP Pyridoxal 5’-phosphate PMP Pyridoxamine 5’-phosphate TA Transaminase TEMPO 2,2,6,6-Tetramethylpiperidine 1-oxyl THF Tetrahydrofurane VAHK Vanillin-acetone hydroxy ketone

viii

Table of contents

Abstract ...... i Populärvetenskaplig sammanfattning ...... ii List of appended papers ...... iv Contributions to appended papers ...... v Papers not included in this thesis...... vi List of abbreviations ...... vii Table of contents ...... ix 1 Introduction ...... 1 1.1 Amines, their importance and how they are made ...... 1 1.2 Amine transaminases ...... 5 1.3 Amine transaminases and green chemistry ...... 10 2 Aim ...... 13 3 Present investigation ...... 15 3.1 Preparation of pharmaceutical synthons and natural products (Papers I and II) ...... 15 3.2 Alternative starting materials for amine transaminases (Papers II and III) ...... 23 3.3 Amine transaminases in one-pot amination/kinetic resolution reactions (Paper IV) ...... 30 4 Discussion and conclusion ...... 41 5 Acknowledgements ...... 47 6 Bibliography ...... 48

Introduction |1

1 Introduction

1.1 Amines, their importance and how they are made

The amine functional group is of great importance in nature. The simplest amines, primary amines, have many different functions and can serve as building blocks for other functional groups and larger molecules (Figure 1). Dopamine, a primary amine, is an important neurotransmitter. The amino acid lysine has a primary amine side chain, which can act as an acid/base catalyst. Primary amines can also form amides together with carboxylic acids; this is how amino acids are linked together to form proteins. Capsaicin, a compound responsible for the spicy taste of chili peppers, is an amide formed from a primary amine and a fatty acid. Alkylations of primary amines give secondary amines such as the hormone adrenaline, tertiary amines such as the analgesic morphine or even quaternary ammonium cations such as the neurotransmitter acetylcholine.

Figure 1: A selection of important natural amines and amine-derived compounds.

2| Introduction

Given the diverse functions of naturally occurring amines, it is not surprising that many synthetic bioactive compounds and pharmaceuticals contain the amine functionality as well. A large number of the top 200 best- selling drugs in 2013 are either primary amines or use primary amines as building blocks1. Many of these amines are also chiral. The synthesis of primary amines, especially chiral ones, is thus of great importance.

Non-chiral primary amines are accessible through reductive amination of aldehydes, which can be accomplished for example by formation of an intermediate oxime with hydroxylamine which can be subsequently reduced to the corresponding amine product2 (Figure 2). If the starting material is a prochiral ketone instead of an aldehyde, the product is a chiral amine. However, in most cases a single product enantiomer is desired, and the challenge is then to find a method that gives a high enantiomeric excess of the product. Typical such methods are enantioselective hydrogenations of imines, enamines or enamides, although many other methods exist3-6. Though these methods are widely used, they are not without drawbacks. The hydrogenation step requires hydrogen gas under high pressure as well as metal catalysts such as ruthenium, rhodium or iridium which have to be removed from the final product. Also, the enantiomeric excess obtained is often not high enough and additional steps are then required.

Figure 2: General synthesis of primary amines from carbonyl compounds.

Introduction |3

Biocatalysis7-15 is an interesting alternative to metal-catalyzed hydrogenation methods for the synthesis of primary amines, since it has the potential to be both more enantioselective and more environmentally friendly. Kinetic resolution of racemic amines using lipases is a well- established method which has been used in industry for many years now15- 18. Starting from a prochiral ketone, the racemic amine can be obtained for example by reductive amination. Subsequent kinetic resolution with lipases gives the amide, which can be converted to the desired amine enantiomer by a deprotection step (Figure 3). An alternative is to use a transaminase (TA)14-15, 19-26 to resolve the racemic amine. Transaminases catalyze the transfer of an amine group to a carbonyl compound. With this

Figure 3: Schematic representation of biocatalytic primary amine synthes is with either lipases or transaminases (TAs) starting from carbonyl compounds.

4| Introduction

method, one enantiomer of the racemic amine is selectively converted back to the ketone, and there is no need for a deprotection step to obtain the final product (Figure 3). However, a major drawback of these kinds of kinetic resolution processes is that they are limited to a maximum theoretical yield of 50%. There are ways of circumventing this: for example, the ketone coproduct obtained in the transaminase process can be reused to make more racemic amine, or a dynamic kinetic resolution process could be used instead, but the most efficient method would be asymmetric synthesis of the desired amine enantiomer directly from the corresponding prochiral ketone (Figure 3). A highly impactful such method was demonstrated in 2010, where the pharmaceutical Sitagliptin (ranked number 17 in worldwide drug sales in 2013 1) was synthesized using a transaminase6 (Figure 4). Sitagliptin contains a chiral primary amine function, which was previously made by asymmetric hydrogenation of the corresponding enamine with a rhodium catalyst and high pressure hydrogen gas, resulting in an enantiomeric excess of 97%. With the transaminase, enantiomeric excess was increased to 99.95%, the yield and productivity was higher, the total amount of waste was reduced and there was no longer any need for heavy metals or high pressure hydrogen gas6. This example clearly demonstrates the usefulness of transaminases.

Figure 4: Sitagliptin, an important primary amine pharmaceutical.

Introduction |5

1.2 Amine transaminases

Transaminases catalyze the transfer of an amino group from an amine (the amine donor) to a carbonyl compound (the amine acceptor). These enzymes are dependent on the cofactor pyridoxal 5'-phosphate (PLP, the active form of vitamin B6) (Figure 5), and the transamination follows a ping-pong bi-bi reaction mechanism26-29 (Figure 6). In the first half of the reaction, the amine donor enters and forms an imine with PLP. The imine tautomerizes, assisted by a catalytic lysine residue and the pyridine ring of PLP acting as an electron sink. Hydrolysis of this second imine gives the ketone product and leaves the cofactor in its amine form, pyridoxamine 5'- phosphate (PMP). In the second step, the amine acceptor enters and the same steps take place in reverse: an imine is formed between PMP and the amine acceptor, the imine tautomerizes and is attacked by the catalytic lysine residue to give the amine product and the cofactor in its original form, PLP, covalently bound to the lysine.

Figure 5: The structure of the transaminase cofactor pyridoxal 5'-phosphate (PLP).

Transaminases are classified as EC 2.6.1.X, where the last number indicates what kind of substrate each transaminase acts on. Many transaminases such as aspartate transaminase (EC 2.6.1.1) and alanine transaminase (EC 2.6.1.2) act specifically on α-amino acids, and due to their limited substrate scope these α-transaminases are not very useful for synthetic applications. Transaminases which accept substrates where the carboxyl group is more distant from the amine or which lack a carboxyl group altogether are much more interesting: These enzymes are called ω- transaminases (for their ability to accept ω-amino acids as substrates) or amine transaminases (ATAs, for their ability to accept amine substrates as opposed to only amino acids)14-15, 19-26.

6| Introduction

Figure 6: The mechanism for a transaminase half reaction: An amine donor (left) enters and the amine group is transferred to PLP to give PMP. The second half of the reaction follows the same steps in reverse: The amine group of PMP is transferred to an amine acceptor to give PLP (covalently bound to lysine) and the amine product.

Amine transaminases often show excellent enantioselectivity, making them very attractive for use in synthetic applications6, 30-34. Until recently most known amine transaminases were (S)-selective, but now a number of

Introduction |7

(R)-selective transaminases have been discovered and made commercially available35. The enantioselectivity can be explained by the layout of the enzyme active site, which is typically composed of one large and one small binding pocket36-41, as shown in Figure 7 for an (S)-selective amine transaminase. The group in the small binding pocket should preferably not be larger than methyl while the large pocket generally prefers aromatic or carboxyl groups36-41. (S)-1-Phenylethylamine thus fits very well into this active site. (R)-1-Phenylethylamine on the other hand does not fit: either the amine group would be pointing in the wrong direction, or the phenyl group would need to go in the small binding pocket. This explains the high selectivity shown by amine transaminases. The small binding pocket does however limit the substrate scope of these enzymes, but this can be solved with enzyme engineering. An example of this is the previously mentioned process for synthesis of Sitagliptin. This molecule has large groups on both sides of the amine, but the enzyme was engineered to accept it as a substrate through several rounds of mutations6.

Figure 7: Schematic representation of the binding pockets of an ( S)-selective amine transaminase. The figure shows the substrate (S)-1-phenylethylamine bound to the cofactor PLP.

Synthesis of amines with amine transaminases is in theory very simple. The carbonyl starting material reacts with a preferably cheap amine donor to give the amine and a carbonyl coproduct. The reactions can be run in water and room temperature which is excellent from an environmental point of view. However, in reality things are not always that simple. Since the transamination reaction is reversible, it is important that the equilibrium is shifted towards the products in order to give a high yield.

8| Introduction

Unfortunately, for many synthetically useful transaminations the reactants are heavily favored. This is especially problematic when the starting material is a conjugation stabilized ketone such as acetophenone; The equilibrium constant for the transamination of alanine and acetophenone to form 1-phenylethylamine has been reported42-43 to be 8.8*10-4 (Figure 8).

Figure 8: The equilibrium for amine transaminase-catalyzed asymmetric synthesis often favors the starting materials. This is especially true when the starting material is a conjugated ketone such as acetophenone: In this example the equilibrium constant was reported 42- 43 to be 8.8*10 - 4 .

If the desired product is an enantiomerically pure chiral amine, an alternative is to run the reaction in the thermodynamically favored direction as a kinetic resolution. While the maximum theoretical yield for such a reaction is only 50%, it can still be useful if the equilibrium heavily favors the starting material in the asymmetric synthesis direction. Also, the ketone coproduct formed in a kinetic resolution can be reused to make more amine34. However, a functioning asymmetric synthesis is the more efficient method, and there are a number of ways to overcome the equilibrium problems14-15, 21, 24. One of the simplest ways is to add a large excess of the amine donor, but this approach leads to poor atom economy. There is also the possibility to remove the formed coproduct. For example if isopropylamine is used as amine donor, the coproduct is acetone which can be removed from the reaction by evaporation. A more elegant solution to the equilibrium problem is to use an enzyme cascade, adding another enzyme to remove the coproduct (Figure 9). For example, if the amine

Introduction |9

donor is alanine, the keto acid pyruvate will be formed as a coproduct. Pyruvate can then be decarboxylated by the enzyme pyruvate decarboxylase (PDC) thus shifting the reaction equilibrium towards product formation44. In a similar fashion, a dehydrogenase enzyme (such as lactate dehydrogenase (LDH) for pyruvate or alcohol dehydrogenase for acetone) can be added to convert the coproduct to the corresponding alcohol42, 45. This reaction requires stochiometric amounts of the reducing agent nicotinamide adenine dinucleotide (NADH), and due to NADH being relatively expensive another enzyme is usually added to regenerate it. Examples of such enzymes are formate dehydrogenase (FDH), which regenerates NADH by oxidizing formate to carbon dioxide, and glucose dehydrogenase (GDH), which regenerates NADH by oxidizing glucose45-47.

Figure 9: Unfavorable equilibria in amine transaminase (ATA) catalyzed asymmetric syntheses can be shifted towards the product side by the use of additional enzymes to remove the coproduct. In this example the coproduct is pyruvate, which can be removed with enzymes such as pyruvate decarboxylase (PDC), L-alanine dehydrogenase ( L-ADH) or lactate dehydrogenase (LDH).

10| Introduction

A third enzymatic method to shift the equilibrium is to use a cascade where the amine donor is recycled. For example, if the amine donor is L-alanine, the pyruvate coproduct can be converted back to L-alanine with L-alanine dehydrogenase (L-ADH) and ammonia46-47. This enzyme also requires NADH and is therefore usually combined with one of the above mentioned enzymes for NADH regeneration. Thus the overall reaction works as a reductive amination, where a ketone is converted to an amine with ammonia and formate or glucose as reducing agent.

Recently, methods have been developed where the equilibrium is displaced simply by a smart choice of amine donor48-52. For example, when diamines are used as amine donors the co-products formed can spontaneously cyclize and form aromatic compounds, thus shifting the overall equilibrium to the product side49-52. These methods are very promising since neither a large excess of amine donor nor additional enzymes are needed.

1.3 Amine transaminases and green chemistry

Amine transaminase catalysis (and biocatalysis in general) is often advertised as being green or environmentally friendly. Indeed, looking at the twelve principles of green chemistry amine transaminases seem like an attractive alternative53-56. The transaminases are non-toxic, biodegradable and made from renewable resources. The reactions can be run at ambient temperature and pressure in water at close to neutral pH. Also, the enantiomeric excess of the product amine is often very high, so no further steps are required to enhance the quality of the product.

However, amine transaminase reactions come with environmental drawbacks as well57-58. Many interesting amine transaminase substrates are poorly soluble in water, leading to very low substrate concentrations in these reactions. Low substrate concentration leads to large reaction volumes and thus large amounts of waste, and while much of the waste is water, it still needs to be treated if it has been contaminated with other compounds. In addition, as described previously, the unfavorable equilibrium in many amine transaminase-catalyzed reactions necessitates the use of excess amine donor, and this leads to poor atom economy. If further steps are required after the transamination to reach the final product these will often require non-aqueous solvents. This is unfortunate

Introduction |11

when aqueous conditions are used for the transamination reaction since water has a high heat of vaporization and boiling point compared to other common solvents, making it complicated and costly to remove15. Some of these problems could potentially be circumvented by performing amine transaminase reactions in organic solvents. As with most enzymes this has a negative effect on the activity59-61, but with reaction engineering or immobilization methods amine transaminases can be made more active and stable under such conditions62-64. Still, this removes the positive aspects of using water as a non-toxic and renewable solvent65-66. It also complicates matters if an enzyme cascade is used, as all the enzymes involved would need to be active in the organic solvent. The above- mentioned enzymes for equilibrium displacement (Figure 9) also require nicotinamide cofactors and/or amino acid substrates which would not be soluble under such conditions. Overall, running amine transaminase reactions in organic solvents offers some advantages, but there are clearly some drawbacks as well. Finally, when evaluating the environmental aspect of using amine transaminase catalysis it is important to consider the enzyme production process57. Enzymes are made by fermentation, which requires heating and generates large amounts of liquid waste contaminated with microorganisms and antibiotics. Purification of the enzyme requires chromatography which generates even more liquid waste. To sum up, one should be careful when calling an amine transaminase- catalyzed reaction green or environmentally friendly before the environmental impact of the process has been quantified by for example a life-cycle assessment57.

12| Aim

Aim |13

2 Aim

As shown in the Introduction, amine transaminases are attractive catalysts for use in synthetic chemistry. However, the green chemistry aspect is one of the major selling points for enzyme catalysis, and as described in Section 1.3 there are some serious concerns when using amine transaminases, especially with waste formation. Our research group aimed to improve amine transaminase reactions from a green chemistry point of view, by designing the reactions to prevent unnecessary formation of waste. We were mainly interested in using amine transaminase catalysis for the synthesis of valuable products such as pharmaceuticals and other biologically active compounds. These kinds of compounds are often synthesized in several steps, where only one step would be catalyzed by the amine transaminase. The intermediates in these kinds of multi-step reactions are typically isolated after each step to change solvent and avoid compatibility issues, but this results in yield loss, additional work and the formation of unnecessary waste. However, if it is possible to perform several reaction steps in the same reaction vessel without isolation of intermediates, this problem can be circumvented57. The use of enzymes in such processes has been a hot research topic in the last few years, and many examples have been demonstrated, both where the reaction steps are performed simultaneously (cascades)67-100 or where the steps are performed in sequence95-119. However, at the time we began our work only a few such examples involving amine transaminases were known34, 60, 120- 122. For this reason, our goal was to develop methods for performing multi- step reactions involving amine transaminase catalysis in one pot. Such methodology would both prevent the formation of waste and make the overall process more efficient by removing the need for unnecessary isolation steps, and thus the use of amine transaminases in multi-step one- pot reactions would be a valuable tool in synthetic chemistry.

14| Present investigation

Present investigation | 15

3 Present investigation

3.1 Preparation of pharmaceutical synthons and natural products (Papers I and II)

We identified two groups of compounds as interesting targets for multi- step one-pot processes involving amine transaminase catalysis: Chiral 1,3- amino alcohols, which are interesting synthons for pharmaceuticals, and capsaicinoids, the compounds responsible for the spicy taste of chili peppers.

Chiral 1,3-amino alcohols are compounds that contain both an alcohol and an amine stereocenter; important examples of such compounds include Ritonavir and Lopinavir which are used for treatment of HIV123-124. Assembly of the chiral 1,3-amino alcohol motif requires two important steps, synthesis of the chiral amine group and synthesis of the chiral alcohol group. This can be accomplished for example by asymmetric synthesis of amino ketones or hydroxy imines followed by stereoselective reduction of either the ketone or imine125-128, although many other methods exist129-131. We envisioned an alternative synthesis route where an amine transaminase is instead used to form the amine stereocenter, since this could in theory give access to chiral 1,3-amino alcohols in high enantiomeric excess using mild reaction conditions, without the need for protecting groups, harmful reagents, metal catalysts or high pressure hydrogen gas. In line with our overall goal of improving the efficiency and preventing the formation of waste in amine transaminase processes, we also wanted to combine the amination step with the alcohol forming step in one pot if possible. Such a method was recently demonstrated for the synthesis of chiral 1,2-amino alcohols30. Here, the authors used the enzyme pyruvate decarboxylase for asymmetric synthesis of hydroxy ketones, which were subsequently converted to the corresponding chiral 1,2-amino alcohols with an amine transaminase.

Our initial idea was to use an organocatalyzed aldol reaction for the asymmetric synthesis of hydroxy ketones. Such reactions can be performed with amino acid catalysts such as alanine under mild conditions132-134 compatible with amine transaminases which could make it possible to form the chiral amine group in the same pot. Several amine transaminases were screened for activity towards the racemic benzaldehyde-acetone hydroxy

16| Present investigation

ketone (BAHK) and vanillin-acetone hydroxy ketone (VAHK) shown in Figure 10, which were prepared with an aldol reaction, but unfortunately no conversion to amino alcohol was observed for any of these enzymes. We hypothesized that the reaction equilibrium might be heavily shifted towards the hydroxy ketone side due to a stabilizing intramolecular hydrogen bond between the alcohol hydrogen and the keto group. The amine donor alanine was used in large excess to try to shift the equilibrium, but it was found to promote the retro-aldol reaction, degrading the hydroxy ketones to benzaldehyde and vanillin respectively. Since these compounds are substrates for the amine transaminases, the products obtained from these reactions were benzylamine and vanillylamine. The same results were obtained when an enzyme cascade system with an alanine dehydrogenase was used for equilibrium displacement. To avoid the retro- aldol reaction the amine donor was changed to 1-phenylethylamine, but still no amino alcohol was formed. Our next idea was to destabilize the hydroxy ketone by protecting the alcohol with an acetyl group, but the protected hydroxy ketone was not converted to the amine either. Finally, we decided to try the reaction in the thermodynamically favored direction. We synthesized the amino alcohol corresponding to BAHK (Figure 10) and attempted to convert it to the hydroxy ketone with amine transaminase catalysis, but this was also unsuccessful and we concluded that the investigated compounds simply were not accepted as substrates for the enzymes tested.

We decided to investigate the phenylacetaldehyde-acetone hydroxy ketone (PAHK) shown in Figure 10 as an alternative substrate, and this time we found several transaminases which were able to convert it to the corresponding amino alcohol. The most promising enzymes were the (S)- selective amine transaminase from Chromobacterium violaceum (Cv- ATA) and the (R)-selective amine transaminase ATA-025 (from Codexis®). The fact that these two enzymes are enantiocomplementary means that both the (S)- and (R)-amine products can be obtained with this method. Unfortunately we were not able to synthesize PAHK with an aldol reaction, and thus another method was needed to prepare this compound in high enantiomeric excess.

Ketoreductases (KREDs) are enzymes which catalyze the reduction of ketones to alcohols, or if the reaction is run in the opposite direction, the oxidation of alcohols to ketones. Thus with an enantioselective KRED, the

Present investigation | 17

Figure 10: Three different hydroxy ketones (top) were investigated as starting materials for the synthesis of 1,3-amino alcohols (middle) (Paper I). Important intermediates and byproducts are also shown (bottom). enantiomers of PAHK could be obtained by kinetic resolution of the racemate, or by asymmetric synthesis starting from the corresponding diketone (shown as intermediate in Figure 10). However, the KRED needs to act selectively on the group closest to the ring to avoid formation of the diol or the hydroxy ketone regioisomer byproducts shown in Figure 10. We screened 22 KREDs from Codexis®, and five of these enzymes were able to oxidize the alcohol group of PAHK. All these enzymes were (S)-selective, and thus in a kinetic resolution of the racemate the (S)-enantiomer was consumed, leaving behind (R)-PAHK as the product with an enantiomeric excess of 89% at 50% conversion for the best enzyme (KRED-P1-B10). The (S)-enantiomer was available through asymmetric synthesis with the same enzyme starting from the diketone. The enantiomeric excess of (S)-PAHK was 86%, and only trace amounts of the hydroxy ketone regioisomer (Figure 10) was detected, indicating high regioselectivity. However, if the reaction was left for too long (S)-PAHK was further reduced to the diol.

18| Present investigation

Figure 11: Starting from racemic PAHK (top), all four diastereomers of the 1,3-amino alcohol were synthesized using the ketoreductase KRED -P1-B10 together with either an (S)-selective (Cv-ATA) or an (R)-selective (ATA-025) amine transaminase (Paper I).

By using the KRED and the amine transaminases we were able to synthesize all four stereoisomers of the amino alcohol product shown in Figure 11. First, racemic PAHK was prepared with a Grignard reaction

Present investigation | 19

followed by Wacker oxidation. The hydroxy ketone was then resolved with KRED-P1-B10 to give (R)-PAHK (86% ee) and the diketone, which were isolated with column chromatography. The diketone was then reduced to (S)-PAHK (71% ee) with KRED-P1-B10. Finally, the amine transaminases Cv-ATA and ATA-025 were used to prepare the four amino alcohol stereoisomers, as shown in Figure 11, with an enantiomeric excess of >98% for the amine group in all four cases.

In summary, we were able to prepare all four stereoisomers of a chiral 1,3- amino alcohol by combining amine transaminases with a ketoreductase. Starting from a racemic hydroxy ketone, only 2-3 steps were required to reach the final products. The reaction conditions were mild and no protecting groups or metal catalysts were needed. However, there is a need for separation of the hydroxy ketone and the diketone after the kinetic resolution step, and thus all the reaction steps can unfortunately not be performed in the same pot.

Next, we turned our attention to our second group of target compounds: capsaicinoids, the compounds responsible for the hot and spicy taste of chili peppers (Figure 12). These compounds are used for analgesic products and pepper sprays, and also have a variety of other physiological effects135- 137. While capsaicinoids can be extracted from pepper fruits, many of them are present in very low amounts and thus chemical synthesis can be a more efficient method for obtaining these compounds138-139. Chemical synthesis also gives access to non-natural capsaicinoids such as phenylcapsaicin which can be used as an anti-fouling agent in for example boat paint applications140 (Figure 12). These compounds can be synthesized in two steps from vanillin through an amination- followed by an amidation step (Figure 13). For example, metal-catalyzed reductive amination of vanillin gives vanillylamine, which can be further converted to the amide product with acyl chlorides (Schotten–Baumann process) or in a lipase-catalyzed reaction with fatty acids141-142.

Amine transaminases are known to convert vanillin to vanillylamine40, so this reaction could be used as an alternative to metal-catalyzed reductive amination in the synthesis of capsaicinoids. This method also opens up the possibility of forming the amide in the same pot to give capsaicinoids in a two-step one-pot process. The Schotten-Baumann process takes place in a water/organic solvent two phase system. It could thus be possible to first

20| Present investigation

convert vanillin to vanillylamine with an amine transaminase in water, and then add solvent and acyl chloride to form the final amide product in the same pot. Alternatively, addition of a lipase and a fatty acid could also give the final amide product in one pot.

Figure 12: The structures of important natural and non -natural capsaicinoids.

Figure 13: General steps for the synthesis of capsaicinoids.

Our initial experiments showed that it was possible to convert vanillin to vanillylamine with the amine transaminase from Chromobacterium violaceum (Cv-ATA), but the equilibrium for this reaction was found to

Present investigation | 21

heavily favor the starting materials. A large excess (50 equivalents) of the amine donor L-alanine was used to try to circumvent this, but still the conversion to vanillylamine was only 25%. We thus decided to employ an enzyme cascade system to shift the equilibrium towards the product side. In addition to the amine transaminase, an L-alanine dehydrogenase was used to regenerate the amine donor L-alanine and a glucose dehydrogenase was used to regenerate the NADH cofactor needed for this reaction. With this system we were able to reach 95% conversion to the product vanillylamine in analytical scale. Next, we explored methods for further converting vanillylamine to capsaicinoids. By using acyl chlorides and Schotten-Baumann conditions we were able to prepare capsaicin and nonivamide from vanillylamine with isolated yields of 91% and 94% respectively.

Next, we wanted to prepare capsaicinoids from vanillin in one pot by combining the two methods described above. By scaling up the amine transaminase-catalyzed reaction, we were able to prepare 100 mg of vanillylamine with over 99% of the vanillin starting material converted to the product. The vanillylamine was not isolated; instead we added sodium biocarbonate followed by chloroform and acyl chloride to form the final product capsaicinoid directly in the same pot. With this method we were able to prepare nonivamide from vanillin with an overall isolated yield of 92% (Figure 14).

Our newly developed method for synthesis of capsaicinoids seemed very promising due to the high yield and the fact that all the steps could be run in one pot without any isolation of intermediates. However, the acyl chloride and chloroform used in the second step are harmful reagents which reduce the overall sustainability of the process. For this reason we wanted to explore the possibility of using a lipase for the second step instead of the Schotten-Baumann conditions. This would give us an overall process catalyzed entirely by enzymes, like the one developed in Paper I for synthesis of chiral 1,3-amino alcohols. We started as before by preparing vanillylamine from vanillin with the amine transaminase system. The reaction mixture was lyophilized to remove water which would otherwise interfere with the lipase reaction. Next, fatty acid in 2-methyl-2-butanol was added together with lipase B acrylic resin from Candida antarctica (Novozymes®) to perform the second reaction step in the same pot. The product nonivamide was isolated with an overall yield of 52% (Figure 14),

22| Present investigation

which was disappointing compared to the 92% yield achieved with the Schotten-Baumann method. However, the lipase method could still be interesting due to the elimination of harmful chloroform and acyl chlorides from the process.

Figure 14: Synthesis of nonivamide from vanillin in a two -step one-pot process (Paper II). Vanillin is first aminated with an amine transaminase, followed by an amidation step with either an acyl chloride or a lipase and a fatty acid.

Present investigation | 23

3.2 Alternative starting materials for amine transaminases (Papers II and III)

To increase the applicability of our newly developed methodology for one- pot synthesis of capsaicinoids (Paper II), we wanted to broaden the substrate scope and investigate alternative starting materials. We wanted to explore the possibility of starting the synthesis from alcohols instead of aldehydes, while still performing all steps in the same pot. Such a method would make it possible to prepare capsaicinoids in one pot starting from vanillyl alcohol which occurs naturally and can be derived from lignin. Our goal was to find a method for oxidation of alcohols to aldehydes that would allow for us to proceed with the amination reaction directly in the same pot. Such processes were recently demonstrated, using alcohol dehydrogenases for oxidation of alcohols74-75, 121-122, 143. An alternative could be to use a metal catalyst to oxidize the alcohol. Another interesting idea for possible alternative starting materials was inspired by the work described in Paper I. During our work with the hydroxy ketones BAHK and VAHK (Figure 10), we discovered that alanine catalyzed the retro-aldol degradation of these compounds into the corresponding aldehydes, benzaldehyde and vanillin respectively. In the presence of an amine transaminase, these aldehydes were further converted to the amines using alanine as the amine donor. This means that these kinds of hydroxy ketones could be used as alternative starting materials in the process for synthesis of amides such as capsaicinoids. The reason this is interesting is because structures similar to VAHK can be derived from lignin144. With methods for starting the synthesis of capsaicinoids from either vanillin, vanillyl alcohol or vanillin aldol adducts, all of which can be derived from lignin, it could be possible to use lignin as renewable raw material for the process.

We attempted the synthesis of vanillylamine with VAHK as starting material (Figure 15). The conditions used were the same as when vanillin was used as starting material, but in this case L-alanine was acting as both amine donor and catalyst for the retro-aldol reaction. Unfortunately this reaction suffered from side-product formation, as dehydration of VAHK gives a heavily resonance stabilized cinnamyl ketone. When all VAHK had been consumed, only 40% had been converted to vanillylamine. No 1,3- amino alcohol side-product was observed which was in line with our results from Paper I. Similar results were obtained when BAHK (Figure 10, page

24| Present investigation

17) was used as starting material for the synthesis of benzylamine under the same conditions – after all of the hydroxy ketone had been consumed only 52% of it had been converted to benzylamine.

Figure 15: Synthesis of vanillylamine in a one -pot cascade process starting from VAHK (Paper II). Alanine catalyzes the retro -aldol transformation of the hydroxy ketone to vanillin, which is subsequently aminated with an amine transaminase (ATA).

Next, we wanted to investigate an enzymatic method for conversion of vanillyl alcohol to vanillylamine in one pot. The alcohol dehydrogenase (ADH) from horse liver was found to catalyze the oxidation of vanillyl alcohol to vanillin alongside reduction of the cofactor NAD+. We decided to add the amine transaminase Cv-ATA, along with the amine donor L- alanine, to further convert the formed vanillin to vanillylamine in the same pot. These two enzymes were combined with an L-alanine dehydrogenase which reduces the pyruvate co-product back to L-alanine while simultaneously oxidizing NADH to NAD+, thus regenerating the cofactor needed for the alcohol dehydrogenase reaction (Figure 16). Unfortunately we were only able to convert 61% of the vanillyl alcohol starting material to vanillylamine with this method.

Present investigation | 25

Figure 16: Synthesis of vanillylamine from vanillyl alcohol in a one -pot cascade process (Paper II). An alcohol dehydrogenase (ADH) catalyzes the oxidation of vanillyl alcohol to vanillin, which is subsequently aminated with an amine transaminase (ATA).

We decided to try heterogeneous palladium(0) nanoparticles as catalysts for the aerobic oxidation of alcohols145-147. These catalysts were previously shown to be stable and easily recyclable without leaching145-147, and could thus prove to be a sustainable option despite being based on a transition metal. Our initial experiments showed that it was possible to fully oxidize vanillyl alcohol to vanillin (over 99% conversion) with either palladium(0) on controlled pore glass (Pd(0)-CPG) or palladium(0) on mesocellular foam (Pd(0)-MCF) as catalyst. In comparison, we were only able to reach a conversion of 43% with commercially available palladium(0) on carbon (Pd(0)/C). With these promising oxidation results, we wanted to investigate if we could further convert the formed vanillin to vanillylamine in the same pot (Figure 17). The crude reaction mixture containing vanillin, toluene and palladium(0) nanoparticles was thus used as starting material

26| Present investigation

for a transamination reaction with the same enzyme system as described earlier. The experiments were performed in analytical scale with an initial vanillin concentration of 1.5 mM after dilution of the crude oxidation reaction mixture with aqueous buffer solution. Under these conditions, the relative amount of toluene was low and no phase separation was observed. The reaction was found to be slower than when pure vanillin was used as starting material, but we were still able to reach a high overall conversion of 87% from vanillyl alcohol to vanillylamine.

Figure 17: One-pot amination of vanillyl alcohol by palladium -catalyzed aerobic oxidation followed by amination with an amine transaminase (Paper II and Paper III). The enzymes L-alanine dehydrogenase and glucose dehydrogenase were used together with the transaminase for equilibrium displacement.

Our initial results for conversion of alcohols to amines with a combination of palladium(0) nanoparticle and amine transaminase catalysis seemed promising, and thus we wanted to further develop this system. We decided to run reactions with 0.1 mmol of starting material in a reaction volume of 5 mL for the transamination, giving us a substrate concentration of 20 mM for this step. Under these conditions, the toluene from the oxidation step (0.25-0.33 mL) formed a separate phase, unlike what we observed for our initial small scale reactions. The ability of the enzymes to operate in this two-phase system was investigated with benzaldehyde dissolved in toluene as the starting material. We found that the toluene phase was harmful to the enzymes: When these were dissolved in buffer solution and added drop-wise to the reaction vessel containing the two phases, they had to pass through the toluene (the top phase in this case) which caused denaturation. However, this problem was easily circumvented by first forming the two phases and then carefully adding the enzymes to the aqueous bottom phase. Mixing of the system was also found to be important. With no mixing the conversion to benzylamine was low, but by mixing the reactions

Present investigation | 27

on an orbital shaker we were able to convert 90% of benzaldehyde to benzylamine. The same results were obtained when running the reactions without toluene, and thus the two-phase system did not seem to negatively affect the performance of the enzymes, as long as these were added directly to the aqueous phase.

Next, we applied the above described method to the one-pot synthesis of benzylamine and vanillylamine from the corresponding alcohols (Figure 18, Table 1). The alcohols were first oxidized in toluene by the palladium catalyst Pd(0)-MCF, followed by addition of buffer solution and the enzymes to give benzylamine or vanillylamine with overall conversions of 75% and 70%, respectively. Unfortunately these conversions were a bit lower than what we had hoped for, and we decided to try the reaction with copper(I)-TEMPO as alternative oxidation catalyst148-149. Using either 2,2- bipyridyl or DABCO as ligand (Cu(I)-TEMPO-bipyridyl and Cu(I)- TEMPO-DABCO, respectively), the copper catalyst was able to fully oxidize benzyl alcohol to benzaldehyde. Buffer solution and enzymes were added as before, and we were able to prepare benzylamine with a high overall conversion of 92% by using the Cu(I)-TEMPO-DABCO oxidation catalyst. Use of Cu(I)-TEMPO-bipyridyl resulted in a much lower overall conversion of 35%, and thus this catalyst was not used for further studies (Table 1).

We performed the one-pot synthesis of a range of different benzylamines by using Cu(I)-TEMPO-DABCO as oxidation catalyst (Figure 18, Table 1). The reactions generally reached high conversions of at least 90% to the product amines, however notable exceptions were the phenolic products 4- hydroxybenzylamine (e) and vanillylamine (b) for which no conversion to the amine could be observed. For this reason the Cu(I)-TEMPO-DABCO catalyst cannot be used for the synthesis of capsaicinoids from vanillyl alcohol, but luckily this reaction was possible with the palladium catalyst as shown earlier.

Next, we wanted to investigate the synthesis of amines other than benzylamine derivatives. The Cu(I)-TEMPO-DABCO catalyst could successfully oxidize cinnamyl- and furfuryl alcohol to give the corresponding amines (d and g respectively, Figure 18) with overall conversions of above 80% after addition of the enzymes. Unfortunately, none of the oxidation catalysts investigated so far were able to successfully oxidize secondary- or aliphatic alcohols. Instead, we found that it was

28| Present investigation

possible to oxidize these kinds of alcohols by using a metal-free system where oxidation took place in a dichloromethane/water two-phase system with TEMPO, sodium hypochlorite and sodium bromide (NaOCl- TEMPO)149-152. The metal-free oxidation system could successfully oxidize the investigated aliphatic and secondary alcohols, but when the enzyme system was added to convert the intermediate carbonyl compounds to the amine products (i, j and k, Figure 18, Table 1) the conversions were lower than expected: The aliphatic amine products j and k were obtained with conversions of 23% and 31% respectively, while no conversion at all was observed to the chiral amine i. We tried removing the salts from the oxidation products by extraction prior to addition of the enzyme system, but unfortunately this did not lead to improved conversions.

Figure 18: Reaction scheme for the one -pot amination of alcohols (Paper III). Results for the synthesis of amine products a-k are shown in Table 1.

Finally, to demonstrate the scaleability of our chemoenzymatic method for one-pot amination of alcohols we performed preparative synthesis of benzylamine and chlorobenzylamine (a and c in Figure 18) in 0.8 mmol scale from the corresponding alcohols, using the Cu(I)-TEMPO-DABCO

Present investigation | 29

catalyst with the same conditions as before. The products were isolated with good yields of 69% and 73% for benzylamine and chlorobenzylamine, respectively. Thus, we had shown that our chemoenzymatic one-pot two- step methodology could efficiently convert a range of primary alcohols to amines. However, the drawbacks are lower conversions for aliphatic amines, and the fact that chiral amines cannot be obtained with this method.

Table 1: Results for the one-pot amination of alcohols (Paper III). Various oxidation catalysts were used for oxidation of alcohols to carbonyl compounds, which were further aminated with an amine transaminase together with the enzymes L-alanine dehydrogenase and glucose dehydrogenase for equilibrium displacement. The conversion shown is conversion to the amine product, and the different products are shown in Figure 18.

Entry Oxidation catalyst Product Conversion (%) 1 Pd(0)-MCF a 75 2 Cu(I)-TEMPO-bipyridyl a 35 3 Cu(I)-TEMPO-DABCO a 92 4 Pd(0)-MCF b 70 5 Cu(I)-TEMPO-DABCO b 0 6 Cu(I)-TEMPO-DABCO c 92 7 Cu(I)-TEMPO-DABCO d 82 8 Cu(I)-TEMPO-DABCO e 0 9 Cu(I)-TEMPO-DABCO f 90 10 Cu(I)-TEMPO-DABCO g 84 11 Cu(I)-TEMPO-DABCO h 95 12 Cu(I)-TEMPO-DABCO i 0 13 NaOCl-TEMPO i 0 14 NaOCl-TEMPO j 23 15 NaOCl-TEMPO k 31

30| Present investigation

3.3 Amine transaminases in one-pot amination/kinetic resolution reactions (Paper IV)

As described in the previous section, we found metal catalysts which were compatible with an amine transaminase in two-step one-pot reactions. We wanted to further explore the possibilities of such metal/ATA combinations by using them for different kinds of reactions. Since we had found palladium catalysts in our previous work which were compatible with amine transaminases, we decided to investigate if we could use palladium to form racemic amines and then perform an amine transaminase-catalyzed kinetic resolution in the same pot to get enantiomerically pure chiral amines (Figure 19).

Figure 19: Proposed one-pot two-step method for the synthesis of enantiomerically pure chiral amines by palladium -catalyzed reductive amination followed by amine transaminase -catalyzed kinetic resolution.

We set out to develop a method for preparation of racemic amines by palladium-catalyzed reductive amination using ammonium formate as both the nitrogen and hydride source, as such a method would be both step and atom efficient. Our initial experiments revealed chemoselectivity issues when the reductive amination of vanillin was catalyzed by Pd(0)- MCF at room temperature: The reaction reached a total conversion of 90%, but conversion to the amine product was only 34%, while 56% of the starting material was further reduced to creosol (Table 2). However, we found that this problem could be circumvented by increasing the reaction temperature. At 80°C the reaction reached full conversion, where 94% of the vanillin starting material was converted to vanillylamine. The reaction was also possible with Pd(0)-CPG as catalyst, although conversion to the amine was slightly lower (77%). We also investigated a number of commercially available palladium catalysts, but the reactions with those resulted in low chemoselectivity (see Table 2 for details), and thus we

Present investigation | 31

decided to use Pd(0)-MCF and Pd(0)-CPG for further studies. The recyclability of these catalysts showed to be excellent and no leaching was observed when used for reductive amination with the above described conditions.

Table 2: Results for the amination of vanillin with various palladium catalysts (Paper IV).

Entry Catalyst Temp (°C) Conversion (%) Ratio (A:B:C) 1 Pd(0)-MCF 22 90 38:62:0 2 Pd(0)-MCF 60 >99 86:14:0 3 Pd(0)-MCF 80 >99 94:6:0 4 - 80 <1 - 5 Pd(0)-CPG 80 >95 79:14:7

6 Pd(PPh3)4 80 <1 -

7 Pd(OH)2/C 80 >99 55:45:0 8 Pd/C 80 >99 34:60:6

9 Pd(OAc)2 80 >99 39:38:23

We were able to successfully convert a range of aldehydes to amines using Pd(0)-MCF as catalyst (Figure 20), but in order to reach chiral amines the starting material must be a prochiral ketone. Thus we tried the reaction with the simple ketone acetophenone as starting material, but unfortunately the product obtained was the alcohol and not the amine. However, we found that we could switch the chemoselectivity by changing

32| Present investigation

solvent from toluene to methanol. With this method we were able to prepare a range of racemic amines in high yields (Figure 21). Next, we wanted to perform an amine transaminase-catalyzed kinetic resolution in the same pot to give the enantiomerically pure chiral amines. From our work with Paper II and Paper III we knew that the amine transaminase from Chromobacterium violaceum (Cv-ATA) is compatible with the palladium catalyst, but in order to access both enantiomers of the product amine a complementary enzyme with the opposite enantiopreference was

Figure 20: Results for the amination of aldehydes with the Pd(0) -MCF catalyst (Paper IV).

Present investigation | 33

needed. By using the amine transaminase ATA117 from Codexis® we were able to prepare (S)-amines with an excellent enantiomeric excess of >99% by palladium-catalyzed reductive amination followed by ATA-catalyzed kinetic resolution in one pot (Figure 22). Unfortunately, when we changed enzyme to Cv-ATA to get the (R)-amines we were not able to convert all the unwanted enantiomer, and as a result the enantiomeric excess of this product was below 90%. Similar results were obtained with the (S)- selective amine transaminase ATA113 from Codexis® (Figure 22).

Figure 21: Results for the synthesis of amines with the Pd(0) -MCF catalyst (Paper IV). The yield presented is the yield after amidation and isolation of the product, and the products were racemic (except for the cyclohexylamine in the bottom right which is achiral ).

34| Present investigation

Figure 22: Results for the synthesis of chiral amines with a one-pot two-step process (Paper IV). Starting from a prochiral ketone, palladium-catalzyed reductive amination gives an intermediate racemic amine which is subsequently subjected to kinetic resolution catalyzed by an amine transaminase to give the final amine product in high enantiomeric excess. The kinetic resolution converts the other amine enantiomer back to the ketone starting material, so it is not wasted. The yield presented is based on consumed ketone.

Present investigation | 35

It has been shown that Lipase B from Candida antarctica can be combined with palladium catalysts for the dynamic kinetic resolution of amines153-155. Starting from a racemic amine, the lipase selectively converts one enantiomer to the corresponding amide while the palladium catalyzes the continuous racemization of the starting material, thus making it possible to reach yields higher than 50%. This enzyme is (R)-selective, and since we were unable to reach enantiomeric excesses of over 90% for (R)-amines with our amine transaminase-catalyzed kinetic resolution the use of a lipase could be an interesting alternative. We started as before with palladium-catalyzed reductive amination of ketones in methanol, and since methanol is a substrate for the lipase it had to be evaporated before the second step. Next, the lipase was added together with the acyl donor: Here we chose to use ethyl methoxyacetate as methoxyacetate esters have previously been shown to be effective for similar reactions156. Toluene was added as the new solvent along with hydrogen gas to promote the racemization. With this method we were able to prepare (R)-amides with isolated yields of around 60% and enantiomeric excesses of over 90%, the best result being an ee of >99% for the vanillyl compound (Figure 23).

By combining palladium and lipase catalysis we had found a new way of converting carbonyl compounds to amides. We decided to try this method for the preparation of capsaicinoids, and compare the performance to that of our previously developed amine transaminase-based method (Paper II). In the first step, palladium-catalyzed reductive amination in toluene converted vanillin to vanillylamine. Next, the lipase was added to the same pot together with fatty acid to give capsaicin, nonivamide or phenylcapsaicin with isolated yields of above 70%. To further explore the possibilities of this method, we also converted a range of different aldehydes to the corresponding amides (Figure 24). Finally, we wanted to see if we could use this method to prepare capsaicinoids from alcohols in one pot, by employing the oxidation conditions used in Paper II and Paper III. Vanillyl alcohol was first oxidized to vanillin, followed by the reductive amination step in the same pot to give vanillylamine. After adding the lipase and performing the amidation step, nonivamide was isolated with a yield of 49% (Figure 25).

36| Present investigation

Figure 23: Results for the synthesis of chiral amides with a one -pot two-step process (Paper IV). Starting from a ketone, palladium-catalyzed reductive amination gives an intermediate racemic amine which is subsequently subjected to dynamic kinetic resolution. The palladium catalyst catalyzes the racemization of the intermediate amine, while a lipase is used for amidation to give the final amide product.

Present investigation | 37

Figure 24: Results for the synthesis of amides with a two -step one-pot process (Paper IV). Starting from an aldehyde, palladium-catalyzed reductive amination gives an intermediate amine which is subsequently amidated with a lipase to give the final amide product.

38| Present investigation

Figure 25: Synthesis of nonivamide from vanillyl alcohol with a one-pot three- step process (Paper IV). Palladium-catalyzed oxidation of vanillyl alcohol gives vanillin, which is further converted to vanillylamine through reductive amination, also catalyzed by palladium. Lipase-catalyzed amidation then gives the final product nonivamide.

With the results so far in this section, we have demonstrated a palladium- catalyzed amination method and how it can be combined with either amine transaminases or lipases for the one-pot synthesis of chiral amines or amides. We decided to further explore the possibilities of this methodology by applying it to the synthesis of the pharmaceutical compound Rivastigmine31, 33, 157 (Figure 26) (unpublished data), which is used for the treatment of Alzheimer’s and Parkinson’s diseases and was one of the 200 most sold pharmaceuticals worldwide in 2013 1. This compound can be accessed from (S)-3-(1-aminoethyl)phenol, a chiral primary amine (as shown in Figure 26), and our plan was to prepare this amine in high enantiomeric excess with amine transaminase catalysis and then perform the other steps required to reach the final product. Also, the ketone starting material is stabilized by conjugation with the ring system, and thus the equilibrium for transamination can be expected to heavily favor the ketone as previously described in Figure 8 (page 8). This problem can conveniently be circumvented by use of our newly developed methodology to first form the racemic amine with palladium catalysis and then proceed with amine transaminase-catalyzed kinetic resolution in the same pot to give the chiral amine. The drawback of kinetic resolution being limited to 50% yield can be avoided by reusing the ketone byproduct as starting material for the racemic amine synthesis.

Present investigation | 39

Figure 26: Structures of the pharmaceutical compound Rivastigmine and the compound (S)-3-(1-aminoethyl)phenol it can be prepared from.

In our initial analytical scale experiments, we were able to prepare the (S)- 3-(1-aminoethyl)phenol (Figure 26) from the corresponding ketone with an enantiomeric excess of >99% by using ATA117 and the same conditions as described in Figure 22. Next, we investigated methods for converting the amine intermediate to the final product Rivastigmine. Di-methylation of the amine was possible with formic acid and formaldehyde at 110°C, and subsequent carbamoylation gave the final product Rivastigmine (Figure 27). To demonstrate the scalability of the process, we performed the one- pot synthesis of the (S)-3-(1-aminoethyl)phenol intermediate starting from 1 mmol of ketone, resulting in an enantiomeric excess of 97% (Figure 27). Work to further improve this process is currently ongoing.

Figure 27: Synthesis of Rivastigmine (unpublished data). Starting from a ketone, palladium-catalyzed reductive amination gives an intermediate amine which is subjected to kinetic resolution with an amine transaminase in the same pot to give the (S)-3-(1-aminoethyl)phenol in high enantiomeric excess. Methylation and carbamoylation gives the final product Rivastigmine.

40| Discussion and conclusion

Discussion and conclusion | 41

4 Discussion and conclusion

The overall goal of this work has been to explore the possibilities of using amine transaminase catalysis in multi-step one-pot reactions, and the results presented in this thesis clearly show that such reactions are feasible. Capsaicinoids, the spicy compounds from chili peppers, were successfully synthesized from vanillin by combining an amine transaminase with either a lipase or acyl chlorides. Amine transaminases were also successfully combined with metal catalysts for the amination of alcohols. Furthermore, by combining both these methods capsaicinoids can be obtained from vanillyl alcohol. Amine transaminases were also employed together with metal catalysts for the synthesis of chiral amines through a reductive amination/kinetic resolution sequence, which was applied for the synthesis of the pharmaceutical compound Rivastigmine. All of these reactions were successfully performed in a multi-step one-pot fashion. Additionally, amine transaminases were used together with ketoreductases for the synthesis of chiral 1,3-amino alcohols. While this synthesis was not performed in one pot, it should be possible to do so in the future with some modifications such as using another method for diketone preparation.

A major concern when performing multi-step one-pot reactions is that the components of the different reaction steps need to be compatible with each other. It is worth noting that in several of the methods presented here such as the synthesis of capsaicinoids and the amination of alcohols, amine transaminases have been used in combination with two other enzymes (L- alanine dehydrogenase and glucose dehydrogenase) for equilibrium displacement. In order for such a system to function, all of these enzymes need to be compatible not only with each other, but also with components such as organic solvents and metal catalysts used in the other steps. This also means that when results are not as good as expected, as for the amination of aliphatic and secondary alcohols (Section 3.2), it is difficult to immediately tell which part of the complex system that is the problem. Using a simpler method for equilibrium displacement, such as an excess of amine donor or a “smart” amine donor (as described in Section 1.2) could possibly lead to a system with fewer compatibility problems and a system which is easier to evaluate.

One of the main reasons for performing several reaction steps in one pot is the green chemistry aspect as described in Section 1.3. When intermediates

42| Discussion and conclusion

do not need to be isolated in between steps, formation of waste is prevented. With the work presented in Section 3.2, we aimed to further improve the sustainability of our methods by the use of renewable resources in the form of lignin derived compounds. We were able to access capsaicinoids from either vanillin or vanillyl alcohol, but when a slightly more complicated starting material in the form of a vanillyl hydroxy ketone was used, conversions were lower than desired. The structure of lignin is very complicated giving rise to a large number of possible degradation products144, and since there were already complications with very simple lignin-like compounds, we decided to not continue this approach. However, this work might be interesting to follow up in the future, especially if new methods for lignin degradation are developed.

The reactions for aminations of alcohols presented in this thesis were performed in water/organic solvent two-phase systems. The reason for this was that organic solvent was needed for the oxidation step and aqueous buffer solution was needed for the amination step, and when these two steps were combined in the same pot the result was a two-phase system, but the use of a system like this might be convenient also for other reasons. As many of the aldehyde or ketone starting materials for these reactions are more soluble in organic solvents than water, an organic phase can act as a substrate reservoir, allowing for higher substrate loadings in the reactions which will in turn allow for lower total reaction volumes and thus less waste generated57. However, in amine transaminase reactions the product amine will be protonated and be more water soluble than the substrate aldehyde or ketone, which might lead to equilibrium problems as the product will accumulate in the water phase with the transaminase, while the substrate is mostly located in the organic phase. This might have been a contributing factor to the difficulties we had with the amination of ketones in Section 3.2, as the already unfavorable equilibrium becomes even more shifted towards the starting materials by the use of a separate organic phase.

With the palladium-catalyzed amination method presented in Section 3.3, we were able to access chiral amines through kinetic resolution with either amine transaminases or a lipase. The lipase method has the disadvantage that a deprotection step is needed to convert the amide product to the corresponding amine, but a major advantage is the possibility of performing a dynamic kinetic resolution, thus circumventing the fact that

Discussion and conclusion | 43

kinetic resolutions are limited to a yield of 50%. Unfortunately, we were only able to reach yields of about 60% with the lipase-catalyzed dynamic kinetic resolution, which is not much higher than the close to 50% yields obtained with the amine transaminase-catalyzed kinetic resolution. The overall yield can also be increased by reusing the racemic amine left from the lipase reaction, or the ketone formed in the amine transaminase reaction, making both options more attractive. Overall, it is difficult to tell which method is more efficient without further scale-up and optimization of the reactions.

The first method for capsaicinoid synthesis presented in this thesis (Section 3.1) is a combination of an amination reaction with an amine transaminase and an amidation reaction with either a lipase or acyl chlorides. However, since the product is not chiral, the enantioselectivity of the amine transaminase is not needed in this case, and thus amination could also be performed with a non-selective catalyst. For this purpose, we tried replacing the amine transaminase-catalyzed step with the palladium- catalyzed reductive amination method presented in Section 3.3. Out of these different methods, the best yield (92%) was obtained with the amine transaminase/acyl chloride method. However, this method suffers from the use of harmful reagents in the form of violently reactive acyl chlorides which are in turn made from toxic thionyl chloride, as well as the carcinogenic solvent chloroform. Slightly lower yields (around 70%) were obtained with the palladium/lipase method, but this method uses a more environmentally benign solvent in the form of toluene54, and the transition metal catalyst can be recycled. The yield for the amine transaminase/lipase method was even lower (52%), but this method has the advantage of being entirely enzymatic and thus has the least harmful reagents and catalysts among the three methods.

Overall, the work presented in this thesis has touched upon most of the principles of green chemistry53-56. The combination of multiple reaction steps in one pot prevents the formation of waste and avoids unnecessary solvent usage. The amine transaminase reactions take place in water, a non-toxic solvent, at ambient temperature and pressure which makes them energy efficient. Catalysis has been used for all reaction steps, and no protecting groups have been needed. The amine transaminases and other enzymes used are non-toxic and biodegradable, as are many of the substrates used such as the amine donor alanine. As for the metal-

44| Discussion and conclusion

catalyzed steps, the palladium nanoparticles used are heterogeneous and recyclable, and oxygen was used as a non-toxic and atom-efficient oxidizing agent.

A few of the green aspects of the processes presented in this thesis do however need to be further considered. The use of renewable resources in the form of lignin derived compounds was investigated, but further development is needed before this concept can be of more general use. Analysis of the reactions does require sample preparation, which leads to unnecessary waste. However, a reverse phase HPLC method without the need for product derivatization was used for monitoring conversion and enantiomeric excess in the amine transaminase reactions, and thus the use of additional organic solvents and reagents could be avoided. Finally, the atom economy of amine transaminase reactions is a problem, as the amine donor or acceptor is wasted. The use of additional enzymes to regenerate these only leads to other compounds being wasted, such as glucose when L-alanine dehydrogenase was used together with glucose dehydrogenase. Also, the waste formation in the enzyme production process has not been addressed in this work.

Even if the work presented in this thesis appears to be in accordance with most of the principles of green chemistry, it is important to note that the actual sustainability or environmental impact of the processes presented has not actually been quantified. In order to truly see how green these reactions are they would need to be scaled up, conditions such as enzyme loading and substrate concentration would need to be optimized and then parameters such as energy consumption and amount of waste formed would need to be analyzed - time and resource consuming work which might be better suited for industry than academia. Nevertheless, we think that the concepts demonstrated in this thesis are a step in the right direction, and could serve as a basis for future work on green chemistry.

In conclusion, the work presented in this thesis shows how amine transaminase catalysis can be applied in multi-step one-pot reactions. In these kinds of reactions there is no need for isolation of intermediates, which prevents the formation of unnecessary waste. However, further scale-up and optimization of these reactions is needed before the environmental impact of these processes can be quantified to determine how green they really are. We have shown how amine transaminases in

Discussion and conclusion | 45

multi-step one-pot reactions can be used for the synthesis of valuable compounds such as chiral amines and amino alcohols, natural products such as capsaicinoids and pharmaceuticals such as Rivastigmine. In the last few years other research groups have also been working on similar processes, and there are now a number of publications related to amine transaminases in multi-step one-pot reactions30, 143, 158-162. Hopefully, in the future some of these concepts can be scaled up to industrial conditions and contribute to making the chemical industry more green.

46| Acknowledgements

Acknowledgements |47

5 Acknowledgements

First of all, I would like to thank Per Berglund for being my supervisor during these years, and for giving me lots of freedom and responsibility for my own work. Big thanks also to the KTH School of Biotechnology for making this all possible by funding my studies through an excellence PhD student position.

Next, I would like to thank my collaborators from other universities who were involved in the work presented in this thesis. Special thanks to Armando Córdova, who has been involved in all the included papers – without you and your many great ideas this thesis would not have been possible. Thanks also to Armando's research group, Samson, Carlos, Luca, Rana and Guangning for working with me on these projects. I would also like to thank my collaborators from the University of Greifswald: Hannes Kohls, Uwe T. Bornscheuer and Matthias Höhne, for working with me on the synthesis of amino alcohols and making that publication possible.

Big thanks also to all the people who have been working with me in the biocatalysis group during these years. Special thanks to Henrik, who supervised me during my master thesis project and taught me how to work in the lab. Thanks to Henrik and Maja for teaching the biocatalysis course with me and making it both appreciated among the students and fun for us as teachers. Thanks to Maja, Peter, Stefan and Fabian for hanging out with me during the conferences in Manchester and Vienna. And finally, to everyone in the group, also including Lisa, Federica, Shan, Fei, Maria, Mats and Kalle: Thanks for interesting and fruitful discussions during group meetings, in the office and during lunches. Special thanks to Kalle for reviewing my thesis and for being a great source of inspiration during these years.

I would also like to thank my other co-workers at floor 2 and at the division of Industrial Biotechnology, especially Nina, Ela and Caroline for being very helpful.

Finally, I would like to thank the Roos foundation and Klasons foundation for funding my participation in the conferences Biotrans2013 and Biotrans2015.

48| Bibliography

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