Amine Transaminases in Multi-Step One-Pot Reactions1064519/... · 2017. 1. 12. · 1.1 Amines,...

Amine Transaminases in Multi-Step One-Pot

Reactions

Mattias Anderson

KTH Royal Institute of Technology

School of Biotechnology

Stockholm 2017

© Mattias Anderson

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

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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.

i

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

ii

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.

iv

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.

v

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.

vi

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

ix

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

In t roduc t ion |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 select ion of important natural amines and amine-derived compounds.

2 | In t roduc t ion

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.

In t roduc t ion |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 representat ion of bioca talyt ic pr imary amine synthes is with either l ipases or transaminases (TAs) start ing from carbonyl compounds.


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: Sitagl ipt in, an important primary amine pharmaceutical.

In t roduc t ion |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.


Figure 6: The mechanism for a transaminase half react ion: An amine donor (left) enters and the amine group is transferred to PLP to give PMP. The second half of the react ion fol lows the same steps in reverse: The amine group of PMP is transferred to an amine acceptor to give PLP (covalent ly 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

In t roduc t ion |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 representat ion of the binding pockets of an ( S )-select ive amine transaminase. The f igure 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.


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 equil ibrium for amine transaminase-catalyzed asymmetric synthesis often favors the start ing materials. This is especial ly true when the start ing material is a conjugated ketone such as acetophenone: In this example the equil ibrium constant was reported 4 2 - 4 3 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

In t roduc t ion |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 equi l ibria in amine transaminase (ATA) catalyzed asymmetric syntheses can be shif ted towards the product side by the use of addit ional 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 | I n t roduc t ion

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

In t roduc t ion |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 | A im

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 | P resent inves t igat i on

Present i nves t igat i on | 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


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


Figure 10: Three dif ferent hydroxy ketones (top) were invest igated as start ing 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.


Figure 11: Start ing from racemic PAHK (top), al l four diastereomers of the 1,3-amino alcohol were synthesized using the ketoreductase KRED -P1-B10 together with either an (S)-select ive (Cv -ATA) or an (R)-select ive (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


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


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


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),


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 vanil l in in a two -step one-pot process (Paper I I). Vanil l in is f i rst aminated with an amine transaminase, fol lowed by an amidat ion step with either an acyl chloride or a l ipase and a fatty acid.


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


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 vani l lylamine in a one -pot cascade process start ing from VAHK (Paper I I). Alanine catalyzes the retro -aldol transformation of the hydroxy ketone to vani l l in, 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.


Figure 16: Synthesis of vani l lylamine from vanil lyl alcohol in a one -pot cascade process (Paper I I) . An alcohol dehydrogenase (ADH) catalyzes the oxidat ion of vani l lyl alcohol to vani l l in, 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


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 aminat ion of vani l lyl alcohol by pal ladium -catalyzed aerobic oxidat ion fol lowed by aminat ion with an amine transaminase (Paper I I and Paper I I I ) . The enzymes L-alanine dehydrogenase and glucose dehydrogenase were used together with the transaminase for equi l ibrium 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


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


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: React ion scheme for the one -pot aminat ion of alcohols (Paper I I I ) . 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


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 aminat ion of alcohols (Paper I I I ). Various oxidat ion catalysts were used for oxidat ion of alcohols to carbonyl compounds, which were further aminated with an amine transaminase together with the enzymes L-alanine dehydrogenase and glucose dehydrogenase for equi l ibr ium displacemen t. The conversion shown is conversion to the amine product, and the dif ferent 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


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 enantiomerical ly pure chiral amines by pal ladium -catalyzed reduct ive aminat ion fol lowed by amine transaminase -catalyzed kinet ic resolut ion.

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


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 aminat ion of vani l l in with various pal ladium 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


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 aminat ion of aldehydes with the Pd(0) -MCF catalyst (Paper IV).


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 amidat ion and isolat ion of the product, and the products were racemic (except for the cyclohexylamine in the bottom right which is achiral ).


Figure 22: Results for the synthesis of chiral amines with a one-pot two-step process (Paper IV). Start ing from a prochiral ketone, pal ladium-catalzyed reduct ive aminat ion gives an intermediate racemic amine which is subsequently subjected to kinet ic resolut ion catalyzed by an amine transaminase to give the f inal amine product in high enantiomeric excess. The kinet ic resolut ion converts the other amine enantiomer back to the ketone start ing material, so i t is not wasted. The yield presented is based on consumed ketone.


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).


Figure 23: Results for the synthesis of chiral amides with a one -pot two-step process (Paper IV). Start ing from a ketone, pal ladium-catalyzed reduct ive aminat ion gives an intermediate racemic amine which is subsequently subjected to dynamic kinet ic resolut ion. The pal ladium catalyst catalyzes the racemizat ion of the intermediate amine, while a l ipase is used for amidat ion to give the f inal amide product .


Figure 24: Results for the synthesis of amides with a two -step one-pot process (Paper IV). Start ing from an aldehyde, pal ladium-catalyzed reduct ive aminat ion gives an intermediate amine which is subsequently amidated with a l ipase to give the f inal amide product .


Figure 25: Synthesis of nonivamide from vanil lyl alcohol with a one-pot three-step process (Paper IV). Pal ladium-catalyzed oxidat ion of vani l lyl alcohol gives vani l l in, which is further converted to vani l lylamine through reduct ive aminat ion, also catalyzed by pal ladium. Lipase-catalyzed amidat ion then gives the f inal 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.


Figure 26: Structures of the pharmaceutical compound Rivast igmine and the compound (S)-3-(1-aminoethyl)phenol i t 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 Rivast igmine (unpublished data). Start ing from a ketone, pal ladium-catalyzed reduct ive aminat ion gives an intermediate amine which is subjected to kinet ic resolut ion with an amine transaminase in the same pot to give the (S)-3-(1-aminoethyl )phenol in high enantiomeric excess. Methylat ion and carbamoylat ion gives the f inal product Rivast igmine.

40 | D iscuss ion and conc lus ion

Discuss ion and conc lus ion | 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


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


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-


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


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 | B ib l iography

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