Discovery, Evolution and Synthetic Applications of

1

Discovery, Evolution and Synthetic Applications of

Phenylalanine Ammonia-Lyase

Thesis submitted to the University of Manchester for the degree of

Doctor of Philosophy

Faculty of Science and Engineering

2017

Syed T. Ahmed

School of Chemistry

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Table of Contents

Thesis Abstract ..................................................................................................................... 7

Declaration and Copyright Statement .................................................................................... 8

Acknowledgements ............................................................................................................... 9

1.1 Abstract .................................................................................................................... 11

1.2 Introduction .............................................................................................................. 11

1.3 Classification of ammonia-lyases.............................................................................. 12

1.3.1 Aspartate ammonia-lyases ..................................................................................... 12

1.3.2 Methylaspartate ammonia-lyase ............................................................................. 13

1.3.3 Aminoacyl-CoA ammonia lyase .............................................................................. 14

1.3.4 Hydroxy amino acid dehydratase/deaminases ........................................................ 15

1.3.5 Ethanolamine ammonia-lyase ................................................................................. 16

1.3.6 Amino acid Cylodeaminases ................................................................................... 16

1.3.7 Aromatic amino acid ammonia-lyase ...................................................................... 17

1.4 Structure and mechanism of arylalanine ammonia-lyase (PALs, HALs and TALs) ........ 18

1.4 Synthetic applications of phenylalanine ammonia-lyase ........................................... 27

1.4.1 Synthesis of L-arylalanines ................................................................................ 27

1.4.2 Synthesis of D-arylalanines ............................................................................... 37

1.4.3 Enzymatic and chemo-enzymatic cascade applications .................................... 39

1.5 Conclusion and perspectives ........................................................................................ 47

1.6 References.................................................................................................................... 47

2.1 Abstract .................................................................................................................... 60

2.2 Introduction .............................................................................................................. 60

2.3 Results and discussion ............................................................................................. 61

2.3.1 PAL and DAADH optimization ................................................................................ 61

2.3.2 Suzuki-Miyaura optimization step ........................................................................... 62

2.3.3 Chemo-enzymatic route optimization ...................................................................... 64

2.4 Conclusions .................................................................................................................. 66

2.5 References ............................................................................................................... 66

3.1 Abstract ......................................................................................................................... 69

3.2 Introduction ................................................................................................................... 69

3.3 Results and discussion ................................................................................................. 71

3.3.1 Preliminary feasibility experiments .......................................................................... 71

3.2.2 Optimization of the Knoevenagel-Doebner condensation step ................................ 73

3.2.3 Optimisation of the PAL-mediated hydroamination step .......................................... 75

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3.2.4 Extension towards phenylalanine analogues .......................................................... 77

3.3 Conclusion .................................................................................................................... 79

3.4 References.................................................................................................................... 79

4.1 Abstract ......................................................................................................................... 83

4.2 Introduction ................................................................................................................... 83

4.3 Results and Discussion ................................................................................................. 85

4.3.1 Reaction optimization and preparative scale synthesis of compounds 3a-3k .......... 86

4.3.2 Electronic effect on reaction rate and chiral purity ................................................... 87

4.3.3 Chemo-enzymatic deracemization of compounds 3b-j ........................................... 89

4.3.4 Extending the synthetic strategy to heterocyclic compounds L-5a-f ........................ 89

4.4 Conclusion .................................................................................................................... 90

4.5 References.................................................................................................................... 90

5.1 Abstract ......................................................................................................................... 94

5.2 Introdcution ................................................................................................................... 94

5.2.1 Therapeutic application of PALs ............................................................................. 95

5.2.2 Application and limitation of PAL catalyzed biotransformation ................................ 96

5.3 Results and discussion ................................................................................................. 97

5.3.1 Elucidating the structure AvPAL and analysis of the active site .............................. 97

5.3.2 Identification of new PAL enzymes ......................................................................... 98

5.3.3 Genomic context analysis of new PALs ................................................................ 100

5.3.4 PAL-TAL discrimination ........................................................................................ 100

5.3.4 Isolation of newly discovered PAL enzymes ......................................................... 101

5.3.5 pH and temperature stability test of new enzymes ................................................ 101

5.3.6 Substrate scope evaluation ................................................................................... 102

5.3.7 Conclusion ............................................................................................................ 105

5.3.8 References ........................................................................................................... 105

6.1 Abstract ....................................................................................................................... 109

6.2 Introduction ................................................................................................................. 109

6.3 Results and discussion ............................................................................................... 110

6.3.1 Wild-type enzyme screening ................................................................................. 110

6.3.2 PbPAL engineering strategy ................................................................................. 111

6.3.3 Analysis of enzyme mutants ................................................................................. 112

6.3.4 Formation of β-amino acids side-product .............................................................. 116

6.3.5 Preparative scale synthesis of compounds 2c-h and 2j-m .................................... 116

6.3.6 Conclusion ............................................................................................................ 117

6.4 References.................................................................................................................. 117

7.1 General methods......................................................................................................... 120

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7.1.1 NMR spectroscopy ............................................................................................... 120

7.1.2 High-resolution mass spectrometry ....................................................................... 120

7.1.3 Strain and plasmid ................................................................................................ 120

7.1.4 Transformation into competent cells ..................................................................... 120

7.1.5 Biocatalyst production ........................................................................................... 121

7.1.6 Lyophilization procedure of wet cells .................................................................... 121

7.1.7 Ion-exchange purification of amino acids .............................................................. 121

7.2 Chapter 2 supporting information ................................................................................ 122

7.2.1 Co-solvent tests for the DAADH reaction .............................................................. 122

7.2.3 Chemicals ............................................................................................................. 122

7.2.4 Specific optical rotation ......................................................................................... 122

7.2.5 TLC analysis ......................................................................................................... 122

7.2.6 Flash chromatography .......................................................................................... 122

7.2.7 Microwave reactions ............................................................................................. 122

7.2.8 Non-chiral HPLC analysis and conversion ............................................................ 123

7.2.9 Enantiomeric excess of L- and D-2 ....................................................................... 124

7.2.10 Site-directed mutagenesis .................................................................................. 125

7.2.11 D-amino acid dehydrogenase gene (DAADH) ..................................................... 125

7.2.12 Biotransformation with lyophilized PAL enzyme .................................................. 125

7.2.13 Biotransformation with DAADH (cell-free extract) ............................................... 125

7.2.14 Synthesis of Palladium catalyst 6 ........................................................................ 126

7.2.15 General procedure for the optimization of the Suzuki-Miyaura cross-coupling

reaction ......................................................................................................................... 126

7.2.16 Erlenmeyer-Plöchl synthesis of 4-bromophenylpyruvic acid 4 ............................. 126

7.2.17 General procedure for Suzuki-Miyaura cross-coupling reaction to synthesise

standards of compounds L-1a-k .................................................................................... 127

7.2.18 General procedure (A) for one-pot N-Boc protection and Suzuki-Miyaura cross-

coupling reaction (from biotransformation product L-11 to L-1-a-k) ................................ 127

7.2.19 General procedure (B) for the one-pot DAADH reductive amination, N-Boc

protection and Suzuki-Miyaura coupling (from ketoacid 4 to D-1a-k) ............................. 127

7.2.20 Compound characterization data ........................................................................ 128

7.2.21 Synthesis of amide 15 from L-1b ........................................................................ 132

7.2.22 References ......................................................................................................... 132


7.3.1 Chemicals ............................................................................................................. 133

7.3.2 Non-chiral HPLC analysis ..................................................................................... 133

7.3.3 Chiral HPLC analysis ............................................................................................ 134

7.3.4 Knoevenegal-Doebner condensation for the synthesis of cinnamic acids ............. 135

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7.3.5 Knoevenagel-Doebner optimization in microwave reactor .................................... 135

7.3.6 PAL amination screening ...................................................................................... 135

7.3.7 Preparative scale telescopic condensation-hydroamination procedure. ................ 135

7.3.8 Characterization data for compounds 2j-l, 2o, 2n and 3j-l, 3o and 3n. ..................... 136


7.4.1 Chemicals and enzymes ....................................................................................... 139

7.4.2 NMR time-course experiment for compounds 3g-l ................................................ 139

7.4.3 Telescopic synthesis of L-5a-e ............................................................................. 145

7.4.4 Unsuccessful biotransformation/synthesis ............................................................ 146



7.4.7 Knoevenagel-Doebner synthesis of acrylic acids 2a-l ........................................... 150

7.4.8 Telescopic synthesis of amino acid 3a-l ................................................................ 150

7.4.9 Deracemization cascade of compounds 3a-l using DAAO .................................... 150

7.4.10 Characterization data for compounds 2a-l, 3a-l, 4a-f and 5a-e ........................... 151


7.5.1 Supplementary figures .......................................................................................... 159

7.5.1 Molecular visualization and modelling ................................................................... 166

7.5.2 X-ray crystallography ............................................................................................ 167

7.5.3 Database searches ............................................................................................... 168

7.5.4 Cloning of Wild-type BlPAL and SrPAL sequences ............................................... 168

7.5.5 Purified enzyme plate reader assay ...................................................................... 169

7.5.6 Purified enzyme assay conditions ......................................................................... 169


7.5.7 Whole-cell biotransformation ................................................................................ 170


7.5.9 Preparative scale reaction and characterization of (S)-1l ...................................... 171


7.6.1 Chemicals and enzymes ....................................................................................... 172

7.6.2 PbPAL mutants exhibiting β-lyase activity............................................................. 172

7.6.3 Site-directed mutagenesis .................................................................................... 172

7.6.4 Non-chiral HPLC conversions ............................................................................... 174

7.6.5 Chiral HPLC conversions ...................................................................................... 174

7.6.6 Knoevenegal-Doebner synthesis of acrylic acids of 1d-q ...................................... 175

7.6.7 Analytical scale biotransformation ......................................................................... 175

7.6.8 Preparative scale biotransformation ...................................................................... 175

7.6.9 Characterisation data of compounds 1c-q and L-2c-m ......................................... 175

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Thesis Abstract

The emergence of biocatalysis and biotechnology in the pharmaceutical and fine chemical

industry has transformed the way we manufacture high value chemicals. In an era of geo-

political uncertainty, dwindling natural resources and political pressure to combat climate

change, the wider chemical industry has invested in biocatalysis where it has played a vital

role in developing sustainable manufacturing processes. Other factors such as regulatory

hurdles, driving down costs, societal forces, where consumers are demanding products

produced from natural sources has played a key role in the evolution of this field. Perhaps the

biggest influence came from the scientific community, with decreasing cost of gene synthesis

and sequencing (a bottleneck for many years in this field) following Moore’s law has expedited

development in this area of research and has led to several commercial applications in the

pharmaceutical and fine chemical industry.

The purpose of this thesis is to highlight the synthetic application of Phenylalanine Ammonia-

Lyase and its potential in the sustainable manufacturing of amino acid API’s. This report will

cover extensively the background of this enzyme, its uses in chemoenzymatic processes and

in synthetic biology followed by recent work on expanding the substrate scope through enzyme

discovery, evolution and synthetic biochemistry.

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Declaration and Copyright Statement

Except where stated, the work referred to in the thesis has not been submitted in support of

an application for another degree or qualification of this or any other university or other institute

of learning.

i) The author of this thesis (including any appendices and/or schedules to this thesis)

owns certain copyright or related rights in it (the”Copyright”) and he has given The

University fo Manchester certain rights ti use such Copyright, including for

administrative purposes.

ii) Copies of this thesis, either in full or in extracts and whether in hard or electronic

copy, may be made only in accordance with the Copyright, Designs and Patents

Act 1988 (as amended) and regulations issued under it or, where appropriate, in

accordance with licensing agreements which the University has from time to time.

This page must form part of any such copies made.

iii) The ownership of certain Copyright, patents, designs, trademarks and other

intellectual property (the “Intellectual Property”) and any reproductions of copyright

works in the thesis, for example graphs and tables (“Reproductions”), which may

be described in this thesis, may not be owned by the author and may be owned by

third parties. Such Intellectual Property and Reproductions cannot and must not be

made available for use without the prior written permission of the owner(s) of the

relevant Intellectual Property and/or Reproductions.

iv) Further information on the conditions under which disclosure, publication and

commercialisation of this thesis, the Copyright and any Intellectual Property and/or

Reproductions described in it may take place is available in the University IP Policy

(seehttp://documents.manchester.ac.uk/DocuInfo.aspx?DocID=24420), in any

relevant Thesis restriction declarations deposited in the University Library, The

University Library’s regulations (see

http://www.library.manchester.ac.uk/about/regulations/) and in The University’s

policy on Presentation of Theses

9

Acknowledgements

I would like to begin by thanking my family especially my Mum and Dad for the continued

support throughout my Ph.D. I would like to thank my supervisors Professor Nicholas J. Turner

and Professor Sabine L. Flitsch for giving me the opportunity to work on this wonderful project

and their support, guidance and encouragement throughout what has been a challenging yet

rewarding 4 years in their group.

Special thanks go to Dr. Fabio Parmeggiani and Dr. Nicholas J. Weise (PAL team) for all the

help and training in molecular biology from setting up transformation to designing primers for

mutagenesis and of course the most important skillset of all, how to use a pipette. It has not

been the easiest project, with several hurdles over the years, but as a team we came through

and their input has been invaluable for my Ph.D.

I would like to thank Fabio, Dr. James Galman (John), Iustina Slabu (Rustina), Ulrike

Klemstein, Dr. William Birmingham (William of Chorlton), Dr. Jane Kwok, Dr. Joanne Porter,

Kun Huang and Dr. Cunyu Yan for the awesome company during our lunch breaks, hiking

days out and gaming sessions over the years. I can be slow picking up rules for new games

so I would like to thank Joanne for the continued patience showed during those difficult times

(Saboteur).

Thanks must also go to Paula Tipton and Emma Mellor for all their help on booking flights,

hotels and other logistics and to Dr. Mark Corbett, Dr. Hannah Roberts and Dr. Claire Doherty

for the support and collaboration. I would like to thank Dr. Ian Rowles for his role in managing

my project, making sure we were fed during project meetings and to all the friendly banter we

had over the years.

And finally, I would like to give my deepest thanks and appreciation to the Turner-Flitsch group,

whom I have enjoyed every moment with. All the conversation, our almost daily cake breaks

to the sometimes crazy and hilarious PJR sessions has made my time here with everyone

amazing (although I would not do it again). I wish everyone including my fellow Ph.D. students

who will be finishing soon all the best in their research and a successful career whichever field

you go into and good luck to the new starters.

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Chapter 1

Synthetic and therapeutic applications of ammonia-Lyase

and aminomutases

Fabio Parmeggiani, Nicholas J. Weise, Syed T. Ahmed and Nicholas J. Turner

School of Chemistry, Manchester Institute of Biotechnology, University of

Manchester, 131 Princess Street, M1 7DN, Manchester, United Kingdom.

Published in Chemical Reviews DOI: 10.1021/acs.chemrev.6b00824

Publication date: May 12th, 2017.

Acknowledgements: The review article was a collaborative effort by the authors. The doctoral

candidate wrote the section on synthetic applications of phenylalanine ammonia-lyase

including enzymatic and chemo-enzymatic cascade applications. In addition, this chapter will

introduce synthetic applications of PAL which will be covered in more detail in chapters 2-4.

Sections on aminomutases, synthetic applications of other ammonia-lyase and therapeutic

applications of PALs have been omitted and can be found online.

AMMONIA-LYASES

AMINOMUTASES

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1.1 Abstract

Ammonia-lyases are mechanistically and structurally diverse enzymes which catalyze the

deamination of amino acids in nature by cleaving the C-N bond. Of the many protein families

in which this enzyme activities are found, only a subset has been employed in the synthesis

of optically pure fine chemicals or in medical applications. This review covers the natural

diversity of this enzymes, highlighting particular enzyme classes that are used within industrial

biotechnology. These highlights details of the discovery and mechanistic investigations of

these commercially-relevant enzymes, along with comparisons of their various applications as

stand-alone catalysts, components of artificial biosynthetic pathways and in

biocatalytic/chemo-enzymatic cascades. This chapter will provide a brief introduction to

ammonia-lyase enzymes, its classification system and highlighting past and current

applications of phenylalanine ammonia-lyase (PAL) in organic synthesis. Therefore, this

chapter will also introduce synthetic applications of PAL which will be covered in more detail

in chapters 2-4.

1.2 Introduction

During the past decade, biocatalysis has had an increasing impact on manufacturing in the

chemical industry, leading to the development of greener, more efficient and more sustainable

synthetic processes. This impact has been especially prominent in the preparation of

pharmaceuticals, agrochemicals and fine chemicals, as well as in the development of new

biopharmaceuticals for the treatment of several diseases. However, even though the

industrial-scale use of enzymes is now well established, its application is somewhat limited to

specific enzyme classes (e.g., lipases, proteases, dehydrogenases and more recently

transaminases, aldolases, nitrilases, etc.). At the fundamental level, biocatalysis is a rapidly

expanding area that offers novel catalysts, processes and technologies at an ever-

accelerating pace. Some of these new biocatalysts present great potential for stereoselective

synthesis, but currently they are not commonly applied in large scale manufacturing.

The enzymes belonging to the structurally and mechanistically diverse families of ammonia-

lyases exemplify this situation perfectly. These proteins catalyze a range of transformations

based upon α-amino acid scaffolds (and rarely upon other amine derivatives), with excellent

stereoselectivity and often perfect atom economy. In common with many other classes of

biocatalysts, the toolbox of ammonia-lyase is rapidly expanding because of recent

developments in bioinformatics, protein engineering and directed evolution, which have led to

a significant increase in the number and range of enzymes available for screening and

application in organic synthesis. Furthermore, most often the reactions catalyzed by this

enzyme family do not have a counterpart in traditional synthetic organic chemistry. This makes

12

their implementation in synthetic processes highly desirable for preparative applications at any

scale, but specifically with a view to industrial production of value-added chemicals that are

hard to access stereoselectively otherwise.

1.3 Classification of ammonia-lyases

Ammonia-lyases (EC 4.3.1.X, defined as carbon-nitrogen lyases that release ammonia as one

of the products) catalyze the reversible cleavage of C-N bonds, typically of α-amino acids,

generating ammonia and an unsaturated or cyclic derivative. Despite this apparently specific

and well-defined transformation, the differences in structure, function and mechanism are

remarkable, giving rise to 31 different EC subclasses (Table 1). The vast majority of these

enzymes (definitely all the most relevant and better studied subclasses) fall into one of seven

main groups based on the specific analogies in the reaction they catalyze and the types of

substrates they accept (Scheme 1).

Scheme 1. Representative reactions catalyzed by the most important classes of ammonia-lyases.

1.3.1 Aspartate ammonia-lyases

Aspartate ammonia-lyases (DALs), also known as aspartases, catalyze the reversible

deamination of L-aspartic acid to fumaric acid.1–3 They play a crucial role in bacterial nitrogen

metabolism and they belong to a broad and well-characterized superfamily (the

“aspartase/fumarase” superfamily) with a characteristic tertiary and quaternary structure, as

well as a similar active site architecture.4 However, other members of this superfamily are

known to catalyze different C-N lyase reactions, C-O lyase reactions and other unrelated

transformations. The vast majority of the practical applications of aspartase exploit the reverse

13

of the natural reaction for the asymmetric synthesis of L-aspartic acid. Aspartase is among the

most specific enzymes known, with multiple attempts of engineering failing to afford more

versatile biocatalysts.

1.3.2 Methylaspartate ammonia-lyase

3-Methylaspartate ammonia-lyases, or methylaspartases (MALs) share mechanistic and

structural features with the enolase superfamily.2,5,6 They were first identified in anaerobic

bacteria but later proven to be insensitive to oxygen and found in a number of non-anaerobic

species. MALs catalyze the deamination of (2S,3S)-threo-3-methylaspartate to 2-

methylfumaric acid (i.e., mesaconic acid) and require no specific cofactor, only divalent (Mg2+)

and monovalent (K+) cations. In spite of the similarity of the transformation performed, MALs

are structurally very different from DALs, being more related to the versatile superfamily of

enolases, that catalyze a broad range of reactions (e.g. dehydrations, racemizations and

cycloisomerizations).7 Also, again in contrast with aspartases, the substrate tolerance of

methylaspartases is much broader and it has been expanded further by structure-based

protein engineering, leading to the synthesis of a large variety of substituted aspartic acid

derivatives.

Table 1. Updated EC classification of ammonia-lyases (obsolete classes are shown in red).

EC number Enzyme Acronym Created Last

modified

4.3.1.1 aspartate ammonia-lyase DAL 1961

4.3.1.2 methylaspartate ammonia-lyase MAL 1961

4.3.1.3 histidine ammonia-lyase HAL 1961 2008

4.3.1.4 formimidoyltetrahydrofolate

cyclodeaminase

1961 2000

4.3.1.5 phenylalanine ammonia-lyase

(deleted and subdivided into EC 4.3.1.23-

25)

PAL 1965 deleted 2008

4.3.1.6 β-alanyl-CoA ammonia-lyase βACAL 1965

4.3.1.7 ethanolamine ammonia-lyase EAAL 1972

4.3.1.8 hydroxymethylbilane synthase

(transferred to EC 2.5.1.61)

1972 deleted 2003

4.3.1.9 D-glucosaminate ammonia-lyase DGDD 1972 2004

4.3.1.10 L-serine-O-sulfate ammonia-lyase 1972

4.3.1.11 dihydroxyphenylalanine ammonia-lyase

(deleted for lack of experimental evidence)

1972 deleted 2007

14

4.3.1.12 ornithine cyclodeaminase OCD 1976

4.3.1.13 carbamoylserine ammonia-lyase 1976

4.3.1.14 3-aminobutyryl-CoA ammonia-lyase ABCAL 1999

4.3.1.15 diaminopropionate ammonia-lyase 1999

4.3.1.16 threo-3-hydroxy-L-aspartate ammonia-lyase 2001 2011

4.3.1.17 L-serine ammonia-lyase LSDD 2001 2014

4.3.1.18 D-serine ammonia-lyase DSDD 2001

4.3.1.19 threonine ammonia-lyase TDD 2001 2014

4.3.1.20 erythro-3-hydroxy-L-aspartate ammonia-

lyase

2001 2011

4.3.1.21 aminodeoxygluconate ammonia-lyase

(deleted, identical to EC 4.3.1.9)

2002 deleted 2004

4.3.1.22 3,4-dihydroxyphenylalanine reductive

deaminase

2007

4.3.1.23 tyrosine ammonia-lyase TAL 2008

4.3.1.24 phenylalanine ammonia-lyase PAL 2008

4.3.1.25 phenylalanine/tyrosine ammonia-lyase PAL/TAL 2008

4.3.1.26 chromopyrrolate synthase

(transferred to EC 1.21.3.9)

2010 deleted 2013

4.3.1.27 threo-3-hydroxy-D-aspartate ammonia-

lyase

2011

4.3.1.28 L-lysine cyclodeaminase KCD 2012

4.3.1.29 D-glucosaminate-6-phosphate ammonia-

lyase

2013

4.3.1.30 dTDP-4-amino-4,6-dideoxy-D-glucose

ammonia-lyase

2011

4.3.1.31 L-tryptophan ammonia-lyase 2016

1.3.3 Aminoacyl-CoA ammonia lyase

The fermentation of β-alanine, performed only by very few organisms, proceeds via activation

to the CoA thioester, followed by the elimination of ammonia by a β-alanyl-CoA ammonia-

lyase (βACAL), producing acryloyl-CoA. Two enzymes of this class have been identified in

Clostridium propionicum, and one of them (Cp-βACAL-2) was isolated, purified and

demonstrated to be active.8 The reversibility of the reaction and the tolerance towards different

derivatives of the substrate (e.g., crotonyl-CoA, methacryloyl-CoA) have also been

demonstrated, illustrating a possible application in the production of small β-amino acids. More

15

recent structural studies on Cp-βACAL-2, including a crystal structure in complex with

propionyl-CoA, led to the proposal of a mechanism based on the formation of an enolate

intermediate and retro-Michael elimination of an ammonium ion.9 A similar activity is that of L-

3-aminobutyryl-CoA ammonia-lyase (ABCAL), first identified in Clostridium subterminale,10

and later in other organisms such as Brevibacterium.11 This enzyme converts 3-aminobutyryl-

CoA into crotonyl-CoA and ammonia, and it has been postulated to follow the same

mechanism as βACAL. Interestingly, it has been demonstrated that ammonia can be replaced

with hydroxylamine in the ABCAL amination reaction, leading to the heterocycle 3-methyl-5-

isoxazolidinone, after cyclization of the expected 3-hydroxyaminobutyryl-CoA product and loss

of CoA.10 However, due to the difficulty of accessing CoA-derivatives as substrates, these

enzymes are unlikely to find widespread synthetic use in large scale applications.

1.3.4 Hydroxy amino acid dehydratase/deaminases

A large family of phylogenetically related enzymes dependent on pyridoxal 5’-phosphate (PLP)

catalyze reversible reactions where the C-N bond of a β-hydroxy-α-amino acid is replaced with

a C=O bond, with concomitant elimination of ammonia and water. These enzymes belong to

class II of the PLP-dependent enzymes family, and their mechanism involves binding to PLP

as an imine (through the α-amino group) and elimination of water from the β-position of the

substrate, yielding an enamine that tautomerizes to the imine and is rapidly hydrolyzed non-

enzymatically in the aqueous environment, yielding the corresponding α-keto acid and

ammonia. These enzymes are often referred to as ammonia-lyases because of the C-N bond

cleavage, but naming them dehydratases/deaminases reflects more clearly the steps of the

mechanism (noting that a water molecule is lost in the first step, but one is required for imine

hydrolysis). Examples are L- or D-serine dehydratase/deaminase (L- or D-serine to

pyruvate),12,13 L-threonine dehydratase/deaminase (L-threonine to 2-oxoglutarate),14,15 L-

threo-3-hydroxyaspartate dehydratase/deaminase (L-threo-3-hydroxyaspartate to

oxaloacetate)16 and D-glucosaminate dehydratase/deaminase (D-glucosaminate to D-2-

dehydro-3-deoxygluconate).17 One interesting exception is diaminopropionate ammonia-lyase

(L-α,β-diaminopropionate to pyruvate),18 since its substrate bears an amino group instead of

a hydroxyl group at the β-position, and therefore the α,β-elimination results in a loss of

ammonia instead of water. Another related exception is carbamoylserine ammonia-lyase, that

cleaves the carbon-oxygen bond of carbamoylserine, releasing CO2 and a first molecule of

ammonia, followed by the usual tautomerization and hydrolytic deamination to form pyruvate

with elimination of a second molecule of ammonia.19 No preparative application of these

enzymes is known, even though a few have been used in the design of assays or

biosensors.20,21

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1.3.5 Ethanolamine ammonia-lyase

Even though the overall reaction of ethanolamine ammonia-lyase (EAAL) resembles closely

that of dehydratases/deaminases, this enzyme performs the conversion with a completely

different mechanistic pathway. EAAL reversibly converts ethanolamine into acetaldehyde and

ammonia, an activity first discovered in choline-fermenting Clostridium sp.22 and later identified

in several other organisms, including Salmonella, Klebsiella and Bacillus. Unlike any other

ammonia-lyase, EAALs require the cofactor adenosylcobalamin (AdoCbl or coenzyme B12).23

The availability of crystal structures of the enzyme with cofactor analogues bound24 supported

mechanistic studies and gave a precise view of the key binding interactions. The mechanism

has been extensively studied with native and recombinant enzymes and found to be more

similar to that of AdoCbl-dependent aminomutases. The Co-C bond of AdoCbl is cleaved

homolytically, forming the 5’-deoxyadenosyl free radical, which removes a proton radical from

C1 of the substrate. The ethanolamine-derived radical thus generated undergoes a

rearrangement with migration of the amine from C2 to C1, yielding a hemiaminal radical, which

abstracts a hydrogen radical from 5’-deoxyadenosine, to regenerate the 5’-deoxyadenosyl

radical. The resulting hemiaminal rapidly decomposes to the products ammonia and

acetaldehyde.25,26 In spite of the very extensive body of work on the mechanistic aspects of

these fascinating enzymes, alternative substrates have not been found and the applications

of EAALs remain rather limited.

1.3.6 Amino acid Cylodeaminases

The first member of this class to be identified and isolated was L-ornithine cyclodeaminase

(OCD) from Clostridium sporogenes PA 3679, which converts L-ornithine to L-proline and

ammonia.27 Early mechanistic studies suggested that the reaction involves the initial

deamination of the α-amino group of the substrate to give 2-oxo-5-aminopentanoate, which

cyclizes spontaneously to Δ1-pyrroline-2-carboxylate, followed by reduction of the latter to L-

proline.28 These two complementary redox reactions are carried out by a tightly bound

nicotinamide adenine dinucleotide (NAD+) cofactor molecule in the active site, via hydride

abstraction and donation. Thus, amino acid cyclodeaminases are the only nicotinamide

cofactor-dependent ammonia-lyases known to date. In fact, this mechanism resembles closely

that of amino acid dehydrogenases (classified as oxidoreductases); however, the

cyclization/reduction steps make the whole process redox-neutral, thus cyclodeaminases are

better regarded to as ammonia-lyases. The first X-ray crystal structure of an OCD (from

Pseudomonas putida)29 allowed the definition of substrate binding residues and the proposal

of an alternative mechanism, in which the imine formed by dehydrogenation of the substrate

undergoes attack by the free amine to give an α-aminoproline intermediate, from which

ammonia is eliminated. The resulting imine is then reduced as before. In a subsequent attempt

17

to elucidate the biosynthesis of pipecolate-containing macrolide immunosuppressants such

as rapamycin, the analogous cyclodeamination reaction of L-lysine to L-pipecolic acid was

also identified.30,31 This reaction is catalyzed by L-lysine cyclodeaminases (KCDs), with strong

mechanistic similarities to OCDs.32 Recombinant expression and purification of a few

cyclodeaminases has been achieved, opening up the way to practical applications. For

instance, very recently, a simple and highly efficient process for the conversion of L-lysine to

L-pipecolic acid was demonstrated using whole cells of E. coli overproducing KCD as the

biocatalyst, up to a 17.25 g L–1 product concentration.33 Another example is the synthetic

biology approach to the production of L-proline by expression of the OCD gene from P. putida

in an ornithine-producing Corynebacterium glutamicum strain, with yields of up to 0.36 g L-

proline per gram of glucose.34 These results already showed the great synthetic potential of

cyclodeaminases, and intensification of these processes could offer significantly improved

industrial routes to cyclic amino acids in the years to come.

1.3.7 Aromatic amino acid ammonia-lyase

Phenylalanine ammonia-lyase (PAL), histidine ammonia-lyase (HAL) and tyrosine ammonia-

lyase (TAL) catalyze the reversible elimination of ammonia from the corresponding aromatic

L-amino acids, yielding cinnamic acid, urocanic acid and p-coumaric acid, respectively.35–38

Only one report is available in the literature about the conversion of L-tryptophan to 3-

indoleacrylic acid by a putative tryptophan ammonia lyase (WAL) from Rubrivivax

benzoatilyticus,39 however the gene has not been conclusively identified and further work will

be necessary before the existence of such enzyme in nature can be confirmed. The

characteristic feature of arylalanine ammonia-lyases is the presence of the unusual highly

electrophilic heterocycle 4-methylideneimidazol-5-one (MIO), formed by post-translational

cyclization and dehydration of a tripeptide in the active site, which mediates these challenging

amination/deamination reactions.37,40 This functionality is often improperly called “MIO

cofactor”, especially in the older literature, but this is not formally correct because cofactors

are non-protein-derived molecules (or ions). Similarly, it is often referred to as “MIO prosthetic

group”, but this is also incorrect, since it implies the separate biosynthesis and subsequent

post-translational attachment of this group to the apo-enzyme. Given that the formation of the

MIO occurs from nothing but the folded polypeptide chain, it would be better described just as

a modified active site residue or catalytic moiety, instead. In recent years, aromatic amino acid

ammonia-lyases (particularly PALs) have become popular as excellent and versatile catalysts

for the synthesis of substituted amino acids, and a plethora of applications have been

described with these enzymes alone or in the context of chemo-enzymatic cascade

catalysis.41–44 The remainder of this chapter will primarily focus on the structure, mechanistic

studies and synthetic applications of phenylalanine ammonia-lyase.

18

1.4 Structure and mechanism of arylalanine ammonia-lyase (PALs, HALs and TALs)

The non-oxidative deaminations of L-phenylalanine, L-tyrosine and L-histidine (mediated by

PALs, TALs and HALs, respectively) to form the corresponding α,β-unsaturated acids

(cinnamic acid, p-coumaric acid and urocanic acid) have different metabolic and catabolic

functions in various organisms.

HALs play a major role in amino acid degradation. While catabolic pathways of most amino

acids usually start with the oxidative removal of the α-amino group by transaminases, in the

case of histidine this process begins with the HAL mediated non-oxidative deamination,

yielding urocanate that is further converted to N-formiminoglutamate. The latter is either used

for the synthesis of 5-formiminotetrahydrofolate (in mammals), or is further degraded to

glutamate (in bacteria and some fungi). Interestingly, the groups of eukaryotes found not to

employ HALs in primary metabolism (Dikarya and land plants) are composed of organisms

known to encode polypeptides with PAL activity, acquired through horizontal gene transfer.45

PALs, on the other hand, are needed for biosynthetic purposes. The conversion of

phenylalanine into cinnamic acid is the first step of phenylpropanoid biosynthesis in plants,

which affords a stunningly broad collection of polyhydroxylated aromatic compounds (e.g.,

lignans, coumarins, flavonoids, stilbenes, anthocyanins).46 By siphoning the primary

metabolite phenylalanine into polyphenol synthetic pathways, PAL effectively acts as a

gateway enzyme between primary and secondary metabolism. TALs serve the same role

(mainly as an alternative pathway to p-coumaroyl-CoA), but they are involved in more specific

pathways, possibly adding evidence to the speculation that they are rarer than PALs.47

However, considering that the p-coumaric acid product of the TAL reaction can also be derived

from the cinnamic acid product of the PAL reaction by means of cinnamic acid 4-hydroxylase

(CAH), the metabolic functions of PALs and TALs are strongly related.

Arylalanine ammonia-lyases, generally known as class I lyase-like enzymes are ubiquitous in

nature with PALs and TALs being less common in bacteria. A large number of those have

been characterized, produced recombinantly in E. coli and purified to homogeneity. The most

studied enzymes of this family are RgPAL from Rhodotorula glutinis,48 RtPAL from

Rhodosporidium toruloides (formerly known as Rhodotorula gracilis),49 PcPAL from

Petroselinum crispum (parsley),50,51 AvPAL from Anabaena variabilis,52 RsTAL from

Rhodobacter sphaeroides, SxTAL (BagA) from Streptomyces sp.,53 PfHAL from

Pseudomonas fluorescens,54 and PpHAL from Pseudomonas putida.55

The reactions catalyzed by arylalanine ammonia-lyases are chemically more challenging than

those of DALs and MALs, since the abstraction of a proton from the β-carbon is unfavorable

due to the very low acidity (pKa ~ 40) of the β-protons. Indeed, it is quite surprising that these

19

enzymes do not require complex cofactors and radical mechanisms as seen, for instance, with

some aminomutases. This has posed a considerable mechanistic challenge to biochemists

and, after half a century of research, the answer is still not completely clear.

The very first mechanistic studies performed on HAL identified a catalytically essential

electrophile, derived either from a tightly bound cofactor or from active site functionalization.56

Inactivation by the radioactively labelled nucleophiles [3H]-borohydride and [14C]-cyanide

afforded, upon complete hydrolysis, [3H]-alanine and [14C]-aspartate, respectively, suggesting

that the electrophilic group was dehydroalanine (acting as a Michael acceptor).48,45,57,58 It was

also recognized that the nitrogen of dehydroalanine formed a Schiff base with a carbonyl

compound that could not be identified.57 Site-directed mutagenesis studies in PpHAL,59

PcPAL60 and RsTAL61 demonstrated that an active site serine residue is the precursor of the

catalytically essential dehydroalanine (Ser143 in PpHAL, Ser102 in PcPAL, Ser150 in RsTAL).

Interestingly, the HAL and PAL mutants where the serine was replaced with a cysteine showed

identical catalytic properties, suggesting that the dehydroalanine-derived functionality could

be formed by post-translational modification via elimination of either H2O or H2S.62,63

Unexpectedly, with the first X-ray crystallographic study of PpHAL, Schulz and co-workers

showed that the electron density around the predicted dehydroalanine residue was not

consistent with any of the standard proteinogenic amino acids, and the electrophilic

functionality in the active site was more complex than expected. The observed structure was

the five-membered heterocycle 4-methylideneimidazol-5-one (MIO, formally 5-methylene-3,5-

dihydro-4H-imidazol-4-one).64 The MIO moiety, very often inappropriately called “MIO

cofactor” or “MIO prosthetic group” (as briefly discussed in Section 1.1.4), is in fact a

dehydroalanine derivative formed by cyclization and double dehydration of an active site

tripeptide, typically Ala-Ser-Gly and, less frequently, Thr/Ser/Cys-Ser-Gly (Scheme 2).37,40

Scheme 2. Mechanism of formation of the MIO electrophile by cyclization and double dehydration of the active site

tripeptide Ala-Ser-Gly.

The mechanism of formation of the MIO electrophile is post-translational and autocatalytic,

since heterologous expression of prokaryotic and eukaryotic PALs and HALs results in fully

20

active enzymes. It closely resembles the formation of the fluorophore of green fluorescent

protein (GFP), discovered in 1996,65 but, in contrast with arylalanine ammonia-lyases, the

cyclizing tripeptide in GFP is Ser/Thr-Tyr-Gly, and the final step is an oxidation by molecular

oxygen instead of the second elimination of water.66 A very recent molecular dynamics

simulation study provided great insight into the process of MIO formation in PpHAL, supporting

the idea that mechanical compression from neighboring residues is required to prevent the

formation of stabilizing hydrogen bonds and to enforce the correct alignment of donor and

acceptor orbitals.67

The crystal structure of PpHAL also showed for the first time the general structural features of

arylalanine ammonia-lyases, homotetramers mainly built of α-helices of varying lengths.68

Each subunit contains a globular N-terminal domain and an elongated C-terminal domain with

five parallel α-helices, surrounded by six more (Figure 1). The tetrameric structure contains

four identical active sites, each built of residues belonging to three different subunits.64 Certain

plant and fungal PALs have been shown to bear an additional C-terminal multi-helix domain,

which may play a role in the fine regulation of enzyme activity in these organisms via substrate

channeling and/or overall destabilization of the protein.50,69 Structural alignments of these

domains point to their origins as partial duplication or gene fusion events within the

evolutionary history of plant and fungal PALs.70 Other than the presence of this additional

domain, the general structural features of arylalanine ammonia lyases are found invariably in

all the known crystal structures of the members of this family, e.g., RtPAL,49 PcPAL50 and

AvPAL.52 All of the enzymes that have been structurally characterized were found to contain

the MIO moiety (already demonstrated by spectroscopic methods131) and to have almost

identical active site geometry (Scheme 3a).

Figure 1. X-ray crystal structure of PpHAL (PDB ID: 1B8F), showing the homotetramer and the domains of each

subunit. The MIO heterocycles are shown in red.

C-terminal

domain

N-terminal

domain

21

Just a few years later, the first crystal structure of a TAL was reported (RsTAL from

Rhodobacter sphaeroides),72 demonstrating that only one residue (His89 in RsTAL) is

responsible for its selectivity for tyrosine. The single point mutation His89Phe resulted in a

complete selectivity switch from TAL to PAL activity, as was also demonstrated independently

and simultaneously by Schmidt-Dannert and co-workers.47 Analogously, the Phe144His

mutation introduced in AtPAL1 from Arabidopsis thaliana gave the opposite switch from PAL

to TAL activity.47 These remarkable examples of modulation of substrate specificity are rather

rare; an earlier attempt to turn PcPAL into a HAL by replacing the only two non-conserved

active site residues (Leu138His-Gln488Glu) gave a mutant much less active on phenylalanine

and with a KM for histidine almost identical to that of HAL, albeit with much lower kcat.73 These

results indicate the subtle influence residues far from the active site can have, and also

reinforce the idea of the active site glutamate at the second position being a determining

feature of HALs (as glutamine is found in all other family members).73,74 More recently,

examples of enzymes with TAL activity (e.g., SxTAL/BagA from Streptomyces)113 or dual

phenylalanine-tyrosine specificity (e.g., ZmPAL1 from Zea mays)75 have been identified with

the non-homologous histidine active site residues being seemingly responsible for TAL

activity, presumably due to the interaction of these two residues with the para-substituent of

the substrate. The existence of these enzymes, combined with the known bifunctionality of

some fungal enzymes point to multiple convergent evolution events resulting in TAL activity

within different clades of the class I lyase-like enzyme family. As variation at either position

from hydrophobic residues to histidine is shown to confer tyrosine or histidine acceptance,

these two active site positions act as “selectivity residues” (Scheme 3b).

22

Scheme 3. Schematic representation of the active site of PcPAL (a) and putative roles of the so-called selectivity

residues in substrate recognition (b). Selectivity residues are shown in blue.

In particular, for PAL activity hydrophobic interactions are predominant, for TAL activity the

histidine nitrogen interacts with the para-hydroxy functionality of the substrate, and for HAL

activity (recognized in early studies to depend on Zn2+ ions76) the histidine residue and the

histidine substrate are proposed to coordinate the divalent cation, as shown by detailed

computational simulations.77 Examples of the most typical selectivity residue combinations

seen in the aromatic binding pocket, and the corresponding substrate specificities, are shown

in Table 2.

23

Table 2. Examples of ammonia-lyase substrate specificities and selectivity residues.

Activity Selectivity residues Examples

PAL Phe-Leu PcPAL, AvPAL, AtPAL1

TAL Tyr-His BagA

TAL His-Leu RsTAL

PAL/TAL His-Leu ZmPAL1

PAL/TAL His-Gln RgPAL, RtPAL

HAL Ser-His PpHAL, PfHAL

Mutagenesis studies on PpHAL,78 PcPAL73 and RsTAL61 revealed also the presence of an

essential catalytic tyrosine residue (Tyr53 in PpHAL, Tyr110 in PcPAL, Tyr60 in RsTAL) on a

flexible loop covering the binding site. In contrast with PpHAL, the first crystal structure of a

PAL did not show a complete picture of the active site, since the flexible loop carrying the

catalytic tyrosine was either absent (RtPAL) or locked in the inactive open conformation

(PcPAL), as shown in Figure 4. By homology modelling, using the closed-loop structure of

PpHAL as a guide, a closed loop structure of PcPAL was built, along with those of two bacterial

homologs.69 Dynamic simulations led to the hypothesis that the additional C-terminal domain

of eukaryotic PALs (absent in bacterial PALs) destabilizes the enzyme, thus decreasing its

lifetime, a potentially important feature in the regulation of phenylpropanoid biosynthetic

pathways. Further focused directed evolution work around the carboxylate/amine binding sites

only led to variants with decreased activity, highlighting the presence of a complex and highly

mutation-sensitive network of hydrogen bonds.79

Figure 2. Overlay of the X-ray crystal structures of PpHAL (PDB ID: 1B8F) and PcPAL (PDB ID: 1W27). The loop

bearing the catalytic tyrosine is locked in the closed conformation in the structure of PpHAL (green), but in an

inactive open conformation in the structure of PcPAL.

open loop (PcPAL)

closed loop (PpHAL)

MIO

24

It is worth mentioning that the crystallization of PpHAL required the mutagenesis of a surface

cysteine to alanine (Cys273Ala) in order to obtain high quality crystals reproducibly.64 A similar

strategy, applied to the recently discovered and characterized AvPAL from Anabaena

variabilis,52 allowed researchers to obtain the first crystal structure of a PAL with all the

residues in the catalytically active closed conformation. In this case, mutation of two surface

cysteine residues was required (AvPAL-Cys503Ser-Cys565Ser) and the solution of the

structure afforded the first complete picture of the active site of a PAL.70

The mechanism of MIO-dependent ammonia-lyases has been debated for many decades,

and is still considered partly controversial. The most likely mechanistic paths proposed are

summarized in Scheme 4.

Scheme 4. Mechanisms proposed for the deaminations mediated by arylalanine ammonia-lyases.

The first proposal by Hanson and Havir,58 later revised by Cleland and co-workers,80 was

formulated even before the structure of the MIO had been fully elucidated. This reaction

mechanism (Scheme 4, path A) involved a direct interaction of the electrophilic functionality

with the deprotonated amino group of the substrate, forming a covalent amino-MIO

intermediate. This step facilitates the reaction by forming a secondary ammonium ion, a better

leaving group than less substituted species. Then, the catalytically essential enzymatic base

abstracts the pro-S proton from the β-position, forming a carbanion intermediate, immediately

followed by elimination of the MIO-NH2 leaving group (E1cB mechanism). Lastly, reprotonation

of the MIO-NH2 moiety and elimination of ammonia completes the cycle. The β-proton

abstraction and the MIO-NH2 elimination can, in principle, occur according to three different

25

pathways: E1 (via a carbocation intermediate), E2 (concerted) and E1cB (via a carbanion

intermediate), as shown in Scheme 4. However, the E1 mechanism is highly unlikely, not only

due to the high energy of the α-carbocation intermediate, but also because of the

incompatibility with the orientation of helix dipoles, as observed, for instance, in RtPAL.49 On

the basis of the kinetic isotope effects observed with 15N-labelled and β-2H2-labelled

phenylalanines, the concerted E2 mechanism was also originally excluded.80 However, this

statement is strictly valid only if the elimination step is rate-limiting, and a detailed

computational study on the mechanism of RsTAL reactions indicated that this is not

necessarily the case.81 In this work, Poppe and co-workers revealed a tandem nucleophilic

and electrophilic enhancement by a proton transfer from the protonated amino group of the

zwitterionic substrate (increasing the nucleophilicity of the nitrogen) to the nitrogen of the MIO

ring (increasing the electrophilicity of the double bond). This step is mediated by Tyr300, a

residue found to be fully conserved not only in all known 3D structures of MIO-enzymes, but

also across the gene sequences (e.g., Tyr351 in PcPAL, Scheme 3a). The calculations

suggested that such proton transfer is the rate-determining step of the reaction, possibly

justifying why no significant 2H or 15N isotope effects could be observed experimentally.

In 1995, a completely different mechanistic pathway was proposed by Rétey and co-

workers.82,83 This mechanism (Scheme 3, path B) involves first a Friedel-Crafts-like attack of

the MIO electrophile with formation of a covalent ring-MIO intermediate, which renders the β-

proton more acidic by generating a positively charged complex. The Friedel-Crafts-like

complex then decomposes with elimination of an ammonium ion, to restore the aromaticity of

the system and regenerate the MIO.84,85 This mechanism is plausible for electron-rich aromatic

rings such as histidine or tyrosine, but less plausible for the PAL reaction on unsubstituted

substrates. Evidence to support this mechanism includes the relative rates of different

substrates due to resonance effects (e.g., much higher reaction rates of m-tyrosine with PAL

compared to phenylalanine),83 and the stereoelectronic viability observed in the reaction of a

suitably-designed synthetic model compound.86

A very recent computational study also highlighted the possibility of a single-step mechanism

for the TAL reaction, without the formation of covalent bonds with the MIO.87 This hypothesis,

however, was considered unlikely on the basis of serious contradictions with experimental

data, such as the kinetic isotope effects,80 the stepwise release of the arylacrylate prior to that

of ammonia,88 and the reversibility even at relatively low ammonia concentrations.89

The amino-MIO mechanism is now generally accepted as the most plausible, primarily based

on structural data (e.g., RsTAL with the inhibitor 2-aminoindan-2-phosphonate bound,90

computational simulations81 and evidence of conversion of substrates that cannot react via a

26

Friedel-Crafts-like mechanism (e.g., pentafluorophenylalanine91 or propargylglycine89). The

key problem is the difficulty of abstracting a non-acidic β-proton in the E1cB mechanism, with

no convincing explanation (although examples of abstraction of an apparently non-acidic

proton vicinal to a good leaving group performed by hydroxide ions are known). On the other

hand, the E2 mechanism overcomes this difficulty, and computational data also showed that

substrate binding to form the amino-MIO intermediate caused a weakening of the Cα-N bond

and, at the same time, increased the acidity of the β-proton, potentially enabling a smooth E2

elimination.81 The debate between the E1cB and the E2 mechanisms will probably continue

until conclusive experimental evidence is found.

The conversion of arylalanines bearing a strongly electron-withdrawing group (EWG) is also

worth mentioning because of its mechanistic implications. The conversion of 4-nitrohistidine

with HAL was reported by Klee et al. in 1979.92 In addition, no deuterium kinetic isotope effect

was observed with β-2H2-4-nitrohistidine, as opposed to unsubstituted β-2H2-histidine. This can

be explained by considering that the decrease in electron-density of the ring leads to an

increased acidity of the β-protons, thus making the abstraction of the pro-S hydrogen no longer

the rate limiting step. More recently, Rétey and co-workers demonstrated the conversion of

nitrophenylalanine with PcPAL, in both the deamination and the amination directions, showing

also that the MIO-less mutant PcPAL-Ser202Ala is active on such substrates, even with

considerably higher specific activities than the natural substrates.93 This was thought to be

further evidence of a Friedel-Crafts-like mechanism, since the strong electron-withdrawing

effect of the nitro group provides the same activation as that provided by the MIO moiety after

the Friedel-Crafts-type attack. Very recently, however, we showed evidence of a MIO-

independent competing reaction pathway with highly activated substrates (i.e., those carrying

strong EWGs), that proceeds non-stereoselectively and more slowly.154 With isotopic labelling

and mutagenesis studies it was shown that this pathway is consistent with an E1cB

mechanism and proceeds via stereoselective deprotonation of the pro-R β-proton of D-

phenylalanine, followed by either non-selective reprotonation of the carbanion, or elimination

of ammonia. This remarkable MIO-independent activity could be increased by engineering95

(and exploited in biocatalytic applications, see Section 1.4.2), but attempts to engineer a truly

D-selective PAL are currently unsuccessful.

Despite the ongoing mechanistic debate, which will certainly promote more discussion and

research in the future, aromatic amino acid ammonia-lyases (PALs in particular) have been

employed very effectively in multiple practical applications, ranging from the synthesis of

amino acids, to assays and biosensors, to the development of new therapeutic strategies.

27

1.4 Synthetic applications of phenylalanine ammonia-lyase

The past few decades have seen an exponential rise in the use of biocatalysts to convert

readily available and inexpensive starting materials to value-added products. This has been

credited to several factors, including the advancement in recombinant DNA technologies, the

development of suitable high-throughput screening systems, the improvement of

bioinformatics for the discovery of new enzymes, and the reduction in the cost of DNA

synthesis and sequencing. This has led to the identification and development of enzymes with

unique and novel chemistry better suited for sustainable manufacturing. Perhaps the greatest

influence came from the fine chemical and pharmaceutical industry at a time when strict rules

and regulations were enforced to reduce the carbon footprint in order to combat climate

change. This led to the adoption of greener strategies to mitigate current problems the industry

was facing, including the need for harmful organic solvents, the generation of toxic waste, the

poor atom economy of most fine chemical processes and the reliance on non-renewable

feedstocks. The concept of “greenness” of chemical processes was already considered

relevant in the 1960s, however, it was only in 1998 when Anastas and Warner developed the

12 principles of green chemistry. Biocatalytic processes typically adhere to most (if not all) of

these principles, contributing to the ever-increasing popularity of enzymatic reactions in

industrial processes.

1.4.1 Synthesis of L-arylalanines

Derivatives of L-phenylalanine are ubiquitous in nature, involved in many metabolic processes

or pathways and are chiral structural units of a number of chemical messengers, including

hormones and neurotransmitters. Non-natural L-arylalanines are also crucial in a myriad of

chemical processes and are incorporated as key pharmacophores in many active

pharmaceutical ingredients (APIs) and drug candidates. Whilst D-phenylalanine is much rarer

in nature, the D-enantiomers of many arylalanines are almost equally common and useful

building blocks in synthetic chemistry. A representative selection of APIs, drug candidates or

precursors based on L- and D-arylalanine synthons is presented in Scheme 5.

28

Scheme 5. Representative examples of APIs, drug candidates or precursors containing L- and D-arylalanine

derivatives (the arylalanine moieties are highlighted in blue).

Due to the reversibility of the enzymatic reactions of arylalanine ammonia-lyases, first alluded

to by Williams and Hiroms in 1967 for HALs,96 and Hanson and Havir in 1968 for PALs,97 it is

synthetically more useful to drive the reaction in the amination direction to produce the higher

value L-arylalanine from the inexpensive arylacrylic acid substrate. Several methods already

exist for the chemical synthesis of enantiopure amino acids, for example, the asymmetric

hydrogenation of dehydroamino acids,98 a process well known for the synthesis of L-DOPA,

or the classical Strecker synthesis99 (either performed in an asymmetric fashion100 or followed

by chemical or enzymatic resolution). The ammonia-lyase reaction, however, has a number

of unique advantages over other methods: 100% atom economy, inexpensive and easily

accessible substrates, and no requirement for expensive cofactors or regeneration systems.

Furthermore, the direct addition of ammonia to the α-position of the acrylic acid occurs with

anti-Michael regioselectivity, a reaction which cannot be emulated with current synthetic

methods, highlighting the synthetic utility of these biocatalysts.

In order to favor the equilibrium position towards the amination direction, the biotransformation

step is carried out in the presence of high concentrations of ammonia. The potential industrial

application of PAL was first demonstrated in 1976, in a patent filed by Pfizer using RtPAL from

Rhodosporidium toruloides (formerly known as Rhodotorula gracilis) covalently immobilized

onto a solid support.100 Unsubstituted cinnamic acid with a concentration of 500 mM in the

presence of concentrated buffered ammonia at pH 9.5 gave 90% yield of L-phenylalanine after

29

18 h incubation at 37°C. Yamada et al. soon published the first paper with full optimization

parameters of RgPAL from Rhodotorula glutinis for the hydroamination of cinnamic acid to L-

phenylalanine.102 A number of factors were tested, such as optimal growth conditions, pH,

ammonia concentration and substrate loading. The best growth conditions were found with

1% yeast extract, 1% peptone, 0.05% L-phenylalanine and 0.5% sodium chloride. Substrate

loading experiments showed 60 mM to be the ideal concentration for amination

(concentrations higher than this resulted in substrate inhibition). Optimum ammonia

concentration was determined to be 9 M at pH 10.0, yielding 70% conversion to L-

phenylalanine.

Earlier work on the synthesis of L-phenylalanine opened up new avenues towards the

synthesis of non-natural amino acids. It was a decade later when RgPAL was screened

against a comprehensive panel of substituted cinnamic acid derivatives, highlighting the scope

and limitations of this transformation (Scheme 6).103 Under similar conditions previously

reported with this enzyme,102 a number of fluoro-, chloro, bromo-, methyl-, methoxy- and

dihalogenated compounds gave moderate to very good conversion. Interestingly, for

monosubstituted substrates, a general correlation between the conversion and the position of

the substituent could be observed: the rate of amination was found to decrease in the order

ortho- > meta- > para-. A difference of >35% in conversion was observed between the three

isomers of fluoro-, chloro- and bromo- substrates, for instance. This was ascribed to the steric

clash between the substituent and active site residues, as also postulated by the authors.

Unsurprisingly, increasing further the size of the aromatic ring either dramatically reduced the

conversion values (e.g., 1-naphthyl) or afforded no conversion at all (e.g., indol-3-yl, 3,4-

methylenedioxyphenyl). On the other hand, substrates where the phenyl ring was replaced

with a short aliphatic chain (e.g., trans-2-butenoic acid) gave no conversion either. Smaller

thienylacrylic acids were accepted, albeit with low conversion to the corresponding amino acid.

To probe further the substrate tolerance of PAL, α- and β-substituted fluoro- and

methylcinamic acid were tested under amination condition. No conversion was observed with

these substrates, and they were in fact found to be mildly inhibitory to the enzyme. It is worth

mentioning that up to now (more than 25 years later), no wild-type or mutant PAL active on a

cinnamic acid derivative bearing any substituent other than H (or 2H and 3H)88,94,104 on the C=C

bond has been identified. The same situation applies to the attempts to expand the nucleophile

scope: even though deamination of N-substituted amino acids has been reported (e.g., N-

methylphenylalanines105), amination with methylamine or any other nitrogen nucleophile has

not been successful, as of yet. Such extremely challenging targets would represent a major

breakthrough and a very valuable addition to the ammonia-lyase toolbox for biocatalysis.

30

Scheme 6. RgPAL catalyzed synthesis substituted phenylalanine analogues (percentage values refer to

conversions).

Bromophenylalanines are particularly valuable products for further chemical manipulations to

afford structurally diverse L-phenylalanine analogs (see chapter 2). However, RtPAL and

RgPAL suffer from narrow substrate specificity and poor conversions with bromocinnamic

acids (particularly the p-bromo isomer) limiting their use as industrial biocatalysts. Recent

engineering efforts by Rowles et al. successfully improved substrate tolerance of a related

PAL (RgrPAL from Rhodotorula graminis) towards p-bromocinnamic acid.106 The design and

screening of five CASTing libraries (each covering two spatially close positions) in the active

site allowed the identification of the RgrPAL-His143Cys-Gln144Asn variant (RgrPAL-31E) with

an impressive 28-fold improvement in activity compared to the wild-type. Even though AvPAL

was found to be more tolerant towards that substrate (71% conversion), similar engineering

work on AvPAL by Ahmed et al. yielded a modest improvement in activity with the single point

mutation Phe107Ala (80% conversion).107

The discovery, cloning and screening of new arylalanine ammonia-lyase enzymes from

different sources proceeded at a much faster rate in recent years, occasionally stumbling into

remarkable new properties. In 2008, DSM patented the discovery and expression of new PAL

genes from several organisms (Shewanella oneidensis, Idiomarina lohiensis, Fibrobacter

succinogenes and Vibrio vulnificus) with increased expression levels and very high relative

activities on o-bromocinnamic acid, compared to RgPAL.108 Besides their increased activity,

however, the striking feature of this set of new biocatalysts (in particular the PAL from I.

31

lohiensis) was the extraordinary tolerance to high substrate concentrations, even at elevated

pH values where the solubility is higher. The combination of high activity and absence of

substrate inhibition is extremely appealing to increase the productivity of industrial biocatalytic

processes.

As a further demonstration of the industrial viability of PALs, the intensified synthesis of a

number of non-natural halophenylalanine derivatives was reported by Weise et al., in

collaboration with the chemical company Johnson Matthey.109 Using ammonium carbamate

as the ammonia source instead of the more conventional pH-adjusted ammonia solutions or

other ammonium salts, considerable improvement in conversions could be obtained, along

with a much simpler downstream processing. The superior conversion was attributed to

ammonium carbamate releasing two equivalents of ammonia whilst maintaining a lower ionic

strength. Several important parameters including temperature, pH, catalyst formulation,

substrate loading and incubation time were optimized to improve the efficiency of the

biocatalytic process. Preparative scale synthesis of a number of amino acids was carried out

yielding full conversion in each case with a space-time yield (STY) in the range of 5-20 g L–1

d–1 (Scheme 7). The use of ammonium carbamate improved the isolation efficiency by

evaporation of the buffer at its sublimation temperature, alleviating many problems

encountered using previous methods, such as the requirement for ion-exchange purification

(with additional costs and sometimes low recovery). Optimized conditions for 3-fluorocinnamic

acid led to an impressive STY of 237.6 g L–1 d–1 with excellent enantiopurity. To demonstrate

the modularity of this process, the method was also integrated in two chemo-enzymatic

processes (synthesis of biarylalanine derivatives107 and telescopic one-pot

condensation/hydroamination.110

Scheme 7. Panel of non-natural L-halophenylalanines produced with an intensified AvPAL mediated

hydroamination using ammonium carbamate as the ammonia source (space-time yields are reported).

Previous work has alluded to the fact that no modification of the carboxylic acid portion of the

substrate is tolerated by the enzyme. Remarkably, Nair and co-workers reported the PAL

catalyzed hydroamination of methyl trans-cinnamate to L-phenylalanine methyl ester using

32

RgPAL in an organic-aqueous biphasic mixture, due to the poor solubility of the substrate

(Scheme 8).111 A solvent screen showed n-heptane to be the best co-solvent, giving the

highest product yield. Under the best conditions, with a substrate loading of 100 mM, only 7%

conversion could be achieved. While the potential applications (e.g., for aspartame synthesis)

are exciting, the low conversions obtained from the biotransformation are far from ideal for

industrial application. Unfortunately, no further work through reaction optimization or enzyme

engineering was reported with this substrate, which suggests the extreme difficulty in

engineering PAL enzymes to accept substrates lacking the carboxylate moiety essential for

substrate binding.

Scheme 8. RgPAL catalyzed synthesis of L-phenylalanine methyl ester in a biphasic system.

Several phenylalanine analogues with non-aromatic cyclic systems were explored by different

groups, either for mechanistic investigation or in the attempt to understand and expand the

substrate spectrum. Ressler et al. demonstrated promising activity towards the deamination

of L-(2,5-dihydrophenyl)alanine, to afford the corresponding acrylic acid with RgPAL. Kinetic

data revealed 10-fold decrease in kcat but similar binding affinity to L-phenylalanine, yielding

85% conversion after 4 days (Scheme 9a),112 disputing earlier works suggesting that PAL

enzymes do not accept non-aromatic substrates. A tentative explanation for the deamination

was provided two decades later by Retéy and co-workers using the Friedel-Crafts

mechanism.113 They also postulated the regioisomer L-(1,4-dihydrophenyl)alanine to be a

competitive inhibitor of PAL, and this was proved through inhibition studies with PcPAL

(Scheme 9a). Gloge et al., in pursuit of unique substrates for potential novel applications, tried

unsuccessfully the hydroamination of cyclooctatetraenylacrylic acid (Scheme 9b).114 The lack

of activity can be attributed to two key factors: the non-aromatic character, the size and the

conformation of the ring. Earlier work on PALs highlighted the requirement of substrates to

bear a planar 6-membered aromatic ring for optimal substrate binding and catalysis. The

increased size of the 8-membered ring could lead to a clash with the active site residues lining

the hydrophobic pocket, although the thermodynamically favored puckered conformation

adopted by the non-aromatic ring might be the key factor that prevents the substrate from

binding effectively in the active site.

33

Scheme 9. Early studies of amination/deamination of non-conventional PAL substrates: dihydrophenylalanine

isomers (a), cyclooctatetraenylacrylic acid (b), pyrimidinylalanines (c).

Additional mechanistic work on PcPAL was carried out testing the deamination of two aromatic

pyrimidine substrate analogues: L-(pyrimidin-2-yl)alanine and L-(pyrimidin-5-yl)alanine

(Scheme 9c).91 The pyrimidin-2-yl regioisomer was determined to be a competitive inhibitor,

forming a strong irreversible covalent bond with the MIO functionality leading to a stable

pyrimidinium complex. This was interpreted as possible supporting evidence for the Friedel-

Crafts-type mechanism, even though the formation of this inert complex does not in itself

disprove the E1cB mechanism. On the other hand, the pyrimidin-5-yl isomer was accepted by

PcPAL to give the acrylic acid with a respectable 57% yield.

Nonetheless, these fundamental studies led also to the exploration of the substrate scope of

PcPAL, highlighting its broad substrate tolerance and its industrial value in biocatalytic

synthesis of non-natural amino acids, mainly through the work of Rétey, Poppe and co-

workers. Several polyfluoro- and monochloro- phenylalanine analogues were synthesized with

moderate to good isolated yields (37-88%) with high enantiopurity using purified enzyme

(Scheme 10). Nitrogen-containing heteroaromatic substrates were also tested, affording

excellent conversion for all three regioisomers of pyridylacrylic acid and (pyrimidin-5-yl)acrylic

acid. The substrate scope of PcPAL was then probed with furan and thiophene systems,

including larger fused aromatic rings (Scheme 29).115 Many of these substrates were

converted efficiently with good isolated yields, even though some were previously shown to

34

give poor or no activity with fungal PALs.105 PcPAL failed to convert only the substrates

containing 3-substituted furans and thiophenes. The lack of activity towards these substrates

was ascribed to the unfavorable binding conformation the substrates adopted in the active

site, seen in the light of the Friedel-Crafts-like mechanism.84 However, the 3-thienylacrylic acid

substrate is accepted to some extent by RtPAL,103 pointing towards either a different

mechanism or an altered substrate binding mode in a different enzyme. The substrate

tolerance of PcPAL was further challenged with 5-arylfuran-2-yl derivatives, affording the

corresponding biarylalanines in 49-66% isolated yields after 36-48 h (Scheme 10).116 Nitro-

substituted phenylalanines were also obtained in very good yields.79,93 Access to such a broad

variety of compounds through one simple biocatalytic method (with excellent enantiomeric

purity in all cases) opened up potential sustainable routes to the manufacture of countless

non-natural amino acid derivatives for the pharmaceutical and agrochemical industry.

Furthermore, such a broad substrate scope facilitated the development of PAL-specific assay

methods using modified or functionalized cinnamic acid analogues as molecular probes.117,118

Scheme 10. Production of a broad panel of aromatic and heteroaromatic amino acids by PcPAL (percentage values

refer to isolated yields).

The discovery of the cyanobacterial AvPAL from Anabaena variabilis52 led to an expansion of

the substrate scope of PAL aminations for the production of non-natural amino acids. When

comparing its catalytic activity with that of PALs from yeasts (RgPAL) and plants (PcPAL),

AvPAL presented an even broader substrate scope towards several non-natural substrates.119

This was highlighted with the synthesis of L-(4-trifluoromethyl) phenylalanine (a substrate not

35

accepted by RgPAL and PcPAL) on a preparative scale, with excellent conversion and

enantiopurity, in a moderate isolated yield of 42% (Scheme 11).

Scheme 11. Preparative scale synthesis of L-(4-trifluoromethyl) phenylalanine with AvPAL.

As a further illustration of the synthetic utility and the robust activity of PAL enzymes, Barron

et al. investigated the synthesis of L-phenylalanines by RgPAL in ionic liquids (ILs). Four

commonly used ILs were tested: 1-butyl-3-methylimidazolium methylsulfate ([BMIM][MeSO4]),

1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]), 1-butyl-3-methylimidazolium

hexafluorophosphate ([BMIM][PF6]) and 1-butyl-3-methylimidazolium lactate

([BMIM][lactate]). Ionic liquids tend to infer thermal/operational stability and generally show

reduced enzyme inhibition/deactivation compared to organic solvents. [BMIM][PF6] was found

to be the best IL for this application, giving a maximum conversion of 59% in the amination

direction using 1 M ammonium hydroxide adjusted to pH 9.0.120

Enzyme immobilization, a widely used method to improve the lifespan and operational stability

of enzymes for industrial applications, has been recently demonstrated with PALs. The ability

to recycle enzymes after each biotransformation is an attractive strategy to improve overall

efficiency and to reduce waste and costs. The advent of microreactor technology has rekindled

efforts in developing innovative processes with immobilized enzymes. Pioneering work

conducted by Poppe and co-workers demonstrated the potential application of immobilized

arylalanine ammonia-lyases.121 PcPAL was covalently attached to carboxylated single-walled

carbon nanotubes (SwCNT), through either a direct link or a glycerol diglycidyl ether (GDE)

linker (Scheme 12). To determine the stability of the covalently immobilized enzyme,

optimization tests were carried out with 2-thienylacrylic acid as a model substrate, under harsh

amination conditions (6 M NH4OH, pH 10.0). The directly linked enzyme suffered significant

loss of activity after three cycles, while the GDE linked enzyme showed greater tolerance,

retaining 98% activity after three cycles and 50% after five cycles. Optimal amination

conditions in continuous flow mode were found to be 2 M NH4OH, pH 10.0. The difference in

conversion between immobilized and free enzyme (58% compared to 89%) is due to the low

ammonia concentration, in order to prevent hydrolysis and degradation of the covalent linker.

Immobilized PcPAL showed remarkable thermal stability compared to the free enzyme: no

significant loss of activity at 70°C for 38 h, with an optimum temperature found to be around

60°C (1.5-fold increase in temperature tolerance compared to the free enzyme). This method

positively affects two key parameters of the biocatalyst, its reusability over several cycles and

its thermal tolerance, both important for industrial manufacturing. A very recent report from the

36

same group demonstrated even higher stability and durability of PcPAL immobilized on

aminated SwCNTs compared to carboxylated SwCNTs.122 Experiments aimed at

demonstrating the recyclability of the immobilized biocatalyst highlighted an unusual

inactivation profile, with a relatively long stable plateau, followed by rapid inactivation. This led

to the interesting hypothesis that the deactivation is caused by the slow (and reversible)

replacement of structural water with ammonia, leading to a sudden loss in activity only when

the inner regions close to the active site are affected. This hypothesis seems to be confirmed

by experimental results, since the original activity could be restored multiple times by

incubating the deactivated immobilized biocatalyst with a slightly acidic reconditioning buffer

after each run.122

Scheme 12. Immobilization of PcPAL on a carboxylated single-walled carbon nanotube (SwCNT) and application

to the synthesis of L-arylalanines using microreactor technology.

Additional work carried out by the same group demonstrated the immobilization of PcPAL on

magnetic nanoparticles (MNPs).89 The MNPs-immobilized enzyme was retained by means of

permanent magnets in several small cells within a microfluidic reactor. Remarkably, the

enzyme in the microfluidic system managed to carry out successfully the synthesis of L-

propargylglycine from the corresponding acrylic acid, a substrate which was thought to be

incompatible with PAL enzymes, affording 14% isolated yield (Scheme 13). This contradicts

earlier works which suggested the substrates require an aromatic ring to enable binding to the

active site, and also disfavors the Friedel-Crafts mechanism. More importantly, Poppe and co-

workers have highlighted new opportunities towards catalyzing the conversion of acyclic

acrylic acid analogues, broadening the substrate scope and potential applications of

ammonia-lyases.

Scheme 13. Synthesis of L-propargylglycine using PcPAL (on MNPs) in a microfluidic system.

37

1.4.2 Synthesis of D-arylalanines

The biocatalytic synthesis of the non-proteinogenic enantiomer of amino acids represents an

attractive strategy in industrial processes compared to conventional chemical synthesis.

Where direct asymmetric synthesis is not possible, two main approaches exist to produce

optically pure D-arylalanines: kinetic resolution (stereoselective removal of the L-enantiomer)

or deracemization (two-step conversion of the L-enantiomer to either the D-enantiomer or the

racemic mixture, through a non-chiral intermediate). Both processes have typical advantages

and disadvantages. For example, while kinetic resolution might be a much simpler and

cheaper process, the maximum conversion is limited to 50%, making the process intrinsically

wasteful. Employing a deracemization cascade can mitigate this problem by recycling the L-

enantiomer, with theoretical conversion >99%, although the process is usually more complex,

requiring several components for the reaction. PALs have been used for a long time in the

synthesis of D-arylalanines by stereoselective deamination of the L-enantiomer in a chemically

synthesized racemic mixture. The acrylic acid can be removed easily (e.g., by ion-exchange

chromatography or recrystallization), leaving behind the unconverted D-product. An additional

advantage of this method is that the PAL reaction (in the natural direction) proceeds more

efficiently without the need for high pH and ammonia concentrations. PAL-based

deracemization approaches were developed only very recently.

An early study reported the synthesis of a range of D-arylalanines with >99% ee and moderate

to good isolated yields using a crude homogenate of Rhodotorula graminis cells.123 Even

though the method may be considered rudimental since the lysate contains a number of

uncharacterized enzymes (including, most likely, RgrPAL), it highlights the simplicity and low

cost of the process, which led to the resolution and isolation of several substituted

phenylalanine analogs (Scheme 14).

Scheme 14. Isolation of enantiopure D-phenylalanines from racemic mixtures using Rhodotorula graminis

homogenate (percentage values refer to isolated yields).

A similar strategy was also reported by Shibatani et al. exploiting the HAL activity of

Achromobacter liquidum cells, for the production of urocanic acid and D-histidine.124 The

conversion rates were improved by addition of triethanolamine lauryl sulfate (TEALS), a

38

surfactant to increase the permeability of the membrane. No inhibition of HAL was observed

in the presence of TEALS and 45% isolated yield of D-histidine and 46% isolated yield of

urocanic acid was obtained from racemic histidine (Scheme 15).

Scheme 15. Synthesis of D-histidine and urocanic acid from the enantioselective deamination of rac-histidine.

Rétey and co-workers93,115,116 and later Poppe and co-workers 89,121 employed isolated PcPAL

for the production of a very broad panel of enantiomerically pure D-heteroarylalanines via the

consumption of the L-enantiomer (Scheme 16). Furthermore, Zhu et al. demonstrated the

large scale resolution of racemic phenylalanine in a packed-bed reactor using covalently

immobilized RgPAL on a mesoporous silica support, to give >99% conversion with a space-

time yield of 7.2 g L–1 h–1 and perfect enantioselectivity.246

Scheme 16. Isolation of enantiopure D-arylalanines from racemic mixtures using PcPAL (percentage values refer

to isolated yields).

To overcome the 50% theoretical yield limitation of kinetic resolution approaches, Parmeggiani

et al. recently proposed a chemo-enzymatic amination-deracemization strategy for the

synthesis of D-phenylalanines directly from cinnamic acids (Scheme 17).95 Firstly, a PAL

converts the cinnamic acid into L-phenylalanine (enantiopure or containing small amounts of

D-enantiomer, depending on substituents), then the L-amino acid deaminase from Proteus

mirabilis (PmLAAD) selectively oxidizes the L-enantiomer, rapidly followed by its non-selective

chemical reduction by ammonia-borane to afford rac-phenylalanine. Repeated cycles of L-

selective oxidation and non-selective reduction result in accumulation of the D-enantiomer in

very high ee. Solid-phase screening of a very large site-saturation mutagenesis library of

AvPAL afforded variants with improved activity towards D-phenylalanine production (AvPAL-

His359Tyr found to be the most efficient). Even though the PAL variants did not afford

39

enrichment in the D-enantiomer, they allowed the racemic composition to be reached more

quickly, and performed better in the cascade system. This procedure allowed the production

of enantiopure D-amino acids with >98% ee and conversions between 62-80%. A similar

system, employing a D-amino acid oxidase (DAAO) for selective removal of D-product in place

of the LAAD, allowed to improve to >99% ee the optical purity of those L-arylalanines that

were not obtained in enantiopure form with PAL mediated aminations.95,126

Scheme 17. Chemo-enzymatic amination-deracemization cascade for the synthesis of D-phenylalanines from

cinnamic acids (percentage values refer to isolated yields).

1.4.3 Enzymatic and chemo-enzymatic cascade applications

The exponential rise of enzymatic reactions in industrial synthesis is set to rival traditional

chemical multi-step syntheses in the near future. Nonetheless, while biocatalysis has gained

enormous attention in the context of synthetic applications, it is unlikely to replace completely

traditional chemical synthesis. By harnessing the robustness of chemical catalysis with the

elegance of biocatalysis, new modular and efficient routes towards highly functionalized

molecular scaffolds from simpler starting materials can be attained.

This has been demonstrated with PAL enzymes by developing telescopic routes to aromatic

amino acids starting from the corresponding aromatic aldehydes. Successful synthesis of a

range of halophenylalanines and arylalanines was carried out by Rétey and co-workers with

a three-step chemo-enzymatic process (Scheme 18a).127 First, a Wittig-type reaction (Horner-

Wadsworth-Emmons) in water using ethyl (triphenylphosphoranylidene)acetate converted the

aldehyde to the corresponding ethyl cinnamate. Ester hydrolysis using pig liver esterase (PLE)

followed, to afford the cinnamic acid. Lastly, after addition of ammonia and pH adjustment,

purified PcPAL was added for the amination step. Full conversion to ethyl cinnamate was

afforded from the Wittig reaction with a 95:5 mixture of (E) and (Z) diastereoisomers. While

PLE successfully hydrolyzed both isomers, PAL, on the other hand, was specific for the (E)-

isomer, leaving the (Z)-isomer untouched, thus leading to slightly decreased conversions.

Furthermore, a major by-product of this process is triphenylphosphine oxide, notoriously

difficult to remove from organic reactions. By employing an ion-exchange purification,

halophenylalanines and heteroarylalanines were obtained with 72-91% isolated yields and

>98% ee over a period of 48-96 h.

40

2

Scheme 18. One-pot chemo-enzymatic syntheses of arylalanines from the corresponding aldehydes via Wittig-

type reaction, hydrolysis and amination (a) or Knoevenagel-Doebner condensation and amination (b).

We recently reported an alternative to this process, in this case using a Knoevenagel-Doebner

condensation reaction for the synthesis of the cinnamic acid starting material (Scheme 18b),110

with improved atom economy and reduced waste generation (i.e., no formation of the (Z)-

isomer or triphenylphosphine oxide). The reaction parameters for both the chemical and

biocatalytic reactions were optimized for one-pot two-step operation; in particular switching

the solvent from pyridine to DMSO proved crucial to avoid severe inhibition of the enzyme

(AvPAL and RgPAL) in the biotransformation step. For the chemical step, microwave

irradiation was employed to reduce reaction time and to afford greater control over the

temperature. Experimental data revealed quantitative yields of the cinnamic acid only in an

intermediate range of temperatures (with higher values consistently leading to lower

conversions, likely due to DMSO decomposition in the presence of bases). The activity of

RgPAL and AvPAL was tested towards halocinnamic acid analogs in the presence of varying

DMSO concentrations, revealing a surprisingly high tolerance of PALs for this co-solvent (no

significant inhibition was observed up to 10% v/v and reasonable activity was still present at

40% v/v). This enabled substantially increased substrate loadings and productivities, leading

to the one-pot telescopic synthesis of a number of halogenated amino acids in good to

excellent conversions with high enantiopurity. RgPAL showed preference towards the mono-

substituted substrates whilst AvPAL showed higher tolerance for bulkier dihalogenated

substrates. Preparative scale synthesis of five amino acid precursors of different APIs afforded

71-84% isolated yields with >98% ee.110

By implementing the same telescopic process, novel ring-substituted heteroarylalanines were

synthesized, including products containing EDGs.126 A panel of pyridylalanines was

synthesized from the corresponding aldehydes with isolated yields of 32-65% with high

enantiopurity. Several aldehydes from the picoline (ortho-), nicotine (meta-) and isonicotine

(para-) series were incubated with AvPAL affording full conversion after 2-30 h (Scheme 19).

41

While the conversion of each compound was high, the enantiopurity differed greatly between

the three series. Low ee was obtained for the picoline and isonicotine isomers while the

nicotine isomers generally gave high ee. The difference in optical purity was reported to be

dependent on the electronic density of the aromatic system: electron-poor rings gave low ee

compared to neutral or electron-rich ring systems, consistently with the alternative MIO-

independent PAL mechanism postulated to afford the D-enantiomer.94 The addition of a

strongly electron-donating methoxy substituent did not suppress the reactivity (EDG-

substituted cinnamates are very poorly accepted by PALs), even though the observed ee

profile suggests the electron-withdrawing power of the pyridine nitrogen is surpassed by the

electron-donating ability of the methoxy group. This work sheds new light on the

enantioselective addition of ammonia in the PAL catalyzed amination reaction, since electronic

effects were shown to play an important role in determining the final enantiopurity of the

product. In light of these results, we reported the optimum conditions to afford the highest

enantiopurity by monitoring the biotransformation giving the best compromise between

conversion, ee and purity, essential for the synthesis of chiral APIs. The demonstrated cost-

saving measures of adopting chemo-enzymatic telescopic routes shows great potential for

industrial application.

Scheme 19. Telescopic synthesis of a panel of heteroarylalanines (percentage values refer to isolated yields).

The benefits of chemo-enzymatic and multi-enzymatic catalysis for PAL reactions are not

limited to the in situ synthesis of cinnamic acid precursor. Combining the PAL reaction with

further downstream manipulations allows diversification of the range of non-natural amino acid

derivatives, with the advantage of reducing the purification efforts and total process cost.

Scheme 20 shows several examples of molecules of relevance for the pharmaceutical and

42

agrochemical industry that have been synthesized with one-pot cascade/stepwise processes

involving PALs. Except for the cascade synthesis of D-arylalanines (described in Section

1.4.2), the others will be described in the following.

Scheme 20. Chemo-enzymatic one-pot stepwise/cascade processes involving PALs for the synthesis of

industrially relevant phenylalanine derivatives.

Exploiting the reaction scope of metal catalysis in combination with stereospecific biocatalytic

steps is a powerful strategy to access complex chiral scaffolds. However, the implementation

of chemo-enzymatic processes usually requires a trade-off in the choice of solvent and

conditions (chemical steps are normally run in organic solvents at high temperatures, while

biocatalytic steps proceed in aqueous buffers close to room temperature), sometimes leading

to one-pot two-step strategies where the conditions are modified in between, without

purification of the intermediate. However, in the attempt to adhere even more to the principles

of green chemistry, next-generation chemo-enzymatic processes for industrial production of

fine chemicals will attempt to eliminate completely the use of organic solvents. This has been

demonstrated by the chemical company DSM in one of the most frequently cited examples of

industrial application of ammonia-lyases: the integration of the PAL reaction with a water-

compatible metal catalyzed cyclization reaction in the conversion of 2-chlorocinnamic acid to

(S)-indoline-2-carboxylic acid,128 a precursor of antihypertensives such as perindopril and

indolapril (Scheme 21). The cinnamic acid was added to the biotransformation mixture in

portions to prevent product inhibition, leading to 91% conversion and >99% ee. To avoid

possible racemization in the cyclization step and to reduce the overall cost of the process,

copper was used instead of palladium as the metal catalyst. The best reaction parameters

were found in neat water using 2 mol% CuCl giving full conversion to the cyclized product,

affording (S)-indoline-2-carboxylic acid with 60% isolated yield from 2-chlorocinnamate with

no loss of optical purity from the enzymatic step.

43

Scheme 21. PAL mediated chemo-enzymatic synthesis of (S)-indoline-2-carboxylic acid.

With a similar approach, we recently reported the chemo-enzymatic synthesis of (S)-1,2,3,4-

tetrahydroisoquinoline-3-carboxylic acid via a PAL mediated biotransformation followed by a

Pictet-Spengler reaction.129 The crude PAL mixture was lyophilized to remove water and

ammonium carbonate, and the crude material used directly for the Pictet-Spengler reaction

with concentrated aqueous formaldehyde in acidic conditions, to afford 53% isolated yield of

the enantiopure tetrahydroisoquinoline scaffold (Scheme 22).

Scheme 22. Synthesis of (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid by combining PAL biocatalytic step

and a Pictet-Spengler reaction.

Another example from our group that highlights the power of chemo-enzymatic approaches,

is the synthesis of biarylalanine API precursors by integrating the PAL mediated amination of

p-bromocinnamic acid with the palladium catalyzed Suzuki-Miyaura coupling, a key chemical

transformation widely used in medicinal chemistry (Scheme 23).130 To maximize the

conversion of the biotransformation step the AvPAL-Phe107Ala variant was used, with a larger

active site cavity to accommodate the bulky para-substituent. The palladium coupling reaction

was optimized in neat water by screening different water-soluble palladium catalysts under

microwave irradiation. However, a one-pot telescopic route was found not to be feasible, due

to the high ammonia concentration inhibiting the palladium catalyzed step. Thus, the amino

acid was isolated by adsorption on ion-exchange resin, before submitting it to a one-pot N-

protection and Suzuki coupling. Employing a panel of arylboronic acids, a broad range of N-

Boc-protected L-biarylalanines could be obtained with >99% ee, in 33-65% isolated yield from

the starting p-bromocinnamic acid (and with a different biocatalytic strategy the corresponding

D-biarylalanines were accessed as well).107 The synthetic value of this process was

exemplified by synthesizing a DPP-IV inhibitor starting via the biarylalanine intermediate in 5

steps, with an overall isolated yield of 30% from p-bromocinnamic acid (Scheme 23).

44

Scheme 23. Chemo-enzymatic synthesis of biarylalanines by coupling PAL biocatalytic synthesis with Suzuki-

Miyaura coupling (percentage values refer to isolated yields).

While a huge amount of work on PALs was conducted in the reverse (non-natural) direction

to synthesize high-value chiral amino acids, the natural reaction has not been overlooked for

its synthetic utility, especially in the context of synthetic biology. The synthesis of cinnamic

acid analogues from phenylalanine can provide a range of commercially relevant compounds

by incorporating additional chemical and/or enzymatic steps. For example, (S)-styrene oxide,

a key fine chemical towards functionalized chiral diols, was produced in vivo from glucose.

PAL, trans-cinnamic acid decarboxylase (CADC) and styrene monooxygenase (SMO) were

coexpressed in E. coli to afford the product epoxide from glucose via the primary metabolite

phenylalanine (Scheme 24).274

Scheme 24 In vivo production of (S)-styrene oxide from glucose via co-expression of PAL, CADC and SMO.

Using a similar strategy Kroutil and co-workers demonstrated the in vivo synthesis of a panel

of vinylated phenols starting from simple substituted phenols.132 This multi-enzymatic cascade

involved co-expression of tyrosine phenol-lyase from Citrobacter freundii (CfTPL), tyrosine

ammonia-lyase from Rhodobacter sphaeroides (RsTAL) and ferulic acid decarboxylase from

Enterobacter sp. (ExFAD). The first step of the cascade involves a C-C bond formation

reaction between phenol and pyruvate catalyzed by a CfTPL variant with a relaxed substrate

specificity to obtain L-tyrosine derivatives. RsTAL catalyzes the deamination of L-tyrosine to

coumaric acid followed by decarboxylation by EsFAD to produce several different substituted

vinylated phenols with >99% conversion and isolated yields varying from 65-83% (Scheme

25).

Scheme 25. Multi-enzymatic synthesis of vinylated phenols (percentage values refer to isolated yields).

45

A remarkable eight-step ten-enzyme cascade was reported by Li and co-workers,133 with PAL

as the gateway to a variety of valuable chemical intermediates including styrene oxide, diols,

α-hydroxyacids and phenylglycine derivatives (Scheme 26). The enzymes involved are PAL,

phenylacrylic acid decarboxylase (PAD), styrene monooxygenase (SMO), epoxide hydrolase

(EH), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), hydroxymandelate

oxidase (HMO), transaminase (TA), glutamate dehydrogenase (GluDH) and catalase (CAT).

The enzymatic synthesis of enantiopure (S)-phenylglycine (eight enzymes for functional group

manipulations, GluDH for cofactor regeneration and CAT for H2O2 removal) was achieved with

85% conversion and >99% ee. The products were obtained from biomass feedstock, since

bio-derived L-phenylalanine was used as the starting material and the reactions were run in a

biphasic mixture using bio-derived ethyl oleate from renewable resources as the organic

solvent.

Scheme 26. Eight-step cascade for the synthesis of (S)-phenylglycine starting from the primary metabolite L-

phenylalanine.

The advent of synthetic biology has revolutionized the in vivo production of fine chemicals,

particularly in the areas of metabolic engineering and synthetic biology, and PALs/TALs show

great potential towards the industrial scale production of secondary metabolites in microbial

cell factories. The PAL reaction can be seen as the gatekeeping step to the phenylpropanoid

biosynthetic pathway, thus a substantial amount of work has been carried out on the

integration of these enzymes into different pathways and hosts to produce bio-derived fine

chemicals from phenylalanine and tyrosine. A few representative examples are shown in

Scheme 27. Katsuyama et al. co-produced PAL from Rhodotorula rubra, 4-coumaryl ligase

from Lithospermum erythrorhizon and stilbene synthase from Arachis hypogaea in E. coli,

affording 20 mg L–1 of pinosylvin and 37 mg L–1 resveratrol from 490 mg L–1 phenylalanine and

540 mg L–1 tyrosine respectively.134 A similar approach was employed by Nielsen and co-

workers by introducing the phenylpropanoid biosynthetic pathway into Saccharomyces

cerevisiae. By controlling the metabolic fluxes through overexpression of the genes for the

enzymes involved in malonyl-CoA synthesis and the feedback-inhibition resistant versions of

3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase and chrorismate mutase, a

20,000-fold improvement in resveratrol yield from glucose was obtained.135 To improve the

46

conversion further, Li et al. incorporated the resveratrol biosynthetic pathway from Arabidopsis

thaliana and resveratrol synthase from Vitis vinifera in S. cerevisiae, producing 800 mg L–1 of

resveratrol.136 Styrene was also produced by employing a similar method, using hybrid PAL

isoforms from Populus kitakamiensis.137 Moriguchi and co-workers reported on the production

of 9.4 mg L–1 chrysin from tyrosine in E. coli.138 Ververidis and co-workers highlighted the

production of quercetin by coexpressing eight genes in yeast. The strain was tested for its

ability to produce quercetin by supplementing the growth media with intermediate substrates

in the phenylpropanoid pathway; naringenin, p-coumaric acid and phenylalanine. Naringenin

and p-coumaric afforded 0.38 mg L–1 and 0.26 mg L–1 of quercetin respectively while

phenylalanine gave no traceable amount of product. The authors postulated that a suboptimal

spatial arrangement of metabolic enzymes (referred to as a metabolon) may have been

present in this system, hence affording poor yield of the final product.139 Furthermore, to

counteract the lack of highly active and specific TALs for metabolic engineering applications,

a recent enzyme discovery and comparative in vivo study was carried out, leading to a panel

of new TALs that show potential for production of p-coumarate and related phenylpropanoids

in different strains of bacteria and yeasts.140 Indeed, by harnessing the power of metabolic

engineering and synthetic biology, these and a large number of other challenging target

molecules could be obtained with PALs/TALs in vivo, as extensively covered in a very recent

review.43

Scheme 27. PAL/TAL mediated production of secondary metabolites through metabolic engineering and synthetic

biology.

47

1.5 Conclusion and perspectives

In view of the importance of amino acids as pharmaceutical and agrochemical intermediates,

methods for their synthesis, particularly on scale, are likely to increase in demand in the future.

In many cases the production of these building blocks will need to be carried out in an

economic and sustainable manner, delivering products of high stereochemical purity at low

cost. This challenge provides significant opportunities for the development of engineered

biocatalysts, and in particular ammonia-lyases which, as demonstrated above, possess the

potential to catalyze production of this class of molecules with high atom efficiency and

enantioselectivity starting from inexpensive raw materials. Although some industrial processes

based upon ammonia-lyases are starting to emerge, more research is needed to expand the

substrate scope of this class of enzymes as well increases in stability and activity, so that they

are able to tolerate the high substrate concentrations that are required on scale.

Ammonia-lyase has been shown to be suitable for cascade reactions, enabling sequential

transformations to be carried out in a one-pot manner. The rapid developments in synthetic

biology and metabolic pathway engineering are likely to witness greater application of this

enzyme as key component of multi-step biocatalytic processes, in order to either create non-

natural amino acids in vivo, or alternatively use amino acids as renewable feedstocks for

conversion to other high value products.

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L-Arylalanines from Arylaldehydes. Eur. J. Org. Chem. 2006, 1113–1116.

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133. Zhou, Y.; Wu, S.; Li, Z. Cascade Biocatalysis for Sustainable Asymmetric Synthesis :

From Biobased L-Phenylalanine to High-Value Chiral Chemicals. Angew. Chem. Int.

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Methyl Ethers in Escherichia coli. Biotechnol. J. 2007, 2, 1286–1293.

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Metabolism in Yeast (Saccharomyces cerevisiae): Implications for Control of Metabolic

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Pathway Leading to Heterologous Biosynthesis of Various Flavonoids and Stilbenoids

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59

Chapter 2

Chemo-enzymatic Synthesis of Optically Pure L- and D-

Biarylalanines through Biocatalytic Asymmetric Amination

and Palladium-Catalysed Arylation

Syed T. Ahmed, Fabio Parmeggiani, Nicholas J. Weise, Sabine L. Flitsch, Nicholas

J. Turner



Published in ACS Catalysis, 2015, 5, pp 5410–5413

Publication date: August 10th, 2015.

Acknowledgements: This chapter contains work which was a collaborative effort by the

authors. The doctoral candidate was involved in the experimental design, synthesis and writing

of the research paper.

Pd

PAL

Suzuki coupling

DAADH

>99% ee (L) >99% ee (D)

60

2.1 Abstract

A chemo-enzymatic approach was developed and optimised for the synthesis of a range of N-

protected non-natural L- and D-biarylalanine derivatives. Starting from 4-bromocinnamic acid

and 4-bromophenylpyruvic acid using a phenyl-alanine ammonia lyase (PAL) and an evolved

D-amino acid dehydrogenase (DAADH), respectively, both enantiomers of 4-

bromophenylalanine were obtained and subsequently coupled with a panel of arylboronic

acids to give the target compounds in high yield and optical purity, under mild aqueous

conditions.

2.2 Introduction

An increasing number of drugs in development contain non-natural amino acids in their core

motif, in particular, the biarylalanine moiety (Figure 1). For example, L-biarylalanines have

been incorporated in dipeptidyl-peptidase-4 (DPP IV)1 inhibitors, α4β7 integrin inhibitors2, viral

3C-protease inhibitors3 and endothelin-converting enzyme inhibitors,4 while D-enantiomers

feature in botulinum toxin inhibitors,5 amyloid-β peptide aggregation inhibitors,6 kinesin-14

motor protein KIFC1 inhibitors7 and reverse cholesterol transport facilitators.8

Figure 1. Patented pharmaceuticals containing biarylalanines as chiral building blocks.

Although a number of chemical methods have been reported in the literature for the synthesis

of non-natural amino acids,9 to date no chemo-enzymatic approach to biarylalanines has been

reported. Herein we present two efficient biocatalytic strategies (hydroamination of 4-

bromocinnamic acid 3 and reductive amination of 4-bromophenylpyruvic acid 4) for the

preparation of both enantiomers of 4-bromophenylalanine 2, followed by protection and

61

Suzuki-Miyaura coupling with a panel of arylboronic acids, to afford the target compounds L-

and D-1 (Scheme 1).

Scheme 1. Chemo-enzymatic approach to L- or D-1.

One of the most attractive biocatalytic routes to optically pure L-arylalanines is the asymmetric

hydroamination of arylpropenoic acids catalysed by phenylalanine ammonia lyases (PALs),10

with 100% atom economy and no need for cofactor regeneration systems. In nature, PAL

catalyses the deamination of L-phenylalanine to cinnamic acid,11 while the reverse reaction

has been exploited in the synthesis of novel L-phenylalanine analogues from substituted

cinnamic acids.12

2.3 Results and discussion

2.3.1 PAL and DAADH optimization

The biotransformation of 4-bromocinnamic acid 3 was tested under the standard conditions

for PAL aminations (10 mM of 3, 5 M aqueous ammonia, pH 9.6) using three different wild-

type PALs overproduced in E. coli whole cells: PcPAL from Petroselinum crispum (parsley),

RgPAL from the red yeast Rhodotorula glutinis and AvPAL from the cyanobacteria Anabaena

variabilis. The conversions obtained are reported in Table 1 with the best results being

obtained with AvPAL, which has recently emerged as a promising candidate for preparative-

scale biotransformations because of its broader substrate scope.13

Figure 2. Active site model of 3 bound to AvPAL (MIO = 4-methylideneimidazol-5-one).

Analysis of the active site of AvPAL suggested that the conversion might be improved by

reducing the steric clash between the bromine atom and the residue F107 in the aromatic

62

binding pocket (Figure 2). We therefore designed variants where F107 was mutated to smaller

hydrophobic residues, i.e., F107I, F107L and F107A. For all three variants the conversions

were found to be higher compared to the WT (Table 1) and the highest value was obtained

with the variant F107A. HPLC analysis on a chiral stationary phase showed that the

phenylalanine product L-2 was obtained with >99% ee.

Table 1. PAL amination of 3. Expt. cond.: 25 mg mL–1 lyophilized E. coli cells producing PAL, 5 mM 3, 5 M NH4OH,

pH 9.6, 37°C, 18 h. [a]Determined by HPLC.

PAL variant Conv. (%)a

PcPAL 44 RgPAL 67 AvPAL 71

AvPAL-F107L 72 AvPAL-F107I 75 AvPAL-F107A 80

To access D-2 we selected an NADPH-dependent D-amino acid dehydrogenase (DAADH,

engineered from a meso-diaminopimelate dehydrogenase from Corynebacterium

glutamicum), which has been previously reported to be a very effective catalyst for the

reductive amination of a wide range of aliphatic and aromatic α-ketoacids.14 The reductive

amination of 4-bromophenylpyruvic acid 4 was tested with DAADH cell-free extract (10 mM of

4, 200 mM NH4+, 100 mM glucose, 100 mM carbonate buffer, pH 9), using methanol as a co-

solvent and the glucose / glucose dehydrogenase (GDH) system for the regeneration of the

NADPH cofactor, giving complete conversion to D-2 with >99% ee by HPLC.

2.3.2 Suzuki-Miyaura optimization step

Having access to both enantiomers of 2 through biocatalytic routes, we turned our attention to

the development of the arylation step. Three palladium catalysts (5-7) were shown to be active

under aqueous conditions (Table 2)15-17 and in order to investigate their suitability, a model

reaction was set up between 4-bromobenzoic acid 4 and phenylboronic acid 9a.

Scheme 2. Catalysts for aqueous Suzuki coupling.

Catalyst 5, in water/ethanol (2:1) mixture as solvent, gave a good yield of compound 10 (Table

2, entry 1), while in neat water the conversion dropped considerably to 24% (entry 2). Phenol

and biphenyl were identified as the predominant side-products, however with exclusion of

oxygen the conversion increased to 36% (entry 3). In an oxidative environment palladium can

react with oxygen to form a palladium-peroxo intermediate, which catalyses the homo-

63

coupling and the oxidation of the boronic acid.18 By increasing catalyst concentration and

temperature, up to 86% yield of 10 in neat water was obtained (entry 6), without the need for

a nitrogen atmosphere. Davis et al. have reported the use of catalyst 6 in the bio-conjugation

of proteins and peptides via a Suzuki-Miyaura cross-coupling reaction19. However, the reaction

with 6 was slow and a maximum yield of 41% was obtained (entry 7). We applied the best

conditions from catalyst 5 to catalyst 6 (entry 8): although the yield increased, it was still lower

than with 5. Catalyst 7 was completely insoluble under the conditions tested (entry 9), so it

was not pursued any further. Na2CO3 as a cheaper alternative to Cs2CO3 (entry 10) gave lower

conversion. In order to reduce the reaction times (24 h), microwave irradiation was employed

affording similar yields after only 20 min (entry 11).

Table 2. Optimisation of the reaction between 8 and 9a under aqueous conditions. [a] Determined by HPLC.

Entry Cat.

(mol%) Solvent Cond. Temp.

(°C) Base Conv.

(%)a

1 5 (2) H2O/EtOH 2:1 4 h, air 50 Cs2CO3 62 2 5 (2) H2O 4 h, air 50 Cs2CO3 24 3 5 (2) H2O 4 h, N2 50 Cs2CO3 36 4 5 (10) H2O 4 h, N2 50 Cs2CO3 71 5 5 (10) H2O 4 h, air 50 Cs2CO3 43 6 5 (10) H2O 24 h, air 80 Cs2CO3 86 7 6 (4) H2O 4 h, air 50 KPi buffer pH 7.5 41 8 6 (10) H2O 24 h, air 80 Cs2CO3 56 9 7 (2-10) H2O 4 h, air 50 Cs2CO3 <5

10 5 (10) H2O 24 h, air 80 Na2CO3 68 11 5 (10) H2O 20 min, air, MW 120 Cs2CO3 84

The optimised conditions from the model reaction (entry 11) were applied to the coupling of

N-Boc-4-bromo-L-phenylalanine L-11 with 9a, affording the final biaryl product L-1a in 93%

conversion and >99% ee by HPLC (Scheme 3).

Scheme 3. One-pot protection and arylation of L-2.

64

2.3.3 Chemo-enzymatic route optimization

Addition of Boc2O after the PAL biotransformation formed the desired product L-11, but also

side-products tert-butyl carbamate 12 (by reaction with free ammonia) and 4-phenylcinnamic

acid 13 (from the coupling between 3 and 9a). The coupling reaction performed in the

presence of varying amounts of 12 and 3 led to lower yields (Figure 3a). Furthermore,

protection and coupling attempts on the crude PAL biotransformation mixture yielded no

product, due to the high ammonia concentration in the buffer, as demonstrated by control

experiments with increasing NH4+ concentration (Figure 3b).

Figure 3. Control experiments for the cross-coupling of L-11 and 9a in the presence of varying concentrations of

impurities 3 and 12 (a) and ammonia (b).

Therefore, for the PAL mediated synthesis of L-1a, the removal of ammonium salts and

unreacted 3 from the reaction mixture was performed by adsorption on an ion-exchange resin,

affording quantitative recovery of pure L-2 ready for use in the following step. Boc-protection

and cross-coupling could be performed in one-pot to afford compound L-1a (>99% ee) in 74%

isolated yield resulting in an overall yield of 54% from 3 (Table 3).

In the case of the DAADH biotransformation, the substantially lower ammonia concentration

and the complete consumption of the starting material prevent competing side reactions and

catalyst deactivation. Therefore, it was possible to successfully run the whole sequence as

65

one-pot system, giving D-1a (>99% ee) with overall conversion of 86% and isolated yield of

57% from 4 (Table 3).

Table 3. Chemo-enzymatic synthesis of compounds L-1a-k (via hydroamination followed by one-pot protection and

coupling) and D-1a-k (via one-pot reductive amination, protection and coupling). [a] Conversion of L-2 to L-1

determined by HPLC. [b] Isolated yield of L-1 from L-2. [c] Overall isolated yield of L-1 from 3. [d] Conversion of 4 to

D-1 determined by HPLC. [e] Overall isolated yield of D-1 from 4.

L-1a-k D-1a-k

Product R Conv. from L-2

(%)a

Isol. yield from L-2

(%)b

Overall isol. yield

from 3 (%)c

Overall conv. from 4 (%)d

Overall isol. yield

from 4 (%)e

1a H 75 74 54 86 57 1b 2-F 99 86 62 99 56 1c 3-F 65 58 43 99 66 1d 4-F 85 70 40 99 64 1e 4-Cl 95 85 63 98 66 1f 3-Cl 80 60 35 74 47 1g 2-MeO 65 64 47 95 62 1h 3-MeO 99 89 65 99 61 1i 4-MeO 99 90 64 97 68 1j 3,4-methylenedioxy 99 86 61 99 70 1k 4-Ph 82 55 40 95 40

In order to demonstrate the generality of our approach, using the optimised conditions for the

chemo-enzymatic synthesis of L- and D-1a, we employed a panel of substituted phenylboronic

acids 9b-k, to afford L- and D-biarylalanine derivatives L- and D-1b-k in high yield and >99%

ee (Table 3).

As an example of the practical relevance of these building blocks, we exploited our chemo-

enzymatic approach to L-1b in the synthesis of the DPP IV inhibitor 151 (Scheme 4) in 30%

overall yield from 3.

Scheme 4. Chemo-enzymatic synthesis of DPP IV inhibitor 15.

66

2.4 Conclusions

In summary, we designed a green, efficient route to a range of biarylalanines through the

marriage of two enantio-complementary enzymatic transformations with a combinatorial

chemo-catalytic coupling. The modular independence of the bio- and chemo-catalytic

conversions shown here may be more broadly applicable in the field of medicinal chemistry,

allowing similar expansion to the product range of other biocatalysts.

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18 Adamo, C.; Amatore, C.; Ciofini, I.; Jutand, A.; Lakmini, H. J. Am. Chem. Soc. 2006, 128,

6829-6836.

68

Chapter 3

Telescopic one-pot condensation-hydroamination strategy

for the synthesis of optically pure L-phenylalanines from

benzaldehydes

Fabio Parmeggiani, Syed T. Ahmed, Nicholas J. Weise, Nicholas J. Turner



Published in Tetrahedron, 2016, 72, pp 7256–7262

Publication date: November 17th, 2016.

Acknowledgements: This chapter contains work which was submitted as part of a special

issue on modern developments in biotransformation. The doctoral candidate was involved in

the synthesis, reaction optimization and contribution to the results and discussion section. .

69

3.1 Abstract

A chemo-enzymatic telescopic approach was designed for the synthesis of L-arylalanines in

high yield and optical purity, starting from commercially available and inexpensive substituted

benzaldehydes. The method exploits a chemical Knoevenagel-Doebner condensation

(optimised to give complete conversions in a short reaction time, employing microwave

irradiation) and a biocatalytic phenylalanine ammonia lyase mediated hydroamination (for the

stereoselective addition of ammonia). The two reactions can be run sequentially in one pot,

bringing together the advantages of chemical and biological catalysis. The preparative

applicability was demonstrated with the synthesis of five L-dihalophenylalanines (71-84%

yield, 98-99% ee) of relevance as molecular probes, for medicinal chemistry and for the

synthesis of pharmaceutical ingredients.

3.2 Introduction

Non-natural amino acid analogues are widespread chiral building blocks implicated in the

production of various natural and synthetic bioactive compounds. Of particular interest are

ring-substituted phenylalanine analogues which can be incorporated into natural polypeptides

to enable novel chemical biological studies1 or used as intermediates for peptidomimetic and

small-molecule pharmaceuticals.2

Biocatalysis offers many alternatives for the synthesis of these useful building blocks.3 For L-

phenylalanine analogues, one of the best strategies is the stereoselective hydroamination of

cinnamic acid derivatives mediated by phenylalanine ammonia lyases (PALs, EC 4.3.1.24-

25).4 In nature, these enzymes catalyse the abstraction of ammonia from the primary

metabolite L-phenylalanine to yield the corresponding acrylic acid. This can then be

incorporated into various natural products such as antibiotic compounds in bacteria5 and

phenylpropanoids (including lignin) in terrestrial plants.6 The discovery of the reversibility of

the natural reaction in the presence of high concentrations of ammonia,7 has enabled an easy

and efficient approach to L-arylalanines, starting from a range of simple prochiral arylacrylic

acids.

The vast majority of cinnamic acid derivatives both on industrial and laboratory scale are

synthesised through the simple Knoevenagel condensation8 as modified by Doebner,9 based

on the reaction of an aromatic aldehyde and malonic acid in the presence of a base (usually

a secondary amine such as piperidine or pyrrolidine) in refluxing pyridine. The

benzylidenemalonic acid intermediate decarboxylates in situ, affording the corresponding

cinnamic acid, which can be isolated in high yield by diluting the mixture with ice, acidifying it

and filtering the crude product. The reaction is very efficient, tolerates a wide range of

70

substituted aryl groups, and affords the (E)-stereoisomer of the product with perfect

diastereoselectivity.

In spite of the simplicity of this synthesis, in order to reduce the overall processing time,

minimise waste and increase the productivity of the system, it would be desirable to combine

the two synthetic steps in a simple one-pot strategy, especially for those amino acid derivatives

where the corresponding cinnamic acids are not commercially available, but the aldehyde is.

Indeed, from a fine chemical manufacturing perspective it is generally appealing to run more

than one reaction concurrently in the same vessel in a so-called “one-pot cascade”

combination, wherever possible. Biocatalysis is particularly suited for this purpose, since many

enzymes are compatible with each other and work under similar mild conditions: various

successful multi-enzymatic cascade systems appeared in the literature in the last decade,10

only rarely involving PALs.11 Equally advantageous has been the integration of chemical and

biocatalytic steps,12 successfully exploited for a range of different purposes, such as to shift

the equilibrium of thermodynamically limited enzymatic reactions, to synthesise the enzyme

substrate in situ from a simpler precursor, or to diversify and further manipulate the product of

a biotransformation.

In some instances, though, the extreme difference in conditions (e.g., temperature and

solvent) make the cascade strategy not feasible. In those situations, similar advantages can

still be achieved with a so-called “telescopic” approach, where the reagents for the second

step are added only when the first one is completed, and the second reaction is run under

conditions that are independent on the previous.

Since the conditions for the Knoevenagel-Doebner condensation and for the PAL-mediated

amination are incompatible, we ruled out the possibility to combine them as a cascade system,

but we envisaged that a telescopic strategy could be feasible. Herein, we report on our

attempts to combine the two reactions in a one-pot strategy for the conversion of a range of

available and inexpensive arylaldehydes 1 to the corresponding L-phenylalanines L-3 in good

yield and excellent optical purity (Scheme 1).

Scheme 1. Telescopic synthesis of L-phenylalanines from benzaldehydes.

71


3.3.1 Preliminary feasibility experiments

A typical Knoevenagel-Doebner condensation procedure involves heating under reflux for a

few hours the aromatic aldehyde (1 equiv.), malonic acid (3 equiv.) and a base such as

piperidine or pyrrolidine (2-10 mol %) in pyridine as a solvent. Such a procedure, applied to

benzaldehyde 1a (as a model substrate, used throughout the optimisation described below)

affords quantitative conversion to trans-cinnamic acid 2a (Scheme 2).

Scheme 2. Classical Knoevenagel-Doebner condensation of 1a.

The substrate/product concentrations for the Knoevenagel-Doebner reaction are in the molar

range, while for the biocatalytic amination (Scheme 3) concentrations of 20-50 mM are rarely

exceeded. Therefore, we expected that the PAL-mediated amination step could be

conveniently telescoped by diluting the reaction mixture with buffered ammonia solution and

adding the biocatalyst.

Scheme 3. Classical PAL-mediated hydroamination of 2a.

For this purpose, we tested the crude Knoevenagel-Doebner reaction mixture (1:50 dilution in

the reaction buffer, giving a 20 mM final concentration of 2a) as a substrate for the amination

mediated by PAL under standard conditions (5 M aqueous ammonia solution, pH adjusted to

9.6 with H2SO4, 37°C, 24 h). Two PALs from different kingdoms overproduced in E. coli

BL21(DE3) were tested as a whole-cell biocatalyst: RgPAL from the red yeast Rhodotorula

glutinis13 and AvPAL from the cyanobacterium Anabaena variabilis.14 No conversion to L-3a

was observed, even after prolonged incubation time or with increased biocatalyst loadings.

This could be attributed to the inhibitory effect of residual pyridine: indeed, control experiments

showed that 2.5% v/v of pyridine completely suppresses the activity of both the enzymes

tested, and concentrations as low as 1.0% v/v can considerably reduce it (Table 1).

72

Table 1. Effect of increasing pyridine concentrations on the amination of cinnamic acid 2a mediated by RgPAL

and AvPAL. Experimental conditions: 5 mM 2a, 40 mg mL-1 PAL biocatalyst (wet cells), 5 M NH4OH, pH 9.6 (adj.

with H2SO4), 37ºC, 24 h. [a] Calculated by HPLC analysis on a non-chiral stationary phase.

Pyridine conc. [% v/v]

Conv. RgPAL [%]a

Conv. AvPAL [%]a

0 61 47 1.0 7 4 2.5 <1 <1 5.0 <1 <1 10.0 <1 <1

A variation of the Knoevenagel-Doebner condensation in the literature replaces pyridine with

DMSO,15 a safer and more environmentally friendly solvent that is also well-known for being

much better tolerated by enzymes.16 PAL aminations generally do not require co-solvents due

to the good solubility of cinnamates in the alkaline reaction medium, therefore little attention

in the previous literature has been devoted to the co-solvent tolerance of this class of

enzymes. Thus, we first investigated the influence of varying amounts of DMSO on the

conversion of cinnamic acid 2a to phenylalanine 3a by RgPAL and AvPAL under standard

conditions (Figure 1).

Remarkably, both enzymes appeared to tolerate considerable amounts of this co-solvent: high

concentrations lead to lower conversions but the reaction still proceeded with a significant

drop only over a concentration of 20% v/v. For RgPAL, addition of DMSO up to 10% v/v did

not change the overall reaction rates appreciably. For AvPAL, the initial rates did not seem to

drop up to 20-30% v/v, giving after 2 h almost the same conversions as achieved with no co-

solvent. On the other hand, the stability of the biocatalyst seemed to be affected, since the

final conversions after 24 h became progressively lower with 20-40% v/v of co-solvent.

Nevertheless, the modest decrease in activity of both PALs in the presence of substantial

amounts of DMSO (as opposed to what observed with pyridine) proved that the telescopic

approach outlined before could, in principle, be feasible.

73

Figure 1. Effect of increasing DMSO concentrations on the amination of cinnamic acid 2a mediated by RgPAL and

AvPAL. Experimental conditions: 5 mM 2a, 40 mg mL-1 PAL biocatalyst (wet cells), 5 M NH4OH, pH 9.6 (adj. with

H2SO4), 37ºC.

To test this strategy, the same Knoevenagel-Doebner condensation was repeated in the

presence of DMSO instead of pyridine, according to literature conditions,15 affording again

quantitative conversion of 1a to 2a. The resulting mixture was then diluted with ammonia

solution and incubated with PAL as described above for the reaction in pyridine. In this case,

PAL activity was almost fully retained, affording 58 and 51% conversion to L-3a (over 24 h)

with RgPAL and AvPAL, respectively. Having thus proven the practical applicability of our

system, we then turned to the optimisation of each step individually, in order to maximise the

overall efficiency of the process.

3.2.2 Optimization of the Knoevenagel-Doebner condensation step

With the aim of reducing the reaction times as much as possible, we attempted to optimise

the conditions for cinnamic acid synthesis by employing microwave heating. The uniform and

rapid heating of reaction mixtures allows faster reaction times to be achieved and in some

cases even higher yields.

A series of optimization reactions were set up to probe the efficiency of the Knoevenegel-

Doebner condensation under microwave conditions, using 1a as the model substrate (Table

2). Initially we tested three temperatures (60, 90, 120°C) with a reaction time of 5 min.

Surprisingly, the highest conversion was seen with the lowest temperature (entry 1) and a

0

20

40

60

80

0 2.5 5 10 20 30 40

Co

nv

. [%

]

DMSO conc. [% v/v]

2 h

24 h

0

20

40

60

80

0 2.5 5 10 20 30 40

Co

nv

. [%

]

DMSO conc. [% v/v]

2 h

24 h

AvPAL

RgPAL

74

similar trend was observed when the reaction time was increased to 20 minutes (entries 4-6).

In both cases, a higher temperature resulted in lower conversion, which we postulate is due

to the thermal decomposition of DMSO under microwave irradiation, evident from the

darkening of the reaction mixtures at higher temperatures. The instability of DMSO in the

presence of acids or bases at high temperature is known in the literature, with side products

identified as dimethyl sulphide, bis(methylthio)methane and methyl disulphide.17 The highest

temperature where no decomposition was observed was 60°C, with a reaction time of 45

minutes giving 93% conversion (entry 8). Another possibility is that, at higher temperatures,

the rate of decarboxylation of malonic acid to give acetic acid (observed by 1H NMR) and

carbon dioxide, is increased more than that of the desired reaction, thus resulting in overall

lower consumption of the starting aldehyde.

In light of our initial temperature/time optimisation study, two further experiments were carried

out: increasing the base from 2 mol% to 6 mol% (entry 10) and using 2 equivalents of malonic

acid (entry 11) to improve reaction time and drive it to completion. The former gave lower

conversion of 81%, while the latter yielded full conversion. By shortening the reaction time to

20 min, the yield did not drop (entry 12), but reducing it to 10 min the reaction was no longer

complete (entry 13), even with 3 equivalents of malonic acid (entry 14). This indicates the

reaction must be performed for at least 20 min under these conditions.

Table 2. Optimization of the Knoevenagel-Doebner reaction for the synthesis of 2a employing microwave heating.

[a]Calculated by 1H NMR (by integration of the singlet at 9.82 ppm for 1a and the doublet at 6.27 ppm for 2a);

[b]Considerable darkening and formation of subproducts was observed; cFormation of β-phenylalanine was

observed.

Entry Time [min]

Temp. [ºC]

Base Malonic acid [equiv.]

Conv. [%]a

1 5 60 2 mol% piperidine 1.1 75 2 5 90 2 mol% piperidine 1.1 51b 3 5 120 2 mol% piperidine 1.1 57b 4 20 60 2 mol% piperidine 1.1 87 5 20 70 2 mol% piperidine 1.1 76 6 20 90 2 mol% piperidine 1.1 52b 7 30 60 2 mol% piperidine 1.1 89 8 45 60 2 mol% piperidine 1.1 93 9 60 60 2 mol% piperidine 1.1 93

10 30 60 6 mol% piperidine 1.1 81 11 30 60 2 mol% piperidine 2.0 >99 12 20 60 2 mol% piperidine 2.0 >99 13 10 60 2 mol% piperidine 2.0 91 14 10 60 2 mol% piperidine 3.0 92 15 20 60 2 mol% Et2NH 2.0 57 16 20 60 2% v/v conc. NH4OH 2.0 77c 17 20 60 5% v/v conc. NH4OH 2.0 97c

75

We also considered the use of an alternative base in our screening. Diethylamine (also a

secondary amine similar to piperidine) gave a substantially lower conversion of 57% under the

same conditions of entry 12 (entry 15), possibly because of its higher volatility. Employing

ammonia as the base was also very attractive due to it being already a component required

for the second step of the telescopic process: a conversion of 97% was measured using

concentrated ammonium hydroxide instead of piperidine (entry 17). However, in this case, we

also observed the formation of a white precipitate (not seen with diethylamine or piperidine),

that was isolated and found to be mostly composed of β-phenylalanine (confirmed by 1H NMR

and HPLC), drastically lowering the overall yield of cinnamic acid. Indeed, a high-yield

synthesis of aromatic β-amino acids from the same starting materials uses similar conditions

with ammonium formate or acetate as the ammonia source.18 Employing ammonium

hydroxide, it is not surprising to observe this competing side reaction. Thus, in order to achieve

complete conversions and avoid the formation of side-products such as the β-amino acid, we

reverted back to piperidine as the base, leading to the following optimised conditions: 60°C,

20 min, 2 mol% piperidine, 2.0 equiv. malonic acid.

3.2.3 Optimisation of the PAL-mediated hydroamination step

The standard buffer for PAL-mediated aminations consists of aqueous ammonia solution (5

M) brought to pH 9.6 with sulphuric acid. In the attempt to maximise the conversion of the

biocatalytic reaction on the raw mixture from the previous step in DMSO (>99% conv. to 2a),

we screened several other ammonium sources at different concentrations and pH values

(Table 3). Even though good results were obtained with the standard buffer, considerably

higher conversions were achieved with a more concentrated solution (13% w/v, approximately

7 M) adjusted to pH 10.0 with CO2 (previously employed in a large-scale application of PAL

reactions7c). In contrast, saturated or highly concentrated solutions of ammonium salts (used

for similar reactions with related aminomutase enzymes19) perfomed poorly, likely due to the

combined effect of the higher ionic strength and the lower pH values.

Table 3. Screening of different buffers for the PAL-mediated hydroamination of 2a in the raw Knoevenagel-Doebner

condensation mixture. Experimental conditions: 1:50 dilution of Knoevenagel-Doebner mixture (20 mM final conc.

2a), 40 mg mL-1 PAL biocatalyst (wet cells), 37ºC, 24 h. [a]Calculated by HPLC analysis on a non-chiral stationary

phase.

Reaction buffer

Conv. RgPAL [%]a

Conv. AvPAL [%]a

5 M NH4OH (adj. with H2SO4), pH 9.6 59 48 7 M NH4OH (adj. with CO2), pH 10.0 81 55

sat. (NH4)2SO4, pH 8.2 3 7 sat. NH4Cl, pH 8.4 16 13

2.5 M (NH4)2CO3, pH 9.0 56 37

76

In addition, we investigated the maximum substrate loading practically feasible for the

hydroamination to occur in good yields: different volume ratios of the condensation mixture

and ammonia buffer were combined and incubated with different concentrations of AvPAL

(Figure 2). RgPAL afforded high conversions up to 1:10 dilution (100 mM 2a), while AvPAL

showed a gradual decrease in efficiency with increasing ratios. In order to avoid a significant

drop in conversions and to keep the substrate loading as high as possible, a 1:20 dilution ratio

was chosen as a trade-off.

Figure 2. Effect of increasing the condensation mixture/buffer ratio on the amination of cinnamic acid 2a mediated

by RgPAL and AvPAL. Experimental conditions: 40 mg mL-1 PAL biocatalyst (wet cells), 7 M NH4OH, pH 10.0 (adj.

with CO2), 37ºC, 24 h.

To conclude our optimisation study, we performed a time course experiment to select the most

reasonable biotransformation time for further screening (Figure 3). RgPAL reached the

thermodynamic limit for the reaction in approximately 2 h, while AvPAL did not get to the same

level even after 24 h. Unsubstituted 2a is known to be a better substrate for RgPAL than for

AvPAL, but the latter enzyme showed a much broader substrate scope and higher activity on

more interesting non-natural substrates.20 Therefore, we decided to perform the screening-

scale biotransformations with both enzymes for 24 h.

Figure 3. Time course of the amination of cinnamic acid 2a (from Knoevenagel-Doebner condensation) mediated

by RgPAL and AvPAL. Experimental conditions: 1:20 dilution of condensation mixture, 40 mg mL-1 PAL biocatalyst

(wet cells), 7 M NH4OH, pH 10.0 (adj. with CO2), 37ºC.

0

20

40

60

80

100

1:50 1:20 1:10 1:5

Co

nv

. [%

]

Volume ratios

RgPAL

AvPAL

Vol. ratio

Subs. conc.

DMSO conc.

1:50

20 mM

2% v/v

1:20

50 mM

5% v/v

1:10

100 mM

10% v/v

1:5

200 mM

20% v/v

0

20

40

60

80

100

0 5 10 15 20 25

Co

nv

. [%

]

Time [h]

RgPAL

AvPAL

77

The enantiomeric excess value of L-3a was >99% in both cases (by HPLC on a chiral

stationary phase), proving that DMSO and the other additional components do not affect the

enantioselectivity of PAL amination of 2a.

3.2.4 Extension towards phenylalanine analogues

The general applicability of our strategy was demonstrated by testing a panel of halogenated

aldehydes 1b-p as starting materials, to afford the corresponding halocinnamic acids 2b-p,

that were converted in a telescopic fashion into the corresponding L-halophenylalanines L-3b-

p (Table 4). Aldehydes 1b-i were employed to validate our methodology, since the

corresponding cinnamic acids are known to be substrates for PALs,7,11 while aldehydes 1j-p

were investigated to expand the substrate scope (the amination of several difluorocinnamic

acids with a plant PAL has been reported in the literature,21 but substrates 2j-p have not been

tested before, to the best of our knowledge). Quantitative conversions were obtained in all

cases for the Knoevenagel-Doebner step, and good to very high conversions with almost all

substrates for the PAL hydroamination step (indeed the latter were almost identical to those

achieved with the pure halocinnamic acid as substrate under co-solvent-free conditions).

Mono-halogenated substrates gave similar results to those previously reported with other

PALs (some with higher conversion), and all the new substrates were converted to some

extent by at least one of the enzymes with good yields. In general, higher conversions were

more often obtained with AvPAL, proving once again its broad substrate tolerance and its

better suitability as a biocatalyst for synthetic applications.

It should be mentioned that cinnamic acids 2n-p are not commercially available (from major

suppliers), and those are the cases where our strategy turns out to be more valuable, since it

easily allows access to optically pure L-phenylalanines without the need to isolate the

intermediate.

78

Table 4. Evaluation of the substrate scope of the telescopic chemo-enzymatic synthesis of L-phenylalanines L-3a-

p from benzaldehydes 1a-p. Experimental conditions: 1:20 dilution of condensation mixture (optimised conditions),

40 mg mL-1 PAL biocatalyst (wet cells), 7 M NH4OH, pH 10.0 (adj. with CO2), 37ºC, 12 h. [a] Calculated by HPLC

analysis on a non-chiral stationary phase; [b] Calculated by HPLC analysis on a chiral stationary phase; c - = not

determined.

Substrate R RgPAL AvPAL Conv. [%]a

ee L-3 [%]b

Conv. [%]a

ee L-3 [%]b

1a H 84 >99 41 >99 1b 2-F 93 >99 93 >99 1c 3-F 92 >99 93 >99 1d 4-F 39 >99 65 >99 1e 2-Cl 97 98 96 98 1f 3-Cl 94 98 70 >99 1g 4-Cl 21 >99 88 98 1h 2-Br 96 98 69 >99 1i 4-Br 4 -c 41 >99 1j 3,4-F2 22 99 93 99 1k 2,4-Cl2 <1 -c 95 98 1l 3,4-Cl2 43 98 95 99

1m 3,5-Cl2 73 >99 55 99 1n 2-Cl-4-F 10 -c 97 97 1o 3-Cl-4-F 90 >99 94 >99 1p 4-Cl-3-F 25 -c 94 97

To demonstrate the practical applicability of our strategy, we performed a preparative scale

synthesis of five non-commercially available dihalophenylalanines (L-3j-k, n and o), which

were isolated in pure form by adsorption on ion-exchange resin in 72-84% overall yield starting

from the aldehyde (Scheme 4). These compounds, besides being useful molecular probes for

CH/π interactions22 are relevant building blocks for medicinal chemistry and drug discovery,

and have been used in the synthesis of peptidomimetic or small-molecule pharmaceuticals.

For instance, L-3j has been incorporated in selective inhibitors of individual nuclear hormone

receptors,23 L-3k in cholecystokinin B antagonists for the treatment of CNS disorders24 and L-

3o in indolizinone antivirals.25

79

Scheme 4. Preparative scale telescopic chemo-enzymatic conversion of five dihalobenzaldehydes into L-

dihalophenylalanines for medicinal chemistry applications.

Furthermore, by monitoring the course of the reactions over time, we observed that a reaction

time of 2-4 h was sufficient to achieve conversion values similar to those obtained on analytical

scale after 24 h. This demonstrates that halogenated substrates are turned over much more

quickly than 2a by AvPAL, with clear benefit for the productivity of the process, since the

preparative scale reactions from the aldehyde to the isolated pure L-amino acid product could

be run in less than 6 h.

3.3 Conclusion

Telescoping reactions steps has several advantages in chemical synthesis, from the reduction

of processing time to the minimisation of waste effluents. In this chapter, we proposed an

efficient route to enantiopure non-natural L-amino acid derivatives from simple precursors

(benzaldehydes, malonic acid and ammonia) using a tandem chemical condensation and

asymmetric biocatalytic hydroamination. The apparent incompatibility of the differing reaction

conditions required has been addressed via cosolvent tolerance studies of the candidate

phenylalanine ammonia lyase biocatalysts. This has subsequently enabled optimisation of

both steps for a telescoped synthesis via dilution of the crude chemical reaction mixture in the

biotransformation buffer. The ease and generality of this method allows the expansion of the

L-amino acids range attainable through PAL-mediated amination, by accessing the diversity

of commercially available substituted benzaldehyde starting materials. As a representative

example, we provided a high-yield optimised route to five optically pure L-

dihalophenylalanines that are incorporated in several candidate active pharmaceutical

ingredients.

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17. Head, D. L.; McCarty, C. G. Tetrahedron Lett. 1973, 16, 1405-1408.

18. (a) Rodionow, W. M.; Postovskaja, E. A. J. Am. Chem. Soc. 1929, 51, 841-847: (b)

Rodionow, W. M. J. Am. Chem. Soc. 1929, 51, 847-852; (c) Tan, C. Y. K.; Weaver, D.

F. Tetrahedron 2002, 58, 7449-7461

19. Weise, N. J.; Parmeggiani, F.; Ahmed, S. T.; Turner, N. J. J. Am. Chem. Soc. 2015,

137, 12977-12983.

20. Lovelock, S. L.; Turner, N. J. Bioorg. Med. Chem. 2014, 22, 5555-5557.

21. Gloge, A.; Zon, J.; Kovari, A.; Poppe, L.; Retey, J. Chem. Eur. J. 2000, 6, 3386-3390.

22. Fujita, T.; Nose, T.; Matsushima, A.; Okada, K.; Asai, D.; Yamauchi, Y.; Shirasu N.;

Honda, T.; Shigehiro, D.; Shimohigashi, Y. Tetrahedron Lett.. 2000, 41, 923-927.

23. Geistlinger, T. R.; Guy, R. K. J. Am. Chem. Soc. 2003, 125, 6852-6853.

24. Augelli-Szaffan, C. E.; Horwell, D. C.; Kneen, C.; Ortwine, D. F.; Pritchard, M. C.;

Purchase, T. S.; Roth, B. D.; Trivedi, B. K.; Hill, D.; Suman-Chauhan, N.; Webdale, L.

Bioorg. Med. Chem. 1996, 4, 1733-1745.

25. Ruesbam, F.; Dragovich, P., U.S. Patent 0227774 A1, 2008.

82

Chapter 4

Synthesis of enantiomerically pure ring-substituted

L-pyridylalanines by biocatalytic hydroamination

Syed T. Ahmed, Fabio Parmeggiani, Nicholas J. Weise, Sabine L. Flitsch and

Nicholas J. Turner



Published in Organic Letters, 2016, 18, pp 5468–5471

Publication date: October 21st, 2016.

Acknowledgements: This chapter highlights work conducted by the doctoral candidate on

expanding the telescopic strategy (discussed in the previous chapter) and substrate scope of

the enzymes. The doctoral candidate took a leading role in the experimental design,

compound synthesis/isolation and characterization including manuscript preparation in

collaboration with the authors.

83

4.1 Abstract

Current routes to nitrogen-containing hetero-arylalanines involve complex multi-step synthesis

and are often reliant on protection/deprotection steps and wasteful chromatographic

purifications. In order to complement existing methodologies, a convenient telescopic strategy

was developed for the synthesis of L-pyridylalanine analogues (12 examples) and other L-

heteroarylalanines (5 examples) starting from the corresponding aldehydes. A phenylalanine

ammonia lyase (PAL) from Anabaena variabilis was used as the biocatalyst, to give

conversions ranging between 88-95%, isolated yields of 32-60% and perfect enantiopurity

(>99% ee) by employing an additional deracemisation cascade where necessary.

4.2 Introduction

Non-natural heteroaromatic amino acids and their derivatives have gained great attention in

medicinal chemistry as chiral building blocks and as drug candidates.1 For example, L-α-

azatyrosine, a natural product isolated from Streptomyces chibansis2 and actinobacteria has

been shown to possess antibiotic and anti-cancer properties3 and is implicated in the

biosynthesis of kedarcidin, an anti-tumour antibiotic.4 The biosynthetic origin of L-α-

azatyrosine is still unclear and, as a result, access to these compounds and their derivatives

from biological sources is limited.

Currently, there are four major synthetic routes to these compounds: via classical resolution5

chiral auxiliaries,6 asymmetric hydrogenation7 and the “chiral pool” approach8 (Scheme 1).

Scheme 1. Currently employed strategies for the synthesis of substituted L-pyridylalanines.

The classical resolution approach is one of the earliest routes proposed, based on selective

hydrolysis of racemic pyridylalanine esters by hydrolases such as subtilisin5a and α-

chymotrypsin.5b The nature of this method limits the theoretical yield to 50%, of which 35%

could be achieved in practice. The chiral auxiliary approach consists of a diastereoselective

alkylation of the Schöllkopf reagent (2,5-diketopiperazine derivative) and subsequent

hydrolysis of the resulting aza-enolate. Multiple chromatography steps are required to access

84

the amino acids, resulting in poor atom efficiency with moderate to good yield.6 Asymmetric

hydrogenation of N-protected dehydroamino acid over a rhodium catalyst is one of the most

common routes to enantiopure amino acids,7 a process well-known for the synthesis of L-

DOPA.9 However, due to catalyst poisoning attributed to the nitrogen on the pyridine ring, the

synthesis of pyridylalanines with this method requires first a protection step by forming the N-

oxide, then a reductive deprotection after the asymmetric hydrogenation step. Another

strategy includes addition of a non-complexing acid such as HBAF to protonate the nitrogen

to prevent catalyst deactivation.7a Finally, a recently developed synthesis employed palladium

catalysed Negishi coupling between a suitable bromopyridine and protected L-β-iodoalanine,

obtained from L-serine (“chiral pool” approach).8 All of the synthetic strategies reported to date

require lengthy multistep processes, multiple chromatographic stages, protection/deprotection

steps and hence result in relatively low yields for the synthesis of these valuable pyridylalanine

API building blocks.

With the advent of biotechnology and the use of enzymes in organic synthesis, the need for

expensive catalysts, toxic solvents, heavy metals and protecting groups have been mitigated.

In many cases, biocatalysis has become the strategy of choice for the regioselective and

stereoselective synthesis of high value chiral building blocks for pharmaceutical and

agrochemical applications. Previous attempts to develop asymmetric biocatalytic approaches

to pyridylalanines (to circumvent the limitation of 50% maximum theoretical yield of enzymatic

resolution) have had limited success. For example, tyrosine phenol lyase (TPL), a PLP-

dependent enzyme, has been used to synthesise L-azatyrosine isomers after incubation with

hydroxypyridines and ammonium pyruvate for 5 days with isolated yields of 10-13%.10

The enzyme phenylalanine ammonia lyase (PAL) has recently emerged as a biocatalyst for

the synthesis of non-natural amino acids;11 PAL catalyses the highly enantioselective cofactor-

free addition of ammonia to inexpensive arylacrylic acid derivatives. This allows access to

substituted L-phenylalanines with perfect atom economy, no protection/deprotection steps and

without the need for expensive cofactor recycling systems (common for many enzymatic

reactions).12 The anti-Michael addition of a nucleophile to an α,β-unsaturated acid is difficult

to achieve chemically and highlights the synthetic utility of this enzyme. The synthesis of

unsubstituted pyridylalanines has been demonstrated by Retey and co-workers, yielding 60-

75% of the isolated product product using a PAL from Petroselinum crispum (PcPAL).13 Kinetic

data for the natural reaction (amino acid to acrylic acid) have also been reported for pyridine

and pyrimidine substrates.14 However, there are no reports on substituted pyridylacrylic acids,

bearing functional handles for further manipulation, being substrates for PALs in the context

of synthetic applications. Herein, we present an efficient one-pot synthetic route to the target

85

mono- and disubstituted L-pyridylalanines in high yield and ee, based on PAL-mediated

asymmetric hydroamination as summarised in Scheme 2.

Scheme 2. One-pot telescopic synthesis of L-pyridylalanines L-3a-l by Knoevenagel-Doebner condensation and

biocatalytic hydroamination.

4.3 Results and Discussion

Pyridylacrylic acid substrates 2a-l can be synthesised easily from the corresponding

benzaldehydes 1a-l and malonic acid, via the Knoevenagel-Doebner condensation,15 then

submitted to biocatalytic hydroamination to produce L-3a-l. The procedure for the latter step

utilizes the bacterial PAL from Anabaena variabilis (AvPAL), expressed recombinantly in

Escherichia coli BL21(DE3) cells, and an unadjusted NH2COONH4 reaction buffer. The

biocatalyst, used as lyophilized whole cells, can easily be prepared in high yield (6 g L–1 of

culture medium) and has been used in intensified preparative processes. 16

As a substrate panel for our investigation, we selected six bromopyridine carboxaldehydes 1a-

f (Scheme 2), because of the practical applications of the corresponding arylalanines as chiral

building blocks (Scheme 3). For instance, they are found in several classes of

pharmaceuticals, such as anticoagulants,17a DPPI inhibitors,17b leukocyte adhesion

inhibitors,17c azaindoline anticancer agents17d and antidiabetics.17e Furthermore, molecules of

this class have also been used as core structures of organocatalysts.8 In many cases the key

chiral centre is the pyridylalanine motif, highlighting the importance of stereoselective

synthesis of these compounds by employing an inexpensive and simple process.

86

Scheme 3. Examples of synthetic applications of products L-3a, f and e to the synthesis of APIs, natural products

and organocatalysts.

In order to test the robustness of the method, we expanded our panel including chloro- (1g-i),

dichloro- (1j) and methoxy- (1k-l) substituted pyridine carboxaldehydes to provide access to

the novel uncharacterised compounds 3g-l. Since not all the possible aldehydes are

commercially available, we elected to cover all the three isomeric classes of substituted

pyridines: the nicotinaldehyde series (meta-relationship, 1a, b, g), the picolinaldehyde series

(ortho-relationship, 1c and d) and the isonicotinaldehyde series (para-relationship, 1e, f, h-l).

4.3.1 Reaction optimization and preparative scale synthesis of compounds 3a-3k

The Knoevenagel-Doebner condensation carried out in DMSO at 100 °C for 16 h15c afforded

full conversion to acrylic acids 2a-2l (confirmed by 1H NMR). The usual work-up procedure for

the reaction involves quenching the reaction mixture with HCl, however with the presence of

a nitrogen atom in the ring, the isolation proved troublesome due to the high solubility of the

target products 2a-l. Addition of the condensation mixture to deionised water at neutral pH

caused precipitation of cinnamic acids 2a-d, g-j and l in high purity, but with low yield. A

different work-up procedure was adopted by extracting the product with ethyl acetate followed

by purification by chromatography, affording higher yields but unsatisfactory purity (by 1H

NMR). Surprisingly, acrylic acids 2e, f and k partially precipitated out of solution when cooled

to room temperature (without any work-up), with good isolated yields obtained by addition of

water and cooling the solution at 4 °C.

Due to the low yields obtained for the isolation of compounds 2a-l, we envisaged a one-pot

telescopic route18 starting from the aldehydes 1a-l to access pyridylalanines 3a-l. A solution of

the aldehyde (1 M), malonic acid and piperidine in DMSO was heated, the condensation

mixture was diluted in saturated aqueous ammonium carbamate (50 mM final concentration

of 2a-l) and lyophilized E. coli cells producing AvPAL (25 mg mL-1) were added as the

biocatalyst. Biotransformation mixtures were incubated at 37 °C for 2-30 h (Table 1). Very high

conversions were achieved and the products could be readily purified by adsorption on ion-

exchange resin (Dowex® 50WX8) to afford white solids of excellent purity (by 1H NMR and

HPLC) in good yields.

87

Table 1. One-pot telescopic synthesis of compounds L-3a-l. [a] Conversion measured by reverse phase HPLC on

a non-chiral phase (compounds 3a-f) or by 1H NMR (compounds 3g-l). [b] Enantiomeric excess after the telescopic

condensation-hydroamination, measured by reverse phase HPLC on a chiral phase. [c] Enantiomeric excess after

the subsequent deracemization step (Scheme 4), measured by reverse phase HPLC on a chiral phase. [d] Isolated

yield calculated from 1a-l. [e] Not required due to the high optical purity of the product.

prod. time

(h)

conv

(%)a

ee

(%)b

final ee

(%)c

isol. yield

(%)d

3a 22 97 >99 – e 51

3b 6 97 93 >99 47

3c 6 95 90 >99 53

3d 30 88 61 >99 44

3e 4 99 80 >99 52

3f 4 99 84 >99 48

3g 22 99 >99 – e 45

3h 2 99 92 >99 40

3i 2 97 92 >99 60

3j 2 99 96 >99 32

3k 30 99 >99 – e 46

3l 22 99 >99 – e 56

4.3.2 Electronic effect on reaction rate and chiral purity

Compounds with electron-donating groups (EDGs) generally lead to low conversion with PAL

enzymes, whereas those with electron-withdrawing groups (EWGs) are accepted with high

conversion, provided that the substrate is not sterically hindered.9 The presence of an electron-

poor pyridine ring enhances the rate of catalysis, hence leading to higher conversions.

Remarkably, while methoxy-substituted phenylalanines have previously shown no conversion

with PAL enzymes, compounds 3k and l (with a hybrid system of EDG/EWG) gave full

conversion with AvPAL, with the exception of 3m, presumably due to the high electron density

in the ring and steric clash with active site residues. As a general trend, isonicotine substrates

(nitrogen at the para-position) were converted faster compared to the other isomers, while

substrates with bulky substituents at the para-position (2 a, d and g) required longer reaction

times. It is worth noting that for some of the substrates (2h-l) accumulation of small amounts

of impurities was apparent in the 1H NMR after prolonged incubation with the biocatalyst. As

such, biotransformations were monitored by HPLC and/or 1H NMR in order to identify the

optimal reaction time (Table 1), thus eliminating the formation of side products and giving the

best compromise between conversion, ee and purity of compounds L-3a-l.

88

Concerning the optical purity of the products, we observed that substrates with higher rates of

amination by AvPAL, in general, gave imperfect ee. In the bromopyridyl subset, compounds

3c-f (nitrogen in the ortho- or para-position) were found to give a significant drop in ee over

time, while compounds 3a-b (nitrogen in the meta-position) did not show any appreciable

decrease in optical purity. The evolution of ee over time is shown in Figure 1a. A similar trend

was observed with chloro-substituents, with the ee decreasing for the isonicotine isomers (3h-

j) and being constant for the nicotine isomer (3g).

Figure 1. (a) Evolution of the ee over time for products L-3a-f. (b) Mechanism of PAL-mediated hydroamination

and alternative mechanistic pathway accounting for the formation of the D-enantiomer of the product.

This behavior can be rationalized in light of previous work on the mechanism of the PAL

mediated hydroamination reaction (Figure 1b). The stereoselective addition requires a 4-

methylidene-5-imidazolone (MIO) prosthetic group to bind ammonia before transferring it to

the α-position of the substrate, and an essential tyrosine residue to protonate the

intermediate.11 However, with electron-deficient aromatic systems, there is evidence of an

alternative enzyme catalysed MIO-independent pathway to afford the racemic product, as we

have previously reported.19 This alternative pathway has been shown to be more prevalent for

EWG-bearing substrates, due to the higher partial positive charge at the α-position. Therefore,

the caveat when using substrates bearing EWGs is the potential loss of optical purity due to

this alternative pathway (as seen for some of the substrates in this work, Table 1). The extent

of racemization can be linked to the position of the nitrogen atom (o-, p- >> m-): for the picoline

50

60

70

80

90

100

0 5 10 15 20 25 30 35

ee

[%]

Time [h]

3a

3b

3c

3d

3e

3f

(a)

(b)

89

and isonicotine series, the delocalisation of the negative charge of the carbanion-like

intermediate on the nitrogen is more effective, making the substrate more susceptible to

addition of free ammonia.

Interestingly, while halogen substituents enhance this effect by decreasing the electron density

of the ring, EDGs can mitigate it (Table 1): methoxy-substituted compounds (3k and l) gave a

final ee >99%, which indicates the electron-donating power of the methoxy group counteracts

the electron-withdrawing capability of the nitrogen atom.

4.3.3 Chemo-enzymatic deracemization of compounds 3b-j.

In order to obtain high ee values we employed an additional chemo-enzymatic cascade

system to produce enantiomerically pure L-enantiomer in a deracemisation process (Scheme

4).20 D-amino acid oxidase (DAAO) selectively oxidises the D-enantiomer to the imino acid

followed by reduction with ammonia-borane in a non-selective manner. Complete

consumption of the D-amino acid was observed after 2 hours, to give L-3b-j with >99% ee

(Table 1).

Scheme 4. Chiral polishing of products L-3b-j by a chemo-enzymatic deracemisation cascade.

4.3.4 Extending the synthetic strategy to heterocyclic compounds L-5a-f

Finally, to demonstrate the broad applicability of our methodology and to highlight the

substrate scope of the enzyme, we tested a panel of six substituted aromatic aldehydes

bearing different heterocycle moieties (isoxazole, thiophene, quinoline and isoquinoline).

Complete conversions were obtained with all the substrates for the condensation step, while

only isoxazole, thiophene and quinoline derivatives were accepted by the enzyme, yielding

amino acids L-5a-e. The synthesis of the isoquinolinylalanine L-5f was unsuccessful due to

the poor acceptance of the corresponding acrylic acid by AvPAL. The dramatic difference

between quinoline and isoquinoline substrates can be attributed to the different electronic

properties of the two systems, i.e., the position of the nitrogen atom (p- >> m-). Amino acids

L-5a-e were obtained with >99% ee without additional deracemization, in moderate to good

isolated yield (Scheme 5).

90

Scheme 5. Examples of additional heteroarylalanine derivatives obtained by one-pot condensation-

hydroamination.

4.4 Conclusion

In summary, we have developed a one-pot telescopic route to afford L-pyridylalanine

analogues in high conversions, good isolated yields and excellent purity from the

corresponding aldehydes. Novel substrates that were previously unreported with PALs were

used to synthesize ring-substituted pyridylalanine derivatives. The process was made simple

and efficient by utilizing the enzyme in a stable powdered formulation (lyophilized whole cells)

which provided an alternative green route to current synthetic methodologies. Reaction

optimization was carried out to give the highest conversion, ee and purity, implementing an

additional chemo-enzymatic cascade to give >99% ee for compounds which gave low

enantiopurity. This work adds to the current toolbox for the synthesis of non-natural

heteroaromatic amino acids, allowing simpler and more efficient access to complex API

intermediates.

4.5 References

1 (a) Amino Acids, Peptides and Proteins in Organic Chemistry, Volume 4, Protection

Reactions, Medicinal Chemistry, Combinatorial Synthesis; Hughes, A. B., Ed.; Wiley:

Chichester, UK, 2011 (b) Asymmetric Synthesis and Application of α-Amino Acids;

Soloshonok, V. A., Izawa, K., Eds.; American Chemical Society: Washington, DC, 2009.

2 (2) Inouye, S.; Shomura, T.; Tsuruoku, T.; Ogawa, Y.; Watanabe, H.; Yoshida, J.; Niida. T.

Chem. Pharm. Bull. 1975, 23, 2669-2677.

3 Izawa, M.; Takayama, S.; Shindo-Okada, N.; Doi, S.; Kimura, M.; Katsuki, M.; Nishimura,

S. Cancer Res. 1992, 52, 1628-1630.

4 Huang, S-X.; Lohman, J. R.; Huang T.; Shen B. Proc. Natl. Acad. Sci. USA 2013, 110,

8069-8074.

91

5 (a) Folkers, K.; Kubiak, T.; Stepinski, J. Int. J. Pept. Protein Res. 1984, 24, 197-200; (b)

Cooper, M. S.; Seton, A. W.; Stevens, M. F. G.; Westwell, A. D. Bioorg. Med. Chem. Lett.

1996, 6, 2613-2616; (c) Miyazawa, T. Amino Acids 1999, 16, 191-213.

6 Croce, P. D.; Rosa, C. L.; Pizzatti, E. Tetrahedron: Asymmetry, 2000, 11, 2635-2642.

7 (a) Adamczyk, M.; Akireddy, S. R.; Reddy, R. E. Org. Lett., 2001, 3, 3157-3159; (b)

8 (a) Tabanello, S.; Valangogne, I.; Jackson, R. W. F. Org. Biomol. Chem., 2003, 1, 4254-

4261; (b)

9 Vineyard, B. D.; Knowles, W. S.; Sabacky, M. J.; Bachman,G. L. D.; Weinkauff, J. J. Am.

Chem. Soc., 1977, 99, 5946-5952.

10 Watkins, E. B.; Phillips, R. S. Bioorg. Med. Chem. Lett., 2001, 11, 2099-2100.

11 (a) Turner, N. J. Curr. Opin. Chem. Biol. 2011, 15, 234-240; (b) Heberling, M. W.; Wu, B.;

Bartsch, S.; Janssen, D. B. Curr. Opin. Chem. Biol. 2013, 17, 250-260. (c) Poppe, L.

Curr. Opin. Chem. Biol. 2001, 5, 512-524.

12 (a) Yamada, S.; Nabe, K.; Izuo, N.; Nkamichi, K.; Chibata, I. Appl. Environ. Microbiol,

1981, 42, 773-778; (b) Paizs, C.; Katona, A.; Retey, J. Chem. Eur. J. 2006, 12, 2739-

2744; (c) Ahmed, S. T.; Parmeggiani, F.; Weise, N. J.; Flitsch, S. L.; Turner, N. J. ACS

Catal. 2015, 5, 5410-5413.

13 Gloge, A.; Langer, B.; Poppe, L.; Retey, J. Arch. Biochem. Biophys. 1998, 359, 1-7.

14 Lovelock, S. L.; Turner, N. J. Bioorg. Med. Chem., 2014, 22, 5555-2227.

15 (a) Knoevenagel, E. Chem. Ber., 1898, 31, 2596-2619; (b) Doebner, O. Chem. Ber., 1900,

33, 2140-2142; (c) Kingsbury, C. A.; Max, G. J. Org. Chem. 1978, 43, 3131-3139.

16 (a) de Lange, B.; Hyett, D. J.; Maas, P. J. D.; Mink, D.; van Assema, F. B. J.; Sereinig, M.;

de Vries, A. H. M.; de Vries, J. G. ChemCatChem, 2011, 3, 289-292; (b) Weise, N. J.;

Ahmed, S. .T.; Parmeggiani, F.; Siirola, E.; Pushpanath, A.; Schell, U.; Turner, N. J. Cat.

Sci. Tech. 2016, 6, 4086-4089.

17 (a) Mjalli, A.; Andrews, R.; Guo, X-C.; Christen, D. P.; Gohimmukkula, D. R.; Huang, G.;

Rothlein, R.; Tyagi, S.; Yaramasu, T.; Behme, C. US Patent WO 2004/014844 A2, August

8 2003; (b) Cage, P.; Furber, M.; Luckhurst, A. C.; Sanganee, J. H.; Stein, A. L. WO

Patent 2009/074829 A1, 18 June 2009; (c) Konradi, A. W.; Pleiss, M. A.; Thorsett, E. D.;

Ashwell, S.; Welmaker, G. S.; Kreft, A.; Sarantakis, D.; Dressen, D. B.; Grant, F. S.;

Semko, C.; Xu, Y-Z.; Stappenbeck, F. US Patent WO 2002/0052375 A1, May 2 2002; (d)

Hogg, J. H.; Kester, R. F.; Liang, W.; Yun, W. WO Patent 2014/056871 A1, April 17 2014;

(e) Chen, Y.; Goldberg, S. L.; Hanson, R. L.; Parker, W. L.; Gill, I.; Tully, T. P.; Montana,

M. A.; Goswami, A.; Patel, R. N. Org. Process Res. Dev., 2011, 15, 241-248.

18 Parmeggiani, F.; Ahmed, S. T.; Weise, N. J.; Turner, N. J. Tetrahedron, DOI:

10.1016/j.tet.2015.12.063, in press.

19 Lovelock, S. L.; Lloyd, R. C.; Turner, N. J. Angew. Chem. Int. Ed., 2014, 53, 4652-4656.

92

20 Parmeggiani, F.; Lovelock, S. L.; Weise, N. J.; Ahmed, S. T.; Turner, N. J. Angew. Chem.

Int. Ed., 2015, 54, 4608-4611.

93

Chapter 5

Zymophore identification enables the discovery of novel

phenylalanine ammonia lyase enzymes

Nicholas J. Weisea, Syed T. Ahmeda, Fabio Parmeggiania, James L. Galmana, Mark

S. Dunstanb, Simon J. Charnockc, David Leysa,b and Nicholas J. Turnera,b*

a School of Chemistry, Manchester Institute of Biotechnology, University of

Manchester, 131 Princess Street, Manchester, M1 7DN, United Kingdom

b SYNBIOCHEM, Manchester Institute of Biotechnology, University of Manchester,

131 Princess Street, Manchester, M1 7DN, United Kingdom

c Prozomix Ltd., Station Court, Haltwhistle, Northumberland, NE49 9HN, United

Kingdom.

Published in Scientific Reports, 2017, 18, pp 5468–5471

Publication date: October 21st, 2016.

Acknowledgements: This chapter highlights the work on the discovery of five new PAL

enzymes. The doctoral candidate was responsible for the experimental design and data

analysis.

Active site

sequence motifs

New enzymes for

application

Discovery of...

94

5.1 Abstract

The suite of biological catalysts found in Nature has the potential to contribute immensely to

scientific advancements, ranging from industrial biotechnology to innovations in bioenergy and

medical intervention. The endeavour to obtain a catalyst of choice is, however, wrought with

challenges. Herein we report the design of a structure-based annotation system for the

identification of functionally similar enzymes from diverse sequence backgrounds. Focusing

on an enzymatic activity with demonstrated synthetic and therapeutic relevance, five new

phenylalanine ammonia lyase (PAL) enzymes were discovered and characterised with respect

to their potential applications. The variation and novelty of various desirable traits seen in

these previously uncharacterised enzymes demonstrates the importance of effective

sequence annotation in unlocking the potential diversity that Nature provides in the search for

tailored biological tools. This new method has commercial relevance as a strategy for assaying

the ‘evolvability’ of certain enzyme features, thus streamlining and informing protein

engineering efforts.

5.2 Introdcution

Currently, a major challenge in the area of enzyme discovery remains the accurate retrieval

from databases of enzymes with specific traits, from among the vast number related

sequences - an approach hindered by misannotation from sequence curation methods which

are often implemented in the absence of specialist protein functional knowledge.1 This

problem is especially relevant in enzymes which are structurally diverse but have convergently

evolved similar catalytic residue groupings, or in families where the function of choice

constitutes an interspersed minority of available sequences. The former problem has been

addressed in an enzyme class-specific manner by the computational assignment of clusters

of active site residues conferring the desired chemistry on otherwise uncharacterised proteins.

Examples include the mapping of active site ‘constellations’ for ene-reductase activity,2 use of

BioGPS descriptors to aid discovery of promiscuous side-activity3 or the retrieval of catalytic

residue arrangements via the Catalytic Site Atlas or PatternQuery.4,5 Whilst these methods

provide simple means of accessing proteins with desired catalytic activity, their use is mostly

limited to structural databases containing only a fraction of the number of entries that a

sequence database would have. Such constraints are also less amenable to family-specific

catalytic arrangements not documented elsewhere in nature where a wealth of sequence data

can potentially harbour more scope for investigation. The structural methods also do not take

into account other active site residues which differentiate desired and unwanted activity, such

as selectivity and substrate discrimination.

95

Enzymes with phenylalanine ammonia lyase (PAL) activity are able to catalyse non-oxidative

deamination to yield cinnamate (1a) and ammonia from the proteinogenic amino acid L-

phenylalanine (L-Phe, 2a).9 The amine abstraction, in this case, is mediated by adduct

formation with a 4-methylideneimidazole-5-one (MIO) post-translational modification in the

active site.10,11 This in turn promotes elimination in conjunction with proton abstraction via a

catalytic tyrosine residue situated on an inner active site mobile loop lid.11,12 In Nature, this

PAL-mediated reaction often constitutes a gateway from primary metabolism to specialist

compound biosynthesis, with examples including phenylpropanoids in plants13 and various

antibiotics in bacteria (Figure 1a).14–17

Figure 1. The catalytic activity of phenylalanine ammonia lyase (PAL) enzymes as implicated in biosynthesis/biotherapeutics (a) and biocatalysis (b).

5.2.1 Therapeutic application of PALs

In the field of biomedical research, the selective catabolism of L-Phe by PALs has been

exploited as a treatment for a number of diseases. One such example is phenylketonuria, an

inborn error of metabolism in which metabolic dysregulation leads to neurological symptoms

such as microcephaly, seizures and intellectual disabilities.18 As the condition emanates from

aromatic amino acid imbalance - high phenylalanine and low tyrosine levels - removing L-Phe

has been shown to offer great advantages over alternative treatments based on complex

dietary restriction which are currently prescribed.19,20 Essential amino acids, required by all

growing cells, can also be degraded in vivo to restrict cancer tumour growth (amino acid

depletion therapy) - a promising strategy that has been demonstrated with metabolites such

as L-Asn, L-Arg, L-Met, L-Tyr and aptly L-Phe.21 Relevant examples with respect to the last

example in particular include the decrease in metastatic phenotype of B16BL5 melanoma

upon aromatic amino acid depletion22 and the use of PAL-based formulations to prevent

growth of abnormal lymphocytes both in vitro23 and in leukemic mice.24 The commercial

importance of PALs as therapeutic agents is evidenced by various patent applications from

SIPHONING OF PRIMARY METABOLITES

(biosynthetic pathways and medical intervention)

AMINATION OF ARYLACRYLATES

(pharmaceutical and fine chemical synthesis)

PHENYLALANINE AMMONIA LYASE (PAL)

2a 1a

12

4

3

a

b

96

specialist enzyme (Codexis)25 and biopharmaceutical (BioMarin)26 production companies, as

well as from multinational drug and chemical corporations (Novo Nordisk and BASF).27,28

5.2.2 Application and limitation of PAL catalyzed biotransformation

PALs also find use in the area of green chemistry and sustainable pharmaceutical

manufacture as industrial biocatalysts, due to the reversibility of the deamination reaction

(Figure 1b). The use of concentrated yet inexpensive ammonia buffers has been

demonstrated to allow regio- and enantioselective amination of a broad range of readily-

accessible arylacrylic acids, yielding high value unnatural amino acids – a route that is difficult

to emulate via chemical methods. Basic molecular cloning techniques have enabled the simple

preparation of enzyme-based catalysts from renewable feedstocks, allowing the exploitation

of their inherent selectivity and ambient reaction conditions by synthetic chemists. As such,

there are many examples of biocatalytic routes in industry, including PAL-mediated processes

to produce L-Phe,29 unnatural amino acid building blocks of antifungal peptides (e.g., 3),30

and the antihypertensive drug perindopril 4.31,32 The use of PALs for aromatic amino acid

synthesis is particularly attractive due to the 100% atom efficiency and coenzyme

independence of the reaction, mitigating waste production, by-product separation

requirements and mediator supplementation / regeneration costs.30

In spite of the broad scope of application associated with PALs, their use is limited to a handful

of specific enzymes,29,33,34 greatly restricting the variability available in desirable traits. Recent

efforts have focused on modification of the PAL from Anabaena variabilis (AvPAL), via

mutagenesis or chemical conjugation, to overcome characteristics of the enzyme which are

considered detrimental to therapeutic applications.19,20,25 Such traits generally include low

catalytic activity, poor stability at 37°C, accessible proteolytic cleavage sites, low acid

tolerance (if taken orally), aggregation and high immunogenicity. The few PALs used in a

biocatalytic context (of which AvPAL is again the most prominent)34–36 show more immediate

promise, with little or no engineering required for some industrial applications. However, many

pharmaceutically-relevant targets that are potentially within the scope of the synthetic PAL

reaction remain out of reach due to the restricted substrate scope of characterised enzymes.

In the class I lyase-like family, aminomutase and histidine/tyrosine specific enzymes are

reasoned to form the majority of available sequences with PALs constituting the minority.37

We therefore set out to develop a new strategy for the discovery of PAL enzymes based on

the four-step process outlined in Figure 2.

97

Figure 2. Flow diagram of the strategy employed to find new PAL enzymes for synthetic and medical applications.


5.3.1 Elucidating the structure AvPAL and analysis of the active site

Although several crystal structures of PAL enzymes have been solved, none exists with the

natural ligand trans-cinnamic acid bound in the active site, thereby preventing correct and

unambiguous assignment of the phenylalanine-specific MIO-dependent ammonia lyase

zymophore. The gene encoding AvPAL was mutated, incorporating two surface substitutions

(C503S and C565S, known to aid crystallisation38) and replacement of the loop tyrosine with

a catalytically inert but shape-complementary phenylalanine (Y78F). The triple variant was

purified and co-crystallised with the natural deamination product. Following successful crystal

growth, the structure of the AvPAL-ligand complex (PDB ID: 5LTM) was solved by molecular

replacement using the previously obtained ligand-free double variant (PDB ID: 3CZO) as a

search model. Superimposition of these two structures revealed excellent complementarity

with a few small differences between the unoccupied and occupied active sites. The only

backbone displacement was a shift in the outer active site loop, with the fully closed

conformation from the previously reported structure seen to clash with the inner active site

position 78 in the co-crystallised protein. In the case of the unoccupied enzyme, the amino

acid at position 78 is tyrosine which points inward toward the active site, providing no contact

with the outer loop, whereas the new structure features Y78F with a dihedral swing of ~140o

causing the bulky side chain to protrude outwards. Other differences included H359 moving

into the active site sphere in the cinnamate bound structure, along with smaller shifts in the

positions of Q311 and R317 (see chapter 5 supporting information , Figure S1). Presumably

these represent movements which occur upon substrate binding, although they may be

induced instead by the Y78F variation as exchange of polar and non-polar residues in the

inner active site loop of related enzymes has been reported to influence conformational

dynamics.39 Interestingly, these three seemingly mobile positions have been identified as

hotspots in mutagenic studies of various PAL-relatives, with variation at H359 shown to affect

catalytic efficiency36 and the two carboxylate-binding positions (Q311/R317) being

implemented in substrate positioning and selectivity.40,41 This observation sheds light on

possible conformational selection or induced fit models which may help to guide engineering

STRUCTURAL

DETERMINATION

OF ZYMOPHORE

ESTABLISHMENT

OF MOTIF QUERY

POSITIONS

FUNCTIONAL

DISCRIMINATION

OF SEQUENCES

gfhfffghkghjgjhgjhgjghgj

hjhjkghjfhg

TcPAM PcPAL1 RtPAL AvPAL StlA

…DIYGVTTGFGA--… …DSYGVTTGFGA--… …SVYGVTTGFGG--… …PIYGVTSGFGGMA… …VIYGINTGFGGNA… **:.:***.

…QESLIRCLLAG… …QKELIRFLNAG… …QKALLEHQLCG… …QTNLVWFLKTG… …QQNLLTFLSAG… * *: *

…SASGDLIPLAYIA… …TASGDLVPLSYIA… …SASGDLSPLSYIA… …GASGDLVPLSYIT… …GASGDLIPLSYIA… ***** **:**:

…KEGLALVNGT… …KEGLALVNGT… …KEGLGLVNGT… …KEGLAMMNGT… …KEGLALINGT… ****.::***

…KPKQDRYALRSS… …KPKQDRYALRTS… …ILRQDRYPLRTS… …ELIQDRYSLRCL… …DTLQEVYSIRCA… *: *.:*

…DNPLID… …DNPLID… …DNPLID… …DNPLID… …DNPIID… ***:**

…AEQHNQD… …AEQHNQD… …AEMANQA… …AEQFNQN… …TEQYNQD

:* **

gfhfffghkghjgjhgjhgjghgj

hjhjkghjfhg

TcPAM PcPAL1 RtPAL AvPAL StlA

…DIYGVTTGFGA--… …DSYGVTTGFGA--… …SVYGVTTGFGG--… …PIYGVTSGFGGMA… …VIYGINTGFGGNA… **:.:***.

…QESLIRCLLAG… …QKELIRFLNAG… …QKALLEHQLCG… …QTNLVWFLKTG… …QQNLLTFLSAG… * *: *

…SASGDLIPLAYIA… …TASGDLVPLSYIA… …SASGDLSPLSYIA… …GASGDLVPLSYIT… …GASGDLIPLSYIA… ***** **:**:

…KEGLALVNGT… …KEGLALVNGT… …KEGLGLVNGT… …KEGLAMMNGT… …KEGLALINGT… ****.::***

…KPKQDRYALRSS… …KPKQDRYALRTS… …ILRQDRYPLRTS… …ELIQDRYSLRCL… …DTLQEVYSIRCA… *: *.:*

…DNPLID… …DNPLID… …DNPLID… …DNPLID… …DNPIID… ***:**

…AEQHNQD… …AEQHNQD… …AEMANQA… …AEQFNQN… …TEQYNQD

:* **

DISCOVERY

OF DIVERSE

ENZYME CANDIDATES

98

of enzymes in both this class and others by targeting residues which distinguish bound and

unbound conformations.

5.3.2 Identification of new PAL enzymes

Visualisation of the occupied active site allowed the identification of 19 residues within a 6 Å

sphere of the ligand (Figure 3), including the MIO and catalytic amino acid at position 78, the

carboxyl positioning residues, the hydrophobic aryl binding pocket and other assorted

substrate-constraining side chains. As a test of the orthogonality of this PAL zymophore, a

sequence alignment was performed using characterised ammonia lyase and aminomutase

enzymes specific to phenylalanines and/or tyrosine or histidine (see chapter 5 supporting

information, Figure S3). The PALs in the selection all showed conservation at all or all but one

of the residues, while ammonia lyases accepting both L-Phe and L-Tyr and (R)-selective

aminomutases varied at two positions. Bacterial lyases and mutases specific to tyrosine and

histidine ammonia lyases (HALs) showed conservation at only 15 residues with (S)-β-Phe

forming mutases scoring just 13/19. Interestingly, even those enzymes which differed by the

same number of residues, tended to do so at different positions, highlighting the importance

of using as many of the residues as possible in further investigations. As such, the entire motif

was used and a score of 18/19 was deemed to be the threshold for consideration of sequences

as potential PALs.

The 19-amino acid active-site sequence motif, as inferred from the AvPAL-cinnamate

structure, was used to distinguish potentially useful enzyme sequences from other family

members. The sequences were collected using the basic local alignment search tool (BLAST)

to search the entire knowledge database of the universal protein resource (UniProtKB) using

various query sequences.

Figure 3. The zymophore conferring PAL-specific reactivity and selectivity as identified from the co-crystal structure

of the ammonia lyase from Anabaena variabilis (AvPAL) and its deamination product trans-cinnamate (PDB ID:

5LTM). The motif is shown on both structural overlay of the empty and occupied enzyme active sites and the

sequence with each of the 19 amino acids given a number to mark its position.

99

As the most widely reported enzyme of this class in applied research, the protein sequence of

AvPAL was used for the first search and the 100 hits showing the closest identity were

analysed. Of these, 17 were shown to give close agreement with the desired active site, with

the only differences occurring at positions 6 and 7 of the motif if at all. Four of the results,

including the previously characterised PAL from Nostoc punctiforme (NpPAL),42 had

hydrophobic residues at these positions, whereas all others contained a histidine at position

7. This variation is known to be associated with strict tyrosine ammonia lyase (TAL) activity in

the bacterial enzyme BagA.43 The 13 potential TALs were discounted along with NpPAL

leaving a closely related sequence from Oscillatoria sp., and two more distant relatives from

Planctomyces brasiliensis and Methylobacterium sp. Next, the sequence of the

phylogenetically-distinct PAL, StlA from Photorhabdus luminescens,16 was used to search for

PALs in a different part of the family tree. This time, within the first 100 hits the majority were

found to contain YH or FH at positions 6 and 7, indicating possible unwanted TAL activity.

Among the few PAL-like sequences, most were from the related enterobacterial genera

Photorhabdus and Yersinia sharing high sequence identity with the query sequence. The

remaining four PAL sequences were surprising in that they were from the amoebozoa genus

Dictyostelium, whereas all other sequences were of eubacterial origin.

Table 1. The most prominent PAL sequences identified from the universal protein resource knowledge base

(UniProtKB)

Name Origin Motif

match Query Seq. id.

BlPAL Brevibacillus laterosporus 19/19 BagA 58%

SrPAL Streptomyces rimosus 19/19 BagA 54%

MxPAL Methylobacterium sp. 18/19 AvPAL 50%

DdPAL Dictyostelium discoideum 18/19 StlA 48%

PbPAL Planctomyces brasiliensis 19/19 AvPAL 46%

Finally, the bacterial TAL sequence BagA from Streptomyces sp. was used for one final search

to find PAL sequences not closely related to those currently known. It is worth noting that,

although most of the hits were found to be TAL-like, with YH and FH at positions 6 and 7, there

were more notable and diverse examples of active sites matching the AvPAL-derived motif

than with either of the searches using characterised PAL query sequences. These included

the previously reported RxPAL from Rubrobacter xylophilans,44 as well as additional

sequences from Bacillus subtilis and Actinopolyspora erythraea and several from both

Brevibacillus laterosporus and various species of the genus Streptomyces.

100

5.3.3 Genomic context analysis of new PALs

Interestingly the PALs from Dictyostelium discoideum and Brevibacillus laterosporus have

been previously identified by Nielsen et al. using a synteny-based approach to search for class

Iyase-like genes in the putative context of TAL-containing metabolic pathways.45 This is an

interesting finding, given the identification of these enzymes as PALs by their active sites in

this study (rather than TALs based on inference of genetic co-localisation) and also given the

rarity of PAL-like sequences seen in our database searches compared to the abundant TAL-

like sequences. To add further evidence to the assertion that these sequences possessed

PAL activity, genetic context analyses were conducted, revealing association with fatty acid

and polyketide biosynthetic operons as has been reported previously with this class of

enzyme.42 In the case of Brevibacillus laterosporus the enzyme could even be putatively linked

to the production of the cinnamate component of basiliskamides A and B - a link missing from

previous biosynthetic hypotheses (see chapter 5 supporting information, Figure S7).46 Five of

the potential PAL sequences mined from the protein database using the AvPAL zymophore

motif were chosen for characterisation based on their sequence identity to their closest

characterised relative and the results of the genetic context analyses (Table 1).

5.3.4 PAL-TAL discrimination

In all searches using characterised query sequences, evidence was uncovered of multiple

PAL-TAL neofunctionalisation events within certain clades in the family, with indication of

known PAL-active site motifs (with FL at selectivity positions) interspersed among known TAL

motifs (with YH) and a possibly intermediary motif (FH). These findings point to the incidence

of several substrate switching events resulting in distinct and unreported clades of TAL

enzymes. This phenomenon may constitute an evolutionary strategy for the creation of

chemical diversity in related biosynthetic pathways, interchanging between use of L-Phe or L-

Tyr as a starting point, followed by further diversification and mixing of secondary metabolic

enzymes to create new products. The searches also reveal that TAL-like sequences vastly

outnumber PALs in the areas of sequence space targeted in this study, further demonstrating

the importance of robust annotation methods over more random sampling of sequence

diversity with this family of enzymes. The most interesting new sequences from the database

were the PALs from Dictyostelium sp. including DdPAL - an example of an aromatic amino

acid ammonia lyase from the protist kingdom. At around 50% sequence identity to StlA, this

enzyme arises from a fairly recent gene transfer event between bacteria and eukaryotes,

separate to that more widely reported to fungi and plants.37 We suggest that this gene transfer

is the evolutionary result of a sustained endosymbiotic relationship between amoebozoa and

bacteria, similar to that hypothesised for the acquisition of PALs in other kingdoms. This

proposal aligns with reports of primitive agriculture between Dictyostelium discoideum and

101

their bacterial livestock47 - an interaction which could result in xenologous proteins in these

otherwise unrelated organisms.

5.3.4 Isolation of newly discovered PAL enzymes

Of the enzymes selected for further study, BlPAL and SrPAL were cloned from their host

organisms whereas the genes for DdPAL and MxPAL were custom synthesised as codon-

optimised constructs for expression in laboratory strain E. coli. These genes were then

subcloned into expression plasmids. The gene encoding PbPAL was prepared and provided

in an appropriate vector by collaborators at Prozomix Limited. After production of all five

proteins as N-terminal His6-tagged variants, and successful purification of four, each enzyme

was characterised with respect to amino acid specificity, pH optimum and stability at 37oC. To

verify that the enzymes did not possess TAL or HAL activity, the isolated enzymes were

incubated with L-Tyr or L-His and production of the corresponding deamination products

assayed via spectrophotometry. In all cases, significant increases in absorbance, indicating

acrylic acid production, were not seen with either L-Tyr or L-His (see chapter 5 supporting

information, Figure S8). All four enzymes possessing exclusively PAL-activity were also shown

to be misannotated in the universal protein resource UniProtKB, with all designated as HALs

except for BlPAL which was incorrectly assigned TAL function (Table 1). These incorrect

designations, widespread in public databases,1 impair the efforts of scientists in the search

for new disease treatments, biocatalyst templates and components of unknown metabolic

pathways. The case of enzymes with PAL activity illustrates this prominently, as correct

annotation of these enzymes enables advances to be made in all three of these areas (e.g

treatment of cancer / PKU, production of unnatural amino acids, identification of operons for

bioactive molecules like basiliskamides).

5.3.5 pH and temperature stability test of new enzymes

To confirm PAL activity (rather than TAL or HAL) the specific activities with respect to the

deamination of L-Phe were tested with each enzyme. This was performed under different

conditions to assess the natural variation in therapeutically-relevant traits such as turnover

number and stability at blood and / or gastrointestinal temperature and pH (Figure 4). The

specific activity measurements made with each ammonia lyase indicated that SrPAL was the

most active enzyme in the group, with a specific activity of 1102 mU mg–1, a value far higher

than that of the other enzymes which all gave ~500 mU mg–1. SrPAL was also found to retain

this high activity after incubation at 37oC for 1 h, with BlPAL and DdPAL losing ~5% of their

original activity and PbPAL giving just 85% relative specific activity after the same treatment.

The pH for optimal activity among those tested was 10 for all enzymes with decrease in activity

seen as the pH was lowered and only DdPAL retaining any detectable activity at pH 6. Despite

102

tolerance of a lower pH, DdPAL gave a broader pH profile, losing a greater percentage of its

initial activity between pH 10 and 8.

Figure 4. Variation of specific activity (a), pH tolerance (b) and stability at 37°C (c) among four new ammonia

lyases from Streptomyces rimosus, Brevibacillus laterosporus, Planctomyces brasiliensis and Dictyostelium

discoideum which could be isolated for investigation.

The differences in specific activity between the enzymes were striking, particularly as SrPAL

was shown to be far more active than the others with greater retention of activity after

incubation at 37°C. This difference was of particular interest as SrPAL and BlPAL share the

closest sequence identity of any two enzymes in this study (see chapter 5 supporting

information, Figure. S6) and yet have a two-fold difference in turnover rate. These sequences,

although closely related, possess many amino acid substitutions which are likely to influence

the dynamics of the entire protein, possibly accelerating movements constituting the catalytic

cycle or channelling solvent vibrations to the active site to help overcome activation

energy.48,49 The identical active site, yet variable overall sequence of SrPAL compared to

BlPAL, seems to contribute to the former enzyme being a better candidate as a biotherapeutic,

due to its high activity and longevity at average body temperature after 1 hour. However, after

24 hours all enzymes still allowed 50-60% of their initial maximal conversion with only DdPAL

showing a greater loss in activity. SrPAL was also found to be the most acid tolerant of the

enzymes along with DdPAL, which, whilst being less active and less robust initially, still

retained more activity at pH 6 that either BlPAL or PbPAL. This feature may make SrPAL (or

even DdPAL) a good starting point for the development of an acid tolerant enzyme for oral

administration, ensuring function and stability in the digestive tract. These variable findings

with no one enzyme possessing all the most desirable traits, highlight the utility of our simple

method for estimating the viability of engineering by sampling variation capacity in a handful

of diverse but related natural sequences.

5.3.6 Substrate scope evaluation

In order to evaluate the synthetic potential of each enzyme, the E. coli pastes prepared for

characterisation were used as whole cell biocatalysts for the amination of a range of ring-

0

200

400

600

800

1000

1200

SrPAL DdPAL PbPAL BlPAL0

20

40

60

80

100

SrPAL PbPAL BlPAL DdPAL

0 h

1 h

24 h

Sp

ec.

ac

tiv

ity (

mU

mg

–1)

0

20

40

60

80

100

2 4 6 8 10

SrPAL

DdPAL

PbPAL

BlPAL

Re

l. c

on

ve

rsio

n (

%)

Re

l. c

on

ve

rsio

n (

%)

pH

a b c

103

substituted arylacrylates. Through use of a reported method,30 the biotransformations were

run with all five enzymes and a representative panel of 16 cinnamate substrates 1a-p before

isolation of crude product and analysis (Table 2). Interestingly, despite the biocatalysts having

nearly identical active sites, there was evident variability in substrate tolerance and

enantioselectivity, aside from that expected from previous investigations (e.g., low

enantioselectivity with 1n-p).36,50 For instance, both SrPAL and PbPAL were found to give very

high conversions for the majority of substrates but the enantioselectivity of the reaction

catalysed by the former enzyme was superior in most cases. When compared to BlPAL

however, SrPAL gave similarly excellent enantiomeric excess values across the board but

much less variable conversion. A comparison between the three most discernible substrate

profiles of BlPAL, MxPAL and DdPAL revealed that even the relative conversions were not

the same between the enzymes. Whilst BlPAL gave the highest conversion for 1g, followed

by MxPAL and then DdPAL (76%, 56% and 28%) this pattern was reversed with the ortho-

isomer 1e (12%, 63% and 84%) and different again for the meta- 1f (81%, 51% and 57%).

Another interesting feature shared by MxPAL and DdPAL is that they both convert the natural

ligand 1a to a lower extent than most of its ring-substituted derivatives. DdPAL even gave

higher conversion with the seemingly challenging substrate 1l – a compound not even

accepted by BlPAL or MxPAL and poorly converted by the otherwise highly active SrPAL.

Table 2. PAL-catalysed amination of various ring-substituted phenylacrylate derivatives

The discovery that this new panel of enzymes showed high activity with substrates possessing

electron donating substituents was striking, as this class of substrate is not reported to be well

accepted by PAL enzymes. Production of m-methoxy-L-phenylalanine (S)-2l by three of the

SrPAL BlPAL PbPAL MxPAL DdPAL

1 Conv.a

(%) eeb (%)

Conv.a (%)

eeb (%)

Conv.a (%)

eeb (%)

Conv.a (%)

eeb (%)

Conv.a (%)

eeb (%)

1a (H) 92 >99 (S) 80 >99 (S) 91 >99 (S) 11 – 25 95 (S) 1b (2-F) 98 >99 (S) 99 >99 (S) 99 94 (S) 91 >99 (S) 60 88 (S) 1c (3-F) 96 >99 (S) 96 >99 (S) 91 88 (S) 89 >99 (S) 58 82 (S) 1d (4-F) 92 >99 (S) 92 >99 (S) 94 96 (S) 53 96 (S) 33 >99 (S) 1e (2-Br) 99 96 (S) 12 >99 (S) 99 94 (S) 63 82 (S) 84 90 (S) 1f (3-Br) 97 >99 (S) 81 >99 (S) 98 86 (S) 51 >99 (S) 57 82 (S) 1g (4-Br) 96 >99 (S) 76 >99 (S) 97 96 (S) 56 >99 (S) 28 >99 (S) 1h (2-Cl) 99 >99 (S) 6 >99 (S) 99 94 (S) 29 >99 (S) 85 86 (S) 1i (3-Cl) 91 96 (S) 23 >99 (S) 97 91 (S) 34 >99 (S) 74 80 (S) 1j (4-Cl) 82 >99 (S) 35 >99 (S) 70 92 (S) 22 >99 (S) 41 >99 (S)

1k (2-MeO) <1 – <1 – 27 96 (S) <1 – 15 >99 (S)

1l (3-MeO) 19 85 (S) <1 – 92 99 (S) <1 – 40 96 (S) 1m (4-MeO)

<1 – <1 – 8 99 (S) <1 – <1 –

1n (2-NO2) 97 87 (S) 39 88 (S) 99 70 (S) 19 60 (S) 70 58 (S) 1o (3-NO2) 98 88 (S) 54 92 (S) 99 88 (S) 38 86 (S) 68 62 (S) 1p (4-NO2) 92 70 (S) 37 88 (S) 99 32 (S) 36 44 (S) 67 78 (S)

104

new PALs is of particular interest, as this unnatural amino acid is a relevant building block for

pharmaceutical synthesis (Figure 5). Examples of APIs that are be synthesised from this

amino acid are antiviral peptides, such as 5,38 and oxazolidinone antidiabetics, such as 6.39 In

light of this, a preparative scale amination of 1l was performed using PbPAL, due to its superior

enantioselectivity and impressive conversions compared to SrPAL and DdPAL. By re-running

the biotransformation with a 10-fold increase in substrate loading and volume (50 mg mL-1

whole cells) an isolated yield of 61% enantiomerically pure L-amino acid (ee >99%) was

obtained from 89 mg of starting material.

The differences in relative conversion and enantioselectivity in the biocatalysts (selected

based on the similarity of their zymophore) also allude to the often overlooked contribution of

non-active site residues to catalysis. These distal residues could coordinate breathing motions

across the entire enzyme structure which may direct the malleability of the active site and

modulate substrate accommodation. This has already been speculated within this class of

enzymes, with the discovery that active site residues alone cannot discriminate closely related

ammonia lyase and aminomutase enzymes in plants.53 Although protein dynamics are known

to influence enzyme activity,48,49 there is little evidence of their consideration in biocatalyst

research and development. Thus, this method of sampling sequence diversity outside the

active-site of an enzyme could be a crucial step in the investigation of these phenomena. The

varying overall sequence-dynamics relationship of each enzyme may be the reason why, for

example, PbPAL shows excellent activity with the novel substrate 3-methoxycinnamate,

whereas BlPAL, which has an identical active site, shows none. These underinvestigated

considerations of protein dynamics for biocatalysis (also enzyme replacement therapy) will

hopefully benefit from dedicated investigation of these phenomena in future work.

Figure 5. Preparative scale synthesis of m-methoxy-L-phenylalanine (S)-2l by the newly discovered ammonia

lyase from Planctomyces brasiliensis (PbPAL) and examples of antiviral and antidiabetic compounds 5 and 6

which can be synthesised from this building block.

Compounds with electron-donating ring-substituents have been considered difficult for PALs,

presumably due to deactivation of the α-carbon as a result of the relative strengths of the

aromatic ring and carboxyl group of these substrates as opposing electron sinks. This theory

105

is thought to explain the conversely high activity of substrates (such as nitrocinnamates) with

other ammonia lyases.36,50 The discovery of PbPAL emphasises the difficulty with which such

an activity would have been arrived upon using current engineering techniques without

targeting active site residues, especially in the absence of a high throughput enzyme assay.

In this respect, our discovery method offers many advantages over traditional diversification

across an entire parent sequence, which often involves lengthy and iterative mutagenesis

yielding many poorly active variants, few useful hits and inherent problems with screening.6,54

As such, we believe our method provides a complementary strategy to currently available

techniques, particularly for selecting specific catalytic activities from functionally diverse

families, such as other amino acid degrading enzymes, imine reductases / reductive

aminases, esterases / hydrolases and hydroxynitrile lyases.

5.3.7 Conclusion

In summary, we designed a new sequence-based annotation method to discover novel,

uncharacterised and structurally diverse PAL enzymes. We identified 19 key residues involved

in substrate binding and catalysis and our sequence-based annotation system used these

residues (the PAL zymophore) as queries to identify new PAL enzymes. A scoring system was

used to afford five structurally diverse enzymes with identical active sites. Screening of the

newly discovered enzymes revealed unique properties suited for either therapeutic or

biocatalytic applications.

5.3.8 References

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4. Porter, C. T.; Bartlett, G. J; Thornton, J. M. Nucleic Acids Res., 2004, 32, D129-133.

5. Sehnal, D.; Pravda, L.; Svobodová-Vařeková, R.; Ionescu, C.-M.; Koča, J.; Nucleic

Acids Res. 2015, 43, W383-388.

6. Höhne, M.; Schätzle, S.; Jochens, H.; Robins, K.; Bornscheuer, U. T. Nat. Chem. Biol.

2010, 6, 807–813.

7. Turner, N. J. Nat. Chem. Biol. 5, 567–573.

8. Romero, P.; Arnold, F. H. Nat. Rev. Mol. Cell Biol. 2009, 10, 866–876.

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10. Poppe, L. Curr. Opin. Chem. Biol. 2001, 5, 512–524.

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13. Zhang, X.; Liu, C.-J. Mol. Plant. 2015, 8, 17–27.

14. Hill, A. M.; Thompson, B. L.; Harris, J. P.; Segret, R. Chem. Commun., 2003, 44, 1358–

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Chem. Int. Ed. Engl. 2015, 54, 4608–4611.

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108

Chapter 6

Access to Challenging Electron Rich Phenylalanine

Analogues by Engineering a Phenylalanine Ammonia-

Lyase from Planctomyces brasiliensis

Syed T. Ahmed, Fabio Parmeggiani, Nicholas J. Weise, Ulrike Klemstein, Sabine L.

Flitsch and Nicholas J. Turner



Manuscript submitted

Acknowledgements: This chapter highlights the mutagenesis work conducted by the doctoral

candidate on expanding the substrate scope of this newly discovered enzyme with the help

and guidance from the co-authors. The doctoral candidate was involved in the experimental

design and execution of this study including manuscript preparation in collaboration with the

authors.

ACTIVE SITE

ENGINEERING

EDG = MeO, OCH2O, Et, MeS (17 examples)

109

6.1 Abstract

Engineered variants of phenylalanine ammonia lyase from Planctomyces brasiliensis were

developed through rational design efforts focusing on the aryl binding pocket of the active

site, guided by structural and phylogenetic inference. These approaches allowed inherent

problems traditionally associated with the biocatalytic hydroamination of acrylic acids, such

as low conversion and poor regioselectivity with alkyl- and methoxycinnamic acids, to be

overcome. The new suite of PbPAL variants described here represents a valuable addition

to the biocatalytic toolbox, allowing previously inaccessible amino acid building blocks to be

prepared regio- and enantioselectively.

6.2 Introduction

The discovery of phenylalanine ammonia lyase (PAL) enzymes and their application in the

industrial production of L-phenylalanine by biocatalytic hydroamination of cinnamic acid has

led to several successful endeavours to diversify the substrate scope of this enzyme towards

the production of substituted phenylalanine analogues from the corresponding cinnamic

acids.1-5 While the synthetic utility of PALs has been demonstrated by the chemo-enzymatic

synthesis of a range of phenylalanine analogues 6-8 including the production of the non-natural

D-enantiomer,9 the substrate scope of PALs has been limited primarily to compounds with

electron-poor/neutral ring systems, with no general success in engineering PALs to have

broad activity towards electron-rich analogues (Scheme 1a.)

Scheme 1. Lack of suitable PAL catalysts for electron-rich phenylalanines (a). Panel of challenging electron-rich

cinnamic acid substrates (1a-q) selected for this study (b).

110

Recently, we reported on the identification of several new PAL enzymes, including those from

Dictyostelium discoideum (DdPAL) and Planctomyces brasiliensis (PbPAL), exhibiting

variable activity profiles towards a broad panel of simple cinnamic acid derivatives.10

Remarkably, both PbPAL and DdPAL were able to convert m-methoxycinnamic acid (a

substrate shown to be converted poorly - if at all - by other PAL enzymes) to the corresponding

amino acid. While the active site compositions of the PAL enzymes are nearly identical, huge

differences in conversion were observed between the enzymes, demonstrating the

contribution of amino acid residues distal to the active site. Herein we describe our engineering

efforts to generate a panel of PAL biocatalysts with an expanded substrate scope, able to

accept a broad range of demanding electron-rich substrates.


6.3.1 Wild-type enzyme screening

A panel of cinnamic acids bearing electron-donating substituents (Scheme 1b), synthesised

from the corresponding aldehydes by a Knoevenagel-Doebner condensation (see chapter 6

supporting Information), was tested with PbPAL and DdPAL, along with the well-known

biocatalysts AvPAL from Anabaena variabilis and RgPAL from Rhodotorula glutinis for

comparison (Figure 1). Of the four enzymes, PbPAL was the only one to give moderate to

good conversion of substrates 1a-b and 1d-l, even though very low or no activity was

displayed with compounds 1c and 1m-q. It is worth noting that three of the substrates (1b, 1i

and 1l) were seen to afford the corresponding amino acids in high conversion with many of

the enzymes tested. This interesting observation shows that the low activity of most of the

substrates in this panel should not be ascribed only to the presence of electron-donating

substituents, but rather to a complex interplay of stereoelectronic factors and affinity to the

enzyme active site.

111

Figure 1. Screening of four different wild-type PALs against electron-rich substrates, highlighting (substrates 1o-s

are omitted since they did not show any activity with any of the tested enzymes).

6.3.2 PbPAL engineering strategy

In order to increase the conversions of the substrates which displayed poor activity, PbPAL

was selected as a very promising starting point and two separate engineering strategies were

employed to broaden its substrate range. We targeted two key adjacent residues in the aryl

binding pocket of the active site of PbPAL (F113 and L114), also known as “selectivity

residues” because they confer to the enzyme its substrate preference for either phenylalanine,

tyrosine or histidine (PAL, TAL, HAL, respectively).1a The first of these engineering

approaches (Figure 2A) was the replacement of the two residues with those of other members

of the enzyme family known to bind hydroxylated amino acids such as L-tyrosine and L-

DOPA.11,12 Combinations of HL, 11 YH 12 and YL13 were seen, as compared with the

homologous FL in PbPAL (and most PALs), therefore we decided to investigate the behaviour

of mutants F113H, F113Y and L114H. The second approach (Figure 2B) was based on

targeting of these selectivity residues with smaller, hydrophobic amino acids to widen the

active site cavity around the para-position of the substrate, as attempted in previous studies

with AvPAL[8a] and other homologs.14,15 For this design strategy we chose to consider

mutants F113A, F113I and F113V.

Su

bs

trate

Conversion [%]

0 20 40 60 80 100

1a

1b

1c

1d

1e

1f

1g

1h

1i

1j

1k

1l

1m

PbPAL DdPAL AvPAL RgPAL

112

Figure 2. Schematic representation of the substrate binding site of PbPAL, highlighting the positions selected for

engineering.

6.3.3 Analysis of enzyme mutants

All mutations were introduced by site-directed mutagenesis and the variants produced in E.

coli for activity tests under the same conditions used for the WT enzyme (Table 1). No

appreciable differences in the expression levels were observed compared to WT, with the only

exception of L114H that showed very poor gene expression and no activity (see chapter 6

supporting Information). Rewardingly, considerable improvements were seen with almost all

the substrates considered, with the only exception of 1b (already highly converted by WT) and

1i. The most significant increases in conversion were seen with meta- and para- substituted

compounds, and in particular substrates 1c, f, g, h, and m (all bearing a heteroatom at the

para-position) were better accepted by all the mutants tested. As a general trend, the widened

active site variants (strategy B) performed better than the TAL-like variants (strategy A). A

notable exception is 1d, for which both the TAL-like variants gave better yields (presumably

due to hydrogen bonding between the Y/H residue and the fluorine atom), while all the other

variants showed decreased tolerance for this substrate.

Remarkably, significant improvements in activity with the highly deactivated substrates 1m-q

were achieved (e.g., >12-fold for the 3,4-methylenedioxy substrate 1m with F113I), including

the creation of new activity for 1n-q, unreactive with WT but accepted by some of the variants,

albeit with low conversions (3-12%). In an effort to improve conversions further, we also

investigated double variants: F113Y-L114H (for strategy A), F113I-L114A and F113I-L114V

(for strategy B). As observed with L114H, the expression level of F113Y-L114H was

considerably low (see chapter 6 supporting information). Biotransformation results are

reported in Table 1. The switching of L114 to smaller hydrophobic residues in the F113I

variants did not afford any improvement for the substrates tested, and was even detrimental

in some instances (e.g. 1a, c, i).

113

An attempt to combine the two strategies led us to modify further the best-performing strategy

A variant (F113Y) with an additional mutation aimed at reducing the steric hindrance of the

aliphatic side-chain at position 114 (L114I, since a switch from F to A in strategy B mutants

proved less efficient than F to I). Even though the resulting F113Y-L114I mutant proved

successful in improving only a few of the conversions seen with F113Y (e.g. 1g, where the

increased active site volume better accommodates the m-Cl substituent), this double mutant

afforded the highest conversion across the panel for the challenging substrate 1p (18%).

These results highlight the difficulty in engineering a broadly applicable PAL biocatalyst able

accept diverse highly electron-donating and hydrophilic substrates. Even though expanding

the hydrophobic binding pocket afforded increased conversions with many of the substrates

tested, no additional improvement was seen upon further broadening (as demonstrated by the

double variants F113I-L114A and F113I-L114V).

Employing our rational design process, we were able to extend the substrate scope of PbPAL

towards very demanding electron-rich substrates. The engineering success with 1m and 1c,

combined with the existing high activity for the respective isomers 1l and 1b, imply that the

improvements were mainly achieved through the removal of steric hindrance around the para-

position of the substrates. Additionally the conversion of 1l and m but poor activity with the

similarly substituted 1n and p imply that the electron donating potential of methylenedioxy- vs.

dimethoxy-substitution is likely lower. This may be due to the constrained 5-membered ring

system which contains the two oxygens in the latter case, contravening optimal overlap of lone

pairs with the aryl ring system. The clear preferences observed for certain variants towards

specific substrates highlight the flexibility of these engineered biocatalysts, diversifying the

PAL toolbox.

114

Table 1. Biotransformation of electron-rich cinnamic acid substrates by PbPAL variants.[a]

Strategy A

variants

Strategy B variants

Subs.[b] R WT F113H F113Y F113A F113I F113V F113I-

L114A

F113I-

L114V

F113Y-

L114I

Conv.[c]

[%]

Conv.[

c]

[%]

Conv.[c]

[%]

Conv.[c]

[%]

Conv.[c]

[%]

Conv.[c]

[%]

Conv.[c]

[%]

Conv.[c]

[%]

Conv.[c]

[%]

1a 2-MeO 27 7 22 30 8 9 – – 4

1b 3-MeO 92 55 91 89 92 89 4 92 76

1c 4-MeO 8 55 60 40 67 30 3 44 48

1d 4-F-3-MeO 79 91 90 67 30 26 – 24 88

1f 3-F-4-MeO 40 86 87 87 87 86 – 86 87

1g 3-Cl-4-MeO 38 78 42 70 88 77 8 84 73

1h 4-MeS 84 95 94 95 95 96 87 95 94

1i 2-Et 48 9 24 24 11 8 – – 26

1j 3-Et 30 78 – 86 80 88 24 85 4

1k 4-Et 48 – 17 84 75 85 14 84 17

1m 3,4-OCH2O 3 42 27 54 67 48 – 48 11

1n 2,3-(MeO)2 – 3 – 2 10 – – 2 –

1o 2,4-(MeO)2 – – – – 6 – – – –

1p 3,4-(MeO)2 – 4 12 – – – – – 18

1q 2-F-3,4-

MeO

– 3 9 – – – 3 – –

[a] The highest conversion obtained for each substrate is highlighted. [b] Substrates 1e and 1l are omitted since they afforded good conversions with the WT enzyme (91 and 92%,

respectively). [c] Conversion determined by reverse phase HPLC.

116

6.3.4 Formation of β-amino acids side-product

In addition, with some of the variants we detected a side product which upon further

investigation revealed to be the corresponding β-amino acid (see chapter 6 supporting

information) This is a side activity of PALs we have reported previously,16 and to mitigate this,

the biotransformation was monitored over time in order to identify the optimal reaction time to

give maximum yield and minimum side-product formation. The production of the β-regioisomer

is presumably enhanced by the electronic properties of the substrates in this study, as seen

with AvPAL16 and related aminomutase enzymes.17 We infer that, by increasing the activity

(and thus number of turnovers) of an ammonia lyase for substrates with electron-rich ring

systems, more ammonia addition is directed to the β-position, as seen with prolonged time

course experiments in a previous study.16

6.3.5 Preparative scale synthesis of compounds 2c-h and 2j-m

To demonstrate the synthetic utility of these enzymes we performed several preparative scale

biotransformations with the best variant for each substrate (giving the optimal trade-off

between high conversion and low β-amino acid side-product formation). Reactions were

conducted on a 100 mg scale affording the corresponding amino acids 2c-g and 2j-m in

excellent conversions and good isolated yield (Scheme 2), except for 2h, which consistently

gave considerable losses during purification. Most of these methoxyphenylalanine building

blocks have been used in the synthesis of a broad range of APIs and natural products, such

as L-DOPA (from 2m), cryptophycin A (from 2g),18 anticancer bouvardin derivatives (from

2f)[19] and fluoroepinephrine (from 1q). 20

Scheme 2. Preparative scale transformation with engineered PbPAL variants.

117

6.3.6 Conclusion

In conclusion, we have successfully engineered a recently discovered PAL enzyme from P.

brasiliensis to accept electronically and sterically demanding substrates. By using specific

variants, tailored to individual substrates, preparative scale reactions affording excellent

conversion, ee and good isolated yields could be demonstrated. Furthermore, this work

highlighted the importance of implementing enzyme discovery (starting with a different and

unique wild-type template to other PALs) to enable and facilitate engineering strategies.

6.4 References

1. Parmeggiani, F.; Weise N. J.; Ahmed S. T.; Turner, N. J. Chem. Rev. 2017, DOI: 10.1021/acs.chemrev.6b00824.

2. (a) Turner, N. J. Curr. Opin. Chem. Biol. 2011, 15, 234-240; (b) Heberling, M. W.; Wu,

B.; Bartsch, S.; Janssen, D. B. Curr. Opin. Chem. Biol. 2013, 17, 250-260. (c) Poppe,

L. Curr. Opin. Chem. Biol. 2001, 5, 512-524.

3. (a) Yamada, S.; Nabe, K.; Izuo, N.; Nkamichi, K.; Chibata, I. Appl. Environ. Microbiol,

1981, 42, 773-778; (b) Paizs, C.; Katona, A.; Retey, J. Chem. Eur. J. 2006, 12, 2739-

2744.

4. Gloge, A.; Langer, B.; Poppe, L.; Retey, J. Arch. Biochem. Biophys. 1998, 359, 1-7.

5. Lovelock, S. L.; Turner, N. J. Bioorg. Med. Chem., 2014, 22, 5555-2227.

6. Yamada, S.; Nabe, K.; Izuo, N.; Nakamichi, K.; Chibata, I. Appl. Environ. Microbiol.

1981, 42, 773–778.

7. Renard, G.; Guilleux, J.-C.; Bore, C.; Malta-Valette, V.; Lerner, D. A. Biotechnol. Lett.

1992, 14, 673–678.

8. a) Gloge, A.; Zoń, J.; Kövári, Á.; Poppe, L.; Rétey, J. Chem. Eur. J. 2000, 6, 3386–

3390 b) Paizs, C.; Toşa, M. I.; Bencze, L. C.; Brem, J.; Irimie, F. D.; Rétey, J.

Heterocycles 2010, 82, 1217–1228.

9. a) Ahmed, S. T.; Parmeggiani, F.; Weise, N. J.; Flitsch, S. L.; Turner, N. J. ACS Catal.

2015, 5, 5410-5413 b) Parmeggiani, F.; Ahmed, S. T.; Weise, N. J.; Turner, N. J.

Tetrahedron 2016, 72, 7256–7262 c) Ahmed, S. T.; Parmeggiani, F.; Weise, N. J.;

Flitsch, S. L.; Turner, N. J. Org. Lett. 2016, 18, 5468–5471

10. a) Parmeggiani, F.; Lovelock, S. L.; Weise, N. J.; Ahmed, S. T.; Turner, N. J. Angew.

Chem. Int. Ed. 2015, 54, 4608–4611 b) Zhu, L.; Zhou, L.; Huang, N.; Cui, W.; Liu, Z.;

Xiao, K.; Zhou, Z. PLoS One 2014, 9, e108586.

11. Weise N. J.; Ahmed S. T.; Parmeggiani, F.; Galman J. L; Charnock, S. L.; Turner N. J.

Sci. Rep., 2017, revised manuscript submitted.

12. Louie G. V.; Bowman, M. E.; Moffitt, M. C.; Baiga, T. J.; Moore, B. S.; Noel, J. P. Chem.

Biol. 2006, 13, 1327–38.

118

13. Zhu, Y.; Liao, S.; Ye, J.; Zhang, H.; Biotechnol. Lett. 2012, 34, 269–74.

14. Walter, T.; King, Z.; Walker, K. D. Biochemistry 2016, 55, 1–4.

15. Bencze, L. C.; Filip, A.; Banoczi, G.; Tosa, M. I.; Irimie, F. D.; Gellert, A.; Poppe, L.;

Paizs, C. Org. Biomol. Chem. 2017, 15, 3717-3727.

16. Rowles, I.; Groenendaal, B.; Binay, B.; Malone, K. J.; Willies, S. C.; Turner, N. J.;

Tetrahedron 2016, 72, 7343–7347.

17. Weise, N. J.; Parmeggiani, F.; Ahmed S. T.; Turner, N. J. Top. Catal., 2017, revised

manuscript submitted.

18. Weise N. J.; Parmeggiani, F.; Ahmed S. T.; Turner N. J. J. Am. Chem. Soc. 2015, 137,

12977-12983.

19. Janetka, J. W.; Rich, D. H. J. Am. Chem. Soc. 1997, 119, 6488-6495.

20. Trimurtulu, G.; Ohtani, I.; Patterson, G. M. L.; Moore, R. E.; Corbett, T. H.; Valeriote,

F. A.; Demchik, L.; J. Am. Chem. Soc. 1994, 116, 4729-4737.

21. Gladstone, M. N.; Zhang, G.; Sammakia, T.; WO pat. 126617 A1, 29 August 2013.

22. Herbert, B.; Kim, I. H.; Kirk, K. L. J. Org. Chem. 2001, 66, 4892-4897.

119

Chapter 7

General Methods and Supporting Information for

Chapters 2-6

Acknowledgements: This chapter contains experimental methods including molecular

biology protocols and synthetic procedures for chapters 2-6. NMR, HRMS and chiral HPLC

spectrum have been omitted and can be found in the electronic supplementary information

from each respected journal. The chapter is divided into general experimental methods and

chapter specific protocols.

120

7.1 General methods

7.1.1 NMR spectroscopy

1H and 13C NMR spectra were recorded on a Bruker Avance 400 spectrometer (400 MHz) at

298K without additional internal standard. Carbon data for compounds 4b, 4d and 5e in

chapter 4 were recorded on a Bruker Avance III 500 spectrometer. Chemical shifts are

reported as δ in parts per million (ppm) and are calibrated against residual solvent signal.

Synthesized cinnamic acids were measured in either CDCl3 or DMSO-d6. A water suppression

method was used to obtain the 1H NMR spectra of amino acids in water, with D2O as the

residual solvent peak (1H NMR: D2O = 4.79 ppm). 1H NMR data were analysed on MestreNova

and are reported as follows: chemical shift (ppm), multiplicity (s = singlet, d = doublet, dd =

doublet of doublets, t = triplet, q = quartet, m = multiplet), coupling constants (Hz), proton

integration.

7.1.2 High-resolution mass spectrometry

HRMS analysis was performed using a Waters LCT time-of-flight mass spectrometer

connected to a Waters Alliance LC (Waters, Milford, MA, USA). For compounds bearing a

halogen atom, the most abundant isotope was reported; 79Br and 35Cl.

7.1.3 Strain and plasmid

E. coli BL21 (DE3) was used as an expression host for protein production. Work reported in

chapter 2 used a pET16b-AvPAL plasmid containing Anabaena variabilis (codon-optimised

for E. coli) obtained as described previously.1 Ampicillin (Amp) was added where appropriate

to a final concentration of 100 μg mL–1. Solid media were prepared by addition of agar (1.5%

w/v) to liquid media.

For chapters 2-6 a pET-28a plasmid was used with the appropriate enzyme using kanamycin

as the antibiotic at a final concentration of 50 μg mL–1.

7.1.4 Transformation into competent cells

Competent cells (50 µL aliquot) were thawed on ice for 15 min, transferred to a pre-chilled

tube containing plasmid DNA (20-50 ng). The tubes were gently flicked for mixing, incubated

in ice for 30 min, heat-shocked at 42°C for 30 s and transferred back to ice for 5 min. SOC

medium (900 µL) was added to the tube under sterile conditions and incubated at 37°C, 250

rpm for 1 h. The culture was then concentrated to 100 µL (by centrifugation and resuspension)

and spread across LB plates containing suitable antibiotics using a spreader under sterile

condition. Plates were incubated overnight at 37°C and stored at 4˚C.

121

7.1.5 Biocatalyst production

A single colony of E. coli BL21(DE3) carrying the appropriate PAL plasmid was inoculated in

LB medium (8 mL) supplemented with ampicillin or kanamycin and was grown overnight at

37°C at 250 rpm. The starter culture was used to inoculate LB-based auto-induction medium2

(800 mL, Formedium) containing ampicillin or kanamycin and glycerol (0.5% v/v) which was

incubated at 18°C and 180 rpm for 4 days. The cells (10 g) were harvested by centrifugation

(4,000 rpm, 20 min, 4°C) and stored at –20°C.

7.1.6 Lyophilization procedure of wet cells

Harvested wet cells were flash frozen in liquid nitrogen followed by drying under high

vacuum overnight to yield a powdered formulation.

7.1.7 Ion-exchange purification of amino acids

Supernatant from the reaction mixture was acidified to pH < 2 by addition of aqueous H2SO4

(10% w/v) and centrifuged (4000 rpm, 10 min, 4°C) to remove cells and insoluble components.

Dowex® 50WX8 hydrogen form (2.5 g) was washed with deionised water (50 mL) and

aqueous H2SO4 (25 mL, 10% w/v). The acidified supernatant from the biotransformation was

loaded onto the resin (1 mL min–1). The resin was washed repeatedly with deionised water

(until pH ~ 7) and the product was eluted with aqueous NH4OH (30 mL, 10% w/v). Fractions

containing the product were pooled and evaporated in a centrifugal evaporator, to afford amino

acids of interest.

122

7.2 Chapter 2 supporting information

7.2.1 Co-solvent tests for the DAADH reaction

In order to increase the substrate loading in the reductive amination of 4 mediated by DAADH,

the reaction was tested in the presence of a range of co-solvents (5% v/v).

Only methanol and dimethyl sulfoxide afforded complete conversion and for preparative

applications methanol was employed.

7.2.3 Chemicals

Analytical grade reagents and solvents were obtained from Sigma-Aldrich, AlfaAesar or Fisher

Scientific and used without further purification, unless stated otherwise.

7.2.4 Specific optical rotation

Specific rotations were measured on an ADP 440+ polarimeter with a path length of 0.5 dm.

Concentrations (c) are given in g/100 mL and units are in 10–1 deg cm2 g–1.

7.2.5 TLC analysis

Thin layer chromatography (TLC) was carried out on Merck TLC silica 60 F254 aluminium

backed sheets. Plates were dipped into a solution of ninhydrin to visualise spots.

7.2.6 Flash chromatography

Chromatographic separations were performed on silica gel (pore size 60 Å, 220-240 mesh

size, particle size 35-75 µm), using as eluent DCM/MeOH 85:15 for compounds 1a-d and 1g-

k, and pure EtOAc for compounds 1e-f.

7.2.7 Microwave reactions

Reactions were carried out in a CEM Discoverer microwave reactor using sealed reaction

vessels. Maximum power output was set to 200 W and maximum pressure was set to 247 psi.

All microwave reactions were conducted under stirring.

0

20

40

60

80

100

0 5 10 15 20

Co

nve

rsio

n (

%)

Time (h)

MeOH

iPrOH

DMSO

THF

MeCN

Acetone

123

7.2.8 Non-chiral HPLC analysis and conversion

Conversions were measured on a non-chiral reverse-phase Zorbax C-18 Extend column (50

mm × 4.6 mm × 3.5 µm, Agilent). Mobile phase aq. NH4OH 0.1 M pH 10 / MeOH, isocratic

50:50 (5 min), gradient from 50:50 to 10:90 (over 15 min), isocratic 10:90 (5 min), flow rate 1.0

mL min–1, temperature 40°C, detection wavelength 210 nm (retention times are reported in

the following table).

Compound tR (min)

L- and D-2 2.3

3 4.8

4 4.0

8 1.3

9a 1.4

9b 1.3

9c 1.4

9d 1.6

9e 2.0

9f 1.9

9g 1.7

9h 1.7

9i 2.0

9j 1.3

9k 6.5

10 2.2

L- and D-11 4.8

L- and D-1a 6.5

L- and D-1b 6.6

L- and D-1c 7.1

L- and D-1d 6.8

L- and D-1e 9.9

L- and D-1f 9.8

L- and D-1g 7.7

L- and D-1h 7.8

L- and D-1i 8.1

L- and D-1j 7.6

L- and D-1k 11.3

124

7.2.9 Enantiomeric excess of L- and D-2

Chiral analysis of 2 was carried out on a reverse-phase Crownpak CR(+) column (150 x 4 mm

x 3.5 µm, Daicel). Mobile phase aq. HClO4 1.14% w/v / MeOH 96:4, flow rate 1.0 mL min–1,

temperature 40°C, detection wavelength 210 nm. tR (D) = 20.6 min, tR (L) = 27.0 min.

In order to prove that no loss of optical purity is observed in the Suzuki-Miyaura step, the ee

of L-1a was determined by HPLC after a three step derivatisation protocol: (a) esterification of

the carboxylic acid group with trimethylsilyldiazomethane (TMSD); (b) deprotection of the

amine; (c) conversion of the free amino group to a glycosyl isothiocyanate with 2,3,4,6-tetra-

O-acetyl-β-D-glucopyranosyl isothiocyanate (GITC).1,2

To a solution of isolated 1a (20 mg) in MTBE (1 mL), TMSD (20 µL, 1.6 M in hexane) was

added, and the mixture was left to stand at r. t. for 3 h. MTBE was removed in vacuo and the

residue was dissolved in DCM/TFA (3:1, 1 mL) and stirred at r. t. for 24 h. The solvent was

removed and the residue redissolved in MeCN (1 mL). Et3N (1 µL) and GITC (100 µL, 10%

w/v in MeCN) were added and the mixture was left to stand at r. t. for 1 h to give the derivatised

product 1a’. A sample was diluted with MeOH (1:10) and analysed on a non-chiral reverse-

phase Zorbax C-18 Extend column (50 mm × 4.6 mm × 3.5 µm, Agilent). Mobile phase aq.

NH4OH 0.1 M pH 10 / MeOH, isocratic 50:50 (10 min), gradient from 50:50 to 10:90 (over 15

min), isocratic 10:90 (5 min), flow rate 1.0 mL min–1, temperature 40°C, detection wavelength

210 nm. Retention times: tR (D-1a’) = 4.6 min, tR (L-1a’) = 7.2 min.

125

7.2.10 Site-directed mutagenesis

Mutagenesis was carried out using the QuikChange II protocol (Agilent) with the following primers

(mutations are underlined):

F107L_Fw 5’-CCAATCTGGTTTGGCTTCTGAAAACCGGTGCAGG-3’

F107L_Rv 5’-CCTGCACCGGTTTTCAGAAGCCAAACCAGATTGG-3’

F107I_Fw 5’-CCAATCTGGTTTGGATTCTGAAAACCGGTGCAGG-3’

F107I_Rv 5’-CCTGCACCGGTTTTCAGAATCCAAACCAGATTGG-3’

F107A_Fw 5’-CCAATCTGGTTTGGGCGCTGAAAACCGGTGCAGG-3’

F107A_Rv 5’-CCTGCACCGGTTTTCAGCGCCCAAACCAGATTGG-3’

DpnI digest was carried out by addition of DpnI (1 µL) and incubation at 37°C for 1 h. This was then

stored at 4°C for further transformation into ultra-competent E. coli XL10-Gold cells. The presence of

the desired mutation was verified by sequencing of plasmid DNA.

Plasmid DNA samples were used to transform competent E. coli BL21(DE3) cells for expression.

7.2.11 D-amino acid dehydrogenase gene (DAADH)

The daadh gene, evolved from dapdh from Corynebacterium glutamicum,3 was synthesised

by Geneart and cloned in pET-26b (Novagen) at the restriction sites NdeI and XhoI. The

resulting pET26b-DAADH construct, checked by sequencing, was used to transform

competent cells.

7.2.12 Biotransformation with lyophilized PAL enzyme

4-bromocinnamic acid 3 (22.5 mg, final conc. 10 mM) was dissolved in aqueous NH4OH

solution pH adjusted with H2SO4 (5 M, pH 9.6, 10 mL). Lyophilized E.coli BL21(DE3) cells

overproducing the suitable PAL or variant (30 mg mL–1) were added and the suspension was

incubated at 37°C with shaking (250 rpm) for 24 h. Biotransformation samples (300 μL) were

mixed with MeOH (300 μL), thoroughly shaken and centrifuged (13000 rpm, 1 min), then the

supernatant was transferred to a filter vial and used directly for HPLC analysis.

7.2.13 Biotransformation with DAADH (cell-free extract)

4-bromophenylpyruvic acid 4 (12 mg, final conc. 10 mM) was dissolved in MeOH (250 µL, 5%

v/v) and added to the reaction buffer (5 mL, 200 mM NH4Cl, 100 mM D-glucose, 0.2 mM

NADP+, 100 mM Na2CO3, pH 9.0). DAADH cell-free extract (100 µL) and GDH (4 U mL–1) were

added and the mixture was incubated at r. t. with gentle agitation (50 rpm) for 24 h. Samples

were analysed as described for PAL biotransformations.

126

7.2.14 Synthesis of Palladium catalyst 6

2-amino-4,6-dihydroxypyrimidine (13 mg, 0.10 mmol) was added to a solution of NaOH (2 mL,

0.10 M) and stirred for 5 min until homogenous. Pd(OAc)2 (11 mg, 0.05 mmol) was added and

the mixture was heated to 65°C for 45 min to give a yellow solution. The solution was diluted

with deionised water to a final volume of 5 mL to give a catalyst solution (0.01 M final

concentration) that was used without further treatment.

7.2.15 General procedure for the optimization of the Suzuki-Miyaura cross-coupling

reaction

4-bromobenzoic acid 8 (0.4 mmol, 1 eq), phenylboronic acid 9a (0.6 mmol, 1.5 eq), the Pd-catalyst 5-7

(2-10 mol%) and the base (1.2 mmol, 2 eq) were added to the required solvent (10 mL). The mixture

was heated at the required temperature (50-80°C) for 4-24 h. The mixture was acidified with aq. HCl (3

mL, 3 M) and extracted with EtOAc (2 × 10 mL). Combined organic extracts were dried over MgSO4

and concentrated in vacuo.

7.2.16 Erlenmeyer-Plöchl synthesis1 of 4-bromophenylpyruvic acid 4

(Z)-4-(4-bromobenzylidene)-2-methyloxazol-5(4H)-one

To a solution of p-bromobenzaldehyde (7.84 g, 42 mmol, 1.0 equiv.) in

Ac2O (19 mL), were added anhydrous NaOAc (4.50 g, 55 mmol, 1.3

equiv.) and N-acetylglycine (6.45 g, 55 mmol, 1.3 equiv.). The mixture

was heated for 4 h under reflux (sand bath 140°C), then cooled to room

temperature. Cold water (50 mL) was added and the mixture was stirred for 30 min in an ice

bath. The solid was filtered and washed with cold water (3 × 20 mL) and cold Et2O (10 mL) to

afford the corresponding azalactone, (Z)-4-(4-bromobenzylidene)-2-methyloxazol-5(4H)-one,

as a yellow solid of sufficient purity for the next step (9.84 g, 37 mmol, 88% yield). 1H NMR

(400 MHz, chloroform-d) δ 7.95 (d, J = 8.5 Hz, 2H), 7.57 (d, J = 8.6 Hz, 2H), 7.06 (s, 1H), 2.41

(s, 3H); 13C NMR (101 MHz, CDCl3) δ 167.76, 166.80, 133.42, 133.10, 132.18, 132.04, 129.86,

126.00, 15.7

127

(Z)-3-(4-bromophenyl)-2-hydroxyacrylic acid (4)

The crude azlactone (9.00 g, 34 mmol) was suspended in aq. HCl

(30 mL, 3 M) and refluxed for 6 h (sand bath 140°C). The solid was

filtered and washed with cold water (3 × 30 mL) to give pure 4 as an

orange solid (7.55 g, 91% yield). 1H NMR (400 MHz, MeOH-d4) δ 7.51 (d, J = 8.5 Hz, 2H),

7.10 (d, J = 8.6 Hz, 2H), 6.50 (s, 1H); 13C NMR (101 MHz, MeOH-d4) δ 168.02, 143.11, 135.62,

132.339, 132.34, 121.89, 110.02.

7.2.17 General procedure for Suzuki-Miyaura cross-coupling reaction to synthesise

standards of compounds L-1a-k

N-Boc-4-bromo-L-phenylalanine L-11 (0.4 mmol, 1eq), arylboronic acid 9a-k (0.6 mmol, 1.5

eq), Pd-catalyst 5 (0.04 mmol, 10 mol%) and CsCO3 (1.2 mmol, 2 eq) were added to deionised

water (10 mL), then the mixture was microwave heated (200 W, 120°C) for 20 min. The mixture

was acidified with aq. HCl (3 mL, 3 M) and extracted with EtOAc (2 × 25 mL). Combined

organic extracts were dried over MgSO4 and concentrated to give products 1a-k.

7.2.18 General procedure (A) for one-pot N-Boc protection and Suzuki-Miyaura cross-

coupling reaction (from biotransformation product L-11 to L-1-a-k)

4-bromo-L-phenylalanine L-11 (12 mg, 50 µmol) and CsCO3 (55 mg, 150 µmol, 3 eq) was

added to deionised water (5 mL) in a microwave reactor vessel containing a magnetic stirrer

bar. THF (2.5 mL) was added followed by addition of Boc2O (13 mg, 60 µmol, 1.2 eq). The

solution was microwave heated (200 W, 90°C) for 15 min and cooled to room temperature.

Arylboronic acid 9a-k (75 µmol, 1.5 eq) and Pd-catalyst 5 (5.0 µmol, 10 mol%) was added to

the reaction vessel and the mixture was microwave heated (200 W, 120°C) for 20 min. The

reaction was cooled to room temperature and acidified with aq. HCl (3 mL, 3 M) and extracted

with EtOAc (2 × 25 mL). Combined organic extracts were dried over MgSO4 and concentrated

under reduced pressure. The residue was purified by column chromatography to give pure L-

1a-k.

7.2.19 General procedure (B) for the one-pot DAADH reductive amination, N-Boc

protection and Suzuki-Miyaura coupling (from ketoacid 4 to D-1a-k)

4-bromophenylpyruvic acid 4 (12 mg, final conc. 10 mM) was dissolved in MeOH (250 µL, 5%

v/v) and added to the reaction buffer (5 mL, 200 mM NH4Cl, 100 mM D-glucose, 0.2 mM

NADP+, 100 mM Na2CO3, pH 9.0). DAADH cell-free extract (100 µL) and GDH (4 U mL–1) were

added and the mixture was incubated at r. t. with gentle agitation (50 rpm) for 24 h. Deionised

water (5 mL), THF (5 mL) and Boc2O (23 mg, 102 µmol, 5 eq) were added, then the solution

was microwave heated (200 W, 90°C) for 15 min and cooled to room temperature. The mixture

was then processed as described in general procedure A, affording pure D-1a-k.

128

7.2.20 Compound characterization data

(S)-3-(4-bromophenyl)-2-((tert-butoxycarbonyl)amino)propanoic acid (L-11)

4-bromo-L-phenylalanine (250 mg, 1 mmol) was added to 10%

solution of NaOH (1.5 g in 13.5 mL). THF (15 mL) was added to the

mixture followed by the addition of Boc-anhydride (268 mg, 1.2 mmol,

1.2 eq) and left to stir for 24 hours at room temperature. THF was removed in vacuo and the

remaining solution was acidified with 10% HCl (10 mL) and extracted with DCM (2 × 25 mL).

Organic layer was dried with MgSO4 and concentrated to give compound 11 as a white solid

(150 mg, 43%) 1H NMR (400 MHz, chloroform-d) δ 7.46 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.0

Hz, 2H), 7.19 (d, J = 8 Hz, 1H, NH) 4.07 (m, 1H), 2.98 (dd, J = 12.0, 4.0 Hz, 1H), 2.77 (dd, J

= 16.0 Hz, 12.0 Hz, 1H), 1.31 (s, 9H); 13C NMR (101 MHz, chloroform-d) δ 173.35, 155.40,

137.48, 131.37, 130.93, 119.48, 78.05, 54.87, 35.74, 28.06; MS -ESI (m/z) 342.3 [M]–.

3-([1,1'-biphenyl]-4-yl)-2-((tert-butoxycarbonyl)amino)propanoic acid (1a)

White crystals (21 mg, 70% for L-1a, 5 mg, 57% yield for D-1a);

[α]20D= +19.6 (c=1, MeOH) for L-1a, [α]20

D= –21.0 (c=1, MeOH)

for D-1a, vs. lit. [α]20D= +23.8 (c=3.1, MeOH); 1H NMR (400 MHz,

DMSO-d6) δ 7.60 (d, J = 8 Hz, 2H), 7.56 (d, J = 8 Hz, 2H), 7.45 (t,

J = 8 Hz, 2H), 7.35 – 7.30 (m, 3H), 7.00 (brs, 1H, NH) 4.21 – 4.15 (m, 1H), 3.08 (dd, J = 13.7,

4.5 Hz, 1H), 2.85 (dd, J = 13.7, 10.3 Hz, 1H), 1.32 (s, 9H); 13C NMR (101 MHz, chloroform-d)

δ 173.72, 155.29, 140.01, 138.08, 137.52, 129.74, 128.86, 127.18, 126.47, 126.33, 77.43,

55.23, 36.17. 28.13; HRMS-ESI (m/z) [M+Na]+ calcd.for C20H23NO4: 364.1525, found,

364.1522.

2-((tert-butoxycarbonyl)amino)-3-(2'-fluoro-[1,1'-biphenyl]-4-yl)propanoic acid (1b)

Clear oil (33 mg, 62% for L-1b, 5.5 mg, 56% yield for D-1b);

[α]20D= +10.1 (c=1, MeOH) for L-1b, [α]20

D= –9.2 (c=0.8, MeOH)

for D-1b; 1H NMR (400 MHz, DMSO-d6) δ 7.48 – 7.45 (m, 2H),

7.37 (t, J = 7.5 Hz, 3H), 7.32 – 7.25 (m, 2H), 7.19 (d, J = 8.4 Hz,

1H), 4.15 (td, J = 9.5, 8.6, 4.4 Hz, 1H), 3.08 (dd, J = 13.7, 4.3 Hz,

1H), 2.88 (dd, J = 13.7, 10.6 Hz, 1H), 1.32 (s, 9H). 13C NMR (101 MHz, DMSO-d6) δ 173.53,

160.26, 155.46, 137.76, 135.21, 133.06, 130.63, 129.34, 128.49, 124.48, 123.62, 115.92,

78.05, 55.02, 36.08, 28.10; HRMS-ESI (m/z) [M+Na]+ calcd for C20H22FNO4: 382.1431, found,

382.1424.

129

2-((tert-butoxycarbonyl)amino)-3-(3'-fluoro-[1,1'-biphenyl]-4-yl)propanoic acid (1c)

Clear oil (38 mg, 71% for L-1c, 6.5 mg, 66% yield for D-1c);

[α]20D= +9.4 (c=0.8, MeOH) for L-1c, [α]20

D= –9.8 (c=1, MeOH) for

D-1c; 1H NMR (400 MHz, DMSO-d6) δ 7.60 (d, J = 8.2 Hz, 2H),

7.52 – 7.43 (m, 3H), 7.32 (d, J = 8.1 Hz, 2H), 7.16 (m, 1H), 4.07

(m, 1H), 3.07 (dd, J = 13.6, 4.3 Hz, 1H), 2.86 (dd, J = 13.6, 9.7

Hz, 1H), 1.30 (s, 9H) 13C NMR (101 MHz, DMSO-d6) δ 173.62, 161.52, 142.46, 138.12,

136.82, 130.85, 129.83, 126.58, 122.56, 116.93, 113.08, 78.11, 55.19, 36.12, 28.17; HRMS-

ESI (m/z) [M+Na]+ calcd for C20H22FNO4: 382.1431, found, 382.1429.

2-((tert-butoxycarbonyl)amino)-3-(4'-fluoro-[1,1'-biphenyl]-4-yl)propanoic acid (1d)

Clear oil (30 mg, 60% for L-1d, 6.4 mg, 64% yield for D-1d);

[α]20D= +7.49 (c=0.7, MeOH) for L-1d, [α]20

D= –8.1 (c=0.5,

MeOH) for D-1d; 1H NMR (400 MHz, DMSO-d6) δ 7.81 (dd, J

= 8.6, 6.5 Hz, 1H), 7.66 (dd, J = 8.7, 5.5 Hz, 2H), 7.53 (d, J =

8.1 Hz, 2H), 7.31 – 7.22 (m, 2H), 7.16 – 7.09 (m, 1H), 4.06

(m,1H), 3.06 (dd, J = 14.0, 4.7 Hz, 1H), 2.85 (dd, J = 13.6, 9.7 Hz, 1H), 1.30 (s, 9H) 13C NMR

(101 MHz, DMSO-d6) δ 173.56, 160.50, 155.46, 137.28, 136.43, 129.68, 128.35, 126.33,

115.49, 114.12, 78.03, 55.11, 36.00, 28.09; HRMS-ESI (m/z) [M+Na]+ calcd for C20H22FNO4:

382.1431, found, 382.1440.

2-((tert-butoxycarbonyl)amino)-3-(4'-chloro-[1,1'-biphenyl]-4-yl)propanoic acid (1e)

White solid (80 mg, 65% for L-1f, 3.5 mg, 47% yield for D-1f);

[α]20D= +6.5 (c=0.6, MeOH) for L-1f, [α]20

D= –7.2 (c=0.5,

MeOH) for D-1e; 1H NMR (400 MHz, DMSO-d6) δ 7.67 (d, J

= 8.0 Hz, 2H), 7.59 (d, J = 8.0 Hz, 2H), 7.50 (d, J = 8.0 Hz,

2H), 7.34 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 8 Hz, 1H, NH) 4.11

(m, 1H), 3.06 (dd, J = 13.7, 4.4 Hz, 1H), 2.86 (dd, J = 13.7, 10.6 Hz, 1H), 1.31 (s, 9H); 13C

NMR (101 MHz, DMSO-d6) δ 173.54, 155.46, 138.75, 137.78, 136.80, 132.07, 129.78, 128.81,

128.23, 126.34, 78.04, 55.09, 35.94, 28.11; HRMS-ESI (m/z) [M+Na]+ calcd for C20H2235ClNO4:

398.1135, found, 398.1128.

130

2-((tert-butoxycarbonyl)amino)-3-(3'-chloro-[1,1'-biphenyl]-4-yl)propanoic acid (1f)

White solid (73 mg, 65% for L-1f, 3.5 mg, 47% yield for D-1f);

[α]20D= +6.12 (c=0.8, MeOH) for L-1f, [α]20

D= –6.2 (c=1, MeOH)

for D-1f; 1H NMR (400 MHz, DMSO-d6) δ 7.69 (s, 1H), 7.62 (d, J

= 8 Hz, 3H), 7.50 (t, J = 8 Hz, 1H), 7.40 (d, J = 12Hz, 1H), 7.35

(d, J = 8.0, 2H), 7.28 (d, J = 8 Hz, 1H, NH), 4.12 (m, 1H), 3.06 (dd,

J = 13.7, S16 4.3 Hz, 1H), 2.86 (dd, J = 13.6, 10.7 Hz, 1H), 1.32 (s, 9H);13C NMR (101 MHz,

DMSO-d6) δ 173.52, 155.46, 142.12, 138.11, 136.60, 133.68, 130.68, 129.78, 127.04, 126.55,

126.19, 125.16, 77.92, 55.06, 36.09, 28.10; HRMS-ESI (m/z) [M+Na]+ calcd for C20H2235ClNO4:

398.1135, found, 398.1128.

2-((tert-butoxycarbonyl)amino)-3-(4'-methoxy-[1,1'-biphenyl]-4-yl)propanoic acid (1g)

White solid (45 mg, 41% for L-1g, 6.6 mg, 68% yield for D-1g);

[α]20D= +5.82 (c=0.52, MeOH) for L-1g, [α]20

D= –5.5 (c=0.8,

MeOH) for D-1g; 1H NMR (400 MHz, DMSO-d6) δ 7.36 (d, J =

8.1 Hz, 2H), 7.34 – 7.29 (m, 1H), 7.28 – 7.22 (m, 3H), 7.08 (d, J

= 7.9 Hz, 1H), 7.00 (t, J = 7.4 Hz, 1H), 6.85 (brs, 1H, NH) 3.74

(s, 3H), 3.14 – 3.04 (m, 1H), 2.89 (dd, J = 13.4, 9.5 Hz, 1H), 1.33 (s, 9H) 13C NMR (101 MHz,

DMSO-d6) δ 156.00, 155.24, 137.00, 135.92, 130.23, 129.64, 128.82, 128.58, 120.68, 111.61,

77.80, 550.34, 48.56, 36.47, 28.15; HRMS-ESI (m/z) [M+Na]+ calcd for C21H25NO5: 394.1630,

found, 394.1636

1H NMR (400 MHz, DMSO-d6) δ 7.58 (d, J = 8.7 Hz, 2H), 7.52 (d, J = 8.1 Hz, 2H), 7.29 (d, J

= 8.0 Hz, 2H), 7.00 (d, J = 8.7 Hz, 2H), 4.10 (m, 1H), 3.78 (s, 3H), 3.14 – 2.98 (m, 1H), 2.92 –

2.77 (m, 1H), 1.32 (s, 9H) 13C NMR (101 MHz, DMSO-d6) δ 160.82, 158.68, 137.75, 136.67,

135.77, 132.35, 129.64, 127.50, 125.81, 114.26, 112.84, 77.92, 55.09, 36.17, 28.12; HRMS-

ESI (m/z) [M+Na]+ calcd for C21H25NO5: 394.1630, found, 394.1622.

2-((tert-butoxycarbonyl)amino)-3-(3'-methoxy-[1,1'-biphenyl]-4-yl)propanoic acid (1h)

Clear oil (55 mg, 51% for L-1h, 6 mg, 61% yield for D-1h); [α]20D=

+7.8 (c=1.00, MeOH) for L-1h, [α]20D= –7.5 (c=0.80, MeOH) for

D-1h; 1H NMR (400 MHz, DMSO-d6) δ 7.54 (d, J = 8.0 Hz, 2H),

7.35-7.31 (m, 3H), 7.18 (d, J = 7.8Hz, 1H), 7.14 (s, 1H), 6.90 (d,

J = 10.2 Hz, 1H), 6.7 (brs, 1H, NH), 4.09 (s, 1H), 3.80 (s, 3H),

3.12 (d, J = 10.2 Hz, 1H), 2.91 (dd, J = 13.2, 9.1 Hz, 1H), 1.32 (s, 9H) 13C NMR (101 MHz,

DMSO-d6) δ 159.65, 155.10, 141.60, 138.01, 137.80, 129.86, 129.79, 126.30, 118.76, 112.70,

111.92, 77.72, 55.64, 55.01, 36.58, 28.14; HRMS-ESI (m/z) [M+Na]+ calcd for C21H25NO5:

394.1630, found, 394.1627.

131

2-((tert-butoxycarbonyl)amino)-3-(2'-methoxy-[1,1'-biphenyl]-4-yl)propanoic acid (1i)

Clear oil (40 mg, 37% for L-1i, 5.9 mg, 62% yield for D-1i);

[α]20D= +11.1 (c=1.00, MeOH) for L-1i, [α]20

D= –10.9 (c=0.90,

MeOH) for D-1i; 1H NMR (400 MHz, DMSO-d6) δ 7.58 (d, J

= 8.7 Hz, 2H), 7.52 (d, J = 8.1 Hz, 2H), 7.29 (d, J = 8.0 Hz,

2H),7.03 (d, J = 8 Hz, 1H, NH), 7.00 (d, J = 8.7 Hz, 2H), 6.80

4.10 (m, 1H), 3.78 (s, 3H), 3.14 – 2.98 (m, 1H), 2.92 – 2.77 (m, 1H), 1.32 (s, 9H) 13C NMR

(101 MHz, DMSO-d6) δ 160.82, 158.68, 137.75, 136.67, 135.77, 132.35, 129.64, 127.50,

125.81, 114.26, 112.84, 77.92, 55.09, 36.17, 28.12; HRMS-ESI (m/z) [M+Na]+ calcd for

C21H25NO5: 394.1630, found, 394.1622.

3-(4-(benzo[d][1,3]dioxol-5-yl)phenyl)-2-((tert-butoxycarbonyl)amino)propanoic acid

(1j)

White solid (40 mg, 50% for L-1j, 7 mg, 70% yield for D-1j);

[α]20D= +9.15 (c=2.8, MeOH) for L-1j, [α]20

D= –8.5 (c=1.00,

MeOH) for D-1j; 1H NMR (400 MHz, DMSO-d6) δ 7.50 (d, J =

7.9 Hz, 2H), 7.28 (d, J = 7.8 Hz, 2H), 7.21 (s, 1H), 7.11 (d, J

= 8.0 Hz, 1H), 6.97 (d, J = 8.1 Hz, 1H), 6.88 (d, J = 8 Hz, 1H, NH), 6.05 (s, 2H), 4.08 (s, 1H),

3.10 – 3.00 (m, 1H), 2.90 – 2.81 (m, 1H), 1.32 (s, 9H) 13C NMR (101 MHz, DMSO-d6) δ 155.26,

147.88, 146.61, 137.82, 137.05, 134.31, 129.63, 126.05, 119.93, 108.58, 106.91, 101.05,

77.88, 55.39, 18.57, 36.18, 28.14; HRMS-ESI (m/z) [M+Na]+ calcd for C21H23NO6: 408.1423,

found, 408.1424.

3-([1,1':4',1''-terphenyl]-4-yl)-2-((tert-butoxycarbonyl)amino)propanoic acid (1k)

White solid (45 mg, 37 % for L-11a, 4 mg, 40% yield for

D-11a); [α]20D= +16.2 (c=1.00, Acetone) for L-11a, [α]20

D=

–15.9 (c=1.00, Acetone) for D-11a; 1H NMR (400 MHz,

DMSO-d6) δ 7.75 (s, 4H), 7.72 (d, J = 7.2 Hz, 2H), 7.64 (d,

J = 8.2 Hz, 2H), 7.48 (t, J = 7.6 Hz, 2H), 7.41 – 7.32 (m,

3H), 7.20 (d, J = 8 Hz, 1H, NH), 4.13 (td, J = 9.4, 8.6, 4.5 Hz, 1H), 3.07 (dd, J = 13.7, 4.4 Hz,

1H), 2.88 (dd, J = 13.6, 10.4 Hz, 1H), 1.33 (s, 8H) 13C NMR (101 MHz, DMSO-d6) δ

173.57,155.39, 139.55, 138.85, 129.76, 128.94, 127.42, 127.12, 126.96, 126.51, 126.30,

78.16, 48.56, 36.12, 28.12; HRMS-ESI (m/z) [M+Na]+ calcd for C26H27NO4: 440.1838, found,

440.1838

132

7.2.21 Synthesis of amide 15 from L-1b

(R)-1-((S)-2-amino-3-(2'-fluoro-[1,1'-biphenyl]-4-yl)propanoyl)pyrrolidine-2-carbonitrile

(15)

L-1b (150 mg, 0.41 mmol, 1 eq), NHS (97 mg, 0.82 mmol, 2

eq), EDCI (160 mg, 0.82 mmol, 2 eq) and Et3N (290 µL, 2.05

mmol, 5 eq) were added to DMF (10 mL) and stirred at r.t. for

15 min. 2-(S)-cyanopyrrolidine (192 mg, 0.50 mmol, 1.2 eq)

was added and the resulting solution was stirred at r. t. for 20 h. Sat. aqueous NaHCO3 (10

mL) was added and the mixture was extracted with EtOAc (3 × 25 mL). The organic extract

was then washed with brine (30 mL), dried over anhydrous MgSO4 and the solvent was

removed in vacuo to give a crude oil. This was purified by chromatography using PE/EtOAc

1:1 to afford the N-protected amide as a clear oil. The intermediate N-protected amide was

added to DCM/TFA (3 mL, 2:1) and left to stir at r. t. for 1 h, followed by removal of solvent in

vacuo to give a crude residue. Et2O (2 mL) was added and product 15 precipitated out as the

trifluoroacetic acid salt to give white crystals (65 mg, 50%) 1H NMR (400 MHz, MeOH-d4) δ

7.66-7.60 (m, 3H), 7.56-7.51 (m, 2H), 7.36-7.22 (m, 3H), 4.85 (dd, J = 8 Hz, 1H), 4.45 (dd, J

= 12 Hz, 1H), 3.36-3.24 (m, 3H), 2.68 (m, 1H), 2.22 (m, 2H), 1.97 (m, 1H), 1.82 (m, 1H) 13C

NMR (101 MHz, MeOH-d4) δ 168.79, 137.10, 134.47, 131.70, 131.11, 130.86, 130.83, 125.79,

118.81, 117.17, 116.94, 54.01, 47.88, 47.64, 38.83, 30.82, 26.00 HRMS (ESI, m/z) calcd.

mass 338.1669 [M+H]+, found 338.1684 [M+H]+.

7.2.22 References

1. N. Nimura; H. Ogura; T. Kinoshita, J. Chromatogr. 1980, 202, 375-379.

2. W. C. Shieh; J. A. Carlson; J. Org. Chem. 1992, 57, 379-381.

3. K. Vedha-Peters; M. Gunawardana; J. D. Rozzell; S. J. Novick; J. Am. Chem. Soc.

2006, 128, 10923-10929.

133


7.3.1 Chemicals

Analytical grade reagents and solvents were obtained from Sigma-Aldrich, AlfaAesar or

Fisher Scientific and used without further purification, unless stated otherwise.

7.3.2 Non-chiral HPLC analysis

The conversions of the Knoevenagel-Doebner condensations were estimated by 1H NMR,

comparing the peak areas for the signal at δ = 6.0-6.5 ppm (d, =CHCOOH) for 2a-p and the

signal at δ = 9.0-9.5 ppm (s, CHO) for 1a-p. The conversions of the PAL biotransformations

were measured by HPLC on a non-chiral reverse-phase Zorbax C-18 Extend column (50 mm

× 4.6 mm × 3.5 µm, Agilent), flow rate 1.0 mL min–1, temperature 40°C, detection wavelength

210 nm. Method 1 (mobile phase: aq. NH4OH 0.1 M pH 10 / MeOH, 9:1); Method 2 (mobile

phase: aq. NH4OH 0.1 M pH 10 / MeOH, 8:2); Method 3; (mobile phase: aq. NH4OH 0.1 M

pH 10 / MeOH, 7:3); Method 4 (mobile phase: aq. NH4OH 0.1 M pH 10 / MeOH, 6:4).

retention time tR

[min]

2 3 Method

a 2.9 1.3 1

b 4.5 1.7 1

c 4.1 1.6 1

d 3.7 1.6 1

e 4.9 2.0 2

f 3.8 1.6 3

g 6.9 2.9 3

h 6.1 2.5 3

i 8.8 3.6 3

j 5.0 2.1 1

k 7.4 3.1 4

l 8.1 3.5 4

m 10.5 4.4 4

n 7.0 2.9 3

134

o 8.8 3.3 3

p 9.3 3.9 3

7.3.3 Chiral HPLC analysis

The enantiomeric excess values of L-3a-p were measured on a reverse-phase Crownpak

CR(+) column (150 mm × 4 mm × 3.5 µm, Daicel). Flow rate 1.0 mL min–1, temperature 40°C,

detection wavelength 210 nm. Method 1 (mobile phase: aq. HClO4 1.14% w/v / MeOH 96:4);

Method 2 (mobile phase: aq. HClO4 1.14% w/v / MeOH 86:14). Authentic standards for D-

amino acids were obtained commercially or produced using a chemo-enzymatic

deracemization method (see section 7.4.6).

retention time tR

[min]

D-3 L-3 Method

a 5.1 7.9 1

b 6.9 9.2 1

c 7.6 10.9 1

d 8.0 9.9 1

e 10.2 13.0 2

f 17.1 20.9 2

g 13.9 17.6 2

h 13.6 16.4 2

i 18.2 23.7 2

j 10.9 13.2 1

k 33.4 42.1 2

l 46.9 63.8 2

m 48.2 61.6 2

n 12.6 14.7 2

o 17.0 22.8 2

p 17.2 21.5 2

135

7.3.4 Knoevenegal-Doebner condensation for the synthesis of cinnamic acids

The suitable aldehyde (10 mmol), malonic acid (30 mmol, 3.12 g) and piperidine (0.5 mL) were

dissolved in pyridine (20 mL) and the mixture was heated under reflux for 4 h. The solution

was cooled to room temperature and poured in ice-cold aqueous HCl (100 mL, 3 M). The white

solid precipitate was filtered, washed with water (3 × 50 mL), aqueous NaHCO3 (20 mL, 5%

w/v), then again with water (2 × 50 mL) and dried in an oven (60°C). If required, the crude

solid was recrystallysed from EtOH/H2O.

7.3.5 Knoevenagel-Doebner optimization in microwave reactor

Benzaldehyde 1a (0.05 mol) and malonic acid (0.055-0.150 mol) were dissolved in DMSO (50

mL) to make a stock solution (final conc. 1 M 1a). Aliquots (1 mL) were taken, the suitable

base was added and the mixture was heated in a microwave reactor (30-60 min, 60-120°C).

To check the conversion, a sample of the solution (100 μL) was diluted in CDCl3 (600 μL) and

used directly for 1H NMR analysis.

7.3.6 PAL amination screening

Pure 2a or a suitable volume of the Knoevenagel-Doebner condensation mixture in DMSO

was added to the required aqueous NH4OH solution or ammonium salt solution. Wet E.coli

BL21(DE3) cells overproducing the suitable PAL (40 mg mL–1) were added and the

suspension was incubated at 37°C with shaking (180 rpm) for the required time.

Biotransformation samples (10-500 μL depending on substrate concentration) were mixed

with MeOH (500 μL), thoroughly shaken and centrifuged (13000 rpm, 1 min), then the

supernatant was transferred to a 0.45 μm filter vial and used directly for HPLC analysis.

7.3.7 Preparative scale telescopic condensation-hydroamination procedure.

The suitable aldehyde 1j-l, 1n or 1o (0.5 mmol), malonic acid (1.0 mmol) and piperidine (0.01

mmol) were dissolved in DMSO (500 µL) and the mixture was heated in a microwave reactor

(30 min, 60°C). After cooling, ammonia solution (9.0 mL, 13% w/v, approx. 7 M, adjusted to

pH 10.0 by slow addition of dry ice chunks) was added, followed by E. coli BL21(DE3) cells

overproducing the required PAL (400 mg wet cell paste). The suspension was stirred at 37°C

for several hours, monitoring the conversion by HPLC. For product isolation, the reaction

mixture was acidified to pH < 2.0 using aqueous H2SO4 (20% w/v) and centrifuged (8000 rpm,

5 min) to remove cells and precipitated unconverted substrate. Dowex® 50WX8 hydrogen form

resin (5.0 g), packed in a disposable plastic column, was washed with deionised water (20

mL) and aqueous H2SO4 (20 mL, 5% w/v). The supernatant from the biotransformation was

loaded onto the resin (1 mL min–1). The resin bed was washed with deionised water until the

flow-through tested neutral, then the product was eluted with aqueous NH4OH (20 mL, 5%

136

w/v). Fractions containing the product were pooled and dried overnight in a centrifugal

evaporator, to afford the corresponding L-phenylalanine L-3j-l, L-3n or L-3o as a white solid.

7.3.8 Characterization data for compounds 2j-l, 2o, 2n and 3j-l, 3o and 3n.

E)-3-(3,4-difluorophenyl)acrylic acid (2j)

White solid (1.70 g, 92%); 1H NMR (DMSO-d6, 400 MHz), δ 7.85-7.91

(m, 1H), 7.55-7.57 (m, 1H), 7.56 (d, J=16.0 Hz, 1H), 7.43-7.50 (m, 1H),

6.57 (d, J=16.0 Hz, 1H). 13C NMR (DMSO-d6, 101 MHz), δ 167.30,

150.35 (dd, 1JCF=248 Hz, 2JCF=13 Hz), 149.64 (dd, 1JCF=244 Hz, 2JCF=13 Hz), 141.64, 132.03

(dd, 3JCF=6 Hz, 4JCF=4 Hz), 125.82 (dd, 3JCF=7 Hz, 4JCF=3 Hz), 120.68, 117.88 (d, 2JCF=17 Hz),

116.65 (d, 2JCF=18 Hz).

(E)-3-(2,4-dichlorophenyl)acrylic acid (2k)

White solid (2.04 g, 94%); 1H NMR (DMSO-d6, 400 MHz), δ 7.92-7.97

(m, 1H), 7.79 (d, J=15.6 Hz, 1H), 7.69-7.72 (m, 1H), 7.44-7.49 (m,

1H), 6.63 (d, J=15.6 Hz, 1H). 13C NMR (DMSO-d6, 101 MHz), δ

166.96, 137.44, 135.24, 134.32, 130.93, 129.48, 129.36, 127.94, 122.99.

(E)-3-(3,4-dichlorophenyl)acrylic acid (2l)

White solid (1.99 g, 92%; 1H NMR (DMSO-d6, 400 MHz), δ 7.92-7.97

(m, 1H), 7.79 (d, J=16.3 Hz, 1H), 7.69-7.73 (m, 1H), 7.42-7.49 (m,


167.21, 141.20, 135.12, 132.35, 131.70, 130.91, 129.95, 128.10,

121.57.

(E)-3-(2-chloro-4-fluorophenyl)acrylic acid (2n)

White solid (1.87 g, 93%; 1H NMR (DMSO-d6, 400 MHz), δ 7.98-8.03

(m, 1H), 7.80 (d, J=16.0 Hz, 1H), 7.51-7.58 (m, 1H), 7.24-7.32 (m, 1H),

6.59 (d, J=16.0 Hz, 1H). 13C NMR (DMSO-d6, 101 MHz), δ:167.08,

162.64 (d, 1JCF=253 Hz), 137.56, 134.50 (d, 3JCF=11 Hz), 129.99 (d,

3JCF=9 Hz), 128.62 (d, 4JCF=3 Hz), 122.18, 117.14 (d, 2JCF=25 Hz), 115.29 (d, 2JCF=21 Hz).

(E)-3-(3-chloro-4-fluorophenyl)acrylic acid (2o)

White solid (1.94 g, 96%). 1H NMR (DMSO-d6, 400 MHz), δ 7.97-8.02

(m, 1H), 7.70-7.75 (m, 1H), 7.56 (d, J=16.1 Hz, 1H), 7.42-7.48 (m,


167.29, 157.94 (d, 1JCF=249 Hz), 141.36, 132.37 (d, 3JCF=4 Hz), 130.22, 129.01 (d, 3JCF=8 Hz),

120.69 (d, 4JCF=2 Hz), 120.16 (d, 2JCF=18 Hz), 117.30 (d, 2JCF=21 Hz).

137

(S)-2-amino-3-(3,4-difluorophenyl)propanoic acid (L-3j)

White crystals (84 mg, 84%); 1H NMR (D2O+NaOH, 400 MHz), δ 7.03-

7.18 (m, 2H), 6.90-6.99 (m, 1H), 3.36-3.44 (m, 1H), 2.81-2.90 (dd,

J=13.6,6.0 Hz, 1H), 2.70-2.79 (dd, J=13.6,7.2 Hz, 1H). 13C NMR

(D2O+NaOH, 101 MHz), δ 182.06, 150.94 (dd, 1JCF=243 Hz, 2JCF=12 Hz), 150.09 (dd, 1JCF=242

Hz, 2JCF=13 Hz), 135.33 (dd, 3JCF=5 Hz, 4JCF=3 Hz), 125.51 (dd, 3JCF=6 Hz, 4JCF=3 Hz), 117.83

(d, 2JCF=17 Hz), 116.97 (d, 2JCF=17 Hz), 57.33, 39.94; HRMS-ESI (m/z) [M+H]+ calcd for

C9H9F2NO2: 202.0680, found, 202.0689

(S)-2-amino-3-(2,4-dichlorophenyl)propanoic acid (L-3k)

White crystals(88 mg, 75%);1H NMR (D2O+NaOH, 400 MHz), δ 7.45

(d, J=2.0 Hz, 1H), 7.16-7.26 (m, 2H), 3.43-3.52 (m, 1H), 2.95-3.06

(dd, J1=13.4,6.4 Hz, 1H), 2.81-2.90 (dd, J=13.4,7.6 Hz, 1H). 13C NMR

(D2O+NaOH, 101 MHz), δ 182.12, 134.83, 134.53, 132.43, 132.31,

128.90, 127.06, 56.40, 38.13; HRMS-ESI (m/z) [M+H]+ calcd for C9H935Cl2NO2: 234.0089,

found, 234.0093

(S)-2-amino-3-(3,4-dichlorophenyl)propanoic acid (L-3l)

White crystals (97 mg, 83%); 1H NMR (D2O+NaOH, 400 MHz), δ

7.21-7.26 (m, 1H), 7.17 (s, 1H), 6.90-6.98 (m, 1H), 3.21-3.27 (m, 1H),

2.68-2.75 (m, 1H), 2.51-2.62 (m, 1H). 13C NMR (D2O+NaOH, 101

MHz), : 181.88, 138.85, 131.31, 131.00, 130.20, 129.61, 129.11,

57.23, 39.94; HRMS-ESI (m/z) [M+H]+ calcd for C9H935Cl2NO2: 234.0089, found, 234.0083

(S)-2-amino-3-(2-chloro-4-fluorophenyl)propanoic acid (L-3n)

White solid (79 mg, 73%); 1H NMR (D2O+NaOH, 400 MHz) δ 7.18-

7.29 (m, 2H), 6.96-7.05 (m, 1H), 3.42-3.55 (m, 1H), 2.96-3.09 (dd,

J=13.6,6.4 Hz, 1H), 2.84-2.92 (dd, J=13.6,8.0 Hz, 1H). 13C NMR

(D2O+NaOH, 101 MHz), δ 182.26, 160.00 (d, 1JCF=245 Hz), 134.26 (d,

3JCF=11 Hz), 132.46 (d, 3JCF=8 Hz), 132.04 (d, 4JCF= 3 Hz), 116.42 (d, 2JCF=25 Hz), 113.78 (d,

2JCF=21 Hz), 56.51, 37.91; HRMS-ESI (m/z) [M+H]+ calcd for C9H935ClFNO2: 218.0384, found,

218.0397

138

(S)-2-amino-3-(3-chloro-4-fluorophenyl)propanoic acid (L-3o)

White crystals (77 mg, 71%); 1H NMR (D2O+NaOH, 400 MHz), δ

7.23-7.29 (m, 1H), 7.06-7.12 (m, 2H), 3.35-3.42 (m, 1H), 2.81-2.88

(dd, J=13.6,5.6 Hz, 1H), 2.68-2.79 (dd, J=13.6,7.2 Hz, 1H). 13C NMR

(D2O+NaOH, 101 MHz), δ 182.00, 157.79 (d, 1JCF= 245 Hz), 135.45

(d, 4JCF=3 Hz), 131.01, 129.30 (3JCF=7 Hz), 119.70 (d, 2JCF=18 Hz), 116.32 (d, 2JCF=21 Hz),

57.34, 39.77; HRMS-ESI (m/z) [M+H]+ calcd for C9H935ClFNO2: 218.0384, found, 218.0385

139


7.4.1 Chemicals and enzymes

Analytical grade reagents, solvents and aldehydes 1a-l were obtained from Sigma-Aldrich,

Alfa-Aesar or Fluorochem and have been used without further purification. D-amino acid

oxidase (DAAO) from porcine kidney and L-amino acid oxidase (LAAO) from Crotalus

adamanteus were purchased from Sigma-Aldrich.

7.4.2 NMR time-course experiment for compounds 3g-l

Conversion for compounds 3h-l and 5a-e were obtained by 1H NMR, monitoring the

appearance of the ABX system corresponding to the aliphatic portion of the amino acid and

the disappearance of the acrylic acid doublets. A sample of the crude biotransformation

mixture (600 µL) was added to D2O (200 µL), thoroughly mixed and centrifuged to remove

whole cell components and suspended solids. The supernatant was then analysed by NMR

with suppression of the residual water signal. Representative spectra are shown in the

following.

3h

140

3i

3j

141

3k

3l

142

3l

5a

143

5b

5c

144

5d

5e

145

7.4.3 Telescopic synthesis of L-5a-e

Prod. Time (h) Conv. (%) ee (%) Isol. yield (%)

5a 22 >99 >99 40

5b 4 88 >99 35

5c 4 95 >99 54

5d 4 80 >99 44

5e 22 93 >99 37

146

7.4.4 Unsuccessful biotransformation/synthesis

(E)-3-(6-methoxypyridin-3-yl) acrylic acid (2m) was synthesized and tested with AvPAL. No

conversion was observed after 30 h incubation.

Also, the synthesis and isolation of (E)-3-(2-bromopyridin-3-yl)acrylic acid (2n) was

unsuccessful, with no trace of the acrylic acid doublets present whilst following the reaction by

NMR. The reaction mixture turned black (as opposed to being colorless to pale yellow) yielding

a black/grey solid.

147

(E)-3-(isoquinolin-4-yl)acrylic acid (4f) was synthesized successfully and tested with AvPAL

yielding no conversion after 30 h incubation.

148


The conversion of compounds 2a-f to 3a-f was measured on a non-chiral reverse-phase

Zorbax C-18 Extend column (50 mm x 4.5 µm, Agilent). Mobile phase aq. NH4OH 0.1 M pH

10 / MeOH 90:10 (12 min), flow rate 1 mL min-1, temperature 40°C, detection wavelength 210

nm (retention time are reported below).

Retention time (tR)

[min]

3 2

a 2.30 5.00

b 2.37 5.35

c 2.65 7.01

d 3.17 8.53

e 1.80 3.40

f 1.90 3.16


The optical purity of amino acid products 3a-j and 5a-e was measured on a reverse-phase

Crownpak CR(+) column (150 mm x 4 mm x 3.5 µm, Daicel). Mobile phase aq. HClO4 1.14%

w/v / MeOH 96:4, flow rate 1.0 mL min–1, temperature 40°C, detection wavelength 210 nm.

The optical purity of amino acid products 3k-l was measured on a reverse-phase Chirobiotic

T column (250 mm x 46 mm x 5 µM, Supelco). Mobile phase H2O/MeOH 60:40, flow rate 0.8

mL min-1, temperature 40°C, detection wavelength 210 nm (retention times are reported

below).

149

Retention time (tR)

[min]

D-3 L-3

a 5.45 7.20

b 3.58 4.28

c 7.45 9.76

d 7.57 9.06

e 4.01 5.56

f 1.93 2.14

g 3.23 6.79

h 1.84 2.06

i 3.35 4.55

j 6.94 9.21

k –a 8.65

l 6.16 5.59

aD-3k could not be produced with the method described below.

Retention time (tR)

[min]

D-5 L-5

a 2.17 2.54

b 19.80 26.25

c 16.15 22.80

d 14.00 18.50

e –a 2.10

aD-5e could not be produced with the method described below.

Compounds 3a, g, k, l gave >99% L-enantiomer in all cases. In order to verify the separation

capability of the column employed, a deracemisation was performed to produce a small but

measurable amount of the D-enantiomer. This was done with a chemo-enzymatic cascade

identical to the one described in the paper for the chiral polishing of the L-enantiomer (as

150

shown in the scheme below), but using in this case L-amino acid oxidase (LAAO) instead of

DAAO.

7.4.7 Knoevenagel-Doebner synthesis of acrylic acids 2a-l

Aldehyde 1a-l (1 mmol) and malonic acid (3 mmol) were added to DMSO (1 mL). The mixture

(final conc. 1 M 1a-l) was stirred until complete dissolution of the starting materials. Piperidine

(2 mol%) was added, followed by heating at 100°C for 16 h. The mixture was cooled to room

temperature and quenched with distilled water (5 mL) resulting in the precipitation of the acrylic

acid. The solid was centrifuged and the water removed by decanting. The acrylic acid was

washed 3 times by re-suspending in distilled water (5 mL) with decanting the liquid each time.

Methanol (10 mL) was added to transfer the solid to a round-bottomed flask, followed by

concentrating the sample in vacuo to give the title compound as a white solid.

7.4.8 Telescopic synthesis of amino acid 3a-l

The crude condensation mixture (500 µL) was added to a saturated solution of ammonium

carbamate (9.5 mL) to give a final concentration of 50 mM 2a-l. Lyophilized cells producing

AvPAL (250 mg, 25 mg mL–1) were added and the mixture was shaken until complete

resuspension of the biocatalyst. The suspension was incubated at 37°C, 180 rpm for 2-30 h.

Once the biotransformation reached full conversion (by HPLC and/or 1H NMR), the mixture

was centrifuged (4000 rpm, 10 min, 4°C) and the supernatant collected for purification. To

follow the reaction by HPLC analysis, a sample of the biotransformation mixture (50 µL) was

added to MeOH (450 µL), thoroughly mixed and centrifuged (13000 rpm, 1 min, 4°C). The

supernatant was transferred to a filter vial and used directly for analysis.

7.4.9 Deracemization cascade of compounds 3a-l using DAAO

Amino acid 3a-l (10 mM) and borane-ammonia complex (50 mM) were dissolved in phosphate

buffer (2 mL, 100 mM, pH 8.0). DAAO (1 mg mL-1) was added to start the reaction and the

mixture was incubated at 37°C for 1-3 h. To monitor the reaction, samples (200 uL) were taken

151

at regular intervals, diluted with methanol (200 uL), centrifuged (13000 rpm, 1 min, 4°C) and

the supernatant analysed by HPLC on a chiral stationary phase.3

The same method (using LAAO instead of DAAO) was used to produce HPLC standards of

the D-enantiomers of 3a, g, k and l, that were obtained enantiomerically pure from the

biotransformations.

7.4.10 Characterization data for compounds 2a-l, 3a-l, 4a-f and 5a-e

(E)-3-(6-bromopyridin-3-yl) acrylic acid (2a)

White solid (91 mg, 40%); 1H NMR (400 MHz, D2O+NaOH) δ 8.22 (s,

1H), 7.72 (d, J = 8 Hz, 1H), 7.47 (d, J = 8 Hz, 1H), 7.14 (d, J = 16 Hz,

1H), 6.45 (d, J = 16 Hz, 1H); 13C NMR (100 MHz, D2O+NaOH) δ

174.58, 148.72, 140.85, 137.38, 135.24, 130.86, 128.48, 127.34;

HRMS-ESI (m/z) [M+H]+ Calcd for C8H679BrNO2: 227.9655; found, 227.9652.

(E)-3-(5-bromopyridin-3-yl) acrylic acid (2b)


2H), 8.00 (s, 1H), 7.10 (d, J = 16 Hz, 1H), 6.43 (d, J = 16 Hz, 1H); 13C

NMR (100 MHz, D2O+NaOH) δ 174.32, 149.56, 146.28, 137.22,

134.87, 133.00, 128.04, 120.68; HRMS-ESI (m/z) [M+H]+ Calcd for

C8H679BrNO2: 227.9655; found, 227.9652.

(E)-3-(6-bromopyridin-2-yl) acrylic acid (2c)

White solid (52 mg, 23%); 1H NMR (400 MHz, D2O+NaOH) δ 7.58 (t, J =

8 Hz, 1H) 7.40-7.45 (m, 2H), 7.03 (d, J = 16 Hz, 1H), 6.56 (d, J = 16 Hz,

1H); 13C NMR (100 MHz, D2O+NaOH) δ 174.32, 154.84, 141.10, 104.19,


C8H679BrNO2: 227.9655; found, 227.9649.

(E)-3-(5-bromopyridin-2-yl) acrylic acid (2d)

Pale yellow solid (59 mg, 26%); 1H NMR (400 MHz, D2O+NaOH) δ

8.46 (s, 1H), 7.89 (d, J = 8 Hz, 1H), 7.42 (d, J = 8 Hz, 2H), 7.16 (d, J

= 16 Hz, 1H), 6.62 (d, J = 16 Hz, 1H); 13C NMR (100 MHz,

D2O+NaOH) δ 174.62, 151.94, 150.01, 140.48, 137.63, 129.22,

124.53, 120.39; HRMS-ESI (m/z) [M+H]+ Calcd for C8H679BrNO2: 227.9655; found, 227.9652.

152

(E)-3-(2-bromopyridin-4-yl) acrylic acid (2e)

White solid (224 mg, 99%); 1H NMR (400 MHz, D2O+NaOH) δ 8.11 (d, J

= 8 Hz, 1H), 7.53 (s, 1H), 7.33 (d, J = 4 Hz, 1H), 7.04 (s, J = 16 Hz, 1H),

6.55 (d, J = 16 Hz, 1H); 13C NMR (100 MHz, D2O+NaOH) δ 174.00,

149.65, 146.60, 141.28, 135.64, 130.70, 126.17, 121.37; HRMS-ESI

(m/z) [M+H]+ Calcd for C8H679BrNO2: 227.9655; found, 227.9650.

(E)-3-(3-bromopyridin-4-yl) acrylic acid (2f)



1H), 6.51 (d, J = 16 Hz, 1H); 13C NMR (100 MHz, D2O+NaOH) δ 173.85,

151.31, 147.28, 143.38, 135.48, 131.38, 121.85, 121.78; HRMS-ESI

(m/z) [M+H]+ Calcd for C8H679BrNO2: 227.9655; found, 227.9651.

(E)-3-(6-chloropyridin-3-yl) acrylic acid (2g)



1H), 6.49 (d, J = 16 Hz, 1H); 13C NMR (100 MHz, D2O+NaOH) δ

174.73, 150.37, 148.43, 137.90, 135.35, 130.68, 127.19, 124.72; HRMS-ESI (m/z) [M+H]+

Calcd for C8H635ClNO2: 184.0160; found, 184.0158.

(E)-3-(3-chloropyridin-4-yl) acrylic acid (2h)



1H), 6.55 (d, J = 16 Hz, 1H); 13C NMR (100 MHz, D2O+NaOH) δ 173.93,

148.84, 146.77, 141.61, 132.89, 131.33, 131.04, 121.43; HRMS-ESI

(m/z) [M+H]+ Calcd for C8H635ClNO2: 184.0160; found, 184.0158.

(E)-3-(2-chloropyridin-4-yl) acrylic acid (2i)

White solid (79 mg, 43%); 1H NMR (400 MHz, D2O+NaOH) δ 8.19 (s, J

= 8 Hz, 1H), 7.47 (s, 1H), 7.37 (d, J = 8 Hz, 1H), 7.12 (d, J = 16 Hz, 1H),

6.60 (d, J = 16 Hz, 1H); 13C NMR (100 MHz, D2O+NaOH) δ 174.10,

150.80, 149.21, 147.04, 135.80, 130.61, 122.49, 121.05; HRMS-ESI

(m/z) [M+H]+ Calcd for C8H635ClNO2: 184.0160; found, 184.0157.

153

(E)-3-(2,5-dichloropyridin-4-yl) acrylic acid (2j)

White crystals (69 mg, 32%); 1H NMR (400 MHz, D2O+NaOH) δ 8.26 (s,

1H), 7.64 (s, 1H), 7.41 (d, J = 16 Hz, 1H), 6.60 (d, J = 16 Hz, 1H); 13C

NMR (100 MHz, D2O+NaOH) δ 173.59, 148.84, 148.78, 144.42, 132.56,

131.92, 130.05, 121.71; HRMS-ESI (m/z) [M+H]+ Calcd for

C8H535Cl2NO2: 217.9770; found, 217.9793

(E)-3-(2-methoxypyridin-4-yl) acrylic acid (2k)



1H), 6.45 (d, J = 16 Hz, 1H), 3.79 (s, 3H); 13C NMR (100 MHz,

D2O+NaOH) δ 174.68, 152.86, 141.39, 133.24, 132.18, 132.86, 129.53,

121.51, 56.19; HRMS-ESI (m/z) [M+H]+ Calcd for C9H9NO3: 180.0655;

found, 180.0663.

(E)-3-(3-methoxypyridin-4-yl) acrylic acid (2l)


= 4 Hz, 1H), 7.05 (d, J = 16 Hz, 1H), 6.96 (d, J = 4 Hz, 1H), 6.98 (s, 1H),

6.48 (d, J = 16 Hz, 1H), 3.77 (s, 3H); 13C NMR (100 MHz, D2O+NaOH) δ

173.80, 163.47, 146.10, 145.76, 136.45, 128.58, 114.74, 107.83, 53.42;

HRMS-ESI (m/z) [M+H]+ Calcd for C9H9NO3: 180.0655; found, 180.0663.

(E)-3-(6-methoxypyridin-3-yl) acrylic acid (2m)

White solid (86 mg, 48%); 1H NMR (400 MHz, D2O+NaOH) δ 8.03

(s, 1H), 7.85 (d, J = 8 Hz, 1H), 7.19 (d, J = 16 Hz, 1H), 6.76 (d, J =

12 Hz, 1H), 6.32 (d, J = 16 Hz, 1H), 3.83 (s, 3H); 13C NMR (100 MHz,

D2O+NaOH) δ 175.30, 164.11, 146.52, 137.75, 136.66, 125.10,

123.73, 110.60: HRMS-ESI (m/z) [M+H]+ Calcd for C9H9NO3: 180.0655; found, 180.0654.

(S)-2-amino-3-(6-bromopyridin-3-yl) propanoic acid (3a)


1H), 7.51-7.58 (m, 2H), 3.48 (t, J = 8 Hz, 1H), 2.88-2.93 (dd, J = 16,

8 Hz, 1H), 2.80-2.85 (dd, J = 12, 8 Hz, 1H); 13C NMR (100 MHz,

D2O+NaOH) δ 181.18, 149.98, 140.79, 138.82, 133.75, 128.18,

56.90, 37.02; HRMS-ESI (m/z) [M+H]+ Calcd for C8H979BrN2O2: 244.9920; found, 244.9785.

154

(S)-2-amino-3-(5-bromopyridin-3-yl) propanoic acid (3b)


1H), 8.18 (s, 1H), 7.78 (s, 1H), 3.35-3.38 (dd, J = 8, 4 Hz, 1H), 2.80-

2.85 (dd, J = 16, 8Hz, 1H), 2.69-2.75 (dd, J = 16, 8Hz, 1H); 13C NMR

(100 MHz, D2O+NaOH) δ 181.43, 147.90, 147.65, 140.50, 136.50,

120.30, 57.07, 37.56; HRMS-ESI (m/z) [M+H]+ Calcd for C8H979BrN2O2: 244.9920; found,

244.9923.

(S)-2-amino-3-(6-bromopyridin-2-yl) propanoic acid (3c)

White solid (64 mg, 53%); 1H NMR (400 MHz, D2O+NaOH) δ 7.51 (t, J =

8 Hz, 1H), 7.35 (d, J = 8 Hz, 1H), 7.16 (d, J = 8 Hz), 3.42-3.46 (dd, J = 8,

8 Hz, 1H), 2.93-2.98 (dd, J = 16, 8 Hz, 1H), 2.69-2.75 (dd, J = 16, 8 Hz,



C8H979BrN2O2: 244.9920; found, 244.9901.

(S)-2-amino-3-(5-bromopyridin-2-yl) propanoic acid (3d)


1H), 7.90 (d, J = 8 Hz, 1H), 7.21 (d, J = 8 Hz, 1H), 3.53-3.57 (dd, J =

8, 8 Hz, 1H), 3.03-3.08 (dd, J = 16, 8 Hz, 1H), 2.83-2.88 (dd, J = 12,

8 Hz, 1H); 13C NMR (100 MHz, D2O+NaOH) δ 181.73, 156.76,

149.33, 140.31, 125.81, 118.40, 56.74, 42.05; HRMS-ESI (m/z) [M+H]+ Calcd for

C8H979BrN2O2: 244.9920; found, 244.9871.

(S)-2-amino-3-(2-bromopyridin-4-yl) propanoic acid (3e)


= 8 Hz, 1H), 7.40 (s, 1H), 7.17 (d, J = 8 Hz, 1H), 3.39-3.42 (dd, J = 8, 4

Hz, 1H), 2.69-2.75 (dd, J = 16, 8 Hz, 1H); 13C NMR (100 MHz,

D2O+NaOH) δ 181. 27, 152.20, 149.26, 140.80, 129.06, 124.42, 56.73,

39.94; HRMS-ESI (m/z) [M+H]+ Calcd for C8H979BrN2O2: 244.9920; found, 246.9985.

(S)-2-amino-3-(3-bromopyridin-4-yl) propanoic acid (3f)


1H), 8.30 (d, J = 4 Hz, 1H), 7.29 (d, J = 4 Hz, 1H), 3.55 (t, J = 8 Hz, 1H),

3.04-3.09 (dd, J = 16, 8 Hz, 1H), 2.90-2.95 (dd, J = 12, 8 Hz, 1H); 13C

NMR (100 MHz, D2O+NaOH) δ 181.46, 150.74, 148.41, 147.14, 126.64,

123.20, 55.97, 40.39: HRMS-ESI (m/z) [M+H]+ Calcd for C8H979BrN2O2: 244.9920; found,

244.9871.

155

(S)-2-amino-3-(6-chloropyridin-3-yl) propanoic acid (3g)

White solid (45mg, 45%); 1H NMR (400 MHz, D2O+NaOH) δ 8.15 (s,

1H), 7.67 (d, J = 8 Hz, 1H), 7.40 (d, J = 8 Hz, 1H), 3.46 (t, J = 8 Hz,

1H), 2.89-2.94 (dd, J = 16, 8 Hz, 1H), 2.81-2.87 (dd, J = 16, 8 Hz,

1H); 13C NMR (100 MHz, D2O+NaOH) δ 181.12, 149.37, 148.52,

141.04, 133.31, 124.38, 56.95, 36.90; HRMS-ESI (m/z) [M+H]+ Calcd for C8H935ClN2O2:

201.0425; found, 201.0422.

(S)-2-amino-3-(3-chloropyridin-4-yl) propanoic acid (3h)


1H), 8.19 (d, J = 4 Hz, 1H), 7.21 (d, J = 4 Hz, 1H), 3.46 (t, J = 8 Hz, 1H),

2.96-3.01 (dd, J = 12, 8 Hz, 1H), 2.82-2.88 (dd, J = 16, 8 Hz, 1H); 13C

NMR (100 MHz, D2O+NaOH) δ 181.46, 148.13, 146.60, 132.47, 126.42,

55.87, 37.93; HRMS-ESI (m/z) [M+H]+ Calcd for C8H935ClN2O2: 201.0425; found, 201.0421.

(S)-2-amino-3-(2-chloropyridin-4-yl) propanoic acid (3i)


= 4 Hz, 1H), 7.23 (s, 1H), 7.12 (d, J = 4 Hz, 1H), 3.42 (t, J = 8 Hz, 1H),

2.84-2.89 (dd, J = 16, 8 Hz, 1H), 2.71-2.77 (dd, J = 16, 8 Hz, 1H); 13C

NMR (100 MHz, D2O+NaOH) δ 181.07, 152.36, 150.23, 148.73, 125.24,

124.04, 56.66, 39.86; HRMS-ESI (m/z) [M+H]+ Calcd for C8H935ClN2O2: 201.0425; found,

201.0421.

(S)-2-amino-3-(2,5-dichloropyridin-4-yl) propanoic acid (3j)


0.5H), 7.29 (s, 1H), 3.44 (t, J = 8 Hz, 1H), 2.94-2.96 (dd, J = 16, 8 Hz,

1H), 2.78-2.84 (dd, J = 16, 8 Hz, 1H); 13C NMR (100 MHz, D2O+NaOH)

δ 181.17, 149.66, 148.37, 148.07, 131.53, 126.41, 55.71, 37.99; HRMS-

ESI (m/z) [M+H]+ Calcd for C8H835Cl2N2O2: 235.0036; found, 235.0063.

(S)-2-amino-3-(2-methoxypyridin-4-yl) propanoic acid (3k)


1H), 7.93 (d, J = 8 Hz, 1H), 7.07 (d, J = 4 Hz, 1H), 3.78 (s, 3H), 3.43 (t, J

= 8 Hz, 1H), 2.84-2.89 (dd, J = 12, 8 Hz, 1H), 2.69-2.74 (dd, J = 12, 8

Hz, 1H); 13C NMR (100 MHz, D2O+NaOH) δ 181.43, 154.44, 141.44,

136.69, 132.23, 125.89, 55.97, 55.76, 34.58; HRMS-ESI (m/z) [M+H]+

Calcd for C9H12N2O3: 197.0921; found, 197.0920.

156

(S)-2-amino-3-(3-methoxypyridin-4-yl) propanoic acid (3l)


= 4 Hz, 1H), 6.87 (d, J = 8 Hz, 1H), 6.68 (s, 1H), 3.83 (s, 3H), 3.59 (t, J =

8 Hz, 1H), 2.95-2.99 (dd, J = 12, 4 Hz, 1H), 2.81-2.86 (dd, J = 12, 8 Hz,


117.65, 110.48, 56.26, 53.99, 39.12; HRMS-ESI (m/z) [M+H]+ Calcd for C9H12N2O3: 197.0921;

found, 197.0919.

(E)-3-(3-methylisoxazol-5-yl)acrylic acid (4a)

White solid (460mg, 66%) 1H NMR (400 MHz, DMSO-d6) δ 7.39 (d, J =

16 Hz, 1H), 6.74 (s, 1H), 6.66 (d, J = 16 Hz, 1H), 2.41 (s, 3H); 13C NMR

(100 MHz, D2O+NaOH) δ 170.33, 166.55, 159.79, 130.77, 126.94, 99.88,

11.75; HRMS-ESI (m/z) [M+H]+ Calcd for C7H7NO3: 154.0504; found,

154.0507.

(E)-3-(5-bromothiophen-2-yl)acrylic acid (4b)

White solid (265 mg, 57%) 1H NMR (400 MHz, D2O+NaOH) δ 7.33

(d, J = 16 Hz, 1H), 7.05 (d, J = 4 Hz, 1H), 7.00 (d, J = 4 Hz, 1H), 6.14

(d, J = 16 Hz, 1H); 13C NMR (100 MHz, D2O+NaOH) δ 175.12,

141.74, 132.94, 131.26, 130.32, 123.25, 113.78 ; HRMS-ESI (m/z) [M+H]+ Calcd for

C7H579BrO2S: 232.9272; found, 232.9265.

(E)-3-(4-bromothiophen-2-yl)acrylic acid (4c)

White solid (180 mg, 39%) 1H NMR (400 MHz, D2O+NaOH) δ 7.36 (s,

1H), 7.34 (d, J = 16 Hz, 1H), 7.18 (s, 1H), 6.25 (d, J = 16 Hz, 1H); 13C

NMR (100 MHz, D2O+NaOH) δ 174.92, 140.97, 132.30, 131.11, 124.61,

124.08, 109.88; HRMS-ESI (m/z) [M+H]+ Calcd for C7H579BrO2S:

232.9272; found, 232.9262.

(E)-3-(5-chlorothiophen-2-yl)acrylic acid (4d)

White solid (132 mg, 36%) 1H NMR (400 MHz, D2O+NaOH) δ 7.31

(d, J = 16 Hz, 1H), 7.03 (d, J = 4 Hz, 1H), 6.91 (d, J = 4 Hz, 1H), 6.11

(d, J = 16 Hz, 1H); 13C NMR (100 MHz, D2O+NaOH) δ 175.11,

138.92, 133.19, 130.99, 129.57, 127.52, 122.94; HRMS-ESI (m/z) [M+H]+ Calcd for

C7H535ClO2S: 188.9777; found, 188.9764.

157

(E)-3-(quinolin-4-yl)acrylic acid (4e)

White solid (257 mg, 65%) 1H NMR (400 MHz, D2O+NaOH) δ 819 (d,

J = 4 Hz, 1H), 7.57 (t, J = 8 Hz, 2H), 7.48 (t, J = 8 Hz, 1H), 7.41 (d, J =

16 Hz, 1H), 7.31 (t, J = 8 Hz, 1H), 6.98 (d, J = 4 Hz, 1H), 6.22 (d, J =

16 Hz, 1H); 13C NMR (100 MHz, D2O+NaOH) δ 174.21, 148.79, 146.01, 141.09, 133.50,

130.53, 129.90, 127.41, 127.01, 125.19, 123.13, 117.38; HRMS-ESI (m/z) [M+H]+ Calcd for

C12H9NO2: 200.0712; found, 200.0736.

(E)-3-(isoquinolin-4-yl)acrylic acid (4f)

White solid (257 mg, 65%) 1H NMR (400 MHz, D2O+NaOH) δ 8.44 (s,

1H), 7.81 (s, 1H), 7.53 (d, J = 8 Hz, 1H), 7.41-7.48 (m, 2H), 7.28-7.37

(m, 2H), 6.04 (d, J = 16 Hz, 1H); 13C NMR (100 MHz, D2O+NaOH) δ

174.72, 151.68, 138.21, 133.66, 132.76, 131.44, 128.04, 127.72,

127.61, 127.13, 126.05, 121.92; HRMS-ESI (m/z) [M+H]+ Calcd for C12H9NO2: 200.0712;

found, 200.0735.

(S)-2-amino-3-(3-methylisoxazol-5-yl)propanoic acid (5a)

White solid (68 mg, 40%); 1H NMR (400 MHz, D2O+NaOH) δ 6.05 (s, 1H),

3.57-3.60 (dd, J = 8, 4 Hz, 1H), 2.96-3.01 (dd, J = 12, 4 Hz, 1H), 2.86-

2.91 (dd, J = 16, 8 Hz, 1H), 2.35 (s, 3H); 13C NMR (100 MHz, D2O+NaOH)

δ 180.08, 171.03, 161.68, 102.09, 54.66, 30.53, 11.32; HRMS-ESI (m/z)

[M+H]+ Calcd for C7H10N2O3: 171.0770; found, 171.0751.

(S)-2-amino-3-(5-bromothiophen-2-yl)propanoic acid (5b)

White solid (56 mg, 35%); 1H NMR (400 MHz, D2O+NaOH) δ 6.86 (d,

J = 4 Hz, 1H), 6.57 (d, J = 4 Hz, 1H), 3.34 (t, J = 8 Hz, 1H), 2.96 (d,

J = 8 Hz, 2H); 13C NMR (100 MHz, D2O+NaOH) δ 181.53, 141.94,

130.07, 127.02, 109.38, 56.90, 35.16; HRMS-ESI (m/z) [M+H]+ Calcd for C7H879BrNO2S:

249.9537; found, 249.9534.

(S)-2-amino-3-(4-bromothiophen-2-yl)propanoic acid (5c)


1H), 6.75 (s, 1H), 3.36 (t, J = 8 Hz, 1H), 2.99 (d, J = 8 Hz, 2H); 13C NMR

(100 MHz, D2O+NaOH) δ 181.45, 141.69, 128.66, 122.02, 108.48,

56.97, 34.85; HRMS-ESI (m/z) [M+H]+ Calcd for C7H879BrNO2S:

249.9537; found, 249.9537.

158

(S)-2-amino-3-(5-chlorothiophen-2-yl)propanoic acid (5d)


J = 4 Hz, 1H), 6.68 (d, J = 4 Hz, 1H), 3.52 (t, J = 4 Hz, 1H), 2.96 (d,

J = 4 Hz, 2H); 13C NMR (100 MHz, D2O+NaOH) δ 179.99, 138.32,

127.41, 126.31, 126.24, 56.63, 34.38; HRMS-ESI (m/z) [M+H]+ Calcd for C7H835ClNO2S:

206.0043; found, 206.0047.

(S)-2-amino-3-(quinolin-4-yl)propanoic acid (5e)


J = 4 Hz, 1H), 8.00 (d, J = 8 Hz, 1H), 7.83 (d, J = 8 Hz, 1H), 7.63 (t, J

= 8 Hz, 1H), 7.50 (t, J = 8 Hz, 1H), 7.18 (d, J = 4 Hz, 1H), 3.46-3.50

(dd, J = 8, 8 Hz, 1H), 3.25-3.30 (dd, J = 16, 8 Hz, 1H), 3.01-3.07 (dd, J = 16, 8 Hz, 1H); 13C

NMR (100 MHz, D2O+NaOH) δ 181.64, 149.51, 146.53, 146.24, 129.87, 127.97, 127.35,

126.89, 124.00, 122.36, 56.90, 37.33; HRMS-ESI (m/z) [M+H]+ Calcd for C12H12N2O2:

217.0977; found, 217.0973.

159


7.5.1 Supplementary figures

Figure S1. Overlaid structures of the AvPAL triple variant structure with bound cinnamate (solved in this study,

PDB ID: 5LTM) and the ligand-free double variant structure (reported previously, PDB ID: 3CZO).

160

Figure S2. a) the zymophore conferring PAL-specific chemistry and selectivity as identified from the co-crystal

structure of the ammonia lyase from Anabaena variabilis (AvPAL) and its deamination product trans-cinnamate

(PDB ID: 5LTM). b) an overlay of the existing active site structure of AvPAL with the co-crystal structure from this

work. c) the previously reported empty active site structure of AvPAL (PDB ID: 3CZO).

161

SgTAM 2(2) LTVEAVRRVA-EERAT---VDVPAESIAKAQKSREIFEGIAEQNIPIYGVTTGYGEMIYM 75

CmdF 1(1) LSIYDVADVC-MKRAT---VELDPSQLERVAVAHERTQAWGEAQHPIYGVNTGFGELVPV 63

PpHAL 1(1) LTLAQLRAIH-AAPVR---LQLDASAAPAIDASVACVEQIIAEDRTAYGINTGFGLLAST 66

AdmH 2(2) ISLEEIARAA-RDHQP---VTLHDEVVNRVTRSRSILESMVSDERVIYGVNTSMGGFVNY 90

BagA 1(1) LTISQTVAAASREGSE---FAVSEDALRAMNASRNLKLEILATGKPIYGVTTGFGDSVNR 64

StlA 0(0) ISLEDIYDIA-IKQKK---VEISTEITELLTHGREKLEEKLNSGEVIYGINTGFGGNANL 73

AvPAL -(-) LTINDVARVARNGTL--VSLTNNTDILQGIQASCDYINNAVESGEPIYGVTSGFGGMANV 90

TcPAM 1(1) ITVAHVAALARRHDVK--VALEAEQCRARVETCSSWVQRKAEDGADIYGVTTGFGACSSR 92

PcPAL1 1(1) LTISQVAAISARDGSG-VTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHR 122

RtPAL 0(0) LNLGDVVSAARKGRP--VRVKDSDEIRSKIDKSVEFLRSQLSM--SVYGVTTGFGGSADT 122

Sam8 1(1) SSREYLARVVRSAGWDAGLTSCTDEEIVRMGASARTIEEYLKSDKPIYGLTQGFGP--LV 70

SgTAM 2(4) QVDKSKEVELQTNLVRSHSAGV--------------GPLFAEDEARAIVAARLNTLAKGH 121

CmdF 2(3) MIPRQHKRELQENLIRSHAAGG--------------GEPFADDVVRAIMLARLNCLMKGY 109

PpHAL 2(3) RIASHDLENLQRSLVLSHAAGI--------------GAPLDDDLVRLIMVLKINSLSRGF 112

AdmH 2(4) IVPIAKASELQNNLINAVATNV--------------GKYFDDTTVRATMLARIVSLSRGN 136

BagA 2(3) QISPEKTARLQNELIRYHLNGT--------------GQLASDEVVRATVLIRANCLARGN 110

StlA 0(0) VVPFEKIAEHQQNLLTFLSAGT--------------GDYMSKPCIKASQFTMLLSVCKGW 119

AvPAL -(-) AISREQASELQTNLVWFLKT--------------GAGNKLPLADVRAAMLLRANSHMRGA 136

TcPAM 1(2) RTN--RLSELQESLIRCLLAGVFTKG------CAPSVDELPATATRSAMLLRLNSFTYGC 144

PcPAL1 1(1) RTK--QGGALQKELIRFLNAGIFGNG-------SD--NTLPHSATRAAMLVRINTLLQGY 171

RtPAL 2(2) RTE--DAISLQKALLEHQLCGVLPSSFDSFRLGRGLENSLPLEVVRGAMTIRVNSLTRGH 180

Sam8 1(2) LFDADSELEQGGSLISHLGTGQ--------------GAPLAPEVSRLILWLRIQNMRKGY 116

SgTAM 0(4) SAVRPIILERLAQYLNEGITPAIPEIGSLGASGDLAPLSHVASTLIGEG-----YVLR-D 175 CmdF 0(3) SGASVETVKLLAEFINRGIHPVIPQQGSLGASGDLSPLSHIALALIGEG-----TVSF-K 163

PpHAL 0(3) SGIRRKVIDALIALVNAEVYPHIPLKGSVGASGDLAPLAHMSLVLLGEG-----KARYKG 167

AdmH 1(5) SAISIVNFKKLIEIYNQGIVPCIPEKGSLGTSGDLGPLAAIALVCTGQW-----KARY-Q 190

BagA 0(3) SGVSLPVVELLLDFLKHDILPTVPERGSVGASGDLVPLCYLAYALTGQG-----KVRH-R 164

StlA 0(0) SATRPIVAQAIVDHINHDIVPLVPRYGSVGASGDLIPLSYIARALCGIG-----KVYY-M 173

AvPAL -(-) SGIRLELIKRMEIFLNAGVTPYVYEFGSIGASGDLVPLSYITGSLIGLDPSFKVDFNG-- 194

TcPAM 0(2) SGIRWEVMEALEKLLNSNVSPKVPLRGSVSASGDLIPLAYIAGLLIGKPSVIARIGDD-- 202

PcPAL1 0(1) SGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTG-- 229

RtPAL 0(2) SAVRLVVLEALTNFLNHGITPIVPLRGTISASGDLSPLSYIAAAISGHPDSKVHVVHEGK 240

Sam8 0(2) SAVSPVFWQKLADLWNKGFTPAIPRHGTVSASGDLQPLAHAALAFTGVGEAWTRDADG-R 175

SgTAM 0(4) GRPVETAQVLAERGIEP--LELRFKEGLALINGTSGMTGLGSLVVGRALEQAQQAEIVTA 233

CmdF 0(3) GQVRKTGDVLREEGLKP--LELGFKGGLTLINGTSAMTGAACVALGRAYHLFRLALLATA 221

PpHAL 0(3) -QWLPATEALAIAGLEP--LTLAAKEGLALLNGTQASTAYALRGLFQAEDLYAAAIACGG 224

AdmH 0(5) GEQMSGAMALEKAGISP--MELSFKEGLALINGTSAMVGLGVLLYDEVKRLFDTYLTVTS 248

BagA 0(3) GETRPTAEVLAELGLQP--VTLEAKDGLALINGTSFSAAFAVLNTEAAAELADVADICTA 222

StlA 0(0) GAEIDAAEAIKRAGLTP--LSLKAKEGLALINGTRVMSGISAITVIKLEKLFKASISAIA 231

AvPAL -(-) -KEMDAPTALRQLNLSP--LTLLPKEGLAMMNGTSVMTGIAANCVYDTQILTAIAMGVHA 251

TcPAM 0(2) -VEVPAPEALSRVGLRP--FKLQAKEGLALVNGTSFATAVASTVMYDANVLLLLVETLCG 259

PcPAL1 0(1) -VILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSA 288

RtPAL 0(2) EKILYAREAMALFNLEP--VVLGPKEGLGLVNGTAVSASMATLALHDAHMLSLLSQSLTA 298

Sam8 0(2) WSTVPAVDALAALGAEP--FDWPVREALAFVNGTGASLAVAVLNHRSALRLVRACAVLSA 233

...

SgTAM 1(5) GKDVQRSEIYLQKAYSLRAIPQVVGAVRDTLYHARHKLRIEL-NSANDNPLFFEG--KEI 350 CmdF 0(3) GNDVVDTGVYLQDAYTLRAVPQILGPVLDTLDFARKLIEEEL-NSTNDNPLIFDVP-EQT 338

PpHAL 0(3) ----------VQDPYSLRCQPQVMGACLTQLRQAAEVLGIEA-NAVSDNPLVFAAE-GDV 324

AdmH 1(6) LVKAS--NHQIEDAYSIRCTPQILGPVADTLKNIKQTLTNEL-NSSNDNPLIDQTT-EEV 363

BagA 0(3) -RHYQRLTRSIQDRYSLRCAPHVNGVLRDMLDWVRTWMTVEI-NSSSDNPLFDPST-GAV 337

StlA 1(1) HQEITQLNDTLQEVYSIRCAPQVLGIVPESLATARKILEREV-ISANDNPLIDPEN-GDV 347

AvPAL -(-) --------ELIQDRYSLRCLPQYLGPIVDGISQIAKQIEIEI-NSVTDNPLIDVDN-QAS 357

TcPAM 0(2) --------KPKQDRYALRSSPQWLAPLVQTIRDATTTVETEV-NSANDNPIIDHAN-DRA 365

PcPAL1 0(1) --------KPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREI-NSVNDNPLIDVSR-NKA 394

RtPAL 0(2) --------ILRQDRYPLRTSPQWLGPLVSDLIHAHAVLTIEAGQSTTDNPLIDVEN-KTS 407

Sam8 1(3) ----------LQEPYSLRCAPQVLGAVLDQLDGAGDVLAREV-DGCQDNPITYEG---EL 327

SgTAM 1(6) GLHSGFAGAQYPATALVAENRTIG-PASTQSVPSNGDN-QDVVSMGLISARNARRVLSNN 462

CmdF 1(4) GLLCGFEGGQYLATSIASENLDLAAPSSIKSLPSNGSN-QDVVSMGTTSARKSLRLCENV 451

PpHAL 2(5) GVNSGFMIAQVTAAALASENKALSHPHSVDSLPTSANQ-EDHVSMAPAAGKRLWEMAENT 435

AdmH 1(7) GLRLGLMGGQFMTASITAESRASCMPMSIQSLSTTGDF-QDIVSFGLVAARRVREQLKNL 476

BagA 1(4) GLQHGFKGMQIACSSLTAEALKHSGPASTFSRSTEAHN-QDKVSMAPIAARDARTVIELT 456

162

StlA 0(1) GMYQGFKGVQLSQTALVAAIRHDCAASGIHTLATEEYN-QDIVSLGLHAAQDVLEMEQKL 459

AvPAL -(-) KVNMGLKGLQICGNSIMPLLTFYGNSIADRFPTHAEQFNQNINSQGYTSATLARRSVDIF 472

TcPAM 0(2) SVDYGLKGLDIAMAAYSSELQYLANPVTT-HVHSAEQHNQDINSLALISARKTEEALDIL 479

PcPAL1 0(1) SLDYGFKGAEIAMASYCSELQFLANPVTN-HVQSAEQHNQDVNSLGLISSRKTSEAVEIL 508

RtPAL 0(2) SLSYHCKGLDIAAAAYTSELGHLANPVTT-HVQPAEMANQAVNSLALISARRTTESNDVL 520

Sam8 1(4) GRGAGLAGVQISATSFVSRIRQLVFPASLTTLPTNGWN-QDHVPMALNGANSVFEALELG 439

Figure S3. Sections of a sequence alignment of characterised class I lyase-like enzymes linking their known

catalytic activity to the number of conserved / varied amino acids within the active site. Yellow residues show

conservation at these positions whereas blue residues indicate variation and the number of variations in each line

is given on the left hand side (cumulative number in brackets). The enzymes in the alignment are: the (S)-selective

TAM from Streptomyces globisporus (SgTAM),S3 the (R)-selective TAM from from Chondromyces crocatus

(CmdF),S4 the bacterial HAL from Pseudomonas putida (PpHAL),S5 the (S)-selective PAM from Pantoea

agglomerans (AdmH),S6 two bacterial PALs from Photorhabdus luminescens (StlA)S7 and Anabaena variabilis

(AvPAL),S8 the (R)-selective PAM from from Taxus canadensis (TcPAM),S9 one of the PAL paralogues from

Petroselinum crispum (PcPAL1),S10 the bifunctional PAL/TAL from Rhodosporidium toruloides (RtPAL)S11 and two

distinct TALs from from Streptomyces sp. (BagA)S12 and from Saccharothrix espanaensis (Sam8).S13

>DdPAL (Dictyostelium discoideum)

MIETNHKDNFLIDGENKNLEINDIISISKGEKNIIFTNELLEFLQKGRDQLENKLKENVA

IYGINTGFGGNGDLIIPFDKLDYHQSNLLDFLTCGTGDFFNDQYVRGIQFIIIIALSRGW

SGVRPMVIQTLAKHLNKGIIPQVPMHGSVGASGDLVPLSYIANVLCGKGMVKYNEKLMNA

SDALKITSIEPLVLKSKEGLALVNGTRVMSSVSCISINKFETIFKAAIGSIALAVEGLLA

SKDHYDMRIHNLKNHPGQILIAQILNKYFNTSDNNTKSSNITFNQSENVQKLDKSVQEVY

SLRCAPQILGIISENISNAKIVIKREILSVNDNPLIDPYYGDVLSGGNFMGNHIARIMDG

IKLDISLVANHLHSLVALMMHSEFSKGLPNSLSPNPGIYQGYKGMQISQTSLVVWLRQEA

APACIHSLTTEQFNQDIVSLGLHSANGAASMLIKLCDIVSMTLIIAFQAISLRMKSIENF

KLPNKVQKLYSSIIKIIPILENDRRTDIDVREITNAILQDKLDFINLNL

>MxPAL (Methylobacterium sp.)

MNSNQQIIVSGSRLSVDQIVEVGLHKRNLFLTCDPQLRRTINDAADFVYRAVANEEVVYG

INTNFGGMANQVLSLNEVEDLQQNLIWGLKCGVGKKLPAAQVRSAMFIRANMLAKGVSGA

RAELIERYLVFLNAGITPVVRDLGSIGASGDLVPLAQIAGCLIGLGPSFRVERDGEEMDA

LSALSMLNLQPLKLRAKEGLALVNGSSMMSSIGAHCVHDTRHLTRLALHVHAMLIQALNA

SSESFDPFIHQNKPHPGQIAVAAAMRHLLRGSKSLKPNGHRKADGSGSLLQDRYSVRCLP

QYLGPIVDGLHAIEGQIEVEANSVDDNPLIDLENERLLHGGNFFAEYVALGMDQLRTYMA

LLAKHLDVQIAFAVAPEFNSGLPASLVGDQDNRIKFGLKGLQICANSIVPKLLHLSNGIS

VLFPTHAEQFNQNINSQGFNSATLASESVSLFKQYLAISLVFGIQAMDLRARATGGGFDG

RRYLSPTLLPLYETVRALLGRPASDERPLVFRNDEQDLSDHVAAIVADLSRPGGEIIGAM

AAEFAPGAGPAFGSPATRAGGRVAVAP

>PbPAL (Planctomyces brasiliensis)

MLASSPSGHTNPVLSGAPLSINVVADIGRQRLIPSLTDDEQVLNRVHACRDVVQKAVRNN

ERIYGITTGFGGMSDIPIPPQHVAQTQDNLLAFLSTSTGASLDPRHVRAAMALRANVLLQ

GRSGVRLELIERLVEFLRQDAIPVVCDLGSIGASGDLVPLGVIARSIIGHPSTTQVKYQG

EQADSHDVLQQLNYSALQLEAKEGLALVNGTSFSSAIAANCVFESQRLLSLSLVLQSIMV

RALGGHPEAFHPFVDENKPHPGQGWSAQMMRDLLSYSPNDSKRNGDLAQDRYSLRCLAQY

FAPIVEGIAQISQSISTEMNAVSDNPLIDVDTGRFHQSGNFLGQYVAMSMDQLRRHLGLL

AKHLDVQIAQLVAPAFNNGLPASLRGNSSRPFNMGLKGLQITGNSIMPLLTYLGNPLTEH

FPTHAEEFNQNINGLSWGSANLAWRSVQLFQHYLSVASIFAVQAIDLRAGLEADHCDGRE

LLGETATELYETVYDLLERNCGQESPFLFNDDEQSLEVDLQMLNGDLAGAGRMHEAVSSV

TDSFLAEFCE

>SrPAL (Streptomyces rimosus)

MHTMDTALAANDKAELLIDGHTLTVADVVSGARPADTTRVRARLAEGAVQRIEQSLALKN

KVIEAGLPVYGVTSGFGDSNTRQISGLKSEALQTNLIRFLSCGIGPVATPDVIRATMIVR

ANCLARGASGIRTEILELLLDCLNNDVLPPIPERGSVGASGDLVPLSYVAALLTGQGKAL

HQGEEKDASAALADAGLGAVVLGAKEGLALVNGTSFMSGFATLAVHDATELAFAADLSTA

LASQVLQGNPGHFVPFIFDQKPHTGTRTSARTIRELLGNPEDCDPSVDPEGAALTESGFR

QLEEPIQDRYSVRCAPHVTGVLRDTLDWAKNWVEVEINSTNDNPLFDVEAGMVRNGGNFY

163

GGHVGQAMDALKTAVASVGDLLDRQLELIVDEKFNNGLTPNLIPRFDADSWEAGLHHGFK

GMQIAASGLTAEALKNTMPATSFSRSTEAHNQDKVSMATIAARDARTVVELVRQVAAIHL

LALCQAADLRGQECLSAPTRAAYELIRSVSATMDGDRPLARDIELVVGLIASGELRRAVE

DAGRD

>BlPAL (Brevibacillus laterosporus)

MSQVALFEQELMLHGKHTLLLNGNDLTITDVAQMAKGTFEAFTFHISEEANKRIEECNEL

KHEIMNQHNPIYGVTTGFGDSVHRQISGEKAWDLQRNLIRFLSCGVGPVADEAVARATML

IRTNCLVKGNSAVRLEVIHQLIAYMERGITPIIPERGSVGASGDLVPLSYLASILVGEGK

VLYKGEEREVAEALGAEGLEPLTLEAKEGLALVNGTSFMSAFACLAYADAEEIAFIADIC

TAMASEALLGNRGHFYSFIHEQKPHLGQMASAKNIYTLLEGSQLSKEYSQIVGNNEKLDS

KAYLELTQSIQDRYSIRCAPHVTGVLYDTLDWVKKWLEVEINSTNDNPIFDVETRDVYNG

GNFYGGHVVQAMDSLKVAVANIADLLDRQLQLVVDEKFNKDLTPNLIPRFNNDNYEIGLH

HGFKGMQIASSALTAEALKMSGPVSVFSRSTEAHNQDKVSMGTISSRDARTIVELTQHVA

AIHLIALCQALDLRDSKKMSPQTTKIYNMIRKQVPFVERDRALDGDIEKVVQLIRSGNLK

KEIHDQNVND

Figure S4. The full amino acid sequences of the 5 new PALs selected for characterisation in Fasta format.

SrPAL ---MHTMDTALAANDKAELLIDG--HTLTVADVVSGARPADTTRVRARLAEGAVQRIEQS 55

BlPAL MSQVALFEQELMLHGKHTLLLNG--NDLTITDVAQMAK-GTFEAFTFHISEEANKRIEEC 57

DdPAL ---------MIETNHKDNFLIDGENKNLEINDIISISK----GEKNIIFTNELLEFLQKG 47

MxPAL ------------MNSNQQIIVSG--SRLSVDQIVEVGL--HKRNLFLTCDPQLRRTINDA 44

PbPAL -------MLASSPSGHTNPVLSG--APLSINVVADIGR--QRLIPSLTDDEQVLNRVHAC 49

: ::.* * : : . . . :.

SrPAL LALKNKVIEAGLPVYGVTSGFGDSNTRQISGLKSEALQTNLIRFLSCGIGPVATPDVIRA 115

BlPAL NELKHEIMNQHNPIYGVTTGFGDSVHRQISGEKAWDLQRNLIRFLSCGVGPVADEAVARA 117

DdPAL RDQLENKLKENVAIYGINTGFGGNGDLIIPFDKLDYHQSNLLDFLTCGTGDFFNDQYVRG 107

MxPAL ADFVYRAVANEEVVYGINTNFGGMANQVLSLNEVEDLQQNLIWGLKCGVGKKLPAAQVRS 104

PbPAL RDVVQKAVRNNERIYGITTGFGGMSDIPIPPQHVAQTQDNLLAFLSTSTGASLDPRHVRA 109

. : :**:.:.**. :. . * **: *. . * *.

SrPAL TMIVRANCLARGASGIRTEILELLLDCLNNDVLPPIPERGSVGASGDLVPLSYVAALLTG 175

BlPAL TMLIRTNCLVKGNSAVRLEVIHQLIAYMERGITPIIPERGSVGASGDLVPLSYLASILVG 177

DdPAL IQFIIIIALSRGWSGVRPMVIQTLAKHLNKGIIPQVPMHGSVGASGDLVPLSYIANVLCG 167

MxPAL AMFIRANMLAKGVSGARAELIERYLVFLNAGITPVVRDLGSIGASGDLVPLAQIAGCLIG 164

PbPAL AMALRANVLLQGRSGVRLELIERLVEFLRQDAIPVVCDLGSIGASGDLVPLGVIARSIIG 169

: * :* *. * ::. :. . * : **:*********. :* : *

SrPAL QG---KALHQGEEKDASAALADAGLGAVVLGAKEGLALVNGTSFMSGFATLAVHDATELA 232

BlPAL EG---KVLYKGEEREVAEALGAEGLEPLTLEAKEGLALVNGTSFMSAFACLAYADAEEIA 234

DdPAL KG---MVKYNEKLMNASDALKITSIEPLVLKSKEGLALVNGTRVMSSVSCISINKFETIF 224

MxPAL LGPSFRVERDGEEMDALSALSMLNLQPLKLRAKEGLALVNGSSMMSSIGAHCVHDTRHLT 224

PbPAL HPSTTQVKYQGEQADSHDVLQQLNYSALQLEAKEGLALVNGTSFSSAIAANCVFESQRLL 229

. . : : .* . .: * :*********: . *... . . :

SrPAL FAADLSTALASQVLQGNPGHFVPFIFDQKPHTGTRTSARTIRELLGNPEDCDPSVDPEG- 291

BlPAL FIADICTAMASEALLGNRGHFYSFIHEQKPHLGQMASAKNIYTLLEGSQLSKEYSQIVGN 294

DdPAL KAAIGSIALAVEGLLASKDHYDMRIHNLKNHPGQILIAQILNKYFNTSDNNTKSSNITFN 284

MxPAL RLALHVHAMLIQALNASSESFDPFIHQNKPHPGQIAVAAAMRHLLRGSKSLKPNGHRKAD 284

PbPAL SLSLVLQSIMVRALGGHPEAFHPFVDENKPHPGQGWSAQMMRDLLSYS----PNDSKRN- 284

: :: . * . : : : * * * * : : .

SrPAL -AALTESGFRQLEEPIQDRYSVRCAPHVTGVLRDTLDWAKNWVEVEINSTNDNPLFDVEA 350

BlPAL NEKLDSKAYLELTQSIQDRYSIRCAPHVTGVLYDTLDWVKKWLEVEINSTNDNPIFDVET 354

DdPAL ----QSENVQKLDKSVQEVYSLRCAPQILGIISENISNAKIVIKREILSVNDNPLIDPYY 340

MxPAL ----------GSGSLLQDRYSVRCLPQYLGPIVDGLHAIEGQIEVEANSVDDNPLIDLEN 334

PbPAL ------------GDLAQDRYSLRCLAQYFAPIVEGIAQISQSISTEMNAVSDNPLIDVDT 332

. *: **:** .: . : : : . :. * :..***::*

SrPAL GMVRNGGNFYGGHVGQAMDALKTAVASVGDLLDRQLELIVDEKFNNGLTPNLIPRFDADS 410

BlPAL RDVYNGGNFYGGHVVQAMDSLKVAVANIADLLDRQLQLVVDEKFNKDLTPNLIPRFNNDN 414

DdPAL GDVLSGGNFMGNHIARIMDGIKLDISLVANHLHSLVALMMHSEFSKGLPNSLSP------ 394

MxPAL ERLLHGGNFFAEYVALGMDQLRTYMALLAKHLDVQIAFAVAPEFNSGLPASLVG-----D 389

PbPAL GRFHQSGNFLGQYVAMSMDQLRRHLGLLAKHLDVQIAQLVAPAFNNGLPASLRG-----N 387

. .*** . :: ** :: :. :.. *. : : *...*. .*

SrPAL WEAGLHHGFKGMQIAASGLTAEALKNTMPATSFS-RSTEAHNQDKVSMATIAARDARTVV 469

BlPAL YEIGLHHGFKGMQIASSALTAEALKMSGPVSVFS-RSTEAHNQDKVSMGTISSRDARTIV 473

DdPAL -NPGIYQGYKGMQISQTSLVVWLRQEAAPACIHS-LTTEQFNQDIVSLGLHSANGAASML 452

MxPAL QDNRIKFGLKGLQICANSIVPKLLHLSNGISVLFPTHAEQFNQNINSQGFNSATLASESV 449

PbPAL SSRPFNMGLKGLQITGNSIMPLLTYLGNPLTEHFPTHAEEFNQNINGLSWGSANLAWRSV 447

. : * **:** ..: :* .**: . . :: * :

SrPAL ELVRQVAAIHLLALCQAADLRGQ---------ECLSAPTRAAYELIRSVSA--------- 511

BlPAL ELTQHVAAIHLIALCQALDLRDS---------KKMSPQTTKIYNMIRKQVP--------- 515

DdPAL IKLCDIVSMTLIIAFQAISLRMKSIE-----NFKLPNKVQKLYSSIIKIIP--------- 498

MxPAL SLFKQYLAISLVFGIQAMDLRARATGGGFDGRRYLSPTLLPLYETVRALLGRPASDERPL 509

PbPAL QLFQHYLSVASIFAVQAIDLRAGLEADHCDGRELLGETATELYETVYDLLERNCGQESPF 507

164

. :: : ** .** : *. :

SrPAL -TMDGDRPLARDIELVVGLIASGE-------LRRAVEDAGRD---------------- 545

BlPAL -FVERDRALDGDIEKVVQLIRSGN-------LKKEIHDQNVND--------------- 550

DdPAL -ILENDRRTDIDVREITNAILQDK-------LDFINLNL------------------- 529

MxPAL VFRNDEQDLSDHVAAIVADLSRPGGEIIGAMAAEFAPGAGPAFGSPATRAGGRVAVAP 567

PbPAL LFNDDEQSLEVDLQMLNGDLAGAG------RMHEAVSSVTDSFLAEFCE--------- 550

Figure S5. The full multiple sequence alignment of all 5 new PALs with the positions homologous to the AvPAL

zymophore highlighted. Yellow residues show conservation at these positions whereas blue residues indicate

variation.

SrPAL BlPAL DdPAL MxPAL PbPAL

SrPAL 100.00 56.62 30.27 33.08 30.25

BlPAL 56.62 100.00 33.65 32.38 29.62

DdPAL 30.27 33.65 100.00 30.69 30.16

MxPAL 33.08 32.38 30.69 100.00 45.69

PbPAL 30.25 29.62 30.16 45.69 100.00

Figure S6. A percentage identity plot of the 5 new PALs as inferred from the multiple sequence alignment.

Figure S7. Putative link of BlPAL to the biosynthesis of the basiliskamide antifungal secondary metabolites of

Brevibacillus laterosporus.S14

165

Figure S8. Protein expression and purification gel of the newly discovered PAL enzymes.

Ld Sr Bl Dd Pb Mx Sr Bl Dd Pb Mx Ld

170 130 100

70

55

40

35

25

15

10

Ladder Pb

PA

L

SrP

AL

BlP

AL

Dd

PA

L

MxP

AL

166

Figure S9. Spectrophotometric assays of TAL and HAL activity with each of the novel ammonia lyases which could

be purified for isolated enzyme studies.

7.5.1 Molecular visualization and modelling

The protein structure for the ammonia lyase AvPAL was downloaded from the Research

Collaboratory for Structural Bioinformatics’ Protein Data Bank – RCSB PDB – www.rcsb.org

(PDB ID: 3CZO). This was used to form an overlaid visualisation of the occupied and empty

AvPAL active sites with the YASARA molecular modelling software (version 14.7.17).

0

0.005

0.01

0.015

0.02

0 200 400 600 800 1000

Ab

so

rban

ce (

AU

)

Time (s)

Deamination of L-Tyr to coumaric acid

SrPAL BlPAL DdPAL PbPAL

0

0.005

0.01

0.015

0.02

0 200 400 600 800 1000

Ab

so

rban

ce (

AU

)

Time (s)

Deamination of L-His to urocanic acid

SrPAL BlPAL DdPAL PbPAL

http://www.rcsb.org/

167

7.5.2 X-ray crystallography

AvPAL-Y78F-C503S-C565S was purified as described previously for other AvPAL variants,

concentrated in a centrifugal spin concentrator (Vivaspin 30 kDa cut-off) to a final

concentration of 15 mg mL–1. Crystallisation occured in conditions similar to those reported for

the AvPAL-C503S-C565S,39 with rod shaped crystals appearing in a broad range of

PEG1500 concentrations (12-20%) and 100 mM SPG buffer (succinic acid, sodium

dihydrogen phosphate, glycine, pH 7.0). Protein and reservoir solution were mixed in a 1:1

ratio and incubated at 25°C for 3 days. Crystals were cryoprotected in 25% glucose before

being flash-frozen in liquid nitrogen. Data was collected at Diamond Light Source (Didcot, UK)

on station i24, from a single cryofrozen crystal. Data was processed and scaled using XDS

and the structure solved by molecular replacement using PHASER and the AvPAL-C503S-

C565S structure (PDB ID: 3CZO) as a search model. Iterative cycles of rebuilding and

refinement were carried out in COOT and Phenix Refine. The crystal structure of AvPAL-

Y78F-C503S-C565S bound to cinnamate has been deposited in the protein data bank (PDB

ID: 5LTM).

Table S1. Data collection and refinement statistics for crystallographic studies.

AvPAL-Y78F-C503S-C565S

bound to cinnamate

(PDB ID: 5LTM)

Data collection

Space group P 43 2 2

Cell dimensions

a, b, c [Å] 78.1, 78.1, 354.5

α, β, γ [] 90, 90, 90

Wavelength [Å] 0.987

Resolution [Å]* 2.4-29.8 (2.46-2.41)

Rsym or Rmerge* 0.169 (0.047-0.75)

I / σI * 14.9 (3.0)

Completeness [%]* 99.7 (96.6)

CC1/2* 99.7 (85.9)

Redundancy* 12.7 (12.1)

Total observations 552678

Total unique 43577

Refinement

Resolution [Å] 2.41

Rwork / Rfree 0.156 / 0.205

Mean B value [overall Å2] 26.9

r.m.s. deviations

Bond lengths [Å] 0.009

Bond angles [] 1.31

* values in parentheses are for highest resolution shell

168

7.5.3 Database searches

Discovery of uncharacterized. potential enzyme sequences was undertaken using an

appropriate query (protein sequence of ammonia lyases from Anabaena variabilis,

Photorhabdus luminescens and Streptomyces sp.) to perform a sequence similarity search

within the knowledge base of the universal protein resource. In each case the basic local

alignment search tool (BLAST) was used to find regions of sequence similarity between an

amino acid sequence and the in silico translated DNA sequences of all available genomes

and metagenomes (tBLASTn). All searches were gapped and unfiltered with a statistical

significance value threshold of E=10. The choice of protein substitution matrix was set to

automatic and thus assigned computationally based on the length of sequence. Sequences

were downloaded in fasta format for inspection and alignment. All sequence alignments were

performed through use of the W2 command line interface for the Clustal multiple sequence

alignment computer programme, as available online. All alignments made use of the Gonnet

protein weight matrix with the ‘gap open’ penalty score set to 10 in all cases. The initial pairwise

alignment type was set to slow with a gap extension score of 0.1. With subsequent multiple

alignments the gap extension score was set to 0.2 in addition to a gap distance penalisation

value of 5, without end gap penalisation or iteration. Sequences were clustered via the

neighbour-joining method. Alignments of all putative PALs were performed against the primary

sequence of AvPAL to allow accurate mapping of the zymophore motif onto homologous

positions.

7.5.4 Cloning of Wild-type BlPAL and SrPAL sequences

The wild-type sequences for SrPAL and BlPAL were cloned and amplified by colony PCR from

Brevibacillus laterosporus (NCIMB 701124) and Streptomyces risomsus subsp. rimosus

(NCIMB 8229) using the following primers:

SrPAL-Fw:5’-ACCTGCATATGCACACCA-TGGACACTGCCCTGGCAG-CCAACG-3’,

SrPAL-Rv:5’-TCGCACTCGAGTCAGTCCCGCC-

CGGCGTCCTCGACGGCCCGGCGGAGC-3’,

BlPAL-Fw:5’-GCTTACATATGAGTCAAGTAG-CCCTTTTCGAACAAGAGTTGATGC-3’,

BlPAL-Rv: 5’-TCGATGGATCCCTAGTCATTCAC-ATTTTGATCGTGAATTTCTTTC-3’.

The following touchdown PCR protocol was used in the first phase: 3 min initial denaturation

at 98°C and then 15 cycles of 30 s denaturation at 98°C, 30 s annealing at 65°C (-1°C each

cycle) and 2 min elongation at 72°C. The second phase involved: 20 cycles of 30 s

denaturation at 98°C, 30 s annealing at 55°C, and 2 min elongation at 65°C, with 5 min final

extension time at 72°C. After PCR product purification they were subcloned into pET-28b

between the NdeI and XhoI restriction sites.

169

7.5.5 Purified enzyme plate reader assay

A solution containing 0.5 mg mL–1 enzyme (20 µL) was added to a 96-well plate (flat bottom)

followed by addition of substrate solution (L-Tyr or L-His, 180 µL, 5 mM) to a total volume of

200 µL. The assay was performed at 37°C for 20 minutes measuring at 30 s intervals.

Detection wavelengths; coumaric acid, 380 nm; urocanic acid, 320 nm.

7.5.6 Purified enzyme assay conditions

50 uL of enzyme solution (0.5 mg mL–1) was added to 430 uL solution of either pH 8 or 10 100

mM borate buffer or pH 6 100 mM NaPi followed by addition of 20 uL L-phenylalanine (250

mM stock solutions) to a final substrate concentration of 5 mM and a final enzyme

concentration of 0.05 mg mL–1.

Temperature tests were conducted by incubating the enzyme (0.05 mg mL–1) in pH 8 borate

buffer at the specified times 1, 24 and 48 hrs. This was followed by addition of 20 uL L-

phenylalanine detailed above.


Reverse phase HPLC analyses were performed on an Agilent 1200 Series system equipped

with a G1379A degasser, G1312A binary pump, a G1329 autosampler unit, a G1316A

temperature controlled column compartment and a G1315B diode array detector. Where

appropriate, an external column cooling jacket was employed instead of the temperature

controlled column compartment. Conversion and product distribution analyses were

performed on a ZORBAX Extend-C18 column (50 mm × 4.6 mm × 3.5 μm Agilent). Mobile

phase: NH4OH buffer (0.35% w/v, pH 10.0) / MeOH (see Table S2). Flow rate: 1 mL min–1.

Temperature: 40°C. Detection wavelength: 210 nm. Peaks were assigned via comparison with

commercially available standards. Conversions and product ratios were calculated from peak

area integrations with use of appropriate response factors where needed.

170

MeOH

[%]

Temp.

[°C]

Retention time (tR)

[min]

1 2

H 10 40 2.3 5.4

2-F 10 40 2.8 7.2

3-F 10 40 3 7.7

4-F 10 40 2.7 7.1

2-Br 30 40 2.1 4.3

3-Br 30 40 2.8 6.2

4-Br 30 40 2.9 6.5

2-Cl 20 40 3.0 7.4

3-Cl 30 40 2.2 4.8

4-Cl 30 40 2.3 5.0

2-OCH3 20 40 2.3 4.5

3-OCH3 20 40 1.9 4.0

4-OCH3 10 40 2.2 6.4

2-NO2 10 40 3.9 5.9

3-NO2 10 40 3.1 6.8

4-NO2 10 40 2.8 6.1

7.5.7 Whole-cell biotransformation

The relevant arylacrylic acid 1a-p was dissolved at the required concentration (10 mM) in

unadjusted ammonium carbamate solution (4 M, pH ~9.9). Whole cells producing PAL were

added (50 mg mL–1) and the mixture incubated at 37°C with agitation of 250 rpm for 24 h. The

sample was centrifuged (3 min, 13000 rpm) to remove the catalyst, and the supernatant (250

uL) was diluted with MeOH (1:1) and passed through a filter vial for HPLC analysis.


Enantiomeric excesses were measured using a CROWNPAK CR(+) HPLC column, (150 mm

× 4 mm × 5 µm, Daicel). Mobile phase: aq. HClO4 (1.14% w/v, pH 2.0) / MeOH (see Table

S3). Flow rate: 1 mL min–1 for runs at 25oC. Detection wavelength: 210 nm. Peaks were

assigned via comparison with the literatureS1 and with commercially available standards.

Enantiomeric excess values were calculated from peak area integrations. Retention times are

171

given below for separation of authentic standards using methods based on previous

investigations.S2

MeOH

[%]

Temp.

[°C]

Retention time (tR)

[min]

(R)-1 (S)-1

H 4 25 5.0 6.3

2-F 4 25 5.8 7.5

3-F 4 25 6.5 8.3

4-F 4 25 6.6 8.1

2-Br 14 25 12.1 16.5

3-Br 14 25 19.7 24.8

4-Br 14 25 20.3 26.5

2-Cl 14 25 8.1 10.1

3-Cl 14 25 11.1 15.3

4-Cl 14 25 11.4 14.1

2-OCH3 4 25 11.3 14.1

3-OCH3 4 25 13.9 17.1

4-OCH3 4 25 14.1 17.3

2-NO2 4 25 6.5 8.1

3-NO2 4 25 7.4 10.6

4-NO2 4 25 7.4 8.6

7.5.9 Preparative scale reaction and characterization of (S)-1l

Arylacrylic acid 1l was dissolved at the required concentration (50 mM) in unadjusted

ammonium carbamate solution (4 M, pH ~9.9). Whole cells producing PbPAL were added (50

mg mL–1) and the mixture incubated at 37°C with agitation of 250 rpm for 24 h. The sample

was subjected to centrifugation (10 min, 4000 rpm) to remove the catalyst followed by ion

exchange purification as described in section 7.1.7).

(S)-2-amino-3-(3-methoxyphenyl)propanoic acid (1l): White

crystals (60 mg, 61% yield; 1H NMR (400 MHz, D2O+NaOH): 7.24

(t, J = 8 Hz, 1H), 6.81-6.84 (m, 3H), 3.76 (m, 3H), 3.43 (t, J = 8, 1H),

2.88-2.93 (dd, J = 16, 8 Hz, 1H), 2.73-2.78 (dd, J = 12, 8 Hz, 1H); 13C NMR (101 MHz,

D2O+NaOH): 182.30, 158.79, 140.11, 129.70, 122.27, 114.83, 112.22, 57.32, 55.26, 40.74;

HRMS (m/z): [M]+ calcd. for C10H14NO3,196.0895; found 196.1017.

172


7.6.1 Chemicals and enzymes

Analytical grade reagents, solvents and the corresponding aldehydes to compounds 1d-m

were purchased from Sigma-Aldrich, Alfa-Aesar and/or Fluorochem. The wild-type PbPAL

enzyme was kindly donated by Prozomix Ltd.

7.6.2 PbPAL mutants exhibiting β-lyase activity

Substrate F113A Conv. [%]

F113I Conv. [%]

F113V Conv. [%]

F113H Conv. [%]

F113Y Conv. [%]

F113I/L114V Conv. [%]

1c - 14 18 - - 27 1f 4 - - - - - 1g 5 2 2 4 8 4 1h 10 15 15 12 - 15 1m 10 5 6 13 - -

Formation of the β-amino acids after prolonged incubation with PbPAL mutants with a select

few substrates.

7.6.3 Site-directed mutagenesis

Mutagenesis was carried out using the In-Fusion cloning kit (Clontech) with the following

primers (mutations are underlined):

FL-FH_Fw: 5’-CCTGCTGGCATTTCACTCCACATCTACGGGAGCG-‘3

FL-FH_Rv: 5’-CGCTCCCGTAGATGTGGAGTGAAATGCCAGCAGG-‘3

FL-YH_Fw: 5’-CCTGCTGGCATATCACTCCACATCTACGGGAGCG-‘3

FL-YH_Rv: 5’-CGCTCCCGTAGATGTGGAGTGATATGCCAGCAGG-‘3

FL-AL_Fw: 5’-CCTGCTGGCAGCTCTGTCCACATCTACGGGAGCG-‘3

FL-AL_Rv: 5’-CGCTCCCGTAGATGTGGACAGAGCTGCCAGCAGG-‘3

F113NDT_Fw: 5’-CCTGCTGGCANDTCTGTCCACATCTACGGGAGCG-‘3

F113NDT_Rv: 5’-CGCTCCCGTAGATGTGGACAGAHNTGCCAGCAGG-‘3

FL-YA Fw: 5’-CCTGCTGGCATATGCGTCCACATCTACGGGAGCG-‘3

FL-YA Rv: 5’-CGCTCCCGTAGATGTGGACGCATATGCCAGCAGG-‘3

FL-YI Fw: 5’-CCTGCTGGCATATATCTCCACATCTACGGGAGCG-‘3

FL-YI Rv: 5’-CGCTCCCGTAGATGTGGAGATATATGCCAGCAGG-‘3

FL-YV Fw: 5’-CCTGCTGGCATATGTGTCCACATCTACGGGAGCG-‘3

FL-YV Rv: 5’-CGCTCCCGTAGATGTGGACACATATGCCAGCAGG-‘3

FL-YL Fw: 5’-CCTGCTGGCATATCTGTCCACATCTACGGGAGCG-‘3

FL-YL Rv: 5’-CGCTCCCGTAGATGTGGACAGATATGCCAGCAGG-‘3

173

FL-IA Fw: 5’-GATAACCTGCTGGCAATTGCGTCCACATCTACGGGAGCG-‘3

FL-IA Rv: 5’-CGCTCCCGTAGATGTGGACGCAATTGCCAGCAGGTTATC-‘3

FL-IV Fw: 5’-GATAACCTGCTGGCAATTGTGTCCACATCTACGGGAGCG-‘3

FL-IV Rv: 5’-CGCTCCCGTAGATGTGGACACAATTGCCAGCAGGTTATC-‘3

DpnI digest was carried out by addition of DpnI (1 µL) and incubation at 37°C for 1 h

followed transformation into stellar competent cells using standard protocol. Mutations were

verified by sequencing of the plasmid DNA.

PbPAL_WT

PbPAL NDT_IL

PbPAL NDT_VL

PbPAL_AL

PbPAL_FH

PbPAL_YH

PbPAL_YA

PbPAL_YI

PbPAL_YV

PbPAL_YL

PbPAL_IA

PbPAL_IV

174

7.6.4 Non-chiral HPLC conversions

The conversion of compounds 1a-q to 2a-q was measured on a non-chiral reverse-phase

Zorbax C-18 Extend column (50 mm x 4.5 µm, Agilent). Mobile phase aq. NH4OH 0.1 M pH

10 / MeOH, flow rate 1 mL min-1, temperature 40°C, detection wavelength 210 nm (retention

time are reported below).

Retention time (tR) [min]

% Solvent composition

L-2 1 aq. NH4OH MeOH

a 4.5 11 90 10 b 3.7 9.8 90 10 c 3.6 8.4 90 10 d 4.8 13 90 10 e 4.7 14 80 20 f 3.9 9.4 90 10 g 3.7 9 80 20 h 4.7 12 80 20 i 3.5 8.5 70 30 j 4.3 10.4 70 30 k 4.3 10.1 70 30 l 3.7 10.8 90 10

m 3.1 6.9 90 10 n - 15.9 90 10 o 9.3 17.9 90 10 p - 6.2 90 10 q - 10.5 90 10

7.6.5 Chiral HPLC conversions

The optical purity of amino acid products 2c-h and 2j-m was measured on a reverse-phase

Crownpak CR(+) column (150 mm x 4 mm x 3.5 µm, Daicel). Mobile phase aq. HClO4 1.14%

w/v / MeOH 85:15, flow rate 1.0 mL min–1, temperature 40°C, detection wavelength 210 nm.

Retention time (tR) [min]

D-2 L-2

c - 7.17 d - 8.41 e - 21.97 f - 8.21 g - 22.81 h - 21.15 j - 28.79 k - 32.53 l 6.05 7.72

m - 6.61

175

7.6.6 Knoevenegal-Doebner synthesis of acrylic acids of 1d-q

The corresponding aldehydes of compounds 1d-q (1 eq) and malonic acid (3 eq) were added

to Pyridine (100 mL). The mixture was stirred until complete dissolution of the starting

materials followed by addition of Piperidine (2 mol %) and refluxing the solution for 4-6 h. The

solution was cooled to room temperature and quenched with distilled water (100 mL) resulting

in the precipitation of the acrylic acid. The solid was filtered and washed with HCl (1M, 50 mL)

followed by oven drying to afford acrylic acids 1d-q (80-95% isolated yield).

7.6.7 Analytical scale biotransformation

E. coli cells harbouring PbPAL wild-type and mutant variants (50 mg) were added to a solution

ammonium carbamate (4 M, 990uL). Substrates 1a-q (10 uL, 0.5 M stock solution in DMSO)

was added to the biotransformation mixture to a final substrate concentration of 5 mM and

incubated at 37°C for 16 h. The mixture was centrifuged and the supernatant (250 uL) was

diluted with methanol (250 uL) and passed through a filter vial and used directly for HPLC

analysis.

7.6.8 Preparative scale biotransformation

Acrylic acids 1c-m was added to a solution of 4 M ammonium carbamate (10 mL) in the

presence of 10% DMSO (for substrates with poor solubility) to give a final substrate

concentration of 50 mM. Wet E. coli cells (50 mg mL–1) harbouring either PbPAL wild-type or

mutants were added to the solution and incubated at 37°C for 16 h. The biotransformation

mixture was centrifuged and the supernatant was purified using ion-exchange

chromatography.

7.6.9 Characterisation data of compounds 1c-q and L-2c-m

(E)-3-(4-fluoro-3-methoxyphenyl) acrylic acid (1d)

white solid (96%); 1H NMR (DMSO-d6) δ 7.51-7.58 (m, 2H, CH=CH,

ArH), 7.20-7.26 (m, 2H, ArH), 6.56 (d, 1H, J = 16 Hz, CH=CH), 3.38

(s, 3H, OCH3); 13C NMR (DMSO-d6) δ 167.55, 152.57 (d, 1JCF = 248

Hz), 147.39 (d, 2JCF = 11 Hz), 143.08 (d, 4JCF = 1 Hz), 131.32 (d, 3JCF

= 3 Hz), 121.63 (d, 3JCF = 7 Hz), 119.21, 116.08 (d, 2JCF = 19 Hz), 112.79, 56.05.

(E)-3-(4-chloro-3-methoxyphenyl) acrylic acid (1e)

White solid (95%); 1H NMR (DMSO-d6) δ 7.58 (d, 1H, J = 16 Hz,

CH=CH), 7.47 (s, 1H, ArH), 7.43 (d, 1H, J = 8 Hz, ArH), 7.25 (d, 1H,

J = 8 Hz, ArH) 13C NMR (DMSO-d6) δ 167.56, 154.77, 143.01,

134.70, 130.14, 122.94, 121.66, 120.23, 111.80, 56.28.

176

(E)-3-(3-fluoro-4-methoxyphenyl) acrylic acid (1f)

White solid (96%); 1H NMR (DMSO-d6) δ 7.62 (d, 1H, J = 12 Hz,

ArH), 7.51 (d, 1H, J = 16 Hz, CH=CH), 7.14 (t, 1H, J = 8 Hz, ArH),

6.43 (d, 1H, J = 16 Hz, CH=CH), 3.85 (s, 3H, OCH3) 13C NMR

(DMSO-d6) δ 167. 78, 151.63 (d, 1JCF = 243 Hz), 148.88 (d, 2JCF =

11 Hz), 142.87 (d, 4JCF = 2 Hz), 127.54 (d, 3JCF = 7 Hz), 125.99 (d, 3JCF = 3 Hz), 118.19, 114.82

(d, 2JCF = 19 Hz), 113.75, 56.11.

(E)-3-(3-chloro-4-methoxyphenyl) acrylic acid (1g)

White solid (82%); 1H NMR (DMSO-d6) δ 7.83 (s, 1H, ArH), 7.66 (d,

1H, J = 8 Hz, ArH), 7.52 (d, 1H, J = 16 Hz, CH=CH), 7.17 (d, 1H, J

= 8 Hz, ArH), 6.47 (d, J = 16 Hz, CH=CH), 3.89 (s, 3H, OCH3) 13C

NMR (DMSO-d6) δ 167.57, 155.81, 142.36, 129.35, 128.74, 127.86,

121.56, 118.10, 112.87, 56.29.

(E)-3-(4-(methylthio)phenyl) acrylic acid (1h)

White solid (92%); 1H NMR (DMSO-d6) δ 7.62 (d, 2H, J = 8 Hz, ArH),

7.55 (d, 1H, J = 16 Hz, CH=CH), 7.27 (d, 2H, J = 8 Hz, ArH), 6.48

(d, 1H, J = 16 Hz) 13C NMR (DMSO-d6) δ 167.73, 143.43, 141.32,

130.63, 128.69, 125.60, 118.12, 14.21.

(E)-3-(2-ethylphenyl) acrylic acid (1i)

Brown solid (72%); 1H NMR (CDCl3) δ 8.16 (d, 1H, J = 16 Hz, CH=CH),

7.61 (d, 1H, J = 8 Hz, ArH), 7.36 (t, 1H, J = 8 Hz, ArH), 7.23-7.27 (m, 2H,

ArH), 6.41 (d, 1H, J = 16 Hz, CH=CH), 2.79-2.85 (q, 2H, J = 8 Hz,

CH2CH3), 1.25 (t, 3H, J = 8 Hz, CH2CH3) 13C NMR (CDCl3) δ 172.64, 144.56, 144.17, 132.27,

130.70, 129.36, 126.76, 126.44, 118.41, 26.42, 15.95.

(E)-3-(3-ethylphenyl) acrylic acid (1j)

white solid (94%); 1H NMR (CDCl3): δ 7.81 (d, 1H, J = 16 Hz,

CH=CH), 7.39 (m, 2H, ArH), 7.33 (t, 1H, J = 8 Hz, ArH), 7.27 (d, 1H,

J = 8 Hz, ArH), 6.47 (d, 1H, J = 16 Hz, CH=CH), 2.66-2.72 (q, 2H, J

= 8 Hz, CH2CH3), 1.27 (t, 3H, J = 8 Hz, CH2CH3) 13C NMR (CDCl3) δ 172.89, 147.53, 145.12,

134.18, 130.63, 129.07, 128.02, 125.95, 117.19, 28.84, 15.61.

177

(E)-3-(4-ethylphenyl) acrylic acid (1k)

white solid (91%); 1H NMR (DMSO-d6): δ 7.58-7.60 (d, 1H, J = 8 Hz,

ArH), 7.56 (d, 1H, J = 16 Hz, CH=CH), 7.25 (2H, d, J = 8 Hz, ArH),

6.47 (d, 1H, J = 16 Hz, CH=CH), 2.59- 2.64 (q, 2H, J = 8 Hz,

CH2CH3), 1.17 (t, 3H, J = 8 Hz, CH2CH3) 13C NMR (DMSO-d6) δ

167.71, 146.31, 143.95, 131.78, 128.35, 128.30, 118.17, 28.09, 15.39.

(E)-3-(benzo[d][1,3]dioxol-4-yl)acrylic acid (1l)

white solid (97%); 1H NMR (DMSO-d6): δ 7.51 (d, 1H, J = 16 Hz,

CH=CH), 7.11 (d, 1H, J = 8 Hz, ArH), 6.97 (d, 1H, J = 8 Hz, ArH), 6.87

(t, 1H, J = 8 Hz, ArH), 6.55 (d, 1H, J = 16 Hz, CH=CH), 6.14 (s, 2H,

OCH2O) 13C NMR (DMSO-d6) 167.52, 147.62, 146.14, 138.20, 122.29,

121.96, 121.45, 116.73, 109.85, 101.62.(E)-3-(benzo[d][1,3]dioxol-5-yl) acrylic acid (1m)


CH=CH), 7.36 (s, 1H, ArH), 7.15 (d, 1H, J = 8 Hz, ArH), 6.94 (d, 1H,

J = 8 Hz, ArH), 6.39 (d, 1H, J = 16 Hz, CH=CH), 6.07 (s, 2H, OCH2O)

13C NMR (DMSO-d6) 167.85, 149.15, 148.05, 143.88, 128.70,

124.67, 117.10, 108.49, 106.68, 101.57.

(E)-3-(3, 4-dimethoxyphenyl) acrylic acid (1n)


CH=CH), 7.31-7.33 (m, 1H, ArH), 7.11 (s, 1H, ArH), 7.10 (d, 1H, J

= 4 Hz, ArH), 6.51 (d, 1H, J = 16 Hz, CH=CH), 3.82 (s, 3H, OCH3),

3.75 (s, 3H, OCH3) 13C NMR (DMSO-d6) δ 167.61, 152.68, 147.53,

138.01, 127.65, 124.33, 120.18, 118.84, 114.54.

(E)-3-(2, 4-dimethoxyphenyl) acrylic acid (1o)


CH=CH), 7.47 (d, 1H, J = 8 Hz, ArH), 6.52 (d, 1H, J = 8 Hz, ArH), 6.

46 (s, 1H, ArH), 6.45 (d, 1H, J = 16 Hz, CH=CH), 3.88 (s, 3H, OCH3),

3.85 (s, 3H, OCH3) 13C NMR (DMSO-d6) δ 167.61, 160.70, 144.00,

136.24, 119.86, 106.06, 102.42, 55.37.

(E)-3-(3,4-dimethoxyphenyl) acrylic acid (1p)


CH=CH), 7.14 (d, 1H, J = 8 Hz, ArH), 7.08 (s, 1H, ArH), 6.88 (d, 1H,

J = 8 Hz, ArH), 6.33 (d, 1H, J = 16 Hz, CH=CH), 3.93 (s, 6H, OCH3)

13C NMR (CDCl3) δ 172.63, 151.64, 149.37, 147.14, 127.14, 123.29,

114.98, 111.14, 109.85, 56.14, 56.04.

178

(E)-3-(2-fluoro-4,5-dimethoxyphenyl) acrylic acid (1q)


CH=CH), 7.32 (d, 1H, J = 8 Hz, ArH), 6.96 (d, 1H, JHF = 16 Hz, ArH),

6.54 (d, 1H, J = 16 Hz, CH=CH), 3.80 (s, 3H OCH3), 3.79 (s, 3H,

OCH3) 13C NMR (CDCl3) δ 167.71, 155.62 (d, 1JCF = 243 Hz), 151.87

(d, 3JCF = 10 Hz), 145.64, 135.61 (d, 4JCF = 3 Hz), 118.78, 112.63 (d, 3JCF = 13 Hz), 109.77 (d

1JCF = 5 Hz), 100.43 (d, 2JCF = 28 Hz), 56.13.

(E)-3-(4-(dimethylamino)phenyl) acrylic acid (1s)

brown solid (62%); 1H NMR (DMSO-d6): δ 7.45-7.49 (m, 3H, ArH

and CH=CH), 6.69 (d, 1H, J = 12 Hz, ArH), 6.21 (d, 1H, J = 14 Hz,

CH=CH), 2.96 (s, 6H, N(CH3)2); 13C NMR (DMSO-d6): 168.23,

151.60, 144.68, 129.74, 121.56, 112.93, 111.79.

(S)-2-amino-3-(4-methoxyphenyl) propanoic acid (2c)

white solid (36%); 1H NMR (D2O+NaOH) δ 7.22 (d, 2H, J = 8 Hz,

ArH), 6.96 (d, 2H, J = 8 Hz, ArH), 3.83-3.86 (m, 1H, CH2CHNH2),

3.12-3.17 (dd, 1H, J = 16, 4 Hz, CH2CHNH2), 2.97-3.03 (dd, 1H, J =

16, 8 Hz, CH2CHNH2) 13C NMR (D2O+NaOH) δ 175.14, 158.09,

130.58, 129.31, 127.95, 114.41, 56.23, 55.33, 36.04; HRMS-ESI (m/z) [M+H]+ Calcd for

C10H13NO3: 196.0895; found, 196.0989.

(S)-2-amino-3-(4-fluoro-3-methoxyphenyl) propanoic acid (2d)

white solid (63%); 1H NMR (D2O+NaOH) δ 7.07-7.12 (1H, dd, J =

12, 8 Hz, ArH) 6.99-7.02 (dd, 1H, J = 8, 4 Hz, ArH), 6.80-6.84 (m,

1H, ArH), 3.88 (s, 3H, OCH3), 3.69-3.73 (m, 1H, CH2CHNH2), 3.05-

3.10 (dd, 1H, J = 12, 4 Hz, CH2CHNH2), 2.92-2.97 (dd, 1H, J = 12,

8 Hz, CH2CHNH2) 13C NMR (D2O+NaOH) δ 182.20, 150.79 (d, 1JCF = 239 Hz), 146.31 (d, 3JCF

= 11 Hz), 134.76, 121.97 (d, 3JCF = 7 Hz), 115.54 (d, 2JCF = 18 Hz), 114.69, 57.33, 56.07,

40.27; HRMS-ESI (m/z) [M+H]+ Calcd for C10H12FNO3: 214.0801; found, 214.0915.

(S)-2-amino-3-(4-chloro-3-methoxyphenyl) propanoic acid (2e)


ArH), 6.98 (s, 1H, ArH), 6.84 (d, 1H, J = 12 Hz, ArH), 3.88 (s, 3H,

OCH3), 3.66-3.70 (m, 1H, CH2CHNH2), 3.04-3.08 (dd, 1H, J = 12, 4

Hz, CH2CHNH2), 2.91-2.96 (dd, 1H, J = 12, 4 Hz, CH2CHNH2) 13C

NMR (D2O+NaOH) δ 182.12, 153.86, 138.82, 129.70, 122.72, 119.41, 113.80, 57.28, 56.04,

40.52; HRMS-ESI (m/z) [M+H]+ Calcd for C10H12ClNO3: 230.0506; found, 230.0591.

179

(S)-2-amino-3-(3-fluoro-4-methoxyphenyl) propanoic acid (2f)

white solid (55%); 1H NMR (D2O+NaOH) δ 6.99-7.10 (m, 3H, ArH),

3.85 (s, 3H, OCH3), 3.78-3.81 (m, 1H, CH2CHNH2), 3.07-3.12 (dd,

1H, J = 16, 4 Hz, CH2CHNH2), 2.94-3.00 (dd, 1H, J = 16,, 8 Hz,

CH2CHNH2) 13C NMR (D2O+NaOH) δ 175.63, 156.29 (d, 1JCF = 241

Hz), 145.90 (d, 2JCF = 11 Hz), 129.03 (d, 3JCF = 6 Hz), 125.51 (d, 4JCF = 3 Hz), 116.65 (d, 2JCF

= 19 Hz), 114.16, 56.18 (d, 3JCF = 4 Hz), 36.37; HRMS-ESI (m/z) [M+H]+ Calcd for

C10H12FNO3: 214.0801; found, 214.0909.

(S)-2-amino-3-(3-chloro-4-methoxyphenyl) propanoic acid (2g)

white solid (30%); 1H NMR (D2O+NaOH) δ 7.31 (s, 1H, ArH), 7.16-

7.19 (dd, 1H, J = 8, 4 Hz, ArH), 7.07 (d, 1H, J = 8 Hz, ArH), 3.88 (s,

3H, OCH3), 3.74-3.78 (m, 1H, CH2CHNH2), 3.05-3.10 (dd, 1H, J =

16, 8 Hz, CH2CHNH2), 2.92-2.97 (dd, 1H, J = 16, 8 Hz, CH2CHNH2)

13C NMR (D2O+NaOH) δ 182.16, 152.76, 131.87, 130.58, 128.99, 121.08, 112.85, 57.30,

56.11, 39.54; HRMS-ESI (m/z) [M+H]+ Calcd for C10H12ClNO3: 230.0506; found, 230.0575.

(S)-2-amino-3-(4-(methylthio)phenyl) propanoic acid (2h)

white solid (10%); 1H NMR (D2O+NaOH) δ 7.72(d, 2H, J = 8 Hz,

ArH), 7.19 (d, 1H, J = 8 Hz, ArH), 3.49-3.52 (dd, 1H, J = 8, 4 Hz,

CH2CHNH2), 2.93-2.97 (dd, 1H, J = 12, 4 Hz, CH2CHNH2), 2.80-2.85

(dd, 1H, J = 12, 8 Hz, CH2CHNH2), 2.46 (s, 3H SCH3) 13C NMR

(D2O+NaOH) δ 181.26, 135.41, 135.07, 130.07, 126.70, 57.14, 39.61, 14.84; HRMS-ESI (m/z)

[M+H]+ Calcd for C10H13NO2S: 212.0667; found, 212.0750.

(S)-2-amino-3-(3-ethylphenyl) propanoic acid (2j)

white solid (50%); 1H NMR (D2O+NaOH) δ 7.27 (t, 1H, J = 8 Hz,

ArH), 7.12-7.16 (m, 2H, ArH), 7.06 (d, 1H, J = 8 Hz, ArH), 3.44-3.47

(m, 1H, CH2CHNH2), 2.92-2.97 (dd, 1H, J = 16, 8 Hz, CH2CHNH2),

2.75-2.80 (dd, 1H, J = 12, 8 Hz, CH2CHNH2), 2.57-2.63 (q, 2H, J =

8 Hz, CH2CH3), 1.16 (t, 3H, J = 8 Hz, CH2CH3) 13C NMR (D2O+NaOH) δ 182.31, 145.11,

138.41, 128.94, 128.67, 128.69, 126.69, 126.04, 57.35, 40.65, 28.16, 15.10; HRMS-ESI (m/z)

[M+H]+ Calcd for C11H15NO2: 194.1103; found, 194.1176.

180

(S)-2-amino-3-(4-ethylphenyl) propanoic acid (2k)


ArH), 7.17 (d, 2H, J = 8 Hz, ArH), 3.44-3.47 (m, 1H, CH2CHNH2),

2.91-2.96 (dd, 1H, J = 15, 8 Hz, CH2CHNH2), 2.76-2.81 (dd, 1H, J =

12, 4 Hz, CH2CHNH2), 2.56-2.62 (q, 2H, J = 8 Hz, CH2CH3), 1.15 (t,

3H, J = 8 Hz, CH2CH3) 13C NMR (D2O+NaOH) δ 182.08, 143.17, 135.30, 129.52, 127.99,

57.32, 40.08, 27.83, 15.13; HRMS-ESI (m/z) [M+H]+ Calcd for C11H15NO2: 194.1103; found,

194.1174.

(S)-2-amino-3-(benzo[d][1,3]dioxol-4-yl) propanoic acid (2l)

white solid (44%); 1H NMR (D2O+NaOH) δ 6.73-6.87 (m, 3H, ArH),

5.94 (d, 2H, J = 4 Hz, OCH2O), 3.76-3.79 (dd, 1H, J = 8, 4 Hz,

CH2CHNH2), 3.09-3.14 (dd, 1H, J = 12, 4 Hz, CH2CHNH2), 2.91-2.97

(dd, 1H, J = 16, 8 Hz, CH2CHNH2) 13C NMR (D2O+NaOH) δ 177.55,

146.72, 145.78, 123.43, 121.95, 118.06, 107.69, 100.77, 55.56, 32.42; HRMS-ESI (m/z)

[M+H]+ Calcd for C10H11NO4: 210.0688; found, 210.0776.

(S)-2-amino-3-(benzo[d][1,3]dioxol-5-yl) propanoic acid (2m)


ArH), 6.79 (s, 1H, ArH), 6.75 (d, 1H, J = 8 Hz, ArH), 5.93 (s, 2H,

OCH2O), 3.82-3.84 (m, 1H, CH2CHNH2), 3.09-3.14 (dd, 1H, J = 16,

4 Hz, CH2CHNH2), 2.94-2.99 (dd, 1H, J = 12, 8 Hz, CH2CHNH2) 13C

NMR (D2O+NaOH) δ 175.20, 147.43, 146.35, 129.25, 122.71, 109.53, 108.69, 101.06, 56.24,

36.71; HRMS-ESI (m/z) [M+H]+ Calcd for C10H11NO4: 210.0688; found, 210.0799.

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Discovery, Evolution and Synthetic Applications of