Monoamine oxidase: Tunable Activity for Amine Resolution and

Subscriber access provided by Kaohsiung Medical University

Review

Monoamine oxidase: Tunable Activity for Amine Resolution and Functionalization Vasco Figueiredo Batista, James Galman, Diana Cláudia Gouveia Alves Pinto, Artur M. S. Silva, and Nicholas J Turner ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b03525 • Publication Date (Web): 08 Nov 2018 Downloaded from http://pubs.acs.org on November 9, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

Monoamine Oxidase: Tunable Activity for Amine Resolution and Functionalization Vasco F. Batistaα*, James L. Galmanβ, Diana C. G. A. Pintoα, Artur M. S. Silvaα, Nicholas J. Turnerβ αDepartment

of Chemistry & QOPNA, University of Aveiro, Campus Universitário de

Santiago, 3810-193 Aveiro, Portugal. βSchool

of Chemistry, Manchester Institute of Biotechnology, University of Manchester,

131 Princess Street, Manchester, M1 7DN, UK.

ABSTRACT Monoamine oxidases (MAO) are biocatalysts for the oxidation of a wide range of different amines including α-chiral amines. Their high selectivity and activity, along with the environmental advantages inherent to enzymatic synthesis, place MAOs in the spotlight for future application in industrial biocatalytic processes. To date, these enzymes have been used in both amine resolution and amine functionalization. MAO from Micrococcus

luteus was employed in the multi-enzymatic synthesis of benzylisoquinoline alkaloids, and MAO from Aspergillus niger (MAO-N) in deracemisation experiments. MAO-N was also applied to several bio-bio and bio-chemo cascades for amine functionalization, exploring the increased reactivity of the imine/iminium species. MAO-N has been extensive engineered to alter the size and electronic properties of its active site, creating variants capable of oxidizing a broad range of α-aliphatic and aromatic amines.

ACS Paragon Plus Environment

1

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 65

This review provides an in-depth analysis of current research in the biocatalytic applications of MAOs, coupled with available data on the limitations and challenges that still hinder their industrial application. It also highlights the importance of chiral amines and the biochemical importance of Human MAO in metabolism. Finally, the development of alternative amine oxidases, such as CHAO or HLNO/HDNO, is briefly surveyed, along with a discussion on possible future developments on this field. KEYWORDS Biocatalysis,

Green

Chemistry,

Enzyme

engineering,

MAO-N,

Chiral

amines,

Deracemisation.

1. Introduction The growth of the chemical industry, driven by the search for a better standard of living, continues to strain the earth’s resources and ecosystems. This phenomenon is amplified by aggressive product development policies that sometimes neglect process optimization, creating industries with high energy consumption and toxic waste production.1 The environmental impact of such processes is particularly evident in the pharmaceutical industry, where the synthesis of highly complex chiral drugs can drive Efactor values up to 100 (the mass of waste obtained from the process can be 100x the mass of the product).2 In the last decades, however, the concept of Green Chemistry has


2

Page 3 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

gained strength within the scientific community, prompting a change to more efficient and sustainable processes.3 Catalysis can play a major role in this change, activating substrates to drive reactions forward under mild conditions.4 Enzymes emerge as optimal catalysts from a “green” point of view, containing active sites capable of precise reaction chemistry. Their unique three-dimensional profile induces exceptional chemo-, regio- and enantioselectivities, under mild reaction conditions and in water.5,6 Biocatalysis, the application of enzymes and microorganisms in synthesis, is thus rapidly expanding in synthetic chemistry, with notable industrial applications in the production of sitagliptin (1)7, simvastatin (2)8, artemisinin (3)9 and paroxetine (4) (Chart 1).10 Advances in gene mining, heterologous expression and enzyme engineering allow lower production costs and increased enzyme efficiency and versatility, surpassing the goals for industrial implementation .11–13 Within this rapid development of biocatalysis, new methods have been developed for the enantioselective synthesis of amines containing a stereogenic center in an α position, hereafter designated as chiral amines. F HO

H 2N O

F N

N N

F

O O

O O

O

O

O

O H

H

O

N CF3

H N

H

O (1)

(2)


(3)

O

O

F

(4)

3

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 65

Chart 1. Active pharmaceutical ingredients (APIs) produced through biocatalysis with the following enzymes: sitagliptin (1) - (R)-transaminase; simvastatin (2) - acyltransferase; artemisinin (3) multi-enzymatic process in Saccharomyces cerevisiae; paroxetine (4) - hydrolase.

Chiral amines are ubiquitous compounds with widespread use in pharmaceuticals, agrochemicals, resolving agents and chiral catalysts, as seen in the following examples (Chart 2).14,15 As APIs they are commonly found in the form of racemates, but recent regulations on drug toxicity - along with the introduction of the “chiral switch” policy by the US Food and Drug Administration - reinforced the search for its enantiopure analogues.16 Enantiopure chiral amines can be synthetized via asymmetric reduction of imines or enamides - with metal complexes, chiral Lewis bases or organocatalysts - or selective amination of aldehydes or alkenes.17 “Chiral-pool” or resolution alternative approaches are also available.18 These methods present serious limitations concerning versatility, complexity, selectivity and/or yield, resulting in a relevant environmental impact and increased waste treatment cost. Moreover, common product/catalyst separation protocols are not trivial, discouraging industrial application. As such, out-of-date crystallization processes, with maximum theoretical yields of 50%, remain the industrial standard.19 NH2

HO

O

H N O

H

MeOOC

S

O

H N N

N

O

NMe2

COOH (5)

(6)


(7)

4

Page 5 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

Chart 2. Examples of APIs containing chiral amines: amoxicillin, ritalin and rivastigmine, respectively.

Several enzymes were identified and engineered for the production of enantiopure amines, via either asymmetric synthesis with transaminases,20 amine dehydrogenases21, ammonia lyases22 or imine reductases23, or kinetic resolutions with lipases24 or amine oxidases (AOs). AOs, the subject of this review, are oxidoreductases that catalyze the oxidative hydrolysis of biogenic primary amines to aldehydes. Found in most organisms, from bacteria to humans, these enzymes can be subdivided into copper-dependent enzymes and flavin-dependent enzymes.25 The first use copper and pyrroloquinoline quinone (TQQ) as cofactors to form a TQQ-imine intermediate that is only undone through hydrolysis, limiting their applicability in biocatalysis. These enzymes are also not suitable for the oxidation of tertiary amines as the reaction intermediate would be an TQQ-imine species with a tetracoordinated nitrogen atom. However, AOs that use flavin-adenine dinucleotide (FAD) as a cofactor have been explored in biocatalysis for either the in situ generation of imines or the ability to perform deracemisation reactions with an appropriate reductant. The majority of flavin-dependent AOs are Monoamine Oxidases (MAOs), whose relevance is noted in review and opinion articles on oxidative enzymes. In our opinion, a detailed review article on the biocatalytic applications and substrate versatility of MAOs is lacking. To fill this gap, the following review focuses on monoamine oxidases as tunable catalysts for the resolution and functionalization of chiral amines.26


5

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 65

2. Monoamine oxidase: an historic and biological background MAO was discovered by Mary Bernheim, in 1928, as the first enzyme active in the oxidation of tyramine with molecular oxygen; it was consequently named “tyramine oxidase”.27 By 1938, several studies also confirmed its involvement in the metabolism of several monoamine neurotransmitters, expanding its known substrate scope and thus triggering the change in name.28 In humans, it is found in two major isoforms (MAO-A and MAO-B) bound to the outer membrane of mitochondria of several cell types. MAO-A is expressed in the central nervous system (CNS), liver, lungs and gastrointestinal tract, playing an essential role in the catabolism of food-based monoamines. In contrast, MAOB is found almost exclusively in the CNS, where it is responsible for the inactivation of monoaminergic neurotransmitters.29 Due to its role in neurotransmitter regulation, MAO dysfunction has been linked to several neurological disorders, and selective inhibitors of the B isozyme are indeed marketed for the treatment of depression and Alzheimer’s and Parkinson’s diseases.30 Despite their similar structure (~70% sequence identity), these enzymes differ greatly in substrate specificity: MAO-A shows higher affinity towards serotonin and norepinephrine while MAO-B is selective to phenylethylamine and benzylamine. However, both isozymes are responsible for the deamination of dopamine, tyrosine and tyramine.31 To date, enzymes assigned to the MAO family (E.C. 1.4.3.4) [amine: oxygen oxidoreductase (deaminating) (flavin-containing)] have been identified in a broad range


6

Page 7 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

of organisms, from bacteria to mammals, catalyzing the 2e- oxidation of several biogenic primary amines with either aliphatic or aromatic substituents. Hydrolysis thereafter follows

in vivo, forming the aldehyde/ketone and releasing ammonia, as shown in Scheme 1. The catalytic cycle is completed by the regeneration of the FAD cofactor, converting oxygen to hydrogen peroxide.32 NH2 R

FAD

H 2O 2

R

MAO NH R

FADH2

R1

O2

O

H 2O

R

R1

NH3

Scheme 1. MAO as a catalyst for amine oxidation in vivo.

Structural and mechanistic insights The development of heterologous systems for the expression of human MAO, along with the discovery and study of fungal and bacterial variants, uncovered some common structural features within this class. These have been the target of interesting reviews that we strongly recommend for further information.33,34 Briefly, MAOs are globular watersoluble flavoproteins that share more than 20% sequence identity to other flavoproteins, interacting with its dinucleotide cofactor through the common Rossman fold. Also, they


7

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 65

typically present an unusual C-terminal α-helix responsible for anchoring the protein to the outer membrane of the cell. Most present a rather similar active site, a hydrophobic protein region containing an aromatic cage formed by few amino acids; this cage is believed to improve substrate binding through an increase in the amine’s nucleophilicity. The substrate is guided through a narrow entrance channel that may or may not contain “gating residues”, responsible for substrate recognition. Within the active site, MAOs contain a FAD cofactor, usually bound to a cysteine residue through the 8α-methyl position of the flavin; the isoalloxazine ring system is maintained in a strained state, exhibiting a bent of ca. 30º that is thought to facilitate the formation of adducts at either the N(5) or C(4a) positions.35 The mechanism through which the oxidation occurs is subject to controversy, as four distinct pathways are currently under consideration (Scheme 2).35 To further complicate the debate, the specific mechanism is thought to vary according to both the enzyme and the nature of the substrate.36 Sliverman et al. proposed a single-electron transfer from the amine substrate to the flavin, creating an amine radical cation (A).37 However, studies have not been able to detect the presence of any radical species. Alternatively, a stepwise hydride mechanism (B), through cleavage of the αC-H bond, has been proposed by Edmondson as the pathway for MAO-B.36 This hypothesis was later refined to a new twostep hydride mechanism (C), with the formation of a C-N bond followed by water-assisted deprotonation of the nitrogen atom.38 Edmondson also proposed a polar nucleophilic


8

Page 9 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

mechanism (D) in which the addition of the amine substrate to the C(4a) carbon of the flavin is followed by intramolecular deprotonation. In fact, Miller and Edmondson found good correlations between quantum calculations of this mechanism and experimental data for MAO-A, suggesting this may be the correct mechanism for this enzyme.39

S

B

R N

N

R

O

N O

H NH2

R N N H

H

NH

H

H O H

O

O

N

N

NH

H

O

H H

N H H

S N

NH

N

NH

N

O

NH O

H NH2 R

S N

N

NH

R

N

O

H H

O NH

N

O NH2

A

R

O

H H

O

N

N H

N

NH2

D

R

O

R

O

R

O

NH

O

R

N

O NH

N H

NH

N

R

H N

N

S

R

R

S

R

R

S N

S

H O H

R

R

S

NH

NH2

H

O

N

N

NH

N H

C S

NH2 R

Scheme 2. Proposed mechanisms for the MAO-catalyzed oxidation of amines. A – Single-electron transfer mechanism, B – Step-wise hydride mechanism; C – Two-step hydride mechanism; D – Polar nucleophilic mechanism.


9

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 65

Evaluating enzymatic activity

Evaluation of mammalian MAO’s activity is crucial for testing selective inhibitors with pharmacological potential. In result, either colorimetric, fluorometric or luminescent assay kits that can be applied to high-throughput systems are commercially available. In a biocatalytic context, however, reports on improved and high-throughput methods for assaying MAO activity are scarce and fairly limited to MAO from Aspergillus niger. A common method to assess the activity of MAO variants is the solid-phase colorimetric screening assay with the formation of a distinctive red color.40 This occurs when H2O2, the oxidation by-product, is combined with horseradish peroxidase (HRP) and a chromogenic peroxidase substrate. Alternatively, a liquid-phase quantitative colorimetric assay can be used to quickly screen both enzyme or substrate libraries in microtiter plates. This assay quantifies H2O2 formation using HRP to catalyze the oxidative coupling of 2,4,6-tribromo-3-hydroxibenzoic acid and 4-aminoantipyrine to form a highly stable magenta dye. Its concentration is then readily monitored through absorbance measurements at 510 nm, allowing the determination of both the specific activity and kinetic parameters of the enzyme. While labor- and time-efficient, this method has a relatively high error that is usually reduced by performing replicates.41 A very recent article holds the sole proof of concept for high-throughput MAO screening with a biocatalytic goal.42 A novel ultra-high-throughput fluorometric method using a similar principle to that described above was used to screen large variant libraries; a


10

Page 11 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

fluorogenic probe, dihydrofluorescein diacetate, was oxidized by H2O2 in the presence of intracellular peroxidases. The desired enzyme variants were detected and isolated with flow cytometry coupled to a fluorescence-activated cell sorter, allowing the quantification of enzymatic activity and separation of a single cell. The use of flow cytometry allowed the measurement and separation of up to 10000 cells per second, and its relevance was validated by successfully obtaining improved variants to the oxidation of chosen substrates.

Biochemical considerations Monoamine oxidases take part in important catabolic and anabolic processes involving most aromatic amino acids. A detailed representation of these processes, such as the one that follows (Scheme 3), can be useful to evaluate the substrate scope of these enzymes and develop new and improved synthetic routes to access complex chiral compounds. In the metabolism of tyrosine (8), peripheral MAO-A plays a crucial role in the oxidation of the tyramine (9), a metabolite found in several foods. Both isozymes are also responsible for the degradation of tyrosine-derived neurotransmitters such as dopamine (10a) and epinephrine (10b). Remarkably, one of the resulting aldehydes (11a) is the main scaffold for the synthesis of relevant natural isoquinoline alkaloids such as norcoclaurine (12). Likewise, MAO can metabolize the tyrosine precursor phenylalanine (13) to phenylacetaldehyde (14).43


11

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 65

These oxidative enzymes are documented for the degradation of serotonin (15) as part of tryptophan metabolism (16). Less is known, however, about their role in the oxidative cyclization of the hydroxy-kynuramine metabolites (17) to the biologically relevant 4hydroxyquinolines (18). Finally, MAOs are known to participate in the degradation of histidine (19). Histidine metabolism occurs via two distinct enzymes, diamine oxidase (DAO) and histamine methyltransferase (HMT). However, the absence of DAO in the human brain makes enzymatic methylation to (20) the sole metabolic fate of this amino acid. Further degradation occurs via deamination with MAO-B followed by oxidation with an aldehyde dehydrogenase to yield the corresponding acid (21).43


12

Page 13 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

ACS Catalysis

H N

NH2

N

Histidine metabolism

M A O

N N

NH2 (20)

(13) Phenylalanine metabolism

Tyrosine metabolism

OH

(9)

R

H N

R1

OH

(10)

O

CHO OH

(21)

(11)

N

O R1 N H

OH

NH2

NH2

NH2 (17)

R2

O

OH N H

(14)

R1

O

N (18)

R N

R

R OH

(16)

Tryptophan metabolism

(15)

OH R

O

H 2N

NH2

N H

H 2N

(8)

H 2N

N N

OH

H 2N

(19)

COOH

HOOC

HOOC

COOH

OH

O

R COOH OH R = OH, COOH

OH

OH

R

O

O OH

a) R0= H, R1 = H; b) R = OH, R1 = CH3.

N H R = H, OH

OH HN

(12)

OH OH OH

Scheme 3. The biological role of MAO: a metabolic overview. 43


13

OH

R2

a) R1 = H, R2 = OH b) R1 = OH, R2 = H

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

3.

MAO

from

Page 14 of 65

Micrococcus luteus in the artificial synthesis of

benzylisoquinoline alkaloids The search for new bioactive compounds prompted the use of complex natural products as pharmaceuticals. Amongst them, plant alkaloid metabolites are commonly extracted for commercialization, at great cost and effort. Metabolic engineering and total chemical synthesis approaches, though occasionally applied, fail to meet industry standards in sustainability and efficiency. The implementation of enzymatic synthesis, combining microbial pathways with plant-derived genes, allows the fast and environment friendly synthesis of alkaloids in batch; microbes do not need large culture spaces and have a fast growth rate. One such case is found in the reconstruction of the benzylisoquinoline alkaloid biosynthetic pathway in microbial cell cultures by Minami and coworkers (Scheme 4). Firstly, they devised a route for the synthesis of the key intermediate (S)-reticuline (25) from dopamine (22) (up to 55 mg/L), in Escherichia coli, through a norcoclaurine synthase (NCS) catalyzed Pictet-Spengler reaction followed by sequential hydroxylations and methylations. However, while NCS is (S) specific, the high activity of MAO prompted a spontaneous condensation reaction to racemic tetrahydropapaverin (THP) (24), effectively leading to (R,S)-reticulin.44 MAO played a crucial role in this pathway, driving the conversion of dopamine to 3,4-dihydroxiphenylacetaldehyde

(23).

In

particular,

cytosol-soluble

MAO

from

Micrococcus luteus was used to overcome the difficult expression of membrane-bound


14

Page 15 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

eukaryotic enzymes in microbial expression systems. The inter-conversion of these two substrates allowed further development of this pathway via in situ production of dopamine, an expensive and unstable substrate. To do so, an L-tyrosine (8) overproducing E. coli strain was developed and combined with a bacterial tyrosinase and LDOPA specific decarboxylase. Combination of the above pathways and optimization of temperature, time, oxygenation, culture medium and induction parameters allowed reticuline yields up to 282 mg/L from either glycerol or glucose.45–47 The fermentation system implemented to produce reticuline was further coupled with cytochrome-containing

S.

cerevisiae

for

the

synthesis

of

more

complex

benzylisoquinoline alkaloids (BIAs): (S)-scoulerin (26), a precursor to berberine derived probiotics, was obtained through condensation catalyzed by berberine bridge enzyme (BBE)48 and magnoflorine (27) by sequential oxidation/methylation reactions.44 The use of yeast expression systems was indispensable at this point to achieve an efficient production of membrane-bound cytochrome enzymes.


15

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 65

COOH

OH HO

OH

NH2

Tyrosine over-producing HO pathway

Glycerol

TYR

HO

COOH NH2

HO

L-(8)

L-DOPA DODC HO

MeO

HO NH

HO

6OMT

H

HO

NH2

HO (22)

NH

HO

H NCS or Pictet-Spengler

HO OH

OH

3'-Hydroxycoclaurine

(24)

MAO HO O

HO (23)

CNMT MeO

MeO HO

H

N

HO

N

H

4'OMT MeO

HO

OH

OH 3'-Hydroxy-N-methylcoclaurine

MeO HO

(25)

MeO N H

OH OMe

(26)

MeO

MeO

N

HO HO

O

O N

MeO

MeO (27)

N O

(28)

(29)

Scheme 4. Combination of microbial pathways and plant enzymes in the synthesis of benzylisoquinoline alkaloids.


16

Page 17 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

The microbial production of BIAs from (S)-reticulin has been established and presents great potential for future industrial applications. In fact, Kyoto University patented a segment of this pathway in 2015.49 However, the extension of these methods to more complex and widely used opioids, such as morphine and codeine, is hindered by the lack of enzymes capable of inverting the chirality of (S)-reticuline. A recent attempt describes the ground-breaking synthesis of thebaine (28) (2.1 mg/L from glycerol) with a stepwise culture of four engineered strains, using a spontaneous Pictet-Spengler reaction to obtained racemic reticuline as an intermediate. The introduction of two additional enzymes in the thebaine producer allowed further functionalization to hydrocodone (29), thus proving the general applicability of this method for opioid production.50

4. MAO from Aspergillus niger: successful engineering in amine resolution and functionalization In 1991, Legge et al. reported the in situ synthesis of norlaudanosoline from dopamine by induction of endogenous MAO in A. niger.51 Interestingly, the authors transposed Yamada’s earlier findings to this work, concluding that a copper enzyme (currently semicarbazide-sensitive amine oxidase - SSAO) was responsible for dopamine oxidation.52 Nowadays, it is clear that this reaction is catalyzed by flavin-containing MAOs. Only three years later, Schilling and Lerch reported the isolation of two distinct amine oxidases, one SSAO and one MAO, from butylamine-induced cultures of A. niger.53


17

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 65

Expression of these enzymes was highly dependent on the nitrogen source provided, and was suppressed in the presence of ammonia; optimized cell-growth and expression conditions used butylamine or pentylamine, at concentrations of 0.15% (v/v), as sole nitrogen sources. Characterization of the isolated MAO, from now on designated as MAON, revealed a tetrameric enzyme with an absorption spectrum typical of flavin-containing enzymes. Notably, dialysis experiments showed that the flavin cofactor was noncovalently bound. Comparison to mammalian MAO-A and MAO-B showed cumulative substrate specificity and inhibition profiles, suggesting MAO-N as an evolutionary precursor of these isozymes.54 The same author readily cloned this enzyme in E. coli BL21 (DE3), expressing it through the T7 promoter of the pET3a vector.55 Substrate scope studies with recombinant enzyme highlighted MAO-N’s preference for some aliphatic and aromatic amines over most biogenic amines. The crystal structure of wild-type (WT) MAO-N has not yet been reported. However, the Grogan group at the University of York obtained single-crystals of two engineered variants, MAO-N D3 and D5, and solved both structures in 2008.56 Oddly, slight differences cause a significant change in space group, respectively, P21 (a = 103.4, b = 132.6, c = 187.2 A˚, β = 90.1º), with two tetramers in the asymmetric unit, and P41212 or P43212 (a = 108.3, c = 235.7 A˚), with half a tetramer in the asymmetric unit. The MAO-N monomer, however, is similar: it is composed of 495 amino acids with a molecular weight of 55 617 Da. Its tertiary structure, comprised of 17 α-helices and 21 β-sheets, assumes


18

Page 19 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

the p-hydroxybenzoate hydroxylase (PHBH) fold, common in flavoproteins, resembling that of mammalian MAO enzymes. Nevertheless, several differences are important at the enzymes’ termini: MAO-A and MAO-B present ~50 additional amino acids at the Cterminus, forming a protruding helix thought to anchor these enzymes to the mitochondrial membrane, while MAO-N contains the tripeptide Ala-Arg-Leu, the peroxisomal targeting signal; in contrast, this fungal enzyme contains ~40 additional amino acids at the Nterminus whose function is yet to be determined.57 The active site of MAO-N is defined by a large hydrophobic pocket that extends to the protein surface via an entrance channel. In contrast to mammalian MAO, this enzyme does not have either a flexible loop or “gating” residues for entrance regulation but similar functions may still be carried out by protruding hydrophobic residues. Within the active site, Trp430 and Phe466, perpendicular to the flavin moiety, constitute the previously mentioned aromatic cage. The flavin, non-covalently bound, is positioned through a series of hydrogen bonds with water molecules and amine groups in the protein backbone. In particular, electrostatic interactions with guanidinium (Arg77) and carboxylic (Glu69) groups play essential roles in cofactor straining.57 A structural view on MAO-N engineering MAO-N was the subject of several rounds of evolution through discrete mutations close to the enzyme’s active site, here described and summarized on Table 1. These led to a larger hydrophobic pocket and an improved electronic environment, increasing both its


19

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 65

activity and substrate scope. The first improved variant, MAO-N D1 (Asn336Ser), showed increased activity for a variety of bulkier substrates.58 Ser336’s location close to the active site, behind the Trp430 residue of the aromatic cage, suggests the formation of a new weak hydrogen bond with the indole’s nitrogen atom, altering the position of tryptophan within the active site and reducing steric constrains. Evidence showed that this new interaction may affect the dipole moment within the aromatic cage, leading to reduced activity with less nucleophilic substrates. The introduction of two additional mutations, Met348Lys and Ile246Met, created the first variant with enhanced synthetic potential, MAO-N D3.41 The mutation Met348Lys caused an increase in catalytic activity due to unknown conformational changes; the introduction of a side chain amine group created a new hydrogen bond near the protein’s surface. The latter allowed the uptake of sterically demanding bicyclic compounds through increased flexibility of the entrance channel - methionine has a highly flexible side chain. Following these results, MAO-N D5 was engineered to achieve increased activity with both secondary and tertiary amines (Figure 1).59 Two consecutive mutations (Thr384Asn and Asp385Ser), introduced far from the active site, caused a slight change to the protein’s tertiary structure, increasing the active pocket’s size and thus relieving steric constrains.


20

Page 21 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

Figure 1. Active site pocket of MAO-N D5 with bound FAD cofactor. The mutated amino acid residues are highlighted.60(PDB ID: 2vvm)

Rowles et al. further improved MAO-N’s activity through a rational re-design of MAO-N D5 for the oxidation of a dimethoxylated 1,2,3,4-tetrahydroisoquinoline.61 Curiously, the large volume of the substituted aromatic ring caused a shift in the orientation of this substrate within the active site, further protruding into the entrance channel. As a result, steric hindrance with the side-chains of amino acids located in this channel limited the enzyme’s

activity.

Four

target

residues

–

– Phe210Leu/Leu213Thr/Met242Gln/Met246Thr – were identified by molecular modelling and optimized over two rounds of saturated mutagenesis and solid-phase screening, revealing a new variant (MAO-N D9) with up to 990-fold increase in activity for Crispine A.


21

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 65

A final variant, MAO-N D11, was developed by the Turner group for the oxidation of bulky diaryl chiral amines (Figure 2).60 The authors identified the Ala249 and Trp430 residues as optimal targets for site-directed mutagenesis due to their role in defining the limits of the hydrophobic pocket. Indeed, the introduction of an additional Trp430Gly mutation allowed a significant increase in the active pocket’s size - from 140 Â3 (D5) to 464 Â3 (D11) - leading to increased activity with 4-chlorobenzhydrylamine. Note that this change has a substantial impact on the aromatic cage, undermining its central importance to MAO-N’s activity. This variant was also tested for the deracemization of 1benzyltetrahydroisoquinolines, presenting a remarkable selectivity for the (R) isomer. Molecular modelling studies suggest that an inversion in the position of the bicyclic structure within the active site, now almost co-planar to the flavin, is responsible for this stereoinversion.

Figure 2. Active site pocket of MAO-N D11 with bound FAD cofactor. The mutated amino acid residues are highlighted.60 (PDB ID: 3ZDN)


22

Page 23 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

Two independent engineering experiments were reported for the MAO-N-catalyzed deracemization of mexiletine. Firstly, Chen et al. used a semi-rational approach for the further development of MAO-N D5, using docking studies to identify six relevant residues along the hydrophobic pocket – Phe210, Leu213, Met242, Leu245 and Ile367.62 Two new variants with enhanced activity were obtained following several rounds of screening and combinatorial experiments: A-1 (D5+F210V/L213C) and AC-1 (D5+F210V/I367T). The reduction in size from Phe210 to valine effectively widens the hydrophobic pocket while the shift from lysine to cysteine at 213 causes the formation of a new disulfide bond, enlarging the binding pocket. Finally, the I367T mutation reduces the polarity of the entrance channel, further contributing to favorable uptake of hydrophobic substrates. Later, the Reetz group applied iterative saturation mutagenesis (ISM) directly to WT MAO-N, varying residues on both the entrance channel and active site.63 A total of 23 residues were grouped into six randomization sites, subjected to saturation mutagenesis with a reduced NDT alphabet and screened using a solid colorimetric assay. Further grouping and ISM experiments found two relevant variants – LG-I-D11 and LG-J-B4 (see Table 1)– with increased activity towards 1-substituted THIQs. Interestingly, the first enzyme showed reversed stereoselectivity for not only 1-benzyltetrahydroisoquinoline (as seen in MAO-N D11), but also several 1-alkyl THIQs. Molecular dynamics simulations were used to assess the effects of individual changes on the overall enhanced activity of these two variants. The exchange of Trp for smaller amino acids, at positions 230 and


23

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 65

430, caused significant geometrical changes, thus increasing the size of the active site pocket. The C214L mutation appeared to have a similar effect as it occurs in the active site pocket. The remaining mutations occur on the enzyme’s entrance channel, significantly decreasing its polarity and thus enhancing substrate uptake. Table 1. Mutations Present on Known MAO-N Variants Variant/aa F21 L21 C21 W23 M24 I246 R25 R260 N33 M34 T35 Y36 I367 T38 D38 W43 0

3

4

0

2

9

6

8

4

5

4

5

0

D158

-

-

-

-

-

-

-

-

S

-

-

-

-

-

-

-

D341

-

-

-

-

-

M

K

K

S

K

-

-

-

-

-

-

D559

-

-

-

-

-

M

-

-

S

K

-

-

-

N

S

-

D961

K

T

-

-

Q

T

-

-

S

K

-

-

-

N

S

-

D1160

K

T

-

-

Q

T

-

-

S

K

-

-

-

N

S

G

A-162

V

C

-

-

-

M

-

-

S

K

-

-

-

N

S

-

AC-162

V

-

-

-

-

M

-

-

S

K

-

-

T

N

S

-

LG-I-

-

-

L

R

-

-

-

-

-

-

-

-

-

-

-

C

-

-

-

I

R

-

-

-

-

-

S

V

-

-

-

R

D1163 LG-J-B463

Looking through the scope: the versatility of MAO-N The past decade has seen the development of MAO-N enzymes with distinct active sites, progressively larger and more hydrophobic. Subsequently, the range of substrates within their scope shifted, with recent variants - such as MAO-N D9 and D11 - showing activity with secondary and tertiary cyclic amines. This gradual change instigated several studies


24

Page 25 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

on their substrate scope, allowing a proper assessment of MAO-N’s potential for future synthetic applications. Furthermore, it provided a helpful tool to determine the appropriate enzyme for a given biocatalytic experiment. Liquid-phase colorimetric screening assays (see page 7) were applied to a broad range of primary amines bearing alkyl or aromatic substituents in the α carbon; the most relevant, from our perspective, are compiled in Charts 3 and 4, respectively. There is a clear preference for less mutated MAOs to oxidize smaller, less branched substrates, particularly mono α-substituted; note the decrease in activity from the linear amine (30) to branched (31) and (32), both in MAO-N WT and D1. However, unclear steric effects may play a key role through increased hydrophobic interactions, causing the proportional increase in activity with size seen in (38). The larger active pocket of MAO-N D5 and D9 markedly enhances its activity with bulky primary amines, allowing the oxidation of branched amines containing propene (33), cyclohexyl (35) or phenyl (34) substituents. Still, MAO-N D11 shows lower activity than its predecessors with all substrates, as an even larger pocket may reduce hydrophobic interactions.40,41,64


25

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

NH2

NH2

NH2

n

n

Page 26 of 65

NH2

3

H 2N

iBu

(30) 612/100

NH2

(31) (32) a) n = 1: 15/335/10 a) n = 0: 0/8 b) n = 2: 0/6 b) n = 1: 0/26/80/345/85 c) n = 4: 0/3/15/435/10 c) n = 2: 0/20 d) n = 3: 0/2

(33) 10/135/95

(34) 15/110/15

NH2

NH2 Me

NH2

n NH2

(35) 0/3

(36) 9/127/75/1010/270

(37) 55/0/0

(38 a) n = 1: 0/1 b) n = 2: 0/2 c) n = 3: 1/5 d) n = 4: 2/22

(39) a) (2S): 71/25 b) (2R): 0/3

Chart 3. Screening studies on the oxidation of primary amines by MAO-N variants. The values correspond to relative activities (%), taking as reference the activity of MAO-N D5 with αmethylbenzylamine (AMBA). Note that an accurate comparison between different articles was only possible through comparison of the enzyme’s kcat with AMBA: kcatD1/kcatD5 = 6.01 min-1/43.4 min-1 = 7.22. Color mapping was used to identify the enzymes: WT, D1, D5, D9, D11.40,41,64

Similar patterns can be seen with the α-aromatic primary amines shown in Chart 4, where only the D5, D9 and D11 variants display high activities. A clear advantage is shown by the D9 enzyme, with a 5-fold increase in activity from D5 with AMBA (41). The introduction of a larger cyclopropyl α-substituent (42) increases this difference, almost doubling D9’s activity and rendering D5 completely inactive. Comparable behavior is found with bicyclic substrates, as MAO-N D9 is highly active with cyclopentane (44a and 45b), cyclohexane (44b and 45c) and cycloheptane (45d and 46) structures. A single exception is pinpointed by the oxidation of diphenylmethylamine; the two bulky phenyl rings are best


26

Page 27 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

accommodated

by

MAO-N D11’s larger active pocket.40,41,59,64

NH2

NH2

NH2

X

OH n

R

(40) a) X = O: 45/45/15 b) X = S: 70/50/0

NH2 X

NH2

(41) 0/14/14/100/500/325

(42) a) R = Et: 0/170/0 b) R = C3H5: 0/920/210 c) R = CH2CHCH2: 10/25/10 d) R = Ph: 45/50

NH2

NH2

(43) a) n = 1: 0/7 b) n = 2: 0/3

NH2

n n

(44) a) n = 1, X = S: 5/165/5 b) n = 2, X = O: 30/1070/50

X (45) a) n = 1: 100/60/0 b) n = 2: 40/995/825 c) n = 3: 0/0.5/5/460/50 d) n = 4: 0/30/20

(46) 40/70/40

(47) a) X = O: 0/110/0 b) X = NMe: 5/10/0

Chart 4. Screening studies on the oxidation of α-aromatic primary amines by MAO-N variants. Color mapping was used to identify the respective enzymes: WT, D1, D3, D5, D9, D11.40,41,59,64

The oxidation of secondary and tertiary amines with MAO-N variants was also studied with limited results (Chart 5). Both D1 and D3 variants proved effective in the oxidation of

N-methylated benzylamine (48a) but not their α-methylated analog (48b) - D9 is the most active enzyme with this substrate. Moreover, the steric constrains of N,N-dimethyl-αmethylbenzylamine (49) proved too demanding for current enzymes to overcome. The introduction of a N-methoxy substituent has a noticeable negative impact on enzyme activity; the similar activities found for substrates (50) to (53) suggest that electronic


27

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 65

differences caused by the N-O bond may have a bigger impact on activity than any changes to the α substituents.40,41,64,65 NHMe

NMe2

NHOMe

R

(48) a) R = H: 339/170 b) R = Me: 0.5/1/15/70/0

(49) 2/0/0

NHOMe

(50) 13

NHOMe

NHOMe

F (51) 17

(52) 3

(53) 16

Chart 5. Screening studies on the oxidation of secondary and tertiary amines by MAO-N variants. Color mapping was used to identify the respective enzymes: WT, D1, D3, D5, D9, D11.40,41,64,65

Finally, we summarize results on the activity of MAO-N with cyclic amines presenting distinct substitution patterns. Pyrrolidine (54a) and piperidine (56a) analogs bearing a 1-phenyl substitution were readily oxidized by MAO-N D1 enzyme, with a gradual decrease in activity following the degree of mutation. Note the sharp increase in activity from (56a) to (56b), contradictory to previous results from Chart 6 that indicated reduced activity through successive N-methylation. Non-substituted bicyclic amines, on the other hand, seem to favor the use of either WT – (58) – or D3 – (57) – enzymes. Still, the introduction of additional ring systems and/or N-methylation greatly increased steric


28

Page 29 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

constraints,

shifting

optimal

activity

towards

MAO-N D11.40,41,59,64,66 In summary, the evolution of MAO-N has created a versatile enzyme library for amine oxidation, with substrates bearing different scaffolds and substitution patterns. Clearly, a decision is required during the design of new biocatalytic experiments to select the enzyme that best matches with the structural properties of the target substrate. For example, it may be advantageous to use the D11 variant when working with an amine containing two large α-substituents, but the WT enzyme may be more suitable for the oxidation of small linear amines. The section above tries to simplify the laborious search process associated to such a decision, providing a systematic overview of the scope of MAO-N.


29

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

R

H N

R

Page 30 of 65

Me N

R N

R

H N

n

(56)

(57)

a) R = Ph: 565/364/247 a) R = Ph: 134

a) R = H: 1/4

a) R = H: 284/450

b) R = CH2OMe: 26/14

b) R = Me: 565/364/247

b) R = Me: 15/40

(54)

(55) b) R = Py: 83 c) R = CH2OH: 31 d) R = CH2OMe: 26/14/1.5

MeO NH R

(58) 183/10/40

NR1

N R

R1

N R1

(59)

(60)

a) R = H, R1 = H: 0/3/10

a) R = H, R1 = H: 0/29/206 a) R = 7-OMe, R1 = H: 0/0/59

(61)

b) R = 6,7-OMe, R1 = H: 0/1 b) R = H, R1 = OH: 0/0/29 b) R = 7-OMe, R1 = OMe: 29/0/59 c) R = H, R1 = Me: 19 c) R = 7-OH, R1 = OH: 0/0/147 d) R = H, R1 = OH: 0/0/37

Chart 6. Screening studies on the oxidation of cyclic amines by MAO-N variants. Color mapping was used to identify the respective enzymes: WT, D1, D3, D5, D9, D11.40,41,59,64,66

Application of engineered MAONs: deracemization of chiral amines The high versatility and selectivity of MAO-N, supported by thorough substrate scope studies, has led to a range of synthetic applications. One opportunity addresses the challenge of the resolution of α-chiral amines, a common bottleneck in industrial synthesis that required low-yield crystallization or chemical resolution procedures. The Turner’s group embraced this challenge by developing a protocol, in 2002, for the deracemization of AMBA through repeated cycles of MAO-N D1-catalysed oxidation followed by nonselective chemical reduction (Scheme 5A).58 Several reducing agents were screened for


30

Page 31 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

this reaction, including sodium borohydride, Pd/C with ammonium formate, for catalytic transfer hydrogenation, and amine-borane complexes. Ammonia borane was by far the slowest reducing agent tested, requiring over 20 hours to achieve complete reduction. Yet, the increased stability, low cost and the low concentration required (1 equiv.) favored its use. Subsequently, NH3-BH3 was applied to the deracemisation of AMBA (62) with MAO-N D1 - 77% yield and 93% e.e. of (R) – and 1-methyltetrahydroisoquinoline (MTQ) (64) with MAO-N D3 - 71% yield and 99% e.e.. By immobilizing the enzyme on a Eupergit C resin, the authors increased the yield of (R)-MTQ to 95% within 96h.41 Since then, additional deracemization experiments were reported with a wide range of amines, seen in Scheme 5B. (R)-2-phenylpyrrolidine (63) was obtained with excellent e.e. and good yields using immobilized MAO-N D3.40 The nicotine analog 6559 and neurotoxin 6760 were also obtained in a near enantiopure fashion using MAO-N D5.67 On the other hand, the deracemization of mexiletine (71) with MAO-N AC1 proceeded with low yield, perhaps due to either imine hydrolysis or an inefficient extraction procedure.62 MAO-N D9 and D11 were applied to the deracemization of THIQ (66 and 74) and β-carboline (69, 70 and 72) derivatives bearing sterically demanding substituents in the α-carbon; exceptional

e.e.’s were obtained for these substrates, and their highly hydrophobic character allowed an improved extraction yield (>90%).60,61,68 An exception is provided by the diphenylmethylamine derivative 73, whose yield was compromised by fast imine hydrolysis in aqueous medium.60 Note that MAO-N LG-I-D11, not included in the referred


31

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 65

scheme, was also effective in the deracemisation of (R)-1-methyl, ethyl, isopropyl and phenyl THIQs with good e.e. (80-99%) and yields (73-86%).63 A solution was later proposed to overcome the competing hydrolysis of acyclic imines in aqueous medium by coupling a transaminase to the MAO-N/NH3-BH3 system. This approach relied on selective (S)-amine oxidation followed by rapid hydrolysis to its ketone analogue, which then underwent selective reductive amination to afford optically pure (R)amines. This procedure was demonstrated with a range of ring substituted AMBAs, achieving over 80% conversion and >99% e.e. for all compounds. Note, however, that transaminase inhibition by H2O2 required the addition of catalase for the reaction to proceed to completion.69


32

Page 33 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

A

NH2 R1

NH3-BH3

R2

NH2 R1

NH

R2

R1

R2

MAO - N

B NH2

NH2

H N (63)

(62) 77%  93% e.e.

R = H : 80%  98% e.e.

O

NH (64)

(71)

95%  99% e.e.

52%  96% e.e.

MeO

H N

N

MeO (66)

(65) -  97% e.e.

-  >97% e.e. X

N H (67) 85%  90% e.e.

Ar N H (68) X = C, N, O, S

NH N H (69) 93%  99% e.e.

NH2

NH Alk, Ph

N H

Cl

(72)

(73)

-  >92% e.e.

47 %  100% e.e.

HO N

H, OH

OH

N N H H (70) 95%  >99% e.e.

H, OMe (74) 77- 85 %  >97% e.e.

Scheme 5. The MAO-N-catalyzed deracemization of chiral amines: general procedure (A) and examples (B). First value indicates yield, followed by enantiomeric excess of the product.

Two alternative approaches were reported for the deracemisation of chiral amines with MAO-N. Foulkes et al. revisited the use of palladium as catalyst for the transfer


33

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 65

hydrogenation of imines with NH4COOH or H2, supporting Pd(0) in E. coli cells containing MAO-N D5.70 This allowed the combination of both oxidative and reductive catalytic capabilities within a single organism, limiting metal contamination of the final product. Note, however, that the reaction conditions of these two catalysts are not compatible, as MAO-N oxidation requires oxygen and Pd reduction requires a hydrogen gas atmosphere. To circumvent this obstacle, the authors report a one-pot reaction with separated cycles, controlled through flushing with either air or hydrogen, to obtain (R)-MTQ with 96% e.e – a similar performance to conventional Pd/C catalysis – and 10% higher yield. Alternatively, Köhler et al. devised an innovative homogeneous catalyst for this reaction through biomimetic design.71 By coupling a [Cp*Ir(biot-p-L)Cl] complex with streptavidin, a protein with high affinity towards biotin, the authors compartmentalized the metal catalyst within the protein environment, improving its compatibility with the oxidative enzyme. Consequently, its application in the deracemisation of both MTQ and 1cyclohexanepyrrolidine lead to enantiopure compounds within 8 hours of reaction time. It was highlighted that the addition of catalase is of utmost importance in this system, preventing poisoning of the iridium catalyst by H2O2. The kinetic resolution of amines was coupled with additional chemical or enzymatic routes to produce enantiopure amine derivatives. Eve et al. reported, in 2007, the kinetic resolution of N-methoxy-1-cyclohexylethylamine through selective oxidation with MAO-N D5, trapping the remaining (R) amine through reduction and treatment with triflic


34

Page 35 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

anhydride (44% yield and 99% e.e.).65 Remarkably, the oxime was also recovered (46%) and found to be exclusively in (E) configuration, revealing the previously unknown geometric isomer selective properties of this enzyme. Finally, the deracemisation of 1benzyltetrahydroisoquinolines was coupled with BBE to yield enantiopure berberines. By using the redox cycle to feed BBE with the appropriate substrate, enantiomerically pure tetracyclic compounds were obtained in high yields (up to 88%). A change of the reducing agent from ammonia-borane to morpholine-borane was crucial to the success of this cascade reaction, circumventing its incompatibility with the new enzyme.72

A cascade of possibilities: coupling MAO-N oxidation with chemical and enzymatic reactions The MAO-N catalyzed selective oxidation of amines offers chemists an easy tool for the generation of the highly reactive imine or iminium group. Further coupling of chemical and enzymatic methods, thereby allows the synthesis of diverse compounds. The synthesis


35

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 65

of (R)-harmacine (78) through an oxidative Pictet-Spengler reaction followed by deracemisation is a relevant example.60 Incubation of 75 with MAO-N D9 formed an iminium species (76) that underwent spontaneous cyclization to 77. This tetracyclic structure was promptly deracemized in the presence of the MAO-N/NH3-BH3 system, yielding the (R) isomer with 83% conversion and complete enantioselectivity (Scheme 6).

N N H (75)

N

1. MAO-N D9, O2 (1atm), 2. BH3-NH3 KPO4 buffer, 37ºC, 96+6h

H N H (78)

DKR

Oxid. N

N

N H

N H

(76)

(77)

H

Scheme 6. MAO-N catalysis in the synthesis of (R)-Harmacine

More complex systems were also developed using enzymatic cascade reactions. A transaminase (ATA113)/MAO-N D9 system was used for the one-step reductive amination of 1,4-diketones (79), followed by MAO-borane, to yield trans-1-methyl-4arylpyrrolidines (80) with complete conversion and enantio- and diastereoselectivity (Scheme 7).73 To reduce NAD+ cofactor cost, a lactate dehydrogenase/glucose dehydrogenase system was used for its regeneration. Likewise, cis-1-methyl-4phenylpyrrolidine was synthesized with an (R) selective ATA117 (65% yield, >99% de.).


36

Page 37 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

1. ATA113, GDH/LDH L-Alanine 2. MAO-N D5/D9, NH3-BH3 Ar HEPES buffer O 30ºC, 24+24h

O

Ar

(79)

H N

(80)

Scheme 7. Cascade reaction for the enantioselective synthesis of pyrrolidines

In the same year, Bechi et al. described the enzymatic oxidation of simple THIQs to cyclic lactams in a cascade reaction with MAO-N D9 and E. coli xanthine dehydrogenase (Scheme 8).74 This approach proved more favorable than chemoenzymatic MAON/H2O2/CuI catalysis, reaching over 90% conversion for both substrates. Still, xanthine dehydrogenase was not able to catalyze the conversion of THIQs containing NO2 in a

para position to the imine. MAO-N D9 E. coli XDH N

R Phosphate buffer 20ºC, 2h

(81) N R= H, Me

N

R

O (83)

R

(82)

Scheme 8. Formation of lactams by biocatalyzed double-oxidation of lactams from THIQs

Scalacci et al. recently reported a one-pot chemoenzymatic cascade for the synthesis of a diverse range of pyrroles (Scheme 9).75 It was envisioned that the ring-closing metathesis of diallyl amines followed by MAO-N D5 catalyzed oxidation/aromatization,


37

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 65

would obviate the need for expensive palladium or peroxide-based catalysis. A biphasic system was used to synthetize pyrroles with highly variable yields, depending on the size and

electronic

properties

of

the

N-substituent; the use of an aryl substituent seems highly favorable and the presence of a methyl group at C2 did not affect the enzyme’s performance. The authors applied this aromatization strategy to the synthesis of an antitubercular derivative, N-(pfluorophenyl)-3,4-di(N-phenylpiperazine)pyrrole (86).

F R N

Grubbs catalyst, MAO-N D5

R N N

iso-octane/Phosphate buffer 1:4 37ºC, 24h (84)

(85)

PhN

N

N

NPh

(86)

Scheme 9. Synthesis of pyrroles through ring-closing metathesis followed by aromatization.

An extension to this aromatization protocol was later reported by Toscani et al. for the double oxidation of tetrahydropyridines (THPs) (87) with MAO-N variants to form pyridines (89a) or pyridinium ions (89b) (Scheme 10).76 While MAO-N catalyzed the first C-N oxidation to (88), the aromatization could proceed via either O2-mediated oxidation or tautomerization followed by a second MAO-N oxidation. Good yields were obtained for a range of 3 or 4 aryl-substituted THPs when using MAO-N D9 in a two-solvent system


38

Page 39 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

with DMSO or isooctane; the authors suggested that the use of a miscible polar solvent such as DMSO caused slow catalyst inactivation, requiring further addition of enzyme after 9 hours. In particular, five distinct aryl-substituted pyridines (p-Me, OMe, F, Cl and Br) and two pyridinium ions (3-Ph and 4-Ph) were synthesized with yields reaching 99%. 2-substituted THPs were also partially oxidized with this system, with low yields limited by both steric constraints and the enzyme’s enantioselectivity. R N

R N

MAO-N D9, O2 Ar

(87) a) R = H; b) R = Me.

DMSO or Isooctane(1.4%) Phosphate buffer 37ºC, 18-24h

R N Ar

N or Ar

(88) a) R = H; b) R = Me.

Ar (89a) R = H

N Ph (89b) R = Me.

Scheme 10. Synthesis of pyridines and pyridinium ions through oxidation and aromatization of THPs.

Finally, MAO-N has been used to generate reactive imines for more diverse chemistry. The Orru group developed Ugi reactions for the synthesis of highly functionalized 3,4substituted prolyl peptides and N-aryl proline amides. First, desymmetrization of mesoamines with MAO-N D5 produced the corresponding (3S,7R) imines in good yields and near enantiomeric purity, following recrystallization.77 These chiral imines were reacted with different carboxylic acids and isocyanides, following a three-component Ugi reaction,


39

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 65

in overall good yields and diastereomeric ratios (Scheme 11A). Changes to the substituents in both reagents did not significantly impact the reaction’s yield or selectivity, thus validating the versatility of said method for the asymmetric synthesis of proline derivatives. N-aryl proline amides, on the other hand, were obtained through a UgiSmiles MCR, exchanging the carboxylic acid for an electron deficient phenolic compound (Scheme 11B).78 After significant changes to the reaction conditions, amides bearing a wide range of substituents were synthesized with overall excellent diastereomeric ratios and average to good yields. Note that in both cases the increased steric constraint of (93) over (92) lead to an increased selectivity but slightly reduced yield.

R1COOH, R2-NC

O N

DCM, RT, 24-48h MAO-N D5

or N H (90)

A

N H (91)

R1

HN O

R2

or N (92)

N (93)

OH X

X

O

R2-NC

N

R1

DCM, RT, 24-48h

B

X

HN X

R2

R1

Scheme 11. Multicomponent reactions in the synthesis of proline derivatives, through either Ugi (A) or Ugi-Smiles (B) multicomponent reactions.

A similar set of amine substrates was used in an oxidative Aza-Friedel-Crafts one-pot reaction for the synthesis of enantioenriched 2-substituted pyrrolidine derivatives (Scheme 12).79 High diastereoselectivity (>93%) with average to good yields were found


40

Page 41 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

for all the pyrrole derivatives undergoing addition. However, two equivalents of nucleophile were required to drive the reaction to completion. In addition, limitations were identified regarding the amine substrate: the presence of a 5,6-double bond in (97) led to a spontaneous decomposition to form pyrrole and furan, and N-methyl-(98) undergoes racemization through a hydride-transfer mechanism, leading to negligible selectivity in the oxidation step.

X or N H (90)

N H (94) X = C (95) X = O

MAO-N D5

or N (96)

X

pyrrole derivative

N

(one-pot, two-step) 17+24h

N H

Ar

(97) X = C (98) X = O

Scheme 12. C1 functionalization of pyrroles via Aza-Friedel Crafts reactions

Another application of MAO-N catalysis for C-C bond formation derives from a recent partnership between Merck and Codexis®, searching for an improved route for the synthesis of boceprevir (103).80 They developed a one-pot oxidative Strecker reaction based on MAO-N-catalyzed oxidative desymmetrization of (99) followed by selective addition of cyanide. However, the presence of cyanide in the reaction media quickly inactivated the enzyme and the accumulation of product in the reaction media caused irreversible enzyme inhibition. The solution was to “trap” the imine through a reversible addition of hydrogen sulfite, as seen in Scheme 13.


41

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 65

From the perspective of process development, this article provides a unique view on the challenges and opportunities of the scale-up of MAO-N catalysis. First and foremost, the authors highlight limitations in enzyme activity and thermal stability. Four rounds of enzyme engineering were performed, starting from MAO-N WT, to reach a hybrid enzyme (A. niger /A. oryzae) with 7-fold increase in activity and prolonged stability at 50ºC. Optimal reaction conditions were found at a pH value of 7.4, maintained through either NaOH titration or additional free amine, and a pure oxygen atmosphere (207 mmHg). More important, this variant could catalyze the aforementioned reaction at 200mM substrate concentration, as imine capture ensured the enzyme’s activity at high conversions. Continuous addition of substrate over the course of 20 hours lead to complete conversion of the amine, with a remarkable final concentration of 65g/L. Essential to the industrial application of this new biocatalytic route was the ~60% reduction in E-factor, attesting its high environmental sustainability .80 H N

(99)

N NaHSO3

MAO-N

(100)

H N

SO3

(101) O H N

H N

NaCN

H N

N

H N

CN

(102) NH2 O

O O

O (103)


42

Page 43 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

Scheme 13. MAO-N catalysis in the industrial synthesis of boceprevir (103).


43

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 65

Process optimization The sustainable synthesis of enantiopure compounds is still a demanding challenge in current industrial chemistry but the ability of MAO-N to selectively introduce chirality into a variety of amines makes it an attractive target for future industrial applications. Merck and Codexis® have demonstrated the application of this enzyme in the industrial production of an API. In this study, data regarding process development provided valuable knowledge for future scale-up experiments. A schematic of what a future industrial biocatalytic process with MAO-N may look like is also presented for clarification (Figure 3). Ramesh et al. first evaluated the effects of both fermentation and cell-washing on enzyme activity, using 3-azabicyclo[3,3,0]octane as a model substrate.81 They found that a shift in cultivation media from lysogeny broth (LB) to more complex media, such as Terrific Broth (TB), drastically decreased MAO-N’s initial activity. However, a simple wash of TBgrown cells in KPO4 buffer reversed this effect, leading to an over 2-fold increase in activity from LB-grown cells. The increased activity, combined with a higher fermentation yield, justifies the use of TB growth with cell washing in the scale-up of MAO-N expression, thus decoupling the fermentation and biocatalysis steps. Further information regarding optimal oxygenation and induction time was reported by Voulgaris et al. in 2011, showing optimal activity at fermentations reaching rather high levels of acetate (8 g/L).82 This corresponded to a late induction in an aerated reactor,


44

Page 45 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

where oxygen became limited (89%) with up to 50% yield.87 Subsequently, they searched this fungus’ genome for monoamine oxidases, identifying five enzymes with distinct activity, after heterologous expression in

E. coli BL21 (DE3).88 The most active one, MAO5-ZMU6, catalyzed the kinetic resolution of 2-methyltetrahydroquinolines bearing either methyl, methoxide or halogen substituents in the phenyl ring. Although all amines eventually obtained high e.e., the recurring higher than 50% conversion suggests that this enzyme is not completely selective and may slowly oxidize the (R) isomer as well. Interestingly, switching the parent fungal strain to this isolated enzyme also eliminated most selectivity in the oxidation of THQs bearing other 2-substituents. Previous work on fungal monoamine oxidases for the resolution of chiral amines also spurred interest in the development of a bacterial equivalent. Its improved expression in a bacterial vector and distinct substrate profile could provide an added value to a deracemization toolbox. Back in 2012, Leisch et al. made the first step towards this goal


47

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 65

by screening the substrate profile of a cyclohexylamine oxidase from Brevibacterium

oxydans IH-35A (CHAO)).89 This 50 kDa flavoprotein showed promising activity against a range of cycloalkylamine derivatives (104-107), with a remarkable increased activity in the methylated analogue 105b (Chart 7). Furthermore, CHAO was significantly active with some α-arylamines (41 and 42a) but showed limited results with all secondary and tertiary amines (107 and 108), including a tetrahydroquinoline (THQ) derivative (111). Following these results, the authors attempted the kinetic resolution of some amines with CHAO, separating the ketone byproduct via an alkaline extraction. Near enantiomerically pure

(R) isomers of 41, 42a, 109 and 110 were isolated with up to 42% isolated yield. By contrast, amine 109 could not be resolved as both isomers were oxidized at similar rates. The Turner deracemization protocol was applied to racemic 41, 109 and 110 to yield pure

(R) isomer with up to 86% yield; the remaining amine was converted to the alcohol due to competitive reduction of the ketone catalyzed by the amine-borane complex.

NH2

NH2

NH2

N

R H N

R (104) 6

(105) a) R=H: 100 b) R=CH3: 133

(106) 75

NH2

NH2

NH2

R

(41) R=CH3: 48 (42a) R= C2H5: 7

(109) 12

(108) traces

(107) a) R=H: 6 b) R=CH3: 0

(110) 27


H N

(111) traces

48

Page 49 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

Chart 7. The substrate scope of wild-type CHAO. The values correspond to relative activities (%), taking as reference the activity of WT CHAO with cyclohexylamine.

The crystal structure of this CHAO was latter published by Mirza et al., revealing a close similarity with MAO-N and other flavoenzymes; active-site features such as the characteristic aromatic cage are here maintained.90 This enzyme was the subject of mutagenesis to create an industrially viable enzyme. The Zhu group combined mutations on 13 amino acids close to the active site – F88, T198, L199, M226, Q233, Y321, F351, L353, I366, F368, P422 and Y459. The Y321, F368 and Y459 residues form an aromatic cage responsible for substrate binding, alike the one found on MAO-N, while T198, L199, M266 and F351 regulate the access to the active site through a hydrophobic entrance channel. Consequently, several mutants were discovered with distinct substrate profiles and increased activity (up to 30-fold increase from WT); some were applied in the deracemization of pharmaceutically-relevant substrates, represented in Chart 8. (R)-1aminotetraline (45c)91, a norsertraline precursor, as well as (111)92 and (113)93 were obtained with complete selectivity and good isolated yields in a reduced timeframe. However, the deracemization of (112) proceeded with lower e.e. and only moderate yield, possibly due to the increased steric hindrance of this secondary amine and an inefficient extraction protocol. Nonetheless, it stands as the only AO-catalyzed resolution of this irreversible inhibitor of MAO and promising Alzheimer drug. Last of all, D-valine (115) was obtained with exceptional yield and selectivity from its racemic ethyl ester, via


49

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 50 of 65

deracemisation followed by hydrolysis with HCl; this proof of concept opens the way to the development of a wide-ranging resolution of natural and non-natural amino acids.94 NH2

HN

H N

H N MeO

(R)-45c 76% yield (>99% e.e.) CHAO L353M O

(R)-111 76% yield (98% e.e.) CHAO T198F, L199S, M226F HCl (aq.)

OEt NH2 (R)-114 CHAO Y321I, M226T

(R)-112 51% yield (93% e.e.) CHAO Y321I, M226T

(R)-113 78% yield (>99% e.e.) CHAO Y321I

O OH NH2 (115) D-Valine 95% yield (>99% e.e.)

Chart 8. Deracemisation of chiral amines with mutated CHAO enzymes.

Finally, a recent article presented the development of a new library of CHAO variants for the deracemization of 2-substituted THQs; single mutations on 11 active-site amino acids led to the discovery of enantio-complementary variants with distinct activity (Chart 9). (S)THQs bearing isopropyl and cyclopropyl substitutions were obtained with good yields (>89%) using the Q233A variant. Notably, the single mutation of either L225 or Y450 to amino acids with reduced steric hindrance significantly changed the orientation of the bicyclic structure within the active site, leading to a total inversion of selectivity seen in all the structures here shown. This remains the best enzymatic method for the overall deracemization of 2-substituted THQs, precursors to several important natural alkaloids.


50

Page 51 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

Moreover, it reinforces a trend, first seen with MAO-N (see 72 and 74), that stereoinversion can be achieved through a change in the substrate’s orientation relative to the FAD cofactor.95

H N

H N

H N

116 L225A 97% e.e. (R) Q233A 96% e.e. (S)

117 L225A 91% e.e. (R) Q233A >99% e.e. (S)

118 L225A 94% e.e. (R)

H N

4

119 L225A >99% e.e. (R)

OMe H N

H N

120 L225A 99% e.e. (R)

121 Y459T 94% e.e. (R)

Chart 9. Engineered CHAO in the deracemization of 2-substituted THQs.

The stereoinversion of AO mutants via random or site-directed mutagenesis may provide a route to enantio-complementarity in the deracemization of chiral amines. However, one must search for paths of less resistance, namely through the screening and development of natural (S) selective AOs. 6-Hydroxy-nicotine oxidases (HDNO and HLNO) from

Arthrobacter nicotinovorans, key enzymes in the nicotine metabolism, are promising candidates. Although no study has so far focused on the broad substrate scope of the L selective HLNO, HDNO was screened and engineered with such a purpose.96 WT HDNO was active with nicotine analogues bearing hydroxyl substituents, longer N-alkyl chains or a piperidine ring but could not oxidize other common substrates of MAO-N, such as α-


51

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 52 of 65

MBA or THIQ. Heath et al. thus performed combinatorial active site testing to reach an optimal versatility with a E350L/E352D double mutant, applied to the Turner deracemization of a broad range of cyclic amines here shown (Chart 10). Despite its increased substrate scope, this variant was only active with secondary and tertiary amines, in contrast to MAO-N. In a distinct paper, the combination of this double mutant with an imine reductase, replacing the common borane reduction, yielded diverse substituted piperidines, and even some thiomorpholines, with over 99% e.e..67

N R1

N R1

R2

N

(122) a) R1= Me, R2= OH: >99% b) R1= Me, R2= H: >99% c) R1= H, R2= H: >99%

NH N

(123) a) R1= H: >99% b) R1= Me: 84/97%

MeO

MeO

NH

NH

MeO

(125) 84% (R)

(124) >99%

(126) 81%

N

MeO

(66) >99%

Chart 10. HDNO variants in the deracemisation of chiral amines. Color mapping was used to identify the enzymes: WT, E350L/E352D.

6. Future perspectives and final remarks The value of monoamine oxidases as sustainable catalysts for amine oxidation is clearly established throughout this review. These enzymes are highly active with a variety of


52

Page 53 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

primary, secondary and tertiary amines, tolerate the presence of additional functional groups and can be engineered for increased activity or scope. They can also be employed in bio-bio or bio-chemo cascade reactions for the resolution of chiral amines or its Cα-H functionalization, via imine or iminium intermediates. However, we are only at the beginning of the road; the branches continue to multiply and with it the horizon of future MAO applications in biocatalysis. Microbial replication of the metabolic pathways described in Section 2 is still fairly limited to dopamine oxidation and, even then, the goal of an efficient synthesis of morphine remains elusive. The exploration of additional metabolisms can provide efficient synthetic routes for diverse imidazole, indole and quinoline-derived compounds; their importance in pharma was already reviewed97–99 In a similar way, the coupling of amine oxidation with additional chemical routes, particularly in the Cα-H functionalization of amines through metathesis, Ugi or Aza-Friedel-Crafts reactions, remains mostly unexplored. The increased reactivity of the imine group offers countless opportunities for further functionalization through nucleophilic additions to the α carbon; aza-Cope-Mannich (A)100, aza-Heck (C)101, azaPrins (B)102 or aza-Diels-Alder (D)103 reactions are yet to be reported (Scheme 14).


53

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(A)

R2 N

HO

R2 R

N

R2

R

(B)

N

HO

Ph

N

R2

R OHC

N

HO

Ac

Page 54 of 65

(C)

N

TMSO

R2

Ph R1

R

N

(D)

+

R2 R1

R3

O N R3

Scheme 14. Exploring the reactivity of imine derivatives for amine functionalization through the following reactions: A – aza-Cope-Mannich, B – aza-Prins, C – aza-Heck, D – aza-Diels-Alder.

The engineering of MAO-N, in particular, is perhaps the most well-developed aspect of MAO research. Most residues within the active site have been mutated and over twelve major variants were presented in Section 4. Yet, there is room for improvement: the oxidation of tertiary amines is slow and limited to small alkyl chains and the presence of nucleophilic substituents on cyclic aromatic structures severely hinders activity. Breaking these limitations may facilitate the coupling of MAO-N oxidation to chemical reactions that require protected imines and, on the other hand, allow the resolution of α-aromatic amines containing a catechol group, common in bioactive compounds. We hypothesize that these goals may be achieved by either mixing and combining current mutations into novel variants or, more interestingly, engineering MAO-N’s secondary structure to obtain subtle conformational shifts that may alter activity and selectivity. Ultimately, it would be ideal to


54

Page 55 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

apply rational design to this enzyme’s evolution. This would require a tremendous knowledge of the protein structure, substrate orientation and the steric and electronic impact of each mutation in overall activity. The major long-term objective for research in MAO catalysis must be further application towards industrial processes, overcoming current enzyme inactivation and inhibition restraints by both substrate (amine) and product (imine). For this, additional engineering experiments aiming at increased enzyme stability can be made or, more simply, versatile industrial processes developed to circumvent these obstacles; the continuous addition of substrate to the reactor and the removal of product via recirculating resin columns are the most viable options. Ideally, a flow process using MAO immobilized in a column could be also applied to limit local substrate and product concentrations. In summary, it has been shown that MAO catalysis can be successfully applied for the laboratory scale resolution and functionalization of chiral amines; work by Merck and Codexis® has also paved the way towards industrial implementation. It is our belief that the field will continue to expand, and with it the tools available for the development and optimization of MAO-catalyzed processes, leading to a widespread use of these enzymes in industrial amine oxidation. AUTHOR INFORMATION Corresponding Author


55

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 56 of 65

*[email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no conflicting interest. ACKNOWLEDGEMENTS Vasco F. Batista is supported by the Fundação para a Ciência e a Tecnologia (FCT) through the grant PD/BD/135099/2017. J.L.G. thanks the support of the BIOINTENSE project financed through the European Union 7th Framework Programme (Grant agreement no. 312148). Artur M. S. Silva and Diana C. G. A. Pinto are members of the University of Aveiro and belong to the Organic Chemistry Research Unit (QOPNA). QOPNA are subsidized by Portuguese National Funds through the FCT/ Ministério da Educação e Ciência (MEC) (UID/QUI/00062/2013) and are co-financed by FEDER funds under the PT2020 Partnership agreement. N.J.T. acknowledges the ERC for the award of an Advanced Grant. REFERENCES


56

Page 57 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

(1)

Sigman, R. OECD Environmental Outlook; OECD Environmental Outlook; OECD Publishing: Paris, France, 2001.

(2)

Sheldon, R. A. The E Factor: Fifteen Years On. Green Chem. 2007, 9, 1273.

(3)

Sanderson, K. Chemistry: It’s Not Easy Being Green. Nature 2011, 469, 18–20.

(4)

Albini, A.; Protti, S. Activation of Chemical Substrates in Green Chemistry. In Paradigms in Green Chemistry and Technology; Springer International Publishing: London, United Kingdom, 2016; 25–61.

(5)

Bornscheuer, U. T.; Huisman, G. W.; Kazlauskas, R. J.; Lutz, S.; Moore, J. C.; Robins, K. Engineering the Third Wave of Biocatalysis. Nature 2012, 485, 185–194.

(6)

Sheldon, R. A.; Woodley, J. M. Role of Biocatalysis in Sustainable Chemistry. Chem. Rev. 2018, 118, 801–838.

(7)

Savile, C. K.; Janey, J. M.; Mundorff, E. C.; Moore, J. C.; Tam, S.; Jarvis, W. R.; Colbeck, J. C.; Krebber, A.; Fleitz, F. J.; Brands, J.; Devine, P. N.; Huisman, G. W.; Hughes, G. J. Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin Manufacture. Science. 2010, 329, 305–309.

(8)

Xie, X.; Tang, Y. Efficient Synthesis of Simvastatin by Use of Whole-Cell Biocatalysis. Appl. Environ. Microbiol. 2007, 73, 2054–2060.

(9)

Paddon, C. J.; Keasling, J. D. Semi-Synthetic Artemisinin: A Model for the Use of Synthetic Biology in Pharmaceutical Development. Nat. Rev. Microbiol. 2014, 12, 355–367.

(10)

Scott, R.; Neville, S.; Zhao, L.; Ran, N. Compounds and Processes to Prepare Chiral Intermediates for Synthesis of Paroxetine. WO 2009/005647 A2, 2007.

(11)

Gong, J.-S.; Lu, Z.-M.; Li, H.; Zhou, Z.-M.; Shi, J.-S.; Xu, Z.-H. Metagenomic Technology and Genome Mining: Emerging Areas for Exploring Novel Nitrilases. Appl. Microbiol. Biotechnol. 2013, 97, 6603–6611.

(12)

Rosano, G. L.; Ceccarelli, E. A. Recombinant Protein Expression in Escherichia coli: Advances and Challenges. Front. Microbiol. 2014, 5, 1–17.

(13)

Hammer, S. C.; Knight, A. M.; Arnold, F. H. Design and Evolution of Enzymes for NonNatural Chemistry. Curr. Opin. Green Sustain. Chem. 2017, 7, 23–30.

(14)

France, S.; Guerin, D. J.; Miller, S. J.; Lectka, T. Nucleophilic Chiral Amines as Catalysts in Asymmetric Synthesis. Chem. Rev. 2003, 103, 2985–3012.

(15)

Chiral resolving agents http://www.sigmaaldrich.com/chemistry/chemistryproducts.html?TablePage=16265015 (accessed Oct 5, 2017).

(16)

Gellad, W. F.; Choi, P.; Mizah, M.; Good, C. B.; Kesselheim, A. S. Assessing the Chiral Switch: Approval and Use of Single-Enantiomer Drugs, 2001 to 2011. Am. J. Manag. Care 2014, 20, e90–e97.


57

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 58 of 65

(17)

Nugent, T. C.; El-Shazly, M. Chiral Amine Synthesis - Recent Developments and Trends for Enamide Reduction, Reductive Amination, and Imine Reduction. Adv. Synth. Catal. 2010, 352, 753–819.

(18)

Mane, S. Racemic Drug Resolution: A Comprehensive Guide. Anal. Methods 2016, 8, 7567–7586.

(19)

Woelfle, M.; Seerden, J.-P.; de Gooijer, J.; Pouwer, K.; Olliaro, P.; Todd, M. H. Resolution of Praziquantel. PLoS Negl. Trop. Dis. 2011, 5, e1260.

(20)

France, S. P.; Hussain, S.; Hill, A. M.; Hepworth, L. J.; Howard, R. M.; Mulholland, K. R.; Flitsch, S. L.; Turner, N. J. One-Pot Cascade Synthesis of Mono- and Disubstituted Piperidines and Pyrrolidines Using Carboxylic Acid Reductase (CAR), ω-Transaminase (ωTA), and Imine Reductase (IRED) Biocatalysts. ACS Catal. 2016, 6, 3753–3759.

(21)

Ye, L. J.; Toh, H. H.; Yang, Y.; Adams, J. P.; Snajdrova, R.; Li, Z. Engineering of Amine Dehydrogenase for Asymmetric Reductive Amination of Ketone by Evolving Rhodococcus Phenylalanine Dehydrogenase. ACS Catal. 2015, 5, 1119–1122.

(22)

Rowles, I.; Groenendaal, B.; Binay, B.; Malone, K. J.; Willies, S. C.; Turner, N. J. Engineering of Phenylalanine Ammonia Lyase from Rhodotorula graminis for the Enhanced Synthesis of Unnatural L-Amino Acids. Tetrahedron 2016, 72, 7343–7347.

(23)

Wetzl, D.; Gand, M.; Ross, A.; Müller, H.; Matzel, P.; Hanlon, S. P.; Müller, M.; Wirz, B.; Höhne, M.; Iding, H. Asymmetric Reductive Amination of Ketones Catalyzed by Imine Reductases. ChemCatChem 2016, 8, 2023–2026.

(24)

Thalén, L. K.; Zhao, D.; Sortais, J.-B.; Paetzold, J.; Hoben, C.; Bäckvall, J.-E. A Chemoenzymatic Approach to Enantiomerically Pure Amines Using Dynamic Kinetic Resolution: Application to the Synthesis of Norsertraline. Chem. - A Eur. J. 2009, 15, 3403– 3410.

(25)

Mondovì, B.; Agrò, A. F. Structure and Function of Amine Oxidases. In Structure and Function Relationships in Biochemical Systems. Advances in Experimental Medicine and Bioligy; Bossa, F., Chiancone, E., Finnazi-Agrò, A., Eds.; Springer: New York, 1982; 141– 153.

(26)

Busto, E.; Simon, R. C.; Richter, N.; Kroutil, W. Enzymatic Synthesis of Chiral Amines Using ω-Transaminases, Amine Oxidases, and the Berberine Bridge Enzyme. In Green Biocatalysis; John Wiley & Sons, Inc: Hoboken, NJ, 2016; 17–57.

(27)

Hare, M. L. Tyramine Oxidase: A New Enzyme System in Liver. Biochem. J. 1928, 22, 968–979.

(28)

Slotkin, T. A. Mary Bernheim and the Discovery of Monoamine Oxidase. Brain Res. Bull. 1999, 50, 373.

(29)

MAO - The Mother of All Amine Oxidases, 1st ed.; Finberg, J., Youdim, M., Riederer, P., Tipton, K., Eds.; Springer-Verlag Wien GmbH, 1998.


58

Page 59 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

(30)

Tipton, K. F.; Boyce, S.; O’Sullivan, J.; Davey, G. P.; Healy, J. Monoamine Oxidases: Certainties and Uncertainties. Curr. Med. Chem. 2004, 11, 1965–1982.

(31)

Ramsay, R. Monoamine Oxidases: The Biochemistry of the Proteins As Targets in Medicinal Chemistry and Drug Discovery. Curr. Top. Med. Chem. 2012, 12, 2189–2209.

(32)

Gaweska, H.; Fitzpatrick, P. F. Structures and Mechanism of the Monoamine Oxidase Family. Biomol. Concepts 2011, 2, 116–125.

(33)

Edmondson, D. E.; Binda, C. Monoamine Oxidases. In Subcellular Biochemistry; Springer Nature Singapore Pte Lda: Singapore, Singapore, 2018; Vol. 87, 117–139.

(34)

Iacovino, L. G.; Magnani, F.; Binda, C. The Structure of Monoamine Oxidases: Past, Present, and Future. J. Neural Transm. 2018, 125, 1567-1579.

(35)

Chajkowski-Scarry, S.; Rimoldi, J. M. Monoamine Oxidase A and B Substrates: Probing the Pathway for Drug Development. Future Med. Chem. 2014, 6, 697–717.

(36)

Orru, R.; Aldeco, M.; Edmondson, D. E. Do MAO A and MAO B Utilize the Same Mechanism for the C–H Bond Cleavage Step in Catalysis? Evidence Suggesting Differing Mechanisms. J. Neural Transm. 2013, 120, 847–851.

(37)

Silverman, R. B. Radical Ideas about Monoamine Oxidase. Acc. Chem. Res. 1995, 28, 335– 342.

(38)

Vianello, R.; Repič, M.; Mavri, J. How Are Biogenic Amines Metabolized by Monoamine Oxidases? European J. Org. Chem. 2012, 2012, 7057–7065.

(39)

Cakir, K.; Erdem, S. S.; Atalay, V. E. ONIOM Calculations on Serotonin Degradation by Monoamine Oxidase B: Insight into the Oxidation Mechanism and Covalent Reversible Inhibition. Org. Biomol. Chem. 2016, 14, 9239–9252.

(40)

Carr, R.; Alexeeva, M.; Enright, A.; Eve, T. S. C.; Dawson, M. J.; Turner, N. J. Directed Evolution of an Amine Oxidase Possessing Both Broad Substrate Specificity and High Enantioselectivity. Angew. Chemie Int. Ed. 2003, 42, 4807–4810.

(41)

Carr, R.; Alexeeva, M.; Dawson, M. J.; Gotor-Fernández, V.; Humphrey, C. E.; Turner, N. J. Directed Evolution of an Amine Oxidase for the Preparative Deracemisation of Cyclic Secondary Amines. ChemBioChem 2005, 6, 637–639.

(42)

Sadler, J. C.; Currin, A.; Kell, D. B. Ultra-High Throughput Functional Enrichment of Large Monoamine Oxidase (MAO-N) Libraries by Fluorescence Activated Cell Sorting. Analyst 2018, 143, 4747–4755.

(43)

KEGG PATHWAY Database http://www.genome.jp/kegg/pathway.html (accessed Oct 16, 2017).

(44)

Minami, H.; Kim, J.-S.; Ikezawa, N.; Takemura, T.; Katayama, T.; Kumagai, H.; Sato, F. Microbial Production of Plant Benzylisoquinoline Alkaloids. Proc. Natl. Acad. Sci. U. S. A.


59

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 60 of 65

2008, 105, 7393–7398. (45)

Nakagawa, A.; Matsuzaki, C.; Matsumura, E.; Koyanagi, T.; Katayama, T.; Yamamoto, K.; Sato, F.; Kumagai, H.; Minami, H. (R,S)-Tetrahydropapaveroline Production by Stepwise Fermentation Using Engineered Escherichia coli. Sci. Rep. 2015, 4, 6695.

(46)

Nakagawa, A.; Minami, H.; Kim, J.-S.; Koyanagi, T.; Katayama, T.; Sato, F.; Kumagai, H. Bench-Top Fermentative Production of Plant Benzylisoquinoline Alkaloids Using a Bacterial Platform. Bioengineered 2012, 3, 49–53.

(47)

Nakagawa, A.; Minami, H.; Kim, J.-S.; Koyanagi, T.; Katayama, T.; Sato, F.; Kumagai, H. A Bacterial Platform for Fermentative Production of Plant Alkaloids. Nat. Commun. 2011, 2, 326.

(48)

Hawkins, K. M.; Smolke, C. D. Production of Benzylisoquinoline Alkaloids in Saccharomyces cerevisiae. Nat. Chem. Biol. 2008, 4, 564–573.

(49)

Sato, F. .; Minami, H. Method for Producing Alkaloids. US8993280, 2015.

(50)

Nakagawa, A.; Matsumura, E.; Koyanagi, T.; Katayama, T.; Kawano, N.; Yoshimatsu, K.; Yamamoto, K.; Kumagai, H.; Sato, F.; Minami, H. Total Biosynthesis of Opiates by Stepwise Fermentation Using Engineered Escherichia coli. Nat. Commun. 2016, 7, 10390.

(51)

Hoover, L. K.; Moo-Young, M.; Legge, R. L. Biotransformation of Dopamine to Norlaudanosoline by Aspergillus niger. Biotechnol. Bioeng. 1991, 38, 1029–1033.

(52)

Yamada, H.; Adachi, O.; Ogata, K. Amine Oxidases of Microorganisms. Part IV: Further Properties of Amine Oxidase of Aspergillus niger. Agric. Biol. Chem. 1965, 29, 912–917.

(53)

Schilling, B.; Lerch, K. Amine Oxidases from Aspergillus niger : Identification of a Novel Flavin-Dependent Enzyme. Biochim. Biophys. Acta - Gen. Subj. 1995, 1243, 529–537.

(54)

Mitchell, D. J.; Nikolic, D.; Rivera, E.; Sablin, S. O.; Choi, S.; van Breemen, R. B.; Singer, T. P.; Silverman, R. B. Spectrometric Evidence for the Flavin−1-Phenylcyclopropylamine Inactivator Adduct with Monoamine Oxidase N. Biochemistry 2001, 40, 5447–5456.

(55)

Schilling, B.; Lerch, K. Cloning, Sequencing and Heterologous Expression of the Monoamine Oxidase Gene from Aspergillus niger. MGG Mol. Gen. Genet. 1995, 247, 430– 438.

(56)

Atkin, K. E.; Reiss, R.; Turner, N. J.; Brzozowski, A. M.; Grogan, G. Cloning, Expression, Purification, Crystallization and Preliminary X-Ray Diffraction Analysis of Variants of Monoamine Oxidase from Aspergillus niger. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2008, 64, 182–185.

(57)

Atkin, K. E.; Reiss, R.; Koehler, V.; Bailey, K. R.; Hart, S.; Turkenburg, J. P.; Turner, N. J.; Brzozowski, A. M.; Grogan, G. The Structure of Monoamine Oxidase from Aspergillus niger Provides a Molecular Context for Improvements in Activity Obtained by Directed Evolution. J. Mol. Biol. 2008, 384, 1218–1231.


60

Page 61 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

(58)

Alexeeva, M.; Enright, A.; Dawson, M. J.; Mahmoudian, M.; Turner, N. J. Deracemization of α-Methylbenzylamine Using an Enzyme Obtained by In Vitro Evolution. Angew. Chemie Int. Ed. 2002, 41, 3177–3180.

(59)

Dunsmore, C. J.; Carr, R.; Fleming, T.; Turner, N. J. A Chemo-Enzymatic Route to Enantiomerically Pure Cyclic Tertiary Amines. J. Am. Chem. Soc. 2006, 128, 2224–2225.

(60)

Ghislieri, D.; Green, A. P.; Pontini, M.; Willies, S. C.; Rowles, I.; Frank, A.; Grogan, G.; Turner, N. J. Engineering an Enantioselective Amine Oxidase for the Synthesis of Pharmaceutical Building Blocks and Alkaloid Natural Products. J. Am. Chem. Soc. 2013, 135, 10863–10869.

(61)

Rowles, I.; Malone, K. J.; Etchells, L. L.; Willies, S. C.; Turner, N. J. Directed Evolution of the Enzyme Monoamine Oxidase (MAO-N): Highly Efficient Chemo-Enzymatic Deracemisation of the Alkaloid (±)-Crispine A. ChemCatChem 2012, 4, 1259–1261.

(62)

Chen, Z.; Ma, Y.; He, M.; Ren, H.; Zhou, S.; Lai, D.; Wang, Z.; Jiang, L. Semi-Rational Directed Evolution of Monoamine Oxidase for Kinetic Resolution of rac-Mexiletine. Appl. Biochem. Biotechnol. 2015, 176, 2267–2278.

(63)

Li, G.; Yao, P.; Gong, R.; Li, J.; Liu, P.; Lonsdale, R.; Wu, Q.; Lin, J.; Zhu, D.; Reetz, M. T. Simultaneous Engineering of an Enzyme’s Entrance Tunnel and Active Site: The Case of Monoamine Oxidase MAO-N. Chem. Sci. 2017, 8, 4093–4099.

(64)

Herter, S.; Medina, F.; Wagschal, S.; Benhaïm, C.; Leipold, F.; Turner, N. J. Mapping the Substrate Scope of Monoamine Oxidase (MAO-N) as a Synthetic Tool for the Enantioselective Synthesis of Chiral Amines. Bioorg. Med. Chem. 2017, 26, 1338-1346.

(65)

Eve, T. S. C.; Wells, A.; Turner, N. J. Enantioselective Oxidation of O-Methyl-NHydroxylamines Using Monoamine Oxidase N as Catalyst. Chem. Commun. 2007, 2, 1530.

(66)

Schrittwieser, J. H. .; Groenendaal, B. .; Willies, S. C. .; Ghislieri, D. .; Rowles, I. .; Resch, V. .; Sattler, J. H. .; Fischereder, E.-M. .; Grischek, B. .; Lienhart, W.-D. .; Turner, N. J. .; Kroutil, W. . Deracemisation of Benzylisoquinoline Alkaloids Employing Monoamine Oxidase Variants. Catal. Sci. Technol. 2014, 4, 3657–3664.

(67)

Heath, R. S.; Pontini, M.; Hussain, S.; Turner, N. J. Combined Imine Reductase and Amine Oxidase Catalyzed Deracemization of Nitrogen Heterocycles. ChemCatChem 2016, 8, 117– 120.

(68)

Ghislieri, D.; Houghton, D.; Green, A. P.; Willies, S. C.; Turner, N. J. Monoamine Oxidase (MAO-N) Catalyzed Deracemization of Tetrahydro-β-Carbolines: Substrate Dependent Switch in Enantioselectivity. ACS Catal. 2013, 3, 2869–2872.

(69)

O’Reilly, E.; Iglesias, C.; Turner, N. J. Monoamine Oxidase-ω-Transaminase Cascade for the Deracemisation and Dealkylation of Amines. ChemCatChem 2014, 6, 992–995.

(70)

Foulkes, J. M.; Malone, K. J.; Coker, V. S.; Turner, N. J.; Lloyd, J. R. Engineering a Biometallic Whole Cell Catalyst for Enantioselective Deracemization Reactions. ACS Catal. 2011, 1, 1589–1594.


61

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 62 of 65

(71)

Köhler, V.; Wilson, Y. M.; Dürrenberger, M.; Ghislieri, D.; Churakova, E.; Quinto, T.; Knörr, L.; Häussinger, D.; Hollmann, F.; Turner, N. J.; Ward, T. R. Synthetic Cascades Are Enabled by Combining Biocatalysts with Artificial Metalloenzymes. Nat. Chem. 2012, 5, 93–99.

(72)

Schrittwieser, J. H.; Groenendaal, B.; Resch, V.; Ghislieri, D.; Wallner, S.; Fischereder, E.M.; Fuchs, E.; Grischek, B.; Sattler, J. H.; Macheroux, P.; Turner, N. J.; Kroutil, W. Deracemization By Simultaneous Bio-Oxidative Kinetic Resolution and Stereoinversion. Angew. Chemie Int. Ed. 2014, 53, 3731–3734.

(73)

O’Reilly, E.; Iglesias, C.; Ghislieri, D.; Hopwood, J.; Galman, J. L.; Lloyd, R. C.; Turner, N. J. A Regio- and Stereoselective ω-Transaminase/Monoamine Oxidase Cascade for the Synthesis of Chiral 2,5-Disubstituted Pyrrolidines. Angew. Chemie Int. Ed. 2014, 53, 2447– 2450.

(74)

Bechi, B.; Herter, S.; McKenna, S.; Riley, C.; Leimkühler, S.; Turner, N. J.; Carnell, A. J. Catalytic Bio–chemo and Bio–bio Tandem Oxidation Reactions for Amide and Carboxylic Acid Synthesis. Green Chem. 2014, 16, 4524–4529.

(75)

Scalacci, N.; Black, G. W.; Mattedi, G.; Brown, N. L.; Turner, N. J.; Castagnolo, D. Unveiling the Biocatalytic Aromatizing Activity of Monoamine Oxidases MAO-N and 6HDNO: Development of Chemoenzymatic Cascades for the Synthesis of Pyrroles. ACS Catal. 2017, 7, 1295–1300.

(76)

Toscani, A.; Risi, C.; Black, G. W.; Brown, N. L.; Shaaban, A.; Turner, N. J.; Castagnolo, D. Monoamine Oxidase (MAO-N) Whole Cell Biocatalyzed Aromatization of 1,2,5,6Tetrahydropyridines into Pyridines. ACS Catal. 2018, 8, 8781–8787.

(77)

Znabet, A.; Ruijter, E.; de Kanter, F. J. J.; Köhler, V.; Helliwell, M.; Turner, N. J.; Orru, R. V. A. Highly Stereoselective Synthesis of Substituted Prolyl Peptides Using a Combination of Biocatalytic Desymmetrization and Multicomponent Reactions. Angew. Chemie Int. Ed. 2010, 49, 5289–5292.

(78)

Znabet, A.; Blanken, S.; Janssen, E.; de Kanter, F. J. J.; Helliwell, M.; Turner, N. J.; Ruijter, E.; Orru, R. V. A. Stereoselective Synthesis of N-Aryl Proline Amides by Biotransformation–Ugi-Smiles Sequence. Org. Biomol. Chem. 2012, 10, 941–944.

(79)

de Graaff, C.; Oppelaar, B.; Péruch, O.; Vande Velde, C. M. L.; Bechi, B.; Turner, N. J.; Ruijter, E.; Orru, R. V. A. Stereoselective Monoamine Oxidase-Catalyzed Oxidative AzaFriedel-Crafts Reactions of meso-Pyrrolidines in Aqueous Buffer. Adv. Synth. Catal. 2016, 358, 1555–1560.

(80)

Li, T.; Liang, J.; Ambrogelly, A.; Brennan, T.; Gloor, G.; Huisman, G.; Lalonde, J.; Lekhal, A.; Mijts, B.; Muley, S.; Newman, L.; Tobin, M.; Wong, G.; Zaks, A.; Zhang, X. Efficient, Chemoenzymatic Process for Manufacture of the Boceprevir Bicyclic [3.1.0]Proline Intermediate Based on Amine Oxidase-Catalyzed Desymmetrization. J. Am. Chem. Soc. 2012, 134, 6467–6472.

(81)

Ramesh, H.; Zajkoska, P.; Rebroš, M.; Woodley, J. M. The Effect of Cultivation Media and


62

Page 63 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

Washing Whole-Cell Biocatalysts on Monoamine Oxidase Catalyzed Oxidative Desymmetrization of 3-Azabicyclo[3,3,0]Octane. Enzyme Microb. Technol. 2016, 83, 7– 13. (82)

Voulgaris, I.; Arnold, S. A.; Speight, R.; Harvey, L. M.; McNeil, B. Effects of Dissolved Oxygen Availability and Culture Biomass at Induction upon the Intracellular Expression of Monoamine Oxidase by Recombinant E. coli in Fed Batch Bioprocesses. Process Biochem. 2011, 46, 721–729.

(83)

Markošová, K.; Camattari, A.; Rosenberg, M.; Glieder, A.; Turner, N. J.; Rebroš, M. Cloning and Upscale Production of Monoamine Oxidase N (MAO-N D5) by Pichia pastoris. Biotechnol. Lett. 2018, 40, 127–133.

(84)

Ramesh, H.; Woodley, J. M. Process Characterization of a Monoamine Oxidase. J. Mol. Catal. B Enzym. 2014, 106, 124–131.

(85)

Zajkoska, P.; Rosenberg, M.; Heath, R.; Malone, K. J.; Stloukal, R.; Turner, N. J.; Rebroš, M. Immobilised Whole-Cell Recombinant Monoamine Oxidase Biocatalysis. Appl. Microbiol. Biotechnol. 2015, 99, 1229–1236.

(86)

Markošová, K.; Dolejš, I.; Stloukal, R.; Rios-Solis, L.; Rosenberg, M.; Micheletti, M.; Lye, G. J.; Turner, N. J.; Rebroš, M. Immobilisation and Kinetics of Monoamine Oxidase (MAON-D5) Enzyme in Polyvinyl Alcohol Gels. J. Mol. Catal. B Enzym. 2016, 129, 69–74.

(87)

Qin, L.; Zheng, D.; Cui, B.; Wan, N.; Zhou, X.; Chen, Y. Bio-Mediated Oxidative Resolution of Racemic 2-Substituted 1,2,3,4-Tetrahydroquinolines. Tetrahedron Lett. 2016, 57, 2403–2405.

(88)

Deng, G.; Wan, N.; Qin, L.; Cui, B.; An, M.; Han, W.; Chen, Y. Deracemization of PhenylSubstituted 2-Methyl-1,2,3,4-Tetrahydroquinolines by a Recombinant Monoamine Oxidase from Pseudomonas monteilii ZMU-T01. ChemCatChem 2018, 10, 2374–2377.

(89)

Leisch, H.; Grosse, S.; Iwaki, H.; Hasegawa, Y.; Lau, P. C. K. Cyclohexylamine Oxidase as a Useful Biocatalyst for the Kinetic Resolution and Dereacemization of Amines. Can. J. Chem. 2012, 90, 39–45.

(90)

Mirza, I. A.; Burk, D. L.; Xiong, B.; Iwaki, H.; Hasegawa, Y.; Grosse, S.; Lau, P. C. K.; Berghuis, A. M. Structural Analysis of a Novel Cyclohexylamine Oxidase from Brevibacterium Oxydans IH-35A. PLoS One 2013, 8, e60072.

(91)

Li, G.; Ren, J.; Iwaki, H.; Zhang, D.; Hasegawa, Y.; Wu, Q.; Feng, J.; Lau, P. C. K.; Zhu, D. Substrate Profiling of Cyclohexylamine Oxidase and Its Mutants Reveals New Biocatalytic Potential in Deracemization of Racemic Amines. Appl. Microbiol. Biotechnol. 2014, 98, 1681–1689.

(92)

Li, G.; Ren, J.; Yao, P.; Duan, Y.; Zhang, H.; Wu, Q.; Feng, J.; Lau, P. C. K.; Zhu, D. Deracemization of 2-Methyl-1,2,3,4-Tetrahydroquinoline Using Mutant Cyclohexylamine Oxidase Obtained by Iterative Saturation Mutagenesis. ACS Catal. 2014, 4, 903–908.

(93)

Li, G.; Yao, P.; Cong, P.; Ren, J.; Wang, L.; Feng, J.; Lau, P. C. K.; Wu, Q.; Zhu, D. New


63

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 64 of 65

Recombinant Cyclohexylamine Oxidase Variants for Deracemization of Secondary Amines by Orthogonally Assaying Designed Mutants with Structurally Diverse Substrates. Sci. Rep. 2016, 6, 24973. (94)

Gong, R.; Yao, P.; Chen, X.; Feng, J.; Wu, Q.; Lau, P. C. K.; Zhu, D. Accessing D-Valine Synthesis by Improved Variants of Bacterial Cyclohexylamine Oxidase. ChemCatChem 2018, 10, 387–390.

(95)

Yao, P.; Cong, P.; Gong, R.; Li, J.; Li, G.; Ren, J.; Feng, J.; Lin, J.; Lau, P. C. K.; Wu, Q.; Zhu, D. Biocatalytic Route to Chiral 2-Substituted-1,2,3,4-Tetrahydroquinolines Using Cyclohexylamine Oxidase Muteins. ACS Catal. 2018, 8, 1648–1652.

(96)

Heath, R. S.; Pontini, M.; Bechi, B.; Turner, N. J. Development of an R -Selective Amine Oxidase with Broad Substrate Specificity and High Enantioselectivity. ChemCatChem 2014, 6, 996–1002.

(97)

Shalmali, N.; Ali, M. R.; Bawa, S. Imidazole: An Essential Edifice for the Identification of New Lead Compounds and Drug Development. Mini-Reviews Med. Chem. 2018, 18, 142– 163.

(98)

Kaushik, N.; Kaushik, N.; Attri, P.; Kumar, N.; Kim, C.; Verma, A.; Choi, E. Biomedical Importance of Indoles. Molecules 2013, 18, 6620–6662.

(99)

R. Solomon, V.; Lee, H. Quinoline as a Privileged Scaffold in Cancer Drug Discovery. Curr. Med. Chem. 2011, 18, 1488–1508.

(100) Ratmanova, N. K.; Belov, D. S.; Andreev, I. A.; Kurkin, A. V. Synthesis of Non-Natural LAlanine Derivatives Using the Aza-Cope–Mannich Reaction. Tetrahedron: Asymmetry 2014, 25, 468–472. (101) Race, N. J.; Hazelden, I. R.; Faulkner, A.; Bower, J. F. Recent Developments in the Use of Aza-Heck Cyclizations for the Synthesis of Chiral N-Heterocycles. Chem. Sci. 2017, 8, 5248–5260. (102) Liu, X.; McCormack, M. P.; Waters, S. P. An Aza-Prins Cyclization Approach to Functionalized Indolizidines from 2-Allylpyrrolidines. Org. Lett. 2012, 14, 5574–5577. (103) Cao, M.-H.; Green, N. J.; Xu, S.-Z. Application of the Aza-Diels–Alder Reaction in the Synthesis of Natural Products. Org. Biomol. Chem. 2017, 15, 3105–3129.

TABLE OF CONTENTS GRAPHIC


64

Page 65 of 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis


65

Monoamine oxidase: Tunable Activity for Amine Resolution and

Recommend Documents