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Transaminase catalyzed asymmetric synthesis of active pharmaceutical ingredients

Yiman Cui, Yadong Gao, Licheng Yang

PII: S2666-5549(24)00031-0
DOI: https://doi.org/10.1016/j.gresc.2024.03.003
Reference: GRESC 257

To appear in: Green Synthesis and Catalysis

Received Date: 7 November 2023

Revised Date: 29 February 2024
Accepted Date: 11 March 2024

Please cite this article as: Y. Cui, Y. Gao, L. Yang, Transaminase catalyzed asymmetric synthesis
of active pharmaceutical ingredients, Green Synthesis and Catalysis, https://doi.org/10.1016/
j.gresc.2024.03.003.

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Graphical abstract

Transaminase catalyzed asymmetric synthesis of active pharmaceutical ingredients

Yiman Cui, Yadong Gao, Licheng Yang*
a
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academ y of Medical Sciences & Peking
Union Medical College, 100050 Beijing, China.

f
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In this paper, the application of transaminase in the synthesis of important drug molecules containing chiral amine groups was

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reviewed. Through concrete examples, the authors highlight the catalytic capabilities of transaminases in different ways and the
-p
main challenges faced by transaminases, and provide the furture application prospect.
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Review

Transaminase catalyzed asymmetric synthesis of active pharmaceutical

ingredients
Yiman Cui, Yadong Gao, Licheng Yang
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking
Union Medical College, 100050 Beijing, China.

ARTICLE INFO ABSTRACT

Article history: Chiral amine molecules constitute vital components of pharmaceutical ingredients. Recent years
Received have witnessed a growing focus on the efficient synthesis of chiral amines. Transaminases, as
Received in revised form
catalysts, have emerged as green, efficient, and highly selective solutions for substrates
Accepted
Available online containing ketones or aldehydes, demonstrating exceptional performance in the synthesis of

f
active drug molecules and natural products. This review primarily centers on the application of

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transaminases in the synthesis of important drug molecules bearing chiral amine group on acyclic
or cyclic backbones. We delve into specific examples, highlighting the catalytic prowess of the
Keywords: sole transaminase catalyst as well as the combination with other enzymes in cascade

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Transaminase transformations. This review illustrates the primary challenges that transaminases face and
Biocatalysis
Asymmetric synthesis
Chiral amine
-p
provides practical solutions that have been developed in these contexts. These solutions
encompass various strategies and techniques that enhance the applicability and efficiency of
re
Pharmaceutical ingredients transaminase-catalyzed reactions. In closing, we offer an outlook on the future of transaminase
applications, discussing potential developments and emerging areas where this green and
selective catalysis may play a pivotal role.
lP

1 Introduction 1.1 The Types of transaminases

Chiral amine-containing compounds play a pivotal role in the Transaminases can be classified based on substrate type into α-
na

realm of organic active pharmaceutical ingredients. In the retail transaminases and ω-transaminases. The majority of α-
sales data for 2022, it is noteworthy that over 42% of the top 200 transaminases are specific for the (S)-enantiomer of the substrate
drugs encompass chiral amine motifs, underscoring the critical and exhibit a pyridoxal phosphate (PLP) type I folding [7]. These
ur

need for efficient and convenient methods for synthesizing chiral enzymes facilitate the transfer of the amino group from the α
amine compounds [1]. carbon portion of amino acids to the ketone portion of α-ketoacids,
Jo

Transaminases, a class of enzymes proficient in catalyzing the requiring the presence of carboxylic acid groups at the location of
conversion of carbonyl groups into amino groups, are prevalent carbonyl functional groups. While α-transaminases exclusively
in animals, plants, and human bodies [2-4]. Beyond their allow the formation of α-amino acids [8], ω-transaminases exhibit
biological roles, these versatile enzymes find applications in greater versatility by accepting various ketones or amines, making
clinical monitoring and as catalysts in chemical processes [5]. them more useful and universal [9]. ω-Transaminases are further
While some transaminases are employed in their wild-type forms, categorised into β-transaminases and amine transaminases.
the majority are utilized as mutated variants to enhance their Notably, amine transaminases are often synonymous with ω-
utility. transaminases, attracting attention due to their broad scope range
Chiral amine-containing compounds play a pivotal role in the [10,11].
realm of organic active pharmaceutical ingredients. What makes Beyond substrate type, transaminases belong to PLP-
transaminases particularly attractive are their unique attributes, dependent enzymes and can be classified based on the fold-type
such as substrate specificity, the ability to function under simple and alignment of amino acid sequences [12-14]. Specifically, the
and mild reaction conditions, and their environmentally friendly PLP-fold types I (aspartate transaminase superfamily) and IV (D-
nature. These features position transaminases as powerful tools alanine transaminase family) encompass transaminases.
for synthesizing drug molecules and complex natural products. Moreover, transaminases can be grouped into six classes based on
Given their significance, this review aims to provide a structural features and sequence similarity. Subgroups I and II,
comprehensive overview of the pivotal role transaminases play in including L-aspartate transaminase and L-alanine transaminase,
the synthesis of active pharmaceutical ingredients (APIs) [6]. fall under α-transaminases, while subgroup III exclusively
comprises ω-transaminases [15]. Class IV includes D-amino acid
transaminases and branched-chain transaminases (BCATs), class

Corresponding author.
Email address: ylch@imm.ac.cn (L. Yang)

1
V contains L-serine transaminase, and class VI comprises sugar aldimine (Ⅱ); proton transfer occurs under the action of catalytic
transaminases, equivalent to InterPro’s DegT/DnrJ/EryC1 family residue Lys, forming quinone-type structure intermediate (Ⅲ) and
[16]. ketoimine intermediate (Ⅳ) successively. Finally, ketoimine
intermediates are hydrolyzed to produce ketone and 5’-
1.2 The reaction mechanism of transaminase pyridoxamine phosphate (PMP). In the second step, the amino
Transaminases usually don’t exert their function without the group of PMP is transferred to the latent chiral ketone to generate
cofactor PLP [17]. The reaction cycle of transaminases can be chiral amines and complete the regeneration of the coenzyme
divided into two half-reactions: oxidative deamination of an cycle [19]. In this step, S-type amines are mainly generated when
amine donor and reductive amination of a ketone acceptor [18]. the catalytic residue Lys is located on the si plane of the quinone
The specific catalytic mechanism can be represented in Figure 1. intermediate (relative to C4’ of PLP, S-type transaminase), and R-
Firstly, the amino group of the catalytic residue Lysine (Lys) in type amines are mainly generated when the catalytic residue Lys
the active center is connected to the aldehyde group of PLP by is located on the re plane (R-type transaminase). The regeneration
forming an internal aldimine (Ⅰ). Then, the amino group of the of PLP demonstrates that only the catalytic amount of this
amino donor replaces the Lys with the PLP to form an external auxiliary factor is required, and the reaction is reversible.

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Figure 1. General mechanism of transaminase.

1.3 The engineering of transaminase utilizing sacrificial amine donors. These strategies are particularly
Wild-type transaminases have encountered persistent effective for ω-transaminases, such as the cycling or removal of
challenges, including unfavorable reaction equilibrium and pyruvate formed from L-alanine [28].
limited synthesis of sterically hindered substrates due to the Another example is about using “smart” diamine donors for
unique spatial structure of the substrate binding region [20,21]. high conversion rates of chiral amines. In 2014, OReilly’s group
Additionally, issues in stereoselectivity and stability have been reported an efficient process that allows reactions to proceed in
observed [22-24]. In response, researchers have sought to modify high conversion in the absence of by-product removal using only
the selectivity and stability of transaminases through rational or one equivalent of a diamine donor (ortho-xylylenediamine). The
semi-rational design strategies at the molecular level [25]. This incorporation of the spontaneous polymerization of isoindole by-
has resulted in the generation of various mutants, enhancing products into a high-throughput screening platform adds another
substrate promiscuity and meeting the demands of large-scale layer of efficiency, allowing for the identification of the required
industrial production. ω-transaminase activity. This method is not only simple but also
One notable challenge in transamination reactions is the compatible with both (R)- and (S)-selective ω-transaminases,
existence of equilibrium reactions, limiting their application. making it versatile for substrates with unfavorable equilibrium
Strategies have been devised to shift the equilibrium towards positions, such as 1-indanone [29].
product formation, including process control strategies and In addressing challenges related to substrate preference
cascade reactions employing additional biochemical reactions of modification, researchers have employed various methods. One
side products [26,27]. For instance, asymmetric synthesis of approach involves active center modification through computer-
products can be achieved by rapid removal of by-products or by aided analysis and virtual activity screening technology. This


strategy has led to the generation of mutants with significantly Concerning stereoselectivity, achieving practicability for
enhanced catalytic activities for specific substrates. various substrates beyond α-amino acids has been a primary goal.
Transformation of catalytic performance for large, sterically Advances in biotechnology and protein engineering have
hindering substrates has been achieved through rational molecular facilitated the discovery and generation of new transaminases
transformation. By utilizing protein structure analysis, molecular with diverse substrate specificities [30,31].
docking, molecular dynamics simulation, and in vitro screening, Furthermore, studies on the molecular modification of ω-
optimal mutants have been generated with substantially improved transaminase from various sources have contributed significantly
reaction rates for specific catalytic syntheses. to the field. Examples include the enzymatic production of
Stabilizing amine transaminase for enhanced storage and Sitagliptin, a type 2 diabetes treatment drug. Molecular
operational stability has been a focus. Strategies such as protein modification techniques, such as site-specific saturation mutation,
engineering, biocatalyst formulation, and reaction medium have resulted in mutants with significantly enhanced catalytic
engineering have been employed. Notably, efforts to enhance the activities for specific substrates, marking milestones in the
thermostability of specific mutants have been successful, as development of asymmetric catalytic synthesis of chiral amines
demonstrated by the work of Huang’s group [25]. [32,33].

Table 1.
Examples of APIs synthesized via transaminases.

f
Compound
Name Structure Bioactivity Enzyme Reference

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number

Key intermediate related

ATA-117 from Vibrio
1 3-ArylGABA with a variety of psychiatric [38]

r
fluvialis
disorders
-p ArRmut11-A60V/M117F/
re
G279V
2 Pregabalin Nerve pain drug [39]
from Arthrobacter sp.
KNK168
lP
na

3 Niraparib (MK-4827) A drug for ovarian cancer ATA-301 from Codexis [40]
ur

4 Ivabradine A HCN channel blockers WTA740 from Codexis [42]

Jo

Codexis TA-P1-A06
5 AZD1480 A JAK2 kinase inhibitor [43] [44]
from Vibrio fluvialis

A pharmaceutically (S)-selective TA (3FCR,

6 Bulky chiral amine relevant chiral amine belonging to fold class I) [45]
bearing bulky substituents from Vibrio fluvialis

Sedative, anticancer, Chromobacterium

7 Organic fluorides [51]
antibacterial activity violaceum

3
 ArR from Arthrobacter
8 Chiral β,β- difluoroamine \ sp.
[52]

The precursor of prostate ATA-113 from Vibrio

9 Silodosin [53]
hyperplasia drug fluvialis

ATA-117
10 Sitagliptin phosphate Antidiabetic drug [32] [63]
from Arthrobacter sp.

f
roo
Active component of
11 Sacubitril -p angiotensin receptor
neprilysin inhibitor
(Entresto)
CDX-043 (E.C. 2.6.1)
from Codexis
[56] [57]
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lP

12 MK-4305 Orexin receptor CDX-017 from Codexis [62]

na
ur

13 Levofloxacin The antimicrobial agent ATA-256 from Codexis [64]

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A pyruvate TA
14 Levetiracetam An antiepileptic drug [65]
from V. fluvialis JS17

Angiotensin I converting DAPA AT from M.

15 Biotin [66]
enzyme (ACE) inhibitor tuberculosis

A late-stage candidate for

Vfat variant r414 from
16 Imagabalin generalized anxiety [68]
Vibrio fluvialis
disorder


 Dual orexin receptor ATA-117
17 MK-6096 [69][80]
antagonist from Arthrobacter sp.

ArR-TA from
18 (-)-Xenovenine A kinase inhibitor [72]
Arthrobacter sp.

ArS-TA from
19 (+)-Xenovenine Anticancer activity [72]
Arthrobacter sp.

(Cv2025) from

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20 Metaraminol A drug for hypertension Chromobacterium [81]

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violaceum ATCC 12472

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A potential candidate for
21 Methoxsamine -p hypotension and
incontinence, ophthalmic
applications
(S)-selective BmTA from
Bacillus megaterium
[82]
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lP

Engineered ATA-117
22 Florfenicol intermediate An antibacterial drug [83]
from Arthrobacter sp.
na

ATA-117 from Vibrio

23 (R)-Mexiletine An analgesic drug [84]
fluvialis
ur

Vf-ω-TA (ATA-113,
Jo

Drugs for Alzheimer’s

24 (S)-Rivastigmine ATA-114, ATA-117) [85]
disease
from Vibrio fluovibrio

ArR-TA from
A monoterpenoid indole
25 Strictosidine derivatives Arthrobacter sp. [86]
alkaloids

Pd-v-TA from
26 Naftifine An antifungal agent [88]
Paracoccus denitrificans

(R)-6-methylpiperidin-2-
27 A potent antifungal agent ATA-117 from Codexis [89]
one

5
 ATA (EC 2.6.1.18)
Analgesic and anti-cancer
28 Capsaicin analogues from Chromobacterium [91]
activities
violaceum

29 MK-7246 A CRTH2 antagonist CDX-017 from Codexis [92]

Armut11 from (R)-

30 (R)-ramatroban An antiallergy drug [93]
Arthrobacter

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31 SMO inhibitors A potential anticancer drug ATA-036 from Codexis [95]

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32 Vernakalant -p A novel antiarrhythmic
agent
The ATA-013 variant
ATA-303 from Codexis
[97] [98]
re
lP

ArRMut11 from
33 17-α-amino steroids A potent sulfatase inhibitor [99]
Arthrobacter sp.
na

An API molecule for

Vf-TA from Vibrio
34 Ozanimod ulcerative colitis and [100]
fluvialis
ur

multiple sclerosis.
Jo

Amine with Spirocyclic An active motif found in

35 ATA-200 from Codexis [101]
ring natural products

Endocrine therapy drugs

36 CDK 2/4/6 inhibitors ATA-71 from c-LEcta [102]
for breast cancer

An ingredient of TM1040 (3FCR)

37 Bicyclic amine [103]
neuroactive compounds from Ruegeria sp.

A γ secretase inhibitors
38 PF-03084014 ATA-47 from c-LEcta [104]
with antitumor activity


 An EGFR inhibitor for lung ATA-01 from Aspergillus [105]
39 Nazartinib
cancer fumigatus [107]

A potent epidermal growth

ATA-01 from Aspergillus [106]
40 Besifloxacin factor receptor in lung
fumigatus [107]
cancer

A transaminase variant
from
A CGRP antagonist for the
41 Rimegepant Chromobacterium [108]
treatment of migraine
violaceum

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Tert-butyl ((2R, 4R)-2-
An intermediate of API for ATA-P2-B01
42 methyltetrahydro-2H- [109]
the Parkinson’s disease

r
from from Codexis
pyran-4-yl) carbamate
-p
re
A precursor of PARP
43 Rucaparib ATA-302 from Codexis [40]
lP

inhibitors
na

Key structure found in

related bioactive ATA-117 from
44 (-)-Pinidinone [71]
therapeutics and Arthrobacter sp.
ur

agrochemicals
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A key intermediate for the

Armut11 from (R)-
45 Saxagliptin antagonistic drug [93]
Arthrobacter
ramatroban

2 APIs synthesized by transaminases 2.1.1 Transamination reaction of aldehydes involving

transaminases alone
Transaminases possess advantages such as substrate specificity,
simple and mild reaction conditions, and environmentally 3-ArylGABA 1 (γ-aminobutyric acid) derivatives [34-36] are
friendly characteristics. It provides an effective strategy for the integral to various neurological functions [37]. Dysregulation in
synthesis of related drug molecules and complex natural products. the central GABA system can contribute to the onset of
Here we present selected examples of API and natural product psychiatric disorders. The biological activity of 3-aryl GABA
molecules that can be catalyzed by transaminases. (Table 1) derivatives is notably linked to the (R)-enantiomer, with the (S)-
Based on the substrate types that transaminases can catalyze, enantiomer exhibiting lower activity. In 2009, Kroutil’s group
pharmaceutical ingredients can be categorized into acyclic and developed an asymmetric synthesis of the GABA derivative
cyclic amine compounds. In some instances, a single precursor (R)-4-phenylpyrrolidin-2-one using dynamic kinetic
transaminase is sufficient to complete the reaction, while in others, resolution (DKR) with the commercial stereoselective
cascade reactions involving multiple enzymes are employed to transaminase ATA-117 and alanine as an amine donor. This
complete the whole transformation. enzymatic strategy streamlined the process, reducing the number
of steps compared to alternative methods and avoiding
2.1 Synthetic routes to potential acyclic aliphatic amine drug cumbersome racemic production (Scheme 1A) [38].
molecules

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Scheme 1. The synthesis of amines from aldehyde substrates. (A) Synthetic of GABA 1 intermediates involved ω-transaminase; (B)
Production of Pregabalin 2 from intermediate 3-formyl-5-methylhexanoic acid by transaminase; (C) Synthesis of intermediate piperidine
by ATA-301/302; (D) Chemoenzymatic roadmap towards Ivabradine 4.

Pregabalin 2, employed in treating epilepsy, neuropathic pain, avoids inefficient chemical cleavage or chiral chromatographic
or fibromyalgia, was synthesized by Neill’s group in 2014. They methods and eliminates hazardous reactions with diazotizing
introduced a process for manufacturing the formulated compound agents (Scheme 1C) [41].
3-aminomethyl-5-methylhexanoic acid under transaminase In 2017, Sabín’s group synthesized the heart rate-lowering
conditions. Reductive amination with an amine donor, such as agent Ivabradine 4 through a four-step synthesis of N-methylated
isopropylamine (i-PrNH2) or α-amino acids, yields chiral- (S)-amine intermediates [42]. Enzymatic kinetic resolution by
enriched 3-aminomethyl-5-methylhexanoic acid (Scheme 1B) lipase-catalyzed alkoxy carbonylation and subsequent chemical
(Process and Intermediates for the Preparation of Pregabalin. reduction provided the N-methylated (S)-amine, just one step
2014, WO2014155291A1) [39] away from Ivabradine 4. The bioamination of α-aldehydes by ω-
Niraparib 3, a poly (ADP-ribose) polymerase (PARP) inhibitor transaminase, is a dynamic kinetic resolution process, utilizing L-
used in cancer treatment [40], was synthesized using a alanine as an amino donor and yielding the primary amine in 100%
biocatalytic strategy involving commercial transaminases ATA- yield (Scheme 1D) [43-45].
301 and ATA-302. The process includes dynamic kinetic
resolution of the α-chiral aldehyde, leading to an enantiopure 2.1.2 Transamination reaction of acyclic ketone involving
lactam, a precursor of niraparib 3. This transamination strategy transaminases alone

8
 Besides the aldehyde substrates, there are also numerous enzyme TA-P1-A06 for evaluation. The evolved enzyme
examples of ketone substrates participating in transamination demonstrated the ability to aminate the corresponding ketone
catalysis. Some ketone substrates carry aromatic rings at various (450 g, ~50 g/L) in a two-phase (H2O/toluene) reaction mixture,
positions, whereas others just carry fatty side chains. Therefore, producing 1-(5-fluoropyrimidin-2-yl)-ethan-1-one with > 99%
we divided the following transamination reactions into three enantiomeric excess (ee) and 68% yield. The extraction of
subgroups according to the substrate type. acetophenone (from the amine donor 1-phenylethylamine) into
the toluene phase played a crucial role in avoiding reaction
2.1.2.1 Transamination reaction of acetophenone-type inhibition. The best combination of intermediates involved Vibrio
substrates fluvialis ω-transaminase and (S)-α-methylbenzylamine. Toluene
The study conducted by Wells’s group in 2013 involving the was identified as the preferred solvent for this reaction. The
synthesis of AZD1480, an orally active JAK2 kinase inhibitor, success of this study suggests that transaminases have strong
highlights the potential of transaminases in the production of potential and competitiveness in the production of chiral amines.
chiral amines. In this case, the researchers screened 47 However, it’s noted that, at the time, most examples were limited
commercially available transaminases and selected the Codexis to laboratory-scale procedures (Scheme 2A) [46,47].

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Scheme 2. The synthesis of amines from acetophenone-type substrates. (A) Synthesis of key intermediates of AZD1480 5 using
transaminases in a two-phase system; (B) Designed (S)-selective transaminases that catalyze bulky ketone substrates; (C) Asymmetric
biotransamination of fluorinated ketones; (D) Reaction of α,α-difluoroketones with transaminase.

The study conducted by Moody’s group in 2016 addressed the In 2017, Fernández’s group conducted a study focusing on the
challenge of wild-type transaminases having limited activity on synthesis of organic fluorides, specifically compound 7, which
chiral amine substrates with bulky substitutions. Starting with an has gained attention due to its diverse applications in various
amine transaminase from Vibrio fluvialis that lacked catalytic scientific fields such as agrochemicals [49], drugs [50-52], and
activity toward the bulky aromatic ketone 2-acetylbiphenyl, the asymmetric catalysts [53]. The presence of fluorine in organic
researchers aimed to design an (S)-selective transaminase with compounds can significantly influence their physicochemical
enhanced activity. The rational design involved introducing a properties, including acidity, hydrogen bonding, lipophilicity,
minimum of seven mutations to the enzyme. This engineered solubility, and biological properties [54,55], thereby enhancing
variant demonstrated a significant increase in the reaction rate, metabolic stability [56]. In this study, transamination reactions
surpassing a 1716-fold improvement. The outcome was the were employed for the synthesis of fluorinated ketone substrates.
production of an enantiomerically pure (S)-amine product with > The researchers utilized several representative transaminases and
99% ee. Additionally, the variant, which utilized i-PrNH2 as the their variants to perform these reactions, achieving positive
amine donor, maintained 90% activity for 18 hours at 50 oC outcomes with excellent enantioselectivity for the production of
(Scheme 2B) [48]. both amine enantiomers. The ee values obtained were greater than

9
99%. Notably, L-alanine or D-alanine served as the amine donor this transamination process was isopropylamine (i-PrNH2)
in these transamination reactions (Scheme 2C) [57]. (Scheme 2D).
In 2022, Lavandera’s group explored the synthesis of chiral
α,α-difluoroamines, a class of biologically important compounds, 2.1.2.2 Transamination reaction of ketone substrates with non-
using transaminases [58]. The specific compound targeted in this adjacent aromatic groups
study was the β,β-difluramide 8. The researchers achieved a high Besides the acetophenone-type structure, some substrates also
separation yield ranging from 55% to 82% and ensured bear aromatic groups not adjacent to the ketone group.
enantiomeric purity exceeding 99%. The amine donor utilized in

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Scheme 3. The synthesis of amines from acyclic non-acetophenone type substrates (A) Synthesis of Silodosin 9 intermediate using (R)-
ω-transaminase; (B) Biocatalytic amination to prepare Sitagliptin 10 using a transaminase variant from Arthrobacter sp.; (C) Biocatalytic
amination to prepare Sacubitril 11 and related LCZ696 with CFX-043 and CDX-043.

In 2014, Kroutil’s group explored the synthesis of the precursor employed in this process were D-alanine or L-alanine (Scheme
amine of Silodosin 9 (Uroreco® and Rapaflflo®), an (R)- 3A) [59].
enantiomeric prodrug used in the treatment of urinary tract In 2010, Savile’s group reported an efficient biocatalytic
disorders. Silodosin is derived from substrates containing method for the synthesis of Sitagliptin phosphate (Januvia®), an
benzopentamembered heterocycles far from the ketone groups. antidiabetic drug used to treat type II diabetes [60,61]. This
The researchers conducted various kinetic resolution and biocatalytic approach replaced the rhodium-catalyzed asymmetric
asymmetric reductive amination reactions to prepare the enamine hydrogenation with a crucial transamination step. The
Silodosin amine. To achieve this, they screened ω-transaminases enzyme employed for this process was the (R)-selective ω-
from different strains and identified ω-transaminases with transaminase from Arthrobacterium. The reaction was conducted
excellent bioactivity originating from (R)-Arthrobacter sp. The at 45 °C with dimethyl sulfoxide (DMSO) as a cosolvent, and
enzymatic conversion of ketone precursors to amines was carried isopropylamine (i-PrNH2) served as the amine donor. The
out under mild conditions, resulting in high conversion rates (> reaction resulted in the synthesis of 882-922 g of the Sitagliptin
97%) and enantiomeric excess (ee > 97%). The amine donors intermediate with a yield of 88%-92% [32]. The enantiomeric
excess exceeded 99.95%. The transamination reaction

10


demonstrated high stereoselectivity, crucial for the synthesis of concentration of 75 g/L, with a low enzyme loading of 1%, the
chiral pharmaceuticals. One of the notable achievements process achieved a high conversion rate of 90% with the
highlighted in the text is overcoming the limited substrate range enantiomeric purity exceeding 99.9% (Scheme 3C, condition 2)
of transaminases. Transaminases typically face challenges in [63].
catalyzing reactions with relatively large substituents on either The synthesis of Suvorexant (Belsomra® or MK-4305), a first-
side of the ketone group. The rational engineering of natural class orexin receptor antagonist used for the treatment of
transaminases played a crucial role in enabling the industrial insomnia, involves a chiral diazepine ring. Traditionally, this
application of the biocatalytic process (Scheme 3B). chiral diazepine ring was synthesized through an asymmetric
In 2020, Kleinbeck’s group developed a kilo-scale amine reduction process facilitated by a heavy metal ruthenium
transamination process for the synthesis of Sacubitril 11, an catalyst [64]. However, this process was known for being long
enkephalinase inhibitor used in the treatment of hypertension or and cumbersome. In 2016, Wallace’s group proposed a more
heart failure. They used the (S)-selective transaminase CFX-043 environmentally friendly approach using biocatalytic
to convert a γ-keto acid to a crucial (2S, 4R)-amino acid transamination techniques [65-67]. The process involves
intermediate. The reaction was carried out in non-buffered water asymmetric transamination of a ketone with suitable leaving
at a pH of 8.5 at 58 ℃. Isopropylamine was used as the amino groups, leading to the construction of the diazepine structure. The
acid donor (Scheme 3C, condition 1) [62]. process achieved a 71% yield, with product ee value exceeding
In 2021, Novick’s team took the process a step further by 99%. Isopropylamine (i-PrNH2) was used as the amine donor [68].

f
performing 11 rounds of enzyme evolution. They started with the This enantioselective biocatalytic reaction was considered an

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enzyme ATA-217, which had trace activity. Through evolution, environmentally friendly alternative to traditional chemical
they engineered a variant called CDX-043. This variant synthesis methods. Notably, this strategy has already
incorporated 17 amino acid mutations and demonstrated demonstrated its utility in pharmaceutical applications,

r
improved activity. The optimized CDX-043 variant enabled particularly in the biotransformation approach of Sitagliptin 10
efficient and cost-effective large-scale production of the
Sacubitril precursor. The reaction achieved a substrate
-p (Scheme 4A) [69].
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lP
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Scheme 4. (A) The transaminase involved in the synthesis of the MK-4305 12 precursor; (B) Synthesis of levofloxacin 13 intermediates
catalyzed by transaminase.

In the synthesis of Levofloxacin 13 (an antibiotic used to treat was ω-transaminase, which was purified from Vibrio fluvialis
bacterial infections), Fernandez’s group in 2016 employed a JS17. The amine donor utilized in the reaction was benzylamine.
commercially available transaminase, specifically ATA-256. The The researchers overcame the challenge of severe product
process involved the screening of transaminases for the synthesis inhibition by benzaldehyde through the use of an extracted
of a series of racemic amines from corresponding ketones, hexane/water biphasic reaction system (Scheme 5A) [71].
including the precursor of Levofloxacin. With i-PrNH2 as the Resistance to all first-line and second-line antituberculosis
amine donor, the transamination process resulted in a 99% drugs has been successively reported in the last 40 years.
conversion and with greater than 99% ee of the precursor ketone Therefore, new drugs that inhibit new targets in Mycobacterium
to the amine product (Scheme 4B) [70]. tuberculosis are constantly required.
In 2011, Aldrich’s group addressed the urgent need for new
2.1.2.3 Transamination reactions of ketone substrates without drugs to combat drug-resistant strains of Mycobacterium
aromatic rings tuberculosis (Mtb), the causative agent of tuberculosis. They
Transamination reactions of ketone substrates without targeted the Biotin biosynthetic pathway in Mtb, specifically
aromatic rings are also frequently reported. In 2009, Shin’s group focusing on the enzyme 7,8-diaminopelargonic acid transaminase
presented a method for the asymmetric synthesis of non-natural (DAPA AT), which plays a crucial role in the synthesis of Biotin.
amino acids using a single enzyme-catalyzed route. The process Biotin is an essential cofactor for many enzymes, and inhibiting
involved the synthesis of L-2-aminobutyric acid from 2- its biosynthesis can lead to cell death [72-75].
oxobutyric acid. The enzyme employed for this transformation

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 Among the enzymes involved in this biosynthetic route, two a more direct and efficient approach [76]. In 2012, Midelfort et al.
are dependent on PLP: the 8-amino-7-oxononanoate synthase combined several protein engineering approaches to optimize the
(AONS) and the 7,8-diaminopelargonic acid transaminase selectivity and activity of Vibrio fluorogenes ω-transaminase
(DAPA AT). DAPA AT uses PLP as a cofactor to catalyze simple (VfAT) for the synthesis of (3S,5R)-ethyl 3-amino-5-
transamination. However, the amine donor is not an amino acid methyloctanoate, the key intermediate in the synthesis of
but S-adenosyl-L-methionine (AdoMet), which is a unique Imagabalin 16. An improved enzyme variant with a 60-fold
situation in transaminase (Scheme 5B). increase in the initial reaction rate was designed. (S)-
This research not only provides insights into the biochemistry methylbenzylamine (S-MBA) was used as the amine donor in the
of Mtb but also suggests a promising avenue for the development transamination reaction. The researchers solved the crystal
of new drugs to combat tuberculosis, especially drug-resistant structure of the enzyme, providing valuable insights into its
strains. molecular architecture. The work shed light on critical residues
In the development of Imagabalin 16, a late-stage candidate for responsible for substrate specificity in the transamination of (R)-
generalized anxiety disorder, researchers faced challenges with ethyl-5-methyl-3-oxooctanoate and structurally related β-
traditional chemical methods due to cost and prolonged reaction ketoesters (Scheme 5C) [77].
times. To address these issues, they opted for enzyme catalysis as

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Scheme 5. Transamimoniation of non-aromatic ring ketone substrates. (A) Synthesis of Levetiracetam 14 intermediates; (B) Biosynthesis of
Biotin 15 in Mtb; (C) Synthesis of Imagabalin 16 via Vfat ω-transaminase variants.

In 2014, Xiang’s group detailed the asymmetric synthesis of transamination step played a crucial role in shortening the overall
MK-6096 17, a potent dual orexin receptor antagonist used for the reaction routes and improving synthetic efficiency (Scheme 6A)
treatment of sleep disorders. The synthesis involved several key [78].
steps, including the biological transamination of a chiral In 2015, Chung’s group also contributed to the asymmetric
ketodiester. Conversion of keto-dilactone to amino-dilactone was synthesis of the piperidine core of MK-6096 17. This synthesis
achieved using the transaminase ATA-117, with alanine serving involved biocatalytic transamination and crystallization-induced
as the amine donor. The highly enantioselective transamination dynamic resolution. A single enzyme system (CDX-017) was
reaction of chiral α-methylpiperidine was followed by the employed for the synthesis of the important intermediate. i-PrNH2
cyclization of the amino-diester at pH 3.5 to obtain the desired was used as the amine donor (Scheme 6B) [79].
piperidone with an α-methyl stereocenter. The biological

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Scheme 6. Varies routes to access MK-6096 17. (A) Synthesis of MK-6096 17 intermediate using (R)-ω-transaminase variant; (B) synthesis of
-p
MK-6096 17 precursors by transaminase; (C) Preparative scale conversion of (-)-Pinidinone 44 using (R)-Selective ATA117.

In 2016, Ryan et al. conducted a study reporting the large-scale Transaminases also have a wide range of applications in natural
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synthesis of chiral piperidine. The synthesis utilized readily product synthesis. In 2016, Kroutil’s group reported the
available chiral prehensions and employed ω-transaminase, preparation of the pyrrolizidine alkaloid Xenovenine 18/19. The
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demonstrating excellent conversion and isolated yield. The synthesis involved a five-step process, with a key asymmetric step
researchers highlighted the use of a reversible ω-transaminase utilizing transaminases. This step, a regioselective and
reaction coupled with a strong thermodynamic driving force. This stereoselective mono-amination of the tri-one, was crucial for the
approach facilitated the shuttling of the amine functionality across overall synthesis. The transaminases facilitated the regio- and
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a molecular framework to form the desired product. The stereoselective amination of a single ketone moiety out of three,
significance of this work lies in the expansion of ω-transaminase enabling the shortest and highest-yielding total synthesis of the
applications in total synthesis, particularly in the synthesis of bicyclic pyrrolizidine alkaloid without the need for protecting
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more complex alkaloid scaffolds, including MK-6096 17 strategies. Alanine was employed as the amine donor in this
(Scheme 6C) [80]. transformation (Scheme 7) [81].
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Scheme 7. Synthesis of helicoid compounds catalyzed by transaminase.

2.1.3 Cascade reaction involving transaminases applications [86-88]. Here we summarize some interesting
Cascade reactions in synthesis methods have proven to be synthesis designs based on enzyme cascade reactions leading to
highly useful, particularly in the context of constructing complex complex chiral drug molecular intermediates.
molecular scaffolds. These methods offer advantages such as Cascade reactions involving multiple enzymes represent
shortened reaction routes, avoidance of unstable or toxic complex enzymatic processes where multiple substrates are
intermediates, increased atomic efficiency, and reduced waste catalyzed by multiple enzymes to generate products. In this
[82-85]. In the past decade, numerous cascade reactions have intricate network, intermediates formed by one enzyme can
been reported, contributing to the construction of biologically become substrates for the next enzyme, leading to the ultimate
significant molecular structures. This approach enhances the production of the desired final product. While transaminase alone
effective application of transaminases (TAs) in organic synthesis. can catalyze the production of MK-6096 17, cascade reactions
Several reviews have delved into the intricacies of cascade involving multiple enzymes are a relatively rare but powerful
reactions involving TAs, providing detailed insights into their approach.

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Scheme 8. (A) A cascade reaction involving transaminase to synthesize piperidone, a key intermediate of MK-6096 17; (B) Enzymes cascades
in whole cells for the synthesis of piperidine (ATA-117, ω-transaminase from Arthrobacter sp.; NCAR, carboxylic acid reductase from Nocardia
sp.; MCAR, carboxylic acid reductase from Mycobacterium marinum; (S)-IRED, imine reductase from Streptomyces sp. GF3546; (R)-IRED,
imine reductase from Streptomyces sp. GF3587; BsSfp, 4’-phosphopantetheinyl transferase from Bacillus subtilis).

In the kilogram-scale synthesis of the first generation of MK- stereocenter of hydroxymethyl have been encountered. In this
6096 17, the piperidine ring segment plays a crucial role. The cascade system, D-alanine serves as the amine donor for
synthesis involves the formation of a lactam from dimethyl transaminase (Scheme 8A) [39]. One notable advantage of this
malonate and methyl ketone through a Michael addition reaction cascade reaction strategy is its ability to circumvent the need for
followed by transaminase biocatalysis. In the second step, a three- the purification of intermediates, streamlining the overall
enzyme cascade system, including transaminases, is employed to synthesis process.
synthesize chiral precursors. However, challenges such as low The expanding repertoire of biocatalyst-mediated reactions has
yield of the piperidine ring and difficulty in controlling the given rise to the development of multi-step in vitro enzyme

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cascades that operate under universally compatible reaction utilizing Corynebacterium glutamicum. This process yielded
conditions. While this approach has seen considerable progress, pyruvate and L-alanine through the implementation of iterative
the construction of pathways within a single whole-cell system strains and process engineering. Notably, the research group
has been less explored but offers numerous advantages. In 2017, achieved a high conversion of chiral aminoalcohol drugs by
Flitsch’s group reported on whole-cell enzymatic cascade employing an enzyme cascade reaction, involving carboxylation
reactions designed for the allosteric and/or enantioselective and transamination steps. The cascade reaction begins with the
conversion of simple linear keto acids into valuable cyclic amine enzymatic carboxylation of 3-OH-benzaldehyde and
products. The cascade begins with carboxylic acid reduction, acetaldehyde. Thiamine bisphosphate (ThDP)-dependent
initiating transamination, imine formation, and subsequent imine enzymes facilitate the decarboxylation of pyruvate in the initial
reduction. This system is constructed and optimized using step. The resulting 2-hydroxyketone intermediate then undergoes
standard genetic manipulation techniques, and the cascade a reaction with an aromatic aminoalcohol, employing i-PrNH2
necessitates only starting materials, amine donors (specifically D- and a (S)-selective amine transaminase (Scheme 9A) [90].
alanine), and a whole-cell catalyst. The cofactors required for the Methoxamine 21 is a potential candidate for the treatment of
reactions are internally provided through glucose metabolism. A hypotension and incontinence, or for use in ophthalmic
set of synthetic keto acids was employed in the study, yielding applications. In 2019, the Rother group developed a biocatalytic
high conversion rates (up to 93%) and enantiomeric excess (up to approach to produce all four stereoisomers of Methoxamine 21
93%) of piperidine [89]. Piperidine, in turn, can be used as an from readily available pyruvate and 2,5-dimethoxybenzaldehyde.

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intermediate for various APIs, such as MK-6096 17 (Scheme 8B). The synthesis was achieved in a one-pot, two-step sequential

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Due to the lack of readily available material in some cases, cascade reaction without the need for intermediate separation.
ketone substrates were usually generalized in situ through a The transamination reactions crucial to the cascade utilized
cascade reaction from aldehydes, thus transaminases were usually Bacillus megaterium transaminase, with i-PrNH2 serving as the

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found to catalyze transformations in combination with lyases. amine donor. This transaminase demonstrated high activity and
Metaraminol 20 is a crucial active pharmaceutical ingredient
employed in antihypertensive therapy and serves as a precursor
-p selectivity, enabling the efficient synthesis of all four
Methoxamine stereoisomers. The achieved isomeric contents
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for the synthesis of other bioactive compounds. In 2022, Noack’s ranged from 94% to 99%, with total conversions for the two-step
group successfully demonstrated a one-pot fermentation process cascade falling between 59% and 80% (Scheme 9B) [91].
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Scheme 9. The cascades from aldehydes. (A) Enzyme cascade to Metaraminol 20; (B) Sequential cascade reaction to Methoxamine 21; (C)
Cascade reaction to (1R, 2R)-p-Methylsulfonyl phenylserinol.

Florfenicol 22, a 3’-fluorine derivative of thiamphenicol, is transketolase (TK) and ω-transaminase to generate diol
widely used in veterinary medicine due to its high antimicrobial intermediates with two stereocenters. In the transamination
activity and safety. In 2021, Lin’s group addressed the challenge reaction, the evolved ATA117_ACHH mutant was employed,
of developing simplified and environmentally friendly catalytic acting as a ω-transaminase, and various amine donors were used
methods for the stereoselective production of Florfenicol 22. for catalyzing the ketone substrate/amine acceptor (a ketone or
They established a highly stereoselective enzymatic one-pot aldehyde). This innovative approach allowed for the efficient and
reaction for the synthesis of aminophenol containing two stereoselective synthesis of Florfenicol 22 in a one-pot enzymatic
stereocenters of Florfenicol from the industrial raw material 4- reaction (Scheme 9C) [92].
(methylsulfonyl) benzaldehyde. This process involved coupling

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Scheme 10. (A) Deracemization of rac-Mexiletine by a One-Pot/two-step synthesis procedure; (B) An intermediate in the synthesis of (S)-
Rivastigmine 24 involving ω-transaminase; (C) Conversation for Strictosidine derivatives 25 in the one-pot, one step cascade reaction; (D) One-
Pot, two-step cascade reaction involving transaminase to produce intermediate of Naftifine 26; (E) Biocatalytic transamination routes to (R)-6-
methylpiperidin-2-one 27.

Mexiletine 23 is a therapeutically relevant chiral amine used enantiomers through a decyclization method, achieving greater
clinically for its antiarrhythmic, antimuscarinic, and analgesic than 99% enantiomeric excess and isolated yields up to 97%. The
properties. Pharmacological studies have shown that the (R)- enantiomeric isomers of optically pure Mexiletine were generated
enantiomer of Mexiletine is more active than the (S)-enantiomer. by kinetic resolution, combining amino acid oxidase and
Due to the possible side effects of the racemate, there is a demand subsequent stereoselective amidase catalyzed by enantio-
for the development of a pure enantiomer of this drug. complementary ω-transaminases. Importantly, amino acid
In 2009, Kroutil’s group reported a one-pot, two-step method oxidase could be recovered in situ, requiring only catalytic
for the synthesis of Mexiletine from racemic Mexiletine. This amounts of pyruvate for kinetic resolution (Scheme 10A) [93].
process utilized ω-transaminase for the synthesis of the two

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 (S)-Rivastigmine 24 is a highly effective drug for the early secologanin catalyzed by Strictosidine synthase, yielding the final
treatment of Alzheimer’s disease, and it also provides beneficial products. Higher conversions were achieved when running the
effects on dementia in Parkinson’s patients. In 2010, Faber’s two reactions in a stepwise fashion. An appropriate ω-
group developed a chemoenzymatic synthesis of (S)- transaminase was used to obtain the amines with up to > 98% ee.
Rivastigmine using alcohol dehydrogenase and transaminase, The amine donor L-alanine was required for (S)-selective
achieving an overall isolated yield of 71% [94]. The synthesis transaminases, while D-alanine was required for (R)-selective
involved initial screening for 3-hydroxyacetophenone using enzymes (Scheme 10C) [96,97].
seven different ω-transaminases, including four commercial Naftifine 26 is a potent antifungal agent. In 2012, Kroutil’s
enzymes obtained from Codexis (Vibrio fluovibrio ω- group developed a one-pot oxidation-transamination cascade
transaminases, ATA-113, ATA-114, and ATA-117). To balance reaction for the amination of benzyl alcohols, achieving a short
the diversion of the amine product and remove the by-product route suitable for the synthesis of the Naftifine precursor (E)-3-
pyruvate, the researchers chose to combine lactate dehydrogenase phenylprop-2-en-1-amine [98]. The process involved the use of
with a glucose dehydrogenase/glucose recycling system to galactitol oxidase to oxidize benzyl alcohols to the corresponding
regenerate NADH. This highly stereoselective and short aldehydes using O2 as the oxidizing agent. Subsequently, they
chemoenzymatic synthesis of (S)-Rivastigmine was developed employed ω-transaminase for in situ amination, converting the
through a four-step procedure with a total separation rate of 71%. aldehydes to amines. For the transformation of the aldehyde to the
The proposed approach represents the shortest route published at amine, a ω-transaminase was selected, and L-alanine served as the

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that time for the synthesis of (S)-Rivastigmine (Scheme 10B). ideal amine donor. L-alanine could be efficiently recycled from

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In 2016, Kroutil’s group developed a method to prepare pyruvate via reductive amination using alanine dehydrogenase
optically pure C3-methyl-substituted Strictosidine derivatives 25, and NADH recycling, facilitating the completion of the reaction
key intermediates in the biosynthesis of drug-related (Scheme 10D).

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monoterpenoid alkaloids. This was achieved by establishing two In 2014, Xiang’s group achieved the synthesis of (R)-6-
-p
stereocenters in the β-carbon base core through a one-pot cascade
of amidase and Strictosidine synthase [95]. The first step of the
methylpiperidin-2-one 27 from methyl 5-oxohexanoate with a
high yield of 91% and excellent ee (99.7%). The process involved
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reaction sequence involved the reductive amination of prochiral a triple enzyme system, with D-alanine serving as the ammonia
indolyl ketones via transaminases, resulting in the formation of source. The key steps included lactamization at methyl (R)-5-
optically pure α-methyltryptamines. In the second step, these aminohexanoate and subsequent alkaline post-treatment to obtain
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optically pure α-methyltryptamines were condensed with the desired product (Scheme 10E) [99].
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Scheme 11. Idealized ammonium formate-driven system for ω-transaminase-mediated (R)-Amination using inexpensive ammonium formate as
the driving force.

In 2019, Zheng’s group developed an efficient and eco-friendly 2017, researchers embarked on a one-pot total synthesis of
one-pot, three-enzyme cascade method for the synthesis of (R)- capsaicin analogs, specifically 4-(hydroxymethyl)-2-
chiral amines. The cascade system included (R)-specific ω- methoxyphenol analogs 28, extracted from lignin. The synthesis
transaminase, amine dehydrogenase, and formic acid involved a multi-step process utilizing a combination of
dehydrogenase. This approach enabled the amination reactions of heterogeneous metal catalysis, organocatalysis, and enzymatic
various ketones with high conversions and excellent ee (> 99%). cascades. The heterogeneous palladium nanoparticles were
The use of inexpensive ammonium formate as the sole sacrificial employed to oxidize alcohols to aldehydes. Subsequently, an
agent, along with (R)-2-pentanamine as the amine donor, enzymatic cascade system, featuring transaminase with alanine as
contributed to the economic and environmentally friendly aspects the amine donor, converted the aldehydes to amines. Vanillin was
of the synthesis. The cascade system demonstrated its versatility synthesized either through the oxidation of vanillyl alcohol or the
by efficiently transforming prositagliptin ketone and 1- reverse aldol conversion of aldol compounds. The amination of
phenoxypropan-2-one into the corresponding products with high vanillin involved the enzymatic cascade system with
efficiency (Scheme 11) [100]. transaminase, converting aldehydes to amines. Notably, alcohol
Capsaicin stimulates gastric juice production, improves dehydrogenase (ADH) facilitated the oxidation of vanillyl alcohol,
appetite, and enhances gastrointestinal motility. It is one of the contributing to NAD+/NADH regeneration during transamination.
components of chili peppers and has pain-relieving properties. In The last step, amidation, was accomplished using lipase-mediated

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amidation, an environmentally friendly strategy that avoids the solvent removal. This integrated approach, combining various
use of halogenated organics. catalysts and reactions, offered a more efficient and economical
The entire synthesis was conducted in a one-pot fashion, route for the total synthesis of capsaicin analogs (Scheme 12)
eliminating the need for intermediate purification steps except for [101].

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Scheme 12. Enzymatic reaction route for synthesizing capsaicin analogues 28.

2.2 Synthetic routes to potential drug molecules of cyclic isopropylamine (i-PrNH2) as an amine donor, could efficiently

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aliphatic amines
2.2.1 Transamination reaction involving transaminases alone
In 2012, O'Shea’s group developed different synthetic routes
-p convert the ketone intermediate to the corresponding amine with
98%-99% ee and a yield of 81% under optimized reaction
conditions. The total yield through eight steps of production was
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for MK-7246 (29), a potent and selective CRTH2 antagonist for 49%, showcasing the efficiency of this enzymatic route compared
the treatment of respiratory diseases. One of the routes involved to the previous chemical synthesis, which required 18 steps with
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screening against selegiline transaminase, and an off-the-shelf an overall yield of only 10%. This enzymatic approach not only
enzyme variant CDX-017 was identified. This variant, with shortens the synthetic route but also enhances the overall
production of the final product (Scheme 13A) [102].
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Scheme 13. (A) Synthesis of the intermediate of MK-7246 29 with transaminase variant CDX-017; (B) Synthesis of the intermediate of (R)-
ramatroban 30 with transaminase variant ArRmut11.

In 2014, Kroutil’s group reported the enzymatic synthesis of a In 2014, researchers at Pfizer reported a concise, asymmetric
key intermediate of the anti-allergy drug ramatroban 30. They synthesis of an enzymatic transamination reaction for a smooth
utilized the transaminase variant Armut11 ω-transaminase from receptor inhibitor, which demonstrated superior efficacy and
(R)-Arthrobacter to achieve the amination of the ramatroban safety in phase I clinical trials involving patients with various
ketone precursor. Interestingly, in this transamination reaction, it blood-related cancers, including acute myeloid leukemia (AML)
was found that 2-propylamine could be detrimental to ketone [106]. This synthesis featured a one-step creation of the required
precursors, leading to the formation of various by-products. This two stereocenters by dynamic kinetic splitting. The entire
issue was effectively addressed by using (R)-1-phenylethylamine synthesis required only five linear steps and achieved a total yield
as the preferred amine donor, resulting in improved selectivity of 40% without the need for column chromatography, making this
and avoiding unwanted by-products (Scheme 13B) [103-105]. synthetic approach suitable for large-scale commercial supply.
Interestingly, racemic substrates were mutually convertible, while
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the ω-transaminase with i-PrNH2 as an amine donor could then convert it into the desired amino product (Scheme 14A)
selectively catalyze one configuration of the raw material and [107].

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Scheme 14. (A) SMO inhibitor 31 precursor was synthesized by transamination of DKR; (B) DKR transamination reaction to synthesize
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Vernakalant 32 intermediate.

In 2014, Limanto’s group developed an efficient route to In this synthesis, they utilized transaminases from Vibrio fluvialis,
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synthesize the antiarrhythmic drug Vernakalant 32 [108]. The which accepted i-PrNH2 as an amine donor. This approach
approach involved three novel strategies starting from readily enabled the selective synthesis of the key intermediate (S)-4-
available and inexpensive raw materials. The key steps included cyano-1-aminoindane with a satisfactory yield and excellent
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α-etherification modified with zinc chloride and pyrrolidine, enantiomeric excess (99%) (Scheme 15B) [111].
enzymatic dynamic asymmetric transamination (DA-TA) of α- Spirocyclic ring systems 35 are important intermediates for the
substituted ketones, and alkyl B(OH)2-promoted amidation. The design and synthesis of drug-active substances, they are also
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transamination step was optimized by kinetic transamination, common core compounds in natural products. In 2022, Kohrt’s
leading to the synthesis of the (1R, 2R)-trans-aminohexyl ether group utilized a transaminase (ATA-200 from Codexis) with i-
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precursor with ATA-303, which uses i-PrNH2 as the amine donor. PrNH2 as the amine donor to synthesize the enantiomer of a key
This precursor was obtained within 24 hours in > 99.5% ee. This intermediate, boc-protected-1-oxa-8-azaspiro[4.5]decan-3-amine,
work represented the first reported example of enzymatic DA-TA with high enantiomeric excess (> 99%). As the scale of the
of α-substituted ketones, providing the corresponding trans- transaminase reaction increased, they observed that increasing the
amino products in higher stereoselectivity and DMSO content to 8-10% led to improved conversion (99%) and
diastereoselectivity (Scheme 14B) [109]. consistently high enantiomeric excess values (98.6%). Under
Steroids are a wide variety of secondary metabolites that are these optimized conditions, the process was scaled up to 580
essential for controlling various biological processes, having grams at 35 ℃, providing chiral amines effectively with a yield
great potential as drugs. In 2014, Hailes’s group developed a of 82% and 97.8% ee (Scheme 15C) [112].
highly stereoselective, efficient, and sustainable biocatalytic In 2022, Morris’s group employed transaminase ATA-71 from
pathway to synthesize 17-α-amino steroid 33. They used the ω- c-LEcta to synthesize a key intermediate of CDK 2/4/6 inhibitors
transaminase variant ArRMut11 from Arthrobacter sp. in a one- 36, introducing chiral secondary amines with high
step preparation scale with a separation yield of 83%-89%. The diastereoselectivity. To overcome the unfavorable reaction
amine donor in this process is i-PrNH2. This biocatalytic method equilibrium, they utilized (R)-methylbenzylamine (R-MBA) as a
represented the shortest route for 17-α-amino steroids 33 new amine donor, replacing i-PrNH2. Additionally, to remove
published at that time (Scheme 15A) [110]. acetophenone from the reaction mixture, they included 25% v/v
In 2019, the group of Groger proposed a new alternative toluene in the water reaction mixture, achieving optimal
approach for the synthesis of (S)-4-cyano-1-aminoindanones, selectivity. Ultimately, the transamination reaction successfully
which are chiral key intermediates in the production of Ozanimod produced the target cyclic chiral amine intermediate in 91% yield
34, a molecule promising for the treatment of multiple sclerosis. and 96.9% ee (Scheme 15D) [113].

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Scheme 15. (A) Transaminase catalyzes the synthesis of 17-α-amino-steriods 33; (B) Synthesis of Ozanimod 34 intermediates by asymmetric
reductive amination catalyzed by transaminase; (C) ω-TA catalyzed amination of 1-Oxa-8-azaspiro[4.5]decan-3-amine 35; (D) Transaminase
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catalyzes the synthesis of important fragments of CDK 2/4/6 inhibitors 36.

In 2016, Bornscheuer’s group developed a transaminase production of γ-secretase inhibitors with potential antitumor
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variant through protein engineering to enable the selective activity, exemplified by the drug molecule PF-03084014 38.
asymmetric amination of large, site-resistant bridged bicyclic During the initial screening process, the commercial transaminase
rings. This variant, utilizing i-PrNH2 or alanine as amine donors, ATA-47 from c-Lecta emerged as the most promising catalyst.
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facilitated the specific synthesis of target exocyclic amines with > The team successfully demonstrated the conversion of substituted
99.5% ee. This achievement marked the first example of the tetrone with i-PrNH2 to the corresponding (S)-amine using this
transaminase-catalyzed synthesis of amine-containing bicyclic enzyme, achieving excellent selectivity in the reaction conducted
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bridging moiety (Scheme 16A) [114]. at 37-39 °C (Scheme 16B) [115].

In 2017, Pfizer’s researchers outlined a chemo-enzymatic
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synthesis pathway for a crucial chiral intermediate in the

Scheme 16. (A) Selective synthesis of exo-3-amino-8-aza-bicyclo[3.2.1]oct-8-yl-phenyl-methanone via reductive amination of ketone catalyzed
by the ATA 3FCR and its variants. (B) Synthesis of intermediate of PF-03084014 38 catalyzed by transaminase.

In 2017, Wang’s group employed ω-transaminase ATA-01 in growth factor receptor antagonist primarily employed in lung
combination with i-PrNH2 to efficiently establish the stereocenter cancer treatment) [116] and Besifloxacin 40 (a fourth-generation
of (3R)-N-Boc-3-aminoazepane, a crucial precursor in the fluoroquinolone antibacterial agent belonging to the broad-
synthesis of pharmaceutical compounds with therapeutic spectrum class of antibiotics) (Scheme 17A) [117].
significance. This approach not only avoided the use of metal In 2022, Jiao’s team reported a transaminase engineering
catalysts but also resulted in a shortened synthetic route for the program aimed at converting pyridine octadecyclic ketone to
production of this compound, which serves as a building block for pyridine octadecyclic amine, a key intermediate in the synthesis
the synthesis of drugs like Nazartinib 39 (a potent epidermal of Rimegepant 41. Through a rational design method, they

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obtained an active enzyme variant from an initially inactive performance, achieving a conversion rate of 99%, diastereomer
enzyme. This variant, which utilizes i-PrNH2 as an amine donor, excess (de) > 99.5%, and an 80.2% yield in a gram-scale reaction,
was further enhanced in activity through various evolutionary showcasing its potential for industrial applications (Scheme 17B)
strategies. The resulting enzyme variant exhibited impressive [119].

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Scheme 17. (A) Enzymatic amination of important intermediates of Nazartinib 39 and Besifloxacin 40; (B) Enzymatic amination of Rimegepant

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41.

2.2.2 Cascade reactions involving transaminases

-p manner. Initially, the undesired enantiomer was removed through
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Amine transaminases (ATAs) are powerful enzymes for the reduction by ADH (ADH-097) from the racemic carbon-based
stereospecific production of chiral amines. However, the substrate. Subsequently, the transaminase (ATA-P2-B01) with i-
synthesis of amines incorporating more than one stereocenter is PrNH2 as an amine donor was introduced in the reaction vessel to
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still a challenge. perform a stereoselective transamination, yielding the desired (2R,

In 2021, Burns’s group introduced a biocatalytic cascade 4R)-amine 42. This three-step, two-enzyme, one-pot sequence
reaction for the synthesis of tert-butyl ((2R,4R)-2- avoids the use of organic solvents and certain metal oxidation
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methyltetrahydro-2H-pyran-4-yl) carbamate 42, an intermediate steps, contributing to a more environmentally friendly synthetic
designed for the treatment of Parkinson’s disease. The two route (Scheme 18) [120].
biocatalytic reactions were carried out in a sequential one-pot
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Scheme 18. One-pot kinetic resolution by ADH followed by amination of the remaining enantiomer catalyzed by a transaminase.

3 Conclusion The specificity of transaminases is emphasized as a valuable

feature in drug molecule synthesis, allowing for the catalysis of
The review covers a range of synthesis strategies, including
specific sites in drug molecules. However, despite the success
transamination reactions catalyzed solely by transaminases and
of transaminase catalysis, there are limitations, particularly in
more complex cascade reactions involving multiple enzymes.
the synthesis of spatially closed chiral amines. Challenges
Transaminases play a central role in these enzymatic
include issues related to stereoselectivity and stability of
transformations, leading to the creation of diverse amines from
transaminases. The current set of transaminases may also have
ketones or aldehyde precursors, such as chain fatty amines and
limitations in meeting the specific needs of synthesizing active
cyclic amines.
drug molecules, necessitating further screening and
Transaminases, are highlighted for their high specificity,
modification efforts.
catalyzing specific reactions or producing specific
Recent progress in enzyme engineering techniques, coupled
configurations. The efficiency and mild conditions (specific pH
with the integration of artificial intelligence technologies, is
and temperature requirements) make enzymes attractive tools in
noted for significantly improving the efficiency and specificity
drug molecule synthesis.

21
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Natural Science Foundation of China (No. 22371302), the Non-
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[117] Y. Feng, Z. Luo, G. Sun, M. Chen, J. Lai, W. Lin, S. assistant professor. His research
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Yiman Cui received her M.S. degree

from Yunnan University in 2021. In
2022, she joined the Yang group at the
Institute of Materia Medica, Chinese
Academy of Medical Sciences & Peking
Union Medical College for her PhD
studies. Her research interests focus on
the chemoenzymatic synthesis of active
pharmaceutical ingredients.

24


Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☐ The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:

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