Enantioselective synthesis of 1-Aryl-substituted tetrahydroisoquinolines employing imine reductase

Article abstract of DOI:10.1021/acscatal.7b02628

Tetrahydroisoquinolines (THIQs) with a C1-aryl-substituted groups are common in many natural and synthetic compounds of biological importance. Currently, their enantioselective synthesis are primarily reliant on chemical catalysis. Enzymatic synthesis using imine reductase is very attractive, because of the cost-effectiveness, high catalytic efficiency, and enantioselectivity. However, the steric hindrance of the 1-aryl substituents make this conversion very challenging, and current successful examples are mostly restricted to the simple alkyl-THIQs. In this report, through extensive evaluation of a large collection of IREDs (including 88 enzymes), we successfully identified a panel of steric-hindrance tolerated IREDs. These enzymes are able to convert meta- and para-substituted chloro-, methyl-, and methoxyl-benzyl dihydroisoquinolines (DHIQs) into corresponding R- or S- THIQs with very high enantioselectivity and conversion. Among them, the two most hindrance-tolerated enzymes (with different stereospecificity) are also able to convert ortho-substituted chloro-, methyl-, and methoxyl-benzyl DHIQs and dimethoxyl 1-chlorobenzyl-DHIQs with good enantiometric excess. Furthermore, using in silico modeling, a highly conserved tryptophan residue (W191) was identified to be critical for substrate accommodation in the binding cavity of the S-selective IRED (IR45). Replacing W191 with alanine can dramatically increase the catalytic performance by decreasing the K_m value by 2 orders of magnitude. Our results provide an effective route to synthesize these important classes of THIQs. Moreover, the disclosed sequences and substrate binding model set a solid basis to generate more-efficient and broad-selective enzymes via protein engineering.

Full text of DOI:10.1021/acscatal.7b02628

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Letter
Enantioselective Synthesis of 1-Aryl-Substituted
Tetrahydroisoquinolines Employing Imine Reductase
Jinmei Zhu, Hongqun Tan, Lu Yang, Zheng Dai, Lu Zhu,
Hongmin Ma, Zixin Deng, Zhenhua Tian, and Xudong Qu
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b02628 • Publication Date (Web): 15 Sep 2017
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Page 1 of 6
ACS Catalysis
1
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Enantioselective Synthesis of 1-Aryl-Substituted Tetrahydroiso-
quinolines Employing Imine Reductase
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†
Jinmei Zhu, Hongqun Tan, Lu Yang, Zheng Dai, Lu Zhu, Hongmin Ma, Zixin Deng, Zhenhua
‡
,†,§
Tian, and Xudong Qu*
†
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, School of Pharmaceutical Sci-
ences, Wuhan University, Wuhan 430071, China.
‡
Shanghai R& D Center, Abiochem Co. Ltd., 1299 Ziyue Road, Minhang District, Shanghai, 200241, China.
§
Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing, Jiangsu, China.
ABSTRACT: Tetrahydroisoquinolines (THIQs) with a C1-aryl-substituted groups are common in many natural and synthetic
compounds of biological importance. Currently, their enantioselective synthesis are majorly reliant on chemical catalysis. Enzymat-
ic synthesis using imine reductase is very attractive due to the cost-effectiveness, high catalytic efficiency and enantioselectivity.
However the steric-hindrance of the 1-aryl substituents make this conversion very challenging, and current successful examples are
mostly restricted to the simple alkyl-THIQs. In this report, through extensive evaluation of a large collection of IREDs (including
8
8 enzymes), we successfully identified a panel of steric-hindrance tolerated IREDs. These enzymes are able to convert meta-, pa-
ra-substituted chloro-, methyl-, and methoxyl-benzyl DHIQs (dihydroisoquinolines) into corresponding R- or S- THIQs with very
high enantioselectivity and conversion. Among them, the two most hindrance-tolerated enzymes (with different stereo-specificity)
are also able to convert ortho-substituted chloro-, methyl-, and methoxyl-benzyl DHIQ and dimethoxyl 1-chlorobenzyl-DHIQs with
good ees. Further using in silico modelling, a highly conserved tryptophan residue (W191) was identified to be critical for substrate
accommodation in the binding cavity of the S-selective IRED (IR45). Replacing W191 with alanine can dramatically increase the
catalytic performance by decreasing the K value 2 orders of magnitude. Our results provide an effective route to synthesize these
m
important classes of THIQs. Moreover, the disclosed sequences and substrate binding model set a solid basis to generate more effi-
cient and broad-selective enzymes via protein engineering.
KEYWORDS: biocatalysis, imine reductase, tetrahydroisquinolines, hindrance tolerated IRED, solifenacin.
Tetrahydroisoquinolines (THIQs) are a class of important mo-
lecular scaffolds common in natural products and biological
1
,2
active molecules. Compounds with this moiety often possess
a remarkable spectrum of bioactivities, such as multidrug re-
sistance reversal, anti-diabetic properties, bradycardia, and
2
-4
central nervous system (CNS) activities. Therefore, efficient
incorporation of THIQs has become an important synthetic
1
-4
Figure 1. Selected bioactive 1-aryl-THIQs. I, II and III (solifen-
acin) are representative compounds of pharmaceutical interest
orclinical use.
strategy in drug discovery. Within this class of compounds,
the 1-aryl substituted products are of particular interest due to
their broad spectrum of biological activities and significant
5,6
pharmaceutical properties. The representative molecules I,
quinolines (DHIQs) is particularly attractive. These enzymatic
routes are cost-effective and generally able to afford very high
II and solifenacin (III) (Figure 1), have been used as drug
7
28
candidates and medicine for neurological disorders, leukemia
enantioselectivity. However, due to the strong steric-
8
treatment and clinical treatment of overactive bladder syn-
hindrance of aryl substituents in C1 which affects enzyme
activity, these studies are mostly restricted to 1-alkyl substitut-
9,10
drome, respectively.
19-26
ed THIQs.
The only successful attempt to access 1-aryl-
To date, synthetic efforts have been extensively devoted 1to,2
THIQ was very recently reported using the IRED SnIR which
converts the imine substrate (1a, scheme 1) into S-1-phenyl-
the development of efficient methods for THIQs synthesis.
However, despite the high catalytic efficiency of chemical
27
1
,2
THIQ (S-1b) with a moderate e. e. value (51 %). Considera-
tion of the poor stereo-selectivity and limited substrate scope
of SnIR, additional enzymes with more broad specificity and
higher catalytic efficiency and stereo-selectivity are desired.
To solve this bottleneck, we herein performed extensive ex-
ploration of the sequence space of IREDs. Through systemati-
cally exploring the activity of a large collection of IREDs (88
new entities), we successfully identified a
catalysis, their enantioselectivity and cost remains a chal-
1
,2,11
lenge for industrial application.
Additional cost-effective
approaches especially through biocatalysis are thus highly
welcomed. Recently, the application of artificial imine reduc-
1
2
13,14
tases (IREDs), norcoclaurine synthase,
dase,
monoamine oxi-
have been suc-
15-17
18
19-27
reductive aminase and IREDs
cessfully developed for the synthesis of THIQs. Among of
them, employing IREDs to asymmetrically reduce dihydroiso-
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Page 2 of 6
Scheme 1. Asymmetric Reduction of 1-benzyl-DHIQ Derivatives by IREDs.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
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7
8
9
0
a
Table 1. Conversion and Enantioselectivity of Selected IREDs toward 1a-10a [conversion (%); e. e.(%) ].
Sub.
IR2
IR8
99; >99
99; >99
IR17
IR19
IR20
IR32
IR45
99; >99
99; >99
IR71
IR96
IR99
R
R
R
R
R
R
R
R
S
S
R
S
S
1
2
3
a
a
a
100; 96
100; >99
99; >99
99; >99
99; >99
7; 30
78; 90
97, 97
R
R
R
R
R
R
R
R
99; >99
99; >99
100; >99
100; 99
100; >99
4; 84
29; 47
24; 16
R
R
R
R
R
R
R
R
S
R
R
R
R
S
100; 99
100; 97
100; >99
100; >99
99; >99
100; >99
100; >99
99; 98
99; 91
47; 58
26; 19
94; 72
74; 81
50; 21
R
R
R
R
S
S
R
4a
100; >99
100; >99
100; >99
99; >99
100; >99
99; >99
26; 8
16; 2
R
R
R
R
R
S
R
R
5
6
7
8
a
a
a
a
99; >99
99; >99
99; >99
99; >99
99; >99
100; 35
46; 97
24; 91
R
R
R
R
R
R
R
R
S
R
R
99; >99
99; >99
99; >99
100; >99
100; >99
99; >99
79; 36
82; 3
52; 95
27; 34
R
R
R
R
R
S
R
R
R
100; >99
99; >99
100; >99
100; >99
99; >99
99; >99
5; 21
28; 93
4; 97
5; 93
R
S
36; >99
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
7; 26
0
0
0
0
0
0
0
0
0
S
R
9a
0a
23; 5
9; >99
R
R
1
5; >99
8; 98
a
Stereo-specificity are indicated by the superscripts; e. e. values in bold indicate S-configuration. All reactions were performed at
0 °C for 24 hr.
3
group of highly hindrance-tolerated enzymes that can effi-
ciently convert a series of methyl-, chloro-, and methoxyl-
substituted 1-phenyl-DHIQs and 6,7-diomethoxyl-DHIQ into
the corresponding aryl-THIQs with high enantioselectivity and
conversion.
IR71 as bait, a large collection of homologous proteins were
further identified from the Imine Reductase Engineering Data-
base. To ensure the collection contains adequately diverse
specificity, proteins with high amino acid identity to each oth-
er were excluded. Based on these criteria, a total of 95 novel
IREDs were identified (Table S1) including 68 R-type (IR1-3,
29
To identify the sequence that can accept model substrate 1-
phenyl-DHIQ (1a), we initially examined a small collection of
five IREDs (IR4, IR26, IR71, IR82, IR97, see supplementary
information) which previously have been demonstrated to
5
-25, 27-70) and 27 S-type (IR72-81, 83-96, 98-100) IREDs
(
classified based on the residue in 187 according to the refer-
29
ence ). Genes encoding these novel IREDs were synthesized
based on the codon bias of Escherichia coli and cloned into
pET28a for overexpression. Evaluation of their expression in
E. coli revealed that most proteins were well expressed in the
supernatant; 7 IREDs (IR 10, 23, 67, 73, 84, 85, 86) formed
inclusion bodies (Figure S1) and were excluded from further
studies.
efficiently convert the 1-methyl-DHIQ into 1-methyl-
22,25
THIQ.
These proteins were assayed for the asymmetric
reduction activity of the 1a. One enzyme IR71 shows clear
activity (Table 1) although the conversion (7 %) and enanti-
oselectivity (R-selective, 30 % e. e.) are modest. By using
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ACS Catalysis
Table 2. Kinetics Parameters of Selected IREDs.
1
2
3
4
5
6
7
8
9
Sub.
IR2
IR17
IR45
Km
mM)
k
(s )
k / K
(s mM )
Km
(mM)
0.19
k
(s )
0.52
k / K
(s mM )
2.73
Km
(mM)
0.17
k
(s )
k / K
(s mM )
1.88
cat
cat
-1
m
cat
cat
-1
m
cat
cat
-1
m
-
1
-1
-1
-1
-1
-1
(
1
2
3
4
a
a
a
a
0.026
0.048
0.024
0.029
0.022
0.023
1.54
59.23
0.32
0.33
0.041
0.034
-
1.48
9.57
1.54
3.65
11.29
30.83
398.75
53.10
0.59
0.019
0.12
5.93
2.09
0.71
1.40
1.00
10.05
110.00
5.91
0.11
0.031
0.11
-
3.00
1.32
0.31
-
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
5a
166.91
490.87
0.047
0.069
29.78
14.49
6
a
-
-
-
a
Hyphen indicates data not determined.
The soluble IREDs (88 novel entities) were subsequently
poor e. e. values (3-21%) (Table 1). These results indicate that
IREDs, including both of R and S-types, naturally prefer to
produce R-isomers of substituted-benzyl THIQs. It is very
interesting that IR45 retains the S-specificity to most sub-
strates, especially for the meta-substituted benzyl-DHIQs, with
very high conversion (99%) and S-specificity (e. e. 91%-
>99%) (Table 1). For the para-substituted methyl and meth-
oxyl benzyl-DHIQs, IR45 resulted in a low-to-medium S-
selectivity (e. e. 20.9%-35%), and only reversed to R-
selectivity when the para-substituted chlorobenzyl-DHIQ is
employed (e.e. 36%). The activity of IR45 is influenced by the
size of the para-substitute groups (decrease from methyl, chlo-
ro to methoxyl), suggesting existence of steric hindrance in the
binding pocket (see below). Finally, the kinetic parameters of
IR45 and the two other most active R-selective IREDs (IR2
and IR7) were characterized, which confirmed that these pro-
teins are indeed highly efficient to substrates 2a-6a (Table 2).
The identification of efficient IREDs, particularly the rare S-
specific IR45, is very important and can provide an efficient
and cost-effective avenue to generate enantio-pure substituted
benzyl-THIQs.
employed to assay of the model substrate 1a based on the UV-
Vis absorbance of NADPH in 340 nM. Compound 1a was
generally a poor substrate for most of the enzymes, with only
1
0 enzymes (IR2, 8, 17, 19, 20, 32, 45, 96 and 99 as well as
the initially identified IR71) exhibiting obvious activities. Fur-
ther evaluation of the substrate conversions by HPLC (Table 1)
revealed that i) all the new enzymes except for IR96 (78%
conversion) show very good activity, leading to almost com-
plete conversion (97%-100%); ii) IR2, 8, 17, 19, 20, 32 are R-
specific and IR45, 96, 99 are S-specific. These two groups
exhibit very good stereo-selectivity with e. e. values ranging
from 97 % to >99 %, except for the IR96 (e. e.= 90%). Inter-
estingly, unlike other R-type IREDs (IR2, 8, 17, 19, 20, 32)
IR45 shows a complete reversed stereo-specificity. The per-
formance of IR45 (99% conversion and >99% e. e.) and IR97
(
97% conversion and 97% e. e.) are particularly attractive as
their product S-1b is the pivot intermediate of solifenacin
Figure 1). Further analysis of the kinetic parameters of IR45
(
and the two other most active R-selective IREDs (IR2 and
IR7) revealed that their catalytic efficiency are indeed quite
-
1
-1
efficient (k / K = 1.88, 59.23 and 2.73 s mM respectively)
The good tolerance to the steric hindered meta-, para-
substituted benzyl-DHIQs by these enzymes, led us further
interested in their conversion of more challenging substrates,
for instance the ortho-substituted-benzyl DHIQs. Three repre-
sentative methyl, chloro and methoxyl substituted benzyl-
DHIQs (8a-10a, Scheme 1) were prepared and assayed by
above 10 IREDs. Although most of the IREDs are completely
inactive to these substrates, IR2 and IR45 are still able to con-
vert them with a conversion ranging from 5-36% (Table 1).
The stereo-selectivity of these reactions were generally very
high (e. e. 98%- >99%), with the exception of 9a by IR2 (e. e.
5%) and 8a by IR45 (e. e. 26%) generated in low optical puri-
ty (Table 1). IR45 keeps the S-specificity to methyl-substituted
substrate (8a) and reversed to R-specificity for the chloro,
methoxyl benzyl-DHIQs (9a and 10a). The reversed stereo-
specificity toward ortho-chloroniated, methoxylated and para-
chloroniated substrates (also see below 11a) is interesting,
and may result from the both of the steric-hindrance and elec-
trostatic effects between substrate and residues in the catalytic
cavity. In addition to the 1-aryl group, some pharmaceutically
interesting THIQs also have methoxyl groups in the C6 and
cat
m
(Table 2). Moreover, both of IR45 and IR17 show no obvious
or strong substrate inhibition effect (no inhibition for IR17;
weak inhibition for IR45, Figure S2B, C), which makes them
very suitable for large scale production. To demonstrate the
practical utility of these enzymes, preparative scale biotrans-
formations of 1a-7a by IR2, 8, 17 and IE45 were performed
on a 5 mM scale. The complete conversion with 91.2%-96.5%
isolated yields and > 99% e. e. of S-1b-7b (Table S2) confirms
their potential for practical application.
The ability to accept benzyl group in the C1 position of
DHIQ led us to further evaluate their potential to convert sub-
stituted-benzyl DHIQs. Representative substituents, including
methyl, chloro and methoxyl groups in the meta- and para-
position of 1-benzyl-DHIQ (2a-7a, Scheme 1) were prepared
and assayed. Of significance, IR2, 8, 17, 19, 20, 32 exert both
excellent conversion (> 99%) and stereo-selectivity (e.e. >
99%) of the six meta- and para-substituted benzyl-DHIQs to
the corresponding R-substituted benzyl-THIQs (R-2b-7b) (Ta-
ble 1). Contrary to the R-type, the S-type IREDs (IR71, 96 and
9
9) show very diverse conversion of these substrates. More so,
their stereo-specificities are mostly reversed to the R-
configuration and only in a few cases show S-specificity with
2
C7 (Figure 1). The methoxyl groups increase both of the ste-
ric hindrance and electron density which can reduce the imine
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Page 4 of 6
reactivity, thus are even more challenging for enzyme reduc- the R-products in IR2. It is interesting that the residues consti-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
tion. Currently, no results have been reported to convert 1-aryl
dimethoxyl-THIQs by IREDs. To test the activity of our en-
zymes for these kinds of substrates, the model substrate p-
chlorobenzyl-6,7-dimethoxyl-DHIQ (11a) was prepared and
assayed by above ten IREDs (IR2, 8, 17, 19, 20, 32, 45, 71, 96
and 99). As anticipated, most of the enzymes are completely
inactive to this highly steric-hindered substrate.
tuting the upper left cavity in most IREDs are W or Y (corre-
sponding to 191 in IR45) and A or S (corresponding to 254’ in
IR45) (Figure S4), which could be too bulky to accommodate
the substituted benzyl-THIQs. This may explain the reason
why the natural preference of IREDs to substituted benzyl-
THIQs are mostly R-specific. In IR45, the upper left cavity is
composed by the W191 and the smallest residue G254’; to
further expand its space, W191 was changed into L and A
respectively. Kinetic analysis of the resulting mutants IR45-
W191L and IR45-W191A revealed that the Km of both mu-
tants are dramatically decreased, particularly for the W191A
Interestingly, the two most tolerated IR2 and IR45 can still
convert it into R-11b with a medium conversion (62% and 53%
respectively) and high stereo-selectivity (e. e. of 99% for IR2
and 92% for IR45 respectively) (Scheme 2). R-11b is a phar-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
maceutically important intermediate, and these enzymes pro-
whose K value is 2 orders of magnitude (170 fold) lower than
m
vide a simple and efficient route for its synthesis. Both IR2
and IR45 can accommodate diverse substituents in the 1-
benzyl group (meta-, ortho and para-positions), hence it is
also expected for them to convert other dimethoxyl substitut-
ed-benzyl DHIQs. The broad tolerance to steric hindrance of
IR2 and IR45 provides a biocatalytic route to access very ste-
ric-hindered aryl-THIQs, and their sequence is a good starting
point to create more efficient and stereo-specific IREDs via
protein engineering.
the wild-type IR45 (Table 3). This result confirmed its critical
role in substrate accommodation in the upper left binding cavi-
ty. In addition, the decreased kcat value also indicated this con-
served residue is important for maintenance of the protein
confirmation. Further evaluation of other mutations in this
position may be able to identify more balanced mutants. The
overall catalytic efficiency (k / K ) of W191A is about 8 fold
cat
m
better than the wild-type IR45 (Table 3); more so substrate
inhibition in W191A was completely eliminated comparing to
the wild-type IR45 (K = 2.81 mM) (Figure S2C and S7B),
i
Scheme 2. Asymmetric Reduction of p-chlorobenzyl-6, 7-
dimethoxyl-DHIQ (11a) by IR2 and IR45.
Finally, the distinct stereo-specificity of IR45 from other R-
type IREDs led us to analyze their mode of recognition. Con-
sistent to their specificity, IR2, 8, 17, 19, 20 and 32 are phylo-
genetically close to each other. The identity between them
ranges from 70% to 80%, while their identity to IR45 is only
about 39%. To correlate these difference to the substrate speci-
ficity the structures of IR2 and IR45 were modeled based on
3
0
the R-type IRED Q1EQE0 (Figure S3), which is 44% and 64%
identical to the IR2 and IR45 respectively. Docking 1a into the
binding pocket of IR45 revealed that the substrate is closely
surrounded by the conserved aspartic acid (D183) and
NADPH, which fits the catalytic model of nucleophilic addi-
Figure 2. An in silico model of IR2 and IR45 with the substrate
1a. The structures of IR2 (grey) and IR45 (indigo blue) are mod-
30
eled based on Q1EQE0 (pdb: 3zhb) and docked with 1a (yel-
lowish brown) and NADPH (pink); critical residues in the
binding cavity are labeled by corresponding amino acids; resi-
dues from chain B are indicated by quotation marks. To clearly
show the catalytic dyad, both of the proton-donating hydroxyl
group (red) of aspartic acid (D171/183) and hydrogen atom
(grey) of the NADPH are highlighted in round shape.
30
tion by NADPH and protonation by D183 (Figure 2). More-
over, the benzyl group of 1a is pointing to the upper left cavity
which is constituted by the W191 of subunit A and G254’ of
subunit B. This orientation makes present the molecule to the
Si face of NADPH and subsequently yields the S-product.
Interestingly, superimposing IR2 to IR45 revealed some dis-
tinct features: i) residues M186 (chain A), and H221’ and
L225’ (chain B) in the IR45 are replaced by smaller residues
G174, L209’, V213’ in IR2, which forms a large cavity in the
upper right region. ii) The G254’ in IR45 was replaced by
alanine (A242’) in IR2 which can cause a steric hindrance to
the phenyl group of substrate (1.6 Å). These differences to-
gether force the benzyl group of 1a to reside in the upper right
cavity and expose the Re face to the NADPH and to generate
Table 3. Kinetics Parameters of IR45 and its mutants in
the conversion of 1a.
Enz.
IR45
Km
(mM)
k
(s )
k / K
(s mM )
cat
cat
-1
m
-
1
-1
0.17
0.027
0.001
0.32
1.88
IR45-W191L
0.013
0.015
0.48
IR45-
15.00
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W191A

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ACS Catalysis
ACKNOWLEDGMENT
making this mutant even more efficient for practical applica-
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tion. The identification of the significant W191 provides a
promising way to expand (by engineering) the scope of S-
specific 1-aryl-DHIQs processing IREDs. Currently, engineer-
ing the upright binding cavities of IR2 is also in progress to
improve the performance and expand its substrate scope.
This work was financially supported by the NSFC (31322002,
1270119). We thank Wenbo Liu in the College of Chemistry
Wuhan University for providing the help of chiral analysis.
3
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In summary, through identification and evaluation of a large
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stereo-specificity toward 1-ary-THIQs has been identified.
These enzymes are able to efficiently convert para- meta-
substituted methyl-, chloro-, and methoxyl-phenyl DHIQs into
corresponding THIQ products with high conversion and enan-
tioselectivity (both of R- and S-, up to > 99% e. e.). To the
most challenging substrates, including ortho-substituted phe-
nyl-DHIQs and diomethoxyl chloro-benzyl-DHIQ, both IR45
and IR2 maintain activity with good enantioselectivity in most
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1
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IR1-IR100; HPLC analysis of the enzyme conversion; chiral
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AUTHOR INFORMATION
Corresponding Author
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25) Scheller, P. N.; Nestl, B. M. Appl. Microbiol. Biotechnol. 2016,
00, 10509-10520.
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1052-1059.
*Email: quxd@whu.edu.cn
ORCID
Xudong Qu: 0000-0002-3301-8536
Notes
The authors declare no competing financial interest.
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