Monoamine Oxidase (MAO-N) Whole Cell Biocatalyzed Aromatization of 1,2,5,6-Tetrahydropyridines into Pyridines

Article abstract of DOI:10.1021/acscatal.8b02386

A sustainable MAO-N biocatalyzed process for the synthesis of pyridines from aliphatic tetrahydropyridines (THP) has been developed. Pyridine compounds were synthesized under mild reaction conditions and with high conversion, exploiting MAO-N whole cells as aromatizing biocatalysts. The kinetic profile of the whole cell biocatalytic transformation was finally investigated via in situ ¹⁹F NMR.

Full text of DOI:10.1021/acscatal.8b02386

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Article
Monoamine oxidase (MAO-N) whole cells biocatalyzed
aromatization of 1,2,5,6-tetrahydropyridines into pyridines
Anita Toscani, Caterina Risi, Gary W. Black, Nicola L. Brown,
Ali Shaaban, Nicholas J Turner, and Daniele Castagnolo
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b02386 • Publication Date (Web): 07 Aug 2018
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Monoamine oxidase (MAOꢀN) whole cells
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biocatalyzed
aromatization
of
1,2,5,6ꢀ
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tetrahydropyridines into pyridines
Anita Toscani,‡ Caterina Risi,‡ Gary W. Black,† Nicola L. Brown,† Ali Shaaban,‡ Nicholas J.
Turner,± and Daniele Castagnolo‡,*
‡School of Cancer and Pharmaceutical Sciences, King’s College London, 150 Stamford
†
Street, SE1 9NH, London, United Kingdom. Department of Applied Sciences, Northumbria
±
University, Ellison Place, NE1 8ST, Newcastle upon Tyne, United Kingdom. School of
Chemistry, Manchester Institute of Biotechnology, University of Manchester, 131 Princess
Street, M1 7DN, Manchester, United Kingdom.
ABSTRACT. A sustainable MAOꢀN biocatalyzed process for the synthesis of pyridines from
aliphatic tetrahydropyridines (THP) has been developed. Pyridine compounds were
synthesized under mild reaction conditions and with high conversion exploiting MAOꢀN
whole cells as aromatizing biocatalysts. The kinetic profile of the whole cell biocatalytic
transformation was finally investigated via in situ 19F NMR.
KEYWORDS. biocatalysis, MAOꢀN, pyridine, aromatization, tetrahydropyridine, in-situ
NMR
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INTRODUCTION
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The pyridine ring system represents the major class of heterocycles and constitute the key
structural unit of many pharmaceutical, agrochemical and material compounds.1 Natural
molecules such as the vitamins niacin and pyridoxine or the ubiquitous redox system
NADP/NADPH contain a pyridine nucleus as well as many drugs like the antitubercular
agent isoniazid 1, the analgesic etoricoxib 2 or the anticancer agent sorafenib 3.2 Pyridine
derivatives also find application in flavor and fragrance industry due to their peculiar
olfactory properties ranging from the unpleasant fishꢀlike smell of pyridine itself to the fried
chicken smell of 2ꢀpentylpyridine 4 or the peppermint odor of lutidine.3 A plethora of
methodologies for the synthesis of pyridines has been reported in the literature so far.4 The
two most commonly used methods to synthesise substituted pyridines are the Hantzsch5 and
the Chichibabin syntheses.6 Both these approaches show some limitations though, often
leading to desired pyridines in low yields and with the formation of many side products. In
addition, the Hantzsch synthesis of pyridines is a two steps protocol which requires an
oxidation step commonly carried out with nitric acid, metal complexes or high temperatures.7
Due to the versatility of pyridine rings and their broad use in different research fields, new
approaches to access this important class of compounds are constantly developed.8
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In an effort to find new, greener and more sustainable routes to access aromatic nitrogen
heterocycles from aliphatic precursors, we recently discovered that the monoamine oxidase
MAOꢀN, variants from Aspergillus niger, can act as aromatizing biocatalysts and can
catalyze the synthesis of pyrrole derivatives A from 3ꢀpyrrolines B and allylamine substrates9
(Figure 1). In nature, MAO enzymes are known to metabolize the xenobiotic 1,2,5,6ꢀ
tetrahydropyridines (THPs) into pyridine derivatives or pyridinium compounds through an
aromatization biotransformation. The natural human monoamine oxidase MAOꢀB catalyzes
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the oxidation of 1ꢀmethylꢀ4ꢀphenylꢀ1,2,3,6ꢀtetrahydropyridine (MPTP) into the neurotoxin 1ꢀ
methylꢀ4ꢀphenylpyridinium (MPP+), a toxic metabolite involved in Parkinson’s disease,
through a double oxidative process via the 1ꢀmethylꢀ4ꢀphenylꢀ2,3ꢀdihydropyridinium
(MPDP+) intermediate.10 Inspired by this metabolic biotransformation and as a natural
evolution of our previous work, we decided to investigate the possibility to exploit MAOꢀN
as aromatizing biocatalysts in the synthesis of pyridine derivatives and to develop a robust
biocatalytic process to access this important class of compounds.
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Herein, the development of an aromatizing biocatalytic protocol for the synthesis of
pyridines and pyridinium derivatives D from aliphatic THP substrates C using freezeꢀdried
MAOꢀN whole cell biocatalysts under mild conditions is described. In addition, an in-situ
19FꢀNMR kinetic study of the MAOꢀN whole cell biocatalyzed aromatization reaction is
presented. In-situ NMR allows the real time monitoring and analysis of chemical reactions
including biocatalytic processes, as previously described.11 However, to the best of our
knowledge, no examples of in-situ NMR studies on whole cell biocatalyzed transformations
have been reported yet.
RESULTS AND DISCUSSION
The MAOꢀN mediated aromatization of the THP 5a into the pyridine 6a was first
investigated. Freezeꢀdried whole cells containing MAOꢀN variants D5, D9, and D11 were
selected on the basis on their known activity and selectivity toward structurally related
piperidines.12 The THP 5a was suspended in a buffer solution (pH = 7.8) in the presence of
minimum amount of cosolvent (DMSO or isooctane8) and treated with different MAOꢀN
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biocatalysts at 37 C for 18ꢀ24h. The conversion (%) of the biotransformation reaction was
determined by 1HꢀNMR integration of the crude mixture or by HPLC. Results are reported in
Table 1. The treatment of 5a with MAOꢀN variants D5, D9 and D11 in a 1:66 buffer/DMSO
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mixture lead to the desired pyridine 6a with good conversions (entries 1-3) with the best
result obtained with enzyme D9 (76%). Interestingly, when a DMSO/DMF 1:4 mixture was
used as cosolvent (1.6% of the whole reaction mixture) an increase in the yield of 6a was
observed (entry 4). On the other hand, excellent and greater conversions (>90%, entries 5-7)
were obtained when isooctane was employed. In the presence of isooctane, the aromatization
reaction was completed in 18h with all the MAOꢀN variants and, again, the variant D9
proved to be the best biocatalyst. The role of the cosolvent seems to be crucial to some extent
for the outcome of the reaction as shown in our previous work.9 Isooctane is a water nonꢀ
miscible solvent, which, together with the buffer solution, generates a biphasic system with
low mass transfer.13 We hypothesised that, even if in small concentration, a water miscible
solvent like DMSO may have, in the long term, a detrimental effect on the MAOꢀN
biocatalyst leading to its progressive inactivation. On the other hand, in the presence of a
nonꢀwater miscible solvent like isooctane this inactivation could be prevented, thus leading,
as final result, to higher substrate conversion. Although substrate/product phase transfer
mechanisms are not fully clear for this biotransformation, it is reasonable to envisage that the
aromatization of 5a into 6a could occur at or in proximity of the waterꢀsolvent interphase. It
is also possible that different cosolvents affect the permeability of cell membranes of the
whole cell biocatalysts, in turn aiding the substrate uptake and improving the reaction
outcome. Interestingly, as further corroboration of the key role of the cosolvent, when the
biotransformation was carried out in buffer only, the pyridine 6a was obtained with only 80%
conversion (entry 8). The aromatization of 5a with the nicotine oxidase biocatalyst 6ꢀHDNO
E350L/E352D14 was also explored, but no conversion was observed and the THP was fully
recovered from the reaction mixture (entry 9). Finally, as confirmation that the aromatization
of 5a is catalyzed by the MAOꢀN, rather than spontaneously promoted by the oxygen in the
air or other chemicals and side products present in the reaction mixture, a series of blank
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experiments were carried out. When no biocatalyst was added to the reaction mixture (entry
10), no traces of the pyridine 6a were detected after 24h, confirming that the MAOꢀN is
responsible for the aromatization of 5a. Similarly, the treatment of 5a with E. coli
BL21(DE3) cells no harboring MAOꢀN enzymes under optimized conditions did not lead to
any pyridine 6a (entry 11). The MAOꢀN catalyzed biotransformations requires oxygen as coꢀ
substrate for the oxidation reaction, thereby producing H2O2 as a sideꢀproduct. In order to
confirm if the H2O2 formed during the biotransformation affects the aromatization of the
THP, the oxidation of 5a in the presence of the enzyme catalase was investigated. In fact,
catalase acts as H2O2 scavenger, converting the hydrogen peroxide formed during the MAOꢀ
N biocatalyzed aromatization into H2O. When the substrate 5a was treated with MAOꢀN D9
in the presence of catalase, the desired pyridine 6a was formed with 72% conversion after
18h (entry 12), thus confirming that the H2O2 has no effect (or only minor effect) on the
biocatalytic aromatization of THPs into pyridines. As further confirmation that H2O2 was not
responsible for the aromatization of the THP, the substrate 5a was suspended in buffer
solution (pH 7.8) at 37 oC and treated with H2O2 30% w/w. Only traces (<4%) of the pyridine
6a were detected from the reaction mixture after 24h.
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According to previous studies on the MAO metabolism,10 we assume that the biocatalytic
aromatization of THP substrates occurs in two steps as shown in Scheme 1. The enzyme
MAOꢀN catalyzes first the CꢀN oxidation of 5a leading to the 5,6ꢀdihydropyridinium
intermediate 7a. This latter can spontaneously aromatize into 6a in the presence of O2 from
the air or alternatively tautomerize into the intermediate 7b and, eventually, undergoes a
second MAOꢀN catalyzed CꢀN oxidation leading to 6a.
Once the best reaction conditions were identified the scope of the aromatizing
biotransformation was investigated. A series of THPs 5b-f and 14a-b were synthesised as
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reported in Scheme 2.15 The commercial NꢀBocꢀpiperidone was treated with different arylꢀ
Grignard reagents leading to tertiary alcohol derivatives 8b-e, which were in turn treated with
TFA leading to 5b-e through dehydratation and concomitant Bocꢀcleavage (Scheme 2a). The
synthesis of THP 14a-b was carried out according to the protocol described in Scheme 2b.
Benzaldehyde or hexanal and allylamine were reacted to form the imines 11a-b, which were
in turn treated with allylmagnesium bromide in the presence of ZnEt2 and (Boc)2O to afford
the dienes 12a-b.16 Ringꢀclosing metathesis of 12a-b led to the THPs 13a-b which were
finally deprotected with HCl/AcOEt to give desired 14a-b as HCl salts. The THPs 5b-f and
14a-b, together with the NꢀmethylꢀTHP 15a-c17 were then treated with the MAOꢀN whole
cell biocatalysts. The results are reported in Tables 2 and 3. The THP 5b was fully converted
(99%, entry 1) into pyridine 6b by MAOꢀN D9 when the reaction was carried out in
buffer/isooctane mixture. Comparable conversions were observed with MAOꢀN D5 and D11,
whilst slightly lower conversions were obtained when DMSO was used as cosolvent. The
pyridines 6c-d were also obtained with excellent conversions (entries 2-3), in particular when
additional loading of the MAOꢀN biocatalysts was used in the course of the reaction. The
chloroꢀderivative 6e was obtained in slightly lower amount (57%, entry 4),18 while the
bromoꢀderivative 6f was formed with only 34% conversion (entry 5) probably due to the
steric hindrance of the halogen substituent on the phenyl ring. The Nꢀmethyl derivatives 15a-
b were fully converted (99ꢀ87%) into the desired pyridinium ions 16a-b by the MAOꢀN D9
biocatalyst. On the other hand, arecoline 15c, a THP bearing an electron withdrawing
substituent, was not converted into the desired pyridinium 16c even when higher biocatalyst
loading and longer reaction times were employed. The THPs 14a-b bearing a stereocentre at
C6 were only partially converted by MAOꢀN D9 and D11 into the corresponding pyridines
17 and the flavoring agent 4 (Table 3). These results were not unexpected, since it is well
documented that MAOꢀN biocatalysts can stereoselectively oxidize one enantiomer over the
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other in chiral amines. In fact, MAOꢀN enzyme selectively aromatized the enantiomers (S)ꢀ
14a-b into pyridines 17 and 4 (entries 1-2), whilst the opposite enantiomers (R)ꢀ14a-b were
recovered from the reaction mixture as major products with approximately 20% ee.19 It is
also plausible that factors (i.e. steric factors) may contribute to the low conversion observed
for 14a-b. Attempts to increase the conversion of 14a-b by increasing the catalyst loading or
the reaction time were unsuccessful.
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The MAOꢀN whole cell biocatalyzed aromatization of THP 5d was finally investigated by
in situ 19F NMR spectroscopy. The experiment was carried out under standard conditions
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(buffer pH 7.8, isooctane cosolvent, 37 C) using the whole cell biocatalyst MAOꢀN D9.
1
Attempts to monitor the biocatalytic aromatization of THP substrates via in situ H NMR
proved to be tricky due to the interference caused by the cells in the reaction mixtures, as well
as the cosolvent.20 The THP 5d, bearing a fluorine atom, was thus chosen as candidate for the
in situ NMR experiments, as it would allow monitoring of the 19F nucleus with less
interferences arising from the whole cell biocatalyst. The substrate 5d was suspended in
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buffer solution containing 1.6% isooctane and treated with MAO D9 at 37 C. The reaction
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was monitored for 24h during which time F NMR experiments were recorded in sequence
every 15 minutes (number of scans: 128) using the protonꢀdecoupled 19F sequence. A
selected number of 19F NMR spectra is reported in Figure 2a. After approximately 90 min
from the addition of the biocatalysts to the reaction mixture, a signal at ꢀ113.61 pm belonging
to the pyridine 6d appeared in the NMR spectrum and it became clearer after 120 min. The
intensity of the signal at ꢀ115.56 ppm belonging to the substrate 5d start to decrease already
after 30 min and the signal disappear completely after 10h with concomitant increase of the
6d fluorine peak. According to the in-situ NMR experiment the reaction was completed
within 12h. As the integration of the peaks is proportional to the concentration of the species
involved, the kinetic profiles of each species were reported by plotting the integration of each
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peak against the time (Figure 2b). The less sharp and defined signals of 6d, compared to 5d,
are most likely due to residues of the biocatalyst (cells and their organelles present in the
reaction mixture) or affinity of the substrate to the cell membranes, which, limiting the
mobility of the compounds, may be responsible for the broadening of the fluorine peak. To
confirm the full conversion of the biotransformation, the reaction was stopped after 24h,
extracted and the crude mixture was purified affording THP 6d in 87% isolated yield.21
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Interestingly, the F signal of 6d starts to appear after a consistent time (more than 90 min)
while the signal of 5d start to decrease after few minutes. It is likely that during the first
phase of the biotransformation, the THP 5d is converted into the partially oxidized
intermediates 7 (Scheme 1) before being fully aromatized into 6d. A weak signal at around ꢀ
108.75 ppm was detected by 19FꢀNMR spectra after 30 min. This peak disappears completely
after 4h and it might belong to intermediates 7.22 It is plausible that the low concentration and
short halfꢀlife of intermediates 7 over the reaction time, as well as the interference caused by
the whole cell biocatalyst, made their detection not clearly evident.
CONCLUSIONS
In conclusion, an efficient biocatalytic protocol for the synthesis of pyridines from aliphatic
tetrahydropyridine precursors exploiting MAOꢀN whole cell biocatalysts has been described.
The methodology represents a mild, alternative and more sustainable approach to access 6ꢀ
membered nitrogen heterocycles through the aromatization of aliphatic substrates. In
particular, the biocatalyst MAOꢀN D9 was found able to catalyze the biotransformation of a
variety of arylꢀTHPs into aryl pyridines 6 and pyridinium ions 15 with excellent conversions
by mimicking the natural metabolic activity of human MAO enzymes. Also the challenging
chiral THP substrates 14a-b were partially converted into the pyridine 17 and the flavouring
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agent 4. Finally, the first example of in situ 19F NMR study of a whole cell biocatalyzed
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reaction was reported.
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O
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N
N
O
NHNH2
H
Sorafenib 3
Anticancer
Cl
O
O
N
F3C
N
N
Isoniazid 1
Antitubercular
H
H
SO2Me
Cl
N
2ꢀPentylpyridine 4
Flavoring ingredient
(grilled/fried chicken, pork
and beef aroma)
N
N
Etoricoxib 2
anti-inflammatory
COX-2 inhibitor
Previous work
H
N
N
H
N+
MAOꢀN D5
R
R
R
B
A
This work
R
R
R
aromatization
N
N
N
MAOꢀN
H
D
C
Figure 1. Pyridine nucleus containing drugs and flavoring agents
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Figure 2. a) Stacked in situ 19F NMR spectra of the aromatization of 5d using MAOꢀN D9
whole cells. b) The kinetic profile of the reaction shows the disappearance of 5d and the
formation of the pyridine product 6d.
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Scheme 1. Plausible mechanism of MAOꢀN biocatalytic aromatization.
(a) Synthesis of 4-substituted THPs
R
R
R
MgBr
O
HO
TFA
THF,
0 oC to rt
N
N
N
H
Boc
Boc
8bꢀe
(b) Synthesis of 6-substituted THPs
5bꢀe
4 Å MS
DCM
R
N
H2N
+
R
O
9aꢀb
10
11aꢀb
1)
MgBr
ZnEt2, THF
2) Boc2O
Grubbs' cat.
2nd gen.
HCl/AcOEt
R
N
R
N
—HCl
R
N
DCM
H
Boc
Boc
14aꢀb
13aꢀb
12aꢀb
Scheme 2. a) Synthesis of aryl THPs 5b-e. b) Synthesis of chiral THPs 14a-b
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Table 1. Biocatalytic aromatization of THP 5a
5
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∗
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N
N
MAO-N
whole cells
ꢂ H
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Buffer (pH 7.8)
cosolvent,
∗
ꢂ
N
time, 37 oC
N
H
Entry
MAO-Na
D5
Cosolvent (1.4%) Time (h) Conv. (%)b
1
2
DMSO
DMSO
24
24
24
24
18
18
18
15
24
24
24
24
70
76
67
95
92
98f
95
80
0
D9
3
D11
D9
DMSO
4
DMSO/DMF 1:4
isooctane
isooctane
isooctane
ꢀ
5
D5
6
D9
7
D11
D9
8
9
6ꢀHDNO
ꢀ c
DMSO
10
11
12
DMSO
0
ꢀ d
DMSO
0
D9e
isooctane
72
aFreezeꢀdried whole cells (140mg / 0.1mmol of 5a) were used. Conditions are reported in the Supporting
Information. bConversion was determined by HꢀNMR integration of the crude mixture or by HPLC
1
c
d
analysis using a Chiralpak IG column. No biocatalyst was added to the reaction mixture. E. coli
BL21(DE3) cells no harboring MAOꢀN enzymes were used. eCatalase was added to the reaction mixture.
fLarge scale reaction afforded 6a with 99% conv. and 75% isolated yield
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Table 2. Biocatalytic aromatization of THPs 5b-f and 15a-c
5
6
R
R
7
8
9
N
H
5bꢀf
N
6bꢀf
MAOꢀN
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
buffer pH = 7.8
R
R
cosolvent, 37 oC, time
N+
N
16aꢀc
15aꢀc
Cosolvent
Pyridine/
MAO-
Entry THP
R
Time
Conv. (%)b
Na
(1.4%)
pyridinium
D5
D9
DMSO
20h
20h
20h
20h
20h
20h
65
85
72
93
99
95
DMSO
DMSO
D11
D5
6b
1
4ꢀ(4ꢀMeꢀPh)
5b
isooctane
isooctane
isooctane
D9
D11
D9
D9
DMSO
DMSO
DMSO
isooctane
DMSO
DMSO
DMSO
DMSO
isooctane
isooctane
DMSO
24h
48h
24h
24h
24h
24h
48h
24h
24h
24h
24
63
73
98
70
58
50
55
98
54
28
55
2
4ꢀ(4ꢀMeOꢀPh)
D9c
5c
6c
D9
D11
D9
D9
3
4
4ꢀ(4ꢀFꢀPh)
4ꢀ(4ꢀClꢀPh)
D9c
5d
5e
6d
6e
D9
D11
D9
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5
D9c
D9
DMSO
24h
57
20
isooctane
DMSO
24 h
6
7
8
9
D9
D9c
D11
34
30
16
24 h
24 h
24 h
5
4ꢀ(4ꢀBrꢀPh)
5f
6f
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
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60
isooctane
isooctane
6
7
8
4ꢀPh
3ꢀPh
D9
D9
D9
isooctane
isooctane
isooctane
24h
24h
99
87
0
15a
15b
15c
16a
16b
16c
3ꢀCOOMe
24h
aFreezeꢀdried whole cells were used. Conversion was determined by HꢀNMR integration of the crude
b
1
c
mixture or by HPLC analysis using a C18 reverse phase column (for compounds 16a-c). Additional
amount of MAOꢀN D9 (140 mg / 0.1 mmol substrate) was added after 9 h.
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Table 3. Biocatalytic aromatization of THPs 14a-b
5
6
7
8
MAOꢀN
+
9
R
N
buffer pH = 7.8/
isooctane 66:1
37 oC, 24h
R
N
H
R
N
H
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
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42
43
44
45
46
47
48
49
50
51
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53
54
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14a
14b R = Pentyl
R = Ph
17 R = Ph
(R)ꢀ14a R = Ph
(R)ꢀ14b R = Pentyl
4
R = Pentyl
Pyridine/THP
ratio. (%)a
25:75
(R)-14a-b
Entry THP
MAO-N
ee (%)c
D9
D9b
D11
D9
21
ꢀ
1
26:74
14a
24:76
20
ꢀ
18:82
2
D9b
26:74
ꢀ
14b
D11
24:76
ꢀ
aThe ratio between the pyridine product (17 or 4) and the unreacted THP (14a-b) was determined by
b
HPLC using a Chiralpak IG column. Additional amount of MAOꢀN D9 (140 mg / 0.1 mmol substrate)
c
was added after 9h. The ee of unreacted THP was determined by GC using a BetaꢀDEX 325 Capillary
Column.
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AUTHOR INFORMATION
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Corresponding Author
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*Daniele Castagnolo, School of Cancer and Pharmaceutical Sciences, King’s College London,
150 Stamford Street, SE1 9NH, London, United Kingdom. Eꢀmail: daniele.castagnolo@kcl.ac.uk
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Funding Sources
EPSRC King’s College London Strategic Fund
ASSOCIATED CONTENT
Supporting Information. Experimental procedures and full characterization for new compounds
1
13
and substrates are reported in the Supporting Information. Copies of HꢀNMR and CꢀNMR
spectra for new compounds are reported. This information is available free of charge on the ACS
Publications website.
ACKNOWLEDGMENT
We are grateful to EPSRC King’s College London Strategic Fund for financial support. Dr
Richard Lloyd (Glaxo Smith Kline) is gratefully acknowledged for helpful discussions. We
thank the Università degli Studi di Siena for a PhD leave of absence for CR.
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REFERENCES
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Cycloaddition of Enamides and Alkynes for the Synthesis of Highly Substituted Pyridines
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Alkoxy/2ꢀSulfenylpyridines and Dihydrofuro[2,3ꢀb]pyridines by Trapping in Situ Generated
1,4ꢀOxazepine. J. Org. Chem., 2017, 82, 9515ꢀ9524; i) Colby, D. A.; Berman, R. G.; Ellman,
J. A. Synthesis of Dihydropyridines and Pyridines from Imines and Alkynes via CꢀH
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Castagnoli, N. Jr. Studies on the Molecular Mechanism of Bioactivation of the Selective
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a) Lauder, K.; Toscani, A.; Qi, Y.; Lim, J.; Charnock, S.J.; Korah, K.; Castagnolo, D.
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Photoꢀbiocatalytic OneꢀPot Cascades for the Enantioselective Synthesis of 1,3ꢀ
Mercaptoalkanol Volatile Sulfur Compounds. Angew. Chem., Int. Ed. 2018, 57, 5803ꢀ5807. b)
Drexler, M. T.; Foley, D. A.; Ward II, H. W.; Clarke, H. J. IR and NMR Reaction Monitoring
Techniques for Nucleophilic Addition Reactions: In Situ Monitoring of the Addition of
Benzimidazole to a Pyridinium Salt. Org. Process Res. Dev. 2015, 19, 1119–1127. c) Foley,
D. A.; Bez, E.; Codina, A.; Colson, K. L.; Fey, M.; Krull, R.; Piroli, D.; Zell, M. T.; Marquez,
B. L.; NMR Flow Tube for Online NMR Reaction Monitoring. Anal. Chem. 2014, 86, 12008–
12013. d) Buser, J. Y.; McFarland, A. D. Reaction Characterization by Flow NMR:
Quantitation and Monitoring of Dissolved H2 via flow NMR at High Pressure. Chem.
Commun. 2014, 50, 4234ꢀ4237. e) Susanne, F.; Smith, D. S.; Codina, A. Kinetic
Understanding Using NMR Reaction Profiling. Org. Process Res. Dev. 2012, 16, 61–64.
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Herter, S.; Medina, F.; Wagschal, S.; Benhaïm, C.; Leipold, F.; Turner, N. Mapping the
Substrate Scope of Monoamine Oxidase (MAOꢀN) as a Synthetic Tool for the
Enantioselective Synthesis of Chiral Amines. J. Bioorg. Med. Chem. 2018, 26, 1338 – 1346
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a) Denard, C. A.; Hartwig, J. F.; Zhao, H. Multistep OneꢀPot Reactions Combining
Biocatalysts and Chemical Catalysts for Asymmetric Synthesis. ACS Catal. 2013, 3, 2856 −
2864. b) Denard, C. A.; Bartlett, M. J.; Wang, Y.; Lu, L.; Hartwig, J. F.; Zhao, H.
Development of a OneꢀPot Tandem Reaction Combining RutheniumꢀCatalyzed Alkene
Metathesis and Enantioselective Enzymatic Oxidation To Produce Aryl Epoxides. ACS Catal.
2015, 5, 3817−3822. c) Denard, C. A.; Huang, H.; Bartlett, M. J.; Lu, L.;Tan, Y.; Zhao, H.;
Hartwig, J. F. Cooperative Tandem Catalysis by an Organometallic Complex and a
Metalloenzyme. Angew. Chem., Int. Ed. 2014, 53, 465−469.
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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.
15.
See Supporting Information for synthetic procedures. The THPs 5a and 5f are
commercially available and were purchased.
16.
Tait, M. B.; Butterworth, S.; Clayden, J. 2,2ꢀ and 2,6ꢀDiarylpiperidines by Aryl
Migration within Lithiated Urea Derivatives of Tetrahydropyridines. Org. Lett. 2015, 17,
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17.
The THPs 15c is commercially available and was purchased. The THP 15a-b was
5
6
7
8
synthesised according to literature as described in the Supporting Information
18.
Contrary to what observed with substrate 5a, the conversion of THPs 5c-d into 6c-d was
not affected by the nature of the cosolvent. In fact, comparable results were obtained when
both DMSO or isooctane were used.
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19.
The selective oxidation of the (S)ꢀenantiomer of 14a-b was established according to the
known enantioselctivity of MAOꢀN biocatalysts (see reference 11). The ee of (R)ꢀ14a was
confirmed by GC analysis using a BetaꢀDEX 325 Capillary Column. Attempts to separate the
enantiomers of 14b via HPLC or GC were unsuccessful
1
20.
An example of in situ H NMR of the whole cell biocatalyzed aromatization reaction is
reported in the Supporting Information together with further details on the experiment.
21.
Both a 1H NMR and a 19F NMR spectrum in CDCl3 of the crude reaction mixture after in
situ 19F NMR showing full conversion of the biotransformation are reported in the Supporting
Information (Figures S4 and S5).
22.
Information.
A full 19FꢀNMR spectrum showing the peak at ꢀ108.75 is reported in the Supporting
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SYNOPSIS.
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2.0
1.5
1.0
0.5
0
stacked fluorine spectra.1r.esp
-0.5
-1.0
-1.5
-2.0
-112.5
-113.0
-113.5
-114.0
-114.5
Chemical Shift (ppm)
-115.0
-115.5
-116.0
-116.5
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