A biocatalytic cascade for the amination of unfunctionalised cycloalkanes

Article abstract of DOI:10.1039/c7ob02569f

Here we describe a one-pot, three-enzyme, cascade involving a cytochrome P450 monooxygenase, an alcohol dehydrogenase and a reductive aminase for the synthesis of secondary amines from cycloalkanes. Amine product concentrations of up to 19.6 mM were achieved. The preparative scale amination of cyclohexane was also demonstrated with a space-time yield of 2 g L^-1 d^-1.

Full text of DOI:10.1039/c7ob02569f

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A biocatalytic cascade for the amination of unfunctionalised
cycloalkanes
Michele Tavanti,a Juan Mangas-Sanchez,a Sarah L. Montgomery,a Matthew P. Thompsona and
Nicholas J. Turner*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Here we describe a one-pot, three-enzyme, cascade involving a metal complexes in place of the natural heme.22–26 Even
cytochrome P450 monooxygenase, an alcohol dehydrogenase and though these studies have extended the potential for
a reductive aminase for the synthesis of secondary amines from biocatalytic C-H functionalisation remarkably, these novel
cycloalkanes. Amine product concentrations of up to 19.6 mM catalysts either rely on pre-functionalized molecules
were achieved. The preparative scale amination of cyclohexane possessing both the nitrogen functionality and the target C-H
was also demonstrated with a space-time yield of 2 g L-1 d-1.
bond (intramolecular C-H amination), or on pre-activated
aminating agents (e.g., tosyl azides).
A large number of biologically active molecules contain
nitrogen functionalities,1,2 and consequently there is demand
for the development of efficient synthetic routes to access
these valuable chemicals. Biocatalytic routes have been
developed for the synthesis of chiral amines using
transaminases,3,4 ammonia lyases,5,6 amine dehydrogenases,7
monoamine oxidases8,9 and imine reductases.10–12 Recently,
the toolbox of enzymes for amine synthesis has been
expanded by the discovery of an NADP(H)-dependent
reductive aminase (RedAm) from Aspergillus oryzae
(AspRedAm). This enzyme is capable of catalysing not only the
reduction of preformed imines, but also the coupling of
amines and ketones followed by the subsequent reduction of
the imine intermediate.13 All of these enzymes require
electrophilic centres to insert nitrogen atoms, whereas a more
elegant route would be the direct replacement of an
unactivated C-H with a C-N bond or its synthetic equivalent.
In organic chemistry, C-H activation is typically enabled by
metal complexes and requires the presence of auxiliary
groups.14–20 Rare enzymatic counterparts have been found in
nature to carry out this attractive chemistry,21 and only
recently, heme-proteins have been engineered to perform
C(sp3)-H amination reactions. This biocatalytic amination
reaction was achieved through the introduction of mutations
at residues essential for the native mono-oxygenation activity
of a cytochrome P450 or through the installation of synthetic
Oxidative enzymes have previously been successfully
combined with amination biocatalysts to functionalise C(sp3)-H
bonds,28,29 alkenes30 or to convert alcohols to amines.31,32
Moreover, biocatalytic redox-neutral hydrogen-borrowing
processes have been demonstrated for the conversion of
alcohols into the corresponding primary amines.33,34,35 Thus
far, these studies are limited to the synthesis of primary
amines due to the nature and specificity of the amination
biocatalyst. However, recently we have demonstrated the
synthesis of aliphatic and aromatic secondary amines starting
from primary and secondary alcohols via hydrogen-borrowing
employing a single alcohol dehydrogenase (ADH) and the
newly characterised AspRedAm.36
Herein, we report
a new biocatalytic cascade for the
conversion of cycloalkanes to secondary amines employing a
cytochrome P450 mono-oxygenase for the first oxidation step
followed by amination using an ADH in combination with a
RedAm (Scheme 1). As a model substrate, we selected
cyclohexane
1
for P450-catalyzed hydroxylation to the
followed by ADH
and subsequent
corresponding alcohol cyclohexanol
2
mediated oxidation to cyclohexanone
3
amination with AspRedAm.
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In the light of these initial results, we incubated crude enzyme
preparations (the exception being AspRedAm, used in purified
form) with
1
in the presence of 1 mM NAD+ and 1 mM NADP+,
250 mM sodium formate and 250 mM methylamine, but
unfortunately no amine formation was detected. We
speculated that the cofactors required for the hydrogen-
borrowing step (NADP+/NADPH) were intercepted by
endogenous E. coli enzymes, leading to imbalanced cofactor
regeneration.33,34 Thus, the amount of lysate added for the
hydroxylation step was reduced and, gratifyingly, conditions
were found to carry out the reaction as a one-step process (4.5
mM N-methylcyclohexylamine 1b produced, Table S1). Next,
we decided to extend the one-step cascade for the amination
Scheme 1. Biocatalytic cascade for the amination of cycloalkanes.
For the initial C-H activation step, we selected the P450-BM3 of
1 using a panel of six amine nucleophiles at different
mutant R47L/Y51F which was previously shown to have high loadings (250, 100, 50, 25 and 10 mM, as reported in Table S1),
activity towards cyclohexane.37 In particular, it was observed guided by our initial investigation on the effect of the selected
that the hydroxylation of cyclohexane improved when the amines on hydroxylation activity (Figure 2 and Table S1). The
reaction was carried out in a biphasic system, with the outcome of the amination cascade was observed to be a trade-
substrate acting as co-solvent.38 In order to uncouple the off between the inhibitory effect of the amine donor on the
cofactor dependence of the two steps and avoid competition P450 and the reactivity of the same amine nucleophile in the
between the P450 and the RedAm for reducing equivalents, AspRedAm-catalysed reductive amination.13 3.6 mM N-
the two amino acid substitutions R966D and W1046S were propargylcyclohexylamine
introduced in the reductase domain of the self-sufficient P450- cyclopropylcyclohexylamine 1f were produced when
BM3 to increase its affinity towards NADH.38,39 Cofactor propargylamine
and cyclopropylamine were used as
regeneration for the oxyfunctionalisation step was performed reacting partners in the one-step process, with 0.7 mM
by the formate dehydrogenase from Candida boidinii cyclohexanone
left. Interestingly, the highest product
(CboFDH).40 Finally, for the amination step via hydrogen- concentration was observed with
(8.3 mM), although a
borrowing we selected variant of the ADH from considerable amount of
(4.7 mM) and (5.9 mM) remained
Thermoanaerobacter ethanolicus (TeSADH W110A)41 and unreacted. Moreover, 9.6 mM cyclohexanone
accumulated
AspRedAm, as previously reported by our group.36
with just 1 mM NADP+ when
was employed as the amine
1c
and
2.1
mM
N-
c
f
3
e
a
2
3
3
a
In an attempt to exploit all the selected enzymes in a one-step donor, although only a low concentration of cyclohexylamine
process, we initially investigated the effect of amines a–f at 1a was produced (0.2 mM, Table S2). These results suggest
different substrate concentrations on the performance of the that NADP+ regeneration might also be supported by the P450
P450, expressed as residual total turnover numbers (TTNs, (which can still accept the phosphorylated cofactor)38 and/or
Figure 1). The negative effect on P450 performance was most by other enzymes in the lysate. Moreover, coupling efficiency
pronounced at 250 mM amine, with only small primary between cofactor consumption and substrate oxidation in the
amines, such as ammonia
tolerated. At 50 mM amine, P450 hydroxylation activity was To test these hypotheses, experiments with purified proteins
still significantly affected, especially with propargylamine
were performed using as the amine donor, and almost total
conversion of to 1e was achieved (92%). On the one hand,
a and methylamine b
, being hydroxylation step cannot be underestimated.38,42
c
.
e
3
this result indicates that endogenous enzymes in E.coli might
compete with AspRedAm for reducing equivalents. On the
other hand, there was also a substantial amount of
2 left (70%
of total products formed), which might suggest competition
between the P450 and AspRedAm for NADPH, leading to a
buildup of the ketone intermediate when less reactive amine
donors were employed (Figure S1).Taking into account some
of the inherent limitations associated with
a one-step
approach and the application of purified enzymes, we decided
to explore the potential of the one-pot cascade as a two-step
process with crude enzyme preparations. This approach made
it possible to increase the enzyme loading in the first step
without impacting on the presence of the amine donor for the
second step (Table S2). With this setup, production of N-
propargylcyclohexylamine 1c reached 19.6 mM, and good
Figure 1. Effect of various amine donors selected for the amination cascade on the
performance of the P450. See Supporting Information for full experimental conditions.
results were also obtained with allylamine
e
and
cyclopropylamine f (15.4 and 17.1 mM product, respectively),
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with
formed (Figure
improvements were achieved when amines
employed in the two-step process, with 50%-80% products both in terms of yield and conversion of the intermediate
3
accounting for 19%, 35% and 16% of total products detected with both
e
and
f
. We speculate that the reactivity of
2
and Table S2). Less pronounced AspRedAm towards specific carbonyl acceptors and amine
and were partners influences the outcome of the amination cascade
a,
b
d
represented by
3.
ketone to the amine.
Figure 2. One-step and two-step cascade for the amination of cyclohexane using a
panel of primary amines. See Supporting Information for full experimental conditions.
Figure 3. Two-step cascade for the amination of cyclopentane and cycloheptane. See
Supporting Information for full experimental conditions.
The P450 variant employed in this study can accept a variety of To examine the scalability of the process, we selected the two-
cycloalkanes37 and, similarly, TeSADH has already been step cascade for the amination of cyclohexane with
to form
employed with a range of cyclic alcohols.39 AspRedAm can also 1c. A 2-fold improvement in TTN for the hydroxylation step
catalyse the reductive amination of cyclopentanone , albeit was found when the reaction was carried out in a flask with
less efficiently than that of 3 13
Thus, we explored the orbital shaking instead of a round-bottom flask with magnetic
c
8
.
possibility of expanding the scope of the cascade to other stirring (5707 vs. 2976), probably due to a better mass-transfer
cycloalkanes (Figure 3 and Table S3). When employing amine and/or oxygenation (Figure S3).38 Following this, the amination
donors
starting from cyclopentane
cyclooctane 10, even though product formation was detected In summary, we have demonstrated a biocatalytic cascade for
when AspRedAm was incubated with cyclooctanone 11 and the preparation of set of secondary amines from
(40 % conversion, Figure S2), unfortunately, the multi-step cycloalkanes. Unlike chemical approaches for the addition of
c
,
e
and
f
, product formation was detected when step yielded 50 mg of the final product 1c giving a space-time
4
and cycloheptane
7.
For yield of 2 g L-1 d-1 (Figure S4).
e
a
amination of 10 was unsuccessful.
When was reacted with
amines across aliphatic C-H bonds, the presented cascade is
c
4,
10.6 mM N- carried out in aqueous buffer, does not produce any toxic by-
propargylcyclopentylamine 4c was produced (50% of total products and does not require additional deprotection steps to
products formed), whereas and gave only 2.2 mM N- obtain the final amine product. The plethora of information
allylcyclopentylamine 4e and 3.0 mM N- available for oxidoreductases, combined with the broad
e
f
cyclopropylcyclopentylamine 4f, with the accumulation of the substrate tolerance of reductive aminases, will allow for a
intermediate oxidised products, which represented 85 and larger application of the presented cascade to access a variety
79% of total products formed, respectively. Modest product of different amines starting from unfunctionalized compounds.
concentrations and an excess cycloheptanone
products formed) were obtained when the bulkier
cycloheptane was used, with 3.3 mM N-
9 (61% total
7
propargylcycloheptylamine 5c produced with
c
and no product
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M.T. has received funding from the European Union (EU) 18
Project ROBOX (Grant Agreement No. 635734) under EU’s
Horizon 2020 Programme Research and Innovation actions 19
H2020-LEIT BIO-2014-1. J.M.S was funded by grant
BB/M006832/1 from the UK Biotechnology and Biological 20
Sciences Research Council. S.L.M. was supported by a CASE
studentship from Johnson Matthey. We thank the industrial 21
affiliates of the Centre of Excellence for Biocatalysis,
Biotransformations and Biomanufacture (CoEBio3) for 22
awarding a studentship to M.P.T. N.J.T. also acknowledges the
J. L. Roizen, M. E. Harvey and J. D. U. Bois, Acc. Chem. Res.,
2012, 45, 911–922.
D. N. Zalatan and J. Du Bois, J. Am. Chem. Soc., 2008, 130,
9220–9221.
J. A. Labinger and J. E. Bercaw, Nature, 2002, 417, 507–
514.
P. A. Frey and O. T. Magnusson, Chem. Rev., 2003, 103,
2129–2148.
C. C. Farwell, R. K. Zhang, J. A. Mcintosh, T. K. Hyster and F.
H. Arnold, ACS Cent. Sci., 2015, 1, 89–93.
P. Dydio, H. M. Key, H. Hayashi, D. S. Clark and J. F.
Hartwig, J. Am. Chem. Soc., 2017, 139, 1750-1753.
J. A. McIntosh, P. S. Coelho, C. C. Farwell, Z. J. Wang, J. C.
Lewis, T. R. Brown and F. H. Arnold, Angew. Chemie - Int.
Ed., 2013, 52, 9309–9312.
ERC for the award of an Advanced Grant.
23
24
Conflict of interests
There are no conflicts of interest to declare.
25
26
27
R. Singh, J. N. Kolev, P. A. Sutera and R. Fasan, ACS Catal.,
2015, 5, 1685–1691.
Notes and references
C. K. Prier, R. K. Zhang, A. R. Buller, S. Brinkmann-Chen and
F. H. Arnold, Nat. Chem., 2017, DOI: 10.1038/NCHEM.2783
P. R. Ortiz de Montellano, Cytochrome P450: Structure,
Mechanism and Biochemistry, Springer Science & Business
Media, 4th edn., 2015.
1
2
D. Ghislieri and N. J. Turner, Top. Catal., 2014, 57, 284–300.
T. C. Nugent and M. El-Shazly, Adv. Synth. Catal., 2010,
352, 753–819.
3
4
J. L. Galman, I. Slabu, N. J. Weise, C. Iglesias, F.
Parmeggiani, R. C. Lloyd and N. J. Turner, Green Chem.,
2017, 19, 361–366.
28
29
30
31
32
33
M. Schrewe, N. Ladkau, B. Bühler and A. Schmid, Adv.
Synth. Catal., 2013, 355, 1693–1697.
P. Both, H. Busch, P. P. Kelly, F. G. Mutti, N. J. Turner and S.
L. Flitsch, Angew. Chemie - Int. Ed., 2015, 55, 1511–1513.
S. Wu, Y. Zhou, T. Wang, H.-P. Too, D. I. C. Wang and Z. Li,
Nat. Commun., 2016, 7, 11917.
C. K. Savile, J. M. Janey, E. C. Mundorff, J. C. Moore, S. Tam,
W. R. Jarvis, J. C. Colbeck, A. Krebber, F. J. Fleitz, J. Brands,
P. N. Devine, G. W. Huisman and G. J. Hughes, Science,
2010, 329, 305–309.
M. Pickl, M. Fuchs, S. M. Glueck and K. Faber,
ChemCatChem, 2015, 7, 3121–3124.
5
F. Parmeggiani, S. L. Lovelock, N. J. Weise, S. T. Ahmed and
N. J. Turner, Angew. Chemie - Int. Ed., 2015, 54, 4608–
4611.
L. Martínez-Montero, V. Gotor, V. Gotor-Fernández and I.
Lavandera, Green Chem., 2017, 19, 474–480.
K. Tauber, M. Fuchs, J. H. Sattler, J. Pitzer, D. Pressnitz, D.
Koszelewski, K. Faber, J. Pfeffer, T. Haas and W. Kroutil,
Chem. - A Eur. J., 2013, 19, 4030–4035.
6
7
8
N. J. Weise, F. Parmeggiani, S. T. Ahmed and N. J. Turner, J.
Am. Chem. Soc., 2015, 137, 12977–12983.
T. Knaus, W. Böhmer and F. G. Mutti, Green Chem., 2017,
19, 453–463.
34
35
36
F. G. Mutti, M. Breuer and N. J. Turner, Science, 2015,
6255, 1525–1529.
D. Ghislieri, A. P. Green, M. Pontini, S. C. Willies, I. Rowles,
A. Frank, G. Grogan and N. J. Turner, J. Am. Chem. Soc.,
2013, 135, 10863–10869.
M. Thompson and N. J. Turner, ChemCatChem, 2017,
DOI:10.1002/cctc.201701092.
9
R. S. Heath, M. Pontini, B. Bechi and N. J. Turner,
ChemCatChem, 2014, 6, 996–1002.
S. L. Montgomery, M. P. Thompson, G. A. Aleku, B.
Dominguez and N. J. Turner, Angew. Chem. Int. Ed., 2017,
56,10491-10494.
10
11
12
Z. Maugeri and D. Rother, J. Biotechnol., 2017,
DOI:10.1016/j.jbiotec.2017.05.015.
37
38
A. Pennec, C. L. Jacobs, D. J. Opperman and M. S. Smit,
Adv. Synth. Catal., 2014, 357, 118–130.
P. Matzel, M. Gand and M. Höhne, Green Chem., 2017, 19,
385–389.
S. C. Maurer, K. Kühnel, L. A. Kaysser, S. Eiben, R. D. Schmid
and V. B. Urlacher, Adv. Synth. Catal., 2005, 347, 1090–
1098.
J. Mangas-Sanchez, S. P. France, S. L. Montgomery, G. A.
Aleku, H. Man, M. Sharma, J. I. Ramsden, G. Grogan and N.
J. Turner, Curr. Opin. Chem. Biol., 2017, 37, 19–25.
G. A. Aleku, S. P. France, H. Man, J. Mangas-sanchez, S. L.
Montgomery, M. Sharma, F. Leipold, S. Hussain, G. Grogan
and N. J. Turner, Nat. Chem., 2017, 9, 961-969.
C. Liang, F. Collet, F. Robert-Peillard, P. Müller, R. H. Dodd
and P. Dauban, J. Am. Chem. Soc., 2008, 130, 343–350.
D. N. Zalatan and J. Du Bois, Top Curr Chem, 2010, 292,
347–378.
39
40
41
A. Pennec, F. Hollmann, M. S. Smit and D. J. Opperman,
ChemCatChem, 2015, 7, 236–239.
13
H. Slusarczyk, S. Felber, M. Kula and M. Pohl, Eur. J.
Biochem., 2000, 267, 1280–1289.
14
15
16
17
M. M. Musa, R. S. Phillips, M. Laivenieks, C. Vieille, M.
Takahashi and S. M. Hamdan, Org. Biomol. Chem. Chem.,
2013, 11, 2911–2915.
42
S. Staudt, E. Burda, C. Giese, C. A. Müller, J. Marienhagen,
U. Schwaneberg, W. Hummel, K. Drauz, H. Gröger, Angew.
Chemie - Int. Ed. 2013, 52, 2359–2363.
M.-L. Louillat and F. W. Patureau, Chem. Soc. Rev., 2014,
43, 901–910.
K. M. Waltz and J. F. Hartwig, Science, 1997, 277, 211–213.
4 | J. Name., 2012, 00, 1-3
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