Asymmetric synthesis of 3-substituted cyclohexylamine derivatives from prochiral diketones via three biocatalytic steps

Article abstract of DOI:10.1002/adsc.201201057

Prochiral bicyclic diketones were transformed to a single diastereomer of 3-substituted cyclohexylamine derivatives via three consecutive biocatalytic steps. The two chiral centres were set up by a C-C hydrolase (6-oxocamphor hydrolase) in the first step and by an ω-transaminase in the last step. The esterification of the intermediate keto acid was catalysed by a lipase in the second step if possible. For two substrates the C-C hydrolytic step as well as the esterification could be run simultaneously in a one-pot cascade in an organic solvent. In one example, the reaction mixture of the first two steps could be directly subjected to bio-amination in an organic solvent without the need to change the reaction medium. Depending on the choice of the ω-transaminase employed and the substrate the cis- as well as the trans-diastereomers could be obtained in optically pure forms. Copyright

Full text of DOI:10.1002/adsc.201201057

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DOI: 10.1002/adsc.201201057
Asymmetric Synthesis of 3-Substituted Cyclohexylamine Deriva-
tives from Prochiral Diketones via Three Biocatalytic Steps
Elina Siirola,a Francesco G. Mutti,a Barbara Grischek,a Sebastian F. Hoefler,a
Walter M. F. Fabian,a Gideon Grogan,b and Wolfgang Kroutila,*
a
Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria
Fax : (+43)-316-380-9840, phone: (+43)-316-380-5350; e-mail: wolfgang.kroutil@uni-graz.at
York Structural Biology Laboratory, Department of Chemistry, University of York, York, YO10 5YW, U.K.
b
Received: December 4, 2012; Published online: February 22, 2013
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201201057.
Abstract: Prochiral bicyclic diketones were trans-
formed to a single diastereomer of 3-substituted cy-
clohexylamine derivatives via three consecutive bio-
catalytic steps. The two chiral centres were set up
ble building blocks in the synthesis of potential phar-
maceuticals,[5] for example, as modulators for the mul-
tidrug-resistant protein MRP1.[6] However, catalytic
and highly stereoselective methods for the prepara-
tion of 3-substituted cyclohexylamines in general are
rare,[7,8] necessitating even resolution of enantiomers
using chromatography.[9] Only in the cases of 3-amino-
cyclohexanol[10] or 1,3-cyclohexyldiamine[11] are more
options available.
À
by a C C hydrolase (6-oxocamphor hydrolase) in
the first step and by an w-transaminase in the last
step. The esterification of the intermediate keto
acid was catalysed by a lipase in the second step if
À
possible. For two substrates the C C hydrolytic step
as well as the esterification could be run simultane-
ously in a one-pot cascade in an organic solvent. In
one example, the reaction mixture of the first two
steps could be directly subjected to bio-amination
in an organic solvent without the need to change
the reaction medium. Depending on the choice of
the w-transaminase employed and the substrate the
cis- as well as the trans-diastereomers could be ob-
tained in optically pure forms.
We envisaged that prochiral bicyclic diketones (1a–
c) might serve as starting materials for the prepara-
tion of aminocyclohexylacetic acid derivatives 4 via
a three-enzyme reaction sequence. In this sequence,
À
the stereoselective hydrolysis of a C C bond cata-
lysed by a b-diketone hydrolase[12] would be the first
step, followed by an esterification employing a lipase
(Scheme 1). Finally the chiral amine moiety would be
introduced by an w-transaminase (wTA).[13] Thus, the
two chiral centres might be controlled on the one
À
Keywords: asymmetric catalysis; biosynthesis; bio-
hand by the C C hydrolase and on the other hand by
the wTA.
À
transformations; C C hydrolase; w-transaminases
À
Since the C C hydrolysis of substrate 1a in buffer
has been described previously,[14] 1a was used as
a model compound to test the reaction sequence. The
enzyme employed for the first step was the 6-oxocam-
Biocatalytic asymmetric transformations have become phor hydrolase (OCH) from Rhodococcus sp.
key steps for the preparation of optically pure com- NCIMB 9784, which catalyses the hydrolytic retro-
pounds.[1] However, commonly only a single step in Claisen condensation of non-enolisable, symmetrical
the synthetic route is performed by an enzyme. Ex- b-diketones leading to optically pure keto acid 2a.[14]
ceptions are enzymatic cascade reactions,[2,3] where at Since the resulting keto acid 2a was a non-substrate
least two enzymes work beside each other. This ena- for amination by wTAs, whereas the corresponding
bles shorter reaction times, minimises work-up steps methyl ester 3a was well accepted (vide infra), our
À
and reduces waste. Hence applying a sequence of ste- emphasis was to combine the hydrolytic C C cleav-
reoselective enzymatic reactions for the synthesis of age with an esterification step in a simultaneous cas-
compounds with two or more chiral centres[4] is an ap- cade. At a first glance the reaction conditions for
pealing option when starting from prochiral sub- these two steps look incompatible, since water is re-
strates. Recently, it was recognised that 2-(3-aminocy- quired for the hydrolysis, while water is detrimental
clohexyl)acetic acid derivatives (4), unnatural d- for the esterification. Although water is a substrate
amino acids bearing two chiral centres, serve as valua- for the hydrolytic step, it is also a product of the
Adv. Synth. Catal. 2013, 355, 1703 – 1708
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Elina Siirola et al.
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Scheme 1. Three-enzyme sequence for the synthesis of 2-(3-aminocyclohexyl)acetic acid methyl ester derivatives (4a–c) bear-
ing two chiral centres starting from prochiral diketones 1a–c.
esterification step, thus formally, only catalytic H2O:MeOH (97.5/2.5/1 vvÀ1 vÀ1) leading to complete
amounts of water should be required due to its possi- conversion within one hour (Figure 1). It is notable
À
ble internal “reuse”. Methanol was not a nucleophile that the C C cleaving step ran faster in the presence
accepted by the C C hydrolase, which would lead of the lipase-catalysed esterification, thus the inter-
[15]
À
directly to the methyl ester. Therefore it was required mediate keto acid (S)-2a was instantly esterified and
to test the OCH in organic solvents in the presence of was not detected by GC-MS analysis during the time
only a minimum amount of water: sufficient to pro- course of the reaction. Furthermore, the enzymes
mote enzymatic activity[16] and run the C C hydrolysis could be recovered from the organic solvent and recy-
À
but low enough to enable the methyl ester formation. cled without loss of activity or selectivity at least five
OCH was recently shown to tolerate a broad variety times.
of organic cosolvents up to 80% vvÀ1 maintaining in
To test the amination step, enzymatically prepared
most solvents its excellent stereoselectivity.[15] Diiso- (S)-3a was subjected to the wTA catalysed amination
propyl ether (DIPE) was the organic solvent best tol- using l- or d-alanine or 2-propylamine as an amine
erated. In the presence of 80% vvÀ1 of DIPE the donor in aqueous buffer. In aqueous solution most
enzyme still retained 51% residual activity compared wTAs accept alanine as amine donor, and only few 2-
to aqueous buffer system. Methanol, on the other propylamine. Employing alanine as amine donor, the
hand, inactivated the enzyme.
formed coproduct pyruvate needs to removed for
Although in the previous work DIPE was used up thermodynamic reasons either by transforming pyru-
to 80% vvÀ1 as a cosolvent in the C C hydrolysis, the vate to alanine employing an alanine dehydrogenase
À
conditions for the simultaneous one-pot reaction with
the lipase-catalysed esterification needed to be tuned
in order to minimise the competing ester hydrolysis
catalysed by the lipase. Employing 2.5% vvÀ1 (1.4M)
of water in DIPE, OCH (lyophilised crude cell ex-
tract, 20 mgmLÀ1) catalysed the hydrolysis of 1a
(50 mM) to 2a with full conversion and perfect stereo-
selectivity (ee >99%) within three hours.
The esterification of (S)-2a (50 mM) was first stud-
ied separately, thus Candida antarctica lipase B was
tested in DIPE:H2O (97.5:2.5 vvÀ1) with methanol.
Commercial Novozyme 435 (10 mgmLÀ1) enabled
Figure 1. OCH (lyophilised crude extract, 20 mgmLÀ1) and
methyl ester formation leading to (S)-3a with 97%
conversion in the presence of just 1% vvÀ1 of metha-
CAL-B (Novozyme 435, 20 mgmLÀ1) catalysed conversion
À
nol within 3 h. Consequently, the C C hydrolysis of
&
*
of diketone 1a (50 mM) to (S)-3a in DIPE:H2O:MeOH
1a was combined with the esterification in DIPE: (97.5/2.5/1, vvÀ1 vÀ1) at 308C, 120 rpm.
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Asymmetric Synthesis of 3-Substituted Cyclohexylamine Derivatives from Prochiral Diketones
Table 1. Transaminase catalysed reductive amination of (S)-3a.[a]
Entry
w-Transaminase
c [%]
ee [%]
de [%]
Product
1
2
3
4
5
6
7
8
9
Vibrio fluvialis
>99
>99
<1
97
<1
8
96
<1
<1
>99
>99
n.a.
>99
n.a.
>99
>99
n.a.
>99
99
n.a.
>99
n.a.
97
97
n.a.
n.a.
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
Chromobacterium violaceum
Arthrobacter citreus
Bacillus megaterium
Alcaligenes denitrificans
(R)-Arthrobacter
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
(R)-Arthrobacter variant[b]
Aspergillus terreus
n.a.
n.a.
Hyphomonas neptunium
n.a.
[a]
w-Transaminase (lyophilised whole cells, 20 mgmLÀ1) in phosphate buffer (100 mM, pH 7, 1 mM PLP) at 308C, 120 rpm,
24 h. l- or d-alanine (250 mM), LDH (90 U), GDH (30 U), NAD+ (1 mm) and glucose (150 mm).
[b]
2-Propylamine (1M) as amine donor in phosphate buffer (100 mM, pH 8) at 458C, 120 rpm, 24 h. n.a. not applicable.
(AlaDH) or by reduction to lactate with a lactate de- adding the substrate and the amine donor (2-propyl-
hydrogenase (LDH).[13c] The AlaDH as well as the
amine, 3 equivalents). Employing ArRmut11-wTA
AHCTUNGTRENNUNG
LDH requires NADH for reduction which is recycled, the reaction resulted in 91% conversion within 24 h
for example, with glucose and a glucose dehydrogen- leading to the (1S,3R)-4a diastereomer (ee >99%, de
ase (GDH). Various wTAs were tested like the (S)-se- 98%).
lective w-transaminases from Vibrio fluvialis (His-
The His-VF-wTA-catalysed reaction proceeded
VF),[17] Chromobacterium violaceum (CV),[18] Arthro- more slowly, resulting in 71% conversions within 96 h
bacter citreus (ArS),[19] Bacillus megaterium (BM)[20] giving optically pure (1S,3S)-4a (ee >99%, de
and Alcaligenes denitrificans (AD)[21] and (R)-selec- >99%).
tive w-transaminases from Arthrobacter sp. (wild-type
ArR[22] and variant ArRmut11[23]), Aspergillus ter- via reduction of the imine formed between benzyl-
reus[24] and Hyphomonas neptunium.[24] Best results
amine and rac-3a using NaBH(OAc)3, it was noticed
When synthesising racemic reference material 3a
A
ACHTUNGTRENNUNG
were obtained with His-VF-wTA leading to the opti- that the trans-diastereomer (1R*,3R*) (96%) was
cally pure diastereomer (1S,3S)-4a in >99% ee and formed preferentially, while only a minor amount
>99% de (Table 1). For the diastereomer (1S,3R)-4a (4%) of the cis-diastereomers (1R*,3S*)-4a was de-
bearing an (R)-configured amine moiety, the (R)-se- tected (Figure 2, trace 1).
lective enzyme variant from Arthrobacter sp.
ArRmut11-wTA turned out to be best suited.
ArRmut11-wTA was also tested for the amination of
keto acid (S)-2a, however (S)-2a was a non-substrate.
Since the first two steps were performed in an or-
ganic solvent, it would be desirable to run also the
subsequent third step in an organic environment, ide-
ally in the same solvent as employed in the first two
steps. The possibility to carry out the biocatalytic
steps in solvents compatible with upstream and down-
stream chemistry reduces the extraction and solvent
switching steps, which are often necessary for the con-
ventional aqueous biotransformations. Thus keeping
the same solvent can provide more efficient synthetic
routes.[25] Asymmetric bio-amination in organic sol-
vents employing crude cell extracts has been reported
recently.[26]
The same methodology was applied here by sub-
jecting the organic reaction mixture of the first two
steps directly to the transamination step after removal
of the hydrolases by filtration. Lyophilised crude ex-
tracts of His-VF-wTA and ArRmut11-wTA were pre-
Figure 2. GC-trace 1: chemically prepared all-rac-4a (Chira-
sil-DEX CB column) after derivatisation (see the Support-
ing Information). Peaks from left to right: (1S,3S)-4a,
(1R,3R)-4a, (1R,3S)-4a and (1S,3R)-4a. GC-trace 2: enzy-
matically prepared (1S,3S)-4a (His-VF-wTA). GC-trace 3:
treated in water saturated DIPE in order to equili-
brate the amount of water for the catalyst before enzymatically prepared (1S,3R)-4a (ArRmut11-wTA).
Adv. Synth. Catal. 2013, 355, 1703 – 1708
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Elina Siirola et al.
COMMUNICATIONS
Thus, the chemical reductive amination of keto solvent (DIPE), 3b was not accepted as a substrate by
ester 3a preferably led to the trans-diastereomer, these enzymes.
whereas the cis/trans selectivity can be controlled via
Keto ester (S)-3c was very well transformed by the
the biocatalytic approach by the choice of the catalyst wTAs leading to >99% conversion and perfect ste-
employed. When starting from (S)-3a, ArRmut11- reoselectivity (ee >99%, de >99%) with both (S)-
wTA gave access to the cis- while His-VF-wTA pro- and (R)-selective enzymes in aqueous buffer leading
duced the trans-stereoisomer.
to optically pure (1S,5S)-4b and (1S,5R)-4c, respec-
To demonstrate the scope of the synthetic three- tively (entries 5 and 6). ArRmut11-wTA aminated
enzyme strategy for related compounds, substrates 1b (S)-3c also in organic solvent leading to 89% conver-
and 1c were synthesised. Both substrates have never sion within 24 h at 50 mM substrate concentration
been tested before employing OCH. OCH hydrolysed (entry 7).
both diketones to the corresponding d-keto acids with
All products 2–4 were also prepared on a prepara-
perfect and very high stereoselectivity, respectively tive scale using 0.1–0.5 g of substrate. For instance, to
(ee >99% and ee=99% for 2b and 2c, respectively) get sufficient amounts of all above mentioned diaste-
leading to the (S)-enantiomers, whereby 1c was trans- reomers of 4a–c, the aminations of (S)-3a–c were suc-
formed faster than 1b. In case of 1b, >99% conver- cessfully performed on 100-mg scales employing (R)-
sion was reached within four hours using 10 mM sub- and (S)-selective wTAs.
strate concentration, whereas 1c gave >99% conver-
sion with the same biocatalyst loading and reaction preparation of optically pure 3-substituted cyclohexyl-
time at 50 mM substrate concentration. amine derivatives [methyl 2-(3-aminocyclohexyl)ace-
In conclusion, a biocatalytic synthetic route for the
À
The simultaneous one-pot reaction system for C C tates] from prochiral bicyclic b-diketones was estab-
hydrolysis and esterification using OCH and CAL-B lished employing three biocatalytic reaction steps. In
as catalysts led to >99% conversion of 1b to 3b the case of substrates 1a and 1b, the simultaneous cas-
(10 mM) within four hours. Interestingly, 2c was not cade reaction catalysed by two hydrolases, namely
À
a substrate of CAL-B, so in the case of substrate 1c, a C C hydrolase and a lipase, resulted in the corre-
the C C hydrolysis was first carried out in aqueous sponding methyl esters 3a and 3b in organic solvent
buffer, and the methyl ester was formed chemically.
À
(DIPE) containing 2.5% vvÀ1 water and 1% vvÀ1
Reductive amination of (S)-3b was tested with the methanol. The organic phase of the reaction with 1a
transaminases identified as best suitable for (S)-3a could be directly applied in the transaminase-cata-
(Table 1). In comparison to (S)-3a the keto ester (S)- lysed reactions thus giving the diastereomers (1S,3S)-
3b was aminated slower, probably due to increased 4a and (1S,3R)-4a by a one-pot approach. In the case
À
sterical hinderness. Nevertheless, optically pure of substrate 1c, C C-bond hydrolysis was catalysed in
(1S,5R)-4b was obtained in aqueous buffer (ee >99%, buffer, and the methyl ester of the crude keto acid
de >99%) (Table 2, entries 1 and 2). Interestingly, the was prepared chemically followed by transamination,
(S)-selective wTA His-Vf as well as the (R)-selective which could successfully be performed in organic sol-
enzyme ArRmut11 yielded the same trans-product vent as well as in buffer.
(1S,5R)-4b. When performing the reaction in organic
Table 2. Reductive amination of (S)-3b and (S)-3c by His-VF-wTA and ArRmut11-wTA.[a]
Entry
Substrate
wTA
Solvent
c [%]
ee [%]
de [%]
Product
(1S,5R)-4b[e]
1
2
3
4
5
6
7
8
3b
3b
3b
3b
3c
3c
3c
3c
ArRmut11
His-VF
ArRmut11
His-VF
ArRmut11
His-VF
ArRmut11
His-VF
buffer[b]
buffer[c]
DIPE[d]
DIPE[d]
buffer[b]
buffer[c]
DIPE[d]
DIPE[d]
69
25
>99
>99
n. a.
n. a.
> 99
> 99
> 99
n. a.
>99
>99
n. a.
n. a.
>99
99
ACHTUNGTRENNUNG
(1S,5R)-4b[e]
ACHTUNGTRENNUNG
n.c.
n.c.
>99
>99
89
–
–
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
>99
n. a.
ACHTUNGTRENNUNG
n.c.
–
[a]
Substrate concentration 50 mM, enzymes applied as lyophilised crude extracts (20 mgmLÀ1).
2-Propylamine (1M) as amine donor in phosphate buffer (100 mM, pH 8) at 458C, 120 rpm, 24 h.
[b]
[c]
l-Alanine (250 mM) was used as amine donor in phosphate buffer (100 mM, pH 7, 1 mM PLP) at 308C, 120 rpm, 24 h.
LDH (90 U), GDH (30 U), NAD+ (1 mM) and glucose (150 mM).
[d]
[e]
DIPE (H2O sat.) was used as a solvent and 2-propylamine (150 mM) as amine donor. Reactions were shaken at 258C,
750 rpm, 24 h.
Switch in Cahn–Ingold–Prelog priority. n.c. no conversion; n.a. not applicable.
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Asymmetric Synthesis of 3-Substituted Cyclohexylamine Derivatives from Prochiral Diketones
rated NaHCO3 solution (100 mL), and then were extracted
Experimental Section
by ethyl acetate (3ꢁ500 mL). After drying the combined or-
ganic layers over Na2SO4 the reactions were analysed by
GC-FID (see the Supporting Information).
Expression and Preparation of the Enzymes
OCH was expressed in E. coli and the lyophilised crude
extract was prepared as previously described.[15] The
crude extract had
a
specific enzymatic activity of
9.0 mmol1a minÀ1 mgcrude
in buffer.[14] w-TAs were ex-
enzyme
Acknowledgements
pressed in E. coli BL21 (DE3) as previously described.[17b,22]
Lyophilised cells containing overexpressed wTAs were used
as enzyme preparations in buffer. Cell-free extracts of the
w-TAs were prepared as described[26] for the experiments in
organic solvent.
This research was supported by a Marie Curie Networks for
Initial Training fellowship in the project BIOTRAINS (FP7-
PEOPLE-ITN-2008-238531). F. G. M. acknowledges the Eu-
ropean Unionꢀs Seventh Framework Programme FP7/2007-
2013 under grant agreement n8 245144 (AmBioCas). B. G.
received funding from the Austrian BMWFJ, BMVIT, SFG,
Standortagentur Tirol and ZIT through the Austria FFG-
COMET-Funding Program.
À
Simultaneous C C Hydrolysis and Esterification
OCH (lyophilised cell free extract, 20 mgmLÀ1), CAL-B
(Novozyme 435, 20 mgmLÀ1) and the diketone (1a, 50 mM;
1b, 10 mM) were suspended in DIPE/H2O/MeOH mixture
(97.5:2.5:1, 1 mL) in an Eppendorf tube. The water content
of the equilibrated reaction system was 5567 ppm. The sam-
ples were shaken in an orbital shaker at 120 rpm, 308C in
that way that the tubes were in horizontal positions. Samples
(100 mL) were diluted by EtOAc (300 mL) and the conver-
sions were analysed by GC-MS and GC-FID (see the Sup-
porting Information). In case the products 3a–c were
needed for the amination reactions in buffer, the reactions
were stopped by filtering the hydrolases, and the solvents
were removed under air flow to obtain a crude product. In
case of the one-pot approach, hydrolases were removed by
filtration, and the remaining solution was applied directly
for the amination step in organic solvent.
References
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Amination in DIPE with 2-Propylamine as an Amine
Donor
This was carried out as previously described for MTBE as
solvent.[26] w-TAs (lyophilised crude extract, 20 mgmLÀ1)
were shaken in water-saturated DIPE (1 mL) in an Eppen-
dorf tube for 15 min at 258C, 750 rpm in an Eppendorf
shaker. Water (16 mL) was added, and the enzyme prepara-
tion was further shaken for 15 min. Substrate was added as
a crude, purified material, or in a one-pot approach as a solu-
tion in DIPE/MeOH/H2O (97.5:2.5:1), and 2-propylamine
(150 mM) was added. The reactions were shaken at 258C,
750 rpm and samples (100 mL) were taken at intervals. The
samples were diluted by EtOAc (300 mL) and the conver-
sions were analysed by GC-FID as described in the Support-
ing Information.
Amination in Aqueous Buffer with 2-Propylamine as
an Amine Donor
Lyophilised E. coli BL21 (DE3) cells containing the overex-
pressed (R)-selective variant from Arthrobacter sp.
(ArRmut11) were rehydrated in phosphate buffer (100 mM,
pH 8) containing 1M isopropylamine. Since the harvested
E. coli cells were resuspended in PLP (1 mM) containing
phosphate buffer (100 mM, pH 7) before lyophilisation, no
addition of PLP was necessary. After 15 min rehydration at
308C, 120 rpm, the substrate (50 mM) was added. Reaction
mixtures were shaken at 500 rpm, 458C for 16 h in an Ep-
pendorf shaker. The reactions were stopped by adding satu-
[4] For enzymatic cascade reactions leading to products
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