Chemoenzymatic Approaches to the Synthesis of the Calcimimetic Agent Cinacalcet Employing Transaminases and Ketoreductases

Article abstract of DOI:10.1002/adsc.201701485

Several chemoenzymatic routes have been explored for the preparation of cinacalcet, a calcimimetic agent. Transaminases (TAs) and ketoreductases (KREDs) turned out to be useful biocatalysts for the preparation of key optically active precursors. Thus, the asymmetric amination of 1-acetonaphthone yielded an enantiopure (R)-amine, which can be alkylated in one step to yield cinacalcet. Alternatively, the bioreduction of the same ketone resulted in an enantiopure (S)-alcohol, which was easily converted into the previous (R)-amine. In addition, the reduction was efficiently performed with the KRED and its cofactor co-immobilized on the same porous surface. This self-sufficient heterogeneous biocatalyst presented an accumulated total turnover number (TTN) for the cofactor of 675 after 5 consecutive operational cycles. Finally, in a preparative scale synthesis the TA-based approach was performed in aqueous medium and led to enantiopure cinacalcet in two steps and 50% overall yield. (Figure presented.).

Full text of DOI:10.1002/adsc.201701485

Title: Chemoenzymatic Approaches to the Synthesis of the
Calcimimetic Agent Cinacalcet Employing Transaminases and
Ketoreductases
Authors: Lisa Marx, Nicolás Ríos-Lombardía, Judith Farnberger,
Wolfgang Kroutil, Ana Isabel Benítez-Mateos, Fernando
López-Gallego, Francisco Morís, Javier Gonzalez-Sabin, and
Per Berglund
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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content of this Accepted Article.
To be cited as: Adv. Synth. Catal. 10.1002/adsc.201701485
Link to VoR: http://dx.doi.org/10.1002/adsc.201701485

10.1002/adsc.201701485
Advanced Synthesis & Catalysis
FULL PAPER
DOI: 10.1002/adsc.200((will be filled in by the editorial staff))
Chemoenzymatic Approaches to the Synthesis of the Calcimimetic
Agent Cinacalcet Employing Transaminases and Ketoreductases
Lisa Marx,a,b Nicolás Ríos-Lombardía,b Judith F. Farnberger,c Wolfgang Kroutil,d Ana I.
Benítez-Mateos,e Fernando López-Gallego,e,f Francisco Morís,b Javier González-
Sabínb* and Per Berglunda*
a
KTH Royal Institute of Technology, Department of Industrial Biotechnology, SE-106 91 Stockholm, Sweden Phone:
+46 87907037, email: per.berglund@biotech.kth.se
EntreChem, S.L., Vivero Ciencias de la Salud, Santo Domingo de Guzmán, 33011 Oviedo, Spain
b
Phone: +34 985 259 021; email: jgsabin@entrechem.com
Austrian Centre of Industrial Biotechnology, ACIB GmbH, c/o University of Graz, Harrachgasse 21, 8010 Graz,
c
Austria
d
Institute of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Harrachgasse 21, 8010 Graz, Austria
Heterogeneous biocatalysis group, CIC biomaGUNE, Edificio Empresarial "C", Paseo de Miramón 182, 20009,
e
Donostia, Spain
f
IKERBASQUE, Basque Foundation for Science, Bilbao (Spain)
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.200######.((Please
delete if not appropriate))
Abstract. Several chemoenzymatic routes have been
explored for the preparation of cinacalcet, a calcimimetic
agent. Transaminases (TAs) and ketoreductases (KREDs)
turned out to be useful biocatalysts for the preparation of
key optically active precursors. Thus, the asymmetric
amination of 1-acetonaphthone yielded an enantiopure (R)-
amine, which can be alkylated in one step to yield
cinacalcet. Alternatively, the bioreduction of the same
ketone resulted in an enantiopure (S)-alcohol, which was
easily converted into the previous (R)-amine. In addition,
the reduction was efficiently performed with the KRED and
its cofactor co-immobilized on the same porous surface.
This self-sufficient heterogeneous biocatalyst presented an
accumulated total turnover number (TTN) for the cofactor
of 675 after 5 consecutive operational cycles. Finally, in a
preparative scale synthesis the TA-based approach was
performed in aqueous medium and led to enantiopure
cinacalcet in two steps and 50% overall yield.
Keywords: Cinacalcet, Transaminases, Ketoreductases,
Asymmetric synthesis, Biocatalysis
1
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10.1002/adsc.201701485
Advanced Synthesis & Catalysis
Scheme 1. Chemoenzymatic roadmap towards
cinacalcet (1).
Introduction
Cinacalcet
(naphthyl)ethyl]-3-[3-(trifluoromethyl)-
phenyl]propane-1-amine,[1] is
calcimimetic
(1),
chemically
N-[(1R)-1-
Results and discussion
a
5 approved for the treatment of secondary
hyperparathyroidism in patients with chronic
kidney disease (CKD) receiving dialysis and for
the treatment of hypercalcaemia associated to
parathyroid carcinoma.[2] The plethora of reported
10 syntheses are based mainly on the coupling of a
60 IRED-catalyzed reductive amination of 3
Traditionally, chiral amines have been prepared
enzymatically from racemates and by means of a
kinetic resolution based on lipase-catalyzed
65 aminolysis or hydrolysis reactions.[7] However, in
the last decade other enzymatic approaches
converting prochiral carbonyl compounds into
amines have emerged as a valuable alternative: i)
TAs transfer a primary amino group from a donor
70 substrate to an acceptor compound mediated by
chiral
precursor,
namely
(R)-(+)-1-(1-
naphthyl)ethylamine (2), with an achiral moiety by
different strategies: e.g. amide or imine formation
followed
by
reduction,
or
nucleophilic
15 substitution.[3] The key chiral amine was accessed
by classical or enzymatic resolutions which suffer
from the inherent 50% maximum yield[4] or by
several asymmetric synthetic approaches in
organic media.[5] The excellent properties
20 displayed by enzymes in terms of selectivity and
reactivity under mild reaction conditions make
enzymatic catalysis an attractive alternative for the
pyridoxal-5-phosphate
(PLP);[8]
ii)
amino
dehydrogenases (AmDHs) catalyze the NAD(P)H-
dependent reductive amination of carbonyl
compounds with ammonia;[9] iii) IREDs and its
75 recently discovered subclass reductive aminases
(RedAms) accept secondary linear and cyclic
amino donors and give entry to secondary and
tertiary amines which are not accessible by using
the previous enzymes.[10]
production
of
building
blocks
for
pharmaceuticals.[6] Thus, taking the advances in
25 the present biocatalytic toolbox into consideration
as well as the structural features of cinacalcet, we
devised three feasible chemoenzymatic approaches
from 1-acetonaphthone (3, Scheme 1). Specifically,
the asymmetric step would be catalyzed by
30 different enzymes: a) a direct amination of 3 with
3-(3-(trifluoromethyl)phenyl)propan-1-amine (5)
mediated by imine reductases (IREDs), to provide
1 in a single step; b) an asymmetric amination of 3
using transaminases (TAs) to yield the primary
35 amine (R)-2; c) an asymmetric reduction catalyzed
by ketoreductases (KREDs). Then, in the last two
approaches the selective follow-up chemistry of
the resulting optically active compounds would
enable facile entry to cinacalcet according to
40 reported methods. Herein, we report our findings
on the development of novel chemoenzymatic
routes towards this drug.
80 Initially, we focused on the most straightforward
approach to cinacalcet, namely the IRED-catalyzed
reductive amination of 3. To this end, we used the
commercially available amine 5 and screened a set
of IREDs overexpressed in E. coli from the
85 Greifswald University collection (Scheme 2).[11]
Unfortunately, and despite using a high excess of
amine (50 equivalents), this combination of
substrates did not yield detectable product after 48
h. In view of this, we were interested whether the
90 ketone 3 or the amine 5 was causing the limitation.
Actually, whereas ketones of comparable size to 3
have been successfully converted by IREDs, 5
might be a challenging amino donor since it is by
far bigger than reported examples.[10c,d] Besides, a
95 drawback could arise from the unfavorable
equilibrium of imine formation in water for 3 even
though some substantial evidences have recently
supported the imine formation in the active site of
some IREDs with a selection of ketone and amine
100 partners (Scheme 2).[10d] Further attempts
performed with the chemically synthesized imine 6
and IREDs did not show conversion in any case.
45
50
55
105
110
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10.1002/adsc.201701485
Advanced Synthesis & Catalysis
Scheme 2. Proposed IRED-catalyzed synthesis of 1.
170
175
Transaminase catalyzed amination of 3
115
Given the disappointing results displayed by
IREDs, we turned our attention to the amination of
3 employing TAs which would enable the primary
amine 2 in 100% yield,[12] one step away from
c
ee
Entry
TA
DMSO
(%)
(%)[b] (%)[b]
120 cinacalcet. Thus, 3 was screened with a series of
28 commercially available TAs (Codex®
Transaminase Screening Kit) and also with several
TAs overexpressed in E. coli.
1
2[c]
3
ATA-024
ATA-024
ATA-025
ATA-025
ATA-033
ATA-033
ATA-217
ATA-251
ATA-287
10
10
10
10
10
10
10
10
10
20
98
90
90
80
95
76
80
94
92
75
>99 (R)
>99 (R)
>99 (R)
>99 (R)
99 (R)
In a first set of experiments 3 was assayed at a 20
125 mM substrate concentration with the commercial
kit of TAs at 45 ºC in 100 mM phosphate buffer
pH 9.0, supplemented with PLP (1 mM) and
isopropylamine (iPrNH2, 1 M). Additionally, 10 %
v/v of DMSO was added due to the solubility
130 issues of 3. Under these conditions, some of the
TAs were very active and reached high
conversions after 24 h. More interestingly, almost
all of them displayed perfect asymmetry in the
amination of the carbonyl group, leading to the
135 target (R)-2 with >99% ee and also its counterpart
(S)-2 depending on the catalyst. Table 1 shows
selected results of this screening.[13] Further
attempts at higher substrate concentration (50 mM)
resulted in lower conversion, as typically observed
140 with TAs (entries 2, 4 and 6). Concerning the TAs
overexpressed in E. coli, only variants of the R-
selective ArRmut11[14] emerged as active
biocatalysts.
Originally, ArRmut11 has been specifically
145 evolved starting from a wild type enzyme[15] for
ketones bearing to bulky substituents on an
industrial setting.[14] Just recently, the introduction
of further mutations reducing the size of one of the
two binding pockets while maintaining the
150 exceptionally high activity and stability of
ArRmut11 has been reported.[16] Thus, we
4[c]
5
6[c]
7
99 (R)
>99 (S)
>99 (S)
98 (S)
8
9
ArRmut11-
G279A
>99 (R)
10
11
ArRmut11-
20
20
80
65
>99 (R)
>99 (R)
M117F/G279A
ArRmut11-
A60V/M117F/
G279A
12
[a] Reaction conditions for entries 1-9: 3 (20 mM) in 100
mM KPi pH 9.0 (500 L, 1mM PLP, 1 M iPrNH2), TA
(1 mg), DMSO (10% v/v), 24 h at 45 ºC and 250 rpm;
Entries 10-12: 3 (50 mM) in 100 mM KPi pH 9.0 (1 mL,
180 1 mM PLP, 500 mM iPrNH2), TA (20 mg lyophilized
cells), DMSO (20% v/v), 48 h at 45 ºC and 800 rpm. [b]
Measured by HPLC. [c] [3]: 50 mM.
envisioned
variants
ArRmut11-G279A,
and ArRmut11-
In view of the excellent stereoselectivity displayed
185 by the ArRmut11 variants, we took the challenge
of optimizing the biotransformation heading for a
process scale up in a manufacture setting. Hence,
aiming at a conversion enhancement, we tested
other amino donors such as D-alanine, benzylamine
190 and (R)--methylbenzylamine (MBA) in a high
molar excess since the thermodynamic equilibrium
is strongly shifted towards 3. Table 2 collects the
conversion measured at different reaction times
with these reagents. In no case traces of the (S)-
195 enantiomer were detected by HPLC, which
demonstrates the perfect selectivity exerted by the
TAs. On the other hand, D-alanine was not a
suitable amino donor for these enzymes, with
conversions up to 5% due to thermodynamics as
200 one possible reason. Concerning the other reagents,
the three ArRmut11 variants led to similar
ArRmut11-M117F/G279A
A60V/M117F/G279A to be convenient catalysts
155 for the transformation of ketone 3. They were used
as lyophilized cells under slightly different reaction
conditions, namely 45 ºC, 800 rpm, 100 mM
phosphate buffer pH 9.0 (1 mM PLP, 500 mM
iPrNH2) and 20% v/v DMSO. Interestingly, the
160 substrate concentration could be increased to 50
mM. Again, 3 was aminated with perfect
enantioselectivity, (R)-2 being obtained in
enantiopure form in all cases (entries 10-12).
Likewise, the conversion after 48 h was up to 80%.
165 Further attempts at higher concentrations such as
100 mM and 200 mM resulted in a drastic drop in
conversion.
Table 1. Asymmetric amination of 3 employing ATAs.[a]
3
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10.1002/adsc.201701485
Advanced Synthesis & Catalysis
conversions with values around 80%. Therefore,
and attending to its lower price and facility to be
removed, we opted for implementing the amination
205 based on iPrNH2 in the synthetic scheme.
cofactor recycling. Initially, the screening was
225 performed at 27 mM substrate concentration with a
phosphate buffer pH 7.0, IPA (15% v/v) and
DMSO (5% v/v).The reaction mixtures were
incubated at 30 ºC and 250 rpm during 24 h. From
the series of KREDs contained in the kit, it was
230 possible to identify biocatalysts affording both
enantiomers of 4 with complete conversion and
excellent enantioselectivity (Table 3). Actually, the
(S)-enantiomer of 4 is the target compound for our
synthetic purposes towards 1, as discussed later.
235
Table 2. Optimization of the amino donor in the
amination of 3 with variants from ArRmut11.[a]
Conversion (%)[b]
Entry
TA
Amino
donor
5 h 24 h
48 h
75
ArRmut11-
G279A
iPrNH2
iPrNH2
50
67
1
2
Table 3. Asymmetric bioreduction of 3 employing
KREDs.[a]
ArRmut11-
M117F/G27
9A
50
67
80
65
5
240
245
ArRmut11-
A60V/M117
F/G279A
iPrNH2
4
1
62
5
3
4
5
6
7
8
9
ArRmut11- D-alanine
Entry
KRED
Buffer
ee
c
M117F/G27
9A
(%)[b]
(%)[b]
1
2
3
4
5
6
7
NADH110
P1-B02
P1-B10
P1-B12
P1-C01
P2-D11
P3-B03
Mix N
Mix P
Mix P
Mix P
Mix P
Mix P
Mix P
>99
>99
>99
>99
>99
>99
>99
>99 (R)
93 (R)
90 (R)
90 (R)
80 (S)
99 (S)
99 (S)
ArRmut11- D-alanine
A60V/M117
3
3
2
F/G279A
ArRmut11-
M117F/G27
9A
BnNH2
BnNH2
37
51
64
72
64
65
63
78
84
84
ArRmut11-
A60V/M117
F/G279A
[a]
Reaction conditions for entry 1: 3 (27 mM), Mix N
pH 7.0 [900 L, 250 mM KPi, 2 mM MgSO4, 1.1 mM
NADP+, 1.1 mM NAD+, 80 mM D-glucose, 10 U/mL
GDH], KRED (2 mg), i-PrOH (165 µL), DMSO (55
250 µL), 24 h at 30 ºC and 250 rpm. For entries 2-7,
identical conditions but using Mix P: 125 mM KPi, 1.25
mM MgSO4, 1.0 mM NADP+, pH 7.0. [b] Determined by
HPLC.
ArRmut11- (R)-MBA 35
M117F/G27
9A
ArRmut11- (R)-MBA 43
A60V/M117
F/G279A
255 Very recently the co-immobilization of
a
210 [a] Reaction conditions: 3 (50 mM) in 100 mM KPi pH
9.0 (1 mL, 1 mM PLP, 500 mM amino donor), TA
(20 mg lyophilized cells), DMSO (20% v/v), 48 h at 45
ketoreductase and NADPH on agarose beads
activated with tertiary amine groups (AG-DEAE)
has been reported. The resulting catalyst, defined
as self-sufficient heterogeneous biocatalyst, was
260 able to catalyze asymmetric reductions without
exogenous cofactor addition.[18] Moreover, it could
be successfully re-used several batch operational
cycles and operated in a plug-flow reactor. Seeing
as the exquisite selectivity and conversion
265 displayed by the KREDs towards 3, we envisaged
to integrate this technology into the present
synthetic approach.[19] Actually, many promising
enzymatic processes are often hampered by their
[b]
ºC and 800 rpm. Determined by HPLC. The ee was
>99% in all cases for (R)-2.
215
KRED-catalyzed bioreduction of 3
The last approach displayed in Scheme 1 towards
cinacalcet consisted in a stereoselective reduction
220 of 3 to yield (S)-1-(1-naphthyl)ethanol [(S)-4].[17]
For this, the panel of engineered KREDs from
Codexis was tested employing glucose/glucose
dehydrogenase (GDH) or isopropanol (IPA) for
4
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10.1002/adsc.201701485
Advanced Synthesis & Catalysis
cofactor dependency that requires very expensive
270 reagents, endangering the economic viability.[20]
With these premises, KRED-P2-D11 was
immobilized on porous agarose beads activated
with diethyl-aminoethyl groups (AG-DEAE)
(Table 4). 96% of the offered enzyme was
275 immobilized preserving 100% of its specific
activity. The high expressed activity upon the
immobilization is an excellent results compared to
previous results reported for other KREDs.[18,21]
Under the operational conditions tested in Table 3,
280 the immobilized enzyme was 9 times more stable
than its soluble counterpart (Table 4). Both
immobilized and soluble enzyme underwent a first-
order inactivation with residual activity under
320 cofactor. Hence, the immobilized NADPH
accumulated a TTN of 450 whereas the KRED did
3600.On the other hand, we studied the continuous
asymmetric reduction of 3 in a bed-packed flow
reactor using the self-sufficient heterogeneous
325 biocatalyst. Unfortunately, the systems failed
observing conversions < 5% at 50-200 µL x min-1
flow rates. Unlike the enzyme of the original report,
namely KRED P1-A04,[18] KRED P2-D11
immobilized on the same matrix (AG-DEAE) was
330 inefficient recycling the immobilized redox
cofactor which burdened the continuous
production of (S)-4 (see the Supporting
Information for more details).
those
inactivation
conditions
(Supporting
335 Table 5. Recycling of KRED P2-D11 and NADPH co-
immobilized on AG-DEAE for the reduction of 3.[a]
285 information, Figure S1).
Entry
Cycle
t (h)
c (%)[b]
ee (%)[b]
Table 4. Immobilization and stability parameters
of KRED P2-D11 on AG-DEAE.
1
2
3
4
5
1
2
3
4
5
24
24
24
24
24
>99
>99
>99
>99
>99
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
Soluble
Immobilized
0.96
-1
Load (mg x gcarrier
)
-
-
Immobilization yield (%)[a]
Specific Activity (U x mg-1)
Expressed Activity (%)[b]
Inactivation constant (h-1) [c]
Half-life time (h) [c]
96
2.25
105±10
0.07
40.2
2.26
-
0.33
4.47
-
[a]
Stabilization factor[d]
9
Reaction conditions: 3 (27 mM) and 1 mM MgCl2
were incubated in 1.0 mL of 17% IPA in 10 mM Tris-
HCl at pH 7 and 25 ºC with 30 mg of AG-DEAE-P2-
340 D11 (6.5 mgKRED and 10 μmol NADPH/gcarrier) and
290 [a] Immobilization yield () = (immobilized mgKRED x g-
/offered mgKRED x g-1 carrier) x 100.
Expressed
1
[b]
carrier
activity (%) = (Specific activity of immobilized KRED /
Specific activity of soluble KRED) x 100. The specific
activity was calculated with 1 mM ketone 3, 0.25 mM
[b]
maintained under gentle rotational agitation (50 rpm).
Determined by HPLC.
[c]
295 NADPH and 9% Acetonitrile at 25ºC and pH 7.
Chemoenzymatic synthesis of cinacalcet
345
Stability parameters were calculated by non-linear
fitting of inactivation time-courses under 17% IPA,
4.9% DMSO at pH 7 and 30 ºC (See supporting
information Fig S1). [d] Stabilization factor = Half-life
300 time immobilized enzyme / Half-life time soluble
enzyme.
The synthetic sequences towards 1 are depicted in
Scheme 3. The TA-based approach (route A)
started with the amination of 3 with iPrNH2
catalyzed by ArRmut11-M117F/G279A (Table 2,
350 entry 2). This biotransformation was scaled up to
100 mg with 3 at 50 mM substrate concentration to
render enantiopure (R)-2 in 77% yield. Then, and
after extractive work up, this amine was directly
The high activity and stability of the immobilized
KRED P2-D11 encouraged us to co-immobilize
305 higher KRED loads (6.5 mg x gcarrier-1) with
NADPH (10 µmol of NADPH per gram of carrier).
Then, the resulting biocatalyst was tested for the
reduction of 3 using 3% of catalyst load and IPA as
sacrificing reductant. We found a conversion of
310 >99% after 24 h preserving the enantioselectivity
of the KRED [>99% ee for (S)-4] without
exogenous supply of NADPH. Moreover, this self-
alkylated
in
toluene
with
3-[3-
355 (trifluoromethyl)phenyl]propan-1-ol (7) mediated
by the iridium catalyst [Cp*IrCl2]2 (1 mol%).[22]
Thus, 1 was isolated after filtration through silica
gel with a yield of 60% for the overall two-step
sequence.
360 Encouraged by this result, we took the challenge of
developing a one-pot sequential synthesis of
cinacalcet in aqueous medium. Actually, a related
catalyst [Cp*IrI2]2 was reported to promote the
alkylation of equimolar amounts of amine and
365 alcohol in water.[23] Hence, a first issue was
confirming that [Cp*IrCl2]2 catalyzes the coupling
of 2 and 7 in refluxing water. Pleasantly, the
sufficient biocatalyst was reused up in
5
consecutive cycles without losing effectiveness
315 (Table 5) with a catalyst load decreased by a factor
of 9 (0.195 mgKRED/mL) regarding to the free
enzyme (1.79 mgKRED/mL). The recyclability of the
biocatalyst significantly increased the total
turnover number (TTN) of both enzyme and
5
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10.1002/adsc.201701485
Advanced Synthesis & Catalysis
process took place with high yield and water as the
only by-product released. Accordingly, the
study a few years ago showed that the treatment of
405 electron withdrawing group-substituted benzyl
alcohols with tosyl chloride renders chloride
derivatives instead of the expected tosylates.[26] On
the other hand, the fact of obtaining racemic
species reveals the formation of a carbocation, via
410 SN1 mechanism, which would be stabilized by the
phenyl ring and reacted with Cl− to form racemic
8. In a further attempt, the employment of AgNO3
as a scavenger of chloride ions enabled to obtain
the mesylate 9 (route B2). Subsequent nucleophilic
415 substitution with the amine 10 gave entry to 1
although as a racemate. This suggests the lability
of the mesyl derivative and the inconvenience of
this methodology. Finally, the use of a base such as
DBU in the presence of diphenyl phosphorazidate
420 (DPPA) suppressed the SN1 chemistry and
rendered the azide (R)-9 of opposite
stereochemistry with a very slight erosion of the
enantiomeric purity (97% ee, route B3).[27] Then,
LiAlH4 reduction provided the amine (R)-2 which
425 was converted into 1 as described above. The
overall yield of the formal total synthesis of 1 was
65% after four steps. Comparing both
chemoenzymatic approaches, route A (TA-based
approach) involves two less steps and does not
430 require chromatographic purification. Likewise,
and from an environmental point of view, it avoids
the employment of hazardous reagents such as
DPPA and is entirely performed in aqueous
medium.
370 amination of
3
catalyzed by ArRmut11-
M117F/G279A was performed again as described
above. Next, the reaction mixture containing
enantiopure (R)-2 was evaporated to dryness to
remove the excess of iPrNH2, preventing the
375 consumption of alcohol 7 by the amino donor.
Then, the resulting crude product was suspended in
water and treated with 7 and catalyst [Cp*IrCl2]2 (1
mol%) at reflux. Unfortunately, no product was
detected after 12 h which suggested metal
380 inhibition by the enzyme or its cofactor. A further
attempt consisted in testing commercially available
TAs instead of lyophilized whole cells to simplify
the enzymatic milieu. However, the iridium
catalyst was totally inhibited again, precluding the
385 one-pot process.[24] Nevertheless, the only required
setting to solve this drawback was removing the
insolubles (e.g., precipitated protein) by
centrifugation after the enzymatic step, and adding
to the supernatant both [Cp*IrCl2]2 and 7. Under
390 this experimental setting, enantiopure 1 was
produced with an overall yield of 50%.
Concerning the KRED-based approach (route B),
the first step was affording the enantiopure alcohol
(S)-4 with complete conversion by using one of the
395 two last enzymes depicted in Table 3 (entries 6-7).
Then, and following a patented procedure (route
B1), the hydroxyl group was treated with mesyl
chloride to make it a good leaving group for a
further nucleophilic substitution.[25] However, 1-(1-
400 chloroethyl)naphthalene (8) was isolated in
racemic form instead of the expected mesyl
derivative. Similarly, the employment of tosyl
chloride led to identical outcome. In this context, a
435
6
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10.1002/adsc.201701485
Advanced Synthesis & Catalysis
Scheme 3. Chemoenzymatic synthetic approaches towards 1.
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10.1002/adsc.201701485
Advanced Synthesis & Catalysis
extracted with ethyl acetate (2  500 μL). The organic
layers were separated by centrifugation (90 s, 13000 rpm),
combined, and finally dried over Na2SO4. Then, the ee of
the amine was determined by chiral normal phase.
Conclusion
Amination of 3 employing variant ArRmut11-
M117F/G279A (preparative scale)
Two chemoenzymatic syntheses towards cinacalcet,
the only approved drug acting as calcimimetic, have
been developed. In the first route, the key
asymmetric step was an amination of the ketone 3
employing TAs. Further iridium-catalyzed alkylation
of the resulting enantiopure amine (R)-2 led to
cinacalcet in 50% overall yield for the two-step
sequence. Notably, both steps were run in aqueous
medium and the process did not require purification
or isolation of any intermediates. On the other hand,
the bioreduction of the same ketone by KREDs
provided enantiopure alcohol (S)-4 quantitatively.
Then, further follow-up chemistry enabled the amine
(R)-2 via SN2 inversion with DPPA and further
reduction of the resulting azide, to complete the
synthetic scheme towards cinacalcet in four steps and
65% overall yield. Despite the second route requiring
more steps, the KRED was successfully co-
immobilized with NADPH giving the possibility to
be re-used in 5 batch operational cycles without
adding of exogenous NADPH.
Ketone 3 (100 mg, 0.588 mmol) was placed in a bottom
round flask containing 10.0 mL of 100 mM KH2PO4
buffer (pH 9, 0.5 M iPrNH2, 1 mM PLP). Then,
lyophilized whole cells of ArRmut11-M117F/G279A (200
mg) were added and the resulting suspension magnetically
stirred at 800 rpm and 45 ºC for 72 h. To determine the
conversion, 10 µL of the mixture were diluted with 90 µL
of Mili Q water and analyzed by achiral reverse phase with
previous centrifuging and filtering of the sample. After
that time the reaction was basified with a saturated
solution of Na2CO3 (5 mL) and extracted with ethyl
acetate (2  5 mL). The organic layers were separated by
centrifugation (90 s, 13000 rpm), combined, and finally
dried over Na2SO4. Then, the resulting amine (R)-2 was
submitted to the next synthetic step without further
purification (Scheme 3, route A). Eventually, for an
analytical sample the amine 2 was purified by flash
chromatography of by acid-base extraction (78 mg, 77%
yield). The ee of (R)-2 was determined by chiral normal
phase (>99%).
Bioreduction of 3 employing KREDs from the
Codexis’ kit (analytical scale)
In a 2.0 mL eppendorf tube, KRED (2 mg), 3 (27 mM),
DMSO (55 µL) and i-PrOH (165 µL) were added to 900
µL of 125 mM KH2PO4 buffer (1.25 mM MgSO4, 1 mM
NADP+) pH 7.0 or 900 µL of 250 mM KH2PO4 buffer (2
mM MgSO4, 1.1 mM NADP+, 80 mM D-glucose, 10
U/mL glucose dehydrogenase) pH 7.0 in the case of
KRED-101, KRED-119, KRED-130, KRED-NADH-101
and KRED-NADH-110. The reaction was shaken at 250
rpm and 30 ºC for 24 h. To determine the conversion, 10
µL of the mixture were diluted with 90 µL of Mili Q water
and analyzed by achiral reverse phase with previous
centrifuging and filtering of the sample Then the mixture
was extracted with ethyl acetate (2  500 μL), the organic
layers separated by centrifugation (90 sec, 13000 rpm),
combined and dried over Na2SO4. Then, the ee was
measured by chiral normal phase.
Experimental Section
General Remarks
Kits of TAs and KREDs were purchased from Codexis.
The cultures for overexpression of the IREDs were
inoculated from E. coli BL21 (DE3) glycerol stocks from
Greifswald University containing the respective IREDs in
a pET 22b(+) vector. The standard procedure for
expression was followed as described in ref. 11a. TA
variants from ArRmut11 were overexpressed in E. coli and
used as lyophilized cells (for more details about these TAs,
see ref. 16). For the enzymatic reactions, commercially
available solvents were used. Thin-layer chromatography
was performed on precoated TLC plates of Merck silica
gel 60F254, using potassium permanganate as developing
reagent. For column chromatography, Merck silica gel 60
Immobilization of, thermal stability and activity
assay of KRED P2-D11 immobilized on Ag-DEAE
1
(particle size, 40 - 63 µm) was used. H NMR and proton-
decoupled 13C NMR spectra were obtained using a 300
MHz spectrometer using the  scale (ppm) for chemical
shifts; calibration was made on the CDCl3 (13C; 76.95
ppm) or the residual CHCl3 (1H; 7.26 ppm) signals. HPLC
analyses to determine degree of conversions were carried
out in an Agilent RR1200 HPLC system, using a reversed
phase column (Zorbax Eclipse XDB-C18, RR, 18μm, 4.6
x 50 mm, Agilent). HPLC analyses to determine ee were
performed on a Hewlett Packard 1100 LC liquid
chromatograph, using normal phase columns (Chiralcel
OJ-H and Chiralpak IA).
The protein immobilization was carried out with 1 g of
AG-DEAE incubated with 10 mL of 0.1-0.65mg/mL
KRED P2-D11 in 10 mM sodium phosphate pH 7.0 for 1 h
at room temperature. Then,
1
g
of AG-DEAE
immobilizing KRED was incubated with 10 mL of 1mM
NADPH in 10 mM Tris-HCl at pH 7.0. The operational
stability was determined by incubating soluble and
immobilized KRED under the reaction conditions (1mM
MgCl2, 4.9% DMSO and 17% IPA in a 10 mM tris-HCl at
pH 7.0) at 30ºC. Samples were withdrawn at different
incubation times and the residual enzymatic activity was
spectrophotometrically determined. For this purpose, the
activity of KRED was measured by monitoring the
absorbance at 340 nm along the time in a VarioskanTM
Flash Multimode Reader (Thermo Scientific) using 96-
well plates. Briefly, 200 µL of a reaction mixture
containing 1 mM of 3, 1 mM MgCl2, 0.25 mM of NADH
and 9% Acetonitrile in 10 mM sodium phosphate buffer
(pH 7.0) was incubated with 20 µL of enzymatic solution
or suspension at 25 ºC under orbital shaking integrated
into the reader. One unit of activity was defined as the
amount of enzyme that was required to either reduce or
oxidize 1 μmol of NADPH at 25 ºC and pH 7.0.
Amination of 3 employing TAs from the Codexis’
kit (analytical scale)
Reactions were carried out in a 2.0 mL eppendorf tube.
The corresponding TA (1.0 mg) was added to 500 µL of
100 mM KH2PO4 buffer pH 9.0 containing iPrNH2 (1 M)
and the cofactor PLP (1.0 mM). Then, a solution of 3 (20
mM) in DMSO (50 µL) was added and the resulting
mixture was shaken at 250 rpm and 45 °C for 24 h. To
determine the conversion, 10 µL of the mixture were
diluted with 90 µL of Mili Q water and analyzed by
achiral reverse phase with previous centrifuging and
filtering of the sample. Then, the reaction was quenched
by addition of 100 µL of aqueous 10 N NaOH and
Bioreduction of
3
in batch employing an
immobilized KRED
8
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10.1002/adsc.201701485
Advanced Synthesis & Catalysis
In a micro Bio-spinTM chromatographic column (BIO-
RAD), a reaction mixture containing 3 (27 mM) and 1.0
mL of 17% IPA in 10 mM Tris-HCl and 1 mM MgCl2 at
pH 7.0 was incubated with 30 mg of self-sufficient
heterogeneous biocatalysts (6.5 mgKRED P2-D11 and 10 μmol
NADPH/gcarrier) and maintained under gentle rotational
agitation (50 rpm) at 25 ºC. As control reaction, same
amount of KRED immobilized on AG-DEAE and soluble
and exogenous NADPH were mixed in the same
conditions. The reaction was stopped by vacuum filtration
and the conversion and ee determined as described above.
Next, the immobilized biocatalyst was washed with 10
volumes of 10 mM Tris-HCl at pH 7.0 to be re-used in the
next batch cycle.
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1g of self-sufficient heterogeneous biocatalyst (5.85
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Acknowledgements
LM, FM, JGS and PB acknowledge funding from the
European Union’s Horizon 2020 MSCA ITN-EID
program under grant agreement No 634200 (Project
BIOCASCADES). N.R-L acknowledges MINECO for
funding under Torres-Quevedo program (PTQ-12-05 407).
PB, WK, FLG and ABM acknowledge funding from the
Cost Action CM1303 Systems Biocatalysis. FLG also
acknowledges funding from IKERBASQUE. Prof
Matthias Höhne, Greifswald University, DE, is gratefully
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10
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Advanced Synthesis & Catalysis
FULL PAPER
Chemoenzymatic Approaches to the Synthesis of
the Calcimimetic Agent Cinacalcet Employing
Transaminases and Ketoreductases
Adv. Synth. Catal. Year, Volume, Page – Page
Lisa Marx, Nicolás Ríos-Lombardía, Judith
Farnberger, Wolfgang Kroutil, Ana I. Benítez-
Mateos, Fernando López-Gallego, Francisco Morís,
Javier González-Sabín* and Per Berglund*
0
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