Communications
benzyl-substituted derivative 5m and detectable conversion
was also observed with the bulky 1-naphthyl-substituted 5n,
suggestive of an enzyme possessing a large active site. For
substrates bearing phenyl derivatives, the introduction of
a substituent on the ring led to an increase in enantioselectivi-
ty. Both electron-donating and electron-withdrawing groups
were readily accepted, with high conversion and ee observed.
Interestingly, a moderate decrease in selectivity was observed
if moving the position of the methoxy-substituent from para
to meta and ortho, signaling an increasing contribution of
a competing binding mode in which the opposite face of the
imine is exposed for reduction by the NADPH. The (R)-IRED
readily accepted other heterocyclic motifs including a thio-
phene ring (5l).
recycling system, this approach was used for any preparative
biotransformations.
Kinetic parameters were determined for representative sub-
strates (Table 3). Kinetic constants with the previously reported
(S)-IRED from Streptomyces sp. GF3546 have been described
À1
with substrates 1a, 3a, 5a, 7, 7a, and 9 (i.e., k =0.024 s ,
cat
À1
À1
À1
À1
À1
0.039 s , 0.137 s , 0.445 s , 0.040 s , and 0.483 s , respec-
[16]
tively). Excluding 7, significantly higher kcat values and lower
Michaelis constants (K ) were observed for the (R)-IRED de-
m
scribed herein (Table 3). The previously reported substrate 2-
methyl-1-pyrroline 1a had a notably lower k /Km value (cata-
cat
lytic efficiency) than several piperideine substrates tested. In
particular, we observed a greater than sixty-fold increase in
k /K for 2-methyl-1-piperideine 5a over pyrroline 1a. This
cat
m
With simple alkyl and alkenyl substituents, the (R)-IRED
showed excellent enantioselectivity. In addition, reduction of
a,b-unsaturated imine 5j proceeded exclusively at the carbon–
nitrogen double bond with no reduction of the alkene ob-
served. Reduction of n-propyl-substituted piperideine 5i result-
ed in the formation of the more active (R)-enantiomer of the
natural product coniine in >98% ee. To demonstrate the appli-
cation of the (R)-IRED biocatalyst for preparative-scale synthesis
of chiral amines, the reduction of 5j was performed on a 1.0 g
preference for 6- and 7-membered ring systems is shared with
the (S)-IRED and may allude to the natural function of this
class of enzyme. The (R)-IRED also displayed greater activity to-
wards simple alkyl- and alkenyl-substituted imines compared
with those bearing aromatic moieties.
A selection of ketones and oximes were screened for activity
with the (R)-IRED, with no conversion detected (see Supporting
Information). The absence of activity towards ketones suggests
future potential applications of this enzyme for intermolecular
reductive aminations, which remains a significant challenge in
organic synthesis.
(25 mm) scale, yielding (R)-coniine 6i (90% yield, >98% ee),
which was isolated as its hydrochloride salt. In addition to
simple 2-substituted piperideines, 3,4-dihydroisoquinolines 7
and 7a were also confirmed to be substrates for the enzyme.
Significantly, the corresponding N-methyl iminium derivatives
We have recently determined the structure of oxidoreduc-
[22]
tase Q1EQE0 from Streptomyces kanamyceticus, which shares
50% sequence identity with the (R)-IRED, and which also cata-
lyzes the (R)-selective reduction of imine substrates such as 2-
methyl-1-pyrroline 1a. The structure of Q1EQE0 revealed a di-
meric association in which two monomers associate very close-
ly through domain swapping. The active site, containing
NADPH, is a channel that traverses the width of the dimer. The
active site also revealed residues which may be responsible for
catalysis, including Asp187, which was proposed to be the cat-
alytic residue for protonation of the imine in IRED-mediated
9
and 9a were also reduced, with chiral amine 10a produced
with comparable selectivity (74% ee), albeit with reduced con-
version, demonstrating the potential application of this biocat-
alyst for the synthesis of tertiary chiral amines.
Biotransformations of several substrates conducted with the
isolated enzyme, coupled with an NADPH cofactor recycling
system using glucose dehydrogenase 2 from Bacillus megateri-
[
22]
um,
showed no significant change in enantioselectivity if
[22]
compared to their whole-cell equivalents, with the exception
of dihydroisoquinoline 7a for which unusually the selectivity
appeared to diminish to give (R)-8a in 47% ee (see Supporting
Information). As the whole-cell biocatalyst requires only the ad-
dition of glucose for cofactor recycling and negates the neces-
sity of using large amounts of NADPH or an additional cofactor
catalysis. The similarity between the two sequences gave us
confidence to build a model of the (R)-IRED (Figure 1a) using
2
[23]
the Phyre server. In the model, Asp187 in Q1EQE0 is con-
served in the (R)-IRED as Asp172 and many of the other resi-
dues within the region of the active site also appear to be con-
served between the two enzymes (Figure 1b).
The role of Asp172 in the catalytic mechanism was further
probed by generation of the Asp172Ala and Asp172Lys point
mutants by site-directed mutagenesis. Interestingly, both mu-
tants retained catalytic activity although conversions were gen-
erally lower, for example, for 5c the conversion was reduced
from 92% for the wild-type isolated protein to 40% and 72%
for the Asp172Ala and Asp172Lys mutants, respectively. Re-
markably, reduction of 5a by the mutants showed no change
in enantioselectivity whereas for 7a the ee value was increased
to 81% for the Asp172Ala variant (Table 4). These initial studies
highlight the importance of this residue as a hotspot for muta-
genesis to improve conversions and/or enantioselectivity.
In summary, the gene for Streptomyces sp. GF3587 (R)-IRED
was overexpressed in E. coli to produce a recombinant whole-
cell biocatalyst possessing broad substrate scope, suitable for
Table 3. Kinetics data for cyclic imine substrates 1–7.
Substrate
R
V
max
k
cat
À1
K
m
k
cat/K
m
À1
À1
[s
]
[mm]
[s mm
]
1
3
5
5
5
5
5
5
7
7
9
a
a
a
c
e
i
Me
Me
Me
p-MeOPh
cyclohexyl
n-propyl
isopropenyl
2-thienyl
H
0.633
6.57
7.60
0.474
0.0388
8.35
4.24
1.16
0.255
0.340
1.905
0.351
3.64
4.21
0.263
0.0215
4.63
1.88
5.22
0.371
1.05
1.55
0.804
1.77
0.244
0.317
0.155
0.481
0.187
0.698
11.4
0.250
0.0139
5.76
1.33
2.64
0.447
1.22
2.195
j
l
2.35
0.643
0.141
0.189
1.057
a
Me
H
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