Communications
well as secondary amines.[12] However, the reactions presented
so far suffered from high catalyst loadings and low substrate
concentrations. To date, no systematic screening of a significant
number of IREDs for reductive amination has been reported,
and the potential of this enzyme class for this reaction still
needs to be assessed. Recently, we identified and characterized
a set of 22 IREDs that were screened against a panel of cyclic
imines and applied in preparative-scale reductions to the cor-
responding amines.[11f] In this study, we expanded this IRED li-
brary by nine additional enzymes (Table S1, Figure S1; see the
Supporting Information). Three of the formerly reported en-
zymes were omitted from the screening owing to lack of pro-
tein stability.
ered the whole range from moderate to excellent, depending
on the combination of IRED, ketone, and amine (Table S5). Re-
garding the ketones, we found that conversion for acetophe-
none (1) was very poor (<10%) for virtually all tested amines
and IREDs except for IR_32 and IR_33, which transformed
1 with methylamine into 2b with conversions of 36 and 23%,
respectively.
We applied IREDs IR_11 and IR_20 for reductive amination
on a preparative scale: Ketones 3 and 7 were chosen as model
substrates, as both potentially lead to the formation of enan-
tio- and diastereoisomeric products, which would thus readily
allow us to determine the stereochemical preference of the
catalyst. The transformation of 100 mg of 7 to 8a by using
IR_20 yielded 64 mg of (1S,3R)-8a·HCl (50% yield, 94%de, 96%
conversion after 73 h). By converting 150 mg of 7 with methyl-
amine by using IR_11, 156 mg of (1S,3R)-8b·HCl was obtained
after workup (71% yield, 98%de, 98% conversion after 18 h).
IR_20 was also applied to transform 400 mg of 3 with methyl-
amine to yield 330 mg of (R)-4b·HCl (55% yield, 96%ee, 95%
conversion after 48 h).
Thus, we systematically screened 28 IREDs, of which 26 were
in-house enzymes chosen from our in-house database
(Table S1), to identify enzymes suitable for the reductive ami-
nation of ketones on a preparative scale. The aim was to pre-
pare both primary and secondary amines, as depicted in
Scheme 1. We tested acetophenone (1), 2-hexanone (3), cyclo-
hexanone (5), (R)-3-methylcyclohexanone (7), and 2-methoxy-
cyclohexanone (9) as model substrates for the targeted reac-
tion, each with ammonia (a), methylamine (b), or butylamine
(c) (Scheme 2). The screening was performed by using purified
enzymes at 0.6 mgmLÀ1, a ketone concentration of 20 mm,
and amines a–c in 250 mm concentration, adjusted to give
a final reaction pH of 9.3 (as described in the Supporting Infor-
mation).
All preparative-scale reactions were performed at room tem-
perature with substrate loadings of 0.5%w/v ketone for trans-
formations with ammonia and 1%w/v ketone for transforma-
tions with methylamine, as described in detail in the Support-
ing Information. For all these preparative-scale reactions, the
reaction rates and overall conversions were lower than those
for the reduction of cyclic imines.
The reductive amination reactions at a concentration of
1%w/v ketone with a catalyst loading of 0.6 mgmLÀ1 did not
reach full conversion within 24 h. In contrast, 6-methyl-2,3,4,5-
tetrahydropyridine (11) in concentrations up to 4%w/v was
readily converted into (S)-2-methylpiperidine (12) (77% yield,
>98%ee, Figure S42).
The reasons for the lower conversions of the reductive ami-
nations need to be clarified, as little has been reported about
the exact reaction mechanism and the rate-limiting steps for
IRED-catalyzed reductive amination. We hypothesize that the
actual concentration of the intermediate imine in the aqueous
mixture is one of the major rate-limiting parameters. Although
the reactions are performed at high pH values and with an
excess amount of the amine, imine formation in aqueous solu-
tion is highly disfavored owing to the great excess amount of
water present in the mixture.
Scheme 2. Reductive amination reactions tested for a set of model ketones
with ammonia, methylamine, and butylamine by using NADPH-dependent
IREDs.
To elucidate the concentration of the imine in our prepara-
tive reactions, the methylamination reaction of 3 into 4b was
investigated by online NMR spectroscopy. For technical rea-
sons, the pH of the reaction, unlike that for the preparative ex-
amples, was not controlled for the NMR measurement. We
found that the reaction displayed Michaelis–Menten-like be-
havior by following the NMR signal of the methyl group adja-
cent to the respective ketone or product amine (Figure S46).
Imine formation, however, could not be detected during the
course of this reaction. Thus, we performed a NMR experiment
on an aqueous high-pH blank sample (pH>9) with ketone and
amine only, omitting the IRED from the mixture and analyzing
quantitatively the proposed ketone imine equilibrium by
HMBC 2D NMR spectroscopy (Figure S47). Nevertheless, even
All targeted product amines 2a–10b were accessible by at
least one of the tested IREDs. The conversions for the reac-
tions, however, varied from excellent (>90%) to very poor
(<10%) (Table 1) and strongly depended on the amine and
the ketone used.
Regarding the substrate amines, we found that transforma-
tions with methylamine (see 2b—10b) generally led to the
highest conversions; for amines 4b–10b the conversions
exceeded 90% in many cases. The enantiomeric excess (ee) or
diastereomeric excess (de) values of the formed products cov-
ChemCatChem 2016, 8, 2023 – 2026
2024
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Wetzl, Dennis
