FULL PAPER
Cell pellets were resuspended in 0.1 M potassium phosphate
buffer (0.49 mL, pH 7.4) supplemented with 20 mM glucose
monohydrate and transferred into 96 deep well plates furnished
with glass Hirschmann vials (Hirschmann Laborgeräte GmbH
& Co. KG, Eberstadt, DE), enabling reusability of 96 deep well
plates without cross contamination. Reactions were started by
addition of 10 μL of a 0.5 M substrate solution prepared in
ethanol, to a final concentration of 10 mM. Biotransformations
mobile phase for the purification of generated products from
substrates 2 and 3 was water/acetonitrile (55/45), which was
hold isocratically for 22 min. Peaks were detected at 210 nm for
the reaction analysis of 2 and at 310 nm for 3 (Supplementary
section 1, Figs. S6 and S13). The collected fractions of each
product were evaporated in vacuo and analyzed via 1H- and 13C-
NMR (supplementary section 2, Figs. S14–S24).
Chemical synthesis of (-)-(S)-1,2,3,4-Tetrahydroquinoline-4-
ol. The chemical synthesis of (À )-(S)-1,2,3,4-tetrahydroquino-
line-4-ol was done according to Kolcsar and collegues.[35] The
raw product was extracted five times with MTBE, evaporated in
vacuo and dissolved in 4 mL ACN. Preparative HPLC
purification was performed as described in 1.5. A total of
28.1 mg (10% yield) (À )-(S)-1,2,3,4-tetrahydroquinoline-4-ol
was obtained. The enantiomeric excess for raw and purified
product was determined via chiral HPLC-DAD to 81% ee and
35% ee, respectively.
°
were incubated at 30 C and 800 rpm, in the case of substrate
naphthalene 1, only for 0.5 h and in the case of substrates
1,2,3,4-tetrahydroquinoline 2 and 2-phenylpyridine 3 for 20h.
Reactions were stopped by addition of 0.5 mL of the extraction
solvent MTBE. After vigorous mixing and centrifugation
(4000×g at room temperature for 5 min) the organic layer was
removed and analyzed via HPLC-DAD, to quantify the
achieved product formation and selectivity. In addition, HPLC-
ESI-MS analysis was performed to confirm the masses of the
generated products. The whole set of screening results is shown
in supplementary section 4, Figures S25–S27.
HPLC-DAD for quantification. For substrate 1 biotransforma-
tions HPLC-DAD and ESI-MS methods were performed
according to Wissner and colleagues.[17] For the analysis of the
biotransformation products of substrates 2 and 3, an Agilent
1260 Infinity II system (Santa Clara, US), equipped with a C18-
column (Supelco C18 Discovery, 5 μm, 4.0×150 mm, Belle-
fonte, US) and a diode array detector (Agilent 1260 Infinity II
Validation of mutant library hits. Variants fulfilling one or
more of the following criteria were reevaluated with the
corresponding substrate: 1) Variants displaying over 200% total
product formation in comparison with TDO wild type, 2)
Variants showing a switch in product distribution in favor to the
side product, and in addition, at least 50% total product
formation, when compared to TDO wild type, 3) Variants
generating selectively one product, 4) Double variants have to
be at least as active or as selective as the parent single variant
and fulfill at least one of the criteria previously mentioned.
°
DAD HS, Santa Clara, US) was operated isothermally at 30 C
°
and 50 C for 2 and 3, respectively. Measurements were run at a
flow rate of 1.0 mLminÀ 1. For 2, the mobile phase was water/
methanol 55/45 (v/v) hold isocratically for 10 min and 1 μL
sample was injected. For 3, the mobile phase was water/
methanol with a linear gradient of; t=0 min, 55/45 (v/v); t=
4.0 min, 50/50 (v/v); t=8.0 min, 42/58 (v/v); t=11.50 min,
42/58 (v/v); t=11.51 min, 55/45 (v/v); t=13.00 min, 55/45 (v/
v) and 4 μL sample was injected. Peak areas were measured by
the integrator and transformed into concentration using the
corresponding standard curves. For substrates 2 and 3, the
employed wavelength was 210 and 310 nm respectively, to
detect the generated products.
Biotransformations were performed according to 1.3, with the
only difference, that no glass Hirschmann inlets were used. This
increased the total working volume from 1.0 mL to 2.0 mL,
resulting in an increased product formation for all tested
substrates. In addition, the enantiomeric excess of the generated
products was determined via chiral HPLC-DAD. Validation
results of variants fulfilling the established criteria are shown in
Table 1, 2 and 3.
Semi-preparative biotransformations. Semi-preparative bio-
transformations were performed according to 1.4 with the only
exception that for substrate 1,2,3,4-tetrahydroquinoline 2, the
TDOF114H_A223T and TDOF366V catalyzed biotransformations were
performed in TRIS-buffer (0.1 M, pH 8.5) supplemented with
20 mM glucose monohydrate instead of potassium phosphate
buffer (see supplementary section 5). For semi-preparative
biotransformation of 3, TDOM220A_V309G was employed. A
number of 200 simultaneous driven biotransformations were
performed in 96 deep well plates, and combined after reaction
completion, thus representing a total of 100 mL reaction
volume. The combined reaction mixtures were extracted with
MTBE either five times (1:1) for 2, or nine times (1:2) for 3.
The combined organic layers were evaporated in vacuo and
redissolved in 6 mL of an acetonitrile-water mixture. The
purification step was performed by preparative HPLC in an
Agilent 1260 Infinity HPLC-DAD system (Santa Clara, US),
equipped with a preparative C18-column (Supelco, Discovery
C18, 5 μm, 21.2×100 mm, Bellefonte, US) and a fraction
collector (Agilent 1260 Infinity II, Santa Clara, US). Product
purification was performed with a flow rate of 4.0 mLmin-1 and
repeated injections of 70 μL of the crude reaction mixture. The
HPLC-ESI-MS for confirmation. To confirm generation of
the hydroxylated products 2a, 3a and 3b, HPLC-ESI-MS was
employed. An Agilent 6130 Quadrupole LC System (Santa
Clara, US), was operated using an identical HPLC method and
column as described in section 1.7, above. The ESI-MS was run
using the following parameters: API-ES spray chamber with a
drying gas flow of 12 LminÀ 1, nebulizer pressure of 50 psi,
°
drying gas temperature 350 C and a capillary voltage of
3500 V positive and negative. The mass detection for 2a, 3a
and 3b was performed in positive SIM-modus with m/z=150
[M+H], 172 [M+H] and 190 [M+H], respectively.
Chiral HPLC-DAD. Chiral HPLC-DAD measurements for 1a
were performed according to Wissner and colleagues.[17] Chiral
assessments of the products 2a and 3a were performed on an
Agilent 1260 Infinity HPLC-DAD system (Santa Clara, US),
equipped with a normal phase Chiralpak IC column (5 μm,
°
4.6×250 mm, Daicel, Osaka, JP), heated to 30 C. Analysis was
performed with a flow rate of 1.4 mLminÀ 1 and 15 μL sample
injection. For the separation of the 2a enantiomers, n-hexane/
isopropanol (95/5) was used as mobile phase and hold isocrati-
cally for 22 min. For the separation of 3a enantiomers, n-
hexane/isopropanol (90/10) was used and hold for 90 min.
Adv. Synth. Catal. 2021, 363, 1–11
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© 2021 The Authors. Advanced Synthesis & Catalysis
published by Wiley-VCH GmbH
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