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Figure 1. Time course of the cascade reaction for the synthesis of (R)-
phenylglycinol from styrene oxide. Styrene oxide (1, ~), (R)-phenylglycol (2,
♦), 2-hydroxyacetophenone (3, &), (R)-phenylglycinol (4, *). Reaction
conditions (20 mL): styrene oxide 20 mM, L-Ala 200 mM, NH4Cl 250 mM,
VrEH2M263V 4 U/mL, EaGDH 2 U/mL, AlaDH 2 U/mL, ω-TAY150F/V153A 2 U/mL,
NAD+ 2 mM, PLP 0.5 mM, 100 mM glycineÀ NaOH buffer, pH 8.0, 30 C. All
°
biotransformations were performed in triplicate and error bars refer to �s.d.
produced from racemic styrene oxide (20 mM) with 40.5% yield
and 99% ee (Figure 1 and Figure S5, supporting information).
The low yield of target product was mainly due to the
accumulation of (R)-phenylglycol (12 mM). Therefore, the dos-
age of EaGDH and ω-TAY150F/V153A were increased from 2 U/mL to
4 U/mL and the catalyst ratio between EaGDH and ω-TAY150F/
Figure 2. Investigation of the thermodynamic equilibrium of the cascade
reaction. (R)-Phenylglycol (2, ♦), 2-hydroxyacetophenone (3, &), (R)-phenyl-
glycinol (4, *). (A) Time course of the cascade reaction from (R)-phenylglycol
to (R)-phenylglycinol. Reaction conditions (20 mL): (R)-phenylglycol 22.3 mM,
L-Ala 200 mM, NH4Cl 250 mM, EaGDH 2 U/mL, AlaDH 2 U/mL, ω-TAY150F/V153A
2 U/mL, NAD+ 20 mM, NADH 20 mM, PLP 0.5 mM, 100 mM glycineÀ NaOH
was also increased from 1:1 to 1:4 aiming to further
V153A
improve the conversion of (R)-phenylglycol to (R)-phenylglyci-
nol (Table S5, supporting information). However, there was still
about 8–12 mM (R)-phenylglycol remained in the reaction
mixture, indicating that catalyst loading of EaGDH and ω-
TAY150F/V153A was not the predominant reason for the accumu-
lation of such reaction intermediate.
°
buffer, pH 8.0, 30 C. (B) Time course of the cascade reaction from (R)-
phenylglycinol to (R)-phenylglycol. Reaction conditions (20 mL): (R)-phenyl-
glycinol 22.3 mM, L-Ala 200 mM, NH4Cl 250 mM, EaGDH 2 U/mL, AlaDH 2 U/
mL, ω-TAY150F/V153A 2 U/mL, NAD+ 20 mM, PLP 0.5 mM, NADH 20 mM,
°
100 mM glycineÀ NaOH buffer, pH 8.0, 30 C. All biotransformations were
performed in triplicate and error bars refer to �s.d.
Since EaGDH and ω-TAY150F/V153A catalysed reactions are both
reversible, the thermodynamic equilibrium might be the main
reason for the accumulation of the intermediate (R)-phenyl-
glycol. To verify our hypothesis, forward and reverse reactions
of the cascade system employing (R)-phenylglycol or (R)-
phenylglycinol as substrate, respectively, were investigated
(Figure 2). Starting from 22.3 mM (R)-phenylglycol, 7.06 mM (R)-
phenylglycinol was produced with 7.35 mM 2-hydroxyaceto-
phenone, while 7.21 mM (R)-phenylglycol was produced with
6.87 mM 2-hydroxyacetophenone starting from 22.3 mM (R)-
phenylglycinol. The concentrations of three reactants in the
forward and reverse reactions were almost identical, suggesting
that thermodynamic equilibrium of the cascade system caused
the accumulation of intermediate (R)-phenylglycol and there-
fore resulting in low yield of the target product.
could only selectively adsorb (R)-phenylglycinol, while the other
reaction components were not absorbed by HD-8 (Figure S1 &
S2, supporting information). Therefore, resin HD-8 was selected
for the in situ removal of (R)-phenylglycinol to drive the
thermodynamic equilibrium. To this end, a new bio-reactor
system coupling the three-step cascade reaction with the in situ
product removal for the synthesis of (R)-phenylglycinol from
racemic styrene oxide was constructed (Figure S7, supporting
information). As can be seen from Figure 3, the accumulation of
intermediate (R)-phenylglycol was significantly decreased from
60% to 16.5% due to the efficient adsorption of product to the
resin, and no product could be detected in the reaction mixture.
The product was then eluted from the cation exchange resin
HD-8 with 2 M NaOH, and after follow up purification steps, (R)-
phenylglycinol was isolated with 81.9% yield, which is
obviously superior to that without in situ product removal
(40.5%), suggesting that the thermodynamic equilibrium of the
reaction was efficiently driven to the product with in situ
product removal strategy.
In situ product removal by resin adsorption has been proven
to be
a useful strategy in driving the thermodynamic
equilibrium to the product, since the resin is nontoxic,
inexpensive, easy to handle, and recyclable.[21] Three types of
cation exchange resin including HD-8, C160, and C104plus were
subjected to adsorption experiment. Interestingly, macroporous
strong acid cation exchange resin HD-8 was observed to be a
good adsorbent for the target product (R)-phenylglycinol, and
In summary, a new three-step one-pot cascade system
involving enantioconvergent hydrolysis of racemic styrene
ChemCatChem 2019, 11, 1–7
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