Z.-Q. Liu et al. / Biochimie xxx (2014) 1e5
3
3.4. The effect of substrate molar ratio on the asymmetric aldol
reactions of isatins and cyclic ketones
In order to further enhance the yield and enantioselectivity of
the nuclease p1 catalyzed enantioconvergent aldol reactions of
isatins and cyclic ketones, the effect of substrate molar ratio was
investigated on the model reaction. When the reaction solvent was
acetonitrile, the best result was observed with 1 mmol cyclohexa-
none (Fig. 3), just as we described before. Surprisingly, nuclease p1
showed the highest activity and enantioselectivity in 1 mL cyclo-
hexanone (about 9.6 mmol), which substituted acetonitrile as the
solvent, under this condition the highest ee value of 69% and yield
of 99% were got in the enzymatic reaction. In this two-phase
environment, nuclease p1 was in water so that it could maintain
catalytic activity and tertiary structure for stereoselectivity, there-
fore, good enantioselectivity and high yield were obtained.
Consequently, the enzymatic asymmetric aldol addition between
isatin and cyclic ketone proceeded in solvent-free condition, the
best conditions are: isatin (0.2 mmol), cyclohexanone (1 mL), DI
(0.15 mL), nuclease p1 (150 mg), 18 ꢁC.
Fig. 2. Influence of water content on the nuclease p1 catalyzed asymmetric aldol re-
actions of isatins and cyclic ketones. Conditions: isatin (0.2 mmol), cyclohexanone
(1 mmol), acetonitrile (1 mL), enzyme (70 mg), 18 ꢁC, 120 h.
3.5. The reaction time course of the asymmetric aldol reactions of
isatins and cyclic ketones
(Table 2, entry 5), the best activities were observed in DMSO and
DMF but with very poor enantioselectivity (Table 2, entries 1 and
2). Generally, strong polar solvent like DMSO and DMF had big
influence on the tertiary structure of enzymes, this may result in
poor enantioselectivity. Nevertheless, no product was observed in
toluene (Table 2, entry6), this was probably because of low solu-
bility of isatin in toluene. And nuclease p1 showed no activity to
this aldol reaction in THF (Table 2, entry7). Next, some controlled
experiments were evaluated to verify the catalytic specificity of
nuclease p1 on the aldol reaction of isatin and cyclohexanone.
Because nuclease p1 is a zinc-dependent enzyme, EDTA (ethylene
diamine tetraacetic acid) was used to destroy the active site and
denature the enzyme. As we expected, no products were observed
with denatured nuclease p1 or in the absence of enzyme (Table 2,
entries 8 and 9). These results suggested that the tertiary structure
of this nuclease p1 was essential for the reaction.
Finally, we studied the reaction time course of the aldol addition
between isatin and cyclohexanone under solvent-free condition. It
could be seen from Fig. 4, the ee value almost kept constant from
63% to 69% during the whole reaction phase. Nevertheless, the
yields had an obvious increase as the reaction time went by, and
reached the platform after 96 h. This meant that substrates quickly
entered the active site of enzyme and fitted in it, but product
releasing was slow, the reaction completed thoroughly after 4 days.
3.6. Scope and limitation of the enzymatic asymmetric aldol
reactions of isatins and cyclic ketones
With the optimal reaction conditions in hand, we studied the
substrate scope and the generality of the enzymatic asymmetric
aldol reactions of isatins and cyclic ketones, various isatin de-
rivatives and cyclic ketones were investigated. As shown in Table 3,
a wide range of substrates could participate in the reaction to form
3-hydroxyindolin-2-ones in moderate to excellent yields. The
3.3. The influence of water content on the asymmetric aldol
reactions of isatins and cyclic ketones
The role of water is crucial to the enzyme, not only it affects the
enantioselectivity and activity of enzymes, but it acts as “molecular
lubricant” to change the conformational flexibility [55e61].
Therefore, it is very necessary to confirm the optimum water con-
tent in this system. A series of water contents was inspected in
nuclease p1-catalyzed asymmetric aldol reaction in 1 mL MeCN. As
shown in Fig. 2, the yield and ee value were considerably affected by
the water content. Almost no reaction proceeded when the water
content was less than 0.05 (Vwater/VMeCN), which fully illustrated
the vital function of water in this enzymatic reaction. Nuclease p1
simultaneously exhibited the best enantioselectivity (63%) and
yield (18%) at water content 0.15 (Vwater/VMeCN) respectively. As the
water content increased, both the yield and ee value obviously
dropped. Therefore, water content 0.15 (Vwater/VMeCN) was chosen
for the following optimization. We then examined the impact of
enzyme loading on the aldol reaction of isatin (0.2 mmol) with
cyclohexanone (1 mmol) under the water content 0.15 (Vwater
/
VMeCN). When the amount of enzyme increased to 150 mg, a major
enhancement in yield was observed from 18% to 34%. Further in-
crease had no effect on the asymmetric aldol reaction in ee and
yield. So we chose the enzyme loading of 150 mg for the further
studies.
Fig. 3. Influence of cyclohexanone amount on the nuclease p1 catalyzed asymmetric
aldol reaction. Conditions: isatin (0.2 mmol), DI (0.15 mL), enzyme (150 mg), 18 ꢁC,
120 h.
Please cite this article in press as: Z.-Q. Liu, et al., Enzymatic enantioselective aldol reactions of isatin derivatives with cyclic ketones under