3.2 Investigation of the key reaction parameters
The compatibility of all involved catalysts and their reaction
conditions, especially the solvent, is a critical factor that requires a
compatibility window that is often difficult to meet in one pot
(Gómez Baraibar et al., 2016). To construct the tandem reaction, each
sub-step was pre-tested. The kinetics of Knoevenagel condensation in
H2O was investigated. The highest concentration of1b was set as 50 mM due to the tolerance of the enzymes. As
shown in Figure 2 and Table S2,
the
condensation efficiency markedly accelerated with increasing the
concentration of 1b in first 1 hours, and significantly slowed
down and reached equilibrium in 2 hours, due to a decrease in substrate
concentration and reversibility of the
Knoevenagel
condensation. In addition, another possible reason may be because
1,3-diketones tend to form stable six membered cyclic enols resulting in
lower reactivity in water (Hu, Guan, Deng, & He, 2012). To solve this
problem, we considered the solvent in this system. Traditionally,
Knoevenagel condensation is always conducted in DMF and DMSO because
aprotic solvents may lead to the deprotonation of the active hydrogen
substrate, which is why water is seemed as negative factor (Siebenhaar,
Casagrande, Studer, & Blaser, 2001). It was understood that all enzymes
need essential bound water, and that enzymatic activity in organic
solvents depends on water content (C.-H. Wang, Guan, & He, 2011). Thus,
we selected an organic solvent optimized by mixing with water to promote
Knoevenagel condensation. As expected, the species of the solvent
affected the yield obviously (Figure S1 and S2). 30% DMSO promoted the
yield up to 35%, in contrast to the yield of only 20% in water. This
shows that deprotonation is indeed vital in a reaction that is more
facile in non-protonic solvents, as expected according to the mechanism
of Knoevenagel condensation. Meanwhile, compared to classical
Knoevenagel condensation in DMF combined with a molecular sieve, this
sub-step presented a very low yield due to aqueous environment (C.-J. Li
& Chen, 2006).
Although the condensation process is significantly different in presence
and absence of DMSO, the gap may be narrowed when the condensation and
reduction are synergistic. Firstly, the high cost of NADH requiresin situ regeneration, as a common cofactor cycling system, GDH
was selected. 1b and EAA were chosen as substrates to conduct
tandem reactions. As shown in Figure 3A, the addition of GDH greatly
increases the yield. But 3b was not synthesized in GDH alone,
while GDH promotes the Knoevenagel
condensation. Meanwhile, all available NADH were used and 8% yield was
obtained in NerA alone, indicating that NerA may accomplish the
condensation of 1b and EAA and reduction of 2bindependently. To verify this conclusion, the reaction with NerA or GDH
on their own were conducted, shown in Table S3. It was found that NerA
on its own was able to facilitate the reaction, achieving a yield of
84%.
Then, the yield was determined in both water and 30% DMSO. As shown in
Figure 3B, the yield in 30% DMSO achieved 53% in 1 hour, whereas only
reached 36% in aqueous solvent. However, we noticed that the product3b grows linearly in both systems (Figure 3B), prolonging the
reaction time and improving the loading of enzymes may compensate for
the loss of yield due to the removal of organic solvent. To test the
idea, the same amount of NerA (2.4 mg mL-1) was added
in the aqueous phase, along with 30% DMSO and the reaction time was
extended to reach equilibrium. As shown in Figure 3C, the yield in water
reached 89% in 4 hours, still lower than that after 3 hours in 30%
DMSO (90%). There was almost no residue of 2b in water, and
the yield was the same as in 30% DMSO at 5 hours, so that condensation
is a limiting step in water. In fact, some ERs, such as YersER, tend to
reduce the E -isomer and the isomerization is required to reduce
the isomeric mixture (Litman, Wang, Zhao, & Hartwig, 2018). Both theE and Z isomers formed by condensation of aldehydes with
1, 3-dicarbonyl compounds in this reaction can be effectively reduced by
NerA.
Molecular
docking experiments showed that carbonyl oxygen atom in either of the
acetyl or carboxylic acid group of 2b can form hydrogen bonds
with H178 and N181 in catalytic pocket, and the side chain extends into
the hydrophobic cavity formed by Y65 and Y356 (Figure 4). Therefore, the
unsaturated double bond is correctly fixed and efficiently reduced.
Next, 3 mg mL-1 NerA was added with 1b and
EAA in aqueous solvent, as shown in Figure 3D, equilibrium was achieved
in 3 hours in both systems. The yield in water was comparable with that
of 30% DMSO (90% vs. 91%, respectively) while 30% DMSO encumbered
the separation and purification of the products. In addition, using
water as the medium has received considerable attention in organic
synthesis due to its economic, environmental, safety, and other
advantages (Patel, Sharma, & Jasra, 2008). Based on these results, a
neat aqueous solvent was chosen.
Next, the dosage of NerA and the molar ratio of substrates were tested
to maximize the final yield. Shown in Table 1, the yield was promoted by
the addition of NerA. Furthermore, Figures 3C and 3D also showed that
when the amount of NerA was increased to 3 mg mL-1,
the formation of 3b was significantly accelerated compared with
2.4 mg mL-1, and more 2b accumulated in the
first hour. Indicating that the increase of enzymes obviously promotes
the condensation and reduction. However, 3 mg mL-1 of
NerA is sufficient, since the yield of 3b is similar to that of
3.6 mg mL-1. To further optimize the reaction, we
observed that the yield was maximized when the substrate molar ratio was
1:2. Finally, 3 mg mL-1 NerA and a substrate molar
ratio of 1:2 were used.