3.1 Preparation for appropriate catalysts
In 1909, Dakin found that primary amino acids can catalyze Knoevenagel
condensation (DAKIN, 2009). The fact that bovine serum albumin (BSA),
which slightly catalyzed the condensation between benzaldehyde and ethyl
cyanoacetate in water phase (W. Li et al., 2015) encouraged us to
investigate other enzymes which may also possess the ability to promote
Knoevenagel condensation. To test the idea, the enzymes NerA (CAA74280),
OYE2.6 (XP_001384055), IPR
(Q6WAU1), GDH (WP_003246720), and ADH4 (XP_001387122)
were chosen to compare with glycine (Chaudhry et al., 2016), proline,
and lysine (Y. Wang, Shang, Wu, Fan, & Chen, 2006; Y. Zhang, Sun,
Liang, & Shang, 2010), as catalysts in the model reaction (Figure S3).
To date, it hasn’t been recorded in the literature that these enzymes
have a direct role in Knoevenagel condensation and natural amino acids
can promote the reaction obviously. For the pretest, ethyl acetoacetate
(EAA) and valeraldehyde 1b were used as substrates. As shown in
Table S1, both enzymes and amino acids were able to efficiently promote
Knoevenagel condensation in neat aqueous solvent. Interestingly, the
yield of the reaction was not significantly affected by the species of
enzymes (Table S1, list 4-8), but were positively correlated with the
amount of catalyst (Table S1, list 4-8 and 9-13).
According to the literature (W. Li et al., 2015), lipases like PPL, MJL,
YILip2 can also promote Knoevenagel condensation in a water-ethanol
solvent system. 10 mg mL-1 enzymes and 96.7% ethanol
by volume were employed to catalyze the reaction efficiently.
Interestingly, unspecific reaction in lipase-catalyzed Knoevenagel
condensation was also observed in addition to specific reaction. Our
work demonstrated that several enzymes could catalyze the condensation,
with the exception of lipases. 2.4 mg mL-1 enzymes in
neat aqueous solvent maintained the ability to catalyze Knoevenagel
condensation. On the basis of the mechanism as reported by Knoevenagel,
we thought that the hydrophilic
amino acids on the surface of enzymes were associated with the formation
of a Schiff base with aldehyde substrates, before the obtained imine
intermediates condensed with carbanions (Zheng, Li, Tao, & Zhang,
2019).
The Knoevenagel condensation catalyzed by a certain concentration of
enzymes is comparable to that of the amino acids, which inspired us to
consider that a low catalytic turnover would be further promoted if the
condensation products could be converted by the same enzyme.
Ene-reductases, which catalyze the reduction of α, β-unsaturated alkene,
have highlighted the potential reduction of Knoevenagel condensation
products. Subsequently, we tried to construct the tandem reaction using
single ER which catalyzes both
Knoevenagel condensation andin situ reduction of the condensation products to obtain more
stable and valuable products. Several ERs were investigated to ascertain
which one can reduce intermediates 2a -k . Following a
review of the literature, three of ERs (NerA, OYE2.6 and IPR) were
cloned and expressed (Oberdorfer et al., 2013; B. Zhang, Zheng, Lin, &
Wei, 2016). The reduction activity of each catalyst towards
intermediates 2a-k was then tested, shown in Figure 1, except
that IPR had no obvious catalytic activity for any of the products, the
other two reductases had wide substrate ranges. In particular, NerA
presented a broader range of substrate species and higher activity on
nearly all the products we were interested in. It’s worth noting that
the products derived from the condensation of benzaldehyde (2f )
and furan aldehyde (2h , 2i ) with EAA were reduced
effectively. These bulky substrates are often difficult to reduce using
other ERs (Peters, Frasson, Sievers, & Buller, 2019). Phenyl group
bound with the electron withdrawing groups (2g ) was reduced
faster than 2f , which may facilitate hydrogen transfer between
olefin and reduced flavin cofactor (de Paula, Zampieri, Nasário,
Rodrigues, & Moran, 2017). OYE2.6 also had a wide reduction capacity,
but the relative activity on all substrates was at least 40% lower than
that of NerA. OYE2.6 had relatively low reduction activities, especially
towards the large size compounds (2g-2k ), which may attribute
to the smaller activity centre (Oberdorfer et al., 2013; B. Zhang,
Zheng, Lin, & Wei, 2016). In addition, NerA takes NADH as a co-factor
while the co-factor of OYE2.6 is NADPH. Considering economy and
usability, NerA was chosen for the one-pot reaction presented here.