Page 231 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

9.2 Enzyme-Catalyzed Stereoselective Reactions in Continuous-Flow Systems 207

of cell lysate. Results from the microscale experiments were fully consistent with
those obtained from the stirred batch reactor experiments.
A two-step enantioselective synthesis of 2-amino-1,3,4-butanetriol 12 was per-
formed in continuous mode using two serial capillary microreactors with His -
6
tagged transketolase (TK)/ω-transaminase (TAM) bound to the wall via immobilized
Ni-nitrilotriacetic acid (Ni-NTA) complex [78]. The TK-catalyzed conversion of
hydroxypyruvate 8 and glycolaldehyde 9 to l-erythrulose 10 followed by the
TAM-catalyzed amination resulted in the formation of the product. This work
demonstrated the implementation of a dual enzyme microreactor system for the
evaluation of a de novo pathway for an enzyme-catalyzed synthesis.

9.2.2
Stereoselective Processes Catalyzed by Hydrolases

Hydrolases − especially lipases − proved to be versatile biocatalysts for synthetic
biotransformations [79, 80]. The vast majority of the enzymatic stereoselective
processes have been performed so far in batch mode [29, 30, 81]. Very recently,
a review appeared on lipase-catalyzed reactions under continuous-flow conditions
[82], and here we extend this overview with an analysis of the range of selectivities,
effects of reaction conditions and the mode of enzyme immobilization on the
lipase, and in general hydrolase-catalyzed continuous-flow biotransformations.

9.2.2.1 Applicable Types of Selectivities
Enzymes – and thus hydrolases – can realize all kinds of selectivities such as
chemo-, regio-, diastereomer and diastereotopic selectivity, as well as enantiomer
and enantiotopic selectivity [83]. Accordingly, lipases were applied in all possible
kinds of stereoselective biotransformations [29, 30, 79, 81, 83] such as KR [79,
84], deracemization, and dynamic kinetic resolution (DKR) [85]. In this review, we
wish to concentrate on methods enabling the continuous-mode hydrolase-mediated
production of compounds in high enantiomeric purity.

9.2.2.2 Stereoselective Hydrolytic Reactions
Systems for performing hydrolytic reactions were often not real flow-through
systems but contained continuously operated elements with a certain kind of
recirculation.
For example, the enantiomer selective hydrolysis of racemic 3-methylthietan-
2-one was performed in organic media in PBR filled with Pseudomonas cepacia
lipase (PcL) immobilized on Celite to produce enantiopure (R)-3-mercapto-2-
methylpropanoic acid [86]. The product inhibition was successfully overcome by
incorporating an aqueous extraction unit to give the product in 40% yield with
99% ee.
A continuous-flow closed-loop PBR packed with Candida rugosa lipase (CrL) on
Amberlite XAD-7 was applied for enantiomer selective hydrolysis of the racemic
naproxen ethoxyethyl ester to enantiopure (S)-(+)-naproxen [87] on a kilogram scale.