Multistep Continuous Flow Transformations Using Vortex Fluidics

Author: Joshua Britton

Britton, Joshua, 2016 Multistep Continuous Flow Transformations Using Vortex Fluidics, Flinders University, School of Chemical and Physical Sciences

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Nature pieces together complex natural products through enzyme-based assembly lines. These highly

ordered multi-domain proteins perform iterative transformations to a transferable motif, with each protein in

the pathway conducting a specific transformation. As an analogous in vitro approach, multistep continuous

flow systems mimic nature to create organic compounds in the laboratory. Although conventional

microfluidic systems are well studied, their thin film counterparts are relatively new, and have not been

considered for multistep syntheses. Here, the vortex fluidic device (VFD) mediates multistep continuous

flow transformations in thin films. Both conventional organic techniques and biocatalysis were explored.

Both of these approaches afforded efficient multistep systems, displaying the potential for this technology.

The first approach revolved around traditional organic chemistry being confined to a continuous flow thin

film. All operational parameters associated with VFD-mediated continuous flow synthesis were optimized

and explored. The VFD offers a range of benefits over reactions in round bottom flasks. Micromixing,

vibrational contributions, byproduct removal and in situ solvent exchange enhance reaction rates and yields.

Micromixing, for example, improved biodiesel generation by allowing a room temperature continuous flow

system that decreased reagent and catalyst usage with a concomitant shorter reaction time. Furthermore, thesynthesis of an α-aminophosphonate and lidocaine constituted the first examples of multistep reactivity inthin films. Both approaches used a single reactor to create a molecular assembly line synthesis where

intermediate compounds are passed along well-defined reactions zones. These syntheses utilized in situ

solvent exchanges to mediate each step of the reaction sequence in its ideal solvent. This approach can

increase reaction rates whilst providing reaction flexibility in multistep syntheses.

The second approach progressed continuous flow biocatalysis. Initial studies discovered that vibrations

native to the VFD can drive enzymatic catalysis within thin films. In pursuing continuous flow reactivity,

two protein immobilization techniques were developed. The first explored non-specific immobilization via

an APTES treated reactor surface. This method requires minute quantities of protein, is generalizable, and

showed stability for ten hours of processing. The second immobilization strategy revolved around

immobilized metal affinity chromatography. A silica based resin attached proteins to the reactor surface

through polyhistidine tag interactions. This allowed distinct enzyme zones to be created on the reactor

surface for exploration of multistep enzymatic transformations in continuous flow. Furthermore, protein

purification and immobilization can be achieved simultaneously in only ten minutes from complex cell

lysate. This rapid technique allows biochemical pathways to be imprinted on the reactor surface to explore

continuous flow enzymatic syntheses, limited only by imagination.

Overall, multistep continuous flow chemistry in thin films has been developed using both conventional

organic chemistry and biochemical techniques. In developing these concepts, VFD-mediated single and

multistep transformations, biodiesel generation, protein purification, enzyme acceleration, and protein

immobilization were explored and optimized. Multistep continuous flow chemistry has the opportunity to

improve compound generation for this planet, and in remote locations, such as space. The results disclosedwithin this thesis lay the foundation for pursuing such aspirations.

Keywords: Vortex Fluidics, Flow Chemistry, Biocatalysis, Organic Synthesis, Multi-step Synthesis

Subject: Chemistry thesis

Thesis type: Doctor of Philosophy
Completed: 2016
School: School of Chemical and Physical Sciences
Supervisor: Colin L. Raston