Automation and Microfluidics for the Efficient, Fast, and Focused Reaction Development of Asymmetric Hydrogenation Catalysis.

Automation and microfluidic tools potentially enable efficient, fast, and focused reaction development of complex chemistries, while minimizing resource- and material consumption. The introduction of automation-assisted workflows will contribute to the more sustainable development and scale-up of new and improved catalytic technologies. Herein, the application of automation and microfluidics to the development of a complex asymmetric hydrogenation reaction is described. Screening and optimization experiments were performed using an automated microfluidic platform, which enabled a drastic reduction in the material consumption compared to conventional laboratory practices. A suitable catalytic system was identified from a library of RuII -diamino precatalysts. In situ precatalyst activation was studied with 1 H/31 P nuclear magnetic resonance (NMR), and the reaction was scaled up to multigram quantities in a batch autoclave. These reactions were monitored using an automated liquid-phase sampling system. Ultimately, in less than a week of total experimental time, multigram quantities of the target enantiopure alcohol product were provided by this automation-assisted approach.

Synthesis of 4-Azido-N-acetylhexosamine Uridine Diphosphate Donors: Clickable Glycosaminoglycans.

Unnatural chemically modified nucleotide sugars UDP-4-N3-GlcNAc and UDP-4-N3-GalNAc were chemically synthesized for the first time. These unnatural UDP sugar products were then tested for incorporation into hyaluronan, heparosan, or chondroitin using polysaccharide synthases. UDP-4-N3-GlcNAc served as a chain termination substrate for hyaluronan or heparosan synthases; the resulting 4-N3-GlcNAc-terminated hyaluronan and heparosan were then successfully conjugated with Alexa Fluor 488 DIBO alkyne, demonstrating that this approach is generally applicable for labeling and detection of suitable glycosaminoglycans.

Chemoenzymatic Synthesis of 4-Fluoro-N-Acetylhexosamine Uridine Diphosphate Donors: Chain Terminators in Glycosaminoglycan Synthesis.

Unnatural uridine diphosphate (UDP)-sugar donors, UDP-4-deoxy-4-fluoro-N-acetylglucosamine (4FGlcNAc) and UDP-4-deoxy-4-fluoro-N-acetylgalactosamine (4FGalNAc), were prepared using both chemical and chemoenzymatic syntheses relying on N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU). The resulting unnatural UDP-sugar donors were then tested as substrates in glycosaminoglycan synthesis catalyzed by various synthases. UDP-4FGlcNAc was transferred onto an acceptor by Pastuerella multocida heparosan synthase 1 and subsequently served as a chain terminator.

ePathOptimize: A Combinatorial Approach for Transcriptional Balancing of Metabolic Pathways.

The ability to fine tune gene expression has created the field of metabolic pathway optimization and balancing where a variety of factors affecting flux balance are carefully modulated to improve product titers, yields, and productivity. Using a library of isopropyl ß-D-1-thiogalactopyranoside (IPTG)-inducible mutant T7 promoters of varied strength a combinatorial method was developed for transcriptional balancing of the violacein pathway. Violacein biosynthesis involves a complex five-gene pathway that is an excellent model for exploratory metabolic engineering efforts into pathway regulation and control due to many colorful intermediates and side products allowing for easy analysis and strain comparison. Upon screening approximately 4% of the total initial library, several high-titer mutants were discovered that resulted in up to a 63-fold improvement over the control strain. With further fermentation optimization, titers were improved to 1829 ± 46 mg/L; a 2.6-fold improvement in titer and a 30-fold improvement in productivity from previous literature reports.