The development of improved cryopreservative materials is necessary to enable complete recovery of living cells and tissue after frozen storage. Remarkably, poly(vinyl alcohol) (PVA) displays some of the same cryoprotective properties as many antifreeze proteins found in cold tolerant organisms. In particular, PVA is very effective at halting the Ostwald ripening of ice, a process that mechanically damages cells and tissue. Despite the large practical importance of such a property, the mechanism by which PVA interacts with ice is poorly understood, hindering the development of improved cryoprotective materials. Herein, we quantitatively evaluated ice growth kinetics in the presence of PVA at different pH conditions and in the presence of a range of neutral salts. We demonstrated that pH, but not salt identity, alters the ability of PVA to halt ice grain coarsening. These observations are consistent with hydrogen-bonding playing a crucial role in PVA-mediated ice recrystallization inhibition. The evolution of the size distribution of ice crystals with annealing was consistent with incomplete surface coverage of ice with PVA. Binding assay measurements of dissolved fluorescently labeled PVA in an ice slurry showed that PVA interacts with ice through weak adsorption (<9%) to the ice crystal surface, which stands in contrast to fluorescently tagged type III antifreeze peptide, which binds strongly (ca. 64%) under the same conditions.

Recent studies have demonstrated that effective protein production requires coordination of multiple cotranslational cellular processes, which are heavily affected by translation timing. Until recently, protein engineering has focused on codon optimization to maximize protein production rates, mostly considering the effect of tRNA abundance. However, as it relates to complex multidomain proteins, it has been hypothesized that strategic translational pauses between domains and between distinct individual structural motifs can prevent interactions between nascent chain fragments that generate kinetically trapped misfolded peptides and thereby enhance protein yields. In this study, we introduce synthetic transient pauses between structural domains in a heterologous model protein based on designed patterns of affinity between the mRNA and the anti-Shine-Dalgarno (aSD) sequence on the ribosome. We demonstrate that optimizing translation attenuation at domain boundaries can predictably affect solubility patterns in bacteria. Exploration of the affinity space showed that modifying less than 1% of the nucleotides (on a small 12 amino acid linker) can vary soluble protein yields up to ∼7-fold without altering the primary sequence of the protein. In the context of longer linkers, where a larger number of distinct structural motifs can fold outside the ribosome, optimal synonymous codon variations resulted in an additional 2.1-fold increase in solubility, relative to that of nonoptimized linkers of the same length. While rational construction of 54 linkers of various affinities showed a significant correlation between protein solubility and predicted affinity, only weaker correlations were observed between tRNA abundance and protein solubility. We also demonstrate that naturally occurring high-affinity clusters are present between structural domains of β-galactosidase, one of Escherichia coli's largest native proteins. Interdomain ribosomal affinity is an important factor that has not previously been explored in the context of protein engineering.

Herein we demonstrate ap acked bed flow reactor capable of achieving highly regio-and stereoselective CÀH functionalization reactions using an ewly developed Rh 2 (S-2-Cl-5-CF 3 TPCP) 4 catalyst. To optimize the immobilized dirhodium catalyst employed in the flowr eactor,w es ystematically study both (i)t he effects of ligand immobilization position, demonstrating the critical factor that the catalyst-support attachment location can have on the catalyst performance, and (ii)s ilica support mesopore length, demonstrating that decreasing diffusional limitations leads to increased accessibility of the active site and higher catalyst turnover frequency. We employt he immobilized dirhodium catalyst in as imple packed bed flow reactor achieving comparable yields and levels of enantioselectivity to the homogeneous catalyst employed in batch and maintain this performance over ten catalyst recycles.

A tandem system comprising in-line diazo compound synthesis and downstream consumption in a rhodium-catalyzed cyclopropanation reaction has been developed. Passing hydrazone through a silica column absorbed with Cu­(OAc)2-H2O/N,N-dimethylaminopyridine oxidized the hydrazone to generate an aryldiazoacetate in flow. The crude aryldiazoacetate elutes from this column directly into a downstream cyclopropanation reaction, catalyzed by the chiral dirhodium tetracarboxylates, Rh2(R-p-Ph-TPCP)4 and Rh2(R-PTAD)4. This convenient flow to batch method was applied to the synthesis of a range of 1,2-diarylcyclopropane-1-carboxylates in high yields and with high levels of enantioselectivity.

Copper-catalyzed, aerobic hydrazone oxidation to produce synthetically useful diazo compounds is achieved in a continuous, three-phase flow reactor with complete conversion and high selectivity by adapting a previously published batch methodology to a flow system described herein and conducting a parameter space exploration to optimize the process conditions. The robust nature of the process is demonstrated, with complete hydrazone conversion and a 90% steady-state diazo compound selectivity maintained over 11 residence times. Employing the diazo synthesis upstream of a limited scope of dirhodium(II)catalyzed carbene reactions demonstrates the utility of this process in generating on-demand diazo compounds for cycloaddition and activated secondary C−H insertion reactions. The resulting process represents, to the best of our knowledge, the first reported catalytic process for hydrazone oxidation in flow. Additionally, this process, which only generates water as an oxidative byproduct, is an improvement over previous noncatalytic attempts to synthesize diazo compounds in flow, which generate stoichiometric amounts of waste. The increased sustainability and mitigation of safety hazards associated with handling reactive diazo compounds may make this approach suitable for practical applications.