A Design of Experiment (DoE) and kinetic screening study was carried out using an automated reaction screening platform, and as a case study, an early stage in the synthesis of a late phase developmental candidate was investigated. Key impurities were tracked and kinetically modeled, and significant factors impacting impurity formation were identified. In particular, factors that influence the formation of the diastereomer 4, a precursor to an API impurity identified as a Critical Quality Attribute (CQA), were identified and optimized to minimize its formation. Acetic acid, methanesulfonic acid, volumes of solvent, amino alcohol, and reaction B temperature were observed to be the most significant factors along with a factor interaction between methanesulfonic acid and the reaction B temperature. From the experimental data, diastereomer levels of 2.5–5.4 mol % were observed and a kinetic model was developed around the diastereomer formation. Good agreement between the model and experimental data gave confidence in understanding the contributing factors of diastereomer generation, and enabled confirmation of process parameter recommendations to support risk assessments and Quality by Design (QbD) activities. In total, automation provided a 4–5 times savings in FTE hours over a manual process when conducting these experiments and greatly accelerated the generation of supporting information for a drug file.

Real-time monitoring of gene expression dynamics and population levels in a multispecies microbial community could enable the study of the role of changing gene expression patterns on eco-evolutionary outcomes. Here we report the design and validation of a unique experimental platform with an in situ fluorescence measurement system that has high dynamic range and temporal resolution and is capable of monitoring multiple fluorophores for long-term gene expression and population dynamics experiments. We demonstrate the capability of our system to capture gene expression dynamics in response to external perturbations in two synthetic genetic systems: a simple inducible genetic circuit and a bistable toggle switch. Finally, in exploring the population dynamics of a two species microbial community, we show that our system can capture the switch between competitive exclusion and long-term coexistence in response to different nutrient conditions.

High frequencies of mutant mitochondrial DNA (mtDNA) in human cells lead to cellular defects that are associated with aging and disease. Yet much remains to be understood about the dynamics of the generation of mutant mtDNAs and their relative replicative fitness that informs their fate within cells and tissues. To address this, we utilize long-read single-molecule sequencing to track mutational trajectories of mtDNA in the model organism Saccharomyces cerevisiae. This model has numerous advantages over mammalian systems due to its much larger mtDNA and ease of artificially competing mutant and wild-type mtDNA copies in cells. We show a previously unseen pattern that constrains subsequent excision events in mtDNA fragmentation in yeast. We also provide evidence for the generation of rare and contentious non-periodic mtDNA structures that lead to persistent diversity within individual cells. Finally, we show that measurements of relative fitness of mtDNA fit a phenomenological model that highlights important biophysical parameters governing mtDNA fitness. Altogether, our study provides techniques and insights into the dynamics of large structural changes in genomes that we show are applicable to more complex organisms like humans.

The Pd-catalyzed carbonylation of cyclic β-chloro enones using simple phosphine ligands is described. Screening identified P(Me)(t-Bu) 2 as the most general ligand for an array of chloro enone electrophiles. The reaction scope has been evaluated on a milligram scale across 80 examples, with excellent reactivity observed in nearly every case. Carbonylation can be achieved even in the presence of potentially sensitive or inhibitory functional groups, including basic nitrogens as well as aryl chlorides or bromides. Twenty examples have been run on a gram scale, demonstrating scalability and practical utility. Using P(Me)(t-Bu) 2 , the reaction rate depends on both nucleophile and electrophile identity, with completion times varying between 3 and >18 h under a standard set of conditions. Switching to P(t-Bu) 3 for the carbonylation of 3-chlorocyclohex-2-enone with methanol results in a dramatic rate increase, enabling effective catalysis with kinetics consistent with rate-limiting mass transfer. Stoichiometric oxidative addition of 3-chlorocyclohex-2-enone and 3-oxocyclohex-1enecarbonyl chloride to both Pd[P(t-Bu) 3 ] 2 and Pd(PCy 3 ) 2 has enabled characterization and isolation of several potential catalytic intermediates, including Pd−vinyl and Pd−acyl species supported by P(t-Bu) 3 and PCy 3 ligands. Monitoring the oxidative addition of 3-chlorocyclohex-2-enone to Pd(PCy 3 ) 2 by NMR spectroscopy indicates that coordination of the alkene precedes oxidative addition. As a result of these studies, methyl 3-oxocyclohex-1-enecarboxylate has been synthesized via Pd-catalyzed carbonylation of 3-chlorocyclohex-2-enone in 90% yield on a 60 g scale with only 0.5 mol % catalyst loading.

Eukaryotic cells contain numerous copies of mitochondrial DNA (mtDNA), allowing for the coexistence of mutant and wild-type mtDNA in individual cells. The fate of mutant mtDNA depends on their relative replicative fitness within cells and the resulting cellular fitness within populations of cells. Yet the dynamics of the generation of mutant mtDNA and features that inform their fitness remain unaddressed. Here we utilize long read single-molecule sequencing to track mtDNA mutational trajectories in Saccharomyces cerevisiae. We show a previously unseen pattern that constrains subsequent excision events in mtDNA fragmentation. We also provide evidence for the generation of rare and contentious non-periodic mtDNA structures that lead to persistent diversity within individual cells. Finally, we show that measurements of relative fitness of mtDNA fit a phenomenological model that highlights important biophysical parameters governing mtDNA fitness. Altogether, our study provides techniques and insights into the dynamics of large structural changes in genomes that may be applicable in more complex organisms.