Generation and Application of Homoallylic α,α-Diboryl Radicals via Diboron-Promoted Ring-Opening of Vinyl Cyclopropanes: cis-Diastereoselective Borylative Cycloaddition.

Carbon-centered radicals stabilized by adjacent boron atoms are underexplored reaction intermediates in organic synthesis. This study reports the development of vinyl cyclopropyl diborons (VCPDBs) as a versatile source of previously unknown homoallylic α,α-diboryl radicals via thiyl radical catalyzed diboron-directed ring opening. These diboryl stabilized radicals underwent smooth [3+2] cycloaddition with a variety of olefins to provide diboryl cyclopentanes in good to excellent diastereoselectivity. In contrast to the trans-diastereoselectivity observed with most of the dicarbonyl activated VCPs, the cycloaddition of VCPDBs showed a remarkable preference for formation of cis-cyclopentane diastereomer which was confirmed by quantitative NOE and 2D NOESY studies. The cis-stereochemistry of cyclopentane products enabled a concise intramolecular Heck reaction approach to rare tricyclic cyclopentanoid framework containing the diboron group. The mild reaction conditions also allowed a one-pot VCP ring-opening, cycloaddition-oxidation sequence to afford disubstituted cyclopentanones. Control experiments and DFT analysis of reaction mechanism support a radical mediated pathway and provide a rationale for the observed diastereoselectivity. To the authors' knowledge, these are the first examples of the use of geminal diboryl group as an activator of VCP ring opening and cycloaddition reaction of α-boryl radicals.

Alpaca single B cell interrogation and heavy-chain-only antibody discovery on an optofluidic platform.

In vivo VHH discovery approaches have been limited by the lack of methodologies for camelid B cell interrogation. Here, we report a novel application of the Beacon® optofluidic platform to the discovery of heavy-chain-only antibodies by screening alpaca B cells. Methods for alpaca B cell enrichment, culture, IgG2/3 detection, and sequencing were developed and used to discover target-specific VHH from an alpaca immunized with prostate-specific membrane antigen (PSMA) or a second target. PSMA-specific hits were expressed as VHH-Fc and characterized using label-free techniques. Anti-PSMA IgG2/3 titer plateaued on day 153, when on-Beacon IgG2/3 secretion and target binding rates peaked. Of 13 recombinantly expressed VHH-Fc, all but one bound with nanomolar affinity, and five were successfully humanized. Repertoire sequencing uncovered additional variants within the clonal lineages of the validated hits. The establishment of this workflow extends the powerful Beacon technology to enable rapid VHH discovery directly from natural camelid immune repertoires.

Evaluating the solubility of compounds intended for intramammary infusion based upon tests conducted across a range of milk matrices.

The ability to evaluate drug solubility in milk and milk-related products has relevance both to human and veterinary medicine. Model compounds explored in a previous investigation focused on drug solubility assessments when delivered in milk-associated vehicles for administration to human patients. In the current investigation, we focus on the solubility of drugs intended for delivery via intramammary infusion to cattle. Because there are logistic challenges typically associated with obtaining raw milk samples for these tests, there is a need to determine potential alternative media as a substitute for raw bovine milk. Given the complexity of the milk matrix, aqueous media do not reflect the range of factors that could impact these solubility assessments. This led to the current effort to explore the magnitude of differences that might occur when substituting raw bovine milk with off-the-shelf milk products such as whole milk, skim milk, or reconstituted whole milk powder. We considered conclusions based upon the solubility assessments derived from the use of the model compounds studied in our previous report and compared them to conclusions obtained when testing two drugs with differing physicochemical characteristics that are approved for administration via bovine intramammary infusion: cephapirin benzathine and cephapirin sodium. Based upon these results, we recommend that whole milk or reconstituted whole milk can substitute for bovine raw milk for the solubility assessment of compounds intended for administration via intramammary infusion. However, unlike the human drug situation, these tests should be conducted at 38°C.

Accelerated antibody discovery targeting the SARS-CoV-2 spike protein for COVID-19 therapeutic potential.

BACKGROUND: Rapid deployment of technologies capable of high-throughput and high-resolution screening is imperative for timely response to viral outbreaks. Risk mitigation in the form of leveraging multiple advanced technologies further increases the likelihood of identifying efficacious treatments in aggressive timelines. METHODS: In this study, we describe two parallel, yet distinct, in vivo approaches for accelerated discovery of antibodies targeting the severe acute respiratory syndrome coronavirus-2 spike protein. Working with human transgenic Alloy-GK mice, we detail a single B-cell discovery workflow to directly interrogate antibodies secreted from plasma cells for binding specificity and ACE2 receptor blocking activity. Additionally, we describe a concurrent accelerated hybridoma-based workflow utilizing a DiversimAb™ mouse model for increased diversity. RESULTS: The panel of antibodies isolated from both workflows revealed binding to distinct epitopes with both blocking and non-blocking profiles. Sequence analysis of the resulting lead candidates uncovered additional diversity with the opportunity for straightforward engineering and affinity maturation. CONCLUSIONS: By combining in vivo models with advanced integration of screening and selection platforms, lead antibody candidates can be sequenced and fully characterized within one to three months.