The palladium-catalyzed Tsuji–Trost reaction has been extensively studied under synthetically relevant conditions (millimolar concentrations of substrates and catalyst, aprotic solvents, no additives). Despite the increasing use of the Tsuji–Trost reaction in other areas (e.g., chemical biology), the paucity of kinetic studies at micromolar concentrations of substrates in water has impeded progress. Herein, we show that a fluorescence-based high-throughput method provided massive Eyring plot data and revealed three kinetic regimes. The associated turnover-limiting steps (TLSs) were assigned as the oxidative addition (regime 1; ΔH ⧧ > 0), nucleophilic attack (regime 2; ΔH ⧧ ≈ 0), and association (regime 3; ΔH ⧧ < 0, inverse temperature dependence). A kinetic profile under particular conditions depended on the substrate concentration and reaction temperature. Density functional theory calculations supported these findings. This work indicates that a TLS under dilute conditions may be different from that under synthetically relevant conditions and may provide a path toward the development of faster and more reproducible Tsuji–Trost reactions for synthetic, analytical, and biological applications.

The pandemic emergency has brought to light the need for a new generation of rapidly developed vaccines that induce longer-lasting, potent, and broader immune responses. While the mRNA vaccines played a critical role during the emergency in reducing SARS-CoV-2 hospitalization rates and deaths, more efficient approaches are needed.

Exosome based vaccines represent an interesting opportunity in the pandemic time we live. Compared to the available vaccines, an exosome-based vaccine may answer to the need of efficacy and increased safety. Here, we used exosomes to deliver a "cocktail" protein-based vaccine, in which two independent viral proteins are delivered using the exosome membrane as carrier. Cells were engineered to express either SARS-CoV-2 Delta spike (StealthTM X-Spike, STX-S) or nucleocapsid (StealthTM X-Nucleocapsid, STX-N) protein on the surface and facilitate their trafficking to the exosomes. When administered as single product, both STX-S and STX-N induced a strong immunization with the production of a potent humoral and cellular immune response. Interestingly, these effects are obtained with administration of nanograms of protein and without adjuvant. Therefore, we developed a multivalent low dose vaccine, namely STX-S+N, using a teeter-toother dose approach of STX-S and STX-N. In two independent animal models (mouse and rabbit), administration of STX-S+N resulted in increased antibody production, potent neutralizing antibodies with cross-reactivity to other VOC and strong T-cell response. Importantly, no competition in immune response was observed. Our data show that our exosome platform has an enormous potential to revolutionize vaccinology by rapidly facilitating antigen presentation, and for therapeutics by enabling cell and tissue specific targeting.

Exosomes are emerging as potent and safe delivery carriers for use in vaccinology and therapeutics. A better vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to provide improved, broader, longer lasting neutralization of SARS-CoV-2, a more robust T cell response, enable widespread global usage, and further enhance the safety profile of vaccines given the likelihood of repeated booster vaccinations. Here, we use Capricor's StealthXTM platform to engineer exosomes to express native SARS-CoV-2 spike Delta variant (STX-S) protein on the surface for the delivery of a protein-based vaccine for immunization against SARS-CoV-2 infection. The STX-S vaccine induced a strong immunization with the production of a potent humoral immune response as demonstrated by high levels of neutralizing antibody not only against the delta SARS-CoV-2 virus but also two Omicron variants (BA.1 and BA.5), providing broader protection than current mRNA vaccines. Additionally, both CD4+ and CD8+ T cell responses were increased significantly after treatment. Quantification of spike protein by ELISA showed that only nanograms of protein were needed to induce a potent immune response. This is a significantly lower dose than traditional recombinant protein vaccines with no adjuvant required, which makes the StealthXTM exosome platform ideal for the development of multivalent vaccines with a better safety profile. Importantly, our exosome platform allows novel proteins, or variants in the case of SARS-CoV-2, to be engineered onto the surface of exosomes in a matter of weeks, comparable with mRNA vaccine technology, but without the cold storage requirements. The ability to utilize exosomes for cellular delivery of proteins, as demonstrated by STX-S, has enormous potential to revolutionize vaccinology by rapidly facilitating antigen presentation at an extremely low dose resulting in a potent, broad antibody response.