1-Ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) bioconjugations have been utilized in preparing variants for medical research. While there have been advances in optimizing the reaction for aqueous applications, there has been limited focus toward identifying conditions and side reactions that interfere with product formation. We present a systematic investigation of EDC/N-hydroxysulfosuccinimide (sNHS)-mediated bioconjugations on carboxylated peptides and small proteins. We identified yet-to-be-reported side products arising from both the reagents and substrates. Model peptides used in this study illustrate particular substrates are more susceptible to side reactions than others. From our studies, we found that bioconjugations are more efficient with high concentrations of amine nucleophile but not sNHS. Performing bioconjugations on a model affibody protein show that the trends established with model peptides hold for more complex systems.

Rapid development of antisense therapies can enable on-demand responses to new viral pathogens and make personalized medicine for genetic diseases practical. Antisense phosphorodiamidate morpholino oligomers (PMOs) are promising candidates to fill such a role, but their challenging synthesis limits their widespread application. To rapidly prototype potential PMO drug candidates, we report a fully automated flow-based oligonucleotide synthesizer. Our optimized synthesis platform reduces coupling times by up to 22-fold compared to previously reported methods. We demonstrate the power of our automated technology with the synthesis of milligram quantities of three candidate therapeutic PMO sequences for an unserved class of Duchenne muscular dystrophy (DMD). To further test our platform, we synthesize a PMO that targets the genomic mRNA of SARS-CoV-2 and demonstrate its antiviral effects. This platform could find broad application not only in designing new SARS-CoV-2 and DMD antisense therapeutics, but also for rapid development of PMO candidates to treat new and emerging diseases.

The 20S core particle of the proteasome in M. tuberculosis (Mtb) is a promising, yet unconventional drug target. This multimeric peptidase is not essential, yet degrades proteins that have become damaged and toxic via reactions with nitric oxide (and/or the associated reactive nitrogen intermediates) produced during the host immune response. Proteasome inhibitors could render Mtb susceptible to the immune system, but they would only be therapeutically viable if they do not inhibit the essential 20S counterpart in humans. We designed and synthesized selective inhibitors of the Mtb 20S based on both its unique substrate preferences and the structures of substrate-mimicking covalent inhibitors of eukaryotic proteasomes called syringolins. Unlike the parent syringolins, the designed analogs weakly inhibit the human 20S (Hs 20S) proteasome and preferentially inhibit Mtb 20S over the human counterpart by as much as 74-fold. Moreover, they can penetrate the mycobacterial cell envelope and render Mtb susceptible to nitric oxide-mediated stress. Importantly, they do not inhibit the growth of human cell lines in vitro and thus may be starting points for tuberculosis drug development.

A chemistry for the facile two-component macrocyclization of unprotected peptide isocyanates is described. Starting from peptides containing two glutamic acid γ-hydrazide residues, isocyanates can be readily accessed and cyclized with hydrazides of dicarboxylic acids. The choice of a nucleophilic linker allows for the facile modulation of biochemical properties of a macrocyclic peptide. Four cyclic NYAD-1 analogues were synthesized using the described method and displayed a range of biological activities.