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Macrocyclization can confer enhanced stability, target affinity, and membrane permeability to peptide scaffolds, all of which are desirable properties for chemical probes and therapeutics. A wide array of macrocyclization chemistries have been reported over the last few decades; however, these often have limited compatibility with each other and across chemical environments, thus restricting access to specific molecular properties. In an effort to address some of these limitations, we recently described the use of Diels-Alder [4 + 2] cycloadditions for peptide macrocyclization. Among the attributes of this chemistry, we demonstrated that Diels-Alder cyclization can template diverse peptide secondary structures, proceed in organic or aqueous environments, and endow improved pharmacologic properties on cyclized peptides. Here, we present synthetic processes and characterization methods for the synthesis of Diels-Alder cyclized peptides.

Macrocyclization can confer enhanced stability, target affinity, and membrane permeability to peptide scaffolds, all of which are desirable properties for chemical probes and therapeutics. A wide array of macrocyclization chemistries have been reported over the last few decades; however, these often have limited compatibility with each other and across chemical environments, thus restricting access to specific molecular properties. In an effort to address some of these limitations, we recently described the use of Diels-Alder [4 + 2] cycloadditions for peptide macrocyclization. Among the attributes of this chemistry, we demonstrated that Diels-Alder cyclization can template diverse peptide secondary structures, proceed in organic or aqueous environments, and endow improved pharmacologic properties on cyclized peptides. Here, we present synthetic processes and characterization methods for the synthesis of Diels-Alder cyclized peptides.

Chemoproteomic methods can report directly on endogenous, active enzyme populations, which can differ greatly from measures of transcripts or protein abundance alone. Detection and quantification of family-wide probe engagement generally requires LC-MS/MS or gel-based detection methods, which suffer from low resolution, significant input proteome requirements, laborious sample preparation, and expensive equipment. Therefore, methods that can capitalize on the broad target profiling capacity of family-wide chemical probes but that enable specific, rapid, and ultrasensitive quantitation of protein activity in native samples would be useful for basic, translational, and clinical proteomic applications. Here we develop and apply a method that we call soluble activity-dependent proximity ligation (sADPL), which harnesses family-wide chemical probes to convert active enzyme levels into amplifiable barcoded oligonucleotide signals. We demonstrate that sADPL coupled to quantitative PCR signal detection enables multiplexed "writing" and "reading" of active enzyme levels across multiple protein families directly at picogram levels of whole, unfractionated proteome. sADPL profiling in a competitive format allows for highly sensitive detection of drug-protein interaction profiling, which allows for direct quantitative measurements of in vitro and in vivo on- and off-target drug engagement. Finally, we demonstrate that comparative sADPL profiling can be applied for high-throughput molecular phenotyping of primary human tumor samples, leading to the discovery of new connections between metabolic and proteolytic enzyme activity in specific tumor compartments and patient outcomes. We expect that this modular and multiplexed chemoproteomic platform will be a general approach for drug target engagement, as well as comparative enzyme activity profiling for basic and clinical applications.

Phosphorylation at aspartic acid residues represents an abundant and critical post-translational modification (PTM) in prokaryotes. In contrast to most characterized PTMs, such as phosphorylation at serine or threonine, the phosphoaspartate moiety is intrinsically labile, and therefore incompatible with common proteomic profiling methods. Herein, we report a nucleophilic, desthiobiotin-containing hydroxylamine (DBHA) chemical probe that covalently labels modified aspartic acid residues in native proteomes. DBHA treatment coupled with LC-MS/MS analysis enabled detection of known phosphoaspartate modifications, as well as novel aspartic acid sites in the E. coli proteome. Coupled with isotopic labelling, DBHA-dependent proteomic profiling also permitted global quantification of changes in endogenous protein modification status, as demonstrated with the detection of increased E. coli OmpR phosphorylation, but not abundance, in response to changes in osmolarity.

We report a novel photoproximity protein interaction (PhotoPPI) profiling method to map protein-protein interactions in vitro and in live cells. This approach utilizes a bioorthogonal, multifunctional chemical probe that can be targeted to a genetically encoded protein of interest (POI) through a modular SNAP-Tag/benzylguanine covalent interaction. A first generation photoproximity probe, PP1, responds to 365 nm light to simultaneously cleave a central nitroveratryl linker and a peripheral diazirine group, resulting in diffusion of a highly reactive carbene nucleophile away from the POI. We demonstrate facile probe loading, and subsequent interaction- and light-dependent proximal labeling of a model protein-protein interaction (PPI) in vitro. Integration of the PhotoPPI workflow with quantitative LC-MS/MS enabled unbiased interaction mapping for the redox regulated sensor protein, KEAP1, for the first time in live cells. We validated known and novel interactions between KEAP1 and the proteins PGAM5 and HK2, among others, under basal cellular conditions. By contrast, comparison of PhotoPPI profiles in cells experiencing metabolic or redox stress confirmed that KEAP1 sheds many basal interactions and becomes associated with known lysosomal trafficking and proteolytic proteins like SQSTM1, CTSD, and LGMN. Together, these data establish PhotoPPI as a method capable of tracking the dynamic subcellular and protein interaction "social network" of a redox-sensitive protein in cells with high temporal resolution.

Macrocyclization can improve bioactive peptide ligands through preorganization of molecular topology, leading to improvement of pharmacologic properties like binding affinity, cell permeability and metabolic stability. Here we demonstrate that Diels-Alder [4+2] cycloadditions can be harnessed for peptide macrocyclization and stabilization within a range of peptide scaffolds and chemical environments. Diels-Alder cyclization of diverse diene-dienophile reactive pairs proceeds rapidly, in high yield and with tunable stereochemical preferences on solid-phase or in aqueous solution. This reaction can be applied alone or in concert with other stabilization chemistries, such as ring-closing olefin metathesis, to stabilize loop, turn, and α-helical secondary structural motifs. NMR and molecular dynamics studies of model loop peptides confirmed preferential formation of endo cycloadduct stereochemistry, imparting significant structural rigidity to the peptide backbone that resulted in augmented protease resistance and increased biological activity of a Diels-Alder cyclized (DAC) RGD peptide. Separately, we demonstrated the stabilization of DAC α-helical peptides derived from the ERα -binding protein SRC2. We solved a 2.25 Å co-crystal structure of one DAC helical peptide bound to ERα, which unequivocally corroborated endo stereochemistry of the resulting Diels-Alder adduct, and confirmed that the unique architecture of stabilizing motifs formed with this chemistry can directly contribute to target binding. These data establish Diels-Alder cyclization as a versatile approach to stabilize diverse protein structural motifs under a range of chemical environments.