Chemoselective peptide cyclization at tyrosine. With urazole-containing peptides (14) in hand, we next identified mild conditions to access Tyr-linked cyclic peptides (16). We envisioned a one-pot strategy wherein oxidation of urazole peptides ( ) would generate reactive TAD peptides (int-16) in situ that could undergo spontaneous cyclization at an embedded Tyr residue to access Tyr-linked cyclic peptides (16) (Figure ). This strategy presented several compatibility challenges, as the urazole oxidation conditions must be selective and tolerant of native amino acid residue side chain functionalities. At the onset of our investigations, biocompatible electrochemical urazole oxidation methods were unknown. Moreover, there was no information regarding the spontaneity of cyclization events provided TAD peptides (int-16) could be efficiently formed. These challenges necessitated the identification of conditions to, first, generate TAD peptides (int-16) selectively and second, induce cyclization with an embedded Tyr residue to provide cyclic peptides (16). Using 14d as a model sequence, preliminary studies evaluated inorganic and organic nitrite reagents as potentially suitable oxidants. Sodium nitrite (NaNO2) is known to efficiently promote the oxidation of urazoles to TADs and has demonstrated tolerance of amino acid residue functionality. Indeed, when an acidic solution of 14d (0.19 M, 1 equiv) in acetonitrile was treated with NaNO2 (1.0 equiv), the presumed TAD intermediate (see int-16) was readily generated in situ as qualitatively observed by the immediate change from colorless to a carmine red color that has been characteristically attributed to the extended azo chromophore of TADs (UV λmax = 532 nm). Based on conditions developed for protein bioconjugation with N4-aryl TAD derivatives, we attempted to induce cyclization by diluting the presumed TAD-containing solution with a phosphate buffered acetonitrile-water solution (1:1 CH3CN/100 mM phosphate buffer (PB), pH = 7.0). Unfortunately, mass spectrometry analysis indicated that TAD intermediate hydrolysis and Tyr residue nitration occurred alongside a significant amount of unreacted 14d. This result can be partly rationalized by the limited solubility of NaNO2 in organic solvents, which, despite the observable TAD formation, could have suppressed the reaction efficiency. To address this issue, we evaluated the reactivity of tert-butyl nitrite, t-BuNO2, due to its greater solubility in organic solvents. Following the oxidation of 14d (1 equiv) with t-BuNO2 (1.3 equiv) in acidic acetonitrile (1 vol% AcOH in CH3CN), the formed TAD intermediate (int-16d) was subsequently diluted with a phosphate buffered acetonitrile-water solution. Still, products of TAD hydrolysis and oxidation at the Tyr residue remained prevalent. The hydrolysis result is consistent with a recent study from Heise and coworkers that evaluated the rate of N4-alkyl TAD hydrolysis in mixed aqueous-organic solutions. From this, we hypothesized that TAD hydrolysis pathways could be suppressed by decreasing the dilutant water content. Accordingly, a solution of in situ formed TAD intermediate (int-16d) was diluted into a phosphate buffered acetonitrile-water solution (3:2 CH3CN/100 mM PB, pH = 7.0) and analysis of the mixture indicated the formation of a new product as observed by analytical LC with UVdetection. The suspected cyclic peptide (16d) exhibited increased polarity relative to 14d as observed by a shift in retention time. Mass spectrometric analysis of the collected peak revealed a monoisotopic mass of 545.2 Da, consistent with the anticipated [M+H] + ion for cyclic peptide 16d. Encouraged by this result, these conditions were used to synthesize cyclic peptides 16a-16h (Figure 3, using condition A). Nevertheless, we regularly observed incomplete consumption of 14, as well as some undesirable byproducts that we presume to derive from oxidation at Tyr. Therefore, we continued to evaluate aqueous compatible oxidants for the improved in situ formation of TAD-containing peptides (int-16). Chlorinating reagents such as t-butyl hypochlorite and trichloroisocyanuric acid in DMF affected the oxidation, but promiscuous overoxidation resulted in low yields of isolated 16d (Figure , see the Supplementary Information). However, we discovered that oxidations using N-chlorosuccinimide (NCS) in DMF reduced the production of overoxidation products and provided 16d in an improved 54% isolated yield. The comparatively improved yield for the model system supported the general use of NCS and all subsequent cyclizations were conducted using these conditions. Upon establishing conditions to selectively generate TAD-containing peptides (int-16), we focused on solution pH optimization to improve the cyclization event (Figure , see the Supplementary Information). Seminal studies conducted by Barbas and coworkers demonstrated the pH sensitivity of the conjugation of N4-phenyl urazole (PTAD) and a model Tyr residue. This led us to evaluate cyclization efficiencies using phosphate-buffered acetonitrile-water conditions at pH = 7.0, 8.0, 9.0, and 10.0. Ultimately, solution pH = 8.0 was found optimal for cyclization, affording 16d in 64% isolated yield (Figure ). This protocol (condition B: 1.1 equiv NCS and 1.1 equiv pyridine in DMF; dilute into 3:2 CH3CN/100 mM PB at pH = 8.0) was applied to efficiently access 14 size-and sequence-diverse cyclic peptides in good yields (Figure ). Notably, this general procedure exhibits high amino acid residue side chain tolerance, being compatible with native histidine (16o), lysine (16p), serine (16q and 16r), aspartic acid (16s), asparagine (16t), and arginine (16y and 16z, vide infra) residue side chain functionalities. Oxidation of substrates containing Cys or tryptophan (Trp) residues result in complex mixtures, likely the result of thiol (Cys) and indole (Trp) oxidation and their orthogonal nucleophilic reactivity toward-TADs. However, the use of an acetamidomethyl (Acm) protection strategy together with oxidation condition A reduced side reactions for Cys residue-containing substrates, as demonstrated by access to cyclic peptide 16h. Overall, we have developed a procedurally simple peptide cyclization method which provides facile access to a representative collection of Tyr-linked cyclic peptides ( ) from native peptide sequences under aqueous conditions. We anticipate that the use of electrochemical oxidation methods for the in situ generation of TADs will expand the utility of this approach by enabling orthogonality to native, oxidation-prone residues. Such strategies are currently being explored in our laboratory.