Abstracttert-Butyl nitrite was identified as a safe and chemoselective nitrating agent that provides preferentially mononitro derivatives of phenolic substrates in the presence of potentially competitive functional groups. On the basis of our control experiments, we propose that the reaction proceeds through the formation of O-nitrosyl intermediates prior to C-nitration via homolysis and oxidation. The reported nitration method is compatible with tyrosine-containing peptides on solid support in the synthesis of fluorogenic substrates for characterization of proteases.The technique of Fluorescence Resonance Energy Transfer (FRET) is currently a common method for monitoring structural, functional, or aggregation changes in evaluated biomolecules. 1 Real-time monitoring of hydrolase activity is a particularly useful application of FRET, because the substrate can be chemically modified at sites remote to the scissile bond, minimizing probe interferences with substrate recognition. The 2-aminobenzoic acid (2-Abz) fluorophore and 3-nitrotyrosine quencher pair has been utilized extensively in determining proteolytic activities of endoproteases. 2 As amino acids, these agents represent a convenient fluorophore-quencher combination that can be readily incorporated into a conventional solid phase peptide synthesis protocol. As a part of a program in evolution of site-specific proteases as therapeutic agents, we are interested in developing a flexible and practical platform for kinetic and thermodynamic characterization of evolved enzymes. Thus, we envisioned a unified synthetic strategy for accessing both internally quenched substrates, useful for realtime kinetic analysis of active enzymes, as well as unquenched variants, suitable for fluorescence quenching and anisotropy studies with inactive mutants. We were very keen, therefore, to identify a reagent that could perform a nitration reaction on solid phaseimmobilized peptides containing tyrosines with high efficiency and selectivity. Unfortunately, the existing nitrating agents, such as mixed acids, superacids, and metal nitrates, are not suitable for polymer-supported substrates because of their polymer-reactive or heterogeneous nature. 3,4 In addition, many of the existing reagents can lead to over-nitration of phenols 4a,g,h and ssavinov@purdue.edu. In our attempt to perform diazotization of tyrosine with t-BuONO, 6 we observed the formation of unexpected yellow products. To explore this finding further, we subjected Boc-Tyr-OH (1a) to the nitrite treatment in dichloromethane at room temperature (Scheme 1). After 3 hours of agitation, we detected a complete consumption of 1a and quantitative (>95%) formation of Boc-Tyr(3-NO 2 )-OH (2a) and corresponding N-nitroso derivative 3a (2a:3a 57:43). While there have been reports of C-nitro products emerging from the exposure of electron-rich aromatic compounds to inorganic 7 or organic 8 nitrites, no mechanistic or scope studies have been carried out. In 1 H NMR spectum of the crude reaction mixure we also identified a tra...