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Reactions that can selectively modify amino acid sequences within peptides and proteins are important for preparing protein reagents, immobilizing proteins, and making antibody-drug conjugates. The development of new reactions often begins with known chemistries and optimizes yields using a small set of peptide reactants. This article describes the use of peptide arrays and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) to discover and characterize unanticipated sequence-selective reactions of peptides. This work reports the selective acetylation of HY (histidine-tyrosine) and YH (tyrosine-histidine) dyads when treated with acetic anhydride in aqueous conditions. More broadly, this example illustrates the benefits of using peptide arrays and a label-free analysis method to discover peptide-modifying reactions and gain mechanistic insight into their sequence specificity.

Site-specific modification of proteins has important applications in biological research and drug development. Reactive tags such as azide, alkyne, and tetrazine have been used extensively to achieve the abovementioned goal. However, bulky side-chain “ligation scars” are often left after the labeling and may hinder the biological application of such engineered protein products. Conjugation chemistry via dehydroalanine (Dha) may provide an opportunity for “traceless” ligation because the activated alkene moiety on Dha can then serve as an electrophile to react with radicalophile, thiol/amine nucleophile, and reactive phosphine probe to introduce a minimal linker in the protein post-translational modifications. In this report, we present a mild and highly efficient enzymatic approach to incorporate Dha with phosphothreonine/serine lyases, OspF and SpvC. These lyases originally catalyze an irreversible elimination reaction that converts a doubly phosphorylated substrate with phosphothreonine (pT) or phosphoserine (pS) to dehydrobutyrine (Dhb) or Dha. To generate a simple monophosphorylated tag for these lyases, we conducted a systematic approach to profile the substrate specificity of OspF and SpvC using peptide arrays and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry. The optimized tag, [F/Y/W]-pT/pS-[F/Y/W] (where [F/Y/W] indicates an aromatic residue), results in a ∼10-fold enhancement of the overall peptide labeling efficiency via Dha chemistry and enables the first demonstration of protein labeling as well as live cell labeling with a minimal ligation linker via enzyme-mediated incorporation of Dha.

Comparison of methanol photochemistry at three-dimensionally (3D) networked aerogels of TiO2or Au–TiO2reveals that incorporated Au nanoparticles strongly sensitize the oxide nanoarchitecture to visible light. Methanol dissociatively adsorbs at the surfaces of TiO2and Au–TiO2aerogels under dark, high-vacuum conditions. Upon irradiation of either ultraporous material with broadband UV light under anaerobic conditions, adsorbed methoxy groups act as hole-traps and extend conduction-band and shallow-trapped electron lifetimes. A higher excited-state electron density arises for UV-irradiated TiO2aerogel relative to commercial nanoparticulate TiO2, indicating that 3D networked TiO2more efficiently separates electron–hole pairs. Upon excitation with narrow-band visible light centered at 550 nm, long-lived excited-state electrons are evident on CH3OH-exposed Au–TiO2aerogelsbut not on identically dosed TiO2aerogelsverifying that incorporated Au nanoparticles sensitize the networked oxide to visible light. Under aerobic conditions (20 Torr O2) and broadband UV illumination, surface-sited formates accumulate as adsorbed methoxy groups oxidize, at similar rates, on Au–TiO2and TiO2aerogels. Moving to excitation wavelengths longer than ∼400 nm (i.e., the low-energy range of UV light) dramatically decreases methoxy photoconversion for methanol-saturated TiO2aerogel, while Au–TiO2aerogel remains highly active for methanol photooxidation. The wavelength dependence of formate production on Au–TiO2tracks the absorbance spectrum for this material, which peaks at λ = 550 nm due to resonance with the surface plasmon in the Au particles. The photooxidation rate for Au–TiO2aerogel at 550 nm is comparable to that for TiO2aerogel under broadband UV illumination, indicating efficient energy transfer from Au to TiO2in the 3D mesoporous nanoarchitecture.