Facile labeling of proteins of interest is highly desirable in proteomic research as well as in the development of protein therapeutics.

Bioorthogonal conjugation chemistry has enabled the development of tools for the interrogation of complex biological systems. Although a number of bioorthogonal reactions have been documented in literature, they are less ideal for one or several reasons including slow kinetics, low stability of the conjugated product, requirement of toxic catalysts, and side reactions with unintended biomolecules. Herein we report a fast (>103 M−1s−1) and bioorthogonal conjugation reaction that joins semicarbazide to an aryl ketone or aldehyde with an ortho-boronic acid substituent. The boronic acid moiety greatly accelerates the initial formation of a semicarbazone conjugate, which rearranges into a stable diazaborine. The diazaborine formation can be performed in blood serum or cell lysates with minimal interference from biomolecules. We further demonstrate that application of this conjugation chemistry enables facile labeling of bacteria. A synthetic amino acid D-AB3, which presents a 2-acetylphenylboronic acid moiety as its side chain, was found to incorporate into several bacterial species through cell wall remodeling, with particularly high efficiency for Escherichia coli. Subsequent D-AB3 conjugation to a fluorophore-labeled semicarbazide allows robust detection of this bacterial pathogen in blood serum.

Biocompatible and bioorthogonal conjugation reactions have proven to be powerful tools in biological research and medicine. While the advent of bioorthogonal conjugation chemistries greatly expands our capacity to interrogate specific biomolecules in situ, biocompatible reactions that target endogenous reactive groups have given rise to a number of covalent drugs as well as a battery of powerful research tools. Despite the impressive progress, limitations do exist with the current conjugation chemistries. For example, most known bioorthogonal conjugations suffer from slow reaction rates and imperfect bioorthogonality. On the other hand, covalent drugs often display high toxicity due to off-target labeling and immunogenicity. These limitations demand continued pursuit of conjugation chemistries with optimal characteristics for biological applications. A spate of papers appearing in recent literature report the conjugation chemistries of 2-formyl and 2-acetyl phenylboronic acids (abbreviated as 2-FPBA and 2-APBA, respectively). These simple reactants are found to undergo fast conjugation with various nucleophiles under physiological conditions, showing great promise for biological applications. The versatile reactivity of 2-FPBA and 2-APBA manifests in dynamic conjugation with endogenous nucleophiles as well as conjugation with designer nucleophiles in a bioorthogonal manner. 2-FPBA/APBA conjugates with amines in biomolecules, such as lysine side chains and aminophospholipids, in a highly dynamic manner to give iminoboronates. In contrast to typical imines, iminoboronates enjoy much improved thermodynamic stability, yet are kinetically labile for hydrolysis due to imine activation by the boronic acid. Dynamic conjugations as such present a novel binding mechanism analogous to hydrogen bonding and electrostatic interactions. Implementation of this covalent binding mechanism has yielded reversible covalent probes of prevalent bacterial pathogens. It has also resulted in reversible covalent inhibitors of a therapeutically important protein Mcl-1. Such covalent probes/inhibitors with 2-FPBA/APBA warheads avoid permanent modification of their biological target, potentially able to mitigate off-target labeling and immunogenicity of covalent drugs. The dynamic conjugation of 2-FPBA/APBA has been recently extended to N-terminal cysteines, which can be selectively targeted via formation of a thiazolidino boronate (TzB) complex. The dynamic TzB formation expands the toolbox for site-specific protein labeling and the development of covalent drugs. On the front of bioorthogonal conjugation, 2-FPBA/APBA has been found to conjugate with α-nucleophiles under physiologic conditions with rate constant ( k) over 1000 M s, which overcomes the slow kinetics problems and rekindles the interest of using the conjugation of α-nucleophiles for biological studies. With fast kinetics being a shared feature, this family of conjugation chemistries gives remarkably diverse product structures depending on the choice of nucleophile. Importantly, ...

Antibiotic resistance of bacterial pathogens poses an increasing threat to the wellbeing of our society and urgently calls for new strategies for infection diagnosis and antibiotic discovery. The antibiotic resistance problem at least partially arises from extensive use of broad-spectrum antibiotics. Ideally, for the treatment of infection, one would like to use a narrow-spectrum antibiotic that specifically targets and kills the disease-causing strain. This is particularly important considering the commensal bacterial species that are beneficial and sometimes even critical to the health of a human being. In this contribution, we describe a phage display platform that enables rapid identification of peptide probes for specific bacterial strains. The phage library described herein incorporates 2-acetylphenylboronic acid moieties to elicit dynamic covalent binding to the bacterial cell surface. Screening of the library against live bacterial cells yields submicromolar and highly specific binders for clinical strains of Staphylococcus aureus and Acinetobacter baumannii that display antibiotic resistance. We further show that the identified peptide probes can be readily converted to bactericidal agents that deliver generic toxins to kill the targeted bacterial strain with high specificity. The phage display platform described here is applicable to a wide array of bacterial strains, paving the way to facile diagnosis and development of strain-specific antibiotics.

Bioorthogonal fluorogenic reactions serve as enabling tools in research and biotechnology. Herein we describe fluorogenic conjugations of semicarbazide with courmarin derivatives that incorporate a 2-acetylphenylboronic acid motif. These designed courmarins rapidly conjugate with semicarbazide to give diazaborine products with significantly enhanced fluorescence. To demonstrate potential applications of this fluorogenic reaction, we synthesized a semicarbazide-presenting amino acid D-Dap-Scz, which readily incorporates into the cell wall of Staphalococcus aureus and serves as a handle for conjugation with the coumarins. The fluorogenic conjugation of the coumarins to cell surface semicarbazide enables facile visualization of D-Dap-Scz treated bacteria.