Fast Diazaborine Formation of Semicarbazide Enables Facile Labeling of Bacterial Pathogens

Abstract

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^-1 s^-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.

Figure 1.. Diazaborine formation of semicarbazide.

a) Schematic illustration of diazaborine formation of semicarbazide in comparison to that of phenylhydrazine. b) ¹H-NMR spectra of semicarbazide mixed with equimolar 2-FPBA (top) and 2-APBA (bottom) respectively. c) Crystal structure of DAB1, the diazaborine conjugate of semicarbazide with 2-FPBA. d) Crystal structure of DAB3, a diazaborine conjugate of semicarbazide with the synthetic amino acid L-AB3.

Figure 2.. Stability, efficiency and kinetics of formation of diazaborines.

a) ¹H-NMR analysis of DAB2 formation as well as potential decomposition demonstrating the kinetic stability of DAB2. b) Yields of diazaborine formation for various combinations of reactants as determined by ¹H NMR. Due to the fast kinetics, all reactions went to completion within 10 min. c) A representative kinetic profile of DAB1 formation monitored by UV-vis spectroscopy. d) A representative kinetic profile of DAB2 formation monitored by UV-vis spectroscopy. The kinetic measurements were performed three times and the error values for t_1/2 and k₂ were reported as the standard deviations of the three trials. Details of curve fitting are provided in SI.

Figure 3.. Compatibility and orthogonality of the diazaborine chemistry to biological systems.

a) ¹H-NMR spectra illustrating the weak affinity and rapid reversibility of the 2-APBA-cysteine conjugation. b) ¹H-NMR analysis showing that the diazaborine formation of 2-FPBA, but not 2-APBA, is inhibited by the presence of free cysteine. All reactants were used at 0.2 mM in a pH 7.4 buffer. The reaction mixtures were incubated for 40 min before analysis. c) DAB1 and d) DAB2 formation in buffer alone and in presence of FBS (20%, v/v), HEK cell lysate (5 mg/mL), S. aureus cell lysate (5 mg/mL) respectively. For c) and d), the samples were prepared with 25 µM 2-FPBA or 2-APBA and 50 µM Scz-NBD in a pH 7.4 buffer. The LC traces were recorded after 30 min incubation. The relative peak areas of Scz-NBD over the diazaborine product are shown on the LC traces and used to estimate the conversion of the reactions.

Figure 4.. Cytotoxicity studies of the reactants for diazaborine conjugation.

a) Percentage of cell survival determined by colony counting for E. coli and S. aureus. b) Percentage of cell survival determined by the MTT assay for HEK 293T cells. Phenylhydrazine (Phz) was included as a direct comparison to semicarbazide (Scz). Camptothecin (Camp) was used as a positive control in the MTT assay of HEK 293T cells. All compounds were tested at 50 µM concentration.

Figure 5.. Semicarbazide ligation enables bacterial labeling

a) Schematic illustration of the two-step (D-AB3 followed by Scz-FITC) protocol for bacterial cell labeling. b) Microscopic images showing that the D-AB3 and Scz-FITC treatment gives varied degrees of labeling across bacterial species. c) Flow cytometry results corroborating the E. coli selectivity of the D-AB3 and Scz-FITC staining protocol. The concentration dependence data were analyzed according to Michaelis-Menten kinetics (Figure S21) and discussed in the text. d) Flow cytometry histograms showing the semicarbazide-enabled bacterial labeling is unaffected by blood serum or cell lysate. All labeling experiments were performed with 50 µM Scz-FITC.