Fluorogenic diazaborine formation of semicarbazide with designed coumarin derivatives

Abstract

Bioorthogonal fluorogenic reactions serve as enabling tools in research and biotechnology. Herein we describe fluorogenic conjugations of semicarbazide with coumarin derivatives that incorporate a 2-acetylphenylboronic acid motif. These designed coumarins 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.

Fig 1

Schematic of the reaction of a coumarin derivative (AB21) with semicarbazide to generate a highly fluorescent diazaborine conjugate.

Fig 2. Coumarin derivatives display variable fluorescence properties

(a) Structures of the coumarin derivatives AB21, AB22, AB23 and AB24. Numbering of the coumarin core structure is shown on AB21. (b) Coumarin derivatives displaying similar absorbance properties (50 μM, 1 cm cuvette, 1X PBS Buffer, pH 7.4).(c) Fluorescence emission of the coumarin derivatives showing quenched fluorescence of AB21 and AB22 (10 μM, 1X PBS Buffer, pH 7.4, λ_ex= 392 nm (AB22/24), 397 nm (AB21) and 402 nm (AB23)).

Fig 3. Diazaborine formation of AB21/22 with semicarbazide induces an increase in fluorescence

(a) LC traces showing clean and complete conjugation of AB21/22 (200 μM) to semicarbazide (200 μM) in 30 minutes. The identity of the peaks were confirmed with mass-spec data. (b) Conjugation to semicarbazide leading to much enhanced fluorescence of AB21 and AB22 (10 μM, 1x PBS Buffer, pH 7.4, λ_ex= 392 nm (AB22) and 397 nm (AB21)).

Fig 4. APBA coumarin derivatives efficiently label S. aureus membranes without washing

(a) Schematic two-step labelling process involving 1) D-Dap-semicarbazide incorporation into peptidoglycan followed by 2) APBA coumarin conjugation to form a stable diazaborine. (b) S. aureus incubated with 0 μM, 100 μM or 500 μM D-Dap-Semicarbazide for four hours followed by 100 μM AB21, AB22, AB24 or FPBA-FITC incubation for 2 hours. AB21 and AB22 showed dose dependent bacterial membrane labelling, whereas AB24 displayed no fluorescence labelling and FPBA-FITC gave significant background staining.