Rapid formation of a stable boron-nitrogen heterocycle in dilute, neutral aqueous solution for bioorthogonal coupling reactions

Abstract

Combining 2-formylphenylboronic acid with 4-hydrazinylbenzoic acid in neutral aqueous solution at low, equimolar concentrations of the reagents results in a single, stable product, a 1,2-dihydro-1-hydroxy-2,3,1-benzodiazaborine, in a matter of minutes with no side products. Application of this reaction to protein conjugation demonstrates that the reaction is orthogonal to protein functional groups, and the resulting conjugate withstands SDS-PAGE analysis. This reaction should be particularly useful for couplings that must be performed with low concentrations of reagents under physiologically compatible conditions.

Figure 1

Structures of salicyaldehyde (Sal, 1), p-hydrazinylbenzoic acid (HBA, 2), the hydrazone of Sal and HBA (3), 2-formylphenylboronic acid (2fPBA, 4), and the product of 2fPBA and HBA (5)

Figure 2

Absorption difference spectra of fPBA and HBA in 0.1 M phosphate buffer, pH 7, (5 μM each, final concentration) as a function of time. Note that the absorptivity of the low energy band initially increases and then decreases over the course of the reaction.

Figure 3

Kinetic data collected under pseudo-first order conditions. Absorption difference spectra of 50 μM HBA + 2.5 μM fPBA were collected as a function of time. Data were fit to two sequential first order reactions using Olis GlobalWorks software. A plot of the change in absorption at 350 nm vs time is shown (red points), which is proportional to the concentration of the intermediate hydrazone as a function of time. The dashed line is the theoretical curve for k₁ = 0.114 s⁻¹ (pseudo-first order rate constant for the formation of the hydrazone) and k₂ = 0.015 s⁻¹ (first order rate constant for formation of the DAB), which were obtained from the global fit of the spectral data.

Figure 4

Proton NMR spectrum of 5 in immediately after mixing fPBA and HBA to a final concentration of 2.5 mM each in 0.1 M phosphate buffer, pH 7.0, containing 10% D₂O.

Figure 5

Coumarin-fPBA ester conjugate (6), NHS-ester for labeling BSA with HBA (7)

Figure 6

Emission spectra of BSA-hydrazine and BSA reacted with or without probe 6 excited at 320 nm. BSA or BSA-hydrazine (final concentration 25 μM) was allowed to react with or without probe 6 (final concentration 75 μM) at room temperature for 15 min. Excess probe was removed by rapid gel filtration chromatography and emission spectra of equal concentration of each protein sample were obtained.

Figure 7

SDS-PAGE of BSA or BSA-hydrazine after reaction with probe 6.