Three-Component Protein Modification Using Mercaptobenzaldehyde Derivatives

Abstract

A chemoselective primary amine modification strategy that enables the three-component, one-pot bioconjugation is described. The specifically designed, mercaptobenzaldehyde-based bifunctional linker achieves highly selective and robust amine labeling under biocompatible conditions. This linker demonstrates wide functional group tolerance and is simple to prepare, which allowed facile payload incorporation. Finally, our studies have shown that the introduction of linker does not impair the function of modified protein such as insulin.

Figure 1.

Three-component one-pot bioconjugation.

Figure 2.

Three-component conjugation of human insulin. Reagents and conditions: 100 μL THF/PBS (1:4, v/v), pH 5.5, 0.15 equivalent of CuSO₄, 0.3 equivalent of sodium ascorbate, 0.3 equivalent of TBTA and 9 equivalents of AF546 azide were used, final concentration of insulin is 1.72 mM, incubated for 4 h. Examination of modified insulin 17 function on THP1 cells. (a) Bright field and fluorescence images of THP1 cells incubated with 0.06 mM AF546 azide for 4 h. (b) Bright field and fluorescence images of THP1 cells incubated with 0.06 mM modified insulin 17 for 4 h. Scale bar: 20 μm.

Scheme 1.. Synthesis of bicyclic [3.3.1] framework under mild conditions.^a

[a] Reaction conditions: compound 2 (0.364 mmol) was dissolved in THF (0.4 mL), THF/PBS buffer (1: 9, v/v), rt, 10 h.

Scheme 2.. One-pot bioconjugation with different azido compounds.^{a, b}

[a] Reaction conditions: 0.1 equivalent of CuSO₄, 0.2 equivalent of sodium ascorbate, 0.1 equivalent of TBTA and 2 equivalents of 1, 1.2 equivalent of glycine methyl ester hydrochloride and 1.2 or 2.4 equivalent of azide were used. [b] Isolated yields.

Scheme 3.. Second cargo incorporation of mono-clicked products.^{a, b}

[a] Reaction conditions: 0.1 equivalent of CuSO₄, 0.2 equivalent of sodium ascorbate, 0.1 equivalent of TBTA and 1.5 equivalents of azido compound were used. [b] Isolated yields. [c] Reaction was stirred for 30 h.