Glutathione Mediates Control of Dual Differential Bio-orthogonal Labelling of Biomolecules

Abstract

Traditional approaches to bio-orthogonal reaction discovery have focused on developing reagent pairs that react with each other faster than they are metabolically degraded. Glutathione (GSH) is typically responsible for the deactivation of most bio-orthogonal reagents. Here we demonstrate that GSH promotes a Cu-catalysed (3+2) cycloaddition reaction between an ynamine and an azide. We show that GSH acts as a redox modulator to control the Cu oxidation state in these cycloadditions. Rate enhancement of this reaction is specific for ynamine substrates and is tuneable by the Cu:GSH ratio. This unique GSH-mediated reactivity gradient is then utilised in the dual sequential bio-orthogonal labelling of peptides and oligonucleotides via two distinct chemoselective (3+2) cycloadditions.

Keywords: CuAAC; bio-orthogonal chemistry; ligation; oligonucleotide; peptide.

Figure 1

(a) CuAAC as a bio‐orthogonal tool in chemical biology. (b) Design concept: Controlling ynamine reactivity as a function of Cu:GSH ratio. (c) Sequential labelling of biomolecules exploiting conditional ynamine reactivity mediated by GSH:Cu.

Figure 2

(a) GSH acts as a Cu ligand and redox mediator. (b) Formation of thio‐alkene adducts (2–3) by the reaction of ynamine (1 a, (200 μM) with GSH (1–10 mM) in Dulbecco's phosphate‐buffered saline (DPBS) containing 10 % methanol (MeOH). (c) Stability of 1 a–d (200 μM) in the presence of GSH (10 mM). Error bars correspond to the standard deviation of three replicate experiments. All analyses conducted using reverse phase high pressure liquid chromatography (RP‐HPLC).

Figure 3

Influence of [GSH] and NaAsc on the formation of 5 a. Reaction conditions: (black) 1 a (200 μM), 4 a (500 μM), Cu(OAc)₂ (350 μM), 10 % MeOH, 1X DPBS, rt. (red)+GSH (1 mM). (blue)+NaAsc (1 mM). Shaded error bands correspond to the standard deviation of three replicate experiments.

Figure 4

Reaction kinetics of triazole formation using benzyl azide. (a), azidoethanol (b), and picolyl azide (c). Reaction conditions: 1 a–d (200 μM), 4 a–c (500 μM), Cu(OAc)₂ (350 μM), GSH (1 mM), 10 % MeOH, 1X DPBS, rt. * Addition of NaAsc (1 mM). Shaded error bands correspond to the standard deviation of three replicate experiments.

Figure 5

(a) DoE analysis as a function of conversion to triazole (5 a). Reaction conditions: 1 a (200 μM), 4 a (500 μM), Cu(OAc)₂ (100–400 μM), GSH (100 μM‐1 mM), 10 % MeOH in 1X DPBS, rt. (b) Organic co‐solvent effects on the formation of 5 a. Reaction conditions: 1 a (200 μM), 4 a (500 μM), Cu(OAc)₂ (100 μM), GSH (100 μM), 1X DPBS in 10 % organic co‐solvent. Shaded error bands correspond to the standard deviation of three replicate experiments. (c) EPR spectrum of the ynamine‐azide (3+2) cycloaddition in HFIP:H₂O (1 : 9).

Figure 6

(a) CuAAC reaction of azide‐modified CPPs (6–7) and ynamine (8) catalysed by Cu(OAc)₂ and GSH. (b) Reaction profile of the formation of 9 (red) and 10 (grey). Reaction conditions: 8 (200 μM), 6/7 (200 μM), Cu(OAc)₂ (100 μM), GSH (100 μM), 10 % HFIP in 1X DPBS, rt. (c) Reaction profile of the formation of 9 using different co‐solvents (10 % co‐solvent) in 1X DPBS. Shaded error bands correspond to the standard deviation of three replicate experiments.

Figure 7

(a) Sequential and selective modification of a double azide labelled CPP (12). Reaction conditions: (i) 12 (200 μM), 8 (220 μM), 11 (220 μM), Cu(OAc)₂ (500 μM), GSH (500 μM), 20 % HFIP in 1X DPBS, rt, 2 h; (ii) EDTA (5 mM), 4 h. (b) Reaction profile of the formation of mono‐functionalised CPP (13, red), followed by the di‐functionalised CPP (14, blue) from 12 (black). Shaded error bands correspond to the standard deviation of three replicate experiments. (c) RP‐HPLC trace of the reaction mixture after 6 h.

Figure 8

(a) Sequential modification of a dual‐alkyne modified ODN (16). Reaction conditions: (i) 16 (20 μM), 15 (20 μM), 5 % HFIP in 1X DPBS (20 mM MgCl₂), rt, 1 h; (ii) 18 (30 μM), Cu(OAc)₂ (50 μM), GSH (50 μM), 2 h. (b) RP‐HPLC time course of the sequential, dual modification of 16 to form 19 via 17. Shaded error bands correspond to the standard deviation of three replicate experiments. (c) RP‐HPLC trace of the reaction mixture after 3 h.