Tripodal Amine Ligands for Accelerating Cu-Catalyzed Azide-Alkyne Cycloaddition: Efficiency and Stability against Oxidation and Dissociation

Abstract

Ancillary ligands, especially the tripodal ligands such as tris(triazolylmethyl)amines, have been widely used to accelerate the Cu-catalyzed azide-alkyne cycloaddition (CuAAC, a "click" reaction). However, the relationship between the activity of these Cu(I) complexes and their stability against air oxidation and ligand dissociation/exchange was seldom studied, which is critical for the applications of CuAAC in many biological systems. In this work, we synthesized twenty-one Cu(I) tripodal ligands varying in chelate arm length (five to seven atoms), donor groups (triazolyl, pyridyl and phenyl), and steric hindrance. The effects of these variables on the CuAAC reaction, air oxidation, and ligand dissociation were evaluated. Reducing the chelate arm length to five atoms, decreasing steric hindrance, or using a relatively weakly-binding ligand can significantly increase the CuAAC reactivity of the Cu(I) complexes, but the concomitant higher degree of oxidation cannot be avoided, which leads to rapid degradation of a histidine-containing peptide as a model of proteins. The oxidation of the peptide can be reduced by attaching oligo(ethylene glycol) chains to the ligands as sacrificing reagents. Using electrospray ionization mass spectrometry (ESI-MS), we directly observed the tri- and di-copper(I)-acetylide complexes in CuAAC reaction in the [5,5,5] ligand system and a small amount of di-Cu(I)-acetylide in the [5,5,6] ligand system. Only the mono-Cu(I) ligand adducts were observed in the [6,6,6] and [5,6,6] ligand systems.

Keywords: ascorbic acid; copper(I) ligands; copper-catalyzed alkyne-azide cycloaddition; dissociation constant; oxidation.

Fig. 1

Structural formulas of tripodal tris(triazolylmethyl)amine ligands for bioconjugation reaction.

Fig. 2

Structural formulas of Cu(I) ligands (1–21). The ligands are classified according to 1) donor types: [Tr,Tr,Tr] for 1–5, [Tr,Tr,Py] for 8, [Tr,Py,Py] for 9, [Py,Py,Py] for 10 and 11, [Py,Py,Ph] for 12–16, and [Tr,Tr,Ph] for 6 and 17–21, where Tr = triazoyl, Py = pyridyl, and Ph = phenyl; 2) chelate arm lengths: [6,6,6] for 2, 8–10, 12–14, 17 and 18, [5,5,5] for 1, 6, 11 and 21, [5,5,6] for 4 and 20, [5,6,6] for 5, 15, 16 and 19, and [7,7,7] for 3; and 3) methylation as in 13–16 and 18–21 to enhance the steric hindrance and electron donating property.

Fig. 3

Performance of the 21 ligands (1–21) of Cu⁺. (A) Apparent second-order rate constant (k_obs) for the reaction of azido-coumarin AC (100 µM) with propargyl alcohol (50 µM) in the presence of the ligand (100 µM), CuSO₄ (100 µM), and sodium ascorbate (5 mM) in phosphate buffered saline (PBS, pH = 7.4) in air at 24 ± 1 °C during 60 min.^– (B) The initial air-oxidation rate (V_o) of sodium ascorbate (200 µM) by diffused O₂ in the presence of ligand/Cu(I) 1:1 complexes (100 µM) in PBS (pH = 7.4) at 24 ± 1 °C. (C) Dissociation constants of the Cu(I)-ligand complexes measured by competitive binding assay with Bca. (D) The trade-off between CuAAC over oxidation (k_obs/V_o). (E) The trade-off between CuAAC over dissociation constants (k_obs/K_D). Error bars represent the standard deviation from three-repeated measurement. [m,n,o] represents the chelate arm length. Me in [m,n,o-Me] represents the methyl substitution on the methylene group between central amine and phenyl arm, for 13, 15, 18–21. Me2 in [m,n,o-Me2] represents the methyl substitutions on the 6-position of pyridyl arms, for 14 and 16.

Fig. 4

DFT (B3LYP/LANL2DZ)-optimized geometries of the Cu(I) complexes bearing 1, 2, 3, 5, 10, 11 ligands (A–F, respectively), in which the EG₄ group was replaced with a methyl group. Red: Cu, blue: N, gray: C. Hydrogen atoms were omitted for clarity. The average of the three bond angles between N(1)−Cu−N(2), oxidation rate V_o (10⁻⁸ M s⁻¹), and CuAAC second-order rate constants k_obs (M⁻¹s⁻¹) were labeled on each complex.

Fig. 5

Species corresponding to the assigned ESI-MS peaks obtained after mixing the alkyne with the ligand [Tr,Tr,Tr] 1, 2, 4 or 5, and CuSO₄/Na-ascorbate. * and ** were formed by adventitious oxidation of the OEG side chain in the alkyne or the ligand during sample introduction to the ESI-MS. ESI-MS spectra of the Cu(I)-ligand-alkyne 1:1:0.5 mixture in water. CuSO₄ (100 µM), ligand (100 µM), PE (50 µM), Na ascorbate (1 mM). (A) Ligand 1. (B) Ligand 2. (C) Ligand 4. (D) Ligand 5.

Fig. 6

Recovery (%) of the azido-peptide peptides AP (red) and the triazolyl-peptide TP (blue) during CuAAC reaction of AP in air with propargyl alcohol. Reaction conditions: the azido-peptide AP (50 µM), propargyl alcohol (50 µM), ligand (100 µM), CuSO₄ (50 µM), and sodium ascorbate (500 µM) in Milli-Q water at 24 ± 1 °C. There was no ligand added in the (−) control. The red and blue bars represent the % amount of AP and TP relative to the amount of AP starting material, respectively, measured by LC-MS. The total represents the % peptide starting material and product withstanding the oxidation under the CuAAC conditions. Each data point was the mean of three replicates and the error bar represented the standard deviation.

Fig. 7

Total ion mass spectrum of ligand 1 fragments (total ion). The spectrum was obtained from the 60 min CuAAC reaction mixture containing the peptides by zoom in the region of m/z = 520–840 and retention time from 5–7 min. The experimental condition is given in ESI. The oligo ethylene glycol (OEG) unit is designated as the [m,n,l] system where m, n, l = 1–4. The labeled peaks were summarized in Table S4, ESI (Red: alcohol derivatives; Yellow: aldehyde derivatives; Blue: acid derivatives; Green: ether derivatives).^, The single and multiple labeling represents the single and multiple components of the ligand fragments, respectively.

Scheme 1

Reactions for the measurement of (A) CuAAC activity; (B) initial rate of oxidation; and (C) K_D of the Cu(I) complexes with the ligands listed in Fig. 2.

Scheme 2

The CuAAC reaction of the peptide AP (50 µM) with propargyl alcohol (50 µM), ligand (100 µM), CuSO₄ (50 µM), and sodium ascorbate (500 µM) in Milli-Q water at 24 ± 1 °C in air to form the product TP and the oxidation byproducts (2-imidazolidone derivatives) AP^I/II/III, TP^I/II/III as well as a complex mixture of other oxidation byproducts.

Scheme 3

Proposed mechanism of CuAAC and Cu(I) oxidation. L: ligands; L’: ligands or alkyne; Aky: alkyne; ROS: reactive oxygen species; OEG: oligo ethylene glycol side arms from ligands.