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, 2018, 3152870
eCollection

A Simple Approach to Bioconjugation at Diverse Levels: Metal-Free Click Reactions of Activated Alkynes With Native Groups of Biotargets Without Prefunctionalization

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A Simple Approach to Bioconjugation at Diverse Levels: Metal-Free Click Reactions of Activated Alkynes With Native Groups of Biotargets Without Prefunctionalization

Xianglong Hu et al. Research (Wash D C).

Abstract

The efficient bioconjugation of functional groups/molecules to targeted matrix and bio-related species drives the great development of material science and biomedicine, while the dilemma of metal catalysis, uneasy premodification, and limited reaction efficiency in traditional bioconjugation has restricted the booming development to some extent. Here, we provide a strategy for metal-free click bioconjugation at diverse levels based on activated alkynes. As a proof-of-concept, the abundant native groups including amine, thiol, and hydroxyl groups can directly react with activated alkynes without any modification in the absence of metal catalysis. Through this strategy, high-efficient modification and potential functionalization can be achieved for natural polysaccharide, biocompatible polyethylene glycol (PEG), synthetic polymers, cell penetrating peptide, protein, fast whole-cell mapping, and even quick differentiation and staining of Gram-positive bacteria, etc. Therefore, current metal-free click bioconjugation strategy based on activated alkynes is promising for the development of quick fluorescence labeling and functional modification of many targets and can be widely applied towards the fabrication of complex biomaterials and future in vivo labeling and detection.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Schematic illustration for the metal-free click bioconjugation with native groups of biotargets based on activated alkynes. Native functional groups, including amine, thiol, and hydroxyl groups in different targets at diverse levels are employed to achieve on-demand modification of natural/synthetic polymers, peptide, protein, rapid whole cell mapping, and specific staining of Gram-positive bacteria.
Figure 2
Figure 2
Schematic illustration for metal-free amino-yne click bioconjugation between chitosan and PEG with fluorescent activated alkynes. (a) Functionalization of natural polymers via the amino-yne click reaction. (b) Photographs of chitosan, Chit-TPA, and Chit-TPE in the presence of UV irradiation or not. (c) Synthetic rout of PEGylation of triphenylamine (TPA) via the amino-yne click reaction. (d) Hydrodynamic diameter distribution and TEM image (inset) determined for the aqueous dispersion of PEG-TPA. (e) Fluorescent emission spectrum of PEG-TPA aqueous dispersion (λex= 405 nm, insert photographs: the water dispersion under white light and UV irradiation). (f) CLSM images of EMT-6 cells upon incubating with the aqueous dispersion of PEG-TPA at 5 μM for 4 h and cell nucleus were costained with Red dot1.
Figure 3
Figure 3
Schematic illustration for metal-free thiol-yne and hydroxyl-yne reactions to modify synthetic polymer and polysaccharide. (a) Modification of synthetic polymers derived from reversible addition-fragmentation chain-transfer (RAFT) polymerization. General synthetic route for the terminal modification with activated alkynes, as an illustration, affording PDMA-TPE. (b) Typical PL spectra of PDMA-TPE in water-THF mixtures with different water contents and (c) change of PL maximum of PDMA-TPE with water content of the aqueous mixture (insets: photographs of PDMA-TPE dispersion samples under UV illumination). (d) Synthetic route employed for the modification of hydroxypropyl cellulose (HPC) via the hydroxyl-yne reaction.
Figure 4
Figure 4
Metal-free functionalization of peptide and protein. (a) Schematic illustration for the thiol-yne click conjugation of Tat peptide with TPA, affording one cell-penetrating peptide conjugate, Tat-TPA. The sequence of Tat peptide is YGRKKRRQRRR. (b) Fluorescent emission spectrum of Tat-TPA (λex= 405 nm, insert photographs: Tat-TPA aqueous dispersion under white light and UV irradiation). (c) CLSM images of EMT-6 cells upon 30 min incubation with the aqueous dispersion of Tat-TPA and TPA at 2 μM. No detectable fluorescence was observed for cells incubated with TPA. (d) Schematic illustration for the click conjugation of BSA with TPA, affording BSA conjugates, BSA-TPA. (e) Hydrodynamic diameter distribution of BSA and BSA-TPA in water (Inset: TEM image determined for BSA-TPA in water). (f) Photographs of BSA-TPA aqueous dispersion under white light and UV irradiation. (g) SDS-PAGE results recorded for BSA and BSA-TPA, and lower row image was taken under UV irradiation.
Figure 5
Figure 5
Rapid whole-cell labeling and mapping of HeLa cells upon 2 min incubation with activated alkynes. HeLa cells were quickly treated with three small fluorescent molecules, respectively, including TPA, alkyne-TPA, and the addition product of alkyne-TPA and n-butylamine.
Figure 6
Figure 6
Specific labeling of Gram-positive bacteria upon 2 min incubation with activated alkynes. Two kinds of Gram-positive bacteria including Staphylococcus aureus (S. aureus) and Bacillus subtilis (B. subtilis) were incubated with three small fluorescent molecules, respectively, including TPA, alkyne-TPA, and the addition product of alkyne-TPA and n-butylamine.

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