Alkyne PEG

Click Chemistry Protocols

Introduction

Click Chemistry was defined by Sharpless and co-workers in 2001 to describe pairs of functional groups that rapidly and selectively react (“click”) with each other in mild, aqueous conditions. The Click Chemistry concept has been expanded to biolabeling, chemical biology field and pharmaceutical R&D due to its mild condition as well as high selectivity.

There are three types of Click Chemistry in general:

1. Cu(I)- catalyzed Azide - Alkyne Click Chemistry reaction (CuAAC)

Classic Click Chemistry uses copper as a catalyst, which requires an appropriate chelating ligand such as TBTA and THPTA. TBTA covers some of the practical applications for Click Chemistry except for aqueous conjugation reactions and THPTA further simplifies Click Chemistry by allowing the entire reaction to be run in water, affording biological compatibility for Click reactions.

2. Strain-promoted Azide - Alkyne Click Chemistry reaction (SPAAC)

CuAAC reactions are limited in applications due to the requirement of a cytotoxic copper catalyst. A Copper free non-toxic labeling method of Azides is the Strain-Promoted Azide - Alkyne Click Chemistry (SPAAC) reaction. SPAAC reactions has a remarkably decreased activation energy by using strained cyclooctynes without requiring an exogenous catalyst.

3. Tetrazine – Alkene Ligation

The Tetrazine - Alkene Ligation is ideally suited for in vivo cell labeling as well as low concentration applications. A tetrazine functionalized molecule can react with a terminal or strained Alkene molecule to form a stable conjugate via a Dihydropyrazine moiety, which is considered as non-toxic biomolecule labeling method.

Example Protocols:

This section contains some general protocols for click reactions.

Note: These protocols may be referenced as a starting point to further optimize your own Click Chemistry procedures.

Labeling of Antibodies (Copper free)

1.     Prepare the azide-attached antibody in sodium phosphate buffer.

2.     Via SPAAC click chemistry, azide-modified antibody (400 μg in 1 mL TBS buffer) was incubated with DBCO/DIBO/BCN moiety bearing molecule (200 μL of a 2 mM solution in DMSO) for optimized time at room temperature.

3.     The final product can be purified via size exclusion chromatography or other protocols as needed.

Reference: Zeglis et al. (2013), Bioconjugate Chemistry 24(6):1057

Labeling of Oligonucleotides and DNA (Copper catalyzed)

1. Freshly prepare the following ‘Click Solution’:
• 200 mM THPTA ligand in water
• 100 mM CuSO4 in water
• alkyne labeled oligo in water
• 100 mM sodium ascorbate in water
• 10 mM azide in DMSO/tBuOH or water.
2. Before the reaction, incubate CuSO4 with THPTA ligand in a 1:2 ratio for several minutes.
3. To the alkynyl functionalized oligo/DNA solution, add an excess of azide (4-50 eq) and 25 equivalents of THPTA/CuSO4 mixture.
4. Incubate the mixture at room temperature for 30-60 minutes.
5. The final oligo product can be purified by ethanol precipitation or other methods as needed.