Copper-free Click Chemistry to Crosslink Azides and Amines

Quanta BioDesign’s monodispersed, discrete PEG (dPEG®) products include a wide range of useful tools for click chemistry. Most of our reagents are designed to enable copper-free click chemistry, which is essential for biocompatible labeling and crosslinking. Copper-free click chemistry that allows crosslinking of an amine to an azide is the subject of this post.

The Discovery of Copper-Free Click Chemistry

This post provides a short synopsis of the discovery of copper-free click chemistry. For a comprehensive review of click chemistry and its applications with dPEG® compounds.

Copper-free click chemistry is an indispensable tool in the toolbox of biotechnologists worldwide. Carolyn Bertozzi and coworkers discovered copper-free click chemistry (formally known as strain-promoted azide-alkyne cycloaddition, or SPAAC) in 2004. Bertozzi was looking for bioconjugation techniques that were both bioorthogonal and biocompatible (1). The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction independently discovered in 2002 by Meldal’s group (2) and Sharpless and coworkers (3) uses copper(I) salts that are often toxic to live cells. The cytotoxicity of these copper salts spurred the development of less toxic and non-toxic alternatives that are biocompatible.

Bertozzi developed SPAAC after reading a 1961 paper by Wittig and Krebs that reported that phenyl azide and cyclooctyne react violently (4). From that clue, the Bertozzi lab developed and tested cyclooctyne derivatives to find a compound with optimal stability, water solubility, and reactivity with azides. Other labs joined the search, and today several different strained cyclooctyne compounds find use in copper-free click chemistry reactions.

Quanta BioDesign’s Copper-Free Click Chemistry Products for Crosslinking Amines with Azides

To conjugate a dPEG® crossbridge to an azide via copper-free click chemistry, Quanta BioDesign chose the strained cyclooctyne known as DBCO (for dibenzylcyclooctyne; also known as DIBAC, or ADIBO) See Figure 1. Researchers in the laboratory of Floris van Delft at Radboud University Nijmegen, The Netherlands, developed DBCO (5). The compound has broad solubility, high reactivity with azides, and excellent stability. To DBCO, Quanta BioDesign conjugates various lengths of amino-dPEG®-acid spacers to create a water-soluble, amine-reactive, and copper-free click chemistry reagent. We then functionalize the acid with 2,3,5,6-tetrafluorophenol (TFP), forming the active TFP ester for amine reactivity.

Figure 1: Dibenzylcyclooctyne (DBCO) for strain-promoted azide-alkyne cycloaddition (SPAAC), also known as copper-free click chemistry. The formal IUPAC name of this compound is 5-{2-azatricyclo[,9]hexadeca-1(16),4(9),5,7,12,14-hexaen-10-yn-2-yl}-5-oxopentanoic acid. CAS number 1207355-31-4. PubChem compound ID: 85470609. ChemSpider ID: 32741547.

For conjugating dPEG® products to amines, we much prefer TFP esters over N-hydroxysuccinimidyl (NHS) esters based on published performance data. Research shows that TFP esters are more hydrolytically stable and more reactive with amines than NHS esters (6). The use of TFP esters also generally results in higher yields of amide bond formation than NHS esters. We at Quanta BioDesign have also published our findings of TFP esters’ performance compared to NHS esters. Please see TFP Esters Have More Hydrolytic Stability and Greater Reactivity than NHS Esters for information.


Quanta BioDesign currently offers three dPEG® products for crosslinking azides with amines. These three products have progressively longer dPEG® spacers to match the intended application.

Figure 2: General chemical structure of Quanta BioDesign’s DBCO-dPEG® TFP ester products for crosslinking azides and amines. Please click the links below to go to the product pages for the individual compounds.

Product Number 11362, DBCO-dPEG®4-TFP ester

Product Number 11366, DBCO-dPEG®12-TFP ester

Product Number 11370, DBCO-dPEG®24-TFP ester 


Product number 11362 has the shortest dPEG® spacer and, consequently, very slight water solubility because the functional groups on each end are highly hydrophobic. Product number 11366 has an intermediate-length dPEG® spacer and is sparingly soluble in water. Dilute solutions (mM concentrations) in water are possible, but concentrated solutions may prove challenging to achieve. The use of a water-miscible solvent is recommended for both of these products.

In typical applications with these products, the TFP ester end should be reacted with an amine before the DBCO end is reacted with an azide. However, the user must determine the correct order of operations for any crosslinking experiment empirically. Each reaction is unique, and we at Quanta BioDesign have no exact method of predicting what order of operations is optimal for your crosslinked biomolecules.

For your next research project, consider Quanta BioDesign’s copper-free click chemistry products.


(1) Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. A Strain-Promoted [3 + 2] Azide−Alkyne Cycloaddition for Covalent Modification of Biomolecules in Living Systems. J. Am. Chem. Soc. 2004126(46), 15046–15047.

(2) Tornøe, C. W.; Christensen, C.; Meldal, M. Peptidotriazoles on Solid Phase:  [1,2,3]-Triazoles by Regiospecific Copper(I)-Catalyzed 1,3-Dipolar Cycloadditions of Terminal Alkynes to Azides. J. Org. Chem. 200267 (9), 3057–3064.

(3) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective “Ligation” of Azides and Terminal Alkynes. Angewandte Chemie International Edition 200241(14), 2596–2599.<2596::AID-ANIE2596>3.0.CO;2-4.

(4) Wittig, G.; Krebs, A. Zur Existenz niedergliedriger Cycloalkine, I. Chemische Berichte 196194(12), 3260–3275.

(5) Dommerholt, J.; Rutjes, F. P. J. T.; van Delft, F. L. Strain-Promoted 1,3-Dipolar Cycloaddition of Cycloalkynes and Organic Azides. Top Curr Chem (Z) 2016374(2), 16.

(6) (a) Wang, J.; Zhang, R.-Y.; Wang, Y.-C.; Chen, X.-Z.; Yin, X.-G.; Du, J.-J.; Lei, Z.; Xin, L.-M.; Gao, X.-F.; Liu, Z.; Guo, J. Polyfluorophenyl Ester-Terminated Homobifunctional Cross-Linkers for Protein Conjugation. Synlett 201728(15), 1934–1938. (b) Lockett, M. R.; Phillips, M. F.; Jarecki, J. L.; Peelen, D.; Smith, L. M. A Tetrafluorophenyl Activated Ester Self-Assembled Monolayer for the Immobilization of Amine-Modified Oligonucleotides. Langmuir 200824(1), 69–75.


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