Three Click Chemistry Crosslinking Ideas with dPEG®

Have you ever been working your way through a product catalog when you’ve come across a product you weren’t sure how to use? I know I have. In those “What do I do with this?” moments, tips that explain how to use a product effectively really help me. In this post, I offer three ideas on how to use one of our click chemistry crosslinking products in various applications. These suggestions extend to other products in the same line. Also, I hope that it stimulates your thinking about other areas of click chemistry with Quanta BioDesign’s discrete PEG (dPEG®) products.

What is click chemistry?

Barry Sharpless coined the term click chemistry in 1998 (1). He specified that these reactions are:

  • high yielding,
  • stereospecific,
  • modular, and,
  • “wide in scope.”

According to Sharpless, click chemistry reactions must “generate only inoffensive byproducts that can be removed by nonchromatographic methods….” Moreover, such reactions must be simple to perform with readily available starting materials and reagents. Additionally, they must be able to be conducted in benign solvents, such as water, or easily removable solvents. The resulting product “must be stable under physiological conditions” (1). Sharpless identified four classifications of click chemistry reactions. These categories are:

  • cycloadditions, such as the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), but also hetero Diels-Alder cycloadditions;
  • nucleophilic ring openings;
  • non-aldol type carbonyl chemistry, and
  • additions to carbon-carbon multiple bonds, such as thiol-ene and thiol-yne reactions (2-6).

Click chemistry has become enormously important in chemical synthesis since Sharpless’s original paper. In fact, surveys of the chemical literature show that click chemistry has revolutionized drug discovery and development and many other areas of chemistry (see references 2 – 4).

Why use click chemistry?

Click chemistry solves many synthetic chemistry problems. Indeed, numerous compounds and macromolecular constructs can be synthesized via click chemistry. These products include organic and organometallic nanoparticles, dendrimers, small molecule drugs, peptide drugs, and antibody-drug conjugates (ADCs).

Also, because it uses benign or easily removable solvents, click chemistry qualifies as environmentally friendly “green chemistry” (7). As a matter of fact, typical click chemistry reactions run faster in water than in organic solvents (1). Research and experience demonstrate that click chemistry can be a highly useful synthetic process for reactions that require aqueous environments, such as cell-based assays (8). Unfortunately, some types of click chemistry – for example, CuAAC – require cytotoxic metal salts. However, strain-promoted azide-alkyne cycloaddition (SPAAC), avoids these harmful salts.

Click chemistry crosslinking ideas using dPEG® reagents.

Figure 1 shows the structure of PN10524, azido-dPEG®11-amine. Quanta BioDesign manufactures and sells this click chemistry crosslinking product. An azide group and a primary amine reside on opposite ends of a single molecular weight PEG linker (molecular weight of 570.67 Daltons; linker length of 36 atoms, ≈42.7 Å).

Figure 1: PN10524, Azido-dPEG®11-amine, one of Quanta BioDesign’s click chemistry crosslinking reagents.

This product crosslinks carboxylic acids with alkyne-containing compounds. The amine end of the molecule forms amide bonds with carboxyl groups directly using a carbodiimide such as EDC. More commonly, the amide bond forms by reacting the amine with an active ester such as an N-hydroxysuccinimidyl (NHS) or 2,3,5,6-tetrafluorophenyl (TFP) ester. On the opposite end of the linker, the azide group reacts with alkyne-containing molecules via click chemistry reactions such as CuAAC or SPAAC. Please see Scheme 1 below.

Scheme 1: Click chemistry crosslinking reactions using PN10524, azido-dPEG®11-amine. In this scheme, both copper-catalyzed and copper-free click chemistry reactions are shown.

So, how can you use this product effectively? Here are three suggestions.

1. Surface modification

Most scientists have encountered the problem of non-specific interactions. Proteins, peptides, and nucleic acids stick randomly to uncoated metal, plastic, or glass surfaces, and the outcome can be frustrating. This problem complicates Western blots, ELISAs, the analysis of column fractions collected in plastic or glass tubes, and many other areas of study. Several methods exist to reduce or eliminate these non-specific interactions. These methods include coating glass surfaces with sugars (9), silanization (9a), and many types of PEGylation (10, 11).

Suppose, though, that you want to eliminate non-specific interactions and, at the same time, coat your surface with a reactive coating that allows for further modification? Using the example crosslinker, azido-dPEG®11-amine, you functionalize the surface with carboxylic acid groups, activate them, and then react the crosslinker with the activated surface. The result is shown in Figure 2.

Figure 2: Surface coating with PN10524, Azido-dPEG®11-amine.

A dense coating of dPEG® molecules reduces or eliminates non-specific binding. This coating, though, leaves many closely-spaced azide groups sticking up from the surface. These azide groups can react with some target molecules (a small molecule, peptide, or protein into which an alkyne group has been installed). However, steric hindrance (crowding) prevents most of the azide groups from reacting with the target alkyne. The unreacted azides are effectively wasted.

A better way to coat the surface is to mix the crosslinker with a methoxy-terminated dPEG® amine. In this case, I recommend mixing PN10278, m-dPEG®8-amine (Figure 3) in a ratio of >3:1 with azido-dPEG®11-amine crosslinker. At 29.7 Å, m-dPEG®8-amine is shorter than azido-dPEG®11-amine (44.2 Å), resulting in the construct shown in Figure 4.

Figure 3: PN10278, m-dPEG®8-amine.
Figure 4: Mixed surface coating of PN10524, azido-dPEG®11-amine, and PN10278, m-dPEG®8-amine. See the text for a discussion of the rationale for creating this construct. Compared to Figure 2, above, the surface azide groups in this construct are less crowded.

This type of coating reduces or eliminates non-specific binding. Fewer azide groups on the surface result in less crowding of the subsequent alkyne reactants. In turn, reduced steric crowding facilitates the introduction of large molecules such as peptides and proteins onto the surface. Thus, the decreased steric hindrance may prove particularly advantageous when carrying out SPAAC click chemistry with cyclooctyne groups that are often bulky.

2. Protein/Peptide/Small Molecule Crosslinking

Professor Ravi S. Kane, then at Rensselaer Polytechnic Institute, currently Professor, Garry Betty/V Foundation Chair and GRA Eminent Scholar in Cancer Nanotechnology, at Georgia Institute of Technology, used structure-based design to develop a heptavalent anthrax toxin inhibitor (12). The inhibitor consisted of a seven-membered β-cyclodextrin core. The primary hydroxyl groups of the β-cyclodextrin core were then converted to terminal alkyne groups by reaction with propargyl bromide. Using copper-catalyzed click chemistry, Professor Kane’s group reacted the heptavalent alkyne with azido-dPEG®11-amine in high yield. They next reacted the free amines first with chloroacetic anhydride and then with a peptide that modeling studies strongly suggested would inhibit the formation of anthrax toxin. See Figure 5.


Figure 5: The reaction scheme used to develop a heptavalent anthrax toxin inhibitor. See the text for details and a discussion. This image is taken from reference 12, below, and is reprinted by permission, copyright 2011, American Chemical Society.

The result was a well-defined macromolecular structure with precise spatial control that appeared from modeling studies to be able to inhibit the formation of anthrax toxin. Please see Figure 6.

Figure 6: A heptavalent anthrax toxin inhibitor built by structure-based design using Quanta BioDesign’s PN10524, azido-dPEG®11-amine. See the text for a detailed discussion. This image is taken from reference 12, below, and is reprinted by permission, copyright 2011, American Chemical Society.

Indeed, in testing the heptavalent anthrax toxin inhibitor, six out of seven rats treated with the inhibitor and exposed to anthrax did not develop anthrax. Conversely, rats exposed to anthrax and either not treated with inhibitor or treated with a sham inhibitor developed anthrax symptoms and died.

CuAAC is a simple reaction that gives high yields with minimal byproducts. This research shows the power of using click chemistry with a dPEG® reagent to exert spatial control over macromolecular product design. The precise spatial control needed to position the inhibiting peptide from the β-cyclodextrin core would not have been possible with a dispersed PEG. Only with a single molecular weight PEG (that is, a dPEG®) could that kind of control been obtained. Having a single molecular weight PEG product allows greater control over product design and purity. Also, compared to traditional, dispersed PEGs, a dPEG® compound simplifies product analysis. This research (among others) proves that point.

3. Atomic Force Microscopy

Atomic Force Microscopy (AFM) is a type of scanning probe microscopy with a resolution to fractions of a nanometer. One use of AFM measures the force between a probe and a sample. This use of AFM is known as force spectroscopy.

In single-molecule AFM force spectroscopy (SMFS), compounds of interest are attached by linkers to the cantilevers used to measure the force. Each cantilever attaches a single molecule (13), hence the name.

PEG is the most commonly used linker for attaching biomolecules to cantilevers (14-16). However, PEG polymers are dispersed and have varied chain lengths (15, 16). Thus, traditional PEG linkers are less than ideal for SMFS.

A 2012 Master’s Thesis by Jamie Maciaszek in the lab of Yuri L. Lyubchenko reported that linkers containing a single molecular weight and chain length were superior to traditional, dispersed PEG linkers (17). Moreover, using PN10524, azido-dPEG®11-amine, SMFS research in the lab of George Lykotrafitis detected and quantitatively mapped individual calcium-activated small conductance (SK) potassium channels in living neurons. The researchers joined the amine end of the crosslinker to APTES-activated silicon nitride cantilevers. Click chemistry crosslinking then linked the molecules of interest with the resulting azide-coated surface. The dPEG® linker was necessary for the research because it had no chain length heterogeneity (18).


Click chemistry crosslinking using Quanta BioDesign’s dPEG® products improves applications such as surface modification, macromolecular construction, and AFM (including SMFS). In this post, I showed how product number 10524, azido-dPEG®11-amine, is useful for all of these purposes. We make and sell this type of crosslinker in five sizes ranging from dPEG®3 (15.4 Å) to dPEG®35 (129.0 Å). Yo.

Buy the Products Mentioned

Please click the links below to go to the product pages of the products discussed in this post.

PN10278, m-dPEG®8-amine

PN10524, azido-dPEG®11-amine


1. Hartmuth C. Kolb, M. G. Finn, and K. Barry Sharpless. Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angew. Chem. Int. Ed. (2001), 40, 2004-2021.

2. Hartmuth C. Kolb and K. Barry Sharpless. The Growing Impact of Click Chemistry on Drug Discovery. Drug Discovery Today (December 2003), 8(24), 1128-1137.

3. John E. Moses and Adam D. Moorhouse. The growing applications of click chemistry. Chem. Soc. Rev. (2007), 36, 1249-1262.

4. Christopher D. Hein, Xin-Ming Liu, and Dong Wang. Click Chemistry, a Powerful Tool for Pharmaceutical Sciences. Pharm Res (October 2008), 25(10), 2216-2230.

5. Charles E. Hoyle and Christopher N. Bowman. Thiol-Ene Click Chemistry. Angew. Chem. Int. Ed. (2010), 49(9), 1540-1573.

6. Charles E. Hoyle, Andrew B. Lowe, and Christopher N. Bowman. Thiol-click chemistry: a multifaceted toolbox for small molecule and polymer synthesis. Chem. Soc. Rev. (2010), 39, 1355-1387.

7. Chao-Jun Li and Barry M. Trost. Green chemistry for chemical synthesis. Proc. Nat. Acad. Sci. (September 9, 2008), 105(36), 13197-13202.

8. Greg T. Hermanson. “Chemoselective Ligation; Bioorthogonal Reagents,” in Bioconjugate Techniques, 3rd edition. New York: Academic Press, 2013, page 771. We at Quanta BioDesign recommend Greg’s book to all of our customers. You can buy it from us. To get started, please click this link and then click “Add to Cart” to order the book.

9. Gangadhar Jogikalmath. Method for blocking non-specific protein binding on a functionalized surface. US 20080213910 A1, September 4, 2008.

9a.    Nick R. Glass, Ricky Tjeung, Peggy Chan, Leslie Y. Yeo, and James R. Friend. Organosilane deposition for microfluidic applications. Biomicrofluidics (2011), 5(3), 036501–036501-7. DOI: 10.1063/1.3625605. PMCID: PMC3364836.

10. Jacob Piehler, Andreas Brecht, Ramūnas Valiokas, Bo Liedberg , and Günter Gauglitz. A high-density poly(ethylene glycol) polymer brush for immobilization on glass-type surfaces. Biosensors & Bioelectronics (2000), 15, 473–481.

11. Hongwei Chen, Julie Yeh, Liya Wang, Xinying Wu, Zehong Cao, Y. Andrew Wang, Minming Zhang, Lily Yang, and Hui Mao. Reducing Non-Specific Binding and Uptake of Nanoparticles and Improving Cell Targeting with an Antifouling PEO-b-PγMPS Copolymer Coating.. Biomaterials (July 2010), 31(20): 5397–5407. DOI: 10.1016/j.biomaterials.2010.03.036

12. Amit Joshi, Sandesh Kate, Vincent Poon, Dhananjoy Mondal, Mohan B. Boggara, Arundhati Saraph, Jacob T. Martin, Ryan McAlpine, Ryan Day, Angel E. Garcia, Jeremy Mogridge, and Ravi S. Kane. Structure-Based Design of a Heptavalent Anthrax Toxin Inhibitor. Biomacromolecules (2011), 12(3), 791–796. DOI: 10.1021/bm101396u.

13. Keir C. Neuman and Attila Nagy. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nature Methods (June 2008), 5(6), 491-505. DOI: 10.1038/nmeth.1218

14. Bo-Hyun Kim, Nicholas Y Palermo, Sándor Lovas, Tatiana Zaikova, John Keana, and Yuri Lyubchenko. Single molecule atomic force microscopy force spectroscopy study of Aß-40 interactions. Biochemistry (2011), 50(23), 5154–5162. DOI: 10.1021/bi200147a.

15. Timothy V. Ratto, Kevin C. Langry, Robert E. Rudd, Rodney L. Balhorn, Michael J. Allen, Michael W. McElfresh. Force Spectroscopy of the Double-Tethered Concanavalin-A Mannose Bond. Biophysical Journal (2004), 86(4), 2430-2437.

16. Zenghan Tong, Andrey Mikheikin, Alexey Krasnoslobodtsev, Zhengjian Lv, Yuri L. Lyubchenko. Novel polymer linkers for single molecule AFM force spectroscopy. Methods (April 2013), 60(2), 161-168.

17. Jamie L. Maciaszek. Detection of SK2 Channels on Hippocampal Neurons. Master’s Thesis. University of Connecticut Graduate School, 2012.

18. Jamie L. Maciaszek, Heun Soh, Randall S. Walikonis, Anastasios V. Tzingounis, and George Lykotrafitis. Topography of Native SK Channels Revealed by Force Nanoscopy in Living Neurons. The Journal of Neuroscience (2012), 32(33), 11435-11440. DOI:10.1523/JNEUROSCI.1785-12.2012.


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