Have you ever been working your way through a product catalog and encountered a product you weren’t sure how to use? I know I have. It helps in those moments of “What do I do with this?” to have some tips on how to use a product effectively. Today I want to take one of our unique click chemistry discrete PEGylation (dPEG®) reagents, PN10524, azido-dPEG®11-amine, and offer three ideas on how to get the most out of it in an application.
Figure 1 shows the structure of PN10524. An azide group on one end provides the click chemistry function. At the other end, a primary amine reacts with carbonyl groups. Between the two end groups is a single molecular weight (discrete) PEG linker.
What is click chemistry?
Barry Sharpless coined the term click chemistry in 1998 to define high yielding, stereospecific, modular reactions that are “wide in scope” and “generate only inoffensive byproducts that can be removed by nonchromatographic methods….” (1) In Sharpless’ definition, click chemistry reactions must be simple to perform with readily available starting materials and reagents. Moreover, these reactions must use benign or easily removable solvents. Finally, the resulting product “must be stable under physiological conditions.” (1) The four major classifications of click chemistry reactions are
- cycloadditions, such as the Copper(I)-catalyzed azide-alkyne cycloaddition, 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)
Even a casual survey of the chemical literature will show that click chemistry has revolutionized many areas of chemistry research and development, including drug discovery and development (see also, 3).
Why use click chemistry?
Click chemistry solves many (though not all) synthetic chemistry problems. Click chemistry qualifies as environmentally friendly “green chemistry” because of its use of benign or easily removable solvents and the requirements that byproducts be inoffensive and able to be removed by nonchromatographic methods. (7) Moreover, typical click chemistry reactions run faster in water than in organic solvents. (1) This makes click chemistry a highly useful synthetic process for reactions that require aqueous environments, such as cell-based assays. (8)
Click chemistry ideas for dPEG® PEGylation reagents
So, how can you use this product effectively? Here are three suggestions:
I suspect that most scientists who study binding interactions experience “non-specific binding” at times. It is a real headache in Western blots, ELISAs, analyzing column fractions collected in plastic or glass tubes, and many other areas. Proteins, peptides, and nucleic acids stick randomly to uncoated metal, plastic, or glass surfaces. The outcome can be frustrating. In worst-case situations, the data may be incomprehensible due to non-specific binding.
Several methods exist to reduce or eliminate non-specific binding. In particular, silanization is highly popular for coating glass surfaces to get rid of non-specific binding. Moreover, coating glass surfaces with sugars has been patented as a way to eliminate non-specific binding. (9) Finally, PEGylation eliminates non-specific binding. (10, 11)
Suppose, though, that you want to eliminate non-specific binding 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 could functionalize the surface with carboxylic acid groups, activate them, and then react the surface with the crosslinker. The result would be something like Figure 2.
A dense coating of dPEG® molecules will reduce or eliminate non-specific binding. This, however, leaves a large number of azide groups sticking up from the surface. While these azide groups can react with some target molecules (a small molecule, peptide, or protein into which an alkyne group has been installed), not all of them will react with the target due to crowding (steric hindrance). Reducing the number of target molecules will reduce crowding, but the unreacted azide groups 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 would recommend PN10278, m-dPEG®8-amine (Figure 3a), be mixed in a ratio of >2:1 with the azido-dPEG®11-amine crosslinker. The ratio of shorter:longer dPEG® could be as high as 9:1 (19). PN10278 is a shorter molecule (29.7 Å) than PN10524 (44.2 Å), so the result will look something like Figure 3b.
This type of coating will reduce or eliminate non-specific binding. There are fewer azide groups with which to react, but those that react will be much less likely to be sterically hindered. Again, as discussed above, any small molecule, peptide, or protein onto which an alkyne function has been introduced can react with the azide. The reaction can be copper catalyzed or strain catalyzed, depending on the type of alkyne group used. A recently published paper by Yanqiu Du, Jing Jin, and Wei Jiang of the Chinese Academy of Sciences found that PEG backfilling (mixing shorter dPEG® chains with longer chains) formed a stable platform for the immobilization of antibodies (19).
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. Reacting the primary hydroxyl groups of the β-cyclodextrin core with propargyl bromide converted them to terminal alkyne groups. Subsequently, copper-catalyzed click chemistry was used to react the heptavalent alkyne with azido-dPEG®11-amine in high yield. The free amines were reacted with a peptide designed to inhibit the formation of anthrax toxin. See Figure 4.
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.
Indeed, tests showed that the construct effectively inhibited formation of anthrax toxin. Six out of seven rats treated with the inhibitor and exposed to anthrax did not develop anthrax. Conversely, untreated rats died from anthrax exposure. Moreover, rats treated with a sham inhibitor died from exposure to anthrax.
Copper-catalyzed click chemistry gives high yields with minimal byproducts in an easy reaction. This research shows how discrete PEG (dPEG®) coupled with click chemistry exerts 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 a dPEG® permitted the necessary spatial control in the design. Furthermore, dPEG® simplifies product analysis. This research (among others) proves that point.
Atomic Force Microscopy
Atomic Force Microscopy (AFM) is a type of scanning probe microscopy with resolution to fractions of a nanometer. One use of AFM measures force between a probe and a sample. This is known as force spectroscopy. The theory of AFM is explained here and here with very helpful graphics at each link. Additionally, helpful explanations of force spectroscopy can be found here and here.
Single molecule AFM force spectroscopy (SMFS) measures the properties of molecules of interest that are attached via linkers to cantilevers. A single molecule of interest attaches to each cantilever. (13) The most commonly used linker for attaching biomolecules to cantilevers is poly(ethylene glycol). (14-16)
Traditional dispersed PEG reagents are not ideal for SMFS because of the disperse nature of PEG polymers, which includes heterogenous chain lengths (15, 16). For example, Yuri L. Lyubchenko and colleagues used traditional, dispersed PEG linkers and found it necessary to determine all of the different linker lengths in order to calculate force. (14) Notably, research in the lab of George Lykotrafitis used PN10524, azido-dPEG®11-amine in a SMFS application to detect and quantitatively map individual calcium-activated small conductance (SK) potassium channels in living neurons. The amine end of the crosslinker reacted with the APTES-activated silicon nitride cantilevers. This left an azide surface coating that could be reacted with molecules of interest. Furthermore, the dPEG® linker was necessary to the research because it had no chain length heterogeneity. (17, 18)
Click chemistry and dPEG®ylation enable useful and exciting chemistry. For example, these two tools permit surface modification that eliminates non-specific binding and installs helpful functional groups. Moreover, dPEG® products enable precise spatial control in macromolecular construction, because the PEG linkers are of uniform size. Likewise, the absolute uniformity of dPEG® products enables atomic force microscopy applications such as single molecule AFM force spectroscopy. Product number 10524, azido-dPEG®11-amine, proves itself 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). You can find all five these valuable crosslinkers and many other types of crosslinkers on our website: www.quantabiodesign.com/.
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- 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.
- 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.
- John E. Moses and Adam D. Moorhouse. The growing applications of click chemistry. Chem. Soc. Rev. (2007), 36, 1249-1262.
- 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.
- Charles E. Hoyle and Christopher N. Bowman. Thiol-Ene Click Chemistry. Angew. Chem. Int. Ed. (2010), 49(9), 1540-1573.
- 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.
- Chao-Jun Li and Barry M. Trost. Green chemistry for chemical synthesis. Proc. Nat. Acad. Sci. (September 9, 2008), 105(36), 13197-13202.
- Greg T. Hermanson. “Chemoselective Ligation; Bioorthogonal Reagents,” in Bioconjugate Techniques, 3rd edition. New York: Academic Press, 2013, page 771. Quanta BioDesign sells this book! Click here to purchase it!
- Gangadhar Jogikalmath. Method for blocking non-specific protein binding on a functionalized surface. US 20080213910 A1, September 4, 2008.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Jamie L. Maciaszek. Detection of SK2 Channels on Hippocampal Neurons. Master’s Thesis. University of Connecticut Graduate School, 2012.
- 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.
- Yanqiu Du, Jing Jin, and Wei Jiang. A study of polyethylene glycol backfilling for enhancing target recognition using QCM-D and DPI. Journal of Materials Chemistry B (2018), 6, 6217-6224.
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About the Author
Robert H. Woodman, Ph.D. is a Senior Product Development Scientist and the QC Manager for Quanta BioDesign, Ltd. He is on LinkedIn at https://www.linkedin.com/in/roberthwoodman, on Twitter at @RobertHWoodman and @QuantaBioDesign, and on Google+ at https://plus.google.com/+RobertWoodman. Feel free to contact him via social media.
Related Click Chemistry Crosslinkers
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