Conjugation to biomolecules with surface accessible sulfhydryl groups is often preferred because such groups are relatively rare in proteins, making site specific conjugation more likely.[1] The reaction between a sulfhydryl group and a bromoacetyl moiety is a well-known alternative to the more popular thiol-maleimide reaction.
The Thiol-Bromoacetyl Reaction
The thiol-bromoacetyl reaction is used primarily to modify free thiol groups on proteins, peptides, and other biomolecules. Sulfhydryl groups react most readily with bromoacetyl derivatives. However, the reaction is not entirely specific. Bromoacetyl derivatives can also react with other groups found on proteins, including N-terminal α-amines, the ε-amine of lysine, the imidazole side chain nitrogen atoms of histidine, and the thioether linkages of methionine. Nevertheless, the reaction between bromoacetyl derivatives and sulfhydryl groups is favored, and controlling reaction stoichiometry and pH can yield specific conjugates.[2]
The thiol-bromoacetyl reaction rate depends largely on the degree of ionization and, consequently, on the pH at which the reaction is performed.[3] The thiol-bromoacetyl reaction proceeds most favorably in the range of pH 7.5 – 9.0, whereas the thiol-maleimide reaction must be conducted at a much lower pH (6.5 – 7.5) to avoid reactions with primary amines.2,[4]
Advantages Over Maleimide Products
Stability of the conjugated product is the principal advantage of the thiol-bromoacetyl reaction over the thiol-maleimide reaction. That is, the thioether linkage formed by the thiol-bromoacetyl reaction is highly stable. By comparison, the thioether linkage formed from the maleimide reaction is not entirely stable. Under certain conditions, particularly in vivo, the thio-succinimide product will undergo slowly a retro Michael addition to reform the maleimide. Thioethers formed from maleimide conjugation require additional post-conjugation steps to stabilize the thiol ether.4,[5]
Another advantage of the bromoacetyl reactive function is that it can be paired with an azide function on the same linker. Maleimide groups react with azide groups.[6],[7] In contrast, bromoacetamido-dPEG®-azide crosslinkers are stable. This allows for click chemistry crosslinking between alkynes (either terminal aliphatic alkynes or strained cyclooctyne derivatives) and thiols. The orthogonality of the two reactive groups also gives our customers complete control of their bioconjugation reactions.
Other Bromoacetyl Products
Quanta BioDesign manufactures and sells numerous bromoacetyl-functionalized dPEG® products.[8] Because we add the bromoacetyl functional group to our dPEG® products via an amide linkage, we refer to products in this line as bromoacetamido and/or bromoacetamide. A partial list of such products is provided below.
In addition to our bromoacetamido-dPEG®-azide crosslinkers, which are useful for both copper catalyzed and copper free click chemistry, we have bromoacetamido-dPEG®-DBCO crosslinkers that are designed specifically for strain promoted, copper free click chemistry. For crosslinking amines and thiols, we offer bromoacetamido-dPEG®-TFP esters. Although N-hydroxysuccinimidyl (NHS) esters are more popular and more widely used, tetrafluorophenyl (TFP) esters have demonstrated superior hydrolytic stability and amine reactivity compared to NHS esters. Also, we sell homobifunctional and homotetrafunctional bromoacetyl-functionalized dPEG® products for crosslinking thiols.
Bromoacetyl Functionalized dPEG® Products (Partial List)
| PN11217, Bromoacetamido-dPEG®3-azide |
| PN11204, Bromoacetamido-dPEG®11-azide |
| PN11205, Bromoacetamido-dPEG®23-azide |
| PN11221, Bromoacetamido-dPEG®4-DBCO |
| PN11223, Bromoacetamido-dPEG®12-DBCO |
| PN11224, Bromoacetamido-dPEG®24-DBCO |
| PN11200, Bromoacetamido-dPEG®4-TFP ester |
| PN11202, Bromoacetamido-dPEG®12-TFP ester |
| PN11203, Bromoacetamido-dPEG®24-TFP ester |
PN11338, Bis-bromoacetamido-dPEG®11
PN11434, Bromoacetamido-dPEG®12-Tris(-dPEG®11-bromoacetamide)3
References
[1] Hermanson, G. T. Chapter 2, Functional Targets for Bioconjugation. Bioconjugate Techniques, 3rd edition. Academic Press: New York, 2013, 127-228, especially pages 191-193. Want to learn more about Greg’s book? Click here now for a review of Greg’s book and a link to purchase it.
[2] Hermanson, G. T. Chapter 3, The Reactions of Bioconjugation. Bioconjugate Techniques, 3rd edition. Academic Press: New York, 2013, 229-258, specifically pages 240-241, discussing haloacetyl reactions.
[3] Gurd, F. R. N. [62] Carboxymethylation. In Methods in Enzymology; Enzyme Structure; Academic Press, 1967; Vol. 11, pp 532–541. https://doi.org/10.1016/S0076-6879(67)11064-1.
[4] Schelté, Philippe; Boeckler, C.; Frisch, B.; Schuber, F. Differential Reactivity of Maleimide and Bromoacetyl Functions with Thiols: Application to the Preparation of Liposomal Diepitope Constructs. Bioconjugate Chem. 2000, 11(1), 118–123. https://doi.org/10.1021/bc990122k.
[5] Szijj, P. A.; Bahou, C.; Chudasama, V. Minireview: Addressing the Retro-Michael Instability of Maleimide Bioconjugates. Drug Discovery Today: Technologies 2018, 30, 27–34. https://doi.org/10.1016/j.ddtec.2018.07.002.
[6] Varma, I. K.; Choudhary, V.; Gaur, B.; Lochab, B.; Oberoi, S.; Chauhan, R. Curing and Thermal Behavior of Poly(Allyl Azide) and Bismaleimides. Journal of Applied Polymer Science 2006, 101(1), 779–786. https://doi.org/10.1002/app.24001.
[7] Zhu, H.-Z.; Wang, G.; Wei, H.-L.; Chu, H.-J.; Zhu, J. Click Synthesis of Hydrogels by Metal-Free 1,3-Dipolar Cycloaddition Reaction between Maleimide and Azide Functionalized Polymers. Macromol. Res. 2016, 24(9), 793–799. https://doi.org/10.1007/s13233-016-4120-7.
[8] For an excellent overview of Quanta BioDesign’s discrete PEG (dPEG®) products, please see, Hermanson, G. T. Chapter 18, PEGylation and Synthetic Polymer Modification. Bioconjugate Techniques, 3rd edition. Academic Press: New York, 2013, 787-838.