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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.

Figure 1: Reaction between a bromoacetyl group and a free thiol. This stylized reaction represents a generic bromoacetyl group reacting with a free thiol group on a yellow surface (for example, a protein surface).
Figure 1: Reaction between a bromoacetyl group and a free thiol.

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.

Figure 2: Stability of Bromoacetyl-azide. In the upper half of the image, a bromoacetamido-dPEG®-azide is shown. This molecule is stable. In the bottom half of the image a maleimido-dPEG®-azide is shown. This molecular construct is not stable, because maleimides react with azides.
Figure 2: Stability of Bromoacetyl-azide. In the upper half of the image, a bromoacetamido-dPEG®-azide is shown. This molecule is stable. In the bottom half of the image a maleimido-dPEG®-azide is shown. This molecular construct is not stable, because maleimides react with azides.

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)

This image represents the general chemical structure of Bromoacetamido-dPEG®n-azide. This molecule contains a bromoacetyl group joined via an amide bond to a dPEG®-azide. The dPEG® moiety can have 3, 11, or 23 ethylene oxide units in the chain.
PN11217, Bromoacetamido-dPEG®3-azide

PN11204, Bromoacetamido-dPEG®11-azide

PN11205, Bromoacetamido-dPEG®23-azide

This image represents a bromoacetamido-dPEG®n-DBCO product group. The bromoacetyl group is connected via an amide bond to a dPEG®-acid moiety with 4, 12, or 24 ethylene oxide units in the chain. The acid terminus of the dPEG® moiety is amide-bonded to a dibenzylcyclooctyne (DBCO) group that is used for copper-free click chemistry, known by the acronym SPAAC.
PN11221, Bromoacetamido-dPEG®4-DBCO

PN11223, Bromoacetamido-dPEG®12-DBCO

PN11224, Bromoacetamido-dPEG®24-DBCO

This image shows the chemical representation of bromoacetamido-dPEG®n-TFP ester. The bromoacetyl moiety is connected via an amide bond to the TFP ester of a dPEG® acid. The dPEG® moiety may be 4, 12, or 24 ethylene oxide groups long.
PN11200, Bromoacetamido-dPEG®4-TFP ester

PN11202, Bromoacetamido-dPEG®12-TFP ester

PN11203, Bromoacetamido-dPEG®24-TFP ester

This image shows the chemical structure of product number 11338, bis-bromoacetamido-dPEG®11. This molecule is used for cross-linking thiol groups. There is a bromoacetyl group on each end of molecule joined via an amide bond to a dPEG®11 linker.
PN11338, Bis-bromoacetamido-dPEG®11

This image shows the chemical structure of the 4-armed dPEG® product number 11434, which contains a bromoacetyl moiety at the end of each arm. It is used for cross-linking free thiol groups.
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.

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