Maleimide Reaction Chemistry

2022-04-07

巯基马来酰亚胺反应是一种Click化学反应吗?

硫醇与马来酰亚胺快速反应生成硫代丁二酰亚胺(图1)是目前生物偶联中最常用的半胱氨酸残基定点修饰方法之一[1]，[2]虽然在某些条件下，巯基和马来酰亚胺基之间形成的硫醚键是缓慢可逆的(见下面的讨论)，但马来酰亚胺基团本身是相对稳定的[3]，所以适宜于进行生物连接反应。

Figure 1: The reaction between a free thiol and a maleimide produces a thiosuccinimide product. The process is a type of "click chemistry" reaction. On the left-hand side of the image, a free thiol (SH) group is shown with a maleimide group. In the center of the image, an arrow pointing from left to right has the word "click" in it to symbolize a click chemistry reaction. The final product on the right-hand side of the image shows that the maleimide group and the free thiol have reacted to form a thiosuccinimide product.Figure 1: 游离硫醇与马来酰亚胺之间的反应产生硫代丁二酰亚胺产物。这个过程是一种“点击化学”反应。

巯基马来酰亚胺反应是Click化学反应吗?

对这个问题的简短回答是，“是的，硫醇-马来酰亚胺反应是一种click化学反应。”具体来说，游离硫醇与马来酰亚胺基团之间的反应是一种加成反应，称为硫醇-迈克尔加成[4] ，也称作硫醇-马来酰亚胺反应[5]. 许多科学家认为这个反应是一种“点击化学”反应，因为它符合Kolb, Finn和Sharpless定义的“点击化学”的大部分标准[6] (Figure 2).

Figure 2: What makes a reaction "click chemistry"? This slide, which is adapted from Reference 6, vide infra, lays out the criteria for click chemistry reactions as established by Kolb, Finn, and Sharpless.Figure 2: 什么样的反应可以称为“点击化学” 6, vide infra.

巯基马来酰亚胺反应的机理是什么?

Figure 3 shows the general mechanism of the thiol-maleimide reaction. Figure 1 of reference 5 below contains a more detailed reaction scheme. The reason for the high reactivity of the olefin is due primarily to (a) the ring strain arising from the bond angle distortion and (b) the positioning of the carbonyl groups in the cis-conformation. Indeed, in highly polar solvents such as water, dimethyl sulfoxide (DMSO), N,N’-dimethylformamide (DMF), or N,N’-dimethylacetamide (DMAC), the thiol-maleimide reaction proceeds without a catalyst, because the polar solvent forms the thiolate ion, which is the active species for the reaction.4

Figure 3: Simplified general mechanism of the thiol-maleimide reaction, which is a specific type of Michael addition reaction.Figure 3: Simplified general mechanism of the thiol-maleimide reaction, which is a specific type of Michael addition reaction.

From pH 6.5 to pH 7.5, the thiol-maleimide reaction is chemoselective for thiols. At pH 7.0, the reaction rate of maleimide with thiols is about 1,000 times faster than the reaction rate of maleimide with amines. However, above pH 7.5, free primary amines react competitively with thiols at the maleimide C=C bond (Figure 3).1

Figure 4: The thiol-maleimide reaction. (a) The reaction is chemoselective for thiols from pH 6.5 - 7.5. (b) Above, pH 7.5, thiol chemoselectivity is lost and the maleimide moiety begins reacting with free amines (e.g., lysine).Figure 4: The thiol-maleimide reaction. (a) The reaction is chemoselective for thiols from pH 6.5 – 7.5. (b) Above pH 7.5, thiol chemoselectivity is lost, and the maleimide moiety begins reacting with free amines (e.g., lysine).

硫代琥珀酰亚胺共轭物（Thiosuccinimide Conjugate）稳定吗?

In aqueous solution, the maleimide ring can be opened by hydrolysis (Figure 5). This susceptibility to hydrolysis increases with increasing pH. Importantly, if the ring-opening reaction occurs before thiolation, the resultant maleic amide is unreactive to thiols. On the other hand, if thiolation has occurred, the ring-opened succinamic acid thioether is stable. Because of the propensity for ring-opening hydrolysis and inactivation, we at Quanta BioDesign do not recommend aqueous storage for products containing a maleimide, which is intended for future reactions. If solution storage is required, use a dry, water-miscible, biocompatible solvent such as DMSO, DMF, or DMAC (see below for more information).

Figure 5: The reactions of thiol and maleimide. See the text for a complete description of these reactions.Figure 5: The reactions of thiol and maleimide. See the text for a complete description of these reactions.

As shown in the top left portion of Figure 5, the thiosuccinimide product can undergo a retro-Michael reaction to regenerate the maleimide. The reformed maleimide is then free to react with the same or a different thiol. In vivo, this retro-Michael reaction can lead to so-called “payload migration,” as shown by the exchange reaction in Figure 5 on the lower right-hand side.[7] Research into payload migration and the stability of thiosuccinimide groups on antibody-drug conjugates (ADCs) has shown that the stability of the thiosuccinimide ring depends on the site of conjugation on the antibody[8],[9]. The reformed maleimide can then react with other serum proteins such as serum albumin, causing the payload to exert off-target effects. The thiosuccinimide ring should be hydrolyzed after the conjugation reaction and workup of the conjugated product to avoid payload migration when using the thiol-maleimide conjugation.[10],[11],[12],[13],[14]

In contrast, the thiol-bromoacetamide reaction leads to a stable thioether product that is not as susceptible to the reverse reaction as the thiol-maleimide conjugation reaction. However, the thiol-bromoacetamide reaction is slower and requires a higher pH to proceed. Furthermore, it is not as chemoselective as the thiol-maleimide reaction. To learn more about the thiol-bromoacetamide reaction with Quanta BioDesign products, please click here.

Working with Maleimide-Containing Products in Thiol-Maleimide Reactions

Quanta BioDesign’s maleimide-containing dPEG® products are easy to use effectively following our recommended instructions on the product information sheets that are included with every order. The following are some recommended best practices for working with our maleimide-containing dPEG® products.

  • Products should be stored at the recommended temperature except when in use. The recommended storage temperature for almost all products that contain maleimide is -20°C.
  • When a product containing maleimide is removed from storage, it should be allowed to equilibrate fully to ambient temperature before opening the bottle or vial in which the product is stored.
  • Aqueous solutions of maleimide-containing products should be made immediately before use. If the maleimide functional group is to be reacted, the pH should be 6.5 – 7.5, and preferably as low as possible within that range.
  • Aqueous buffers and organic solutions of maleimide-containing products should be free of primary and secondary amines and free of thiols. If a base needs to be used in a reaction with a maleimide-containing product, we recommend a highly hindered organic base such as 2,6-lutidine (CAS number 108-48-5; EC number 203-587-3).
  • Do not store maleimide-containing products in aqueous solutions due to the risk of hydrolysis. Instead, use a dry, biocompatible, water-miscible solvent (g., DMSO, DMF, or DMAC) for the long-term storage of these compounds. These solvents can be dried suitably over 3 Å molecular sieves (8×12 mesh recommended) for 24 – 48 hours at 20 – 25°C. Solubilized maleimide-containing products should be stored at -20°C. Quanta BioDesign does not have information on the stability of maleimide-containing products stored in solution because we explicitly do not recommend such storage for these products.
  • When conjugating a maleimide containing product to a protein, if a stock solution of the maleimide-containing product in an organic solvent (vide supra) is added to the reaction mixture, no more than 10% of the final reaction volume should be the organic solvent, while the rest of the volume should be water or aqueous buffer (for example, PBS). Some sensitive proteins may require that the amount of organic solvent be much less than 10% of the final reaction volume.

参考文献

[1] Hermanson, G. T. Chapter 3, The Reactions of Bioconjugation. Bioconjugate Techniques, 3rd edition. Academic Press: New York, 2013, 229-258, specifically page 241, discussing maleimide reactions. 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] Ravasco, J. M. J. M.; Faustino, H.; Trindade, A.; Gois, P. M. P. Bioconjugation with Maleimides: A Useful Tool for Chemical Biology. Chemistry – A European Journal 2019, 25**(1), 43–59. https://doi.org/10.1002/chem.201803174.

[3] Hermanson, G. T. Chapter 2, Functional Targets for Bioconjugation. Bioconjugate Techniques, 3rd edition. Academic Press: New York, 2013, 127-228, specifically page 148, discussing maleimide reactions. Click here now for a review of Greg’s book and a link to purchase it.!

[4] Nair, D. P.; Podgórski, M.; Chatani, S.; Gong, T.; Xi, W.; Fenoli, C. R.; Bowman, C. N. The Thiol-Michael Addition Click Reaction: A Powerful and Widely Used Tool in Materials Chemistry. Chem. Mater. 2014, 26**(1), 724–744. https://doi.org/10.1021/cm402180t.

[5] Northrop, B. H.; Frayne, S. H.; Choudhary, U. Thiol–Maleimide “Click” Chemistry: Evaluating the Influence of Solvent, Initiator, and Thiol on the Reaction Mechanism, Kinetics, and Selectivity. Polym. Chem. 2015, 6**(18), 3415–3430. https://doi.org/10.1039/C5PY00168D.

[6] Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angew. Chem. Int. Ed. 2001, 40**(11), 2004–2021. https://doi.org/10.1002/1521-3773(20010601)40:11%3C2004::AID-ANIE2004%3E3.0.CO;2-5.

[7] Dong, L.; Li, C.; Locuson, C.; Chen, S.; Qian, M. G. A Two-Step Immunocapture LC/MS/MS Assay for Plasma Stability and Payload Migration Assessment of Cysteine–Maleimide-Based Antibody Drug Conjugates. Anal. Chem. 2018, 90**(10), 5989–5994. https://doi.org/10.1021/acs.analchem.8b00694.

[8] Shen, B.-Q.; Xu, K.; Liu, L.; Raab, H.; Bhakta, S.; Kenrick, M.; Parsons-Reponte, K. L.; Tien, J.; Yu, S.-F.; Mai, E.; et al. Conjugation Site Modulates the in Vivo Stability and Therapeutic Activity of Antibody-Drug Conjugates. Nature Biotechnology 2012, 30 (2), 184–189. https://doi.org/10.1038/nbt.2108.

[9] Zheng, K.; Chen, Y.; Wang, J.; Zheng, L.; Hutchinson, M.; Persson, J.; Ji, J. Characterization of Ring-Opening Reaction of Succinimide Linkers in ADCs. Journal of Pharmaceutical Sciences 2019, 108**(1), 133–141. https://doi.org/10.1016/j.xphs.2018.10.063.

[10] Tumey, L. N.; Charati, M.; He, T.; Sousa, E.; Ma, D.; Han, X.; Clark, T.; Casavant, J.; Loganzo, F.; Barletta, F.; et al. Mild Method for Succinimide Hydrolysis on ADCs: Impact on ADC Potency, Stability, Exposure, and Efficacy. Bioconjugate Chem. 2014, 25**(10), 1871–1880. https://doi.org/10.1021/bc500357n.

[11] Fontaine, S. D.; Reid, R.; Robinson, L.; Ashley, G. W.; Santi, D. V. Long-Term Stabilization of Maleimide–Thiol Conjugates. Bioconjugate Chem. 2015, 26**(1), 145–152. https://doi.org/10.1021/bc5005262.

[12] Christie, R. J.; Fleming, R.; Bezabeh, B.; Woods, R.; Mao, S.; Harper, J.; Joseph, A.; Wang, Q.; Xu, Z.-Q.; Wu, H.; et al. Stabilization of Cysteine-Linked Antibody Drug Conjugates with N-Aryl Maleimides. Journal of Controlled Release 2015, 220, 660–670. https://doi.org/10.1016/j.jconrel.2015.09.032.

[13] Ponte, J. F.; Sun, X.; Yoder, N. C.; Fishkin, N.; Laleau, R.; Coccia, J.; Lanieri, L.; Bogalhas, M.; Wang, L.; Wilhelm, S.; et al. Understanding How the Stability of the Thiol-Maleimide Linkage Impacts the Pharmacokinetics of Lysine-Linked Antibody–Maytansinoid Conjugates. Bioconjugate Chem. 2016, 27 (7), 1588–1598. https://doi.org/10.1021/acs.bioconjchem.6b00117.

[14] 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.