Modulating Thiol pKa Promotes Disulfide Formation at Physiological pH: An Elegant Strategy To Design Disulfide Cross-Linked Hyaluronic Acid Hydrogels
Modulating Thiol pKa Promotes Disulfide Formation at Physiological pH: An Elegant Strategy To Design Disulfide Cross-Linked Hyaluronic Acid Hydrogels
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DOI：
10.1021/acs.biomac.8b01830
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发表时间：
2019-03-01
影响因子：
6.2
通讯作者：
Varghese, Oommen P.
中科院分区：
文献类型：
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作者：
Bermejo-Velasco, Daniel;Azemar, Alice;Varghese, Oommen P.
二硫键在蛋白质生物学中发挥着至关重要的作用，科学家已利用它来开发抗体-药物缀合物、传感器以及将其他生物分子固定到材料表面。尽管其用途广泛，但二硫化物化学受到一些不可避免的限制，例如需要碱性条件（pH > 8.5），强氧化剂和长反应时间。我们在这里证明，在β-位置上含有吸电子基团的巯基底物影响巯基的去质子化，巯基是形成二硫键的关键反应中间体。使用小分子底物如L-半胱氨酸的反应动力学评价表明，在生理pH（pH 7.4）下，与常规硫醇底物即3-巯基丙酸（k（1）= 1.80 X 10（-4）min（-1））相比，二硫化物形成的反应速率高2.8倍（k（1）= 5.04 X 10（-4）min（-1））。有趣的是，当使用N-乙酰-L-半胱氨酸底物（k（1）= 0.51 × 10（-4）min（-1））时，未观察到相同的效果。我们进一步将这种含巯基的分子（半胱氨酸、N-乙酰半胱氨酸和3-巯基丙酸）接枝到生物聚合物即透明质酸（HA）上，并通过分光光度分析确定不同巯基的pK（a）值。β-位的吸电子基团将巯基的pK（a）分别降低至HA-半胱氨酸（HA-Cys）的7.0; N-乙酰半胱氨酸（HA-ActCys）的7.4;和HA-巯基（HA-SH）衍生物的8.1。这些实验进一步证实，硫醇盐（R-S-）离子的浓度可以随着吸电子基团的存在而增加，这可以在生理pH下促进二硫化物交联的水凝胶形成。事实上，在pH 7.4下，用半胱氨酸或N-乙酰基基团接枝的HA分别在3.5分钟或10小时内形成水凝胶。在交联反应完成后，两种凝胶均显示储能模量G'接近3300-3500 Pa，这表明交联水平相当。另一方面，HA-SH凝胶在pH 7.4下甚至在24小时后也不形成任何凝胶。最后，我们证明了新制备的水凝胶具有优异的水解稳定性，但可以通过细胞定向过程（酶促和还原降解）降解。我们相信，我们的研究提供了一个有价值的洞察力的因素，控制二硫化物的形成和我们的结果是有用的开发策略，这将有助于产生稳定的硫醇功能化的生物分子或促进快速硫醇氧化根据生物医学的需要。
The disulfide bond plays a crucial role in protein biology and has been exploited by scientists to develop antibody-drug conjugates, sensors, and for the immobilization other biomolecules to materials surfaces. In spite of its versatile use, the disulfide chemistry suffers from some inevitable limitations such as the need for basic conditions (pH > 8.5), strong oxidants, and long reaction times. We demonstrate here that thiol-substrates containing electron-withdrawing groups at the beta-position influence the deprotonation of the thiol group, which is the key reaction intermediate in the formation of disulfide bonds. Evaluation of reaction kinetics using small molecule substrate such as L-cysteine indicated disulfide formation at a 2.8-fold higher (k(1) = 5.04 X 10(-4) min(-1)) reaction rate as compared to the conventional thiol substrate, namely 3-mercaptopropionic acid (k(1) = 1.80 X 10(-4) min(-1)) at physiological pH (pH 7.4). Interestingly, the same effect could not be observed when N-acetyl-L-cysteine substrate (k(1) = 0.51 X 10(-4) min(-1)) was used. We further grafted such thiol-containing molecules (cysteine, N-acetyl-cysteine, and 3-mercaptopropionic acid) to a biopolymer namely hyaluronic acid (HA) and determined the pK(a) value of different thiol groups by spectrophotometric analysis. The electron-withdrawing group at the beta-position reduced the pK(a) of the thiol group to 7.0 for HA-cysteine (HA-Cys); 7.4 for N-acetyl cysteine (HA-ActCys); and 8.1 for HA-thiol (HA-SH) derivatives, respectively. These experiments further confirmed that the concentration of thiolate (R-S-) ions could be increased with the presence of electron-withdrawing groups, which could facilitate disulfide cross-linked hydrogel formation at physiological pH. Indeed, HA grafted with cysteine or N-acetyl groups formed hydrogels within 3.5 min or 10 h, respectively, at pH 7.4. After completion of cross-linking reaction, both gels demonstrated a storage modulus G' approximate to 3300-3500 Pa, which indicated comparable levels of cross-linking. The HA-SH gel, on the other hand, did not form any gel at pH 7.4 even after 24 h. Finally, we demonstrated that the newly prepared hydrogels exhibited excellent hydrolytic stability but can be degraded by cell-directed processes (enzymatic and reductive degradation). We believe our study provides a valuable insight on the factors governing the disulfide formation and our results are useful to develop strategies that would facilitate generation of stable thiol functionalized biomolecules or promote fast thiol oxidation according to the biomedical needs.