Comparison of hydrazone heterobifunctional cross-linking agents for reversible conjugation of thiol-containing chemistry.
Comparison of hydrazone heterobifunctional cross-linking agents for reversible conjugation of thiol-containing chemistry.
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DOI：
10.1021/bc100049c
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发表时间：
2010-10-20
影响因子：
4.7
通讯作者：
Grainger DW
中科院分区：
文献类型：
--
作者：
Christie RJ;Anderson DJ;Grainger DW
可逆共价偶联化学允许位点和条件特异性偶联和解偶联反应，是纳米技术、生物偶联方法、成像和药物传递系统中有吸引力的组成部分。在这里，我们比较了三种杂双功能交联剂，含有硫醇和胺反应化学，与已知和经常发表的水溶性聚合物聚[N-（2-羟丙基甲基丙烯酰胺）]（pHPMA）的肼衍生物形成ph不稳定的腙，随后通过马来酰亚胺加成将含硫醇的分子偶联到交联剂上。以流行的杂双官能团交联剂琥珀酰亚胺基-4-（n -马来酰亚胺甲基）环己烷-1-羧酸盐（SMCC）为原料制备了两种新型交联剂，该交联剂经修饰后含有末端醛基团（即1-（n -3-丙烷）-4-（n -马来酰亚胺甲基）环己烷羧酰胺，PMCA）或甲基酮基团（即1-（n -3-丁酮）-4-（n -马来酰亚胺甲基）环己烷羧酰胺，BMCA）。第三种交联剂是市售的n -4-乙酰苯基马来酰亚胺（APM）。PMCA和BMCA对酰肼衍生的pHPMA表现出优异的反应性，基本上完全结合了聚合物活性位点，而APM仅偶联了约60%的活性位点，尽管相对于聚合物酰肼基团的摩尔量多了3倍。然后将所有带有这些双功能剂的聚合物腙偶联物与巯基修饰的四甲基罗丹明染料进一步反应，确定了初始腙聚合物偶联后交联剂马来酰亚胺的反应性。将染料标记的聚合物偶联物在37°C的磷酸盐缓冲盐水中孵育，结果表明，APM引起的腙偶联在pH 7.4和5.0之间的稳定性差异最大，在pH 5.0的24小时孵育期间，水解和染料释放增加。由交联剂BMCA形成的聚合物偶联腙具有中等稳定性，早期pH值为5.0时水解程度高得多，但pH值为7.4时5小时后水解显著。在pH 7.4和5.0条件下，PMCA交联剂形成的腙释放速率无显著差异。
Reversible covalent conjugation chemistries that allow site- and condition-specific coupling and uncoupling reactions are attractive components in nanotechnologies, bioconjugation methods, imaging and drug delivery systems. Here, we compare three heterobifunctional crosslinkers, containing both thiol- and amine- reactive chemistry, to form pH-labile hydrazones with hydrazide derivatives of the known and often published water-soluble polymer, poly[N-(2-hydroxypropyl methacrylamide)] (pHPMA), while subsequently coupling thiol-containing molecules to the crosslinker via maleimide addition. Two novel crosslinkers were prepared from the popular heterobifunctional crosslinking agent, succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), modified to contain either terminal aldehyde groups (i.e., 1-(N-3-propanal)-4-(N-maleimidomethyl) cyclohexane carboxamide, PMCA) or methylketone groups (i.e., 1-(N-3-butanone)-4-(N-maleimidomethyl) cyclohexane carboxamide, BMCA). A third crosslinking agent was the commercially available N-4-acetylphenyl maleimide (APM). PMCA and BMCA exhibited excellent reactivity towards hydrazide-derivatized pHPMA with essentially complete hydrazone conjugation to polymer reactive sites, while APM coupled only ~ 60% of available reactive sites on the polymer despite a 3-fold molar excess relative to polymer hydrazide groups. All polymer hydrazone conjugates bearing these bifunctional agents were then further reacted with thiol-modified tetramethylrhodamine dye, confirming crosslinker maleimide reactivity after initial hydrazone polymer conjugation. Incubation of dye-labeled polymer conjugates in phosphate buffered saline at 37°C showed that hydrazone coupling resulting from APM exhibited the greatest difference in stability between pH 7.4 and 5.0, with hydrolysis and dye release increased at pH 5.0 over a 24hr incubation period. Polymer conjugates bearing hydrazones formed from crosslinker BMCA exhibited intermediate stability with hydrolysis much greater at pH 5.0 at early time points, but hydrolysis at pH 7.4 was significant after 5 hrs. Hydrazones formed with the PMCA crosslinker showed no difference in release rates at pH 7.4 and 5.0.
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