Facile and stabile linkages through tyrosine: bioconjugation strategies with the tyrosine-click reaction.
Facile and stabile linkages through tyrosine: bioconjugation strategies with the tyrosine-click reaction.
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DOI：
10.1021/bc300665t
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发表时间：
2013-04-17
影响因子：
4.7
通讯作者：
Barbas, Carlos F., III
中科院分区：
文献类型：
--
作者：
Ban, Hitoshi;Nagano, Masanobu;Gavrilyuk, Julia;Hakamata, Wataru;Inokuma, Tsubasa;Barbas, Carlos F., III
报道了4-苯基-3H-1，2，4-三唑啉-3，5（4 H）-二酮（PTADs）的点击式酪氨酸标记反应的范围、化学选择性和实用性。为了研究PdR衍生物在肽和蛋白质化学中的实用性和化学选择性，我们合成了具有叠氮基、炔基和酮基的PdR衍生物，并研究了它们与氨基酸衍生物和肽的反应。与蛋白质，我们研究了酪氨酸点击反应与半胱氨酸和赖氨酸靶向标记方法的兼容性，并证明，蛋白质的化学选择性三功能化是很容易实现的。在特定情况下，我们注意到Pd 3分解导致形成推定的异氰酸酯副产物，其在标记中是混杂的。然而，通过向反应介质中加入少量的2-氨基-2-羟甲基-丙烷-1，3-二醇（Tris），该副反应产物很容易被清除。为了研究酪氨酸点击反应将聚乙二醇链引入蛋白质（聚乙二醇化）的潜力，我们证明了这种新型试剂提供了胰凝乳蛋白酶原的选择性聚乙二醇化，而传统的基于琥珀酰亚胺的聚乙二醇化靶向赖氨酸残基提供了更多样化的聚乙二醇化产物。最后，我们应用酪氨酸点击反应制备了一种新型的抗体药物偶联物。为此目的，我们合成了一种连接到HIV进入抑制剂aplaviroc的Peptide衍生物。用该试剂标记抗体曲妥珠单抗提供了标记的抗体缀合物，其显示出有效的HIV-1中和活性，证明了该反应在产生具有小分子的蛋白质缀合物中的潜力。酪氨酸点击键证明了对极端pH、温度和暴露于人血浆的稳定性，表明该键比生物缀合中常用的马来酰亚胺型键显著更稳健。这些研究支持了该反应在温和水性条件下在宽pH范围内使用各种生物学上可接受的缓冲液如磷酸盐缓冲盐水（PBS）和2-氨基-2-羟甲基-丙烷-1，3-二醇（Tris）缓冲液以及其它缓冲液和混合缓冲组合物对小分子、肽和蛋白质进行化学选择性修饰中的广泛用途。
The scope, chemoselectivity, and utility of the click-like tyrosine labeling reaction with 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-diones (PTADs) is reported. To study the utility and chemoselectivity of PTAD derivatives in peptide and protein chemistry, we synthesized PTAD derivatives possessing azide, alkyne, and ketone groups and studied their reactions with amino acid derivatives and peptides of increasing complexity. With proteins we studied the compatibility of the tyrosine click reaction with cysteine and lysine-targeted labeling approaches and demonstrate that chemoselective tri-functionalization of proteins is readily achieved. In particular cases, we noted PTAD decomposition resulted in formation of a putative isocyanate by-product that was promiscuous in labeling. This side reaction product, however, was readily scavenged by the addition of a small amount of 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) to the reaction medium. To study the potential of the tyrosine click reaction to introduce poly(ethylene) glycol chains onto proteins (PEGylation), we demonstrate that this novel reagent provides for the selective PEGylation of chymotrypsinogen whereas traditional succinimide-based PEGylation targeting lysine residues provided a more diverse range of PEGylated products. Finally, we applied the tyrosine click reaction to create a novel antibody drug conjugate. For this purpose, we synthesized a PTAD derivative linked to the HIV entry inhibitor aplaviroc. Labeling of the antibody trastuzumab with this reagent provided a labeled antibody conjugate that demonstrated potent HIV-1 neutralization activity demonstrating the potential of this reaction in creating protein conjugates with small molecules. The tyrosine click linkage demonstrated stability to extremes of pH, temperature and exposure to human blood plasma indicating that this linkage is significantly more robust than maleimide-type linkages that are commonly employed in bioconjugations. These studies support the broad utility of this reaction in the chemoselective modification of small molecules, peptides, and proteins under mild aqueous conditions over a broad pH range using a wide variety of biologically acceptable buffers such as phosphate buffered saline (PBS) and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) buffers as well as others and mixed buffered compositions.
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15
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Blackman, Melissa L.;Royzen, Maksim;Fox, Joseph M.
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Fox, Joseph M.
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