Synthesis, In Vitro Evaluation, and In Vivo Metabolism of Fluor/Quencher Compounds Containing IRDye 800CW and Black Hole Quencher-3 (BHQ-3)
Synthesis, In Vitro Evaluation, and In Vivo Metabolism of Fluor/Quencher Compounds Containing IRDye 800CW and Black Hole Quencher-3 (BHQ-3)
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DOI：
10.1021/bc100457s
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发表时间：
2011-07-01
影响因子：
4.7
通讯作者：
Swenson, Rolf E.
中科院分区：
文献类型：
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作者：
Linder, Karen E.;Metcalfe, Edmund;Swenson, Rolf E.
含有合适的荧光剂/猝灭剂（Fl/Q）对的蛋白酶可切割肽在被其靶蛋白酶切割之前是光学暗的，从而产生荧光。这种方法已被用于许多Fl/Q对，但很少有报道与IRDye 800 CW，一种流行的近红外（NIR）荧光。我们探索了含偶氮键的黑洞淬灭剂3（BHQ-3）作为IRDye 800 CW的淬灭剂的用途，并发现IRDye 800 CW/BHQ-3是合适的FI/Q对，尽管缺乏用于荧光共振能量转移（FRET）应用的适当光谱重叠。基质金属蛋白酶（MMP）体外切割IRDye 800 CW-PLGLK（BHQ-3）AR-NH 2（8）及其D-精氨酸（Dare类似物（9））产生预期的切割片段。在注射后3分钟，“不可裂解的”对照IRDye 800 CW-（1，13-二氨基-4，7，10-三氧杂十三烷）-BHQ-3（10）在正常小鼠的血浆中明显分解。主要代谢产物的m/z和UV/维斯光谱与BHQ-3部分中的偶氮键裂解一致。制备该代谢产物的真实标准品（11）证实了归属。尽管IRDye 800 CW/BHQ-3构建体在酶促裂解之前显示出有效的接触淬灭，但由于其偶氮键的不稳定性，BHQ：3在体内应谨慎使用。
Protease-cleavable peptides containing a suitable fluor/quencher (Fl/Q) pair are optically dark until cleaved by their target protease, generating fluorescence. This approach has been used with many Fl/Q pairs, but little has been reported with IRDye 800CW, a popular near-infrared (NIR) fluor. We explored the use of the azo-bond-containing Black Hole Quencher 3 (BHQ-3) as a quencher for IRDye 800CW and found that 1RDye 800CW/BHQ-3 is a suitable Fl/Qpair, despite the lack of proper spectral overlap for fluorescence resonance energy transfer (FRET) applications. Cleavage of IRDye 800CW-PLGLK(BHQ-3)AR-NH2 (8) and its D-arginine (Dare analogue (9) by matrix metalloproteinases (MMPs) in vitro yielded the expected cleavage fragments. In vivo, extensive metabolism was found. Significant decomposition of a "non-cleavable" control IRDye 800CW-(1,13-diamino-4,7,10-trioxatridecane)-BHQ-3 (10) was evident in plasma of normal mice by 3 min post injection. The major metabolite showed a m/z and UV/vis spectrum consistent with azo bond cleavage in the BHQ-3 moiety. Preparation of an authentic standard of this metabolite (11) confirmed the assignment. Although the IRDye 800CW/BHQ-3 constructs showed efficient contact quenching prior to enzymatic cleavage, BHQ:3 should be used with caution in vivo, due to instability of its azo bond.