The clinical success of antibody-drug conjugates and numerous other stimuli-responsive drug delivery systems motivates the need to develop molecular tools to quantitatively study their intracellular processing. To this end, researchers have developed fluorescent-based probes utilizing Förster resonance energy transfer (FRET). Quenched probes have the potential to eliminate fluorescence bleed through, which is common in FRET-based systems. However, the hydrophobicity of many broad-spectrum fluorescence quenchers can complicate the design of protein-based molecular probes. In this work, we investigate the potential for 2,4-dinitroaniline (2,4-DNA) to serve as a hydrophilic fluorescence quencher. A support-free synthesis of oligothioetheramide (oligoTEA) linkers was developed and applied to the design of quenched fluorescence probes. These quenched probes were based on intramolecular static quenching of boron dipyrromethene (BODIPY)-FL by 2,4-DNA. Probes containing a reduction-sensitive disulfide bond and a protease-sensitive valine–citrulline-PABC linker were synthesized using a model protein – human transferrin. Within HeLa cells, the apparent degradation rate of the disulfide bond was greater than the valine–citrulline-PABC linker. This work establishes a versatile method for synthesizing multifunctional crosslinkers and identifies 2,4-DNA as an effective fluorescence quencher for protein-based bioconjuates.

中文翻译：

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基于蛋白质的“开启”分子探针用于细胞内键裂解的设计

抗体-药物偶联物和许多其他刺激反应性药物递送系统的临床成功激发了对开发分子工具以定量研究其细胞内加工的需求。为此，研究人员开发了利用Förster共振能量转移（FRET）的荧光探针。淬灭的探针具有消除荧光渗漏的潜力，这在基于FRET的系统中很常见。但是，许多广谱荧光猝灭剂的疏水性会使基于蛋白质的分子探针的设计复杂化。在这项工作中，我们研究了2,4-二硝基苯胺（2,4-DNA）用作亲水性荧光猝灭剂的潜力。开发了无支持的寡硫醚酰胺（oligoTEA）接头合成方法，并将其用于淬灭荧光探针的设计。这些淬灭的探针基于2,4-DNA对硼二吡咯亚甲基（BODIPY）-FL的分子内静态淬灭。使用模型蛋白-人转铁蛋白合成了含有还原敏感性二硫键和蛋白酶敏感性缬氨酸-瓜氨酸-PABC接头的探针。在HeLa细胞中，二硫键的表观降解速率大于缬氨酸-瓜氨酸-PABC接头。这项工作建立了合成多功能交联剂的通用方法，并确定了2,4-DNA是基于蛋白质的生物缀合物的有效荧光猝灭剂。在HeLa细胞中，二硫键的表观降解速率大于缬氨酸-瓜氨酸-PABC接头。这项工作建立了合成多功能交联剂的通用方法，并确定了2,4-DNA是基于蛋白质的生物缀合物的有效荧光猝灭剂。在HeLa细胞中，二硫键的表观降解速率大于缬氨酸-瓜氨酸-PABC接头。这项工作建立了合成多功能交联剂的通用方法，并确定了2,4-DNA是基于蛋白质的生物缀合物的有效荧光猝灭剂。