Synthesis of Novel Macrocycle-Containing Polymers as Protease Sensors

描述（由申请人提供）：Swager研究小组开发的扩增荧光聚合物显示出作为各种分析物的灵敏和特异性传感器的显著能力。该提案旨在利用放大荧光聚合物的这种传感能力来开发用于蛋白酶检测的新测定法。为此，提出了具有一定百分比的大环单元的放大混合共聚物的合成。这些大环化合物将方酸菁染料包封在聚合物主链附近，并将含有可被靶蛋白酶切割的寡肽接头。在不存在靶蛋白酶的情况下，染料将保持在大环中，并且将发生从聚合物主链到方酸染料的有效的德克斯特型能量转移。聚合物的激发将导致方酸菁染料的选择性放大发射。大环的蛋白酶介导的裂解将释放方酸菁染料，导致聚合物和染料之间的Dexter能量转移的淬灭。德克斯特型能量转移的使用将增加系统的灵敏度，因为方酸染料的发射将在完全黑暗的背景下发生，而没有残留的聚合物荧光。将使用荧光光谱法监测能量转移的效率，以检测方酸菁染料的发射，其发生在近红外光谱区。荧光光谱将进一步用于测量靶蛋白酶切割大环的速率，因为这将导致聚合物和染料之间的Dexter能量转移的淬灭和染料发射的沉默。公共卫生相关性本提案描述了一类新的放大荧光聚合物的合成，其可用于超灵敏蛋白酶检测。蛋白酶活性与包括癌症在内的多种疾病有关;因此，体内蛋白酶的检测对于多种疾病的诊断和新疾病治疗的开发都是至关重要的。所提出的大环聚合物可以很容易地适用于检测几乎任何目标蛋白酶，以及其他生物相关的目标。

DESCRIPTION (provided by applicant): Amplifying fluorescent polymers developed in the Swager research group have shown a remarkable ability to act as sensitive and specific sensors for various analytes. This proposal seeks to harness this sensing ability of amplifying fluorescent polymers for the development of new assays for protease detection. To do so, the synthesis of an amplifying mixed co-polymer with some percentage of macrocyclic units is proposed. These macrocycles will encapsulate squaraine dyes in close proximity to the polymer backbone, and will contain oligopeptide linkers that can be cleaved by a target protease. In the absence of the target protease, the dye will be held in the macrocycle and efficient Dexter-type energy transfer will occur from the polymer backbone to the squaraine dye. Excitation of the polymer will result in selective amplified emission from the squaraine dye. Protease-mediated cleavage of the macrocycle will release the squaraine dye, leading to a quenching of the Dexter energy transfer between the polymer and the dye. The use of Dexter-type energy transfer will increase the sensitivity of the system, as the emission of the squaraine dye will occur on a completely dark background, without residual polymer fluorescence. The efficiency of energy transfer will be monitored using fluorescence spectroscopy to detect the emission of the squaraine dye, which occurs in the near-infrared spectral region. Fluorescence spectroscopy will further be used to measure the rate at which the target protease cleaves the macrocycle, as this will result in a quenching of the Dexter energy transfer between the polymer and a dye and a silencing of the dye's emission. PUBLIC HEALTH RELEVANCE This proposal describes the synthesis of a new class of amplifying fluorescent polymers that can be utilized for ultrasensitive protease detection. Protease activity has been implicated in a variety of diseases, including cancer; the detection of proteases in vivo is therefore critical both for the diagnosis of multiple diseases and for the development of new disease treatments. The proposed macrocyclic polymer can easily be adapted to detect virtually any target protease, as well as other biologically relevant targets.