Ooi Jocelyn M F, Fairhall Jessica M, Spangler Benjamin, Chong Daniel J W, Feng Brian Y, Gamble Allan B, Hook Sarah
School of Pharmacy, University of Otago Dunedin New Zealand
Novartis Institutes for BioMedical Research (NIBR) in Emeryville California USA.
RSC Adv. 2022 May 23;12(25):15631-15642. doi: 10.1039/d2ra02272a.
Bioorthogonal chemistry can facilitate the development of fluorescent probes that can be used to sensitively and specifically detect the presence of biological targets. In this study, such an assay was developed to evaluate the uptake and delivery of antimicrobials into , building on and extending previous work which utilised more resource intensive LCMS detection. The bacteria were genetically engineered to express streptavidin in the periplasmic or cytoplasmic compartments, which was used to localise a bioorthogonal probe (BCN-biotin). Azido-compounds which are delivered to these compartments react with the localised BCN-biotin-streptavidin in a concentration-dependent manner a strain-promoted alkyne-azide cycloaddition. The amount of azido-compound taken up by bacteria was determined by quantifying unreacted BCN-biotin-streptavidin an inverse electron demand Diels-Alder reaction between remaining BCN-biotin and a tetrazine-containing fluorescent dye. Following optimisation and validation, the assay was used to assess uptake of liposome-formulated azide-functionalised luciferin and cefoxitin. The results demonstrated that formulation into cationic liposomes improved the uptake of azide-functionalised compounds into the periplasm of , providing insight on the uptake mechanism of liposomes in the bacteria. This newly developed bioorthogonal fluorescence plate-reader based assay provides a bioactivity-independent, medium-to-high throughput tool for screening compound uptake/delivery.
生物正交化学有助于开发荧光探针，用于灵敏且特异地检测生物靶标的存在。在本研究中，基于并扩展了先前使用资源消耗更大的液相色谱 - 质谱检测的工作，开发了这样一种测定方法来评估抗菌剂进入细菌的摄取和递送情况。对细菌进行基因工程改造，使其在周质或细胞质区室中表达链霉亲和素，用于定位生物正交探针（BCN - 生物素）。递送至这些区室的叠氮化合物以浓度依赖的方式与定位的BCN - 生物素 - 链霉亲和素发生反应——一种应变促进的炔 - 叠氮环加成反应。通过定量未反应的BCN - 生物素 - 链霉亲和素（剩余的BCN - 生物素与含四嗪的荧光染料之间的逆电子需求狄尔斯 - 阿尔德反应）来确定细菌摄取的叠氮化合物的量。经过优化和验证后，该测定方法用于评估脂质体包裹的叠氮功能化荧光素和头孢西丁的摄取情况。结果表明，制备成阳离子脂质体可提高叠氮功能化化合物进入细菌周质的摄取量，为脂质体在细菌中的摄取机制提供了见解。这种新开发的基于生物正交荧光酶标仪的测定方法为筛选化合物摄取/递送提供了一种与生物活性无关的中高通量工具。
