O' Connor Darragh, Byrne Aisling, Keyes Tia E
School of Chemical Sciences, National Centre for Sensor Research, Dublin City University Glasnevin Dublin 9 Ireland
RSC Adv. 2019 Jul 23;9(40):22805-22816. doi: 10.1039/c9ra03905h.
Lipid membrane fluorescent probes that are both domain-selective and compatible with demanding microscopy methods are crucial to elucidate the presence and function of rafts and domains in cells and biophysical models. Whereas targeting fluorescent probes to liquid-disordered (L) domains is relatively facile, it is far more difficult to direct probes with high selectivity to liquid-ordered (L) domains. Here, a simple, one-pot approach to probe-cholesterol conjugation is described using Steglich esterification to synthesise two identical BODIPY derivatives that differ only in the length of the aliphatic chain between the dye and cholesterol. In the first, BODIPY-Ar-Chol, the probe and cholesterol were directly ester linked and in the second BODIPY-Ahx-Chol, a hexyl linker separated probe from cholesterol. Uptake and distribution of each probe was compared in ternary, phase separated giant unilamellar vesicles (GUVs) using a commercial L marker as a reference. BODIPY-Ar-Chol targets almost exclusively the L domains with selectivity of >90% whereas by contrast introducing the C linker between the probe and cholesterol drove the probe to L with excellent selectivity (>80%). The profound impact of the linker length extended also to uptake and distribution in live mammalian cells. BODIPY-Ahx-Chol associates strongly with the plasma membrane where it partitioned preferably into opposing micron dimensioned do-mains to a commercial L marker and its concentration at the membrane was reduced by cyclodextrin treatment of the cells. By contrast the BODIPY-Ahx-Chol permeated the membrane and localised strongly to lipid droplets within the cell. The data demonstrates the profound influence of linker length in cholesterol bioconjugates in directing the probe.
对脂筏和结构域在细胞及生物物理模型中的存在和功能进行阐释时，兼具结构域选择性且能与高要求显微镜方法兼容的脂质膜荧光探针至关重要。虽然将荧光探针靶向液态无序（L）结构域相对容易，但将探针高选择性地导向液态有序（Lo）结构域则困难得多。本文描述了一种简单的一锅法探针 - 胆固醇共轭方法，利用施陶丁格酯化反应合成了两种仅在染料与胆固醇之间脂肪链长度上存在差异的相同硼二吡咯衍生物。第一种是BODIPY - Ar - Chol，探针与胆固醇直接通过酯键相连；第二种是BODIPY - Ahx - Chol，己基连接子将探针与胆固醇分隔开。以市售的L标记物作为参照，比较了每种探针在三元相分离巨型单层囊泡（GUVs）中的摄取和分布情况。BODIPY - Ar - Chol几乎专门靶向L结构域，选择性大于90%；相比之下，在探针与胆固醇之间引入碳连接子则使探针以优异的选择性（>80%）导向Lo结构域。连接子长度的深远影响还延伸至其在活哺乳动物细胞中的摄取和分布。BODIPY - Ahx - Chol与质膜强烈结合，在质膜上它优先分配到与市售L标记物相对的微米尺寸结构域中，并且通过对细胞进行环糊精处理可降低其在膜上的浓度。相比之下，BODIPY - Ahx - Chol可穿透膜并强烈定位于细胞内的脂滴。数据表明胆固醇生物共轭物中连接子长度对探针导向具有深远影响。
