Live-cell imaging of cholesterol trafficking depends on suitable cholesterol analogs. However, existing fluorescent analogs of cholesterol either show very different physicochemical properties compared to cholesterol or demand excitation in the ultraviolet spectral region. We present a strategy to synthesize two novel intrinsically fluorescent sterol probes with a close resemblance of cholesterol. The analogs contain four conjugated double bonds in the ring system and either a keto group (probe 5) or a hydroxy group (probe 6) in the C3 position. The emission of 5 is in the visible range of the spectrum, i.e., red-shifted by 150 nm compared to the widely used dehydroergosterol. Together with its high multiphoton absorption, this allows for imaging of 5 on conventional microscopes, including multicolor 3D and time-lapse microscopy. Molecular dynamics simulations and nuclear magnetic resonance spectroscopy reveal that 5 can condense the fatty acyl chains of phospholipids in model membranes. In giant unilamellar vesicles, 5 partitions equally into the liquid-ordered and disordered phases. In contrast, 6 emits in the ultraviolet range and is unstable in solution, preventing its use in live-cell imaging applications. The good photophysical properties of 5 make it a suitable analogue for improved live-cell imaging of sterol transport.

中文翻译：

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具有改进光物理特性的新型胆固醇本征荧光类似物的合成和表征


胆固醇运输的活细胞成像取决于合适的胆固醇类似物。然而，现有的胆固醇荧光类似物要么表现出与胆固醇截然不同的物理化学性质，要么需要在紫外光谱区域激发。我们提出了一种合成两种与胆固醇非常相似的新型固有荧光甾醇探针的策略。类似物在环系统中包含四个共轭双键，在 C3 位置包含一个酮基（探针 5）或一个羟基（探针 6）。5 的发射在光谱的可见范围内，即与广泛使用的脱氢麦角甾醇相比，红移了 150 nm。再加上其高多光子吸收能力，这允许在传统显微镜（包括多色 3D 和延时显微镜）上对 5 进行成像。分子动力学模拟和核磁共振波谱表明，5 可以浓缩模型膜中磷脂的脂肪酰基链。在巨大的单层囊泡中，5 平均分为液体有序相和无序相。相比之下，6 在紫外范围内发射，在溶液中不稳定，因此无法用于活细胞成像应用。5 的良好光物理特性使其成为改进甾醇转运活细胞成像的合适类似物。