用于研究生物正交钌催化剂在革兰氏阳性细菌体内的内化和作用的荧光探针。

IF 4.2 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Nicole Schubert, James W Southwell, Melissa Vázquez-Hernández, Svenja Wortmann, Sylvia Schloeglmann, Anne-Kathrin Duhme-Klair, Patrick Nuernberger, Julia E Bandow, Nils Metzler-Nolte
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引用次数: 0

摘要

生物正交反应对生物分子的化学修饰非常有用,在哺乳动物细胞中的研究已经非常深入。相比之下,人们很少关注此类反应在细菌中的可行性。在此,我们报告了用于监测生物正交催化剂在革兰氏阳性细菌枯草杆菌中的内化和活性的改性香豆素染料。我们合成并鉴定了两种基于 7-氨基香豆素的荧光团,以确定它们的发光特性。在两种具有不同溶解度的 7-氨基香豆素衍生物的氮原子上引入氨基甲酸烯丙酯(R2N-COOR')基团后,荧光发射强度降低,发射最大值显著蓝移。重要的是,这种氨基甲酸烯丙酯基团可以通过本研究中的生物正交有机金属钌催化剂解笼,从而在生物相关条件下产生荧光产物。ICP-OES 分析证实并量化了这种催化剂的内化作用。在用两种笼型染料培养细菌后,分别对细菌细胞质和细胞外部分进行了调查,这有助于确定它们的位置,以及通过添加催化剂确定它们的非笼型形式。事实上,我们观察到了明显的差异,因为只有亲脂性更强的染料位于细胞内,而且重要的是一直留在细胞内,似乎避免了外流机制。然而,这种染料的非笼型并没有被保留下来,而是主要存在于细胞外空间。最后,我们还研究了催化剂的一系列苷元共轭衍生物,以进行相同的转化。尽管与非共轭型相比,它们的吸附作用并不明显,但无论培养基中的铁含量如何,都能观察到类似的细胞内反应速率,这一事实证明了它们的吸附作用与革兰氏阳性枯草杆菌细胞利用的铁转运体无关。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Fluorescent probes for investigating the internalisation and action of bioorthogonal ruthenium catalysts within Gram-positive bacteria.

Bioorthogonal reactions are extremely useful for the chemical modification of biomolecules, and are already well studied in mammalian cells. In contrast, very little attention has been given to the feasibility of such reactions in bacteria. Herein we report modified coumarin dyes for monitoring the internalisation and activity of bioorthogonal catalysts in the Gram-positive bacterial species Bacillus subtilis. Two fluorophores based on 7-aminocoumarin were synthesised and characterised to establish their luminescence properties. The introduction of an allyl carbamate (R2N-COOR') group onto the nitrogen atom of two 7-aminocoumarin derivatives with different solubility led to decreased fluorescence emission intensities and remarkable blue-shifts of the emission maxima. Importantly, this allyl carbamate group could be uncaged by the bioorthogonal, organometallic ruthenium catalyst investigated in this work, to yield the fluorescent product under biologically-relevant conditions. The internalisation of this catalyst was confirmed and quantified by ICP-OES analysis. Investigation of the bacterial cytoplasm and extracellular fractions separately, following incubation of the bacteria with the two caged dyes, facilitated their localisation, as well as that of their uncaged form by catalyst addition. In fact, significant differences were observed, as only the more lipophilic dye was located inside the cells and importantly remained there, seemingly avoiding efflux mechanisms. However, the uncaged form of this dye is not retained, and was found predominantly in the extracellular space. Finally, a range of siderophore-conjugated derivatives of the catalyst were investigated for the same transformations. Even though uptake was observed, albeit less significant than for the non-conjugated version, the fact that similar intracellular reaction rates were observed regardless of the iron content of the medium supports the notion that their uptake is independent of the iron transporters utilised by Gram-positive Bacillus subtilis cells.

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来源期刊
CiteScore
6.10
自引率
0.00%
发文量
128
审稿时长
10 weeks
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