Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen (Germany).
Chemistry. 2014 Jan 3;20(1):146-57. doi: 10.1002/chem.201303433. Epub 2013 Dec 11.
The synthesis, reactivity, and photophysical properties of new rhodamines with intense red fluorescence, two polar residues (hydroxyls, primary phosphates, or sulfonic acid groups), and improved hydrolytic stability of the amino-reactive sites (NHS esters or mixed N-succinimidyl carbonates) are reported. All fluorophores contain an N-alkyl-1,2-dihydro-2,2,4-trimethylquinoline fragment, and most of them bear a fully substituted tetrafluoro phenyl ring with a secondary carboxamide group. The absorption and emission maxima in water are in the range of 635-639 and 655-659 nm, respectively. A vastly simplified approach to red-emitting rhodamines with two phosphate groups that are compatible with diverse functional linkers was developed. As an example, a phosphorylated dye with an azide residue was prepared and was used in a click reaction with a strained alkyne bearing an N-hydroxysuccinimid (NHS) ester group. This method bypasses the undesired activation of phosphate groups, and gives an amphiphilic amino-reactive dye, the solubility and distribution of which between aqueous and organic phases can be controlled by varying the pH. The presence of two hydroxyl groups and a phenyl ring with two carboxyl residues in the dyes with another substitution pattern is sufficient for providing the hydrophilic properties. Selective formation of a mono-N-hydroxysuccinimidyl ester from 5-carboxy isomer of this rhodamine is reported. The fluorescence quantum yields varied from 58 to 92% for free fluorophores, and amounted to 18-64% for antibody conjugates in aqueous buffers. The brightness and photostability of these fluorophores facilitated two-color stimulated emission depletion (STED) fluorescence nanoscopy of biological samples with high contrast and minimal background. Selecting a pair of fluorophores with absorption/emission bands at 579/609 and 635/655 nm enabled two-color channels with low cross-talk and negligible background at approximately 40 nm resolution.
报告了具有强烈红色荧光、两个极性残基（羟基、伯膦酸基或磺酸基）和改进的氨基反应性位点（NHS 酯或混合 N-琥珀酰亚胺碳酸酯）水解稳定性的新型罗丹明的合成、反应性和光物理性质。所有荧光团都含有 N-烷基-1,2-二氢-2,2,4-三甲基喹啉片段，其中大多数都带有全取代的四氟苯基环和仲羧酰胺基。在水中的吸收和发射最大值分别在 635-639nm 和 655-659nm 范围内。开发了一种大大简化的方法，用于制备具有两个可与各种功能接头兼容的磷酸基团的红色发射罗丹明。例如，制备了带有叠氮基的磷酸化染料，并将其用于带有 N-羟基琥珀酰亚胺（NHS）酯基团的应变炔烃的点击反应中。该方法避免了磷酸基团的非预期活化，并提供了一种两亲性氨基反应性染料，其在水相和有机相之间的溶解度和分布可以通过改变 pH 值来控制。在具有另一种取代模式的染料中，两个羟基和一个带有两个羧基残基的苯基环足以提供亲水性。报道了从这种罗丹明的 5-羧基异构体选择性形成单-N-羟基琥珀酰亚胺酯。对于游离荧光团，荧光量子产率从 58%到 92%不等，对于水相缓冲液中的抗体缀合物，荧光量子产率达到 18%-64%。这些荧光团的亮度和光稳定性促进了具有高对比度和最小背景的生物样品的双色受激发射耗尽（STED）荧光纳米显微镜。选择一对吸收/发射带在 579/609nm 和 635/655nm 的荧光团可以实现两个通道，其具有低串扰和几乎可以忽略不计的背景，分辨率约为 40nm。
