title: Polar red-emitting rhodamine dyes with reactive groups: synthesis, photophysical properties, and two-color STED nanoscopy applications.
abstract: 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.
authors: Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen (Germany).
authors: Kirill
authors: K
authors: Kolmakov
authors: Christian A
authors: CA
authors: Wurm
authors: Dirk N H
authors: DN
authors: Meineke
authors: Fabian
authors: F
authors: Göttfert
authors: Vadim P
authors: VP
authors: Boyarskiy
authors: Vladimir N
authors: VN
authors: Belov
authors: Stefan W
authors: SW
authors: Hell
mesh_terms: D001386:Azides / Q000737:chemistry; D057930:Click Chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D010766:Phosphorylation; D011804:Quinolines; D012235:Rhodamines / Q000138:chemical synthesis / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014867:Water / Q000737:chemistry
chemical_list: D001386:Azides; D005456:Fluorescent Dyes; D011804:Quinolines; D012235:Rhodamines; C053059:1,2-dihydro-2,2,4-trimethylquinoline; D014867:Water
keywords: bioconjugation; chromophores; click chemistry; fluorescent probes; rhodamines