A range of bright and photostable rhodamines and carbopyronines with absorption maxima in the range of λ=500–630 nm were prepared, and enabled the specific labeling of cytoskeletal filaments using HaloTag technology followed by staining with 1 μm solutions of the dye–ligand conjugates. The synthesis, photophysical parameters, fluorogenic behavior, and structure–property relationships of the new dyes are discussed. Light microscopy with stimulated emission depletion (STED) provided one‐ and two‐color images of living cells with an optical resolution of 40–60 nm.

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.

Electronic energy transfer (EET) between chromophores is of fundamental importance for many biological processes and optoelectronic devices. However, common models fall short in fully describing the process, especially in bichromophoric model systems with a donor and acceptor connected by a rigid linker providing perpendicular geometries. Herein, we report a novel strategy for preparing bichromophores containing adamantane or 2-(2-adamantylidene)adamantane as rigid spacers, providing a fixed distance between chromophores, and their parallel or perpendicular arrangement without chromophore rotation. New fluorophores were developed and linked via spiroatoms. Bichromophores with identical (blue-blue) or different (blue-red) chromophores were synthesized, either in orthogonal or parallel geometry. These were characterized by absorption/fluorescence spectroscopy, time-resolved fluorescence anisotropy, and fluorescence antibunching measurements. Based on the Förster point-dipole approximation, EET efficiencies were estimated by using geometrical parameters from (time-dependent) density functional calculations. For bichromophores with parallel geometry, the predicted EET efficiencies were near unity and fit the measurements. In spite of estimated values around 0.4 and 0.5, 100 % efficiency was observed also for bichromophores with orthogonal geometry. The new rigid scaffolds presented here open new possibilities for the synthesis of bichormophores with well-defined parallel or perpendicular geometry.

Helle und photostabile Rhodamine und Carbopyronine mit Absorptionsmaxima im Bereichv on l = 500-630 nm wurden entwickelt, die nachA nwendung von Halo- Tag Hier präsentieren wir eine Reihe zellgängiger Fluoreszenzmarker für lebende Zellen und wenden sie in hochauflçsender Ein-und Zweifarbenfluoreszenzmikroskopie mittels stimulated emission depletion (STED) an.[2] Für eine spezifische Markierung der intrazellulären Proteine in lebenden Zellen verwendeten wir eine gängige und zuverlässige Methode,d ie auf HaloTag-Fusionsproteinen basiert.[3] Dabei wird das Zielprotein genetisch an ein künstliches Enzym (modifizierte Dehalogenase aus Rhodococcus rhodochrous) gebunden (fusioniert). Das Enyzm ist dazu in der Lage,s elektiv und schnell eine kovalente Bindung mit dem Substrat zu bilden.Die Auswahl geeigneter Farbstoffe für intrazelluläre Markierungen von lebenden Zellen ist aufgrund der Anforderungen an die Zellgängigkeit begrenzt. Einige Rhodamine, [4] Carbopyronine [5] und Siliconrhodamine (SiR), [6] deren Helligkeit vor kurzem verbessert wurde, [1h] kçnnen die äußere Plasmamembran intakter Zellen durchdringen. [7] Leider ist die spektrale Vielfalt photostabiler fluoreszierender Farbstoffe,d ie sich für die spezifische Markierung intrazellulärer Strukturen und gleichzeitig für die hochauflçsende Mikroskopie eignen, sehr begrenzt. Weiterhin sind nur wenige dieser Farbstoffe kommerziell erhältlich (siehe Abbildung S1 in den Hintergrundinformationen).In kommerziellen STED-Mikroskopen kommen Laser in drei spektralen Bereichen für die stimulierte Emission zum Einsatz: l = 592/595, 660 und 765/775 nm.[8] Ein roter (l ! 620 nm) oder sogar IR-Laser ist vorteilhaft, da dieser lebende Zellen weniger beeinträchtigt und unerwünschte Autofluoreszenz, Photobleichen und Lichtstreuung vermindert. Deshalb konzentriert sich diese Studie auf die Entwicklung und Charakterisierung von zellgängigen Rhodamin-und Carbopyronin-Farbstoffen für die hochauflçsende Mikroskopie mit dem orangeroten (l = 618 nm) oder IR-STED-Laser (l = 775 nm).Es stellte sich heraus,d ass membrangängige Farbstoffe vorzugsweise elektrisch neutral oder Zwitterionen mit einem kurzen Abstand zwischen den Ladungen sind. Zudem weisen sie eine mçglichst kompakte Struktur,e in relativ geringes Molekulargewicht (M < 700 Da) und einige Heteroatome auf,d ie als Wasserstoffbrückendonoren oder -akzeptoren wirken kçnnen. Diese Merkmale ermçglichten eine Auswahl bekannter [4b, 6] sowie die Synthese neuer grün, gelb und rot emittierender Rhodamine,C arbopyronine und Siliconrhodamine (Schema 1). Die spektralen Eigenschaften der untersuchten Farbstoffe dieser Art sind in Tabelle 1u nd Abbildung S2 der Hintergrundinformationen zusammengefasst. Bisher sind lediglich 6'-Carboxyisomere erfolgreich zur Markierung von Proteinen durch die Halo-oder SNAP-tagbasierte Selbstmarkierungstechnik eingesetzt worden.

Aromatic nucleophilic substitution (SNAr) of fluorine in 9‐(3′‐carboxy‐4′,5′,6′,7′‐tetrafluorophenyl) groups of xanthene dyes constitutes a powerful tool in dye design. Thiols and amines regioselectively replace F‐6′. This approach enables additional hydrophilic residues or functional groups required for bioconjugation to be introduced. By using this methodology, a “bright” and photostable dye for two‐color superresolution microscopy was synthesized (with absorption and emission maxima at 604 and 627 nm, respectively). In the case of red‐emitting rhodamine dyes with 3′‐carboxy‐4′,5′,7′‐trifluorophenyl residues, two‐dimensional NMR techniques and a chemical transformation were used to prove the precise position of the additional substituent – a carboxylic acid group linked through the S‐atom at C‐6′. Furthermore, simple 1H NMR spectra reliably permit the position of the additional carboxy substituent in the 3′‐carboxyphenyl ring (at C‐5′ or C‐6′) to be established. Information on the exact position of this substituent is significant for the design of molecular probes and for the prediction of the properties of their bioconjugates.