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.

Twinkle, twinkle little star: Photoswitchable fluorophores for super‐resolution imaging

“…For instance, the dye Abberior STAR 635P can achieve a resolution in STED which is approximately two times better than the best resolution achievable with any of the fluorescent proteins, presumably due to a high photostability [81,82], and in STORM/PALM, the dye Cy5 gives several times more photons per localization than any of the currently preferred photoswitchable fluorescent proteins [22,[68][69][70]. Because there are a wide array of organic fluorophores spanning the visible and near infrared region of the electromagnetic spectrum, there are many good options for multicolor imaging of cellular structures for both STED and STORM/PALM [22,78,81,83,84]. Notably, the duty cycle of many photoswitchable fluorescent proteins is 10-1000 times lower than many of the currently favored photoswitchable organic fluorophores, and may enable experiments at higher densities with photoswitchable Table 1 A selection of fluorescent proteins used for super-resolution fluorescence imaging.…”

“…These include long-established STED fluorophores such as Chromeo 488, Atto 565, and Atto 647 N, as well the recent rhodamine derivatives, carbopyronine derivatives, or Abberior STAR series fluorophores which are now commercially available (e.g., Abberior STAR 635P) [34,52,81,82,99,100]. Multicolor imaging strategies include the use of common excitation and depletion wavelengths with closely emitting fluorophores (e.g., that can be separated through spectral unmixing techniques, as was described above for two-color fluorescent protein imaging).…”

General and Facile Purification of Dye-Labeled Oligonucleotides by Ph-Controlled Extraction

“…The relationship between the pH of an aqueous solution and the solubility of a molecule in that solution has been noted in an earlier study (4). The pH-dependent solubility can be understood using the Henderson-Hasselbalch equation:…”

Red Emitting Coumarins: Insights of Photophysical Properties with DFT Methods

“…Design and synthesis of red emitting dyes are gaining interest in recent years [34][35][36][37][38]. The molecules of coumarin class are known to be highly fluorescent [39] with moderate to good quantum yields [40] and are used in biological applications such as fluorescent markers for proteins [41], cellular imaging [42] and lasers [43].…”