Synthesis of Carboxy ATTO 647N Using Redox Cycling for Xanthone Access

Bachman, James L; Pavlich, Cyprian I; Boley, Alexander J.; Marcotte, Edward M.; Anslyn, Eric V.

doi:10.1021/acs.orglett.9b03981

Cited by 7 publications

(7 citation statements)

References 43 publications

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“…We first focused on the novel carborhodamine 59 (Scheme ), which is a fluorine-containing analog of ATTO 647N ( 105 ; Figure a). The “famous” ATTO 647N is perhaps the best dye for in vitro single-molecule imaging and has found widespread use as a label for advanced imaging experiments. ,, ATTO 647N conjugates have not been used in live-cell imaging experiments, however, due to structural constraints. A key design principle used in many ATTO dyes involves amidation of the ortho -carboxyl group on the pendant ring with 4-(methylamino)butanoic acid.…”

Section: Resultsmentioning

confidence: 99%

“…We also achieved high-yielding syntheses of the des-fluoro analogs of rhodamines 57 and 59 using this chemistry (Scheme S7). In contrast, all attempts to synthesize 57 or 59 through addition of lithiated 6 to anthrones containing a julolidine motif 57 or via dibromination of 56 or 58 failed to provide appreciable amounts of desired carborhodamine products. Pd-Catalyzed Cross-Coupling to Prepare Unsubstituted and N-Aryl Rhodamines.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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Optimized Red-Absorbing Dyes for Imaging and Sensing

Grimm,

Tkachuk,

Patel

et al. 2023

J. Am. Chem. Soc.

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Rhodamine dyes are excellent scaffolds for developing a broad range of fluorescent probes. A key property of rhodamines is their equilibrium between a colorless lactone and fluorescent zwitterion. Tuning the lactone−zwitterion equilibrium constant (K L−Z ) can optimize dye properties for specific biological applications. Here, we use known and novel organic chemistry to prepare a comprehensive collection of rhodamine dyes to elucidate the structure−activity relationships that govern K L−Z . We discovered that the auxochrome substituent strongly affects the lactone− zwitterion equilibrium, providing a roadmap for the rational design of improved rhodamine dyes. Electron-donating auxochromes, such as julolidine, work in tandem with fluorinated pendant phenyl rings to yield bright, red-shifted fluorophores for live-cell single-particle tracking (SPT) and multicolor imaging. The N-aryl auxochrome combined with fluorination yields red-shifted Forster resonance energy transfer (FRET) quencher dyes useful for creating a new semisynthetic indicator to sense cAMP using fluorescence lifetime imaging microscopy (FLIM). Together, this work expands the synthetic methods available for rhodamine synthesis, generates new reagents for advanced fluorescence imaging experiments, and describes structure−activity relationships that will guide the design of future probes.

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Section: Resultsmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

Optimized Red-Absorbing Dyes for Imaging and Sensing

Grimm,

Tkachuk,

Patel

et al. 2023

J. Am. Chem. Soc.

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“…It is known as KK 114 (see Figure 1 and Ref. [11]) and was supposed to be a better and cheaper alternative for the dye ATTO 647 N. The latter, in turn, was the first dye purposefully designed for STED microscopy by ATTO-TEC GmbH [13]. For this application, photostability and, especially, stability toward photo-induced reactive oxygen species were crucial.…”

Section: Design Of Photostable and Hydrophilic Red-emitting Sted Fluo...mentioning

confidence: 99%

Red-Emitting Fluorophores with Tailored Properties for Microscopy and Nanoscopy

Kolmakov,

Lucidi,

V. Nizovtsev

2024

Dye Chemistry - Exploring Colour From Nature to Lab

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Extended series of red-emitting rhodamine dyes were synthesized and tested. This revealed the real factors determining the quality of STED (stimulated emission depletion) imaging, dye photostability in general, and stability of the corresponding reactive labels. Previously overlooked and underestimated observations are now being highlighted and discussed. So are some very important syntheses-related aspects. The studies were also expanded on far-red emitting oxazine and silicon rhodamine dyes. An intriguing discovery emerged when certain fluorophores, which were originally designed for STED, demonstrated an ability to discern between viable and dead bacterial cells. Recent publications by Lucidi and co-workers showed that certain positively charged dyes allow imaging of bacterial membranes using both conventional techniques, for example, confocal laser scanning microscopy (CLSM), and STED. This opens avenues for investigating membrane biogenesis and diagnostics.

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“…The parent compounds themselves are highly modifiable at the xanthene core and through substituents at the pendant aryl group or aniline. In the case of rhodamine, variants have also been prepared by substituting the central oxygen atom to give fluorophores such as the carbopyronine Atto 647N ( 3 ), − among others. − Xanthene-based dyes generally possess desirable spectral properties, including high extinction coefficients and quantum yields (QYs), fluorescence in the visible region, and overall chemical stability . However, a key drawback to these fluorescent probes is their tendency for fluorescence self-quenching.…”

Section: Introductionmentioning

confidence: 99%

Evaluating the Effect of Dye–Dye Interactions of Xanthene-Based Fluorophores in the Fluorosequencing of Peptides

et al. 2022

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A peptide sequencing scheme utilizing fluorescence microscopy and Edman degradation to determine the amino acid position in fluorophorelabeled peptides was recently reported, referred to as fluorosequencing. It was observed that multiple fluorophores covalently linked to a peptide scaffold resulted in a decrease in the anticipated fluorescence output and worsened the single-molecule fluorescence analysis. In this study, we report an improvement in the photophysical properties of fluorophore-labeled peptides by incorporating long and flexible (PEG) 10 linkers at the peptide attachment points. Long linkers to the fluorophores were installed using copper-catalyzed azide−alkyne cycloaddition conditions. The photophysical properties of these peptides were analyzed in solution and immobilized on a microscope slide at the singlemolecule level under peptide fluorosequencing conditions. Solution-phase fluorescence analysis showed improvements in both quantum yield and fluorescence lifetime with the long linkers. While on the solid support, photometry measurements showed significant increases in fluorescence brightness and 20 to 60% improvements in the ability to determine the amino acid position with fluorosequencing. This spatial distancing strategy demonstrates improvements in the peptide sequencing platform and provides a general approach for improving the photophysical properties in fluorophore-labeled macromolecules.

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Synthesis of Carboxy ATTO 647N Using Redox Cycling for Xanthone Access

Cited by 7 publications

References 43 publications

Optimized Red-Absorbing Dyes for Imaging and Sensing

Optimized Red-Absorbing Dyes for Imaging and Sensing

Red-Emitting Fluorophores with Tailored Properties for Microscopy and Nanoscopy

Evaluating the Effect of Dye–Dye Interactions of Xanthene-Based Fluorophores in the Fluorosequencing of Peptides

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