Optimization and functionalization of red-shifted rhodamine dyes

Abstract

Expanding the palette of fluorescent dyes is vital for pushing the frontier of biological imaging. Although rhodamine dyes remain the premier type of small-molecule fluorophore due to their bioavailability and brightness, variants excited with far-red or near-infrared light suffer from poor performance due to their propensity to adopt a lipophilic, nonfluorescent form. We report a general chemical modification for rhodamines that optimizes long-wavelength variants and enables facile functionalization with different chemical groups.

Molecular Fluorophores for Deep-Tissue Bioimaging

“…The equilibrium exists in almost all X-rhodamines, but it appears that the equilibrium propensity (lactone to zwitterion) is inversely correlated to absorption wavelength; that is, for example, near-infrared rhodamines containing sulfone (−SO 2 −) and phosphorus (−PO(OH)−) groups are easier to be in lactone form. 47 On the basis of azetidine-substituted X-rhodamine structures, Lavis and co-workers have established principles for fine-tuning the fluorogenic properties of various X-rhodamines by exploring substitution patterns on the azetidine rings 48 or the pendant phenyl ring 47 ( Figure 3 e). In general, weakening nucleophilicity of the carboxylate anion or improving electron deficiency of the xanthene scaffold will shift the equilibrium to the open form.…”

“…All these strategies gained positive returns, while a more general strategy with minimal structural change was to use four-membered azetidine rings as donors (Figure c), which preserved the requisite small size and high membrane permeability of fluorophores for use in live cells. Proposed and perfected by Lavis and co-workers, − this general method created a palette of X-rhodamines (Janelia Fluor) with improved quantum efficiencies that span the visible (500–700 nm) range by fine-tuning the substitution patterns on azetidine rings (Figure c). The improved brightness of these rhodamines under 1P excitation also extended to 2P excitation at a wavelength range of 1000–1300 nm (Figure d) .…”

In vivo nerve-specificity of rhodamines and Si-rhodamines

“…All fluorophores used in this study, with the exception of rhodamine B, were synthesized according to previously reported synthetic routes. 19,22 Rhodamine B was purchased from Sigma-Aldrich (St. Louis, MO). All analytical thin layer chromatography (TLC) was performed on ready-to-use plates with silica gel 60 (32-63 μm, EMD Millipore, Burlington, MA) and all flash chromatographic purification was performed by column chromatography using silica gel (Sorbent Technologies Inc., Norcross, GA).…”

“…90 nm relative to their parent structural analogues. [15][16][17][18][19] The study detailed herein represents an effort to investigate whether such a strategy may be applied towards spectrally tuning potential fluorescent candidates to be used as nerve-specific contrast agents for FGS. A comparison was made to determine whether two previously reported compounds, Si-TMR and Si-rhodamine B, would maintain the nerve-specific properties previously observed in structurally analogous rhodamine derivatives.…”

Sulfonamides Are an Overlooked Class of Electron Donors in Luminogenic Luciferins and Fluorescent Dyes

“…Impressively, this is brighter than most rhodols with dialkylamine donors. 19,20 Moreover, the bis-TFP rhodamine 47 is also fluorescent, with a quantum yield of 0.40 and extinction coefficient of 51,000 M −1 cm −1 , suggesting that sulfonamides could also be incorporated into related red-shifted scaffolds such as oxazines 21 and azasilines, 22 as well as sulfone, 23 phosphorus, [24][25][26] and Si-substituted rhodamines 27 and rhodols. 28 In summary, sulfonamide electron donors do not behave like "caged" carboxamides, but instead expand the scope of luminogenic molecules beyond the canonical hydroxyl and amino-substituted analogues.…”