Mild and scalable synthesis of phosphonorhodamines

Abstract

Since their discovery in 1887, rhodamines have become indispensable fluorophores for biological imaging. Recent studies have extensively explored heteroatom substitution at the 10' position and a variety of substitution patterns on the 3',6' nitrogens. Although 3-carboxy- and 3-sulfono-rhodamines were first reported in the 19th century, the 3-phosphono analogues have never been reported. Here, we report a mild, scalable synthetic route to 3-phosphonorhodamines. We explore the substrate scope and investigate mechanistic details of an exogenous acid-free condensation. Tetramethyl-3-phosphonorhodamine (phosTMR) derivatives can be accessed on the 1.5 mmol scale in up to 98% yield (2 steps). phosTMR shows a 12- to 500-fold increase in water solubility relative to 3-carboxy and 3-sulfonorhodamine derivatives and has excellent chemical stability. Additionally, phosphonates allow for chemical derivatization; esterification of phosTMR facilitates intracellular delivery with localization profiles that differ from 3-carboxyrhodamines. The free phosphonate can be incorporated into a molecular wire scaffold to create a phosphonated rhodamine voltage reporter, phosphonoRhoVR. PhosRhoVR 1 can be synthesized in just 6 steps, with an overall yield of 37% to provide >400 mg of material, compared to a 6-step, ∼2% yield for the previously reported RhoVR 1. PhosRhoVR 1 possesses excellent voltage sensitivity (37% ΔF/F) and a 2-fold increase in cellular brightness compared to RhoVR 1.

Scheme 1. Mild synthesis of phosphonorhodamines.

Scheme 2. Stepwise formation of phosTMR.

Scheme 3. Synthesis of triarylmethane intermediates.

Scheme 4. Synthesis of phosphonorhodamines.

Scheme 5. Direct synthesis of a functionalized phosTMR.

Fig. 1. Spectroscopic characterization of tetramethylrhodamines. Normalized absorbance and fluorescence spectra (a–d) in PBS, plots of λ_maxvs. pH (e–h) and corresponding chemical structures (i–l) for carboxyTMR (a, e and i), phosTMR (b, f and j), phosTMR·OEt (c, g and k) and sulfoTMR (d, h and l). pH titrations were performed in 10 mM buffered solutions (see ESI†) containing 150 mM NaCl ranging from pH 2.3 to 9.8 at a final dye concentration of 2 μM. Where applicable, titration curves were fit to sigmoidal dose response curves (solid black) to enable pK_a determination (dashed red). Determined pK_a values are reported next to chemical structures (i–l).

Scheme 6. Synthesis of phosTMR diesters 24 and 25.

Fig. 2. Tunable localization of 3-phosphonoTMRs. Widefield fluorescence (a–d) and transmitted light (e–h) images of HEK293T cells stained with 500 nM dyes 16 (a and e), 23 (b and f), 24 (c and g), or 25 (d and h) in HBSS for 20 min at 37 °C. Quantification (i) of cellular fluorescence. Values are mean fluorescence intensity ± S.E.M. for n = 21, 32, 45, and 19 regions of interest (ROI) for each dye. Each ROI contained between 2 and 20 individual cells. “n.s.” means that the cellular fluorescence values were not above background fluorescence. Coverslips were placed into fresh HBSS prior to imaging. Scale bar is 20 μm.

Fig. 3. Synthesis and characterization of phosRhoVR 1. (a) Synthesis of phosRhoVR 1, 30 from aldehyde 27. (b) Normalized absorbance (solid line) and fluorescence emission (dashed line) spectra of phosRhoVR 1 in ethanol. (c) HEK cells stained with 500 nM phosRhoVR 1. Scale bar is 20 μm. (d) Plot of the fractional change in fluorescence of phosRhoVR 1 vs. time for 100 ms hyper- and depolarizing steps (±100 mV in 20 mV increments) for single HEK cells under whole-cell voltage-clamp mode. (e) Plot of ΔF/F vs. final membrane potential, revealing a voltage sensitivity of approximately 37% per 100 mV. Error bars are ±SEM for n = 6 cells. If not visible, error bars are smaller than the marker.