Asymmetric and Reduced Xanthene Fluorophores: Synthesis, Photochemical Properties, and Application to Activatable Fluorescent Probes for Detection of Nitroreductase

Abstract

Xanthene fluorophores, including fluorescein, rhodol, and rhodamines, are representative classes of fluorescent probes that have been applied in the detection and visualization of biomolecules. "Turn on" activatable fluorescent probes, that can be turned on in response to enzymatic reactions, have been developed and prepared to reduce the high background signal of "always-on" fluorescent probes. However, the development of activity-based fluorescent probes for biological applications, using simple xanthene dyes, is hampered by their inefficient synthetic methods and the difficulty of chemical modifications. We have, thus, developed a highly efficient, versatile synthetic route to developing chemically more stable reduced xanthene fluorophores, based on fluorescein, rhodol, and rhodamine via continuous Pd-catalyzed cross-coupling. Their fluorescent nature was evaluated by monitoring fluorescence with variation in the concentration, pH, and solvent. As an application to activatable fluorescent probe, nitroreductase (NTR)-responsive fluorescent probes were also developed using the reduced xanthene fluorophores, and their fluorogenic properties were evaluated.

Keywords: activatable fluorescent probe; fluorescence; nitroreductase; reduced rhodafluor; xanthene fluorophore.

Figure 1

Structural analysis of typical and reduced xanthene fluorophores. (A) Reactivity of fluorescein and rhodol derivatives in alkylation and amide coupling reactions; and (B) equilibrium between closed and open forms affecting fluorescence.

Figure 2

Strategies for synthesizing asymmetric and reduced xanthene fluorophores.

Scheme 1

Synthesis of reduced fluorescein, rhodol, and rhodamine fluorophores. Reagents and conditions: i) CH₃I or methoxymethyl (MOM)-Cl, K₂CO₃. DMF, rt, 1= 99%, 2 = 89%; ii) LiAlH₄, THF, 0 °C; iii) p-chloranil, MeOH, rt, 3 = 79% over 2 steps, 4 = 88% over 2 steps; iv) Tf₂O, pyridine, CH₂Cl₂, 0 °C, to rt, 5 = 86%, 6 = 86%; v) amine or imine, Pd catalyst (Pd(OAc)₂, Pd₂(dba)₃·CHCl₃ or Pd(PPh₃)₄), ligand (BINAP or Xantphos), Cs₂CO₃, toluene, 105 °C, 7 = 20%, 8 = 41 %, 9 = further reaction without purification, 10 = 100%, 11 = 47%; vi) aq. 1 N HCl, THF, rt, 12 = 55% over 2 steps; vii) TFA, CH₂Cl₂, rt, 13 = 55%, 14 = 84%; viii) N-phenyl-bis-(trifluoromethanesulfonimide), K₂CO₃, CH₃CN, 0 °C, to rt, 15 = 69%, 16 = 57%; ix) benzophenone imine, Pd(OAc)_2, Cs₂CO₃, BINAP, toluene, 105 °C; x) aq. 1 N HCl, THF, rt, 17 = 44% over 2 steps, 18 = 30% over 2 steps.

Scheme 2

Reactivity of reduced fluorescein and typical fluorescein fluorophores on alkylation and amide coupling reactions_. Reagents and conditions: i) CH₃I, K₂CO₃, DMF, rt, 86% for 19, 87% for 24; ii) benzyl bromide, DBU, acetone, rt, 98% for 20, 71% for 25; iii) EDC, HOBt, iPrNEt₂, DMF, rt, 50% for 21, 8% for 26.

Scheme 3

Synthesis of NTR-responsive fluorescent probes with p-nitrobenzyl group_. Reagents and conditions: i) 4-nitrobenzyl bromide, Ag₂O, toluene, reflux, 27 = 54%, 28 = 48%; ii) 4-nitrobenzyl chloroformate, iPrNEt₂, CH₂Cl₂, rt, 29 = 86%, 30 = 47%.

Figure 3

Concentration-dependent fluorescence spectra of asymmetric and reduced xanthene fluorophores in PBS (10 mM, pH 7.4). (A) fluorescein 3 (λ_ex/λ_em = 456/518 nm); (B) rhodol 7 (λ_ex/λ_em = 473/535 nm); (C) rhodol 8 (λ_ex/λ_em = 490/554 nm); (D) rhodol 12 (λ_ex/λ_em = 467/519 nm); (E) rhodol 13 (λ_ex/λ_em = 472/532 nm); (F) rhodol 14 (λ_ex/λ_em = 510/553 nm); (G) rhodamine 17 (λ_ex/λ_em = 490/537 nm); (H) rhodamine 18 (λ_ex/λ_em = 504/556 nm). *Fluorescence of rhodol (13) and rhodamine (17) derivatives with monoethylamino group was estimated in the range 0.5 to 2 μM because fluorescence at 5 μM is over threshold.

Figure 4

pH-Dependent fluorescence spectra of asymmetric and reduced xanthene fluorophores (Normalized fluorescence intensity plotted against pH at 1.0 μM) in buffers with pH ranging from 2 to 13. (A) rhodol 13 (λ_ex/λ_em = 472/532 nm) and rhodol 14 (λ_ex/λ_em = 510/553 nm); (B) fluorescein 3 (λ_ex/λ_em = 456/518 nm), rhodamine 17 (λ_ex/λ_em = 490/537 nm) and rhodamine 18 (λ_ex/λ_em = 504/556 nm); (C) rhodol 7 (λ_ex/λ_em = 473/535 nm), rhodol 8 (λ_ex/λ_em = 490/554 nm) and rhodol 12 (λ_ex/λ_em = 467/519 nm)..

Figure 5

Solvent effect of asymmetric and reduced xanthene fluorophores (1.0 μM) in DMSO, IPA, EtOH, MeOH, and water. (A) fluorescein 3 (λ_ex/λ_em = 456/518 nm); (B) rhodol 7 (λ_ex/λ_em = 473/535 nm); (C) rhodol 8 (λ_ex/λ_em = 490/554 nm); (D) rhodol 12 (λ_ex/λ_em = 467/519 nm); (E) rhodol 13 (λ_ex/λ_em = 472/532 nm); (F) rhodol 14 (λ_ex/λ_em = 510/553 nm); (G) rhodamine 17 (λ_ex/λ_em = 490/537 nm); (H) rhodamine 18 (λ_ex/λ_em = 504/556 nm).

Figure 6

Stability of nitroreductase (NTR)-responsive fluorescent probes (1.0 μM). (A) Effect of variation in pH from 2 to 13 at 25 °C; (B) Effect of variation in temperature (25, 28, 31, 34, 37, 43, and 45 °C) in PBS (10 mM, pH 7.4).

Figure 7

Kinetic study of NTR-responsive fluorescent probes and fluorescence calibration curves of corresponding fluorophores. (A) Plot of fluorescence emission of probes (1.0 μM) with reaction time in the presence of 10 μg/mL of NTR, NADH (500 μM), PBS (pH 7.4), and incubation at 37 °C; (B) Calculated concentrations of the corresponding fluorophores released from NTR-responsive fluorescent probes during NTR reaction; (C–F) Calibration curves at concentrations of 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, and 1.0 μM of fluorophore (Inset: Linear regression graph); Concentration-dependent changes in the fluorescence emission of fluorophore 3 (C), 14 (D), 12 (E), and 18 (F).