Donor-acceptor-donor fluorene derivatives for two-photon fluorescence lysosomal imaging

Abstract

As part of a strategy to achieve large two-photon absorptivity in fluorene-based probes, a series of donor-acceptor-donor (D-A-D) type derivatives were synthesized and their two-photon absorption (2PA) properties investigated. The synthesis of D-A-D fluorophores was achieved by efficient preparation of key intermediates for the introduction of central electron acceptor groups. To accomplish the synthesis of two of the new derivatives, a high-yield method for a one-step direct dibromomethylation of phenyl sulfide was developed. The linear and nonlinear optical properties, including UV-vis absorption, fluorescence emission, fluorescence anisotropy, and two-photon absorption (2PA), of the new D-A-D compounds were measured and compared to their D-A or D-D counterparts. Fully conjugated acceptor moieties in the center of the D-A-D fluorophore led to the greatest increase in the 2PA cross section, while weakly conjugated central acceptors exhibited only a modest increase in the 2PA cross section relative to D-A diploar analogs. Encapsulation of the new probes in Pluronic F 108NF micelles, and subsequent incubation in HCT 116 cells, resulted in very high lysosomal colocalization (>0.98 colocalization coefficient) relative to commercial Lysotracker Red, making the micelle-encapsulated dyes particularly attractive as fluorescent probes for two-photon fluorescence microscopy lysosomal imaging.

Figure 1

Structures of fluorene derivatives for bioimaging.

Figure 2

Structures of probes 1–8.

Figure 3

Synthesis of compounds 1 – 4. a) HNO₃/H₂SO₄, 0 °C, 1.5 h, 41%; b) DMF, NaH, r.t., 20 h, 55– 85%.

Figure 4

Synthesis of 5 and 6. a) Pd(AcO)₂/PPh₃, K₂CO₃, Bu₄NCl, DMF, reflux, 20 h, 70%; b) P(OEt)₃, reflux, 2h; c) 15, DMF, NaH, r.t., 20 h, 37-53%; d) paraformaldehyde, 33% HBr in HOAc, 70 °C, 48 h, 77%; e) H₂O₂, HOAc, 90 °C, 30 min, 68%.

Figure 5

Synthesis of 7 and 8. a) DMF, NaH, r.t., 20 h, 43%; b) Pd(AcO)₂/PPh₃, K₂CO₃, Bu₄NCl, DMF, reflux, 20 h, 71%; c) NMP, 110 °C, 3 h, 70%; d) P(OEt)₃, o-dichlorobenzene, reflux, 16 h, 93%; e) 15, DMF, NaH, r.t., 20 h, 43%.

Figure 6

Absorption (1_ab, 2_ab), fluorescence (1_fl, 2_fl), fluorescence anisotropy (1_aniso, 2_aniso) and 2PA spectra (1_2PA, 2_2PA) of 1 (grey) and 2 (black) in cyclohexane. Fluorescence anisotropy was measured in polyTHF.

Figure 7

Absorption (3_ab, 4_ab), fluorescence (3_fl, 4_fl), fluorescence anisotropy (3_aniso, 4_aniso) and 2PA spectra (3_2PA, 4_2PA) of 3 (grey) and 4 (black) in cyclohexane. Fluorescence anisotropy was measured in polyTHF.

Figure 8

Absorption (5_ab, 6_ab), fluorescence (5_fl, 6_fl), fluorescence anisotropy (5_aniso, 6_aniso) and two-photon absorption spectra (5_2PA, 6_2PA) of 5 (grey) and 6 (black) in cyclohexane. Fluorescence anisotropy was measured in polyTHF.

Figure 9

Absorption (7_ab, 8_ab), fluorescence (7_fl, 8_fl), fluorescence anisotropy (7_aniso, 8_aniso) and two-photon absorption spectra (7_2PA, 8_2PA) of 7 (grey) and 8 (black) in cyclohexane. Fluorescence anisotropy was measured in polyTHF.

Figure 10

Colocalization images of HCT 116 cells co-incubated with probe 6 in Pluronic™ micelles and LysoTracker® Red. Images were taken using a 60×, oil immersion objective. A) DIC, 500 ms; B) One-photon confocal fluorescence image using a custom made filter cube (Ex:377/50; DM: 409; Em:525/40), 4ms. C) One-photon confocal LysoTracker® fluorescence image using a Texas Red filter cube (Ex:562/40; DM: 593; Em:624/40) 350ms. D) Overlapped image of A, B and C (colocalization coefficient 0.99).

Figure 11

Images of HCT 116 cells incubated with fluorescence probe 8 in Pluronic™ micelles (30 μM, 3 h) taken with 60×, oil immersion objective. a) DIC, 400 ms. b) One-photon fluorescence image (filter cube Ex: 377/50 DM: 409 Em: 525/40). c) 3D reconstruction from overlaid 2PFM images (Ex: 700 nm; Em. long-pass filter 690 nm).