Brilliant BODIPY-fluorene Copolymers With Dispersed Absorption and Emission Maxima

Abstract

Four systems 1a – d were prepared to investigate the optical properties of copolymers comprised of polyfluorene doped with BODIPY-based fluors. The underlying hypothesis was energy harvested via the strong absorptivity of the major component, fluorine, would be primarily emitted from the BODIPY parts at much higher wavelengths. Optimization of the polymerization process as a function of the mol % of BODIPY, indicated that the brightest polymers were formed when approximately 4 fluorene units were co-polymerized with every BODIPY precursor. These polymers were cast into nanoparticles of ca 40 nm diameter. Treatment of clone 9 rat liver cells with suspensions of these particles resulted in uptake without encapsulation in lysosomes, or organelle targeting.

A relatively new field of interest in the chemistry of BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) dyes^1–3 is to polymerize them to concentrate multiple fluor units in single molecules,^4–7 hence to form bright particles for materials or for imaging biological systems. Polymers of this kind tend to have desirable properties for those applications because they concentrate several fluorescent groups in one molecule. Brightness-per-fluorophore in homopolymeric-dye systems is not necessarily greater than the free dye, and scope for manipulation of spectroscopic properties is limited; consequently it is logical to research similar polymers from two or more components. This strategy allows donor fragments that absorb light of a desirable wavelength to collect and transfer energy to fluorophores that then fluoresce. In this paradigm, donor:acceptor ratios may be adjusted to optimize the brightness-per-fluorophore (note brightness-per-fluorophore = absorption cross-section of the donor parts × quantum yield of the fluorophore in the polymer).

To the best of our knowledge this has not been attempted; all the BODIPY-containing polymers reported to date are homopolymeric or BODIPY-bridge-BODIPY-bridge... (ie ABAB) composites.^5–15 There are none of the type (donor)_m-BODIPY-(donor)_n-BODIPY-.... in which the acceptor fluorophores is “doped” into the donor-polymer backbone. This is significant because donor-rich polymers could have extremely high UV absorption cross sections, and accommodate fast, efficient, energy transfer to the BODIPY fluorophores, affording bright probes with excellent separations of absorption and emission wavelengths. Significantly, direct covalent linkage of the donor and acceptor fragments facilitates energy transfer through-bonds hence the requirement for overlap of the donor emission with the acceptor absorption (as in through-space energy transfer) no longer applies.

This communication describes donor-rich polymers 1 designed to maximize energy transfer to BODIPY or related acceptor fragments. Fluorene donor parts were chosen because of their photostabilities, large molar absorptivities, high two-photon cross-sections, and ease of syntheses.^16–18 Violet-blue emissions from polyfluorenes are not ideal for cellular or in vivo imaging, but we hypothesized that appropriately designed BODIPY-doped polyfluorene polymers could be excited at the donor parts, and would emit brightly at much longer wavelengths characteristic of the acceptor parts. Another design attribute of these systems is that variations of the acceptors would enable tuning of fluorescence outputs. Data described here shows that in fact such polymers could be made and optimized for emission via the acceptor. Further, representative systems were cast into particles of around 50 nm that were shown to permeate into Clone 9 rat liver cells where they could be imaged.

Scheme 1 outlines the synthetic route that was used to obtain the probes 1 featured in this study. It relies on Suzuki-based polymerizations of two fluorene components and the diiodides 2a–e; the latter 2a–d are all novel compounds, and their syntheses were based on related materials that had previously been prepared but without the two aryl iodide groups or, in some cases, without meso-substituents entirely (see supporting), while AzaBODIPY 2e was previously prepared by our group.²³ Those acceptors have emissions that are dispersed at intervals of about 30 to 40 nm, from 520 to 700 nm (Table 1). BODIPY 2b has an extremely high molar absorptivity and an exceptional quantum yield, and 2c shows an unusually large Stokes’ shift (ie 1565 cm⁻¹). Acceptor-precursors 2a–d have ortho-methoxy-substituents on their meso-aryl groups to minimize radiationless decay via rotation. The polymers were purified by precipitation from the medium used for the Suzuki couplings (toluene) by addition to methanol. Low molecular weight impurities were subsequently removed via multiple acetone washes, each over an extended period.

Scheme 1.

Syntheses of the polymers 1a–e yielding M_w ranging from 7 to 15 kDa (supporting).

Table 1.

Spectroscopic properties of fluorene and acceptors in CH₂Cl₂

	λabs max(nm)	ε × 10−4 (cm−1•M−1)	λem max(nm)	φ	fwhma (nm)	Stokes Shift (cm−1)
fluorene	266	14.7	308	0.28b	26	5126
2a	510	8.0	521	0.36c	26	414
2b	556	12.0	573	1.0d	32	534
2c	556	5.6	609	0.48d	46	1565
2d	667	15.0	685	0.46e	37	394
2e	678	6.0	710	0.05e	47	665

^aFluorescence full width at half maximum peak height

^bNaphthalene in cyclohexane as standard (φ = 0.92)¹⁹

^cFluorescein in 0.1M NaOH as standard (φ = 0.92)²⁰

^dRhodamine B in EtOH as standard (φ = 0.65)²¹

^eZinc phthalocyanine in pyridine as standard (φ = 0.30)²²

Experiments were performed to optimize the brightness of 1a when excited at the fluorene λ_max/abs by varying the fluorene:BODIPY ratios used in the polymerization. These studies (supporting) showed the brightness peaked at ca 4:1 (donor:acceptor). This observation reflects a compromise between effects that increase the brightness (eg the cross section of the donor runs) and those that decrease it (eg acceptor-acceptor quenching, structural perturbations, and average separation of the fluorene units to the next BODIPY in the chain). No significant change in either the absorbance or emission wavelengths was observed for the acceptor when increasing its concentration in the polymer. These changes were monitored via Energy Transfer Efficiency (ETE %) and Energy Transfer (ET %) as indicated in Table 2. We define ET as the percentage of total fluorence of the polymers excited at the donor attributable to the acceptors. ETE % is a measure of the quantum yield of the cassette when irradiated at the donor. It reflects the extent of energy transfer including the negative effects of non-radiative loss in the transfer process.

Table 2.

Photophysical properties of polyfluorenes incorporated with acceptors in CH₂Cl₂

polymer	mol % acceptora	λabs fluorene (nm)	λabs bodipy (nm)	λem (nm)b	ETE %c	ET %d	φacceptor	φpolymerh	molar absorptivity × 10−5 (cm−1•M−1)
1a	19	343	505	517	70.8±0.5	90.5±0.6	0.147±0.002e	0.104±0.002	5.1±0.9
1b	27	343	549	566	60±2	97.2±0.3	0.24±0.01f	0.14±0.05	5.74±0.07
1c	22	344	547	602	48±8	95.9±0.5	0.13±0.02f	0.06±0.02	5.18±0.03
1d	25	347	657	677	37±3	98±1	0.25±0.07g	0.09±0.03	4.3±0.3
1e	40	336	703	753	49.4±0.3	86±2	0.002g	0.001	4.58±0.07

^aEstimated by NMR.

^bEmission maximum when excited at 358 nm (fluorenes)

^cEnergy transfer efficiency (ETE) was calculated by dividing the quantum yield of acceptor when excited at fluorene donor by the quantum yield of acceptor when excited at acceptor.

^dPercent of the total fluorescence being emitted by the acceptor.

^eFluorescein in 0.1M NaOH as standard (φ = 0.92).²⁰

^fRhodamine B in EtOH as standard (φ = 0.65)²¹

^gZinc phthalocyanine in pyridine as standard (φ = 0.30)²²

^hQuantum yield of polymer calculated as the product of ETE and the quantum yield of the acceptor.

$$\text{ETE}\%=\frac{\text{quantum}\text{yield}\text{of}\text{the}\text{acceptor}\text{fragment}\text{in}\text{the}\text{cassette}\text{excited}\text{at}\text{the}\text{donor}}{\text{quantum}\text{yield}\text{of}\text{the}\text{acceptor}\text{fragment}\text{in}\text{the}\text{cassette}\text{excited}\text{at}\text{the}\text{acceptor}}\times 100$$

Overall the observations described above motivated us to make a series of polymers that contained ca 0.19 equivalents of the acceptor monomers; this was achieved using 0.54 equivalents of the BODIPY acceptor. A 48 h polymerization run was used; shorter times gave less brilliant polymers (see supporting). Table 2 gives the essential parameters of polymers 1a – e formed via the conditions mentioned above.

Emissions from the polymers occur mainly from the acceptors with ET % ranging from 86–98 % (Figure 1). The emission maxima of polymers 1a–d are at wavelengths slightly lower to that of the free acceptors 2a–d. For example, polymer 1d has an emission maximum at 685 nm while acceptor 2d has a maximum at 677 nm. Polymer 1e shows a considerable increase (43 nm) in its emission wavelength compared to acceptor 2e. This could be because the phenyl groups on azaBODIPYs in 1e can adopt conformations that are more planar with the heterocyclic core than the ortho-substituted meso-aryl groups of 1a – d. In other words, polymerization with fluorene extends the conjugation of the azaBODIPY.

Figure 1.

Normalized fluorescence of 1 in CH₂Cl₂ at 10⁻⁶ M excited at 358 nm.

Energy transfer efficiencies (ETE) in the series decreased as the λ_max/emiss values for the acceptors increased. Throughout the series, and most markedly for the azaBODIPY systems 1e and 2e, fluorescence quantum yields of the acceptor fragments decreased in the polymers; in fact, the quantum yield of the azaBODIPY-polymer 1e is only 0.21% and even with less acceptor, the same type of quenching was observed, see supporting.

McNeill et al. have reported efficient generation of organic nanoparticles from polyfluorene and similar systems.^24,25 We investigated whether comparable nanoparticles could be constructed from doped polymer 1. In the event, slight modifications were found to be more suitable for these polymers; slower addition rate of a more dilute THF solution of the polymer to water gave particles of 48.4 ± 13.3 nm by dynamic light scattering in the case of 1b. TEM investigations of particles made from 1a–d led to aggregation before the analysis though smaller particles averaging 20 to 60 nm could be seen.

Uptake of the organic nanoparticles from 1 was studied on normal rat liver cells (Clone 9). After 40 h incubation at 37 °C in Ham’s + 5% fetal bovine serum (FBS) culture medium, the nanoparticles were observed as bright dots inside the cells (Figure 2); they did not specifically target organelles. Polymers incubated under same conditions did not enter the cells.

Figure 2.

Confocal imaging of Clone 9 rat liver cells with polymeric nanoparticles 1a–d.

The work reported here features cassettes with donor and acceptor fluorophores joined via linkers that would allow conjugation if the molecules became planar.^26–33 However, the twist between these fragments “insulates” the donor and acceptor parts so that the overall emission characteristics of the composites mirror those of the free acceptor. This makes it possible to design of materials with predictable fluorescence emission wavelengths. This property coupled with the fact that there are several donors per acceptor part, offers potential for fluorescent probe design, OLEDs and lasing materials.