Bacteriochlorin Dyads as Solvent Polarity-Dependent Near-infrared Fluorophores and Reactive Oxygen Species Photosensitizers

Abstract

Symmetrical, near-infrared absorbing bacteriochlorin dyads exhibit gradual reduction of their fluorescence (intensity and lifetime) and reactive oxygen species (ROS) photosensitization efficiency with increasing solvent dielectric constant ε. For the directly-linked dyad significant reduction is observed even in solvents of moderate ε (CH₂Cl₂), while for the dyad containing a 1,4-phenylene linker, reduction is more parallel to an increase in solvent ε. Bacteriochlorin dyads are promising candidates for development of environmentally responsive fluorophores and ROS sensitizers.

Graphical Abstract

Photonic agents with photochemical properties responsive to the local microenvironment are of great interest, since they can function, for example, as fluorescent probes that monitor a variety of biological and biochemical processes¹ or photosensitizers of reactive oxygen species (ROS), selectively activated by a specific microenvironment.^2,3,4 Dielectric properties expressed by the dielectric constant ε vary significantly between intracellular organelles^5,6 and macrobiomolecules.^6–8 Recent findings also suggest that the local polarity (which is a function of ε) in the mitochondria of cancerous cells is lower compared to that of healthy ones.⁹ Therefore, the local ε can be a potential target for activation of imaging or therapeutic agents. Although there is a plethora of solvatochromic fluorophores,^1,9,10 only a few of them have been utilized for determination of intracellular ε,^5,8 and there are only a handful of ROS photosensitizers which respond in predictable manner to the local dielectric properties.^2,3,4 However, the majority of ε–responsive fluorophores and photosensitizers absorb and emit at λ < 600 nm, while these with excitation/emission at λ > 650 nm, suitable for deep-tissue applications, are less common.¹⁰ Moreover, such ε-responsive agents reported so far can usually perform either fluorescence or ROS sensitization functions, separately. Bacteriochlorins, strongly near-infrared (near-IR) absorbing tetrapyrrolic macrocycles, exhibit relatively high both fluorescence quantum yield (Φ_f) and quantum efficiency of ROS photosensitization (Φ_ROS, ROS include singlet oxygen ¹O₂, and superoxide radical O₂^-·).^11,12 The relatively high Φ_ROS for bacteriochlorins originates from high yield of intersystem crossing (Φ_ISC), leading to the long-living triplet state, capable of transferring either energy or electron to oxygen, to produce ¹O₂ or O₂^-·, respectively.¹² Thus, bacteriochlorins are utilized as near-IR fluorophores, ROS photosensitizers for photodynamic therapy, and both simultaneously.^12,13 Recently, we found that strongly conjugated bacteriochlorin arrays exhibit a significant reduction in both Φ_f and τ_f in solvents of high ε, due to the greatly enhanced internal conversion.^14,15 We reasoned that similar dependence should be true for Φ_ROS, thus allowing bacteriochlorin dyads to function as ε-dependent ROS photosensitizers. However, strongly-conjugated bacteriochlorin dyads exhibit a low Φ_ISC (i.e. 0.09 – 0.39)¹⁵ and consequently, low Φ_ROS even in non-polar solvents [see Supporting Information (SI) for an example). Therefore, here we describe a new series of weakly-conjugated bacteriochlorin arrays, where bacteriochlorin subunits are connected either directly (BC1), or through 1,4-phenylene (BC2), or 4,4′-biphenylene (BC3) linkers. Our hypothesis is that such constructs should exhibit a relatively high Φ_f and Φ_ROS in solvents of low ε, and both of them will be reduced with increasing solvent polarity. As benchmarks we include bacteriochlorin monomer BC4, and directly-linked chlorin dyad C1, analogous to BC1, to determine whether observed properties are specific to bacteriochlorin dyads (Figure 1).

Figure 1.

Structures of compounds BC1–4 and C1

Synthesis of BC1 starts from Miyaura borylation of known 15-bromobacteriochlorin BC5,¹⁶ which provides a boronic ester BC6 in excellent yield (92%). Subsequent Suzuki coupling of BC6 with BC5, provides BC1 in 62% yield.¹⁷ Suzuki reaction of BC5 with 1,4-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene or 4,4’-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,1’-biphenyl provides BC2 (88% yield) or BC3 (49% yield), respectively. Monomer BC4 was synthesized in similar Suzuki reaction (Scheme S1, SI). Chlorin dyad C1 was prepared following a published procedure,¹⁸ via PIFA-mediated oxidative coupling of monomer ZnC2, and subsequent demetalation of resulted complex ZnC1 (Scheme 2). All new compounds show ¹H, ¹³C NMR, and MS data consistent with expected structures. Note, that both BC1 and C1 are axially chiral, and accordingly, their ¹H NMR spectra show distinctive resonances of diastereotopic protons.

Scheme 2.

Synthesis of dyad C1

The absorption spectra of BC1-4 (Figure 2, Table 1) contain features typical for synthetic bacteriochlorins reported previously,¹¹ i.e. strong Q_y-type bands in the near-IR spectral window (712-738 nm), Q_x–type bands in the visible window (500-510), and B-type bands in UV range (365-370 nm), Note that the Q_y band for BC1 is split with the second maximum at 706 nm. Emission spectra in toluene consist of narrow 0-0 bands, with small (<7 nm) Stokes’ shift versus the Q_y-type band. The maxima of both Q_y-type absorption and emission for dyads are shifted bathochromically, compared to corresponding bands in monomer BC4; the most pronounced shift is observed for BC1 (365 and 436 cm⁻¹ for absorption and emission, respectively), and is much lower for BC2 (98 cm⁻¹ and 116 cm⁻¹) and BC3 (39 cm⁻¹ for both absorption and emission). The positions of absorption and emission maxima vary only slightly in solvents of different polarities (Table S1, SI). The linker-dependent, bathochromic shifts of both the Q_y absorption and emission bands is indicative of increasing electronic communications between bacteriochlorin subunits in dyads with decreasing distance between them.

Figure 2.

Absorption spectra of BC1–4 in toluene: BC1 (black, solid), BC2 (blue), BC3 (red), and BC4 (black, dotted). All spectra are normalized at the B-bands maxima.

Table 1.

Photophysical properties of BC1–4 and C1 in solvents of different dielectric constants.^a)

Solvent	Φf	τf [ns]	ϕROSb)
BC1: λQy = 706, 731 nm, λem = 738 nm (in toluene)
Toluene	0.28	3.6	1.00
THF	0.20	3.1	0.87
CH2Cl2	0.037	0.48	0.097
PhCN	0.031	< 0.4c)	ndd)
MeOHe)	0.007f)	< 0.4c)	ndd)
DMF	0.003f)	< 0.4c)	0.008
BC2: λQy = 717 nm, λem = 721 nm (in toluene)
toluene	0.22	4.1	1.00
THF	0.20	4.2	1.08
CH2Cl2	0.17	3.6	0.94
PhCN	0.11	2.4	ndd)
MeOHe)	0.069	2.3	ndd)
DMF	0.066	1.6	0.39
BC3: λQy = 714 nm, λem = 717 nm (in toluene)
toluene	0.22	4.1	1.00
THF	0.19	4.1	1.06
CH2Cl2	0.18	3.8	0.91
PhCN	0.18	3.9	ndd)
MeOHe)	0.15	3.7	ndd)
DMF	0.16	3.7	0.81
BC4: λQy = 712 nm, λem = 715 nm (in toluene)
Toluene	0.22	4.4	-
THF	0.19	4.5	-
CH2Cl2	0.19	4.2	-
PhCN	0.22	4.6	-
MeOHe)	0.15	4.2	-
DMF	0.20	4.6	-
C1: λQy = 650 nm, λem = 654 nm (in toluene)
Toluene	0.31	5.9	ndd)
DMF	0.29	6.0	ndd)

^a)All data were determined in air-equilibrated solvents. For Φ_f and τ_f determination samples were excited at the maxima of their Q_x band. Fluorescence quantum yields were determined with respect to meso-tetraphenylporphyrin (TPP, Φ_f = 0.070 in non-degassed toluene).¹¹ The estimated error in Φ_f, τ_f, and ϕ_ROS determination is ±10%.

^b)For details of ϕ_ROS determination see SI.

^c)The τ_f is too short to be accurately determined by our experimental set-up.

^d)Not determined.

^e)5% of THF (v/v) was used as a co-solvent.

^f)The weak emission signal at 736 nm overlaps with the second weak emission peak at 717 nm of unknown origin; therefore the Φ_f is approximate.

Fluorescence properties of BC1-4 and C1 (Table 1, Figures S1–S3, SI) were determined in an array of aprotic and protic solvents of broad ε range: toluene (ε = 2.38), THF (ε = 7.58), dichloromethane (CH₂Cl₂, ε = 8.93), PhCN (ε = 26.0), MeOH (ε = 32.7), and DMF (ε = 36.7). In toluene, BC1 shows markedly higher Φ_f (0.28) than BC4 (0.22), whereas both BC2 and BC3 show Φ_f in toluene comparable with that observed for BC4. For BC1 both Φ_f and τ_f diminish when solvent ε increases, resulting in a significant reduction of fluorescence in solvents of moderate ε (THF, CH₂Cl₂, PhCN) and almost no fluorescence in MeOH and DMF. A similar trend was observed for BC2; however, the reduction of both Φ_f and τ_f is less dramatic and more parallel with increase in ε of the solvents. The τ_f in BC2 decreases nearly linearly when ε of the solvent increases (Figure S5, SI), which suggests that this dyad can be useful as a fluorescence lifetime probe for determination of local dielectric constants. BC3 shows marked reduction of Φ_f in DMF only. Both BC4 and C1 show virtually no dependence of both Φ_f and τ_f on solvents ε.

These results indicate that for bacteriochlorin dyads, a new and efficient process for deactivation of the excited state, which competes with fluorescence, becomes accessible in polar solvents. The efficiency of this process clearly depends on ε of the solvent. Moreover, the efficiency of this new process appears to depend on the electronic conjugation between macrocycles in dyads, as the most extensive reduction of fluorescence in given solvent is observed for BC1 (where electronic communication between macrocycles is presumed to be the strongest)..Importantly, this process is specific only for bacteriochlorin dyads, since fluorescence properties for the monomer BC4 and for the chlorin dyad C1 are nearly solvent-independent. To the best of our knowledge, analogous porphyrin dyads show only slight dependence of their photophysical properties on solvent ε.¹⁹

Next, we determined the influence of solvent ε on ROS photosensitization for BC1-4, using 2,5-diphenylisobenzofuran (DPBF). DPBF reacts with ¹O₂ and O₂^-·, causing a decay of DPBF absorbance at 414 nm.^20,21 The comparison of Φ_ROS for given photosensitizer in different solvents is more complex than in case of Φ_f discussed above. Φ_ROS depends on both the properties of the photosensitizer excited states (i.e. Φ_ISC and triplet state lifetime) as well as solvent properties such as oxygen solubility, rate of oxygen diffusion, rate of bimolecular quenching of triplet state by oxygen, etc.²² Thus, influence of a solvent on Φ_ROS is observed, to some extent, for many photosensitizers.²³ Moreover, rate of DPBF degradation depends not only on the amount of singlet oxygen generated by the photosensitizer, but also on the lifetime of singlet oxygen in given solvents and bimolecular rate constants for reaction of singlet oxygen with DPBF, both of which vary in different solvents.²⁴ Hence, to evaluate the influence of solvent ε on ROS photosensitization of dyads, we determined how different the influence of solvent ε on ROS photosensitization is for given dyad, compared to that of the benchmark monomer BC4. Quantitatively, this influence was expressed through relative quantum yield of ROS photosensitization ϕ_ROS (see SI for the exact definition and measurement methodology). The ϕ_ROS were determined for dyads BC1-3 in four solvents: toluene (as a reference solvent), THF, CH₂Cl₂, and DMF. The plots of rate of DPBF absorbance decays for BC1-4 are shown in Figure S8, SI, and the resulting ϕ_ROS are given in Table 1. For BC1 we observed a dramatic reduction of ϕ_ROS in both CH₂Cl₂ (10-fold) and DMF (125-fold) compared to that in toluene. For BC2 and BC3 ϕ_ROS exhibit a lesser reduction than BC1, with the exception of DMF, for which ϕ_ROS is significantly (2.5-fold) lower for BC2 but only slightly (1.2-fold) lower for BC3, compared to toluene. The influence of solvent ε on ϕ_ROS reduction was further confirmed by determining the rate of DPBF decay in the presence of BC1 in a series of toluene/DMF mixtures with increasing DMF concentration. The ϕ_ROS gradually decreases with increasing ratio of DMF in the mixture (Figure S9, SI).

The quenching of ¹O₂ photosensitization of BC1 in DMF was further confirmed by monitoring ¹O₂ luminescence at 1270 nm, which in toluene is observed for both BC1 and BC4, but in DMF it is observed only for BC4 and is completely absent for BC1 (Figure S7, SI). The quantum yields of ¹O₂ photosensitization (Φ_Δ) in air-equilibrated toluene (determined by comparing the intensity of ¹O₂ luminescence generated by bacteriochlorins, to those generated by TPP) are 65% for both BC1 and BC4. Φ_Δ corresponds well with Φ_ISC determined previously for various meso-substituted bacteriochlorins, (i.e. Φ_ISC = 0.54-0.71).¹¹

In summary, fluorescence and ROS photosensitization ability in bacteriochlorin dyads are reduced when the solvent ε increases. The degree of quenching of photochemical activity in these dyads appears to be dictated by the strength of electronic interaction between bacteriochlorin subunits. This indicates, that the degree of the response of photochemical properties to the solvent ε in dyads can be adjusted for desired applications and polarity ranges, by proper molecular design (i.e. selecting the linker between bacteriochlorin subunits). These properties, along with high Φ_f and relatively long τ_f of near-IR emission, as well as high Φ_Δ in non-polar environment, allow bacteriochlorin dyads to be promising platform for development of smart fluorophores and ROS photosensitizers activated in environment of low ε, and fluorescence lifetime probes for determination of local dielectric properties.

Scheme 1.

Synthesis of dyads BC1–3