Water-soluble Pyrrolopyrrole Cyanine ( PPCy ) near-infrared fluorescent pH indicators for strong acidity †

Water-soluble, fluorescence-switchable derivatives of pyrrolopyrrole cyanines (PPCys) which serve as near-infrared pH indicators have been synthesized. The dyes reported have pKa values of 2.4–3.4 and thus are suitable for sensing in highly acidic environments. The new pH probes were evaluated by fluorescence imaging experiments in a cellular environment.

were evaluated by fluorescence imaging experiments in a cellular environment.
Fluorescence based sensing has become an important technique in biomedicine.A number of these methods, such as ophthalmological angiography based on indocyanine green fluorescence 1 or intrasurgical tumor targeting based on administration of 5-aminolevulinic acid 2 are now routinely used.In general, for all medical applications deep penetration depths of the techniques are important.Therefore, due to the reduced scattering and low absorption at long wavelengths, the 20 application of near-infrared (NIR) fluorophores is desirable. 3onitoring pH is crucial for the understanding of physiological and pathological processes in organisms.Changes in pH values also play a pivotal role on cellular events, such as signal transduction, autophagy or apoptosis. 4While many pH fluorescence indicators have been designed to work in the pH range of roughly 6 to 8 or in the weak acidic range from 4 to 6, 5 hardly any pH-sensitive fluorophores working in the strong acidic range below pH 4 are known. 6Such chromophores would, however, be of great interest for e.g.imaging the gastrointestinal 30 tract. 7n this contribution, we report the development of novel water-soluble NIR fluorophores which exhibit strong fluorescence in the highly acidic range.The dyes are based on the recently developed Pyrrolopyrrole Cyanines (PPCys) which themselves are derived from diketopyrrolopyrroles (DPPs). 8PPCys are highly fluorescent and photostable NIR dyes which allow an easy tailoring of their absorption and emission properties by appropriate substitution patterns. 9Their structureproperty relationships meanwhile are well understood.This allowed e.g. the development of PPCy dyes with an acid/basedependent fluorescence switching by introducing aminophenyl donor groups into the PPCy skeleton. 10Those derivatives, as nearly all PPCy dyes reported to date, however, are only soluble in nonpolar solvents.Only very recently, the synthesis of the first water-soluble PPCy dyes by a postsynthetic modification route was reported, 11 which yielded ionic (tetrasulfonated) PPCys with high water-solubility (mM range).This opens way for integrating the excellent photophysical properties of PPCys as NIR dyes into water-soluble pH indicators.Our first attempt into this direction 50 was the development of such dyes by transferring the established synthetic route for water-solubilisation onto the described fluorescence-switchable derivatives.Yet, this strategy failed (for synthesis scheme of the failed route see Fig. S1 in ESI †).We have engineered a new postmodification route which yields non-55 ionic PPCys with high water-solubility.This synthetic strategy is applicable to the aminophenyl substituted, fluorescenceswitchable PPCys.We describe the synthesis and spectroscopic characterization of four PPCy based near-infrared pH indicators.These dyes exhibit pK a values between 2.4 and 3.4 and thus are 60 applicable in strongly acidic environments.Fluorescence imaging demonstrates their great potential as pH indicators on a cellular level.As other PPCys, 12 we envision that the new dyes can also be employed for live animal imaging.
The synthetic route (Scheme 1; for experimental The obtained PEG-BR 2 -PPCys show a high solubility not only in water, but also in organic solvents like dichloromethane, methanol or DMSO, owing to the nature of the where c (PPCy-NR 2 ) is the concentration of dye with free and c 15 (PPCy-NR 2 H + ) the concentration of the dye with protonated amino group(s).For c (PPCy-NR 2 ) = c (PPCy-NR 2 H + ), which is true for the fluorescence intensity at half its maximum, the pK a equals the pH value.As it was seen in earlier protonation experiments of aminophenyl substituted PPCys, the two amino 20 functions are successively protonated. 10However, the second protonation occurs very rapidly after the first one, so that the two very slightly different pK a values may for pratical purposes be regarded as one.1), respectively, demonstrating that the complexing group has only a weak effect on the pK a value.Regarding these results, we were interested in synthesizing a dye with different heteroaromatic end groups to determine the influence of these groups on the pK a value.40 Therefore, benzimidazole substituted PEG-BPh 2 -PPCy 8'' was synthesized and its pK a value was determined to 3.4.This result shows that the pK a depends only weakly on the substitution pattern of the PPCy and instead is mainly determined by the aminophenyl structural motif.found that unprotonated PEG-BPh 2 -PPCy 8' has a Φ F = 0 in pure water (the same as for 7', 9' and 8''; cf.Table 1), but it was 10 shown in previous work that in biological (cellular) environments, the photophysical properties of PPCys are not corresponding to a purely aqueous surrounding. 11To examine whether unprotonated 8' also shows no fluorescence in a biological environment, fluorescence images were taken on fixed 15 CHO cells incubated with a 1 µM solution of 8' for 30 minutes using a scanning stage confocal fluorescence microscope.Fig. 2a indeed shows a weak emission of 8' in the cytoplasm by excitation with a 690 nm laser, revealing a solvation effect of the PPCy in which is different from that of a purely aqueous 20 surrounding.After permeabilization of the cells and adjusting the external cellular medium to pH = 1, PPCy 8' shows the expected intense fluorescence (Fig. 2b).This result documents a diminished contrast between the two forms of the dye in the cellular environment.Moreover, it is thereby shown that nonionic (PEGylated) PPCys enter the cells without application of any permeability-enhancing techniques. 11The same experiment was performed with PEG-BF 2 -PPCy 7'.Fig. 2c and d show the fluorescence images at neutral pH and pH = 1, respectively.While at low pH there is a similar strong emission as in the case 30 of 8', no fluorescence in the unprotonated state is observed at all, meaning an optimal contrast between the two forms of PPCy 7'.
In fact, it was not possible to locate cells by fluorescence imaging at neutral pH, so the pH was first adjusted to 1 (Fig. 2d) and then readjusted to neutral again (Fig. 2c).This also demonstrates that protonation of aminophenyl substituted PPCys in CHO cells is reversible.
An explanation for the better contrast in the case of PEG-BF 2 -PPCy 7' compared to PEG-BPh 2 -PPCy 8' can be found in the absorption spectra of these compounds (see Fig. S5 and S6 40 in ESI † for absorption spectra of 7', 8', 9' and 8'' in CH 2 Cl 2 and H 2 O, respectively).As has been explained in an earlier publication 10 , the intensity of the charge transfer (CT) transition band is a measure of the coupling strength of the aminophenyl donor substituent to the chromophoric system and hence an indicator for the magnitude of the fluorescence quenching in the unprotonated form.The intensity of the CT band in the unprotonated form is much higher for PEG-BF 2 -PPCy 7' than for PEG-BPh 2 -PPCy 8', resulting in a fluorescence quantum yield of 0 compared to 8% in dichloromethane, respectively (cf.Table 1).

50
Comparing the quantum yields in an organic solvent is sensible since, as described above, the solvation effect observed in cells is different from the pure aqueous milieu (Fig. 2a).For PEG-60 BHex 2 -PPCy 9' the CT band intensity is even lower than in the case of 8', which means that the coupling of the aminophenyl donor to the chromophore is very weak.This results in practically no contrast of the quantum yields between the unprotonated and protonated form of 24% to 25%, respectively.Benzimidazol 65 substituted PPCy 8'' again shows a better contrast in quantum yields of 3% to 29%, as its CT band intensity is higher.
In conclusion, we report the synthesis of four novel Pyrrolopyrrole Cyanine dyes with high water-solubility which act as near-infrared fluorescent pH indicators.The synthesis follows 70 a postmodification of PPCys with terminal alkynes in a Cu (I) catalyzed alkyne azide cycloaddition (CuAAC) with poly(ethyleneglycol)azide. The pK a values of the four PPCys are 2.4, 2.5, 3.2 and 3.4 (water:methanol, 1:1), respectively, thus having a highly acidic working range.PEG-BF 2 -PPCy 7' and 75 PEG-BPh 2 -PPCy 8' have been tested as pH indicators in CHO cells and it was found that 7' shows an optimal contrast between the non-fluorescent (unprotonated) and the fluorescent (protonated) form, excelling this derivative as an ideal nearinfrared pH indicator for strong acidity.80 Financial support from the DFG, SFB 767, TP B3 is gratefully acknowledged.
5 long PEG chains.The reactant PEG-N 3 was commercially available (average M n = 1000 g/mol, PDI < 1.1, Sigma-Adrich) and, according to MALDI mass spectra of the final compounds, PEG-chains with ethyleneglycol repeating units of an average n = 22-24 were incorporated.The pK a values of the amino functions 10 at the DPP core were determined via fluorescence titration experiments, using the Henderson-Hasselbalch equation, ‫۶ܘ‬ ൌ ‫۹ܘ‬ ‫܉‬ ‫ܔ‬ ‫܋‬ ‫܀ۼିܡ۱۾۾‪ሺ‬‬ ሻ ‫܋‬ ‫܀ۼିܡ۱۾۾‪ሺ‬‬ ۶ శ ሻ Fig. 1 shows the pH dependent fluorescence spectra of quinoline substituted PEG-BPh 2 -PPCy 8' in 25 water:methanol (1:1) by protonation through HCl.At λ ୫ୟ୶ = 820 nm the ratio between the fluorescence intensity at a certain pH and the maximum intensity was plotted against the pH and a sigmoidal fit revealed the pK a value of 3.2, meaning PPCy 8' turns on strong emission at low pH values (inset in Fig.1; for 30 fluorescence titration spectra of 7', 9' and 8'' see Fig. S2-S4 in ESI †).The quinoline substituted PEG-BF 2 -PPCy 7' and PEG-BHex 2 -PPCy 9' were synthesized to clarify the different electronic effects of the complexing groups on the pK a values of the amino functions.The likewise determined pK a values of these 35 derivatives were 2.4 and 2.5 (Table

45
Fig. 1 Fluorescence spectra of PEG-BPh2-PPCy 8' in water:methanol (1:1) by titration with HCl (asterisk: stray light of λ ୣ୶ୡ = 804 nm); Inset: Determination of the pKa value by plotting the ratio between the fluorescence intensity at a certain pH and the maximum intensity against the pH and sigmoidal fitting.

Fig. 2
Fig. 2 Confocal fluorescence images of fixed CHO cells after 30 minutes of incubation with a 1µM solution of novel PPCy pH indicators: PEG-BPh2-PPCy 8' at a) neutral pH and b) pH = 1; PEG-BF2-PPCy 7' at c) neutral pH and d) pH = 1.For comparison, grey scales of images a and c 55

Table 1
Spectroscopic data of unprotonated and protonated (in brackets) PEG-BR2-PPCys in dichloromethane and water.λ୫ୟ୶ : wavelength of the absorption maximum, λ ୫ୟ୶: wavelength of the fluorescence maximum, ΦF: fluorescence quantum yield.
a Trifluoroacetic acid (TFA) and conc.HCl were used for protonation in dichloromethane and water, respectively; b pKa values were determined in H2O:MeOH, 1:1; c H-aggregation band (monomer band at 739 nm).