Water-Soluble Trityl Radicals for Fluorescence Imaging

Stable tris(trichlorophenyl)methyl radicals have gained interest as all-organic bioimaging agents combining fluorescent and paramagnetic properties. However, cellular uptake has so far only been reported for nanoparticles, because molecular hydrophobic trityl radicals are not soluble in aqueous media. Here, we report the synthesis and characterization of new water-soluble tris(trichlorophenyl)methyl radical derivatives exhibiting red doublet emission. Solubility in water is achieved through functionalization with oligoethylene glycol (OEG) chains. The emission behavior of OEG functionalized trityl radicals is studied in polar environments. Donor-functionalization with carbazole evokes a charge-transfer excited state that is efficiently quenched in polar solvents. In contrast, click-reaction mediated attachment of OEG-azide and trityl acetylene furnishes a triazole functionalized radical with locally excited states and emission in water. Confocal fluorescence microscopy proves successful uptake of the material by macrophages in cell culture, showing the potential of our water soluble trityl radical for fluorescence bioimaging.


Introduction
The stable tris (2,4,6-trichlorophenyl)methyl radical (TTM) and its derivatives represent a class of fluorescent open-shell molecules with emission in the orange to near-infrared spectrum [1][2][3].Because of their doublet ground and excited state character, these molecules represent interesting candidates to overcome the problem of spin statistics in conventional closed-shell emitters, which is a limiting factor for the efficiency in organic light-emitting devices (OLEDs) [2,[4][5][6].Finland-radicals, a subclass of non-fluorescent trityl radicals that can be rendered water soluble, have been employed as magnetic probes in biomedical EPR imaging.These radical spin labels exhibit low toxicity and a narrow single-line resonance, enabling high contrast and superior image quality [7].In contrast, TTM is paramagnetic as well as fluorescent, and therefore represents a promising candidate for dual-mode bioimaging [8].TTM radicals that are functionalized with charged groups are water soluble; however, these radicals do not exhibit fluorescence [9,10].Because of this quenched fluorescence or the limited solubility in aqueous media, neutral TTM radical-based imaging probes have been limited to nanoparticles that were explored in vitro and in vivo for their optical imaging quality [11][12][13].However, the formulation of small molecular TTM radicals into an inert matrix or the polymerization of the radicals into particles requires large amounts of inert and non-magnetic carrier material.As a result, the nanoparticles are amorphous, and any beneficial coupling between the radical species will only take place, if at all, at very low temperatures.To be able to target certain compartments in a cell and to make use of the unpaired radical electron as spin labels or for magnetic field sensing, molecularly dissolved radicals are required.
Here, we report the synthesis of unprecedented TTM derivatives that are functionalized with oligoethylene glycol (OEG) chains, rendering the molecules soluble in polar solvents and aqueous media.In contrast to previous water-soluble TTM radicals, our derivatives exhibit red emission in water with properties similar to those of TTM in nonpolar solvents.We see that radicals with charge transfer (CT) excited state, which are performant emitters in non-polar solvents, are inferior in polar and aqueous media.In contrast, radicals with a locally excited (LE) state exhibit luminescence in non-polar as well as in polar and aqueous media.

Synthesis
To address the challenge of equipping TTM radicals with water solubility and molecular dissolution, while keeping the molecule charge neutral, we functionalize TTM with OEG chains.OEGylation, as well as functionalization with polyethylene glycol (PEGylation), are typical pathways in biomedical materials science for endowing molecules with water solubility.
TTM-derived radical emitters with reported high photoluminescence efficiency typically possess CT excited states [3,14,15].However, it is known that such polar excited states are prone to fluorescence quenching in polar solvents, as a polar medium stabilizes the CT state.Therefore, we chose to test TTM derivatives that will exhibit CT and LE states.Carbazole (Cz) is a well-established donor substituent for TTM, typically delivering a CT state and high photoluminescence quantum yields (ϕ) [3].To improve the solubility of this TTM-Cz radical, we N-functionalize the Cz with an OEG chain (see Figure 1).Moreover, to compare the photoluminescence character of such a CT emitter to an LE emitter in a polar environment, we employ an electron-deficient triazole linker for OEGylation.Since the emission is expected to not significantly depend on the length of the OEG, we start with a tetraethylene glycol (n = 4), despite the reduced capability to increase the solubility of our non-polar TTMs in water.
solvents and aqueous media.In contrast to previous water-soluble TTM radicals, our derivatives exhibit red emission in water with properties similar to those of TTM in nonpolar solvents.We see that radicals with charge transfer (CT) excited state, which are performant emitters in non-polar solvents, are inferior in polar and aqueous media.In contrast, radicals with a locally excited (LE) state exhibit luminescence in non-polar as well as in polar and aqueous media.

Synthesis
To address the challenge of equipping TTM radicals with water solubility and molecular dissolution, while keeping the molecule charge neutral, we functionalize TTM with OEG chains.OEGylation, as well as functionalization with polyethylene glycol (PEGylation), are typical pathways in biomedical materials science for endowing molecules with water solubility.
TTM-derived radical emitters with reported high photoluminescence efficiency typically possess CT excited states [3,14,15].However, it is known that such polar excited states are prone to fluorescence quenching in polar solvents, as a polar medium stabilizes the CT state.Therefore, we chose to test TTM derivatives that will exhibit CT and LE states.Carbazole (Cz) is a well-established donor substituent for TTM, typically delivering a CT state and high photoluminescence quantum yields (ϕ) [3].To improve the solubility of this TTM-Cz radical, we N-functionalize the Cz with an OEG chain (see Figure 1).Moreover, to compare the photoluminescence character of such a CT emitter to an LE emitter in a polar environment, we employ an electron-deficient triazole linker for OEGylation.Since the emission is expected to not significantly depend on the length of the OEG, we start with a tetraethylene glycol (n = 4), despite the reduced capability to increase the solubility of our non-polar TTMs in water.4) is synthesized via Suzuki coupling of HTTM-I (3) and the boronic ester 2 [16].HTTM functionalized with OEG chains of different length (7 & 8) with triazole as a linker is obtained from alkyne-azide Click-reaction with alkyne-HTTM (6).The latter is obtained from deprotection of 5 which is synthesized from 3 by Sonogashira-coupling using TMS-acetylene.The radicals are obtained from their respective precursors by deprotonation followed by mild oxidation.
OEGylated carbazole 1 is obtained from 3-iodocarbazole, and borylation provides the building block 2 (see Figure 1).The OEGylation of both TTM-derivatives-with Cz or triazole-is achieved by starting from the iodinated HTTM 3 (HTTM-I, see Figure 1) [16].HTTM-I reacts readily with the previously N-OEGylated carbazole 2 in a Suzuki crosscoupling reaction to give the donor-functionalized HTTM 4 in good yields and at mild reaction conditions.Alternatively, 3 can also be functionalized efficiently with an alkyne via Sonogashira cross-coupling to give Compound 5.After cleavage of the TMS protecting group, the resulting HTTM-alkyne 6 is subjected to a copper-catalyzed alkyne-azide Clickreaction, to give the respective triazole-linked radical precursors 7 and 8 in excellent yield.
The compounds 4 and 7 are converted to the respective radicals 9 and 10 by deprotonation, followed by mild oxidation with p-chloranil of the resulting anions [17].The open-shell nature of the resulting molecules is confirmed by EPR spectroscopy.The single signal indicates that the unpaired electron is mainly located at the methine carbon, as previously observed and described for related compounds (see Figure S2) [12,14,16,18].

Optical Characterization of Radicals Carrying OEG Chains in Organic Solvents
Both OEGylated TTM radicals 9 and 10 show weak absorption in the visible and strong absorption bands in the UV spectrum, as is typical for TTM and its derivatives (see Figure 2) [3,12,[14][15][16]18].The absorption in the visible region appears red-shifted with respect to TTM.For carbazole-functionalized TTM derivatives, this bathochromic shift in the absorption is related to a charge-transfer from the carbazole to the TTM moiety [19].The low-energy absorption behavior of 9 is comparable to TTM carrying N-phenyl carbazole, indicating that N-PEGylation does not significantly affect the electronic properties of the donor substituent [20].The low energy absorption of 10 appears to be hypsochromically shifted with respect to 9. The electron deficient triazole attached to TTM will not act as an electron donor to TTM, and therefore the tendency for a CT excitation will be reduced.Still, the absorption is slightly red-shifted (λ max = 582 nm) compared to unfunctionalized TTM (λ max = 540 nm) (see Figure 2b) [17].Despite triazole not acting as an electron-donor, the extension of the aromatic system into the triazole unit might be responsible for lowering of the absorption energy.To check the suitability of our new OEG substituted TTM derivatives as radical emitters in water, we record the photoluminescence spectra in different solvents of increasing polarity (see Figure 3a,b).For 9, the emission maximum is clearly shifting with increasing solvent polarity, as is typical for CT excited states.Such charge-transfer upon excitation can be observed from the dependence of the Stokes shift on the orientation polarizability of the solvent in a Lippert-Mataga plot (see Figure S5).In addition, the spectra are vibrationally broadened, indicating strong interaction with the solvent molecules.For radical 9, we observe a drop in the photoluminescence quantum yield from ϕ = 29% in heptane and 20% in diethyl ether to 4% in ethyl acetate.Quenching of the photoluminescence be-  (10).Absorption (dashed grey) and emission (solid grey) spectra for toluene solution of TTM are shown for comparison.
To check the suitability of our new OEG substituted TTM derivatives as radical emitters in water, we record the photoluminescence spectra in different solvents of increasing polarity (see Figure 3a,b).For 9, the emission maximum is clearly shifting with increasing solvent polarity, as is typical for CT excited states.Such charge-transfer upon excitation can be observed from the dependence of the Stokes shift on the orientation polarizability of the solvent in a Lippert-Mataga plot (see Figure S5).In addition, the spectra are vibrationally broadened, indicating strong interaction with the solvent molecules.For radical 9, we observe a drop in the photoluminescence quantum yield from ϕ = 29% in heptane and 20% in diethyl ether to 4% in ethyl acetate.Quenching of the photoluminescence becomes even more efficient for highly polar solvents, such as acetone, ethanol, or methanol.We do not observe fluorescence of radical 9 for solutions of even higher solvent polarity like water.In contrast, the emission spectrum of 10 is hardly affected by the solvent polarity, which further supports our hypothesis from above that the excited state of radical 10 represents an LE state.The variation in the emission maxima in the different solvents lies within a range of 10 nm, while no clear trend is observable.This is in agreement with the observation from a Lippert-Mataga plot, also indicating the LE state (see Figure S5).Unfortunately, the solubility of 10 in water is too low in terms of studying its electronic properties in aqueous media.The value of ϕ is constantly low at 2% in solvents of different polarity, and also insensitive to the environment.While donor-substituted derivatives reach ϕ values close to unity, such molecules typically undergo CT excitation, such as observed for 9 [1,21].However, for the application in polar environments, less efficient LE emissions, such as from 10, appear more appropriate.

Quantum Chemical Calculations
To get further insight into the different emission behaviors of 9 and 10, and support our hypotheses of CT and LE excited states, we employ density functional theory (DFT) and time-dependent density functional theory (TD-DFT) calculations on our radicals.As the length of the OEG-chain is expected to have a minor effect on the electronic properties of the excited state, it is shortened to a methoxy ethane group to reduce computation time.The molecules are optimized in their ground state geometry on the PBE0-GD3(BJ)/6-311++G(d,p) level of theory.The aqueous environment is simulated using the SCRF-SMD method.The natural transition orbitals (NTOs) for the D1 excitation in the ground state geometry for the radicals are shown in Figure 3c,d.
For most TTM derivatives, the D1 excitation is related to a HDMO-SOMO transition,

Quantum Chemical Calculations
To get further insight into the different emission behaviors of 9 and 10, and support our hypotheses of CT and LE excited states, we employ density functional theory (DFT) and time-dependent density functional theory (TD-DFT) calculations on our radicals.As the length of the OEG-chain is expected to have a minor effect on the electronic properties of the excited state, it is shortened to a methoxy ethane group to reduce computation time.The molecules are optimized in their ground state geometry on the PBE0-GD3(BJ)/6-311++G(d,p) level of theory.The aqueous environment is simulated using the SCRF-SMD method.The natural transition orbitals (NTOs) for the D 1 excitation in the ground state geometry for the radicals are shown in Figure 3c,d.
For most TTM derivatives, the D 1 excitation is related to a HDMO-SOMO transition, while excitation of the unpaired electron appears at much higher energy [4,22,23].Additionally, 9 shows a CT D 1 excitation with a clear charge migration from the carbazole-donor to the TTM-acceptor moiety.Such behavior has been theoretically described for structurally related TTM derivatives connected to the 3-position of carbazole (see Figure 3c,d) [20,24].As expected, there is no involvement of the aliphatic OEG substituent in the excitation, substantiating our omission of this unit for the calculations.The charge separation upon excitation explains the strong interaction of the molecule in the D 1 state with its solvent environment.As the experimental observation of fluorescence quenching indicates, such polar excited states are unfavorable for emission in polar environments like water.The well-established concept of donor-functionalization for improved optical properties of TTM obviously works only for an environment of relatively low polarity [1,3].In contrast, radical 10 shows a LE D 1 excitation.While the NTO of the electron (e − ) in 10 resembles that of radical 9, the NTO of the hole (h + ) is more strongly spread across the TTM moiety in 10 rather than in 9 (see Figure 3c,d).In addition, both hole and electron NTOs occupy the triazole substituent corroborating our hypothesis that the red-shifted absorption and emission spectra in radical 10 could originate from an extended π-conjugated system, with respect to unfunctionalized TTM.Such a π-extension of the conjugated system is supported by a planar configuration of the triazole and the attached phenyl ring of the TTM moiety, as is observed in the DFT optimized geometry of radical 10 (see Figure S4).

Water-Soluble TTM
To render the TTM-triazole-OEG radical water soluble, we increase the OEG chain length from n = 4 to n = 20 (see Figure 1).For the formation of the respective radical 11, we use AgNO 3 as an oxidizing agent instead of p-chloranil.This exchange simplifies the purification of the product by aqueous extraction rather than requiring column chromatography, which becomes increasingly tedious with increasing OEG chain lengths.In contrast to 10, with an OEG chain length of n = 4, radical 11 with an OEG degree of polymerization of n = 20 is very well soluble in the aqueous medium.The solubility of 11 in water is determined to be at least 10 mg•mL −1 , which is far above the concentrations commonly used for fluorescent markers and tags in cell culture experiments.
Equipped with our insight gained from the study of 9 and 10 in polar solvents and the quantum chemical contemplations, we study compound 11 in aqueous solution and cell-culture experiments.Both, absorption and emission spectra for 11 in water resemble those of 10 with the much shorter OEG chain (see Figure 4).This observation confirms the minor impact of the length of the OEG chain on the electronic properties of the radicals, which are mainly governed by the aromatic units.Therefore, it is not surprising that the emission spectrum of 11 is also not greatly affected by solvent polarity, as expected for an emission originating from an LE state.Since many amphiphilic molecules form micelles in aqueous media, which could affect the optical properties, we perform dynamic light scattering (DLS) experiments with the non-emissive closed-shell precursor 8.The latter is chosen to avoid interference of the fluorescent radical with the DLS laser wavelength (632.8 nm).In a concentration of 1 mM of 8 in water, we do not detect any aggregates.This concentration is well above the concentrations used for spectroscopy and cell culture experiments.Therefore, we conclude that the absorption and emission profiles correspond to individually dissolved molecules.The photoluminescence quantum yield of 11 in both toluene and water is ϕ = 2%.This invariance in performance in solvents of different polarity further substantiates our hypothesis of an LE state.These observations lead us to conclude that light emitting radicals for aqueous media should be designed to have an LE state, while CT excited states should be avoided.While ϕ = 2% does not seem to be high, this quantum yield is in the range of those reported for TTM without further functionalization in non-polar solvents [1,[14][15][16].As discussed above, much higher ϕ are possible in donor-functionalized TTM radicals; however, these cannot be transferred into polar media without photoluminescence quenching and loss of performance.To test the applicability of our new and water soluble radical fluorescent dye, we incubate macrophages in 500 µM and 50 µM (see Figure 5a,b) solutions of the radical 11 for one hour.The fluorescent cell media is the removed and fresh media is added (see Section 3 for further details).The macrophages are then subjected to confocal laser scanning microscopy, allowing fluorescence imaging of live cells at high resolution.Within the time of incubation, the macrophages have clearly taken up the fluorescent radical dye, which is red fluorescent, when excited with a laser excitation wavelength of λ = 405 nm (see Figure 5).The cell nuclei and potential organelles inside of the macrophages appear as dark entities, indicating that our OEG-functionalized radical emitter is too hydrophilic to enter the more hydrophobic compartments of the cell.Over the course of the incubation and imaging, the macrophages appeared healthy, indicating that our radicals are suitable for live cell imaging (see Figure 5).While apoptotic cell rounding is not visible in the total period of 1:15 h of exposure of the cells to the radical dye, its toxicity needs to be further investigated and quantified.To test the applicability of our new and water soluble radical fluorescent dye, we incubate macrophages in 500 µM and 50 µM (see Figure 5a,b) solutions of the radical 11 for one hour.The fluorescent cell media is the removed and fresh media is added (see Section 3 for further details).The macrophages are then subjected to confocal laser scanning microscopy, allowing fluorescence imaging of live cells at high resolution.Within the time of incubation, the macrophages have clearly taken up the fluorescent radical dye, which is red fluorescent, when excited with a laser excitation wavelength of λ = 405 nm (see Figure 5).The cell nuclei and potential organelles inside of the macrophages appear as dark entities, indicating that our OEG-functionalized radical emitter is too hydrophilic to enter the more hydrophobic compartments of the cell.Over the course of the incubation and imaging, the macrophages appeared healthy, indicating that our radicals are suitable for live cell imaging (see Figure 5).While apoptotic cell rounding is not visible in the total period of 1:15 h of exposure of the cells to the radical dye, its toxicity needs to be further investigated and quantified.To test the applicability of our new and water soluble radical fluorescent dye, we incubate macrophages in 500 µM and 50 µM (see Figure 5a,b) solutions of the radical 11 for one hour.The fluorescent cell media is the removed and fresh media is added (see Section 3 for further details).The macrophages are then subjected to confocal laser scanning microscopy, allowing fluorescence imaging of live cells at high resolution.Within the time of incubation, the macrophages have clearly taken up the fluorescent radical dye, which is red fluorescent, when excited with a laser excitation wavelength of λ = 405 nm (see Figure 5).The cell nuclei and potential organelles inside of the macrophages appear as dark entities, indicating that our OEG-functionalized radical emitter is too hydrophilic to enter the more hydrophobic compartments of the cell.Over the course of the incubation and imaging, the macrophages appeared healthy, indicating that our radicals are suitable for live cell imaging (see Figure 5).While apoptotic cell rounding is not visible in the total period of 1:15 h of exposure of the cells to the radical dye, its toxicity needs to be further investigated and quantified.

Materials and Methods
Reagents and solvents are used as commercially provided.Unless otherwise stated, all reactions are performed under a nitrogen atmosphere.Silica gel with a particle size of 40-63 µm is used for column chromatography.

Figure 1 .
Figure 1.Synthesis of new OEG-functionalized TTM derivatives.The HTTM precursor carrying the N-OEGylated carbazole (4) is synthesized via Suzuki coupling of HTTM-I (3) and the boronic ester 2[16].HTTM functionalized with OEG chains of different length(7 & 8)  with triazole as a linker is obtained from alkyne-azide Click-reaction with alkyne-HTTM(6).The latter is obtained from deprotection of 5 which is synthesized from 3 by Sonogashira-coupling using TMS-acetylene.The radicals are obtained from their respective precursors by deprotonation followed by mild oxidation.

Figure 3 .
Figure 3. Emission spectra for solutions of radicals 9 (a) and 10 (b) in solvents of different polarity (0.1 ± 0.02 mM).While the emission maxima of 9 is red-shifted, with increasing solvent polarity, the emission of 10 is insensitive to the polarity of the environment.Natural transition orbitals (NTOs) for the D0 → D1 transition in 9 (c) and 10 (d) in their ground state geometry, calculated on the PBE0-GD3(BJ)/6-311++G(d,p), SCRF (SMD, water), level of theory.

Figure 3 .
Figure 3. Emission spectra for solutions of radicals 9 (a) and 10 (b) in solvents of different polarity (0.1 ± 0.02 mM).While the emission maxima of 9 is red-shifted, with increasing solvent polarity, the emission of 10 is insensitive to the polarity of the environment.Natural transition orbitals (NTOs) for the D 0 → D 1 transition in 9 (c) and 10 (d) in their ground state geometry, calculated on the PBE0-GD3(BJ)/6-311++G(d,p), SCRF (SMD, water), level of theory.

Figure 5 .
Figure 5. Confocal laser scanning micrographs of live cell macrophages incubated with radical 11.(a) Overview and (b) closeup on two macrophages with radical fluorescent dye, indicating that the radical 11 can be utilized for live cell staining.

Figure 5 .
Figure 5. Confocal laser scanning micrographs of live cell macrophages incubated with radical 11.(a) Overview and (b) closeup on two macrophages with radical fluorescent dye, indicating that the radical 11 can be utilized for live cell staining.

Figure 5 .
Figure 5. Confocal laser scanning micrographs of live cell macrophages incubated with radical 11.(a) Overview and (b) closeup on two macrophages with radical fluorescent dye, indicating that the radical 11 can be utilized for live cell staining.