Gadolinium(iii)-dota Complex Functionalized with Bodipy as a Potential Bimodal Contrast Agent for Mri and Optical Imaging

The synthesis and characterization of a novel gadolinium(III) DOTA complex functionalized with a boron-dipyrromethene derivative (BODIPY) is described. The assembly of the complex relies on azide diazotransfer chemistry in a copper tube flow reactor. The azide thus formed is coupled directly with an alkyne via click chemistry, resulting into a paramagnetic and luminescent gadolinium(III) complex. Luminescent data and relaxometric properties of the complex have been evaluated, suggesting the potential applicability of the complexes as a bimodal contrast agent for magnetic resonance and optical imaging. The complex displays a bright emission at 523 nm with an absorption maximum of 507 nm and high quantum yields of up to 83% in water. The proton relaxivity of the complex measured at 310 K and at frequencies of 20 and 60 MHz had the values of 3.9 and 3.6 s −1 ·mM −1 , respectively.

MRI excels in its special resolution and depth penetration, but it suffers from a low sensitivity.Combining it with a complimentary imaging technique would allow for obtaining a better diagnostic tool, as it was recently demonstrated by a successful combination of MRI and PET probes [13][14][15].The combined contrast agent gives both insight into the morphology (MRI) and information about biomedical processes (PET) of the human body.Another promising imaging modality complementary to MRI is optical imaging, which has a very good sensitivity, but lacks the spatial resolution and depth penetration.The latter drawback can be circumvented by choosing the absorption and emission wavelength close to the biological window [16].Different optical probes have already been coupled to DTPA or DOTA, such as luminescent polymers in lipophilic aggregates [17], luminescent metal complexes [18][19][20][21][22][23][24][25][26][27], dendrimers [28], and organic dyes such as quinolone [29], rhodamine [30,31], fluoresceine [32], and naphalimide [33,34].
A promising class of organic dyes are 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (abbreviated as BODIPY) and its derivatives.This class of small organic molecules generally has very high extinction coefficients, fairly sharp fluorescence peaks, and high quantum yields.The BODIPY core is pretty stable in physiological conditions and relatively insensitive to the environment.Their emission wavelength is tunable by increasing the resonance within the BODIPY core [35].The BODIPY dyes have been used in a variety of applications such as biological labels and probes [36][37][38], fluorescent probes [39], laser dyes [40,41], light emitting diodes [42], solar cells [43,44], and potential sensitizers in photodynamic therapy [45,46].Although BODIPYs are versatile compounds with good optical properties, their use in developing bimodal contrast agents remain scarce [47][48][49][50][51][52]. 18F labeled BODIPY derivatives have been recently suggested as bifunctional reporters for hybrid optical/positron emission tomography imaging [53,54].An example of DOTA ligand functionalized with BODIPY compound was reported in 2010 by Bernhard et al. [47] with the idea to generate a bimodal imaging agent for optical and nuclear imaging (PET/CT) by introducing In(III), Ga(III) and Cu(II) into the DOTA moiety.In 2012 the same group expanded their compound to a DOTAGA derivative (GA = glutaric acid) which gave more stable complexes with the transition metals [48,51,52].Considering the versatility and favorable optical properties of BODIPY organic dyes, there are surprisingly very few examples of its use as an optical probe in the development of bimodal contrast agents.The main disadvantage of BODIPY dyes is their hydrophobicity, which limits their solubility and therefore hinders their straightforward application as a probe for medical imaging.Although overcoming the solubility issue poses a challenge, it is also beneficial as it was demonstrated that hydrophilic dyes can improve cell permeability [50].
We have recently shown that BODIPY dyes can be functionalized in a copper tube flow reactor via chemistry that converts primary amines into azides using the catalyst generated in situ from the metallic copper [55].In this paper we further expand this strategy for performing azide-alkyne cycloaddition in order to create a Gd-DOTA-BODIPY derivative.We report on the synthesis and characterization of this novel compound and evaluate its potential as a bimodal contrast agent for MRI and optical imaging.

Synthesis of Gd-DOTA-BODIPY Derivative
The main concept used in order to create a Gd-DOTA-BODIPY derivative is based on using click chemistry to couple a BODIPY dye to a DOTA moiety, resulting in a bimodal agent with both optical and paramagnetic entities (Figure 1).The novel BODIPY derivative developed in this work is  3,2]diazaborinin-4-ium-5-uide, presented in Figure 1.The synthesis of the BODIPY core starts with the protection of β-analine with phtalic acid in solvent free conditions.Subsequently reaction with thionyl chloride converts the acid group into an acid chloride, which can react further with two equivalent of 2,4-dimethylpyrrole to form a dipyrromethene derivative.Complexation of this product with borontrifluoride dietherate and a base forms BODIPY-(CH2)2-phtalimide.This product can easily be transformed into BODIPY-(CH2)2-amine by using hydrazine in ethanol, which was clearly demonstrated by the disappearance of the aromatic peaks at 7.77 and 7.88 ppm, and appearance of a new peak at 1.61 ppm in the 1 H NMR spectrum (Scheme 1).The final BODIPY product contains an amine function which can be transformed in a copper tube into an azide via a diazotranfer reagent (ISA•H2SO4) giving BODIPY-(CH2)2-azide shown in Figure 2a [56,57].The propargylated Ln(III)-DOTA complex (Ln = La, Gd) that was used for coupling to BODIPY dye is shown in Figure 2b [58,59].The coupling was performed by using a flow chemistry approach, which can mitigate some safety related issues regarding working with azides [60].Organic azides can be synthesized from primary amines via a diazotransfer reaction.The potential short shelf life and highly explosive nature of some diazotransfer reagents requires their careful consideration of the safety issues for their handling [61].Some safety issues can be circumvented by introduction of imidazole-1sulfonyl azide (ISA) or its hydrogen sulfate salt (ISA•H2SO4) [56,62].The flow reaction was performed by mixing solutions of BODIPY-(CH2)2-amine and ISA•H2SO4, which is graphically represented in Scheme 2. Upon leaving the reactor, the reaction mixture was directly added to a solution of propargylated Ln(III)-DOTA (Ln = La or Gd), resulting in the final product Ln(III)-DOTA-BODIPY.The Ln-DOTA-BODIPY complexes have been isolated and purified (see Figure S13) and the diamagnetic lanthanum(III) complex La-DOTA-BODIPY has been characterized by 1 H NMR spectroscopy in solution (Figure 3).The 1 H NMR spectrum shows a distinct peak of the triazole proton at 7.55 ppm and two protons on the BODIPY core at 6.00 ppm, indicating a successful linkage of the DOTA moiety with the BODIPY derivative.The 1 H NMR spectrum also shows broad peaks in the region from 2.20-3.82ppm which are typical for the protons in the DOTA ring [63].

Photophysical Properties of the Gd-DOTA-BODIPY
The BODIPY derivatives are typically strongly red colored.Most BODIPY derivatives are apolar in nature and dissolve very well in apolar organic solvents, like chloroform or dichloromethane, and will emit a bright green fluorescence.Due to the coupling to hydrophilic Ln-DOTA complex the final Ln-DOTA-BODIPY adducts are water soluble and give bright green fluorescence in aqueous solution.Figure 4 shows a picture of an aqueous solution of the synthesized Gd-DOTA-BODIPY in the absence and in the presence of excitation light at 366 nm.The green fluorescence is clearly visible during excitation.Furthermore, the electronic spectra of Gd-DOTA-BODIPY are given in Figure 4 and depict the characteristic and rather narrow absorption and emission bands which are typical for BODIPY dyes.A main absorption band (blue dotted line) with a maximum λabs(max) at 503 nm is observed for Gd-DOTA-BODIPY in aqueous solution.These visible absorption bands can be assigned to the S0→S1 transition [64].An additional, considerably weaker broad absorption band is observed in the UV-VIS region around 360 nm, and is attributed to the S0→S2 transition.It should be noted that the optical properties of the BODIPY dyes can be tuned by increasing conjugation through placing different substituents on the BODIPY core.Such substitutions increase the resonance of the whole structure and provide a red shift of the main emission band, which is favorable for biological applications.A tradeoff between tissue penetration and image resolution for in vivo imaging can be made in the optical imaging window (from 665 to 900 nm) [65].In this region the extinction coefficients of the main sources of absorption such as hemoglobin, deoxyhemoglobin, and water, are at their minimum.However, it must be taken into account that placing additional substituents on the BODIPY usually renders the system more hydrophobic and decreases the solubility of the entire complex in water.
The emission maximum in water of Gd-DOTA-BODIPY (Figure 4) is observed at 523 nm and is in accordance with the previously reported BODIPY derivatives [66].The excitation maximum (red dashed line) recorded at 560 nm shows a peak at 503 nm.The fluorescent quantum yields QL of La-DOTA-BODIPY and Gd-DOTA-BODIPY were determined upon ligand excitation by a comparative method, using a solution of Rhodamine 6G (Q = 78%) [67] in water as the standard.The advantage of BODIPY dyes is that the quantum yields of their derivatives are generally high, even in water [39].The quantum yield was determined according to the following equation: In this equation the s and x refer to the standard and the unknown sample respectively, I represents the corrected total integrated emission intensity, A: the absorbance at the excitation wavelength, η: the refractive index of the solution (ηwater = 1.33), and Qs: the quantum yield of the standard (Q = 78%) [67].The samples are diluted until the absorbance at the excitation wavelength is between 0.02-0.05.Quantum yield is recorded with ligand excitation wavelength of 490 nm and gave values of 80% and 83% for La-DOTA-BODIPY and Gd-DOTA-BODIPY, respectively.

Relaxivity of Gd-DOTA-BODIPY
The relaxivity r1 is the efficiency of a 1 mM solution of gadolinium(III) agent to shorten the longitudinal relaxation time (T1).According to the Solomon Bloembergen Morgan theory [68,69] several parameters can alter the relaxivity of a contrast agent.High relaxivity can be obtained with a higher amount of water molecules directly bound to the paramagnetic centre (q).Although an easy parameter to adjust, increasing the q value leads to less stable complexes.Other parameters are the distance between gadolinium(III) and water (r), the water residence time (τM), the rotational correlation time of the paramagnetic center (τR), the electronic relaxation time of gadolinium(III) at zero field (τS0) and the correlation time modulating the electronic relaxation (τv).The relaxivity of Gd-DOTA-BODIPY derivative measured at frequencies of 20 and 60 MHz at 310 K and was found to be 3.9 and 3.6 s −1 •mM −1 respectively.These values are in close comparison with previously reported values for Gd(III)-DOTA complex, which gave values of 3.5 and 3.1 s −1 •mM −1 at 20 and 60 MHz respectively [70].A slight increase compared to the parent Gd-DOTA complex can be attributed to the increase in molecular weight of the Gd-DOTA-BODIPY complex, resulting in the increase of the rotational correlation time (τR) and in overall increase of the relaxivity.The limiting factors for the increase of Gd-DOTA-BODIPY relaxivity are probably the presence of an amide bond in the DOTA scaffold which may slow down the water exchange rate, or flexible linker used for attaching the BODIPY core to DOTA, which may result in high internal rotations within the Gd-DOTA-BODIPY.

Characterization
NMR spectra were measured on a Bruker Avance 300 (Bruker, Karlsruhe, Germany) (operating at 300 MHz for 1 H NMR spectra, operating at 75 MHz for 13 C NMR), Bruker Avance 400 (operating at 400 MHz for 1 H NMR spectra, operating at 100 MHz for 13 C NMR spectra).Chemical shifts are reported in parts per million (ppm) and are referenced to the internal standard tetramethylsilane.For 13 C spectra, residual solvent signals are used as the internal standard.Spectra are taken at room temperature unless otherwise stated.Relaxation rates at 20 and 60 MHz were obtained on a Minispec mq-20, and a Minispec mq-60 (Bruker, Karlsruhe, Germany) respectively.
FT-IR spectra were measured by using a Bruker Vertex 70 FT-IR spectrometer (Bruker, Ettlingen, Germany).ESI-MS measurements were taken on a Thermo Electron LCQ Advantage apparatus (Thermo Scientific, Waltham, MA, USA) with Agilent 1100 pump (Agilent, Waldbronn, Germany) and injection system coupled to Xcalibur data analyzing software.Methanol is used as eluent.TXRF measurements were done on a Bruker S2 Picofox (Bruker, Berlin, Germany) by analyzing approximately 100 ppm gadolinium or europium solutions with respect to a Chem-Lab gallium standard solution (500 µg/mL, 2%-5% HNO3).
3.3.4.Synthesis of DO3A-tBu (4) (Figure 8) The synthesis uses an altered synthesis by Viguier et al. [58].To a solution of tetraazacyclodedecaan (1 eq, 5.80 mmol, 1 g), sodium bicarbonate (3.5 eq, 20.3 mmol, 1.71 g) in 150 mL ACN under an argon atmosphere a solution of tert-butyl bromoacetate (3.5 eq, 20.3 mmol, 3.0 mL) in 50 mL ACN was added drop wise.The mixture was refluxed for 17 h.After removing the salts via filtration over Celite ® the solvent was evaporated and the solid was recrystallized from toluene as a white powder, yield: 2.  General procedure for complexation of propargylated DOTA: Product (6) (1 eq) was dissolved in 5 mL water and the appropriate lanthanide(III) chloride hydrate (1,1 eq) was added.The pH was adjusted to 5.5 with a 0.1 M KOH solution and stirred overnight at 60 °C.The solvent was evaporated.The residue was redissolved in 5 mL water and Chelex-100 was added and stirred for 2 h.This last step was repeated until no free lanthanide was found with an Arsenazo III indicator [71].

General Procedure Flow
Stock solution A: a 2 mL solution in a degassed mixture of 10:3:3 MeOH:DCM:H2O of 1 eq BODIPY-amine and 11 equivalents of DIPEA.
Stock solution B: a 2 mL solution of three equivalents of ISA•H2SO4 in a degassed mixture of 10:3:3 MeOH:DCM:H2O.
Both solutions in a separate syringe are combined via a T-mixer in an ice bath before introducing the mixture to the copper tube flow reactor (150 µL) at room temperature.Flow speed is adjusted so the retention time in the reactor is 300 s.The column is stabilized during three reactor volumes.Upon leaving the reactor, the reaction mixture was directly added to a solution of 1.5 equivalent of Ln-DOTApropargyl and 1.5 eq sodium ascorbate.The mixture is left stirring overnight.The aqueous layer is lyophilized, redissolved in distilled water, purified via HPLC and lyophilized.The products are isolated as bright orange powders.
Product 9 (Gd) (Figure 12  After each reaction the copper tube flow reactor is cleansed in a sonication bath for 10 min while introducing a 1:1 mixture of MeOH:triethylamine at 4 mL/min, followed by 20 mL of pentane at 4 mL/min.The reactor is dried with argon and stored under argon.

Conclusions
In this paper a novel Gd-DOTA-BODIPY derivative is synthesized via a diazotransfer reaction under flow conditions in a copper tube.The complex is water-soluble and exhibits favorable fluorescent properties, thus offering the possibility for the use of BODIPY adducts for in vivo optical imaging.High quantum yield of 83% is obtained in water, and bright emission was observed at 523 nm.Shifting of the emission to the more optimal red-IR region of the spectrum will be attempted in future by increasing the electronic resonance of the BODIPY by adding appropriate substituents to the BODIPY core.The complex exhibits relaxivities which are comparable to the parent Gd-DOTA complex and, therefore, holds potential as a bimodal contrast agent for MRI and optical imaging. 5,5-difluoro-1,3,7,9-tetramethyl-10-(2-(4-((2-gadolinium(III)-(4,7,10-tris(carboxylatomethyl)-1,4,7,10tetraazacyclododecan

Figure 3 .
Figure 3. 1 H NMR spectrum of the final clicked La-DOTA-BODIPY derivative measured in deuterated water.

Figure 4 .
Figure 4.The emission (black line), excitation (red dashed line) and absorption (blue dotted line) spectrum of Gd-DOTA-BODIPY in water.