Small BODIPY Probes for Combined Dual19F MRI and Fluorescence Imaging

Article abstract of DOI:10.1002/cmdc.201600120

The combination of the two complementary imaging modalities¹⁹F magnetic resonance imaging (MRI) and fluorescence imaging (FLI) possesses high potential for biological and medical applications. Herein we report the first design, synthesis, dual detection validation, and cytotoxic testing of four promising BODIPY dyes for dual¹⁹F MRI–fluorescence detection. Using straightforward Steglich reactions, small fluorinated alcohols were easily covalently tethered to a BODIPY dye in high yields, leaving its fluorescence properties unaffected. The synthesized compounds were analyzed with various techniques to demonstrate their potential utility in dual imaging. As expected, the chemically and magnetically equivalent trifluoromethyl groups of the agents exhibited a single NMR signal. The determined longitudinal relaxation times T₁and the transverse relaxation times T₂, both in the lower second range, enabled the imaging of four compounds in vitro. The most auspicious dual¹⁹F MRI–fluorescence agent was also successfully imaged in a mouse post-mortem within a 9.4 T small-animal tomograph. Toxicological assays with human cells (primary HUVEC and HepG2 cell line) also indicated the possibility for animal testing.

Full text of DOI:10.1002/cmdc.201600120

DOI: 10.1002/cmdc.201600120
Full Papers
19
Small BODIPY Probes for Combined Dual F MRI and
Fluorescence Imaging
Anh Minh Huynh,[a] Andreas Mꢀller,[b] Sonja M. Kessler,[c] Sarah Henrikus,[a]
Caroline Hoffmann,[a] Alexandra K. Kiemer,[c] Arno Bꢀcker,[b] and Gregor Jung*[a]
The combination of the two complementary imaging modali-
ties 19F magnetic resonance imaging (MRI) and fluorescence
imaging (FLI) possesses high potential for biological and medi-
cal applications. Herein we report the first design, synthesis,
dual detection validation, and cytotoxic testing of four promis-
ing BODIPY dyes for dual 19F MRI–fluorescence detection.
Using straightforward Steglich reactions, small fluorinated alco-
hols were easily covalently tethered to a BODIPY dye in high
yields, leaving its fluorescence properties unaffected. The syn-
thesized compounds were analyzed with various techniques to
demonstrate their potential utility in dual imaging. As expect-
ed, the chemically and magnetically equivalent trifluoromethyl
groups of the agents exhibited a single NMR signal. The deter-
mined longitudinal relaxation times T1 and the transverse relax-
ation times T2, both in the lower second range, enabled the
imaging of four compounds in vitro. The most auspicious dual
19F MRI–fluorescence agent was also successfully imaged in
a mouse post-mortem within a 9.4 T small-animal tomograph.
Toxicological assays with human cells (primary HUVEC and
HepG2 cell line) also indicated the possibility for animal test-
ing.
Introduction
Today, imaging techniques are indispensable tools in any kind
of life science. Each method has its unique strengths and
weaknesses with varying spatial and temporal resolution limits,
so that a single imaging modality can be used with great ad-
vantage for one application, but is poorly suited for other ap-
plications. For this reason, no imaging technique can provide
all information of the object of interest; therefore, dual imag-
ing is highly desired. Dual imaging is defined by the synergistic
combination of two orthogonal imaging modalities which
allows combination of the inherent advantages of each imag-
ing technique. Well-known examples include various combina-
tions between positron emission tomography (PET), single
photoemission computed tomography (SPECT), fluorescence
imaging (FLI), ultrasound imaging, and magnetic resonance
imaging (MRI).[1] One of the most used combinations is dual
MRI and FLI.[2] MRI has high spatial and temporal resolution rel-
ative to other noninvasive imaging modalities as well as deep
tissue penetration, but lacks sensitivity and specificity. In con-
trast, FLI is often used for molecular imaging, for example, in
histology, due to its high sensitivity and specificity for target
detection. The disadvantage of FLI is that quantification is chal-
lenging. Moreover, detrimental light scattering and absorption
restrict its application to low tissue depth.
MRI, as one of the most important noninvasive methods, is
usually based on the H2O proton relaxation times, which may
vary among different tissues and which can be locally acceler-
ated in the presence of contrast agents. These are often para-
magnetic metal ion complexes with the most prominent metal
being gadolinium (Gd3+). In 2006, however, a possible link be-
tween Gd-containing contrast agents and a medical condition
called nephrogenic systemic fibrosis (NSF) was found.[3] There-
fore, it is highly desirable to establish more abundant ions as
[4]
alternatives, such as iron (Fe3+
)
or manganese (Mn2+),[5]
which might overcome these toxic effects. Another way may
1
be to switch the nucleus from H to 19F MRI. The 19F nucleus is
also well suited for MRI, given its natural abundance of 100%,
1
high gyromagnetic ratio, spin of = and high receptivity of
2
0.834 relative to 1H. Additionally, there is no 19F MRI back-
ground in biological systems. Hence, in the last few years nu-
merous papers about 19F MRI have been published.
[a] A. M. Huynh, S. Henrikus, C. Hoffmann, G. Jung
Department of Chemistry, Biophysical Chemistry,
Saarland University, Campus, 66123 Saarbrꢀcken (Germany)
E-mail: g.jung@mx.uni-saarland.de
Currently, most of the 19F MRI agents or tracers are perfluor-
ocarbon emulsions (PFC), which contain a high number of co-
valently bound 19F atoms and are easily modified for further
applications.[6] Unfortunately, PFCs have shown several disad-
vantages such as heterogeneity, instability, multiple 19F signals,
difficult synthesis, and also accumulation or even retention
within organs for a couple of months.[6c,7] These drawbacks are
largely overcome in a second group of 19F MRI tracers based
on small molecules.[8] The as-yet-described molecules are
chemically stable, easy to synthesize, and have shown short
[b] A. Mꢀller, A. Bꢀcker
Clinic of Diagnostic and Interventional Radiology,
Saarland University Medical Center, 66424 Homburg (Germany)
[c] S. M. Kessler, A. K. Kiemer
Department of Pharmacy, Pharmaceutical Biology,
Saarland University, 66123 Saarbrꢀcken (Germany)
Supporting information for this article can be found under http://
dx.doi.org/10.1002/cmdc.201600120.
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residence times in mice.[8a] Furthermore, as a result of the low
quantity of 19F nuclei relative to ubiquitous protons, these trac-
ers possess chemically and magnetically equivalent fluorine
atoms to maximize the generated magnetic resonance signal.
Despite the mentioned advantages, the limitations of small-
molecule tracers are their difficult modification after completed
synthesis and low sensitivity. Furthermore, a relatively high
probe concentration relative to PFCs is still required.
A convenient way to promote 19F MRI is the combination
with FLI for subsequent co-localization and validation of ac-
quired images. Fluorescent dyes are easy to tether covalently
to the MRI contrast agent. Recently, FLI was shown to serve as
a powerful tool in surgery to guide the scalpel.[9] An intriguing
example is the visualization and complete removal of ovarian
cancer with the help of fluorescein-labeled folate.[10] Neverthe-
less, at the moment, dual 19F MRI and fluorescence agents con-
sist of nanoemulsions, nanoparticles, or amphiphiles combined
with a fluorophore.[6f,h,11] For the combination of MRI and FLI,
a brief and compact fluorescent dye as good starting point for
derivatization is mandatory. Among the vast number of fluoro-
phores, the family of boron dipyrromethene dyes, also known
as BODIPY dyes, possess these properties. They show narrow
emission bands, high quantum yields and high photostabili-
ty,[12] are chemically stable, and can be easily modified. Accord-
ingly, BODIPY dyes are widely used as fluorescent switches,
laser dyes, biomolecule markers, and chemosensors.[13]
On account of the recent trends, herein we describe the syn-
theses of five dual imaging probes 1–5 for 19F MRI and fluores-
cence imaging (Figure 1). The fluorescent part consists of the
well-known tetramethyl-BODIPY scaffold (TMBDP) which has
consistently shown very good fluorescence properties in past
applications.[14] BODIPY dyes have already been established as
dual modality agents for 18F PET and FLI.[15] Here, the 19F MRI
part derives from commercially available trifluoroethyl alcohol,
perfluoro-tert-butyl alcohol, pentafluorophenol, and two fur-
ther synthesized alcohols, which contain 18 or 27 19F nuclei.
Subsequently, we characterized the molecules by fluorescence
spectroscopy, 19F NMR spectroscopy, and relaxometry. We suc-
cessfully mapped different concentrations down to the lower-
millimolar range of the synthesized compounds within a 9.4 T
small-animal magnetic resonance tomograph (MRT). Finally, 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT) assays with human umbilical vein endothelial cells
(HUVEC) and human liver carcinoma cells (HepG2) revealed
their low toxicity, making our compounds candidates for fur-
ther in vivo experiments.
Figure 1.
a) Structures of the synthesized dual imaging tracers. b) Crystallographic structure of compound 4.
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Results and Discussion
Table 1. Synthesis of dual imaging tracers 1–5.
The synthesis of the dual imaging contrast agents follows
a simple and straightforward strategy (Table 1). In a standard
Steglich reaction, the carboxylated BODIPY 6, which was syn-
thesized according to a published method,[16] reacts with five
different alcohols to form the corresponding esters. The alco-
hols 7–9 for the compounds 1–3 are commercially available.
However, the perfluorinated alcohol 10 for compound 5,
which was used in other molecules for 19F MRI, and which
therefore has great potential for similar applications,[8a,b] was
synthesized as described (Scheme 1).[17] The standard Steglich
condensation between alcohol 10 and BODIPY 6 turned out to
be too unreactive, likely due to the low nucleophilicity of the
alcohol. The yields were increased from 20% to 99% with a re-
action variation using Sc(OTf)3, in addition to the reagents 4-
(dimethylamino)pyridine (DMAP) and N,N’-diisopropylcarbodii-
mide.[18] Interestingly, during the synthesis of alcohol 10, a re-
markable side product was collected. After isolation, NMR anal-
ysis showed that the side product to be the benzyl- and
acetyl-protected pentaerythritol 11, which can appear during
the cleavage of the orthoacetate protection group
(Figure 2).[19] With this compound, we were able to synthesize
the acetyl-protected alcohol 12 by following the already
known synthesis pattern. The structure of the finally obtained
compound 4 was confirmed by X-ray crystallography (Fig-
ure 1b).
Entry
1[a]
Alcohol
Product
Yield [%]
91
1
2[a]
2
3
88
82
3[a]
4[b]
4
5
99
81
5[b]
After the successful synthesis, we first validated the fluores-
cence properties of the compounds 1–5. As we did not
change the chromophore system, all synthesized fluorescent
dyes maintained the electronic spectra of the parent com-
pound TMBDP (lexc =501 nm, lem =513 nm; Figure 2a).[20] We
therefore assume that the synthesized BODIPY dyes have the
same good optical properties as the parent BODIPY dye, veri-
fied for 4 and 5 by fluorescence microscopy with appropriate
filter combinations (Figure 2b and Supporting Information Fig-
ure S47). Afterward, we measured 19F NMR spectra to verify
their magnetic activity. As expected, the spectra of the four
compounds 1, 2, 4, and 5 showed a strong singlet from the
chemically and magnetically equivalent CF3 groups and a weak
[a] DMAP, DCC, CH2Cl2, 08C for 1 h, then RT, 24 h; [b] DMAP, Sc(OTf)3,
DIPC, CH2Cl2, À88C for 1 h, then RT, 2 d.
quartet signal from the BF2 group (Figures S3, S10, S21, and
S28). In contrast, the 19F NMR spectrum of compound 3
showed multiple weak signals from the different fluorine
atoms next to carbon and the BF2 unit (Figure S17). The
BODIPY 3 mainly was synthesized to study the influence of var-
ious fluorine nuclei on MRI.
Scheme 1. Synthesis of alcohol 10 and 12 after reaction conditions from Jiang and Yu.[17] Reagents and conditions: a) HCl (0.01n), MeOH, RT, 38%;
b) (CF3)3COH, DEAD, Ph3P, 4 ꢂ MS, THF, 458C, ~64–76%; c) AlCl3, anisole, CH2Cl2, 08C, ~64–85%.
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times higher than a nanoemulsion of a small 19F MR reporter[8c]
and 2–3 times higher than PFC nanoparticles.[6g] Nevertheless,
the shortest T1 values were found for the bulky compounds 4
and 5 with 2.2 seconds, which are still short enough for prom-
ising MRI data collection.
With knowledge about the relaxation times, the next step
was to detect compounds 1–5 at various concentrations in vi-
tro inside the 9.4 T small-animal MRT. BODIPYs 1 and 2 re-
quired concentrations of ~60 to 100 mm for clear detection,
which is only slightly diminished relative to the BF2 moiety of
commercial pyrromethene 546 (Figures S7, S14, and S46). Com-
pound 3 was spectroscopically silent, even at concentrations
higher than 100 mm. We explain this finding by the low
number of magnetically equivalent fluorine atoms and the
rapid dephasing. In contrast, BODIPYs 4 and 5, which have 18
and 27 fluorine atoms, respectively, enabled detection of con-
centrations at 20 and 10 mm (Figure 3a,b). These concentra-
tions are in agreement with published detection limits in this
field, which have the same or even higher number of fluorine
atoms per molecule.[8] With the image of the different concen-
trations of compounds 4 and 5, it is possible to verify the line-
arity between signal-to-noise ratio (SNR) and concentration
(Figure 3c), which was shown in previous publications.[8a,c]
Considering the voxel size of 1ꢃ1ꢃ5 mm3 and the minimal de-
tected concentration of 10 mm, the calculated minimal fluorine
atom number required for our setup is 8ꢃ1017 fluorine atoms
per voxel, which is also valid for the fluorine atoms of the BF2
unit under altered resonance conditions (see Figure S46). A key
step for further in vivo applications is visualization of the syn-
thesized agents under a biological environment. As an initial
step in this direction, we injected 100 mL of a 100 mm solution
of compound 5 into the muscle of a mouse post-mortem (Fig-
ure 3d,e). We used black-light excitation for FLI, similarly to the
successful approach in surgery.[9,10] The red shift of the ob-
served fluorescence presumably arises from reabsorption or
aggregation.[21]
Figure 2.
a) Excitation (black) and emission (red) spectra of compound 5 (CH2Cl2,
100 nm). b) Fluorescence micrograph of HepG2 cells after incubation with
compound 4 (50 mm, green) for 1 h and subsequent fixation. Counterstain-
ing of nuclei was achieved with DAPI (blue). Scale bar: 50 mm.
Table 2. Estimated relaxation times for compounds 1–5..
Compound
T1 [s][a]
T2 [s][a]
1
2
3
4
5
4.8
3.9
2.3
2.2
2.1
1.8
1.7
For future medical or biological experiments, it is also crucial
to test the cytotoxicity of the synthesized molecules. HUVEC
isolated from human vessels served as a model with utmost
relevance for the human in vivo situation.[22] Human HepG2 is
a model cell line frequently used for toxicity studies.[23] Both
cell types were used in a metabolic MTT assay, in which con-
version of the added MTT thiazolium into blue formazan
serves as readout for cell viability. The four BODIPYs 1, 2, 4,
and 5, including the precursor molecule 6, were investigated.
In the case of the uptake of the synthesized agents and the
cleavage of the ester functionality, it is important to know if
the decomposition product 6 is toxic. Still, the free carboxyl
group most likely inhibits cellular uptake, which is why we
used the ethanol ester 13 as a reference compound. The re-
sulting IC50 values in HepG2 for BODIPY 6 and 13 show that
these compounds are probably toxic for these cells (Table 3).
Most interestingly, in case of the HepG2 cells, the synthesized
BODIPYs 1, 2, 4, and 5 were found to be less toxic than the
control compounds 6 and 13 despite their cellular uptake (Fig-
ure 2b). We hypothesize that the greater steric hindrance of
the alcohols in 1 and 2 relative to 13 might decrease the ten-
[b]
–
1.5
1.3
[a] Performed in CHCl3 at 308C, determined with a benchtop relaxometer
(B0 =1.41 T); concentrations were in the range of 20–25 mm. [b] Could
not be fitted due to multiple fluorine signals.
Because of the clear 19F NMR signals, we then determined
the longitudinal relaxation times T1 and the transverse relaxa-
tion times T2 with a 1.41 T 19F NMR benchtop device. The relax-
ation times of the synthesized compounds 1–5 were found to
be in the area of several seconds (Table 2) with a definite corre-
lation to the size of the fluorinated alcohol. The larger the side
group, the shorter the T1 value in particular, which is in agree-
ment with the influence of the rotational diffusion on the re-
laxation times. Hence, the ratio of T1 to T2 dropped from 2.7 to
1.5 for the most fluorinated compounds 4 and 5. Only T2 of 3
indicates rapid dephasing and concomitant loss of an FID
signal. The decay times were higher by a factor of 10–30 in
comparison with a similar 19F MR reporter[8a] in micelles, 3–8
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Figure 3.
19F MRI of compound 5 in CH2Cl2 at various concentrations ranging from 10 to 100 mm, measured with a) FLASH and b) RARE sequences. c) Signal-to-noise
ratio (SNR) vs. concentration of compound 5. d) Black-light (lexc =365 nm) excited fluorescence photograph of a mouse post-mortem, injected with com-
1
pound 5 (100 mL of a 100 mm solution in CH2Cl2). e) Overlay of H (grayscale) and 19F MRI (color) images of the same mouse in panel d.
Table 3. IC50 values for dual imaging agents 1, 2, 4, 5, 6, and 13.
bicity, which may withdraw our molecules from metabolism as
well. Hence, the BODIPYs 4 and 5 show nearly no toxicity
against HepG2 cells. A closer look at the IC50 values in HUVEC
shows that the carboxylic acid 6 is nontoxic against these cells.
This fact indicates that compound 6 is probably not absorbed
by HUVEC, whereas HepG2 cells could uptake BODIPY 6 via
anion transporters,[24] which would explain the toxicity of com-
pound 6 therein. Also, compounds 1 and 2 exhibit lower toxic-
ity in HUVEC than in the HepG2 cell line (Figures S5/6 and S12/
13). Moreover, the esters 4 and 5 did not show toxicity at any
of the concentrations tested (Figures S23/S24 and S30/31), pre-
sumably for the same reasons as in HepG2 cells. Taken togeth-
er, comparison of the MTT assays in HUVEC and HepG2 cells re-
vealed lower toxicity for all compounds in the primary cells. In
summary, the dual imaging agents 4 and 5 are promising can-
didates for in vivo tests given the tolerance by living cells.
BODIPY
IC50 [mm][a]
HUVEC
HepG2
1
2
4
5
6
175
n.d.
n.d.
n.d.
n.d.
n.d.
155
94
n.d.
n.d.
40
13
57
[a] See the Supporting Information for concentration–viability curves;
n.d.: no significant toxicity was detected <100 mm.
dency for their cleavage. The steric effect may also be inferred
from comparison with compounds 4 and 5, which possess
even more sterically demanding alcohols. However, decreased
bioavailability due to storage in lipid-rich cellular domains
must also be considered: Preliminary logP values of >2.5 and
>3 for 4 and 5, respectively, indicate pronounced hydropho-
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the chemical shifts recorded as d values in ppm. Multiplicities are
denoted as follows: s=singlet, d=doublet, t=triplet, sxt=sextu-
plet, m=multiplet.
Conclusions
To demonstrate the principle that BODIPY dyes and 19F MRI are
combinable, five dual FLI/19F MRI reporters were synthesized in
this study. The fluorescence properties of the fluorescent dyes
1–5 are typical for the BODIPY class with high intensity at low
concentrations and narrow emission bands. After verifying the
19F MR activity of all synthesized compounds, the concentra-
tion measurements proved that one or two CF3 groups are in-
sufficient to produce a strong 19F magnetic resonance signal.
The detection limit of 8ꢃ1017 spins is best realized by a large
number of chemically and magnetically identical fluorine
atoms in the form of CF3 groups, and may be undercut by an-
other order of magnitude.[8c] Compounds 4 and 5, with six and
nine CF3 groups, respectively, are decorated enough to pro-
duce bright MR signals at low-millimolar concentrations. Im-
provement of the signal may be obtained by optimizing the
image acquisition, which will be described in a forthcoming
publication. It should be admitted that practical administration
in vivo without local accumulation may hardly produce signals
intense enough for 19F MRI due to dilution. We consequently
studied the cytotoxicity at lower concentrations, which will be
met in whole-body applications. It turned out that the cytotox-
icity of the synthesized BODIPYs 1, 2, 4, and 5 depends on the
volume of the synthesized esters, which presumably hinders
ester hydrolysis. Generally, the cytotoxicity was found to be
lower in primary HUVEC, which are closer to the in vivo situa-
tion than a cell line. We conclude that the system consisting of
BODIPY in conjunction with highly fluorinated alcohols is suita-
ble for dual FLI and 19F MRI by demonstrating that BODIPY 5
can be seen in a mouse post-mortem, both by MRI and FLI. Es-
pecially the short relaxation time T1 and the low toxicity of 4
and 5 makes these compounds valuable starting points for
in vivo experiments with water-soluble derivatives. For future
biomedical application, we propose 4 as promising candidate,
as the remaining protected alcohol function might be exploit-
ed for binding the synthesized dual imaging agents covalently
to a target of interest, thus decreasing ubiquitous background
fluorescence from unbound dye.[10] In addition, the hydrolytic
stability and other pharmacologically relevant parameters must
be studied in greater detail before similar compounds can be
transferred to live-animal models. Finally, even further expan-
sion to triple contrast agents by combination with 18F PET be-
comes feasible.[15]
UV/Vis and fluorescence spectroscopy: Absorption spectra were
recorded using a commercial spectrophotometer (Jasco, V-650),
and fluorescence emission and excitation spectra were obtained
with a commercial spectrofluorimeter (Jasco, FP-6500) at micromo-
lar concentrations, if not stated otherwise.
Fluorescence microscopy: For fluorescence microscopy 250000
cells per well were seeded on glass coverslips in a 24-well format.
Cells were incubated with the respective dye at a concentration of
50 mm for 1 h. After washing twice with PBS, cells were fixed for
15 min with 4% PBS-buffered formalin and counterstained with
DAPI (2 mgmLÀ1 in PBS) for 10 min. Coverslips were mounted using
Fluorsaveꢄ mounting medium (Merck Millipore). Images were ob-
tained and analyzed with an Axio Observer Z1 epifluorescence mi-
croscope (Zeiss; DAPI imaging: lexc =335–383 nm, lem =420–
470 nm; FITC imaging: lexc =455–495 nm, lem =505–555 nm).
MTT assays: MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-
um bromide] assays were performed as previously described.[25] Pri-
mary human umbilical vein cells (HUVEC) were isolated from um-
bilical cords by digestion with 0.01% collagenase A solution
(Roche) and grown in ECGM with supplement mix (Promocell) con-
taining penicillin (100 UmLÀ1), streptomycin (100 mgmLÀ1), kanamy-
cin (50 mgmLÀ1), and 10% FCS (Sigma). Umbilical cords were ob-
tained with the consent of patients (permission by the local ethics
committee). For experiments, cells were used at passage 3 or
4.[22,26] HUVEC or HepG2 cells were seeded at a density of 20000 or
10000 cells per well, respectively. Cells were incubated with the
tested compounds for 24 h prior to MTT assay. Compounds were
dissolved in DMSO and used at the following concentrations: 1, 10,
20, 40, 50, and 100 mm. DMSO served as solvent control. The IC50
values were extrapolated from the resulting concentration–viability
curves.
Relaxometry: The relaxation times were measured with a benchtop
device by Bruker (Minispec mq60, 60 MHz, 1.41 T). The longitudinal
relaxation times T1 were recorded with the inversion recovery se-
quence, and the transverse relaxation times T2 with the Car–Pur-
cell–Meiboom–Gill (CPMG) sequence.
1
19F and H MRI: In vitro and ex vivo magnetic resonance imaging
(MRI) was performed with a 9.4 T MRI animal scanner (Biospec
Avance III94/20; Bruker Biospin GmbH, Ettlingen, Germany) with
a maximum field strength of 675 mTmÀ1, linear inductive rise time
of 130 ms, and maximum slew rate of 4673 mTmÀ1 sÀ1 (BGA12S gra-
dient system; Bruker Biospin GmbH, Ettlingen, Germany), run with
ParaVision 5.1 (Bruker Biospin GmbH, Ettlingen, Germany). Meas-
urements were conducted with a linear MRI transceiver tunable
1
both for 19F and H, designed for imaging of rat whole body, with
Experimental Section
an inner diameter of 72 mm. For both in vitro and ex vivo imaging,
1H MRI with fast low angle shot (FLASH) sequences was used for
orientation of samples in the magnet and for 1st- and 2nd-order
shimming, based on previously recorded field maps (ParaVision 5.1,
macro MAPSHIM). In the ex vivo experiments, additional 1H MR
images were recorded with a rapid acquisition relaxation enhanced
(RARE) sequence, for demonstration of animal morphology and
image fusion with 19F MRI data. 19F basic frequency, reference pulse
gain, and receiver gain were set manually employing MR spectros-
copy pulse programs and TopSpin 2.0PV software (Bruker Biospin
GmbH, Ettlingen, Germany). 19F MRI was performed with FLASH
General: Reagents and solvents were used as purchased from
Sigma–Aldrich, Merck, Acros Organics, and Carbolution Chemicals.
The solvents used were dried using common laboratory methods.
All air-sensitive reactions were carried out under an argon atmos-
phere. Analytical thin-layer chromatography (TLC) was performed
on Silica Gel 60 on PET-Foils by Fluka Analytiks. Column chroma-
tography was performed on a silica gel 60 (63–260 mm).
NMR spectroscopy: 1H, 19F, and 13C NMR spectra were recorded
with a Bruker Avance 2 spectrometer (400, 376 or 100 MHZ) at am-
bient temperature with reference to TMS or solvent standard with
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Table 4. Sequence type and settings used for H and 19F MRI.[a]
Sequence
1H RARE
Use
FOV [mm][b]
48ꢃ32
MTX [voxels][c]
480ꢃ320
TR [ms][d]
1647.3
TE [ms][e]
9.7
NA[f]
8
ST [mm][g]
1
Duration
ex vivo
12 min 31 s
in vitro
ex vivo
64ꢃ32
48ꢃ32
64ꢃ32
48ꢃ32
19F FLASH
19F RARE
2000
2400
1.7
32
5
5
17 min 4 s
5 min 7 s
in vitro
ex vivo
64ꢃ32
48ꢃ32
64ꢃ32
48ꢃ32
126.9
128
[a] B0 =9.4 T in CH2Cl2 at room temperature; concentration as indicated in Figure 3. [b] Field of view. [c] Matrix size. [d] Repetition time. [e] Echo time.
[f] Number of acquisitions. [g] Slice thickness.
3
and RARE sequences optimized for high signal-to-noise ratio and
scan times reasonable for in vivo imaging. Sequence details are
summarized in Table 4. For characterization by MRI, compound 5
was dissolved in CH2Cl2 at 10, 20, 50, and 100 mm. For each con-
centration, a volume of 500 mL in a standard plastic reaction vial
was placed in the magnet and submitted to MRI as described. De-
tectability in animals was tested in three mice sacrificed before-
hand by CO2 inhalation, by intramuscular injection of 100 mL of the
100 mm dilution that had been tested before in the in vitro exami-
nation, and subsequent MRI.
2.96 (m, 2H), 2.63 (t, 2H, JH,H =7.28 Hz), 2.45 (s, 6H), 2.34 (s, 6H),
1.94 ppm (m, 2H); 13C NMR (CDCl3): d=169.5, 154.5, 143.9, 140.3,
131.4, 122.0, 60.6, 33.1, 27.1, 26.2, 16.3, 14.5 ppm; 19F NMR (CDCl3):
d=À73.25 (s, 6F, CF3), À146.58 ppm (m, 2F, BF2); HRMS (ESI): calcd
for C20H22BF8N2O2 [M+H] 484.1647, found 485.1642.
BODIPY 3 was synthesized according to general procedure A. The
crude product was purified by column chromatography (silica gel,
PE/CH2Cl2 2:1, Rf =0.19) to give a red solid (0.13 g, 0.25 mmol, yield
82%). 1H NMR (CDCl3): d=5.99 (s, 2H), 3.01 (m, 2H), 2.78 (t, 2H,
3JH,H =7.03 Hz), 2.44 (s, 6H), 2.35 (s, 6H), 2.00 ppm (m, 2H);
13C NMR (CDCl3): d=187.6, 168.6, 154.5, 144.0, 140.3, 134.6, 131.4,
128.2 121.9, 112.8, 33.1, 27.1, 26.3, 16.3, 14.5 ppm; 19F NMR (CDCl3):
3
Syntheses
d=À146.58 (m, 2F, BF2), À152.71 (m, 2F), À157.57 (t, 1F, JF,F
=
21.80), À162.03 ppm (m, 2F); HRMS (ESI): calcd for C23H20BF7N2O2
[M+H] 501.1584, found 501.1580.
General procedure A: BODIPY carboxylic acid and DMAP
(1.0 equiv) were dissolved in CH2Cl2 and cooled to 08C. After stir-
ring for 10 min, DCC (1.0 equiv) was added and stirred for an addi-
tional 1 h. Then the alcohol (1.0 equiv) was added and the reaction
mixture was allowed to warm to room temperature. After stirring
for 24 h, the obtained suspension was filtered over a small silica
gel layer (3 cm). The layer was washed with CH2Cl2, and the filtrate
was evaporated under reduced pressure. The received crude prod-
uct was purified by flash chromatography with silica.
BODIPY 4 was synthesized according to general procedure B. The
crude product was purified by column chromatography (silica gel,
PE/EtOAc 7:3, Rf =0.55) to give a red solid (0.13 g, 0.14 mmol, yield
99%). 1H NMR (CDCl3): d=6.07 (s, 2H), 4.12 (s, 2H), 4.09 (s, 6H),
3
3.02 (m, 2H), 2.53 (s, 6H), 2.51 (t, 2H, JH,H =7.28 Hz), 2.43 (s, 6H),
2.07 (s, 3H), 1.95 ppm (m, 2H); 13C NMR (CDCl3): d=171.3, 169.7,
154.0, 144.2, 140.0, 131.1, 121.5, 121.2, 118.3, 65.7, 60.4, 60.2, 43.7,
33.2, 26.0, 20.0, 15.9, 14.1 ppm; 19F NMR (CDCl3): d=À70.27 (s, 18F,
CF3), À146.72 ppm (q, 2F, BF2); HRMS (ESI): calcd for C32H30BF20N2O6
[MÀH] 929.1878, found 929.1873.
General procedure B: Alcohol, BODIPY
6 (3.0 equiv), DMAP
(3.3 equiv), and Sc(OTf)3 (0.6 equiv) were dissolved in CH2Cl2 and
stirred for 30 min at À88C. After adding DIPC (3.2 equiv), the reac-
tion mixture was stirred 30 min at À88C and an additional 2 d at
room temperature. The obtained suspension was filtered over
a small silica gel layer (3 cm). After washing the layer with CH2Cl2,
the filtrate was washed with HCl (0.1m, 2ꢃ), diluted Na2CO3 solu-
tion (2ꢃ) and distilled water. The organic phase was then dried
over Na2SO4, and the solvent was removed under reduced pres-
sure. The obtained crude product was purified by SiO2 flash chro-
matography.
BODIPY 5 was synthesized according to general procedure B. The
crude product was purified by column chromatography (silica gel,
PE/EtOAc 8:2, Rf =0.31 (PE/EE, 9:1)) to give a red solid (0.25 g,
0.23 mmol, yield 82%). 1H NMR (CDCl3): d=6.07 (s, 2H), 4.10 (s,
3
2H), 4.06 (s, 6H), 3.00 (m, 2H), 2.53 (s, 6H), 2.50 (t, 2H, JH,H
=
7.28 Hz), 2.42 (s, 6H), 1.96 ppm (m, 2H); 13C NMR (CDCl3): d=171.3,
154.4, 144.5, 140.3, 121.9, 121.5, 118.6, 64.9, 59.8, 45.3, 33.3, 27.2,
26.3, 16.1, 14.5 ppm; 19F NMR (CDCl3): d=À70.37 (s, 27F, CF3),
À146.55 ppm (q, 2F, BF2); HRMS (ESI): calcd for C34H27BF29N2O5
[MÀH] 1105.1550, found 1105.1544.
BODIPY 1 was synthesized according to general procedure A. The
crude product was purified by column chromatography (silica gel,
petroleum ether (PE)/CH2Cl2 2:1, Rf =0.10) to give a red solid
(0.11 g, 0.27 mmol, yield 91%). 1H NMR (CDCl3): d=5.99 (s, 2H),
Alcohol 11 was synthesized according to published reaction condi-
tions (yield 57%).[17] 1H NMR (CDCl3): d=7.30–7.19 (m, 5H), 4.44 (s,
2H), 4.13 (s, 2H), 3.62–3.53 (m, 4H), 3.41 (s, 2H), 1.99 ppm (s, 3H);
13C NMR (CDCl3): d=171.8, 137.7, 128.5, 127.9, 127.6, 73.7, 71.1,
63.7, 63.2, 44.9, 20.8 ppm; HRMS (ESI): calcd for C14H19O5 [MÀH]
267.1232, found 267.1227.
3
3
4.42 (q, 2H, JH,F =8.28 Hz), 2.94 (m, 2H), 2.53 (t, 2H, JH,H =7.28 Hz),
2.44 (s, 6H), 2.34 (s, 6H), 1.92 ppm (m, 2H); 13C NMR (CDCl3): d=
170.9, 154.3, 144.4, 140.3, 131.4, 121.9, 60.6, 33.5, 27.2, 26.4, 16.3,
14.4 ppm; 19F NMR (CDCl3): d=À70.27 (s, 3F, CF3), À146.72 ppm
(m, 2F, BF2); HRMS (ESI): calcd for C19H23BF5N2O2 [M+H] 417.1773,
found 417.1768.
Alcohol 12 was synthesized according to published reaction condi-
tions (yield 36% over two steps).[17] 1H NMR (CDCl3): d=4.15 (s,
2H), 4.09 (s, 4H), 3.59 (s, 2H), 2.10 (s, 3H); 13C NMR (CDCl3): d=
171.1, 121.6, 118.7, 66.5, 61.1, 59.7, 45.8, 20.4 ppm; 19F NMR (CDCl3):
d=À70.38 ppm (s).
BODIPY 2 was synthesized according to general procedure A. The
crude product was purified by column chromatography (silica gel
PE/CH2Cl2 2:1, Rf =0.22) to give a red solid (0.11 g, 0.27 mmol, yield
1
3
91%). H NMR (CDCl3): d=6.00 (s, 2H), 5.71 (sxt, 1H, JH,F =6.02 Hz),
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Full Papers
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BODIPY 13 was synthesized according to general procedure A. The
crude product was purified by column chromatography (silica gel,
PE/EtOAc 8:2, Rf =0.71 (PE/EE, 8:2)) to give a red solid (0.10 g,
0.28 mmol, yield 95%). 1H NMR (CDCl3): d=5.98 (s, 2H), 4.09 (q,
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2H), 2.92 (m, 2H), 2.44 (s, 6H), 2.41 (t, 2H, JH,H =7.28 Hz), 2.35 (s,
6H), 1.87 (m, 2H), 1.21 ppm (t, 3H); 13C NMR (CDCl3): d=172.6,
154.2, 145.1, 140.4, 131.5, 121.7, 80.7, 34.4, 29.7, 27.5, 28.8, 16.4
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Acknowledgements
Financial support by the German Science Foundation (DFG),
JU650/3-1 is gratefully acknowledged. We also thank Dr. Yulin Qi
(Saarland University) for recording mass spectra, Theo Ranßweil-
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Keywords: fluorescence
radiopharmaceuticals · toxicology
·
imaging
agents
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Published online on && &&, 0000
&
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FULL PAPERS
Light and magnetism: The combina-
tion of fluorescence and 19F MRI is ex-
emplified in a mouse post-mortem by
a BODIPY dye highly decorated with flu-
orine atoms. Up to nine CF3 groups pro-
viding 27 equivalent fluorine atoms are
introduced by efficient condensation re-
actions. A detection limit below 10 mm
in 19F MRI together with low toxicity
makes the presented compounds
A. M. Huynh, A. Mꢀller, S. M. Kessler,
S. Henrikus, C. Hoffmann, A. K. Kiemer,
A. Bꢀcker, G. Jung*
&& – &&
Small BODIPY Probes for Combined
Dual 19F MRI and Fluorescence
Imaging
a worthy starting point for further de-
velopment. Local accumulation in vivo
will be accomplished by targeting.
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These are not the final page numbers! ÞÞ