DMAP-BODIPY alkynes: A convenient tool for labeling biomolecules for bimodal pet-optical imaging

Article abstract of DOI:10.1002/chem.201402379

Several new boron dipyrromethene/N,N-dimethylaminopyridine (BODIPY-DMAP) assemblies were synthesized as precursors for bimodal imaging probes (optical imaging, OI/positron emission tomography, PET). The photophysical properties of the new compounds were also studied. The first proof-of-concept was obtained with the preparation of several new BODIPY-labeled bombesins and evaluation of the affinity for bombesin receptors by using a competition binding assay. Fluorination reactions were investigated on DMAP-BODIPY precursors as well as on DMAP-BODIPY-labeled bombesins. Chemical modifications on the BODIPY core were also performed to obtain luminescent dyes emitting in the therapeutic window (650-900 nm), suitable for in vivo imaging, making these compounds promising precursors for PET/optical dual-modality imaging agents.

Full text of DOI:10.1002/chem.201402379

DOI: 10.1002/chem.201402379
Full Paper
&
Imaging Agents
DMAP-BODIPY Alkynes: A Convenient Tool for Labeling
Biomolecules for Bimodal PET–Optical Imaging**
Bertrand Brizet,[a, b] Victor Goncalves,[a] Claire Bernhard,[a] Pierre D. Harvey,[b] Franck Denat,*[a]
and Christine Goze*[a]
Abstract: Several new boron dipyrromethene/N,N-dimethy-
laminopyridine (BODIPY-DMAP) assemblies were synthesized
as precursors for bimodal imaging probes (optical imaging,
OI/positron emission tomography, PET). The photophysical
properties of the new compounds were also studied. The
first proof-of-concept was obtained with the preparation of
several new BODIPY-labeled bombesins and evaluation of
the affinity for bombesin receptors by using a competition
binding assay. Fluorination reactions were investigated on
DMAP-BODIPY precursors as well as on DMAP-BODIPY-la-
beled bombesins. Chemical modifications on the BODIPY
core were also performed to obtain luminescent dyes emit-
ting in the therapeutic window (650–900 nm), suitable for in
vivo imaging, making these compounds promising precur-
sors for PET/optical dual-modality imaging agents.
Introduction
the tissues. Nevertheless, recent advances in fluorescence
imaging instrumentation and probe developments promise
new opportunities, highlighting the emerging performances of
this technique, especially for fluorescence guided-surgery with
targeted molecular imaging.[4]
Molecular imaging is a highly promising field of research and
innovation with huge potential in a wide range of applications
including prognostic, diagnostic, drug discovery, and develop-
ment of theranostics. It enables real-time visualization of bio-
chemical events at the cellular and molecular levels within the
cells, tissues, and intact subjects.[1] In this area of research, nu-
clear imaging, namely positron emission tomography (PET), en-
ables non-invasive diagnosis with high sensitivity by providing
in vivo distribution of radiolabeled biovectors, thus facilitating
detection of cancers. Among the radioelements available for
PET imaging, 18F has become the radionuclide of choice, in
a similar way to 99mTc in gamma scintigraphy.[2] This is due to
its optimal nuclear and chemical properties, that is, low
energy, high abundance of positrons, and relatively long half-
life (109.7 min) in comparison with the 11C technique.[3]
Together, PET and fluorescence imaging presents comple-
mentary features, for example, the high penetrability of the
511 keV photons of PET and the high spatial and temporal res-
olution of fluorescence. These are some of the reasons why
dual-modality PET/optical imaging could strongly be beneficial
for preclinical and clinical applications.[5] In this context, the
preparation of MonoMolecular Imaging Agents (MOMIA),
which combine a fluorescent probe and a radioisotope into
a single molecule, showed marked advantages compared with
the conventional approach (i.e., sequential coupling of a chela-
tor and a NIR fluorophore).[6] Notably, the modification of bio-
molecules on one single attachment point simplifies the dual-
labeling process and minimizes the loss of affinity for the tar-
geted receptors.[7]
Fluorescence imaging is a valuable technique for small
animal imaging. The optical agents also provide the opportuni-
ty to perform histological studies based on the fluorescent
signal to evidence molecular targeting. Clinical applications are
unfortunately more limited owing to their poor penetrability in
Among the fluorescent dyes, BODIPY derivatives are highly
suitable candidates for the design of bimodal agents because
they generally provide high fluorescence quantum yields,[8]
high thermal and photochemical stability, and a high degree
of tunable emission into the red-NIR region from a simple var-
iation of the molecular structure.[8b,9] Moreover, the BODIPY
core bears two fluorine atoms onto its boron center (BF2 unit),
an ideal site for radiofluorination, considering the strength of
the BÀF bond (>730 kJmolÀ1), which is one of the most ther-
modynamically stable covalent bonds known.[10] Beyond medi-
cal imaging, the concept of bimodality can be extended to
other important areas. Indeed, promising work on applications
of fluorinated BODIPY systems as bimodal XPS-fluorescence
labels for the detection of amino groups on SiO2 supports has
recently been reported.[11]
[a] Dr. B. Brizet, Dr. V. Goncalves, Dr. C. Bernhard, Prof. F. Denat, Dr. C. Goze
Institut de Chimie Molꢀculaire de l’Universitꢀ de Bourgogne, UMR 6302
9, avenue Alain Savary, 21078 Dijon (France)
Fax: (+33)380-39-61-17
E-mail: franck.denat@u-bourgogne.fr
christine.goze@u-bourgogne.fr
[b] Dr. B. Brizet, Prof. P. D. Harvey
Dꢀpartment de Chimie Universitꢀ de Sherbrooke
Universitꢀ de Sherbrooke, Quꢀbec, J1K 2R1 (Canada)
[**] DMAP=N,N-dimethylaminopyridine; BODIPY=boron dipyrromethene;
PET=positron emission tomography.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201402379.
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The high potential of 18F-BODIPY dyes has recently been in-
vestigated and different methods have been developed for 18F
fluorination of BODIPY.[12] Indeed, rapid nucleophilic [18F] radio-
labeling of BODIPY dyes using a B-OH BODIPY precursor was
previously reported by the group of Gabba¨ı and Conti.[13] B-OH
BODIPY was pretreated with trimethylsilyl trifluoromethanesul-
fonate (TMSOTf) as an activating/water removing agent, fol-
lowed by radiofluorination.[14] This convenient approach was
also exploited by the group of Weissleder and Mazitschek who
employed a BF2-BODIPY dye as starting material.[15] The group
of Weissleder also performed a radiofluorination of N,N-dime-
thylaminopyridine (DMAP)-BODIPY precursors.[15,16] More re-
cently, Lewis acids such as SnCl4 were used to promote the 18F
labelling through a 19F-18F exchange reaction.[12,17] Additionally,
18F labelling without using any additional reagent, such as
a Lewis acid, considering the fragility of biomolecules. To fulfil
these conditions, we used Weissleder’s procedure, which repla-
ces a DMAP group by 18F on BODIPY without any other re-
agent (Figure 1).[15]
Figure 1. Synthesis of [18F]-BODIPY through DMAP-activated BODIPY.
18
in vivo stability of the BÀ F bond was investigated. No release
of free 18FÀ was detected, proving the metabolic stability of
the BF2 core.[14–15]
We report the synthesis and characterization of various
DMAP-BODIPY precursors bearing activated functional groups
for bioconjugation along with their photophysical properties.
These DMAP-BODIPY assemblies have been attached to
a bombesin derivative and in vitro tests towards the bombesin
receptors are presented. Finally, far-red-emitting BODIPY com-
pounds have been prepared to shift both the absorption and
emission bands towards the therapeutic region for in vivo
imaging.
Our research group has explored the possibility to expand
the library of potential bimodal imaging probes based on
[18F]ÀBODIPY derivatives by using our long-standing experi-
ence in the field of BODIPY synthesis, including far-red emit-
ting systems.[18] Moreover in our previous reports, we investi-
gated the synthesis and the performances of BODIPY-chelators
systems for bimodal single photon emission computed tomog-
raphy (SPECT)/optical imaging.[19] In these systems, the radio-
labeling was performed after bioconjugation, in the last syn-
thetic step. We were thus very interested in the possibility of
using a similar strategy, that is, the introduction of 18F atoms
during the final step.[20]
Results and Discussion
The bifunctional target agent 6 was first prepared (Scheme 1).
This molecule bears a DMAP ligand on the boron atom as
a leaving group, which was previously validated as an ade-
quate strategy for the incorporation of 18F.[16] The compound is
further activated using an isothiocyanate functional group for
Such an approach has never been explored with [18F]-
BODIPY derivatives before. It requires the identification of
a BODIPY probe bearing a living group that: 1) Could be stable
under the bioconjugation coupling conditions; 2) Could allow
Scheme 1. Synthesis of compound 6.
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its conjugation with amines. Compound 6 was obtained in
four steps starting from the known precursor 1 (Scheme 1).[21]
A nitrophenyl function was introduced by peptide coupling
between 1 and 2-(4-nitrophenyl)ethanamine to obtain 2 in
76% yield. The nitro group was reduced into an amine func-
tion to yield 4 in almost quantitative yield.[18b] The next step in-
volved the introduction of DMAP by first replacing one of the
fluorine atoms by the leaving group TMSOTf and then adding
DMAP in situ. Finally, the activation of the amine into an iso-
thiocyanate was performed thus giving access to the desired
compound 6. Noteworthy, the introduction of the DMAP (i.e.,
formation of 3) followed by the reduction of the nitro into an
amine, as an alternative synthetic approach, was not successful
because of the undesired loss of the DMAP group during the
hydrogenation process.
The feasibility of a subsequent radiofluorination on the
DMAP prosthetic compound was successfully verified by reac-
tion of 3 with KF in the presence of the commonly used kryp-
tofix 222 (KF [222]).[25] Compound 2 could then be obtained
from 3 in 10 min, in 50% yield. The fluorination product can
easily be monitored and characterized by using mass spec-
trometry and 11B NMR spectroscopy. The 11B NMR spectrum of
the DMAP-BODIPY product exhibits the expected doublet,
whereas the BF2-BODIPY compound exhibits the typical triplet
Figure 2. Comparison of the 11B NMR spectra of compounds 2 and 3
(192.5 MHz, CDCl3).
characteristic of the 11B-19F couplings (I=1= ). A typical example
2
is shown in Figure 2.
The feasibility of the bioconju-
gation step was evaluated by
treating a slight excess of acti-
vated
DMAP-BODIPY
6
(1.4 equiv) with propylamine, to
mimic the actual conditions of
bioconjugation
(Scheme 2).
However, the DMAP group
proved to be very reactive to-
wards amine, and yielded to
some degradation products.
Scheme 2. Coupling test of 6 with propylamine.
Considering these difficulties,
the substitution of the isothio-
cyanate by another activated
function was investigated.
synthesized in two steps, starting from 1 with, first, the intro-
duction of a triple bond on the BODIPY using a peptidic cou-
pling with prop-2-yne-1-amine, followed by the incorporation
of the DMAP ligand (Scheme 3).
Click chemistry provides a number of avenues for the design
of complex structures and multicomponent functionalized sys-
tems.[22] In particular, the copper-catalyzed azide–alkyne cyclo-
addition has already become a powerful technique in peptide
science due to its simplicity and bioorthogonality. It has been
utilized in the cyclization of peptides, ligation of two or more
peptide fragments, and conjugation to biomolecules, nanopar-
ticles, polymers, and other chemical entities.[23] The impact of
click chemistry on conjugation methods, especially bioconju-
gation, has been extensive in recent years. Indeed, azide func-
tions can be easily introduced in peptides, and even in pro-
teins at an adequate position. Such an approach allows for the
rapid and site-selective labeling of biomolecules without modi-
fying their binding properties to biological targets. However,
implementing such a strategy requires first to functionalize the
BODIPY with an alkyne function. Compound 9 was therefore
Click reaction tests were optimized starting from 9. The cou-
pling reaction was successful when the click conditions pre-
cluded any amine, such as diisopropylethylamine, as a base.
Compound 10 was then obtained by treating 9 with one
equivalent of azidomethylbenzene using CuSO4 as catalyst and
sodium ascorbate (Scheme 4). Noteworthy, the DMAP group
desirably remained anchored on the BODIPY in these condi-
tions. Substitution of the DMAP with one equivalent of FÀ in
the presence of kryptofix 222 could then be performed on 10,
yielding 11 in 42% yield.
A click reaction was also performed on a bombesin deriva-
tive, N3-AEEAc-bombesin(7–14) (N3-BBN). Bombesin is a 14-mer
peptide that binds to three G-protein-coupled receptors (GRP-
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bation in the excited state is
minimal. This observation is cor-
roborated by the fact that the
excitation spectra superpose
well with the absorption ones.
The absorption features located
in the 200–300 nm range are
due to the absorption of trypto-
phan side chain of the bombesin
derivative.
Interestingly, the
DMAP loss going from 12 to 13
is accompanied by the disap-
pearance of its characteristic ab-
sorption band at 279 nm.
Scheme 3. Synthesis pathway of 9.
R, NMB-R, and BRS-3) overexpressed in a wide variety of tumor
tissues. Bombesin and its analogues have been extensively
studied as targeting agents for the detection of cancer in pre-
clinical and clinical studies using numerous imaging modali-
ties.[24] Here, the widely used 8-mer peptide bombesin(7–14),
which contains the minimal sequence required for high affinity
binding to its receptors, was chosen as model. The peptide
was acylated on its N-terminal with an N-(2-(2-(2-azidoeth-
oxy)ethoxy))acetyl chain (N3-AEEAc) to introduce the azide
function and to provide flexibility and water solubility to the
conjugate. The coupling reaction, monitored by HPLC, provid-
ed 12 in 59% yield (Scheme 5).
The fluorination reaction was then performed by treating
compound 12 with one equivalent of KF in dry acetonitrile
(Scheme 6), yielding the desired product 13 in 35% yield after
10 min. This fluorination reaction was competitive with the hy-
drolysis of the B-DMAP bond, which yielded the side product
14. Traces of water were probably present in the KF salt.
The fluorination should be performed in strictly anhydrous
conditions to optimize the yield of formation of BF2-BODIPY.[25]
An interesting point is that the BÀOH function slowly reacts
with FÀ, and could be radiofluorinated using acidic conditions,
even if these conditions are not ideal for BODIPY.[14]
Figure 3 shows the absorption, fluorescence, and excitation
spectra of compounds 12 (A) and 13 (B). Both compounds ex-
hibit two absorption bands characteristic of the BODIPY signa-
ture, which consists of the S0–S1 feature placed near 520 nm,
and the S0–S2 one located near 380 nm. Both bands are readily
assigned to spin-allowed p–p* transitions. The emission bands
are observed at ꢀ540 nm and exhibit a mirror-image relation-
ship with the absorption, indicating that the structural pertur-
Figure 3. Absorption, emission (lex =470 nm) and excitation (lex =590 nm)
spectra of 12 and 13 in 2-MeTHF.
Scheme 4. Synthesis and fluorination of compound 10.
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in 79% yield by treating 8 with
four equivalents of 4-methoxy-
benzaldehyde, in the presence
of piperidine and p-toluenesul-
fonic acid (PTSA; Scheme 7). The
DMAP was then introduced onto
15 and the resulting activated
precursor 16 was subsequently
attached to N3-BBN in 53%
yield.
The formation of the mono-
styryl derivative is also interest-
ing because the dye exhibits an
emission in the red region (at
around 600 nm). In addition, it
may offer the possibility of
a second Knoevenagel function-
alization using a selected alde-
hyde (bearing a grafting func-
tion for example).[28] Thus, the
monofunctionalized compound
19 was synthesized by reacting
18 with 4-(prop-2-yn-1-yloxy)-
benzaldehyde (Scheme 8). The
low reaction yield is explained
by the difficulty to control the
mono- versus di-condensation
and problems with the purifica-
tion. As previously shown for
other derivatives, the DMAP
could be anchored and the re-
sulting assembly 20 was at-
tached to the bombesin deriva-
tive in 31% yield.
Scheme 5. Click reaction of 9 with N3ÀBBN.
Scheme 6. Fluorination test on compound 12.
Biological testing
1
The H NMR spectrum of compound 20 is shown in Figure 5.
Each substituent in the a- and b-pyrrolic positions displays one
signal. The substituents located on the side of the styryl group
are the most deshielded. For instance, the two methyl groups
The affinity of the bioconjugate 13 for bombesin receptors (on
rat cerebral cortex membranes) was evaluated on a radioactive
binding assay using [125I]-[Tyr4]bombesin(1–14) as a ligand.[26]
Under these assay conditions, compound 13 was able to com-
pete for the binding of the radioligand with a Ki of (0.33Æ
0.45) nm, whereas the unlabeled peptide N3-BBN exhibited a Ki
of (0.36Æ0.19) nm (Figure 4). This result illustrates that, despite
its size, conjugation of the BODIPY does not modify significant-
ly the binding affinity of a small peptide such as BBN(7–14) for
its receptors.
Towards the therapeutic region for in vivo imaging
Dyes active in the so-called therapeutic window (650–900 nm)
are more adapted for in vivo applications, such as molecular
imaging. Advantages include minimal interfering absorption
and fluorescence from biological samples, reduced scattering,
and enhanced tissue penetration depth. One simple way to
extend the p-conjugation and to shift the absorption and
emission bands to the NIR region is to perform a Knoevenagel
condensation on the methyl groups at the a-pyrrolic posi-
tions.[27] Indeed, the target distyryl compound 15 was prepared
Figure 4. Displacement of radioligand [125I]-[Tyr4]bombesin(1–14) binding at
rat cerebral cortex membranes by 13 and N3-BBN. Non-specific binding was
defined by 1 mm cold bombesin (1–14).
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in b-pyrrolic positions display two singlets at d=1.34 and
1.36 ppm. The tetrahedral geometry of the boron atom indu-
ces that the DMAP ligand is orthogonal to the indacene plane.
Consequently, the two faces of the indacene are dissymmetric
and the boron atom in BODIPY 20 is a chiral center. Therefore,
protons carried by the CH2 groups in b-pyrrolic positions are
diastereotopic. Notably, protons 7 and 7’, which are on the
side of the styryl group, display each a quartet (3J=7.3 Hz) at
d=2.43 and 2.45 ppm, split by a geminate coupling constant
1J of 12.2 Hz. Additionally, as the methyl in b-pyrrolic position
impedes the rotation of the aromatic ring at the meso position,
the four protons of the aromatic ring display four signals,
whereas they display two doublets for a symmetrical BF2-
BODIPY. Each proton displays a doublet of doublets (3J=
7.9 Hz, 4J=1.5 Hz).
Photophysical studies
Table 1 summarizes the absorption and steady-state fluores-
cence data of the different BODIPY-containing compounds.
Noteworthy, the photophysical data of 12, 13, and 21 were
measured using DMSO/PBS 1:1 as a solvent mixture due to
lack of solubility in PBS (phosphate buffered saline). The emis-
sion lifetimes, tF (3.9<tF <6.9 ns), and the small Stokes shifts,
Dn, confirm the presence of fluorescence. The fluorescence
quantum yields, fF, vary from 35 to 87% and are considered
large enough for imaging applications.
Scheme 7. Synthetic pathway of 17.
Generally, the introduction of DMAP group onto
the BODIPY chromophore induces a bathochromic
shift of the absorption and emission maxima. More-
over, the Stokes shifts (Dn in cmÀ1 units; a linear
scale of energy) of the DMAP-containing BODIPYs are
larger than in their BF2 analogues. For instance, com-
pounds 15 and 16 display fluorescence maxima at
681 and 703 nm, respectively, with Stokes shifts of
610 and 950 cmÀ1. The introduction of styryl substitu-
ents induces a bathochromic shift of the absorption
and fluorescence bands respectively by ꢀ60 and
130 nm for the mono- and distyryl-BODIPY deriva-
tives, and does not affect their fluorescence quantum
yields. Finally, compounds 12, 13, and 21 display
high fluorescence quantum yields (0.35<fF <0.40),
confirming that the introduction of bombesin on
dyes 8, 9, and 20 does not affect the intense lumi-
nescence properties of BODIPY.
Conclusion
Several new potential bimodal agents for PET–optical
imaging have been synthesized and characterized.
The photophysical study demonstrated that these
BODIPY-containing systems exhibit high fluorescence
quantum yields. Bioconjugation on the bombesin de-
rivative N3-BBN and a competitive binding assay with
BF2-BODIPY-BBN were performed evidencing that the
presence of the bimodal agent does not affect the af-
finity of the peptide for the receptors. Thus, the use
Scheme 8. Synthesis pathway of 21.
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Figure 5. 1H NMR spectrum and assignments of 20 (CDCl3, 300 MHz, 298 K).
Table 1. Photophysical properties of selected BODIPY derivatives.[a]
Experimental Section
Materials
Dye
labs
lem
Dn
[cmÀ1
fF
tF
Solvent
[nm]
[nm]
]
[ns]
Unless otherwise noted, all chemicals and solvents were of analyti-
cal reagent grade and were used as received. Absolute dichlorome-
thane (CH2Cl2) was obtained from Carlo Erba. Silica gel (Merck; 70–
120 mm) was used for column chromatography. Analytical thin-
layer chromatography was performed with Merck 60 F254 silica gel
(precoated sheets, 0.2 mm thick). Reactions were monitored by
thin-layer chromatography, RP-HPLC, UV/Vis spectroscopy, and
MALDI/TOF mass spectrometry.
2
3
6
8
526
527
528
525
528
525
527
654
659
654
587
594
592
540
544
544
543
543
542
542
681
703
–
490
590
560
630
520
600
530
610
950
–
0.59[a]
0.54[a]
0.37[a]
0.59[a]
0.64[a]
0.35[a]
0.40[a]
0.42[b]
0.51[b]
–
3.9
6.2
5.6
4.6
6.9
–
2-MeTHF
2-MeTHF
2-MeTHF
2-MeTHF
9
2-MeTHF
12
13
15
16
17
19
20
21
DMSO/PBS 1:1
DMSO/PBS 1:1
2-MeTHF
2-MeTHF
CH3CN
2-MeTHF
2-MeTHF
DMSO/PBS 1:1
–
6.3
4.7
–
5.4
4.7
–
603
617
615
450
630
630
0.76[c]
0.87[c]
0.51[c]
Instrumentation
1
The H, 11B, and 13C NMR spectra were recorded at room tempera-
ture on a Bruker Avance II 300 (300 MHz) or on a Bruker Avance
DRX 600 (600 MHz) spectrometer at the “Welience, Pꢁle Chimie
Molꢂculaire de l’Universitꢂ de Bourgogne (WPCM)”. Chemical shifts
(1H NMR spectra) are expressed in ppm relative to chloroform (d=
7.26 ppm). The UV/Visible spectra were recorded on a Varian Cary
1 spectrophotometer. The mass spectra and accurate mass meas-
urements (HRMS) were obtained on a Bruker Daltonics Ultraflex II
spectrometer in the MALDI/TOF reflectron mode using dithranol as
a matrix or on a LTQ Orbitrap XL (Thermo Scientific) instrument in
ESI mode. Both measurements were registered at the WPCM. The
steady-state fluorescence emission and excitation spectra were ob-
tained on a Fluorolog SPEX 1680 0.22 m double monochromator
spectrometer using quartz cuvettes (1 cm, 3 mL). All fluorescence
spectra were corrected for apparatus response. The fluorescence
lifetimes were measured in 2-MeTHF or DMSO/PBS 1:1 using
a Timemaster Model TM-3/2003 apparatus from PTI incorporating
a nitrogen laser as the source and a high-resolution dye laser (full
width at half maximum, fwhm=1.4 ns). Fluorescence lifetimes
were obtained from high-quality decays and deconvolution or dis-
tribution lifetime analysis. The uncertainties were 100 ps on the
[a] Stokes shift, Dn, was calculated using the Equation: 1/labsÀ1/lem. The
quantum yields, fF, were measured at 298 K, using rhodamine 6G (fF =
0.94 in methanol), rhodamine 101 (fF =1.00 in methanol) and cresyl
violet (fF =0.54 in methanol) as references.[29,31]
of [18F]-BODIPY, instead of bulky multimodal chelation plat-
forms, could be a relevant alternative for the labeling of small
biovectors such as peptides. Additionally, the fluorination of
the labeled biovectors was investigated and showed the possi-
bility of introducing 18F. Finally, several monostyryl and distyryl
derivatives were synthesized, showing the high potential of
these systems for future in vivo imaging, even though some
issues concerning their hydrosolubility will have to be
addressed.
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basis of multiple measurements, which is also the limit of accuracy
of this system.
tion electrospray mass spectrometry (HRMS; Orbitrap, Thermo
Scientific).
N3-BBN: The peptide (N-(2-(2-(2-azidoethoxy)ethoxy))acetyl-
bombesin(7–14)) was obtained by manual solid-phase peptide syn-
thesis (0.48 mmol scale) on a Rink Amide AM resin (Iris Biotech,
0.48 mmolgÀ1). All amino acids, from Iris Biotech or Novabiochem,
were N-a-terminal-protected by the fluorenylmethyloxycarbonyl
group (Fmoc). 2-(2-(2-azidoethoxy)ethoxy))acetic was synthesized
according to the literature.[34] Briefly, the resin (1.0 g) was swelled
in DMF for 30 min and Fmoc was removed by treating the resin
twice with 20% piperidine, 0.1m HOBt in DMF (10 mL) for 10 min.
After the second treatment, the resin was washed with DMF (3ꢃ
10 mL) and CH2Cl2 (3ꢃ10 mL). Coupling reactions were performed
by adding to the resin a pre-made coupling cocktail of N,N-diiso-
propylethylamine (DIPEA, 3 equiv), 2-(1H-Benzotriazole-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, 3 equiv)
and the required amino acid or 2-(2-(2-azidoethoxy)ethoxy))acetic
acid (3 equiv) in DMF (10 mL). The suspension was left shaking for
1 h, drained and washed with DMF (3ꢃ10 mL) and CH2Cl2 (3ꢃ
10 mL). The final peptide was cleaved from the resin (200 mg) with
simultaneous removal of the side-chain protecting groups by treat-
ment with trifluoroacetic acid/triisopropylsilane/3,6-dioxa-1,8-octa-
nedithiol/water (TFA/TIS/DODT/H2O, 94:2.5:2.5:1 v/v; 5 mL) at
room temperature for 1.5 h. The filtrate from the cleavage mixture
was concentrated, precipitated in cold Et2O and collected by cen-
trifugation (twice), and lyophilized to afford crude peptide.
Binding assays
Compounds 13 and N3-BBN were tested by Cerep (Poitiers, France)
on a radioactive binding assay. Briefly, rat cerebral cortex mem-
branes, expressing GPR-R, NMB-R, and BRS-3 receptor subtypes,
were incubated in the presence of 0.01 nm [125I]-[Tyr4]bombesin
ligand (KD =0.71 nm) and a range of concentrations of tested com-
pounds for 60 min at room temperature. Non-specific binding was
determined in presence of an excess (1 mm) of cold bombesin(1–
14). The results are expressed as a percent of control specific bind-
ing. The IC50 values (concentration causing a half-maximal inhibi-
tion of control specific binding) were determined by non-linear re-
gression analysis of the competition curves generated with dupli-
cate values using GraphPad Prism version 6.00 for Mac. The corre-
sponding inhibition constants (Ki) were calculated using the
Cheng–Prusoff equation.[30] Bombesin(1–14) was used as a reference
compound (Ki =0.17 nm). Further details of the methodology for
the assay can be found at http://www.cerep.fr.
Quantum yield measurements
Measurements were performed in distilled 2-MeTHF or in DMSO/
PBS (1:1). Quartz cuvettes of 3 mL with path length of 1 cm
equipped with a septum were used, and all solutions were Ar-de-
gassed prior to measurements. Three different measurements (i.e.,
different solutions) were performed for each quantum yield. The
sample concentrations were chosen to obtain an absorbance of
about 0.05. The fluorescence quantum yield (fF) measurements
were performed with the slit width of 0.5–1.5 nm for both excita-
tion and emission. Relative quantum efficiencies were obtained by
comparing the areas under the corrected emission spectra of the
sample relative to a known standard and the following Equation
was used to calculate fF: fF(sample)=fF(standard)ꢃ(I(sample)/
I(standard))ꢃ(A(standard)/A(sample))ꢃ(h(sample)2/h(standard)2), in
which fF(standard) is the reported quantum yield of the standard, I
is the integrated emission spectrum, A is the absorbance at the ex-
citation wavelength and h is the refractive index of the solvents
used. Rhodamine 6G (fF =0.94 in methanol),[29a] Cresyl violet (fF =
0.94 in methanol),[29c] and Rhodamine 101 (fF =1.00 in methanol)[31]
were used as standards.[32] In all fF determinations, correction for
the solvent refractive index (h) was applied (2-MeTHF: h=1.406,
DMSO/PBS 1:1: h=1.416 methanol: h=1.328).[33]
The product was purified by semi-preparative RP-HPLC from 20 to
50% of B over 30 min to give N3-BBN as a white solid (TFA salt,
25 mg, 36%, purity:>97%, tR =2.33 min). ESI-MS: m/z=1111.6
[M+H]+; 1133.55 [M+Na]+; HRMS (ESI): m/z calcd for
C49H75N16O12S+: 1111.54656; found: 1111.54399.
Compound 1: 2,4-Dimethylethylpyrrole (10.3 g, 83.9 mmol) and 4-
carboxybenzaldehyde (6.30 g, 42.0 mmol) were dissolved in di-
chloromethane (2.60 mL). Trifluoroacetic acid (328 mL) was added
and the mixture was stirred at room temperature for 48 h. A solu-
tion of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 9.53 g,
42.0 mmol) in dichloromethane was added and the mixture was
stirred for 50 min. Triethylamine (84.0 mL, 630 mmol) and BF3·Et2O
(85.0 mL, 672 mmol) were added and the solution turned purple.
After 2 h of stirring, the reaction mixture was washed with water,
dried over magnesium sulfate, and the solvent was evaporated.
The residue was purified by silica gel column chromatography
(AcOEt/hexane, 90:10). Recrystallization in a mixture of dichlorome-
thane and hexane gave 1 as a red solid (8.40 g, 47%). 1H NMR
3
(300 MHz, CDCl3): d=0.92 (t, 6H, J=7.5 Hz), 1.21 (s, 6H), 2.24 (q,
3
3J=7.5 Hz, 4H), 2.47 (s, 6H), 7.39 (d, J=8.4 Hz, 2H), 8.18 ppm (d,
3J=8.4 Hz, 2H).
Purification and analysis of BODIPY-peptides
Compound 2: N-Hydroxybenzotriazole (640 mg, 4.70 mmol), diiso-
propylamine (1.15 mL, 7.10 mmol), 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (900 mg, 4.70 mmol), and 4-nitro-
phenethylamine hydrochloride (0.49 g, 2.40 mmol) were added
dropwise to a solution of 1 (1.00 g, 2.40 mmol) in dimethylforma-
mide (50 mL) and the solution was stirred at room temperature.
After total consumption of the starting material (2 h) monitored by
TLC, the solvent was evaporated. The resulting solid was washed
with water (2ꢃ100 mL) and extracted with dichloromethane. The
organic layer was dried over magnesium sulfate and the solvent
was evaporated to give a red oil. The crude product was purified
by column chromatography on silica gel (ethyl acetate/hexane,
60:40) followed by recrystallization in a mixture of dichlorome-
thane and hexane to give pure 2 as a reddish solid (1.21 g, 76%
The crude products were purified by semi-preparative RP-HPLC on
a Dionex Ultimate 3000 system (Thermo Scientific) equipped with
a Nucleodur C18ec column (Macherey–Nagel, 250ꢃ10 mm, 5 mm)
with a gradient program (solvent A is water with 0.1% TFA and sol-
vent B is acetonitrile with 0.1% TFA) at a flow rate of 2 mLminÀ1
with UV detection at 254, 530, 590, and 650 nm. Fractions were an-
alyzed by using RP-HPLC on a Dionex Ultimate 3000 system, with
a photodiode array detector, on a Chromolith High-Resolution
C18ec column (Merck, 50ꢃ4.6 mm), equipped with
a guard
column (5ꢃ4.6 mm), at a flow rate of 3 mLminÀ1 and the pure frac-
tions were collected and lyophilized to yield the final compounds
as highly purified (>95%) white, red, purple, or blue solids. The
purity of the compounds was determined by RP-HPLC at 254 nm.
The identity of the compounds was checked by low-resolution
electrospray mass spectrometry (ESI-MS; Bruker) or by high-resolu-
1
3
yield). H NMR (300 MHz, CDCl3): d=0.95 (t, J=7.5 Hz, 6H), 1.22 (s,
3
3
6H), 2.27 (q, J=7.5 Hz, 4H), 2.51 (s, 6H), 3.10 (t, JA =7.1 Hz, 2H),
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3
3.78 (td, JA =7.1 Hz, JB =6.0 Hz, 2H), 6.27 (t, JB =6.0 Hz, 1H), 7.37
column chromatography (CH2Cl2/MeOH, 99:1) to give 5 as a red
solid (105 mg, 64%). 1H NMR (300 MHz, CDCl3): d=0.91 (t, 3J=
3
3
3
(d, J=8.3 Hz, 2H), 7.43 (d, J=8.7 Hz, 2H), 7.83 (d, J=8.3 Hz, 2H),
8.19 ppm (d, 3J=8.7 Hz, 2H).
3
7.5 Hz, 6H), 1.31 (s, 6H), 2.13 (s, 6H), 2.20 (q, J=7.5 Hz, 4H), 3.13
3
(t, 3JA =7.0 Hz, 2H), 3.21 (s, 6H), 3.68 (td, 3JA =7.0 Hz, JB =5.7 Hz,
Compound 3: Trimethylsilyl trifluoromethanesulfonate (TMSOTf,
1.05 mmol, 190 mL) and dimethylaminopyridine (DMAP, 1.05 mmol,
128 mg) were added to a solution of 2 (300 mg, 0.524 mmol) in
dry toluene (15 mL), and the mixture was stirred for 30 min at
808C. The solvent was evaporated; the residue was washed with
water (3ꢃ20 mL) and dried over magnesium sulfate. The solution
was concentrated to dryness and the residue was purified by silica
gel column chromatography (CH2Cl2/MeOH, 97:3) to give 3 as
a red solid (397 mg, 92%). 1H NMR (300 MHz, CDCl3): d=0.91 (t,
3
3
3
2H), 6.52 (t, JB =5.7 Hz, 1H), 6.68 (d, J=8.3 Hz, 2H), 6.93 (d, J=
7.6 Hz, 2H), 7.05 (d, 3J=8.3 Hz, 2H), 7.34 (dd, 3JC =8.0 Hz, 4JD =
1.7 Hz, 1H), 7.68 (dd, 3JC =8.0 Hz, 4JD =1.7 Hz, 1H), 7.84 (dd, 3JC =
8.0 Hz, 4JD =1.7 Hz, 1H), 8.00 (d, 3J=7.6 Hz, 2H), 8.01 ppm (dd,
3JC =8.0 Hz, 4JD =1.7 Hz, 1H); 11B NMR (192.5 MHz, CDCl3): d=
1
1.04 ppm (d, J=41.9 Hz); ESI-MS: m/z=523.3 [MÀC7H10N2ÀOTf]+,
645.4 [MÀOTf]+; HRMS (ESI): m/z calcd for C39H47BFN6O+:
645.38897; found: 645.38897.
3J=7.5 Hz, 6H), 1.30 (s, 6H), 2.13 (s, 6H), 2.20 (q, J=7.5 Hz, 4H),
3
Compound 6: Compound 5 (105 mg, 163 mmol) was placed in a so-
lution of chloroform (15 mL), triethylamine (46 mL), and thiophos-
gene (37 mL) were added under nitrogen. The mixture was stirred
1 h at room temperature and the solvent was evaporated. The re-
sulting solid was purified by column chromatography on silica gel
(dichloromethane/methanol, 96:4) followed by recrystallization in
a mixture of dichloromethane and hexane to give pure 6 (63.0 mg,
3.13 (t, 3JA =7.5 Hz, 2H), 3.21 (s, 6H), 3.78 (td, 3JA =7.5 Hz, 3JB =
5.9 Hz, 2H), 6.86 (d, 3J=7.7 Hz, 2H), 7.34 (dd, 3JC =8.0 Hz, 4JD =
1.8 Hz, 1H), 7.47 (d, 3J=8.7 Hz, 2H), 7.55 (dd, 3JC =8.0 Hz, 4JD =
3
3
1.8 Hz, 1H), 7.71 (t, JB =5.9 Hz, 1H), 7.98 (d, J=7.7 Hz, 2H), 8.11
3
4
3
(dd, JC =8.0 Hz, JD =1.8 Hz, 1H), 8.12 (d, J=8.7 Hz, 2H), 8.13 ppm
(dd, JC =8.0 Hz, JD =1.8 Hz, 1H); 13C{1H} NMR (75 MHz, CDCl3): d=
12.2 (ꢃ2), 12.5 (ꢃ2), 14.0 (ꢃ2), 17.0 (ꢃ2), 29.7 (ꢃ2), 35.6, 40.1,
107.6–123.7–128.0–128.3–128.5–128.9–129.9–130.9–134.8–135.4–
3
4
1
3
39%). H NMR (300 MHz, CDCl3): d=0.91 (t, J=7.5 Hz, 6H), 1.32 (s,
3
3
6H), 2.13 (s, 6H), 2.21 (q, J=7.5 Hz, 4H), 3.00 (t, JA =7.4 Hz, 2H),
3.22 (s, 6H), 3.72 (td, 3JA =7.4 Hz, 3JB =5.7 Hz, 2H), 6.90 (d, 3J=
7.7 Hz, 2H), 7.03 (t, JB =5.7 Hz, 1H), 7.16 (d, J=8.4 Hz, 2H), 7.28
137.1–141.0–141.7–142.0–146.7–147.5–154.0–156.2
(ꢃ28),
166.7 ppm; 11B NMR (192.5 MHz, CDCl3): d=1.08 ppm (d, 1J=
37.4 Hz); UV/Vis (THF): l (e): 527 (62), 498 (21), 377 (8), 285 (33),
3
3
3
3
4
(d, J=8.4 Hz, 2H), 7.35 (dd, JC =8.1 Hz, JD =1.7 Hz, 1H), 7.68 (dd,
(35ꢃ10À3 LmolÀ1 cmÀ1);
ESI-MS:
m/z=553.3
3JC =8.1 Hz, JD =1.7 Hz, 1H), 8.01 (dd, JC =8.1 Hz, JD =1.7 Hz, 1H),
4
3
4
242 nm
[MÀC7H10N2ÀOTf]+, 675.3 [MÀOTf]+. HRMS (ESI): m/z calcd for
8.03 (d, 3J=7.7 Hz, 2H), 8.06 ppm (dd, 3JC =8.1 Hz, 4JD =1.7 Hz,
C39H45BF1N6O3+: 675.36315; found: 675.36018.
1H); 11B NMR (192.5 MHz, CDCl3): d=1.07 ppm (d, J=37.3 Hz); ESI-
1
MS: m/z=565.35 [MÀC7H10N2ÀOTf]+, 687.36 [MÀOTf]+; HRMS
(ESI): m/z calcd for C40H45BFN6OS+: 687.34541; found: 687.34293;
UV/Vis (THF): l (e): 528 (65), 498 (22), 380 (9), 285 (41), 274 (35),
243 (36), 224 nm (47ꢃ10À3 LmolÀ1 cmÀ1).
Fluorination of compound 3: 13,16,21,24-hexaoxa-1,10-diazabicy-
clo-(8,8,8)-hexacosane (23.0 mg, 61.0 mmol) and potassium fluoride
(4.00 mg, 69.0 mmol) were added to a solution of 50 mg of 3
(61 mmol) in acetonitrile (2.5 mL). The solution was heated to 708C
and the reaction was monitored by TLC. After 10 min, the solvent
was removed under reduced pressure. Water (5 mL) was added
and the mixture was extracted with dichloromethane (3ꢃ5 mL).
The organic layer was dried over magnesium sulfate and the sol-
vent was evaporated. The residue was purified by silica gel column
chromatography (dichloromethane/hexane, 70:30) followed by re-
crystallization in a mixture of dichloromethane and hexane to give
pure 2 as a red solid (17.0 mg, 50%).
Compound 7: Compound 6 (10.0 mg, 11.0 mmol) was dissolved in
DMF (600 mL) and propylamine (0.70 mL, 9.00 mmol) was added.
The mixture was stirred during one hour at room temperature and
solvent was removed under reduced pressure. The resulting solid
was purified by column chromatography on silica gel (dichlorome-
thane/methanol, 97:3) followed by a recrystallization in a mixture
of dichloromethane and hexane to give pure 7 (2.00 mg, 24%).
3
3
1H NMR (300 MHz, CDCl3): d=0.91 (t, JA =7.4 Hz, 3H), 0.91 (t, J=
3
3
7.5 Hz, 6H), 1.31 (s, 6H), 1.65 (qt, JA =7.4 Hz, JB =7.3 Hz, 2H), 2.15
(s, 6H), 2.22 (q, 3J=7.5 Hz, 4H), 2.96 (t, 3JC =6.8 Hz, 2H), 3.21 (s,
Compound 4: Compound 2 (300 mg, 524 mmol) was placed in
a mixture of dichloromethane and ethanol (45 mL, 1:1), and 10%
Pd/C (5.00 mg, 24.0 mmol) was added under H2. After total con-
sumption of the starting material (1 h) followed by TLC, the sus-
pension was eliminated by filtration on Clarcel and the solvent was
evaporated. The crude product was recrystallized in a mixture of
dichloromethane and hexane to give 4 as a red solid (277 mg,
3
3
3
6H), 3.57 (t, JB =7.3 Hz, 2H), 3.72 (td, JC =6.8 Hz, JD =5.7 Hz, 2H),
3
3
3
6.84 (d, J=7.3 Hz, 2H), 7.03 (t, JD =5.7 Hz, 1H), 7.22 (d, J=8.2 Hz,
2H), 7.34 (dd, JE =8.1 Hz, JF =1.4 Hz, 1H), 7.39 (d, J=8.2 Hz, 2H),
7.47 (dd, 3JE =8.1 Hz, 4JF =1.4 Hz, 1H), 7.78 (dd, 3JE =8.1 Hz, 4JF =
1.4 Hz, 1H), 7.96 (d, J=7.3 Hz, 2H), 8.09 ppm (dd, JE =8.1 Hz, JF =
1.4 Hz, 1H); 11B NMR (192.5 MHz, CDCl3): d=1.12 (d, 1J=36.2 Hz);
ESI-MS: m/z=624.14 [MÀC7H10N2ÀOTf]+, 746.22 [MÀOTf]+.
3
4
3
3
3
4
1
3
97%). H NMR (300 MHz, CDCl3): d=0.94 (t, J=7.5 Hz, 6H), 1.21 (s,
3
3
6H), 2.26 (q, J=7.5 Hz, 4H), 2.50 (s, 6H), 2.83 (t, JA =7.1 Hz, 2H),
3.69 (td, JA =7.1 Hz, JB =6.0 Hz, 2H), 6.36 (t, JB =6.0 Hz, 1H), 6.65
(d, J=8.3 Hz, 2H), 7.02 (d, J=8.3 Hz, 2H), 7.31 (d, J=8.2 Hz, 2H),
7.81 ppm (d, J=8.2 Hz, 2H); 13C{1H} NMR (75 MHz, CDCl3): d=12.1
3
3
3
Compound 8: N-Hydroxybenzotriazole (959 mg, 7.10 mmol), diiso-
propylamine (995 mL, 7.10 mmol), 1-(3-dimethylaminopropyl)-3-eth-
ylcarbodiimide hydrochloride (1.35 g, 7.10 mmol), and propargyla-
mine (227 mL, 3.50 mmol) were successively added to a solution of
compound 1 (1.53 g, 3.50 mmol) in dimethylformamide (100 mL)
and the solution was stirred at room temperature. After total con-
sumption of starting material (2 h) monitored by TLC, the solvent
was evaporated. The resulting solid was washed with water (3ꢃ
100 mL) and extracted with dichloromethane. The organic layer
was dried over magnesium sulfate and the solvent was evaporated
to give a red oil. The crude product was purified by column chro-
matography on silica gel (dichloromethane/heptane, 60:40) fol-
lowed by a recrystallization in a mixture of dichloromethane and
hexane to give 8 as reddish solid in 72% yield (1.15 g, 2.49 mmol).
3
3
3
3
(ꢃ2), 12.8 (ꢃ2), 14.8 (ꢃ2), 17.3 (ꢃ2), 35.0, 41.7, 115.7 (ꢃ2), 127.8 (ꢃ
2), 128.7 (ꢃ2), 129.0 (ꢃ2), 129.9, 130.6, 133.3, 135.2 (ꢃ2), 138.4 (ꢃ
2), 139.0, 139.4 (ꢃ2), 145.2, 154.4 (ꢃ2), 166.8 ppm; 11B NMR
(192.5 MHz, CDCl3): d=0.79 ppm (t, 1J=33.4 Hz); ESI-MS: m/z=
523.3 [MÀF]+, 545.3 [MÀHÀF+Na]+, 565.3 [M+Na]+; HRMS (ESI):
m/z calcd for C32H37BF2N4ONa+: 565.29263; found: 565.28996.
Compound 5: TMSOTf (75.0 mL, 412 mmol) and DMAP (128 mg,
412 mmol) were added to a solution of 4 (112 mg, 206 mmol) in dry
toluene (7 mL), and the mixture was stirred for 30 min at 808C. The
solvent was evaporated, the residue was washed with water (3ꢃ
20 mL) and dried over magnesium sulfate. The solution was con-
centrated to dryness and the residue was purified by silica gel
3
1H NMR (300 MHz, CDCl3): d=0.96 (t, J=7.5 Hz, 6H), 1.22 (s, 6H),
Chem. Eur. J. 2014, 20, 1 – 13
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2.29 (q, J=7.5 Hz, 4H), 2.30 (t, J=2.5 Hz, 1H), 2.51 (s, 6H), 3.78
0.95 (t, 6H, J=7.5 Hz), 1.21 (s, 6H), 2.27 (q, J=7.5 Hz, 4H), 2.51 (s,
4
3
3
3
3
3
(dd, J=2.5 Hz, J=5.2 Hz, 2H), 6.27 (t, J=5.2 Hz, 1H), 7.38 (d, J=
8.4 Hz, 2H), 7.91 ppm (d, 3J=8.4 Hz, 2H); 13C{1H} NMR (75 MHz,
CDCl3): d=11.9, 12.5, 14.6, 17.1, 30.9, 72.1, 79.3, 127.8, 128.9, 130.4,
133.1, 134.0, 138.1, 138.5, 139.7, 154.3, 166.2 ppm; 11B NMR
6H), 4.70 (d, J=5.4 Hz, 2H), 5.50 (s, 2H), 7.20 (t, J=5.4 Hz, 1H),
3
3
7.27 (d, J=8.3 Hz, 2H), 7.34 (m, 5H), 7.56 (s, 1H), 7.91 ppm (d, J=
8.3 Hz, 2H); 13C{1H} NMR (75 MHz, CDCl3): d=11.9, 12.5, 14.6, 17.0,
35.5, 54.3, 77.2, 122.3, 127.8, 128.2, 128.8, 128.9, 129.2, 130.4, 133.0,
134.4, 138.1, 138.7, 139.5, 144.7, 144.8, 154.2, 166.7 ppm; 11B NMR
(192.5 MHz, CDCl3): d=0.77 ppm (t, 1J=67.0 Hz); ESI-MS: m/z=
595.12 [M+H]+, 617.08 [M+Na]+; HRMS (ESI): m/z calcd for
C34H37BF2N6ONa+: 617.29881; found: 617.29663,.
1
(192.5 MHz, CDCl3): d=0.78 ppm (t, J=33.4 Hz).UV/Vis (THF): lmax
:
(e): 238 (39), 494 (27), 525 nm (90ꢃ10À3 LmolÀ1 cmÀ1); MS (ESI): m/
z=460.39 [M+H]+, 484.34 [M+Na]+; HRMS (ESI): m/z calcd for
C27H30BF2N3ONa: 484.23470; found: 484.23559.
Compound 12: 20.6 mL of a 100 mm solution of N3-BBN (DMF,
2.10 mmol), 20.6 mL of a 100 mm solution of compound 8 (DMF,
2.10 mmol), 11.3 mL of a 200 mm solution of CuSO4·5H2O (DMF,
2.30 mmol), and sodium ascorbate (1.23 mg, 6.20 mmol) were mixed
together. The mixture was stirred at room temperature for 1 h and
reaction was monitored by HPLC. The crude product was purified
by HPLC from 30 to 60% of B over 30 min to give 12 as a red solid
(1.21 mg, 31%, purity=98.6%, tR =2.89 min). ESI-MS: m/z=
1552.77 [MÀC7H10N2ÀOTfÀTFA]+, 1674.78 [MÀOTfÀTFA]+; HRMS
(ESI): m/z calcd for C83H114BFN21O13S+: 1674.871021; found:
674.86882.
Compound 9: Trimethylsilyl trifluoromethanesulfonate (TMSOTf,
86.0 mL, 480 mmol) and dimethylaminopyridine (DMAP, 58.0 mg,
480 mmol) were added to a solution of 8 (110 mg, 238 mmol) in dry
toluene (10 mL), and the mixture was stirred for 30 min at 808C.
The solvent was evaporated, the residue was washed with water
(3ꢃ20 mL) and dried over magnesium sulfate. The solution was
concentrated to dryness and the residue was purified by silica gel
column chromatography (CH2Cl2/MeOH, 99:1) to give 9 as a red
solid (118 mg, 69%). 1H NMR (300 MHz, CDCl3): d=0.89 (t, 3J=
3
7.5 Hz, 6H), 1.30 (s, 6H), 2.20 (s, 6H), 2.12 (q, J=7.5 Hz, 4H), 2.22
4
4
3
(t, JA =2.5 Hz, 1H), 3.21 (s, 6H), 4.25 (dd, JA =2.5 Hz, JB =5.4 Hz,
3
3
4
2H), 6.89 (d, J=7.6 Hz, 2H), 7.36 (dd, JC =8.5 Hz, JD =1.8 Hz, 1H),
Fluorination of compound 12: 5.60 mL of a 0.03m solution of 12
(CH3CN/DMF (1:1), 0.17 mmol), 5.60 mL of a 0.03m solution of
13,16,21,24-hexaoxa-1,10-diazabicyclo-(8,8,8)-hexacosane (CH3CN,
0.17 mmol), and 0.01 mg of potassium fluoride (0.17 mmol) were
mixed together. The solution was heated at 708C during 40 min
and monitored by HPLC. The formation of compound 13 was char-
acterized by ESI-MS and HPLC (Conversion=42% after 10 min of
reaction).
3
3
4
7.48 (t, JB =5.4 Hz, 1H), 7.61 (dd, JC =8.5 Hz, JD =1.8 Hz, 1H), 7.98
3
3
4
(d, J=7.6 Hz, 2H), 8.11 (dd, JC =8.5 Hz, JD =1.8 Hz, 1H), 8.12 ppm
(dd, JC =8.5 Hz, JD =1.8 Hz, 1H); 13C{1H} NMR (75 MHz, CDCl3): d=
12.4 (ꢃ2), 12.8 (ꢃ2), 14.5 (ꢃ2), 17.1 (ꢃ2), 29.9, 40.2 (ꢃ2), 71.4, 80.0,
107.8–119.8–122.3–128.4–128.7–128.8–129.0–131.0–134.9–137.8–
140.8–141.9–154.4–156.8 (ꢃ14), 166.5 ppm; 11B NMR (192.5 MHz,
3
4
1
CDCl3): d=1.07 ppm (d, J=36.1 Hz); UV/Vis (THF): l (e): 528 (68),
498 (23), 381 (8), 287 (26), 244 nm (33 mÀ1 cmÀ1); ESI-MS: m/z=
442.4 [MÀC7H10N2ÀOTf]+, 564.4 [MÀOTf]+; HRMS (ESI): m/z calcu-
lated for C34H40BFN5O+: 564.33104; found: 564.32907.
Compound 13: 14.0 mL of a 100 mm solution of N3-BBN (DMF,
1.40 mmol), 14.0 mL of a 100 mm solution of compound 8 (DMF,
1.40 mmol), 7.7 mL of a 200 mm solution of CuSO4·5H2O (DMF,
1.54 mmol), and 0.83 mg of sodium ascorbate (4.19 mmol) were
mixed together. The mixture was stirred at room temperature for
1 h and reaction was monitored by HPLC. The crude product was
purified by HPLC from 30 to 60% of B over 30 min to give 13 as
a red solid (0.955 mg, 41%, purity=98.4%, tR =3.23 min). ESI-MS:
m/z: 1572.80 [MÀTFA+H]+, 1594.77 [MÀTFA+Na]+; HRMS (ESI):
m/z calcd for C76H105BF2N19O13S+: 1572.792111; found: 572.9795; m/
z calcd for C76H104BF2N19O13SNa+: 1594.774711; found: 594.77718.
Compound 10: Benzyl azide (11.0 mL, 84.0 mmol) and compound 8
(60.0 mg, 84.0 mmol) were solubilized in DMF (1.5 mL). CuSO4·5H2O
(21.0 mg, 84.0 mmol) and sodium ascorbate (33.0 mg, 117 mmol)
were added. The mixture was stirred under nitrogen at room tem-
perature for 1 h and the solvent was evaporated under reduced
pressure. The crude mixture was dissolved in dichloromethane and
filtered over Celite. The organic layer was washed with water (3ꢃ
20 mL), dried over magnesium sulfate and the solvent was re-
moved under reduced pressure. The residue was purified by silica
gel column chromatography (dichloromethane/MeOH, 99:1) fol-
lowed by recrystallization in a mixture of dichloromethane and
Compound 15: Compound 8 (500 mg, 1.08 mmol) and p-anisalde-
hyde (316 mL, 2.59 mmol) were dissolved in a mixture of dry tolu-
ene (40 mL), p-toluenesulfonic acid (PTSA, 40.0 mg, 0.23 mmol) and
piperidine (4 mL). The mixture was heated at reflux during 2 h in
a Dean–Stark apparatus and the solvent was removed in situ. Dry
toluene (40 mL) and piperidine (4 mL) were added, and the mix-
ture was heated at reflux for another 2 h. After total consumption
of the starting material (monitored by UV/Vis), the solvent was
evaporated. The resulting solid was washed with water (3ꢃ
100 mL) and extracted with dichloromethane. The organic layer
was dried over magnesium sulfate and the solvent was evaporated
to give a blue solid. The crude product was purified by column
chromatography on silica gel (heptane/dichloromethane, 80:20)
followed by a recrystallization in a mixture of dichloromethane and
1
hexane to give 15 as a red solid (36.0 mg, 51%). H NMR (300 MHz,
3
CDCl3): d=0.91 (t, 6H, J=7.5 Hz), 1.30 (s, 6H), 2.12 (s, 6H), 2.21 (q,
3
3J=7.5 Hz, 4H), 3.22 (s, 6H), 4.74 (d, J=5.2 Hz, 2H), 5.51 (s, 2H),
3
3
6.92 (d, J=7.5 Hz, 2H), 7.29 (d, J=7.2 Hz, 1H), 7.32 (m, 5H), 7.34
3
3
3
(t, J=5.2 Hz, 1H), 7.62 (s, 1H), 7.68 (d, J=7.2 Hz, 1H), 8.00 (d, J=
3
3
7.5 Hz, 2H), 8.01 (d, J=7.2 Hz, 1H), 8.03 ppm (d, J=7.2 Hz, 1H);
11B NMR (192.5 MHz, CDCl3): d=1.05 ppm (d, J=39.5 Hz); ESI-MS:
1
m/z=575.14 [MÀC7H10N2ÀOTf]+, 697.22 [MÀOTf]+; HRMS (ESI): m/
z calcd for C41H47ON8BF+: 697.39515; found: 697.39581.
Compound 11: 13,16,21,24-hexaoxa-1,10-diazabicyclo-(8,8,8)-hexa-
cosane (8.90 mg, 23.6 mmol), and potassium fluoride (1.40 mg,
23.6 mmol) were added to a solution of 10 (20.0 mg, 23.6 mmol) in
acetonitrile (1.5 mL). The solution was heated at 708C and the re-
action was monitored by TLC. After 15 min, the solvent was re-
moved under reduced pressure. Water (3 mL) was added and the
mixture was extracted with dichloromethane (3ꢃ3 mL). The organ-
ic layer was dried over magnesium sulfate and the solvent was
evaporated. The residue was purified by silica gel column chroma-
tography (dichloromethane/hexane, 80:20) followed by recrystalli-
zation in a mixture of dichloromethane and hexane to give pure
11 as a red solid (6.00 mg, 42%). 1H NMR (300 MHz, CDCl3): d=
hexane to give 15 as
a blue solid in 79% yield (600 mg,
0.86 mmol). 1H NMR (300 MHz, CDCl3): d=1.14 (t, 3J=7.5 Hz, 6H),
4
3
1.28 (s, 6H), 2.32 (t, J=2.5 Hz, 1H), 2.58 (q, J=7.5 Hz, 4H), 3.84 (s,
6H), 4.30 (dd, 4J=2.5 Hz, 3J=5.2 Hz, 2H), 6.36 (t, 3J=5.2 Hz, 1H),
6.93 (d, 3J=8.7 Hz, 4H), 7.20 (d, 3J=16.7 Hz, 2H), 7.43, (d, 3J=
8.3 Hz, 2H), 7.56 (d, 3J=8.7 Hz, 4H), 7.65 (d, 3J=16.7 Hz, 2H),
3
7.93 ppm (d, J=8.3 Hz, 2H); 13C{1H} NMR (75 MHz, CDCl3): d=11.8,
14.1, 18.4, 55.4, 72.2, 79.3, 114.3, 127.8, 128.9, 129.4, 130.3, 132.5,
133.9, 134.1, 135.8, 136.1, 138.3, 140.1, 150.9, 160.3, 166.3 ppm;
11B NMR (192.5 MHz, CDCl3): d=1.23 ppm (t, 1J=34.2 Hz); UV/Vis
&
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Chem. Eur. J. 2014, 20, 1 – 13
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10
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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(THF): lmax (e): 250 (21), 334 (31), 368 (68), 608 (37), 654 nm (93ꢃ
10À3 LmolÀ1 cmÀ1); MS (ESI): m/z=720.35 [M+Na]+; HRMS (ESI): m/
z calcd for C43H42BF2N3O2Na: 720.31869; found: 720.32039.
The crude product was purified by column chromatography on
silica gel (dichloromethane/heptane, 40:60) followed by a recrystal-
lization in a mixture of dichloromethane and hexane to give 19
(73.0 mg, 125 mmol) in 5% yield. Distyryl-substituted BODIPY was
Compound 16: TMSOTf (60.0 mL, 330 mmol) and DMAP (4.0 mg,
330 mmol) were added to a solution of 15 (115 mg, 165 mmol) in
dry toluene (10 mL), and the mixture was stirred for 30 min at
808C. The solvent was evaporated, the residue was washed with
water (3ꢃ20 mL) and dried over magnesium sulfate. The solution
was concentrated to dryness and the residue was purified by silica
gel column chromatography (dichloromethane/MeOH, 97:3) to
give 16 as a red solid (103 mg, 66%). 1H NMR (300 MHz, CDCl3):
d=1.06 (t, 3J=7.5 Hz, 6H), 1.36 (s, 6H), 2.29 (t, 4JA =2.5 Hz, 1H),
2.46 (q, 3J=7.5 Hz, 4H), 3.01 (s, 6H), 3.85 (s, 6H), 4.30 (dd, 4JA =
2.5 Hz, 3JB =5.1 Hz, 2H), 6.66 (d, 3J=6.4 Hz, 2H), 6.85 (d, 3J=
1
also obtained as subproduct in 3% yield. H NMR (300 MHz, CDCl3):
3
3
d=(ppm): d=0.97 (t, J=7.5 Hz, 3H), 1.12 (t, J=7.5 Hz, 3H), 1.25
3
4
(s, 3H), 1.27 (s, 3H), 2.29 (q, J=7.5 Hz, 2H), 2.52 (t, J=2.4 Hz, 1H),
2.56 (s, 3H), 2.56 (q, 3J=7.5 Hz, 2H), 3.97 (s, 3H), 4.71 (d, 4J=
2.4 Hz, 2H), 6.97 (d, J=8.8 Hz, 2H), 7.16 (d, J=16.8 Hz, 1H), 7.41
(d, 3J=8.3 Hz, 2H), 7.54 (d, 3J=8.8 Hz, 2H), 7.60 (d, 3J=16.8 Hz,
1H), 8.16 ppm (d, J=8.3 Hz, 2H); 13C{1H} NMR (75 MHz, CDCl3): d=
3
3
3
11.8, 12.1, 13.0, 14.3, 14.7, 17.3, 17.5, 18.5, 52.6, 56.1, 75.9, 78.6,
115.4, 118.6, 128.8, 129.1, 130.5, 130.9, 131.3, 131.6, 133.4, 134.0,
135.1, 138.0, 141.1, 150.0, 155.8, 158.2, 166.8 ppm; UV/Vis (THF):
lmax: (e) 238 (19), 319 (17), 339 (29), 389 (8), 448 (27), 587 nm (81ꢃ
10À3 LmolÀ1 cmÀ1); 11B NMR (192.5 MHz, CDCl3): d=0.98 ppm (t,
1J=33.8 Hz); MS (ESI): m/z=603.4 [M+Na]+; HRMS (ESI): m/z calcd
for C35H35BF2N2O3Na: 603.26071; found: 603.26233.
3
3
16.7 Hz, 2H), 6.94 (d, J=8.6 Hz, 4H), 7.04 (t, JB =5.1 Hz, 1H), 7.05
(d, 3J=16.7 Hz, 2H), 7.37 (d, 3J=8.6 Hz, 4H), 7.47 (d, 3J=7.9 Hz,
3
3
3
1H), 7.77 (d, J=7.0 Hz, 1H), 7.89 (d, J=6.4 Hz, 2H), 8.08 (d, J=
7.0 Hz, 1H), 8.17 ppm (d, 3J=7.9 Hz, 1H); 11B NMR (192.5 MHz,
1
Compound 20: TMSOTf (22.0 mL, 120 mmol) and DMAP (15.0 mg,
120 mmol) were added to a solution of 19 (35.0 mg, 60.0 mmol) in
dry toluene (2 mL), and the mixture was stirred for 30 min at 808C.
The solvent was evaporated, the residue was washed with water
(3ꢃ10 mL) and dried over magnesium sulfate. The solution was
concentrated to dryness and the residue was purified by silica gel
column chromatography (CH2Cl2/MeOH, 98:2) to give 20 as
CDCl3): d=1.45 ppm (d, J=35.5 Hz); UV/Vis (THF): l (e): 659 (59),
440 (14), 376 (39), 292 nm (33 mÀ1 cmÀ1); ESI-MS: m/z=678.4
[MÀC7H10N2ÀOTf]+, 800.4 [MÀOTf]+; HRMS (ESI): m/z calcd for
C50H52BFN5O3+: 800.41502; found: 800.41267.
Compound 17: 5.20 mL of a 100 mm solution of N3-BBN (DMF,
0.52 mmol), 5.20 mL of a 100 mm solution of compound 16 (DMF,
0.52 mmol), 2.80 mL of a 200 mm solution of CuSO4·5H2O (DMF,
0.57 mmol), and 0.30 mg of sodium ascorbate (1.51 mmol) were
mixed together. The mixture was stirred at room temperature for
1 h and the reaction was monitored by HPLC. The crude product
was purified by HPLC from 40 to 70% of B over 30 min to give 17
as a red solid (conversion (HPLC)=41%, purity=98.4%, tR =
3.57 min). Conversion was determined by relative integration of
the chromatogram of the crude compound, at 254 nm; ESI-MS: m/
z=1788.79 [MÀC7H10N2ÀOTfÀTFA]+; 1910.85 [MÀOTfÀTFA]+;
HRMS (ESI): m/z calcd for C99H126BFN21O15S+: 1910.95495; found:
1910.96003,.
1
a purple solid (36.0 mg, 50%). H NMR (300 MHz, CDCl3): d=0.94 (t,
3J=7.5 Hz, 6H), 1.05 (t, 3J=7.5 Hz, 6H), 1.34 (s, 6H), 1.36 (s, 6H),
2.15 (s, 3H), 2.24 (q, 3J=7.5 Hz, 2H), 2.43 (qd, 3JA =7.3, 1JB =
12.2 Hz, 1H), 2.45 (qd, 3JA =7.3 Hz, 1JB =12.2 Hz, 1H), 2.54 (t, 4J=
2.5 Hz, 1H), 3.01 (s, 6H), 3.85 (s, 6H), 3.97 (s, 3H), 4.74 (d, 4J=
3
3
2.5 Hz, 2H), 6.78 (d, J=7.6 Hz, 2H), 6.79 (d, J=16.7 Hz, 1H), 7.01
(d, 3J=8.9 Hz, 2H), 7.03 (d, 3J=16.7 Hz, 1H), 7.37 (d, 3J=8.9 Hz,
2H), 7.40 (dd, 3JC =7.9 Hz, 4JD =1.5 Hz, 1H), 7.78 (dd, 3JC =7.9 Hz,
3
4JD =1.5 Hz, 1H), 7.94 (d, 3J=7.6 Hz, 2H), 8.20 (dd, JC =7.9 Hz, 4JD =
1.5 Hz, 1H), 8.28 ppm (dd, 3JC =7.9 Hz, 4JD =1.5 Hz, 1H); 11B NMR
(192.5 MHz, CDCl3): d=1.18 ppm (d, 1J=37.9 Hz); UV/Vis (THF)
Compound 18: 2,4-Dimethylethylpyrrole (7.50 g, 60.9 mmol) and
methyl 4-formylbenzoate (5.00 g, 30.5 mmol) were dissolved in di-
chloromethane (2 L). Trifluoroacetic acid (140 mL) was added and
the mixture was stirred at room temperature for 24 h. A solution of
2,3-dichloro-5,6-dicyano-p-benzoquinone (6.90 g, 30.5 mmol) in di-
chloromethane was added to the mixture and the solution was fur-
ther stirred for 50 min, triethylamine (60 mL) and BF3·Et2O (60 mL)
were added, the reaction mixture turned purple. After stirring
during 1 h, the reaction mixture was washed with water, dried over
magnesium sulfate and the solvent was evaporated. The residue
was purified by silica gel column chromatography (dichlorome-
thane/hexane 40:60) followed by recrystallization in a mixture of
dichloromethane and hexane to give pure 18 as a red solid (6.00 g,
l
(e): 594 (56), 402 (9), 345 (18), 291 (25), 242 nm (20ꢃ
10À3 MÀ1 cmÀ1); ESI-MS: m/z=561.4 [MÀC7H10N2ÀOTf]+, 683.4
[MÀOTf]+; HRMS (ESI): m/z calcd for C42H45BFN4O3+: 683.35705;
found: 683.35465.
Compound 21: 5.20 mL of a 100 mm solution of N3-BBN (DMF,
0.52 mmol), 5.20 mL of a 100 mm solution of compound 20 (DMF,
0.52 mmol), 2.80 mL of a 200 mm solution of CuSO4·5H2O (DMF,
0.57 mmol), and sodium ascorbate (0.30 mg, 1.51 mmol) were mixed
together. The mixture was stirred at room temperature for 1 h and
the reaction was monitored by HPLC. The crude product was puri-
fied by HPLC from 40 to 70% of B over 30 min to give 21 as a red
solid (conversion (HPLC)=59%, purity=95.4%, tR =3.37 min). ESI-
MS: m/z: 1671.78 [MÀC7H10N2ÀOTfÀTFA]+, 1793.76 [MÀOTfÀT-
FA]+; HRMS (ESI): m/z calcd for C91H119BFN20O15S+: 1793.89700;
found: 1793.90512.
1
3
45%). H NMR (300 MHz, CDCl3): d=0.90 (t, J=7.6 Hz, 6H), 1.18 (s,
3
3
6H), 2.22 (q, J=7.6 Hz, 4H), 2.46 (s, 6H), 3.90 (s, 3H), 7.33 (d, J=
3
8.4 Hz, 2H), 8.10 ppm (d, J=8.4 Hz, 2H).
Compound 19: Compound 18 (1.10 g, 2.51 mmol) and 4-(prop-2-
yn-1-yloxy)benzaldehyde (442 mg, 2.76 mmol) were dissolved in
a mixture of dry toluene (50 mL), PTSA (53.0 mg, 0.31 mmol), and Acknowledgements
piperidine (3.30 mL, 33.0 mmol). The mixture was heated at reflux
during 2 h in a Dean–Stark apparatus and the solvent was re-
The Ministꢄre de l’Enseignement et de la Recherche, the Uni-
moved in situ. Dry toluene (50 mL) and piperidine (3 mL) were
added and the mixture was heated at reflux for another 2 h. After
consumption of approximately a third of starting material (moni-
tored by UV/Vis spectroscopy), the solvent was evaporated. The re-
sulting solid was washed with water (3ꢃ100 mL) and extracted
with dichloromethane. The organic layer was dried over magnesi-
um sulfate and the solvent was evaporated to give a blue solid.
versitꢂ de Bourgogne and the Conseil Rꢂgional de Bourgogne
through the 3MIM Project are acknowledged. P.D.H. thanks the
Natural Sciences and Engineering Research Council for funding.
B.B. thanks the Ministꢄre de l’Enseignement supꢂrieur et de la
Recherche for Ph.D. grants. Yoann Rousselin is warmly ac-
knowledged for the determination of the X-ray structure of
Chem. Eur. J. 2014, 20, 1 – 13
www.chemeurj.org
11
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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&
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Keywords: alkynes · click chemistry · fluorine · imaging
agents · luminescence · radiochemistry
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Published online on && &&, 2014
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Chem. Eur. J. 2014, 20, 1 – 13
www.chemeurj.org
12
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
FULL PAPER
Probing further: Different boron dipyr-
romethene/N,N-dimethylaminopyridine
(BODIPY-DMAP) compounds were syn-
thesized as precursors for positron emis-
sion tomography (PET)-optical imaging
(see figure). Biolabeling was performed
on a bombesin derivative, while pre-
serving the DMAP leaving group on the
boron atom and the affinity for bombe-
sin receptors. Extension of the conjuga-
tion on the BODIPY core was also inves-
tigated, for future in vivo imaging appli-
cations.
&
Imaging Agents
B. Brizet, V. Goncalves, C. Bernhard,
P. D. Harvey, F. Denat,* C. Goze*
&& – &&
DMAP-BODIPY Alkynes: A Convenient
Tool for Labeling Biomolecules for
Bimodal PET–Optical Imaging
Chem. Eur. J. 2014, 20, 1 – 13
www.chemeurj.org
13
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ