First bodipy-DOTA derivatives as probes for bimodal imaging

Article abstract of DOI:10.1039/c0cc02749a

The synthesis and the photophysical studies of the first bodipy-DOTA and its In(iii), Ga(iii) and Cu(ii) complexes are reported. The introduction of an isothiocyanate handle generates a new bimodal imaging agent capable of both optical and nuclear imaging

Full text of DOI:10.1039/c0cc02749a

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First bodipy–DOTA derivatives as probes for bimodal imagingw
Claire Bernhard, Christine Goze, Yoann Rousselin and Franck Denat*
Received 23rd July 2010, Accepted 1st September 2010
DOI: 10.1039/c0cc02749a
The synthesis and the photophysical studies of the first
bodipy–DOTA and its In(III), Ga(III) and Cu(II) complexes are
reported. The introduction of an isothiocyanate handle generates
a new bimodal imaging agent capable of both optical and nuclear
imaging.
for both diagnosis (SPECT or PET) and therapeutic
purposes.8
Among fluorescent probes, bodipy derivatives represent a
promising family due to their exceptional properties.
They exhibit high stability, high extinction coefficients, sharp
emission bands and high quantum yields.9 Bodipys are
involved in many applications such as donor–acceptor
systems, artificial light harvesters, fluorescent sensors, laser
dyes, electron-transfer reagents and light emitting devices.10
Despite all the advantages of this dye, bimodal imaging agents
containing a bodipy moiety have never been synthesized and
studied. This is probably due to one major drawback which
has to be solved before involving the bodipy moiety in bimodal
imaging systems. Indeed, most bodipys suffer from intrinsic
hydrophobicity that limits their usefulness as labels for
biomolecules. While a few teams are working on the synthesis
of hydrophilic bodipys, this interesting field remains largely
unexplored.11
Multimodal imaging agents can provide complementary
information improving the accuracy of disease diagnosis and
enhancing patient management.1 Many clinical and preclinical
imaging applications are based on positron emission
tomography (PET), single-photon emission computed tomography
(SPECT), fluorescence imaging, bioluminescence, magnetic
resonance imaging (MRI), and ultrasound. Each imaging
modality has its own strengths and weaknesses, and thus,
combining different and complementary systems can over-
come inherent limitations associated with any one individual
technique. In particular dual-modality optical/nuclear imaging
may find important preclinical and clinical applications. For
example, the radionuclide component can provide early
quantitative data in a target tissue, and subsequent events
can be monitored longitudinally by an optical method. In
intraoperative procedure, diseased tissues can be localized by
nuclear imaging (ex: PET/CT) which are then biopsied for
histologic validation by an optical method. Recently, elegant
molecular designs have been developed to take full advantage
of the unique high detection sensitivity of both optical and
nuclear imaging methods. One possible approach seeks to fuse
the two imaging systems into one molecule (MonOmolecular
Multimodality Imaging Agent [MOMIA])2 in order to ensure
the same biodistribution of the two probes.
In this communication, we report the synthesis of new
bodipy DOTA derivatives. Interestingly, the coupling of the
azamacrocycle to the bodipy induced the complete solubilization
of the molecule in water. The bodipy–DOTA derivative was
then metallated with Cu(II), In(III) and Ga(III). Complexation
of In(III) and Ga(III) didn’t affect the fluorescence of the bodipy
component of the molecule, while Cu(II) partially quenched
the fluorescence of the ligand.
Bodipy 1 containing an activated ester12 and ethylene
diamine-functionalized cyclen 213 were synthesized by adapting
literature procedures (ESIw). Deprotection of the tert-butyl
ester groups in 2 to give cyclen derivative 3 followed by
coupling to the activated bodipy 1 (Scheme 1) afforded the
desired water-soluble DOTA–bodipy 4. Here the order of
deprotection followed by coupling was essential to synthetic
success. Indeed, the boron part of the bodipy didn’t survive the
tert-butyl ester group deprotection conditions. The synthesis
of the In(III), Ga(III) and Cu(II) complexes was achieved by
addition of one equivalent of the metal salt to bodipy–DOTA
4 (Scheme 2). The three neutral complexes are soluble in water.
All compounds present a strong absorption transition in the
522–527 nm range, which can be attributed to S0–S1 transition
of the bodipy (Table 1).
Despite the high sensitivity and complementary nature of
radionuclear and optical methods, combinations of these two
imaging modalities are rare.3 This is attributable to a variety of
reasons, one of them being the difficulty to synthesize and
characterize species capable of this type of bimodal imaging.4
DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid) is one of the most important chelators in radiochemistry
as it forms very stable complexes with a large number of metal
ions. It thus finds wide application in medical imaging.5 This
azamacrocycle is commonly used for in vivo applications such
as cancer therapy and clinical diagnosis.6 More precisely, the
properties of DOTA have been explored as MRI contrast
agents,7 or for labeling biomolecules using metal radioisotopes
The molar absorption coefficients (e) for this transition are
relatively high, in the 30 000–75 000 Mꢀ1 cmꢀ1 range, and are
strongly dependent on the solvent. A second broader and
weaker band at around 375 nm can be attributed to the
S0–S2 transition of the bodipy fragment. All the compounds
exhibit the typical emission features of bodipy, that is, narrow,
slightly Stokes shifted bands of mirror image shape, and
relatively high quantum yields. In all compounds, the excitation
spectrum of the emissive species matches the absorption,
confirming that the emission comes only from the S1 energy
´
´
Institut de Chimie Moleculaire de l’Universite de Bourgogne,
UMR CNRS 5260, Univ. Bourgogne, 9 Avenue Alain Savary,
21078 Dijon Cedex, France. E-mail: Franck.Denat@u-bourgogne.fr;
Fax: +33-3-80396117; Tel: +33-3-80396115
w Electronic supplementary information (ESI) available: Synthetic
procedures, crystal and photophysical data. CCDC 785489. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c0cc02749a
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 8267–8269 8267

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Scheme 1
Scheme 2
Table 1 Spectroscopic data of bodipy–DOTA derivatives in several
solvents at 298 K
redox active Cu(II) cation. However, the quantum yield of
the Cu(II) complex in water remains 21%, which is still
reasonably high for fluorescent probes.
Bodipy Solvent
l l
abs/nm e/Mꢀ1 cmꢀ1 em/nm Dn/cmꢀ1 Ff (%)a,b
With the model bimodal imaging agent 5–7 in hand we
sought to add a handle that could be used to conjugate the
bodipy–DOTA derivatives to a biorelevant species of interest,
such as an antibody or a peptide. The isothiocyanate
functional group was chosen for this purpose due to its
synthetic accessibility and more importantly its proven track
record in biomolecule conjugation. For instance, the isothio-
cyanate group reacts with free N-terminal lysine residues
under mild conditions forming a thiourea linker.16 Bodipy
1
4
MeCN 525
MeCN 522
MeOH 524
73 000
31 000
59 500
31 000
35 000
32 000
36 000
34 000
75 000
30 400
60 300
542
536
536
536
538
540
538
540
538
539
536
597
400
427
427
460
457
460
602
460
567
391
64
42
58
50
65
48
63
50
61
21
23
H2O
DMF 525
H2O 527
DMF 525
H2O 523
DMF 525
H2O 523
524
5
6
7
DMF 525
a
b
lexc = 488 nm. Using Rhodamine 6G as reference, Ff = 0.78 in
water, lexc=488 nm.14 All Ff are corrected for changes in refractive
index.
state of the bodipy system in the different molecules. For all
molecules, the Stokes shifts (Dn = 534–542 cmꢀ1) are in
good agreement with those reported elsewhere for meso-aryl
substituted bodipy dyes.15
Bodipy–DOTA 4 presents a quantum yield of 50% in water.
The fluorescence is not perturbed upon complexation of
Ga(III) and In(III) which fit well in the cavity of DOTA ligands
(Fig. 1). Fluorescence of the Cu(II) complex 7 is only 50% as
intense as the non-metalated ligand DOTA–bodipy 4. This
quenching is probably due to a partial PET (photoinduced
electron transfer) mechanism between the bodipy and the
Fig. 1 Emission spectra of the bodipy–DOTA 4 and its corresponding
complexes in water at 298 K.
c
8268 Chem. Commun., 2010, 46, 8267–8269
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Notes and references
z The macrocycle DOTA NHS ester was obtained from CheMatech
(www.chematech-mdt.com).
1 J. R. Allport and R. Weissleder, Exp. Hematol., 2001, 29,
1237–1246; P. Sabbah, H. Foerenbach, G. Dutertre, C. Nioche
and O. DeBreuille, Clinical Imaging, 2002, 26, 6–12.
2 C. Li, W. Wang, Q. Wu, S. Ke, J. Houston, E. Sevick-Muraca,
L. Dong, D. Chow, C. Charnsangavej and J. G. Gelovani, Nucl.
Med. Biol., 2006, 33, 349–358.
3 H. Lee, W. J. Akers, P. P. Cheney, W. Barry Edwards, K. Liang,
J. P. Culver and S. Achilefu, J. Biomed. Opt., 2009, 14(4),
0405071–0405073; W. Wang, S. Ke, S. Kwon, S. Yallampalli,
A. G. Cameron, K. E. Adams, M. E. Mawad and E. M. Sevick-
Muraca, Bioconjugate Chem., 2007, 18, 397–402.
4 J. Culver, W. Akers and S. Achilefu, J. Nucl. Med., 2008, 49, 169–172.
5 N. Viola-Villegas and R. P. Doyle, Coord. Chem. Rev., 2009, 253,
1906–1925.
6 A. Otte, E. Jermann, M. Behe, M. Goetze, H. C. Bucher, H. W. Roser,
A. Heppeler, J. Mueller-Brand and H. R. Maecke, Eur. J. Nucl. Med.,
1997, 24, 792–795; M. de Jong, W. H. Bakker, E. P. Krenning, W. A.
P. Breeman, M. E. van der Plujim, B. F. Bernard, T. J. Visser,
´ ´
E. Jermann, M. Behe, P. Powell and H. R. Maecke, Eur. J. Nucl.
Med. Mol. Imaging, 1997, 24, 368–371; R. E. Mewis and
S. J. Archibald, Coord. Chem. Rev., 2010, 254, 1686–1712.
7 P. Caravan, Chem. Soc. Rev., 2006, 35, 512–523; P. Caravan,
J. J. Ellison, T. J. Mc Murry and R. B. Lauffer, Chem. Rev.,
1999, 99, 2293–2352; P. Hermann, J. Kotek, V. Kubicek and
I. Lukes, Dalton Trans., 2008, 3027–3047; A. E. Merbach and
E. Toth, The Chemistry of Contrast Agents in Medical Magnetic
Resonance Imaging, Wiley, Chichester, 2001.
Fig. 2 ORTEP view of compound 11, showing thermal ellipsoid at
the 50% probability level. Disordered ending amine group and
dichloromethane are omitted for clarity.
derivative 812 was coupled with the 4-nitrophenylalanine
methylester 9 to give compound 10 (Scheme 2), which was
then reacted with an excess of ethylenediamine to give primary
amine containing bodipy derivative 11. Red crystals of bodipy
11 suitable for structure determination via X-ray diffraction
were obtained from dichloromethane (Fig. 2).
8 B. B. Azad, V. A. Rota, D. Breadner, S. Dhanvantari and L. G. Luyt,
Bioorg. Med. Chem., 2010, 18, 1265–1272; I. Velikyan, A. Sundin,
Coupling of the bodipy amine 11 with the activated DOTA
NHS esterz 12 gave nitro-bodipy DOTA 13 (Scheme 2). The
advantage of this approach is the possibility to introduce at
this stage any kind of macrocyclic chelating agent. Indeed,
tacn or cross-bridged cyclam derivatives, which are more
adapted to Ga(III) and Cu(II), respectively,17 can be used
instead of the DOTA compound 12. Finally, the nitro
functional group was converted into isothiocyanate via
reduction to the amine and reaction with thiophosgene to give
the bimodal imaging agent 15 containing a handle for
biomolecules.
B. Eriksson, H. Lundqvist, J. Sorensen, M. Bergstrom and
¨
F. Forrer, F. Bruchertseifer, A. Morgenstern, C. Apostolidis,
¨
B. Langstrom, Nucl. Med. Biol., 2010, 37, 265–267; D. Cordier,
¨
S. Good, J. Muller-Brand, H. Macke, J. C. Reubi and A. Merlo,
¨
¨
Eur. J. Nucl. Med. Mol. Imaging, 2010, 37, 1335–1344.
9 R. P. Haughland, The Handbook. A Guide to Fluorescent Probes
and Labeling Technologies, Molecular Probes Inc., Eugene,
Oregon, 10th edn, 2005; G. Ulrich, R. Ziessel and A. Harriman,
Angew. Chem., Int. Ed., 2008, 47, 1184–1201.
10 A. Coskun and E. U. Akkaya, J. Am. Chem. Soc., 2006, 128,
14474–14475; C. Goze, G. Ulrich, L. Charbonniere, M. Cesario,
T. Prange and R. Ziessel, Chem.–Eur. J., 2003, 9, 3748–3755;
F. Lopez Arbeloa, J. Banuelos Prieto, V. Martinez Martinez,
T. Lopez Arbeloa and I. Lopez Arbeloa, ChemPhysChem, 2004,
5, 1762–1771; M. Sartin, F. Camerel, R. Ziessel and A. Bard,
J. Phys. Chem. C, 2008, 112, 10833–10841.
11 N. J. Meltola, R. Wahlroos and A. E. Roini, J. Fluoresc., 2004, 14,
635–647; L. Li, J. Han, B. Nguyen and K. Burgess, J. Org. Chem.,
2008, 73, 1963–1870; S. Lin Niu, G. Ulrich, R. Ziessel, A. Kiss,
P. Y. Renard and A. Romieu, Org. Lett., 2009, 11, 2049–2052;
S. L. Niu, G. Ulrich, P. Retailleau, J. Harrowfield and R. Ziessel,
Tetrahedron Lett., 2009, 50, 3840–3844.
12 M. Brellier, G. Duportail and R. Baati, Tetrahedron Lett., 2010,
51, 1269–1272.
13 J. P. Andre, C. F. G. C. Geraldes, J. A. Martins, A. E. Merbach,
´
In conclusion, the first DOTA–bodipy system and its
corresponding In(III), Ga(III) and Cu(II) complexes were
prepared. The complexes exhibit promising fluorescent
properties and are water-soluble, thus solving one of the
problems preventing the use of bodipy agents for in vivo optical
imaging. A second generation bodipy–DOTA containing an
isothiocyanate function was prepared for use as a tag
capable of simultaneous optical imaging and PET or SPECT
scintigraphy. Further studies (electrochemistry, complexation
constants determination, and modification of the bodipy
skeleton by derivatization reaction with benzaldehyde
derivatives to reach the near-IR range)18 are currently being
undertaken in our laboratory.
M. I. M. Prata, A. C. Santos, J. J. P. de Lima and E. Toth,
Chem.–Eur. J., 2004, 10, 5804–5816.
14 J. Olmsted, J. Phys. Chem., 1979, 83, 2581–2582.
15 A. Cui, X. Peng, J. Fan, X. Chen, Y. Wu and B. Guo,
J. Photochem. Photobiol., A, 2007, 186, 85–92.
16 G. T. Hermanson, Bioconjugate Techniques, Academic Press,
London, 1996.
17 S. Liu, Adv. Drug Delivery Rev., 2008, 60, 1347–1370.
18 Z. Ekemekci, M. D. Yilmaz and E. U. Akkaya, Org. Lett., 2008,
10, 461–464.
Support was provided by the CNRS, the University of
´
Burgundy and the Conseil Regional de Bourgogne through
the 3MIM integrated project. C.B. thanks the French Ministry
for Research for PhD grant.
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 8267–8269 8269