Re and 99mTc complexes of BodP3-multi-modality imaging probes

Article abstract of DOI:10.1039/c4cc06367h

A fluorescent tridentate phosphine, BodP₃ (2), forms rhenium complexes which effectively image cancer cells. Related technetium analogues are also readily prepared and have potential as dual SPECT/fluorescent biological probes.

Full text of DOI:10.1039/c4cc06367h

Showcasing the research of the multidisciplinary team of Benny,
Higham and Pascu.
As featured in:
Re and 99mTc complexes of BodP3 – multi-modality imaging
probes
Shine a light! The fields of organometallic synthesis,
fluorescence cell imaging and radiochemistry come together to
develop complexes which are capable of probing biologically
systems in two complementary ways.
See Paul D. Benny,
Sofia I. Pascu, Lee J. Higham et al.,
Chem. Commun., 2014, 50, 15503.
www.rsc.org/chemcomm
Registered charity number: 207890

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
Re and 99mTc complexes of BodP3 – multi-modality
imaging probes†
Laura H. Davies,a Benjamin B. Kasten,b Paul D. Benny,*b Rory L. Arrowsmith,c
Haobo Ge,c Sofia I. Pascu,*c Stan W. Botchway,d William Clegg,a
Ross W. Harringtona and Lee J. Higham*a
Cite this: Chem. Commun., 2014,
50, 15503
Received 13th August 2014,
Accepted 5th September 2014
DOI: 10.1039/c4cc06367h
www.rsc.org/chemcomm
A fluorescent tridentate phosphine, BodP3 (2), forms rhenium com-
plexes which effectively image cancer cells. Related technetium
analogues are also readily prepared and have potential as dual
SPECT/fluorescent biological probes.
99mTc–phosphine complexes are used in approved and emerging
Single-Photon Emission Computed Tomography (SPECT) imaging
agents due to the attractive nuclear properties of 99mTc (g = 140 keV,
t1/2 = 6 h) and to the facile formation of inert Tc–P coordinate
bonds.1 Resolving the cellular fate of such radiopharmaceuticals
remains challenging due to the spatial limitations of SPECT, thus
there is a drive to develop novel probes with fluorescent tags in
order to facilitate high-resolution imaging by fluorescence micro-
scopy.2 Such a species has to be (i) kinetically inert, (ii) highly
fluorescent upon metal ligation, and (iii) resistant to degrada-
tion by biological molecules. Here we report our work on
developing phosphorus-based probes for this and related appli-
cations, including therapeutics. We recently described the
synthesis of tridentate phosphine 2, BodP3, (Scheme 1) from
air-stable 1, with both retaining the attractive photophysical
properties common to Bodipy.3 Bianchini reported the synth-
Scheme 1 The hydrophosphination reaction between primary phosphine
1 and vinyldiphenylphosphine, to produce the tridentate derivative 2, BodP3.
Fig. 1 The three isomers isolated from the reaction of the tridentate
phosphine 2 and [ReCl(CO)3(PPh3)2] in refluxing toluene.
Bis(diphenylphosphinoethyl)phenylphosphine (triphos-Ph)
esis of the single isomer cis, fac-[ReCl(CO)2(triphos-Me)] by reacts with [ReCl(CO)5] in refluxing toluene to form the related
refluxing mer-[ReCl(CO)3(PPh3)2] with 1,1,1-tris(diphenylphos- isomers reported here, but on prolonged reaction times (424 h)
phinomethyl)ethane.4 With this in mind, 2 was reacted with at 170 1C, only the cis,mer(1) isomer was produced.5 Phosphine 2
mer-[ReCl(CO)3(PPh3)2] under similar conditions; however, a was therefore reacted with mer-[ReCl(CO)3(PPh3)2] in refluxing
mixture of three stereoisomers was generated, 3a–c.
These were separated via chromatography and all three were showed only 3a. In comparison, the radioactive synthon fac-
mesitylene and after 4 h, 31P{1H} NMR spectroscopy indeed
then characterised by X-ray crystallography (Fig. 1 and ESI†).
[
99mTc(CO)3(OH2)3]+ is readily prepared in situ from an IsoLinks
kit.6 Substitution of the aqua ligands under aqueous or mixed
polar organic conditions readily occurs, requiring the prepara-
tion of rhenium analogues under more polar conditions, to
better correlate with the 99mTc experiments. Thus triphos-Ph
was reacted with [Re(CO)5][OTf] in refluxing ethanol to give fac-
[Re(CO)3(triphos-Ph)][OTf], 4; a sample suitable for X-ray crystal-
lographic analysis was obtained by pentane diffusion into a
tetrahydrofuran solution (Fig. 2). To synthesise a fluorescent
analogue, 2 was reacted with [Re(CO)5][OTf] to give exclusively
fac-[Re(CO)3(2)][OTf], 5, characterised by X-ray diffraction (Fig. 3).
a School of Chemistry, Newcastle University, Bedson Building, Newcastle upon Tyne,
NE1 7RU, UK. E-mail: lee.higham@ncl.ac.uk
b Department of Chemistry, Washington State University, Pullman, WA 99164, USA
c Chemistry Department, University of Bath, Bath, BA2 7AY, UK
d Central Laser Facility, STFC, Rutherford Appleton Laboratory,
Harwell Science and Innovation Campus, Oxfordshire, OX11 0QX, UK
† Electronic supplementary information (ESI) available: Spectroscopic, crystallo-
graphic and photophysical data, cell imaging and cytotoxicity experiments. CCDC
849753 and 1001449–1001452. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/c4cc06367h
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 15503--15505 | 15503

View Article Online
Communication
ChemComm
Fig. 4 Absorption and emission spectra of 2 and its metal complexes
cis,mer-[ReCl(CO)2(2)][OTf] 3a and fac-[Re(CO)3(2)][OTf] 5 in THF. For 3b
and 3c see ESI.†
Fig. 2 X-ray crystallographic structure of 4 with 50% probability displace-
ment ellipsoids. Hydrogen atoms and the anion are omitted for clarity.
Selected bond distances [Å] and angles [1]: Re–P1 2.4680(12), Re–
P2 2.4313(13), Re–P3 2.4666(12), Re–C35 1.957(5), Re–C36 1.971(6), Re–
C37 1.954(6); P1–Re–P2 81.88(4), P1–Re–P3 95.12(4), P2–Re–P3 80.92(4),
P1–Re–C35 169.49(16), P2–Re–C36 173.86(14), P3–Re–C37 174.70(16).
attributed to the S0–S2 (p–p*) transition of the Bodipy core;7 the
absorption profiles are displayed in Fig. 4. Phosphine 2 and its
complexes all exhibit emission at room temperature in tetrahydro-
furan, dichloromethane and methanol, on excitation at 485 nm. The
emission maximum (lem) is seen at 527 nm in tetrahydrofuran for 2,
and is shifted to lower wavelengths in dichloromethane and metha-
nol. Upon complexation, no or very little change is observed in the
emission maxima. For all the complexes, when the solvent is
changed from dichloromethane to methanol, the emission maxima
are slightly blue-shifted. The Stokes shift for the complexes are small
(14–15 nm in THF), suggesting negligible structural change on
excitation. The fluorescence quantum yield (FF) for 2 is 0.34 in
tetrahydrofuran, which is comparable to the parent primary phos-
phine 1 (FF = 0.33).3 This is important, as it shows that the two
additional phosphorus groups in this tridentate derivative do not
impact negatively on the fluorescence, and indicates reductive-PeT is
not occurring.
On coordination, the fluorescence quantum yields are slightly
lowered for all the complexes (Table 1). One explanation for this
may be the heavy atom effect, causing spin–orbit coupling and
giving rise to intersystem crossing to the triplet state.8
The FF values are high in comparison to tridentate quinoline-
derived nitrogen-based rhenium complexes, which have FF of
0.003–0.015, and therefore these phosphorus-based probes may
be even more sensitive in vitro cell imaging agents.2
Fig. 3 X-ray structure of 5. Hydrogen atoms, the anion and solvent
molecules are omitted for clarity. Selected bond distances [Å] and angles
[1]: Re–P1 2.4544(13), Re–P2 2.4230(13), Re–P3 2.4831(13), Re–
C54 1.926(6), Re–C55 1.975(6), Re–C56 1.944(6); P1–Re–P2 81.44(5),
P1–Re–P3 91.64(4), P2–Re–P3 81.11(4), P1–Re–C56 167.69(18), P2–Re–
C55 173.27(17), P3–Re–C54 174.80(17).
The Re–P and Re–C bond lengths and angles are typical for such
complexes.4,5 The photophysical properties of 2, 3a–c and 5 were
then measured (Table 1). The absorption spectra of 2 and the
complexes showed a strong S0–S1 (p–p*) transition with a max-
imum of 512 or 513 nm, assigned to the Bodipy core.7
Compound 2 has a typically high molar absorption coefficient of
90 000 MÀ1 cmÀ1 which is lowered for the associated metal com-
plexes (60 000–64 000 MÀ1 cmÀ1). A lower-intensity, broader absorp-
tion band between 370 and 380 nm (e = 2500–4200 MÀ1 cmÀ1) is
The corresponding technetium complexes were first investi-
gated using triphos-Ph as a mimic for 2, to establish the general
labelling conditions. Reacting fac-[99mTc(CO)3(OH2)3]+ with 1 Â
10À5 M triphos-Ph in a pH 7.2 sodium phosphate buffer–ethanol
solution at 85 1C for 1 h resulted in quantitative radiochemical
conversion to 6. A single HPLC peak for 6 closely matched that of
fac-[Re(CO)3(triphos-Ph)]+ 4 (Scheme 2 and Fig. 5). Under the same
Table 1 Photophysical data for phosphine 2 and its Re complexes
a
a
c
d
labs
nm
/
ea/
MÀ1 cmÀ1 nm
lem
/
lem
nm
/
lem /
nm
a,b
b,c
b,d
FF
FF
FF
2
513
90 000
64 000
63 000
—
60 000
527
527
527
526
528
0.34
0.28
0.26
0.18
0.24
525
529
528
527
531
0.40
0.26
0.26
0.24
0.18
523
526
525
524
527
0.39
0.27
0.27
0.25
0.20
3a 513
3b 513
3c 512
e
5
513
a
b
In degassed tetrahydrofuran at room temperature. Measured with
respect to 4,4-difluoro-8-phenyl-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-
c
3a,4a-diaza-s-indacene; dyes were excited at 485 nm. In degassed
Scheme 2 Synthesis of fac-[99mTc(CO)3(triphos-Ph)]+
99mTc(CO)3(2)]+ 7.
6
and fac-
d
dichloromethane at room temperature. In degassed methanol at room
e
temperature. Insufficient quantity of sample isolated for measurement.
[
15504 | Chem. Commun., 2014, 50, 15503--15505
This journal is ©The Royal Society of Chemistry 2014

View Article Online
ChemComm
Communication
Fig. 6 Epi-fluorescence imaging of PC-3 living cells with compounds 3a
and 5. Top: cis,mer-[ReCl(CO)2(2)], 3a, 100 mM, 1% DMSO, 15 minutes.
Bottom: fac-[Re(CO)3(2)]+, 5, 50 mM, 2% ethanol, 15 minutes. (A) Brightfield
image, (B) green channel lex = 460–500 nm, long pass filtered at 510 nm,
(C) overlay of A and B.
Fig. 5 Overlay of HPLC chromatograms from the crude reactions between
triphos-Ph or 2 and [Re(CO)5]+ (UV, 220 nm) or fac-[99mTc(CO)3(OH2)3]+
(NaI, g-detector) to yield fac-[Re(CO)3(triphos-Ph)]+ (4) at 22.9 min, fac-
[
99mTc(CO)3(triphos-Ph)]+ (6) at 23.0 min, fac-[Re(CO)3(2)]+ (5) at 24.4 min
and fac-[99mTc(CO)3(2)]+ (7) at 24.5 min.
in PC-3 cells, which indicated an MI50 (the concentration
required to reduce mitochondrial metabolism to 50%) of
45 mM Æ 5 mM for complex 5, confirming its cytotoxic effect
(ESI†). Furthermore, MTT assays indicated that complex 3a was
innocuous up to 250 mM after 48 h incubation (ESI†). Moreover,
both 3a and 5 possess negligible cytotoxicity at the concentra-
tions required for detection via SPECT, confirming that they are
highly appropriate for use as imaging probes,11 and our work is
now focused in this direction.
conditions, the Bodipy phosphine, 2, produced fac-[99mTc(CO)3(2)]+
7 in similar conversion, giving a single major HPLC peak that
correlated with the rhenium analogue 5 (Fig. 5). The stabilities of 6
and 7 were examined by radio-HPLC using competitive amino acid
challenge assays (1 mM histidine or cysteine) to simulate an in vivo
environment ([PO4]3À 10 mM, pH 7.2, 37 1C). Analysis by HPLC
indicated that both complexes remained 497% stable up to 18 h;
no trans-chelation of the fac-[99mTc(CO)3]+ core in 6 or 7 with either
amino acid was observed during the study. The radiolabelling
yields, purity and stability of 6 and 7 indicate that the tridentate
phosphine ligand system is comparable for fac-[99mTc(CO)3]+ to
other potent tridentate chelates (e.g. histidine, bis(2-pyridylmethyl)-
amine [DPA]).9 Therefore, to the best of our knowledge, complex 7
represents the first example of a phosphine-based, multi-functional
imaging tool, combining (i) a tridentate phosphine for kinetic
stability, (ii) a fluorophore for in vitro imaging, and (iii) a radioactive
metal for in vivo imaging via g-detection by SPECT.
We thank Dr Dyszlewski at Covidien for providing the Isolinks
kits, the NIH/NIGMS (Institutional Award T32-GM008336) and
EPSRC for funding (EP/G005206/1) and the NMSSC, Swansea
for Mass Spectra. SIP and SWB thank the Royal Society, MRC
and STFC for support. We also thank Prof. Jon Dilworth for
rhenium salts and advice.
Notes and references
Radiolabelling of the complexes constitutes the first, crucial
step towards their use in nuclear medicine for in vivo tissue
imaging, but SPECT does not provide information at the
subcellular level due to resolution limitations (1–2 mm). In
contrast, optical imaging methods allow for the direct visuali-
sation of the uptake and localisation of complexes within cells
which often contributes to the understanding of the mecha-
nism of action of such probes in cellular environments, due to
the sub-micron resolution level.10 Thus, in a preliminary
screening, the rhenium complexes 3a and 5 were imaged in
prostate carcinoma (PC-3) cells (cultured as described in the
ESI†), by epi-fluorescence microscopy using single-photon exci-
tation between 460 and 500 nm and an emission filtered at
510 nm (Fig. 6). Remarkably, exchanging a chloride for a
carbonyl ligand renders the cellular behaviour of 3a and 5 very
different; whereas [ReCl(CO)2(2)]+ allows for high-resolution
imaging and enables visualisation of organelles without any
apparent cytotoxicity, fac-[Re(CO)3(2)]+, 5, on the other hand
causes some morphological changes. Further investigations
would elucidate the sub-cellular localisation of the rhenium
1 S. Liu, Chem. Soc. Rev., 2004, 33, 445; I. Santos, A. Paulo and
J. D. G. Correia, Top. Curr. Chem., 2005, 252, 45; P. D. Benny and
A. L. Moore, Curr. Org. Synth., 2011, 8, 566.
2 K. A. Stephenson, S. R. Banerjee, T. Besanger, O. O. Sogbein, M. K.
Levadala, N. McFarlane, J. A. Lemon, D. R. Boreham, K. P. Maresca,
J. D. Brennan, J. W. Babich, J. Zubieta and J. F. Valliant, J. Am. Chem.
Soc., 2004, 126, 8598.
3 L. H. Davies, B. Stewart, R. W. Harrington, W. Clegg and L. J.
Higham, Angew. Chem., Int. Ed., 2012, 51, 4921.
4 C. Bianchini, A. Marchi, L. Marvelli, M. Peruzzini, A. Romerosa,
R. Rossi and A. Vacca, Organometallics, 1995, 14, 3203.
5 A. M. Bond, R. Colton, R. W. Gable, M. F. Mackay and J. N. Walter,
Inorg. Chem., 1997, 36, 1181.
6 R. Alberto, R. Schibli, A. Egli, A. P. Schubiger, U. Abram and
T. A. Kaden, J. Am. Chem. Soc., 1998, 120, 7987.
7 A. Loudet and K. Burgess, Chem. Rev., 2007, 107, 4891.
8 J. C. Koziar and D. O. Cowan, Acc. Chem. Res., 1978, 11, 334.
9 A. Egli, R. Alberto, L. Tannahill, R. Schibli, U. Abram, A. Schaffland,
R. Waibel, D. Tourwe, L. Jeannin, K. Iterbeke and P. A. Schubiger,
J. Nucl. Med., 1999, 40, 1913; G. Liu, S. Dou, J. He, J.-L. Vanderheyden,
M. Rusckowski and D. J. Hnatowich, Bioconjugate Chem., 2004, 15, 1441.
10 P. A. Waghorn, M. W. Jones, M. B. M. Theobald, R. L. Arrowsmith,
S. I. Pascu, S. W. Botchway, S. Faulkner and J. R. Dilworth, Chem. Sci.,
2013, 4, 1430; B. Wang, Y. Liang, H. Dong, T. Tan, B. Zhan, J. Cheng,
K. K.-W. Lo, Y. W. Lam and S. H. Cheng, ChemBioChem, 2012, 13, 2729;
S. J. Butler, L. Lamarque, R. Pal and D. Parker, Chem. Sci., 2014, 5, 1750.
complexes into specific organelles. MTT assays were carried out 11 T. Skotland, Contrast Media Mol. Imaging, 2012, 7, 1.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 15503--15505 | 15505