A Novel Organometallic ReI Complex with Favourable Properties for Bioimaging and Applicability in Solid-Phase Peptide Synthesis

Article abstract of DOI:10.1002/cbic.201000576

Organometallic complexes possess great potential for imaging applications in biology, due to their kinetic stability and often favourable intrinsic properties. In this work we present a new class of Re^I-tricarbonyl complexes with a substituted bis(phenanthridinylmethyl)amine (bpm) ligand. The complex Re(CO)₃(R-bpm) could be conveniently prepared by microwave synthesis from [Re(CO)₃(H₂O)₃]Br and a suitably substituted bis(phenanthridinylmethyl)amine (R-bpm). Complex 5, with R=CH₂-CO₂-CH₃, was characterized by a single-crystal X-ray structure. Complex 6 (R=CH₂-C₆H₄-CO₂H) was used in solid-phase peptide synthesis (SPPS) to label the neurotensin(8-13) (NT) fragment N-terminally. The complexes show luminescence emission with large Stokes shifts (λ_ex=350 nm, λ_em=570 nm). Cellular uptake and intracellular localization studies in several cell lines demonstrate the utility of the new Re(CO)₃(R-bpm) complexes for fluorescence imaging and reveal significant differences between the simple methyl ester 5 and the NT bioconjugate 7. Imaging with metal complexes: The synthesis and characterization of a novel bis(phenanthridinylmethyl) (bpm) ligand and its rhenium tricarbonyl complexes are described. Moreover it is shown that the advantageous photophysical properties of these complexes could be exploited for application in cellular imaging experiments.

Full text of DOI:10.1002/cbic.201000576

DOI: 10.1002/cbic.201000576
A Novel Organometallic ReI Complex with Favourable Properties for
Bioimaging and Applicability in Solid-Phase Peptide Synthesis
Lukasz Raszeja,[a] Abdelouahid Maghnouj,[b] Stephan Hahn,[b] and Nils Metzler-Nolte*[a]
Organometallic complexes possess great potential for imaging
applications in biology, due to their kinetic stability and often
favourable intrinsic properties. In this work we present a new
class of ReI-tricarbonyl complexes with a substituted bis(phe-
nanthridinylmethyl)amine (bpm) ligand. The complex
Re(CO)3(R-bpm) could be conveniently prepared by microwave
synthesis from [Re(CO)3(H2O)3]Br and a suitably substituted
bis(phenanthridinylmethyl)amine (R-bpm). Complex 5, with R=
CH2-CO2-CH3, was characterized by a single-crystal X-ray struc-
ture. Complex 6 (R=CH2-C6H4-CO2H) was used in solid-phase
peptide synthesis (SPPS) to label the neurotensin(8–13) (NT)
fragment N-terminally. The complexes show luminescence
emission with large Stokes shifts (lex =350 nm, lem =570 nm).
Cellular uptake and intracellular localization studies in several
cell lines demonstrate the utility of the new Re(CO)3(R-bpm)
complexes for fluorescence imaging and reveal significant
differences between the simple methyl ester 5 and the NT bio-
conjugate 7.
vestigate the cellular uptake. Beside requiring additional syn-
thetic steps, the dye itself might influence the uptake behav-
iour of the conjugate, or alter its intracellular distribution as re-
cently shown by Puckett and Barton.[18] We have already
reported on the analysis of the cellular uptake of label-free
metal complexes and bioconjugates by atomic absorption
spectroscopy (AAS)[19] and Raman microspectroscopy,[20] there-
by promoting alternative methods. However, fluorescence mi-
croscopy is the method of choice for examination of the intra-
cellular distribution, identification of intracellular targets or in-
vestigation of uptake mechanisms. One way to avoid the use
of additional fluorescent labels is to use luminescent metal
complexes. Whereas the use of luminescent lanthanide com-
plexes in optical imaging techniques is already well estab-
lished, transition metal complexes have been attracting in-
creasing attention in this field.[2,21–24] Luminescent transition
metal complexes are often based on d6-ReI, RuIII or IrIII centres,
the fluorescence properties of which are modified and opti-
mised by variation of the ligand systems.[25] In general, these
complexes benefit from low rates of ligand exchange, large
Stokes shifts, long luminescence lifetimes, enhanced photosta-
bilities and easy excitation.[25] The use of rhenium-based com-
plexes offers the additional possibility to exchange the rheni-
um centre with the radioisotope 99mTc and thus to obtain g-
emitting radiomarkers. Most luminescent rhenium complexes
with potential imaging applications contain the fac-Re(CO)3
core and are based on bisimine ligands such as bipyridine or
phenanthroline[26–30] or on tridentate chelates such as bis(pyri-
dylmethyl)amine or bis(quinolinylmethyl)amine.[31–36]
Introduction
A recent focus of bioinorganic chemistry is the development
of novel metal-based compounds for medicinal and biological
applications. Beside their use as therapeutics for the treatment
of diseases, metal complexes can also act as diagnostic agents
in various biomedical imaging techniques.[1] A useful strategy
to enhance the bioavailability and target specificity of poten-
tially active compounds is to link them to biological vectors
such as peptides, carbohydrates or hormones.[2] In the past we
have reported on the successful synthesis of peptide bioconju-
gates for such purposes, in which we have used a range of dif-
ferent transition metal complexes based on iron,[3–5] copper,[6]
zinc,[6] cobalt,[4,7–9] ruthenium,[10,11] rhenium,[12] platinum[12] and
molybdenum.[13] One major advantage of peptide-based conju-
gates is their variability. Depending on the application, the
peptide unit can have different functions, such as improve-
ment of solubility, enhancement of cellular uptake or targeting
of specific cell types and/or organelles.[14–17] In order to estab-
lish whether the bioconjugate has entered the cell or even
specific cellular organelles it is necessary to visualize the com-
pounds, usually with organic dyes, and then to monitor the
uptake and distribution within the cell by fluorescence micros-
copy. A major disadvantage in this scenario is that a fluores-
cent dye must be attached to the compounds in order to in-
[a] L. Raszeja, Prof. Dr. N. Metzler-Nolte
Lehrstuhl fꢀr Anorganische Chemie I–Bioanorganische Chemie
Fakultꢁt fꢀr Chemie und Biochemie, Ruhr-Universitꢁt Bochum
Universitꢁtsstrasse 150, 44801 Bochum (Germany)
Fax: (+49)234-3214378
Here we report on the synthesis, characterization and photo-
physical behaviour of a novel chelating tridentate ligand based
on bis(phenanthridinylmethyl)amine (bpm), its rhenium com-
plexes and peptide conjugates. The complexes described
below have more favourable photophysical properties than
the above ligand systems. Moreover, we present initial results
on their application in cellular imaging and verify the suitabili-
ty of the Re(bpm) complexes in fluorescence microscopy.
E-mail: nils.metzler-nolte@rub.de
[b] Dr. A. Maghnouj, Prof. Dr. S. Hahn
Abteilung fꢀr Molekulare Gastroenterologische Onkologie
Fakultꢁt fꢀr Medizin, Ruhr-Universitꢁt Bochum
Universitꢁtsstrasse 150, 44801 Bochum (Germany)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201000576: full experimental procedures and
characterizing data for all new compounds; HPLC and MS data for the bio-
conjugate 7; HPLC data for the Cys challenging experiments.
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371

Synthesis
The bisphenanthridinylmethyl (bpm) rhenium(I) tricarbonyl
complexes 5 and 6 were synthesised by two different methods
(Scheme 2). In the first method, the bpm ligand was heated at
reflux together with [Re(CO)3(H2O)3]Br in methanol for 2 h.
More conveniently, we obtained the complexes by heating the
ligands in the presence of [Re(CO)3(H2O)3]Br in a microwave
reactor in methanol for 10 min at 1108C. In both cases the re-
action mixture was filtered and concentrated to half volume.
Crystallization of the rhenium complexes occurred through
slow diffusion of diethyl ether into the methanolic solution.
The product was isolated by filtration and dried in air.
6-(Chloromethyl)phenanthridine (2, Scheme 1) was prepared in
satisfactory yields in a three-step synthesis and used for the
preparation of the chelating ligands 3 and 4. In the first step,
Next, we were curious to explore the potential of the
Re(bpm) complexes in solid-phase peptide synthesis (SPPS)
and biological testing. We thus synthesised a peptide conju-
gate based on neurotensin(8–13), a shortened version of the
native neuropeptide neurotensin,[39] and complex 6. Firstly, the
NT(8–13) sequence (RRPTIL) was synthesised by automated
SPPS on a Wang resin, followed by overnight coupling of the
complex
6 in the presence of O-(7-azabenzotriazol-1-yl)-
N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU) and
N,N-diisopropylethylamine (DIPEA) in a molar ratio of 5:4.5:10
(6/HATU/DIPEA). After completion of the synthesis the conju-
gate was cleaved from the resin and simultaneously side-chain
deprotected with trifluoroacetic acid (TFA; 95%). Precipitation
with cold diethyl ether and centrifugation gave the peptide
bioconjugate 7. LC-MS analysis of the SPPS crude product
Scheme 1. Synthesis of bis(phenanthridinylmethyl)amine ligands. a) Chloroa-
cetyl chloride, pyridine, CH2Cl2, 08C; b) POCl3, 1308C, 20 h; c) glycine methyl
ester, K2CO3, CH3CN, reflux, 20 h; d) 4-(aminomethyl)benzoic acid methyl
ester, K2CO3, CH3CN, reflux, 20 h; then KOH (1m), CH3OH, reflux, 4 h.
biphenyl-2-amine was treated with chloroacetyl chloride; small
amounts of diacetylated byproduct could be removed by
recrystallization from n-hexane. We next cyclised compound 1
under dehydrative conditions in a Bischer–Napieralski reaction
to afford the phenanthridine derivative 2.[37] Finally, the chelat-
ing tridentate ligands 3 and 4 were synthesised through nucle-
ophilic substitution by treatment of 6-(chloromethyl)phenan-
thridine (2) with the corresponding amines.
For the synthesis of the metal complexes 5 and
6
(Scheme 2) we used the rhenium precursor [Re(CO)3(H2O)3]Br,
which was prepared from the rhenium(I)pentacarbonyl bro-
mide by heating at reflux in water.[38]
showed complete conversion to the desired conjugate
through the appearance in the chromatogram of a single
peak, attributable to the bioconjugate 7 by its mass (see the
Supporting Information). We could thus verify that the rheni-
um complexes are stable to SPPS methodology and even to
the harsh cleavage conditions.
After complexation of the bpm ligands with the Re(CO)3
1
core, the H NMR shows relevant changes both in the chemical
shift and also in the multiplicity of the methylene units in the
b-position relative to the heteroaromatic nitrogen of the bpm
moiety. Whereas in the bpm ligand these protons appeared as
a broad singlet at about 4.5 ppm, after complexation they
were split into two set of doublets and shifted downfield to
about 5.7 to 6.2 ppm. The coupling constants of 18.5 Hz indi-
cate geminal couplings, which are due to the conformational
rigidity in these complexes. These observations are consistent
with those made for similar bis(quinolinoyl) rhenium(I) com-
plexes.[36] Both the rhenium complexes 5 and 6, as well the
Scheme 2. Synthesis of the Re(CO)3(R-bpm) complexes. a) [Re(CO)3(H2O)3]Br,
CH3OH, reflux, 2 h; b) [Re(CO)3(H2O)3]Br, CH3OH, microwave 1108C, 10 min.
372
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ChemBioChem 2011, 12, 371 – 376

bioconjugate 7, show the intense characteristic vibrational
bands of the fac-Re(CO)3 unit of A and E symmetry at about
2025 cmꢀ1 and at 1930 cmꢀ1 to 1900 cmꢀ1, respectively. In 5
and 6, the latter band is even split into two bands due to local
symmetry breaking. In comparison with the [Re(CO)3(H2O)3]Br
precursor, the vibrational stretching bands of which appear at
2018 cmꢀ1 and at 1880 cmꢀ1, we find a blue shift in the case of
the rhenium compounds 5, 6 and 7. The X-ray structural results
confirm the fac-{Re(CO)3} core and the coordination of the
three bpm nitrogen atoms (Figure 1). The structure shows a
Photophysical properties
After the successful synthesis of the Re(CO)3(R-bpm) complexes
we investigated their photophysical behaviour to judge their
suitability for cellular imaging applications. The UV–visible
spectra of the bpm ligands in acetonitrile each show two
characteristic absorption maxima at 330 nm and 345 nm, both
with moderate extinction coefficients of about 3000 to
4300mꢀ1 cmꢀ1. The rhenium complexes each differ in the pres-
ence of a typical broad absorption band between 310 nm and
380 nm and a three- to fourfold enhanced extinction coeffi-
cient of about 13000mꢀ1 cmꢀ1 at the absorption maximum.
The fluorescence spectra of the bpm ligands in acetonitrile
(Figure 2) each show an emission maximum, at 410 (3) and
445 nm (4), upon excitation at 350 nm. On changing the sol-
vent to more polar ones such as ethylene glycol (EG) or water,
distinct blue shifts occur and the emission wavelengths move
to about 375 nm in the case of 3 and 380 to 400 nm in that of
4.
Figure 1. ORTEP plot (50% probability, hydrogen atoms and bromide coun-
terion omitted for clarity) of the rhenium complex 5. Selected bond lengths
[ꢁ]: Re1–C1 1.91(1), C1–O1 1.15(2), Re1–C2 1.91(1), C2–O2 1.16(1), Re1–C3
1.909(8), C3–O3 1.16(1), Re1–N3 2.239(9), Re1–N1 2.249(7), Re1–N2 2.202(6).
Selected angles [8]: N1-Re1-N2 84.1(3), N1-Re1-N3 72.8(3), N2-Re1-N3 78.5(3),
C1-Re1-C2 86.1(4), C1-Re1-C3 85.3(4), C2-Re1-C3 89.0(4), N1-Re1-C2 176.0(4),
N2-Re1-C3 172.9(4), N3-Re1-C1 169.6(4).
distorted octahedral geometry, in which the three Re–CO in-
teratomic distances are equal and the short CꢀO bond lengths
further confirm the expected multiple bond character. The Re–
N distances are longer and show single bond character both
for the sp3-hybridized amine nitrogen [Re(1)–N(3) 2.239(9) ꢁ]
and for the sp2-hydridized phenanthridine nitrogen atoms
[Re1–N1 2.249(7) ꢁ and Re1–N2 2.202(6) ꢁ]. In addition, the
distortion of the ideal octahedral geometry is reflected in the
steric arrangement of the five-membered chelate rings [Re1-
N3-C31-C30-N2 and Re1-N3-C17-C16-N1]. In order to verify the
kinetic stabilities of the [Re(CO)3(bpm)]+ complexes, they were
incubated with cysteine and examined afterwards by HPLC-MS.
Incubation of the complexes with a tenfold excess of cysteine
(c=2 mm) at 378C over a period of nine days resulted in a
completely unchanged HPLC-MS analysis (see the Supporting
Information). Consequently, one can conclude that these
Re(bpm) complexes exhibit high kinetic stabilities and low ten-
dencies towards ligand exchange.
Figure 2. Normalized emission spectra of compounds 3, 4, 5, 6 and 7 upon
excitation at 350 nm.
For the rhenium complexes, the fluorescence spectra in ace-
tonitrile with excitation at 350 nm each show a red-shifted
emission of more than 100 nm relative to the free ligand: that
is, at 575 nm for 5, at 570 nm for 6 and at 560 nm for the bio-
conjugate 7. This situation can be explained in terms of a trip-
let metal-to-ligand charge transfer (3MLCT) luminescence,
which is characterized by large Stokes shifts and is already well
known for rhenium(I) tricarbonyl bis(imine)[40,41] and bis(quino-
line)[42] complexes. With use of water as a solvent, the emission
wavelengths of compounds 5 and 6 shift further into the red.
On switching from degassed to non-degassed acetonitrile,
3
we noticed only a minor change in the MLCT emission of 5,
unlike the situation reported for similar rhenium tricarbonyl
complexes based on bis(quinolinylmethyl)-aminoacetic acid
methyl ester.[31] At exactly the same concentrations of 5 we
observed an increase in fluorescence intensity of only about
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373

5% on switching from non-degassed to degassed acetonitrile
(Figure 3). Complex 5 is thus less sensitive to quenching pro-
cesses caused by the presence of dissolved dioxygen. Because
of the large Stokes shift we gain two significant advantages.
Firstly, self-quenching processes, which occur through the re-
absorption of emitted light by adjacent fluorophores are avoid-
ed. Secondly, autofluorescence, which is often caused by en-
dogenous molecules, can easily be suppressed by use of suita-
ble filters.
Figure 4. Fluorescence images (artificial colour) of living cancer cells after
24 h incubation with 7 (50 mm) or 5 (5 mm). A) 7, IMIM-PC2, 200ꢂ. B) 7, HT-
29, 400ꢂ. C) 5, IMIM-PC2, 200ꢂ. D) 5, HT-29, 400ꢂ.
for most organic dyes,[43] even during extended exposure
times.
Figure 4 displays some illustrative false-colour images. Pic-
tures A (IMIM-PC2, 200ꢂ magnification) and B (HT-29, 400ꢂ
magnification) demonstrate the results with bioconjugate 7
after 24 h incubation at a concentration of 50 mm, whereas C
(IMIM-PC2, 200ꢂ magnification) and D (HT-29, 400ꢂ magnifi-
cation) show the results after 24 h incubation of complex 5 in
5 mm concentration. In general, both tested compounds are
taken up well und distributed in the cytosol, whereas the nu-
clear membrane seems to be impermeable to our compounds.
A qualitative comparison between complex 5 and the biocon-
jugate 7 indicates that the organometallic compound is inter-
nalized better and more rapidly than the peptide conjugate,
especially when we consider that complex 5 was incubated at
concentrations ten times lower. For the peptide conjugate 7,
on the other hand, the difference between the two cell lines is
far more pronounced, possibly indicating some selectivity in
the uptake mechanism as a consequence of the attached pep-
tide. The results of the incubation experiments on the HeLa
and the PT-45 cell lines with 5 and 7 look qualitatively similar
to the results discussed above with regard to the uptake ef-
ficiency and the cellular distribution, and are therefore not
shown here.
Figure 3. Emission spectra of complex 5 in degassed (black line) and non-
degassed (grey line) acetonitrile on excitation at 350 nm.
Cellular uptake studies
Encouraged by the promising photophysical properties of the
rhenium complexes described above, we investigated the suit-
ability of these compounds as fluorophores for cellular imag-
ing experiments. Both the rhenium complex 5 and the biocon-
jugate 7 were used for preliminary cell microscopy studies on
several different cell lines: HeLa (cervical cancer), HT-29 (colon
cancer), IMIM-PC2 and PT-45 (both pancreatic cancers). Incuba-
tion of our substances was carried out on adherent cells,
which were seeded on 8-well plates 24 h prior to the experi-
ment. Concentrated DMSO stock solutions of our compounds
were diluted with cell culture medium to final concentrations
of 5 mm to 50 mm. The total amount of DMSO in the dilutions
used was under 0.5% in every case in order to ensure full via-
bility of the cells. After an incubation time of 24 h the cells
were washed with PBS und covered with medium for the
microscopic investigations. The examinations were performed
with an Olympus IX81 fluorescence microscope with use of a
filter system consisting of an excitation filter for excitation
between 330 nm and 385 nm and a long-pass emission filter
for wavelengths higher than 420 nm for detection.
Conclusions
A novel tridentate chelating ligand system based on the bis-
(phenanthridinylmethyl) (bpm) moiety has been developed
and successfully synthesized. The corresponding rhenium(I) tri-
carbonyl complexes were prepared by two different methods
from the [Re(CO)3(H2O)3]Br precursor. Finally, the rhenium com-
plex 6 was used successfully in SPPS for the synthesis of the
neurotensin bioconjugate 7, thereby demonstrating full com-
patibility and stability of the Re(CO)3(R-bpm) complexes to the
SPPS conditions. Photophysical investigations of the rhenium
We can first confirm the suitability of our rhenium com-
plexes for cell microscopy applications. The fluorescence
images (Figure 4) clearly demonstrate a nonhomogenous dis-
tribution of the exogenous Re compound. During our micro-
scopic investigations we did not observe any photobleaching
effects or loss of fluorescence intensity, which are well known
374
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ChemBioChem 2011, 12, 371 – 376

Synthesis of the rhenium compounds—fac-[Re(CO)3((bis(6-phe-
nanthridinylmethyl)amino)acetic acid methyl ester)] bromide (5)
complexes afforded promising results. Red-shifted absorption
maxima, lower excitation energies, large Stokes shifts, reduced
sensitivity towards dioxygen quenching and emission in the
orange range of the visible spectrum provide opportunities for
imaging applications in cell microscopy. Live-cell fluorescence
microscopy of the new compounds verified their potential as
fluorescent dyes. Both the rhenium complex 5 and the biocon-
jugate 7 were internalized by various cell lines and could be
detected under a fluorescence microscope, due to the new lu-
minescent Re(CO)3(R-bpm) core. Whereas the lipophilic methyl
ester 5 is probably internalized by passive diffusion, more pro-
nounced differences across different cell lines can possibly be
attributed to a receptor-mediated uptake mechanism for the
neurotensin conjugate 7. Further investigations to exploit the
favourable properties of the new Re(CO)3(R-bpm) complexes
are currently underway in our laboratory.
Route A: [Bis(6-phenanthridinylmethyl)amino]acetic acid methyl
ester (3, synthesis described in the Supporting Information,
47.1 mg, 100 mmol) was dissolved in methanol (15 mL) under
reflux. A solution of [Re(CO)3(H2O)3]Br[38] (40.2 mg, 100 mmol) in
methanol (2 mL) was then added and heating at reflux was contin-
ued for 2 h. After cooling to room temperature the reaction mix-
ture was filtered. The orange brownish solution was concentrated
to a volume of about 5 mL and filtered again. Slow diffusion of di-
ethyl ether into the methanolic solution afforded prismatic, brown-
ish crystals. These were filtrated and air-dried. The fac-[Re(CO)3-
((bis(6-phenanthridinylmethyl)amino)acetic acid methyl ester)] bro-
mide (5) was obtained in 43% yield (38 mg, 46 mmol).
Route B: [Bis(6-phenanthridinylmethyl)amino]acetic acid methyl
ester (3, 23.6 mg, 50 mmol), together with [Re(CO)3(H2O)3]Br[38]
(20.2 mg, 50 mmol), were suspended in methanol (3 mL). The mix-
ture was then stirred in a CEM discovery microwave reactor for
10 min at 1108C. The resulting dark red solution was filtered. Slow
diffusion of diethyl ether into the methanolic solution afforded
brownish crystals. These were filtrated and air-dried. The fac-
[Re(CO)3((bis(6-phenanthridinylmethyl)amino)acetic acid methyl
ester)] bromide (5) was obtained in 56% yield (23 mg, 28 mmol).
Experimental Section
General remarks: Commercially purchased reagents were used
without any further purification. Solvents needed for synthesis
were of analytical grade, for spectroscopic investigations of spec-
troscopic grade and for HPLC analysis of HPLC grade containing
TFA (0.1%). Degassed acetonitrile was obtained by the freeze-
pump-thaw procedure, applied for at least three cycles. All report-
ed compounds were confirmed to be pure by NMR, HPLC, HRMS
or elemental analysis. NMR spectra were recorded with a Bruker
1H NMR (400 MHz, DMSO): d=9.07–8.84 (m, 4H), 8.50–8.32 (m,
2H), 8.24 (d, J=8.3 Hz, 2H), 8.13 (t, J=7.4 Hz, 2H), 7.89 (t, J=
7.5 Hz, 2H), 7.88–7.81 (m, 4H), 6.18 (d, J=18.9 Hz, 2H), 5.99 (d, J=
18.8 Hz, 2H), 5.06 (s, 2H), 3.71 ppm (s, 3H); 13C NMR (101 MHz,
DMSO): d=195.9, 194.0, 168.96, 167.8, 141.2, 134.5, 132.4, 130.4,
129.3, 128.9, 128.8, 127.9, 124.8, 124.2, 123.1, 122.7, 69.5, 65.7,
52.4 ppm; IR (ATR): n˜ =2023, 1907, 1894 (fac-Re(CO)3), 1759 cmꢀ1
(COꢀOCH3); UV/Vis (CH3CN): lmax (e)=310 (13000), 342 (10600),
360 (8400), 430 nm (415 mꢀ1 cmꢀ1); MS (ESI+): m/z: 741.87 [M]+.
1
DRX 400 (400 MHz for H, 101 MHz for 13C) or a Bruker DPX 200
1
(200 MHz for H, 50 MHz for 13C) spectrometer, with the deuterat-
ed solvent used as internal standard. IR spectra were recorded
with a Bruker Tensor 27 instrument, ESI mass spectra with a Bruker
Esquire 6000 instrument and EI high-resolution mass spectra with a
Fisons VG Autospec instrument. Elemental analysis was performed
with a Foss Heraeus Vario EL instrument. UV/Vis spectra were re-
corded with a JASCO V-670 spectrophotometer and a 1 cm cuv-
ette. Uncorrected steady state emission spectra were acquired with
a PTI Quantamaster QM4 spectrofluorimeter and 1.0 cm quartz cuv-
ettes at 298 K. The excitation light source was a continuous xenon
short arc lamp (75 W). Emission spectra were collected at 908 to
the excitation beam with use of a PTI R928 photomultiplier tube as
the detector. HPLC MS was performed with an Agilent 1100 instru-
ment and a Zorbax SB-C18 column (1.8 mm particle size) coupled
to the Bruker Esquire 6000.
fac-[Re(CO)3((bis(6-phenanthridinylmethyl)aminomethyl)benzoic
acid)] bromide (6): Compound 6 was prepared in the same way as
described above for compound 5 by route B on a 140 mmol scale.
4-[Bis(6-phenanthridinylmethyl)aminomethyl]benzoic acid (4, syn-
thesis described in the Supporting Information) was used instead
of 3 for the synthesis. fac-[Re(CO)3((bis(6-phenanthridinylmethyl)-
aminomethyl)benzoic acid)] bromide (6) was obtained in 56% yield
1
as small, light beige crystals. H NMR (400 MHz, DMSO): d=13.17
(s, 1H), 9.00–8.88 (m, 4H), 8.50–8.40 (m, 2H), 8.19 (s, 4H), 8.16 (d,
J=8.2 Hz, 2H), 8.11 (t, J=7.4 Hz, 2H), 7.90–7.80 (m, 6H), 5.99 (d,
J=18.5 Hz, 2H), 5.69 (d, J=18.6 Hz, 2H), 5.33 ppm (s, 2H); 13C NMR
(101 MHz, DMSO): d=196.2, 194.3, 167.6, 167.0, 141.2, 136.8,
134.5, 133.1, 132.4, 131.7, 130.4, 129.7, 129.4, 128.9, 128.9, 128.0,
124.8, 124.1, 123.0, 123.0, 68.5, 66.8 ppm; IR (ATR): n˜ =2027, 1898
(fac-Re(CO)3), 1699 cmꢀ1 (COOH); UV/Vis (CH3CN): lmax (e)=280
(22000), 310 (12400), 342 (10200), 360 (8200), 450 nm
(290 mꢀ1 cmꢀ1); MS (ESI+): m/z: 803.87 [M]+.
Cell culture and imaging: Dulbecco’s Modified Eagle’s Medium
(DMEM) supplemented with foetal bovine serum (10%), penicillin
and streptomycin was used as growth medium. HeLa, HT-29, IMIM-
PC and PT-45 cells were detached from the wells with trypsin and
EDTA, harvested by centrifugation (300 g, 3 min) and resuspended
again in cell culture medium. The cells were seeded on 8-well ibidi
m-Slide plates at a concentration of 30000 cells per well (120000
cells per mL) and incubated for 24 h. The cells were washed with
PBS (pH 7.4) prior to incubation with the rhenium compounds.
After incubations the cells were washed with PBS (pH 7.4) and cov-
ered with medium for the microscopic examinations. The imaging
experiments were carried out with an OLYMPUS IX81 fluorescence
microscope and use of a filter system consisting of an excitation
filter for excitation between 330 nm and 385 nm and a long pass
emission filter for wavelengths higher than 420 nm for detection.
The platform used was cellM 3.2 (build 1700).
NT(8–13) conjugate 7: Peptide synthesis was carried out with pep-
tide grade reagents and solvents. The following amino acids were
used: Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH and
Fmoc-Ile-OH. NT(8–13) was prepared with a CEM microwave-assist-
ed peptide synthesiser. The sequence (RRPTIL) was synthesised
with a Fmoc-Leu-Wang (0.68 mmolgꢀ1) resin, commercial Fmoc
amino acids (aas), HOBt/TBTU as activator (act) and DIPEA as acti-
vator base in a ratio of 5:4.5:10 (aa/act/base) in the coupling step.
Fmoc removal was carried out with piperidine in DMF (20%). After
completion of the automated NT(8–13) synthesis, the rhenium
complex 6 was coupled overnight with use of HATU as activator
and DIPEA in a ratio of 5:4.5:10 (6/HATU/DIPEA). Final cleavage
ChemBioChem 2011, 12, 371 – 376
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
375

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(ESI+): m/z: 801.69 [M+H]2+
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534.81 [M+2H]3+
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CCDC 793511 (5) contains the supplementary crystallographic data
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Keywords: bioorganometallic chemistry
microscopy · imaging agents · microwave chemistry · rhenium
·
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Received: September 23, 2010
Published online on January 11, 2011
376
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ChemBioChem 2011, 12, 371 – 376