Bridging the gap between in vitro and in vivo imaging: Isostructural Re and 99mTc complexes for correlating fluorescence and radioimaging studies

Article abstract of DOI:10.1021/ja047751b

A bifunctional ligand that is capable of forming Re and ^99mTc complexes as complementary fluorescent and radioactive probes was developed. The tridentate bis(quinoline) amine ligand, which is referred to as the SAACQ system, was prepared in a single step from Fmoc protected lysine in high yield. Reaction of the SAACQ ligand with [Re(CO)₃Br₃]^2- resulted in the formation of the SAACQ-(Re(CO)₃)⁺complex which exhibits favorable fluorescence properties including a long lifetime and a large Stoke's shift. Because the SAACQ ligand is derived from an amino acid, it can readily be linked to or incorporated within peptides as a means of targeting the probe to specific receptors. To demonstrate this feature, the SAACQ ligand and the SAACQ-Re complex were incorporated into fMLFG, a peptide that binds to the formyl peptide receptor (FPR). Uptake of the fMLF[(SAACQ-Re(CO)₃)⁺]G conjugate into human leukocytes in vitro was visualized by fluorescence microscopy, and the observed distribution of the peptide was similar to that of a well-established fluorescent FPR probe. The corresponding Tc complex, fMLF[(SAACQ-^99mTc(CO)₃)⁺]G, was prepared in excellent yield from [^99mTc(CO)₃(OH₂)₃]⁺, which affords the opportunity to correlate the results of the microscopy experiments with in vivo radioimaging studies because the probes are isostructural. Copyright

Full text of DOI:10.1021/ja047751b

Published on Web 06/26/2004
Bridging the Gap between in Vitro and in Vivo Imaging: Isostructural Re and
99mTc Complexes for Correlating Fluorescence and Radioimaging Studies
Karin A. Stephenson,† Sangeeta Ray Banerjee,‡ Travis Besanger,† Oyebola O. Sogbein,†
Murali K. Levadala,‡ Nicole McFarlane,† Jennifer A. Lemon,† Douglas R. Boreham,†
Kevin P. Maresca,§ John D. Brennan,† John W. Babich,§ Jon Zubieta,*,‡ and John F. Valliant*,†
Department of Chemistry, McMaster UniVersity, Hamilton, Ontario, Canada L8S 4M1, The Medical Physics and
Applied Radiation Sciences Unit, McMaster UniVersity, Hamilton, Ontario, Canada L8S 4M1,
Department of Chemistry, Syracuse UniVersity, Syracuse, New York 13244-4100, and
Molecular Insight Pharmaceuticals Inc., Cambridge, Massachusetts 02142
Received April 19, 2004; E-mail: valliant@mcmaster.ca
Scheme 1 a
Two of the most widely employed techniques for visualizing
specific biological processes are fluorescence microscopy1 and
radioimaging.2 Fluorescence microscopy is a powerful tool for
looking at the distribution of fluorescent probes in vitro and for
studying the dynamics of protein motion. The high spatial resolution
of this technique allows accurate depiction of processes at the
subcellular level and of complex systems such as protein trafficking
within cells. Imaging with gamma-emitting radiopharmaceuticals,
using nuclear medicine scanners, allows for sensitive detection of
biological processes deep within the body at resolutions of a few
a Conditions: a) NaBH(OAc)3, Q2A. b) [NEt4]2[Re(CO)3Br3].
millimeters.
reacted with quinoline-2-aldehyde (Q2A) in the presence of Na-
A constraint in the development of radiopharmaceuticals is the
(OAc)3BH to give the bifunctional ligand 2 (Scheme 1). The desired
product, which can be produced in multigram quantities, was
inability to image the behavior and fate of these compounds at the
cellular and subcellular level, in real-time, to rapidly elucidate
isolated in excellent yield (86%) following column chromatography.
mechanisms responsible for cellular specificity and localization.
The Re complex 3 was synthesized by reacting 2 with [NEt4]2-
Substitution of the radiolabel with a fluorescent dye3 may allow
for visualization of the vector compound in vitro, but will likely
alter the physicochemical properties sufficiently to render such
substitution of little value. The optimal system would be one in
which the fluorescent and radioactive prosthetic groups are iso-
structural.
[Re(CO)3Br3] in methanol. The complex was isolated in 80% yield
as the bromide salt.
Compound 3 displays an appreciable absorbance in the UV and
blue regions of the UV-visible spectrum with a peak absorbance
at 301 nm. The extinction coefficients for 3 were 13200 M-1 cm-1
at 301 nm and 2250 M-1 cm-1 at 366 nm. The emission spectrum
It is conceivable that complimentary pairs of fluorescent and
shows that the rhenium probe has two distinct transitions giving
radioactive probes can be prepared using technetium and rhenium.
rise to peak fluorescence intensities at 425 and 580 nm. Compound
99mTc is the most widely used radionuclide in diagnostic medicine,
3 has a lifetime of 4.31-9.76 µs, depending upon the solvent and
owing to its ideal nuclear properties, low cost, and widespread
availability.4 Re(I) complexes on the other hand, have been used
amount of oxygen present, which is sufficiently prolonged to enable
time-gating techniques to be used during in vitro imaging studies
to prepare luminescent probes.5 Fluorescent Re complexes are
to overcome interferences from endogenous fluorescence. Com-
particularly useful for studying biological processes in vitro because
of their long-lifetime, polarized emission, and large Stoke’s shift.
On the basis of the fact that the coordination chemistry of the two
congeners is similar, it should be possible to design a ligand that
forms a fluorescent Re complex and a stable 99mTc complex. Such
a system, which should also possess the ability to be linked to a
pound 3 shows fluorescence anisotropy with a limiting anisotropy
of 0.35 at 424 nm (at -20 °C), which indicates that the probe can
also be used to study small-molecule-receptor or protein-protein
interactions in which the rotational mobility of the probe is
monitored as a function of binding. The quantum yield for 3, which
ranges from 0.003 in chloroform in the presence of air to 0.015 in
targeting agent, would allow images obtained on a fluorescent
ethylene glycol under nitrogen, is low but not significantly different
microscope to be directly correlated with in vivo radioimaging
from the quantum yields reported for other transition-metal based
studies because the structures of the two probes would be effectively
identical.
Recently, we reported the synthesis of a Tc(I)/Re(I) binding
fluorescence probes.5a,7
Having established that 3 has the necessary properties to be used
as a fluorescent probe, the subsequent objective was to develop a
ligand based a lysine-derived bis(pyridyl) amine which was referred
method for preparing bioconjugates that can deliver the ligand to
to as a single amino acid chelate (SAAC).6 The SAAC forms an
specific receptors. Not coincidently, the SAACQ ligand and the
inert complex with the M(CO)3+ core (M ) Re, Tc), and it can be
incorporated into peptides as if it were a natural amino acid. To
prepare a SAAC-type ligand whose Re complex is fluorescent,
while retaining the ability to bind 99mTc, N-R-Fmoc-L-lysine was
SAACQ-Re complex are amino acid analogues which enables
them to be linked to, or incorporated within, peptide-based targeting
agents using a conventional automated synthesizer. To demonstrate
this feature, the SAACQ and SAACQ-Re complexes were
integrated within fMLF, a targeting sequence which has been used
to guide radionuclides to the formyl peptide receptor (FPR), which
† McMaster University.
‡ Syracuse University.
§ Molecular Insight Pharmaceuticals Inc.
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J. AM. CHEM. SOC. 2004, 126, 8598-8599
10.1021/ja047751b CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
(OH2)3]+; a precursor that can be prepared in any radiopharmacy
.
from 99mTcO4- 13 The desired product, whose HPLC retention time
matched that of 5a, was isolated in high yield (90%) via a simple
solid-phase extraction procedure. The stability of the 99mTc-labeled
peptide was subsequently tested by incubating two separate samples
of 5b with an excess of cysteine and histidine in PBS buffer heated
to 37 °C. After 24 h there was no dissociation of the metal which
indicates that the SAACQ ligand forms a Tc-complex that is suitably
robust for use in vivo.
The work reported here demonstrates that: (1) The Re SAACQ
complex has the appropriate fluorescence properties to be used for
in vitro microscopy studies, (2) the SAACQ ligand and its Re
complex can be easily incorporated into peptides using a conven-
tional automated synthesizer, and (3) the SAACQ ligand can be
labeled with 99mTc in high yield and the resulting complex is robust.
In light of these findings it is now possible to use the SAACQ
synthon to prepare complementary fluorescent and radioactive
peptide probes. This offers the unique opportunity to directly
correlate in vitro and in vivo imaging studies, which goes a long
way toward bridging the gap between work in isolated cells and
studies carried out in living subjects.
Figure 1. fMLF[(SAACQ-M(CO)3)+]G containing all L-isomers.
Figure 2. Fluorescence microscopy images of human leukocytes incubated
with 1 nM 5a (left), 50 nM fluorescein-labeled fNLFNTK (middle), and
50 nM 5a (right).
is expressed on neutrophils, as a means of imaging the trafficking
of white blood cells.8 The peptides fMLF(SAACQ)G (4) and fMLF-
[(SAACQ-Re(CO)3)+]G (5a, Figure 1) were prepared, using
compounds 2 and 3, respectively, following a conventional
automated Fmoc synthesis method using glycine-loaded SASRIN
resin and HBTU as the coupling agent. The peptides were isolated
using a standard cleavage cocktail and the products purified by
HPLC. The HPLC purification was needed as a result of epimer-
ization of the formyl-methionine residue which is known to occur
under normal solid-phase synthesis conditions.9 The affinities of 4
and 5a for human FPRs were determined by flow cytometry using
fluorescein-labeled fNLFNTK as the reference ligand.10 Compounds
4 and 5a show Kd values of 11 ( 3 and 27 ( 13 nM, respectively,
which is comparable to that for the parent targeting agent.
To demonstrate that the Re-peptide complex can be used as a
probe for fluorescence microscopy studies, a 1 nM solution of
compound 5a was incubated with human leukocytes and its uptake
observed using a fluorescence microscope (Figure 2). Samples were
excited by a mercury lamp using a commercially available filter
set consisting of a dichroic 360 nm excitation filter, a 400 nm
dichroic long-pass filter, a and 550 nm long-pass emission filter.
At low temperatures, compound 5a could readily be seen bound to
the periphery of the leukocytes but internalized into the cytoplasm
of the cells when samples were allowed to warm to room
temperature. These observations match the results reported for
microscopy studies of other fluorescently labeled chemotactic
peptides.11 Along those lines, a 1:1 mixture of the peptide and a
well-established fluorescent FPR probe, fluorescein-labeled fN-
LFNTK, were incubated with the same cells and microscopy images
taken at different excitation and emission wavelengths so that only
one probe was visualized at a time.12 The images (Figure 2), in
qualitative terms, are effectively identical, which strongly suggests
that the two probes are targeting the same cell populations. A more
quantitative head-to-head comparison of the two probes is currently
underway
Acknowledgment. We acknowledge NSERC of Canada, The
Canadian Foundation for Innovation, The Ontario Innovation Trust,
The Ontario Research Challenge Fund for funding, and Dr. Karen
Gulenchyn and Mr. Ferid Rashid from the Department of Nuclear
Medicine, Hamilton Health Sciences, for providing access to
-
99mTcO4
.
Supporting Information Available: Synthetic procedures for 2,
3, 5a, 5b and fluorescence data and procedures. This material is
available free of charge via the Internet at http://pubs.acs.org.
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compound 4 (500 µg) with 703 MBq (19 mCi) of [99mTc(CO)3-
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