Metal complex mediated conjugation of peptides to nucleus targeting acridine orange: A modular concept for dual-modality imaging agents

Article abstract of DOI:10.1021/bc2000269

To target the nucleus of specific cells, trifunctional radiopharmaceuticals are required. We have synthesized acridine orange derivatives which comprise an imidazole-2-carbaldehyde function for coordination to the [Re(CO)₃]+ or [^99mTc(CO)₃]+ core. Upon coordination, this aldehyde is activated and rapidly forms imines with amines from biological molecules. This metal-mediated imine formation allows for the conjugation of a nuclear targeting portion with a specific cell receptor binding function directly on the metal. With this concept, we have conjugated the acridine orange part to a bombesin peptide directly on the ^99mTc core and in one step. In addition, a linker containing an integrated disulfide has been coupled to bombesin. LC/MS study showed that the disulfide was reductively cleaved with a 60 min half-life time. This concept enables the combination of a nucleus targeting agent with a specific cell receptor molecule directly on the metal without the need of separate conjugation prior to labeling, thus, a modular approach. High uptake of the BBN conjugate into PC-3 cells was detected by fluorescence microscopy, whereas uptake into B16BL6 cells was negligible.

Full text of DOI:10.1021/bc2000269

ARTICLE
pubs.acs.org/bc
Metal Complex Mediated Conjugation of Peptides to Nucleus
Targeting Acridine Orange: A Modular Concept for
Dual-Modality Imaging Agents
Karel Zelenka,† Lubor Borsig,‡ and Roger Alberto*,†
†Institute of Inorganic Chemistry and ‡Institute of Physiology, University of Z€urich, Winterthurerstrasse 190, 8057 Z€urich, Switzerland
S Supporting Information
b
ABSTRACT: To target the nucleus of specific cells, trifunc-
tional radiopharmaceuticals are required. We have synthesized
acridine orange derivatives which comprise an imidazole-2-
carbaldehyde function for coordination to the [Re(CO)3]þ or
[
99mTc(CO)3]þ core. Upon coordination, this aldehyde is
activated and rapidly forms imines with amines from biological
molecules. This metal-mediated imine formation allows for the
conjugation of a nuclear targeting portion with a specific cell
receptor binding function directly on the metal. With this concept, we have conjugated the acridine orange part to a bombesin
peptide directly on the 99mTc core and in one step. In addition, a linker containing an integrated disulfide has been coupled to
bombesin. LC/MS study showed that the disulfide was reductively cleaved with a 60 min half-life time. This concept enables the
combination of a nucleus targeting agent with a specific cell receptor molecule directly on the metal without the need of separate
conjugation prior to labeling, thus, a modular approach. High uptake of the BBN conjugate into PC-3 cells was detected by
fluorescence microscopy, whereas uptake into B16BL6 cells was negligible.
’ INTRODUCTION
nucleus. Confirmation of compound accumulation in the target
is preferentially received from optical imaging since this modality
allows nanometer resolution in cells. In vitro knowledge about
site specificity of a compound can then be extended to the second
modality, namely, in vivo radioimaging. For a successful combi-
nation of optical and radioimaging, chemical identification of the
cold, luminescent, and radioactive compound is mandatory. An
increasing number of publications are emphasizing the impor-
tance of single-compound-based dual-modality imaging based on
a combination of subcellular luminescence and in vivo imaging
from γ-emission.12ꢀ14 One main strategy to target nuclei of
living cells with radiolabeled compounds is based on conjugation
of a suitable chelator to NLS (nucleus localizing signal)
peptides.15 Such cell and nucleus penetrating peptides can cargo
various molecules such as simple metal complexes,16 peptides,
proteins, antisense PNA, or plasmid DNA17 or are labeled with
radioisotopes to quantify in vitro and in vivo biodistribution.18ꢀ22
In our own endeavors to target the nucleus of specific cells, we
combined 99mTc as a favorable SPECT radionuclide with fluo-
rescence markers such as acridine orange or pyrene.21,23 The
rationale behind these studies is to benefit from the Auger
electrons of 99mTc to double-strand break DNA of, e.g., cancer-
ous cells.24ꢀ26 Along this strategy, a bioconjugate consists of a
nuclear targeting agent (with luminescence properties) and a cell
Molecular imaging modalities such as single photon emission
computed tomography (SPECT) and positron emission tomo-
graphy (PET) are major methods for noninvasive diagnosis of
organ malfunctions or diseases.1 Over the past years, the
combination of different imaging modalities has become a major
focus in advanced imaging. By using different regions of the
electromagnetic spectrum for imaging, complementary informa-
tion about, e.g., physiology and metabolism of particular sites in
cells or even in entire organisms may be received concertedly.2,3
Combination of SPECT or PET and CT, for instance, is at the
stage of clinical routine and allows for combining radionuclide
distribution in tissue with anatomical information.4,5 Although
both SPECT and PET have achieved millimeter resolution, it
would be desirable to combine either of these two methods with
optical imaging techniques. Luminescence-based imaging allows
at least on the in vitro level following in real time the compound in
the intracellular space. Highly accurate sensing allows assessment
of the particular intracellular compartment in which the com-
pound accumulates. Numerous examples for fluorescence ima-
ging appeared in the literature with organic or metal-based
fluorescence markers.6ꢀ11 Ideally, optical imaging would also
be possible in vivo; however, many fluorescence markers are not
suitable for deep-body penetration visualization of biological
events. In vitro optical imaging does allow, however, localization
of a compound on the subcellular level. This is particularly
important if a diagnostic or therapeutic radionuclide-based agent
is designed to target a particular cell compartment such as the cell
Received: January 14, 2011
Revised:
March 15, 2011
Published: April 12, 2011
r
2011 American Chemical Society
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receptor specific compound such as a peptide (Scheme 1). The
peptide moiety (e.g., bombesin) targets cells with a specific,
strongly (over)expressed receptor (GRP receptor for bombesin).
Active, receptor-mediated uptake results in site-specific accumu-
lation of the conjugate. Degradation of the peptide in lysosomes
sets the intercalator (fluorescent marker) free, which often
exhibits strong interaction with and uptake into the DNA. Such
fluorescent carriers enhance the accumulation of complexes in
the cell nucleus. Optionally, a cleavable linker ensures the cleavage of
the receptor targeting agent from the nucleus targeting complex.
This bifunctional bioconjugate is then radiolabeled for in vivo
imaging (Scheme 1).
The synthesis of such constructs is demanding, and any new
combination entails a new “total synthesis” of DNA targeting
agent, cell receptor specific moiety, and chelator. Lengthy
procedures are not very convenient for efficient drug finding or
development. Conjugating these two principles directly on the
SPECT imaging modality (99mTc) introduces an in situ building
block concept. The metal complex fragment does mediate the
conjugation of the nuclear targeting and the cell targeting
molecules. Accordingly, each individual vector can be replaced
without the need of new additional syntheses.
activated aldehyde is susceptible for imine formation with the
H2N-terminus of a peptide (Scheme 2). Fluorescence micro-
scopy studies confirm highly selective uptake of this bioconjugate
in the targeted cells.
’ RESULTS AND DISCUSSION
Syntheses. In the building block approach, the combination
of a nucleus targeting function and a cell targeting vector is bound
to the [99mTc(CO)3]þ core via a bidentate imine or hydrazone
chelator. The in situ formation of these bidentate ligands from an
amine and aldehyde represents the base for the building block
concept, since each part can be altered while maintaining an
identical synthetic procedure. The resulting complex is cationic
and of the general form [Re(OH2)(L2)(CO)3]þ. An anion
replaces the coordinated water ligand, yielding an overall neutral
complex. Due to its strongly fluorescent properties, we have
selected acridine orange (AO) as the intercalating nucleus
targeting agent and imidazole-2-carboxyaldehyde (ima) as bi-
dentate ligand. We and others have shown previously that
heteroaromatic aldehydes are strong ligand groups for coordina-
tion to the [99mTc(CO)3]þ moiety.27ꢀ31 Compound 1 was
prepared according to a published procedure32 followed by
alkylation of the imidazole-2-carboxyaldehyde to yield 2 in
reasonable yield. Coupling of a hydrazine model compound gave
the corresponding hydrazone 4, which now contains a strong
bidentate chelator. Reaction with [Re(OH2)3(CO)3]þ in metha-
nol/water gave the model complex 6. Complex 7 which contains
both the nucleus targeting agent and a receptor-specific bombe-
sin peptide (BBN) was prepared similarly. The reaction of 2 with
the bombesin peptide 3 afforded the hydrazone 5. Subsequent
addition of [Re(OH2)3(CO)3]þ gave the complex 7. Complex 7
is now a model for trifunctional radiopharmaceuticals and
comprises the nucleus targeting agent, the cell targeting
vector, and the metal center (Scheme 3). Since the hydrazones
4 and 5 are water stable, they can directly be subjected to
labeling studies.
We present in this study the metal-mediated conjugation of a
nuclear targeting agent (violet in Scheme 1) with cell receptor
targeting peptides (green) and model molecules. In this approach,
the aldehyde coordinates to the [99mTc(CO)3]þ moiety. The
Scheme 1. Concept of a Cell-Specific, Nuclear Targeting
Trifunctional Bioconjugate
A further building block for a final conjugate is the cleavable
linker. Disulfide bonds are known to be cleaved by thiols via thiol/
disulfide exchange.33 They are frequently used in prodrug designs
for pharmaceuticals. In the intracellular space, they are cleaved by
reduction with GSH which sets the active drug free.34ꢀ36 For our
purposes, the disulfide containing cleavable linker 10 was prepared
Scheme 2. Building Block Concept for the Metal-Mediated Formation of Cell-Specific, Nucleus Targeting Radiopharmaceuticals
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Scheme 3a
a (i) K2CO3, DMF; (ii) phenyl-hydrazine, EtOH or 3, PBS; (iii) [Re(OH2)3(CO)3]þ, KI, MeOH/H2O or MeCN/PBS.
Scheme 4. Synthesis of the Orthogonal, Disulfide-Based Cleavable Linker 10 and Its Conjugate to a Peptide 15 a
a (i) Boc2O, NaOH, dioxane/water; (ii) HSCH2CH2COOH, Et3N, CHCl3; (iii) DIPC, TFP, DMF; (iv) Et3N, DMF; (v) TFA/CH2Cl2. 12, 14: R =
NH(CH2)2Ph; 13, 15: R2 = -BBN(8-14)NH2.
Scheme 5a
a (i) PhCH2CH2NH2, MgSO4, CHCl3; (ii) [Re(OH2)3(CO)3]þ, KI, MeOH/PBS; (iii) [Re(OH2)3(CO)3]þ, H2O; (iv) 14 or 15 PBS, MeCN.
in a three-step synthesis. The amino groups of cysteamine were
BOC-protected to give 8. Thiolysis of the disulfide with mercapto-
propionic acid afforded the acid 9. The activated TFP ester 10 for
coupling to terminal amines was prepared along standard proce-
dures. The TFP ester 10 was then used for the preparation of
compounds 12 and 13. The free amines 14 and 15 were obtained
after BOC deprotection (Scheme 4).
by the reaction of imine 16 with [Re(OH2)3(CO)3]þ. Isolation
of the free imines from the reaction of the aldehyde 2 with 14 or
15 was not possible due to their fast hydrolysis. To receive the
true trifunctional compounds 19 and 20, the aldehyde 2 was
reacted in water with an excess of [Re(OH2)3(CO)3]þ. The orange
compound 18 precipitated from solution. In 18, the aldehyde is
1
coordinated to the rhenium center. H NMR confirmed the
Hydrazones such as 4 or 5 are relatively stable toward hydro-
lysis, but imines are not. The model complex 17 was first prepared
presence of the coordinating aldehyde rather than a coordinated
semiacetal.27
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Compound 18 is the key model compound for labeling
reactions and therefore for the metal-mediated assembly of two
different types of targeting agents. Compound 18 reacted with 14
or 15 under imine formation to give the trifunctional compounds
19 and 20, respectively (Scheme 5). Important for the later
labeling with 99mTc, the complexes 19 or 20 were also obtained
from a “one-pot” reaction. Compound 2, 14, or 15 together with
[Re(OH2)3(CO)3]þ was dissolved in a MeOH/PBS mixture
(1:1). After 12 h at 50 °C, compound 19 or 20 could be purified
by preparative HPLC chromatography. The yields after purifica-
tion for both procedures were comparable.
trifunctional complexes 19 and 20 are rhenium-based and can be
used for fluorescence imaging (see later). Fluorescence microscopy
allows for following the biological pathways of these compounds
on the subcellular level, whereas the 99mTc homologues can be
used for in vivo imaging. The unprecedented advantage of this
building block principle is the fact that not only 14 and 15 but
principally any amines can be selected to complete the trifunctional
conjugates. Coordination of the aldehyde in 2 to the [Re(CO)3]þ
center activates the carbonyl carbon. Although nucleophilic attack of
water competes for imine formation, once formed, the imine
complex (e.g., 17) is so strong that the reaction is irreversible and
runs to completion. This is crucial for the 99mTc experiments, since
quantitative formation of the radiopharmaceutical is mandatory.
Cleavage Studies. Compounds 19 and 20 comprise the
disulfide-cleavable linker which is expected to be reduced in
the intracellular space and to release the nucleus targeting
function. In vitro studies should elucidate rate and mechanism
of glutathione GSH-mediated cleavage of the disulfide bond.
Compound 19 (in PBS) was added to a freshly prepared GSH
solution (in PBS). The final concentrations of the GSH and
complexes were 0.5 mM and 0.01 mM, respectively. This GSH
concentration is normally found in cells.37 The solution was
incubated at 37 °C and samples were taken every 26 min and
analyzed with HPLC-MS (see Supporting Information). Figure 1
shows the progress of the cleavage reaction. The integrated
chromatograms of total ion counts (TIC) of one of the cleavage
products (21 m/z: 515.3[M]þ) is plotted versus the starting
materials (sum of the intensities of 19 m/z 1066.4 [M]þ, 19a m/z
988.4 [M-TFAþCl]þ, and 19b m/z 476.7 [M-TFA]2þ are
plotted). Compounds 19a and 19b are formed in solution via
slow anion exchange and change over time.
Complex 20 now comprises the nucleus targeting agent, the
cell targeting vector with a disulfide cleavable linker, and the
metal center (Scheme 6).
Ideally, complete complex 20 is taken up by the cell through
receptor-mediated endocytosis, thus ensuring cell specificity.
Inside the cell, the disulfide linker is cleaved with the reduced
form of GSH, cleaving the peptide vector from the metal-nucleus
targeting moiety complex. Since the remaining complex is
relatively small, it can permeate the nuclear membrane without
necessity of active transport. Intercalation with DNA proved to
be a key process for accumulation of such conjugates in the cell
nucleus.24 The cleavage of the disulfide bond also prevents the
exclusion of the complex from the cell through a receptor. The
Scheme 6. Complex 20 Comprising Nucleus Targeting
Agent (Red), Cell Targeting Vector (Green) with a Disulfide
Cleavable Linker (Blue), and Hexacoordinated Metal Center
The TIC of complex 22a (m/z: 781.3 [M]þ, major product)
exhibits the same growing trend as 21. The different physico-
chemical properties of 22a influenced the MS measurement (ion
trapping, ejection, and detection). The maximum of the 22a
signal was about three times below the trace of 21. The disulfide
bridge in 19 can be cleaved by GSH on the peptide side, resulting
in 21, or on the complex side resulting in 24. HPLC-MS
evidenced that the formation of undesired 24 (or 24a and 24b
Figure 1. Plot of the time dependence of TIC intensities of 21 (red line) and 19 (sum of 19, 19a, and 19b; black line).
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Scheme 7. Cleavage Products after Reaction of 18 or 19 with GSH
Figure 2. Left: radioactive HPLC traces of [99mTc(OH2)3(CO)3]þ (dotted line), 25 (dashed line), and 26 (solid line). Right: UVꢀvis trace of
corresponding Re complex 17a (X = Clꢀ; see ESI); Gradient D.
after exchange of X) was negligible as compared to 22a or 21.
The half-life of disulfide cleavage with the GSH was about 60
min. Due to “steady state” intracellular GSH concentration,37,38
cleavage inside the cells could be even faster. A similar study was
done with complex 20. The formation of the cleavage products
22a and 23 displayed similar trends as for 19 (Figure 1.). An
overview for the cleavage products is given in the Scheme 7.
99mTc-Labeling Studies. The precursor [99mTc(OH2)3-
(CO)3]þ was prepared from [99mTcO4]ꢀ according to literature
procedures or with the commercially available Isolink kit
(Covidien, Tyco-Mallinckrodt Med. B.V. Petten, NL).39,40 It
should be emphasized that 99mTc concentrations are in the range
of 10ꢀ6 to 10ꢀ9 M. After preparation, the solution was buffered
with 0.1 M phosphate buffer to pH 7.4. Conversions to products
were quantified by HPLC analyses with γ-detection. Reaction
times, concentrations, and temperature varied.
Depending on the ligand, the synthesis of complexes of the
general type [99mTc(X)(L2)(CO)3]þ can be done in one or two
steps. Solvent or an anion X is coordinated to the sixth available
coordination site on the fac-[99mTc(CO)3]þ. Labeling was first
tested with a model amine and amino acids under various conditions
to confirm the feasibility of the process with 99mTc. The preparation
of complex 26 was done in two steps. The reaction of aldehyde 2
(0.1 mM, 30 min, 90 °C) with [99mTc(OH2)3(CO)3]þ (Figure 2,
Rt = 19.2 min dotted line) gave the intermediate 25 (dashed line,
Rt = 28 min). After addition of the phenylethylamine (0.2 mM),
the mixture was heated to 90 °C for 45 min to yield the complex
26 (Rt = 29.5 min, solid line). The same result was also obtained
from the “one-pot” preparation, mixing all components together
and heating at 90 °C for 45 min. The formation of complex 26
was confirmed by comparing the retention time of with the Re
analogue 17a (Figure 2, also see ESI).
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Figure 3. Left: radioactive HPLC traces of [99mTc(OH2)3(CO)3]þ (dashed line) and 27 (solid line). Right: UV trace of corresponding Re complex 7
(Gradient D).
Figure 4. Uptake of the complex 20 into PC-3 cells by fluorescence microscopy. Cells were incubated with 20 μM complex 20 for 6 h, washed, then an
additional 18 h in the fresh medium, followed by fixation of cells and nuclear staining by DAPI. (A) PC-3 cells, loaded with 20 in the cytoplasm (green),
clear structured staining. (B) DAPI staining of nucleus (blue). (C) Merged image (overlay).
The same approach was used to prepare the 99mTc analogue
of 20. However, even by variation of gradients and solvents, the
aldehyde complex 25 and resulting imine complex (25 þ 15)
were hardly distinguishable and displayed comparable reten-
tion times. For detailed labeling study and comparison with
corresponding Re complex 20, see ESI. The labeling of 5
(0.22 mM) with [99mTc(OH2)3(CO)3]þ at 90 °C after 60 min
provided product 27 with a retention time of 27.6 min (Figure 3,
solid line).
Cell Uptake Studies. We selected a human prostate adeno-
carcinoma cell line, PC-3, which does express the GRP receptor
and B16-BL6 mouse melanoma cell line with little or no
expression of the GRP receptor. The cell uptake of complex 20
was monitored in the green fluorescence filter as described in the
Experimental Section. To localize the compound in the cell, we
stained nuclei with DAPI (40,6-diamidino-2-phenylindol), routi-
nely used in cell biology. The PC-3 cells were exposed to 20
(20 μM) and the uptake was followed by fluorescence micro-
scopy (Figure 4). Complex 20 clearly accumulated in the cell. Green
fluorescence was mainly detected in the cytoplasm, exhibiting
substructured distribution, probably in lysosomes. There is no
enhanced accumulation of fluorescence in the nucleus. Complex
7 was studied in a similar manner to 20. The accumulation in the
cell was also observed with comparable intensities to those for 20.
The trifunctional complex 20 contains the cell-specific vector
(truncated 8ꢀ14 BBN peptide). Thus, only cells with the
corresponding receptor should accumulate 20. To confirm such
a specific uptake, cell studies were performed on a B16-BL6
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Figure 5. Uptake of complex 20 by B16BL6 cells, performed as described for PC-3. (A) Green channel image. (B) DAPI nucleus staining (blue). (C)
Merged image (overlay).
mouse melanoma cell line which does not express the GRP
receptor. The difference between the PC-3 (Figure 4) and B16-
BL6 (Figure 5) is significant. While PC-3 cells were loaded with
20 in the cytoplasm, there was virtually no accumulation in the
B16BL6 cell line. The uptake of complex 20 by PC-3 cells was
consistent with the presence of the GRP receptor, indicating a
receptor-mediated uptake.32
Gradient A: 0ꢀ5 min 90% A, 5ꢀ17 min 90ꢀ0% A, 17ꢀ25 min
0% A, 25ꢀ27 min 0ꢀ90% A, 27ꢀ35 min 90% A, flow 0.3 mL.
minꢀ1; Gradient B: 0ꢀ2 min 90% A, 2ꢀ12 min 90ꢀ0% A,
12ꢀ17 min 0% A, 17ꢀ18 min 0ꢀ90% A, 18ꢀ25 min 90% A,
flow 0.25 mL.minꢀ1. HPLC analyses were performed on a Merck
L7000 system using a Macherey-Nagel EC 250/3 Nucleodur C18
Gravity 5 μm for radioactive compounds. HPLC solvents were
0.1% trifluoroacetic acid (solvent A) and MeOH or MeCN HPLC
grade (solvent B). Gradient C (MeOH): 0ꢀ5 min 90% A, 5ꢀ
15 min 90ꢀ0% A, 15ꢀ20 min 0% A, 20ꢀ22 min 0ꢀ90% A, 22ꢀ
30 min 90% A. Gradient D (MeCN): 0ꢀ5 min 90% A, 5ꢀ20 min
90ꢀ60% A, 20ꢀ23 min 60% A, 23ꢀ28 min 0% A, 28ꢀ33 min
0% A, 33ꢀ34 min 0ꢀ90% A, 34ꢀ40 min 90% A. Flow rates:
0.5 mL/min, detection with a γ-detector for the radiolabeled
compounds. Preparative HPLC was performed on a Varian Pro
Star system by using either a Macherey-Nagel VP 250/21
Nucleodur C18 Gravity 5 μm or a Macherey-Nagel VP 250/40
Nucleosil 100ꢀ7 C18 column with a flow rate of 12 mL.minꢀ1
and 40 mL.minꢀ1 respectively. The solvents were 0.1% trifluor-
oacetic acid (solvent A) and methanol or acetonitrile (solvent B).
Cell Culture and Complex Uptake. Human prostate cancer
cells, PC-3 were incubated in F12K medium supplemented with
10% fetal calf serum. For microscopy, cells were cultured over-
night on four-chamber slides (Nunc) in which ca. 50 000 cells
were plated. Next day, cells were washed and fresh media
containing complexes 20 or 7 (20 μm) were added. Cells were
incubated with the complex for 6 h. After the loading with a
complex, the fresh medium was added and the cells were
incubated for additional 18 h, washed with phosphate buffer
saline (PBS), and fixed in 4% paraformaldehyde for 10 min in at
r.t. After three washings with PBS, cells were incubated in 1 mg.
mLꢀ1 40,6-diamidino-2-phenylindole (DAPI) for nuclear stain-
ing for 10 min at r.t. Cells were washed three times with
phosphate-buffered saline (PBS), chambers from the slides were
detached, and cells were covered with coverslips in Prolong
Mounting medium (Invitrogen). Slides were evaluated on Zeiss
Axiovert 200 m with the corresponding fluorescence filters for
DAPI (λex = 360 nm, λem = 420 nm) and acridine orange (λex =
496 nm, λem = 525 nm).
’ CONCLUSIONS
The efficient development of imaging probes containing a
therapeutic modality demands a flexible building block concept.
Herein, we presented an approach in which the conjugation of a
nucleus targeting part with a cell-specific vector is metal-
mediated, yielding a trifunctional diagnostic and potentially
therapeutic radiopharmaceutical. A disulfide-based cleavable
linker was introduced on the cell receptor targeting part. In vitro,
the disulfide linker was cleaved with GSH at a reasonable rate.
Fluorescence microscopy with rhenium analogues using GRP/
non-GRP expressing cell lines confirmed the cell specificity
concept. However, studies with complex 20 did not display
increased uptake in the nucleus but stained mainly substructures
of the cytoplasm for so far unknown reasons. Detailed cell studies
with the trifunctional complex 20 and similar systems, prepared
according to the presented building block concept, are currently
under investigation. The approach represents a very general method
for the in situ preparation of strong bidentate ligands and its use for
further development of functional multimodality systems.
’ EXPERIMENTAL SECTION
General Information. The commercially available reagents
were used as received without further purification. Analytical
thin-layer chromatography (TLC) was carried out with alumi-
num-based plates (silica gel 60 F254) from Merck. Plates were
visualized under UV light (λ = 254 nm). Flash chromatography
was carried out on Flashmaster Solo (Argonaut) by using Merck
silica gel 60 (0.040ꢀ0.063 mm). Samples were applied as almost
saturated solutions in the appropriate solvent. 1H NMR and 13
C
NMR spectra were performed on a Bruker 400 and 500 spectro-
meters at 400/500 and 100/125 MHz, respectively. The re-
ported chemical shifts (in δ) are relative to the solvent protons
and carbons as a reference. HPLC-MS (ESI) spectra were
measured on a Bruker HCT spectrometer with Aquinity UPLC
(Waters) with alternating polarity ion detection, using a Macher-
ey-Nagel EC 250/3 Nucleodur C18 Gravity 5 μm. HPLC
solvents were 0.1% formic acid (solvent A) and methanol
(solvent B). The gradients used for analyses were as follows:
General Remarks to the Re Complexes. Under common
HPLC or HPLC-MS conditions, typically two peaks with a ratio
of 1:10 (respectively 1:1 with TFA anion) were observed. The
first peak represents the complex, where the anion is replaced
with a solvent, while the second peak belongs to the original
(whole) complex. During the MS acquisition, the coordinated
solvent is usually lost. For complexes containing peptides, the
sixth coordination position is probably occupied by imidazole
from histidine. Both double- and single-charged species were
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observed under HPLC-MS conditions. The elementary analyses
were not performed due to the various content of TFA after
purification with the preparative HPLC. Thus, to be consistent,
the M.W. and concentrations of the compounds were calculated
without counterions (e.g., as drawn in the schemes). If not
specified in the scheme, the counterions are trifluoroacetates.
3,6-Bis(dimethylamino)-10-(4-(2-formyl-1H-imidazol-1-yl)-
butyl)acridinium (2). A suspension of 1H-imidazole-2-carbal-
dehyde (11 mg, 0.11 mmol) and K2CO3 (100 mg, 0.72 mmol) in
DMF (10 mL, dry) was stirred at r.t. under N2. After 10 min, 1
(70 mg, 0.14 mmol) was added, and the mixture stirred and
heated to 50 °C under N2. After 16 h, the solvent was removed
under HV. The residue was dissolved in 15 mL of CH2Cl2,
filtered over Celite, and dried again. Purification by preparative
HPLC afforded 36 mg 2 (75%) as an orange powder. 1H NMR
(CD3CN, 400 MHz) δ 9.65 (d, 4J = 0.8 Hz, 1H), 8.49 (s, 1H),
7.79 (d, 3J = 9.3 Hz, 2H), 7.31 (bs, 1H), 7.18 (d, 4J = 0.7 Hz, 1H),
7.14 (dd, 3J = 9.3 Hz, 4J = 2.2 Hz, 2H), 6.45 (d, 4J = 1.8 Hz, 2H),
4.53 (bt, 3J = 8 Hz, 2H), 4.43 (t,3J = 6.9 Hz, 2H), 3.20 (s, 12H),
2.07ꢀ1.97 (m, 2H), 1.93ꢀ1.83 (m, 2H). MS (ESI, Gradient B):
tR 9.3 min; positive mode m/z 448.2 (100) [M þ MeOH]þ,
416.2 (33) [M]þ. MS (ESI) m/z (%) 416.2 (100) [M]þ,
448.2(17) [M þ MeOH]þ; calcd for C25H30N5Oþ: 416.24
(100.0).
[Re(I)(4)(CO)3]þ (6). The hydrazone 4 (8 mg, 0.016 mmol)
and KI (50 mg, 0.3 mmol) were dissolved in the methanol/water
mixture (6 mL, 2:1), then [NEt4]2[ReBr3(CO)3] (15 mg,
0.019 mmol) was added. The mixture was stirred 3 h at 60 °C.
The solvents were removed in vacuo, and the crude product was
purified by preparative HPLC to give 6 as dark orange powder in
a yield of (7 mg, 49%).1H NMR (DMSO-d6, 400 MHz): δ 10.40
(s, 1H), 8.77 (s, 1H), 8.57 (s, 1H), 7.90 (d, 3J = 9.4 Hz, 2H), 7.59
(d, J = 1.4 Hz, 1H), 7.45 (d, J = 1.4 Hz, 1H), 7.24 (dd, 3J = 9.4 Hz,
4J = 2.0 Hz, 2H), 7.18ꢀ7.12 (m, 2H), 7.05ꢀ7.00 (m, 3H),6.54
(bs, 2H), 4.78ꢀ4.63 (m, 2H), 4.32 (t, 3J = 6.6 Hz, 2H), 3.20 (s,
12H), 2.03ꢀ1.93 (m, 2H), 1.77ꢀ1.67 (m, 2H). HPLC-MS (ESI,
Gradient B): tR 12.8 min; positive mode m/z 904.3 (100), 902.3
(58) [M]þ; calcd fro C34H36IN7O3Reþ: 904.15 (100.0), 902.15
(57.0).
[Re(5)(CO)3]þ (7). To a solution of 5 (10 mg, 0.007 mmol)
dissolved in the acetonitrile/PBS mixture (4 mL, 1:1, pH = 7.4),
[NEt4]2[ReBr3(CO)3] (20 mg, 0.026 mmol) was added. The
mixture was stirred under N2 at 40 °C. After 2 d, the solvents
were removed and the crude product purified by preparative
HPLC to give 7 as orange powder in a yield of (4 mg, 33%).
HPLC-MS (ESI, Gradient B): tR 11.2 min; positive mode m/z
776.4 (100) [M]2þ, 1551.5 (10) [M-H]þ; calcd for C68H89-
N18O11ReS2þ: 1552.62 (100.0).
NH2NH2CH2CO-Bombesin WAVGHLM (3). Truncated hydra-
zinoacetyl-bombesin 3 (sequence: WAVGHLM) was synthe-
sized SPPS according to the procedure reported previously for
hydrazino derivatives of peptides.41 The product was then
purified by reverse-phase HPLC on a preparative C18 column
(Macherey-Nagel VP 250/40 Nucleosil 100ꢀ7 C18 column),
fractions with the peptide were lyophilized, and pure peptide was
kept under N2 in the freezer at ꢀ25 °C. 3 was obtained as a white
solid. HPLC-MS (ESI, Gradient A): tR 13.4 min; positive mode
m/z 884.5 (100) [MþH]þ; calcd for C40H61N13O8S: 883.45
(100.0); negative mode m/z 882.6 (60) [M-H]ꢀ, 996.6 (100)
[MþTFA]ꢀ.
N,N0-Bis-tert-butoxycarbonylcystamine (8). Compound 8
was prepared according to the published procedure and the
analysis was similar to the literature.42
3-((2-(tert-Butoxycarbonylamino)ethyl)disulfanyl)propanoic
acid (9). Compound 9 was prepared according to the published
procedure43 using the 3-mercaptopropanoic acid instead of
2-mercaptoacetic acid. 300 mg (yield 30%) of the acid 9 were
obtained. TLC Rf = 0.26 (CHCl3/MeOH, 9/1).1H NMR
(CDCl3, 400 MHz): δ 4.93 (bs, 1H), 3.46 (bs, 2H), 2.95 (bt,
3J = 6.9 Hz, 2H), 2.84ꢀ2.77 (m, 4H), 1.46 (s, 9H). MS (ESI)
m/z (%) 304.1 (100) [M þ Na]þ; 316.1(100) [M þ Cl]ꢀ, 280.4
(90) [M - H]ꢀ; calcd for C10H19NO4S2: 281.08 (100.0).
2,3,5,6-Tetrafluorophenyl 3-((2-(tert-butoxycarbonylami-
no)ethyl)disulfanyl)propanoate (10). A solution of 9 (170 mg,
0.6 mmol) in DMF (1 mL, dry) was cooled to 4 °C and
diisopropylcarbondiimid (93 μL, 76 mg, 0.6 mmol) was added.
The mixture was stirred 15 min under N2, and then 2,3,5,6-
tetrafluorophenol (100 mg, 0.6 mmol) in DMF(0.5 mL, dry) was
slowly added and reaction was stirred at r.t. After 12 h, CH2Cl2
(3 mL) was added, precipitate was filtered, and filtrate was
evaporated to dryness. Purification by flash chromatography
(gradient, hexane to hexane:MTBE 6:1) afforded compound
10 (100 mg, 38%) as a white solid. 1H NMR (CDCl3, 500 MHz)
δ 7.05ꢀ7.00 (m, 1H), 4.90 (bs, 1H), 3.46 (bd, 3J = 6.0 Hz, 2H),
3.13 (t, 3J = 6.9 Hz, 2H), 3.05 (t, 3J = 6.9 Hz, 2H), 2.83 (t, 3J = 6.7
Hz, 2H), 1.45 (s, 9H). MS (ESI) positive mode m/z (%) 452.0
(100) [M þ Na]þ, 467.9 (80) [M þ K]þ; negative mode 464.0
(100) [M þ Cl]ꢀ; calcd 429.07 (100.0). Elemental analysis (%)
calcd for C16H19F4NO4S2: C 44.75, H 4.46, N 3.26, S 14.93;
found C 44.52, H 4.47, N 3.00.
3,6-Bis(dimethylamino)-10-(4-(2-((2-phenylhydrazono)-
methyl)-1H-imidazol-1-yl)butyl)acridinium (4). The aldehyde
2 (26 mg, 0.062 mmol) was dissolved in the EtOH (1 mL, dry),
and phenyl hydrazine (7 mg, 6.3 μL, 0.062 mmol) was added to
the mixture. Reaction was stirred and heated to 80 °C for 4 h; the
solvent was then removed in vacuo. The crude product 4 (31 mg,
98%) was pure enough for use in the next step without further
purification. 1H NMR (CD3CN, 400 MHz): δ 10.00 (bs, 1H),
8.42 (s, 1H), 7.94 (s, 1H), 7.70 (d, 3J = 9.3 Hz, 2H), 7.12ꢀ6.93
(m, 8H), 6.75 (t, 3J = 7.3 Hz, 1H), 6.40 (d, 4J = 1.7 Hz, 2H), 4.55
3
(t, J = 7.4 Hz, 2H), 4.35 (t,3J = 6.6 Hz, 2H), 3.08 (s, 12H),
2.10ꢀ2.01 (m, 2H), 2.01ꢀ1.92 (m, 2H). HPLC-MS (ESI,
Gradient B): tR 10.8 min; positive mode m/z 506.4 (100)
[M]þ, [MþH]þ 253.1 (40) [MþH]2þ; calcd for C31H36N7
:
þ
506.3 (100).
3,6-Bis(dimethylamino)-10-(4-(2-((2-(2-(Bombesin 8ꢀ14)-
2-oxoethyl)hydrazono)methyl)-1H-imidazol-1-yl)butyl)-
acridinium (5). The aldehyde 2 (8 mg, 0.019 mmol) and
hydrazino bombesin 3 (25 mg, 0.028 mmol) were dissolved in
the PBS (15 mL, pH = 6.5). The mixture was stirred at r.t under
N2 for 3 days. The orange precipitate was filtered and washed
with water and PBS. The crude product was purified by pre-
parative HPLC to give 5 as orange powder in a yield of (12 mg,
48%). HPLC-MS (ESI, Gradient B): tR 10.0 min; positive mode
m/z 641.6 (100) [MþH]2þ, 1281.7 (75) [M]þ; calcd for
C65H89N18O8Sþ: 1281.7 (100.0).
Bombesin WAVGHLM (11). Truncated bombesin 11 (sequence:
WAVGHLM) was synthesized SPPS and purified according to the
procedure reported previously24).
tert-Butyl 2-((3-oxo-3-(phenethylamino)propyl)disulfanyl)-
ethylcarbamate.TFA (14). A solution containing 10 (10 mg,
0.023 mmol) and Et3N (6.4 μL, 4.7 mg, 0.046 mmol) in 0.3 mL
of DMF was stirred for 30 min at room temperature. After addition
of 2-phenylethanamine (3.2 μL, 3.1 mg, 0.025 mmol), the solution
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Bioconjugate Chemistry
ARTICLE
was further stirred for 12 h. The removal of the solvent in vacuo
afforded crude product 12. The residue was dissolved in 1.5 mL of
the mixture of dichloromethaneꢀTFA (1:0.5). The mixture was
stirred for 3 h under N2 at r.t. After removal of the solvent in vacuo,
the crude product was purified by preparative HPLC to afford 3 mg
(31%) of compound 14 as a white solid. 1H NMR (CDCl3, 400
MHz): δ 8.31 (bs, 2H), 7.29ꢀ7.12 (m, 5H), 6.20 (t, 3J = 5.6 Hz,
1H), 3.46 (q, 3J = 6.5 Hz, 2H), 3.28 (bt, 3J = 5.5 Hz, 2H), 2.94 (bt,
3J = 6.0 Hz, 2H), 2.90 (t,3J = 6.6 Hz, 2H), 2.77 (t, 3J = 7.0 Hz, 2H),
2.49 (t, 3J = 6.7 Hz, 2H). HPLC-MS (ESI, Gradient B): tR 10.2 min;
positive mode m/z: 285.3 (100) [MþH]þ; calcd for C13H20N2-
OS2: 284.11 (100.0).
The orange solution was added to the flask containing the amine
14 (3 mg, 0.007 mmol) and the mixture was stirred at r.t for 16 h;
HPLC-MS showed the product formation. The product was
purified by preparative HPLC to afford 1 mg (18%) of com-
1
pound 18 as an orange powder. H NMR (DMSO-d6, 400
3
MHz): δ 9.09 (s, 1H), 8.77 (s, 1H), 7.94 (t, J = 5.5 Hz, 1H),
7.90 (d, 3J = 9.4 Hz, 2H), 7.78 (d, J = 1.3 Hz, 1H), 7.58 (d, J = 1.2
Hz, 1H), 7.27ꢀ7.07 (m, 7H), 6.54 (bs, 2H), 4.75ꢀ4.65 (m, 2H),
4.50ꢀ4.36 (m, 2H), 4.24ꢀ4.14 (m, 1H), 4.14ꢀ4.04 (m, 1H),
3.21ꢀ2.14 (m, 2H), 3.18 (s, 12H), 3.08ꢀ2.91 (m, 2H), 2.70 (t,
3J = 7.0 Hz, 2H), 2.63ꢀ2.58 (m, 2H). 2.33ꢀ2.28 (m, 2H),
2.11ꢀ2.01 (m, 2H), 1.81ꢀ1.61 (m, 2H). HPLC-MS (ESI,
Gradient B): tR 12.5 min; positive mode m/z 1066.2 (100)
[M]þ, 1064.3 (60) [M]þ; calcd for C43H48F3N7O6ReS2þ: 1066.26
(100.0), 1064.26 (58.6); tR 11.0 min; positive mode m/z 476.6
(100) [M-TFA]2þ; calcd for C41H48N7O4ReS22þ: 953.28 (100).
[Re(15)(CO)3] (20)þ. The aldehyde 2 (4.2 mg, 0.01 mmol) and
the [NEt4]2[ReBr3(CO)3] (22 mg, 0.028 mmol) were dissolved
in PBS (5 mL, pH = 6.5). The mixture was stirred and heated
under N2 at 50 °C for 15 min. The formed precipitate was spun
down, and supernatant removed. The remaining precipitate was
dissolved in the mixture of MeCN:PBS (10 mL, 1:1, pH = 7.4).
The orange solution was added to the flask containing the
peptide 15 (30 mg, 0.03 mmol) and the mixture was stirred at
66 °C for 24 h. The crude product was purified by preparative
HPLC to afford 4 mg (24%) of complex 19 as an orange powder.
HPLC-MS (ESI, Gradient A): tR 15.7 min; positive mode m/z
821.9 (100) [M]2þ, 1642.4 (50) [M-H]þ; calcd for C71H94N17-
O11ReS32þ: 1643.60 (100.0), 1644.61 (75.1).
Bombesin (15). Truncated bombesin 11 (40 mg, 0.049 mmol,
prepared according to the literature) was dissolved in the DMF
(dry, 0.5 mL) with Et3N (20 μL, 15 mg, 0.148 mmol). The
mixture was stirred at r.t under N2. After 15 min, the solution of
10 (24 mg, 0.056 mmol) in DMF (0.5 mL) was added and
mixture was stirred at r.t under N2 for 20 h. The solvent was
removed in vacuo to afford crude product 13. The residue was
dissolved in 1 mL of the mixture of dichloromethaneꢀTFA
(1:1). The mixture was stirred for 12 h under N2 at r.t. After
removal of the solvent in vacuo, the crude product was purified
by preparative HPLC to afford 30 mg (62%) of compound 15 as
a white solid. HPLC-MS (ESI, Gradient A): tR 13.8 min; positive
mode m/z 975.4 (100) [MþH]þ, 488.2 (66) [Mþ2H]2þ
[M-H]ꢀ; calcd for C43H66N12O8S3: 974.43 (100.0).
;
negative mode m/z 1087.5 (100) [MþTFA]ꢀ, 973.4 (15)
3,6-Bis(dimethylamino)-10-(4-(2-((phenethylimino)methyl)-
1H-imidazol-1-yl)butyl)acridinium (16). 2-Phenylethanamine
(6.12 μL, 5.9 mg, 0.048 mmol) was added to the suspension of 2
(8 mg, 0.019 mmol) with dry MgSO4 (50 mg) in CHCl3 (1 mL,
dry). Reaction mixture was stirred at 60 °C for 12 h, filtered over
Celite, and evaporated to dryness to give imine 16 (9.8 mg, 99%).
1H NMR (CDCl3, 400 MHz): δ 8.41 (s, 1H), 8.18 (s, 1H), 7.76
(d, 3J = 9.3 Hz, 2H), 7.15ꢀ7.08 (m, 3H), 7.08ꢀ7.00 (m, 6H),
6.49 (bs, 2H), 4.62 (bt, 3J = 7.8 Hz, 2H), 4.55 (t,3J = 6.8 Hz, 2H),
3.74 (t,3J = 7.2 Hz, 2H), 3.22 (s, 12H), 2.76 (t,3J = 7.2 Hz, 2H),
2.11ꢀ2.01 (m, 2H), 1.95ꢀ1.84 (m, 2H). MS (ESI) m/z (%)
519.5 (100) [M]þ; calcd for C33H39N6þ: 519.32 (100.0).
[Re(I)(16)(CO)3]þ (17). Imine 16 (10 mg, 0.019 mmol), KI
(50 mg, 0.3 mmol), and [NEt4]2[ReBr3(CO)3] (20 mg,
0.026 mmol) were consecutively dissolved in 4 mL of the mixture
of MeOH and PBS (1:1, pH = 6.4). Reaction was stirred for 4 h at
40 °C under N2. HPLC-MS analysis showed complete product
formation. The solvent was removed in vacuo and the product
was dried at high vacuum. The crude product was purified by
preparative HPLC to afford 8 mg (45%) of compound 17 as a
bright orange powder. 1H NMR (DMSO-d6, 400 MHz): δ 8.94
(s, 1H), 8.78 (s, 1H), 7.90 (d, 3J = 9.4 Hz, 2H), 7.70 (d, J = 1.3 Hz,
1H), 7.59 (d, J = 1.2 Hz, 1H), 7.24 (dd, 3J = 9.4 Hz, 4J = 2.0 Hz,
2H), 7.21ꢀ7.14 (m, 4H), 7.10ꢀ7.04 (m, 1H), 6.54 (bs, 2H),
’ ASSOCIATED CONTENT
S
Supporting Information. Detailed materials and methods.
b
This material is available free of charge via the Internet at http://
pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: ariel@aci.uzh.ch.
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4.78ꢀ4.73 (m, 2H), 4.36 (t, J = 6.8 Hz, 2H), 4.19ꢀ4.09 (m,
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