Clickable, hydrophilic ligand for fac-[MI(CO)3] + (M = Re/99mTc) applied in an S-functionalized α-MSH peptide

Article abstract of DOI:10.1021/bc5000115

The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction was used to incorporate alkyne-functionalized dipicolylamine (DPA) ligands (1 and 3) for fac-[M^I(CO)₃]⁺ (M = Re/ ^99mTc) complexation into

Full text of DOI:10.1021/bc5000115

Article
pubs.acs.org/bc
Clickable, Hydrophilic Ligand for fac-[MI(CO)3]+ (M = Re/99mTc)
Applied in an S‑Functionalized α‑MSH Peptide
Benjamin B. Kasten,† Xiaowei Ma,‡ Hongguang Liu,‡ Thomas R. Hayes,† Charles L. Barnes,§ Shibo Qi,‡
Kai Cheng,‡ Shalina C. Bottorff,† Winston S. Slocumb,† Jing Wang,∥ Zhen Cheng,*,‡
and Paul D. Benny*,†
†Department of Chemistry, Washington State University, Pullman, Washington 99164, United States
‡Molecular Imaging Program at Stanford (MIPS), Department of Radiology, Bio-X Program and Canary Center at Stanford for
Cancer Early Detection, Stanford University, Stanford, California 94305, United States
§Department of Chemistry, University of Missouri, Columbia, Missouri 65211-7600, United States
∥Department of Nuclear Medicine, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi, 710032, China
S
* Supporting Information
ABSTRACT: The copper(I)-catalyzed azide−alkyne cyclo-
addition (CuAAC) click reaction was used to incorporate
alkyne-functionalized dipicolylamine (DPA) ligands (1 and 3)
for fac-[MI(CO)3]+ (M = Re/99mTc) complexation into an α-
melanocyte stimulating hormone (α-MSH) peptide analogue.
A novel DPA ligand with carboxylate substitutions on the
pyridyl rings (3) was designed to increase the hydrophilicity
and to decrease in vivo hepatobiliary retention of fac-
[
99mTcI(CO)3]+ complexes used in single photon emission
computed tomography (SPECT) imaging studies with
targeting biomolecules. The fac-[ReI(CO)3(3)] complex (4)
was used for chemical characterization and X-ray crystal
analysis prior to radiolabeling studies between 3 and fac-
[
99mTcI(OH2)3(CO)3]+. The corresponding 99mTc complex (4a) was obtained in high radiochemical yields, was stable in vitro for
24 h during amino acid challenge and serum stability assays, and showed increased hydrophilicity by log P analysis compared to
an analogous complex with nonfunctionalized pyridine rings (2a). An α-MSH peptide functionalized with an azide was labeled
with fac-[MI(CO)3]+ using both click, then chelate (CuAAC reaction with 1 or 3 followed by metal complexation) and chelate,
then click (metal complexation of 1 and 3 followed by CuAAC with the peptide) strategies to assess the effects of CuAAC
conditions on fac-[MI(CO)3]+ complexation within a peptide framework. The peptides from the click, then chelate strategy had
different HPLC tR’s and in vitro stabilities compared to those from the chelate, then click strategy, suggesting nonspecific
coordination of fac-[MI(CO)3]+ using this synthetic route. The fac-[MI(CO)3]+-complexed peptides from the chelate, then click
strategy showed >90% stability during in vitro challenge conditions for 6 h, demonstrated high affinity and specificity for the
melanocortin 1 receptor (MC1R) in IC50 analyses, and led to moderately high uptake in B16F10 melanoma cells. Log P analysis
of the 99mTc-labeled peptides confirmed the enhanced hydrophilicity of the peptide bearing the novel, carboxylate-functionalized
DPA chelate (10a′) compared to the peptide with the unmodified DPA chelate (9a′). In vivo biodistribution analysis of 9a′ and
10a′ showed moderate tumor uptake in a B16F10 melanoma xenograft mouse model with enhanced renal uptake and surprising
intestinal uptake for 10a′ compared to predominantly hepatic accumulation for 9a′. These results, coupled with the versatility of
CuAAC, suggests this novel, hydrophilic chelate can be incorporated into numerous biomolecules containing azides for
generating targeted fac-[MI(CO)3]+ complexes in future studies.
into polypeptides range from standard solid and solution phase
techniques to biological incorporation using engineered codons
during recombinant expression of proteins.6−8 Due to these
benefits, the number and types of biological applications using
CuAAC have grown tremendously in recent years. In the field
INTRODUCTION
■
The copper(I)-catalyzed azide−alkyne cycloaddition (CuAAC)
reaction has become the icon of click chemistry since its
introduction by the Sharpless and Meldal laboratories.1,2
Numerous scientific disciplines (e.g., biology, chemical syn-
thesis, materials science, etc.) have utilized the rapid kinetics,
mild conditions, and orthogonality of CuAAC reactions for
applications ranging from bioconjugation to polymer syn-
thesis.3−5 Methods of introducing the azide or alkyne moiety
Received: January 8, 2014
Revised: February 7, 2014
Published: February 25, 2014
© 2014 American Chemical Society
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Scheme 1. Reaction Schemes and Structures of Compounds
of radiopharmaceuticals, CuAAC has been harnessed for
labeling a variety of biomolecules (e.g., small molecules,
peptides, antibodies, etc.) with short-lived radionuclides during
targeted nuclear imaging studies.9,10 The diagnostic radio-
nuclide 99mTc [t1/2 = 6.02 h, 140 keV γ (89%)] has nearly ideal
nuclear properties for single photon computed emission
tomography (SPECT). The fac-[99mTcI(OH2)3(CO)3]+ core
developed by Alberto has gained recognition for SPECT
applications due to its small molecular volume, versatile
chelation chemistry, straightforward preparation from an
aqueous-based kit, and amenability for readily labeling a wide
variety of biomolecules.11−14
(e.g., high temperature or pH) to drive complexation kinetics
with the fac-[99mTcI(CO)3]+ core.
Tridentate ligands containing aromatic nitrogen donors, such
as histidine or 2,2′-dipicolylamine (DPA), have been shown to
form kinetically and thermodynamically inert fac-
[
99mTcI(CO)3]+ complexes due to the chelate effect (tridentate
as opposed to mono- or bidentate ligands) and the low-spin d6
metal center.12,14,18−21 Despite the high in vitro and in vivo
stabilities of these complexes, their formation often requires
high temperatures (>90 °C) to achieve quantitative radio-
labeling yields in acceptable time frames (30−60 min).
Additionally, the lipophilic fac-[99mTcI(CO)3(DPA)]+ com-
plexes can lead to undesirable uptake and retention of the
labeled radiopharmaceutical in nontarget tissues such as the
liver and gastrointestinal tract.22,23 In an effort to generate a
versatile fac-[99mTcI(CO)3]+ complex with increased hydro-
philicity and high stability, we installed carboxylate groups on
the aromatic rings of an alkyne-functionalized DPA ligand.
Using CuAAC would allow the ligand or radiolabeled complex
to be easily incorporated into a wide spectrum of targeting
molecules containing an azide group. An azide-functionalized α-
melanocyte stimulating hormone (α-MSH) analogue was
chosen as a model targeting biomolecule to test this new
chelate system with fac-[99mTcI(CO)3]+.
CuAAC has been used to incorporate ligands for the fac-
[
99mTcI(CO)3]+ core into biomolecules by several strategies.15
In the click, then chelate strategy, a biomolecule functionalized
with an azide or an alkyne undergoes cycloaddition with a
bifunctional chelator containing the orthogonal coupling
partner prior to complexation with fac-[99mTcI(CO)3]+. The
click to chelate strategy developed by Schibli and Mindt is a
variant of this method where the 1,2,3-triazole generated by the
click reaction acts as an integral ligand donor for the fac-
[
99mTcI(CO)3]+ core.16,17 In an alternative chelate, then click
strategy, a bifunctional chelator is first complexed with 99mTc
and then joined to a biomolecule via CuAAC to generate the
final radiopharmaceutical. As the click reaction can be
accomplished under physiological temperature and pH, this
radiolabeling strategy enables the pairing of sensitive
biomolecules with ligands that require harsh labeling conditions
Malignant melanoma remains the deadliest form of skin
cancer due to its high metastatic rate and to challenges in
detecting and treating these metastases at early stages.24−27
Development of targeting peptides labeled with SPECT
isotopes to identify metastatic melanoma at early stages
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continues to be an active area of medical research.28−35 The
majority of these peptides are α-MSH analogues which bind to
the melanocortin-1 receptor (MC1R), a biomarker that is
overexpressed on primary and metastatic melanoma cells
(∼400 to 22 000 receptors/cell) compared to normal
cells.26,36−39 Several α-MSH analogues labeled with fac-
center following complexation as has been observed with other
fac-[ReI(CO)3(DPA)]+ complexes.47,49,50
The absolute coordination environment of 4 was confirmed
through single-crystal X-ray analysis (Figure 1). Crystallo-
[
99mTcI(CO)3]+ have shown promising results for detecting
models of melanoma in vivo during preclinical stud-
ies.22,32,33,40−42 Utilizing various chelates and attachment
strategies with α-MSH peptides has been shown to modulate
the in vivo properties of the ensuing fac-[99mTcI(CO)3]-labeled
peptides. While many conjugates have demonstrated undesired
uptake in the liver and gastrointestinal tract,22,32,40 introducing
carboxylates on a pyrazolyl-based ligand has recently been
shown to markedly reduce the hepatobiliary retention of α-
MSH peptide analogues after labeling with the fac-
[
99mTcI(CO)3]+ core.33
We hypothesized that the addition of carboxylates on a DPA
chelate would increase the hydrophilicity and decrease the
nonspecific hepatobiliary uptake of the resulting fac-
[
99mTcI(CO)3]+ complex compared to the original DPA ligand
while maintaining complex stability. To test this hypothesis, we
employed a previously characterized DPA-alkyne ligand, 1, and
the new, carboxylate-functionalized DPA-alkyne ligand, 3, in
parallel studies to incorporate the fac-[MI(CO)3]+ (M =
Re/99mTc) core into an α-MSH peptide by convergent
pathways (click, then chelate and chelate, then click) as shown
in Scheme 1. The in vitro stabilities of the complexed ligands
and peptides were determined, and the melanoma targeting
potentials of the labeled peptides were evaluated through (1) in
vitro MC1R affinity, (2) in vitro B16F10 cell uptake assays, and
(3) in vivo biodistribution analyses in B16F10 xenograft
models.
Figure 1. X-ray crystal structure of 4 with thermal ellipsoids shown at
the 50% probability level. Hydrogen atoms were omitted for clarity.
Atoms are colored as follows: C, gray; N, light blue; O, red; Re, dark
blue.
graphic details, bond lengths, and bond angles are given in the
Supporting Information (Tables S5−S7). Crystals of 4 packed
in the P21/c space group with four molecules in the unit cell.
The bond lengths and angles between the carbonyl ligands and
the metal center in 4 (1.918−1.939 Å and 89.18−91.16°,
respectively) were as expected for distorted octahedral fac-
[ReI(CO)3(DPA)]+ complexes.47,49 The bond lengths between
rhenium and the central nitrogen (2.227 Å) or the pyridal
nitrogens (2.163 and 2.176 Å) in 4 were also similar to those in
complex 2 (2.223 and 2.171 Å, respectively) as previously
characterized.47 This suggests that including the carboxylates
did not significantly perturb complexation with the fac-
[ReI(CO)3]+ core. The alkyne was pointed away from the
metal center as would be necessary for participation in CuAAC
reactions with azides.
RESULTS AND DISCUSSION
■
In an effort to generate a versatile ligand for the fac-
[MI(CO)3]+ core with enhanced hydrophilicity, we synthesized
a DPA chelate with carboxylate substitutions on the pyridal
rings and a terminal alkyne on the central amine. Reductive
amination between propargyl amine and methyl 6-formylnico-
tinate using sodium triacetoxyborohydride followed by basic
hydrolysis of the methyl ester groups provided the desired
ligand, 3, in moderate overall yield (55%) following isolation
and purification. Standard characterization techniques were
used to confirm the structure of the isolated material as the
desired compound. The IR spectrum contained a band at 3244
cm−1 which suggested a terminal alkyne group (CH stretching),
although the corresponding expected absorption in the 2000−
2200 cm−1 region (CC stretching) was not present.
However, 1H NMR analysis indicated the presence of a
propargyl-type system due to the resonances at 3.21 and 4.06
ppm for the terminal and methylene protons, respectively.
Reaction of 3 with fac-[ReI(OH2)3(CO)3](SO3CF3) at 70 °C
in mildly acidic (pH 6−6.5) aqueous conditions produced the
desired complex in moderate yield (66%) following purification
Having established that 3 coordinates fac-[ReI(CO)3]+ as
desired, we assessed its labeling efficiency with fac-
[
99mTcI(CO)3]+ to determine its potential for use in SPECT
imaging applications. Radiolabeling 3 with fac-
[
99mTcI(OH2)3(CO)3]+ at 70 °C yielded a single peak with a
similar RP-HPLC tR (19.2 min) as complex 4 (18.9 min),
indicating successful formation of the desired product, 4a
(Figure 2). After determining that complexation occurred as
expected, decreasing concentrations of 3 were used to examine
the effects of the carboxylate substitutions on the radiolabeling
efficiency compared to the original DPA-alkyne chelate, 1.
Quantitative labeling was achieved in 30 min at 70 °C when 3
was employed at 1 × 10−4 M with decreasing yields at lower
concentrations (Table S1). While extending the reaction time
to 1 h increased yields of 4a, labeling efficiency with 3 was
lower than with 1 to produce 2a when identical experimental
conditions were used.47 High decay corrected yields of 4a
(>91%) could be achieved following RP-HPLC purification. A
major objective in preparing the new ligand was to increase the
1
by preparatory RP-HPLC. As anticipated, H NMR analysis of
the isolated product, 4, showed the methylene protons of the
DPA moiety as an AB quartet (5.03/4.96 ppm) with a
downfield shift from their original appearance as a singlet in the
spectrum of 3 (4.62 ppm). ESI-MS analysis confirmed a
ligand:Re(CO)3 ratio of 1:1 in complex 4 as expected for
tridentate coordination at the DPA chelate site. IR analysis
indicated a facial arrangement of the carbonyls on the metal
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to incorporate the ligand or fac-[MI(CO)3]+ complex in a site-
specific manner. NAPamide was chosen because of its high
MC1R affinity and in vivo stability as demonstrated in previous
studies;39,52,53 we have recently employed a similar peptide with
fac-[MI(CO)3]+ using an alternative chelate.41 The thiol group
of cysteine presents an attractive moiety for functionalization
due to its lower pKa compared to other nucleophiles in
biomolecules (e.g., amines, alcohols). Native cysteine residues
are absent from many targeting biomolecules (e.g., peptides,
affibodies) or are not accessible due to disulfide bridge
formation (e.g., proteins, antibodies).54,55 Therefore, synthetic
or recombinant techniques can be employed to install a
nonessential cysteine residue into these scaffolds for selective
functionalization at locations that would not impair affinity for
the biological target. Cysteine was utilized to append an azide
functional group for attaching the alkyne-functionalized
chelates to the peptide via CuAAC. The 2-bromo-N-(3-
azidopropyl)-acetamide azide linker was chosen because it is
stable to hydrolysis in aqueous solution, and it has previously
been used for selective S-alkylation of proteins to incorporate
an azide functionality for subsequent CuAAC reactions.56
The Cys-NAPamide peptide, 5, was prepared using solid
phase peptide synthesis with Fmoc-protected amino acids in
high purity as indicated by RP-HPLC analysis (Figure S1).
Following TCEP reduction of the peptide, alkylation was
performed overnight at room temperature with a slight excess
of 2-bromo-N-(3-azidopropyl)-acetamide in a PBS/MeCN
mixture. These conditions ensured maximum alkylation of the
starting peptide with minimal side product (e.g., dialkylation)
formation. The alkylated peptide, 6, was isolated in moderately
high yields (>70%) and high purity (>99% by RP-HPLC,
Figure S2) following RP-HPLC purification. ESI-MS analysis
confirmed the identity of the product as the desired
monoalkylated species (1341.1 m/z, Table 1). IR analysis
showed a prominent absorption at 2103 cm−1 as anticipated for
the presence of an azide group. Evaluation of 6 with Ellman’s
reagent confirmed that that the azide linker had been
incorporated at the cysteine residue as no free thiols (<1%)
were detected during analysis.
Figure 2. Normalized and offset RP-HPLC chromatograms of 4 (UV
absorbance, 254 nm; lower blue trace) at tR 18.9 min and 4a
(radiodetector, counts per minute (cpm); higher red trace) at tR 19.2
min.
hydrophilicity of the resulting metal complex and to decrease
hepatobiliary retention of radiopharmaceuticals with this
complex compared to the regular DPA complex. Log P analysis
(octanol/PBS partition coefficient) of complexes 2a and 4a
yielded values of −0.23 0.01 and −1.85 0.02, respectively,
confirming that the carboxylate-functionalized DPA complex
was more hydrophilic than the regular DPA complex at
physiological pH.
Targeted radiopharmaceuticals should maintain high bio-
logical stability with the radionuclide to avoid nonspecific
uptake or retention of radioactive species in nontarget tissues
during in vivo applications. Serum stability analyses and amino
acid challenge assays were performed with purified solutions of
4a to assess its resistance to transchelation or oxidation under
biologically relevant conditions (Table S2). As expected from
previous studies employing DPA complexes with fac-
[
99mTcI(CO)3]+,20,21,23,51 4a was >98% stable in serum and in
Following incorporation of the azide linker, two pathways
were examined to label the peptide with fac-[MI(CO)3]+: click,
then chelate and chelate, then click. Both pathways have
previously been utilized with DPA ligands to form metal
complexes with biologically relevant small molecules through
CuAAC reactions.47,51 The click, then chelate route is analogous
to the prevailing strategy in the literature where a targeting
species is functionalized with a bifunctional chelator prior to
the presence of 1 mM histidine or cysteine up to 24 h. These in
vitro results suggested that the chelate would be suitable for
further exploration within a radiopharmaceutical construct for
in vivo applications.
The encouraging radiolabeling results with the novel chelate
prompted its evaluation in a targeting peptide framework. A
NAPamide α-MSH peptide containing a nonessential cysteine
residue (Cys-NAPamide) was chosen as an initial model system
Table 1. Characterization Results for Non-Radioactive Peptides
calcd MW (m/z)
obsvd MW (m/z)
RP-HPLC tR (min)
IC50 (nM)
a
5
6
7
[M+2H]2+: 601.8
[M+2H]2+: 671.8
[M+2H]2+: 790.4,
[M+2H+Cu]2+: 821.9
[M+2H]2+: 834.4,
[M+2H+Cu]2+: 865.8
[M+H]2+: 925.4
602.1
672.0
790.7,
821.2
834.7,
866.2
925.7
968.7
925.5
1936.3
19.0
2.19 0.54
9.60 1.74
--
a
18.7
b
25.2
b
8
25.7
--
c
9
14.6
--
Click, then chelate
Chelate, then click
b
c
10
[M+H]2+: 968.9
14.4
--
c
9′
10′
[M+H]2+: 925.4
[M−H]−: 1936.7
14.9
3.57 1.15
3.13 0.29
c
15.3
a
c
HPLC method 3. HPLC method 4. HPLC method 6.
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complexation with the metal. The chelate, then click approach
was used as an alternative strategy to generate the metal-
complexed peptides to determine if the presence of the fac-
[MI(CO)3]+ core prior to the CuAAC reaction affected
formation of the desired products. CuAAC reactions were
performed to append ligands 1 or 3 to peptide 6 in order to
yield constructs 7 and 8 containing the standard DPA chelate
and the new DPA chelate bearing carboxylates, respectively.
Due to the likelihood of copper coordination by the DPA
chelates,51,57 excess Cu(OAc)2 was used in the CuAAC
reactions to ensure that enough copper(I) would be available
to catalyze the desired cycloaddition reactions. After RP-HPLC
analysis indicated completion of the reactions, EDTA or
sodium sulfide was added to remove copper and the crude
peptides were purified to a single peak by RP-HPLC (Table 1,
Figures S3 and S4). ESI-MS analysis indicated that the peptides
were produced as desired due to the resonances at 790.7 m/z
and 834.7 m/z for the [M+2H]2+ ions of 7 and 8, respectively
(Table 1), although copper complexes of these peptides were
also apparent as minor species in the MS spectra (821.2 m/z
and 866.2 m/z for [M+2H+Cu]2+ ions of 7 and 8, respectively,
Table 1). This result is similar to what has been observed
previously with the DPA-alkyne ligand, 1, following CuAAC
reaction with a functionalized azide.51 Using excess sodium
sulfide to remove residual copper from the peptides by forming
CuS was only partially successful; employing precoordinated
copper complexes (e.g., tris-(benzyl-triazolylmethyl)amine)
during the CuAAC reactions also failed to yield 7 and 8
without residual copper present (data not shown). Despite the
presence of copper in the peptides, subsequent reactions of 7
and 8 with fac-[ReI(OH2)3(CO)3](SO3CF3) yielded the
rhenium-complexed peptides 9 and 10, respectively, following
RP-HPLC purification (Figures S5 and S6). ESI-MS analysis of
9 and 10, which showed the anticipated [M+H]2+ ions at 925.7
m/z and 968.7 m/z, respectively, confirmed coordination of the
fac-[ReI(CO)3]+ core to the peptides (Table 1), and no copper
was evident during analysis following fac-[ReI(CO)3]+ complex-
ation. Overall yields of 9 and 10 from the starting azide-
functionalized peptide 6 via the click, then chelate strategy were
50% and 39%, respectively.
desired coordination of the fac-[ReI(CO)3]+ core in the click,
then chelate strategy. In subsequent reactions, the peptides
obtained by the chelate, then click strategy (9′ and 10′) were
hypothesized to possess the desired N,N,N coordination mode
exclusively at the chelate position because metal complexation
with the DPA ligands, which possess well-defined coordination
and high stability with the fac-[ReI(CO)3]+ core,47,49,58 was
performed prior to attachment to the peptide. The rhenium-
complexed peptides produced by the click, then chelate pathway
(9 and 10) were likely coordination isomers of the desired
products and were not pursued in the biological assays below.
Peptides 5, 6, 9′, and 10′ were used in B16F10 melanoma
cell competitive binding assays with a standard radiolabeled
MC1R binding peptide, 125I-(Tyr2)-NDP, to confirm that the
modifications made to the peptides through linker attachment
and metal complexation did not abrogate their affinity for the
MC1R. These assays yielded IC50 values in the low nanomolar
range for the peptides (Table 1), although the IC50 values were
higher than that of the previously reported NAPamide
sequence when tested with B16F1 melanoma cells (0.27
nM).59 The high affinity of the metalated analogues supported
further exploration of the peptides with 99mTc for targeting
melanoma in SPECT applications.
Both synthetic pathways used with the rhenium analogues
were explored for producing the 99mTc-complexed peptides. In
the click, then chelate pathway, peptides 7 and 8 containing the
DPA chelate and the carboxylate-functionalized DPA chelate,
respectively, were directly complexed with fac-
[
99mTcI(OH2)3(CO)3]+ at 70 °C for 1 h in PBS. RP-HPLC
analysis showed similar tR’s for these 99mTc-labeled peptides,
designated 9a and 10a (Table 2, Figures S5 and S6), as was
Table 2. RP-HPLC tR’s and Isolated Radiolabeling Yields of
Peptides Labeled with 99mTc Following RP-HPLC
Purification
Click, then chelate
Chelate, then click
9a′ 10a′
9a
10a
a
a
a
a
RP-HPLC tR (min)
14.6
14.4
47%
15.1
42%
15.5
71%
isolated, decay-corrected yields
84%
a
The chelate, then click route was then examined to compare
overall reaction yields from 6 and to determine if the products
obtained corresponded to those via the click, then chelate route.
A particular advantage of the chelate, then click synthetic strategy
is that the ligand is complexed with the fac-[MI(CO)3]+ center
prior to cycloaddition with the azide on the peptide, assuring
that the final metal-complexed product is obtained as desired
without the possibility of mixed coordination species.
Surprisingly, RP-HPLC analysis of the chelate, then click
reactions between peptide 6 and complexes 2 and 4 indicated
that species with slightly different tR’s were formed compared to
those from the click, then chelate pathway (Table 1). ESI-MS
analysis of these new species, designated 9′ for the analogue
with the regular DPA complex and 10′ for the analogue with
the carboxylate-functionalized DPA complex, following RP-
HPLC purification of the reactions confirmed that the fac-
[ReI(CO)3]+-labeled peptides were successfully obtained with-
out residual copper via this synthetic pathway (925.5 m/z for
[M+H]2+ ions of 9′ and 1936.3 m/z for [M−H]− ions of 10′,
Table 1). Yields for 9′ and 10′ obtained by the chelate, then click
strategy from peptide 6 were 47% and 70%, respectively. The
different tR’s of 9/9′ and 10/10′ suggest that the presence of
copper prior to rhenium complexation negatively affected the
HPLC method 6.
observed for the respective rhenium analogues, 9 and 10 (Table
1), produced by the click, then chelate strategy. This suggested
that similar coordination modes were obtained with both fac-
[MI(CO)3]+ cores using these peptides. Radiolabeling yields of
9a and 10a after 1 h at 70 °C were greater than 93%
(conversion from fac-[99mTcI(OH2)3(CO)3]+) when the
peptides were used at ∼5 × 10−5 M with declining yields at
shorter times and lower concentrations as was observed with
the ligands (1 and 3) before attachment to the peptides.
Purification of the products by RP-HPLC yielded 9a and 10a in
moderate decay-corrected yields (Table 2).
The chelate, then click pathway was next explored to produce
the 99mTc-complexed peptides 9a′ and 10a′. Performing the
CuAAC reactions with ∼5 × 10−5 M 6 and crude reaction
mixtures of 2a or 4a at 50 °C in PBS yielded 9a′ and 10a′
quantitatively (>98% conversion from 2a or 4a) in as little as
30 min, although decay-corrected yields of 9a′ and 10a′ were
moderate following RP-HPLC isolation (Table 2). Comparison
of the RP-HPLC tR’s of the Re and 99mTc analogues produced
by the chelate, then click route validated the identities of 9a′ and
10a′ (Tables 1 and 2, Figures 3 and 4). As was observed with
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following purification (data not shown). This suggests that
residual copper which was apparent in peptides 7 and 8 prior to
complexation with the fac-[99mTcI(CO)3]+ core led to less
stable coordination modes or may have negatively impacted the
integrity of the radiolabeled peptides (e.g., through metal-
mediated peptide hydrolysis60,61). Employing the precom-
plexed ligands in the chelate, then click strategy did not suffer
these drawbacks, thus highlighting the benefit of this synthetic
pathway for generating the desired fac-[99mTcI(CO)3]+-
complexed peptides, 9a′ and 10a′. Having determined which
species were stable in solution, the effects of the two chelates
(regular DPA vs carboxylate-functionalized DPA) on the overall
hydrophilicity of the peptides was examined. Under the acidic
conditions (0.1% TFA) used for RP-HPLC analysis, the
analogues bearing the novel chelate (8, 10′, 10a′) had slightly
longer tR’s compared to their counterparts with the unmodified
DPA chelate (7, 9′, 9a′). This observation could be explained
by protonation of one of the carboxylates on the modified DPA
chelate at low pH, leading to a zwitterionic form of the chelate
in 8 and an overall neutral charge for the metal complexes in
10′ and 10a′. The DPA ligand in 7, on the other hand, was
protonated at low pH; complexation with the metal in 9′ and
9a′ retained the positive charge from the fac-[MI(CO)3]+ core
regardless of pH. Thus, the tR’s of the compounds under the
acidic RP-HPLC conditions did not adequately correlate to the
anticipated hydrophilicity of the compounds at physiological
pH. Log P analysis at neutral pH confirmed the increased
hydrophilicity of 10a′ (−2.03 0.01) compared to 9a′ (−0.68
0.04) as would be desired for generating a targeted peptide
with less hepatobiliary retention compared to the more
hydrophobic analogue.
Figure 3. Normalized and offset RP-HPLC chromatograms of 9′ (UV
absorbance, 220 nm; lower blue trace) at tR 14.9 min and 9a′
(radiodetector, counts per minute (cpm); higher red trace) at tR 15.1
min produced by the chelate, then click route.
Following the radiolabeling and characterization studies
above, the in vitro B16F10 melanoma cell uptake and MC1R
specificity of 9a′ and 10a′ were explored at 0.5, 1, and 2 h
(Figure 5). 9a′ exhibited moderate accumulation in B16F10
melanoma cells at 37 °C, reaching 2.71 0.18%, 3.30 0.44%,
and 5.86
1.07% of applied activity at 0.5, 1, and 2 h,
Figure 4. Normalized and offset RP-HPLC chromatograms of 10′
(UV absorbance, 238 nm; earlier blue trace) at tR 15.3 min and 10a′
(radiodetector, counts per minute (cpm); later red trace) at tR 15.5
min produced by the chelate, then click route.
the rhenium-complexed peptides, different RP-HPLC tR’s of
9a/9a′ and 10a/10a′ were observed for the products of the
chelate, then click route compared to the click, then chelate route
(Table 2).
The in vitro stabilities of the radiolabeled peptides were
examined prior to testing their in vivo properties. It was likely
that the stabilities of 9a and 10a would differ from 9a′ and 10a′
due to the potentially different fac-[99mTcI(CO)3]+ coordina-
tion modes resulting from the click, then chelate and chelate, then
click strategies. Peptide 9a′ was >99% stable in the presence of
either histidine or cysteine at 37 °C for 4 h with slightly lower
stability (84%) in cysteine at 18 h (Table S3). Peptide 10a′
exhibited >99% stability under these conditions for 18 h (Table
S3). Both 9a′ and 10a′ were >90% stable for 6 h at 37 °C in
serum (Table S4) which indicated they would be candidates for
further biological analysis. RP-HPLC analysis of 9a and 10a
produced via the click, then chelate route, however, showed
multiple species at times beyond 2 h in the above challenge
conditions and when incubated in PBS at room temperature
Figure 5. In vitro B16F10 cell uptake assay of 9a′ and 10a′. B16F10
cells were incubated with 1 μCi of 9a′ or 10a′ for 0.5, 1, and 2 h at 37
°C with or without NDP (3 μM) as a blocking agent. Data represent
the amount of activity associated with the cells as a percentage of the
total activity applied standard deviation and are the average of two
individual experiments performed in quadruplicate. 9a′ only (black
squares with solid black line), 9a′ with NDP (black circles with dotted
black line), 10a′ only (red triangles with solid red line), and 10a′ with
NDP (red triangles with dotted red line).
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Figure 6. Biodistribution (A) and tumor-to-normal tissue ratios (B) of 9a′ and 10a′ in B16F10 tumor-bearing C57BL/6 mice 1 and 2 h after
injection of approximately 100 μCi of 9a′ and 10a′ via the tail vein. Data are expressed as % ID/g standard deviation (n = 4 per group).
respectively. Interestingly, increased cell uptake values were
observed for 10a′, reaching 2.75 0.72%, 4.01 0.47%, and
9.40 1.08% at 0.5, 1, and 2 h, respectively. The significantly
higher uptake of 10a′ compared to 9a′ at 2 h (P < 0.05) in the
melanoma cells was surprising considering that the rhenium-
complexed analogues exhibited similar IC50 values in the MC1R
binding assays (3.57 1.15 nM for 9′ and 3.13 0.29 for 10′).
To confirm whether the binding of 9a′ and 10a′ with B16F10
cells was due to specific interactions with MC1R, the cells were
coincubated with solutions containing 9a′ or 10a′ and excess
amounts of unlabeled NDP for 0.5, 1, and 2 h at 37 °C. As
shown in Figure 5, these blocking conditions caused a decrease
in the uptake of the radiolabeled peptides at 2 h from 5.86
1.07% to 1.42 0.08% (P < 0.05) for 9a′ and from 9.40
1.08% to 1.20 0.22% (P < 0.05) for 10a′. Thus, unlabeled
NDP significantly reduced the binding of 9a′ and 10a′ to
B16F10 cells, indicating that the uptake of the radiolabeled
peptides was mediated by the MC1R.
and 0.49
0.15% for blood, lung, pancreas, and intestine,
respectively). The different accumulations of 9a′ and 10a′
observed in the liver and kidneys confirmed that incorporation
of the carboxylates on the DPA complex in 10a′ altered the
properties of the resulting radiopharmaceutical in vivo
compared to the original DPA complex in 9a′. However, the
amount of 10a′ observed in the intestines suggests the desired
goal of decreasing hepatobiliary retention by using the
hydrophilic complex was not achieved. The lower level of 9a′
in the intestines at 2 h (0.49 0.15%) compared to 10a′ (2.87
0.63%) suggests that the carboxylate substitutions may have
increased the rate of clearance through the liver without
significantly modifying total hepatobiliary uptake. Although the
greater levels of 10a′ in the kidneys and in the blood and could
be due to its increased hydrophilicity (compared to 9a′),
prolonged blood retention of the radionuclide using this
analogue would adversely affect signal-to-noise ratios of MC1R-
expressing tissues during SPECT imaging.
The above in vitro stability and cell uptake results
encouraged the investigation of 9a′ and 10a′ as potential
SPECT peptides for targeting melanoma in vivo. The
biodistribution patterns of 9a′ and 10a′ were examined in
B16F10 melanoma mouse xenograft models at 1 and 2 h to
quantify the uptake of these peptides following intravenous
administration. The results are presented in Figure 6. Peptide
9a′ accumulated to the greatest extent in the liver and kidneys
The two different chelates did not appear to affect MC1R
affinity in vivo as tumor uptake was comparable for both
peptides, reaching 0.79 0.19% for 9a′ and 1.06 0.16% for
10a′ at 1 h p.i. and declining to 0.73 0.05% for 9a′ and 0.71
0.14% for 10a′ at 2 h p.i. The tumor-to-blood ratio (Figure
6) slightly increased for 9a′ at 2 h p.i. (1.2 0.13) compared to
1 h p.i. (0.7 0.17) as would be desired for obtaining SPECT
images of target tissues with low background levels at later time
points. Although the amount of 10a′ in the blood also
decreased over time, its higher overall blood retention led to
lower tumor-to-blood ratios (∼0.4 at both 1 and 2 h p.i.)
compared to 9a′. Many tissues (stomach, brain, muscle, bone,
skin) exhibited lower uptake and retention of 9a′ and 10a′
compared to the melanoma tumors (Figure 6) as would be
desired for diagnostic imaging of malignant melanoma.
However, due to the rates of melanoma metastases in the
hepatobiliary and gastrointestinal systems,65 additional mod-
ifications to 9a′ and 10a′ would be required to minimize the
nonspecific retention of these peptides in soft tissues prior to
further development in SPECT imaging applications.
(5.92
0.87% and 2.28
0.18% at 2 h p.i., respectively),
although retention in the spleen and lung was also observed at
2 h p.i. (1.24 0.35% and 1.00 0.53%, respectively). Other
peptides and small molecule targeting ligands with fac-
[
99mTc(CO)3(DPA)]+ complexes have shown similar biodis-
tribution patterns with high uptake in the hepatobiliary
system.23,62−64 Using the carboxylate-substituted DPA complex
in peptide 10a′ was anticipated to decrease the hepatobiliary
retention of this analogue relative to 9a′ due to the increased
hydrophilicity of the novel chelate. Compared to 9a′, peptide
10a′ exhibited greater renal (3.69 0.40%) than hepatic (2.52
0.61%) accumulation at 2 h p.i.; retention of 10a′ in the
blood (1.91 0.33%), lung (1.50 0.48%), pancreas (0.91
0.25%), and intestine (2.87 0.63%) was also higher compared
to 9a′ at 2 h p.i. (0.61 0.10%, 1.00 0.53%, 0.07 0.03%,
A variety of chelates for the fac-[MI(CO)3]+ core have been
designed with modifications to increase the hydrophilicity and
renal clearance of the resulting complexes.21,23,33,64,66 Several
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have employed the single amino acid chelate (SAAC) approach
which is useful for inserting ligands at nearly any desired
location in peptides made by solid phase synthesis.21,23,58
Pyrazolyl chelates modified with carboxylate groups have also
been employed in α-MSH peptides for targeting melanoma.33
Both strategies have successfully produced peptides with lower
soft tissue retention and greater excretion through renal rather
than hepatobiliary pathways compared to unmodified chelate
structures. The DPA chelates used in the present study were
intended to build on the above concepts by employing a
versatile linker strategy that could be utilized with peptides or
even larger macromolecules produced by both synthetic and
recombinant techniques. Such techniques could be used to
insert azide-bearing residues (or orthogonal reactive groups for
selectively incorporating azides) for chelate attachment via
CuAAC at potentially at any location in the sequence of the
biomolecule, thus promoting the flexibility of the chelation
strategy used here compared to the other strategies mentioned
above.
through the click, then chelate route produced peptides with
inferior in vitro properties compared to those generated by the
chelate, then click strategy. The fac-[MI(CO)3]+-complexed
analogues generated by the chelate, then click strategy were
stable to transchelation, possessed high affinity for the MC1R,
and demonstrated encouraging uptake values in B16F10
melanoma cells in vitro. Biodistribution analysis of the peptides
bearing the two different DPA chelates demonstrated
moderately low uptake in melanoma xenografts in vivo.
While the α-MSH peptide with the new carboxylate-function-
alized complex showed slightly increased renal accumulation
and less hepatic retention than the alternative hydrophobic
analogue, the desired goal of decreasing hepatobiliary retention
by increasing the hydrophilicity of this analogue was not
obtained. However, the versatility of the chelate, then click
strategy with either of the chelates used here maintains the
potential to incorporate the fac-[MI(CO)3]+ core into a wide
variety of biomolecules bearing azide moieties for generating
targeted radiopharmaceuticals via CuAAC.
While the chelates and CuAAC strategies used here are
expected to be generally applicable for labeling biomolecules
with the fac-[MI(CO)3]+ core, several limitations are worth
noting. First, it is possible that incorporating modified residues
within highly optimized targeting ligands (e.g., small molecules,
short peptides) could negatively affect their binding affinities or
pharmacokinetic properties compared to the original targeting
moiety. Second, the unstable nature of the metal-complexed α-
MSH peptides generated through the click, then chelate route in
this study suggests that residual Cu from the CuAAC reaction
can potentially lead to undefined fac-[MI(CO)3]+ complexes
during applications with complex biomolecules. This drawback,
along with other limitations associated with CuAAC click
reactions (e.g., purification requirements to avoid Cu toxicity,
transchelation, etc.) must be considered during future
applications with the click, then chelate strategy in particular.
Additionally, using the carboxylate-modified DPA complex with
the α-MSH peptide in the present study did not lead to the
desired decrease in hepatobiliary retention compared to the
unmodified DPA complex. Despite these observations, the
hydrophilic ligand for the fac-[MI(CO)3]+ core in the chelate,
then click strategy confirmed that its incorporation into a
targeting peptide did not significantly impair peptide function
when compared with a known DPA complex containing the fac-
[MI(CO)3]+ core. The chelate, then click strategy prevents the
possibility of undefined or unstable coordination isomers of the
metal core at undesired sites in targeting biomolecules and can
allow even temperature-sensitive biomolecules to be rapidly
labeled with the fac-[MI(CO)3]+ core under mild conditions.47
Therefore, utilizing this strategy with either of the metal
complexes from the present study would be suitable for
generating a wide variety of targeting radiopharmaceuticals in
future applications.
EXPERIMENTAL PROCEDURES
■
All reagents and solvents were of reagent grade or higher from
commercial suppliers (Aldrich, Fluka, Acros, Fisher) and used
as received unless noted otherwise. fac-[ReI(OH2)3(CO)3]-
(SO3CF3),43 methyl 6-formylnicotinate,44 2-bromo-N-(3-azido-
propyl)-acetamide,45 N,N-bis(pyridine-2-ylmethyl)prop-2-yn-1-
amine46 (ligand 1), and fac-[ReI(CO)3(1)](SO3CF3)47 (com-
plex 2) were prepared from literature procedures. [99mTcO4]−
was obtained from Cardinal Health (Spokane, WA) or from
Stanford Nuclear Medicine Clinic and was used to prepare fac-
[
99mTcI(OH2)3(CO)3]+ via commercially available Isolink kits
(Tyco, Inc.) as previously described.41 125I-(Tyr2)-[Nle4,D-
Phe7]-α-MSH [125I-(Tyr2)-NDP] was purchased from Perkin-
Elmer (Waltham, MA). Rink Amide LS resin, hydroxybenzo-
triazole (HOBt), and 9-fluorenylmethoxycarbonyl (Fmoc)
protected amino acids were purchased from Advanced
ChemTech (Louisville, KY). Elemental analysis was performed
by Intertek Pharmaceutical Services, Inc. (Whitehouse, NJ).
Nuclear magnetic resonance (NMR) spectra were recorded at
293 K on 300 or 400 MHz Varian Mercury Vx spectrometers
1
using 5 mm NMR tubes. H and 13C NMR spectra peak
positions were referenced using residual solvent signals, and
spectra were processed using Varian VNWR 6.1 software. Mass
spectra were obtained on a Thermo-Finnigan LCQ Advantage
instrument for electrospray ionization mass spectrometry (ESI-
MS). Infrared (IR) spectra were recorded on a Thermo
Nicolett 6700 FTIR with an ATR cell and analyzed with
OMNIC 7.1a software. UV/vis spectra were recorded on a
Varian Cary 50 Bio spectrophotometer and analyzed with Cary
WinUV 3.00 software. Solutions of the compounds were
prepared in UV/vis quality methanol and measured in 1 cm
path length quartz cuvettes. Analytical separation and
identification of small molecules and peptides by reversed-
phase high performance liquid chromatography (RP-HPLC)
were conducted using a Varian Pursuit XRs column (C18, 5
μm, 4.6 × 250 mm) with a Phenomenex security guard
cartridge (C18, 4.0 × 3.0 mm). A Perkin-Elmer Series 200
analytical chromatography system equipped with a Perkin-
Elmer Radiomatic 610TR detector and a Hitachi D-7000 series
analytical chromatography system (L-7100 pump, L-7400 UV
detector) equipped with a Berthold FlowStar LB 513
radiodetector were used for the above compounds. Unless
noted otherwise, elutions performed on these systems used a
CONCLUSIONS
■
In summary, a novel chelate based on DPA with enhanced
hydrophilicity has been developed and utilized with the fac-
[MI(CO)3]+ core. Structural assessment using Re and radio-
labeling studies with 99mTc demonstrated that this chelate’s
performance was comparable to a well characterized DPA
ligand. An α-MSH analogue with an S-functionalized azide
linker was used as a model biomolecule to incorporate the
alkyne-functionalized chelates and fac-[MI(CO)3]+ complexes
into the peptides via CuAAC reactions. Metal complexation
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gradient profile (HPLC method 1, previously described as
“Gradient 1”)48 with 0.1% TFA in water (solvent A) and
MeOH (solvent B) as solvents. Chromatograms were plotted
using OriginPro 8.5.1 (OriginLab Corporation, MA). Prepar-
atory RP-HPLC separations and purifications of small
molecules used a Phenomenex Gemini-NX column (C18, 5
μm, 21.2 × 250 mm) with a Phenomenex security guard
cartridge (C18, 21.2 × 15 mm) on a Hitachi D-7000 series
semipreparative chromatography system (L-7150 pump, L-
7400 UV detector). Chromatographic analysis and purification
of the starting peptide (5; Scheme 1) was performed on a
Dionex Ultimate 300 HPLC system (Dionex Corp.) using both
semipreparative (Vydac; 218TP510-C18, 5 μm, 10 × 250 mm)
and analytic (Vydac; 214TP54-C18, 5 μm, 4.6 × 250 mm)
peptide RP-HPLC columns. The mobile phase gradients used
0.1% TFA in water (solvent A) and 0.1% TFA in acetonitrile
(MeCN, solvent C). Radio-HPLC purification and analysis
with the radiolabeled peptides used for biological analysis (9a′
and 10a′; Scheme 1) was conducted with a semipreparative RP-
HPLC Vydac peptide column on a Dionex 680 chromatog-
raphy system with a UVD 170U absorbance detector and
model 105S single-channel radiation detector (Carroll &
Ramsey Associates, Berkley, CA) using a gradient method
(HPLC method 2: 1 mL/min flow rate; 0−3 min 95% A/5% C,
3−8 min linear gradient to 72% A/28% C, 8−33 min linear
gradient to 67% A/33% C, 33−36 min linear gradient to 5% A/
95% C, 36−39 min hold 5% A/95% C, return to 95% A/5% C
after 39 min and equilibrate). The recorded data were
processed with use of Chromeleon v 6.50 software (Sunnyvale,
CA) and were plotted using Origin 8.0 (MicroCal). The
B16F10 murine melanoma cell line was obtained from
American Type Culture Collection (Manassas, VA). Female
C57BL/6 mice were purchased from Charles River Laboratory
(Wilmington, MA).
Small Molecule Synthesis and Complexation Reac-
tions. 6,6′-((Prop-2-yn-1-ylazanediyl)bis(methylene))-
dinicotinic Acid, 3. Methyl 6-formylnicotinate (0.250 g, 1.514
× 10−3 mol) was dissolved in 1,2-dichloroethane (10 mL) while
stirring under N2. Propargyl amine (0.042 mL, 6.58 × 10−4
mol) was added followed by sodium triacetoxyborohydride
(0.349 g, 1.645 × 10−3 mol) and the resulting mixture was
stirred for 3 h. Water (10 mL) was added to quench the
reaction and the solution was stirred for an additional 5 min.
The solution was diluted with 20 mL CH2Cl2 and 20 mL 1 M
NaOH. The organic layer was collected and the aqueous layer
was washed with CH2Cl2 (2 × 20 mL). The combined organic
layers were dried with MgSO4, filtered, and concentrated to
dryness. The resulting yellow oil was dissolved in MeOH and a
1 M solution of NaOH (0.724 mL, 7.24 × 10−4 mol) was
added. The reaction was heated to reflux for 2 h and then
concentrated to dryness under vacuum. The crude residue was
redissolved in H2O and brought to pH 2−3 with 1 M HCl. The
precipitate that formed during acidification was collected by
filtration and rinsed with ice-cold H2O to yield 41.3 mg of the
desired product. The remaining filtrate was purified by
preparatory RP-HPLC using a gradient method (prep-HPLC
method 1) with the following conditions: 10 mL/min flow rate;
0−3 min 100% A, 3−9 min 75% A/25% B, 9−25 min linear
gradient to 100% B, 25−32 min 100% B, 32−33 min linear
gradient to 100% A, 33−38 min equilibrate in 100% A; UV at
220 nm. Product peak tR: 20.6 min. Purified portions were
combined and concentrated to dryness under vacuum to yield
an additional 104 mg of the desired product as a brown
powder. Combined yield, 55%. Anal. Calcd for C17H15N3O4·
2.25 HCl: C, 50.12; H, 4.27; N, 10.32. Found: C, 50.05; H,
1
3.61; N, 10.06. H NMR [δ (ppm), 400 MHz, CD3OD]: 9.20
(d, CPyH, 2 H), 8.50 (dd, CPyH, 2 H), 7.72 (d, CPyH, 2 H), 4.62
(s, Py-CH2-N, 4 H), 4.06 (d, N-CH2-C, 2 H), 3.21 (dd, CCH, 1
H). 13C NMR [δ (ppm), 100 MHz, D2O]: 164.9 (COO), 155.4
(Ar-C), 146.2 (d, 32.8 Hz, Ar-C), 143.7 (d, 28.8 Hz, Ar-C),
129.4 (Ar-C), 127.0 (t, 36.2 Hz, Ar-C), 76.8 (d, 73.2 Hz, N-
CH2), 75.6 (d, 14.8 Hz, N-CH2), 55.0 (t, 25.7 Hz, CH2-C
CH), 42.6 (t, 14.1 Hz, CCH). MS (+ESI): 326.1 m/z; calcd
for [M+H]+: 326.1 m/z. IR (solid, cm−1): 3244, 2716, 1738,
1607, 1194, 1121. UV/vis εmax (223 nm): 27 500 M−1 cm−1.
fac-[ReI(CO)3(N,N,N-3)], 4. Ligand 3 (4.9 × 10−5 mol) was
suspended in 2 mL H2O and dissolved by adding 1 M NaHCO3
until pH ∼7.5 was achieved. The pH was adjusted to 6.5−7
with 0.1 M HCl, and a 0.1 M solution of fac-
[ReI(OH2)3(CO)3](SO3CF3) (0.5 mL, 5 × 10−5 mol) was
added. The solution was stirred at 70 °C for 6.5 h while
maintaining pH 6.5−7 by addition of 1 M NaHCO3 or 0.1 M
HCl as needed. The reaction was then cooled, filtered, and
purified by preparatory RP-HPLC using a gradient method
(prep-HPLC method 2) with the following conditions: 10 mL/
min; 0−3 min 100% A, 3−10 min 75% A/25% B, 10−32 min
linear gradient to 100% B, 32−40 min 100% B, 40−45 min
equilibrate in 100% A; UV at 220 nm. Product peak tR: 26.6
min. Purified portions were combined and concentrated to
dryness under vacuum to yield 19 mg (66%) of the desired
product as a grayish-brown powder. X-ray quality crystals were
obtained by vapor diffusion of ethyl acetate into a solution of 4
dissolved in EtOH. Anal. Calcd for C20H14N3O7Re·H2O: C,
1
39.22; H, 2.63; N, 6.86. Found: C, 39.77; H, 2.40; N, 6.47. H
NMR [δ (ppm), 300 MHz, CD3OD]: 9.33 (d, CPyH, 2 H), 8.48
(dd, CPyH, 2 H), 7.71 (d, CPyH, 2 H), 5.03 (d, J = 18.0 Hz, Py-
CH2A-N, 2 H), 4.96 (d, J = 17.7 Hz, Py-CH2B-N, 2 H), 4.64 (d,
N-CH2-C, 2 H), 3.41 (dd, CCH, 1 H). 13C NMR [δ (ppm), 75
MHz, CD3OD]: 165.3 (COO), 165.1 (Ar-C), 154.1 (Ar-C),
142.2 (Ar-C), 130.7 (Ar-C), 124.7 (Ar-C), 80.8 (N-CH2), 77.3
(N-CH2), 69.1 (CH2-CCH), 58.9 (CCH). MS (+ESI):
596.1 m/z; calcd for [M]+: 596.1 m/z. IR (solid, cm−1): 2033,
1937, 1678, 1183, 1124. UV/vis εmax (213 nm): 19 600 M−1
cm−1.
Cys-NAPamide Analogue Synthesis, Functionaliza-
tion, and Complexation Reactions. Ac-Cys-Nle-Asp-His-D-
Phe-Arg-Trp-Gly-Lys-NH2, 5. The starting peptide 5 (also
abbreviated as Cys-NAPamide) was synthesized on an
automated CS Bio CS336 peptide synthesizer (CS Bio
Company, Inc., Menlo Park, CA) using standard solid-phase
techniques with Fmoc-protected amino acids. Briefly, Rink
Amide LS resin (220 mg, 0.2 mmol, 0.44 mmol/g loading
capacity) was presuspended and swollen in N,N-dimethylfor-
mamide (DMF) for 30 min. Fmoc groups on the resin beads
were deprotected with 20% piperidine in DMF. For every step,
each Fmoc-protected amino acid (1 mmol) was activated in a
solution containing 1 mmol HOBt and 1 M diisopropylcarbo-
diimide in DMF. After deprotection of the Fmoc protection
group of the last amino acid residue, the N-terminal residue was
acetylated by acetic anhydride with HOBt/diisopropylethyl-
amine. The resultant peptide was cleaved and deprotected by a
3 h incubation in a mixture of TFA/triisopropylsilane/1,2-
ethanedithiol/H2O (94:2:2:2). The crude peptide was filtered,
and then precipitated with ice-cold anhydrous diethyl ether.
The resulting peptide pellet was washed four times with ice-
cold anhydrous diethyl ether, dried under a flow of N2, and
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dissolved in a 1 mM dithiothreitol solution. The crude peptide
was purified by semipreparative RP-HPLC with solvents A
(0.1% TFA in H2O) and C (0.1% TFA in MeCN) using a
linear gradient from 95% A/5% C to 35% A/65% C over 45
min at a flow rate of 4 mL/min. Purified fractions were
collected and lyophilized to yield the desired peptide, 5, in 83%
yield and >95% purity by analytical RP-HPLC using HPLC
method 3 (gradient system with solvents A and C; 1 mL/min
flow rate; 0−3 min 95% A/5% C, 3−33 min linear gradient to
35% A/65% C, 33−36 min linear gradient to 15% A/85% C,
hold 15% A/85% C from 36 to 39 min, return to 95% A/5% C
at 39 min and equilibrate in 95% A/5% C; UV at 238 nm). MS
(+ESI): 602.1 m/z, 1202.5 m/z; calcd for [M+2H]2+: 601.8 m/
z; calcd for [M+H]+: 1202.6 m/z.
Ac-S-(3-Azidopropyl)-acetamido-Cys-Nle-Asp-His-D-Phe-
Arg-Trp-Gly-Lys-NH2, 6. Peptide 5 (2.1 mg, 1.36 × 10−6 mol)
was dissolved in 1.2 mL 0.1 M pH 7.4 PBS and mixed with 1.2
mL TCEP gel (tris-2-carboxyethyl phosphine, Pierce) in 0.1 M
pH 7.4 PBS at room temperature for 1.75 h. The gel was
removed by filtration, and the filtrate was immediately frozen
and lyophilized overnight. N2-sparged water (1 mL), N2-
sparged MeCN (0.35 mL), and 2-bromo-N-(3-azidopropyl)-
acetamide (0.45 mg, 2.0 × 10−6 mol) were added to the
reduced peptide, and the solution was stirred at room
temperature for 12 h. The reaction mixture was subsequently
concentrated by rotary evaporation and purified by RP-HPLC
using HPLC method 3. MeCN was removed from the purified
product fractions under vacuum, and the resulting purified
solution was frozen and lyophilized overnight to yield 1.6 mg
(70%) of 6. MS (+ESI): 672.0 m/z, 1343.1 m/z; calcd for [M
+2H]2+: 671.8 m/z; calcd for [M+H]+: 1342.7 m/z. IR
(lyophilized solid, cm−1): 3271, 3206, 2103, 1659, 1642, 1536,
1427, 1181, 1130.
Ellman’s Test for Thiol Content. Peptide 6 was analyzed by
a 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB, Ellman’s
reagent) assay according to instructions from Pierce, with
slight modifications. Briefly, triplicate solutions of 6 at 3.5 ×
10−4 to 4 × 10−4 M in 50 μL H2O were combined with 500 μL
Ellman’s buffer (0.1 M pH 8 sodium phosphate with 1 mM
EDTA) and 10 μL DTNB dissolved at 4 mg/mL in Ellman’s
buffer. The solutions were vortexed and allowed to sit at room
temperature for 15 min, and the UV absorbance of the
solutions at 412 nm was determined. The amount of free thiol
remaining in the peptide solutions was quantified by comparing
the absorbance values at 412 nm to a standard curve generated
by performing the assay with freshly prepared solutions of L-
cysteine at known concentrations.
CuAAC Conjugate of Peptide 6 and Ligand 1, 7. Peptide 6
(300 μg, 1.78 × 10−7 mol) in 70 μL H2O, 1 (2.29 × 10−7 mol)
in 10.9 μL MeOH, sodium ascorbate (6.4 × 10−7 mol),
copper(II) acetate (Cu(OAc)2, 3.2 × 10−7 mol), and 100 μL of
0.1 M pH 7.4 PBS were combined, and the reaction was stirred
under argon at room temperature for 2 h. A solution of 0.1 M
EDTA (100 μL, 1 × 10−5 mol) was added and the solution was
stirred at room temperature overnight. The reaction mixture
was then acidified with 10% TFA in H2O and purified by RP-
HPLC using a gradient method (HPLC method 4: 1 mL/min
flow rate; 0−3 min 95% A/5% B, 3−33 min linear gradient to
35% A/65% B, 33−36 min linear gradient to 1% A/99% B, hold
1% A/99% B from 36 to 39 min, return to 95% A/5% B at 39
min and equilibrate in 95% A/5% B; UV at 238 nm). MeOH
was removed from the purified product fractions under vacuum,
and the resulting purified solution was frozen and lyophilized
overnight to yield 200 μg (51%) of 7. MS (+ESI): 790.7 m/z,
527.6 m/z, 821.2 m/z; calcd for [M+2H]2+: 790.4 m/z; calcd
for [M+3H]3+: 527.3 m/z; calcd for [M+2H+Cu]2+: 821.9 m/z.
This reaction was repeated several times to obtain additional
product for reactions below.
CuAAC Conjugate of Peptide 6 and Ligand 3, 8. Peptide 6
(420 μg, 2.49 × 10−7 mol) in 70 μL 0.1 M pH 7.4 PBS, 3 (3 ×
10−7 mol) in 32.5 μL H2O, sodium ascorbate (9 × 10−7 mol),
Cu(OAc)2 (4.5 × 10−7 mol), and 20 μL of 0.1 M pH 7.4 PBS
were combined, and the reaction was stirred under argon at
room temperature for 3 h. Sodium sulfide (3 mg, 1.25 × 10−5
mol) was added and the solution was stirred at room
temperature for 2.75 h. The reaction mixture was then acidified
with 10% TFA in H2O, centrifuged, and the supernatant was
purified by RP-HPLC with HPLC method 4. Methanol was
removed from the purified product fractions under vacuum, and
the resulting purified solution was frozen and lyophilized
overnight to yield 350 μg (61%) of 8. MS (+ESI): 834.7 m/z,
557.0 m/z, 866.2 m/z, 1667.6 m/z; calcd for [M+2H]2+: 834.4
m/z; calcd for [M+3H]3+: 556.6 m/z; calcd for [M+2H+Cu]2+:
865.8 m/z; calcd for [M+H]+: 1667.8 m/z. This reaction was
repeated several times to obtain additional product for
reactions below.
Click, then Chelate Route for Generating Conjugate of
Peptide 7 and fac-[ReI(CO)3]+, 9. Peptide 7 (200 μg, 9.5 ×
10−8 mol) in 300 μL 0.1 M pH 7.4 PBS and 1 μL 0.1 M fac-
[ReI(OH2)3(CO)3](SO3CF3) (1 × 10−7 mol) were combined
in a vial and stirred under argon at 60 °C for 1.75 h. Following
the reaction, the solution was acidified with 0.1% TFA and
purified by RP-HPLC with solvents A and B using a gradient
method (HPLC method 5: 1 mL/min flow rate; 0−15 min 62%
A/38% B, 15−40 min linear gradient to 100% B, 40−45 min
hold 100% B, return to 62% A/38% B at 34 min and
equilibrate; UV at 220 nm). Methanol was removed from the
purified product fractions under vacuum, and the resulting
purified solution was frozen and lyophilized overnight to yield
200 μg (9.3 × 10−8 mol, 98%) of 9. MS (+ESI): 617.7 m/z,
925.7 m/z; calcd for [M+2H]3+: 617.2 m/z; calcd for [M+H]2+:
925.4 m/z.
Chelate, then Click Route for Generating CuAAC
Conjugate of Peptide 6 and Complex 2, 9′. Peptide 6 (300
μg, 1.78 × 10−7 mol) in 25 μL H2O and 2 (1.81 × 10−7 mol) in
MeOH were combined, and the MeOH was removed by rotary
evaporation. Sodium ascorbate (3.8 × 10−7 mol) in 3.55 μL 0.1
M pH 6 PBS and Cu(OAc)2 (1.9 × 10−7 mol) in 3.33 μL 0.1 M
pH 6 PBS were added, and the reaction vial was purged with N2
for 2 min. The reaction was stirred at room temperature
overnight and then purified by RP-HPLC with HPLC method
5. Methanol was removed from the purified product fractions
under vacuum, and the resulting purified solution was frozen
and lyophilized overnight to yield 200 μg (8.3 × 10−8 mol,
47%) of 9′. MS (+ESI): 617.7 m/z, 925.5 m/z; calcd for [M
+2H]3+: 617.2 m/z; calcd for [M+H]2+: 925.4 m/z.
Click, then Chelate Route for Generating Conjugate of
Peptide 8 and fac-[ReI(CO)3]+, 10. Peptide 8 (300 μg, 1.37 ×
10−7 mol) in 200 μL H2O, 100 μL 0.1 M pH 7.4 PBS, and 1.5
μL 0.1 M fac-[ReI(OH2)3(CO)3](SO3CF3) (1.5 × 10−7 mol)
were combined in a vial and stirred under argon at 60 °C for
2.25 h. Following the reaction, the solution was acidified with
0.1% TFA and purified by RP-HPLC with HPLC method 5.
Methanol was removed from the purified product fractions
under vacuum, and the resulting purified solution was frozen
and lyophilized overnight to yield 200 μg (8.8 × 10−8 mol,
588
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Bioconjugate Chemistry
Article
64%) of 10. MS (+ESI): 968.7 m/z; calcd for [M+H]2+: 968.9
m/z.
determined by incubating solutions of HPLC-purified 4a (200
μL) in mouse serum (200 μL, clarified by centrifugation) in 5
mL labeling vials. Vials were sealed, purged with N2 for 5 min,
and incubated at 37 °C for a total of 24 h. At periodic time
points, aliquots were removed, counted, and added to an equal
volume of ice-cold ethanol to precipitate proteins from the
solution. This mixture was centrifuged and the supernatants
were counted and analyzed by radio-HPLC using HPLC
method 1. Serum stability of the peptides was analyzed by
incubating solutions of HPLC-purified 9a, 9a′, 10a, or 10a′
(100 μCi) in 50 μL of 10 mM pH 7.4 PBS with mouse serum
(500 μL) at 37 °C for a total of 6 h. At periodic time points, the
solutions were filtered through a centrifugal filter (10 K;
Millipore Corp.) and the filtrate was analyzed by radio-HPLC
using HPLC method 2.
Log P analysis. Equal volumes of 10 mM pH 7.4 PBS and 1-
octanol (500 μL each) were vortexed (30 s) in centrifuge tubes
and allowed to separate (2 min). HPLC-purified solutions of
2a, 4a, 9a′, or 10a′ (0.1 μCi in 2−15 μL, MeOH removed
under a stream of N2 following isolation) were added to
triplicate sets of tubes after an equal volume of the aqueous
phase was removed, and the tubes were vortexed (1 min) and
centrifuged (2000 × g, 5 min). Aliquots (50 μL) from each
layer were removed and counted to determine the ratio of
radioactivity present in the octanol layer compared to the
aqueous layer.
In Vitro and In Vivo Biological Evaluations. Cell Culture
and Animal Model. Cell growth and tumor implantation
procedures were performed as previously described.41 Briefly,
B16F10 murine melanoma cells were cultured in Dulbecco’s
modified Eagle’s high-glucose medium (GIBCO, Carlsbad, CA)
and supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin−streptomycin in a humidified incubator containing
5% CO2 at 37 °C. A 70−80% confluent monolayer was
detached with 0.25% trypsin−EDTA and dissociated into a
single-cell suspension for further cell culture and assays. All
animal studies were carried out in compliance with federal and
local institutional rules for the conduct of animal experimenta-
tion. Approximately 1 × 106 cultured B16F10 cells were
suspended in 100 μL of PBS and subcutaneously implanted in
the right shoulders of C57BL/6 mice. Tumors were grown to a
size of 0.5−1 cm in diameter (1−2 weeks) prior to
biodistribution studies.
In Vitro IC50 Cell Binding Analysis. The MC1R binding
affinity of 5, 6, 9′, and 10′ in B16F10 cells was performed as
previously described.41 Briefly, B16F10 cells (5 × 105)
suspended in Dulbecco’s Modified Eagle’s Medium (DMEM)
containing 25 mM N-(2-hydroxyethyl)piperazine-N′-(2-etha-
nesulfonic acid), 0.2% bovine serum albumin, and 0.3 mM 1,10-
phenanthroline were seeded at a density of 0.3 million per well
in 96-well plates and allowed to attach overnight. The cells
were then incubated at 37 °C for 2 h with 125I-(Tyr2)-NDP (20
000 counts per minute) and 5, 6, 9′, or 10′ (peptide
concentration varying from 10−11 to 10−5 M). Cells were
washed three times with ice-cold 10 mM pH 7.4 PBS and lysed
in 1 mL of 1.0 M NaOH containing 0.1% sodium dodecyl
sulfate. The radioactivity of the lysed cells was measured by a γ-
counter (Perkin-Elmer model 1470). The experiment was
performed in quadruplicate wells. The IC50 values (the
concentration of competitor required to inhibit 50% of the
radioligand binding) of the peptides were calculated by using
Origin 8.0 (MicroCal).
Chelate, then Click Route for Generating CuAAC
Conjugate of Peptide 6 and Complex 4, 10′. Peptide 6
(200 μg, 1.19 × 10−7 mol), 4 (1.6 × 10−7 mol), sodium
ascorbate (2.55 × 10−7 mol), and Cu(OAc)2 (1.28 × 10−7 mol)
were dissolved in 100 μL of 0.1 M pH 6 PBS; the reaction vial
was purged with N2 for 2 min and then stirred at room
temperature for a total of 48 h with the addition of more
sodium ascorbate (2.55 × 10−7 mol) and Cu(OAc)2 (5.8 ×
10−8 M) after the first 24 h. After a total reaction time of 48 h,
EDTA (2 × 10−6 mol) was added and the reaction was stirred
for 1 h. The reaction was then acidified with TFA (2.5 μL), and
the solution was centrifuged to pellet the resulting precipitate.
The supernatant was collected and purified by RP-HPLC with
HPLC method 5. Methanol was removed from the purified
product fractions under vacuum, and the resulting purified
solution was frozen and lyophilized overnight to yield 200 μg
(8.8 × 10−8 mol, 70%) of 10′. MS (-ESI): 1936.3 m/z; calcd for
[M-H]−: 1936.7 m/z.
99mTc Reactions with Small Molecules and Peptides.
General Procedures for 99mTc Reactions with Small Molecule
Ligands. Solutions of 1 or 3 in 10 mM pH 7.2 PBS (400−450
μL) were sealed in 5 mL labeling vials and the vials were purged
with N2 for 5−10 min. Solutions of fac-[99mTcI(OH2)3(CO)3]+
(50−100 μL, 0.98−1.37 mCi) were added to bring the final
ligand concentrations to 1 × 10−4 to 1 × 10−6 M in final
volumes of 500 μL, and the vials were heated at 70 °C for 30−
60 min. The vials were cooled prior to analysis and purification
by radio-HPLC using HPLC method 1.
General Procedures for 99mTc Reactions with Peptides.
Click, then chelate route: Solutions of peptides 7 or 8 (2.5 ×
10−8 mol) dissolved in 400 μL 10 mM pH 7.4 PBS were sealed
in 5 mL labeling vials and purged with N2 for 5−10 min. 100
uL of fac-[99mTc(OH2)3(CO)3]+ (0.76−0.81 mCi) was added
to yield final peptide concentrations of 5 × 10−5 M. The
reactions were heated at 70 °C for a total of 1 h with
monitoring by RP-HPLC using HPLC method 1 or HPLC
method 6 (solvents A and B; 1 mL/min flow rate; 0−8 min
62% A/38% B, 8−26 min linear gradient to 100% B, 26−30
min hold 100% B, return to 62% A/38% B after 30 min and
equilibrate; UV at 220, 238, or 254 nm). Peptides were purified
by HPLC method 1 or HPLC method 6. Chelate, then click
route: Crude reaction mixtures of either 2a or 4a (200 μL,
0.34−0.60 mCi) containing ligands 1 or 3 at 5.25 × 10−5
M
were added to a solution containing peptide 6 (2.6 × 10−8 mol)
and sodium ascorbate (2 × 10−6 mol) in 10 mM pH 7.4 PBS
(275 μL) in 5 mL labeling vials. After the vials were sealed and
purged with N2 for 5 min, Cu(OAc)2 (1 × 10−6 mol) was
added to give a final volume of 500 μL with peptide 6 at 5.2 ×
10−5 M, ligands 1 or 3 at 2.1 × 10−5 M, sodium ascorbate at 4 ×
10−3 M, and Cu(OAc)2 at 2 × 10−3 M. The vials were then
heated with stirring at 50 °C for a total of 1 h with periodic
monitoring by radio-HPLC using HPLC method 6. Peptides
were purified by either HPLC method 1 or HPLC method 6.
Stability Analysis. Solutions of HPLC-purified 4a, 9a, 9a′,
10a, or 10a′ (200 μL, MeOH removed under a stream of N2
following isolation) were combined with solutions containing L-
histidine or L-cysteine at 2 × 10−3 M in 10 mM pH 7.4 PBS
(200 μL) in 5 mL labeling vials. Vials were sealed, purged with
N2 for 5 min, and incubated at 37 °C for a total of 18 h (9a,
9a′, 10a, and 10a′) or 24 h (4a) with periodic analysis by radio-
HPLC using HPLC methods 1 or 6. Serum stability of 4a was
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In Vitro B16F10 Melanoma Cell Uptake Assays. Cell uptake
studies of 9a′ and 10a′ were performed as previously
described.41 Briefly, B16F10 cells (3 × 105) were seeded in
12-well tissue culture plates and incubated at 37 °C overnight.
The cells were washed with 10 mM pH 7.4 PBS and then
incubated with 9a′ or 10a′ (1 μCi per well, in DMEM) with or
without NDP (3 μM/well) at 37 °C for 0.5, 1, and 2 h. The
cells were then washed 3 times with 10 mM pH 7.4 PBS and
lysed in 1 mL of 1.0 M NaOH containing 0.1% sodium dodecyl
sulfate and transferred to γ-counter tubes. Radioactivity was
measured by a γ-counter (Perkin-Elmer model 1470). Cell
uptake was expressed as the percentage of added radioactivity.
Experiments were performed twice with quadruplicate wells.
In Vivo B16F10 Melanoma Xenograft Mouse Biodistribu-
tion Analysis. For biodistribution studies, female C57BL/6
mice bearing B16F10 xenografts (n = 4 per group) were
injected via the tail vein with approximately 100 μCi of 9a′ or
10a′ and were euthanized at 1 and 2 h post injection (p.i.).
Tumor and normal tissues of interest were removed and
weighed, and their radioactivity was measured using a γ-
counter. Radioactivity uptake was expressed as a percentage of
the injected radioactive dose per gram of tissue (% ID/g).
Statistical Methods. Statistical analysis was performed
using the Student’s t test for unpaired data. A 95% confidence
level was chosen to determine the significance between groups,
with P < 0.05 being designated as significantly different.
bis-(2-nitrobenzoic acid); EDTA, ethylenediaminetetraacetic
acid; ESI-MS, electrospray ionization mass spectrometry;
EtOH, ethanol; Fmoc, 9-fluorenylmethoxycarbonyl; HOBt,
hydroxybenzotriazole; HPLC, high performance liquid chro-
matography; 125I-(Tyr2)-NDP, 125I-(Tyr2)-[Nle4,D-Phe7]-α-
MSH; % ID/g, percent injected dose per gram of tissue; IR,
infrared; MC1R, melanocortin 1 receptor; MeCN, acetonitrile;
MeOH, methanol; α-MSH, alpha-melanocyte stimulating
hormone; NAPamide, Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-
Lys-NH2; NMR, nuclear magnetic resonance; PBS, phosphate
buffered saline; p.i., post injection; RP-HPLC, reversed-phase
HPLC; SPECT, single photon emission computed tomog-
raphy; TCEP, tris-2-carboxyethyl phosphine; TFA, trifluoro-
acetic acid
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ASSOCIATED CONTENT
■
S
* Supporting Information
Radiolabeling yields for 4a. In vitro stability of 4a, 9a′, and
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: zcheng@stanford.edu. Phone: (650) 723-7866. Fax:
(650) 736-7925.
*E-mail: bennyp@wsu.edu. Phone: (509) 335-3858. Fax: (509)
335-8867.
Author Contributions
Benjamin B. Kasten and Xiaowei Ma contributed equally to this
work.
Notes
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ABBREVIATIONS
■
cpm, counts per minute; CuAAC, copper(I)-catalyzed azide−
alkyne cycloaddition; Cu(OAc)2, copper(II) acetate; Cys-
NAPamide, Ac-Cys-Nle-Asp-His-D-Phe-Arg-Trp-Gly-Lys-NH2;
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