Triazine-based tool box for developing peptidic PET imaging probes: Syntheses, microfluidic radiolabeling, and structure-activity evaluation

Article abstract of DOI:10.1021/bc500034n

This study was aimed at developing a triazine-based modular platform for targeted PET imaging. We synthesized mono- or bis-cyclo(RGDfK) linked triazine-based conjugates specifically targeting integrin ?± _v?₃ receptors. The core molecules could be easily linked to targeting peptide and radiolabeled bifunctional chelator. The spacer core molecule was synthesized in 2 or 3 steps in 64-80% yield, and the following conjugation reactions with cyclo(RGDfK) peptide or bifunctional chelator were accomplished using a€?clicka€? chemistry or amidation reactions. The DOTA-TZ-Bis-cyclo(RGDfK) 13 conjugate was radiolabeled successfully with ⁶⁴Cu(OAc)₂ using a microfluidic method, resulting in higher specific activity with above 95% labeling yields compared to conventional radiolabeling (SA ca. 850 vs 600 Ci/mmol). The dimeric cyclo(RGDfK) peptide was found to display significant bivalency effect using I¹²⁵-Echistatin binding assay with IC₅₀ value as 178.5 ± 57.1 nM, which displayed a 3.6-fold enhancement of binding affinity compared to DOTA-TZ-cyclo(RGDfK) 14 conjugate on U87MG human glioblastoma cell. Biodistribution of all four conjugates in female athymic nude mice were evaluated. DOTA-a€?Clicka€?-cyclo(RGDfK) 15 had the highest tumor uptake among these four at 4 h p.i. with 1.90 ± 0.65%ID/g, while there was no clear bivalency effect for DOTA-TZ-BisRGD in vivo, which needs further experiments to address the unexpected questions.

Full text of DOI:10.1021/bc500034n

Article
pubs.acs.org/bc
Triazine-Based Tool Box for Developing Peptidic PET Imaging
Probes: Syntheses, Microfluidic Radiolabeling, and Structure−
Activity Evaluation
Hairong Li,† Haiying Zhou,† Stephanie Krieger,‡ Jesse J. Parry,‡ Joseph J. Whittenberg,§ Amit V. Desai,§
Buck E. Rogers,‡ Paul J. A. Kenis,§ and David E. Reichert*,†
†Radiological Sciences Division, Mallinckrodt Institute of Radiology, Washington University School of Medicine, 510 South
Kingshighway Boulevard, St. Louis, Missouri 63110, United States
‡Department of Radiation Oncology, Washington University School of Medicine, 4511 Forest Park Boulevard, St. Louis, Missouri
63108, United States
§Department of Chemical & Biomolecular Engineering, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United
States
S
* Supporting Information
ABSTRACT: This study was aimed at developing a triazine-
based modular platform for targeted PET imaging. We
synthesized mono- or bis-cyclo(RGDfK) linked triazine-
based conjugates specifically targeting integrin αvβ3 receptors.
The core molecules could be easily linked to targeting peptide
and radiolabeled bifunctional chelator. The spacer core
molecule was synthesized in 2 or 3 steps in 64−80% yield,
and the following conjugation reactions with cyclo(RGDfK)
peptide or bifunctional chelator were accomplished using
“click” chemistry or amidation reactions. The DOTA-TZ-Bis-
cyclo(RGDfK) 13 conjugate was radiolabeled successfully with
64Cu(OAc)2 using a microfluidic method, resulting in higher specific activity with above 95% labeling yields compared to
conventional radiolabeling (SA ca. 850 vs 600 Ci/mmol). The dimeric cyclo(RGDfK) peptide was found to display significant
bivalency effect using I125-Echistatin binding assay with IC50 value as 178.5 57.1 nM, which displayed a 3.6-fold enhancement of
binding affinity compared to DOTA-TZ-cyclo(RGDfK) 14 conjugate on U87MG human glioblastoma cell. Biodistribution of all
four conjugates in female athymic nude mice were evaluated. DOTA-“Click”-cyclo(RGDfK) 15 had the highest tumor uptake
among these four at 4 h p.i. with 1.90 0.65%ID/g, while there was no clear bivalency effect for DOTA-TZ-BisRGD in vivo,
which needs further experiments to address the unexpected questions.
based scaffold as a toolkit for producing targeted positron
emission tomography (PET) imaging probes with a variety of
choices for receptor targeting ligands and different nuclear or
optical imaging groups. The main goal of this study was to
demonstrate the synthetic feasibility of a triazine-based modular
platform, in which the spacer core molecule could be
conveniently linked to targeting groups (multimeric or
monomeric) and bifunctional chelator labeled with radiometal
using either “click chemistry” or standard amidation reactions
from an activated NHS ester. The “mix and match” concept
allows the rapid synthesis of preclinical drug candidates for PET
or optical imaging.
INTRODUCTION
■
The integrin αvβ3 receptor has been widely studied due to its
major role in tumor angiogenesis and metastasis, and osteoclast
mediated bone resorption.1 DOTA conjugated cyclic arginine-
glycine-aspartic acid (RGD) peptides have been synthesized as
molecular imaging agents of integrin αvβ3 expression,2 and
several research groups have reported multimeric cyclo-
(RGDfK) imaging probes with higher tumor uptake and
improved pharmacokinetic profiles.3 “Click chemistry” utilizing
the reactions of organic azides and terminal alkynes to form
1,2,3-triazoles by Huisgen 1,3-dipolar cycloaddition reactions
catalyzed by Cu (I)4 provides an attractive route for the
preparation of radiopharmaceuticals, especially those utilizing
biomolecules due to its bioorthogonal nature. There are quite a
few examples demonstrating the application of “click chemistry”
in radiopharmaceuticals, e.g., 18F-labeled RGD peptides,5 111In-
DOTA-conjugated octreotide,6 99mTc-galacto-RGD dimer
peptide,7 or 64Cu(68Ga)-DOTA-conjugated RGD peptides,8
and so forth. We have developed a multifunctional triazine-
Herein, we successfully prepared the triazine-based spacer
core, in a stepwise manner under temperature-controlled or
microwave conditions with high synthetic yields. We selected
Received: January 24, 2014
Revised: March 18, 2014
Published: March 24, 2014
© 2014 American Chemical Society
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Scheme 1. Synthesis of Triazine Spacers 8 and 9
Scheme 2. Synthesis of DOTA Conjugated cyclo(RGDfK) Peptides 13−15
the cyclo(RGDfK) peptide for targeting integrin αvβ3 receptors
as a proof-of-principle demonstration. Radiolabeling with
copper-64 was achieved on microfluidic chips with repeatable
excellent radiolabeling yields and high specific activity. We also
evaluated in vitro receptor binding affinity and in vivo
biodistribution.
Fmoc-Arg(Pbf)-OH, Fmoc-Lys(N3)-OH, Fmoc-(D)Phe-OH,
and Fmoc-Asp(OtBu)-OH (3 equiv for each) were coupled to
the resin sequentially by using HBTU/DIPEA coupling agents
after deprotection of the Fmoc group from the C-terminus on
the resin using piperidine. After the synthesis of the linear
peptide on the peptide synthesizer, the desired cyclic peptide
was synthesized by following the cleavage, cyclization, and
deprotection steps developed by Dai et al.11 The overall yield
from the starting Fmoc-Gly-2Cl-Trt resin was 31% yield after
purification by reverse-phase high performance liquid chroma-
tography.
The use of a triazine molecule as a molecular core has been
reported for a dendrimer construct for viral transfection,12 drug
delivery,13 antibody targeting,14 or gadolinium based MRI
agents.15 Cyanuric chloride was used as a core molecule for
sequential modifications using reported temperature controlled
methods.16 There are two routes to synthesize the
RESULTS AND DISCUSSION
■
Chemistry. It has been recognized that cyclo(-Arg-Gly-Asp-
D-Phe-Val-) is a targeting peptide for integrin αvβ3 receptors
and inhibits tumor angiogenesis.9 It was reported that lysine
(K) or glutamic acid (E) could replace the valine (V) residue at
the fifth position without affecting the integrin binding
affinity.9a,10 Since the amino group on lysine amino acid
could be easily modified, we synthesized azido-functionalized
cyclo(RGDfK) peptide starting from Fmoc-Gly-2Cl-Trt resin
using standard Fmoc solid-phase peptide synthesis chemistry.
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Figure 1. Chemical structures of DOTA conjugated triazine spacers 17 and 18.
trisubstituted triazine spacers (8, 9) (Scheme 1). The cyanuric
chloride was first reacted with tert-butyl-(2-(2-(2-
aminoethoxy)ethoxy)ethyl)carbamate linker (SI) at 0 °C to
afford intermediate spacer 1 in the presence of DIPEA in 78%
yield. Spacer 1 then reacted with 2-(2-(prop-2-ynyloxy)-
ethoxy)ethan-1-amine (SI) under microwave irradiation at
140 °C to afford bis-PEG-alkyne-substituted triazine spacer 2 in
82% yield. Mono-PEG-alkyne-substituted triazine spacer 4
could be afforded with second substitution of 2-(2-(prop-2-
ynyloxy)ethoxy)ethan-1-amine linker by heating at 55 °C in
93% yield. Spacer 4 then reacted with 2-ethoxylethylamine to
afford spacer 5 under microwave irradiation at 140 °C in 95%
yield. The t-butyloxycarbonyl (BOC) protecting groups on
both spacers 2 and 5 linker ends were removed by
trifluoroacetic acid/dichloromethane (v/v:1/1) solution at
room temperature to afford pure amino-ended PEG linker
substituted triazine spacer which were functionalized with one
or two acetylene moieties (8, 9) in ca. 90% yield after HPLC
purification. An alternate route to synthesize spacer 8, which
avoids the deprotection step and tedious HPLC purification,
starts with the reaction of cyanuric trichloride and 2-(2-(prop-
2-yn-1-yloxy)ethoxy)ethan-1-amine (2.6 equiv) in the presence
of DIPEA (2 equiv) to afford the stable monochloro-bis-PEG-
acetylene substituted triazine spacer 3 in 90% yield. Spacer 3
reacts with large excess of 2,2′-(ethylenedioxy)bis(ethylamine)
(40 equiv) and DIPEA (20 equiv) in anhydrous acetonitrile to
afford pure triazine spacer 8 in 89% yield after flash
chromatography on silica gel. A similar method was applied
to synthesize triazine spacer 9 containing one acetylene moiety
in three steps. In summary, the construction of triazine spacer
containing acetylene and amino functionalities could be
achieved in three or four steps using amino-BOC protected
PEG linker in 56−69% total yield. Avoiding the use of a
protection−deproctection strategy, the total synthesis of the
spacer molecule could be accomplished in two or three steps of
80% total yield for spacer 8 containing two acetylene moieties,
or 64% total yield for spacer 9 containing one acetylene moiety.
Triazine spacers 8 and 9 reacted with 2.7 or 1.3 equiv azido-
functionalized cyclo(RGDfK) peptide using “click chemistry” in
the presence of TBTA-Cu (I) catalyst complex (13%) at 100
°C under microwave irradiation for 40−60 min (Scheme 2).
The bis or monocyclo(RGDfK) conjugated triazine spacers 10
and 11 were obtained in 90% and 79% yield after HPLC
purification. Different solvent systems for the reaction were
attempted. The protic solvent combination methanol:water
(1:1) was found to have the best yield for the “click” reaction
under microwave irradiation. The peptide conjugates 10 and 11
reacted with DOTA-NHS ester in anhydrous DMF in the
presence of 6 equiv DIPEA to afford the final DOTA
conjugated bis- or mono- cyclo(RGDfK) peptides in ca. 45%
yield after purification. The DOTA-cyclo(RGDfK) standard
peptide17 was synthesized via common amidation reaction from
DOTA-NHS ester and amino-functionalized cyclo(RGDfK)
peptide (Supporting Information). DOTA-“Click”-cyclo-
(RGDfK) 15 peptide was synthesized from “click” reaction
between azido modified cyclo(RGDfK) and propargylamine,
which was followed by amidation reaction with DOTA-NHS
ester.
The “click chemistry” between the acetylene group(s) of the
core molecules and cyclo(RGDfK)-N3 was challenging. First,
we synthesized DOTA-conjugated triazine precursors 17 and
18 (Figure 1) through amidation reaction from activated
DOTA-NHS ester and triazine spacer 8 and 9. Then, we were
unable to obtain the “click” product from free DOTA-
conjugated triazine and cyclo(RGDfK)-N3 using TBTA-Cu
(I) catalyst under various reaction conditions such as
microwave irradiation or heating at high temperature (e.g.,
100 °C) overnight. Subsequent investigation determined that
TBTA-Cu (I) was not stable in the presence of free DOTA,
which would chelate copper as a DOTA-Cu (II) complex.
Starting from chelated DOTA-Cu (II)-conjugated triazine
spacer, the “click” reaction with cyclo(RGDfK)-N3 peptide
was complete in 1 h under microwave irradiation at 100 °C
with 100% conversion yield, while under conventional heating
at 80 °C for 18 h we only obtained 50% conversion yield
(Supporting Information). Starting from the chelated DOTA-
Gd (III)-conjugated triazine spacer, the product could be
obtained under microwave irradiation at 100 °C for 1 h with ca.
30% conversion yield. The chelated Cu (II) would decrease the
Cu (II)−Cu (I) redox couple efficiency to generate active Cu
(I) catalyst.8a As a result, there is insufficient Cu (I) to serve as
catalyst for the click reaction.
These results implied that free DOTA was more likely to
access copper to form DOTA-Cu (II) complex than TBTA
ligand (TBTA-Cu (I) complex). To further validate the
process, we performed a DOTA-TBTA competitive chelation
study using carrier-added 64Cu. Equal moles DOTA-tetra-
carboxylic acid and TBTA-64Cu (carrier added) were mixed for
15 min at ambient temperature. We observed that DOTA-64Cu
(II) was produced from TBTA-64Cu (I) in 100% yield after 15
min, as determined by radio-TLC (Supporting Information).
Since DOTA could quench the copper catalyst during the
“click” reaction and the chelated DOTA-Cu (II) would also
decrease the radiolabeling yield in a later step, the synthesis of
cyclo(RGDfK)-“click”-triazine process was performed prior to
the DOTA conjugation reaction via amidation. The “click”
reaction could be catalyzed with 13% copper(I)-TBTA complex
under microwave irradiation at 100 °C for 40 min to afford
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Table 1. Microfluidic (MF) and Conventional (CV) Radiolabeling of Peptide 13 with 64Cu(OAc)2
radiolabeling
method
temp
(°C)
reaction scale/
[ligand] (μM)
reaction volume total ligand mass
experimental
L/M ratio
time
(min)
HPLC labeling
yield (%)
calculated SA (Ci/
mmol)
(μL)
(μg)
MF
MF
CV
37
37
90
4.5
4.6
9.1
52.6
52.6
192
0.5
0.5
3.7
2.1
7.5
10
10
30
97.4
2
867.1 88
850.9 84.9
535.5
97.8 0.8
99.0
62.3
NH2-TZ-cyclo(RGDfK) 11 in 79% yield. NH2-TZ-Bis-cyclo-
(RGDfK) 10 containing two cyclo(RGDfK) peptides was easily
synthesized under the same microwave conditions for 60
min in 90% yield. When using conventional heating conditions,
extended reaction time was needed. Only monocyclo(RGDfK)
conjugated triazine spacer was observed from MALDI-TOF
spectroscopy after the reaction of spacer 8 and cyclo(RGDfK)-
N3 peptide under conventional heating at 80 °C for 18 h. No
bis-cyclo(RGDfK)-conjugated triazine was observed even after
42 h in the presence of 25% Cu (I)-TBTA catalyst complex.
Thus, microwave heating was found to be the preferred method
to synthesize the “click” peptide product, especially the bis-
peptide conjugated compound. The “click” reaction could be
monitored by MALDI-TOF mass spectroscopy at 1084.6 m/z
(11, [M]+) or 1768.9 (10, [M+H]+). Both the “click” product
and the DOTA conjugate could be purified with HPLC on
reverse phase (C18) column in reasonable yields.
achieve higher specific activity with above 95% labeling yields
(ca. 850 vs 600 Ci/mmol); (d) it could perform reliable
batchwise reactions with repeatable results (n = 4 or 5); (e) it
provides efficient mixing by serpentine mixing channel which
improves the reaction kinetics to shorten the reaction time
(e.g., 10 min); (f) it provides fine control over reaction
conditions due to automated control; (g) it requires a much
smaller overall footprint of the system which minimizes the
amount of space requiring shielding.
In Vitro Study. The cell binding study was performed using
I125-Echistatin binding assay. The results indicated that addition
of the DOTA chelator, triazole moiety from “click” reaction,
and triazine spacer into the molecule decreased the cyclo-
(RGDfK) binding affinity to αvβ3 integrin receptors by
increments of ca. 100 nM of IC50 value for each compound
(Figures 2). The IC50 values were 421.9 90.9, 509.7 203.3,
Conventional and Microfluidic Radiolabeling on Chip.
The microfluidic radiolabeling of DOTA-TZ-Bis-cyclo-
(RGDfK) 13 containing two cyclo(RGDfK) motifs was
achieved with ligand to metal ratios of 2−7 for each reaction
(Table 1). The ligand concentration was 4.5 μM and total mass
of the ligand was 0.5 μg. The radiolabeling process was
performed on chip at 37 °C for 10 min. The radiolabeling yield
was >97% evaluated by radio-HPLC. The specific activity
ranged from 850.9 84.9 to 867.1 88 Ci/mmol (n = 4 or 5).
The conventional radiolabeling was performed under normally
reported condition.2c,d The reaction scale was about 3.7 μg
peptide per mCi 64Cu in 200 μL buffer solution (NaOAc, 0.1
N, pH = 6.8). The reaction was heated at 90 °C for 30 min to
obtain 64Cu-labeled peptide with 99% radiolabeling yield. The
specific activity was 535.5 Ci/mmol.
Figure 2. Competitive inhibition of IC50 toward I125-Echistatin with
DOTA-cyclo(RGDfK) (black, IC50 = 421.9
90.9 nM), DOTA-
“Click”-cyclo(RGDfK) 15 (red, IC50 = 509.7 203.3 nM), DOTA-
TZ-cyclo(RGDfK) 14 (green, IC50 = 637.6 113.5 nM), and DOTA-
TZ-Bis-cyclo(RGDfK) 13 (blue, IC50 = 178.5 57.1 nM). (n = 3,
standard error of mean.)
Microfluidic radiolabeling was achieved on PDMS/glass
radiolabeling chips. Ligand to metal ratio (2−7) was calculated
based on the estimated specific activity of copper-64
production, which differed from batch to batch. Normally,
the products from the first two runs of labeling reaction on
microchip were discarded in order to establish the optimal
labeling condition. After that, four to five repeated runs could
be performed with consistent radiolabeling yields (>95%). The
product was collected in an Eppendorf tube and examined by
radio-HPLC. The delivered activity of the product was
observed to drop with decreasing radiolabeling yields due to
increased dose absorbed into the PDMS material. As expected,
we observed about 45% retention of 64Cu on the microchip
after the experiment, which was consistent with our previous
reports.23
Overall, the microfluidic radiolabeling has several advantages
over conventional radiolabeling method: (a) microfluidic
radiolabeling enables better handling of small reagent volumes
(e.g., 4.5 μM/50 μL); (b) microfluidic radiolabeling requires a
much lower ligand to metal ratio (2−7 vs 60), and only one-
seventh of the ligand mass used in conventional labeling for
each run (0.5 vs 3.7 μg), which greatly reduces the expensive
cost of biomolecules such as peptides or antibodies; (c) it could
and 637.6 113.5 nM for compounds DOTA-cyclo(RGDfK),
DOTA-“Click”-cyclo(RGDfK) 15, and DOTA-TZ-cyclo-
(RGDfK) 14, respectively, while plain cyclo(RGDfK) has
IC50 value of 111.1 nM (data not shown).
The DOTA-TZ-Bis-cyclo(RGDfK) 13 conjugated with two
cyclo(RGDfK) peptides had the strongest binding affinity to
the integrin αvβ3 receptors among this series of compounds
with IC50 value of 178.5
57.1 nM. The low IC50 value of
DOTA-TZ-Bis-cyclo(RGDfK) 13 might be due to the
bivalency effect of the compound,3c compared to DOTA-TZ-
monocyclo(RGDfK). It had significantly increased integrin
αVβ3 binding affinity, similar to the reported HYNIC-dimer
(112
21 nM),18 and FPTA-RGD2 which contains two
cyclo(RGDfK) peptide and one triazole moiety (144
6.5
nM).5
Serum Stability Study. Both triazine-based compounds
conjugated with one or two cyclo(RGDfK) peptides showed
higher stability than DOTA-cyclo(RGDfK) or DOTA-“Click”-
cyclo(RGDfK) 15 in rat serum. Compound DOTA-TZ-
cyclo(RGDfK) 14 was observed to be stable in rat serum for
the first 24 h, and then the stability decreased significantly to ca.
55% at 48 h (Figure 3). Compound DOTA-TZ-Bis-cyclo-
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triazole moiety made DOTA-“Click”-cyclo(RGDfK) 15 have
higher specific targeting, also increased uptake in liver, kidney,
and pancreas compared to DOTA-cyclo(RGDfK) (Figure 5).
Figure 3. Serum stability study of peptide 64Cu-DOTA-cyclo(RGDfK)
(inverted pyramid), 64Cu-DOTA-“Click”-cyclo(RGDfK) (diamond),
64Cu-DOTA-TZ-cyclo(RGDfK) (triangle), and 64Cu-DOTA-TZ-Bis-
cyclo(RGDfK) (square).
(RGDfK) 13 was stable for the first 4 h (>90%), and then the
percentage intact decreased to 60% over the next 44 h.
Compounds DOTA-cyclo(RGDfK) and DOTA-“Click”-cyclo-
(RGDfK) 15 demonstrated lower stability than the triazine-
based conjugates in rat serum. The DOTA-“Click”-cyclo-
(RGDfK) 15 conjugate had slightly lower stability than DOTA-
TZ-Bis-cyclo(RGDfK) 13 for the first 4 h, and then it turned
out to be unchanged for the next 44 h (76%). DOTA-
cyclo(RGDfK) conjugate showed the lowest serum stability,
but nevertheless very close to the behavior of DOTA-“Click”-
cyclo(RGDfK) 15 in rat serum. Serum stability study showed
that triazine-based compounds containing both triazole and
triazine aromatic moieties were fairly stable in the rat serum
environment for at least 4 h, then the stability dropped over
time. These expected phenomena might be due to the
transchelation challenge from copper-binding biomolecules
present in the extracellular and intracellular environments.19
Biodistribution Study and Structure−Activity Evalua-
tion. All four peptide conjugates had higher tumor uptake at 4
h than that at 1 h on homozygous Nu/Nu female athymic nude
mice implanted with 5 × 106 U87MG cells. Compound DOTA-
“Click”-cyclo(RGDfK) 15 had the highest tumor uptake among
these four at 4 h p.i. with 1.90
DOTA-cyclo(RGDfK) 1.65
0.65%ID/g, followed by
0.40%ID/g, DOTA-TZ-Bis-
cyclo(RGDfK) 13 1.15
0.10%ID/g, and DOTA-TZ-cyclo-
(RGDfK) 14 0.86 0.17%ID/g (Figure 4). The insertion of a
Figure 5. Biodistribution study of (A) 64Cu-DOTA-cyclo(RGDfK);
(B) 64Cu-DOTA-“Click”-cyclo(RGDfK); (C) 64Cu-DOTA-TZ-cyclo-
(RGDfK); (D) 64Cu-DOTA-TZ-Bis-cyclo(RGDfK).
We observed that DOTA-TZ-cyclo(RGDfK) 14 had lower
liver, kidney, and pancreas uptake than DOTA-cyclo(RGDfK)
at all time points studied. It can be inferred that the triazine
moiety in DOTA-TZ-cyclo(RGDfK) 14 did not increase the
nontumor organ uptake, although it had the lowest tumor
uptake among these four. The liver washout of DOTA-TZ-
cyclo(RGDfK) 14 was faster than DOTA-“Click”-cyclo-
(RGDfK) 15: the prior compound had decreasing liver uptake
after 1 h p.i., while the latter one had highest liver uptake at 4 h
Figure 4. Tumor uptake of (A) 64Cu-DOTA-cyclo(RGDfK); (B)
64Cu-DOTA-“Click”-cyclo(RGDfK); (C) 64Cu-DOTA-TZ-cyclo-
(RGDfK); (D) 64Cu-DOTA-TZ-Bis-cyclo(RGDfK) p.i.: 1 h (black);
4 h (red); 24 h (green).
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p.i. This was consistent with the measured logD values: DOTA-
“Click”-cyclo(RGDfK) peptide (logD −3.3) had the highest
lipophilicity among this series of peptide conjugates (Support-
ing Information Table S2), which increased the overall hepatic
uptake and hepatobiliary excretion of the peptide conjugate or
its radiocatabolites by specific biochemical pathway.20 DOTA-
TZ-Bis-cyclo(RGDfK) 13 (logD −4.1) displayed a similar
trend of organ uptake over time to DOTA-TZ-cyclo(RGDfK)
14 (logD −3.6). However, contrary to our expectations,
DOTA-TZ-Bis-cyclo(RGDfK) 13 had significantly increased
liver uptake (e.g., 15.86 2.71%ID/g at 1 h p.i.), and slightly
increased kidney and pancreas uptake compared to DOTA-
cyclo(RGDfK) (logD −4.3). A simple lipophilicity argument is
obviously unable to explain this observation. The high liver
uptake is similar to that observed with many RGD targeted
nanoparticles and could be indicative of RES involvement
perhaps through a specific bivalent binding to Kupffer cells; the
monomeric RGD compounds are of much lower affinity and do
not display this high uptake. Shuhendler et al. postulated that
such an effect could explain the high liver uptake of RGD
targeted nanoparticles.21
in reducing nonspecific targeting and consequently increase the
tumor/background ratio because of their hydrophilicity, acidity,
or basicity;18b,22 we did not observe significant advantages of
short PEG linker (two CH2CH2O moieties) in our study.
Longer hydrophilic PEG linkers might be a good choice in our
case. Further studies are ongoing to understand the excretion
pathway of the triazine-based conjugate and if any other factors
play a role in the bivalency effect.
The 64Cu labeled conjugate using the microfluidic method
had moderately higher specific activity than that from using
conventional radiolabeling method (850.9 84.9 to 867.1
88.0 Ci/mmol vs 535.5 to 612.5 Ci/mmol). The biodistribution
results by injecting 64Cu-labeled DOTA-TZ-Bis-cyclo(RGDfK)
13 conjugate demonstrated little differences in mice models
using conventional or microfluidic radiolabeling method
(Supporting Information Figure S4). It indicated that using
microfluidic radiolabeling method could achieve at least the
similar effect to conventional radiolabeling method while
maintaining its intrinsic advantages.
CONCLUSIONS
■
For the design of triazine-based cyclo(RGDfK) dimer
conjugate, the two peptide motifs are separated by 22 bonds,
which should be capable of binding two integrin αvβ3 receptors
simultaneously.3c However, we were surprised to observe only a
20% increase of tumor uptake for dimer conjugate compared to
the monomeric peptide conjugate based on triazine core at 1 h
p.i. (0.81 0.12 vs 0.67 0.21) and 34% increase at 4 h p.i.
We have developed a tool kit of triazine-based scaffold for
diagnostic imaging purposes. We have demonstrated the
synthesis of triazine-based monomeric and dimeric cyclo-
(RGDfK) peptide conjugates containing the bifunctional
chelator DOTA using “click chemistry”. The methodology
and techniques are versatile and provide a convenient method
to switch different functionalities, e.g., PET imaging, optical
imaging, targeting, or therapeutic group in the scaffold. In this
report, we used cyclo(RGDfK) specifically targeting integrin
αvβ3 as proof of principle. A dimeric cyclo(RGDfK) peptide
was found to display significant bivalency effect in cellular
study. The peptide was radiolabeled with 64Cu using two
different methods, microfluidic on-chip radiolabeling and
conventional radiolabeling. The 64Cu-DOTA-TZ-Bis-cyclo-
(RGDfK) 13 using the microfluidic radiolabeling method had
higher specific activity than the one using the conventional
radiolabeling method. Biodistribution of all four conjugates in
female athymic nude mice were evaluated. DOTA-“Click”-
cyclo(RGDfK) 15 had the highest tumor uptake among these
four at 4 h p.i. Further investigations need be done to re-
evaluate the bivalency effect for the dimeric peptide conjugate
in vivo.
(1.15
0.10 vs 0.86
0.17). We did not observe a clear
bivalency effect for DOTA-TZ-BisRGD in the biodistribution
study, although the in vitro study found a 3.6-fold enhancement
of binding affinity on U87MG human glioblastoma cell. The
observed bivalency effect shown in vitro was probably due to
the high density of the integrin αvβ3 receptors on the
glioblastoma tumor whole cell in vitro. A partial blocking effect
was observed in the tumor with coinjection with unlabeled
cyclo(RGDfK) (20 mg/kg body weight). The coinjected cold
peptide blocked 41.7% tumor uptake of 64Cu-DOTA-TZ-
cyclo(RGDfK) which was radiolabeled by conventional method
(0.35 0.04%ID/g) and blocked 36.2% tumor uptake of 64Cu-
DOTA-TZ-Bis-cyclo(RGDfK) 13, which was radiolabeled by
microfluidic method (0.63 0.16%ID/g).
Interestingly, DOTA-TZ-cyclo(RGDfK) 14 conjugate had
the highest tumor to bone uptake ratio at all time points
studied: 1.1 at 1 h p.i., 2.1 at 4 h p.i., and 3.5 at 24 h p.i. among
all four compounds (Supporting Information Figure S3).
Tumor to muscle uptake ratios for all four compounds were
in the range of 2.3−3.0 at 1 h p.i. DOTA-cyclo(RGDfK) had
the highest tumor/muscle uptake ratio as 6.1 at 4 h p.i. For the
comparison of tumor to blood uptake ratio, DOTA-“click”-
cyclo(RGDfK) 15 had highest value as 2.8 at 1 h p.i., while
DOTA-cyclo(RGDfK) had the highest value as 5.3 at 4 h p.i.
DOTA-TZ-Bis-cyclo(RGDfK) 13 had the lowest tumor/
nontumor uptake ratio among these four compounds, probably
due to unexpected low tumor uptake. DOTA-“click”-cyclo-
(RGDfK) 15 and DOTA-TZ-cyclo(RGDfK) 14 conjugates had
rapid liver clearance for the first 4 h after the injection of 64Cu
labeled conjugates, while DOTA-cyclo(RGDfK) and DOTA-
TZ-cyclo(RGDfK) 14 conjugates had slow liver clearance for
the first 4 h (Supporting Information Figure S5). All the
monomeric peptide conjugates had fast kidney clearance for the
first 4 h, and then the uptake level became steady over next 20
h. Spacer linkers have been reported to play an important role
EXPERIMENTAL SECTION
■
General. Peptide synthesis was performed on a Focus Xi
peptide synthesizer (AAPPTec LLC, Louisville, KY). Micro-
wave synthesis was performed on a Biotage Initiator Classic
Microwave Synthesizer (Biotage LLC, Charlotte, NC). 1H
NMR and 13C NMR spectrometry was performed on I400
1
Varian Inova (400 MHz for H NMR and 100.5 MHz for 13C
NMR). MALDI-TOF mass spectrometry was performed on
Voyager-DE STR BioSpectrometry Workstation (Applied
Biosystems, San Francisco, CA). Accurate mass measurements
were conducted on a Thermo Scientific (San Jose, CA) LTQ
Orbitrap Velos mass spectrometer with Xcalibur operating
system. The resolution of the instrument was set at 100,000
(m/z 400), and the instrument was externally calibrated before
the electrospray ionization mass spectrometry (ESI-MS)
analysis. Three microliter flow modular pump components
(syringe pump, a pump driver circuit, and a power supply) were
obtained from Harvard Apparatus (Holliston, MA). A Kapton-
766
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insulated thin film heater (2 in. × 2 in.), Omega CN740
temperature controller, and an Omega SA 1-RTD probe were
obtained from Omega Engineering (Stamford, CT). The
BioScan AR-2000 radio-TLC plated reader was purchased
from BioScan Inc. (Washington, DC). The ThermoMixer C
was purchased from Eppendorf North America (Hauppauge,
NY). Microliter syringes were obtained from Hamilton Co.
(Reno, NV). A Capintec CRC-712 M radioisotope dose
calibrator (Ramsey, NJ) and PerkinElmer 1480 Wizard 3″
Automatic Gamma Counter (Waltham, Massachusetts) were
used for the measurement of radioactivity.
Spacer 2. Spacer 1 (30 mg, 0.08 mmol), 2-(2-(prop-2-yn-1-
yloxy)ethoxy)ethan-1-amine (65 mg, 0.23 mmol), and DIPEA
(79 μL, 0.23 mmol) were dissolved in acetonitrile (2.5 mL),
and charged into a thick-walled vessel. The reaction was heated
at 140 °C for 90 min until the reaction was completed under
microwave irradiation. The reaction solution was cooled to
room temperature, and concentrated under vacuum. The
product was further purified with silica chromatography
(methanol/dichloromethane 5:95) to afford a viscous liquid
1
(38 mg, 82%). H NMR (400 MHz, CDCl3): δ 4.18 (s, 4H),
3.66−3.53 (m, 26H), 3.29 (s, 2H), 2.43 (s, 2H), 1.42 (2, 9H).
13C NMR (100.5 MHz, CDCl3): 165.75, 161.11, 156.06, 79.56,
74.58, 70.11, 69.01, 58.35, 40.26, 28.39. MALDI-TOF: calcd for
C28H47N7O8 [M]+: 609.35; found: 609.51. HRMS (ESI): calcd
for C28H48N7O8 [M+H]+: 610.3559; found: 610.3560.
All solvents and reagents were purchased from commercial
sources and used without further purification. The protected
amino acids including Fmoc-Arg(Pbf)-OH, Fmoc-Lys(N3)-
OH, Fmoc-(D)-Phe-OH, and Fmoc-Asp(OtBu)-OH; cyclo-
(RGDfK) peptide; and Fmoc-Gly-2Cl-Trt resin (200−400
mesh, 1% DVB, 0.77 meg/g) were purchased from AAPPTec
LLC (Louisville, KY). DOTA-NHS-ester was purchased from
Macrocyclics (Dallas, TX). All the other chemicals were
purchased from Sigma-Aldrich (St. Louis, MO) or Fisher
Scientific (Pittsburgh, PA). Deionized water (DI-H2O) was
produced in-house using a Millipore Milli-Q water system.
64CuCl2 was produced at Washington University in St. Louis
School of Medicine, and was obtained in a 0.1 M HCl solution.
Silica TLC plates and C18 TLC silica plates were purchased
from Sorbent Technologies (Norcross, GA). Analytical and
semipreparative reverse phase high performance liquid
chromatography (HPLC) was accomplished on a Hewlett-
Packard 1050 series HPLC (Model 35900E) equipped with a
single-channel high-sensitivity radiation detector (model 105s-
1, Carroll & Ramsey Associates, Berkley, CA) and analyzed on
Chem Station IC software (Agilent Technologies, Santa Clara,
CA). The HPLC analytical column is an Econosil C18 reverse
phase column (10 μm, 250 mm) from Alltech Associates, Inc.
(Deerfield, IL). The HPLC semipreparative C4 reverse phase
column (10 μm, 250 × 10 mm) was purchased from Higgins
Analytical Inc. (Mountain View, CA). The flow rate was 1 mL/
min for analytical HPLC and 2.5 mL/min for semipreparative
HPLC, with the mobile phase of solvent A (0.1% TFA in
water) and solvent B (0.1% TFA in acetonitrile). The UV
detector was set at 220 nm. The gradient analytical HPLC
method A (analytical) was starting from 10% B (0−2 min) to
90% B at 24 min (24−28 min), and returning to 10% B (30−32
min). Method B (analytical) was starting from 5% B (0−2 min)
to 100% B (60 min), and finally to 5% (62 min). Method C
(semipreparative) was starting from 5% B to 22% B (0−17
min), 25% B (47 min), 80% B (102−132 min), and finally to
5% (150 min).
Spacer 3. Cyanuric chloride (100 mg, 0.55 mmol) was
dissolved in anhydrous acetonitrile (2 mL), and the vessel was
charged with nitrogen and cooled in ice bath. 2-(2-(Prop-2-yn-
1-yloxy)ethoxy) ethan-1-amine (204 mg, 1.43 mmol) and
DIPEA (247.6 μL, 1.43 mmol) were added, and then the ice
bath was removed. The reaction solution was stirred at room
temperature for another 5 h. The solution was concentrated
and the crude product was purified with flash column
chromatography (methanol/dichloromethane 1:99) to afford
a white waxy solid (0.20 g, 90%). 1H NMR (400 MHz, CDCl3):
δ 4.09 (d, J = 2.0 Hz, 4H), 3.58−3.51 (m, 16H), 2.39 (t, J = 2.2
Hz, 2H). 13C NMR (100.5 MHz, CDCl3): 169.8, 165.7, 79.60,
74.86, 70.29, 69.46, 69.07, 58.44, 40.62. MALDI-TOF: calcd for
C17H24ClN5O4 [M]+: 397.15; found: 397.39. HRMS (ESI):
calcd for C17H25ClN5O4 [M+H]+: 398.1590; found 398.1585.
Spacer 4. Spacer 1 (40 mg, 0.10 mmol) was dissolved in
acetonitrile (2.5 mL) under inert atmosphere. A solution of
linker 3 (18.4 mg, 0.12 mmol) in acetonitrile (1 mL) and
DIPEA (28.7 μL, 0.15 mmol) was added to the reaction
solution in two portions with a 30 min interval. The reaction
was heated to 55 °C for 3 h and allowed to react until the
reaction was completed. The solvent was removed under
vacuum and the residue was purified with silica chromatog-
raphy (methanol/dichloromethane 5:95) to afford the product
1
as a white waxy solid (48 mg, 93%). H NMR (400 MHz,
CDCl3): δ 4.20 (s, 2H), 3.67−3.54 (m, 18H), 3.33 (s, 2H),
2.45 (s, 1H), 1.43 (s, 9H). 13C NMR (100.5 MHz, CDCl3): δ
169.17, 168.43, 165.56, 156.04, 79.43, 74.69, 70.38, 70.23,
69.40, 68.99, 58.44, 40.60, 40.32, 28.36. HRMS (ESI): calcd for
C21H36ClN6O6 [M+H]+: 503.2379; found: 503.2382.
Spacer 5. Spacer 4 (20 mg, 0.04 mmol), 2-ethoxyethylamine
(21 μL, 0.20 mmol), and DIPEA (35 μL, 0.20 mmol) were
dissolved in acetonitrile (4 mL), and charged into a thick-
walled vessel. The reaction was heated at 140 °C for 90 min
under microwave irradiation until the reaction was completed.
The reaction solution was cooled to room temperature, and
concentrated under vacuum. The product was further purified
with silica chromatography (methanol/dichloromethane 5:95)
to afford a viscous liquid (21.6 mg, 95%). 1H NMR (400 MHz,
CDCl3): δ 4.17 (s, 2H), 3.66−3.45 (m, 24H), 2.85 (s, 2H),
2.43 (s, 1H), 1.41 (s, 9H), 1.16 (t, J = 6.8 Hz, 3H). 13C NMR
(100.5 MHz, CDCl3): 165.90, 156.05, 79.54, 79.05, 74.56,
70.19, 70.08, 69.32, 68.98, 66.31, 58.33, 53.36, 40.36, 40.23,
28.36, 15.07. MALDI-TOF: calcd for C25H45N7O7 [M]+:
555.34; found: 554.97. HRMS (ESI): calcd for C25H46N7O7
[M+H]+: 556.3453; found 556.3454.
Synthesis. Spacer 1. Cyanuric chloride (25 mg, 0.14 mmol)
and tert-butyl-(2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate
(33.50 mg, 0.15 mmol) were dissolved in anhydrous THF (1
mL) at 0 °C. N,N-Diisopropylethylamine (DIPEA, 52 μL, 0.30
mmol) was added to the reaction solution slowly at 0 °C under
inert atmosphere. The reaction solution was stirred for 1.5 h at
0 °C. White solid was removed by filtration, and the solvent
was removed under vacuum. The crude product was purified by
silica chromatography (methanol/dichloromethane 5:95) and
1
obtained as a yellowish viscous liquid (83 mg, 78%). H NMR
(400 MHz, CDCl3): δ 6.80 (br, 1H), 3.66−3.54 (m, 10H), 3.34
(s, 2H), 1.43 (s, 9H). 13C NMR (100.5 MHz, CDCl3): δ
169.78, 165.61, 155.96, 79.12, 70.32, 41.22, 40.23, 28.30.
HRMS (ESI): calcd for C14H24Cl2N5O4 [M+H]+: 396.1200;
found: 396.1199.
Spacer 6. Cyanuric chloride (157.7 mg, 0.86 mmol) and 2-
(2-(prop-2-ynyloxy)ethoxy)ethan-1-amine (123.3 mg, 0.86
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mmol) were dissolved in dry THF at 0 °C under nitrogen flow.
Then DIPEA (200 μL, 1.10 mmol) was added to the reaction
solution slowly. The reaction solution was stirred for 1 h and
monitored by TLC. The solvent was removed under vacuum
and the residue was purified with silica chromatography
(methanol/dichloromethane 2:98) to afford a white waxy
sat. sodium bicarbonate. Dichloromethane was used to extract
the product, and water (1 mL × 3) to wash the organic phase
until the pH 7. The organic layer was dried over anhydrous
sodium sulfate. The solution was filtered and concentrated. The
residue was dissolved in acetone and filtered over a Celite cake.
The product was further purified by HPLC (Method A) to
afford a viscous liquid (24 mg, 90%).
1
solid (0.19 g, 76%). H NMR (400 MHz, CDCl3): δ 6.16 (br,
1H), 4.19 (d, J = 2.4 Hz, 2H), 3.66 (d, J = 4.8 Hz, 8H), 2.45 (s,
1H). 13C NMR (100.5 MHz, CDCl3): 170.18, 166.04,
79.63,75.19, 70.61, 69.31, 69.23, 58.74, 41.53. HRMS (ESI):
calcd for C10H12Cl2N4O2 [M+H]+: 291.0410; found: 291.0410.
Spacer 7. A mixture of spacer 6 (0.13 g, 0.45 mmol), 2-
ethoxyethylamine (0.06 g, 0.65 mmol), and DIPEA (0.06 g,
0.45 mmol) were mixed in anhydrous acetonitrile (1 mL). The
reaction solution was stirred at room temperature. After 2 h,
the reaction was complete as determined by TLC. The reaction
solution was concentrated and the residue was purified by
chromatography with silica gel eluted by 2% dichloromethane/
methanol. The product was obtained as a viscous liquid (0.14 g,
Method 2. Spacer 7 (0.14 g, 0.41 mmol), 2,2′-
(ethylenedioxy)bis(ethylamine) (2.36 mL, 16.10 mmol), and
DIPEA (1.42 mL, 8.20 mmol) were mixed in anhydrous
acetonitrile (10 mL) under nitrogen flow. The reaction mixture
was heated at 80 °C for 3 h until the reaction was found
complete by TLC. The reaction solution was concentrated to
dryness and redissolved in dichloromethane (50 mL). The
organic layer was washed with DI-H2O (10 mL × 1), sodium
bicarbonate (10 mL × 2), and DI-H2O (10 mL × 2). The
organic layer was dried over anhydrous sodium sulfate. The
solvent was removed under vacuum and the residue was
purified with flash column chromatography (silica, methanol/
dichloromethane 5:95, then triethylamine/methanol/dichloro-
methane 2:10:88). The product was obtained as a viscous liquid
1
93%). H NMR (400 MHz, CDCl3): δ 4.11 (d, J = 2.0, 2H),
3.61−3.42 (m, 14H), 2.40 (t, J = 2.4 Hz, 1H), 1.10 (t, J = 7.0
Hz, 3H). 13C NMR (100.5 MHz, CDCl3): 167.81, 165.39,
165.35, 70.02, 69.19, 68.78, 68.31, 66.25, 58.16, 40.39, 14.87.
MALDI-TOF: calcd for C14H22ClN5O3 [M]+: 343.14; found:
343.69. HRMS (ESI): calcd for C14H23ClN5O3 [M]+:
344.1484; found: 344.1478.
Spacer 8. Method 1. Spacer 3 (33 mg, 49.2 mmol) was
dissolved in dichloromethane/TFA (1/1, 2 mL) and the
solution was stirred for 1 d at room temperature. The solvent
was removed under vacuum and the residue was redissolved in
sat. sodium bicarbonate. The product was extracted by
dichloromethane (10 mL × 3), and the organic phase was
washed by water (1 mL × 3) until its pH is neutral. The organic
layer was dried over anhydrous sodium sulfate. The solution
was filtered and concentrated. The residue was dissolved in
acetone and filtered over a Celite cake. The product was further
purified by HPLC (Method A) to afford a viscous liquid (22
mg, 88%).
1
(0.16 g, 86%). H NMR (400 MHz, CDCl3): δ 5.84−5.18 (br,
3H), 4.17 (s, 2H), 3.67−3.44 (m, 24H), 2.84 (t, J = 5.0 Hz,
2H), 2.43 (t, J = 2.4 Hz, 1H), 1.97 (br, 2H), 1.16 (t, J = 6.8 Hz,
3H). 13C NMR (100.5 MHz, CDCl3): 166.30, 79.87, 74.93,
73.68, 70.57, 70.55, 70.49, 70.46, 70.41, 69.70, 69.31, 66.66,
58.67, 41.98, 40.66, 40.55. MALDI-TOF: calcd for C20H37N7O5
[M]+: 455.29; found: 455.22. HRMS (ESI): calcd for
C20H38N7O5 [M+H]+: 455.2929; found: 455.2926.
Cyclo(RGDfK)-N3. The Fmoc-Gly-2Cl-Trt resin (0.40 g, 0.31
mmol) was loaded on Focus Xi peptide synthesizer. Protected
amino acid Fmoc-Arg(Pbf)-OH (0.60 g, 0.93 mmol), Fmoc-
Lys(N3)-OH (0.37 g, 0.93 mmol), Fmoc-(D)Phe-OH (0.36 g,
0.93 mmol), and Fmoc-Asp(OtBu)-OH (0.38 g, 0.93 mmol)
was coupled onto the resin subsequently with 3 mol equiv
relative to the resin by using HBTU/DMF (0.4 M) and
DIPEA/DMF (2 M). Piperidine/DMF (20%) was used to
deprotect the Fmoc group from C-terminal after each coupling
reaction. The Kaiser test was performed after each coupling
reaction. After automated synthesis, the peptide was cleaved
from the resin using a prepared cocktail (30 mL, acetic acid:
2,2,2-trifluoroethanol:dichloromethane 6:6:18). The resin was
suspended in the cocktail solution and the solution was shaken
for 1 h. The resin was then filtered on a fine frit funnel. The
liquid was concentrated under vacuum and the acetic acid was
removed by azeotroping with toluene (∼200 mL). The
concentrated peptide residue was added dropwise to cold
ethyl ether solution (∼100 mL). A light yellow solid formed
and was filtered through a frit funnel. The solid was washed
with cold ether 3 times, and dried in vacuo for 2 h to afford a
crude linear peptide (0.23 g). A 500 mL 2-neck round-bottom
flask was filled with dichloromethane (150 μL), 4-dimethyla-
minopyridine (18.2 mg, 0.15 mmol), propylphosphonic
anhydride (≥50 wt %, in ethyl acetate, 1.3 mL, 2.18 mmol),
and triethylamine (1.5 mL, 10.70 mmol). The linear peptide
was dissolved in dichloromethane (8 mL) and the solution was
added dropwise through an addition funnel for 4 h. The
reaction solution was stirred overnight. The solvent was
removed under vacuum and the residue was dried under
vacuum for 3 h. The crude cyclized peptide was put into a
round-bottom flask, and a solution of deprotection cocktail of
trifluoroacetic acid (14 mL, 0.18 mmol, 95%), water (1 mL),
and DL-dithiothreitol (0.32 g, 2.0 mmol) was added to the flask.
Method 2. Spacer 3 (0.18 g, 0.45 mmol), 2,2′-
(ethylenedioxy)bis(ethylamine) (2.64 mL, 18.12 mmol), and
DIPEA (1.58 mL, 9.06 mmol) were mixed in anhydrous
acetonitrile (50 mL) under nitrogen flow. The reaction mixture
was heated at 80 °C for 3 h until the reaction was complete as
determined by TLC. The reaction solution was concentrated to
dryness and redissolved in dichloromethane (100 mL). The
organic layer was washed with DI-H2O (20 mL × 1), sodium
bicarbonate (20 mL × 2), and DI-H2O (20 mL × 2). The
organic layer was dried over anhydrous sodium sulfate. The
solvent was removed under vacuum and the residue was
purified with flash column chromatography (silica, methanol/
dichloromethane 5:95 then triethylamine/methanol/dichloro-
methane 2:10:88). The product was obtained as a viscous liquid
1
(0.21 g, 89%). H NMR (400 MHz, CDCl3): δ 5.19 (br, 3H),
4.17 (s, 2H), 4.12 (d, J = 2.4 Hz, 4H), 3.61−3.47 (m, 26H),
2.93 (s, 2H), 2.41 (t, J = 2.4 Hz, 2H). 13C NMR (100.5 MHz,
CDCl3): 165.69, 79.42, 74.56, 69.96, 69.94, 69.86, 69.66, 68.83,
58.17, 45.80, 40.42, 40.05. MALDI-TOF: calcd for C23H39N7O6
[M]+: 509.30; found: 509.23. HRMS (ESI): calcd for
C23H40N7O6 [M+H]+: 510.3035; found: 510.3036.
Spacer 9. Method 1. Spacer 5 (33 mg, 59.40 mmol) was
dissolved in dichloromethane/TFA (1:1, 2 mL) and the
solution was stirred for 1 d at room temperature. The solvent
was removed under vacuum and the residue was redissolved in
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The reaction solution was stirred for 30 min at room
temperature. Then the solution was diluted with ice-cold
diethyl ether solution. A white solid was precipitated and
filtered with a fine fritted funnel. The solid was washed with
cold ether and dried overnight. The crude solid was further
purified with semipreparative HPLC (reverse phase C18,
Method C), the fractions were collected and lyophilized to
afford a white fluffy solid (60.10 mg, overall yield 31%). HRMS
(ESI): calcd for C27H39N11O7 [M+H]+: 630.3104; found:
629.3034.
NH2-TZ-Bis-cyclo(RGDfK) 10. Copper sulfate (10 mM, 42
uL) was added to a sodium ascorbate solution (50 mM, 42 μL).
The copper(I) solution was vortexed and allowed to sit for 5
min. The TBTA acetonitrile solution (18.9 mM, 56 μL) was
added to the copper(I) solution to make a TBTA-Cu (I)
complex solution. Spacer 8 (1.5 mg, 2.90 μmol) was added to
the Cu (I)-ligand solution. After 2 min, cyclo(RGDfK)-N3
peptide (5.0 mg, 7.90 μmol) was added to a methanol/water
solution (1/1, 0.8 mL). The whole reaction was heated at 100
°C with microwave irradiation for 1 h. The reaction solution
was concentrated and the residue was purified by HPLC
(Method B, tR = 21.47 min), and lyophilized to afford the
product (4.5 mg, 90%). MALDI-TOF: calcd for C77H117N29O20
[M]+: 1767.90; found: 1766.04. HRMS (ESI): calcd for
C77H118N29O20 [M+H]+: 1768.9102, found: 1768.9077; [M+
H]2+: 884.9588; found: 884.9587.
NH2-TZ-cyclo(RGDfK) 11. Copper sulfate (10 mM, 28 uL for
0.28 μmol) was added to a sodium ascorbate solution (50 mM,
28 μL for 1.40 umol). The copper(I) solution was vortexed and
allowed to sit for 5 min. The TBTA acetonitrile solution (18.9
mM, 37 μL for 0.70 μmol) was added to the Cu (I) solution to
make a TBTA-Cu (I) complex solution. Spacer 9 (0.95 mg,
2.10 μmol) was added to the Cu (I)-ligand solution. After 2
min cyclo(RGDfK)-N3 peptide (1.8 mg, 2.80 μmol) was added
to the reaction solution of methanol/water (1:1, 0.5 mL). The
whole reaction was heated at 100 °C with microwave
irradiation for 40 min. The reaction solution was concentrated
and the residue was purified by HPLC (Method A, tR = 11.48
min), and lyophilized to afford the product (1.8 mg, 79%).
MALDI-TOF: calcd for C47H76N18O12 [M]+: 1084.59, found:
1084.16. HRMS (ESI): calcd for C47H76N18O12K [M+K]+:
1123.5522; found: 1123.4601.
NH2-“Click”-cyclo(RGDfK) 12. Copper sulfate (10 mM, 110
uL for 1.1 μmol) was added to a sodium ascorbate solution (50
mM, 110 μL for 5.50 umol). The Cu (I) solution was vortexed
and sit for 5 min. The TBTA acetonitrile solution (18.9 mM,
145 μL for 2.75 μmol) was added to the copper(I) solution to
make the TBTA-Cu (I) complex solution. Propargyl amine (0.6
mg, 0.01 mmol) was added to the Cu (I)-ligand solution. After
2 min cyclo(RGDfK)-N3 peptide (7.6 mg, 0.01 mmol) was
added to the reaction solution of methanol/water (1:1, 3 mL).
The whole reaction was heated under 100 °C microwave
condition for 20 min. The reaction solution was concentrated.
The residue was purified by HPLC (Method A, tR = 9.71 min),
and lyophilized to afford the product (4.3 mg, 54%). MALDI-
TOF: calcd for C30H44N12O7 [M+H]+: 685.35; found: 685.13;
[M+H2O]+: 702.36, found: 702.03. HRMS (ESI): calcd for
C30H44N12O7 [M+H]+: 685.3529; found: 685.3525.
solution, and the reaction was monitored by MALDI mass
spectrometer. After 24 h, DI-H2O (200 μL) was added to the
reaction solution, and the solution was stirred for 30 min. After
that, the whole solution was filtered through a syringe filter
(0.24 μm). The product was purified by HPLC (Method B, tR
= 19.52 min), and dried by lyophilization to afford a white fluffy
solid (2.5 mg, 48%). HRMS (ESI): calcd for C93H143N33O27 [M
+H+K]2+: 1097.0268; found: 1097.0223.
DOTA-TZ-cyclo(RGDfK) 14. DOTA-NHS ester (5.2 mg, 6.80
μmol) was added to a 1 mL vessel. The vessel was charged with
nitrogen and NH2-TZ-cyclo(RGDfK) 11 (3 mg, 2.70 μmol)
was dissolved in anhydrous DMF (200 μL). DIPEA (7.6 μL,
43.50 μmol) was added to the solution, and the reaction was
monitored by MALDI mass spectrometer. After 24 h, DI-H2O
(150 μL) was added to the reaction solution, and the solution
was stirred for 30 min. After that, the whole solution was
filtered through a syringe filter (0.24 μm). The product was
purified by HPLC (Method B, tR = 18.76 min), and dried by
lyophilization to afford a white fluffy solid (1.8 mg, 45%).
MALDI-TOF: calcd for C63H102N22O19 [M+H]+: 1471.78;
found: 1471.71. HRMS (ESI): calcd for C63H102N22O19 [M-2H
+K]−: 1507.7178; found: 1507.7144.
DOTA-“Click”-cyclo(RGDfK) 15. DOTA-NHS ester (3.6 mg,
4.70 μmol) was added to a 1 mL vessel. The vessel was charged
with nitrogen and NH2-“Click”-cyclo(RGDfK) 12 (2.7 mg,
3.90 μmol) dissolved in anhydrous DMF (200 μL) was added
to the solution. DIPEA (6.6 μL, 37.80 μmol) was added to the
solution, and the reaction solution was stirred at room temp
overnight. After the reaction, DI-H2O (100 μL) was added to
the reaction solution, and the solution was stirred for 30 min.
After that, the whole solution was filtered through a syringe
filter (pore size 0.24 μm). The product was purified by HPLC
(Method B, tR = 15.51 min) and dried by lyophilization to
afford a white fluffy solid (2.7 mg, 64%). MALDI-TOF: calcd
for C46H70N16O14 [M]+: 1070.53, found: 1070.63; [M+H2O]+:
1088.54, found: 1088.56. HRMS (ESI): calcd for C46H70N16O14
[M-2H+K]−: 1107.4743; found: 1107.4651.
PDMS/Glass Chip Fabrication. The radiolabeling chip is a
PDMS-based microreactor. The design of the chip contains
three major components: (1) a staggered herringbone mixing
channel which passively mixes reagents; (2) five incubation
reservoirs for the radiometal−ligand mixture; (3) a thin-film
heater for heating the reaction mixture. The fabrication of the
chip was described in detail in our previous publication.23
Conventional and Microfluidic 64Cu2+ Radiolabeling.
Conventional Radiolabeling. 64CuCl2 in 0.1 M HCl was added
to a sodium acetate buffer (0.1 M, pH 6.8). DOTA conjugated
cyclo(RGDfK) peptides (0.0017 μmol) were mixed with
64Cu(OAc)2 stock solution (1 mCi) in sodium acetate buffer
solution (∼200 μL in total volume). The reaction solution was
incubated on a ThermoMixer (700 rpm) at 90 °C for 30 min.
No further purification was performed. The radiolabeling yield
was determined by radio-TLC and radio-HPLC (>95%). The
specific activity of radiolabeled compounds was ca. 550 Ci/
mmol.
Microfluidic Radiolabeling. The radiometal, ligand, and
buffer solution were pumped into the microreactor through
three separate inlets. The solutions were mixed in the staggered
herringbone mixing channels by chaotic advection induced by
the geometry of the herringbones.24 The mixed reagents then
filled a series of hexagonal reservoirs and the reaction mixture
was incubated at a desired temperature for a specified time. The
reaction product was flushed out by injecting a sodium acetate
DOTA-TZ-Bis-cyclo(RGDfK) 13. DOTA-NHS ester (4.7 mg,
6.20 μmol) was added to a 1 mL vessel. The vessel was charged
with nitrogen and NH2-TZ-Bis-cyclo(RGDfK) 10 (4.3 mg, 2.4
μmol) dissolved in anhydrous DMF (250 μL) was added to the
solution. DIPEA (6.6 μL, 37.80 μmol) was added to the
769
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Article
buffer solution (0.1 M, pH 6.8) into the microreactor. The
syringe pumps were controlled by a LabVIEW virtual
instrument (VI). The flow rates, incubation time, desired
volume of the product (52.6 μL for the total volume of the
holding tanks), and the stoichiometric molar ratios of buffer,
radiometal, and ligand were controlled through the VI. A thin-
film heater was placed underneath the glass surface of the
microreactor. A temperature controller and a resistance
temperature detector (RTD) probe were used to maintain
the temperature within 1 °C of the desired temperature. The
radiometal syringe pump, heater, and microreactor were
shielded with lead shielding (2″ thick). The syringes were
connected to the reactor with Hamilton Luer Lock 30 gauge
PTFE tubing of one foot length.
The PDMS/glass radiolabeling chip was first washed with
diluted nitric acid solution (1 N, 1 mL × 3) at a flow rate 30
μL/min, to remove any metal ions bound nonspecifically to the
chip surfaces. Then the chip was washed with water and sodium
acetate buffer (0.1 N, pH 6.8) separately (1 mL × 3). All
Hamilton glass syringes (250 and 500 μL) and disposable
plastic syringes (1 mL × 3) were rinsed with nitric acid (1 N, 1
mL × 3), water, and sodium acetate buffer (0.1 N, pH 6.8, 1
mL × 3) subsequently. 64Cu(OAc)2 solution (5 mCi, 250 μL),
DOTA-TZ-Bis-cyclo(RGDfK) 13 peptide stock solution (13.5
μM, 250 μL), and sodium acetate buffer (0.1 M, 1 mL) were
loaded into syringes and installed on the syringe pumps. The
radiometal, ligand, and buffer solutions were controlled to mix
in the chip with different volume ratio using LabVIEW interface
software. The total volume was set to 52.6 μL and the total flow
rate was set to 25 μL/min. After each reaction, three chip
volumes of buffer solution were pumped into the chip to wash
the holding tanks thoroughly. The reaction temperature was set
to 37 °C and the residence time was set to 10 min. After
collecting the product, the activity was measured by dose
calibrator (∼200 μCi, Capintec Inc. NJ), and the yield of
radiolabeling reaction was determined by radio-TLC and radio-
HPLC. The first two on-chip reactions were discarded in order
to stabilize the chip condition. Radiolabeling was repeated for
4−5 times under the same condition. The radiolabeling yield
was determined by radio-TLC and radio-HPLC (>97%). The
specific activity of radiolabeled compounds was calculated ca.
850 Ci/mmol.
Cell Culture and in Vitro Binding Assay. The in vitro cell
binding assay was performed with U87MG human glioblastoma
cell line. U87MG human glioblastoma cells were grown in
Dulbecco’s medium (Gibco) supplemented with 10% fetal
bovine serum (FBS). The affinities of cyclo(RGDfK)
conjugates for αvβ3 integrin were examined via competitive
cell binding assay using 125I-echistatin as the integrin αvβ3-
specific radioligand. To determine the in vitro binding affinity of
cyclo(RGDfK) peptide conjugates, U87MG cells were
harvested for membrane preparations as previously described.25
Protein concentrations were determined using the Pierce Non-
Reducing Agent Compatible Kit (Rockford, IL). Briefly, 25 μg
of membrane protein in 100 μL binding buffer (10 mM
HEPES, 5 mM MgCl2, 1 mM EDTA, 0.1% BSA, 10 μg/mL
leupeptin, 10 μg/mL pepstatin, 0.5 μg/mL aprotinin, and 200
μg/mL bacitracin, pH 7.4) was applied to 0.1% polyethylenei-
mine-pretreated wells of a 96-well Multiscreen Durapore
filtration plate (Millipore Corp., Bedford, MA) via vacuum
manifold aspiration. The wells were then washed three times
with washing buffer (10 mM HEPES, 1 mM EDTA, 5 mM
MgCl2, 0.1% BSA). Increasing concentrations of cyclo(RGDfK)
peptide (0−91 μM) were added in a volume of 10 μL to
triplicate wells. To each well, 0.279 kBq of 125I-Echistatin
(PerkinElmer, Boston, MA) in a volume of 100 μL was added.
The plate was incubated at room temperature for 1 h, after
which the wells were washed twice with washing buffer. The
membranes were dried, removed, and placed in separate tubes
for determination of bound radioactivity. The membrane-
associated radioactivity was determined using a Packard II γ
counter (PerkinElmer, Waltham, MA). The IC50 values were
calculated by fitting the data using nonlinear regression in
GraphPad Prism Software (San Diego, CA).
Octanol/Water Partition Coefficient. The octanol and
buffer solution (0.01 M ammonium acetate, pH 6.8) were
mixed and saturated for 1 d. The partition coefficients were
determined by adding 8 μL of 64Cu-labeled peptide conjugate
(20 μCi) to a solution containing 500 μL sat. octanol and 492
μL sat. buffer. The mixed solutions were vortexed for 5 min,
and then centrifuged for 5 min at 3000 rpm. Aliquots (100 μL)
of octanol and buffer were removed, weighed, and counted by
gamma counter. The partition coefficient was calculated as a
ratio of counts in the octanol fraction of unit mass to counts in
the buffer fraction of unit mass. The experiments were
performed in triplicate, and the average LogD value was
reported.
Serum Stability Study. The in vitro serum stabilities of the
64Cu-cyclo(RGDfK) conjugated compounds were evaluated by
incubation with freshly isolated lean rat serum. Each 64Cu-
radiolabeled cyclo(RGDfK) conjugated compound (9 μM, 100
μCi) was mixed with rat serum (100 μL) in a 1.5 mL centrifuge
tube. The reaction was incubated on an Eppendorf
ThermoMixer C (Hauppauge, NY) at 37 °C (300 rpm).
Aliquots were withdrawn at 0, 1, 4, 24, and 48 h, and evaluated
by radio-TLC on a BioScan AR-2000 Imaging Scanner
(Washington, DC). The radio-TLC was analyzed on reverse
phase C18 silica plates using 1:1 methanol/5% ammonium
formate. The reactions were repeated in triplicate.
Animal Model and Biodistribution Studies. Animal
studies were performed in accordance with the guidelines for
the care and use of research animals as defined by the
Washington University Animal Studies Committee. Homo-
zygous Nu/Nu female athymic nude mice at 6−7 weeks of age
were implanted subcutaneously on the rear flank with 5 × 106
U87MG cells in a volume of 100 μL saline. Tumors were
allowed to grow for 14 d, upon which the mice (n = 3) were
injected intravenously with 10 μCi of respective 64Cu-labeled
cyclo(RGDfK) peptide conjugates. One group was coinjected
with 350 μg of cold cyclo(RGDfK) peptide to do the blocking
study. Mice were sacrificed at 1 and 4 h post injection. Blood,
lung, liver, kidney, muscle, bone, pancreas, and tumor were
collected for determination of tissue-associated radioactivity.
Counts were obtained using a γ-counter. The percent injected
dose per gram (%ID/g) for each tissue was calculated to
normalization from a standard dose.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis, TBTA, and DOTA competitive binding study,
reactions of “clickable” DOTA-triazine spacer, determination
of partition coefficient, and in vivo study. This material is
available free of charge via the Internet at http://pubs.acs.org.
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Bioconjugate Chemistry
Article
Christensen, C., and Meldal, M. (2002) Peptidotriazoles on solid
phase: 1,2,3 -triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar
cycloadditions of terminal alkynes to azides. J. Org. Chem. 67 (9),
3057−3064.
(5) Li, Z. B., Wu, Z., Chen, K., Chin, F. T., and Chen, X. (2007)
Click chemistry for F-18-labeling of RGD peptides and microPET
Imaging of tumor integrin alpha(v)beta(3) expression. Bioconjugate
Chem. 18 (6), 1987−1994.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 314-362-8461, Fax: 314-362-9940, E-mail:
ReichertD@wustl.edu.
Notes
The authors declare no competing financial interest.
(6) Yim, C. B., Boerman, O. C., de Visser, M., de Jong, M., Dechesne,
A. C., Rijkers, D. T. S., and Liskamp, R. M. J. (2009) Versatile
conjugation of octreotide to dendrimers by cycloaddition (″click″)
chemistry to yield high-affinity multivalent cyclic peptide dendrimers.
Bioconjugate Chem. 20 (7), 1323−1331.
ACKNOWLEDGMENTS
■
We acknowledge financial support from National Institutes of
Health (5R01CA161348). We also thank Dr. Fong-Fu Hsu at
WUSTL-School of Medicine Biomedical Mass Spectrometry
Facility for the assistance of high resolution electrospray
ionization mass spectrometry. Part of this work made use of the
facilities in the Frederick Seitz Materials Research Laboratory
Central Facilities and Micro-Nano-Mechanical Systems Clean-
room at University of Illinois at Urbana−Champaign, which is
partially supported by the U.S. Department of Energy under
grants DE-FG02-07ER46453 and DE-FG02-07ER46471.
(7) Ji, S. D., Czerwinski, A., Zhou, Y., Shao, G. Q., Valenzuela, F.,
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