Solid-phase synthetic strategy and bioevaluation of a labeled δ-opioid receptor ligand Dmt-Tic-Lys for In Vivo imaging

Article abstract of DOI:10.1021/ol900200k

A general solid-phase synthetic strategy is developed to prepare fluorescent and/or lanthanide-labeled derivatives of the δ-opioid receptor (δOR) ligand H-Dmt-Tic-Lys(R)-OH. The high δ-OR affinity {K _i = 3 nM) and desirable in vivo characterist

Full text of DOI:10.1021/ol900200k

ORGANIC
LETTERS
2009
Vol. 11, No. 12
2479-2482
Solid-Phase Synthetic Strategy and
Bioevaluation of a Labeled δ-Opioid
Receptor Ligand Dmt-Tic-Lys for In Vivo
Imaging
Jatinder S. Josan, David L. Morse,† Liping Xu,† Maria Trissal, Brenda Baggett,
Peg Davis, Josef Vagner, Robert J. Gillies,† and Victor J. Hruby*
Departments of Chemistry, Radiology, Biochemistry & Molecular Biophysics, and
Pharmacology and BIO5 Institute, The UniVersity of Arizona, Tucson, Arizona 85721
hruby@email.arizona.edu
Received January 30, 2009
ABSTRACT
A general solid-phase synthetic strategy is developed to prepare fluorescent and/or lanthanide-labeled derivatives of the δ-opioid receptor
(δOR) ligand H-Dmt-Tic-Lys(R)-OH. The high δ-OR affinity (Ki ) 3 nM) and desirable in vivo characteristics of the Cy5 derivative 1 suggest its
usefulness for structure-function studies and receptor localization and as a high-contrast noninvasive molecular marker for live imaging ex
vivo or in vivo.
Labeling of opioid peptides remains an active area of research
as pharmacological tools to study opioid receptor structure
and function, as well as for imaging.1 Most labeled opioids
for in vivo imaging have been designed to be lipophilic to
permit passage across the blood-brain barrier (BBB). How-
ever, opioid receptors have also been implicated to play a
role in a variety of cancers,2 cardiovascular diseases,3 and
gastrointestinal disorders.4 Further, recent research promises
newer paradigms of opioid analgesia acting outside the
central nervous system.5 Therefore, there is an increasing
need to develop labeled opioid ligands and establish synthetic
strategies, especially solid-phase approaches, for in vivo
imaging of peripherally as well as centrally restricted opioid
receptors.6 These ligands could also be useful for in vitro
studies such as altered opioid receptor expression profiles
observed in patients with morphine dependence and in
hypertrophic scars with associated nociceptive pain.7
(2) (a) Zagon, I. S.; McLaughlin, P. J.; Goodman, S. R.; Rhodes, R. E.
J. Natl. Cancer Inst. 1987, 79, 1059–1065. (b) Schreiber, G.; Campa, M. J.;
Prabhakar, S.; O’Briant, K.; Bepler, G.; Patz, E. F. Anticancer Res. 1998,
18, 1787–1792. (c) Fichna, J.; Janecka, A. Cancer Metastasis ReV. 2004,
23, 351–366. (d) Debruyne, D.; Oliveira, M. J.; Bracke, M.; Mareel, M.;
Leroy, A. Int. J. Biochem. Cell Biol. 2006, 38, 1231–1236.
(3) Villemagne, P. S.; Dannals, R. F.; Ravert, H. T.; Frost, J. J. Eur.
J. Nucl. Med. Mol. Imaging 2002, 29, 1385–1388.
† Current address: Department of Molecular and Functional Imaging,
H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida 33612.
(1) (a) Lipkowski, A. W.; Misicka, A.; Kosson, D.; Kosson, P.; Lachwa-
From, M.; Brodzik-Bienkowska, A.; Hruby, V. J. Life Sci. 2002, 70, 893–
897. (b) Navratilova, E.; Waite, S.; Stropova, D.; Eaton, M. C.; Alves, I. D.;
Hruby, V. J.; Roeske, W. R.; Yamamura, H. I.; Varga, E. V. Mol.
Pharmacol. 2007, 71, 1416–1426. (c) Lever, J. R. Curr. Pharm. Des. 2007,
13, 33–49.
(4) Pol, O.; Palacio, J. R.; Puig, M. M. J. Pharmacol. Exp. Ther. 2003,
306, 455–462.
(5) Stein, C.; Schafer, M.; Machelska, H. Nat. Med. 2003, 9, 1003–
1008.
(6) Ryu, E. K.; Wu, Z.; Chen, K.; Lazarus, L. H.; Marczak, E. D.; Sasaki,
Y.; Ambo, A.; Salvadori, S.; Ren, C.; Zhao, H.; Balboni, G.; Chen, X.
J. Med. Chem. 2008, 51, 1817–1823.
10.1021/ol900200k CCC: $40.75
Published on Web 05/15/2009
 2009 American Chemical Society

Among the wide variety of opioid ligands known, the
dipeptide Dmt-Tic represents the minimal peptide sequence
that selectively interacts with δ-opioid receptor as a potent
antagonist (Kiδ ) 0.022 nM; pA2 ) 8.2).8 Numerous
derivatives of Dmt-Tic have been reported with either agonist
or antagonist properties, µ- or δ-opioid selectivities, or mixed
µ,δ activities, which makes it an ideal candidate for labeling.9
For example, H-Dmt-Tic-Lys(Ac)-OH exhibits high δ-recep-
tor affinity (Ki ) 0.047 nM) in radioligand binding assays.10
Opioid peptide ligands with fluorescent functionalities such
as rhodamine, pyrene, dansyl, and fluorescein have been
described before.1,11 We chose a cyanine (Cy) dye as it is
highly fluorescent and water-soluble, as well as providing a
significant advantage over other optical labels for in vivo
imaging. For example, the Cy5 fluoresces in the far-red
region of the visible spectrum and thus is ideal for minimiz-
ing background artifacts. Labeling of opioid peptides gener-
ally cannot involve the NR-terminus since this is critical for
the opioid receptor affinity (“message region”).12 Further, a
free carboxylic function at the C-terminus is important to
maintain high δ-receptor selectivity.8b,10,12 Thus, the label
must be attached on the side chain, often a C-terminal
residue, in a manner that has minimal influence on the ligand-
binding domain. Here, we describe a solid-phase strategy to
prepare Dmt-Tic ligands and their labeled analogues linked
at the C-terminus lysine side chain via small linkers. In this
context, hydrophilic components such as spacers and labels
were employed for peripheral retention, lower nonspecific
uptake, and faster blood clearance of the ligand.13 The
bioevaluation of the Cy5 probe 1 as a representative example
is described, and the flexibility of the synthetic scheme to
prepare dual-modality agents is highlighted.
mercially available labeling moieties contain an activated
carboxylic acid (e.g., N-hydroxy succinimide (NHS) ester
derivative) or a maleimide that readily react with an amine
or a thiol, respectively. Therefore, the synthetic scheme was
designed for labeling H-Dmt-Tic-Lys(R)-OH by a maleimide
derivative of Cy5 and an NHS ester of the lanthanide chelator
DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetrace-
tic acid). For optimal spacing between the ligand and the
probe, small linkers such as 3-mercaptopropionyl (Mpr) and
8-amino-3,6-dioxaoctanyl (Ado) were employed. The syn-
thesis was started with esterification of NR-Fmoc-Lys(Nε-
Mtt)-OH onto Wang resin (Scheme 1). This was achieved
Scheme 1.
Synthesis of Resin Intermediate 5a
a Abbreviations: Mtt: methyltrityl; Pip. ) piperidine; HOCt ) 6-chloro-
1-hydroxybenzotriazole; DIC ) diisopropylcarbodiimide HBTU: 2-(1H-
benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate).
An easy, robust, and scaleable synthetic route to label Dmt-
Tic ligands was developed based only on commercially
available reagents and a labeling scheme that could be
performed on-resin or in solution as desired. Many com-
from Wang resin, which was mesylated using an 8-fold
excess of MsCl at 0 °C to activate OH groups, followed by
coupling with Fmoc-Lys(Mtt)-OH.14 The NR-Fmoc protec-
tion from 3 was removed with piperidine in DMF, and Fmoc-
Tic-OH was coupled using standard NR-Fmoc/tBu strategy
of solid-phase peptide synthesis to give intermediate 4.15 For
final coupling, Boc-Dmt-OH was used since a free N-
terminal peptide can be directly obtained after final acidic
cleavage. Additionally, a choice of NR-terminal Boc protec-
tion prevents against premature Fmoc deprotection by free
NH2 groups released on the side chain of lysine and any
consequent cyclative elimination (dioxopiperazine) of Dmt-
Tic from Dmt-Tic-Lys(R)-resin.16 Lastly, Boc is smaller than
(7) (a) Narita, M; Funada, M.; Suzuki, T. Pharamaol. Ther. 2001, 89,
1–15. (b) Cheng, B; Liu, H. W.; Fu, X. B.; Sheng, Z. Y.; Li, J. F. Br. J.
Dermatol. 2008, 158, 713–720.
(8) (a) Dmt: 2′,6′-dimethyl-L-tyrosine; Tic: 1,2,3,4-tetrahydroisoquino-
line-3-carboxylic acid. (b) Salvadori, S.; Attila, M.; Balboni, G.; Bianchi,
C.; Bryant, S. D.; Crescenzi, O.; Guerrini, R.; Picone, D.; Tancredi, T.;
Temussi, P. A.; Lazarus, L. H. Mol. Med. 1995, 1, 678–689.
(9) Bryant, S. D.; Jinsmaa, Y.; Salvadori, S.; Okada, Y.; Lazarus, L. H.
Biopolymers (Pept. Sci.) 2003, 71, 86–102.
(10) (a) Balboni, G.; Onnis, V.; Congiu, C.; Zotti, M.; Sasaki, Y.; Ambo,
A.; Bryant, S. D.; Jinsmaa, Y.; Lazarus, L. H.; Trapella, C.; Salvadori, S.
J. Med. Chem. 2006, 49, 5610–5617. (b) Balboni, G.; Salvadori, S.; Guerrini,
R.; Negri, L.; Giannini, E.; Bryant, S. D.; Jinsmaa, Y.; Lazarus, L. H. J. Med.
Chem. 2004, 47, 4066–4071.
(11) (a) Hazum, E.; Chang, K-J.; Shechter, Y.; Wilkinson, S.; Cuatre-
casas, P. Biochem. Biophys. Res. Commun. 1979, 88, 841–846. (b) Mihara,
H.; Lee, S.; Shimohigashi, Y.; Aoyagi, H.; Kato, T.; Izumiya, N.; Costa,
T. FEBS Lett. 1985, 193, 35–38. (c) Berezowska, I.; Chung, N. N.; Lemieux,
C.; Zelent, B.; Szeto, H. H.; Schiller, P. W. Peptides 2003, 24, 1195–1200.
(d) Kumar, V.; Aldrich, J. V. Org. Lett. 2003, 5, 613–616. (e) Balboni, G.;
Salvadori, S.; Piaz, A. D.; Bortolotti, F.; Argazzi, R.; Negri, L.; Lattanzi,
R.; Bryant, S. D.; Jinsmaa, Y.; Lazarus, L. H. J. Med. Chem. 2004, 47,
(14) In a 50 mL bottle containing 1 g of Wang resin (0.93 mmol/g)
with a magnetic stir bar, dry CH2Cl2 was added to swell the resin for 1 h.
The solvent was removed, the bottle closed with a septum and flushed with
nitrogen, and iPr2NEt (9 equiv, 1.4 mL) in 15 mL of CH2Cl2 was added.
The resin slurry was cooled to 0 °C followed by dropwise addition of MsCl
(8 equiv, 0.57 mL) in 2 mL of CH2Cl2. The reaction was stirred for 20
min, the ice-bath was removed, and the stirring was continued for another
20 min (rt). The resin was then transferred to a syringe reactor and washed
with dry CH2Cl2 and dry DMF. Fmoc-Lys(Mtt)-OH (2 equiv, 1.2g), CsI (2
equiv, 0.5g), iPr2NEt (2 equiv, 0.32 mL) in ca. 10 mL of dry DMF were
added, and the reaction was stirred overnight at rt.
6541–6546
.
(12) (a) Hruby, V. J.; Gehrig, C. A. Med. Res. ReV. 1989, 9, 343–401.
(b) Aldrich, J. V.; Vigil-Cruz, S. C. Burger’s Medicinal Chemistry and
Drug DiscoVery, 6th ed.; Abraham, D. J., Ed.; Wiley & Sons: New York,
2003; Vol. 6,Chapter 7, p 329.
(15) The resin was NR-Fmoc deprotected with piperidine/DMF (1:4) and
then washed with DMF, CH2Cl2, 0.2 M HOBt/DMF, and DMF. Fmoc-
Tic-OH (3 equiv), HOCt (3 equiv), and DIC (6 equiv) in DMF were then
added, and the reaction was stirred for 2 h.
(13) (a) Calculated log D of compound 1 reveals a value of 1.01 at pH
7.4 (log D: 2.6, 1.35, 1.16 at pH 1.5, 5.0, 6.5, respectively). (b) Duval,
R. A.; Allmon, R. L.; Lever, J. R. J. Med. Chem. 2007, 50, 2144–2156.
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Org. Lett., Vol. 11, No. 12, 2009

Fmoc, facilitating a faster coupling rate. Despite that, Boc-
Dmt coupling to the sterically hindered Tic-Lys(R)-resin was
challenging. The coupling has to be mediated via strong
HBTU activation accelerated by microwave.17 Also, there
is a conceptual disadvantage of using an unprotected phenolic
group on Dmt. The reaction leads to Dmt self-condensation,
forming small amounts of Dmt-oligomers. Nonetheless, the
formed phenolic esters are susceptible to mild aminolysis
and can be selectively removed by treatment with 50%
piperidine in CH2Cl2:MeOH (5:1) before acidic cleavage.
Following Dmt coupling to give intermediate 5, the
coupling of dyes and chelating agents can be performed on
the resin or in solution. For Cy5 labeling, more cost-effective
conjugation of dye in solution was preferred via a thiol-
maleimide reaction. 3-Mercaptopropionyl was chosen as a
small linker for C-terminal attachment as the dye possesses
a 12-atom linker with maleimide at the end. Thus, Trt-S-
CH2CH2COOH was coupled to 5 using HOCt/DIC protocol
and then cleaved with acidic cocktail (82.5% TFA, 5% H2O,
5% iPr3SiH, 5% thioanisole, and 2.5% ethanedithiol) to give
ligand 6 (Scheme 2). The compound was purified by
Scheme 3.
Syntheses of Eu-DOTA Labeled Ligand 2a
a Abbreviations: Mmt ) methoxytrityl.
Scheme 2. Synthesis of Mpr Derivative 6 and Cy5 Ligand 1
bifunctional handle to investigate its utility for coupling
commonly available labeling moieties, for dual-modality
labeling (e.g., optical/magnetic), for coupling to nanoparticles
with lanthanide labels, and for preparing dimeric ligands at
a later stage (unpublished data). For this purpose, the
synthetic scheme was designed to incorporate orthogonally
protected Fmoc-Cys(Mmt)-OH at the end of the Ado linker
and coupled using standard NR-Fmoc/tBu strategy to give
intermediate 7. The amine group of Cys was then chosen to
couple DOTA on-resin using DOTA-NHS ester (2 equiv)
and iPr2NEt (8 equiv) in DMF for overnight to give 8.19
The peptide was then cleaved from the resin, and europium
chelation was carried out in solution to yield ligand 2.20 The
purified peptides were dissolved in DMSO/H2O (3:2) for
bioassay purposes.21
Purified ligands 1 and 6 were evaluated for their binding
affinity for the δOR in a competitive binding assay using
HCT116 colon carcinoma cells engineered to express the
δOR (Figure 1). Europium-labeled opioid peptide DPLCE
was used as a competing ligand in a time-resolved fluores-
cence (TRF) assay of our own design.22 Peptide 6 retained
high δ receptor affinity (Ki ) 2.5 ( 0.8 nM), which was
preparative HPLC, and the Cy5 dye was conjugated to the
peptide in solution to give ligand 1.18 The on-resin labeling
was tested by synthesizing a DOTA chelate as shown in
Scheme 3. The Mtt protection on lysine was removed with
3% TFA and 5% iPr3SiH in CH2Cl2. Here we employed a
(19) Note that commercially available DOTA-NHS (Macrocyclics, TX)
contains an estimated 3 equiv of TFA by weight. Also, DOTA can
alternatively be coupled using a maleimide derivative, following selective
cleavage of Mmt group with 3% TFA, 5% iPr3SiH in CH2Cl2.
(20) The purified compound was dissolved in (NH4)2CO3/NH4OAc buffer
(pH 8) and 3 equiv of EuCl3·6H2O was added. The reaction stirred overnight
in inert atmosphere (to prevent disulfide formation by air oxidation). The
excess salts were then removed by solid-phase extraction (SPE) (see
Supporting Information).
(16) Caspasso, S.; Sica, F.; Mazzarella, L.; Balboni, G.; Guerrini, R.;
Salvadori, S. Int. J. Pep. Protein Res. 1995, 45, 567–573.
(17) Boc-Dmt-OH (3 equiv), HBTU (3 equiv), and iPr2NEt (6 equiv)
in DMF were added to the resin, and the reaction was heated in a household
microwave for 3 s. The reaction was stirred until it cooled to rt; the heating
was repeated (5x), and the resin was stirried for another 2 h.
(18) Ligand 8 was dissolved in HEPES buffer (pH 7.2), and 1.3 equiv
of Cy5-maleimide was added in aliquots until full conversion was achieved
as monitored on analytical HPLC. The labeled ligand was then separated
with SPE (see Supporting Information).
(21) No diketopiperazine formation was observed in this solvent system
for 1 month; see: Carpenter, K. A.; Weltrowska, G.; Wilkes, B. C.; Schmidt,
R.; Schiller, P. W. J. Am. Chem. Soc. 1994, 116, 8450–8458.
Org. Lett., Vol. 11, No. 12, 2009
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only slightly lower than that reported for Dmt-Tic-Lys-NH2
(Ki ) 0.71 nM) and significantly lower than for Dmt-Tic-
Lys(Ac)-OH (Ki ) 0.047 nM).3,4 However, a direct com-
parison between these TRF results and reported radioligand
binding assays is not wholly instructive because of inherent
difference in the assay methods. Ligand 1, with a Cy5 label,
exhibited a similar bioactivity profile as ligand 6 and retained
high δOR affinity (Ki ) 3 ( 0.1 nM), which is equipotent
to its unlabeled counterpart. Thus, attachment of the Mpr
spacer and the Cy5 label did not interfere in any significant
way in the ligand-receptor interaction. This is in sharp
contrast to many labeled opioid peptides (including Tic-based
analogues) with remarkably high loss in affinity for δOR.23
Further, ligand 1 exhibited high inhibitory potency (Ke )
37 ( 9 pM for n ) 8) in the mouse-isolated vas deferens
(MVD) assay against δ-agonist DPDPE (Figure 1B),24 which
clearly demonstrates it as one of the best labeled δOR
ligands.
Figure 2. Fluorescence imaging of targeted agent. Mice bilaterally
implanted with HCT116 xenografts of (right) control cells, and (left)
cells overexpressing δOR. (A) Mouse imaged 72 h postintravenous
(iv) injection of 100 µg of ligand 1. (B) Mouse imaged 24 h post-
iv injection of 10 µg of ligand 1. (C) Fluorescence intensity values
over whole tumor regions of interest after 72 h of clearance of 100
µg of ligand 1 and after 24 h of clearance of 10 µg of ligand 1.
regions-of-interest (ROIs) over each tumor and noninvolved
muscle tissue. Histograms were generated for each ROI, and
mean fluorescence intensities were determined for each time
point. After 72 h, all δOR(+) tumors had elevated fluores-
cence intensities compared to the corresponding δOR(-)
tumors (Figure 2C). Notably, this intensity differential was
independent of dose, although higher contrast was observed
at low dose (10 µg) after 24 h (cf. high dose (100 µg)
animals). The Cy5-labeled opioid peptide provided a high-
constrast noninvasive molecular marker for live imaging of
cultured cells or in vivo imaging.
Figure 1. (A) Competitive binding assay of Cy5 labeled ligand 1
(Ki ) 3 nM; R2 ) 0.96). (B) Mean concentration-response data
for DPDPE from MVD assay (n ) 4 shown here).
In summary, we have described a solid-phase synthetic
methodology for derivatization of the highly potent δ-opioid
ligand Dmt-Tic-Lys(R)-OH. We sought easy modification
with fluorescent dyes and/or chelating labels either on-resin
or in solution. The applicability of this synthetic approach
was demonstrated by derivatizing a Dmt-Tic ligand with the
lanthanide chelator on a solid-phase support and the Cy5
label in solution. Finally, bioevaluation of the Cy5-labeled
compound demonstrated its potential utility in in vitro studies
and in vivo imaging of the peripherally expressed δ-opioid
receptor.
For in vivo studies, SCID mice were xenografted bilater-
ally with HCT116/δOR and parental HCT116 tumors, which
do not express δOR. Mice then were tail vein injected with
10 µg of ligand 1 and images were acquired at different times
postinjection using a VersArray 1300B cooled CCD camera,
a filtered fiberoptic light source and a tunable emission filter
(CRI, Inc.) (see Supporting Information for methods). After
15 min postinjection, the fluorescence intensity maps indi-
cated systemic presence of the compound with high intensity
throughout the animal (not shown). At 24 h, the compound
was systemically cleared and retained in the δOR-containing
but not the parental tumors, as shown in Figure 2 (A and
B). Images were analyzed using Image-Pro Plus 5 by drawing
Acknowledgment. This research was supported by NCI
grants R01 CA123547 and R01 CA097360 and ABRC Grant
ABRC06-006.
(22) (a) Handl, H. L.; Vagner, J.; Yamamura, H. I.; Hruby, V. J.; Gillies,
R. J. Anal. Biochem. 2005, 343, 299–307. (b) Handl, H. L.; Vagner, J.;
Yamamura, H. I.; Hruby, V. J.; Gillies, R. J. Anal. Biochem. 2004, 330,
242–250.
Supporting Information Available: Experimental details,
characterization of compounds/intermediates, bioassay and
imaging discussed. This material is available free of charge
via the Internet at http://pubs.acs.org.
(23) Arttamangkul, S.; Alvarez-Maubecin, V.; Thomas, G.; Williams,
J. T.; Grandy, D. K. Mol. Pharmacol. 2000, 58, 1570–1580.
(24) Kramer, T. H.; Davis, P.; Hruby, V. J.; Burks, T. F.; Porreca, F.
J. Pharmacol. Exp. Ther. 1993, 266, 577–584.
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