Stabilized 111In-labeled sCCK8 analogues for targeting CCK2-receptor positive tumors: Synthesis and evaluation

Article abstract of DOI:10.1021/bc900465y

Radiolabeled cholecystokinin-8 (CCK8) peptide analogues can be used for peptide receptor radionuclide imaging and therapy for tumors expressing CCK2/gastrin receptors. Earlier findings indicated that sulfated CCK8 (sCCK8, Asp-Tyr(OSO₃H)-Met-Gly-Trp-Met-Asp-Phe-NH₂) may have better characteristics for peptide receptor radionuclide therapy (PRRT) than gastrin analogues. However, sCCK8 contains an easily hydrolyzable sulfated tyrosine residue and two methionine residues which are prone to oxidation. Here, we describe the synthesis of stabilized sCCK8 analogues, resistant to hydrolysis and oxidation. Hydrolytic stability was achieved by replacement of the Tyr(OSO₃H) moiety by a robust isosteric sulfonate, Phe(p-CH ₂SO₃H). Replacement of methionine by norleucine (Nle) or homopropargylglycine (HPG) avoided undesired oxidation side-reactions. The phenylalanine analogue Phe(p-CH₂SO₃H) of l-tyrosine, synthesized by a modification of known synthetic routes, was incorporated in three peptides: sCCK8[Phe²(p-CH₂SO₃H),Met ^3,6], sCCK8[Phe²(p-CH₂SO₃H),Nle ^3,6], and sCCK8[Phe²(p-CH₂SO ₃H),HPG^3,6]. All peptides were N-terminally conjugated with the macrocyclic chelator DOTA (1,4,7,10-tetraazacyclododecane-N,N,N,N- tetraacetic acid) and radiolabeled with In-111. In vitro binding assays on CCK2R-expressing HEK293 cells revealed that all three peptides showed specific binding and receptor-mediated internalization, with binding affinity values (IC₅₀) in the nanomolar range. In vitro oxidation studies demonstrated that peptides with Nle or HPG indeed were resistant to oxidation. In vivo targeting studies in mice with AR42J tumors showed that tumor uptake was highest for ¹¹¹In-DOTA-sCCK8 and ¹¹¹In-DOTA- sCCK8[Phe²(p-CH₂SO₃H),Nle^3,6] (4.78 ± 0.64 and 4.54 ± 1.15%ID/g, respectively, 2 h p.i.). The peptide with the methionine residues replaced by norleucine (¹¹¹In-DOTA- sCCK8[Phe²(p-CH₂SO₃H), Nle^3,6]) showed promising in vivo characteristics and will be further investigated for radionuclide imaging and therapy of CCK2R-expressing tumors.

Full text of DOI:10.1021/bc900465y

Bioconjugate Chem. 2010, 21, 663–670
663
Stabilized 111In-Labeled sCCK8 Analogues for Targeting CCK2-Receptor
Positive Tumors: Synthesis and Evaluation
Susan Roosenburg,#,‡ Peter Laverman,# Lieke Joosten,# Annemarie Eek,# Wim J. G. Oyen,# Marion de Jong,§
Floris P. J. T. Rutjes,‡ Floris L. van Delft,‡,* and Otto C. Boerman#
Department of Nuclear Medicine, Radboud University Nijmegen Medical Center, The Netherlands, Institute for Molecules and
Materials, Radboud University Nijmegen, Nijmegen, The Netherlands, and Department of Nuclear Medicine, Erasmus MC,
Rotterdam, The Netherlands. Received October 25, 2009; Revised Manuscript Received January 15, 2010
Radiolabeled cholecystokinin-8 (CCK8) peptide analogues can be used for peptide receptor radionuclide imaging
and therapy for tumors expressing CCK2/gastrin receptors. Earlier findings indicated that sulfated CCK8 (sCCK8,
Asp-Tyr(OSO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2) may have better characteristics for peptide receptor radionuclide
therapy (PRRT) than gastrin analogues. However, sCCK8 contains an easily hydrolyzable sulfated tyrosine residue
and two methionine residues which are prone to oxidation. Here, we describe the synthesis of stabilized sCCK8
analogues, resistant to hydrolysis and oxidation. Hydrolytic stability was achieved by replacement of the
Tyr(OSO3H) moiety by a robust isosteric sulfonate, Phe(p-CH2SO3H). Replacement of methionine by norleucine
(Nle) or homopropargylglycine (HPG) avoided undesired oxidation side-reactions. The phenylalanine analogue
Phe(p-CH2SO3H) of L-tyrosine, synthesized by a modification of known synthetic routes, was incorporated in
three peptides: sCCK8[Phe2(p-CH2SO3H),Met3,6], sCCK8[Phe2(p-CH2SO3H),Nle3,6], and sCCK8[Phe2(p-
CH2SO3H),HPG3,6]. All peptides were N-terminally conjugated with the macrocyclic chelator DOTA (1,4,7,10-
tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid) and radiolabeled with In-111. In vitro binding assays on
CCK2R-expressing HEK293 cells revealed that all three peptides showed specific binding and receptor-mediated
internalization, with binding affinity values (IC50) in the nanomolar range. In vitro oxidation studies demonstrated
that peptides with Nle or HPG indeed were resistant to oxidation. In vivo targeting studies in mice with AR42J
tumors showed that tumor uptake was highest for 111In-DOTA-sCCK8 and 111In-DOTA-sCCK8[Phe2(p-
CH2SO3H),Nle3,6] (4.78 ( 0.64 and 4.54 ( 1.15%ID/g, respectively, 2 h p.i.). The peptide with the methionine
residues replaced by norleucine (111In-DOTA-sCCK8[Phe2(p-CH2SO3H), Nle3,6]) showed promising in vivo
characteristics and will be further investigated for radionuclide imaging and therapy of CCK2R-expressing tumors.
targeting characteristics as compared to nonsulfated CCK8
(nsCCK8). Behr et al. found that minigastrin analogues
showed extremely high kidney uptake, whereas CCK8
exhibited low kidney uptake (5). The high kidney uptake was
shown to be related to the pentaglutamate sequence of
minigastrin, and was found to be reduced by coinjection of
poly-Glu-containing peptides (6, 7). Unlike gastrin, sCCK8
includes a sulfated tyrosine (Tyr) residue that is easily
hydrolyzable. Moreover, an Fmoc-Tyr(SO3H)-OH building
block is difficult to use in solid-phase peptide synthesis
(SPPS), as it is acid labile (8). Another pitfall of sCCK8 is
the presence of two methionines (Met) that are prone to
oxidation to sulfoxides during radiolabeling and potentially
also in vivo, leading to loss of receptor binding (9, 10).
Therefore, we aimed to develop new and stabilized sCCK8
analogues, resistant to both hydrolysis and oxidation. To this
end, we envisaged replacement of the Tyr sulfate moiety by
a robust isosteric sulfonate. Since sulfonate isosteres of
tyrosine sulfate are not commercially available, synthetic
preparation of the phenylalanine analogue Phe(p-CH2SO3H)
from L-tyrosine was required, to afford a Phe(p-CH2SO3H)
building block that can be incorporated in peptide sequences
under normal coupling conditions in SPPS without additional
side chain protection (11). Second, to prevent oxidation, two
sCCK8 analogues were designed with the Met residues
replaced by norleucine (Nle) or homopropargylglycine
(HPG) (12, 13). All peptides were N-terminally conjugated
with the macrocyclic chelator DOTA (1,4,7,10-tetraazacy-
clododecane-N,N′,N′′,N′′′-tetraacetic acid) to allow radiola-
INTRODUCTION
Cholecystokinin (CCK), a functional peptide hormone in the
gastrointestinal tract and the brain, mediates a variety of
hormonal and neuromodulatory functions mediated by two
receptors, CCK1R and CCK2R (1). In addition, a splice variant
of the CCK2R was identified, CCK2i4svR, which is expressed
at relatively low levels in human colorectal cancer and in
pancreatic cancer, but not in normal tissue (2). All three
receptors belong to the superfamily of G-protein coupled
receptors. Several tumor types, such as small cell lung cancers
(SCLC) and medullary thyroid carcinomas (MTC), abundantly
express CCK2/gastrin receptors (CCK2R) (3).
Since both gastrin and CCK8 display a high affinity for
these receptors (3), radiolabeled analogues of these peptides
may be considered for peptide receptor imaging and peptide
receptor radionuclide therapy (PRRT). We have previously
shown that sCCK8 (Asp-Tyr(OSO3H)-Met-Gly-Trp-Met-Asp-
Phe-NH2) has good characteristics for PRRT compared to
gastrin analogues (4). Sulfated CCK8 has affinity for both
the CCK1 and CCK2 receptor, and shows better CCK2R
* Author for correspondence: Dr. F.L. van Delft, Institute for
Molecules and Materials, Radboud University Nijmegen, Heyendaalseweg
135, 6525 AJ Nijmegen, The Netherlands, e-mail: F.vanDelft@
science.ru.nl.
# Department of Nuclear Medicine, Radboud University Nijmegen
Medical Centre.
‡ Institute for Molecules and Materials, Radboud University Nijmegen.
§ Department of Nuclear Medicine, Erasmus MC.
10.1021/bc900465y  2010 American Chemical Society
Published on Web 03/19/2010

664 Bioconjugate Chem., Vol. 21, No. 4, 2010
Roosenburg et al.
beling. Finally, the three sCCK8 analogues were characterized
both in vitro and in vivo and compared to DOTA-sCCK8.
with EtOAc/heptane (1/1, 3 × 20 mL). Organic layers were
dried on MgSO4 and concentrated in vacuo. The crude product
was purified by flash column chromatography (CH2Cl2, EtOAc/
heptane 1/1). Yield: 6.12 g (19.8 mmol, 68%) colorless oil. RF
EXPERIMENTAL PROCEDURES
1
(EtOAc/heptane 1/3) 0.42; H NMR (CDCl3) δ 1.42 (s, 9H,
Synthesis of Fmoc-Phe(p-CH2SO3Na)-OH. General
Methods. Solvents were distilled from appropriate drying agents
prior to use and stored under nitrogen. Chemicals were
purchased from Sigma-Aldrich and used as received, unless
stated otherwise. Reactions were carried out under inert
atmosphere of dry nitrogen or argon. Standard syringe tech-
niques were applied for the transfer of dry solvents and air- or
moisture-sensitive reagents. Reactions were followed, and RF
values were obtained using thin-layer chromatography (TLC)
on silica gel-coated plates (Merck 60 F254) with the indicated
solvent mixture. Detection was performed with UV-light. NMR
spectra were recorded on a Bruker DMX 300 (300 MHz)
spectrometer in CDCl3 solutions (unless otherwise reported).
Chemical shifts are given in ppm with respect to tetramethyl-
silane (TMS) as internal standard. Coupling constants are
reported as J-values in Hz. Mass was determined by LCQ
measurement. Column or flash chromatography was carried out
using ACROS silica gel (0.035-0.070 mm, and ca. 6 nm pore
diameter).
-C(CH3)3), 3.05-3.14 (m, 2H, Ar-CH2-CH), 3.71 (s, 3H,
-OCH3), 4.58 (m, 1H, R-CH), 4.97 (m, 1H, -NH), 5.22 (d, J )
10.88, 1H, CHdCHH), 5.71 (d, J ) 17.61, 1H, CHdCHH),
6.68 (dd, J ) 17.61, 1H, CHdCH2), 7.07 (d, J ) 9.17, 2H,
Ar-H), 7.33 (d, J ) 8.06, 2H, Ar-H).
N-tert-Butyloxycarbonyl-(p-hydroxymethyl)-L-phenylalanine
Methyl Ester (4). (11) Ozone was bubbled through a solution
of the vinyl compound 3 (4.26 g, 13.96 mmol) in 60 mL MeOH
at -60 °C until the solution turned blue. The mixture was stirred
under N2 for 10 min at -60 °C and 10.3 mL (139.6 mmol, 10
equiv) of Me2S was added. The mixture was stirred for 1 h at
-60 °C. Then, NaBH4 (2.64 g, 69.8 mmol, 5 equiv) was added.
The mixture was stirred at -60 °C for 10 min, then allowed to
warm to 0 °C and stirred for an additional 45 min. Saturated
NH4Cl (60 mL) was added, and the product was extracted with
EtOAc (2 × 100 mL). The organic layers were dried on MgSO4,
filtered, and concentrated to give a yellow-brown oil. The crude
product was purified by flash column chromatography (EtOAc/
heptane 2/3 f EtOAc 100%). Yield: 8.18 g (26.5 mmol, 50.4%
over 2 steps, 74%) of white/yellow crystals. RF (EtOAc/heptane
1/1) 0.31; mp 81 °C; 1H NMR (CDCl3) δ 1.42 (s, 9H,
-C(CH3)3), 3.08 (dd, J ) 10.36, 2H, Ar-CH2-CH), 3.72 (s, 3H,
-OCH3), 4.57 (m, 1H, R-CH), 4.67 (d, J ) 5.85, 2H, -CH2OH),
4.97 (m, 1H, -NH), 7.11 (d, J ) 8.02, 2H, Ar-H), 7.29 (d, J )
8.02, 2H, Ar-H). MS(ESI-TOF): 332.2 (M+Na+), calcd 332.16
(M+H+).
N-tert-Butyloxycarbonyl-L-tyrosine Methyl Ester (1). 30.0 g
(165.55 mmol) L-tyrosine was dissolved in 1.1 L MeOH and
cooled on an ice bath. 15.65 mL (215.24 mmol, 1.3 equiv)
SOCl2 was added dropwise and the reaction mixture was
refluxed at 75 °C overnight. The solvent was evaporated, and
the crude product (38.2 g white solid) was used in the next
1
step. H NMR (D2O) δ 3.25 (dq, 2H, Ar-CH2-CH), 3.86 (s,
3H, -OCH3), 4.39 (t, 1H, R-CH), 6.91-7.21 (dd, 4H, Ar-H).
Next, 38.2 g crude product was dissolved in 500 mL dioxane/
H2O (1/1). 34.65 g (412.5 mmol, 3 equiv) NaHCO3 and 43.21
g (198 mmol, 1.2 equiv) Boc2O were added and the mixture
was stirred at RT for 2 h. Dioxane was removed in vacuo and
EtOAc was added. The organic layer was washed with water
and brine, dried on MgSO4, filtered, and concentrated to yield
an orange oil, that crystallized overnight, yielding 44.52 g (150.7
mmol, 91% over 2 steps) of white crystals. RF (EtOAc/heptane
1/1) 0.49; mp 95 °C; 1H NMR (CDCl3) δ 1.42 (s, 9H,
-C(CH3)3), 3.02 (dq, 2H, Ar-CH2-CH), 3.71 (s, 3H, -OCH3),
4.53 (m, 1H, R-CH), 4.98 (m, 1H, -NH), 5.50 (bs, 1H, -OH),
6.72-6.99 (dd, 4H, Ar-H).
(p-Chloromethyl)-L-phenylalanine Methyl Ester (5). (16)
Compound 4 (10.03 g) was dissolved in 350 mL of CH2Cl2
and SOCl2 (25-30 equiv) was added dropwise, before refluxing
overnight. After evaporation to dryness, the remaining solid was
rinsed twice with Et2O and dried. The product was used in the
next step without further purification. Yield: 4.59 g (62%) of
an off-white solid. RF (CH2Cl2/MeOH/AcOH 9/1/0.1) 0.34; mp
1
178 °C (lit. 180 °C); H NMR (MeOD) δ 3.15-3.32 (dq, 2H,
Ar-CH2-CH), 3.81 (s, 3H, -OCH3), 4.33 (m, 1H, R-CH), 4.65
(s, 2H, -CH2Cl), 7.28 (d, 2H, Ar-H), 7.43 (d, 2H, Ar-H);
MS(ESI-TOF): 228.0 (M+H+), calcd 228.07 (M+H+).
(p-Sulfomethyl)-L-phenylalanine (6). (11, 16) Compound 5
(4.59 g, 20.2 mmol) was dissolved in 200 mL H2O and 20.3 g
(161.6 mmol, 8 equiv) Na2SO3 was added. The mixture was
refluxed at 100 °C for 3 h. The solvent was removed in vacuo
and the crude product was washed with EtOH, yielding 32.25
g of a white solid after drying in vacuo. The crude product was
N-tert-Butyloxycarbonyl-O-trifluoromethylsulfonyl-L-ty-
rosine Methyl Ester (2). (14, 15) 1.0 g (3.39 mmol) of
compound 1 was dissolved in 40 mL CH2Cl2 and cooled on an
ice bath. Then, 720 µL (4.23 mmol, 1.25 equiv) DIPEA and
1.45 g (4.23 mmol, 1.25 equiv) phenyltriflimide were added,
before stirring the mixture at RT overnight. Solvent was
removed in vacuo, and the remaining colorless oil was dissolved
in 40 mL EtOAc. The solution was washed with H2O, 0.1 M
HCl, and brine, dried on MgSO4, and concentrated. The crude
product was purified by flash column chromatography (EtOAc/
heptane 15/85). Yield: 1.095 g (2.56 mmol, 79%) white crystals
(after drying in vacuo). RF (EtOAc/heptane 1/1) 0.65; mp 48
°C (lit.48-49 °C); 1H NMR (CDCl3) δ 1.41 (s, 9H, -C(CH3)3),
3.00-3.20 (dq, 2H, Ar-CH2-CH), 3.71 (s, 3H, -OCH3), 4.61
(m, 1H, R-CH), 5.00 (m, 1H, -NH), 7.22 (s, 4H, Ar-H). MS(ESI-
TOF): 392.0 (M+H+), calcd 392 (M+H+).
1
used in the next step. H NMR (D2O) δ 3.08-3.12 (dq, 2H,
Ar-CH2-CH), 3.97 (m, 1H, R-CH), 4.20 (s, 2H, -CH2SO3Na),
7.33 (d, 2H, Ar-H), 7.43 (d, 2H, H). MS(ESI-TOF): 280.3
(M+H+), calcd 280.3 (M+H+).
N-Fluorenyl-9-methoxycarbonyl-(p-sulfomethyl)-L-phenylalanine
(7). (16) The sulfonate product 6 (5 mmol) was dissolved in 40
mL H2O, cooled on an ice-bath and pH was set to 9.0 with 0.1
M NaOH. Next, 2.11 g (6.25 mmol, 1.25 equiv) Fmoc-OSu
was dissolved in 50 mL dioxane and added to the mixture. The
mixture was stirred on an ice-bath for 30 min, then at RT for
6 h while maintaining the pH between 8.5 and 9.0 with an
automatic titrator filled with 0.1 M NaOH. Dioxane was
evaporated and the excess Fmoc-OSu was extracted with diethyl
ether. The aqueous solution was acidified to pH 5 with 1 M
HCl and concentrated to dryness. The crude product was
dissolved in methanol, the insoluble material was removed by
filtration, and the methanol was evaporated. The residue was
dissolved in water and was washed with ethyl acetate before
concentration. This product was purified by column chroma-
N-tert-Butyloxycarbonyl-p-Vinyl-L-phenylalanine
Methyl
Ester (3). 1.43 g (30.4 mmol, 1.2 equiv) of LiCl in a 250 mL
flask was flame-dried in vacuo. 100 mL of dry DMF, 10.95 g
(25.6 mmol) of compound 2, and 8.23 mL (28.2 mmol, 1.1
equiv) tri-n-butyl vinyl tin were added. The mixture was stirred
at RT for 10 min. (PPh3)2PdCl2 (0.91 g, 3 mol %) was added,
and the mixture was heated up to 90 °C and stirred overnight
in sealed flask. Water was added and the product was extracted

Stabilized 111In-Labeled sCCK8 Analogues
Bioconjugate Chem., Vol. 21, No. 4, 2010 665
tography (MeCN/H2O 9/1), yielding 1.04 g of white solid (2.07
MA, USA). The log P value was calculated with the following
formula:
mmol, 41.4% over 2 steps). RF (ACN/H2O 9/1) 0.24; mp 185
1
°C (lit. 184 °C); H NMR (MeOD) δ 2.93-3.18 (dq, 2H, Ar-
CH2-CH), 3.99 (m, 1H, R-CH), 4.20 (s, 2H, -CH2SO3Na),
7.15-7.41 (m, 8H, Ar-H (Fmoc)), 7.61 (d, 2H, Ar-H), 7.78 (d,
2H, Ar-H); MS(ESI-TOF): 480.1 (M-H+), calcd 480.11
(M-H+).
Log Poctanol/water ) log(cpmoctanol/cpmwater
)
Oxidation Experiments. To study the susceptibility of the
peptide analogues to oxidation, the 111In-labeled peptides were
incubated with oxidation buffer (H2O2/acetic acid/NH4OAc 1/3/
36) for 10 min at RT. Before and after incubation, the peptides
were analyzed using RP-HPLC.
Stability Experiments. To study the stability of the peptide
analogues, the 111In-labeled peptides were incubated in human
serum for 4 h at 37 °C. The samples were centrifuged at 3000
g for 5 min. The plasma was removed and mixed with
acetonitrile. Before and after incubation, the peptides were
analyzed using RP-HPLC.
Synthesis of DOTA-Peptides. Peptides were synthesized
on a peptide synthesizer (Peptide Synthesizer SP 4000-LAB,
Labortec AG) by applying the Fmoc-strategy. In all coupling
steps, DIPCDI and HOBt were used, except for coupling of
Fmoc-Phe(p-CH2SO3H) and DOTA(t-Bu)3, which was per-
formed in the presence of HBTU and DIPEA. After completion
of the synthesis of the peptide on the resin, the peptide was
cleaved off by stirring in TFA/ethanedithiol (EDT)/triisopropyl
silane (TIS)/H2O (92.5/2.5/2.5/2.5) for 3-5 h. The mixture was
filtered, the filtrate centrifuged with Et2O. The precipitated
peptide was washed with Et2O and centrifuged several times.
Formation of the correct product was corroborated with mass
spectrometry (ESI-TOF).
Internalization Experiments. The internalization of the
radiolabeled DOTA-peptides was investigated using HEK293-
CCK2R cells (obtained from M. Hellmich, Univ. of Texas
Medical Branch, Galveston, USA). Cells were cultured in
Dulbecco’s Modified Eagle’s medium (DMEM) and supple-
mented with 10% fetal calf serum (FCS) and G418 (400 µg/
mL). Cells were grown in a humidified atmosphere with 5%
CO2 at 37 °C. Cells were grown to confluency in 6-well plates.
Radiolabeled peptide was added at a concentration of 4.4 ×
10-2 nM (12 000 dpm/well). Cells were incubated for 0.5-2 h
at either 0 or 37 °C, medium was removed, and cells were
washed with ice-cold binding buffer (DMEM supplemented with
0.5% w/v bovine serum albumin (BSA)). To remove receptor-
bound radiolabeled peptide, cells were incubated with acid buffer
(0.1 M acetic acid, 154 mM NaCl, pH 2.0) for 10 min at 0 °C.
After washing the cells twice with ice-cold PBS, the internalized
fraction was determined by counting the cells in a gamma
counter. The receptor-bound activity was also determined by
counting the acid wash fractions.
Determination of IC50 Values. The 50% inhibitory concen-
tration (IC50) of the peptides for binding the CCK2R was
determined on HEK293-CCK2R cells in a competitive binding
assay. Cells were grown to confluency in 6-well plates. Cells
were washed with binding buffer and incubated at RT for 10
min in binding buffer. Subsequently, unlabeled peptide was
added in the range 0.1-100 nM together with a trace amount
of 111In-DOTA-sCCK8[Phe2(p-CH2SO3H), HPG3,6] (12 000
dpm). After incubation at RT for 1 h, binding buffer was
removed and cells were washed twice with binding buffer. Cell-
associated radioactivity was determined. The IC50 was defined
as the peptide concentration at which 50% of binding without
competitor was reached. IC50 values (including standard devia-
tions) were calculated using GraphPad Prism software (version
4.00 for Windows, GraphPad Software, San Diego, CA, USA).
Biodistribution Studies. The in vivo tumor targeting potential
of the 111In-DOTA-peptides was investigated in female athymic
BALB/c mice. Subcutaneous tumors were induced by inocula-
tion with CCK2R expressing AR42J cells (rat tumor of the
exocrine pancreas). Cells were cultured in DMEM with 4500
mg/L D-glucose (Gibco, Invitrogen, Breda, The Netherlands),
supplemented with 10% FCS and 1% penicillin/streptomycin.
Mice were inoculated s.c. with 4 × 106 AR42J cells (200 µL)
in the left flank. When tumors had reached a weight of 0.2 g,
mice were divided in groups of 5 mice each and 370 kBq 111In-
DOTA-peptide (0.1 µg) was injected intravenously. Specificity
was studied in groups (n ) 5) which received a 1000-fold molar
excess of unlabeled sCCK8. Mice were killed 2 h postinjection
(p.i.), a blood sample was drawn, and tissues of interest were
dissected, weighed, and counted in a gamma-counter along with
a standard of the injected activity to allow calculation of the
injected dose per gram tissue (% ID/g). Animal experiments
The remaining t-Bu protecting groups of the chelator were
removed by stirring the product in TFA in the presence of a
few drops of concentrated HCl, leading to complete removal
of all protecting groups within 1 h. Three peptides were
synthesized: DOTA-sCCK8[Phe2(p-CH2SO3H)] (DOTA-D-Asp-
Phe(CH2SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2),
DOTA-
sCCK8[Phe2(p-CH2SO3H), Nle3,6] (DOTA-D-Asp-Phe(CH2SO3H)-
Nle-Gly-Trp-Nle-Asp-Phe-NH2), and DOTA-sCCK8[Phe2(p-
CH2SO3H), HPG3,6] (DOTA-D-Asp-Phe(CH2SO3H)-HPG-Gly-
Trp-HPG-Asp-Phe-NH2).
Peptides were analyzed by analytical HPLC (Alltima C18 5
µm, 3.2 × 150 mm, Alltech, USA) with 5-75% ACN 0.1%
TFA in H2O 0.1% TFA as an eluent. DOTA-sCCK8[Phe2(p-
CH2SO3H)] and DOTA-sCCK8[Phe2(p-CH2SO3H), Nle3,6] were
further purified by SP RP-HPLC (ReproSil 100 C18 250 mm
× 10 mm, Dr. A. Maisch HPLC-GmbH, Germany), using ACN
0.1% TFA (5-75% over 30 min) in H2O 0.1% TFA as an
eluent. Peptide-containing fractions (detected by UV-light) were
lyophilized and analyzed by HPLC and mass spectrometry.
DOTA-sCCK8[Phe2(p-CH2SO3H)] ) 1525.7 (M-H+), calcd )
1526.55. DOTA-sCCK8[Phe2(p-CH2SO3H), Nle3,6] ) 1489.8
(M-H+) calcd ) 1490.64. DOTA-sCCK8[Phe2(p-CH2SO3H),
HPG3,6] ) 1481.8 (M-H+) calcd ) 1482.58.
DOTA-sCCK8 was prepared as described before (17).
Radiolabeling. The DOTA-conjugated peptides were radio-
labeled with 111InCl3 (Covidien, Petten, The Netherlands) in 0.25
M ammonium acetate buffer, pH 5.0, for 30 min at 95 °C.
Labeling efficiency and radiochemical purity were checked by
HPLC (1100 series LC system, Agilent Technologies, Palo Alto,
CA, USA, RP-C18 column 5 µm, 4.6 × 250 mm) with a
gradient of H2O containing 0.1% TFA and ACN containing
0.1% TFA, and ITLC on ITLC-SG strips (Pall Life Sciences,
Milford, MA, USA) with 0.1 M NH4OAc/0.1 M EDTA (1/1
v/v) (RF labeled peptide ) 0, RF unbound 111In ) 1), as well as
MeOH/3.5% NH3 (1/1 v/v) (RF colloid ) 0, RF unbound 111In
or labeled peptide >0.8) as eluents. The radiochemical purities
of peptides used in the studies described here were always above
95%. Maximum specific activity was 5.5 GBq/µmol.
Determination of Partition Coefficient (log P). To deter-
mine the lipophilicity of the radiolabeled peptides, approximately
60 000 dpm 111In-DOTA-peptide was diluted in 3 mL PBS. An
equal volume of 1-octanol was added to obtain a binary system.
The system was vortexed for 10 s and mixed more gently for
another 4 min. The two layers were separated by centrifugation
(3000 g, 5 min). Three samples of 250 µL were taken of both
layers and the radioactivity was determined in a 3″ well-type
gamma-counter (Wallac 1480-Wizard 3, Perkin-Elmer, Boston,

666 Bioconjugate Chem., Vol. 21, No. 4, 2010
Roosenburg et al.
Scheme 1. Synthesis Route of Fmoc-Phe(p-CH2SO3H)-OH
were approved by the local animal welfare committee and
carried out according to national regulations.
latter coupling reaction was found to proceed optimally under
the action of HBTU and DIPEA. After cleavage from the resin
with TFA/H2O (92.5/2.5, v/v) in the presence of ethanedithiol
(EDT) and triisopropyl silane (TIS), with simultaneous protec-
tive group removal, the peptides were purified by RP-HPLC.
Molecular structures of the peptides are depicted in Figure 1.
Partition Coefficient. For 111In-DOTA-sCCK8[Phe2(p-
CH2SO3H] and 111In-DOTA-sCCK8[Phe2(p-CH2SO3H,Nle3,6],
the log P values were -2.97 ( 0.02 and -2.93 ( 0.13,
respectively. The log P value for 111In-DOTA-sCCK8[Phe2(p-
CH2SO3H,HPG3,6] was -2.86 ( 0.14. These values were
comparable to that of the lead compound, 111In-DOTA-sCCK8:
-2.91 ( 0.20. All compounds were relatively hydrophilic, and
the molecular changes did not affect the hydrophilicity of the
peptide significantly.
Statistical Analysis. All mean values are expressed as mean
( standard deviation (SD). Statistical analysis was performed
using one-way analysis of variance using GraphPad InStat
software (version 3.10, Graphpad software). The level of
significance was set at p < 0.05.
RESULTS
Synthesis of Phe(p-CH2SO3H) and Peptides. Since sul-
fonate building blocks are not commercially available, Fmoc-
Phe(p-CH2SO3H)-OH was synthesized starting from L-tyrosine,
by modification of known routes (Scheme 1). First, the amino
group and the acid functionality were protected by a Boc- and
a methyl group, respectively. The hydroxy group of 1 was
activated by triflation using phenyltriflimide and DIPEA, giving
the triflate 2 in 79% yield (14). The next step, a Stille
vinylation (14, 18), was performed under strictly dry conditions
and required careful workup and rapid purification to obtain
the vinylated product 3. Ozonolysis and reduction of the vinyl
compound was performed immediately to obtain the hydroxy-
methylated compound 4 (15, 18). The chloromethyl derivative
5, obtained from alcohol 4 by treatment with thionyl chloride,
was converted into the sulfonate 6 by refluxing in water in the
presence of a large excess of Na2SO3 (9). NMR spectra showed
that the methyl group was concomitantly removed in this
reaction, thereby obviating a separate deprotection step. The
final step in the synthesis of the sulfonate building block was
Fmoc-protection of the amine under basic conditions (16). The
overall yield of the synthesis starting from L-tyrosine was 9%
(19).
Having the phenylalanine sulfonate building block 7 at hand,
we investigated the synthesis of sCCK8 analogues by solid-
phase peptide synthesis. Initial application of standard coupling
conditions (HOBt, DIPCDI) to Fmoc-Phe(p-CH2SO3H)-OH
failed to afford incorporation of Phe(p-CH2SO3H)-OH in the
peptide. Fortunately, switching of reagents to HBTU and DIPEA
led to the desired octapeptide uneventfully. To obtain other more
stable sCCK8 peptide analogues not susceptible to oxidation,
methionine residues were replaced by either norleucine (Nle)
or homopropargylglycine (HPG) residues. After incorporation
of the final amino acid, the macrocyclic chelator DOTA was
coupled on the resin as tri-tert-butyl protected derivative. The
Oxidation Experiments. After 10 min incubation in oxida-
tion buffer (containing hydrogen peroxide and acetic acid), about
66% of the 111In-labeled DOTA-sCCK8[Phe2(p-CH2SO3H),
Met3,6] was oxidized, as judged by RP-HPLC. Both 111In-DOTA-
sCCK8[Phe2(p-CH2SO3H),Nle3,6] and 111In-DOTA-sCCK8[Phe2(p-
CH2SO3H,HPG3,6] did not show any oxidation.
Stability Experiments. In vivo, the peptide analogues should
remain intact long enough to target the gastrin/CCK2 receptors.
After 4 h incubation in human serum at 37 °C, peptides remained
intact. Of the 111In-labeled DOTA-sCCK8[Phe2(p-CH2SO3H),
Met3,6] 2.3% was oxidized after incubation in the serum.
Internalization Experiments. Internalization of the peptides
by HEK293-CCK2R cells was investigated in vitro. After 2 h
incubation at 37 °C, 35.2 ( 2.1% of the added 111In-DOTA-
sCCK8 was internalized by the cells, most of it during the first
30 min (Figure 2). Receptor bound fraction after 2 h was 11.6
( 0.7%. Of both 111In-DOTA-sCCK8[Phe2(p-CH2SO3H)] and
111In-DOTA-sCCK8[Phe2(p-CH2SO3H,HPG3,6], about 13% of
the added radiolabeled compound was internalized by the
CCK2R after 2 h at 37 °C, while 10-12% was still receptor-
bound. The internalized fraction of 111In-DOTA-sCCK8[Phe2(p-
CH2SO3H,Nle3,6] after 2 h was 26.8 ( 1.4% with a receptor-
bound fraction of 9.4 ( 0.6% (Figure 2). Internalization kinetics
of 111In-DOTA-sCCK8 and 111In-DOTA-sCCK8[Phe2(p-
CH2SO3H,Nle3,6] showed to be similar, although the percentage
of internalized fraction was about 7% lower for the synthetic
peptide (P < 0.05). Similar data were found using AR42J cells
(data not shown).

Stabilized 111In-Labeled sCCK8 Analogues
Bioconjugate Chem., Vol. 21, No. 4, 2010 667
Figure 1. Molecular structures of (a) DOTA-sCCK8 and (b) synthetic DOTA-peptides.
Figure 2. Internalization and receptor bound fractions in HEK293-CCK2R cells. Closed symbols indicate internalized fraction and open symbols
indicate receptor-bound fraction. Values are expressed as percentage of the total amount added.
Determination of IC50 Values. The apparent IC50 values for
binding CCK2R were determined for all peptides, using
HEK293-CCK2R cells in a solid-phase competitive binding
assay (Figure 3). All peptides showed an IC50 in the low
nanomolar range, although the apparent IC50 values of the three
synthesized peptides were approximately 10-fold higher than
that of the lead compound. The apparent IC50 value for sCCK8
was 1.16 ( 0.06 nM, for DOTA-sCCK8[Phe2(p-CH2SO3H)]
27.2 ( 1.44 nM, for DOTA-sCCK8[Phe2(p-CH2SO3H), Nle3,6]
11.6 ( 1.06 nM, and for DOTA-sCCK8[Phe2(p-CH2SO3H),
HPG3,6] 13.2 ( 1.12 nM.
Biodistribution. The potential of the radiolabeled DOTA-
peptides for in vivo targeting of CCK2R expressing tumors was
investigated in athymic mice with AR42J tumors. The biodis-
tribution 2 h p.i. for each of the four 111In-labeled peptides is
summarized in Figure 4.
Tumor uptake of 111In-DOTA-sCCK8 and 111In-DOTA-
sCCK8[Phe2(p-CH2SO3H,Nle3,6] were comparable (4.78 ( 0.64
and 4.54 ( 1.15% ID/g, respectively). The radioactivity
concentration of these peptides in the tumor was the highest of
all tissues studied (P < 0.001). For both other compounds, the tumor
uptake was significantly lower (2.18 ( 0.18% ID/g for 111In-
DOTA-sCCK8[Phe2(p-CH2SO3H], P < 0.001) and 1.92 ( 0.31%
ID/g for 111In-DOTA-sCCK8[Phe2(p-CH2SO3H,HPG3,6], P < 0.001),
and pancreatic concentration for these peptides was at the same
level as the concentration in the tumor. Tumor uptake of all
compounds could be blocked by coinjection of an excess of
unlabeled sCCK8, indicating receptor-mediated uptake. For all
compounds tested, the uptake in the normal tissues, such as the
blood, lungs, muscle, spleen, and small intestine was low. Specific
uptake was also found in the pancreas and, to a lesser extent, in
the stomach. Most likely, this is due to relatively high CCK2R

668 Bioconjugate Chem., Vol. 21, No. 4, 2010
Roosenburg et al.
the characterization of three DOTA-conjugated analogues of
the sCCK8 peptide.
A promising method to avoid the instability of the sulfate
ester is the replacement of the sulfated tyrosine by a phenyl-
alanine sulfonate isostere, featuring a covalent C-S bond that
is fully resistant to water. However, lack of commercial
availability of such an unnatural amino acid necessitated a
suitable synthetic preparation.
Synthesis started with a tyrosine triflate derivative (2), which
is readily available from the corresponding protected tyrosine
1 by reaction with phenyl triflimide. For the synthesis of the
hydroxymethylphenylalanines, we investigated different routes:
the palladium-catalyzed coupling of the tyrosine triflate with
tert-butyl acrylate (Heck reaction) and the coupling of the
tyrosine triflate to vinyltributylstannane in the presence of
metallic palladium and lithium chloride (Stille reaction), leading
to styrene derivative 3. The latter reaction was found to give
the best results, which is similar to the published data (14, 18).
Both Tilley et al. and the group of Larsen described this reaction
with slightly different tyrosine derivatives and they could obtain
the allylated compounds in good yields (65-82%) after
purification (14, 18). We found that careful workup and
immediate ozonolysis and reduction were essential to obtain
the hydroxymethylated product 4 in acceptable yields. We also
attempted to follow the four-step synthesis route described by
Miranda et al. (16), but the chloromethylation of phenylalanine,
the first step in this route, also failed to succeed in our hands,
as reported earlier (19). In addition, a third attempt involved
synthesis of the hydroxymethylphenylalanine directly by a Stille
reaction with (tributylstannyl)methanol (20). Several palladium
complexes and solvents were investigated, but the desired
compound could not be produced.
Figure 3. Competition binding analyses (IC50) of four peptides using
HEK293-CCK2R cells.
expression in murine pancreatic tissue en stomach. Kidney uptake
of all peptides was low (<1.5% ID/g).
DISCUSSION
In earlier studies, we as well as others have shown that
radiolabeled CCK8 peptide analogues show promise for peptide
receptor radionuclide targeting of small cell lung cancers and
medullary thyroid carcinomas (4, 5, 8, 12). In particular, sCCK8
was found to possess better CCK-receptor targeting behavior
(4). An important drawback of the peptides used to date,
however, is the intrinsic hydrolytic instability of the tyrosine
sulfate ester. Moreover, since CCK8 contains two methionine
residues, radiolabeling inevitably leads to varying amounts of
thioether oxidation, which negates the binding affinity of the
peptide to CCKR. Having these drawbacks in mind, we aimed
for sCCK8 peptide analogues with improved stability as
compared to the lead compound sCCK8. This study describes
The chloromethyl derivative 5 was obtained from the alcohol
4 by treatment with thionyl chloride. We found that during this
Figure 4. Biodistribution of (a) DOTA-sCCK8, (b) DOTA-sCCK8[Phe2(p-CH2SO3H)], (c) DOTA-sCCK8[Phe2(p-CH2SO3H), Nle3,6], and (d) DOTA-
sCCK8[Phe2(p-CH2SO3H), HPG3,6] in athymic mice bearing CCK2R expressing tumors in the left flank. Values are expressed as percentage of the
injected dose per gram tissue (n ) 5 mice/group). Blocking was performed by coinjection of a 1000-fold molar excess of unlabeled sCCK8. Mice
were dissected at 2 h p.i.

Stabilized 111In-Labeled sCCK8 Analogues
Bioconjugate Chem., Vol. 21, No. 4, 2010 669
reaction the Boc-protective group was conveniently removed
as well, probably due to the formation of HCl in this reaction.
The next step, conversion to the sulfonate 6, was also ac-
companied by an unintended but useful side reaction, i.e.,
hydrolysis of the methyl ester, thereby saving another depro-
tection step. On the basis of the whole sequence, it can be
concluded that a practical, multigram-scale procedure has been
developed in only 8 steps starting from L-tyrosine, leading to
the final Fmoc-protected sulfonate building block 7 in an
acceptable yield (9%).
pancreas expresses the CCK-receptor, whereas in human
pancreatic tissue, the CCK2 receptor is not expressed. Therefore,
in a clinical setting, pancreatic uptake of radiolabeled sCCK8
analogues is not expected.
In summary, DOTA-sCCK8[Phe2(p-CH2SO3H), Nle3,6] is a
peptide with receptor affinity and tumor uptake comparable to
sCCK8, but with an increased stability and therefore is a
promising peptide for use in PRRT.
CONCLUSION
The Fmoc-Phe(p-CH2SO3H)-OH building block was smoothly
incorporated in the peptide sequence on solid phase, but required
replacement of the typical DIPCDI/HOBt mixture with HBTU
and DIPEA as coupling reagents. Three peptides were synthe-
sized with the sulfonate building block, differing in the presence
of Met, Nle, or HPG residues on both positions 3 and 6. Finally,
all peptides were N-terminally conjugated with the macrocyclic
chelator DOTA, also requiring HBTU/DIPEA for activation.
A successful synthetic route was developed to a tyrosine
isostere that is not susceptible to hydrolysis of the sulfate ester
linkage and therefore is a valuable building block for the
preparation of medicinally relevant peptides. A series of
radiolabeled sCCK8 analogues with increased stability over the
natural peptide was successfully synthesized by solid-phase
peptide synthesis. The peptide with methionine residues replaced
by norleucine (DOTA-sCCK8[Phe2(p-CH2SO3H), Nle3,6]) showed
promising characteristics for CCK2R targeting and will be
further investigated for its potency in imaging and therapy.
Receptor binding studies and internalization studies were
performed on HEK293 cells stably expressing the CCK2
receptor. Substituting the tyrosine sulfate by its phenylalanine
isostere resulted in sCCK8 analogues with apparent affinities
for the CCK2 receptor in the low nanomolar range. When the
Met residues were replaced by Nle residues, a more stable
peptide was obtained with comparable tumor uptake in CCK2R
positive tumors. Reubi et al. also found that replacement of
methionine by norleucine in a DTPA-conjugated nonsulfated
CCK8-peptide does not affect the binding affinity for the CCK2
receptor, but increases the plasma stability of the compound
(12). In contrast, Mather et al. found that substitution of the
methionine residue with norleucine in gastrin peptide analogues
lowered both in vitro binding affinity and tumor uptake in vivo.
As Mather synthesized gastrin analogues and not CCK8
analogues, these peptides may be affected differently by
substitution of methionine by norleucine. This difference may
also be due to the two additional histidines on the N-terminus
(9). The group of Aloj synthesized and evaluated a series of
cyclic sCCK8 peptide analogues, in which the tyrosine sulfate
and both methionines were preserved. They found that cycliza-
tion of the peptides caused a 1000-fold decrease in affinity for
both the CCK1 and CCK2 receptor (21, 22).
Homopropargylglycine has not earlier been applied in tumor-
targeting peptides, but has been generally shown to be a suitable
and stable substitute for methionine (13). We found that
replacement of methionine by homopropargylglycine caused
reduction of CCK2R affinity resulting in reduced uptake of the
radiolabeled peptide in the AR42J tumor.
The CCK2 receptor belongs to the family of G-protein-
coupled receptors. When gastrin or CCK8 binds to the receptor,
the complex is internalized, after which the receptor can be
recycled or metabolized. After binding and internalization of
111In-labeled peptides, the radiometal is retained intracellularly
in the lysozomes. The peptides in this study all showed time-
dependent internalization. About 27% of 111In-DOTA-
sCCK8[Phe2(p-CH2SO3H), Nle3,6] was internalized after 2 h,
where 111In-DOTA-sCCK8 showed 35% internalization (P <
0.05).
Supporting Information Available: NMR-spectra, HPLC-
spectra, biodistribution studies. This material is available free
of charge via the Internet at http://pubs.acs.org.
ACKNOWLEDGMENT
Bianca Lemmers is kindly acknowledged for her assistance
with the animal experiments. The study was financially sup-
ported by the Dutch Cancer Society grant KUN 2006-3575.
LITERATURE CITED
(1) Noble, F., Wank, S. A., Crawley, J. N., Bradwejn, J., Seroogy,
K. B., Hamon, M., and Roques, B. P. (1999) International Union
of Pharmacology. XXI. Structure, distribution and functions of
cholecystokinin receptors. Pharmacol. ReV. 51, 745–781.
(2) Hellmich, M. R., Rui, X. L., Hellmich, H. L., Fleming, R. Y. D.,
Evers, B. M., and Townsend, Jr., C. M. (2000) Human colorectal
cancers express a constitutively active cholecystokinin-B/gastrin
receptor that stimulates cell growth. J. Biol. Chem. 275, 32122–
32128.
(3) Reubi, J. C., Schaer, J. C., and Waser, B. (1997) Cholecysto-
kinin(CCK)-A and CCK-B/gastrin receptors in human tumors.
Cancer Res. 57, 1377–1386.
(4) Laverman, P., Be´he´, M., Oyen, W. J. G., Willems, P. H. G.,
Corstens, F. H. M., Behr, T. M., and Boerman, O. C. (2004)
Two technetium-99m-labeled cholecystokinin-8 (CCK8) peptides
for scintigraphic imaging of CCK receptors. Bioconjugate Chem.
15, 561–568.
(5) Behr, T. M., Jenner, N., Be´he´, M., Angerstein, C., Gratz, S.,
Raue, F., and Becker, W. (1999) Radiolabeled peptides for
targeting cholecystokinin-B/gastrin receptor-expressing tumors.
J. Nucl. Med. 40, 1029–1044.
(6) Be´he´, M. P., Kluge, G., Becker, W., Gotthardt, M., and Behr,
T. M. (2005) Use of polyglutamic acids to reduce uptake of
radiometal-labeled minigastrin in the kidneys. J. Nucl. Med. 46,
1012–1015.
(7) Nock, B. A., Maina, T., Be´he´, M. P., Nikolopoulou, A.,
Gotthardt, M., Schmitt, J. S., Behr, T. M., and Maecke, H. R.
(2005) CCK-2/gastrin receptor-targeted tumor imaging with
99mTc-labeled minigastrin analogs. J. Nucl. Med. 46, 1727–1736.
(8) De Luca, S., and Morelli, G. (2004) Synthesis and characteriza-
tion of a sulfated and a non-sulfated cyclic CCK8 analogue
functionalized with a chelating group for metal labeling. J. Pept.
Sci. 10, 265–273.
(9) Mather, S. J., McKenzie, A. J., Sosabowski, J. K., Morris,
T. M., Ellison, D., and Watson, S. A. (2007) Selection of
radiolabeled gastrin analogs for peptide receptor-targeted radio-
nuclide therapy. J. Nucl. Med. 48, 615–622.
The peptides in this study showed very low kidney retention
(1.5% ID/g, 2 h p.i.) and display also low uptake in the main
peripheral tissue. This is in contrast with 111In-labeled mini-
gastrin analogues, which display a very high kidney uptake,
40-60% ID/g (5). Derivatives of minigastrin, lacking the
pentaglutamate sequence, showed a comparably low kidney
uptake compared to CCK8 analogues (23). Low kidney and
peripheral tissue retention are prerequisites for receptor-targeted
imaging and therapeutic agents. The observed specific uptake
in the pancreas can be explained by the fact that in rodents the

670 Bioconjugate Chem., Vol. 21, No. 4, 2010
Roosenburg et al.
(10) Breeman, W. A. P., Fro¨berg, A. C., de Blois, E., van Gameren,
A., Melis, M., de Jong, M., Maina, T., Nock, B. A., Erion, J. L.,
Maecke, H. R., and Krenning, E. P. (2008) Optimised labeling,
preclinical and initial clinical aspects of CCK-2 receptor-targeting
with 3 radiolabeled peptides. Nucl. Med. Biol. 35, 839–849.
(11) Gonzalez-Muniz, R., Cornille, F., Bergeron, F., Ficheux, D.,
Pothier, J., Durieux, C., and Roques, B. P. (1991) Solid phase
synthesis of a fully active analogue of cholecystokinin using the
acid-stable Boc-Phe(p-CH2SO3H) as a substitute for Boc-
Tyr(SO3H) in CCK8. Int. J. Pept. Protein Res. 37, 331–340.
(12) Reubi, J. C., Waser, B., Schaer, J. C., Laederach, U., Erion,
J., Srinivasan, A., Schmidt, M. A., and Bugaj, J. E. (1998)
Unsulfated DTPA- and DOTA-CCK analogs as specific high-
affinity ligands for CCK-B receptor-expressing human and rat
tissues in vitro and in vivo. Eur. J. Nucl. Med. 25, 481–490.
(13) van Hest, J. C. M., Kiick, K. L., and Tirrell, D. A. (2000)
Efficient incorporation of unsaturated methionine analogues into
proteins in vivo. J. Am. Chem. Soc. 122, 1282–1288.
Delft, F. L., and Boerman, O. C. (2007) Targeting of a CCK2
receptor splice variant with 111In-labelled cholecystokinin-8
(CCK8) and 111In-labelled minigastrin. Eur. J. Nucl. Med. Mol.
Imaging 35, 386–392.
(18) Larsen, S. D., Barf, T., Liljebris, C., May, P. D., Ogg, D.,
O’Sullivan, T. J., Palazuk, B., Schostarez, H. J., Craig Stevens,
F., and Bleasdale, J. E. (2002) Synthesis and biological activity
of a novel class of small molecular weight peptidomimetic
competitive inhibitors of protein tyrosine phosphatase 1B. J. Med.
Chem. 45, 598–622.
(19) Herzner, H., and Kunz, H. (2007) (p-Sulfomethyl)phenylala-
nine as a mimic of O-sulfatyl-tyrosine in synthetic partial
sequences of P-Selectin glycoprotein ligand 1 (PSGL-1). Tet-
rahedron 63, 6423–6436.
(20) Danheiser, R. L., Romines, K. R., Koyama, H., Gee, S. K.,
Johnson, C. R., and Medich, J. R. (1998) A hydroxymethyl anion
equivalent: tributyl[(methoxymethoxy)methyl]stannane. Org. Synth.
9, 704–708.
(21) De Luca, S., Saviano, M., Della Moglie, R., Digilio, G.,
Bracco, C., Aloj, L., Tarallo, L., Pedone, C., and Morelli, G.
(2006) Conformationally constrained CCK8 analogues obtained
from a rationally designed peptide library as ligands for chole-
cystokinin type B receptor. ChemMedChem 1, 997–1006.
(22) De Luca, S., De Capua, A., Saviano, M., Della Moglie, R.,
Aloj, L., Tarallo, L., Pedone, C., and Morelli, G. (2007) Synthesis
and biological evaluation of cyclic and branched peptide
analogues as ligands for cholecystokinin type 1 receptor. Bioorg.
Med. Chem. 15, 5845–5853.
(23) Good, S., Walter, M. A., Waser, B., Wang, X., Mu¨ller-Brand,
J., Be´he´, M. P., Reubi, J.-C., and Maecke, H. R. (2008)
Macrocyclic chelator-coupled gastrin-based radiopharmaceuticals
for targeting of gastrin receptor-expressing tumours. Eur. J. Nucl.
Med. Mol. Imaging 35, 1868–1877.
(14) Tilley, J. W., Sarabu, R., Wagner, R., and Mulkerins, K. (1990)
Preparation of carboalkoxyalkylphenylalanine derivatives from
tyrosine. J. Org. Chem. 55, 906–910.
(15) Chen, P., Cheng, P. T. W., Alam, M., Beyer, B. D., Bisacchi,
G. S., Dejneka, A. T., Evans, A. J., Gerytok, J. A., Hermsmeier,
M. A., Humphreys, W. G., Jacobs, G. A., Kocy, O., Lin, P.-F.,
Lis, A., Marella, M. A., Ryono, D. E., Sheaffer, A. K., Spergel,
S. H., Syn, C.-Q., Tino, J. A., Vite, G., Colonno, R. J., Zahler,
R., and Barrish, J. C. (1996) Aminodiol HIV protease inhibitors
Synthesis and structure-activity relationships of P1/P1’ com-
pounds: correlation between lipophilicity and cytotoxicity.
J. Med. Chem. 39, 1991–2007.
(16) Miranda, M. T. M., Liddle, R. A., and Rivier, J. E. (1993)
Synthesis of human CCK26-33 and CCK-33 related analogues
on 2,4-DMBHA and TMBHA. J. Med. Chem. 36, 1681–1688.
(17) Laverman, P., Roosenburg, S., Gotthardt, M., Park, J., Oyen,
W. J. G., de Jong, M., Hellmich, M. R., Rutjes, F. P. J. T., van
BC900465Y