N-terminal sugar conjugation and C-terminal Thr-for-Thr(ol) exchange in radioiodinated Tyr3-octreotide: Effect on cellular ligand trafficking in vitro and tumor accumulation in vivo

Article abstract of DOI:10.1021/jm040794i

For effective targeting of somatostatin receptor (sst) expressing tumors by radiolabeled octreotide analogues, high ligand uptake into sst-positive cells is mandatory. To optimize it, two modifications have been introduced into [ ¹²⁵I]Tyr³

Full text of DOI:10.1021/jm040794i

2778
J. Med. Chem. 2005, 48, 2778-2789
N-Terminal Sugar Conjugation and C-Terminal Thr-for-Thr(ol) Exchange in
Radioiodinated Tyr3-octreotide: Effect on Cellular Ligand Trafficking in Vitro
and Tumor Accumulation in Vivo
Margret Schottelius,*,† Jean Claude Reubi,‡ Veronique Eltschinger,‡ Markus Schwaiger,† and
Hans-Ju¨rgen Wester†
Nuklearmedizinische Klinik und Poliklinik, Klinikum rechts der Isar, Technische Universita¨t Mu¨nchen,
81675 Mu¨nchen, Germany, and Institut fu¨r Pathologie, Universita¨t Bern, 3010 Bern, Switzerland
Received February 23, 2004
For effective targeting of somatostatin receptor (sst) expressing tumors by radiolabeled
octreotide analogues, high ligand uptake into sst-positive cells is mandatory. To optimize it,
two modifications have been introduced into [125I]Tyr3-octreotide ([125I]TOC): C-terminal Thr-
for-Thr(ol) exchange (leading to Tyr3-octreotate (TOCA)) and N-terminal derivatization with
different carbohydrates. Both have significant impact on radioligand uptake into sst2-expressing
cells in vitro and in vivo. Glucose conjugation via Amadori reaction by itself led to improved
tumor uptake of [123I]Gluc-TOC in vivo, which is based on an enhancement of peptide
internalization despite a reduction in receptor affinity. In the case of the doubly modified
analogues [123I]Gluc-TOCA, [123I]Gluc-S-TOCA, and [123I]Gal-S-TOCA, a cumulative effect of
both structural modifications was observed, leading up to a 5-fold increased uptake of these
compounds in sst-expressing tumors compared to [125I]TOC. Thus, glycosylation with small
carbohydrates was found to be a suitable tool to enhance receptor-mediated uptake of
radiolabeled octreotide analogues into sst-positive malignancies, leading to tracers with excellent
characteristics for in vivo sst-imaging applications.
Introduction
intracellular trafficking of both receptor and ligand has
been extensively studied.24,25
A variety of human tumors overexpress high-affinity
receptors for small peptide hormones such as soma-
tostatin (sst),1 vasoactive intestinal peptide (VPAC),2-4
neurotensin (NT),5 substance P,6 gastrin-releasing pep-
tide (GRP),7 cholecystokinin (CCK),8 oxytocin,9,10 vaso-
pressin,11 and R-melanocyte stimulating hormone (R-
MSH),12 providing the opportunity for in vivo receptor
scintigraphy and endoradiotherapy with radiolabeled
neuropeptides. So far, only somatostatin receptor ligands
such as [111In]octreoscan13,14 or [90Y]DOTATOC15-17
have found widespread application in nuclear oncology.
In recent years, however, intensive research has been
directed toward the optimization of other radiolabeled
neuropeptide receptor ligands with respect to labeling
methodology, in vivo stability, and pharmacokinetic
properties, yielding promising candidates for in vivo
VPAC,18 NT,19 GRP, and CCK20-23 receptor imaging.
Generally, the targeting efficiency of neuropeptide
analogues to tumor cells relies on the overexpression
of their cognate receptors on the cell surface as well as
on the extent of their internalization into receptor-
expressing cells. The latter process is of particular
relevance for in vivo tumor targeting, since it leads to
intracellular accumulation of the radionuclide and thus
enhances the scintigraphic signal and/or the therapeutic
ratio. Because of their predominant clinical role, the
internalization of radiolabeled somatostatin analogues
into sst-receptor expressing cells and the subsequent
Upon ligand binding, all five human somatostatin
receptor subtypes (hsst1-5) are internalized in a time-,
temperature-, and subtype-dependent manner (hsst3 >
hsst5 > hsst4 > hsst2 > hsst1).26
The amount of internalized receptor-agonist complex
can be influenced by the level of cellular receptor
regulation27,28 and by the structure of the radioligand.
The latter has been demonstrated by Koenig et al., who
found substantially increased internalization of the
synthetic somatostatin analogue [125I]BIM-23027 com-
pared to that of [125I]SRIF-14 in CHO cells stably
transfected with hsst2, suggesting that the structure of
the radioligand may have a significant impact on the
extent of endocytosis.24 Furthermore, comparative stud-
ies revealed that internalization of radiolabeled oct-
reotide analogues can be substantially increased by
Tyr3-for-Phe3 substitution and even more so by C-
terminal Thr(ol)8-for-Thr8 exchange.29,30 This finding
constitutes the basis of endoradiotherapeutic strategies
applying Tyr3-octreotate (TOCA) analogues labeled with
low-energy â emitters such as 153Sm 31 and 177Lu.32,33
Despite comparably lower internalization, promising
therapeutic results have also been obtained in clinical
studies with [111In]DTPA-octreotide.34,35 The success of
these endoradiotherapeutic strategies, especially when
low-energy particle emitting radionuclides are applied,
crucially depends on the amount of radioligand ac-
cumulated inside the tumor cells.
* To whom correspondence should be addressed. Phone: 49 89 4140
4554/6340. Fax: 49 89 4140 4897. E-mail: M.Schottelius@lrz.tum.de.
† Technische Universita¨t Mu¨nchen.
This quantity, however, is determined not only by the
extent of ligand internalization but also by the rates of
externalization, recycling, and/or degradation of both
‡ Universita¨t Bern.
10.1021/jm040794i CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/01/2005

Conjugation and Exchange
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2779
radioligand and receptor. Both the somatostatin recep-
tor25 and associated ligands24 were shown to be mainly
externalized after dissociation of the receptor-ligand
complex in the early endosomal compartment. While the
rate of externalization of intact radioligand was found
to be agonist-independent, the degree of ligand degrada-
tion and particularly of intracellular activity retention
strongly depends on the nature of the radioligand used
and on the radiolabeling method.24 Radioiodinated
SRIF-14 showed rapid and extensive intracellular deg-
radation and subsequent release of [125I]iodotyrosine
into the external medium,24 whereas radiometalated
octreotide analogues showed significantly reduced in-
tracellular degradation rates. However, substantial
residualization of radioactivity inside the cells after
longer incubation times (∼40 h)36 has been observed,
which has been explained by the trapping of the charged
radiometal-chelate-containing fragments in the lysoso-
mal compartments.37,38
In previous studies, we have investigated the effect
of N-terminal glycosylation of 125I-,39-41 111In-,42
99mTc-,43 and 18F-labeled44,45 octreotide and/or octreotate
derivatives on their pharmacokinetics and tumor ac-
cumulation in vivo. Biodistribution studies in AR42J
tumor-bearing nude mice showed that glucose conjuga-
tion of radioiodinated Tyr3-octreotide (TOC) leads to
increased tumor accumulation.39 Furthermore, the com-
bination of C-terminal substitution of Thr(ol) by Thr
with sugar derivatization as in [123I]maltotriose octreo-
tate ([123I]Mtr-TOCA) was shown to yield radioiodinated
somatostatin analogues with excellent excretion char-
acteristics and high tumor accumulation suitable for in
vivo sst receptor imaging.40 Recently, initial patient PET
studies were performed using [18F]Gluc-Lys(FP)-TOCA
for the localization of sst-positive tumors.44 As found for
Figure 1. Structure of the carbohydrate analogues of radio-
iodinated TOC and TOCA investigated in this study.
(epitope tagged at the N-terminal end) and in the rat
pancreatic tumor cell line AR42J.
Results
Peptide Synthesis. Solid-phase peptide synthesis
(SPPS) of TOC(Dde), I-TOC(Dde), TOCA(Dde), and
I-TOCA(Dde) was carried out using a standard Fmoc
protocol and HOBt and TBTU as coupling agents. Yields
ranged from 85% to 97% based on resin-bound amino
acid or alcohol. Cyclization of the freshly cleaved pep-
tides using H2O2 in an aqueous THF solution buffered
to pH 7 proceeded smoothly within less than 30 min.
While UV purities (220 nm) of crude cyclized TOC(Dde)
and TOCA(Dde) usually exceeded 93%, more side prod-
ucts were observed for both 3-iodo-Tyr3-containing pep-
tides (up to 20%). However, all peptides were used for
the following reaction steps without further purification.
The N-terminal glycosylation of the parent peptides
with glucose, maltose, or maltotriose was achieved
within 16-20 h in yields up to 85% (value based on the
integration of product and precursor peaks in the
reaction control RP-HPLC chromatograms (220 nm)).
Synthesis of the pentafluorophenyl esters of 1-S(2,3,4,6-
tetraacetylglucopyranosyl)-3-mercaptopropionate and
the corresponding galactose analogue from the per-
acetylated sugar precursors was achieved in two steps
in 68-78% overall yield. In contrast to the long reaction
times and elevated temperatures needed for Amadori
glycosylation, N-terminal conjugation of TOCA(Dde)
with the aforementioned thioglycoside active esters was
quantitative within 30-45 min at room temperature.
After subsequent Dde deprotection (and deacetylation
in the case of Gluc-S- and Gal-S-TOCA) and preparative
RP-HPLC purification, all glycosylated TOC and TOCA
derivatives as well as their 3-iodo-Tyr3 counterparts
[
123I]Mtr-TOCA, very high tumor-to-background ratios
were obtained at early time points (<60 min) with
negligible accumulation in excretion organs, demon-
strating the compatibility of the “glycosylated octreo-
tate” concept with different labeling methodologies.
To quantify the effects of glycosylation and C-terminal
Thr(ol)-by-Thr substitution on ligand uptake into sst-
expressing tissues on a cellular level, we performed
detailed in vitro studies with two series of radioiodi-
nated, glycosylated TOC-analogues: [125I]TOC and its
glucose- ([123I]Gluc-TOC) , maltose- ([123I]Malt-TOC),
and maltotriose- ([123I]Mtr-TOC) Amadori derivatives
as well as [123I]TOCA and its corresponding sugar
analogues
123I]Mtr-TOCA. Additionally, the effect of an alternative
[ [
123I]Gluc-TOCA, 123I]Malt-TOCA, and
[
glycosylation method via a mercaptopropionic acid
linker was investigated using [123I]Gluc-S-TOCA and
[
123I]Gal-S-TOCA (Figure 1).
Three major determinants for radioligand accumula-
tion in tumor cells were investigated: (a) the sst-
receptor affinity and specificity, (b) the internalization,
and (c) the extent of ligand externalization and recy-
cling. Additionally, the EC50 of unlabeled TOC for ligand
internalization was determined for all compounds in-
vestigated. Experiments were performed as dual tracer
experiments (123I-labeled compound of interest +
[
125I]TOC as an internal reference) both in CHO (chinese
hamster ovary) cells stably transfected with hsst2

2780 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Schottelius et al.
Affinity Profiles to hsst. In vitro binding properties
of I-TOC, I-TOCA, and their corresponding carbohy-
drated analogues were investigated using cells stably
expressing sst1-sst5 in displacement experiments using
[
125I][Leu8,D-Trp22,Trp25]somatostatin 28 as radioligand.
Results are summarized in Table 1 and are compared
to the control peptide native somatostatin 28, which
binds with high affinity to all five sst subtypes.
All octreotide analogues investigated showed no af-
finity to sst1, very high affinity to sst2, and moderate or
low affinity to sst3-5. Generally, substitution of Thr(ol)8
by Thr8 (I-TOC f I-TOCA) resulted in an increase in
sst2 and sst4 affinity. The N-terminal glycosylation led
to decreased sst2 affinities (except I-Malt-TOC and
I-Mtr-TOC) and sst3 affinities compared to nonmodified
I-TOC and I-TOCA, respectively. Moreover, all sugar
analogues of I-TOCA, independent of the glycosylation
method, showed decreased sst3 and sst5 affinity com-
pared to their corresponding I-TOC-counterparts and
thus enhanced sst2 specificity. Of the compounds inves-
tigated, I-Malt-TOCA and I-Mtr-TOCA demonstrated
the highest sst2 specificity.
Figure 2. Preparative RP-HPLC (A, isocratic eluent of 28%
EtOH (0.5% AcOH) in water (0.5% AcOH)) and analytical
quality control chromatogram (B, isocratic eluent of 29% EtOH
(0.5% AcOH) in water (0.5% AcOH)) of [125I]TOC.
Internalization and EC50,R in CHO Cells. The
amount of internalized [123I]Gluc-TOC, [123I]Malt-TOC,
and [123I]Mtr-TOC and of [123I]TOCA and its five sugar
analogues into CHO cells normalized to the value found
for [125I]TOC from the same experiment in the absence
of unlabeled competitor is shown in Figure 3. While
conjugation of [125I]TOC with maltose and maltotriose
had a comparably small effect on ligand internalization
(109.7 ( 5.5% for [123I]Malt-TOC and 93.0 ( 5.0% for
were obtained in purities of g98%. Overall yields of all
glycosylated peptides based on nonglycosylated precur-
sor ranged from 15% to 34%.
Radiolabeling. The efficiency of radioiodide incor-
poration usually exceeded 95%. However, because of the
formation of radioiodinated side products, radiochemical
yields of the 125I- and 123I-labeled peptides after
RP-HPLC isolation only ranged from 45% to 85%. All
radioiodinated peptides were obtained in high radio-
chemical purity (>99%).
Since the HPLC conditions applied allowed very
efficient separation of the radioiodinated product from
the unlabeled precursor (as shown for [125I]TOC in
Figure 2, ∆tR g 5 min) for all radioligands investigated
and since no coeluting carrier peak was observed in the
labeled peptide peak on the quality control UV chro-
matogram, the specific activity of the labeled peptides
was assumed to be that of the radioiodide used for their
preparation (g74 GBq/µmol for 125I, ∼185 GBq/µmol for
123I).
[
123I]Mtr-TOC), the internalization of [123I]Gluc-TOC
was increased by 28.8 ( 9.4% compared to that of
[
125I]TOC. For [123I]TOCA, a significant increase in
ligand internalization due to C-terminal Thr8-for-Thr-
(ol)8 exchange was found (178.8 ( 6.9% of [125I]TOC).
As observed for [125I]TOC, both the maltose and mal-
totriose derivatives as well as the Gal-S derivative of
[
125I]TOCA also showed nearly unchanged internaliza-
tion compared to their nonmodified analogue. [123I]Gluc-
TOCA, however, showed higher internalization than
[
125I]TOCA (193.1 ( 12.4% of [125I]TOC). For [123I]Gluc-
S-TOCA, the highest internalization among the com-
Table 1. Affinity Profiles (IC50) to the Human sst1-5 Receptors Determined for Somatostatin 28 (SS 28) and the Compounds
Investigated Using [125I][Leu8,D-Trp22,Tyr25]Somatostatin 28 as the Radioligand (Values Are IC50 ( SD [nM], and Number of
Experiments Are in Parentheses)a
peptide
hsst1
hsst2
hsst3
hsst4
hsst5
SS-28
3.6 ( 0.3 (5)
>1000 (2)
>1000 (5)
>1000 (3)
>10000 (3)
>10000 (3)
>10000 (3)
>1000 (3)
>10000 (3)
>10000 (3)
>1000 (3)
>1000 (3)
>10000 (7)
>10000 (3)
>10000 (4)
>10000 (3)
>10000 (6)
>10000 (3)
2.1 ( 0.3 (5)
3.1 ( 1.8 (2)
1.6 ( 0.4 (5)
2.2 ( 0.7 (3)
1.2 ( 0.2 (3)
1.1 ( 0.2 (3)
0.47 ( 0.2 (3)
2.0 ( 0.5 (3)
0.78 ( 0.2 (3)
0.95 ( 0.3 (3)
2.0 ( 0.7 (3)
2.0 ( 0.8 (3)
14 ( 2.6 (6)
1.5 ( 0.4 (3)
11 ( 1.7 (6)
1.6 ( 0.4 (3)
2.5 ( 0.5 (7)
0.2 ( 0.04 (3)
3.2 ( 0.2 (5)
330 ( 146 (2)
186 ( 52 (5)
357 ( 22 (3)
243 ( 47 (3)
210 ( 15 (3)
187 ( 38 (3)
>1000 (3)
757 ( 169 (3)
823 ( 67 (3)
398 ( 19 (3)
491 ( 63 (3)
880 ( 324 (4)
>1000 (3)
3 ( 0.3 (5)
2.5 ( 0.2 (5)
10.8 ( 1.8 (2)
65 ( 17 (5)
TOC
346 ( 114 (2)
729 ( 87 (5)
837 ( 154 (3)
>1000 (3)
I-TOC
I-Gluc-TOC
I-Malt-TOC
I-Mtr-TOC
64 ( 24 (3)
75 ( 19 (3)
>1000 (3)
110 ( 35 (3)
50 ( 5.8 (3)
521 ( 269 (3)
210 ( 46 (3)
327 ( 93 (3)
310 ( 156 (3)
413 ( 167 (3)
393 ( 84 (6)
547 ( 160 (3)
114 ( 29 (5)
187 ( 50 (3)
73 ( 21 (6)
I-TOCA
337 ( 57 (3)
1033 ( 142 (3)
797 ( 61 (3)
823 ( 87 (3)
356 ( 60 (3)
482 ( 134 (3)
>1000 (6)
I-Gluc-TOCA
I-Malt-TOCA
I-Mtr-TOCA
I-Gluc-S-TOCA
I-Gal-S-TOCA
DOTA-TOCb
DOTA-TOCAb
Y-DOTA-TOCb
Y-DOTA-TOCAb
Ga-DOTA-TOCb
Ga-DOTA-TOCAb
453 ( 176 (3)
>10000 (5)
523 ( 239 (3)
>1000 (6)
389 ( 135 (5)
>1000 (3)
613 ( 140 (7)
>1000 (3)
300 ( 140 (3)
377 ( 18 (3)
a Cell membrane pellets were prepared from CHO-K1 cells (sst1 and sst5) and CCL39 cells (sst2-4).48 b Cited from ref 48.

Conjugation and Exchange
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2781
Table 2. Relative EC50 Values (EC50,R) for the Internalization
of Glycosylated [123I]TOC and [123I]TOCA Derivatives
Determined Using CHO Cells Stably Expressing hsst2 (n ) 3 in
Two (/) or Three Separate Determinations, Mean ( SD)
EC50,R
[
[
[
[
[
[
[
[
[
[
125I]TOC
1
123I]Gluc-TOC
123I]Malt-TOC*
123I]Mtr-TOC*
123I]TOCA
1.43 ( 0.06
1.12 ( 0.05
1.01 ( 0.12
1.70 ( 0.11
2.51 ( 0.12
2.13 ( 0.19
1.81 ( 0.07
2.96 ( 0.14
2.62 ( 0.07
123I]Gluc-TOCA
123I]Malt-TOCA
123I]Mtr-TOCA
123I]Gluc-S-TOCA
123I]Gal-S-TOCA
Thr(ol)8 counterparts (P < 0.001). Of the Amadori
analogues investigated, [123I]Gluc-TOCA had the high-
est EC50,R. Both thioglycoside-modified TOCA ana-
logues, however, showed even higher EC50,R values of
up to 2.96 ( 0.14 ([123I]Gluc-S-TOCA).
Figure 3. Internalization of peptides 1B-2F in CHO cells
(hsst2) as percent of the internalization of the reference [125I]-
TOC (1A) found in the same experiment (mean ( SD; n ) 3
in two (/) or three separate determinations, 10 min incuba-
tion).
Externalization and Recycling in CHO Cells. To
assess the extent of ligand externalization of the car-
bohydrated [123I]TOC and [123I]TOCA analogues from
CHO cells compared to [125I]TOC and to investigate the
role of ligand recycling (reuptake after externalization),
two experiments were performed. After an initial incu-
bation for 10 min, allowing the internalization of the
radioligands, cells were washed and then incubated with
radioligand-free medium containing either no unlabeled
competitor (conditions allowing recycling) or 5 µM TOC
(conditions inhibiting recycling) for 30 and 60 min. To
ensure receptor integrity, no acid wash was performed
after the internalization incubation. Thus, externaliza-
tion data are given as the percent of the cellularly
located activity (internalized + receptor bound) at t )
0 of the externalization incubation.
Figure 5A depicts a typical externalization profile of
a glycosylated *I-TOC-analogue compared to [125I]TOC,
in this case shown for [123I]Gluc-TOCA. Both under
conditions allowing and inhibiting ligand recycling, all
glycosylated [125I]TOC and [123I]TOCA analogues showed
nearly identical externalization profiles (data not shown).
When ligand recycling was allowed, 90-93% of the
cellular radioactivity at t ) 0 were still found inside the
cells after 30 min ([125I]TOC: 82-86%) for all sugar
analogues. After 60 min, the amount of intracellularly
located radioactivity was reduced to 82-86% of the
initial activity in the case of the carbohydrated peptides
analogues and to 79-83% for [125I]TOC. Under condi-
tions inhibiting recycling, only 4-8% of the initial
cellularly located radioactivity was found to remain
internalized after 60 min for all carbohydrated peptides
as well as for the reference [125I]TOC.
Figure 4. Determination of the EC50 of unlabeled TOC for
the internalization of [125I]TOC (full circles) and [123I]Gluc-
TOCA (hollow circles) in CHO (hsst2) (A) and AR42J (rsst2)
(B) cells (data are the mean ( SD; n ) 3) and calculation of
the EC50,R for [123I]Gluc-TOCA.
Internalization and EC50,R in AR42J Cells. To
investigate to what extent the internalization of glyco-
sylated [125I]TOC analogues is dependent on the cell line
used, a comparative EC50 determination was performed
in AR42J cells using [123I]Gluc-TOCA as an exemplary
carbohydrated radioligand (Figure 4B). In AR42J-cells,
pounds investigated in this study was found (264.7 (
20.3% of [125I]TOC).
The EC50,R values obtained for [125I]TOC, [123I]TOCA,
and their corresponding glucose, maltose, and mal-
totriose conjugates using CHO cells (Figure 4 A) are
summarized in Table 2. In both the [125I]TOC and the
[
123I]Gluc-TOCA showed 277.9 ( 10.5% of the internal-
ization found for [125I]TOC (vs 193.1 ( 12.4% in CHO
cells). However, the EC50,R found for [123I]Gluc-TOCA
in AR42J cells was unexpectedly low (1.14 ( 0.04 vs
2.51 ( 0.12 in CHO cells).
[
123I]TOCA series, the same pattern in the EC50,R was
found: Gluc > Malt > Mtr ∼ nonglycosylated analogue.
For all [123I]TOCA derivatives, values were significantly
higher than those found for their corresponding
Externalization and Recycling in AR42J Cells.
The externalization of [123I]Gluc-TOCA from AR42J cells

2782 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Schottelius et al.
Table 3. In Vivo Tumor Accumulation of [125I]TOC,
[
125I]TOCA, and Their Glycosylated Analogues in AR42J
Tumor-Bearing Nude Mice 60 min p.i. (/, 30 min p.i.)a
tumor accumulation [% ID/g]
[
[
[
[
[
[
[
[
[
[
125I]TOC
5.5 ( 1.1
8.7 ( 1.5
123I]Gluc-TOC
123I]Malt-TOC
123I]Mtr-TOC*
123I]TOCA
5.2 ( 0.6
4.9 ( 2.2
21.2 ( 9.7
20.5 ( 11.1
11.2 ( 2.4
25.1 ( 4.4
26.2 ( 7.5
22.6 ( 2.2
123I]Gluc-TOCA
123I]Malt-TOCA
123I]Mtr-TOCA
123I]Gluc-S-TOCA
123I]Gal-S-TOCA
a Data are % ID/g (n ) 3-5, mean ( SD).
Thr(ol)8 exchange (TOC f TOCA) resulted in dramati-
cally increased uptake of the radioligands in sst-
expressing tumor tissue. In the [125I]TOC series, only
glucose conjugation led to a significant increase in tumor
uptake of approximately 160% (P < 0.001), while the
[
125I]Malt-TOC and [125I]Mtr-TOC showed an accumula-
tion comparable to [125I]TOC. In contrast, in the [125I]TO-
CA-series, [125I]Gluc-TOCA showed a tumor uptake
comparable to that of [125I]TOCA, whereas tumor ac-
cumulation of [125I]Mtr-TOCA was higher. Of all sugar
analogues of [125I]TOCA investigated, [125I]Gluc-S-
TOCA showed the highest uptake in sst-expressing
tumor tissue.
Discussion
In Figure 1, the structures of radioiodinated TOC and
TOCA and their respective analogues obtained from the
Amadori reaction with glucose (Gluc-TOC/TOCA), mal-
tose (Malt-TOC/TOCA), and maltotriose (Mtr-TOC/
TOCA) are shown. It is important to note that only one
of four possible conformations of the sugar moiety is
shown. Besides the â-pyranoid structure (Figure 1), the
R- and â-furanoid and the open-chain conformations of
the deoxyketoses are possible and may be present in
equilibrium mixtures.46 However, during preparative
and analytical RP-HPLC using isocratic conditions, only
one peak was observed for all Amadori compounds (data
not shown). Therefore, it seems probable that in the case
of all Amadori analogues one sugar conformer repre-
sents the predominant species.
The major advantage of glycosylation via thioglycoside
conjugation versus Amadori reaction consists of the
known conformation of the sugar residue in [125I]Gluc-
and [125I]Gal-S-TOCA. As determined by 1H NMR, only
the â-thioglycoside is produced during the reaction
between the sugar 1,2-trans peracetate precursors and
3-mercaptopropionic acid. Furthermore, compared to
glycosylation via Amadori reaction, peptide acylation
with S-glycosylated 3-mercaptopropionic acid results in
a much greater ease of synthesis, i.e., shorter reaction
times (30 min vs 16 h), higher glycosylation yields
(>99% vs ∼80%) and a significantly lower proportion
of side product formation.
Figure 5. Externalization kinetics of [123I]Gluc-TOCA (circles)
and [125I]TOC (down triangles) under conditions allowing
(ligand-free medium; solid icons, solid lines) and inhibiting (5
µM TOC; hollow icons, dashed lines) ligand recycling in CHO
(hsst2) (A) and AR42J (rsst2) (B) cells. Data are given as
percent of the cellularly located activity (internalized +
receptor bound ligand) at t ) 0 of the externalization experi-
ment (after a 10 min internalization incubation). Data are the
mean ( SD (n ) 3).
compared to the externalization from CHO cells is
shown in Figure 5B. Under conditions allowing ligand
recycling, 72.6 ( 0.9% and 70.8 ( 0.3% of the cellularly
located activity (t ) 0) remained internalized in the case
of [123I]Gluc-TOCA after 30 and 60 min, respectively.
For [125I]TOC, only 51.1 ( 1.0% and 46.9 ( 0.3% of the
initial activity were found inside the cells. When ligand
recycling was inhibited by 5 µM TOC, however, the
fraction of both [123I]Gluc-TOCA and [125I]TOC remain-
ing internalized after 60 min was, like in CHO cells,
comparable ([123I]Gluc-TOCA, 33.4 ( 1.2%; [125I]TOC,
29.1 ( 1.6%). However, in contrast to the data obtained
with CHO cells (4-8%), a significantly higher amount
of activity remained inside the AR42J cells under these
conditions.
For the in vitro assays, the experimental setup chosen
for the comparative evaluation of the carbohydrated
Tumor Accumulation in Vivo. Tumor accumula-
tion of [125I]TOC, [125I]TOCA, and their respective sugar
conjugates in nude mice bearing AR42J tumor with high
sst2 expression 60 min p.i. is summarized in Table 3.
As observed in the internalization studies, Thr8-for-
[
123I]TOC and [123I]TOCA derivatives was modified
compared to previous studies. In earlier internalization
experiments with AR42J cells,47 both the compound of
interest and the reference TOC had been labeled with
iodine-125 and assayed in separate wells. Under these

Conjugation and Exchange
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2783
conditions, limited reproducibility of the internalization
data (i.e., internalization of the compound of interest
as the percent of the internalization found for [125I]TOC
in the same experiment) was found. Small variations
in the specific activities of the compared compounds as
well as differences in cell count and cell viability
between wells might have been responsible for the
discrepancy in the outcome of the experiments. To
eliminate these potential sources of error, a dual-tracer
protocol was established. In the experiments performed
in this study, cells were always coincubated with the
123I-labeled compound of interest and the reference
compound [125I]TOC. This experimental setup guaran-
tees identical (radio)ligand concentrations in all wells
and additionally eliminates potential effects of cell count
and cell viability on the internalization data.
The experiments were carried out using two different
cell lines, CHO cells stably transfected with hsst2 and
AR42J rat pancreatic tumor cells endogenously express-
ing rsst2. Since the rate and extent of ligand accumula-
tion (per 100 000 cells) into the cells varies significantly
between these cell lines, experimental conditions were
adjusted such that for both cell lines absolute uptake
of the reference [125I]TOC ranged between 15% and 20%
of the applied activity. While an incubation time of only
10 min was sufficient to achieve this in the case of the
CHO cells (approximately 100 000 cells per well), AR42J
cells (approximately 250 000 cells per well) required an
incubation time of 30 min to obtain comparable intra-
cellular activity levels. This adjustment of conditions
ensures comparability of data between the cell lines,
since consequently radioligand concentrations in the
internalization medium were comparable for both cell
lines.
Of the compounds investigated, those with the lowest
sst2 affinity compared to their non-carbohydrated coun-
terparts showed the highest increase in internalization,
namely, [123I]Gluc-TOC (in the TOC series) and [123I]G-
luc-TOCA, [123I]Gluc-S-TOCA, and [123I]Gal-S-TOCA (in
the TOCA series). In the case of the TOCA analogues,
a cumulation of the effects of glycosylation and C-
terminal Thr(ol)-by-Thr substitution on internalization
was observed. Thus, conjugation with small sugar
residues, independent of the presence or absence of a
linker unit between peptide and sugar, substantially
enhances internalization of the sst ligands investigated,
whereas larger carbohydrates such as maltose and
maltotriose do not trigger the same effect.
It might be assumed that variations in agonistic
potency among the peptides modified with mono- versus
di- or trisaccharides may be responsible for the above
observation. In a previous study, however, a variety of
Amadori analogues of octreotide (OC) including the
Gluc, Malt, and Mtr derivatives have been evaluated.50
While the receptor affinity of the peptides decreased
only marginally in the order Gluc-OC > OC > Mtr-OC
> Malt-OC, the IC50 [nM] of the peptides for in vitro
growth hormone secretion was substantially and gradu-
ally decreased by peptide glycosylation with carbohy-
drates of increasing size.
That the improved internalization of [123I]Gluc-TOC,
[
123I]Gluc-TOCA, [123I]Gluc-S-TOCA, and [123I]Gal-S-
TOCA is indeed induced by an effect of radioligand
structure on the internalization of the receptor-ligand
complex and not by transport of the glycopeptides via
membrane-associated carbohydrate transporters has
been verified by a competition experiment. Both in the
absence and in the presence of different glucose and/or
mannose concentrations in the assay medium, the ratio
of the amount of internalized glycosylated radiopeptide
to the amount of [125I]TOC (see Figure 3) remained
unchanged (data not shown). Absolute cellular uptake
as a percentage of the applied activity of both the
glycosylated compound of interest and [125I]TOC, how-
ever, was increased in the presence of increasing
amounts of glucose, indicating the energy dependence
of the internalization process.
Ligand internalization of all compounds investigated
correlates well with the corresponding EC50,R (Figure
6). The latter value represents a measure of the radio-
ligand’s ability to compete with unlabeled TOC for
receptor binding and subsequent endocytosis into the
cell; the higher this value is, the more unlabeled
competitor is necessary to inhibit radioligand internal-
ization and thus the better the radioligand’s internal-
ization potency. It is therefore not surprising that the
absolute amount of cellular activity uptake should
correlate with this quantity. As demonstrated in Figure
6, the EC50,R therefore may represent an accurate
predictive value for the ability of a new radioligand to
be efficiently internalized into hsst2-expressing cells.
As expected from previous findings with radiometal-
labeled sst ligands (Table 1),48 in vitro sst2 affinity of
[
125I]TOC was markedly improved by C-terminal Thr(ol)-
by-Thr substitution leading to [125I]TOCA, whereas
affinity to all other sst subtypes remained nearly
unaffected by this modification. The improvement of sst2
affinity of [125I]TOCA vs [125I]TOC by a factor of ∼3,
however, was not as drastic as observed for the respec-
tive radiometalated and nonradiometalated DOTA ana-
logues (factor of up to 10). As anticipated for the
N-terminal introduction of a relatively bulky residue
and as already demonstrated for the modification of
TOC with DOTA, carbohydration of [125I]TOC and more
notedly of [125I]TOCA generally led to reduced sst2
affinity (Table 1). Interestingly, this effect was more
pronounced in the case of the monosaccharides Gluc-
(P < 0.001) as well as Gluc-S- (0.001 < P < 0.0027) and
Gal-S- (P < 0.001) than in the case of the even bulkier
di- and trisaccharides Malt- and Mtr- (0.01 < P < 0.1).
Basically, all tested compounds with a high sst2
affinity also showed a high internalization rate. Within
this group of rapidly internalized compounds with very
high sst2 affinity, one can, however, observe that the
amount of glycosylated radioligand internalized into
sst2-expressing CHO cells (as percent of the reference
Interestingly, the above correlation was only observed
in CHO cells stably transfected with hsst2. In AR42J
cells from a rat pancreatic acinar tumor cell line
expressing rsst2, the internalization of [123I]Gluc-TOCA
compared to [125I]TOC is increased by a factor of 2.8
(CHO cells: 1.9), while the increase in EC50,R is negli-
gible (Figure 4B). Thus, the enhanced internalization
[
125I]TOC) (Figure 3) does not necessarily correlate with
their respective binding affinity to hsst2 (Table 1). Some
previous studies have also demonstrated that octreotide
analogues with comparable binding affinity exhibited
considerable differences in internalization efficiency.28,29,49

2784 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Schottelius et al.
externalization experiments in both CHO and AR42J
cells (Figure 5). Under conditions allowing recycling, i.e.,
reinternalization after externalization, both [125I]TOC
and [123I]Gluc-TOCA show comparable and low exter-
nalization from CHO cells within 60 min. In previous
studies it has been demonstrated in detail that the
externalization rate of intact radioligand from sst2-
expressing cells is independent of radioligand struc-
ture.24 The same has been observed in this study,
independent of the cell line used, when ligand recycling
was inhibited by an excess of cold TOC in the external
medium (Figure 5). Thus, the amount of [125I]TOC and
[
123I]Gluc-TOCA being externalized can be assumed to
be comparable at all time points. Furthermore, since
radioligand concentrations in the external medium are
very low under the given experimental conditions, no
competition for receptor binding between externalized
[
125I]TOC and [123I]Gluc-TOCA and hence no effect of
an enhanced EC50,R of [123I]Gluc-TOCA vs [125I]TOC can
be expected. Thus, the major factor determining the
radioligand levels remaining inside the cells is the
recycling rate, which in the case of CHO cells is shown
to be equally fast for both peptides. When AR42J cells
are used, the overall extent of ligand externalization
under conditions allowing recycling is enhanced com-
pared to CHO cells, and externalization of [123I]Gluc-
TOCA is apparently significantly slower than that of
[
125I]TOC. On the basis of the argument above, this
observation may be explained by faster reinternaliza-
tion, i.e., a higher internalization rate, of [123I]Gluc-
TOCA compared to the nonmodified peptide, which is
in accordance with data from the EC50,R and internal-
ization studies.
Other important factors influencing ligand external-
ization and recycling, however, are intracellular traf-
ficking of the ligand-receptor complex and the extent
of ligand degradation. Upon internalization of the
ligand-receptor complex, it can be targeted either to
endosomes for externalization of both receptor and
ligand or to lysosomes for their degradation. Which of
the two routes is prefentially taken depends mainly on
the nature of the receptor ligand. In the case of δ-opioid
receptors it has been demonstrated that upon binding
of peptidic receptor ligands, the receptors are mainly
targeted toward lysosomal degradation, whereas almost
no degradation, but only ligand and receptor reexter-
nalization and recycling, occurred when an alkaloid
agonist was used.52 In the case of the externalization
study using CHO cells, the experiment under conditions
inhibiting ligand recycling (Figure 5A) showed that
more than 94% of the intracellularly accumulated
ligand, independent of its structure ([123I]Gluc-TOCA vs
Figure 6. (Top) EC50,R vs internalization (as percent of
[
125I]TOC) of all compounds investigated (CHO cells, 10 min
incubation). (Bottom) EC50,R vs relative hsst2 affinity of all
compounds investigated (COI ) compound of interest).
of [123I]Gluc-TOCA in AR42J cells does not seem to be
the result of more efficient receptor binding of the
radioligand compared to [125I]TOC and thus more ef-
ficient ligand internalization, which had been observed
for [123I]Gluc-TOCA in CHO cells. In contrast, data
indicate that in the case of AR42J cells sugar conjuga-
tion and C-terminal oxidation lead to a dramatically
enhanced internalization rate of [123I]Gluc-TOCA. Since
[
123I]Gluc-TOCA and [125I]TOC bind to the receptor with
similar efficiency, as reflected by the EC50,R close to 1
(Figure 4B), the observed drastic increase in internal-
ization of [123I]Gluc-TOCA by a factor greater than 2.5
compared to [125I]TOC can only be explained by an
accelerated radioligand uptake into the cell. That the
rate of receptor internalization and receptor desensiti-
zation induced by the same receptor ligand in different
cell lines can vary substantially has already been
demonstrated for δ-opioid receptors.51 Results differed
significantly between nonneuronal cells transfected with
the respective receptor (corresponding to CHO cells
transfected with hsst2 used in this study) and cells of
neuronal phenotype, which endogenously express δ-o-
pioid receptors at physiological levels (corresponding to
AR42J tumor cells that endogenously express rsst2).
That the internalization rate of [123I]Gluc-TOCA is
indeed enhanced compared to [125I]TOC in AR42J cells
is further supported by data from the comparative
[
125I]TOC), was externalized within 60 min, indicating
an almost exclusive role of receptor and ligand exter-
nalization and recycling. In contrast, 35-40% of inter-
nalized [123I]Gluc-TOCA and [125I]TOC remain inside
the AR42J cells in the corresponding experiment. At
first glance, this finding indicates a higher fraction of
lysosomal targeting of receptor and radioligand in this
cell line compared with CHO cells. The apparent “trap-
ping” of radioactivity in AR42J cells, however, needs
further investigation. So far, data (Figure 5) indicate a
reduced rate of externalization from AR42J cells. It
seems probable that longer observation periods (up to

Conjugation and Exchange
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2785
modified analogues [123I]Gluc-TOCA, [123I]Gluc-S-TOCA
and [123I]Gal-S-TOCA, a cumulative effect of both
structural modifications was observed, leading to up to
5-fold uptake of these compounds in sst-expressing
tumors compared to [125I]TOC. Thus, in this study
glycosylation with small carbohydrates, especially with
the Gluc-S moiety, was found to be a suitable tool to
enhance receptor-mediated uptake of radiolabeled oct-
reotide analogues into sst-positive malignancies. The
availability of different and chemically straightforward
glycosylation methods not only allows further “fine-
tuning” of ligand characteristics but also makes this
approach easily adaptable to a variety of other neu-
ropeptide analogues. Since the limited residualization
of radioiodinated receptor ligands in their target cells
still hampers their application for therapeutic interven-
tions in nuclear medicine, further efforts are directed
toward the combination of the beneficiary effects of
C-terminal oxidation and N-terminal glycosylation with
labeling methods leading to higher retention of radio-
activity in the target cells.
Materials and Methods
1. Peptide Synthesis. 1.1. General Conditions. TCP
(trityl chloride polystyrene) resin was obtained from PepChem
(Tu¨bingen, Germany). DHP-HM (3,4-dihydro-2H-pyran-2-yl-
methoxymethylpolystyrene) resin, Fmoc amino acid, and other
amino acids were supplied by Novabiochem (Bad Soden,
Germany). IodoGen (1,3,4,6-tetrachloro-3R,6R-diphenylgly-
coluril) came from Pierce (Rockford, IL). All other reagents
were purchased from VWR (Darmstadt, Germany), Alexis
(Gru¨nberg, Germany), and Aldrich and Fluka (Neu-Ulm,
Germany). Solvents were used without further purification.
Solid phase peptide synthesis was carried out manually
using a flask shaker (St. John Associates Inc.).
Figure 7. (Top) Internalization (as percent of [125I]TOC, CHO
cells, 10 min incubation) of all compounds investigated vs
tumor accumulation in AR42J tumor bearing nude mice 60
min p.i. (Bottom) hsst2 affinity (IC50 [nM]) of all compounds
investigated vs tumor accumulation in AR42J tumor-bearing
nude mice 60 min p.i.
Analytic RP-HPLC was performed on Nucleosil 100 C18 (5
µm, 250 mm × 4 mm or 125 mm × 4.0 mm) columns (CS
GmbH, Langerwehe, Germany) using a Sykam gradient HPLC
system (Sykam, Fu¨rstenfeldbruck, Germany). The peptides
were eluted applying various gradients of 0.1% TFA in H2O
(solvent A) and 0.1% TFA in acetonitrile (solvent B) in 30 and
15 min, respectively, at a constant flow of 1 mL/min. UV
detection was performed at 220 nm in a 206 PHD UV-vis
detector (Linear Instruments Corporation, Reno, NV). For
radioactivity measurement, the outlet of the UV photometer
was connected to a NaI(Tl) well-type scintillation counter from
EG&G Ortec (Mu¨nchen, Germany).
Preparative RP-HPLC was performed on the same HPLC
system using a Multospher 100 RP 18-5 (250 mm × 10 mm)
column (CS GmbH, Langerwehe, Germany) at a constant flow
of 5 mL/min.
ESI mass spectra were recorded on an LCQ LC-MS system
from Finnigan (Bremen, Germany) using the Hewlett-Packard
series 1100 HPLC system.
240 min) may demonstrate the expected washout of
radioiodinated fragments such as [*I]iodotyrosine from
the cells.
As demonstrated in Figure 7, in vivo tumor ac-
cumulation of [125I]TOC, [125I]TOCA, and their glyco-
sylated analogues in AR42J tumor-bearing nude mice
parallels the results obtained in the in vitro studies
using CHO cells. While tumor accumulation clearly
correlates with the internalization of the respective
radiopeptide and thus also is a function of the ligand’s
EC50,R (see Figure 6), it does not correlate with the hsst2
affinity of the ligand. This finding again illustrates a
special feature of some of the radioiodinated carbohy-
drated somatostatin analogues investigated in this
study, i.e., [123I]Gluc-TOC, [123I]Gluc-TOCA, [123I]Gluc-
S-TOCA, and [123I]Gal-S-TOCA: the enhancement of
peptide accumulation in receptor expressing cells, in
vitro and in vivo, despite reduced receptor affinity
compared to their noncarbohydrated analogues.
1.2. Precursors for Radioiodination. 1.2.1. Tyr3-Lys5-
(Dde)-octreotide (TOC(Dde)). The synthesis of TOC(Dde)
was performed as described previously for TOC.39 Briefly,
freshly prepared Fmoc-Thr(tBu)-ol39,53 was anchored to DHP-
HM resin in the presence of pyridinium p-toluenesulfonate at
80 °C in dry 1,2-dichloroethane. Assembly of the peptide
sequence H2N-dPhe-Cys(Trt)-Tyr-dTrp-Lys(Dde)-Thr-Cys(Trt)
on the resin-bound amino alcohol was performed according to
standard Fmoc protocol using 1.5 equiv of HOBt (1-hydroxy-
benzotriazole) and TBTU (O-(1H-benzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium tetrafluoroborate) as coupling reagents
and N-ethyldiisopropylamine (DIEA) as a base. The peptide
was cleaved from the resin using a 1:1 mixture (v/v) of (95%
TFA, 2.5% TIBS (triisobutylsilane), 2.5% H2O (v/v/v)) and DCM
(dichloromethane). Disulfide bridge formation was achieved
using H2O2 in a THF (tetrahydrofurane)/5 mM NH4OAc
mixture buffered to pH 7 with saturated NaHCO3. TOC(Dde)
Conclusion
Both modifications introduced into [125I]TOC, C-
terminal Thr-for-Thr(ol) exchange, and N-terminal de-
rivatization with different carbohydrates have signifi-
cant impact on radioligand uptake into sst2-expressing
cells, both in vitro and in vivo. Glucose conjugation via
Amadori reaction alone already leads to improved tumor
uptake of [123I]Gluc-TOC in vivo, which is based on an
enhancement of peptide internalization despite a reduc-
tion in receptor affinity. In the case of the doubly

2786 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Schottelius et al.
was obtained in 86% yield based on the resin-bound amino
alcohol. HPLC (30% f 80% in 15 min): tR(cyclic peptide) )
8.0 min; K′ ) 4.30. Calculated monoisotopic mass for of
TOC(Dde) (C59H78N10O13S2) ) 1198.5. Found: m/z ) 1199.3
[M + H]+, m/z ) 1221.3 [M + Na]+
(4Ac)-S-OPfp). The respective 1-S-(2,3,4,6-tetraacetylglyco-
syl)-3-mercaptopropionate (1 equiv) was dissolved in 30 mL
of THF, and N,N′-diisopropylcarbodiimide (1.1 equiv), followed
by pentafluorophenol (1.1 equiv) in 3 mL of THF, was added.
After 24 h, reaction control via TLC (AcOEt/hexane: 4/6 (v/
v)) revealed complete disappearance of the starting material.
The solvent was evaporated, and the crude product was
purified using column chromatography (AcOEt/hexane: 4/6),
yielding the respective pentafluorophenyl active esters in 80-
89% yield (Gluc-S-OPfp, white solid; Gal-S-OPfp, clear oil). 1H
NMR Gluc-S-OPfp (CDCl3, 250 MHz): δ (ppm) 2.04, 2.06, 2.09,
2.11 (4 s, each 3 H, 4 Ac), 3.03-3.14 (m, 4 H, SCH2CH2COOH),
3.74-3.81 (m, 1 H, H-5), 4.15-4.31 (m, 2H, H-6), 4.6 (d, 1 H,
J ) 10 Hz, H-1), 5.09 (t, 1 H, J ) 10, 9.25 Hz, H-2), 5.13 (t, 1
H, J ) 10, 9.25 Hz, H-4), 5.25 (d, 1 H, J ) 9.5 Hz, H-3).
Gluc-S-TOCA and Gal-S-TOCA. TOCA(Dde) (50-100 mg,
1 equiv) and Gluc/Gal(4Ac)-S-OPfp (1.1 equiv) were dissolved
in 1-2 mL of DMF and stirred at room temperature for 2 h in
the presence of 1 equiv of DIEA. After product precipitation
using Et2O and washing of the precipitate with Et2O, the
respective glycosylated peptides were obtained in 46-67%
yield. (Gluc(4Ac)-S-TOCA(Dde): HPLC (30 f 80% B in 15 min)
tR ) 12.1 min; K′ ) 6.31. Gal(4Ac)-S-TOCA(Dde): HPLC (30
f 80% B in 15 min) tR ) 12.0 min; K′ ) 6.42)
1.2.2. Tyr3-Lys5(Dde)-octreotate (TOCA(Dde)). Fmoc-
Thr(tBu)-OH (596 mg, 1.5 mmol) was dissolved in 15 mL of
dry DCM, and an amount of 214 µL of DIEA (1.25 mmol) was
added. Dry TCP resin (1.0 g, 1.0 mmol of trityl chloride groups)
was suspended in this solution and stirred at room tempera-
ture for 5 min. Another quantity of 428 µL of DIEA (2.5 mmol)
was added, and stirring was continued for 90 min. Then 1 mL
of MeOH (methanol) was added to cap unreacted trityl chloride
groups. After 15 min the resin was filtered off, washed twice
with DCM, DMF (N,N-dimethylformamide), and MeOH, and
dried in vacuo. The final load of resin-bound Fmoc-Thr(tBu)-
OH was calculated to be 0.74 mmol/g using the following
formula:
1000(m2 - m1)
load [mmol/g] )
(MW - 36.461 g/mol)m2
m1 ) mass of the dry TPC resin before coupling [g]
m2 )mass of thedried resin after the coupling reaction [g]
MW ) molecular weight of the Fmoc-amino acid [g/mol]
After Dde deprotection for 10 min at room temperature, the
peptides were precipitated using Et2O. For subsequent sugar
deacetylation, peptides were redissolved in 1 mL of MeOH
containing 0.5 equiv of KCN. Deacetylation was complete after
stirring at room temperature for 4-48 h. The product was then
precipitated using Et2O and purified via preparative HPLC,
yielding Gluc-S-TOCA and Gal-S-TOCA in yields of 12-15%
(based on TOCA(Dde)).
After SPPS (see TOC(Dde)), the peptide was cleaved from the
solid support using 95% TFA, 2.5% TIBS, and 2.5% H2O (v/
v/v). Cyclization of the crude peptide was performed in analogy
to TOC(Dde). Yield based on resin-bound Thr: 97%. HPLC
(30% f 80% in 15 min): tR(cyclic peptide) ) 8.4 min; K′ )
4.45. Calculated monoisotopic mass for of TOCA(Dde) (C59H76-
N10O14S2) ) 1212.5. Found: m/z ) 1213.6 [M + H]+, m/z )
1235.7 [M + Na]+.
1.2.3. Peptide Glycosylation via Amadori Reaction. On
the basis of a previously reported method,50 1 equiv of
Lys5(Dde)-protected peptide and 10 equiv of the respective
aldose (glucose, maltose, maltotriose) were dissolved in 1 mL
of MeOH (5% AcOH (acetic acid)) per 100 mg of peptide, and
the reaction mixture was stirred at 60 °C for 16-48 h. The
crude products were precipitated using Et2O (diethyl ether)
and dried in vacuo.
1.2.4. Dde Deprotection. To a solution of 50-200 mg of
peptide in 980 µL of DMF, an amount of 20 µL of hydrazine
hydrate was added. After 10 min at room temperature, the
deprotected product was precipitated using Et2O, washed with
Et2O, and dried in vacuo. All deprotected peptides were
subsequently purified using preparative gradient HPLC.
1.2.5. NR-(1-S-(r-D-Glucopyranosyl)-3-mercaptopropio-
nyl)-D-Phe1-Tyr3-octreotate and NR-(1-S-(r-D-Galactopy-
ranosyl)-3-mercaptopropionyl)-D-Phe1-Tyr3-octreotate
(Gluc-S-TOCA and Gal-S-TOCA). 1-S-(2,3,4,6-Tetraacetyl-
glucopyranosyl)-3-mercaptopropionate and 1-S-(2,3,4,6-
Tetraacetylgalactopyranosyl)-3-mercaptopropionate.54
To a solution of peracetylated sugar (3 mmol, 1.17 g) and
mercaptopropionic acid (12 mmol, 1.04 mL) in 20 mL of dry
dichloromethane, BF3‚Et2O (4.5 mmol, 565 µL) was slowly
added. Product formation was monitored via TLC (AcOEt/
hexane/AcOH: 10/10/1 (v/v/v)). The solution was stirred at
room temperature for 4 h, diluted with dichloromethane, and
extracted with 1 M HCl. The aqueous phase was then
extracted once with dichloromethane. The combined organic
phases were dried over MgSO4, filtered, and evaporated to
dryness. After purification via column chromatography (AcOEt/
hexane/AcOH: 10/10/1 (v/v/v)), 1.01 g (77%) of 2,3,4,6-tet-
raacetyl-1-S-mercaptopropionylglucose were obtained as a
white solid, whereas 2,3,4,6-tetraacetyl-1-S-mercaptopropio-
nylgalactose was obtained as a clear oil in 88% yield.
1-S-(2,3,4,6-Tetraacetylglucopyranosyl)-3-mercapto-
propionic Acid Pentafluorophenyl Ester and 1-S-(2,3,4,6-
Tetraacetylgalactopyranosyl)-3-mercaptopropionic Acid
Pentafluorophenyl Ester (Gluc(4Ac)-S-OPfp and Gal-
Synthetic data for all radioiodination precursors are sum-
marized in Table 4.
1.3. 3-Iodo-Tyr3 Reference Compounds. For the synthe-
sis of the 3-iodo-Tyr3 reference compounds, Fmoc-Tyr(tBu)-
OH was replaced by Fmoc-3-iodo-Tyr(tBu)-OH during SPPS
of TOC(Dde) and TOCA(Dde). All other reaction steps were
performed analogously to the syntheses presented in the
previous section. Yields for all glycosylated peptides after
preparative HPLC (based on the respective nonglycosylated
Dde-protected precursors) were 18-24%.
Synthetic data for all 3-iodo-Tyr3 reference compounds are
summarized in Table 4.
2. Peptide Radioiodination. Radioiodination was gener-
ally performed using the Iodogen method.
A solution of 100-200 µg of peptide in 200 µL of PBS
(phosphate buffered saline, 0.1M, pH 7.4) was transferred to
an Eppendorf tube coated with 30 µg of Iodogen (Pierce,
Rockford, IL). After the addition of 5-20 µL of solution of
radioiodide (Amersham, Buckinghamshire, U.K.) in 0.05 M
NaOH ([125I]NaI (n.c.a.), 18-74 MBq; [123I]NaI (c.a.), 37-185
MBq), the cap was vortexed and the labeling reaction was
allowed to proceed for 20 min at room temperature. The
peptide solution was then removed from the insoluble oxidizing
agent.
The radioiodinated peptides were purified via RP-HPLC
(Nucleosil 100 C18 column (5 µm, 125 mm × 4.0 mm) at a
constant flow of 1 mL/min using an isocratic solvent mixture
of 25% [123I]Mtr-TOCA, 26% [123I]Gluc-TOC, 27% [123I]Malt-
TOC, [123I]Mtr-TOC, and [123I]Malt-TOCA, 28% [125I]TOC,
[
123I]TOCA, and [123I]Gluc-TOCA, or 33% [123I]Gluc-S-TOCA
and [123I]Gal-S-TOCA, and EtOH (0.5% AcOH) in water (0.5%
AcOH). Radiochemical yields after purification ranged from
45% to 85%. HPLC data for all radioiodinated peptides and
the solvent composition applied for quality control HPLC are
given in Table 5.
For the biodistribution experiments, an excess of absolute eth-
anol was added to the collected fraction, and the solvents were
evaporated to dryness. The radioiodinated product was redis-
solved in PBS to yield a solution of radiolabeled peptide with
an activity concentration of approximately 370 kBq/100 µL.
For the paired-label internalization experiments, [125I]TOC
and the respective 123I-labeled radioligand were each redis-

Conjugation and Exchange
Table 4. Synthetic Data of All Radioiodination Precursors and Their Corresponding 3-Iodo-Tyr3 Analogues
HPLC characterization ESI mass spectrometry characterization
calcd MW [g/mol] m/z [M + H]+ m/z [M + Na]+
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2787
peptide
gradient
tR [min]
K′
sum formula
TOC
10% f 60% B (30 min)
10% f 60% B (30 min)
12.9
13.1
12.6
13.1
16.3
15.4
14.8
14.5
8.2
4.48
C
49H66N10O11S2
1034.4
1196.5
1358.5
1520.6
1048.4
1210.5
1372.5
1534.6
1298.5
1298.5
1160.3
1322.4
1484.4
1646.5
1174.3
1336.4
1498.4
1660.5
1424.4
1424.4
1035.2
1197.2
1359.2
1521.2
1049.1
1211.5
1373.1
1535.2
1299.3
1299.3
1161.1
1323.1
1485.2
1647.1
1175.0
1337.0
1499.2
1661.2
1425.6
1425.6
1057.3
1219.3
1381.3
1543.3
1071.2
1233.5
1395.6
1557.2
1321.3
1321.3
1183.1
1345.2
Gluca-TOC
Maltb-TOC
Mtrc-TOC
4.13 C55H76N10O16S2
3.85 C61H86N10O21S2
3.78 C67H96N10O26S2
10% f 60% B (30 min)
10% f 60% B (30 min)
TOCA
10% f 60% B (30 min)
5.54
4.68
4.59
4.48
3.99
3.96
5.79
5.58
5.54
5.33
5.08
5.17
5.69
6.33
4.72
4.79
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
49H64N10O12S2
55H74N10O17S2
61H84N10O22S2
67H94N10O27S2
58H78N10O18S3
58H78N10O18S3
49H65N10O11S2I
55H75N10O16S2I
61H85N10O21S2I
67H95N10O26S2I
49H63N10O12S2I
55H73N10O17S2I
61H85N10O21S2I
67H93N10O27S2I
58H77N10O18S3I
58H77N10O18S3I
Gluc-TOCA
Malt-TOCA
Mtr-TOCA
Gluc-S-TOCA
Gal-S-TOCA
I-TOC
10% f 60% B (30 min)
10% f 60% B (30 min)
10% f 60% B (30 min)
20% f 70% B (15 min)
20% f 70% B (15 min)
8.3
1A 10% f 60% B (30 min)
1B 10% f 60% B (30 min)
1C 10% f 60% B (30 min)
1D 10% f 60% B (30 min)
2A 10% f 60% B (30 min)
2B 10% f 60% B (15 min)
2C 10% f 60% B (30 min)
2D 10% f 60% B (15 min)
16.3
15.8
15.7
15.2
17.0
11.1
16.1
11.2
9.2
I-Gluc-TOC
I-Malt-TOC
I-Mtr-TOC
I-TOCA
I-Gluc-TOCA
I-Malt-TOCA
I-Mtr-TOCA
1669.3
1359.3
1683.2
1447.6
1447.6
I-Gluc-S-TOCA 2E 20% f 70% B (15 min)
I-Gal-S-TOCA 2F 20% f 70% B (15 min)
9.1
a NR-(1-Deoxy-D-fructosyl). b NR-(R-D-Glucopyranosyl-(1-4)-1-deoxy-D-fructosyl). c NR-(O-R-D-Glucopyranosyl-(1-4)-O-R-D-glucopyranosyl)-
(1-4)-1-deoxy-D-fructosyl).
Table 5. HPLC Data for All Radioiodinated Peptides
centrations of the unlabeled peptide ranging from 0.1 to 1000
nM. Somatostatin 28 was run in parallel as control using the
same increasing concentrations. IC50 values were calculated
using a computer-assisted image-processing system. Tissue
standards (autoradiographic [125I]microscales Amersham), con-
taining known amounts of isotopes and cross-calibrated to
tissue equivalent ligand concentrations, were used for quan-
tification.48
4.2. Internalization and Determination of EC50,R (CHO
and AR42J). One day prior to the experiment, cells were
harvested using Trypsin/EDTA (0.05% and 0.02%) in PBS,
centrifuged, and resuspended with culture medium. Concen-
tration of the suspension ranged from 40 000 to 60 000 cells/
mL in the case of CHO cells and from 180 000 to 250 000 cells/
mL in the case of AR42J cells. The suspension was transferred
into 24-well plates (1 mL/well) and placed in the incubator
overnight.
On the day of the experiment (cell count for CHO, 90000-
120000 cells/well; cell count for AR42J, 200000-250000 cells/
well), the culture medium was removed and the cells were
washed once with 250 µL of medium (unsupplemented DMEM
(CHO cells) or RPMI-1640 (AR42J cells)) before being left to
equilibrate in 190 µL of medium at 37 °C for a minimum of 15
min before the experiment. Then 50 µL (per well) of medium
(5% BSA) containing increasing concentrations of unlabeled
TOC were added, followed by the addition of approximately
100 000 cpm of both the respective 123I-labeled peptide and of
the reference [125I]TOC in 10 µL of medium (5% BSA). Final
TOC concentrations in the incubation medium were 0.1, 0.2,
0.5, 0.8, 1, 2, 4, 6, 8, 10, 12, 15, 20, 50, 100, 500, and 1000 nM.
In a control experiment (n.c.a. conditions), ligand-free medium
(5% BSA) was added. Nonspecific internalization was deter-
mined by including 5 µM unlabeled TOC. Experiments were
carried out in triplicate for each concentration.
% EtOH in
isocratic eluent
tR [min]
K′
[
[
[
[
[
[
[
[
[
[
125I]TOC
29
27
27
27
28
29
27
25
32
33
9.5
10.2
7.2
5.52
8.14
5.70
6.71
10.10
7.80
7.78
7.30
9.36
7.95
123I]Gluc-TOC
123I]Malt-TOC
123I]Mtr-TOC*
123I]TOCA
8.1
10.2
8.0
10.1
7.5
8.7
7.5
123I]Gluc-TOCA
123I]Malt-TOCA
123I]Mtr-TOCA
123I]Gluc-S-TOCA
123I]Gal-S-TOCA
solved in assay medium (containing 5% BSA) and diluted to
an activity concentration of approximately 200 000 cpm/10 µL.
A 1:1 (v/v) mixture of both solutions containing approximately
100 000 cpm/10 µL of each radioligand was then used for the
internalization experiment.
3. Cell Culture. AR42J cells were obtained from ECACC
(European Collection of Cell Cultures, Salisbury, U.K.). Cells
were maintained in RPMI 1640 (Seromed, Berlin, Germany)
supplemented with 10% FCS (Seromed) and 2 mM L-glutamine
(Gibco BRL Life Technologies, Karlsruhe, Germany). CHO
cells stably transfected with human sst2 (epitope tagged at the
N-terminal end) were kindly provided by Dr. Jenny Koenig,
University of Cambridge, U.K. Cells were grown in DMEM/
nutrition mix F-12 with Glutamax-I (1:1) (Gibco BRL) supple-
mented with 10% FCS and 500 mg/L Geneticin (Gibco BRL).
Cultures were maintained at 37 °C in a 5% CO2/humidified
air atmosphere.
In the assay medium used for internalization studies, FCS
was replaced by 1% BSA (Sigma, St. Louis, MO).
For cell counting, a CASY1-TT cell counter and analyzer
system (Scha¨rfe System GmbH, Reutlingen, Germany) was
used.
After the addition of the radioligands, the cells were
incubated for 10 min (CHO) or 30 min (AR42J) at 37 °C. To
ensure a constant incubation temperature, the plates were
placed into a sand bath inside the incubator.
Incubation was terminated by placing the plate on an ice
pack for approximately 1 min and by subsequent removal of
the incubation medium. Cells were thoroughly rinsed with 250
µL of fresh medium. The wash medium was combined with
the supernatant of the previous step. This fraction represents
the amount of free radioligand. To remove surface-bound (acid
releasable) radioactivity, the cells were then incubated with
250 µL of ice-cold acid wash buffer (0.02 M NaOAc buffered
with AcOH to pH 5). After removal of the acid wash buffer,
the cells were thoroughly rinsed with another 250 µL of ice-
4. In Vitro Studies. 4.1. sst-Receptor Binding Affinities
and Subtype Specificities (CHO-K1 and CCL39 Cells).
Cells stably expressing human sst1, sst2, sst3, sst4, and sst5
(sst1 and sst5, CHO-K1 cells; sst2-4, CCL39 cells) were grown
as described previously.48 Cell membrane pellets were pre-
pared, and receptor autoradiography was performed on pellet
sections (mounted on microscope slides), as described in detail
previously.48 For each of the tested I-TOC and I-TOCA
derivatives, complete displacement experiments were per-
formed with the universal somatostatin radioligand [125I]-
[Leu8,D-Trp22,Tyr25]somatostatin 28 48 using increasing con-

2788 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Schottelius et al.
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Gugger, M. Bombesin receptor subtypes in human cancers:
detection with the universal radioligand 125I-[D-TYR6,beta-
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cold acid wash buffer. Both acid wash fractions were combined.
The internalized activity was released by incubation with 250
µL of 1 N NaOH, tranferred to vials, and combined with 250
µL of PBS used for rinsing the wells. Quantification of the
amount of free, acid-releasable, and internalized activity was
performed in a γ-counter.
For numerical analysis of the EC50 of TOC for internaliza-
tion, data for both the 123I-labeled compound of interest and
for the reference [125I]TOC in the same experiment were first
corrected by the amount of nonspecific internalization, respec-
tively, and then each was normalized to the amount of
internalized ligand in the absence of unlabeled competitor
(100%). Data were fitted with a weighted two-parameter
logistic function using SigmaPlot. To eliminate the influence
of cell count and cell viability on the absolute EC50 values, data
are expressed as the ratio (EC50,R) of the EC50 observed for
the compound of interest (COI) to the EC50 found for [125I]TOC
in the same experiment (EC50,R ) EC50(COI)/EC50([125I]TOC)).
4.3. Externalization and Recycling (CHO and AR42J).
As in the previous experiment, cells were incubated with the
respective 123I-labeled peptide and the reference [125I]TOC at
37 °C for 10 (30) min and washed with medium. To ensure
receptor integrity, no acid wash was performed after this initial
internalization incubation. Then, to determine the extent of
ligand recycling, two different experiments were performed.
In the experiment allowing ligand recycling, 200 µL of medium
and 50 µL of medium (5% BSA) were added to each well. In
the experiment inhibiting ligand recycling, 200 µL of assay
medium and 50 µL of medium (5% BSA) containing 25 µM
TOC were added to each well. Experiments were carried out
in triplicate for both experimental conditions. Subsequently,
the cells were incubated at 37 °C for 30 and 60 min. The
supernatant was removed and combined with 250 µL of
medium used for rinsing the cells. This fraction represents the
amount of externalized ligand. The subsequent steps, i.e., acid
wash and lysis of the cells, were performed as described for
the internalization experiment.
5. In Vivo Biodistribution Studies. 5.1. Animal Model.
For all in vivo experiments, nude mice (Swiss nu/nu, female,
6-8 weeks) were used. To establish tumor growth, AR42J cells
were treated with 1 mM EDTA in PBS, suspended, centri-
fuged, and resuspended in serum-free RPMI 1640. Mice were
inoculated subcutaneously in the flank with (2.5-5) × 106 cells.
Ten days after inoculation, all mice showed solid palpable
tumor masses (tumor weight of 150-500 mg) and were used
for the experiments.
5.2. Biodistribution Studies. About 370 kBq (10 µCi) of
the 125I-labeled peptides in 100 µL of PBS (pH 7.4) were
injected intravenously (iv) into the tail vein of nude mice
bearing an AR42J tumor. The animals were sacrificed 60 min
postinjection (p.i.) (n ) 5), and the organs of interest were
isolated. The radioactivity was measured in weighed tissue
samples using a γ-counter. Data are expressed as % ID/g tissue
(mean ( SD).
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