Labeling and glycosylate of peptides using click chemistry: a general approach to18F-glycopeptides as effective imaging probes for positron emission tomography

Article abstract of DOI:10.1002/anie.200904137

(Figure Presented) Click for PET: An efficient strategy based on click chemistry has been developed for ¹⁸F-labeling alkyne-bearing peptides with concomitant glycosylation. The mild conditions and general applicability of this reliable reaction gives access to a new class of ¹⁸F- glycopeptide radiopharmaceuticals with improved biological properties for in vivo imaging studies by positron emission tomography (PET).

Full text of DOI:10.1002/anie.200904137

Communications
DOI: 10.1002/anie.200904137
Molecular Imaging
Labeling and Glycosylation of Peptides Using Click Chemistry: A
General Approach to 18F-Glycopeptides as Effective Imaging Probes
for Positron Emission Tomography**
Simone Maschauer, Jꢀrgen Einsiedel, Roland Haubner, Carsten Hocke, Matthias Ocker,
Harald Hꢀbner, Torsten Kuwert, Peter Gmeiner, and Olaf Prante*
In the field of molecular imaging, positron emission tomog-
raphy (PET) has emerged as an imaging modality with
excellent sensitivity for in vivo studies.[1] PET labeling is
challenging since short-lived positron-emitting isotopes such
as 18F and 11C are used as labeling agents.[2] The optimization
and efficient application of rapid and reliable labeling
strategies are prerequisites for obtaining access to new
radiopharmaceuticals for both research and clinical trials.
Bioactive peptides that specifically address molecular
targets in vivo represent an important class of PET tracers to
facilitate predictive imaging and PET-guided therapy. Diverse
strategies for the synthesis of peptide-based radiopharma-
ceuticals using 18F-labeled prosthetic groups have been
elaborated, including chemoselective oxime conjugation[3]
and the use of 18F-labeled maleimide derivatives as cysteine-
reactive reagents.[4,5] Following the concept of click chemistry
introduced by Sharpless et al.,[6] the Huisgen [3+2] azide–
alkyne cycloaddition has been adapted to 18F-radiosynthetic
methods in order to take advantage of its selectivity,
reliability, and speed under aqueous mild CuI-promoted
reaction conditions.[7]
Synthetic approaches to RGD tracers targeting avb3
integrin, which plays a key role in angiogenesis, capitalize
on the pioneering studies by Kessler et al., who successfully
developed cyclic pentameric RGD peptides that selectively
recognize integrin avb3.[9] Various radiolabeled cyclic RGD
peptides have been described.[10] Among these, [18F]galacto-
RGD[11] has been extensively evaluated in clinical studies.
Since glycosylation of peptides is known to frequently
improve the biokinetic and in vivo clearance properties,
[18F]galacto-RGD and further radiopeptides have been
approached.[12,13] However, the multistep radiosynthesis of
[18F]galacto-RGD is time-consuming and laborious. In pro-
posals to overcome this drawback, 18F-labeling by 2-deoxy-2-
[18F]fluoroglucose (FDG) has been discussed.[5,14,15]
The major disadvantages of the 18F-peptide-labeling
strategies currently used are 1) harsh reaction conditions,
2) laborious multiple-step syntheses with a limited decay-
uncorrected radiochemical yield (RCY), which would com-
plicate the automation for large-scale production, and 3) lip-
ophilic derivatization, which impair the biokinetic properties
of the tracer.
The versatility of peptide imaging agents is frequently
hampered by their instability in vivo because of rapid
degradation by endogenous peptidases. As an example, the
synthesis of radiolabeled peptide-based imaging agents for
the neurotensin receptor-1 (NTR-1), which is overexpressed
in a number of human cancers, requires modifications to
improve the metabolic stability.[8]
Based on our previous work on click chemistry in drug
discovery[16] and the synthesis of b-mannosyl azides,[15] we
herein present an efficient strategy toward 18F-labeling with
concomitant glycosylation for the synthesis of 18F-glycopep-
tides as imaging agents for PET. We combined this strategy
with the development of a metabolically stable glycopeptoid
analogue of NT(8–13), which is the highly potent C-terminal
hexapeptide of the natural agonist neurotensin (NT). As a
proof of concept, two 18F-glycopeptides derived from NT(8–
13) and c(RGDfPra), respectively, were applied to biodistri-
bution studies and mPET for imaging NTR and avb3-integrin
expression in vivo using xenograft nude mice models.
In detail, 2-deoxy-2-fluoroglucosyl azide (3) could be
obtained starting from tetraacetylated 2-deoxy-2-fluoroglu-
cose.[15] The glucosyl azide 3 was applied for the CuI-catalyzed
azide–alkyne coupling with a series of alkyne-functionalized
peptides to evaluate the influence of the appended glycosyl
residue on receptor recognition. Commercially available
propargylglycine (Pra) was introduced by solid-phase-sup-
ported synthesis at position X into the sequence of the
bioactive peptide c(RGDfX) and at the N terminus of NT(8–
13) and metabolically stabilized derivatives thereof. Consid-
ering our studies on the influence of peptide backbone
modifications and ligand conformation on affinity changes for
a series of NT(8–13) analogues,[17] metabolic stabilization was
envisioned by alteration of three amino acids in the sequence
[*] Dr. S. Maschauer, Dr. C. Hocke, Prof. T. Kuwert, Dr. O. Prante
Nuklearmedizinische Klinik, Labor fꢀr Molekulare Bildgebung
Friedrich-Alexander-Universitꢁt Erlangen-Nꢀrnberg
Schwabachanlage 6, 91054 Erlangen (Germany)
Fax: (+49)9131-853-4325
E-mail: olaf.prante@uk-erlangen.de
Dr. M. Ocker
Universitꢁtsklinikum Erlangen (Germany)
Dr. J. Einsiedel, Dr. H. Hꢀbner, Prof. P. Gmeiner
Emil Fischer Center
Friedrich-Alexander-Universitꢁt Erlangen-Nꢀrnberg (Germany)
Dr. R. Haubner
Medizinische Universitꢁt Innsbruck (Austria)
[**] This work was supported by the Deutsche Forschungsgemeinschaft
(DFG, MA 4295/1-1) and the BMBF (01EZ0808). We thank B. Weigel
and PET Net GmbH for technical support.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200904137.
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Chemie
Table 1: Receptor binding data of NT analogues and of the reference NT(8–13) as determined by
of NT(8–13) (Table 1). Receptor
binding studies using CHO cells,
stably expressing the human NTR-
1,[18] provided evidence that intro-
duction of Pra at the N terminus
(NT1) did not significantly influ-
ence the NTR-1 affinity, whereas
b-homo-Tyr in position 11 (NT2
and NT3) of the peptoid-like N-(4-
aminobutyl)Gly-Lys (= NLys-Lys)
derivative strongly decreased the
affinity. The replacements of Ile to
Tle in position 12 and Arg-Arg to
NLys-Lys (NT4) were motivated by
an anticipated stabilization towards
peptidase-catalyzed degradation.
The ligation of NT1, NT3, NT4,
and c(RGDfPra) with 2-deoxy-2-
fluoro-b-glucopyranosyl azide (3)
was accomplished in the presence
of CuSO4 and sodium ascorbate to
give the desired peptide conjugates
FGlc-NT1, FGlc-NT3, FGlc-NT4,
and FGlc-RGD in yields of 80–
90% and purities of > 98%. The
retention of the b-anomeric config-
uration of the glycosyl moiety of
FGlc-NT1 was elucidated by
19F NMR spectroscopy.[15]
displacement of [3H]NT using hNTR-1 expressing CHO cells.[a]
Peptide
Sequence
Ki [nm]
NT(8–13)
0.23Æ0.042[b]
NT1
0.29Æ0.046[b]
FGlc-NT1
0.32Æ0.11[c]
NT2
NT3
130Æ140[c]
350Æ78[c]
FGlc-NT3
NT4
400Æ21[c]
5.2Æ0.92[b]
16Æ2.8[c]
FGlc-NT4
[a] Ki values are based on the means of two to seven individual experiments each done in triplicate. [b] Ki
Æstandard error of the mean (SEM) [nm]. [c] Ki ÆSD [nm].
According to our ligand binding
experiments,
the
glycosylated
NTR-1 ligands proved to be almost equipotent to the
respective nonglycosylated congeners (Table 1). In particular
FGlc-NT4 revealed an excellent inhibition constant in the low
double-digit nanomolar range, thereby offering promise for
18F-labeling.
For the radiosynthesis of the desired 18F-labeled glyco-
peptides, the prosthetic group 2-deoxy-2-[18F]fluoroglucosyl
azide [18F]3[15] was prepared by cryptate-mediated 18F-for-
triflate substitution in the mannosyl triflate 1 followed by
basic hydrolysis with NaOH (Scheme 1). Subsequent glyco-
sylation was realized in the same reaction vial in a mixture of
phosphate-buffered saline (PBS)/ethanol (10:1) at 608C; only
100 mg of the alkyne-bearing peptide was required in a total
volume of 500 mL. Starting from [18F]fluoride, high RCYs
were achieved for this three-step, two-pot procedure, and the
18F-glycopeptides [18F]FGlc-NT1, [18F]FGlc-NT3, [18F]FGlc-
NT4, and [18F]FGlc-RGD were obtained in excellent purities
and 17–20% uncorrected RCY (n = 71 individual experi-
ments) with adequate specific activities (55–210 GBqmmolÀ1)
in a total synthesis time of 75 min. Thus, this protocol for the
introduction of an 18F-glucopyranoside label into peptides is
unique and well-suited for the 18F-labeling of peptides since it
was performed 1) in an aqueous environment, 2) with high
chemoselectivity, 3) in reliably high RCY, and 4) at low
reactant concentrations. Notably, the ultimate reliability of
the “18F-click” glycosylation was reflected by a record of 71
Scheme 1. a) K18F, Kryptofix 2.2.2, K2CO3, KH2PO4, acetonitrile,
2.5 min, 858C, 71% RCY; b) NaOH, 5 min, 608C; c) 1. HCl (0.1m);
2. alkynyl peptide, PBS (10% ethanol), CuSO4, sodium ascorbate,
20 min, 608C, 70–80% RCY.
successive radiosyntheses without
attempt.
a single unsuccessful
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Communications
The stability of the 18F-labeled NT analogues was inves-
The successful application of our sequential strategy of
18F-labeling and glycosylation was also demonstrated by the in
vitro and in vivo data on [18F]FGlc-RGD. In vitro binding
experiments were conducted with the disintegrin
tigated in human serum at 378C. In contrast to [18F]FGlc-
NT1, which was completely degraded after 30 min (see
Figure S2 in the Supporting Information), the peptoids
[18F]FGlc-NT3 and [18F]FGlc-NT4 showed dramatically
enhanced metabolic stability of > 90% (Figure 1a). Satura-
[
125I]echistatin using a solid-phase avb3 binding assay (Fig-
ure 2b) and avb3-expressing human U87MG glioblastoma
Figure 2. Purity, avb3-binding in vitro, metabolic stability in vivo, and
mPET imaging of avb3 expression with [18F]FGlc-RGD. a) Purity of FGlc-
RGD and [18F]FGlc-RGD, and HPLC analysis of radioactivity extracted
from mouse organ homogenates (40 min p.i.). b) Competition binding
curve between [125I]echistatin and FGlc-RGD, c(RGDfPra), and
c(RGDfV). The fraction of bound radioactivity to avb3 integrin (B in%)
is shown. Data are expressed as mean values Æstandard deviation
(SD) from three experiments each performed in triplicate. c) Time–
radioactivity curves of [18F]FGlc-RGD in tumor, blood, and muscle
tissue of U87MG-bearing nude mice. Tissue radioactivity is expressed
as the percentage of injected dose per gram of tissue (D, mean value
ÆSD, n=3–6). d) mPET using [18F]FGlc-RGD at 30–40 min p.i.: Trans-
axial images from U87MG-bearing mice injected with the tracer alone
(control) and coinjection of c(RGDfV) (12.5 mgkgÀ1, blocking).
Figure 1. Purity, stability, and specific binding in vitro and mPET imag-
ing with [18F]FGlc-NT4. a) Radio-HPLC traces showing the RCY of
[18F]FGlc-NT4 after 20 min, the radiochemical purity after isolation by
HPLC, the purity of the reference FGlc-NT4 (UV), and the stability of
[18F]FGlc-NT4 in human serum (3 h) and in mouse blood (30 min p.i.).
b) Saturation binding curve of [18F]FGlc-NT4 in HT29 cells in vitro.
c) Static mPET images (left: transaxial, middle: coronal) of nude mice
bearing HT29 tumors at 45–65 min p.i. of 5.7 MBq [18F]FGlc-NT4 with
(bottom) and without (top) coinjection of NT4 (100 mg per mouse).
tion binding and internalization studies with [18F]FGlc-NT3
and [18F]FGlc-NT4 were performed using NTR-expressing
HT29 cells. Specific binding was demonstrated for [18F]FGlc-
NT4 with a Bmax value of 403 fmolmgÀ1, a Kd value of (8.5 Æ
2.9) nm (Figure 1b), and an internalization rate of 60–80%
after 30 min. In vitro autoradiography using rat brain slices
further demonstrated specific binding of [18F]FGlc-NT4 in
NTR-rich areas, which was completely inhibited in the
presence of 1 mm NT (see Figure S6 in the Supporting
Information).
cells, confirming a double-digit nanomolar affinity of FGlc-
RGD for avb3 (see Table S2 in the Supporting Information).
Biodistribution experiments with [18F]FGlc-RGD in
U87MG-bearing mice showed a similar blood clearance, but
a threefold increased uptake in liver and kidney (see
Figure S9 in the Supporting Information), when compared
to [18F]galacto-RGD.[13] The high hepatic and kidney uptake
could not be explained by differences in lipophilicity between
[18F]FGlc-RGD and [18F]galacto-RGD, since their
lgD7.4 values were comparable (lgD7.4 = À3.8 (n = 3) vs.
À3.2[13]). The specific tumor uptake of [18F]FGlc-RGD was
0.49%IDgÀ1 (60 min p.i.). Our studies confirmed excellent in
vivo metabolic stability of [18F]FGlc-RGD and fast tumor
accumulation combined with non-variant tumor/blood and
tumor/muscle ratios of 2.3 and 4.4, respectively, early at
30 min p.i. (Figure 2a,c). This allowed successful imaging of
avb3-integrin expression in vivo (Figure 2d), indicating high
potential of [18F]FGlc-RGD for future application in PET
imaging studies.
We then investigated the biodistribution of [18F]FGlc-NT4
in HT29 xenografted nude mice at 10, 30, and 65 min p.i.
(postinjection; see Figure S7 in the Supporting Information).
[18F]FGlc-NT4 demonstrated metabolic stability in vivo
(>98%, Figure 1a), fast blood clearance, high uptake in the
kidneys, and low uptake in other organs. [18F]FGlc-NT4
uptake in HT29 tumors was 2.0 and 1.2%IDgÀ1 (ID =
injected dose) after 30 and 65 min, respectively, with tumor/
blood ratios rapidly increasing from 1.7 to 3.5 (see Figure S8
in the Supporting Information); this suggests a suitable signal/
noise ratio for PET imaging at early time points after
injection. In fact, mPET studies successfully demonstrated
specific visualization of NTR expression by [18F]FGlc-NT4 in
vivo (Figure 1c).
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Applying our click-chemistry-based glycosylation method
for 18F-labeling, we considerably improved the overall yield of
[18F]FGlc-RGD (20%) and the total synthesis time (70 min)
in comparison with the clinically applied [18F]galacto-RGD
(10%, 200 min[13]). Moreover, we successfully developed
[18F]FGlc-NT4 as the first peptoidic tracer for imaging NTR
expression in vivo by PET.
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Experimental Section
Typical procedure for 18F-labeling with concomitant glycosylation of
peptides: The mannosyl precursor 1[15] (9 mg, 15 mmol) in anhydrous
acetonitrile (450 mL) was added to the dried K+/Kryptofix 2.2.2/18FÀ
complex and the solution was stirred for 2.5 min at 858C. [18F]2 was
isolated by semipreparative HPLC (Kromasil C8, 125 ꢀ 8, 4 mLminÀ1
,
acetonitrile (0.1% trifluoroacetic acid (TFA)/water (0.1% TFA)
30:70) and trapped on a C18 cartridge (Lichrosorb, Merck, 100 mg).
After elution with ethanol (0.8 mL) and evaporation of the solvent, a
solution of NaOH (10% ethanol, 60 mm, 250 mL) was added. After
5 min at 608C (formation of [18F]3), HCl (0.1m, 13.5 mL) was added,
followed by a solution of alkyne-functionalized peptide (100 mg, see
the Supporting Information) dissolved in 250 mL PBS (10% ethanol),
sodium ascorbate (0.6m, 10 mL), and CuSO4 (0.2m, 10 mL). After the
reaction mixture had been stirred for 20 min at 608C, the 18F-
glycopeptides were isolated by semipreparative HPLC and subse-
quent SPE (solid phase extraction), allowing formulation in PBS for
in vitro and in vivo use.
Received: July 27, 2009
Published online: December 22, 2009
Keywords: click chemistry · glycosylation · peptides ·
.
positron emission tomography · radiopharmaceuticals
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