The efficiency of 18F labelling of a prostate specific membrane antigen ligand via strain-promoted azide-alkyne reaction: reaction speed versus hydrophilicity

Article abstract of DOI:10.1039/c8cc03999b

Here we report the ¹⁸F labeling of a prostate specific membrane antigen (PSMA) ligand via a strain promoted oxa-dibenzocyclooctyne (ODIBO)- or bicyclo[6.1.0]nonyne (BCN)-azide reaction. Although ODIBO reacts with azide 20 fold faster than BCN, in vivo PET imaging suggests that ¹⁸F-BCN-azide-PSMA demonstrated much higher tumor uptake and a much higher tumor to background contrast.

Full text of DOI:10.1039/c8cc03999b

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The efficiency of 18F labelling of prostate specific membrane
antigen ligand via strain-promoted azide-alkyne reaction: reaction
speed versus hydrophilicity.
Received 00th January 20xx,
Accepted 00th January 20xx
Mengzhe Wang,a Christopher D. McNitt,b Hui Wang,a Xiaofen Ma, a,c* Sarah M. Scarry,d
DOI: 10.1039/x0xx00000x
Zhanhong Wu,a Vladimir V. Popik, b, * and Zibo Li a,
*
www.rsc.org/
Here we report the 18F labeling of prostate specific membrane azide to phosphines and strain-promoted cycloaddition of
antigen ligand (PSMA) via strain promoted oxa- azide to alkynes (such as cyclooctynes, dibenzocyclooctynes,
dibenzocyclooctyne (ODIBO)- or bicyclo[6.1.0]nonyne (BCN)-azide azadibenzocylcooctynes and
thia-cycloalkynes).10-14
reaction. Although ODIBO reacts with azide 20 fold faster than Unfortunately, phosphines suffered from oxygen sensitivity,
BCN, in vivo PET imaging suggests 18F-BCN-Azide-PSMA while some commonly used cyclooctynes showed slow
demonstrated much higher tumor uptake and tumor to kinetics,
and required lengthy synthetic routes for
15-17
Previously the Popik group reported the
background contrast.
preparation.12,
synthesis of oxa-dibenzocyclooctynes (ODIBO), which is one of
the most reactive cyclooctynes (>45 M-1 s-1) for strain
promoted azide cycloaddition.18 This ultrafast reaction was
also converted to an attractive 18F labelling method that allows
extremely fast conjugation of 18F-ODIBO to azide-tagged
peptides and proteins.19 In this study, we perform side by side
Positron emission tomography (PET) is a powerful imaging
technology that enables the visualization and quantification of
target expression, metabolic perturbations, and many other
biological processes in vivo.1 Of the commonly used PET
radionuclides, 18F is the most broadly utilized due to its ideal
chemical, physical and nuclear properties.2 Despite the great
promise, the short half-life of 18F (~110 min) and the poor
nucleophilicity of fluoride make it difficult to directly
incorporate 18F into complex molecules. Significant amount of
effort was therefore devoted to the development of highly
efficient 18F labelling methods for PET probe construction.3
In the past decades, bioorthogonal reactions have become
a unique tool in diverse fields including nuclear medicine.4, 5 In
particular, copper(I) (Cu(I)) catalysed azide-alkyne reactions
was found to be rapid and clean which has also been
successfully adapted to 18F radiolabelling of various biologically
active agents.6 However, the use of cytotoxic copper catalyst in
reaction complicated quality control process since Cu(I) could
lead to oligonucleotide and polysaccharide degradation in
vivo.7-9 To address this limitation, azide based metal-free click
reactions have been developed including covalent ligation of
comparison between 18F-ODIBO and 18F-bicyclo[6.1.0]nonyne
20, 21
(18F-BCN)
on PET probe construction. Both reaction rate
and probe hydrophilicity are explored for 18F labelling of PSMA
ligands. The obtained information may provide guidance on
selecting appropriate labelling method for PET probe
construction.
As shown in Scheme 1, 18F-ODIBO (18F-
2) was obtained in
Scheme 1. Labeling scheme for 18F-ODIBO (18F-2) and 18F-BCN (18F-4)
5.6 ± 1.1% non-decay corrected isolation yield with >99%
radiochemical purity according to
a
previously reported
standard
protocol (Figure 1a).18 The co-injection with 19F-
2
confirmed its identity (Figure S1). Although 18F-labeled BCN
has been reported before,20, 21 we use a modified method to
achieve 18F-
4 bearing an extra ethylene glycol unit to enhance
BCN’s aqueous solubility. In brief, starting from the bromo-
precursor, nucleophilic substitution was performed using
different solvents, temperature and time. As shown in Table 1,
DMSO and THF result in lower labelling yield compared with
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MeCN. The optimal reaction temperature is 80oC within the much slower with
Journal Name
5
. Five-fold larger amount of 5, longer
tested conditions. Significant amount of unreacted bromo reaction time (15 min) and higher temperature (80oC) are
precursor was observed at room temperature and 60oC, needed to obtain reasonable yield (Table 2). Both reactions
indicating insufficient labelling reaction rate. Further increase could proceed at pH 5.5, 7.0 and pH 8.5. In order to determine
the temperature to 100oC lead to decreased labelling yield the effect of prosthetic group on the lipophilicity of the final
which could be caused by the increased side reaction of Br- PET agents, octanol-water partition coefficient of 18F-
6
and 18F-
elimination and decomposition of BCN motif. We would like to
7
was first evaluated. The log P values of 18F- and 18F-7 18are -
6
point out that the yield determined here are isolation yield, 2.03 ± 0.01 and -2.62 ± 0.04, respectively. This correlates well
which is more relevant to real practice, but much lower than with our expectation that the more hydrophobic ODIBO will
yield determined by radio TLC: the non-specific bindings of lead to more lipophilic product. In addition to the difference in
product to HPLC lines and HPLC columns during purification hydrophilicity, the size of the hydrophobic motif and the
will lower the yield. A 99% radiochemical purity could be length of hydrophilic polyethylene glycol chain may also lead
obtained after purification (Figure 1b).
Table 1 Labelling conditions for 18F-4
to difference in the amphiphilic properties of the two
compounds and further affect their biodistribution in vivo.
Table 2 Labelling conditions for 18F-7
Solvent
DMSO
THF
T(oC)
80
Time/min
Isolation Yield%
T(oC)
Amount of
pH
Isolation Yield%
1
2
3
4
5
6
7
8
10
10
10
10
10
10
20
30
5
1
0
0
8
4
8
9
PSMA-N3/μg
80
1
2
3
4
5
6
7
r.t.
40
60
80
40
40
40
50
50
50
50
10
50
50
7.0
7.0
7.0
7.0
7.0
5.5
8.5
5
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
r.t.
60
26
32
80
36
100
80
Not Detected
21
26
80
Scheme 2. Labelling scheme for 18F-ODIBO-PSMA(18F-6) and 18F-BCN-PSMA (18F-7)
Recognizing the short half-life of 18F, recent effort on new
labelling method development has been mainly focused on
2 4 on hand, we
improving reaction rate. With 18F- and 18F-
explore the factors that should be considered when selecting
appropriate methods for PET probe construction. The major
difference between 18F-
azide 20-fold faster than BCN; and 18F-
than 18F-
(Figure 1). The target of interest in our approach is
2
and 18F-
4
are ODIBO reacts with
is more hydrophilic
4
Figure 1 Radio HPLC profile of (a) 18F-2 and 18F-6. (b) 18F-4 and 18F-7
2
In order to confirm that target binding affinity was still
prostate specific membrane antigen (PSMA), which was found
to be over expressed in prostate cancer with limited
maintained after the peptide modification, we compared the
in vitro cell binding affinity of 19F- and 19F-
with that of the
expression in healthy tissues.22-29 As shown in Scheme 2, 18F-
2
6
7
and 18F-
PSMA ligands.
Due to the fast kinetic of the reaction between the 18F-
and azide(
), we were able to obtain 18F-
4
could react with azide-PSMA ligand to achieve 18F-
clinically used PSMA-617 via competitive cell binding assay. As
shown in Figure 2, all the compounds inhibited the binding in a
dose-dependent manner. As the reference, PSMA-617 had the
50% inhibitory concentration (IC50) as 144.6 nM while the IC50
2
5
6
in 20 ± 1% isolated
6 7 are 108.9 nM and 156.4 nM,
values of 19F- and 19F-
yield with only 10 μg of 5 at neutral condition, room
temperature and within seconds. In contrast, the 18F-
4 reacts
respectively. The comparable IC50 values demonstrated that
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both 19F- and 19F-
6
7 have similar binding affinity compared was 2.49 ± 0.34 % ID/g and 2.24 ± 0.03 % ID/g at 40 min and
with PSMA-617 and could be further evaluated in vivo.
120 min post injection, which was significantly higher than
those of 18F-
at both time points (p=0.01 for 40 min and
6
p=0.0001 for 120 min time points). Although uptake was still
visible in gallbladder and intestine, kidney is the organ with
higher tracer uptake. This observation could be attributed to
the more hydrophilic BCN motif of 18F-
7 and the background
PSMA expression in kidneys.30, 31 Quantitative analysis of major
organs are shown in Figure 4. At 120 min p.i., the tumor to
liver, tumor to muscle ratios are 0.24 ± 0.01 and 2.47 ± 0.90 for
18F-
1.5 and 69.59 ± 7.23 for 18F-
PET images and high tumor to background ratio.
6
respectively. On the other hand, the contrast are 5.83 ±
respectively. This led to clean
7
Figure 2 Competitive binding assay of 19F-6 and 19F-7 compared with PSMA-617 as
reference
Figure 4 Quantitative uptake of major organs in LNCap xenografts post injection of (a)
18F-6 and (b) 18F-7
The targeting specificity of 18F-
7 was confirmed by a
blocking study in which excess amount of unradiolabeld PSMA
ligand was coinjected with the tracer. As shown in Figure 5, the
tumor uptake in blocking group was significantly reduced
compared with that of in the normal group (p=0.04). This
demonstrated that the unradiolabeled PSMA ligand
successfully blocked the targeting sites and reduced the tracer
uptake in the blocking group.
Figure 5 Representative PET/CT images of LNCap xenograft at 120min post injection
of 18F-7 (a) without and (b)with a blocking dose. Quantitative uptake of major organs
derived from PET images.
Figure 3 Representative PET/CT images of (a)(b) LNCap xenograft at 40min and 120min
post injection of 18F-6, respectively and (c)(d) LNCap xenograft at 40min and 120min
post injection of 18F-7, respectively
Conclusion: Due to the short half-life of 18F, significant
amount of effort has recently been devoted to the
development of ultrafast labelling reactions. In this study, we
The targeting efficiency of 18F- and 18F-
6 7 was evaluated by
performing multiple time-point static microPET scans in PSMA
positive LNCap tumor-bearing mice (n=3). After administration
used a highly reactive but more hydrophobic 18F-
reactive but more hydrophilic 18F-
to construct PSMA
2 and a less
4
of 3.7 MBq of either 18F-
6
or 18F-
7
via tail vein, animals were
scanned at 40 min and 120 min post injection. As shown in
Figure 3a and 3b, although 18F-
demonstrated good PSMA
targeting PET probes via azide-alkyne click reaction. Both
agents could be efficiently prepared and demonstrated
6
comparable target binding affinity in vitro. However, 18F-
6
affinity in binding assay, very low uptake can be seen in the
tumor region in vivo. The tumor uptake was only 0.46 ± 0.02 %
ID/g and 0.30 ± 0.01 % ID/g at 40 min and 120 min post
injection, respectively. Radio signals were mainly localized at
gallbladder and intestines, which could be caused by the
failed to provide reasonable tumor to background contrast
potentially due to the hydrophobicity of ODIBO motif. The
more hydrophilic BCN derived tracer showed much higher
tumor uptake and tumor to background ratio. The information
obtained here suggest both reaction speed and hydrophilicity
should be considered when selecting appropriate labeling
relatively more hydrophobic ODIBO motif of 18F-
stability of the reagent. On the contrary, tumor can be clearly
visualized by 18F-
PET (Figure 3c and 3d). The tumor uptake
6 or the
methods for PET probe construction. 18F-
2 might be more
7
suitable for protein labeling which require fast reaction rate
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Journal Name
17.
18.
19.
J. C. Jewett, E. M. Sletten and C. R. Bertozzi, Journal of the
American Chemical Society, 2010, 132, 3688-3690.
C. D. McNitt and V. V. Popik, Org Biomol Chem, 2012, 10,
8200-8202.
M. Boudjemeline, C. D. McNitt, T. A. Singleton, V. V. Popik
and A. P. Kostikov, Organic & Biomolecular Chemistry,
2018, DOI: 10.1039/C7OB02532G.
but may be less affected by hydrophobicity of labeling motif
due to the large molecular weight. Nonetheless, other factors
including position of modification, degree of modification, and
charge change could all affect the distribution of the final
agents.
20.
21.
X.-G. Li, C. Hagert, R. Siitonen, H. Virtanen, O. Sareila, H.
Liljenbäck, J. Tuisku, J. Knuuti, J. Bergman, R. Holmdahl
and A. Roivainen, ACS Medicinal Chemistry Letters, 2016,
7, 826-830.
X. G. Li, A. Roivainen, J. Bergman, A. Heinonen, F. Bengel,
T. Thum and J. Knuuti, Chem Commun (Camb), 2015, 51,
9821-9824.
Conflicts of interest
There are no conflicts to declare.
Notes and references
‡ This work was supported by UNC Chapel Hill Department of
Radiology and BRIC, NSF grant (CHE-1565646), and the Science
and Technology Program of Guangzhou, china (grant number:
201804010448). Financial support for the PSMA ligand synthesis
in the form of start-up funding awarded from the UNC-CH to
Professor Jeffrey Aubé is gratefully acknowledged. We thank the
UNC-CH Department of Chemistry Mass Spectrometry Core Lab,
especially Brandie M. Ehrmann and Karl M. Koshlap in the UNC
ESOP NMR Core Facility (PSMA ligand data).
22.
23.
H. J. K. Ananias, M. C. van den Heuvel, W. Helfrich and I. J.
de Jong, The Prostate, 2009, 69, 1101-1108.
S. Minner, C. Wittmer, M. Graefen, G. Salomon, T.
Steuber, A. Haese, H. Huland, C. Bokemeyer, E. Yekebas, J.
Dierlamm, S. Balabanov, E. Kilic, W. Wilczak, R. Simon, G.
Sauter and T. Schlomm, The Prostate, 2011, 71, 281-288.
M. Rybalov, H. J. K. Ananias, H. D. Hoving, H. G. van der
Poel, S. Rosati and I. J. de Jong, International Journal of
Molecular Sciences, 2014, 15, 6046-6061.
A. Afshar-Oromieh, H. Hetzheim, C. Kratochwil, M.
Benesova, M. Eder, O. C. Neels, M. Eisenhut, W. Kübler, T.
Holland-Letz, F. L. Giesel, W. Mier, K. Kopka and U.
Haberkorn, Journal of Nuclear Medicine, 2015, 56, 1697-
1705.
A. Afshar-Oromieh, T. Holland-Letz, F. L. Giesel, C.
Kratochwil, W. Mier, S. Haufe, N. Debus, M. Eder, M.
Eisenhut, M. Schäfer, O. Neels, M. Hohenfellner, K. Kopka,
H.-U. Kauczor, J. Debus and U. Haberkorn, European
Journal of Nuclear Medicine and Molecular Imaging, 2017,
44, 1258-1268.
C. Cui, M. Hanyu, A. Hatori, Y. Zhang, L. Xie, T. Ohya, M.
Fukada, H. Suzuki, K. Nagatsu, C. Jiang, R. Luo, G. Shao, M.
Zhang and F. Wang, American Journal of Nuclear Medicine
and Molecular Imaging, 2017, 7, 40-52.
24.
25.
1.
2.
3.
S. M. Ametamey, M. Honer and P. A. Schubiger, Chem
Rev, 2008, 108, 1501-1516.
Z. Li and P. S. Conti, Adv Drug Deliv Rev, 2010, 62, 1031-
1051.
H. S. Krishnan, L. Ma, N. Vasdev and S. H. Liang,
Chemistry, 2017, 23, 15553-15577.
26.
4.
5.
K. Lang and J. W. Chin, ACS Chem Biol, 2014, 9, 16-20.
J. P. Meyer, P. Adumeau, J. S. Lewis and B. M. Zeglis,
Bioconjug Chem, 2016, 27, 2791-2807.
6.
7.
M. Meldal and C. W. Tornøe, Chemical Reviews, 2008,
108, 2952-3015.
J. Gierlich, G. A. Burley, P. M. E. Gramlich, D. M.
Hammond and T. Carell, Organic Letters, 2006, 8, 3639-
3642.
27.
28.
8.
E. Lallana, E. Fernandez-Megia and R. Riguera, Journal of
the American Chemical Society, 2009, 131, 5748-5750.
A. J. Link, M. K. S. Vink and D. A. Tirrell, Journal of the
American Chemical Society, 2004, 126, 10598-10602.
G. de Almeida, E. M. Sletten, H. Nakamura, K. K.
Palaniappan and C. R. Bertozzi, Angewandte Chemie
International Edition, 2012, 51, 2443-2447.
J. Dommerholt, S. Schmidt, R. Temming, L. J. A. Hendriks,
F. P. J. T. Rutjes, J. C. M. van Hest, D. J. Lefeber, P. Friedl
and F. L. van Delft, Angewandte Chemie International
Edition, 2010, 49, 9422-9425.
B. Grubmüller, R. P. Baum, E. Capasso, A. Singh, Y.
Ahmadi, P. Knoll, A. Floth, S. Righi, S. Zandieh, C. Meleddu,
S. F. Shariat, H. C. Klingler and S. Mirzaei, Cancer
Biotherapy and Radiopharmaceuticals, 2016, 31, 277-286.
C. Uprimny, Wien Med Wochenschr, 2017, DOI:
10.1007/s10354-017-0569-z.
Y. Kinoshita, K. Kuratsukuri, S. Landas, K. Imaida, P. M.
Rovito, Jr., C. Y. Wang and G. P. Haas, World J Surg, 2006,
30, 628-636.
9.
10.
29.
30.
11.
31.
S. O'Keefe Denise, J. Bacich Dean and D. W. Heston
Warren, The Prostate, 2003, 58, 200-210.
12.
13.
14.
M. Köhn and R. Breinbauer, Angewandte Chemie, 2004,
116, 3168-3178.
A. Kuzmin, A. Poloukhtine, M. A. Wolfert and V. V. Popik,
Bioconjugate Chemistry, 2010, 21, 2076-2085.
X. Ning, J. Guo, M. A. Wolfert and G.-J. Boons,
Angewandte Chemie (International ed. in English), 2008,
47, 2253-2255.
15.
16.
J. A. Codelli, J. M. Baskin, N. J. Agard and C. R. Bertozzi,
Journal of the American Chemical Society, 2008, 130,
11486-11493.
M. F. Debets, S. S. van Berkel, S. Schoffelen, F. P. J. T.
Rutjes, J. C. M. van Hest and F. L. van Delft, Chemical
Communications, 2010, 46, 97-99.
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