1
6 (0.5–1.0 mg) for hot labeled runs. For example, when the
amount of 16 was reduced to 0.5 mg (5.6 mM), the conjugation
took 1 h at room temperature (ESIw). The experiments were
18
reproducible and in each case the resultant conjugate [ F]-17 was
18
the only radioactive product. The identity of [ F]-17 was confirmed
by comparison with a reference sample obtained independently
from the conjugation between 16 and 5-FDR. Thus bioconjugation
to this PAR-2 agonist proved very straightforward.
1
8
1
9
In summary it is demonstrated that [ F]-FDR 10 is an efficient
Fig. 1 (a) Proposed forms of
[
F]-15 in aqueous solution;
1
8
bioconjugation ligand for [ F]-fluoro-labelling of suitably
1
modified peptides. An attractive aspect of [ F]-FDR 10 over
1
b) H-NMR signals representing the proton at the imine bond carbon
(
8
19
of E- and Z-[ F]-15, respectively.
1
8
[
F]-FDG utilising protocols, there is no requirement for
1
8
harsh conditions or pre-derivatisation of the [ F]-sugar, and
labelling can occur at pH 4.6–7.0 in one step. The ligation is
extremely rapid relative to FDG due to the location of the
fluorine at C-5 and the increased reactivity of 5-membered
over 6-membered aldol sugars with alkoxyamines. So there are
special electronic features of FDR that render it particularly
suitable in this context. Additionally an automated production
1
8
method for [ F]-FDR 10 is developed by adapting a standard GE
1
8
s
TRACERLab module used for routine [ F]-FDG synthesis.
18
Scheme 4
F-labelling of peptide 16: (a) efficient reaction under mild
18
18
With its ease of synthesis and its conjugation efficiency [ F]-FDR
conditions; (b) radioactive detection of labelled product [ F]-17.
1
0 emerges as a very attractive bioconjugation tool for clinical and
was explored. Accordingly, compound 14 (2 mg) was incubated
preclinical peptide PET imaging studies.
18
with [ F]-FDR 10 (8–12 MBq) in sodium acetate buffer (110 mL,
0 mM, pH 4.6) in an Eppendorf tube at room temperature. Now
We thank Mr Stuart Craib (University of Aberdeen) for
9
1
8
[
F]-fluoride production. X.-G. Li thanks the Academy of
the molar ratio of 14 relative to 10 is in large excess, and the
reaction proceeded extremely rapidly (B10 min) and to comple-
tion (495%, n = 3) as determined by radiochemical conversion
Finland for the research funding (Grant 133127).
Notes and references
1
8
(
HPLC). The identity of product [ F]-15 was confirmed by
19
1
P. W. Miller, N. J. Long, R. Vilar and A. D. Gee, Angew. Chem.,
Int. Ed., 2008, 47, 8998, and references therein.
(a) D. E. Olberg and O. K. Hjelstuen, Curr. Top. Med. Chem.,
comparison with a reference sample of [ F]-15. During the
18
production of [ F]-FDR 10, D-ribose was generated as a side
product, just as glucose is generated during FDG production.
Based on the results in Table 1 (entries 1 and 2), the oxime
formation with D-ribose is anticipated to be much slower in the
2
´
2010, 10, 1669; (b) B. Kuhnast and F. Dolle, Curr. Radiopharm.,
2010, 3, 174–201.
3
(a) M. Simpson, L. Trembleau, R. W. Cheyne and T. A. D. Smith,
Appl. Radiat. Isot., 2011, 69, 418; (b) F. Wuest, C. Hultsch, M. Berndt
and R. Bergmann, Bioorg. Med. Chem. Lett., 2009, 19, 5426;
(c) M. Namavari, Z. Cheng, R. Zhang, A. De, J. Levi, J. K. Hoerner,
S. S. Yaghoubi, F. A. Syud and S. S. Gambhir, Bioconjugate Chem.,
1
8
presence of [ F]-FDR 10 as a competing conjugation ligand.
1
8
Also there was distinct separation of conjugate [ F]-15 from
unreacted peptide 14 on HPLC such that it is not a contaminant
in the radiolabelled product (ESIw).
2009, 20, 432; (d) C. Hultsch, M. Schottelius, J. Auernheimer, A. Alke
and H.-J. Wester, Eur. J. Nucl. Med. Mol. Imaging, 2009, 36, 1469.
F. Wuest, M. Berndt, R. Bergmann, J. van den Hoff and
J. Pietzsch, Bioconjugate Chem., 2008, 19, 1202.
C. P. Phenix, B. P. Rempel, K. Colobong, D. J. Doudet,
M. J. Adam, L. A. Clarke and S. G. Withers, Proc. Natl. Acad.
Sci. U. S. A., 2010, 107, 10842.
A peptide of clinical relevance was then explored for bioconju-
4
5
1
8
gation with [ F]-FDR 10. The hexapeptide H-SLIGKV-OH is a
well-characterized agonist of the human protease activated receptor
1
2
2
(PAR-2). PAR-2 is involved in a number of pathological
13
processes, including its role in tumor progression. Recent studies
have shown that PAR-2 is involved in the cell invasion of
hepatocellular carcinoma (HCC). HCC is a metastatic malignancy
6 O. Boutureira, F. D’Hooge, M. Ferna
M. Sanchez-Navarro, J. R. Koeppe and B. G. Davis, Chem. Commun.,
010, 46, 8142.
dez-Gonzalez, G. J. L. Bernardes,
´ ´
´
2
7
(a) M. Onega, J. Domarkas, H. Deng, L. F. Schweiger, T. A. E.
Welch, C. Plisson, A. D. Gee and D. O’Hagan, Chem. Commun.,
2010, 46, 139; (b) H. Deng, S. L. Cobb, A. D. Gee, A. Lockhart,
L. Martarello, R. P. McGlinchey, D. O’Hagan and M. Onega,
Chem. Commun., 2006, 42, 654.
14
and its early diagnosis remains a challenge. Peptide 16, which is
commercially available, has an aminoxyacetyl (Aoa) group attached
to the N-terminus of the H-SLIGKV-OH peptide (Scheme 4).
18
Conjugation between 16 (2 mg, 22.6 mM) and [ F]-FDR 10 in
8
K. Hamacher, H. H. Coenen and G. Stocklin, J. Nucl. Med., 1986,
2
sodium acetate buffer was again very rapid (B95% conversion in
7, 235.
9 J. W. Haas, Jr. and R. E. Kadunce, J. Am. Chem. Soc., 1962, 84, 4910.
10 M. Berndt, J. Pietzsch and F. Wuest, Nucl. Med. Biol., 2007, 34, 5.
1
0 min) at room temperature. In the hot labeling experiments,
1
peptide 16 was used in excess of [ F]-FDR 10. Analytical
8
1
1
1 P. M. Moyle and T. W. Muir, J. Am. Chem. Soc., 2010, 132, 15878.
2 M. Molino, E. S. Barnathan, R. Numerof, J. Clark, M. Dreyer,
A. Cumashi, J. A. Hoxie, N. Schechter, M. Woolkalis and
L. F. Brass, J. Biol. Chem., 1997, 272, 4043.
radio-HPLC purification was performed to demonstrate a
1
clear separation of labeled product [ F]-17 from unreacted
8
1
peptide 16. Thus [ F]-17 was obtained in high radiopurity
8
1
3 R. Kaufmann, C. Oettel, A. Horn, K. Halbhuber, A. Eitner and
R. Krieg, Carcinogenesis, 2009, 30, 1487.
(
499%) and was free of precursor 16. The analytical method
did not allow an accurate determination of the specific activity.
Additionally, it is satisfactory to use a reduced amount of peptide
1
4 R. Kaufmann, F. Mubbach, P. Henklein and U. Settmacher,
J. Cancer Res. Clin. Oncol., 2011, 137, 965.
This journal is c The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 5247–5249 5249