Fluoride-18 radiolabeling of peptides bearing an aminooxy functional group to a prosthetic ligand via an oxime bond

Article abstract of DOI:10.1016/j.bmcl.2011.09.124

We have developed a novel F-18 prosthetic ligand named fluoro-PEG- benzaldehyde (FPBA) 1. [¹⁸F]-FPBA 1 is formed in situ from its radiolabeled precursor [¹⁸F]6. Compound 6 is efficiently synthesized in four steps starting from commer

Full text of DOI:10.1016/j.bmcl.2011.09.124

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6992–6995
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fluoride-18 radiolabeling of peptides bearing an aminooxy functional group
to a prosthetic ligand via an oxime bond
Patrick Carberry a, Alan P. Carpenter b, Hank F. Kung a,c,
⇑
a Department of Radiology, University of Pennsylvania, 3700 Market St., Room 305, Philadelphia, PA 19104, USA
b Avid Radiopharmaceuticals, Inc., (Eli Lilly) 3711 Market St., 7th Floor, Philadelphia, PA 19104, USA
c Department of Pharmacology, University of Pennsylvania, 3700 Market St., Room 305, Philadelphia, PA 19104, USA
a r t i c l e i n f o
a b s t r a c t
We have developed a novel F-18 prosthetic ligand named fluoro-PEG-benzaldehyde (FPBA) 1. [18F]-FPBA
1 is formed in situ from its radiolabeled precursor [18F]6. Compound 6 is efficiently synthesized in four
steps starting from commercially available 6-bromo-3-pyridine carbaldehyde 2. [18F]-FPBA was evalu-
ated as a prosthetic ligand to radiolabel three cyclic peptides bearing an aminooxy functional group at
the N-terminus position. Acetal [18F]6 is purified by either solid-phase extraction (SPE) or reverse-phase
HPLC with the overall radiochemical yields (RCY) and radiochemical purity (RCP) in very close agreement.
The SPE purification process has the advantage of shorter reaction times (71–87 min for entire reaction
sequence), while the use of the reverse-phase HPLC purification process allows the use of up to fifty times
less of the expensive synthetic peptides (ꢀ50 nmol) in the oxime coupling reaction. We have demon-
strated an efficient methodology in the production of [18F]-FPBA 1 and demonstrated its use as a pros-
thetic ligand for the labeling of peptides possessing an aminooxy functional group.
Article history:
Received 27 June 2011
Revised 26 September 2011
Accepted 28 September 2011
Available online 5 October 2011
Keywords:
Fluorine-18
Prosthetic ligand
Aminooxy
Oxime
Peptide
PET
Ó 2011 Elsevier Ltd. All rights reserved.
Positron emission tomography (PET) increasingly plays a major
role in clinical diagnosis and basic research.1 PET images can quan-
titatively detect changes in cellular functions and physiological
pathways. Various PET ligands labeled with F-18 have been used
to study specific receptors and transporter sites. Once bound to
the target sites, the decay of positrons produces two high-energy
photons (511 keV, 180° apart), which are detected by coincidence
circuitry of a PET imaging scanner. PET imaging agents can be used
on the picomolar scale while still maintaining high-resolution
images.
Fluorine-18 is one isotope that is widely used for PET imaging
due to its physical and nuclear characteristics.2 There are two
labeling methods used to substitute the fluorine-18 atom into a
molecule. This process is traditionally accomplished by either elec-
trophilic substitution or nucleophilic displacement.1–5 The use of
the fluoride anion [18F]FÀ, that is, nucleophilic displacement, has
the advantages of high specific activity3,4 and more control over
the site at which the fluoride anion will react. The conditions used
for the SN2 type reaction of [18F]FÀ are strongly basic, and are
therefore not suitable for compounds containing acidic protons,
such as those functional groups found in proteins and peptides.
Prosthetic ligands, compounds containing a pre-existing fluorine-
18 labeled moiety, are typically used to indirectly attach the fluo-
rine-18 radionuclide onto a desired protein or peptide.1,5
There have been recent advances in the direct F-18 labeling of
peptides6–10 that do not include the use of a prosthetic ligand. In
one such case, Chen and co-workers were able to directly label a
modified peptide in one step.10 The precursor contains a 4-NO2-
3-CF3 arene moiety, with the nitro group acting as a leaving group
and the 18F-fluoride displacing it through nucleophilic aromatic
substitution. Although the reaction takes place in a timely manner
(40 min), there are some drawbacks to this methodology, which in-
clude harsh reaction conditions that can denature a protein or pep-
tide, incompatibility with certain functional groups (particularly
alcohols), and the use of a relatively large amount (10Â molar ex-
cess) of starting material compared to our current method. Re-
cently,
a novel in situ labeling of peptides, based on the
formation of Al–F bond via 1,4,7-triazacyclononane–Al peptide
complexes, have been reported.11 The Al–F bond is readily formed
under a mild condition. Especially useful and unique, is the fact
that the peptide-Al–18F complexes can be prepared without drying,
that is, directly in an aqueous solution.
The approach of anchoring a prosthetic ligand by way of an
oxime bond has been previously reported in the literature with
the use of aldehydes.12–14 This methodology takes advantage of
the highly chemoselective nature of the oxime bond formation be-
tween aminooxy groups and aldehydes or ketones under an aque-
ous media. Several groups have quite efficiently used [18F]FB–CHO
as their prosthetic ligand to be anchored on to a target molecule by
way of an oxime bond.15–17 Although it is only one-step to produce
⇑
Corresponding author. Tel.: +1 215 662 3096; fax: +1 215 349 5035.
E-mail address: kunghf@sunmac.spect.upenn.edu (H.F. Kung).
[
18F]FB–CHO in high RCY, the compound is volatile and can in-
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.124

P. Carberry et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6992–6995
6993
[
18F]-AV-45, florbetapir.23,24 These oximes showed promising
zresults, but even with their larger size, they still retained some
capability to cross the blood–brain barrier (BBB). We have demon-
strated a general protocol for the fluoride-18 labeling with a new
prosthetic ligand that is tolerant towards several functional groups
and is formed via a chemoselective oxime coupling.25,26 We herein
report our findings on the use of our novel fluorine-18 prosthetic
ligand, named [18F]-fluoro-PEG-benzaldehyde ([18F]-FPBA) 1, to-
wards labeling peptides bearing and aminooxy functional group.
The peptides chosen for this study are designed based on the ami-
no acid sequence found in a known fibrin-binding MRI contrast-
imaging agent, EP-2104R.27
Figure 1. Novel prosthetic ligand [18F]-fluoro-PEG-benzaldehyde ([18F]-FPBA) 1.
The synthesis of prosthetic ligand [18F]-FPBA 1 has been previ-
ously reported by our group.22 The synthesis begins with the pro-
tection of the aldehyde functionality of 6-bromo-3-pyridine
carbaldehyde to afford acetal 228 (Scheme 1). A pegylated chain
is then added onto the molecule and the alcohol function group
is transformed into tosylate 5. The ‘standard’ fluorine-19 prosthetic
precursor 6 is then prepared with the use of tetra-n-butylammo-
nium fluoride hydrate in refluxing tetrahydrofuran. Fluorine 6
was used to determine the formation of the radiolabeled derivative
by HPLC analysis.
Three cyclic peptides were purchased from Bachem (Fig. 2).
Each peptide has similarities to the reported fibrin-binding MRI
contrast agent, EP-2104R.27 All three of these synthetic peptides
possess the aminooxy functionality at the N-terminus of the pep-
tide sequence.
Scheme 1. Synthetic pathway for the preparation of ligand 6. Reagents and
conditions: (i) ethylene glycol, p-TsOH, benzene, reflux (98%); (ii) triethylene glycol,
Cs2CO3, DMF, microwave, 150 °C, 50 min (61%); (iii) p-TsCl, Et3N, DMAP, CH2Cl2
(82%); (iv) TBAF.H2O, THF, reflux (57%).
crease lipophilicity in the target ligand, altering the ligand’s phar-
macokinetics.18 Recently [18F]FDG has been reported to chemose-
lectively form an oxime bond with aminooxy groups,19–21 taking
advantage of the fact that, at elevated temperature, FDG exists in
equilibrium between its cyclic and linear forms. The 18F-FDG con-
jugated peptides have been successfully reported.
Previously, we described the synthesis and optimization of a
novel F-18 prosthetic ligand 1 (Fig. 1).22 Compound 1 was used
to radiolabel two model aminooxy complexes, by way of oxime
coupling, to provide the desired fluorine-18 radiotracers. The bio-
distribution in male ICR mice for both oxime labeled complexes
were compared to that of the known b-amyloid plaque indicator,
Each peptide was subjected to an acidic media (TFA) in the pres-
ence of ligand 6 (Scheme 2). The acidic media served to facilitate
the de-blocking of the acetal functional group as well as promoting
the formation of the oxime bond between the aldehyde and amino-
oxy groups. The reactions were monitored and purified by HPLC.
Figure 2. Peptides 7, 8, and 9. AOAC-Tyr-Asp-Cys3-Hyp-Tyr(3-Cl)-Gly-Leu-Cys8-Tyr-Ile-OH, disulfide bond: 3?8 (7); AOAC-Arg-Hyp-Cys3-Asp-Tyr(3-Cl)-Tyr(3-Cl)-Gly-Thr-
Cys8-Phe-Asp-OH, disulfide bond: 3?8 (8); AOAC-Leu-Hyp-Cys3-Asp-Tyr(3-Cl)-Tyr(3-Cl)-Gly-Thr-Cys9-Leu-Asp-OH, disulfide bond: 3?9 (9).

6994
P. Carberry et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6992–6995
Table 1
The purified oxime was characterized by MALDI mass spectrome-
try (see Supplementary data). It is important to note that the reac-
tions conditions were tolerant of the disulfide bridge found in each
cyclic peptide as determined from mass acquired from the MALDI
mass spectrometry and the same reaction conditions were used to
form the radiolabeled peptides.
With the formation of the desired oximes in hand, we next
turned our attention to the labeling of these synthetic peptides
with the use of our prosthetic ligand [18F]-FPBA 1. The prosthetic
precursor [18F]6 was formed in one step from tosylate 5 (Scheme
3). It is extremely crucial in the radiolabeling step to have the alde-
hyde protected as the acetal to avoid side products.22 The aldehyde
functionality is then de-blocked in situ during the oxime coupling
reaction rendering [18F]-FPBA 1.
Comparison of the formation of oximes using different purification methods
Entry Peptide Quantity
(mg)
Purification RCYa
(%)
RCPa
(%)
Time elapseda
(min)
1
2
3
4
5
6
7
8
9
8
8
8
7
7
9
8
7
9
2.5
3.0
5.0
3.0
5.0
3.0
<0.1
<0.1
<0.1
SPE
21
28
32
25
35
26
24
25
21
97
96
98
97
93
90
>99
>99
>99
79
74
80
71
78
SPE
SPE
SPE
SPE
SPE
87
HPLC
HPLC
HPLC
137
127
135
0.8 mg of tosylate 5 used.
a
RCY, RCP and time for entire radiolabeling sequence.
The prosthetic ligand, compound 1, was successfully coupled to
each peptide (Scheme 4). The reaction conditions appear compati-
ble with the rest of the functional groups located on the peptide,
including the disulfide bridge. The reaction occurs in an ethanol/
aqueous media facilitate with the use of trifluoroacetic acid
(TFA). The acidic media de-blocks the acetal group and enables
the oxime coupling reaction to occur. The final product is purified
using an activated OASIS HLB 3 cc cartridge (SPE). The crude sam-
ple is loaded onto the cartridge via an aqueous media. The car-
tridge is then flushed with ethyl acetate. This washing removes
any of the un-reacted [18F]-FPBA 1. The OASIS HLB 3 cc cartridge
is finally eluted with 1% TFA/ethanol solution to provide the de-
sired radiolabeled oxime peptide.
should remove other organic materials which have similar func-
tional groups found in [18F]-FPBA 1 that would compete in the
oxime coupling reaction. After the reaction was complete and
cooled to room temperature, it was diluted in 2.5 mL of acetonitrile
and injected onto a semi-prep HPLC (for conditions see Supple-
mentary data). The desired samples were collected and concen-
trated to a smaller volume (with the use of an OASIS HLB 3 cc
cartridge) in 1% TFA/ethanol solution. As little as 0.1 mg of peptide
(50Â less) could then be used in the coupling reaction of prosthetic
ligand [18F]-FPBA 1. From the data collected, similar RCY were
achieved using the HPLC purification process as compared to the
SPE purification (see Table 1). Excellent RCP were achieved with
the use of a semi-prep HPLC; however the overall reaction time se-
quence had doubled, due to the use of an HPLC.
Similar to EP-2104R, the peptides selected for this conjugation
studies have demonstrated high binding affinity to the fibrin clots.
We expect that the 18F-labeled peptides will be useful for PET
imaging of specific binding of atherosclerosis in the myocardial
blood vessels. Biological evaluations of these 18F-peptides reported
in this paper are currently ongoing.
The prosthetic precursor [18F]6 was tested in the coupling reac-
tion with each peptide using two different purification methods. In
the first purification process, the crude reaction mixture was
loaded onto an OASIS HLB 3 cc cartridge via water. This was then
washed with water (3Â), leaving the organics loaded onto the OA-
SIS cartridge. The cartridge was then eluted with ethanol (0.7 mL)
to provide [18F]6. This solution was then subjected to an aqueous
acidic media (TFA/H2O) at elevated temperatures (70 °C) using
varying amounts of peptides, ranging from 2.5 to 5.0 mg, to form
the desired radiolabeled oxime peptides (Table 1, entries 1–6).
The overall reaction is very efficient and as depicted in Table 1,
increasing the amount of peptide increases the overall RCY.
Due to the large amount of peptide needed with SPE purifica-
tion for the oxime coupling, it was suggested to purify the pros-
thetic precursor through a reverse-phase HPLC. This method
In conclusion, we have demonstrated an effective method of
radiolabeling aminooxy-functionalized peptides with
a newly
developed prosthetic ligand,
[
18F]-fluoro-PEG-benzaldehyde
([18F]-FPBA) 1. The radiolabeled ligand, [18F]6, was successfully
synthesized in a total of four steps, starting with readily available
6-bromo-3-pyridine carbaldehyde 2. The radiolabeling of tosylate
5 was achieved in high radiochemical yields (71 2%, n = 3) with
excellent radiochemical purity (>99%, based on HPLC analyses) in
short reaction time (10 min). With the use of semi-preparative
HPLC, small amounts (as little as 0.1 mg) of the peptide are needed
to form the desired radiolabeled oxime in RCY similar to those ob-
tained with the use of SPE purification on the prosthetic precursor,
Scheme 2. General coupling between ligand 6 and peptides 7, 8, and 9. Reagents
and conditions: (i) peptide 7, 8, or 9, TFA, EtOH/H2O, 70 °C, 30 min.
[
18F]6. The final radiolabeled peptide is purified by SPE, making
this methodology very efficient and practical. The overall reaction
sequence ranged from 71 to 137 min over two steps, depending on
the purification method used for the ligand [18F]6.
Acknowledgements
Scheme 3. Radiolabeling of ligand [18F]6. Reagents and conditions: (i) K[222]18F,
K2CO3, ACN, 110 °C, 10 min (RCY = 71 2%; RCP = 99 1%; n = 3, for SPE
purification).
We would like to thank Dr. Craig N. Streu and Dr. Kathleen Mol-
nar of the DeGrado Laboratory at the University of Pennsylvania
School of Medicine, Department of Biochemistry and Biophysics
for the use and their aid in acquiring data from the MALDI mass
spectrometry. We would also like to thank Avid Radiopharmaceu-
ticals, Inc., (Eli Lilly) for funding this project.
Supplementary data
Scheme 4. General coupling between ligand [18F]6 and peptides [18F]7-ox, [18F]8-
ox, and [18F]9-ox. Reagents and conditions: (i) TFA, EtOH/H2O, 70 °C, 2 min, then
peptide 7, 8, or 9, EtOH/H2O, 70 °C, 30 min.
Supplementary data for the synthesis, HPLC, and MALDI of
oximes 7-ox, 8-ox, and 9-ox along with the radiosynthesis of

P. Carberry et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6992–6995
6995
prosthetic ligand [18F]6, oximes [18F]7-ox, [18F]8-ox, and [18F]9-ox,
and the corresponding HPLCs associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2011.09.124.
13. Glaser, M.; Morrison, M.; Solbakken, M.; Arukwe, J.; Karlsen, H.; Wiggen, U.;
Champion, S.; Kindberg, G. M.; Cuthbertson, A. Bioconjug. Chem. 2008, 19, 951.
14. Carrasco, M. R.; Alvarado, C. I.; Dashner, S. T.; Wong, A. J.; Wong, M. A. J. Org.
Chem. 2010, 75, 5757.
15. Poethko, T.; Schottelius, M.; Thumshirn, G.; Herz, M.; Haubner, R.; Henriksen,
G.; Kessler, H.; Schwaiger, M.; Wester, H.-J. Radiochim. Acta 2004, 92, 317.
16. Poethko, T.; Schottelius, M.; Thumshirn, G.; Hersel, U.; Herz, M.; Henriksen, G.;
Kessler, H.; Schwaiger, M.; Wester, H.-J. J. Nucl. Med. 2004, 45, 892.
17. Harki, D. A.; Satyamurthy, N.; Stout, D. B.; Phelps, M. E.; Dervan, P. B. Proc. Natl.
Acad. Sci. U.S.A. 2008, 105, 13039.
References and notes
1. Tolmachev, V.; Stone-Elander, S. Biochem. Biophys. Acta, Gen. Subj. 2010, 1800,
487.
2. Cherry, S. R. J. Clin. Pharmacol. 2001, 41, 482.
3. Ametamey, S. M.; Honer, M.; Schubiger, P. A. Chem. Rev. 2008, 108, 1501.
4. Mach, R. H.; Schwarz, S. W. PET Clin. 2010, 5, 131.
5. Kuhnast, B.; Dolle, F. Curr. Radiopharm. 2010, 3, 174.
6. Hohne, A.; Mu, L.; Honer, M.; Schubiger, P. A.; Ametamey, S. M.; Graham, K.;
Stellfeld, T.; Borkowski, S.; Berndorff, D.; Klar, U.; Voigtmann, U.; Cyr, J. E.;
Friebe, M.; Dinkelborg, L.; Srinivasan, A. Bioconjug. Chem. 2008, 19, 1871.
7. Becaud, J.; Mu, L.; Karramkam, M.; Schubiger, P. A.; Ametamey, S. M.; Graham,
K.; Stellfeld, T.; Lehmann, L.; Borkowski, S.; Berndorff, D.; Dinkelborg, L.;
Srinivasan, A.; Smits, R.; Koksch, B. Bioconjug. Chem. 2009, 20, 2254.
8. Mu, L.; Hohne, A.; Schubiger, P. A.; Ametamey, S. M.; Graham, K.; Cyr, J. E.;
Dinkelborg, L.; Stellfeld, T.; Srinivasan, A.; Voigtmann, U.; Klar, U. Angew. Chem.,
Int. Ed. 2008, 47, 4922.
9. Roehn, U.; Becaud, J.; Mu, L.; Srinivasan, A.; Stellfeld, T.; Fitzner, A.; Graham, K.;
Dinkelborg, L.; Schubiger, A. P.; Ametamey, S. M. J. Fluorine Chem. 2009, 130, 902.
10. Jacobson, O.; Zhu, L.; Ma, Y.; Weiss, I. D.; Sun, X.; Niu, G.; Kiesewetter, D. O.;
Chen, X. Bioconjug. Chem. 2011, 22, 422.
18. Colombo, M.; Bianchi, A. Molecules 2010, 15, 178.
19. Namavari, M.; Cheng, Z.; Zhang, R.; De, A.; Levi, J.; Hoerner, J. K.; Yaghoubi, S. S.;
Syud, F. A.; Gambhir, S. S. Bioconjug. Chem. 2009, 20, 432.
20. Wuest, F.; Hultsch, C.; Berndt, M.; Bergmann, R. Bioorg. Med. Chem. Lett. 2009,
19, 5426.
21. Hultsch, C.; Schottelius, M.; Auernheimer, J.; Alke, A.; Wester, H.-J. Eur. J. Nucl.
Med. Mol. Imaging 2009, 36, 1469.
22. Carberry, P.; Lieberman, B. P.; Ploessl, K.; Choi, S. R.; Haase, D. N.; Kung, H. F.
Bioconjug. Chem. 2011, 22, 642.
23. Choi, S. R.; Golding, G.; Zhuang, Z.; Zhang, W.; Lim, N.; Hefti, F.; Benedum, T. E.;
Kilbourn, M. R.; Skovronsky, D.; Kung, H. F. J. Nucl. Med. 2009, 50, 1887.
24. Lin, K.-J.; Hsu, W.-C.; Hsiao, I.-T.; Wey, S.-P.; Jin, L.-W.; Skovronsky, D.; Wai, Y.-
Y.; Chang, H.-P.; Lo, C.-W.; Yao, C. H.; Yen, T.-C.; Kung, M.-P. Nucl. Med. Biol.
2010, 37, 497.
25. Adams, J. P. J. Chem. Soc., Perkin Trans. 2000, 125.
26. Singh, Y.; Edupuganti, O. P.; Villien, M.; Defrancq, E.; Dumy, P. C.R. Chim. 2005,
8, 789.
27. Overoye-Chan, K.; Koerner, S.; Looby, R. J.; Kolodziej, A. F.; Zech, S. G.; Deng, Q.;
Chasse, J. M.; McMurry, T. J.; Caravan, P. J. Am. Chem. Soc. 2008, 130, 6025.
28. Zoppellaro, G.; Ivanova, A.; Enkelmann, V.; Geies, A.; Baumgarten, M.
Polyhedron 2003, 22, 2099.
11. McBride, W. J.; Sharkey, R. M.; Karacay, H.; D’Souza, C. A.; Rossi, E. A.;
Laverman, P.; Chang, C.-H.; Boerman, O. C.; Goldenberg, D. M. J. Nucl. Med.
2009, 50, 991.
12. Wescott, C. R.; Nair, S. A.; Kolodziej, A.; Beltzer, J. P. Application: WO; WO
Patent 2000-US20612; 2001009188; 2001, 114