2-Fluoropyridine prosthetic compounds for the 18F labeling of bombesin analogues

Article abstract of DOI:10.1016/j.bmcl.2013.04.060

Acetylene-bearing 2-[¹⁸F]fluoropyridines [¹⁸F]FPy5yne and PEG-[¹⁸F]FPyKYNE were prepared via efficient nucleophilic heteroaromatic [¹⁸F]fluorination of their corresponding 2-trimethylammoniumpyrdinyl precursors. The prosthetic groups were conjugated to azide- and PEG₃-modified bombesin(6-14) analogues via copper-catalyzed azide-alkyne cycloaddition couplings to yield mono- and di-mini-PEGylated ligands for PET imaging of the gastrin- releasing peptide receptor. The PEG₃- and PEG₂/PEG₃-bearing ¹⁸F peptides showed decreased lipophilicity relative to an analogous non-mini-PEGylated ¹⁸F peptide. Assessment of water-soluble peptide pharmacokinetics and tumour-targeting capabilities in a mouse model of prostate cancer is currently underway.

Full text of DOI:10.1016/j.bmcl.2013.04.060

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3920–3926
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Fluoropyridine prosthetic compounds for the 18F labeling
of bombesin analogues
c
d
d
c
James Inkster a,b, , Kuo-Shyan Lin , Samia Ait-Mohand , Simon Gosselin , François Bénard ,
⇑
Brigitte Guérin d, Maral Pourghiasian c, Thomas Ruth a,c, Paul Schaffer a, Tim Storr b
a TRIUMF, Nuclear Medicine Division, 4004 Wesbrook Mall, Vancouver, Canada BC V6T 2A3
b Simon Fraser University, Dept. of Chemistry, 8888 University Drive, Burnaby, Canada BC V5A 1S6
c BC Cancer Agency, Dept. of Molecular Oncology, 675 West 10th Avenue, Vancouver, Canada BC V5Z 1L3
d Université de Sherbrooke, Department of Nuclear Medicine and Radiobiology, 3001 12e Avenue Nord, Sherbrooke, Canada QC J1H 5N4
a r t i c l e i n f o
a b s t r a c t
Acetylene-bearing 2-[18F]fluoropyridines [18F]FPy5yne and PEG-[18F]FPyKYNE were prepared via efficient
nucleophilic heteroaromatic [18F]fluorination of their corresponding 2-trimethylammoniumpyrdinyl pre-
cursors. The prosthetic groups were conjugated to azide- and PEG3-modified bombesin(6–14) analogues
via copper-catalyzed azide–alkyne cycloaddition couplings to yield mono- and di-mini-PEGylated ligands
for PET imaging of the gastrin- releasing peptide receptor. The PEG3- and PEG2/PEG3-bearing 18F peptides
showed decreased lipophilicity relative to an analogous non-mini-PEGylated 18F peptide. Assessment of
water-soluble peptide pharmacokinetics and tumour-targeting capabilities in a mouse model of prostate
cancer is currently underway.
Article history:
Received 6 March 2013
Revised 12 April 2013
Accepted 22 April 2013
Available online 30 April 2013
Keywords:
Bombesin
Positron emission tomography
Fluorine-18
Ó 2013 Elsevier Ltd. All rights reserved.
CuAAC chemistry
Neuropeptides
Positron emission tomography (PET) is a nuclear diagnostic
technique in which radiolabeled probes are used to non-invasively
image disease-associated molecular targets in vivo. 18F (t1/
2 = 109.8 min) is widely considered the optimal radioisotope for
PET;1 however, the challenges associated with the synthesis of
18F tracers are numerous, particularly when the targeting agent
is a sensitive, functionally complex biomolecule. The basic, anhy-
drous, high temperature reaction conditions typically required to
label with [18F]FÀ usually necessitates the use of a small molecule
prosthetic group, which is radio-synthesized prior to bioconjuga-
tion (i.e., pre-labeling). We previously described the 18F-labeling
of model azide-modified peptide2 and oligonucleotide3 sequences
with prosthetic [18F]FPy5yne ([18F]-1, Fig. 1) by way of an efficient
CuI-catalyzed azide–alkyne cycloaddition (CuAAC)4,5 reaction. This
chemoselective variant of the Huisgen 1,3-dipolar cycloaddition
reaction has become synonymous with the ‘click chemistry’ con-
cept,6 owing to its facility, regioselectivity, and tolerance of water.
Marik and Sutcliffe were the first to utilize CuAAC chemistry for
the preparation of 18F peptides.7 Since then, a number of [18F]fluo-
roaromatic8,9 and [18F]fluoroaliphatic10–14 prosthetic groups bear-
ing azide or acetylene moieties have been developed for this
purpose. The primary technical advantage of [19F]-1- and closely
related analogue [18F]FPyKYNE15 ([18F]-2)- relative to many other
‘click’-able 18F prosthetic groups lies in the outstanding capacity
of the 2-substituted pyridine system to incorporate [18F]FÀ by
way of nucleophilic heteroaromatic substitution.16,17 Furthermore,
[
18F]-1 and [18F]-2 can be prepared in a single radiochemical step
and, like other ortho-[18F]fluoropyridines,18 are likely resistant to
in vivo defluorination.
Bombesin (BBN)19 is an amphibious 14- amino acid analogue of
the gastrin-releasing peptide (GRP). BBN shares a homologous C
terminus with GRP [Trp-Ala-Val-Gly-His-Leu-Met(NH2)], and the
two peptides display similar biologic function.20 As such, bombesin
and bombesin fragments show high affinity for the gastrin-releas-
ing peptide receptor (GRPr).4 GRPr (otherwise known as BB2r) is
one of four receptor subtypes which make up the bombesin recep-
tor superfamily (BB1r–BB4r)21 and is distributed predominantly
throughout the mammalian brain and gut. Apart from a host of
normal physiological functions, GRP acts as a mitogen, morphogen,
and proangiogenic factor of receptive tissues,22 and as such GRP
receptors are found overexpressed in a variety of cancer types.23
In particular, Reubi and co-workers found GRPr expressed in 65%
of a total of 77 excised breast tumors24 as well as 100% (30:30)
of a population of invasive prostatic neoplasms.25
Early attempts at exploiting the BBN/GRPr ligand/receptor pair
for molecular SPECT imaging utilized
[
125I]Tyr-modified BBN
analogues.26,27
A
BBN(7–13) bioconjugate labeled with an
⇑
Corresponding author. Tel.: +1 604 222 1047x6544; fax: +1 604 222 1074.
E-mail address: james_inkster@alumni.sfu.ca (J. Inkster).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.060

J. Inkster et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3920–3926
3921
The following work describes the synthesis of [18F]-1- labeled
BBN analogues with the potential to be used for the detection
and assessment of GRPr-expressing lesions with PET. In addition,
straightforward protocols for the radiosynthesis and CuAAC conju-
gation of a hydrosoluble derivative of [18F]-1 and [18F]-2 (PEG-
[
18F]FPyKYNE; [18F]-3)52 are described here in detail for the first
time. These methods will be of general interest to those seeking
to 18F-label azide-modified biomolecules.
The potential GRPr-targeting vectors utilized were based on
BBN(6–14) [Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met(NH2)] (Fig. 2).
Truncation at the N-terminus of native bombesin yields a less po-
tent peptide than the native form, but additional modifications can
restore binding affinity for GRPr.53 For this work, the following
Figure 1. Acetylene-bearing 2-[18F]fluoropyridines discussed in this work. F = 19
F
or 18F.
alterations relative to bombesin were introduced: D ,
-Tyr6, bAla11
125I-m-iodobenzoate functionality was introduced with increased
stability and improved uptake into ovarian tumors relative to
Thi13 and Nle14. It is assumed that these changes also serve to en-
hance the stability of the peptide in vivo. We prepared this se-
quence on a continuous flow peptide synthesizer using standard
Fmoc chemistry (see Supplementary data). An N3–CH2C(O)-residue
was introduced to the terminus for CuAAC conjugation with acet-
ylene-bearing pendant groups (N3-BBN; Fig. 2). 19F standard 1 and
the radiosynthetic precursor of [18F]-1 (4; see Scheme 2) were pre-
pared via separate syntheses as reported previously.2 The coupling
[
125I]Tyr4-bombesin.28 A variety of BBN derivatives have been la-
beled with 99mTc for SPECT,29–34 with attempted optimizations
focusing on the diminution of the hepatobiliary clearance that is
common to 99mTc-labeled peptides. For PET, a sizable number of
64Cu- labeled BBN analogues have been introduced. Most deriva-
tives reported thus far have employed DOTA as the chelating
agent,35–38 although the observed decupration of this complex
in vivo has prompted the investigation of alternative chelates.39,40
Recently, the synthesis and pre-clinical evaluation of some 64Cu-
NOTA-bearing BBN antagonist ligands was described.41 In addition,
DOTA-modified [b-Ala11, Thi13, Nle14]BBN(6–14) peptide has been
labeled with 68Ga (68Ga-BZH3)42 and evaluated for the delineation
of gastrointestinal stromal tumors in humans.43 Of interest to this
work (vide infra), the chelator was tethered to the targeting agent
by way of a PEG2 chain.
of N3-BBN and
1 under straightforward CuAAC conditions
[Cu(CH3CN)4PF6, TBTA, DMSO, 1.5–2 h] yielded 19F peptide stan-
dard F-ALK-BBN (see Supplementary data).
We predicted that radioactive [18F]F-ALK-BBN might exhibit
unfavourable pharmacokinetic behaviour in vivo due to its high
lipophilicity. Polyethylene glycol (PEG) spacers can serve as a
means to enhance the hydrophilicity of peptide-based molecular
imaging agents which might otherwise undergo undesirable
hepatobiliary clearance as a result of chemical modification (e.g.,
truncation, amino acid replacement, and/or introduction of pen-
dant groups). Thus alternative water-soluble 18F analogues of
BBN(6–14) were envisioned in which mini-PEG moieties could be
introduced both through further modification of the N3 peptide
precursor, as well as through conjugation to a mini-PEGylated 18F
prosthetic. The latter strategy offers an opportunity to introduce
two beneficial characteristics at the same time (i.e., 18F and hydro-
philicity). This will be of general utility when a potential targeting
agent cannot be easily modified- or exhibits diminished bioactiv-
ity- with two pendant groups. To this end, we explored the use
of a ‘2nd generation’ [18F]FPy5yne derivative (PEG-[18F]FPyKYNE;
An 18F BBN derivative was first prepared through the use of
acylating
prosthetic
N-succinimidyl
4-[18F]fluorobenzoate
([18F]SFB); tumour visualization was achieved in small animal
PET experiments, but uptake was moderate and the clearance oc-
curred primarily through the undesirable hepatobiliary route.44
Subsequently, BBN–RGD heterodimers were labeled with [18F]SFB
and used to prove the viability of multi-peptide receptor PET imag-
ing.45 Schirrmacher et al. prepared a formylated silicon-fluoride
acceptor (SiFA)-based prosthetic [p-(di-tert-butyl[18F]fluorosi-
lyl)benzaldehyde] and coupled it to a N-aminooxy-derivatized
BBN analogue.46 Finally, a dibenzocyclooctyne moiety was conju-
gated to [Lys3]BBN to serve as reactive dipolarophile for the cop-
[
18F]-3; Fig. 1), which bears a PEG2 spacer between its [18F]fluori-
per-free CuAAC coupling of
a
series of azide- bearing 18F
nated and conjugating functionalities. An abstract reporting the
radiosynthesis of [18F]-3 was reported in 2009.52 The authors 18F-
labeled 2-bromo and 2-nitro precursors in radiochemical yields
of 650% by radio-TLC (165 °C, 5 min). The TMA moiety is the pre-
mier leaving group for the K[18F]/K2.2.2. fluorination of homo- and
hetero- aromatic systems, both in terms of [18F]FÀ incorporation16
and ease of precursor removal.54 Therefore, we agreed with the ori-
ginal inventors of [18F]-3 that TMA- bearing precursor 5 (Scheme 1)
might offer a superior route to this 18F prosthesis.
The synthesis of precursor 5 and non-radioactive PEG-FPyKYNE
(3) is shown in Scheme 1. Mitsunobu coupling of 2-fluoro-3-
hydroxypyridine55 (6) and alcohol 7 was used to afford 19F stan-
dard 3. Then, a nucleophilic 2-F for 2-NMe2 substitution under
aqueous conditions was employed to furnish 2-dimethylaminopyr-
idine 8 from 3. This approach obviated the need to synthesize 2-
dimethylamino-3-hydroxypyridine, which we have yet to prepare
in yields higher than 52%.2,15 Finally, the mixing of 8 with one
equivalent of methyl triflate yielded PEG-NMe3-KYNE (5) precursor
salt.
prosthetics (including N3-(CH2)4-PEG3-[18F]F).47
Apart from pre-labeling approaches, the relatively robust nat-
ure of many modified BBN fragments has permitted the exploita-
tion of direct 18F-labeling technologies. Trimethylammonium
triflate (TMA)-bearing benzonitrile pendant moieties have been
used to 18F-label BBN-based precursors. Additional lysine48 and
L-
cysteric acid49,50 residues were introduced in an attempt to dimin-
ish peptide lipophilicity and thus favorably modulate tracer biodis-
tribution. In addition, BBN derivatives modified with SiFA (di-tert-
butylarylsilyl) moieties were directly 18F-labeled in acidic DMSO
solutions.51 The resulting di-tert-butyl[18F]fluorosilyl group is sta-
ble in PBS and plasma over 2 h. An 18F BBN analogue of this type
(bearing an isolated lysine residue to improve water-solubility)
showed poor and non-specific uptake into PC3 tumor xenografts
but high and specific uptake into GPRr-rich murine pancreatic tis-
sue. Notably, the log D value of the peptide was high (1.3 0.1,
n = 5), which suggests that the ligand was still rather lipophilic.
The primary advantage of these post-labeling technologies lies in
their remarkable radiosynthetic simplicity. However, it should be
noted that both employ traditional K2.2.2./M[18F]F/M2CO3 condi-
tions at elevated temperatures, and thus do not represent general
approaches for the direct 18F-labeling of biological molecules.
PEG3-bearing N3-BBN derivative N3-BBN-PEG was prepared
by way of similar solid-phase synthetic techniques (see Supple-
mentary data). In addition to the azide functionality, a pendant
15-amino-4,7,10,13-tetraoxapentadecaamide moiety was affixed

3922
J. Inkster et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3920–3926
Figure 2. Peptides referenced in this work. F = 19F and 18F.
to an extraneous Lys residue using EDC-promoted acylation chem-
istry. Subsequent CuAAC coupling of the free peptide with 1 and 3
yielded non-radioactive peptide standards F-ALK-BBN-PEG and F-
PEG-BBN-PEG respectively (see Supplementary data). Only a small
difference in retention time was observed between F-PEG-BBN-
PEG and N3-BBN-PEG upon semi-preparative HPLC separation
(DRt = 0.3 min). Nevertheless, F-PEG-BBN-PEG could be reliably
obtained in chemically pure form after careful HPLC purification
as determined by HPLC and MALDI-TOF.
Average decay corrected (DC) yield of [18F]-1 was 47 6% (n = 5)
from start-of-synthesis.
The reaction conditions used to prepare PEG-[18F]FPyKYNE
([18F]-3) were identical to those used for synthesis of [18F]-1
(Scheme 4). Incorporation of [18F]FÀ during three separate experi-
ments was achieved in yields of 80 10% (3 traces from 3 experi-
ments, n = 9 total), as determined by the gamma counting of
fractionated radioTLC plates. HPLC purification of [18F]-3 was
straightforward and collected yields were satisfactory and repro-
ducible (45–64% DC, n = 4). However, in contrast to [18F]-1, which
could be efficiently extracted from HPLC eluent using tC18 SPE sor-
bent after dilution with 50 mL of water, [18F]-3 was not well-suited
for this approach. The decay- corrected trapping efficiency was 27–
42% (n = 4 experiments) when a small (‘light’) tC18 SPE cartridge
was used.a Not surprisingly, final preparative radiochemical yields
of [18F]-3 were only 16 2% DC (n = 4). However, the efficiency of
the extraction step was significantly improved by employing two
full-size (‘Plus’) tC18 SPE columns. The radio-compound was eluted
from the SPE cartridge with MeOH, then the solvent was evaporated
(90 °C) under a stream of He (73 2% efficiency, DC, n = 3). It is
worth noting that this approach would likely not be possible for
the radiosynthesis of [18F]-1 owing to its low boiling point (69–
70 °C).2 Solvent removal by evaporation resulted in longer protocol
times, but higher collected yields (39 9% DC, n = 4) versus tC18
‘light’ extraction. An attempt to concentrate the solvent mixture at
110 °C resulted in significantly more product loss of [18F]-3 by
Radiosyntheses of [18F]FPy5yne ([18F]-1) followed a protocol
that was introduced2 and later improved3 by us previously. Effi-
cient incorporation of anhydrous [18F]FÀ was achieved by way of
a nucleophilic heteroaromatic substitution reaction from a 2-
TMA triflate precursor (4) in DMSO (0.5 mL), in the presence of
K2CO3 and K2.2.2. phase transfer catalyst at 110 °C (Scheme 2). We
have now further optimized the isolation of [18F]-1. First, it was
established that extraction of [18F]-1 from DMSO reaction solvent
(0.5 mL) was not required prior to HPLC purification. Instead, the
reaction mixture was diluted with an equal amount of water and
injected directly onto a semi-preparative HPLC column. Under
these conditions, successful HPLC separations could only be
achieved if the reaction mixture was transferred from the original
vessel to another one before dilution with H2O. We hypothesize
that this step serves to remove any basic material (K2.2.2./K2CO3/
[
18/19F]FÀ) affixed to the reaction vessel, which would otherwise
be completely solubilized if placed in contact with water. Second,
after HPLC purification and immobilization of [18F]-1 onto a tC18
solid-phase extraction (SPE) cartridge (Sep-pakÒ, Waters), the
radio-compound was eluted directly into a microcentrifuge tube
containing N3-peptide precursor using bioconjugation solvent
(DMF or DMSO). In this fashion, losses associated with the volatil-
ization of [18F]-1 during evaporation of HPLC eluent were obviated.
a
Trapping the 18F prosthetic molecule on a ‘light’ SPE column allowed for elution
with a small volume of organic solvent (0.3 mL). In this way, direct elution into the
bioconjugate reaction mixture was possible, without the need for
concentration step.
a secondary

J. Inkster et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3920–3926
3923
peptide) was accompanied by a marked increase in radiochemical
yield as observed by HPLC, such that complete consumption of
[
18F]-1 starting material could be achieved under 30 min at room
temperature. Second, the removal of the 18F-labeled peptide from
HPLC eluent was achieved by way of tC18 ‘light’ trapping, rather
than evaporation of MeCN/0.1% TFA at 80 °C. Apart from improved
radiosynthetic efficiency, the obviation of this evaporative step
contributes to the general utility of this procedure because many
biomolecules are sensitive to excessive heating. Third, as [18F]F-
ALK-BBN-PEG is soluble in most aqueous mixtures, this radio-
tracer could be formulated in 10% EtOH in isotonic saline, which
is a well-established solvent system for peptide-based PET ligands.
Average collected yield after bioconjugate synthesis was 31 19%
DC (n = 11). The apparent specific activity of [18F]F-ALK-BBN-
PEG in four separate experiments was found to be 42 25 GBq/
lmol (1.2 0.7 Ci/
l
mol) at end-of-synthesis.b
Based on our observations during the HPLC purification of non-
radioactive F-PEG-BBN-PEG, it was expected that the clean separa-
tion of N3-BBN-PEG and [18F]F-PEG-BBN-PEG would be challeng-
ing. In a bid to minimize chemical and radiochemical impurities,
straightforward
CuAAC
conditions
were
employed
[Cu(CH3CN)4PF6/TBTA, RT, DMSO, 30 min; Scheme 4]. Under these
conditions, the radio-bioconjugation did not proceed to comple-
tion. Nevertheless, the collected yield from start of bioconjugation
was 25 13% DC (n = 4), and tracer was obtained in amounts
amendable for in vivo study.c The apparent specific activity of
[
18F]F-PEG-BBN-PEG at end-of-synthesis was found to be
78 38 GBq/lmol (2.1 1 Ci/lmol; n = 7).
During HPLC purification of [18F]F-PEG-BBN-PEG, a radio-
impurity was consistently observed that was notable in terms of
amount (7–17% of total radioactivity) and polarity (Fig. 3, Rt
=
12.3 min). In an effort to establish the nature of this chemical spe-
cies, small molecule prosthetic group [18F]-3 was synthesized and
incubated in a similar CuAAC reaction mixture, but without N3
peptide (Fig. 4). The radio-impurity was observed in this case as
well, although the percent yield by HPLC was smaller than ex-
pected (4%). It is hypothesized that this radio-peak represents
Cu-chelated [18F]-3. The variability in observed yield during 18F
peptide preparations may be the result of small but significant dif-
ferences in free copper in each reaction mixture. Alternatively, the
specific activity of [18F]-3 could be a factor, as the radiochemical
yield of any association between Cu and 18F small molecule should
be negatively correlated with the amount of non-radioactive chela-
tor present. Furthermore, a dependence on high specific activity
conditions does much to explain why this species does not form
in detectible quantities during non-radioactive syntheses of F-
PEG-BBN-PEG.
Scheme 1. Synthesis of the radiosynthetic precursor (5; PEG-NMe3-KYNE) to PEG-
18F]FPyKYNE and its 19F standard (3).
[
evaporation (41% efficiency, DC). Measured apparent specific activity
at end-of-synthesis was 180 9 GBq/ mol (4.9 0.3 Ci/ mol; n = 3).
l
l
Earlier syntheses of 18F-ALK-BBN focused on shortening and
simplifying the general protocol for the preparation of [18F]-1- la-
beled peptides as reported in Inkster et al.2 In the aforementioned
report, HPLC-purified 18F peptide was concentrated by azeotropic
distillation of the collected HPLC eluent (4–8 mL). However, this
method typically takes between 25 and 40 min and does not assure
complete removal of trifluoroacetic acid (TFA) from the final for-
mulation. Thus, this drydown step was avoided in favor of immo-
bilization of the 18F peptide on tC18 sorbent (Scheme 2). Ideally
the tracer could then be eluted from the SPE column with a solvent
amendable for pre-clinical use. The choice of final formulation de-
manded significant attention because [18F]F-ALK-BBN is only spar-
ingly soluble in aqueous solutions. Benzoic acid (10%) in PBS
resulted in acceptable levels of solvation (81% DC), but a signal
associated with BzOH co-eluted with [18F]F-ALK-BBN, thus convo-
luting HPLC assay of the final formulation. A summary of solvent
systems tested can be found in the Supplementary data. In the
end, a 9:1 mixture of (2-hydroxypropyl)-b-cyclodextrin56,57 solu-
tion (10% HPbCD in PBS) and DMSO was employed (88% solvated,
DC). The average preparative bioconjugate yield of [18F]F-ALK-
BBN was 34 17% DC (n = 8).
The lipophilicity of [18F]F-ALK-BBN was assessed relative to its
mono- and di-mini-PEGylated derivatives ([18F]F-ALK-BBN-PEG
and [18F]F-PEG-BBN-PEG) by measuring their coefficients of distri-
bution into equal parts octanol and sodium phosphate buffer
(20 mM, pH 7.4). As expected, logD[7.4] values decreased with an
increase in ethylene oxide groups in the targeting vectors
(1.40 0.01, À0.92 0.03 and À1.12 0.01 respectively, n = 4).
The relatively large logD[7.4] found for
[
18F]F-ALK-BBN
(1.40 0.01) is consistent with the qualitative observation that this
modified peptide is not soluble in purely aqueous solutions. The
difference in logD values between
[
[
18F]F-ALK-BBN-PEG and
18F]F-PEG-BBN-PEG (0.20) is similar to the observed shift in logD
[
18F]F-ALK-BBN-PEG was synthesized in a manner similar to
b
The specific activities were determined to be 14, 39, 43, and 74 GBq/lmol. The
the synthesis of [18F]F-ALK-BBN (Scheme 3). However, three
important improvements were introduced. First, inclusion of
zsodium ascorbate to the reaction mixture (15 equiv. relative to
relationship between these values and murine PET results (PC3 lesions) will be
detailed in a later manuscript.
c
A maximum of 19.9 mCi [
18F]F-PEG-BBN-PEG was prepared starting from a
single batch of [18F]FÀ
.

3924
J. Inkster et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3920–3926
Scheme 2. Radiosynthesis of [18F]-1 and [18F]F-ALK-BBN-PEG.
Scheme 3. Radiosynthesis of [18F]F-ALK-BBN-PEG. [18F]-1 was prepared as in Scheme 2.
Scheme 4. Radiosynthesis of [18F]-3 and [18F]F-PEG-BBN-PEG.
between two [18F]SFB-modified c(RGD)2 peptides prepared with
and without PEG2 tethering groups (0.18).58
evaporation. When compared with other pre-labeled 18F prosthe-
ses in use today, the simplicity of this approach stands out; indeed,
only two radiochemical transformations and two SPE concentra-
tion steps are required to obtain the final radio-bioconjugate. On
the other hand, the need for two HPLC purifications to obtain 18F
peptide of high specific activity remains a significant downside
to the use of [18F]FPy5yne. However, others in our group have
The straightforward preparation of [18F]-1 as described here is a
clear improvement on earlier protocols.2,3 In particular, it was
determined that [18F]-1 need not be extracted out of the reaction
solvent prior to HPLC purification, and that the removal of
HPLC eluent could be achieved by SPE extraction rather than

J. Inkster et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3920–3926
3925
Figure 3. Preparative HPLC of [18F]F-PEG-BBN-PEG synthesis. Top: radioactive detection (arbitrary units). Bottom: UV, 280 nm (absorbance units). Rt of N3-BBN-
PEG = 13.2 min. Rt of [18F]F-PEG-BBN-PEG = 13.8 min. Rt of [18F]-3 = 18.1 min. Note the radiochemical impurity at 12.3 min. HPLC System: 7 (see Supplementary data).
Figure 4. Radiotrace of purified [18F]-3 (Rt = 18.1 min) after incubation in Cu(CH3CN)4PF6/TBTA in DMSO (RT, 30 min). Note the radiochemical impurity at 12.4 min. HPLC
System: 7 (see Supplementary data).
recently established a protocol whereby [18F]-1 can be efficiently
separated from precursor 4 and other interfering species using only
C18 SPE methods.59 This is possible because unusually small
amounts of precursor are used during radiosyntheses (100 nmol).
It is the excellent capacity of 2-substituted pyridines to incorporate
We have committed to repeated syntheses of [18F]F-ALK-BBN,
18F]F-ALK-BBN-PEG, and [18F]F-PEG-BBN-PEG for the determina-
[
tion of certain key molecular imaging parameters of these radio-
peptides in prostate cancer-bearing mice.60,61 A complete sum-
mary of in vivo and ex vivo results will be reported in due course.
In addition, the radio-protocols outlined here make up a viable
general strategy for the [18F]fluorination/post-synthetic PEGylation
of other, less-established biological targeting vectors.
[
18F]FÀ that makes this approach viable.
Potential GRPr-based tumour-targeting agent [18F]F-ALK-BBN
was produced in preparative yields amendable for in vivo study
via CuAAC coupling to [18F]-1, but this peptide required a non-
standard solvent system for formulation (1:1 10% HPbCD in
PBS:DMSO). However, efforts to 18F- label mini-PEGylated precur-
sor peptide N3-BBN-PEG with [18F]-1 resulted in a water- soluble
tracer of enhanced hydrophilicity relative to [18F]F-ALK-BBN
([18F]F-ALK-BBN-PEG). The similarity of PEG-[18F]FPyKYNE to
Acknowledgments
The authors would like to thank the TRIUMF TR13 and BC Can-
cer Cyclotron Operations groups for producing the [18F]FÀ used in
this work. We would also like to thank Suzanne Perry (Michael
Smith Proteomics Unit, UBC) for the MALDI-TOF cited in the Sup-
plementary data section, as well as UBC Mass Spectrometry and
NMR Facilities for their contributions. This work was supported
by a Canadian Institutes of Health Research Operating Grant. T.S.
would like to thank NSERC for a Discovery Grant.
[
18F]FPy5yne ensured that the radiosynthesis of this mini-PEGylat-
ed derivative was straightforward, although a large amount of SPE
sorbent and an additional solvent evaporation step was required to
obtain CuAAC-ready [18F]-3 after HPLC purification and SPE. In this
regard, the reduced volatility of [18F]-3 relative to [18F]-1 proved
advantageous. The coupling of [18F]-3 and N3-BBN-PEG yielded
di-mini-PEGylated ligand [18F]F-PEG-BBN-PEG in radiochemical
yields and specific activities sufficient for further biological assay.
logD[7.4] measurements suggests that this peptide is significantly
more hydrophilic than [18F]F-ALK-BBN and slightly more hydro-
philic than [18F]F-ALK-BBN-PEG.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.04.
060.
The overall preparative yields of [18F]F-ALK-BBN-PEG [14 13%
DC (n = 4) from EOB] and [18F]F-PEG-BBN-PEG [10 7% DC (n = 4)
from EOB] compare reasonably well with yields obtained through
the direct labeling of GRPr ligands which bear SiFA precursors
[13 3% DC (n = 4)]51 and TMA–benzonitrile groups (ꢀ15% DC).50
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