J. A. H. Inkster et al.
approximately 0.9. It should be noted that individual [18F]-
fluorinated peptide degradation products cannot be distin-
guished with this technique. Thus, the observed radioactivity
present at 0.8–1.0 Rf after 15 min in serum [43(Æ4)% (n=3
traces) and 35(Æ1)% (n=3 traces) in two separate experi-
ments] represents only an estimation of the percent remain-
ing of sulfonyl [18F]fluoride-bearing species relative to the
total radioactivity in the serum solution.
the sample was diluted with acetonitrile and let stand 15 min, then a por-
tion (75 mL) was injected into HPLC for analysis. HPLC parameters used
were as follows: compound 4, program 1a. Compounds 1 and 3, pro-
gram 1b. Compounds 2 and 9, program 1c (see the Supporting Informa-
tion). An estimation of percent stability was calculated as follows: ab-
sorbance units of the peak of interest/(total absorbanceÀabsorbance
units of a blank sample)ꢃ100. This approach assumes that the difference
in extinction coefficients between a sulfonyl fluoride and its correspond-
ing sulfonic acid is negligible. The experiment was performed three times
for each compound.
Preparation of sulfonyl [18F]fluorides for radio-TLC analysis; general
procedure: No-carrier-added [18F]FÀ was produced by 13 MeV proton
bombardment of [18O]H2O [18O(p,n)18F reaction]. Typical production was
ACTHNUTRGNEUNG
Summary
1.5–2.6 GBq of [18F]FÀ at end of bombardment for a 10 mA, 5 min irradia-
tion. [18F]Fluoride in [18O]H2O was immobilised on an anion exchange
18F trap-and-release column (ORTG, Inc), then eluted into a 5 mL coni-
cal vial with a solution of caesium carbonate (10 mgmLÀ1, 400 mL). From
this bulk solution, smaller aliquots were removed for further use. An ali-
quot (100 or 200 mL) of [18F]FÀ in aqueous Cs2CO3 was pipetted into a re-
action vessel containing sulfonyl chloride (5–8) or sulfonyl chloride in or-
ganic solvent (100 mL; Table 1). Reaction volume was 0.2 mL and precur-
sor concentration was 30 mm. The reaction was shaken and let stand for
15 min, then the reaction mixture was spotted on 3 or 4 TLC plates. The
plates were 20 cm long, with the origin at 2 cm. Pyridine (80 mL) was
added and the mixture was shaken and let stand for another 15 min
before TLC sampling in the same fashion. The TLC eluent was ethyl ace-
tate. Alternately, pyridine (80 mL) was added immediately after addition
of [18F]FÀ and the reaction sampled after 15 min (Table 2).
Procedure for the purification of [18F]2: An aliquot (100 mL) of [18F]FÀ in
aqueous Cs2CO3 (10 mgmLÀ1) was pipetted into a reaction vessel con-
taining sulfonyl chloride 6 in tBuOH (100 mL). Immediately afterward,
pyridine (80 mL) was added and the mixture was vortexed thoroughly
and let stand for 15 min. The reaction solution was transferred into an
open syringe containing water (20 mL) and immobilised on a Sep-pakꢁ
tC18 light column. The column (activated previously with 2 mL EtOH
and 6 mL water) was washed with water (5 mL), and dried with air
(15 mL). Product benzenesulfonyl [18F]fluoride ([18F]2) was eluted from
the column with either DMSO (300 mL) for further bioconjugation ex-
periments or acetonitrile (1 mL) for determination of specific activity by
HPLC (program 4; see the Supporting Information).
Four 18F labelled arylsulfonyl fluorides bearing 4-formyl-, 3-
formyl-, 4-maleimido- and 4-oxylalkynl moieties were radio-
synthesised in high yields in equal volumes of basic cyclo-
tron target [18O]H2O and organic co-solvent at room tem-
perature. Preparation of anhydrous K2.2.2./KACTHNUTRGNEUNG
[18F]F complex
was not required. In most cases, sulfonyl chloride precursor
could be selectively converted upon addition of pyridine to
a reactive N-sulfonylpyridinium chloride intermediate. In
addition, we synthesised a N-sulfonyl-DMAP chloride salt
(14) and used it to prepare both 19F- and 18F-labelled 4-
(prop-2-ynyloxy)benzenesulfonyl fluoride (4) in excellent
chemical and radiochemical yields.
The in situ degradation of benzenesulfonyl chloride pre-
cursor in the presence of pyridine permitted the efficient
preparative synthesis of 3-formyl-2,4,6-trimethylbenzenesul-
fonyl [18F]fluoride ([18F]2) without the need for time-con-
suming HPLC purification of the prosthetic. Compound
[18F]2 was further coupled to an oxyamino-bearing analogue
of bombesin (BBN-OX-MESIT-SO2ACTHNUTRGNEUNG
[18F]F) via oxime link-
age as proof-of-principle. The overall radiosynthesis proto-
col can be completed in less than 110 min. The sulfonyl fluo-
rinated peptide analogue was found to be stable in DMSO
(10%) in PBS over 2 h at physiological temperature and
pH. Under similar conditions in mouse serum, however,
Radiobioconjugate synthesis of BBN-OX-MESIT-SO2ACTHNUTRGNEUNG
[18F]F: Purified
[18F]2 in DMSO (300 mL) was added to BBN-ONH2 peptide (0.5 mg) in
a microcentrifuge tube and AcOH (5%, 100 mL) was added. The reaction
mixture was vortexed, centrifuged briefly, and placed in a bed of heated
beads (378C) for 30 min. The 18F-labelled peptide was diluted with water
(600 mL) and purified by reverse phase HPLC (program 2b; see the Sup-
porting Information). The collected portion was diluted to 20 mL with
water and trapped on Sep-pakꢁ tC18 light column (activated previously
with 2 mL EtOH and 6 mL water). The column was washed with water
BBN-OX-MESIT-SO2ACTHNUTRGNEUNG
[18F]F showed signs of defluorination.
An accurate estimation of the rate of sulfonyl [18F]fluoride
hydrolysis was complicated by the fact that enzymatic modi-
fication of the 18F-peptide and complexation with serum
protein also occurs. The interrelation between these three
processes remains to be determined, but further insights
might be gleaned through the labelling of alternative target-
ing vectors.
(5 mL) and dried with air (10 mL). BBN-OX-MESIT-SO2ACTHNUTRGNEUNG
[18F]F was usu-
ally eluted from the column with EtOH (300 mL) and diluted with 0.9%
saline solution (2.7 mL). However, for serum stability experiments, the
18F-peptide was eluted from the column with DMSO (400 mL).
Serum stability study: BBN-OX-MESIT-SO2ACTHNUTRGNEUNG
[18F]F in DMSO (34.4–
Experimental Section
44.4 MBq) was diluted 1:9 with fresh mouse serum and heated to 378C.
As control, a second portion of 18F-peptide was treated in an identical
fashion with PBS (150 mm, pH 7.2) in place of mouse serum. The samples
had aliquots (150–500 mL) removed after 15, 60 and 120 min. The aliquots
were quenched with equal amounts of MeCN, chilled at 48C for 15 min,
centrifuged for 3 min (15668g), then the supernate was removed and
counted. After being spotted on three separate silica gel plates (iTLC-S,
Gelman Sciences; 20 cm), the remainder of the 18F-peptide was assayed
by HPLC (program 2b; see the Supporting Information). After drying,
radio-TLC was performed by using 1:1 MeOH/MES buffered saline
(100 mm, pH 4.7) as mobile phase.
Synthesis details and reaction schemes related to the preparation and
characterisation of non-radioactive small molecules 1–4, 6–9, 11–14 and
16, along with peptides BBN-ONH2 and BBN-OX-MESIT-SO2F, can be
found in the Supporting Information.
Assessing hydrolytic stability of small molecule standards 1–4 and 9 in
buffered solution; general procedure: Arylsulfonyl fluoride standards in
MeOH (1, 3 and 4) or diethyl ether (2, 9) were prepared (9.8 mm) and ali-
quots (50 mL) were removed and concentrated in microcentrifuge tubes
over a stream of helium. Each aliquot was incubated in PBS (150 mm,
pH 7.2, 75 mL) at 378C for 15, 70 or 150 min. After the appointed time,
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