C-terminal 18F-fluoroethylamidation exemplified on [Gly-OH9] oxytocin

Article abstract of DOI:10.1002/jlcr.547

The no-carrier-added (n.c.a.) ¹⁸F-fluoroethylamidation of the acid function of the protected nonapeptide Boc-Cys-Tyr(tBu)-Ile-Gln(Mtt)-Asn(Mtt)-Cys-Pro-Leu-Gly-OH forming the labelled peptide hormone derivative [Gly-(2-[¹⁸F]fluoroeth

Full text of DOI:10.1002/jlcr.547

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 217–229.
DOI: 10.1002/jlcr.547
Research Article
18
C-Terminal F-fluoroethylamidation
exemplified on [Gly-OH9] oxytociny
M. Jelinski, K. Hamacher* and H. H. Coenen
Institut fu.r Nuklearchemie, Forschungszentrum Ju.lich GmbH, D-52425 Ju.lich,
Germany
Summary
The no-carrier-added (n.c.a.) 18F-fluoroethylamidation of the acid function of
the protected nonapeptide Boc–Cys–Tyr(tBu)–Ile–Gln(Mtt)–Asn(Mtt)–Cys–
Pro–Leu–Gly–OH forming the labelled peptide hormone derivative [Gly-(2-
[18F]fluoroethyl)NH9]-oxytocin is described. The labelling conditions were
elaborated using a protected tripeptide, identical to the C-terminal sequence of
oxytocin. The prosthetic group n.c.a. 2-[18F]fluoroethylamine was synthesised via
cryptate mediated n.c.a. 18F-fluorination of N-Boc-2-(p-toluenesulfonyloxy)
ethylamine in DMSO (RCY: ca. 60%) and subsequent deprotection with a
radiochemical yield of 46 Æ 5%. [18F]Fluoroethylamine was reacted with
Z–Pro–Leu–Gly–OH in presence of the coupling reagent TBTU or with
activated esters of the model-tripeptide. The activated ester method as well as
the condensation in presence of TBTU yielded 590% of the 18F-fluoroethyl-
amidated tripeptide. TBTU-mediated condensation of n.c.a. 2-[18F]fluoro-
ethylamine with the C-terminal free acid group of protected oxytocin
gave the radiochemical yield of about 75%. Deprotection under acidic
conditions led to the formation of [Gly–(2-[18F]fluoroethyl)NH9]oxytocin
within 75 min with a radiochemical yield of about 30% as measured by
analytical HPLC. Copyright # 2002 John Wiley & Sons, Ltd.
Key Words: nucleophilic 18F-fluorination; 18F-prosthetic group; n.c.a. 2-
[18F]fluoroethylamine; 18F-fluoroethylamidation; oxytocin
.
*Correspondence to: K. Hamacher, Institut fur Nuklearchemie, Forschungszentrum Julich GmbH,
D-52425 Julich, Germany. E-mail: k.hamacher@fz-juelich.de
.
.
yIn parts, this is the diploma work of M. Jelinski
Copyright # 2002 John Wiley & Sons, Ltd.
Received 6 April 2001
Revised 15October 2001
Accepted 27 October 2001

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M. JELINSKI ET AL.
Introduction
Endogenous oxytocin acts as labourinducing and milk ejecting peptide
hormone and it is the most significant pharmaceutical in obstetrics.1 It
plays an important role in the sexual and social behaviour of both sexes.
Besides its responsibility for sexual receptivity, maternal behaviour and
care of the brood, it also influences the choosing of a sexual partner and,
in a general way, the development of social bindings.2
Ouraim was to develop an efficient labelling method of this hormone
by using a small prosthetic group and a labelling position outside the
pharmacophoric group. A future goal will be to study non-invasively
the pharmacokinetics and in vivo distribution of this hormone,
especially the possibility of its uptake in the brain.
It is known, that a derivatisation of the N-terminus of the peptide
results in a significant change of the biological activity3 and that the
cyclic ring system, generated by a disulfide bridge between the two
cysteine-residues, is essential for the biological activity (see Figure 1).4
Consequently a modification of the C-terminal amide function of
oxytocin by a small prosthetic group appeared as the only attractive
alternative for avoiding a major change of its biochemical behaviour.
Therefore, it was decided to substitute the amide function by a
[18F]fluoroethylamide group. N-methylation with [11C]methyl iodide
was not chosen due to the short half-life of carbon-11 and the resulting
limited time frame for pharmacokinetic studies. The radioiodination of
Figure 1. Formula of oxytocin
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FLUOROETHYLAMIDATION OF OXYTOCIN
219
the tyrosine-residue was avoided since [o-iodo-Tyr2]oxytocin is known
as an inhibitor of the oxytocin-receptor.4,5
N-Boc-2-[18F]fluoroethylamine has previously been synthesised by
Guhlke6 using N-Boc-2-bromoethylamine as precursor and by Gilissen
et al.7 using N-Boc-2-(p-toluenesulfonyloxy)ethylamine. Whereas Gilis-
sen reached 2% of 2-[18F]fluoroethylamine by deprotecting N-Boc-2-
[18F]fluoroethylamine and distilling the product, he achieved 39 Æ 6%
by producing the amine from N-(2-[18F]fluoroethyl)phthalimide.7
Tewson reported the 18F-fluoroethylamidation of 2-nitroimidazole
acetic acid via its tetrafluorophenyl ester with a radiochemical yield of
95%, whereas the reaction of the N-hydroxy succinyl ester of 2-
nitroimidazole acetic acid resulted only in low radiochemical yields.8
Unfortunately, no information was given about the radiochemical yield
of 2-[18F]fluoroethylamine.
In this work, the radiochemical yield of N-Boc-2-[18F]fluoroethyla-
mine via 18F-fluorination of N-Boc-2-(p-toluenesulfonyloxy)ethylamine
was optimised. Before labelling oxytocin, 18F-fluoroamidation was
performed using the model-tripeptide Z–Pro–Leu–Gly–OH with an
identical C-terminal amino acid sequence. In context with this
developmental work in the MBq range all radiochemical yields were
determined on an analytical scale.
Experimental
Materials
DMSO (dimethyl sulfoxide), DMF (N,N-dimethylformamide),
DMAP (4-dimethylaminopyridine), DMAA (N,N-dimethylacetamide)
p-nitrophenol, pentafluorophenol, TBTU (O-(benzotriazole-1-yl)-N,N,
N0,N0-tetramethyluronium tetrafluoroborate), BOP (benzotriazole-1-yl-
oxy-tris-(dimethylamino)-phosphonium-hexafluorophosphate), WSC
(1-ethyl-3-(30-dimethylaminopropyl)carbodiimide), HOBT (N-hydroxy-
benzotriazole) and TFA (trifluoroacetic acid) were purchased from
Fluka, N-hydroxysuccinimide, DCC (dicyclohexylcarbodiimide), N-
Boc-2-aminoethanol and 2-fluoroethylamine hydrochloride from Al-
drich. Acetonitrile, diethylether, dioxane, Kryptofix1 2.2.2. and
triethylamine were delivered by Merck. Z–Pro–Leu–Gly–OH
(Z: benzyloxy-carbonyl), Boc–Cys–Tyr(tBu)–Ile–Gln(Mtt)–Asn(Mtt)–
Cys–Pro–Leu–Gly–OH (Boc: tert.-butoxycarbonyl, tBu: tert.-butyl,
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M. JELINSKI ET AL.
Mtt: 4-methyltrityl) and Cys–Tyr–Ile–Gln–Asn–Cys–Pro–Leu–Gly–N–
(2-fluoroethyl)amide were obtained from Bachem (Heidelberg, Ger-
many).
Dioxane was refluxed over lithium aluminum hydride for 30 min and
distilled afterwards. Triethylamine was also purified by distillation.
Pentafluorophenol was dissolved in DMF and dried over sodium
sulfate. Thin layer chromatography was carried out on precoated plates
of silica gel 60 F 254 (Merck). 1H- and 19F-NMR spectra were recorded
on a Bruker Avance 200 apparatus at 200 MHz. Chemical shifts are
reported in ppm relative to TMS (d=0). Mass spectra were performed
with a Finnigan mass spectrometer using Atmospheric Pressure Chemical
Ionisation (APCI). IR spectra (KBr pellet) were recorded using a
Shimadzu (IR-440) spectrometer. Analytical HPLC was performed on
the following system: HPLC-pump (Merck, Hitachi 655A-11), UV/VIS-
detector (Merck, Hitachi 655A) with variable wavelength and a radio-
activity detector (EG&G Ortec, model 276). Melting points were
determined on a Gallenkamp apparatus and are reported uncorrected.
N-Boc-2-(p-toluenesulfonyloxy)ethylamine7
N-Boc-2-ethanolamine (1.10 g, 6.80 mmol) was reacted with p-toluene-
sulfonyl chloride (1.41 g, 7.40 mmol). After stirring for 6 h at 08C the
reaction mixture was kept in the refrigerator overnight before mixing
with 30 ml of ice-cold water. The aqueous phase was extracted with
dichloromethane, the combined organic layers were washed with 0.25 N
hydrochloric acid (2 Â 10 ml) and water(2 Â 10 ml) and dried over sodium
sulphate. The solvent was evaporated and the residue was purified by
column chromatography with hexane/diethylether (1 : 2 v/v) yielding
1.13 g (52.7%, Lit. 30.7%) of N-Boc-2-(p-toluenesulphonyloxy)ethyla-
mine.
mp: 658C; Lit. 63–658C
1H-NMR: s. Reference 7
IR (KBr, cmÀ1): n(N–H) 3410 (s); n(H–C arom.) 3055 (w);n(C¼O)
1712 (s); n(C¼C) 1597 (m); nas (-SO2O-) 1354 (s); n(CH3 Boc) 1267 (s);
n
sym(-SO2O-) 1177 (s)
Mass spectrum (m/z): 216 [M-Boc+H]+.
N-Boc-2-fluoroethylamine
Di-tert.-butyl dicarbonate (1.47 g, 6.78 mmol) was added to a solution
of 2-fluoroethylamine hydrochloride (0.5 g, 5.02 mmol) and triethyla-
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FLUOROETHYLAMIDATION OF OXYTOCIN
221
mine (1.53 g, 14.15 mmol) in DMF (20 ml). After stirring for 6 h at 608C
the reaction mixture was diluted with water (50 ml) and extracted with
diethylether(4 Â 20 ml). The combined organic layers were washed with
0.25 N hydrochloric acid (2 Â 15 ml) and water(2 Â 15 ml) and dried
over sodium sulphate. The solvent was evaporated and the residue
purified on silica gel with hexane/diethylether (1 : 1 v/v) to yield an oil,
which was kept in the refrigerator over night for crystallization (0.62 g,
76.0%, Lit. 77%).
mp=54–568C
1H-NMR: s. Reference 7
2
1
19F-NMR (CDCl3): dÀ224.89 (tt, 1F); J(1H/19F)=47 Hz, J(1H/19F)
=28 Hz
IR (KBr, cmÀ1): n(N–H) 3440 (s); n(C¼O) 1714 (s); n(C–F) 1368 (s);
n(CH3 Boc) 1250 (s)
Z–Pro–Leu–Gly–N–succinimidyl ester
N-Hydroxysuccinimide (90 mg, 0.72. mmol) was added to a solution
of Z–Pro–Leu–Gly–OH (300 mg, 0.72 mmol) in dioxane (4 ml).
While the solution was cooled with ice, dicyclohexylcarbodiimide
(155 mg, 0.79 mmol) was added. After15 min at 0 8C stirring was
continued at room temperature overnight. The dicyclohexylurea was
filtered off, and the solvent was removed under reduced pressure. The
colourless oil was crystallized from dichloromethane/petroleumether
(250 mg, 67.7%).
mp: 1848C
1H-NMR (CDCl3): d 0.93 (s, 6H), 1.60 (m, 2H), 2.20 (m, 4H), 2.85
(s, 4H, succinimidyl), 3.60 (m, 4H), 3.90 (m, 2H), 4.38 (m, 3H), 4.25
(m, 1H), 5.18 (s, 2H), 6.70 (m, 2H), 7.39 (s, 5H)
IR (KBr, cmÀ1): n (N–H) 3495 (m); nas(N–H) 3285 (s); n(C–H arom.)
3065 (w); n(C¼O, Ester) 1730 (s); n(C¼O, Pro) 1692 (s); n(C¼O, Gly)
1644 (s); n(C–O) 1425 (s); n(C–N) 1359 (s); nas(C–O–N) 1202 (s)
Mass spectrum (m/z): 517 [M+H]+.
Z–Pro–Leu–Gly–p–nitrophenyl ester
Dicyclohexylcarbodiimide (165 mg, 0.80. mmol) was added to a cooled
solution of Z–Pro–Leu–Gly–OH (300 mg, 0.72 mmol) and p-nitrophe-
nol (110 mg, 0.80 mmol) in tetrahydrofuran (4 ml). After cooling for
15 min at 08C stirring was continued at room temperature overnight.
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M. JELINSKI ET AL.
The suspension was filtered, and the solvent was removed under reduced
pressure. The product was crystallized from DMF/water (104 mg, 65%).
mp: 1648C
1H-NMR (CDCl3): d 0.93 (s, 6 H), 1.60 (m, 2H), 2.20 (m, 4H), 3.60
(m, 4H), 3.90 (m 2H), 4.38 (m, 3H), 4.25 (m, 1H), 5.18 (s, 2H), 6.70 (m,
2H), 7.37 (s, 7H), 8.30 (m, 2H);
IR (KBr, cmÀ1): nas(N–H) 3285 (s); n(C–H, arom.) 3070 (w); n(C¼O,
Ester) 1764 (m); n(C¼O) 1704 (s); n(C¼O 1643 (s, Gly); n(N–H) 1545 (s);
n(C–O) 1422 (s)
mass spectrum (m/z): 540 [M+H]+.
Z–Pro–Leu–Gly–pentafluorophenyl ester
Dicyclohexylcarbodiimide (0,1 g, 0.49 mmol) was added to an ice cold
solution of Z–Pro–Leu–Gly–OH (0.2 g, 0.48 mmol) and pentafluoro-
phenol (95 mg, 0.52 mmol) in 1 ml DMF, and the reaction mixture was
stirred for 1 h at room temperature. After filtration the solvent was
removed under reduced pressure. The product was crystallized from
hexane (243 mg, 87%).
mp=1388C
1H-NMR (CDCl3): d 0.93 (s, 6 H), 1.60 (m, 2H), 2.20 (m, 4H), 3.60
(m, 4H), 3.90 (m 2H), 4.38 (m, 3H), 4.25 (m, 1H), 5.18 (s, 2H), 6.70
(m, 2H), 7.38 (s, 5H)
19F-NMR (CDCl3): dÀ152.44 (m, 2F), À157.82 (m, 2F), À162.41
(m, 1F)
IR (KBr, cm-1): nas(N–H) 3290 (s); n(C–H, arom.) 3065 (w); n(C¼O,
Ester) 1796 (s); n(C¼O) 1706 (s); n(C¼O) 1639 (s, Gly); n(N–H) 1543 (s);
n(C–O) 1418 (s); n(C–F) 1311 (w)
mass spectrum (m/z): 586 [M+H]+.
Z–Pro–Leu–Gly–2–fluoroethylamide
Z–Pro–Leu–Gly–OH (300 mg, 0.72 mmol) and TBTU (235 mg,
0.73 mmol) were dissolved in 1 ml DMF. Ethyldiisopropylamine (370
ml) and 2-fluoroethylamine hydrochloride (72 mg, 0.72 mmol) were
added. The reaction mixture was stirred for 16 h at room temperature.
The solvent was removed under reduced pressure and the oily residue
was purified by preparative HPLC (RP-18, 15 Â 230 mm, acetonitrile/
water(acetic acid 0.1%, pH 5) (35/65) (v/v), flow: 10 ml/min). The
solvent was removed under reduced pressure, and the product was
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FLUOROETHYLAMIDATION OF OXYTOCIN
223
extracted with glacial acid and precipitated with diethylether to yield
191 mg (58%).
mp: 154–1568C
1H-NMR (DMSO-d6): d–0.90 (m, 6H), 1.50 (m, 3H), 1.83 (m, 3H),
2.20 (m, 1H), 3.45 (m, 6H), 3.70 (m, 2H), 4.28 (m, 2H), 4.5 (m, 1H), 5.08
(s, 2H), 7.35 (s, 5H), 8.10 (m, 2H)
2
1
19F-NMR (DMSO-d6): d–221.89 (tt, 1F); J(1H/19F)=47 Hz, J(1H/
19F)=28 Hz
IR (KBr, cm-1): nas(N–H) 3300 (s); n(C–H, arom.) 3065 (w); n(C¼O)
1710 (s); n(C¼O) 1649 (s, Gly); n(N–H) 1543 (s); n(C–O) 1415 (s);
n(C–F) 1306 (w)
mass spectrum (m/z): 465 [M+H]+.
N.c.a.2-[18F]fluoroethylammonium trifluoroacetate
N.c.a. [18F] fluoride was produced by standard procedures at the JSW-
9,10
.
BC1710 Cyclotron of FZ-Julich.
After azeotropic drying of the
[18F]fluoride containing the aminopolyether complex Kryptofix1
[2.2.2.]/K2CO3 by anhydrous acetonitrile,11 a solution of N-Boc-2-
(p-toluenesulfonyloxy)ethylamine (5 mg) in anhydrous DMSO (250 ml)
was added. The solution was heated 10 min at 1408C before adding
DMAP (10 mg) in anhydrous DMSO (100 ml). The quaternisation was
performed at 1008C for 15 min. After cooling at room temperature, the
reaction mixture was diluted with 0.1 N hydrochloric acid (3.15 ml) and
then passed through a C18 Sep Pak column (Waters). The column was
first rinsed with 0.01 N hydrochloric acid (10 ml) and then washed with
water (5 ml). The radioactive product was eluted with ether (2 ml). The
solvent was removed under reduced pressure, trifluoroacetic acid
(0.3 ml) was added and evaporated after 2 min reaction time.
N.c.a. Z–Pro–Leu–Gly–(2–[18F]fluoroethyl)NH
Method A.
A 0.095 N solution (250 ml) of one of the above mentioned active esters
of Z–Pro–Leu–Gly–OH in DMF and triethylamine (10 ml) was added to
n.c.a. 2-[18F]fluoroethylammonium trifluoroacetate and stirred at room
temperature for 1 min.
Method B.
A solution of Z–Pro–Leu–Gly–OH (2.5. mg, 6 mmol), TBTU (2 mg,
6 mmol) and triethylamine (3 ml) in DMF (250 ml) was added to n.c.a.
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M. JELINSKI ET AL.
2-[18F]fluoroethylamine trifluoroacetate and stirred at room tempera-
ture. For optimising the reaction parameters, the type of coupling
reagent, the concentration of the peptide and the reaction time were
varied.
N:c:a: [Gly-(2-[18F] fluoroethyl)NH9]oxytocin
A given amount of Boc–Cys–Tyr(tBu)–Ile–Gln(Mtt)–Asn(Mtt)–Cys–
Pro–Leu–Gly–OH (optimally 5 mg, 3 mmol), TBTU (1 mg, 3 mmol) and
triethylamine (2 ml) were dissolved in DMF (250 ml), added to n.c.a. 2-
[18F]fluoroethylammonium trifluoroacetate and stirred at room tem-
perature. After a given time the reaction mixture was diluted with 0.1 N
hydrochloric acid and passed through a C-18 Sep Pak cartridge
(Waters). The cartridge was washed with 0.01 N hydrochloric acid and
water in order to remove residual 2-[18F]fluoroethylamine. The labelled
peptide was eluted with acetonitrile and evaporated to dryness.
Deprotection of the oxytocin derivative was subsequently performed
with trifluoroacetic acid (0.2 ml) within 1 min at room temperature.
HPLC of Z–Pro–Leu–Gly–2-[18F] fluoroethylamide and
[Gly-(2-[18F] fluoroethyl)–NH9]oxytocin
The following HPLC-conditions were used for Z–Pro–Leu–Gly–(2-
[18F]fluoroethyl-amide: LiChrosorb RP 8À5m, 250 Â 4 mm, acetonitrile/
phosphate-bufferpH7 (40/60) (v/v), 1 ml/min, k0 (Z–Pro–Leu–Gly–2–
[18F]fluoroethylamide)=2.13, k0 (2-[18F]fluoroethylamine)=0.36. In the
case of [Gly–(2-[18F]fluoroethyl)–NH9]oxytocin the trifluoroacetic acid
was evaporated after the deprotection step and the residue dissolved in
0.2 ml of acetonitrile/water (10/90). An aliquot (50 ml) was analysed by
gradient HPLC: Nucleosil 120À5m C4, 250 Â 4 mm, gradient: 1 min
acetonitrile/water (10/90), 20 min 50% acetonitrile, k0[Gly-(2-[18F]fluor-
oethyl)-NH9]oxytocin=4.24, k0 (free acid)=3.63.
Results and Discussion
Formation of 2-[18F] fluoroethylamine
The preparation of n.c.a. 2-[18F]fluoroethylamine was based on the
cryptate mediated nucleophilic 18F-fluorination11 of N-Boc-2-(p-tolue-
nesulfonyloxy)ethylamine with subsequent deprotection under acidic
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FLUOROETHYLAMIDATION OF OXYTOCIN
225
Scheme 1. Radiosynthesis of 2-[18F]fluoroethylammonium trifluoroacetate
conditions (Scheme 1). The labelling of this precursor, when first
described by Gilissen et al.,7 has been performed in acetonitrile leading
to a radiochemical yield of only about 20% of N-Boc-2-[18F]fluoroethyl-
amine. Here, the yield could be increased up to 30% at 908C by
increasing the precursor concentration up to 0.4 mol/l and up to 61 Æ 6
% when DMSO was used as solvent. With DMF a radiochemical yield
of only about 15% was obtained even at higher temperatures. For
comparison, the nucleophilic 18F-fluorination of N-Boc-2-bromoethyl-
amine in acetonitrile led to a radiochemical yield of 38 Æ 9% at a
temperature of 908C.6
For simpler isolation of the product, the excess of precursor was
reacted with DMAP after 18F-exchange forming a quaternary
ammonium salt (Scheme 2), as Guhlke has demonstrated in the case
of the bromo-precursor.6 This watersoluble salt togetherwith the
Scheme 2. Derivatisation of the excess precursor with DMAP
cryptate and residual [18F]fluoride could easily be separated from the
hydrophobic Boc-protected [18F]fluoroethylamine via solid phase
extraction using a C-18 Sep-Pak cartridge. The N-Boc-2-[18F]fluoro-
ethylamine was obtained by subsequent elution with etherand was
deprotected quantitatively with trifluoroacetic acid at room temperature
within 2 min after evaporation of the solvent. This resulted in a
somewhat improved radiochemical yield of 46 Æ 5% of 2-[18F]fluoro-
ethylamine compared to 39 Æ 6% reached by producing the amine from
N-(2-[18F]fluoroethyl)phthalimide.7
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M. JELINSKI ET AL.
C-Terminal 18F-fluoroethylation of the tripeptide Z-Pro-Leu-Gly-OH
The tripeptide Z–Pro–Leu–Gly–OH represents the C-terminal amino acid
sequence of oxytocin. Accordingly, it is a useful model for developing and
optimising the 18F-fluoroethylamidation of the neuropeptide either based
on the condensation with active esters or starting with the corresponding
free acid. The reaction of 2-[18F]fluoroethylamine with various activated
esters, i.e. the N-succinimidyl-, p-nitrophenyl- and pentafluorophenylester
of Z–Pro–Leu–Gly–OH, could be performed in DMF at room
temperature in the presence of triethylamine. In each case radiochemical
yields 590% were obtained within 1 min.
However, due to the very expensive nonapeptide oxytocin and the
losses when converting to the corresponding activated esters (chemical
yields of esters were 65, 67, 87%, respectively) an attempt was made to
perform the amidation starting with the free acid of the tripeptide in
presence of a coupling reagent such as TBTU, BOP, WSC or HOBT (in
situ activation). The synthesis of Z–Pro–Leu–Gly–(2-[18F]fluoroethyla-
mide in presence of an activating agent and a base such as triethylamine
was performed in DMF at room temperature. Within 10 min and a
precursor concentration of 0.024 mol/l, TBTU supported amidation
gave a radiochemical yield of 91 Æ 2%, which was comparable to those
obtained by condensation using 2-[18F]fluoroethylamine and the active
esters. With the same reaction conditions a good radiochemical yield of
84 Æ 8% was also obtained with BOP, whereas the other two coupling
reagents gave much lower radiochemical yields: 27 Æ 4% (WSC) and
only 1% with HOBT.
In further optimisation experiments the dependence of the radio-
chemical yield on reaction time and precursor concentration was
examined. The results are graphically depicted in Figure 2. The diagram
shows, that a reaction time of 1–2 min is sufficient and that the
maximum radiochemical yield of 90 Æ 7% is achieved with a precursor
concentration of 50.024 mol/l.
Thus, the best reaction conditions found for the synthesis of Z–Pro–
Leu–Gly–2-[18F]fluoroethylamide using the free acid and a coupling
reagent are a precursor concentration of 0.024 mol/l, a reaction
temperature of 208C, a reaction time of 1 min and TBTU as
activating agent. Compared with the active ester method, the synthesis
via TBTU under comparable conditions results in the same radio-
chemical yield of 590% with high reliability. However, the expensive
synthesis of the activated esterof a peptide is avoided when using a
coupling reagent.
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FLUOROETHYLAMIDATION OF OXYTOCIN
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Figure 2. Time dependence of 18F-fluoroethylamidation of Z–Pro–Leu–Gly–OH
conditions: 250 ml DMF, 208C, n=3À4, Z–Pro–Leu–Gly–OH/TBTU/
TEA=1/1/2
[Gly-(2-[18F] fluoroethyl)NH9]oxytocin
Based on the model-studies, [Gly-(2-[18F]fluoroethyl)NH9]oxytocin with
protected functional groups, i.e. Boc–Cys–Tyr(tBu)–Ile–Gln(Mtt)–
Asn(Mtt)–Cys–Pro–Leu–Gly–(2-[18F]fluoroethyl)NH, was correspond-
ingly synthesised via condensation of [18F]fluoroethylamine in presence
of TBTU (Scheme 3).
Results of optimisation experiments with variation of reaction time
and precursor concentration are summarised in Figure 3. Radio-
chemical yields of 75 Æ 5% were obtained after 2 min with a peptide
concentration of 50.01 mol/l, whereas at 0.006 mol/l a radiochemical
yield of about 60% was achieved.
Deprotection of the 18F-fluoroamidated peptide with trifluoroacetic
acid was quantitative at room temperature within 1–2 min. Attempts to
remove the protecting groups after absorption of the 18F-labelled
peptide on a solid phase ordissolved in DMF failed. Thus, the solvent
DMF had to be removed before treatment with TFA. The radio-
chemical yield of [Gly–(2-[18F]fluoroethyl)-NH9]oxytocin was deter-
mined at the end of deprotection with TFA via analytical HPLC to
29 Æ 5% (based on starting [18F]fluoride). The results now encourage
the development of a large scale preparation for pharmacokinetic PET-
studies, including determination of actual specific activity and
examination of the biochemical properties of derivatized oxytocin.
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M. JELINSKI ET AL.
Scheme 3. Reaction sequence of the synthesis of [Gly-(2-[18F]fluoroethyl)NH9]-
oxytocin
Figure 3. Time dependence of 18F-fluoroethylamidation of protected [Gly–
OH9]oxytocin in presence of TBTU, n=3À4, conditions: 250 ml DMF, 208C,
[Gly-OH9]oxytocin/TBTU/TEA=1/1/2
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 217–229

FLUOROETHYLAMIDATION OF OXYTOCIN
229
Conclusion
Since oxytocin has a C-terminal amide function, 18F-labelling of the
peptide hormone was performed via the 18F-fluoroethylamidation of the
free acid of oxytocin with the hope of no significant alteration of the
biochemical properties. The radiochemical yield of the intermediate 2-
[18F]fluoroethylamine was somewhat increased in comparison to
preparations previously described.6,7 With the most suitable coupling
reagent TBTU, 2-[18F]fluoroethylamine can be efficiently condensed
with the peptide acid to give [Gly–(2-[18F]fluoroethyl)NH9]oxytocin.
Thus, expensive synthesis of activated esters can be avoided.
Acknowledgements
We would like to thank Dr. D.H. Hellhammer, University of Trier,
Germany, for support and the team of the JSW BC 1710 cyclotron, FZ-
Julich, forproducing [ 18F]fluoride.
.
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