Reaction of [18F]4-fluorobenzenediazonium cations with cysteine or the cysteinyl group: Preparation of 18F-labeled S-aryl-cysteine and a radiolabeled peptide

Article abstract of DOI:10.1002/jlcr.635

A reaction route for the preparation of no-carrier-added (n.c.a.) [¹⁸F]S-4-fluorophenylcysteine 7 via the [¹⁸F]-4-fluorobenzenediazonium ion 4 is described. The key step in this radiosynthesis is the reaction of 4 with cysteine forming [¹⁸F]4-fluorophenyldiazocysteine 6, which is subsequently converted into 7 by irradiation with 366 nm light. 4 was synthesized by reacting 1,4-dinitrobenzene 1 with [¹⁸F]-fluoride in acetonitrile in a PEEK-capillary in a microwave oven. After dilution of the reaction mixture with methanol, the resulting [¹⁸F]4-fluoro-1-nitrobenzene 2 was submitted to reduction by means of H₂ with Pd on C catalyst. The resulting [¹⁸F]4-fluoroaniline 3 was purified by HPLC and diazotized to 4. The preparation of 4 was optimized with regard to yield and purity. The radiochemical yield of 6 was > 90% (based on 3) while after UV irradiation and HPLC purification 45% of 7 (based on 3) was obtained (yields corrected for decay). The suitability of this method for labeling peptides with fluorine-18 was demonstrated by application to the tripeptide, glutathione (GSH; γ-L-glutamyl-L-cysteinyglycine) 8. Copyright

Full text of DOI:10.1002/jlcr.635

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 1229–1238.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.635
Research Article
18
Reaction of [ F]4-fluorobenzenediazonium
cations with cysteine or the cysteinyl group:
18
preparation of F-labeled S-aryl-cysteine
and a radiolabeled peptide
1
J. T. Patt * and M. Patt
,
2
1
Institut f u. r Interdisziplin a. re Isotopenforschung e.V., Permoser Str. 15,
D-04318 Leipzig, Germany
2
Klinik und Poliklinik f u. r Nuklearmedizin, Liebigstrasse 20a, D-04103, Leipzig,
Germany
Summary
1
A reaction route for the preparation of no-carrier-added (n.c.a.) [ F]S-
8
1
-fluorophenylcysteine 7 via the [ F]-4-fluorobenzenediazonium ion 4 is
8
4
described. The key step in this radiosynthesis is the reaction of 4 with cysteine
1
forming [ F]4-fluorophenyldiazocysteine 6, which is subsequently converted
8
into 7 by irradiation with 366 nm light. 4 was synthesized by reacting
1
,4-dinitrobenzene 1 with [ F]-fluoride in acetonitrile in a PEEK-capillary in a
8
1
microwave oven. After dilution of the reaction mixture with methanol, the
1
resulting [ F]4-fluoro-1-nitrobenzene 2 was submitted to reduction by means
8
18
of H with Pd on C catalyst. The resulting [ F]4-fluoroaniline 3 was purified
2
by HPLC and diazotized to 4. The preparation of 4 was optimized with regard
to yield and purity. The radiochemical yield of 6 was >90% (based on 3) while
after UV irradiation and HPLC purification 45% of 7 (based on 3) was
obtained (yields corrected for decay). The suitability of this method for labeling
peptides with fluorine-18 was demonstrated by application to the tripeptide,
glutathione (GSH; g-l-glutamyl-l-cysteinyglycine) 8. Copyright # 2002 John
Wiley & Sons, Ltd.
Key Words: fluorine-18; diazonium ion; S-aryl-cysteine; cysteinyl; peptide
*
Correspondence to: J. T. Patt, Institut f u. r Interdisziplin a. re Isotopenforschung, Permoser Str. 15,
D-04318 Leipzig, Germany. E-mail: jpatt@iif-leipzig.de
Copyright # 2002 John Wiley & Sons, Ltd.
Received 4 February 2002
Revised 5 June 2002
Accepted 10 July 2002

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J. T. PATT AND M. PATT
Introduction
Metabolic imaging in oncology is an important application of positron
emission tomography (PET). Some amino acids, labeled with positron-
11
18
emitters, such as C and F (t1=2 ¼ 20:3 and 109.7 min, respectively),
are useful tracers in oncology for reporting protein synthesis and/or
1
amino acid transport (for a review see Reference 1). Thus, the F-
8
2
18
labeling of S-aryl substituted cysteine via F-labeled diazonium ions,
as described below, may result in new tracers for tumor imaging with
PET and provide a new method for radiolabeling peptides.
1
8
Feliu described the radiosynthesis of [ F]WIN 44577 via the
3
production of [ F]4-fluorophenylhydrazine and diazonium coupling.
18
18
In this procedure [ F]4-fluorophenylalanine (3) was obtained and
diazotized in high yield, as shown by monitor reactions. Although
8
1
diazothio-compounds are note suitable for a direct introduction of
into an aromatic ring, the high reactivity of the sulfhydryl-group of
F
4
,5
cysteine with diazonium ions to form diazothio-compounds
and
6–8
arylthioethers by thiodediazonation may provide a possible reaction
1
8
pathway for the synthesis of radiopharmaceuticals containing the F-
aryl thioether moiety. In this paper, we describe an adaptation of the
synthesis of the diazonium ion 4 and its application to the synthesis of
1
8
F-labeled 7 (Figure 2). Furthermore, we describe the application of
this method to the radiolabeling of the model tripeptide, glutathione
GSH; g-l-glutamyl-l-cysteinylglycine), 8.
(
Results and discussion
3
The method for the production of 4 described by Feliu was modified in
certain respects (Figure 1): acetonitrile was used instead of dimethyl
1
Figure 1. Modified procedure for the preparation of [ F]4-fluorobenzenediazo-
8
3
nium ions (Feliu)
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REACTION OF [ F]4-FLUROBENZENEDIAZONIUM CATIONS
1231
sulfoxide as solvent. Acetonitrile could be easily removed by evapora-
tion and did not impair the reduction step. Thus the SepPak purification
of 2, which leads to losses in yield due to incomplete trapping of 2 on the
stationary phase of the cartridge, could be avoided. For the catalytic
reduction of 2, hydrogen was used instead of sodium borohydride to
avoid contaminating the product with boron compounds that may
interfere with the chromatographic separation of product. 3 was
purified by HPLC to improve the purity of the diazonium salt solution.
A simplified one-pot procedure without HPLC purification of 3 was
shown to give moderate radiochemical yields of 6 (60% based on 3), but
after irradiation only a rather low amount of the desired product 7 was
obtained. The low UV intensity (UV hand lamp 3 W) and the high
energy absorption by other substances in the reaction mixture may
explain this observation.
1
8
The microwave procedure for NO for F exchange is superior
2
compared to thermal heating procedures, because a considerably lower
amount of precursor can be used with a shorter reaction time. In the
polar aprotic solvent, acetonitrile, the main effects of microwave power
may be superheating and creation of hot spots. Nevertheless, other
effects like stabilization of the transition state of the reaction are
9–11
possible (for a review see References
). Performing the reaction
within PEEK-tubing in a domestic microwave oven is a low-cost
alternative to dedicated radiochemistry microwave units and allows the
usage of low boiling solvents like acetonitrile. Consecutive intervals of
3
0 s full microwave power (30 s, 750 W) followed by 30 s cooling time
each, resulted in radiochemical yields >70% (2 ꢀ 30 s, 29%; 4 ꢀ 30 s,
9%; 6 ꢀ 30 s, 73%). For reduction of the nitro-compound, the crude
4
reaction mixture was diluted with methanol. Complete reduction of 2
was achieved within 10 min by passing a stream of hydrogen through
the reaction mixture in the presence of Pd/C catalyst. It was found, that
acetonitrile does not impair the reduction. After filtration of the
mixture, purification by normal phase HPLC, and evaporation of the
solvent, 3 was obtained in >60% overall radiochemical yield. Figure 2
shows the reaction scheme of the following process. Diazotization of
purified 3 and subsequent coupling to cysteine gave 6 in quantitative
radiochemical yield. The retention time of this radioactive product
agreed with that of the reference compound, as detected by MS
(m=z ¼ 244:06 molecular ion) (Figure 3). After irradiation with 366 nm
UV light and HPLC purification the cysteine conjugate 7 was obtained
in 45% radiochemical yield (based on 3). The retention time of this
Copyright # 2002 John Wiley & Sons, Ltd.
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J. T. PATT AND M. PATT
1
Figure 2. Preparation of [ F]4-fluorophenyl-cysteine by reacting cysteine with
8
1
8
[
F]4-fluorobenzenediazonium ion and subsequent irradiation with 366 nm light
Figure 3. HPLC–MS chromatogram (A,C) and radio-chromatogram (B,D) of 6
and 7. (A) extracted ion chromatogram (m/z=244.1 ꢁ 0.2) of 6, (B) radio
chromatogram of 6, (C) extracted ion chromatogram (m/z=216.1 ꢁ 0.2) of 7,
(D) radio chromatogram of 7
radioactive product on HPLC agreed with that of the authentic
reference compound, as detected by MS ðm=z ¼ 216:05Þ (Figure 3). The
reference compound 7 was purified by HPLC and characterized by
1
19
H-NMR and F-NMR spectroscopy. The melting point of the
product was 179–1818C after HPLC purification and in agreement
2
with a melting point of 185–1868C reported by Goodman et al.
The reaction of arenediazonium ions with thiolate ions is a
well-known procedure for the preparation of arylthioethers. The
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REACTION OF [ F]4-FLUROBENZENEDIAZONIUM CATIONS
1233
mechanism of thiodediazonation is considered to consist of four
7,8
reaction steps:
1
2
. the reaction of an electron donor with the aryldiazothio-compound to form
a radical anion,
. the cleavage of the C–N and N–S bonds in the radical anion to form aryl
radical, nitrogen, and thiolate anion,
3
4
. the recombination of aryl radical and thiolate anion and
. the reoxidation of the radical anion to the arylthioether product.
Our results with n.c.a. 6 show that the propagation step (electron
transfer from the radical anion of step 4 to the aryldiazothio-compound
in step (1)) is not crucial for the reaction. The rapid recombination of
the aryl radical with the thiolate anion has been thoroughly investigated
7
by Dell’Erba et al.. To explain the minor influence of solvent changes
ꢂ
or the presence of a competing nucleophile (CN ) on the reaction,
Dell’Erba et al. proposed a ‘cage effect’. Because of the high reactivity
and the mechanism, this reaction also appears useful for the
radiolabeling of peptides and other biologically active compounds. To
test the applicability of this method for the radiolabeling of peptides, 8
1
8
was reacted with 4. A radiochemical yield of 75% (based on 3) of [ F]
S-4-fluorophenyldiazo-GSH 9 was obtained for an amount of 0.5 mmol
of 8 in the reaction mixture. Higher amounts of precursor peptide 8
resulted in quantitative conversion. The thiodediazonation step to
1
8
[
F]S-4-fluorophenyl-GSH 10 could be performed with 33% yield. This
step is not optimized yet, and might be improved by use of a dedicated
UV-irradiation apparatus. These results illustrate the suitability of this
labeling procedure for peptides.
Experimental
General: Solvents for HPLC and LC–MS and chemicals were purchased
in gradient grade quality or analytical quality by Merck, Germany
unless otherwise stated. HPLC chromatograms were recorded on an
Agilent Chemstations for LC systems equipped with an Nal/Tl detector
connected to the HPLC system via a Dual Channel Interface HP35900E
(Agilent). The low pressure gradient system used for semipreparative
separations consisted of quaternary HPLC pump and vacuum
degasser, autosampler and diode array detector. The high pressure
gradient system consisted of vacuum degasser, binary HPLC pump,
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J. T. PATT AND M. PATT
autosampler, multi wavelength detector and was connected to the mass
spectrometer. TLC were exposed to BAS-MS2325 imaging plate and
read out with the BAS-1800II IP-Reader (Fujifilm). The image was
evaluated with AIDA software (raytest isotopenmessger a. te, Strauben-
hardt, Germany). Mass spectra and LC-MS chromatograms were
obtained using a Mariner Biospectrometry Workstation (Applied
Biosystems) (MS-TOF, positive ion mode, nozzle potential 100 V,
skimmer 1 potential 7.5 V). NMR spectra were recorded on a BRUKER
19
DRX-400 (AVANCE), F-NMR spectra were measured at room
temperature in D O with trifluroacetic acid as internal reference
2
1
(
d(CF COOH)=0 ppm). H-NMR spectra were recorded in D O with
3
2
the HDO peak as reference (d(HDO)=4.67 ppm). For UV-irradiation
of the sample a UV-lamp for TLC-detection (3 W, 366 nm) was used,
which was positioned 4–5 cm above the sample surface. The sample was
irradiated in a small glass beaker. Radiochemical yields were corrected
for decay.
Synthesis of 4-fluorophenylcysteine (7)
A solution of 0.01 M 4-fluoroaniline (for synthesis) in 1 M HCl (1 ml)
diazotized at 08C with 0.1 M sodium nitrite solution (0.1 ml). The
solution of the diazonium salt was added to a stirred solution of cold
(08C) 0.1 M l-cysteine (for biochemistry) in 1 M NaOH (1 ml). The pH
was adjusted to a slightly alkaline value (8–9). The 4-fluorophenyldia-
zocysteine was not isolated but an aliquot of the mixture was filtered
and analyzed by HPLC-MS (2 Zorbax XDB-C8, 4.6 mm ꢀ 15 cm
columns in series, acetonitrile 15%, water (2.5% acetic acid) 85%,
5
min, gradient to acetonitrile 75% at 15 min, 1 ml/min, retention time
for 6 6.4 min). The remaining mixture was irradiated with UV light for
0 min. The mixture was diluted with eluent, filtered and the product
purified by HPLC (Supelcosil LC-ABZ+, 250 ꢀ 7.6 mm, acetonitrile
3
1
5%, water containing 2.5% acetic acid 85%) (injection volume 500 ml,
ml/min, retention time for 7 4.3 min, 6 6.9 min). The separated product
5
1 18
was analyzed by H-NMR, F-NMR mass spectrometry and melting
point.
1
H-NMR (D O) d ppm: 7.6 (2 H, m); 7.21 (2 H, m); 3.85 (1 H, dd; 3.9 Hz;
2
8
.3 Hz); 3.59 (1 H,dd, 3.9 Hz, 14.9 Hz); 3.35 (1 H, dd, 8.3 Hz, 14.9 Hz).
F-NMR (D O): ꢂ39 ppm.
1
9
2
+
m=z: 216.05 (M+H) (Turbo Ion Spray).
Melting point: 179–1818C (decomposition).
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REACTION OF [ F]4-FLUROBENZENEDIAZONIUM CATIONS
1235
18
Synthesis of n.c.a. [ F]1-fluoro-4-nitrobenzene (2)
Heating-block procedure. Aminopolyether 2.2.2 (Kryptofix 222) (10 mg,
8
1
2
6 mmol), K CO (13 mmol) and n.c.a. [ F]fluoride (100–1000 MBq,
2
3
1
8
18
obtained via the O(p,n) F nuclear reaction) in water and acetonitrile
1 ml) were mixed in a 5 ml reaction vial (Alltech, Germany). The water
(
was evaporated under a stream of nitrogen (heating block 110–1208C).
Acetonitrile (1 ml) was added and re-evaporated three times. A solution
of 1 (for synthesis) in acetonitrile (5 mg in 500 ml) was added and the
mixture was refluxed for 30 min. Radio-TLC-analysis of the reaction
mixture showed 50–80% conversion to product (Polygram SIL-G/
UV254 (Macherey-Nagel, Germany); petroleum ether–ethyl acetate
(
4/1, v/v), R ¼ 0:7).
f
For purification of the product, the solvent was evaporated and the
residue dissolved in ethanol (200 ml) and diluted with water (10 ml). The
mixture was passed through an RP-18 cartridge (Sep Pak light, pre-
conditioned with 5 ml ethanol and 5 ml water). An additional amount of
water (5 ml) was passed through the cartridge, which was subsequently
dried with a nitrogen gas stream. The product was eluted with methanol
and used without further purification (radiochemical yield: 30–50%).
Microwave procedure. The reaction was performed in a microwave oven
(for household use, 750 W). The reaction mixture was enclosed in a
PEEK-capillary (0.5 mm inner diameter, completely filled with reaction
mixture, manually closed with 2 PEEK fittings and a PEEK union) and
placed on the glass tray in the microwave oven. Before performing a
radioactive production, a bursting experiment was performed with
inactive reaction mixture in a bended PEEK-tube. The 750 W on time
was chosen well below the time recorded for capillary rupture in this
experiment. The reaction mixture was prepared as described above. The
amount of 1 in acetonitrile was reduced to (0.75 mg in 500 ml). The
reaction mixture was treated with several intervals (30 s) of microwave
energy followed by 30 s cooling time each.
18
Production of [ F]4-fluoroaniline (3) by catalytic reduction of 2
The acetonitrile solution (microwave method) containing 2 was
transferred with methanol (2 ml) from the PEEK loop into a reaction
vessel containing Pd/C (10% Pd; 1–2 mg). A stream of hydrogen was
passed through the mixture for 10 min. The progress of the reduction
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J. T. PATT AND M. PATT
was monitored by TLC (Polygram SIL-G/UV254, Macherey-Nagel,
Germany), petroleum ether-ethyl acetate, 2/1 (v/v), Rf (3)=0.45,
R (2)=0.9). The catalyst was filtered off, the filtrate washed with ethyl
f
acetate and the solution evaporated. The residue was redissolved in
eluent and injected on a semipreparative HPLC column (LiChrosorb Si
6
0, 250 ꢀ 10 mm, ethyl acetate/methanol, 80/20, v/v, 4 ml/min, retention
time for 3 5.0 min) (1 4.2 min, 2 4.3 min, 4-nitroaniline 4.5 min,
,4-phenylenediamine 19 min). The product peak was collected, 1 M
1
HCl (100 ml) was added and the eluent evaporated off. The product was
dissolved in 1 M HCl (1–2 ml).
18
Synthesis of n.c.a. [ F]S-4-fluorophenyldiazocysteine (6)
3
in 1 M hydrochloric acid (100 ml) was treated with 1 M sodium nitrite
solution (10 ml) at 08C for 2 min. Ammonia solution (25%; 30 ml) was
added followed by 0.1 M cysteine (5) solution in 1 M NaOH (100 ml).
An aliquot of the reaction mixture was analyzed by HPLC (2 Zorbax
XDB-C8, 150 ꢀ 4.6 mm columns in series, acetonitrile 15% water (2.5%
acetic acid) 85% 5 min, gradient to acentonitrile 75% at 15 min, 1 ml/
min, retention time for 6 6.4 min) (Figure 3).
18
Synthesis of n.c.a. [ F]S-4-fluorophenylcysteine (7)
The reaction mixture containing 6 was irradiated for 20 min with 366 nm
light. The solution was filtered and 7 was separated by HPLC
(
Supelcosil LC-ABZ+, 250 ꢀ 7.6 mm, acetonitrile 15%, water contain-
ing 2.5% acetic acid 85%) (5 ml/min, retention time for 7 4.3 min, 6
.9 min). The yield (46%, corrected for decay) was determined by
6
collecting the appropriate product fraction and counting product
fraction and aliquot sample. The radiochemical purity of the product
was determined by radio-HPLC, 2 Zorbax XDB-C8, 150 ꢀ 4.6 mm
columns in series, acentonitrile 15%, water (2.5% acetic acid) 85%,
5
min, gradient to acetonitrile 75% at 15 min, 1 ml/min (Figure 3).
18
Synthesis of n.c.a. [ F]S-4-fluorophenyl-GSH (10)
3
in 1 M hydrochloric acid (100 ml) was treated with 1 M sodium nitrite
solution (10 ml) at 08C for 2 min. Hydroxylamine solution (1 M, 20 ml)
was added (1 min). A solution (200 ml) containing ammonia (0.5 M),
sodium acetate (0.5 M) and 8 (154 mg, 0.5 mmol) was added and the
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REACTION OF [ F]4-FLUROBENZENEDIAZONIUM CATIONS
1237
mixture was neutralized by addition of 1 M ammonia (40 ml). An aliquot
of the reaction mixture was analyzed by radio-HPLC, 2 Zorbax
XDB-C8, 150 ꢀ 4.6 mm columns in series, acetonitrile 15% water (2.5%
acetic acid) 85%, 5 min, gradient to acetonitrile 75% at 15 min, 1 ml/
min, to determine the amount of 9 formed. The mixture was diluted
with 400 ml water and irradiated for 10 min with 366 nm UV-light. The
yield of 10 was determined by radio-HPLC. The synthesis procedure
described above was repeated with 100 ml of a 0.002 M solution of 3 in
1
M hydrochloric acid which was reacted with a solution (200 ml)
containing ammonia (0.5 M), sodium acetate (0.5 M) and 8 (307 mg,
mmol). An aliquot of the reaction mixture was analyzed before and
1
after irradiation with UV-light by the HPLC method described above
with the HPLC–MS–TOF connected to the system.
+
m/z: 430.11 D, retention time=12.2 min, (m+H) of 9.
+
m/z: 402.11 D, retention time=11.2 min, (m+H) of 10, after UV
irradiation.
Conclusion
18
The reaction of F-labeled diazonium ions with the amino acid,
cysteine, is a versatile reaction route for the production of non-naturally
occurring amino acids with biological activity in good radiochemical
yields. Furthermore, the procedure described might be useful for
labeling peptides containing a cysteinyl residue. Further improvement
of this method should be possible by optimization of the UV-irradiation
step.
Acknowledgements
The authors would like to thank Dr D. Sorger (Klinik und Poliklinik
f u. r Nuklearmedizin, Leipzig) for performing first cell uptake studies and
Dr M. Findeisen (Institut f u. r Analytische Chemie, Universit a. t Leipzig)
for recording NMR-spectra.
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