4-[18F]Fluoro-N-methyl-N-(propyl-2-yn-1-yl)benzenesulfonamide ([18F]F-SA): A versatile building block for labeling of peptides, proteins and oligonucleotides with fluorine-18 via Cu(I)-mediated click chemistry

Article abstract of DOI:10.1007/s00726-012-1450-4

Cu(I)-mediated [3+2]cycloaddition between azides and alkynes has evolved into a valuable bioconjugation tool in radiopharmaceutical chemistry. We have developed a simple, convenient and reliable radiosynthesis of 4-[ ¹⁸F]fluoro-N-methyl-N-(propyl-2-yn-1-yl)benzenesulfonamide ([ ¹⁸ F]F-SA) as a novel aromatic sulfonamide-based click chemistry building block. [ ¹⁸ F]F-SA could be prepared in a remotely controlled synthesis unit in 32 ± 5 % decay-corrected radiochemical yield in a total synthesis time of 80 min. The determined lipophilicity of [ ¹⁸ F]F-SA (logP = 1.7) allows handling of the radiotracer in aqueous solutions. The versatility of [ ¹⁸ F]F-SA as click chemistry building block was demonstrated by the labeling of a model peptide (phosphopeptide), protein (HSA), and oligonucleotide (L-RNA). The obtained radiochemical yields were 77 % (phosphopeptide), 55-60 % (HSA), and 25 % (L-RNA), respectively. Despite the recent emergence of a multitude of highly innovative novel bioconjugation methods for ¹⁸F labeling of biopolymers, Cu(I)-mediated click chemistry with [ ¹⁸ F]F-SA represents a reliable, robust and efficient radiolabeling technique for peptides, proteins, and oligonucleotides with the short-lived positron emitter ¹⁸F.

Full text of DOI:10.1007/s00726-012-1450-4

Amino Acids (2013) 44:1167–1180
DOI 10.1007/s00726-012-1450-4
ORIGINAL ARTICLE
4-[18F]Fluoro-N-methyl-N-(propyl-2-yn-1-yl)benzenesulfonamide
([18F]F-SA): a versatile building block for labeling of peptides,
proteins and oligonucleotides with fluorine-18 via Cu(I)-mediated
click chemistry
•
Theres Ramenda Jorg Steinbach
•
¨
Frank Wuest
Received: 6 September 2012 / Accepted: 21 December 2012 / Published online: 10 January 2013
Ó Springer-Verlag Wien 2013
Abstract Cu(I)-mediated [3?2]cycloaddition between
azides and alkynes has evolved into a valuable bioconjugation
tool in radiopharmaceutical chemistry. We have developed a
simple, convenient and reliable radiosynthesis of 4-[18F]fluoro-
N-methyl-N-(propyl-2-yn-1-yl)benzenesulfonamide ([18F]F-
SA) as a novel aromatic sulfonamide-based click chemistry
building block. [18F]F-SA could be prepared in a remotely
controlled synthesis unit in 32 5 % decay-corrected radio-
chemical yield in a total synthesis time of 80 min. The deter-
minedlipophilicity of [18F]F-SA (logP = 1.7)allows handling
of the radiotracer in aqueous solutions. The versatility of
[18F]F-SA as click chemistry building block was demonstrated
by the labeling of a model peptide (phosphopeptide), protein
(HSA), and oligonucleotide (L-RNA). The obtained radio-
chemical yields were 77 % (phosphopeptide), 55–60 % (HSA),
and 25 % (L-RNA), respectively. Despite the recent emergence
of a multitude of highly innovative novel bioconjugation
methods for 18F labeling of biopolymers, Cu(I)-mediated click
chemistry with [18F]F-SA represents a reliable, robust and
efficient radiolabeling technique for peptides, proteins, and
oligonucleotides with the short-lived positron emitter 18F.
Keywords Cu(I)-mediated click chemistry ꢀ
Fluorine-18 ꢀ Peptides ꢀ Proteins ꢀ Oligonucleotides ꢀ
Positron emission tomography (PET)
Introduction
Positron emission tomography (PET) is a non-invasive
molecular imaging modality to obtain functional information
of physiological, biochemical and pharmacological processes
in living subjects at the cellular and molecular level (Czernin
and Phelps 2002; Paans et al. 2002; Phelps 2000). The majority
of PET studies utilizes compounds labeled with the short-lived
positron emitter fluorine-18 (18F, t1/2 = 109.8 min) with
2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) as the most
prominent and most frequently used PET radiotracer.
Over the last decades, numerous methods have been
developed for efficientincorporation of 18F into a broad variety
of structurally diverse compounds. Most methods rely on
nucleophilic aliphatic or nucleophilic aromatic substitution
reactions starting from no-carrier-added (n.c.a.) [18F]fluoride.
However, incorporation of [18F]fluoride into higher molecular
weightcompoundslikepeptides, proteins and oligonucleotides
represents a special challenge. Peptides and other biopolymers
can usually not be labeled directly with n.c.a. [18F]fluoride due
to required harsh reaction conditions like elevated tempera-
tures and the use of organic solvents and basic conditions.
Those reaction conditions are not compatible with the struc-
tural and functional integrity of many biopolymers, especially
proteins. Therefore, 18F labeling of biopolymers is frequently
accomplished by means of prosthetic groups. This approach
involves formation of 18F-labeled small organic molecules
capable of being linked to peptides, proteins, and oligonucle-
otides under mild conditions (Kuhnast and Dolle 2010; Liu
et al. 2011; Olberg and Hjelstuen 2010; Okarvi 2001; Wuest
Preliminary results on the synthesis and use of the click chemistry
building block [18F]F-SA have already been published (Ramenda
et al. 2009).
T. Ramenda ꢀ J. Steinbach
Institute of Radiopharmacy, Helmholtz-Zentrum
Dresden-Rossendorf, Dresden, Germany
T. Ramenda
CUP Laboratorien Dr. Freitag GmbH, Radeberg, Germany
F. Wuest (&)
Department of Oncology, University of Alberta,
Edmonton, AB T6G 1Z2, Canada
e-mail: wuest@ualberta.ca
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1168
T. Ramenda et al.
2005). Such linkage is traditionally accomplished via acylation
(Vaidyanathan and Zalutsky 1992; Wester et al. 1996; Garg
et al. 1991; Guhlke et al. 1994; Richter et al. 2009), imidation
(Kilbournetal. 1987), alkylation (Toyokuni et al. 2003; Berndt
et al. 2007; De Bruin et al. 2005), photochemical conjugation
(Wester et al. 1996), and oxime formation (Poethko et al.
2004). More recent developments have also demonstrated
direct incorporation of [18F]fluoride into peptides and proteins
based on highly efficient fluoride-acceptor chemistry exploit-
ing formation of stable Si–F, B–F, or Al–F bonds (Schirrm-
acher et al. 2006; McBride et al. 2012). However, every
method has advantages and limitations, and the development
of rapid, clean and mild synthesis techniques for the radiola-
beling of biopolymers continues.
compounds as building blocks for Cu(I)-mediated
[3?2]cycloadditions. [18F]Fluoroaryl compounds as dis-
played in Scheme 1 contain an aromatic C–F bond which is
known to be stable in vivo towards radiodefluorination.
Preparation of alkyne group-containing compounds
depicted in Scheme 1 was accomplished either via single
step nucleophilic aromatic substitution with n.c.a.
[18F]fluoride in the case of [18F]FPyKYNE, [18F]FPy5yne
and prop-2-yn-1-yl 4-[18F]fluorobenzoate (Kuhnast et al.
2008; Inkster et al. 2008; Vaidyanathan and Zalutsky
1992), or via acylation reaction of propargyl amine with
[18F]SFB in the case of 4-[18F]fluoro-N-(propyl-2-
ynyl)benzamide (Ramenda et al. 2007). 4-[18F]Fluoro-N-
(propyl-2-ynyl)benzamide, [18F]FPyKYNE, [18F]FPy5yne
and prop-2-yn-1-yl 4-[18F]fluorobenzoate were used in
conjugation reactions with azide-functionalized peptides
(Ramenda et al. 2007; Inkster et al. 2008; Vaidyanathan
and Zalutsky 1992). [18F]FPy5yne was also used for click
chemistry labeling of oligonucleotides (Inkster et al. 2009).
Azidomethyl-[18F]fluorobenzene was prepared in a multi-
step reaction sequence starting from 4-[18F]fluorobenzalde-
hyde (Thonon et al. 2009). 4-[18F]Fluorobenzaldehyde was
reduced to 4-[18F]fluorobenzyl alcohol, which was converted
into 4-[18F]fluorobenzyl bromide. In the last reaction step,
azidomethyl-[18F]fluorobenzene was obtained through treat-
ment of 4-[18F]fluorobenzyl bromide with an azide exchange
resin. Azidomethyl-[18F]fluorobenzene was conjugated to an
alkyne-modified model neuropeptide using click chemistry in
high radiochemical yields of 90 %.
In recent years, click chemistry in radiopharmaceutical
chemistry has emerged as a versatile and generic method for
rapid and selective radiolabeling of biomolecules. Especially
the Cu(I)-catalyzed 1,2,3-triazole formation between azides
and alkynes according to a 1,3-dipolar [3?2]cycloaddition
was shown to be a particularly powerful reaction due to its
highdegreeofspecificityandbioorthogonalityofthereactants
(Kolb et al. 2001; Rostovtsev et al. 2002). Moreover, triazoles
are stable under physiological conditions, and they may
associate with potential biological targets through hydrogen
bonding and dipole interactions.
Adversely, the need of using cytotoxic copper has
somewhat hampered the general use of this reaction,
especially for its use in various click chemistry applications
in vivo. As a consequence, several alternative copper-free
methods have been developed recently. Most prominent
strategies are strain-promoted azide-alkyne cycloadditions
(Debets et al. 2011; van Hest and van Delft 2011), Stau-
The preferred single step radiosynthesis of 18F-labeled
aromatic compounds starting from n.c.a. [18F]fluoride
according to a nucleophilic aromatic substitution reaction
requires activated electron-deficient aromatic systems con-
taining a suitable leaving group as labeling precursors.
Prominent examples include electron-deficient aryl com-
pounds like benzaldehydes, benzonitriles, benzoic acid esters,
nitrobenzenes or pyridines with nitro or trimethylammonium
groups as leaving groups.
¨
dinger ligations (van Berkel et al. 2011; Kohn and Bre-
inbauer (2004), and inverse-electron-demand Diels–Alder
reactions (Devaraj et al. 2010; Liu et al. 2012).
However, Cu(I)-mediated [3?2]cycloaddition between
azides and alkynes is a highly effective method for the radiola-
beling of a broad range of biopolymers and small molecules with
radionuclides like 18F. Various 18F-labeled azides and alkynes
and their subsequent use in click chemistry have been described
Surprisingly, only little work has been reported on using aryl
sulfonamides as electron-deficient aromatic systems for nucle-
ophilic aromatic substitution reactions with n.c.a. [18F]fluoride.
Some prominent examples include synthesis of carbonic anhy-
drase inhibitors, a2 adrenergic receptor ligands, and inhibitors
for the serotoninergic 5-HT6 receptor which could be prepared
in a single step radiofluorination reaction with n.c.a. [18F]fluo-
ride using the corresponding aryl sulfonamide containing a nitro
¨
in the literature (Mamat et al. 2009; Wangler et al. 2010).
Most 18F-labeled click chemistry building blocks reported
in the literature represent volatile aliphatic [18F]fluoroalkynes
or [18F]fluoroazides [Marik and Sutcliffe 2006; Glaser and
¨
Arstad 2007; Wangler et al. 2010). Only few examples
describe alkyne- or azide-containing [18F]fluoroaryl
Scheme 1 Various
[
O
O
18F]fluoroaryl compounds for
copper (I)-assisted
[3?2]cycloaddition reactions
O
N3
O
N
H
n
18F
18F
18F
18F
N
4-[18F]Fluoro-N-(prop-
[
18F]FPyKYNE (n=0)
18F]FPy5yne (n=1)
Prop-2-yn-1-yl
4-[18F]fluorobenzoate
Azidomethyl-
18F]fluorobenzene
[
2-ynyl)benzamide
[
123

([18F]F-SA): a versatile building block
1169
or trimethylammonium group as labeling precursors (Enas et al.
1997; Supuran et al. 1998; Tang et al. 2007).
yield 1.46 g (99 %) of the desired compound F-SA as yellow
crystals. 1H-NMR (400 MHz, CDCl3), d 2.06 (t, J = 2.6 Hz,
1 H, C:CH), 2.84 (s, 3 H, CH3), 4.05 (d, J = 2.2 Hz, 2 H,
In this work, we describe the radiosynthesis of
4-[18F]fluoro-N-methyl-N-(propyl-2-yn-1-yl)benzene sul-
fonamide ([18F]F-SA) as a versatile building block for
Cu(I)-assisted [3?2]cycloaddition reactions. The one-step
radiosynthesis was performed in an automated synthesis
unit to allow handling of large 18F activity amounts. The
suitability of using [18F]F-SA in click chemistry reactions
with various azide-functionalized biopolymers was dem-
onstrated with a model peptide, a protein (HSA), and a
17-mer L-RNA as an example for oligonucleotides.
1
CH2), 7.17–7.22 (m, 2 H), 7.83–7.87 (m, 2 H). C-NMR
(100 MHz, CDCl3), d 166.1 (1J(C,F) = 239 Hz), 135.3
(4J(C,F) = 2.9 Hz),
130.7
(3J(C,F) = 9.0 Hz),
115.8
(2J(C,F) = 19.7 Hz), 78.0, 73.2, 39.8, 35.1. LR–MS (ESI
positive): m/z = 228 ([M?H]?). Anal. Calculated for
C10H10FNO2S: C 52.85, H 4.44, N 6.16, O 14.08, S 14.11;
found: C 51.63, H 4.01, N 5.86, O 13.88, S 13.65.
4-Nitro-N-methyl-N-(propyl-2-yn-1-yl)benzene
sulfonamide (4)
Materials and methods
N-Methylpropargyl amine 3 (0.5 g, 7.2 mmol) and triethyl-
amine (2 mL) were dissolved in dry CH2Cl2 (10 mL). At
0 °C, a solution of 4-nitrobenzene-1-sulfonyl chloride 2
(1.5 g,6.8 mmol)inCH2Cl2 (2 mL)wasaddeddropwise. The
reaction mixture was stirred for 1 h at room temperature. 1 N
HCl (100 mL) was added, and the mixture is extracted with
EtOAc. The organic layer was filtered through a silica gel
plug, and the solvent is removed under reduced pressure to
yield 1.58 g (96 %) of the desired compound 4 as yellow
crystals. 1H-NMR (400 MHz, CDCl3), d 2.06 (t, J = 2.6 Hz,
1 H, C:CH), 2.90 (s, 3 H, CH3), 4.13 (d, J = 2.2 Hz, 2 H,
CH2), 8.01–8.05 (m, 2 H), 8.35–8.38 (m, 2 H).
General
All reagents and solvents were purchased from commercial
suppliers and used without further purification unless otherwise
specified. Nuclear magnetic resonance spectra were recorded
on a Varian Unity 400 MHz spectrometer. 1H and 13C chem-
ical shifts are given in ppm and were referenced with the
residual solvent resonances relative to tetramethylsilane
(TMS). Mass spectra were obtained on a Quattro/LC mass
spectrometer (MICROMASS) by electrospray ionization
(ESI). MALDI-TOF mass spectra were recorded on a Bruker
Autoflex II TOF/TOF mass spectrometer. Elemental analyses
were conducted using a LECO CHNS-932 apparatus. Column
chromatography was performed on Merck silica gel (mesh size
230-400 ASTM) according to literature procedure (Still et al.
1978). Reactions were monitored by thin-layer chromatogra-
phy (TLC) on Merck silica gel F-254 aluminium plates, with
visualization under UV (254 nm). Active ester 1-succinimidyl-
5-azidopentanoate 8 was prepared according to literature pro-
cedure (Seo et al. 2003). Amine-functionalized L-RNA 11 was
obtained from NOXXON Pharma AG, Berlin, Germany.
Lipophilicity (logP) of [18F]F-SA was determined at pH
7.4 according to the method reported by Wilson et al. (2001).
4-Amino-N-methyl-N-(prop-2-yn-1-yl)benzene
sulfonamide (5)
Nitro compound 4 (0.5 g, 2.0 mmol) and SnCl292H2O (2.25 g,
10 mmol) were refluxed in EtOH (10 mL) for 3 h. The reaction
mixturewas cooled inan icebath, and the pHwasadjustedto 7–8
by the addition of 5 %-Na2SO4 solution. The formed tin salts
were filtered off, and the residue was extracted with EtOAc. The
combined organic layers were washed with brine, dried, and the
solvent was removed under reduced pressure. Compound 5
(0.28 g, 62 %) was obtained as a yellowish-brown solid.
1H-NMR (400 MHz, DMSOd6), d 2.62 (s, 3 H, CH3), 3.17 (t,
J = 2.5 Hz, 1 H, C:CH), 3.86 (d, J = 2.4 Hz, 2 H, CH2), 6.06
(s, 2 H, NH2), 6.59–6.63 (m, 2 H), 7.37–7.41 (m, 2 H).
Chemical syntheses
4-Fluoro-N-methyl-N-(propyl-2-yn-1-yl)benzene
sulfonamide (F-SA)
4-Trimethylammonium-N-methyl-N-(prop-2-yn-1-
yl)benzene sulfonamide triflate (6)
N-Methylpropargyl amine 3 (0.5 g, 7.2 mmol) and trieth-
ylamine (2 mL) were dissolved in dry CH2Cl2 (10 mL). At
0 °C, a solution of 4-fluorobenzene-1-sulfonyl chloride 1
(1.5 g, 7.7 mmol) in CH2Cl2 (2 mL) was added dropwise.
The reaction mixture was stirred for 1 h at room temperature.
1 N HCl (100 mL) was added, and the mixture is extracted
with EtOAc. The organic layer was filtered through a silicagel
plug, and the solvent is removed under reduced pressure to
Compound 5 (0.25 g, 1.1 mmol) and 2,6-di-tert-butyl-4-pyri-
dine (0.46 g, 0.2 mmol) were dissolved in CH2Cl2 (20 mL).
After 5 min, methyl triflate (0.36 g, 2.2 mmol) was added and
the reaction mixture was stirred at room temperature for 12 h.
The white precipitate was filtered off and discarded. The
remaining solution was treated with additional methyl triflate
(0.18 g, 1.1 mmol) under an argon atmosphere. The solvent
was removed under reduced pressure and the resulting brown
123

1170
T. Ramenda et al.
oil was re-dissolved in CH2Cl2 (5 mL). The solution was
cooled in an ice bath, and diethyl ether was carefully added to
formawhiteprecipitate. Thewhiteprecipitate wasfilteredoff,
washedwithdiethylether,anddriedtoafford0.44 g(97 %)of
25 lmol) in CH3CN (300 lL) for 6 h at 40 °C. Purification
of azido-peptide 9 was achieved with HPLC using the
following gradient: 0 min 10 % CH3CN/0.1 % TFA, 90 %
0.1 % TFA; at 30 min 40 % CH3CN/0.1 % TFA; at 45 min
30 % CH3CN/0.1 % TFA. The peak eluting at 24 min was
collected, and subsequent lyophilisation gave pure azido-
peptide 9 as a white solid (7 mg, 64 %). m/z (ESI, positive)
C33H57N10O14PS calcd 880, found 903 [M?Na]?.
1
compound 6 as a light brown solid. H-NMR (400 MHz,
DMSOd6), d 2,80 (s, 3 H, NCH3), 3.11 (t, J = 2.3 Hz, 1 H,
C:CH), 3.64 [s, 9 H, N(CH3)3], 4.08 (d, J = 2.3 Hz, 2 H,
CH2), 8.04–8.07 (m, 2 H), 8.20–8.23 (m, 2 H). m/z (ESI,
positive) C14H19F3N2O5S2, calcd. 267, found 267 [M-OTf]?.
F-SA-triazole-H-Met-Gln-Ser-pThr-Pro Leu-OH (10)
H-Met-Gln-Ser-pThr-Pro Leu-OH (7)
Azido-peptide 9 (1.2 mg, 1.4 lmol), CuSO495 H2O
(0.25 mg, 1 lmol), sodium ascorbate (2 mg, 10 lmol), and
F-SA (1 mg, 4.2 lmol) in borate buffer (500 lL, pH 8.4)
were stirred for 24 h at 40 °C. Purification of F-SA-triazole-
peptide 10 was performed by HPLC using the following
gradient: 0 min 10 % CH3CN/0.1 % TFA, 90 % 0.1 % TFA;
at30 min40 %CH3CN/0.1 % TFA; at45 min30 %CH3CN/
0.1 % TFA. The peak eluting at 27 min was collected, and
subsequent lyophilisation gave pure peptide 10 as a white
solid (0.6 mg, 36 %). m/z (ESI, positive) C43H67FN11O16PS2
calcd 1,107, found 1,108 [M?H]?, 1,130 [M?a]?.
Peptide synthesis was performed on peptide synthesizer (Syro
I, MultiSynTech, Germany) using standard Fmoc chemistry.
Analytical and semi-preparative HPLC was performed on a
Hewlett Packard Series 1100systemusing a Macherey–Nagel
HPLC column (VP250/10, Nucleosil 100-5 C18, Nautilus)
and eluents of CH3CN/0.1 % TFA (eluent A) and H2O/0.1 %
TFA (eluent B) at a flow rate of 2 ml/min. UV detection was
carried out at a wavelength of 220 nm. Phosphopeptide 7 was
prepared 50 mg of the Fmoc-Leu-preloaded resin. After
swelling the resin with 1 ml of DMF for 10 min and 5-times
DMF washing cycles the Fmoc-protecting group was
removed by treatment of 40 % piperidine in DMF for 15 min
and followed by 20 % piperidine in DMF for another 15 min.
A washing step with 500 ll of DMF was repeated 5 times.
Activation and coupling of Fmoc-protected amino acids
(5 equiv.) were achieved using HBTU (5 equiv.) and HOBt
(5 equiv.) dissolved in DMF and DIPEA dissolved in NMP.
An amino acid/HBTU/HOBt/DIPEA ratio of 1/1/1/3 was
applied, and the coupling time for incorporating Fmoc-
Thr(PO(OBzl)–OH)–OH was 2 and 1 h for all other amino
acids. The removal of the Fmoc group with 40 % piperidine in
DMF for 15 min wasembeddedintoa 5-times cycle washstep
with DMF. The complete resin-bound peptide was washed
with EtOH in a last step. The peptide was deprotected and
cleaved from the solid phase with trifluoroacetic acid/water/
thioanisole/1,2-ethanedithiol (87/5/5/3) at room temperature
for 5 h. The peptide was separated from the resin through a
syringe filter and precipitated by the addition of ice-cold
diethylether. The crude peptide was obtained by removing the
residual ether using a syringe filter. Purification by HPLC
using the following gradient: 0–10 min 10 % eluent A, at
20 min 20 % eluent A, at 40 min 30 % eluent A, at 45 min
50 % eluent A, and subsequent lyophilisation gave pure
peptide 7 as a white solid (19 mg, 15.4 lmol, 78 %).
Azide-functionalization of HSA
Human serum albumin (20 mg, 0.3 lmol) was dissolved in
1 ml of 0.15 M phosphate buffer (PBS, pH = 7.5).
1-Succinimidyl-5-azidopentanoate 8 (9 mg, 36 lmol) was
dissolved in DMSO (20 ll) and added to the protein
solution. The mixture was incubated at room temperature
for 2 h. The product was purified by size exclusion chro-
matography using a HiTrap desalting column (5 mL, GE
Healthcare Europe GmbH, Munich, Germany). A sub-
sequent dialysis step against water was performed for 24 h
at 4 °C (cutoff-value 14,000 Da). The resulting solution
was lyophilized and analyzed by MALDI-TOF MS. The
product was obtained as a white solid (5.7 mg, 29 %).
The number of introduced azide residues was determined
by enzymatic digest of the azide-functionalized HSA fol-
lowed by MALDI-TOF MS analysis of the protein fragments
according to literature procedure (Wa et al. 2006). The
enzymes trypsin, endoproteinase Lys-C and endoproteinase
Glu-C were used for enzymatic digest. Each enzyme was used
for a single digest of azide-modified HSA and native HSA as a
control. The resulting solutions were processed according to
the delayed extraction procedure22 prior to MALDI-TOF MS
analysis. Seven modified lysine residues could be detected
after enzymatic digest with trypsin and Glu-C, respectively,
whereas 22 modified lysine residues were determined after
enzymatic digest with Lys-C. Taking into account multiple
detections of modified lysine residues in the different enzy-
matic digests, the total number of azide-modified lysine resi-
dues was determined to be 27.
HPLC analysis: (tR = 21.4 min). m/z (ESI, positive)
C28H50N7O13PS calcd 755.77, found 756.60 [M?H]?.
Azido-H-Met-Gln-Ser-pThr-Pro Leu-OH (9)
Phosphopeptide 7 (9.4 mg, 12.4 lmol) in borate buffer
(700 lL, pH 8.4) was treated with active ester 8 (6 mg,
123

([18F]F-SA): a versatile building block
1171
Azido-L-RNA (12)
Radiochemical syntheses
Amine-containing L-RNA 11 (200 lL, 50 lM solution
of 1 M NaHCO3, 10 nmol) was reacted with active ester
8 (1 mg, 4.2 lmol) for 2 h at 10 °C. The product
was purified by size exclusion chromatography using
a HiTrap desalting column (5 mL, GE Healthcare
Europe GmbH, Munich, Germany) at a flow rate of
0.5 mL/min using 0.1 M NH4OAc as the eluent. Yield:
96 %. m/z (MALDI-TOF MS) calcd 5,662, found 5,663
[M?H]?.
No-carrier added aqueous [18F]fluoride ion was produced in a
IBA CYCLONE 18/9 cyclotron by irradiation of [18O]H2O
via the 18O(p,n)18F nuclear reaction. Syntheses of [18F]F-SA
wascarriedout inanautomatednucleophilicradiofluorination
module (GE TRACERlabÒ FX-FN). The synthesis module
was modified in terms of program and hard-ware. The syn-
thesis module consists of two main parts: reactor 1 with res-
ervoir vessels 1–6 and reactor 2 with reservoir vessels 7–12.
The reservoir vessels contain the starting materials and are
sealed by septa. The reaction vessels are made from glassy
carbon and sealed with a PEEK cover. The reactors are con-
nected to each other. All connections were made via Teflon
tubings and valves. All reagents and solvents are manipulated
within the module by vacuum or auxiliary gas (nitrogen). The
reactionmixtures in the reactionvessels can be stirred, heated,
cooled, evaporated and pressurized. Evacuation of the reac-
tors is performed with a vacuum pump via a cooling trap filled
with liquid nitrogen. Volatile radioactive substances are thus
trapped. Radioactivity detectors monitor the radioactivity
level in the reactors and in the vessel for the final product.
Before starting the synthesis, a cleaning program for the
module was carried out. Then, the reservoir vessels were
loaded with the required reagent solutions or solvents.
F-SA-triazole-L-RNA (13)
Azido-L-RNA 12 (14.2 lg, 2.5 nmol) and F-SA (1 mg,
4.2 lmol) were dissolved in 1 M NaHCO3 (190 lL) and
50 lL of CH3CN. CuBr (0.1 mg) and TBTA (0.4 mg)
dissolved in DMSO (10 lL) were added, and the reac-
tion mixture was stirred 90 min at 10 °C. The product
was purified by size exclusion chromatography using
a HiTrap desalting column (5 mL, GE Healthcare
Europe GmbH, Munich, Germany) at a flow rate of
0.5 mL/min using 0.1 M NH4OAc as the eluent. Yield:
28 %. m/z (MALDI-TOF MS) calcd 5,888, found 5,889
[M?H]?.
Scheme of the automated synthesis unit for the synthesis of 4-[18F]fluoro-N-methyl-N-(propyl-2-yn-1-yl)
benzene sulfonamide ([18F]F-SA).
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T. Ramenda et al.
4-[18F]Fluoro-N-methyl-N-(propyl-2-yn-1-yl)benzene
sulfonamide ([18F]F-SA)
Radiolabeling of azide-modified HSA
Azide-modified HSA (0.1–0.4 mg, 2–6 nmol), cop-
per(I) bromide (0.2 mg, 1.4 lmol), and TBTA (1 mg,
1.9 lmol) were dissolved in a mixture of phosphate buffer
(140 ll, pH = 7.5) and DMSO (10 ll). The solution was
added to a vial containing [18F]F-SA. The mixture was
incubated for 20 min at 30 °C. The product was purified by
size exclusion chromatography using a HiTrap desalting
column (5 mL, GE Healthcare Europe GmbH, Munich,
Germany) and PBS as eluent. In a typical experiment,
starting from 450 MBq of [18F]F-SA, 250 MBq of radio-
labeled HSA could be obtained, representing a radio-
chemical yield of 55 % based upon [18F]F-SA.
The synthesis started with the elution of [18F]fluoride from the
anion exchange cartridge into the reactor 1 using a solution of
Kryptofix 222 (15 mg; 40 lmol) and potassium carbonate
(2.77 mg; 20 lmol) in aqueous CH3CN (1.5 ml; 66 %
CH3CN). The eluate was dried by means of a vacuum and a
nitrogen stream at 95 °C. The reaction mixture was carefully
dried by addition and evaporation of anhydrous CH3CN
(1 9 3 ml). 4-Trimethylammonium-N-methyl-N-(prop-2-
yn-1-yl)benzene triflate 6 (2.5 mg, 6 lmol) in sulfolane
(1 mL) was added to dried [18F]fluoride in reactor 1. The
mixture was heated at 80 °C for 10 min. The mixture was
cooled to 50 °C, and 1 N HCl was added. The solution was
passed through a C18 cartridge (SepPakÒPlus, Waters). The
cartridge was washed with water, and the product was eluted
with CH3CN into a vessel containing 3 mL of water. The
mixture is loaded onto a semi-preparative HPLC for purifi-
cation [Phenomenex RP-18 column (LUNA C18(2)
10 9 250 mm, 10 lm] column using isocratic elution with
CH3CN/0.1 M TFA (40/60) at a flow rate of 2.5 mL/min) The
peak eluting at 28–30 min was collected in a vial containing
25 mL of water. The mixture was passed through a C18 car-
tridge (SepPakÒPlus, Waters), and the product was eluted
with ethyl ether (1 mL). Ethyl ether was removed in a stream
of nitrogen. In a typical experiment, starting from 16 GBq of
[18F]fluoride, 6.5 GBq (32 % decay-corrected) of compound
[18F]F-SA could be obtained in a total synthesis time of
80 min, including HPLC purification.
[18F]F-SA-triazole-L-RNA ([18F]13)
Azido-L-RNA 12 (0.1 mg) was dissolved in 1 M NaHCO3
(100 lL). A solution of TBTA (0.4 mg) in DMSO (10 lL)
and CuBr (0.2 mg) in 1 M NaHCO3 (40 lL) was added to
the oligonucleotide solution, and the combined solution
was added to [18F]F-SA in CH3CN (50 lL). The reaction
mixture was kept at 10 °C for 20 min. The reaction mix-
ture was analyzed with HPLC (Waters XTerraÒMS C18,
2.5 lm, 3.0 9 50 mm) using gradient elution with CH3CN
(A) and 50 mM TEAA at a flow rate of 0.3 mL/min.
Gradient: 0 min 5 % A, 95 % B; 30 min 50 % A, 50 % B.
Radiochemical yield: 30 % (based upon radio-HPLC
analysis tR = 16.4 min.
Radio-TLC (SiO2, petroleum ether/ethyl acetate 2:1)
Rf = 0.4. Radio-HPLC-analysis (Phenomenex RP-18 col-
umn (LUNA C18(2) 4.6 9 250 mm, 5 lm, 1 mL/min,
CH3CN/0.1 M TFA (40/60), tR = 10.4 min.
Results and discussion
Synthesis of 4-[18F]fluoro-N-methyl-N-(propyl-2-yn-1-
yl)benzene sulfonamide [18F]F-SA
[18F]F-SA-triazole-H-Met-Gln-Ser-pThr-Pro Leu-OH
([18F]10)
Literature reports describing radiosynthesis of 18F-labeled
sulfonamides clearly demonstrate the benefit of using ter-
tiary sulfonamides over primary and secondary sulfona-
mides to achieve reasonable radiochemical yields. Primary
and secondary sulfonamides contain acidic protons at the
sulfonamide nitrogen which can quench the reactivity of dried
[18F]fluoride when exposed to the typical basic reaction con-
ditions of nucleophilic radiofluorinations. Consequently, only
very low radiochemical yields (0.5–2 %) were obtained with
primary and secondary sulfonamides as reported by Hocke
et al. (2005), Shiue et al. (1999), Supuran et al. (1998). Sig-
nificantly higher radiochemical yields of 23–32 % were
obtained when tertiary sulfonamides were used as shown by
Enas et al. (1997), Tang et al. (2007).
In a typical reaction, a solution of azido-peptide 9 (0.3 mg,
0.34 lmol), CuSO495 H2O (0.3 mg, 1.2 lmol), sodium
ascorbate (2.4 mg, 12 lmol) in borate buffer (150 lL, pH 8.4)
was added to [18F]F-SA (50–150 MBq) in borate buffer
(100 lL, pH 8.4). The reaction was kept at 40 °C for 10 min.
Radiolabeled peptide [18F]10 was purified using semi-pre-
parative HPLC as described for reference compound 10. The
peak eluting at 27 min was collected, and the solvent was
evaporated under reduced pressure. The residue was re-dis-
solved in saline (1 mL).
Radiochemical yield: 42 % (isolated, after semi-pre-
parative HPLC), 73 % (based upon radio-HPLC analysis).
Radio-HPLC-analysis (Phenomenex RP-18 column (LUNA
C18(2) 4.6 9 250 mm, 5 lm, 1 mL/min, CH3CN/0.1 M
TFA (40/60), tR = 4.8 min.
Based on these findings and the aim to create a meta-
bolically stable C–F bond, we set up the synthesis of a
18F-labeled alkyne group-containing aromatic tertiary
123

([18F]F-SA): a versatile building block
1173
sulfonamide as click chemistry building block for sub-
sequent copper (I)-assisted [3?2]cycloaddition reactions.
The synthesis of respective 4-fluoro-N-methyl-N-(pro-
pyl-2-yn-1-yl)benzene sulfonamide F-SA as reference
compound and 4-trimethylammonium-N-methyl-N-(pro-
pyl-2-yn-1-yl)benzene sulfonamide triflate 6 as labeling
precursor is illustrated in Scheme 2.
potassium carbonate. Other solvents like DMF and acetoni-
trile gave lower radiochemical yields of 15–20 %.
Labeling precursor 6 in sulfolane was transferred from
reservoir 3 of the synthesis unit into the reactor vial which
contained dried [18F]fluoride. The reaction vial was sealed
and heated at 80 °C for 10 min. After cooling the reaction
vial to 50 °C, 1 N HCl (10 mL) was added from reservoir
4, and the reaction mixture was passed through a C18
cartridge (SepPakÒPlus, Waters). The cartridge was
washed with water (3 mL) from reservoir 5, and the
product was eluted with acetonitrile (1 mL) from reservoir
6. The eluate was subjected to HPLC purification, and
compound [18F]F-SA was isolated in 32 5 % decay-
corrected radiochemical yield at high radiochemical purity
of greater 95 %. The total synthesis time was 80 min
including HPLC purification. The specific activity was
determined in the rage of 120–570 GBq lmoL-1. The use
of an automated synthesis unit allowed the handling of
large starting activities of [18F]fluoride. In a typical
experiment, starting from 16 GBq of n.c.a. [18F]fluoride,
6.5 GBq of isolated [18F]F-SA could be prepared. The
obtained large activity amounts of [18F]F-SA allow
numerous applications of this building block within various
click chemistry reactions.
Reference compound F-SA was prepared in almost
quantitative yield of 99 % in a single step through acyla-
tion reaction of N-methylpropargyl amine with commer-
cially available 4-fluorobenzene-1-sulfonyl chloride 1 in
CH2Cl2 in the presence of TEA as base. Labeling precursor
6 was obtained in a three step synthesis sequence in overall
yield of 58 %.
The first reaction step involved acylation of N-methyl-
propargyl amine with 4-nitrobenzene-1-sulfonyl chloride 2 to
affordcompound4 in97 %yield.Reductionofthenitrogroup
with SnCl2 gave primary amine 5 in 62 % yield. Primary
amine 5 was then treated with an excess of MeOTf in CH2Cl2
in the presence of 2,6-di-tert-butyl-4-methylpyridine (DBMP)
to afford labeling precursor 6 in 97 % yield.
The radiosynthesis of 4-[18F]fluoro-N-methyl-N-(pro-
pyl-2-yn-1-yl)benzene sulfonamide [18F]F-SA was per-
formed in a remotely controlled synthesis unit (GE
TRACERlabÒ FX-FN). The radiosynthesis of [18F]F-SA is
displayed in Scheme 3.
Lipophilicity (logP) of [18F]F-SA
Based on the results of various manual syntheses to opti-
mize reaction conditions, it was shown that reaction of 2.5 mg
of labeling precursor 6 in sulfolane as the solvent at 80 °C for
10 min afforded best radiochemical yields for the radiofluo-
rination step with powerful nucleophilic radiofluorination
agent [18F]KF in the presence of Kryptofix K222 and
Radiolabeling of biopolymers like peptides, proteins, and
oligonucleotides should preferentially occur under physi-
ological condition to avoid decomposition and alteration or
even loss of biological activity of delicate biopolymers.
This especially includes reactions in aqueous media at
Scheme 2 Synthesis of
reference compound F-SA and
labeling precursor 6
18F]KF, K222
sulfolane
Scheme 3 Radiosynthesis of
[
O
O
O
O
4-[18F]fluoro-N-methyl-N-
(propyl-2-yn-1-yl)benzene
sulfonamide [18F]F-SA
OTf
S
S
N
N
80 oC, 10 min
CH3
Me3N
18F
CH3
18F]F-SA
6
[
123

1174
T. Ramenda et al.
physiological pH at ambient temperature. Reactions in
aqueous media require good solubility of all reaction
partners in water. Lipophilicity (logP) is an important
parameter of a molecule which contributes to its solubility
in aqueous media and, therefore, its reactivity. This is of
special importance for radiolabeled compounds at high
specific activity as found for click chemistry building block
[18F]F-SA. However, recently Kostikov et al. (2012)
showed that also lipophilic labeling synthons can be used
in aqueous solutions.
The lipophilicity of compound [18F]F-SA was deter-
mined to be 1.70 0.11 at pH 7.4 using the shake flask
method. This value is comparable to that of prominent
prosthetic group [18F]SFB (logP = 1.75 0.17), which is
frequently used for peptide and protein labeling under
aqueous conditions (Wuest et al. 2009). The determined
logP value of 1.70 0.11 for [18F]F-SA supports appli-
cation of this click chemistry building block under desir-
able aqueous conditions as typically employed for
copper(I)-assisted [3?2]cycloaddition reactions.
functionalized phosphopeptide 9 and peptide triazole 10 is
depicted in Scheme 4.
Azide-modified phosphopeptide 9 was used for cop-
per(I)-assisted [3?2]cycloaddition reaction with [18F]F-
SA. The reaction conditions were adapted from our pre-
vious work describing the 18F-labeling of neuroten-
sin(8–13) through Cu(I)-mediated click chemistry, except
for the amount of azide-modified peptide (Ramenda et al.
2007). In this previous work using Cu(I)-mediated click
chemistry, we could prepare 18F-labeled neurotensin(8–13)
in 66 % radiochemical yields starting from 1 mg of cor-
responding azide-modified peptide.
In a series of experiments using click chemistry with
[18F]F-SA, we tested different amounts of azide-modified
peptide, which were in all cases lower compared to the
used 1 mg of peptide as reported for the click chemistry-
based radiosynthesis of 18F-labeled neurotensin(8–13). The
progress of the click chemistry reaction between azide-
modified phosphopeptide 9 and [18F]F-SA depending on
the amount of used peptide was monitored by radio-HPLC
analysis. The obtained radiochemical yields are summa-
rized in Table 1.
Radiolabeling of azide-functionalized phosphopeptide
9 with [18F]F-SA
The results in Table 1 clearly demonstrate the impor-
tance of the amount of azide-functionalized peptide 9 upon
the radiochemical yield during the Cu(I)-mediated click
chemistry reaction with [18F]F-SA to afford radiolabeled
peptide [18F]10. The highest radiochemical yield of 77 %
was obtained when 0.4 mg of peptide was used (entry 4).
Lowering the peptide amount to 0.3, 0.2, and 0.1 mg
resulted also in lower radiochemical yields of 73, 64, and
49 %, respectively (entries 1–3). These results indicate, that
Cu(I)-mediated click chemistry between [18F]F-SA and
azide-functionalized peptide 9 proceeds in moderate to high
radiochemical yields even at very low peptide amounts of
0.1 mg. This represents an important improvement compared
to previously reported Cu(I)-mediated click chemistry reac-
tions with various peptides using [18F]fluoroaryl compounds
as summarized in Scheme 1. In these reports, typical peptide
amountswereintherangeof1–2 mgleadingtoradiochemical
yields of 37–90 % (Inkster et al. 2008; Kuhnast et al. 2008;
Ramenda et al. 2007; Thonon et al. 2009; Vaidyanathan and
Zalutsky 1992).
Recently, we have described the radiolabeling of a small
phosphopeptide as ligand for the polo-box domain (PBD) of
polo-like kinase 1 (Plk1) with acylation agent [18F]SFB
(Richter et al. 2009). Radiolabeling was accomplished via
conjugation of the N-terminus of PBD-binding phosphopep-
tide H-Met-Gln-Ser-pThr-Pro-Leu-OH 7 with [18F]SFB.
For the labeling of phosphopeptide H-Met-Gln-Ser-pThr-
Pro-Leu-OH 7 with [18F]F-SA according to a copper (I)-
assisted [3?2]cycloaddition, we had to introduce an azide
group into the peptide. Introduction of azide group at the
N-terminal end of phosphopeptide 7 was achieved by the
reactionof1-succinimidyl-5-azidopentanoate8 (2.1 equiv)in
asolutionofCH3CN/Kolthoffbuffer(pH 8.4)(Kolthoff1925)
[3/7 (v/v)] at 40 °C for 6 h. Active ester 8 was recently used in
our group for functionalization of neurotensin(8–13) and
subsequent click chemistry reactions (Richter et al. 2010).
Azide-modified peptide 9 was obtained in 64 % yield
after purification of the reaction mixture by means of
HPLC and subsequent lyophilisation. The purity of peptide
9 exceeded 95 % as confirmed by HPLC analysis, and the
identity was determined by means of electron spray ioni-
zation mass spectrometry (ESI–MS) showing the expected
mass peak at m/z = 903 [M?Na]?. Subsequent click
chemistry reaction of peptide 9 with F-SA in the presence
of CuSO4 and sodium ascorbate as the reducing agent in
Kolthoff’s borate buffer (pH 8.4) gave triazole 10 as ref-
erence compound in 36 % isolated yield after HPLC
purification. Identity of compound 10 was confirmed by
ESI–MS (m/z = 1,108 [M?H]?). Synthesis of azide-
In this work, the obtained high radiochemical yields
(73–77 %) at peptide concentrations as little as 0.3–0.4 mg
make the described Cu(I)-mediated click chemistry reac-
tion with [18F]F-SA a highly promising and suitable bio-
conjugation tool for labeling of peptides with 18F. The use
of a delicate phosphopeptide 9 for Cu(I)-mediated click
chemistry reaction further demonstrates the compatibility
of the labeling method with even highly sensitive com-
pounds. This makes the presented click chemistry reaction
highly versatile and broadly applicable for wide variety of
azide-modified peptides.
123

([18F]F-SA): a versatile building block
1175
Scheme 4 Synthesis of
functionalized phosphopeptides
9 and 10
Table 1 Dependency of the radiochemical yield on the amount of
azide-modified phosphopeptide 9 used in the Cu(I)-mediated click
chemistry reaction with [18F]F-SA
The amino acid sequence of HSA contains 59 lysine
residues which can be used for the introduction of azide
groups via acylation reaction with active ester 1-succini-
midyl-5-azidopentanoate 8. HSA in PBS was treated with
a 10 to 12-fold molar excess of active ester 8 to give
azide-functionalized HSA in 29 % yield after purification
with size exclusion chromatography (SEC). The number
of introduced azide residues was determined by enzymatic
digest of azide-functionalized HSA followed by MALDI-
TOF MS analysis of the protein fragments according to
literature procedure (Wa et al. 2006). The enzymes tryp-
sin, endoproteinase Lys-C and endoproteinase Glu-C were
used for enzymatic digest. Each enzyme was used for a
single digest of azide-modified HSA and native HSA as a
control. The resulting solutions were processed according
to the delayed extraction procedure prior to MALDI-TOF
MS analysis (Wa et al. 2006). Seven modified lysine
residues could be detected after enzymatic digest with
trypsin and Glu-C, respectively, whereas 22 modified
lysine residues were determined after enzymatic digest
with Lys-C. Taking into account multiple detections of
modified lysine residues in the different enzymatic
digests, the total number of azide-modified lysine residues
in HSA was determined to be 27. All these azide groups
Entry
Peptide amount 9 (mg)
Radiochemical yield (%)a
1
2
3
4
0.1
0.2
0.3
0.4
49
64
73
77
All reactions were carried out using CuSO4 (0.3 mg), Na-ascorbate
(2.4 mg) in borate buffer (300 lL, pH 8.6) at 40 °C for 10 min
a
Radiochemical yield was determined by radio-HPLC referring to
the percentage of the radioactivity area of radiolabeled peptide
[
18F]10 related to the total radioactivity area
Radiolabeling of azide-functionalized human serum
albumin (HSA) with [18F]F-SA
The success of using [18F]F-SA for radiolabeling of sen-
sitive compounds like phosphopeptides prompted us to test
the suitability of [18F]F-SA for Cu(I)-mediated click
chemistry reactions with proteins. We chose human serum
albumin (HSA) as model peptide. The 67 kDa protein HSA
is the most abundant protein in human blood plasma.
123

1176
T. Ramenda et al.
are potentially available for Cu(I)-mediated click chem-
istry reaction.
reactions. This general procedure should also be applicable
for the radiolabeling of other proteins with 18F.
First experiments to apply Cu(I)-mediated click chem-
istry to the radiolabeling of azide-HSA failed when in situ
reduction of Cu(II) salts like CuSO4 into Cu(I) in the
presence of Na-ascorbate was used. This procedure led to
significant degradation of azide-HSA presumably due to
Na-ascorbate induced partly or complete reduction of the
17 disulfide bridges abundant in the protein. However,
direct application of Cu(I) salts such as CuBr or CuI would
avoid the use of detrimental reducing agents. Various
multidentate triazoles are known as excellent chelates for
stable complex formation of Cu(I) preventing re-oxidation
of Cu(I) into Cu(II) (Chan et al. 2004; Donnelly et al.
2008). Therefore, we used oligotriazole tris[(1-benzyl-1H-
1,2,3-triazol-4-yl)-methyl]-amine (TBTA) as suitable che-
lator for stabilizing Cu(I). Cu(I)-TBTA complex was
applied to the click chemisty reaction between azide-
functionalized HSA and [18F]F-SA. The reaction was
performed in phosphate buffer (pH 7.5) at 30 °C for
20 min. The product was purified by SEC to give 18F-
labeled HSA in 55–60 % decay-corrected radiochemical
yield based upon [18F]F-SA.
Radiolabeling of azide-functionalized oligonucleotide
with [18F]F-SA
In the last decade, several oligonucleotide probes have
been developed for molecular targeting and imaging of
disease-specific mRNA, and various radiolabeled antisense
oligonucleotides were used for imaging endogenous gene
expression at the transcriptional level (Tavitian 2003;
Roivainen et al. 2004). Moreover, the development of the
systematic evolution of ligands by exponential enrichment
(SELEX) process enabled isolation of oligonucleotide
sequences, also termed as aptamers, capable of targeting
virtually any class of target molecules with high affinity
and specificity (Ellington and Szostak 1990).
18F labeling reactions of oligonucleotides were accom-
plished via various bioconjugation reactions involving
thiourea formation, photoconjugation, acylation reactions
(e.g. [18F]SFB) and alkylation reactions (e.g. [18F]FPy-
BrA). However, only very few reports describe the use of
Cu(I)-mediated click chemistry as a bioconjugation method
for oligonucleotides. This also includes radiolabeling
reactions, and only one report describes the use of Cu(I)-
mediated click chemistry for the radiolabeling with 18F based
on [18F]FPy5yne in the presence of Cu(I) stabilizing ligand
TBTA (Inkster et al. 2009). More recently, a click chemistry-
based approach was also reported for the radiolabeling of
siRNA with fluorine-18 (Flagothier et al. 2012).
Figure 1 shows the results of SEC purification and
subsequent polyacrylamide gel electrophoresis with
sodium dodecylsulfate (SDS-PAGE) for analysis of the
reaction mixture and purified fractions.
This results clearly demonstrate the principle feasibility
to use Cu(I)-mediated click chemistry for the labeling of
proteins with 18F. The reaction conditions are compatible
with the short half-life of 18F. However, no further exper-
iments were carried out to reduce the number of azide
groups in the protein to further optimize the click chemistry
reaction. The application of Cu(I)-TBTA complex provides
a reliable and stable source of Cu(I) for the click chemistry,
and it circumvents in situ reduction of Cu(II) salts as typ-
ically employed for Cu(I)-mediated [3?2]cycloaddition
The reason that only very few applications are known
using Cu(I)-mediated click chemistry for bioconjugation
reactions with oligonucleotides may mainly stem from the
destructive effects of Cu(I)-mediated generation of highly
reactive hydroxyl radicals upon nucleobase oxidation. This
effect has been reviewed previously (Burrows and Muller
1998).
Fig. 1 SEC profile of the
reaction mixture (left) and
SDS-PAGE analysis of reaction
mixture (lane 1) and purified
fractions (lanes 2–5) (right)
123

([18F]F-SA): a versatile building block
1177
Scheme 5 Functionalization of
oligonucleotide 11 with an azide
group
O
O
N3
O N
8
O
5'-CCGCACCGCACAGCCGC-3'
H2N
5
1M NaHCO3
2 h, 10 °C
11
O
5'-CCGCACCGCACAGCCGC-3'
N3
N
5
H
12
However, based on our promising results using [18F]F-
SA for Cu(I)-mediated click chemistry reactions with
peptides and proteins, we wanted to expand the scope of
this reaction also to oligonucleotides.
This described procedure represents an useful alterna-
tive to the recently reported click chemistry-based radio-
synthesis of an 18F-labeled oligonucleotide by Inkster et al.
(2009). The authors could isolate a 18F-labeled oligonu-
cleotide in 25 % radiochemical yields (decay-corrected),
but the total synthesis time was more than 4.5 h including
the synthesis of click chemistry building block
[18F]FPy5yne and purification of radiolabeled oligonu-
cleotide. Considering a synthesis time for click chemistry
building block [18F]F-SA of 80 and a 20 min optimal
synthesis time for the [3?2]cycloaddition between azide-
functionalized oligonucleotide and [18F]F-SA, we could
reduce synthesis time of radiolabeled oligonucleotide to
100 min, excluding purification.
For this purpose we modified a model 17-mer L-RNA
(50-CCGCACCGCACAGCCGC-30) 11 which contains an
aminohexyl spacer at the 50-end for functionalization with
active ester 1-succinimidyl-5-azidopentanoate 8. The
acylation reaction is depicted in Scheme 5.
Acylation reaction of amine-containing oligonucleotide
11 (NOXXON Pharma AG, Berlin, Germany) with an
excess of 1-succinimidyl-5-azidopentanoate 8 had to be
performed at low temperatures of 10 °C to avoid decom-
position. The reaction was completed after 2 h, and only
traces of oligonucleotide 11 as starting material was
detectable as indicated by HPLC analysis of the reaction
mixture. Purification of azide-functionalized oligonucleo-
tide 12 was achieved through SEC to remove the excess of
active ester 8.
Our method and the recently reported synthesis by
Inkster et al. demonstrate that Cu(I)-mediated click
chemistry is a suitable method for the radiolabeling of
oligonucleotides with 18F. This method should be appli-
cable to other nucleic acid-based compounds such as
peptide nucleic acids (PNAs) or aptamers. Current methods
for the 18F-labeling of nucleic acid-based compounds is
mainly limited to alkylation reactions using prosthetic
group 4-[18F]fluorobenzyl-2-bromo-acetamide ([18F]FBBA)
and related compounds.
Azide-containing oligonucleotide 12 was treated with F-
SA in the presence of CuBr as source for Cu(I) and TBTA
as ligand to stably complex Cu(I). The stable Cu(I)-TBTA
complex allows for the [3?2]cycloaddition reaction
between azide-modified oligonucleotide and F-SA while
avoiding oxidative decomposition of the oligonucleotide
through hydroxyl radicals generated in the presence of
in situ formed Cu(I) ions.
In this line, Cu(I)-mediated click chemistry has the
potential to further expand the opportunities to prepare
nucleic acid-based probes labeled with 18F for molecular
imaging applications.
Treatment of azide-modified oligonucleotide 12
(14.2 lg, 2.5 nmol) with F-SA (1 mg, 4.4 lmol) in the
presence of CuBr and TBTA at 10 °C for 90 min in 1 M
NaHCO3/DMS) (19/1, 200 lL) afforded 28 % of desired
triazole 13 after HPLC purification. Identity of triazole 13
was confirmed by MALDI-TOF MS showing a single mass
peak at m/z 5,889 ([M?H]?) which corresponds with the
expected mass. Radiosynthesis was performed under
comparable conditions, and 18F-labeled oligonucleotide
[18F]13 could be prepared in 44 % as determined by radio-
HPLC analysis of the reaction mixture. Extension of the
reaction time from 20 min to up to 80 min did not result in
an increase of radiochemical yield. The radiosynthesis of
18F-labeled oligonucleotide [18F]13 is given in Scheme 6.
Summary and conclusion
Cu(I)-mediated [3?2]cycloaddition between azides and
alkynes has evolved into a valuable bioconjugation tool in
radiopharmaceutical chemistry. The reaction proved to be a
valuable tool for the incorporation of various radionuclides,
including 18F, into a broad variety of structural and func-
tional diverse compounds under mild reaction conditions.
Radiolabeling of bipolymers like peptides, proteins and
oligonucleotides with the short-lived positron emitter fluo-
rine-18 represents a special challenge. Most bioconjugation
123

1178
T. Ramenda et al.
Scheme 6 Cu(I)-mediated
click chemistry for the synthesis
of 18F-labeled oligonucleotide
[
18F]13
methods still rely on the use of various prosthetic groups for
acylation reactions (e.g. [18F]SFB), alkylation reactions
(e.g. [18F]FBAM), and oxime formation (e.g. 4-[18F]fluo-
robenzaldehyde). The list of novel bioconjugation methods
is constantly growing, and examples for Si–F and Al–F
chemistry, as well as examples for copper-free click
chemistry were recently added to the list.
chemistry can also be expanded to the 18F labeling of
peptides as exemplified for HSA. This result has further
expanded the scope of Cu(I)-mediated click chemistry with
18F as a versatile bioconjugation tool in radiopharmaceu-
tical chemistry. In combination with recent developments
to incorporate diverse side chains, including azides, in pre-
defined positions of a given protein, the presented holds
promise for future site-specific labeling reactions of pro-
teins with 18F (de Graaf et al. 2009; Liao 2007; Voloshchuk
and Montclare 2010). Our work has also confirmed a recent
report on the principle feasibility to apply Cu(I)-mediated
click chemistry for the radiolabeling of oligonucleotides
with 18F, while achieving further improvement of the
radiolabeling method.
The application of Cu(I)-mediated [3?2]cycloadditions
as an innovative and versatile bioconjugation tool for 18F
labeling reactions was convincingly demonstrated for a
broad variety of peptides. Most reports used various vola-
tile [18F]fluoroalkyl-alkynes or -azides as click chemistry
building blocks, whereas only few examples dealt with
metabolically more stable [18F]fluoroaryl compounds.
In this report we have described the simple, convenient
and reliable radiosynthesis of [18F]F-SA as an aromatic
sulfonamide-based click chemistry building block. [18F]F-
SA could be prepared in a remotely controlled synthesis
unit to provide sufficient amounts of the radiotracer at high
specific activity for subsequent Cu(I)-mediated click
chemistry reactions. The determined lipophilicity of
[18F]F-SA (logP = 1.7) allows handling of the radiotracer
in aqueous solutions as preferred for click chemistry
reactions and for the use of delicate biopolymers like
peptides, proteins and oligonucleotides. The versatility of
[18F]F-SA as click chemistry building block was demon-
strated by the labeling of a model peptide (phosphopep-
tide), protein (HSA), and an oligonucleotide (L-RNA).
Especially the radiolabeling of phosphopeptide 9 with
[18F]F-SA proved to be a superior 18F labeling method for
peptides compared to commonly employed acylation
reactions with [18F]SFB. The reaction proceeds in good
radiochemical yields (49–77 %) despite the use of only
very little peptide amounts (0.1–0.4 mg). This is a signif-
icant improvement compared to the required typical min-
imum peptide amounts of 1–2 mg for popular [18F]SFB
labeling reactions to achieve comparable radiochemical
yields. We could demonstrate that Cu(I)-mediated click
In conclusion, despite the recent emergence of a multi-
tude of highly innovative novel bioconjugation methods for
18F labeling of biopolymers, Cu(I)-mediated click chem-
istry with [18F]F-SA continues to represent a reliable,
robust and efficient radiolabeling technique for peptides,
proteins, and oligonucleotides with the short-lived positron
emitter 18F.
Acknowledgments The authors are grateful to Mareike Barth, Inge
¨
Kozle, Stephan Preusche and Tilow Krauss for their excellent tech-
nical assistance.
Conflict of interest The authors declare that they have no conflict
of interest.
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