Synthesis and reactivity of a bis-sultone cross-linker for peptide conjugation and [18F]-radiolabelling via unusual "double click" approach

Article abstract of DOI:10.1039/c1ob06600e

A novel homobifunctional cross-linker based on a bis-sultone benzenic scaffold was synthesised. The potential utility of this bioconjugation reagent was demonstrated through the preparation of an original prosthetic group suitable for the [¹⁸F]-labelling of peptides. The labelling strategy is based on the nucleophilic fluorination via the ring-opening of a first sultone moiety followed by the nucleophilic ring-opening of the second remanent sultone by a reactive amine of the biopolymer. Beyond the one-step radiolabelling of the peptide, the second main advantage of this strategy is the release of free sulfonic acid moieties making the separation of the targeted [¹⁸F]-tagged sulfonated compound from its non-sulfonated precursor easier and thus faster. This first report of the successful use of a bis-sultone moiety as a versatile bioconjugatable group was demonstrated through a comprehensive reactivity study involving various nucleophiles, especially those commonly found in biopolymers. An illustrative example, highlighting the potential of this unusual and promising "double click" conjugation approach, was devoted to the radiolabelling of a biological relevant peptide.

Full text of DOI:10.1039/c1ob06600e

View Article Online / Journal Homepage / Table of Contents for this issue
Organic &
Biomolecular
Chemistry
Dynamic Article Links
Cite this: Org. Biomol. Chem., 2012, 10, 1068
www.rsc.org/obc
PAPER
Synthesis and reactivity of a bis-sultone cross-linker for peptide conjugation
and [18F]-radiolabelling via unusual “double click” approach†
Thomas Priem,a,b Ce´dric Bouteiller,*a David Camporese,a Anthony Romieub,c and Pierre-Yves Renardb,c,d
Received 19th September 2011, Accepted 27th October 2011
DOI: 10.1039/c1ob06600e
A novel homobifunctional cross-linker based on a bis-sultone benzenic scaffold was synthesised. The
potential utility of this bioconjugation reagent was demonstrated through the preparation of an
original prosthetic group suitable for the [18F]-labelling of peptides. The labelling strategy is based on
the nucleophilic fluorination via the ring-opening of a first sultone moiety followed by the nucleophilic
ring-opening of the second remanent sultone by a reactive amine of the biopolymer. Beyond the
one-step radiolabelling of the peptide, the second main advantage of this strategy is the release of free
sulfonic acid moieties making the separation of the targeted [18F]-tagged sulfonated compound from its
non-sulfonated precursor easier and thus faster. This first report of the successful use of a bis-sultone
moiety as a versatile bioconjugatable group was demonstrated through a comprehensive reactivity
study involving various nucleophiles, especially those commonly found in biopolymers. An illustrative
example, highlighting the potential of this unusual and promising “double click” conjugation approach,
was devoted to the radiolabelling of a biological relevant peptide.
requires the prior incorporation of a suitable precursor within the
Introduction
peptidyl scaffold, which must be highly reactive toward fluoride-
In the context of positron emission tomography (PET) imaging, it
18 anion through an SN2 or SNAr reaction, especially to avoid
is generally accepted that fluorine-18 (18F) is one of the widely used
side-reactions leading to the decomposition of biopolymer. The
radioisotopes because it possesses unique physical and nuclear
second one, which is the most commonly used because of the
characteristics (i.e., ease of formation, half-life of 109.7 min, high
limitations and drawbacks of the direct labelling approach (vide
resolution, and relative minor radiation dose to patients) suitable
supra), is based on the use of pre-designed prosthetic groups.
for challenging biomedical diagnostic applications frequently in-
Thus, a large number of prosthetic groups allowing the mild
volving peptide-based probes.1,2,3 Two main conjugation strategies
are theoretically possible for the radiolabelling of complex and
fragile biomolecules/biopolymers (e.g., peptides or proteins) with
18F.
The first one is based on the direct no-carrier-added [18F]-
labelling. Indeed, despite the publication of a review claiming that
the direct nucleophilic fluorination of biopolymers is impossible
due to the presence of numerous H-acidic functions in such
biomacromolecules,4 there have been recently several attempts
to implement this strategy to peptides.5 Thus, some studies have
shown that the direct [18F]-labelling of peptides is feasible but
and site-specific introduction of 18F into bioactive molecules have
been developed.4 However, only a few of them have emerged as
universal [18F]-labelling reagents because specifications required
for PET imaging applications are numerous and highly restrictive:
1) a limited number of reaction steps for its [18F]-labelling and
its subsequent introduction onto the biomolecule/biopolymer,
2) short overall reaction time, 3) high [18F]-labelling yield, 4)
high efficiency of the subsequent conjugation to the targeted
biomolecule/biopolymer, 5) good chemoselectivity of the conju-
gation step, 6) ease of purification of the intermediates and final
products from the precursors, and 7) no or limited influence of the
introduced prosthetic group on ligand pharmacokinetics. Most
of the prosthetic groups are designed to selectively react with a
specific functional group of targeted peptide/protein. Thus, they
are often classified according to the type of reaction leading to their
covalent coupling to the biomolecular target. The most frequently
used reactions are [18F]-fluoroacylation, [18F]-fluoroalkylation,
[18F]-fluoroamidation, thiol-alkylation (through Michael addition
or SN2 reaction)6 or photochemical conjugation.3,4 Theoretically,
for an extensive and optimal use of the radioisotope, the key [18F]-
labelling step should occur at a late stage of the synthesis. Despite
this general rule, most of the reported prosthetic groups involve
aAdvanced Accelerator Applications, 20 Rue Diesel, 01630, Saint-Genis-
Pouilly, France. E-mail: cedric.bouteiller@adacap.com; Fax: +33-4-50-99-
30-71; Tel: +33-4-50-99-30-70; Web: http://www.adacap.com
bEquipe de Chimie Bio-Organique, COBRA-CNRS UMR 6014 & FR 3038,
Rue Lucien Tesnie`re, 76131, Mont-Saint-Aignan, France
cUniversite´ de Rouen, Place Emile Blondel, 76821, Mont-Saint-Aignan,
France. E-mail: http://ircof.crihan.fr
dInstitut Universitaire de France, 103 Boulevard Saint-Michel, 75005, Paris,
France
† Electronic supplementary information (ESI) available: Spectroscopic
data of key bis-propanesultone 3 and bis- or monosultone ring-opening
products. See DOI: 10.1039/c1ob06600e
1068 | Org. Biomol. Chem., 2012, 10, 1068–1078
This journal is
The Royal Society of Chemistry 2012
©

View Article Online
one or more steps after the [18F]-labelling for their subsequent
covalent attachment to the targeted peptide/protein. For instance,
this is particularly the case with the very popular amine-reactive
[18F]-labelling agent N-succinimidyl-4-[18F]-fluorobenzoate ([18F]-
SFB).7 Indeed, after the [18F]-fluorination step, two steps are
required to get the activated ester which is able to react with the
selected peptide. Thus, current efforts of radiochemists are now
devoted to the dramatic reduction in the number of steps between
the [18F]-labelling step and the final isolation of the radiolabelled
probe. One-step [18F]-labelling protocol and full-automation of
the process remain two main challenges that would speed up the
development of valuable PET-imaging probes.
Recently, “click chemistry” has emerged as a valuable and
promising tool to reach such challenging goals.8 All too frequently,
the practical implementation of this approach is only focused on
the use of the very popular azide–alkyne Huisgen cycloaddition9
and more recently Diels–Alder type reactions (e.g., tetrazine-trans-
cyclooctene ligation)10 and the Staudinger ligation.11 However,
“click chemistry” refers to a series of reactions that obey certain
criteria, such as being modular, stereospecific, high-yielding and
involving simple experimental procedures. They should also make
use of readily available starting materials, environmentally friendly
conditions (water as (co)solvent, or solvent-free conditions), and
should avoid chromatographic isolations.12 Usually, reactions
included in such a group have a large thermodynamic driving
force and involve the formation of a carbon–heteroatom bond.
Thus, nucleophilic opening reactions of strained rings (e.g.,
aziridines and epoxides) are often considered as belonging to the
“click chemistry” repertoire.13 Such reactions have been already
used for [18F]-labelling of biomolecules through fluoride-induced
ring-opening of cyclic sulfamates and 2,3¢-anhydronucleosides.14
These pioneering works led us to consider the radiolabelling and
bioconjugation ability of sultones (i.e., internal esters of hydroxyl
sulfonic acids), especially those with 5-membered rings (e.g., 1,3-
propanesultone).
In this article, we report the synthesis and reactivity stud-
ies of a bis-sultone cross-linking reagent which can be easily
converted into an original [18F]-prosthetic group meeting most
of the above-cited criteria required for convenient and efficient
radiolabelling of biopolymers.15 Interestingly, the reactivity of
the 1,3-propanesultone moiety toward various nucleophiles (i.e.,
amines, alcohols, oxyamines, halides and thiols) under mild
conditions constitutes the cornerstone of this novel strategy of
labelling. Indeed, the mono-fluorinated labelling reagent can be
directly obtained through the 18F anion-mediated nucleophilic
ring-opening of the first sultone moiety of this homobifunctional
cross-linker, and does not require further activation before its
final coupling with the targeted amine-containing bio-compound
(amino acids and related peptides) through nucleophilic ring-
opening of the remaining sultone (Fig. 1).
Results and discussion
Synthesis and bioconjugation ability of model [18F]-reactive
1,3-propanesultones
Before starting the synthesis of the unusual bis-sultone cross-
linker 3 theoretically suitable for radiolabelling of biologically
active peptides and proteins by way of two sequential nucleophilic
ring-opening reactions, we decided to first evaluate the reactivity
of cognate compounds bearing a single 1,3-propanesultone moi-
ety towards various nucleophiles (especially those found within
biopolymers) and under mild conditions fully compatible with
the stability of most (bio)molecules and biopolymers. Thus, the
synthesis of 1-phenylacyl-1,3-propanesultone 1 was first consid-
ered (Scheme 1). This compound was readily obtained through
Fig. 1 Claimed reactivity of bis-sultone cross-linker 3 for bioconjugation applications.
The Royal Society of Chemistry 2012 Org. Biomol. Chem., 2012, 10, 1068–1078 | 1069
This journal is
©

View Article Online
followed by KBr, KI and finally KF (Table 1, entries 1–4). These
results are in good agreement with those from previous studies
related to reactions of g and d sultones with nucleophiles.16a
Since the ability of tertiary amines to readily react with either
1,3-propanesultone or 1,4-butanesultone has been also reported
by Ward et al.,16b it is important to check the lack of reactivity
of 1 toward Kryptofix[K222] alone. Indeed, this latter cryptand
possesses two tertiary amine moieties which may compete with
the halide anions and thus lead to a wrong interpretation of the
reactivity scale. Fortunately, we observed the formation of the
non-desired quaternary alkylammonium sulfobetaine only after
a prolonged reaction time (ca. 40% after 20 h), and the rate
of its formation has dramatically dropped down to less than
5% in the presence of KI, KBr or KCl, and to 10% for the
competitive reaction with KF. To suppress this side-reaction, the
nucleophilic ring-opening of the sultone 1 with sodium halides
without using cryptand was also investigated (Table 1, entries 5–
8). Interestingly, the fastest ring-opening reaction of the sultone
moiety was observed with NaI (entry 8). This latter reaction
was found to be instantaneous and quantitative, and a gradual
decrease in reactivity was observed from NaI to NaCl, with NaF
no reaction was observed. To complete our comparative reactivity
study in the halide series, we finally try to improve the fluorination
rate of 1 by using alternative fluoride ion sources such as
Scheme 1 Reagents and conditions: (a) n-BuLi, THF -78 ◦C, 1 h; (b)
methyl benzoate, THF, -78 ◦C, 150 min then acetic acid, THF, -78 ◦C to
rt, (60%); (c) benzyl bromide, THF, -78 ◦C, 150 min then acetic acid, THF,
-78 ◦C to rt, (15%).
metalation of 1,3-propanesultone and its subsequent acylation
with methyl benzoate. Thereafter, this compound was subjected
to reaction with various nucleophiles: several potassium halides in
the presence of Kryptofix[K222] at room temperature (Table 1).
Among inorganic halides, KCl was found to react the fastest,
Table 1 Reactivity of model [18F]-reactive 1,3-propanesultones 1 and 2 toward various nucleophilesa
Conversion rate
(HPLC) for 1 (%)
Conversion rate
(HPLC) for 2 (%)
Entry
Nucleophile
1b
2b
3b
4b
5c
6c
7c
8d
9d
KF
5
0
27.5
17
14.5
—
—
—
—
5.5
KCl
KBr
KI
NaF
NaCl
NaBr
NaI
65.5
50.5
22.5
0
1.5
25
100
18
10d
11d
12d
0
—
—
—
0
76
13d
6
—
a All reactions were conducted at rt with 1 equiv. of sultone and nucleophile; HPLC analysis was performed after 45 min of stirring except for entries 12
and 13 (6 h). b Ring-opening reaction in the presence of 1 equiv. of Kryptofix[K222] in CH3CN. c Ring-opening reaction in CH3CN–water (10 : 1, v/v).
d Ring-opening reaction in CH3CN.
1070 | Org. Biomol. Chem., 2012, 10, 1068–1078
This journal is
The Royal Society of Chemistry 2012
©

View Article Online
tetra-n-butylammonium fluoride (TBAF) and CsF. Reac-
tions involving these two species were found to be slightly
slower than the reaction using the standard “naked” fluoride
(KF/Kryptofix[K222]) but no by-products derived from cryptand
were observed. Finally, the optimal fluorination conditions se-
lected for 1 were: 1 equiv. of KF, 1.2 equiv. of Kryptofix[K222]
in a mixture of CH3CN and water (20 : 1, v/v) and gave the
monofluorosulfonic acid in a 38% isolated yield. Iodination of the
sultone 1 using NaI in dry CH3CN afforded the desired coupling
product in a quantitative yield and was recovered by simple
filtration. Bromination and chlorination of 1 were best performed
using the potassium salt (KBr or KCl) and Kryptofix[K222]
in dry CH3CN (36% and 77% isolated yield after 6 h and 2
h 30 min reaction time respectively). The moderate yield for
the bromo derivative is explained by the fact that a part of
product was not recovered because it was not adequately resolved
from Kryptofix[K222] through the RP-HPLC purification. The
structure of the resulting halogeno-sulfonic acids were confirmed
by detailed measurements including ESI mass spectrometry and
NMR analyses, and their high purity was confirmed by HPLC
analyses (see ESI†).
To get further insights on the reactivity of sultone 1, especially
toward representative nucleophiles found in biopolymers (i.e.,
amino, hydroxyl and sulfhydryl groups), nucleophilic ring-opening
reactions were also conducted with propylamine, propanol,
propanethiol (Table 1, entries 9–11) and various L-amino acids
(i.e., cysteine ethyl ester (H-Cys-OEt), Boc-Lys-NH2, tyrosine
ethyl ester (H-Tyr-OEt) and valine methyl ester (H-Val-OMe),
see Table 2). Such reaction was found to occur rapidly with
propylamine (22% isolated yield) whereas no coupling products
were observed with propanol and propanethiol under the mild
experimental conditions we used (i.e., rt and without any base).
This point is of great interest, since it opens an avenue for
selective labelling of peptides through their amine function at
the N-terminal end or a lysine residue. This valuable chemical
behavior was also confirmed by reactions involving amino acids.
Indeed, free a- or e-amino acids such as H-Val-OMe and Boc-
Lys-NH2 were readily reacted with 1 (Table 2, entries 2 and 4).
The superior nucleophilicity of the e-NH2 group was illustrated
by the fact that the reaction with Boc-Lys-NH2 was not univocal
with the significant formation of bis-alkylated lysine derivative
resulting from the competitive reaction with a second equiv.
of sultone 1. For the ambident nucleophile H-Cys-OEt, two
products were isolated, identified as the N- and the S-alkylated
in 12% and 28% isolated yield, respectively (Table 2, entry 1).
The superior reactivity of –SH group was explained by the use of
K2CO3, an additive essential to solubilise and release the amino
group of commercially available amino acid chlorhydrate, which
generates the unprotonated thiolate. Finally, in agreement with
the theory of nucleophilicity, H-Tyr-OEt was found to exclusively
react with 1 via its a-amino group (Table 2, entry 3). Since the
recent use of “super-nucleophiles” (i.e., a-nucleophiles such as
oxyamine, hydrazines and related compounds) constitutes a major
breakthrough in the field of bioconjugation chemistry, especially
to have access to sophisticated biomolecular assemblies through
chemoselective oxime/hydrazone ligations,17 it seemed particu-
larly relevant for us to compare the reactivity oxyamine/amine
toward sultone 1. Thus, two independent reactions with 1 equiv.
of decyloxyamine and decylamine, respectively, in dry CH3CN
at rt have clearly shown that the primary amine reacted faster
than the oxyamine: a conversion rate of 76% (against only 6% for
oxyamine) was obtained after 6 h (Table 1, entries 12 and 13). A
more satisfying conversion rate was obtained with decyloxyamine
only after adding 1 equiv. of K2CO3 (22% after 2 h).
Additionally, the influence of the adjacent carbonyl on the nucle-
ophilic ring-opening efficacy was examined. In this aim, 1-benzyl-
1,3-propanesultone 2 was synthesised from benzyl bromide and
1,3-propanesultone according to a literature protocol18 (Scheme
1). Subsequent reactions with potassium halides and propylamine
were performed under the same conditions than described for
1 (Table 1, entries 1–4 and 9) and conversion rates are always
significantly lower than those observed with the keto derivative.
However, the halogeno-sulfonic acids were isolated in moderate
to good yields (39% for iodo, 45% for bromo and 83% for chloro
derivative) after a prolonged reaction time (> 24 h).
As a result of this comprehensive reactivity study, the sultone
1 bearing a carbonyl group on the a-position of the sultone
ring seemed to afford the best compromise between stability and
reactivity, with a pronounced “hard” electrophilic character for its
reactive centre. Furthermore, experimental conditions, reliability
Table 2 Reactivity of model [18F]-reactive 1,3-propanesultone 1 toward nucleophilic amino acidsa
Isolated yield
(N-alkylation) (%)
Isolated yield
(X-alkylation) (%)
Entry
Amino acid
1
2
3
4
H-Cys-OEt
Boc-Lys-NH2
H-Tyr-OEt
H-Val-OMe
12%
22%
50%
95%
28%
—
b
0
—
b
a All reactions were conducted at rt with 1 equiv. of sultone and amino acid, isolated yields after RP-HPLC purification and subsequent lyophilisation.
b Non-ambident nucleophile.
This journal is
The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 1068–1078 | 1071
©

View Article Online
Scheme 2 Reagents and conditions: (a) n-BuLi, THF -78 ◦C, 1 h; (b) methyl terephthalate, THF, -78 ◦C, 150 min then acetic acid, THF, -78 ◦C
to rt, (40%); (c) KF, Kryptofix[K222], CH3CN (containing 3% water), rt, 20 h, 50% (after RP-HPLC purification and subsequent lyophilisation); (d)
Boc-Lys-NH2, DMF, rt, 5 h, 7.5% (after RP-HPLC purifications, desalting over Dowex H+ resin and subsequent lyophilisation); (e) dodecapeptide
(TFA salt), DIEA, DMF, rt, 8 h, 5% (after RP-HPLC purification and subsequent lyophilisation). Some modifications have been made to these “cold”
conditions for the [18F]-radiosynthetic steps: time 20–30 min, temperature 80–100 ◦C and dry solvents (CH3CN and DMF) were used.
and efficiency of the majority of investigated reactions support
the fact that nucleophilic opening of sultones is a “click” reaction
with a great potential in the field of bioconjugate chemistry.
conjugation to biomolecule/biopolymer under mild conditions
(heating is avoided). Treatment of 3 with KF/Kryptofix[K222]
in CH3CN (containing 3% of water) at rt for 20 h gave the
monofluoropropanesultone 4 which was isolated in pure form by
RP-HPLC (50% isolated yield). Surprisingly, we have observed
that this reaction is favoured by the presence of a small amount
of water (30% isolated yield for the reaction conducted in dry
CH3CN). Instead of a one-pot, two-step reaction, which would
certainly lead to a better yield, we have deliberately chosen to
purify the intermediate 4 before its subsequent final coupling
reaction with the targeted amine-containing compound. That
allowed a reduction of the amount of excess peptide, which
otherwise would have reacted with starting bis-sultone 3 or its
mono-hydrolysed derivative to give a complex mixture difficult to
rapidly resolve. Thus, the fluorinated propanesultone 4 recovered
after lyophilisation was directly subjected to a second ring-opening
reaction upon treatment with Boc-Lys-NH2 or a biological active
dodecapeptide (whose sequence is confidential and not disclosed
within this article) bearing a primary amino group (i.e., e-NH2
from a lysine residue).19 These reactions were performed at rt in dry
DMF (and in the presence of DIEA as a base for dodecapeptide).
Purification was achieved by RP-HPLC to give the corresponding
fluoro-conjugates 5 and 6 in modest isolated yields (7.5% and 5%
respectively). Analysis of 5 and 6 using ESI mass spectrometry
indicated the presence of fluorine and confirmed the integrity
Synthesis and chemical behavior of [18F]-reactive bis-sultone
cross-linker
First, bis-propanesultone 3, the precursor of the targeted pros-
thetic group, was synthesised from dimethyl terephthalate and
1,3-propanesultone according to a similar protocol to the one
designed for 1 (Scheme 2). All spectroscopic data, in particular
NMR and mass spectrometry, are in agreement with the structure
assigned (see ESI†). The stability of this homobifunctional
cross-linker was assessed in different solvents currently used
in radiolabelling protocols (i.e., CH3CN, deionised water and
0.1% aq. TFA). Compound 3 was found to be fully stable in
CH3CN even after several days of incubation. Conversely, storage
in water led to a gradual degradation of 3 through the slow
hydrolysis of its sultone moieties (Fig. 2). Such a phenomenon
was more pronounced in acidic water. However, the moderate
stability of 3 in neutral aq. buffers is not detrimental for its
successful use in bioconjugation reactions involving nucleophiles
more reactive than water. In the context of “cold” fluorine-
labelling, the competitive water-mediated ring-opening reaction
was prevented by performing both fluorination and subsequent
1072 | Org. Biomol. Chem., 2012, 10, 1068–1078
This journal is
The Royal Society of Chemistry 2012
©

View Article Online
Fig. 2 RP-HPLC elution profiles (system A)a of bis-sultone 3 after incubation in deionised water: after 140 min (top, hydrolysis rate = 10%), 270 min
(middle, hydrolysis rate = 50%) and 465 min (bottom, hydrolysis rate = 70%). a Doublet peaks were observed because compounds (3 and its mono-hydrolysed
derivative) are mixtures of two racemic diastereomers.
of the labelled biomolecule/biopolymer (see Fig. 3 for ESI mass
spectrum of 6). Although the use of RP-HPLC for purifications
(followed by lyophilisation) and the small-scale chosen for these
nucleophilic ring-opening reactions (2–5 mmol) induced significant
losses of material, these reactions were found to give satisfying
conversion rates around 30–40%. For further application in
radiolabelling, these modest yields are not so detrimental, since
the unreacted mono-sultone 4 can be easily removed taking into
account the high difference in polarity compared with the desired
conjugate bearing the two free sulfonate groups. However, to speed
up the two-step bioconjugation methods involving the use of bis-
sultone cross-linker 3, further studies will be needed to find suitable
conditions to perform the two derivatisation reactions through a
one-pot procedure and so avoiding any intermediate RP-HPLC
purification.20
sultone 3. Thanks to the difference of polarity between these two
molecules, this was easily performed by solid-phase extraction
using an HLB-SPE cartridge instead of the more conventional
and time-consuming RP-HPLC. Finally, the conjugation of the
peptide with the [18F]-fluorinated propanesultone 7 was carried
out in a mixture of dry CH3CN-DMF (10 : 1, v/v) containing
DIEA for 30 min at 80 ◦C. The reaction leading to [18F]-labelled
peptide 9 proceeded with a satisfying conversion (55%, for the
radio-HPLC elution profile of the crude labelling mixture, see Fig.
4). Its identity was unambiguously confirmed by comparison of the
RP-HPLC retention time with the corresponding “cold” reference
previously synthesised. The [18F]-prosthetic group 7 was also
successfully coupled to Boc-Lys-NH2 (conversion rate 58%, data
not shown). Thus, these results clearly show the main advantage
of the present labelling strategy compared with many published
procedures involving the use of a prosthetic group, namely that
the radiochemically feasible reagent is easily reachable through
a one-step radiosynthesis which is actually the radiolabelling
step of the targeted peptide. Furthermore, the high reactivity
of the propanesultone moiety toward other halogens (especially
iodine)21 should enable rapidly expansion of the radioisotope–
substrate scope of this radiosynthetic procedure, especially for
multi-labelling purposes of selected bioactive peptides (e.g., 76Br,
77Br, 123I and 124I for PET/SPECT imaging, . . . ).
Radiolabelling
[18F]-fluorinated propanesultone 7 was prepared from the bis-
sultone precursor 3 under the conditions described for the prepa-
ration of the “cold” standard (Scheme 2). The fully-automated
synthesis and purification of 7 was performed using a TRACERlab
FX device. An additional step of concentration of the resulting
[18F]-tagged product in CH3CN was added to remove trace
amounts of water which could lead to the premature hydrolysis
of the second sultone moiety. Typically, 0.5–1.8 GBq of purified
[18F]-sultone 7 was obtained within 40 min, starting from 3–6
GBq of [18F]-fluoride (25% average decay-corrected radiochemical
yield for n = 25 and > 95% radiochemical purity, Fig. 4). It is
essential to purify 7 before submitting it to the second ring-opening
reaction, particularly to remove the large excess of starting bis-
Conclusions and future work
In this article, we have described for the first time the synthesis
and a challenging bioconjugate application of an unusual homob-
ifunctional cross-linking reagent whose the two propanesultone
moieties are able to readily and sequentially react with two distinct
This journal is
The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 1068–1078 | 1073
©

View Article Online
Fig. 3 ESI mass spectrum of the [19F]-labelled dodecapeptide 6. Both the doubly charged ions [M + 2H]2+ and [M - H2O + 2H]2+ (calcd mass: 1865.89
and 1847.88) were observed under our ionisation conditions. The second molecular ion was assigned to the cyclic enamine derivative resulting from an
intramolecular nucleophilic addition (of the secondary amine to the ketone moiety) and a subsequent dehydration reaction which were occurred into the
ESI probe.
nucleophiles under mild conditions. Such valuable reactivity has
been built on the development of an original [18F]-propanesultone-
base amine-selective reagent suitable for the prosthetic labelling
with fluorine-18 of amino acids and peptides. Furthermore,
we have demonstrated that this archetypal example of “click”
nucleophilic ring-opening reaction is highly versatile but that its
selectivity toward amines and oxyamines (compared to alcohols,
phenols and thiols) can be easily tuned by using mild reaction
conditions. Thus, this method could be potentially used to label
a wide scope of peptides without requiring a previous chemical
modification of the biopolymer with a specific and complementary
functional group to the targeted prosthetic group. Interestingly,
the two-step sequential derivatisation of bis-sultone cross-linker 3
leads to the post-synthetic introduction of two sulfonate groups
within the (bio)molecular architecture which may be beneficial for
the solubility of the resulting bioconjugate in water and related
aq. buffers. Thus, further applications of this reagent devoted
to the water-solubilisation of highly hydrophobic nanotools such
as fluorophores22 and organic supramolecular compounds (e.g.,
fullerene and related compounds) are expected.
Experimental section
General
Unless otherwise noted, all other commercially available reagents
and solvents were used without further purification. CH3CN
was dried through distillation over CaH2. THF was dried
through distillation over Na/benzophenone. Kryptofix[K222]
(4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane)
and anhydrous DMF (99.8%) were purchased from Sigma–
Aldrich. TLC was carried out on Merck DC Kieselgel 60
1074 | Org. Biomol. Chem., 2012, 10, 1068–1078
This journal is
The Royal Society of Chemistry 2012
©

View Article Online
Wissembourg, France). Chemical shifts are reported in parts per
million (ppm) downfield from residual solvent peaks (acetone-
d6 (dH = 2.05, dC = 29.84 and 206.26), CDCl3 (dH = 7.26, dC
= 77.16), DMSO-d6 (dH = 2.50, dC = 39.52) or CD3OD (dH
=
3.31, dC = 49.0)25 and coupling constants are reported as Hertz
(Hz). Splitting patterns are designated as singlet (s), doublet (d),
double doublet (dd), double double doublet (ddd) and triplet (t).
Splitting patterns that could not be interpreted or easily visualised
are designated as multiplet (m). 13C substitutions were determined
with JMOD experiments, differentiating signals of methyl and me-
thine carbons pointing “up” (+) from methylene and quaternary
carbons pointing “down” (-). Melting points were determined on
a Kofler bench device (Wagner and Munz). Infrared (IR) spectra
were recorded with a universal ATR sampling accessory on a
Perkin Elmer FT-IR Spectrum 100 spectrometer with frequencies
given in reciprocal centimeters (cm-1). The elemental analyses
were carried out with a Flash 2000 Organic Elemental Analyzer
(Thermo Scientific). Analytical HPLC was performed on a
Thermo Scientific Surveyor Plus instrument equipped with a PDA
detector. Semi-preparative HPLC was performed on a Thermo
Scientific SPECTRASYSTEM liquid chromatography system
(P4000) equipped with a UV-visible 2000 detector. Mass spectra
were obtained with a Finnigan LCQ Advantage MAX (ion trap)
apparatus equipped with an electrospray (ESI) source. Fluoride-
18 was produced by the 18O[p,n]18F nuclear reaction using a
GE Medical Systems PETtrace cyclotron [18 MeV proton beam]
(Advanced Accelerator Applications, Saint-Genis-Pouilly, France)
and 18O-enriched water purchased from Marshall Isotopes Ltd.
(98%, Tel Aviv, Israel). Solid-phase extraction (SPE) cartridges
(SepPak QMA Light, Oasis HLB and CM) were obtained from
ABX advanced biochemical compounds (Radeburg, Germany) and
Waters (Guyancourt, France). The HLB cartridges were always
pre-conditioned with ethanol (5 mL), water (5 mL) and dried
with air. Radiosyntheses were performed on a TRACERlab FX
(GE Medical Systems, Buc, France) automated synthesis unit in a
shielded hot cell (8 cm lead, Comecer, Castel Bolognese, Italy). A
flow-count radio-HPLC detector system from Bioscan was used
Fig.
4
Radio-HPLC elution profiles (system A) of purified
[18F]-fluorinated propanesultone 7 (top) and crude of [18F]-labelled do-
decapeptide 9.
F-254 aluminium sheets. The spots were visualised by illu-
mination with a UV lamp (l = 254 nm) and/or staining
with KMnO4 solution. Flash column chromatography purifi-
cations were performed on Geduranꢀ Si 60 silica gel (40–63
R
mm) from Merck. Na-(tert-Butyloxycarbonyl)-L-lysine carboxam-
ide (Boc-Lys-NH2) was prepared from commercially available
Na -(tert-butyloxycarbonyl)-Ne -(benzyloxycarbonyl)-L -lysine
(Boc-Lys(Z)-OH, Bachem or Fluka), by using a conventional
two-step synthetic procedure: aminolysis of mixed anhydride
formed in situ by short treatment with isobutyl chloroformate
and N-methylmorpholine (NMM), followed by hydrogenolysis to
remove the Z protecting group.23 The synthesis of dodecapeptides
(Na-Ac-lysine- or aminooxyacetic acid-terminated) was carried
out on an Applied Biosystems 433A peptide synthesizer using
standard Fmoc/tBu chemistry24 and Wang resin (Iris Biotech,
loading 0.9 mmol g-1) on a scale of 0.25 mmol. The HPLC-
gradient grade acetonitrile (CH3CN) and methanol (CH3OH)
were obtained from VWR. Aq. mobile phases for HPLC were
prepared using water purified with a Milli-Q system (purified
to 18.2 MX cm). Triethylammonium acetate (TEAA, 2.0 M)
and triethylammonium bicarbonate (TEAB, 1.0 M) buffers were
prepared from distilled triethylamine and glacial acetic acid or CO2
gas. Ion-exchange chromatography (for desalting disulfonated
R
only for HPLC analyses (performed on a Dionex UltiMateꢀ 3000
LC system) of reactions involving 18F.
HPLC separations
Several chromatographic systems were used for the analytical
experiments and the purification steps: System A: RP-HPLC
(Thermo Hypersil GOLD C18 column, 5 mm, 4.6 ¥ 100 mm) with
CH3CN and 0.1% aq. trifluoroacetic acid (aq. TFA, 0.1%, v/v,
pH 2.0) as eluents [100% TFA (5 min), linear gradient from 0% to
80% (40 min) of CH3CN] at a flow rate of 1.0 mL min-1. Dual UV
detection was achieved at 254 and 265 nm. System B: RP-HPLC
(Thermo Hypersil GOLD C18 column, 5 mm, 21.2 ¥ 250 mm)
with CH3CN and 0.1% aq. TFA as eluents [100% TFA (10 min),
linear gradient from 0% to 5% (2 min) and 5% to 70% (65 min)
of CH3CN] at a flow rate of 15.0 mL min-1. Dual UV detection
was achieved at 254 and 265 nm. System C: RP-HPLC (Thermo
Hypersil GOLD C18 column, 5 mm, 4.6 ¥ 100 mm) with CH3CN
and aq. triethylammonium acetate buffer (TEAA, 25 mM, pH
7.0) as eluents [100% TEAA (10 min), linear gradient from 0% to
80% (40 min) of CH3CN] at a flow rate of 1.0 mL min-1. UV-vis
detection with the “Max Plot” (i.e., chromatogram at absorbance
R
derivatives 5) was performed with an Econo-Pacꢀ Disposable
chromatography column (Bio-Rad, #732-1010) filled with an
aq. solution of Dowexꢀ 50WX8-400 (Alfa Aesar, ca. 5 g, 15
¥ 35 mm bed), regenerated using aq. 10% HCl solution and
equilibrated with deionised water. NMR spectra (1H, 13C and
19F) were recorded on a Bruker DPX 300 spectrometer (Bruker,
R
This journal is
The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 1068–1078 | 1075
©

View Article Online
maximum for each compound) mode (220-450 nm). System D: RP-
HPLC (Thermo Hypersil GOLD C18 column, 5 mm, 10 ¥ 250 mm)
with CH3CN and aq. triethylammonium bicarbonate (TEAB, 50
mM, pH 7.5) as eluents [100% TEAB (10 min), linear gradient
from 0% to 5% (2 min), 5% to 18% (13 min), 18% to 25% (14 min),
25% to 31% (6 min) and 31% to 61% (15 min) of CH3CN] at a
flow rate of 4.0 mL min-1. Dual UV detection was achieved at 220
and 254 nm. System E: System D with Thermo Hypersil GOLD
C18 column (5 mm, 10 ¥ 100 mm). System F: RP-HPLC (Thermo
Hypersil GOLD C18 column, 5 mm, 10 ¥ 100 mm) with CH3CN
and 0.1% aq. TFA as eluents [100% TFA (10 min), linear gradient
from 0% to 5% (2 min) and 5% to 70% (65 min) of CH3CN] at a
flow rate of 4.0 mL min-1. Dual UV detection was achieved at 220
and 254 nm.
Bis-propanesultone (3)
To a stirred solution of 1,3-propanesultone (447 mg, 3.7 mmol,
2 eq◦uiv.) in dry THF (5 mL), under an argon atmosphere, at
-78 C, was added dropwise n-BuLi (1.3 M in hexane, 2.82 mL,
3.67 mmol, 2 equiv.). After 1 h at -78 ◦C, a solution of dimethyl
terephthalate (358 mg, 1.84 mmol, 1 equiv.) in dry THF (10 mL)
was added dropwise to the previous vigorously stirred mixture.
The resulting reaction mixture was stirred at -78 ◦C for 2 h
30 min, then kept at -78 ◦C and quenched by adding 1 mL
of glacial acetic acid dissolved in dry THF (3 mL). Thereafter,
the mixture was slowly warmed up to rt then diluted with
brine (20 mL) and EtOAc (50 mL). The formation of a solid
residue was observed at the interface of the organic and aq.
phases and was removed by filtration. The aq. phase was washed
with EtOAc (30 mL). The combined organic phases were dried
over anhydrous MgSO4, filtered and then concentrated under
reduced pressure. The resulting crude product was then purified
by flash-chromatography on a silica gel column using a mixture of
cyclohexane–acetone (3 : 2, v/v) as the mobile phase. The desired
bis-propanesultone 3 was isolated as a beige solid (270 mg, yield
1-Phenylacyl-1,3-propanesultone (1)26
To a stirred solution of 1,3-propanesultone (502 mg, 4.1 mmol,
1 eq◦uiv.) in dry THF (7 mL), under an argon atmosphere, at
-78 C, was added dropwise n-BuLi (1.3 M in hexane, 3.15 mL,
4.1 mmol, 1 equiv.). After stirring at -78 ◦C for 1 h, a solution
of methyl benzoate (0.51 mL, 4.1 mmol, 1 equiv.) in dry THF
(5 mL) was added dropwise to the vigorously stirred mixture. The
resulting reaction mixture was stirred at -78 ◦C for 2 h 30 min, then
kept at -78 ◦C, and quenched by adding 1 mL of glacial acetic acid
dissolved in dry THF (3 mL). Thereafter, the mixture was slowly
warmed up to rt. The organic phase was diluted with CH2Cl2
(35 mL) and washed with deionised water (10 mL) and brine
(10 mL). The organic phase was dried over anhydrous MgSO4
and concentrated under reduced pressure. The resulting residue
was then purified by flash-chromatography on a silica gel column,
using a mixture of cyclohexane–EtOAc (7 : 3, v/v) as the mobile
phase, to give the desired product as a white powder (560 mg,
yield 60%). Rf 0.46 (cyclohexane–EtOAc, 3 : 2, v/v); mp 67 2 ◦C;
40%). Rf 0.42 (cyclohexane–acetone, 3 : 2, v/v); mp 210
2
◦C
(dec.); nmax/cm-1 3350, 3013, 2978, 1682, 1508, 1442, 1407, 1343,
1300, 1227, 1191, 1137, 1091, 974, 948, 888, 861, 820, 777; dH
(300 MHz, acetone-d6) 8.20 (s, 4H), 5.63–5.57 (m, 2H), 4.59–4.49
(m, 4H), 3.17–3.08 (m, 2H), 2.80–2.71 (m, 2H); dC (75.5 MHz,
acetone-d6) 189.0, 140.4, 130.4 (2C), 69.6, 61.3, 27.7; MS (ESI-):
m/z 373.27 [M - H]-, calcd for C14H14O8S2 374.01; HPLC (system
A): tR = 26.7 min (purity >96%); lmax (recorded during the HPLC
analysis)/nm 265; elemental analysis (%) calcd: C, 44.91; H, 3.77;
S, 17.13. Found: C, 43.10; H, 3.86; S, 17.50.
Monofluoropropanesultone (4)
200 mL of an aq. solution of KF (62.2 mg mL-1) (12.44 mg,
0.21 mmol, 1 equiv.), Kryptofix[K222] (95 mg, 0.25 mmol,
1.2 equiv.) and 1 mL of CH3CN were mixed in a 25 mL single-neck
round-bottom flask. A solution of bis-propanesultone 3 (80 mg,
0.21 mmol, 1 equiv.) in dry CH3CN (5.5 mL) was then added. The
resulting reaction mixture was stirred at rt for 20 h and checked
for completion by RP-HPLC (system A). Finally, the reaction
mixture was quenched by dilution with aq. TFA and CH3CN
and purified by RP-HPLC (system B, 1 injection, tR = 31.2–35.0
min). The product-containing fractions were lyophilised to give the
monofluoropropanesultone 4 as a beige solid (27.4 mg, yield 32%,
mixture of two racemic diastereomers). dH (300 MHz, CD3OD)
8.16–8.09 (m, 4H), 5.53 (ddd, J 2.5, 6.6, 8.7, 1H), 5.04 (ddd, J 1.0,
4.0, 9.4, 1H), 4.61–4.46 (m, 2H), 4.45–4.37 (m, 1H), 4.29–4.21 (m,
1H), 3.15–3.04 (m, 1H), 2.71–2.58 (m, 1H), 2.55–2.33 (m, 2H). dF
(282.5 MHz, CD3OD) -217.6 (m, –CH2F); MS (ESI-): m/z 393.13
n
max/cm-1 3063, 2963, 1690, 1449, 1342, 1291, 1235, 1181, 1149,
998, 974, 874, 836, 783; dH (300 MHz, CDCl3) 8.02 (d, J 7.2, 2H),
7.58 (t, J 7.3, 1H), 7.48 (t, J 7.9, 2H), 5.08–5.03 (m, 1H), 4.62–4.55
(m, 1H), 4.52–4.44 (m, 1H), 3.27–3.16 (m, 1H), 2.72–2.60 (m, 1H);
dC (75.5 MHz, CDCl3) 187.5, 135.1, 134.8, 129.2, 129.1, 68.2, 59.7,
26.9; MS (ESI-): m/z 225.02 [M - H]-, calcd for C10H10O4S 226.03.
HPLC (system A): tR = 24.2 min (purity 99%); lmax (recorded
during the HPLC analysis)/nm 254; elemental analysis (%) calcd:
C, 53.09; H, 4.45; S, 14.17. Found: C, 53.18; H, 4.18; S, 14.11.
1-Benzyl-1,3-propanesultone (2)15
Monosultone 2 was prepared from benzyl bromide and 1,3-
propanesultone, according to a literature protocol.18 The product
was isolated as a colourless oil (yield 15%). Rf 0.41 (cyclohexane–
EtOAc, 7 : 3, v/v); dH (300 MHz, CDCl3) 7.39–7.24 (m, 5H), 4.49–
4.31 (m, 2H), 3.59–3.48 (m, 1H), 3.42 (dd, J 5.5, 14.0, 1H), 2.89
(dd, J 9.8, 14.0, 1H), 2.56–2.31 (m, 2H); dC (75.5 MHz, CDCl3)
136.1, 129.2, 128.9, 127.6, 67.0, 56.7, 34.6, 29.2; MS (ESI-): m/z
229.27 [M + H2O - H]- (hydration of sultone moiety occurred
during the ionisation process), calcd for C10H12O3S 212.27; HPLC
(system A): tR = 23.6 min (purity 99%); lmax (recorded during the
HPLC analysis)/nm 254.
[M - H]-, calcd for C14FH15O8S2 394.02; HPLC (system A): tR
18.8 and 19.1 min (two racemic diastereomers, purity 92%); lmax
=
(recorded during the HPLC analysis)/nm 265.
Monofluoro-Boc-L-lysine-NH2 conjugate (5)
Monofluoropropanesultone 4 (31.5 mg, 80 mmol, 1 equiv.) was
dissolved in dry DMF (1.8 mL) and Boc-Lys-NH2 (21.5 mg,
80 mmol, 1 equiv.) dissolved in dry DMF (0.8 mL) was added.
The resulting reaction mixture was stirred at rt for 5 h and
1076 | Org. Biomol. Chem., 2012, 10, 1068–1078
This journal is
The Royal Society of Chemistry 2012
©

View Article Online
checked for completion by RP-HPLC (system C). The crude
product was then dissolved in aq. TEAB and twice purified by RP-
HPLC (system D followed by system E). The product-containing
fractions were lyophilised to give the desired lysine conjugate,
which was rapidly desalted by ion-exchange chromatography (to
remove triethylammonium salts) and again lyophilised to give the
acid form of disulfonated monofluoro-Boc-L-lysine derivative 5.
Finally, a third RP-HPLC purification (system F) was performed
to remove the free-amino lysine derivative formed during the ion-
exchange chromatography through the premature cleavage of the
Boc protecting group. Finally, the desired product was obtained
as a white solid (2.8 mg, yield 7%). dH (300 MHz, DMSO-d6)
8.18 (m, 2H), 7.79 (t, J 7.9, 2H), 4.93–4.91 (m, 1H), 4.51–4.42
(m, 1H), 4.24 (t, J 8.6, 2H), 3.77 (t, J 7.4, 2H), 2.67–2.60 (m,
2H), 1.77–1.74 (m, 2H), 1.65–1.55 (m, 2H), 1.37 (s, 9H), 1.31–
1.24 (m, 2H); dF (282.5 MHz, DMSO-d6) -216.7 (m, –CH2F); MS
(ESI-): m/z 620.07 [M - H2O - H]- (cyclic enamine formation
occurred during the ionisation process), 638.07 [M - H]-, calcd
for C25FH38N3O11S2 639.19; HPLC (system C): tR = 15.3 and 15.9
min (four diastereomers, purity 98%); lmax (recorded during the
HPLC analysis)/nm 265.
with a 95% radiochemical purity. HPLC (system A): tR = 18.2 min
(two racemic diastereomers).
[18F]-Labelled dodecapeptide (9). Dodecapeptide and a large
excess of DIEA were put into a reaction vial and dry DMF
(100 mL) was added. Then, 1 mL of the CH3CN solution [18F]-
fluoropropanesultone 7 (vide supra) was added. The vial was put on
a hotplate and heated at 80 ◦C for 30 min. Thereafter, the reaction
was stopped and directly analysed by RP-HPLC (system A with
radioactivity detection). HPLC (system A): tR = 19.8 min (two
racemic diastereomers). The retention time difference between
the UV and radio traces (ca. 1 min) was caused by the serial
arrangement of the detectors.
Acknowledgements
Financial support from La Re´gion Haute-Normandie (for ac-
quiring HPLC instruments and peptide synthesizer), Institut Uni-
versitaire de France (IUF) and a Ph.D. grant from L’Association
Nationale de la Recherche et de la Technologie (ANRT)toThomas
Priem are greatly acknowledged. We thank Annick Leboisselier
(INSA de Rouen) for the determination of elemental analyses and
Ce´drik Massif (Ph.D., Bioorganic Team, Universite´ de Rouen) for
IR measurements.
Monofluoro-peptide conjugate (6)
Monofluoropropanesultone 4 (1.6 mg, 4.06 mmol, 1.6 equiv.) was
dissolved in dry DMF (0.6 mL) and a dodecapeptide (its sequence
is confidential and not disclosed within this article but it contains a
single reactive lysine residue, 5.0 mg, 2.6 mmol, 1 equiv.) previously
dissolved in dry DMF (0.6 mL) and DIEA (10 mL, 57.4 mmol,
15 equiv.) was added. The resulting reaction mixture was stirred
at rt for 8 h and checked for completion by RP-HPLC (system A).
The crude product was then dissolved with aq. TFA and purified
by RP-HPLC (system F). The product-containing fractions were
lyophilised to give the desired monofluoro-peptide conjugate as a
white solid (250 mg, yield 5%). MS (ESI+): m/z 925.40 [M - H2O
+ 2H]2+ (cyclic enamine formation occurred during the ionisation
process), 934.33 [M + 2H]2+, calcd for C79FH128N23O24S2 1865.89;
HPLC (system A): tR = 20.8 min (four diastereomers, purity 94%);
lmax (recorded during the HPLC analysis)/nm 266.
Notes and references
1 A. P. Schubiger, L. Lehmann and M. Friebe, PET Chemistry: The
Driving Force in Molecular Imaging, Springer, Berlin Heidelberg,
2007.
2 (a) S. M. Ametamey, M. Honer and P. A. Schubiger, Chem. Rev., 2008,
108, 1501; (b) S. L. Pimlott and A. Sutherland, Chem. Soc. Rev., 2011,
40, 149.
3 S. M. Okarvi, Eur. J. Nucl. Med., 2001, 28, 929.
4 H. J. Wester and M. Schottelius, in PET Chemistry: The Driving Force
in Molecular Imaging, ed. A. P. Schubiger, L. Lehmann and M. Friebe,
Springer, Berlin Heidelberg, 2007, vol. 62, p. 79 and references cited
therein.
5 (a) J. Becaud, L. Mu, M. Karramkam, P. A. Schubiger, S. M. Ametamey,
K. Graham, T. Stellfeld, L. Lehmann, S. Borkowski, D. Berndorff,
L. Dinkelborg, A. Srinivasan, R. Smits and B. Koksch, Bioconjugate
Chem., 2009, 20, 2254; (b) A. Hohne, L. Mu, M. Honer, P. A. Schubiger,
S. M. Ametamey, K. Graham, T. Stellfeld, S. Borkowski, D. Berndorff,
U. Klar, U. Voigtmann, J. E. Cyr, M. Friebe, L. Dinkelborg and A.
Srinivasan, Bioconjugate Chem., 2008, 19, 1871; (c) O. Jacobson, L.
Zhu, Y. Ma, I. D. Weiss, X. Sun, G. Niu, D. O. Kiesewetter and X.
Chen, Bioconjugate Chem., 2011, 22, 422; (d) L. Mu, A. Hohne, P. A.
Schubiger, S. M. Ametamey, K. Graham, J. E. Cyr, L. Dinkelborg, T.
Stellfeld, A. Srinivasan, U. Voigtmann and U. Klar, Angew. Chem., Int.
Ed., 2008, 47, 4922; (e) U. Roehn, J. Becaud, L. Mu, A. Srinivasan, T.
Stellfeld, A. Fitzner, K. Graham, L. Dinkelborg, A. P. Schubiger and
S. M. Ametamey, J. Fluorine Chem., 2009, 130, 902.
6 For selected examples, see: (a) B. De Bruin, B. Kuhnast, F. Hinnen,
L. Yaouancq, M. Amessou, L. Johannes, A. Samson, R. Boisgard, B.
Tavitian and F. Dolle, Bioconjugate Chem., 2005, 16, 406; (b) F. Wuest,
M. Berndt, R. Bergmann, J. van den Hoff and J. Pietzsch, Bioconjugate
Chem., 2008, 19, 1202; (c) I. Koslowsky, J. Mercer and F. Wuest, Org.
Biomol. Chem., 2010, 8, 4730.
7 P. Johnstro¨m, J. C. Clark, J. D. Pickard and A. P. Davenport, Nucl.
Med. Biol., 2008, 35, 725.
8 (a) M. Glaser and E. G. Robins, J. Labelled Compd. Radiopharm., 2009,
52, 407; (b) C. Mamat, T. Ramenda and F. R. Wuest, Mini-Rev. Org.
Chem., 2009, 6, 21; (c) T. L. Ross, Curr. Radiopharm., 2010, 3, 202; (d) C.
Waengler, R. Schirrmacher, P. Bartenstein and B. Waengler, Curr. Med.
Chem., 2010, 17, 1092.
9 For selected examples, see: (a) T. Ramenda, T. Kniess, R. Bergmann, J.
Steinbach and F. Wuest, Chem. Commun., 2009, 7521; (b) D. Thonon,
C. Kech, J. Paris, C. Lemaire and A. Luxen, Bioconjugate Chem., 2009,
Radiosyntheses
Mono-[18F]-fluoropropanesultone (7). Following delivery of
[18F]-fluoride to the synthesizer module, the radioactivity was
isolated on a QMA cartridge, allowing recovery of [18O]-H2O. The
[18F]-fluoride was eluted with a mixed solution of Kryptofix[K222]
(19.8 mg) in CH3CN (550 mL) and of K2CO3 (3.25 mg) in deionised
water (50 mL), and transferred to the reaction vial. After azeotropic
evaporation of water (3 ¥ CH3CN, 95 ◦C, with a stream of N2
gas), bis-propanesultone 3 (3.5 mg) in dry CH3CN (1 mL) was
added. The radiolabelling was conducted in the reaction vial at
100 ◦C over 20 min. After dilution in a large volume of deionised
water (40–50 mL), the reaction mixture was then loaded onto
a HLB cartridge. After washing of the reactor vial with a 3%
solution of CH3CN in deionised water (10 mL), the product was
finally eluted from the cartridge using a 17% solution of CH3CN
in deionised water (15 mL). Mono-[18F]-fluoropropanesultone 7
was obtained within 40 min with a moderate 25% decay-corrected
radiochemical yield (average value from n = 25 preparations) and
This journal is
The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 1068–1078 | 1077
©

View Article Online
20, 817; (c) F. Mercier, J. Paris, G. Kaisin, D. Thonon, J. Flagothier,
N. Teller, C. Lemaire and A. Luxen, Bioconjugate Chem., 2011, 22,
108; (d) T. Kuboyama, M. Nakahara, M. Yoshino, Y. Cui, T. Sako,
Y. Wada, T. Imanishi, S. Obika, Y. Watanabe, M. Suzuki and H. Doi,
Bioorg. Med. Chem., 2011, 19, 249.
(d) M. Karskela, M. Helkearo, P. Virta and H. Lo¨nnberg, Bioconjugate
Chem., 2010, 21, 748; (e) M. Rashidian, J. K. Dozier, S. Lenevich and
M. D. Distefano, Chem. Commun., 2010, 46, 8998; (f) A. Dirksen and
P. E. Dawson, Bioconjugate Chem., 2008, 19, 2543; (g) C. Olivier, D.
Hot, L. Huot, N. Ollivier, O. El-Mahdi, C. Gouyette, T. Huynh-Dinh,
H. Gras-Masse, Y. Lemoine and O. Melnyk, Bioconjugate Chem., 2003,
14, 430.
10 Z. Li, H. Cai, M. Hassink, M. L. Blackman, R. C. D. Brown, P. S.
Conti and J. M. Fox, Chem. Commun., 2010, 46, 8043.
11 (a) A. Gaeta, J. Woodcraft, S. Plant, J. Goggi, P. Jones, M. Battle, W.
Trigg, S. K. Luthra and M. Glaser, Bioorg. Med. Chem. Lett., 2010,
20, 4649; (b) M. Pretze, F. Wuest, T. Peppel, M. Koeckerling and C.
Mamat, Tetrahedron Lett., 2010, 51, 6410; (c) L. Carroll, S. Boldon, R.
Bejot, J. E. Moore, J. Declerck and V. Gouverneur, Org. Biomol. Chem.,
2011, 9, 136; (d) C. Mamat, M. Franke, T. Peppel, M. Koeckerling and
J. Steinbach, Tetrahedron, 2011, 67, 4521.
18 M. B. Smith and J. Wolinsky, J. Org. Chem., 1981, 46, 101.
19 Such labelling reaction was also performed with the oxyamine-
terminated dodecapeptide (see ESI† for experimental details).
20 For recent examples of such one-pot multi-step bioconjugation proto-
cols, see: (a) M. Galibert, O. Renaudet, P. Dumy and D. Boturyn,
Angew. Chem., Int. Ed., 2011, 50, 1901; (b) R. Mhidia, A. Vallin,
N. Ollivier, A. Blanpain, G. Shi, R. Christiano, L. Johannes and O.
Melnyk, Bioconjugate Chem., 2010, 21, 219.
21 For the synthesis of the “cold” mono-iodo propanesultone, see ESI†.
22 For the use of poly-sulfonated linkers as efficient water-solubilising
moieties of various fluorescent organic dyes, see: (a) A. Romieu, D.
Brossard, M. Hamon, H. Outaabout, C. Portal and P.-Y. Renard,
Bioconjugate Chem., 2008, 19, 279; (b) A. Romieu, T. Bruckdorfer,
G. Clave´, V. Grandclaude, C. Massif and P.-Y. Renard, Org. Biomol.
Chem., 2011, 9, 5337; (c) A. Romieu, D. Tavernier-Lohr, S. Pellet-
Rostaing, M. Lemaire and P.-Y. Renard, Tetrahedron Lett., 2010, 51,
3304.
12 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., Int. Ed.,
2001, 40, 2004.
13 M. V. Gil, M. J. Arevalo and O. Lopez, Synthesis, 2007, 1589.
14 L. Cai, S. Lu and V. W. Pike, Eur. J. Org. Chem., 2008, 17, 2853.
15 This work is partially based on the preliminary works done by Ce´dric
Bouteiller during his Ph.D. thesis under the supervision of Drs. Louisa
Barre´ and Ce´cile Perrio (Cyceron, team GDM-TEP), and was recently
published as a communication, see: S. Schmitt, C. Bouteiller, L. Barre´
and C. Perrio, Chem. Commun., 2011, 47, 11465.
16 (a) D. W. Roberts and D. L. Williams, Tetrahedron, 1987, 43, 1027;
(b) R. S. Ward, J. Davies, G. Hodges and D. W. Roberts, Synthesis,
2002, 243.
23 G. Clave´, P.-Y. Renard, A. Romieu and H. Volland, WO Pat.,
2009043986, 2009.
17 For selected examples of bioconjugations using oxime and hydrazone
ligations, see: (a) G. Clave´, H. Volland, M. Flaender, D. Gasparutto,
A. Romieu and P.-Y. Renard, Org. Biomol. Chem., 2010, 8, 4329; (b) M.
Galibert, L. Sancey, O. Renaudet, J.-L. Coll, P. Dumy and D. Boturyn,
Org. Biomol. Chem., 2010, 8, 5133; (c) A. Meyer, N. Spinelli, P. Dumy,
J.-J. Vasseur, F. Morvan and E. Defrancq, J. Org. Chem., 2010, 75, 3927;
24 G. B. Fields and R. L. Noble, Int. J. Pept. Protein Res., 1990, 35, 161.
25 G. R. Fulmer, A. J. M. Miller, N. H. Sherden, H. E. Gottlieb,
A. Nudelman, B. M. Stoltz, J. E. Bercaw and K. I. Goldberg,
Organometallics, 2010, 29, 2176.
26 H. R. W. Ansink and H. Cerfontain, Phosphorus, Sulfur Silicon Relat.
Elem., 1995, 101, 295.
1078 | Org. Biomol. Chem., 2012, 10, 1068–1078
This journal is
The Royal Society of Chemistry 2012
©